具有降低的吸附作用和增加的水解作用的卡氏枯草杆菌蛋白酶变异体的制作方法

专利名称：具有降低的吸附作用和增加的水解作用的卡氏枯草杆菌蛋白酶变异体的制作方法
技术领域：
本发明涉及新的用于各种洗涤组合物的酶变异体和编码这种酶变异体的DNA序列。
背景技术：
酶构成了最大种类的天然产生的蛋白质。每一类酶通常催化(加速反应而不被消耗)各种化学反应。称为蛋白酶的一种酶已知能够水解(分解化合物成两个或更多个更简单的化合物，在断裂的化学键的两侧接受水分子的H和OH部分)其它蛋白质。将天然产生的和蛋白质设计的蛋白酶作为添加剂加入洗衣洗涤剂制剂中即利用了水解蛋白质的这种能力。在衣服上的许多污垢是蛋白质的，广泛特效的蛋白酶可显著改善这种污垢的除去。
不幸的是，这些蛋白质在其天然的，细菌的环境中的功效水平通常不能转移至相对非天然的洗涤环境。尤其是蛋白酶特性，例如热稳定性、pH稳定性、氧化稳定性和底物特性对于酶的天然环境之外的应用未必达到最佳。
蛋白酶的氨基酸序列决定了蛋白酶的特征。根据氨基酸序列中改变的位点、性质和/或幅度，改变蛋白酶的氨基酸序列可改变酶的特性至各不相同的程度，甚至可失活酶。人们已做了许多尝试以改变蛋白酶的野生型氨基酸序列，以改善它们的性质，从而增加蛋白酶在洗涤环境中的效率。这些尝试包括改变氨基酸序列以便在相当不同的条件下提高热稳定性和改善氧化稳定性。
尽管在现有技术中描述了各种各样的尝试，但人们仍不断需要用于洗涤各种表面的蛋白酶的新有效变异体。发明目的本发明的目的在于提供与野生型酶相比较具有改善的水解作用的卡氏(Carlsberg)枯草杆菌蛋白酶酶变异体。
本发明的另一个目的在于提供含有这些枯草杆菌蛋白酶变异体的洗涤组合物。
发明概述本发明涉及具有野生型卡氏枯草杆菌蛋白酶氨基酸序列的修饰氨基酸序列的卡氏枯草杆菌蛋白酶变异体，野生型氨基酸序列包括第一环区域、第二环区域、第三环区域、第四环区域和第五环区域；其中，修饰的氨基酸序列在一个或多个环区域中的特定标记的位点上含有与野生型卡氏枯草杆菌蛋白酶不同的氨基酸(即取代)，与野生型卡氏枯草杆菌蛋白酶相比，卡氏枯草杆菌蛋白酶变异体具有对不溶解底物降低的吸附作用和增加的水解作用。本发明还涉及编码这种卡氏枯草杆菌蛋白酶变异体的基因。本发明还涉及含有这种卡氏枯草杆菌蛋白酶变异体的用于洗涤各种表面的组合物。
发明详述I.枯草杆菌蛋白酶变异体本发明涉及枯草杆菌蛋白酶，尤其是卡氏枯草杆菌蛋白酶，它们通过突变编码酶的各种核苷酸顺序，从而改变酶的氨基酸序列而被修饰。本发明的修饰枯草杆菌蛋白酶(下文称为“卡氏枯草杆菌蛋白酶变异体”)与野生型卡氏枯草杆菌蛋白酶相比，具有对不溶解底物降低的吸附作用和增加的水解作用。本发明还涉及编码这种卡氏枯草杆菌蛋白酶变异体的突变基因。
本发明的枯草杆菌蛋白酶属于称为蛋白酶的酶的种类。蛋白酶是断裂肽键的催化剂。一种类型的蛋白酶是丝氨酸蛋白酶。丝氨酸的特征在于在活性位点上存在必需的丝氨酸残基。
随着酶浓度的增加溶解底物的酶水解速率增加的现象已经作了充分报导。因而，对于表面结合的底物，例如在许多洗涤应用中遇到的情况，水解速率随着表面浓度的增加而增加似乎是可能的，这已由事实说明。(Brode，P.F.III和D.S.Rauch，LANGMUIR，“枯草芽孢杆菌BPN’。固定化底物上的活性”，卷8，1325-1329页(1992))。事实上，当酶的表面浓度变化时，对于不溶解底物已经发现了表面浓度与速率的直线关系。(Rubingh，D.N.和M.D.Bauer.“在蛋白质溶液界面上的蛋白酶的催化水解”，多聚物溶液，混合和界面(POLYMER SOLUTIONS，BLENDSAND INTERFACES)，by I Noda and d.N.Rubingh，Elsevier编，第464页(1992))。出乎意料的是，当试图将这种原理用于研究得到较好洗涤性能的变并体蛋白酶时，我们不能发现吸附更多的酶得到较好的性能。事实上，我们惊奇地发现与之相反的事实酶与底物的下降的吸附导致增加的底物水解(即，较好的洗涤性能)。
尽管不希望限制于任何理论，但我们相信，当将变异体相互比较时，改善的性能是如下事实的结果，即吸附较少的酶也不太紧密地结合，因而在欲除去不溶解蛋白质底物的表面更易移动。在可比的酶溶液浓度下，增加的移动能力足以胜过通过向表面提供较高浓度酶而给予的任何优点。
本发明描述的突变用于改变(即降低)酶与表面结合的污垢的吸附。在卡氏枯草杆菌蛋白酶中，某些氨基酸在酶分子上形成外环。为了讨论，这些环可称为第一、第二、第三、第四和第五环区域。具体地说，位点58-65形成第一环区域；位点94-106形成第二环区域；位点125-132形成第三环区域；位点153-166形成第四环区域；位点186-190形成第五环区域；和位点199-219形成第六环区域(位点编号类似于野生型枯草杆菌蛋白酶即卡氏枯草杆菌蛋白酶(SEQ ID NO1)的氨基酸序列)。
我们相信，这些环区域在酶分子与表面结合的肽的吸附中起着重要的作用，在一个或多个环区域中具体的突变对吸附具有明显的效果。尽管不希望限制于任何理论，但我们相信出于至少两个原因，这些环区域对卡氏枯草杆菌蛋白酶分子的吸附是重要的。首先，含有环区域的氨基酸能够密切地与分子暴露的任何表面接触。其次，卡氏枯草杆菌蛋白酶分子的环区域与活性位点的接近和结合能使得它们在酶与表面结合的底物(肽/蛋白质污垢)的催化产生的吸附中起作用。
本文使用的术语“变异体”是指含有不同于野生型氨基酸序列的酶。
本文使用的术语“突变卡氏枯草杆菌蛋白酶基因”指的是编码卡氏枯草杆菌蛋白酶变异体的基团。
本文使用的术语“野生型卡氏枯草杆菌蛋白酶”指的是由SEQ IDNO1表示的酶。该卡氏枯草杆菌蛋白酶的氨基酸序列在Smith，E.L.，Delanfe，R.J Evans，W.H.，Landin，M.和Markland，F.S.，生物化学杂志.，卷243，第2184-2191页(1968)(列为参考文献)中进一步描述。
本文使用的术语“卡氏枯草杆菌蛋白酶野生型氨基酸序列”包括SEQID NO1以及除位点58-65、94-106、125-132、153-166、186-190和199-219之外具有修饰氨基酸序列的SEQ ID NO1。
本文使用的术语“更亲水的氨基酸”指的是参考下表中的亲水性值比目标氨基酸具有更大亲水性值的任何其它氨基酸。如下亲水性值表(表1)以增加的亲水性值的下降顺序列出氨基酸(参见Hopp，T.P.，和Woods，K.R.，“由氨基酸序列判断蛋白质抗原决定簇”，美国国家科学院院刊，卷78，3824-3828页，1981，列为参考文献)。
表1氨基酸 亲水性值Trp -3.4Phe -2.5Tyr -2.3Leu，Ile-1.8Val -1.5Met -1.3Cys -1.0Ala，His-0.5Thr -0.4Pro，Gly-0.0Gln，Asn 0.2Ser 0.3Arg+，Lys+，Glu-，Asp-3.0表1还说明了带有电荷(该特征基于约8-9的pH)。正电荷的氨基酸是Arg和Lys，负电荷氨基酸是Glu和Asp，其余的氨基酸是中性的。在本发明的优选实施方案中，取代氨基酸是中性的或负电荷的，更优选负电荷(即Glu或Asp)。
因而，例如说明“用相等或更亲水的中性或带有负电荷的氨基酸取代的Gln”指的是用Asn(它与Gln的亲水性相等)或Ser、Glu或Asp(它们比Gln更亲水)取代的Gln。它们每个均是中性或具有负电荷，与Gln相比较，它们具有较大的亲水性值。同样，说明“用更亲水的中性或带有负电荷的氨基酸取代的Pro”指的是用Gln、Asn、Ser、Glu或Asp取代的Pro。
在本发明的一个实施方案中，卡氏枯草杆菌蛋白酶变异体具有卡氏枯草杆菌蛋白酶野生型氨基酸序列的修饰氨基酸序列，其中野生型氨基酸序列含有在第一环区域、第二环区域、第三环区域、第四环区域、第五环区域或第六环区域的一个或多个中的一个或多个位点上的取代基；从而卡氏枯草杆菌蛋白酶变异体与野生型卡氏枯草杆菌蛋白酶相比较，对不溶解底物具有降低的吸附作用和增加的水解作用。
在本发明的优选实施方案中，在一个或多个环区域中一个或多个位点取代氨基酸，参考表1，是中性的或负电荷的，与在野生型氨基酸序列中目标位点的氨基酸相比较，是相等或更亲水性的，优选是更亲水性的。A.第一环区域中的取代当取代发生在第一环区域中时，取代发生在位点58、59、60、61、62、64或65的一个或多个上。
当取代发生在位点58时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
当取代发生在位点59时，取代氨基酸是Glu。
当取代发生在位点60时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在位点61时，取代氨基酸是Asp、Gln、Glu或Ser。
当取代发生在位点62时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在位点64时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在位点65时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。B.第二环区域的取代当取代发生在第二环区域中时，取代发生在位点94、95、96、97、98、99、100、101、102、103、104、105或106的一个或多个上。
当取代发生在位点94时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Met、Pro、Ser或Thr。
当取代发生在位点95时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Met、Pro、Ser、Thr或Val。
当取代发生在位点96时，取代氨基酸是Asp、Gln、Glu、Ser。
当取代发生在位点97时，取代氨基酸是Asp或Glu。
当取代发生在位点98时，取代氨基酸是Asp或Glu。
当取代发生在位点99时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在位点100时，取代氨基酸是Asp或Glu。
当取代发生在位点101时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在位点102时，取代氨基酸是Asp或Glu。
当取代发生在位点103时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr或Val。
当取代发生在位点104时，取代氨基酸是Asp或Glu。
当取代发生在位点105时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在位点106时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Leu、Met、Pro、Ser、Thr或Val。C.第三环区域中的取代当取代发生在第三环区域中时，取代发生位点125、126、127、128、129、130、131或132的一个或多个上。
当取代发生在位点125时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Met、Pro、Ser、Thr或Val。
当取代发生在位点126时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在位点127时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在位点128时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、His、Pro、Ser或Thr。
当取代发生在位点129时，取代氨基酸是Asp或Glu。
当取代发生在位点130时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在位点131时，取代氨基酸是Asp或Glu。
当取代发生在位点132时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。D.第四环区域中的取代当取代发生在第四环区域中时，取代发生在位点153、154、155、156、157、158、159、160、161、162、163、164、165或166的一个或多个上。
当取代发生在位点153时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在位点154时，取代氨基酸是Asp、Gln、Glu或Ser。
当取代发生在位点155时，取代氨基酸是Asp或Glu。
当取代发生在位点156时，取代氨基酸是Ash、Asp、Gln、Glu、Pro或Ser。
当取代发生在位点157时，取代氨基酸是Asp、Gln、Glu、Ser。
当取代发生在位点158时，取代氨基酸是Asp或Glu。
当取代发生在位点159时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在位点160时，取代氨基酸是Asp或Glu。
当取代发生在位点161时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
当取代发生在位点162时，取代氨基酸是Asp、Gln、Glu或Ser。
当取代发生在位点163时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
当取代发生在位点164时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Met、Pro、Ser、Thr或Val。
当取代发生在位点165时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。和当取代发生在位点166时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr或Val。E.第五环区域中的取代当取代发生在第五环区域中时，取代发生在位点186、187、188、189或190一个或多个上。
当取代发生在位点186时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、His、Pro、Ser或Thr。
当取代发生在位点187时，取代氨基酸是Asp或Glu。
当取代发生在位点188时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr、Tyr或Val。
当取代发生在位点189时，取代氨基酸是Asp或Glu。
当取代发生在位点190时，取代氨基酸是Asn或Glu。F.第六环区域中的取代当取代发生在第六环区域中时，取代发生在位点199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218或219的一个或多个上。
当取代发生在位点199时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、His、Pro、Ser或Thr。
当取代发生在位点200时，取代氨基酸是Asn、Asp、Gln、Glu、Gly或Ser。
当取代发生在位点201时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在位点202时，取代氨基酸是Asp、Asp、Gln、Glu、Gly、His、Pro、Ser或Thr。
当取代发生在位点203时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在位点204时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Met、Pro、Ser或Thr。
当取代发生在位点205时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr或Val。
当取代发生在位点206时，取代氨基酸是Asp或Glu。
当取代发生在位点207时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
当取代发生在位点208时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr或Val。
当取代发生在位点209时，取代氨基酸是Asn、Asp、Gln、Glu、Gly或Ser。
当取代发生在位点210时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
当取代发生在位点211时，取代氨基酸是Asp、Gln、Glu或Ser。
当取代发生在位点212时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
当取代发生在位点213时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr或Val。
当取代发生在位点214时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、His、Pro、Ser或Thr。
当取代发生在位点215时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
当取代发生在位点216时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Met、Pro、Ser、Thr或Val。
当取代发生在位点217时，取代氨基酸是Asp、Gln、Glu或Ser。
当取代发生在位点218时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在位点219时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
G.酶变异体的制备实施例1突变体卡氏枯草杆菌蛋白酶基因构建含有野生型卡氏枯草杆菌蛋白酶基因的噬菌粒(“TP”)。将质粒pUC119的2.8Kbp PvuII限制酶片段(Vieira，J.和Messing，J.，“单链的质粒DNA的制备”，153酶学方法3-11(1989))克隆至质粒pUB110(Bacillus Genetic Stock Center，Columbus，OH 1E9)的PvuII位点。pUC119-pUB110杂交质粒被称为pJMA601。在pJMA601的BamH1限制位克隆入具有TP的聚合酶链反应扩增的产CP的卡氏枯草杆菌蛋白酶基因。噬菌粒TP被转化入大肠杆菌Ung-菌株CJ236，用VCSM13辅助噬菌体(Kunkel，T.A.，J.D.Roberts和R.A.Zakour，“无需表型选择的快速和有效的位点专一诱变”，酶学方法，卷154，367-382页(1987)；由Yuckenberg，P.D.，F.Witney，J.Geisselsoder和J.McClary改进，“使用含尿嘧啶的DNA和噬菌粒载体的体外定点诱变”，定向突变，操作指南，M.J.McPherson编，第27-48页(1991)；均列为本文参考文献)制备单链的含尿嘧啶DNA模板。使用Zoller和Smith方法的单引物定点诱变修饰方法(Zoller，M.J.和M.Smith，“使用M13衍生的载体的寡核苷酸定向诱变一种在DNA任何片段中制备点突变的有效和常用方法”，核酸研究，卷10，6487-6500页(1982)，列为本文参考文献)制备所有突变体(基本上如上述Yuckenberg等人，1991中说明)。寡核苷酸使用Applied BiosystemInc.380B DNA合成仪制备。诱变反应产物转化入大肠杆菌菌株MM 294(American Type Culture Collection E.Coli.33625)中。所有突变体由DNA测序证实，分离的DNA转化入枯草芽孢杆菌表达菌株BG2036(Yang，M.Y.，E.Ferrari和D.J.Henner，(1984)，“枯草芽孢杆菌的中性蛋白酶基因的克隆和克隆的基因用于产生体外得到的缺失突变”，细胞学杂志，卷160，15-21页)。对于某些环突变体，用在相应环中移码终止的密码子突变的修饰TP制备尿嘧啶模板。寡核苷酸用来恢复合适的阅读框和编码在位点58、59、60、61、62、64、65、94、95、96、97、98、99、100、101、102、103、104、105、106、125、126、127、128、129、130、131、132、153、154、155、156、157、158、159、160、161、162、163、164、165、166、186、187、188、189、190、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218和/或219的随机取代(在这些密码子中对于所有三种碱基所有四种寡核苷酸的等摩尔和/或可变混合物)。校正移码终止和制备功能酶的突变由它们消化酪蛋白的能力说明。随机取代用DNA测序确定。
实施例2发酵将含有感兴趣的枯草芽孢杆菌突变体的枯草芽孢杆菌细胞(BG2036)在1升LB-葡萄糖肉汤培养基中的mid-log相中生长，在总体积10升的Biostat ED发酵器(B.Braun Biotech，Inc.，Allentown，Pennsylvania)中培养。发酵介质含有酵母提取物、淀粉、消泡剂、缓冲剂和痕量矿物质(参见发酵操作指南B.McNeil和L.M.Harvey编，1990)。在发酵试验过程中，肉汤保持恒定pH7.0。加入氯霉素以进行诱变质粒的抗菌选择。细胞在37℃生长过夜至A600约60，并收获。
实施例3提纯将发酵肉汤进行如下步骤以得到纯的酶。通过离心除去肉汤中的枯草芽孢杆菌细胞，通过用100K截断膜除去细的粒子而澄清。然后用10K截断膜上浓缩，进行流体透析以降低离子强度，用0.025M MES缓冲剂(2-(N-吗啉代)乙磺酸)调节pH至5.5。通过将酶加入阳离子交换层析柱或亲合吸附层析柱，由柱中用氯化钠或丙二醇梯度洗脱而进一步纯化(参见，Scopes，R.K.，蛋白质纯化理论和操作，Springer-Verlag，NewYork(1984)，列为本文参考文献)。
使用pNA试验(DelMar，E.G.，C.Largman，J.W.Brodrick和M.C.Geokas，生物化学年鉴，卷99，316-320页(1979)，列为本文参考文献)测定在梯度洗脱过程中收集的级分的酶浓度。该试验测量当酶水解溶解的合成底物，琥珀酰-丙氨酰-丙氨酰-脯氨酰-苯基丙氨酰-对-硝基苯胺(sAAPF-pNA)时释放对-硝基苯胺的速率。用分光光度计在410nm测量水解反应产生黄色的速率，与活性酶浓度成比例。此外，在280nm的吸附测量用来测定总蛋白质浓度。活性酶/总蛋白质比率得到酶的纯度，并用于鉴定用作存贮溶液的收集的级分。
为避免在贮存过程中酶的自解，在由层析柱得到的收集级分中加入相等重量的丙二醇。在提纯过程完成后，存贮酶溶液用SDS-PAGE(十二烷基磺酸钠聚丙烯酰胺凝胶电泳)校验，用胰蛋白酶抑制剂型II-T由Sigma Chemical Company(St.Louis，Missouri)购买的火鸡卵白经活性位滴定法测定绝对酶浓度。测量的转化因子将说明在酶分子中不同位点发生的改变使得酶变异体与野生型相比较对溶解底物pNA具有增加的活性。
在使用的制剂中，酶存贮溶液通过Sephadex-G25(Pharmacia，Piscataway，New Jersey)大小排除柱以除去丙二醇和交换缓冲剂。用含有0.01M氯化钙的0.1M Tris缓冲剂(三(羟基甲基-氨基)甲烷)代替在酶存贮溶液中的MES缓冲剂，用盐酸调节pH至8.6。在恒温25℃下在Tris缓冲剂中在pH8.6下进行所有试验。H酶变异体的特性实施例4模型表面制备由CPG Inc.(Fairfield，New Jersey)购买的氨基丙基控制孔玻璃(CPG)用作共价连接由Bachem，Inc.(Torence，California)购买的sAAPD-pNA底物的载体。反应在二甲基亚砜中进行，用(1-乙基-3-[3-(二甲基氨基)丙基]碳化二亚胺盐酸盐)(EDC)作偶合试验。在反应完成后(用pNA试验监测)，除去过量的溶剂，用二甲基亚砜(DMSO)和双蒸馏水漂洗CPGsAAPF-pNA。然后在烘箱中氮气吹扫在约70℃下干燥。反应方案和固定的底物的制备方法如Brode，P.F.III，和D.S.Rauch，“枯草杆菌蛋白酶BPN’在固定底物上的活性”，LANGMUIR，卷8，1325-1329页(1992)(列为本文参考文献)中描述进行。
CPG表面含有62000±7000pNA分子/μm2。从CPG Inc.报导的CPG的50.0m2/g的值来看，表面积保持未变。这说明用于在CPG中加入sAAPf-pNA的方法未破坏孔结构(平均直径486_)。
实施例5表面水解试验使用CPGsAAPf-pNA，酶变异体的吸附和CPG-结合的肽的水解可在同一试验中测量。在含有Tris缓冲剂和已脱气的CPGsAAPF-pNA的烧瓶中加入少量的酶变异体存贮溶液。烧瓶在活塞作用的振荡器中振荡90分钟，在此期间，以不同的时间间隔停止振荡器(例如，在吸附水解作用的最初阶段，例如最初20分钟为每2分钟-在实验结束时为每10分钟)。使CPGsAAPF-pNA静置，取样溶液。如上述Brode等人，1991中所述进行吸附和水解的实验过程和计算。
测定所有的酶的自溶稳定性，在实验的时间内，应未显示明显的自溶损失。因此，酶吸附可通过测量溶液消耗测定。最初酶变异体浓度与在每个间隔的时间点测量的浓度之间的差值得到吸附的酶变异体的量。由表面水解的pNA的量由在410nm时试样的吸附读数测定。水解的pNA的总量由取样的量加上残余在烧瓶中的量计算。该值通过减去当不存在酶时在pH8.6下由Tris缓冲剂水解的pNA的量而校准。根据酶的效率，基准水解范围为总水解的7-29％。
实施例6溶解底物的动力学分析溶解底物sAAPF-pNA的水解速率通过用DU-70分光光度计在410nm下测量作为时间函数的吸附增量而监测。酶浓度保持恒定，并制备成6-10纳摩尔的范围，而对于每次动力学测定，底物浓度在90-700μM sAAPF-pNA之间变化。在900秒的时间内，每秒收集吸附数据点，数据传递至LOTUSTM记录纸(Lotus Development Corporation，Cambridge，Massachusetts)。用标准Linewweaver Burk分析法进行动力学参数的分析，其中在试验最初(通常为最初几分钟)的数据符合于线性回归曲线以得到v0。以标准倒数方式标绘的v0和s0数据得到KM和Kcat。I.卡氏枯草杆菌蛋白酶变异体的实例降低表面结合的底物的吸附作用和增加的水解作用的本发明卡氏枯草杆菌蛋白酶变异体在如下表3-36中说明。在描述的具体突变中，首先给出野生型中出现的原始氨基酸，其次是位点编号，第三是取代氨基酸。
表2环1-单突变变异体Thr58AsnThr58AspThr58GlnThr58GluThr58GlyThr58ProThr58SerAsp59GluGly60AsnGly60AspGly60GlnGly60GluGly60ProGly60SerAsn61AspAsn61GlnAsn61GluAsn61SerGly62AsnGly62AspGly62GlnGly62GluGly62ProGly62SerGly64AsnGly64AspGly64GlnGly64GluGly64ProGly64SerThr65AsnThr65AspThr65GlnThr65GluThr65GlyThr65ProThr65Ser表3环1-双突变变异体Thr58Gln + Asn61GluAsn61Asp + Thr65Pro
Thr58Gln + Asp59GluThr58Gln + Gly62SerGly62Asn + Thr65ProThr58Gln + Gly60AspThr58Pro + Gly60GluAsn61Asp + Thr65SerAsp59Glu + Gly60GlnAsp59Glu + Gly60ProGly62Asp + Gly64GlnAsn61Ser + Thr65ProThr58Asp + Gly60GlnGly60Ser + Gly62GlnAsp59Glu + Gly62ProGly62Asp + Thr65GlyAsn61Gln + Thr65ProAsn61Gln + Gly64AsnGly60Gln + Gly62GlnThr58Gly + Asn61AspThr58Gln + Gly62GlnGly60Asn + Gly62GlnGly60Asn + Gly62AspThr58Glu + Gly62ProGly64Asp + Thr65ProAsp59Glu + Thr65SerGly60Gln + Thr65GlnAsp59Glu + Gly62SerAsn61Glu + Gly64GlnAsp59Glu + Gly60SerGly60Ser + Gly62SerThr58Gly + Gly64GluThr58Asn + Asn61SerAsp59Glu + Thr65AsnGly60Asn + Asn61SerGly60Glu + Gly62GlnThr58Pro + Gly62AspThr58Ser + Asp59GluThr58Asp + Gly62ProAsn61Asp + Gly62ProThr58Ser + Gly62SerGly60Asn + Gly62SerGly64Pro + Thr65AsnGly64Asp + Thr65GlnGly64Glu + Thr65ProGly64Asn + Thr65ProAsn61Ser + Gly64Asp表4
环1-三突变变异体Thr58Pro + Gly64Ser + Thr65GlyAsn61Glu + Gly62Gln + Thr65GlnThr58Gln + Gly62Ser + Gly64ProAsp59Glu + Gly62Asn + Thr65ProThr58Pro + Gly60Glu + Gly64AsnThr58Asn + Gly64Ser + Thr65GlyAsp59Glu + Asn61Gln + Gly64SerThr58Asn + Asn61Glu + Thr65ProGly60Pro + Asn61Asp + Thr65ProThr58Glu + Gly62Gln + Gly64AsnThr58Asn + Gly62Glu + Gly64ProThr58Ser + Asp59Glu + Thr65AsnThr58Glu + Gly62Asn + Gly64GlnThr58Pro + Asp59Glu + Gly62SerGly60Asn + Asn61Asp + Gly62AsnGly62Asp + Gly64Ser + Thr65ProAsp59Glu + Gly60Asn + Thr65AsnThr58Gly + Gly60Pro + Thr65GlyThr58Gln + Gly60Glu + Asn61GlnGly60Pro + Asn61Gln + Gly64AspThr58Asn + Gly62Asn + Thr65ProThr58Pro + Asp59Glu + Gly64AsnThr58Pro + Ash61Ser + Gly62AspThr58Asp + Gly50Pro + Gly62AsnThr58Ser + Gly60Pro + Asn61AspGly60Gln + Gly62Gln + Gly64ProAsp59Glu + Asn61Gln + Thr65AsnGly60Asp + Asn61Gln + Thr65ProAsn61Glu + Gly62Glu + Thr65ProThr58Asp + Asp59Glu + Thr65GlnThr58Glu + Asp59Glu + Thr65SerThr58Glu + Asp59Glu + Thr65Gly表5环1-四突变变异体Asp59Glu + Gly60Pro + Gly62Asn + Gly64GlnThr58Pro + Gly60Pro + Gly62Glu + Thr65GlnThr58Ser + Gly60Pro + Gly64Gln + Thr65ProThr58Pro + Asp59Glu + Asn61Ser + Gly62AsnThr58Glu + Gly60Asn + Gly64Ser + Thr65ProThr58Glu + Gly60Ser + Asn61Gln + Thr65GlnThr58Pro + Asp59Glu + Gly60Asn + Asn61GlnThr58Gly + Gly60Pro + Gly62Glu + Thr65SerGly60Asp + Gly62Ser + Gly64Gln + Thr65GlyThr58Asn + Gly60Pro + Gly62Ser + Gly64Asp
Thr58Pro + Asp59Glu + Gly60Asn + Gly62AsnAsn61Asp + Gly62Gln + Gly64Pro + Thr65AsnThr58Asp + Gly60Pro + Gly62Asn + Gly64SerThr58Asp + Gly60Gln + Gly62Pro + Thr65AsnThr58Asn + Asn61Asp + Gly62Glu + Thr65GlyThr58Ser + Gly60Ser + Asn61Asp + Gly62GluThr58Glu + Asp59Glu + Asn61Gln + Thr65SerThr58Asp + Asp59Glu + Gly62Pro + Thr65GlyGly60Asp + Asn61Asp + Gly62Ser + Thr65GlyGly60Glu + Asn61Glu + Gly62Gln + Gly64ProThr58Pro + Gly60Asp + Asn61Asp + Gly64AsnAsp59Glu + Gly60Asp + Gly64Gln + Thr65SerAsp59Glu + Gly60Asp + Gly64Gln + Thr65ProThr58Pro + Asp59Glu + Gly60Asp + Gly64AsnThr58Ser + Asp59Glu + Gly60Asp + Gly62AsnAsp59Glu + Gly60Glu + Asn61Glu + Gly62SerThr58Glu + Asp59Glu + Gly60Asp + Thr65ProAsp59Glu + Gly60Asp + Gly62Glu + Thr65ProThr58Ser + Asp59Glu + Asn61Asp + Gly62Asp表6环2-单突变变异体Val 94AlaVal 94AsnVal 94AspVal 94CysVal 94GlnVal 94GluVal 94GlyVal 94HisVal 94MetVal 94ProVal 94SerVal 94ThrLeu 95AlaLeu 95AsnLeu 95AspLeu 95CysLeu 95GlnLeu 95GluLeu 95GlyLeu 95HisLeu 95IleLeu 95MetLeu 95ProLeu 95Ser
Leu 95ThrLeu 95ValAsn 96AspAsn 96GlnAsn 96GluAsn 96SerSer 97AspSer 97GluSer 98AspSer 98GluGly 99AsnGly 99AspGly 99GlnGly 99GluGly 99ProGly 99SerSer100AspSer100GluGly101AsnGly101AspGly101GlnGly101GluGly101ProGly101SerSer102AspSer102GluTyr103AlaTyr103AsnTyr103AspTyr103CysTyr103GlnTyr103GluTyr103GlyTyr103HisTyr103IleTyr103LeuTyr103MetTyr103ProTyr103SerTyr103ThrTyr103ValSer104AspSer104GluGly105AsnGly105AspGly105GlnGly105Glu
Gly105ProGly105SerIle106AlaIle106AsnIle106AspIle106CysIle106GlnIle106GluIle106GlyIle106HisIle106LeuIle106MetIle106ProIle106SerIle106ThrIle106Val表7环2-双突变变异体Val 94Gln + Ser100GluAsn 96Ser + Gly 99GlnGly 99Glu + Ile106MetSer 97Asp + Gly105GlnSer102Glu + Tyr103AsnSer100Glu + Ile106GlyAsn 96Asp + Ile106SerGly 99Ser + Ser100GluGly101Gln + Gly105SerAsn 96Asp + Gly 99ProAsn 96Gln + Ser104AspLeu 95Glu + Tyr103ProAsn 96Ser + Gly101AspLeu 95Val + Ser 97GluLeu 95Ala + Gly 99ProVal 94Thr + Gly 99GluLeu 95Gln + Ile106AlaLeu 95Pro + Ile106AlaLeu 95Val + Ser100GluGly101Gln + Tyr103GlyAsn 96Asp + Ile106ProLeu 95Ser + Ser100AspGly 99Pro + Ser100AspVal 94Thr + Leu 95AspSer100Glu + Ile106GlnSer100Asp + Gly101ProLeu 95Cys + Gly105Asp
Ser 98Glu + Gly101SerGly101Asn + Tyr103GlnVal 94Met + Ser102GluGly 99Pro + Gly105AspVal 94Asn + Ser102AspVal 94Cys + Ile106ThrVal 94Gly + Ser104AspVal 94Pro + Gly101ProVal 94Ser + Gly105GluVal 94Met + Ser 98AspVal 94Gln + Ser104AspAsn 96Ser + Ser 98AspSer104Glu + Ile106GlnVal 94Asn + Tyr103ThrTyr103Ala + Ser104AspGly 99Pro + Ser104GluLeu 95Cys + Tyr103LeuVal 94Gly + Gly 99SerGly101Gln + Tyr103SerSer 97Asp + Gly105ProSer 98Asp + Gly105AsnGly 99Gln + Ser100GluGly 99Pro + Ser100GluTyr103Ile + Gly105SerSer100Glu + Gly105ProAsn 96Glu + Tyr103GlyVal 94Met + Gly105AsnVal 94His + Leu 95GluSer100Asp + Tyr103MetLeu 95Met + Gly101GlnGly 99Pro + Gly101AspAsn 96Asp + Gly101AsnVal 94Ala + Gly 99Ser表8环2-三突变变异体Val 94Gln + Leu 95Thr + Ser100GluSer100Glu + Tyr103Met + Ile106ThrSer 97Asp + Gly101Gln + Ile106AsnLeu 95Gln + Gly105Ser + Ile106GlnLeu 95His + Gly 99Gln + Ser100GluVal 94His + Leu 95Met + Asn 96GlnVal 94Asn + Ser 97Glu + Tyr103ProSer 97Asp + Gly101Ser + Tyr103MetSer 97Glu + Tyr103Met + Ile106GlnVal 94Thr + Gly 99Pro + Gly105Asp
Leu 95Gly + Ser 98Glu + Tyr103CysLeu 95Pro + Gly101Asp + Tyr103GlyLeu 95Val + Gly 99Pro + Ser104AspLeu 95Ile + Gly105Ser + Ile106MetSer 97Glu + Gly101Asn + Ile106ThrAsn 96Ser + Gly101Asp + Tyr103AsnVal 94Pro + Ser 98Glu + Ile106AsnGly 99Pro + Tyr103Glu + Ile106SerAsn 96Asp + Gly 99Asn + Tyr103ProLeu 95Gln + Ser 97Asp + Ile106ValVal 94Pro + Ser 98Glu + Tyr103ValLeu 95Asn + Gly101Pro + Ile106HisVal 94Ala + Leu 95His + Ser100GluVal 94Asn + Gly101Ser + Ser104AspVal 94Cys + Gly105Ser + Ile106ThrLeu 95Gly + Gly101Asp + Ile106CysSer102Glu + Tyr103Ile + Ile106GlyVal 94Pro + Leu 95Asp + Ile106ProVal 94Pro + Leu 95Ile + Ile106CysSer100Asp + Gly101Asp + Ile106AsnLeu 95Glu + Asn 96Glu + Tyr103ProGly101Glu + Ser102Asp + Tyr103LeuVal 94Pro + Gly101Asp + Ser102GluAsn 96Asp + Ser 97Asp + Gly 99ProSer 97Asp + Ser 98Asp + Gly 99GluAsn 96Glu + Ser 97Asp + Gly 99AspGly 99Glu + Ser100Asp + Gly101AspGly 99Glu + Ser100Glu + Gly101GluLeu 95Gln + Ser102Asp + Gly105AspAsn 96Ser + Ser102Glu + Ser104GluSer102Asp + Ser104Glu + Gly105ProSer102Asp + Tyr103Ser + Ser104GluVal 94Cys + Ser 97Glu + Gly 99AspAsn 96Asp + Ser 98Glu + Ile106ThrAsn 96Asp + Ser 98Asp + Ile106CysAsn 96Glu + Ser 98Glu + Gly105AsnSer 98Glu + Ser100Glu + Ile106SerSer 98Asp + Ser100Asp + Gly101AsnGly101Asp + Ser102Asp + Ser104GluLeu 95Glu + Ser 98Asp + Gly101AspVal 94Ser + Ser100Glu + Ser102AspLeu 95Val + Ser 97Glu + Ser100GluAsn 96Gln + Ser 97Asp + Ser100AspAsn 96Ser + Ser 97Asp + Ser100AspVal 94Asp + Gly 99Asn + Ser102GluVal 94Asp + Ser102Glu + Ile106HisLeu 95Asp + Gly 99Glu + Ser102Asp
Val 94Thr + Gly101Asp + Ser104GluGly 99Pro + Gly101Asp + Ser104AspSer 98Asp + Gly101Asp + Tyr103Pro表9环2-四突变变异体Val 94Met + Leu 95Asn + Ser 98Asp + Gly105SerAsn 96Ser + Gly101Glu + Tyr103Ile + Gly105AsnLeu 95Val + Ser 97Asp + Gly 99Asn + Tyr103IleLeu 95Ile + Asn 96Gln + Gly 99Asn + Tyr103AspVal 94His + Ser100Glu + Tyr103Ile + Ile106MetSer 97Glu + Tyr103Ile + Gly105Gln + Ile106AlaVal 94Thr + Leu 95Ile + Ser 97Asp + Gly99SerVal 94Thr + Leu 95Cys + Gly 99Asp + Ile106ValLeu 95Cys + Ser 98Glu + Gly 99Asn + Ile106MetAsn 96Gln + Ser 98Asp + Gly105Asn + Ile106GlyLeu 95Thr + Ser100Glu + Tyr103Val + Ile106ProVal 94Thr + Leu 95Val + Asn 96Glu + Tyr103AsnVal 94His + Leu 95Asn + Gly 99Gln + Gly105GlnVal 94Ala + Leu 95Thr + Ser 98Asp + Tyr103IleLeu 95Val + Gly101Gln + Tyr103Cys + Ile106GluGly 99Pro + Ser100Glu + Gly101Ser + Tyr103ThrLeu 95Ala + Gly101Asp + Tyr103Ser + Gly105AsnGly 99Asn + Tyr103Asp + Gly105Asn + Ile106ThrAsn 96Asp + Ser 97Asp + Gly101Ser + Tyr103ThrVal 94Ala + Asn 96Glu + Ser 97Asp + Gly105AsnVal 94Cys + Leu 95Gln + Asn 96Glu + Ser 97GluVal 94Gly + Asn 96Gln + Ser104Glu + Gly105GluSer 97Asp + Ser 98Glu + Gly101Asn + Tyr103ValSer 97Glu + Ser 98Asp + Gly 99Gln + Gly101GlnLeu 95Gly + Asn 96Ser + Ser 97Glu + Ser 98GluAsn 96Glu + Ser 97Glu + Ser 98Asp + Gly101ProAsn 96Asp + Ser 97Asp + Ser 98Asp + Tyr103LeuAsn 96Asp + Ser 97Asp + Ser 98Glu + Tyr103GlyLeu 95Glu + Asn 96Glu + Ser100Asp + Ile106AlaSer 97Asp + Gly 99Glu + Tyr103Cys + Gly105AsnVal 94Gly + Asn 96Ser + Ser 97Glu + Gly 99AspAsn 96Ser + Tyr103Leu + Ser104Asp + Ile106GluLeu 95Ala + Asn 96Glu + Gly 99Ser + Ser100AspAsn 96Asp + Ser 97Glu + Ser100Glu + Ile106ValVal 94Cys + Ser 97Asp + Ser 98Asp + Ser100AspSer 97Asp + Ser 98Asp + Ser100Glu + Tyr103ThrAsn 96Glu + Gly 99Glu + Gly101Asp + Tyr103AlaVal 94Asp + Leu 95Ala + Gly101Glu + Ser102GluSer 98Glu + Gly 99Pro + Ser100Asp + Gly101GluVal 94Glu + Asn 96Gln + Gly101Glu + Ile106Thr
Asn 96Ser + Ser102Asp + Tyr103Asn + Ile106AspLeu 95Cys + Ser 98Asp + Gly 99Asp + Gly101AspSer100Glu + Gly101Pro + Ser102Glu + Ile106AlaVal 94Cys + Ser100Glu + Gly101Pro + Ser102GluVal 94Gln + Ser100Asp + Ser102Glu + Tyr103IleSer 97Asp + Ser100Glu + Gly101Glu + Tyr103AsnVal 94Glu + Asn 96Glu + Gly 99Glu + Gly105ProAsn 96Gln + Ser100Glu + Gly101Asp + Ile106GluVal 94Pro + Leu 95Ala + Ser 97Asp + Ser100AspLeu 95Glu + Gly101Asn + Ser102Glu + Tyr103IleVal 94Asn + Asn 96Glu + Gly101Asp + Gly105AspVal 94Asp + Gly 99Asn + Ser100Asp + Ser102AspVal 94Asp + Leu 95Val + Ser102Asp + Tyr103AlaLeu 95Glu + Gly 99Pro + Ser102Asp + Ser104GluLeu 95Ser + Asn 96Gln + Gly101Glu + Ser104AspVal 94Gln + Asn 96Gln + Gly101Glu + Ser104GluGly101Asp + Tyr103Val + Ser104Glu + Ile106ValAsn 96Asp + Ser102Asp + Tyr103Gly + Gly105AspSer100Glu + Ser102Asp + Ser104Asp + Gly105SerVal 94Asp + Ser 97Asp + Ser100Asp + Gly105Gln表10环3-单突变变异体Leu125AlaLeu125AsnLeu125AspLeu125CysLeu125GlnLeu125GluLeu125GlyLeu125HisLeu125IleLeu125MetLeu125ProLeu125SerLeu125ThrLeu125ValGly126AsnGly126AspGly126GlnGly126GluGly126ProGly126SerGly127AsnGly127AspGly127Gln
Gly127GluGly127ProGly127SerAla128AsnAla128AspAla128GlnAla128GluAla128GlyAla128HisAla128ProAla128SerAla128ThrSer129AspSer129GluGly130AsnGly130AspGly130GlnGly130GluGly130ProGly130SerSer131AspSer131GluThr132AsnThr132AspThr132GlnThr132GluThr132GlyThr132ProThr132Ser表11环3-双突变变异体Leu125Gln + Ser129GluGly130Gln + Thr132AsnGly126Asp + Gly127ProLeu125Ile + Ser131GluGly126Gln + Gly130SerGly126Pro + Thr132ProGly126Asp + Thr132SerAla128Gly + Thr132SerLeu125Ala + Gly126GluLeu125Gln + Ser129AspGly126Pro + Ser131AspGly127Ser + Ser129GluGly130Asn + Ser131GluLeu125Glu + Ala128Gln
Gly126Ser + Thr132SerLeu125Ala + Ser129AspGly127Pro + Thr132AspGly126Pro + Gly130SerLeu125Ala + Ala128AspGly127Gln + Ser131AspLeu125Ala + Thr132SerGly127Pro + Thr132GlyGly126Glu + Thr132AsnLeu125Gly + Ser129GluAla128Thr + Gly130AspAla128His + Thr132AspLeu125Gly + Gly126AsnGly126Ser + Ser129GluLeu125Asn + Thr132AsnGly126Glu + Ala128GlySer131Asp + Thr132GlnSer129Glu + Gly130ProGly127Asn + Ser129AspLeu125Glu + Thr132AsnGly126Gln + Gly127GluAla128Thr + Ser131AspLeu125Pro + Ser131GluGly126Pro + Thr132GlyGly126Gln + Ser129AspLeu125Gly + Gly126SerGly127Gln + Ser129AspGly126Gln + Gly130GluGly127Asp + Gly130AsnLeu125Glu + Gly126SerLeu125Ile + Thr132AspGly126Asn + Gly127AspLeu125Cys + Gly126GlnLeu125Glu + Ala128AsnGly126Gln + Ala128AsnGly126Asp + Gly130AsnLeu125Ser + Gly130SerGly126Asn + Ser129AspLeu125Ser + Ser131AspSer129Asp + Thr132AsnGly127Glu + Ala128ThrLeu125Thr + Gly130ProAla128Pro + Thr132AspGly127Ser + Ser131GluGly126Asn + Thr132GluGly130Asp + Thr132Asn
表12环3-三突变变异体Leu125Asn + Gly126Pro + Ser129GluGly126Asn + Ser129Asp + Gly130ProLeu125Ala + Gly126Ser + Ser131GluGly127Gln + Ala128Thr + Ser129GluLeu125Thr + Ser129Asp + Gly130ProGly126Pro + Ala128Thr + Thr132AspGly126Asn + Gly130Asn + Thr132GlnLeu125Thr + Gly130Gln + Thr132GlnAla128Gly + Ser129Asp + Thr132AsnLeu125Met + Ala128Gln + Ser129AspLsu125Gly + Ala128Asp + Gly130ProLeu125Ile + Gly127Ser + Thr132AspLeu125Val + Gly127Glu + Gly130SerGly127Ser + Gly130Asn + Thr132AsnLeu125Thr + Gly126Ser + Ala128GluLeu125Gln + Gly126Pro + Ser131GluLeu125Glu + Gly130Asn + Thr132AsnGly126Asn + Gly127Pro + Ser129AspGly126Pro + Gly127Pro + Ala128HisGly127Asn + Ala128His + Ser129AspLeu125Thr + Gly130Pro + Thr132SerLeu125Cys + Gly127Gln + Ser129AspGly127Asn + Gly130Glu + Thr132GlyGly127Gln + Gly130Ser + Thr132AspLeu125Thr + Ser129Asp + Thr132ProLeu125Cys + Gly127Glu + Gly130ProAla128Gly + Ser129Glu + Gly130GlnGly127Asp + Gly130Ser + Thr132ProLeu125Pro + Gly126Asn + Gly127AsnGly126Glu + Ala128Ser + Gly130SerLeu125Gly + Gly127Pro + Ser129AspGly126Gln + Ala128Glu + Gly130GlnAla128Gly + Gly130Asn + Thr132AspLeu125Met + Gly126Pro + Gly127GlnLeu125Gln + Ala128Asn + Ser129AspGly127Ser + Ala128Asn + Thr132GlyGly126Gln + Gly127Asp + Gly130SerGly127Asp + Ala128Pro + Thr132ProLeu125His + Ser129Asp + Thr132ProLeu125Ile + Gly126Gln + Thr132GlnGly126Asp + Gly130Ser + Thr132AsnGly127Asn + Ser129Glu + Gly130ProLeu125Pro + Gly127Asp + Gly130AsnLeu125Ile + Ala128His + Ser131Asp
Gly126Pro + Ala128His + Thr132ProLeu125Val + Ala128Gln + Ser129GluLeu125Ser + Gly126Asp + Thr132GlyLeu125Pro + Gly126Asp + Gly127AspAla128Ser + Gly130Asp + Ser131AspGly130Asp + Ser131Glu + Thr132AsnGly126Asn + Gly130Asp + Ser131GluGly126Gln + Gly130Asp + Ser131GluGly130Gln + Ser131Glu + Thr132GluAla128Thr + Ser131Glu + Thr132AspAla128Thr + Ser131Asp + Thr132GluAla128Pro + Ser131Asp + Thr132AspGly127Asp + Ala128Asp + Thr132GlnLeu125Glu + Gly126Glu + Gly130AsnAla128Asp + Ser129Asp + Gly130AsnSer129Asp + Gly130Glu + Thr132Pro表13环3-四突变变异体Leu125Asn + Ala128Asn + Ser131Glu + Thr132ProLeu125Ala + Gly126Gln + Gly127Pro + Gly1305erLeu125Pro + Gly126Asn + Gly127Gln + Ala128ProLeu125Gly + Gly126Asn + Ser129Glu + Gly130AsnAla128Gly + Gly130Asn + Ser131Asp + Thr132SerLeu125Ile + Gly126Pro + Ala128Gly + Thr132GluLeu125Thr + Gly130Pro + Ser131Asp + Thr132GlyLeu125Asp + Gly126Pro + Gly127Ser + Thr132GlnLeu125Gly + Gly126Pro + Gly127Asp + Thr132ProLeu125Val + Gly127Glu + Gly130Asn + Thr132AsnLeu125Cys + Ala128Pro + Gly130Ser + Ser131GluGly126Gln + Gly127Ser + Ala128Thr + Gly130GluLeu125Gly + Gly127Ser + Gly130Ser + Thr132GlyLeu125Pro + Gly126Ser + Ala128His + Thr132AsnLeu125Val + Gly126Gln + Gly127Pro + Ser129AspLeu125Cys + Gly130Pro + Ser131Asp + Thr132SerLeu125Gly + Gly127Gln + Ser129Glu + Thr132SerLeu125Met + Ala128Ser + Gly130Pro + Thr132GlyLeu125Ala + Gly126Asp + Ala128His + Thr132SerGly127Gln + Gly130Glu + Ser131Asp + Thr132AsnGly126Ser + Gly127Gln + Gly130Glu + Ser131GluGly127Pro + Gly130Asp + Ser131Glu + Thr132ProAla128Gly + Gly130Pro + Ser131Asp + Thr132GluGly126Asn + Ala128Pro + Ser131Asp + Thr132AspGly126Gln + Ala128His + Ser131Asp + Thr132AspLeu125Thr + Gly127Glu + Ala128Asp + Gly130GlnGly126Asn + Ala128Gln + Ser129Glu + Gly130Asp
Ala128Gly + Ser129Glu + Gly130Glu + Thr132GlnGly126Gln + Ser129Asp + Gly130Glu + Thr132AsnGly126Asn + Ala128Thr + Ser129Glu + Gly130GluGly126Ser + Gly127Asn + Ser129Glu + Gly130GluLeu125His + Gly127Glu + Ala128Glu + Ser129AspLeu125Thr + Gly127Glu + Ala128Asp + Ser129GluGly126Pro + Gly127Glu + Ala128Asp + Ser129GluAla128Gly + Ser129Glu + Gly130Asp + Ser131AspGly126Gln + Ser129Asp + Gly130Asp + Ser131AspGly127Ser + Ser129Glu + Gly130Glu + Ser131GluAla128Gly + Ser129Asp + Gly130Asp + Ser131GluGly126Asn + Ser129Asp + Gly130Glu + Ser131AspAla128Asn + Ser129Asp + Gly130Asp + Ser131AspGly127Pro + Ser129Glu + Gly130Asp + Ser131GluGly126Ser + Gly130Asp + Ser131Asp + Thr132GluLeu125Met + Gly127Glu + Ser129Asp + Gly130AspGly126Pro + Gly127Asp + Ser129Glu + Gly130AspLeu125Val + Gly127Glu + Ser129Asp + Gly130AspGly127Asp + Ala128Asn + Ser129Asp + Gly130GluGly127Glu + Ala128Thr + Ser129Glu + Gly130SerGly127Asp + Ala128His + Ser129Glu + Gly130SerGly126Ser + Gly127Glu + Ala128Thr + Ser129GluGly126Pro + Gly127Asp + Ala128Asp + Gly130GluLeu125Gly + Gly126Asp + Gly127Glu + Ser129GluGly126Asp + Gly127Glu + Ala128Ser + Ser129AspGly127Ser + Ser129Asp + Gly130Gln + Ser131AspAla128Gly + Ser129Glu + Gly130Asn + Ser131GluLeu125Val + Ser129Asp + Ser131Asp + Thr132SerLeu125Gly + Ala128Gly + Ser129Glu + Ser131AspLeu125Cys + Gly126Pro + Ser129Glu + Ser131GluGly127Ser + Ala128Asn + Ser129Asp + Ser131AspGly126Asn + Ser129Asp + Ser131Glu + Thr132GlnGly126Pro + Ala128Pro + Ser129Asp + Ser131Glu表14环4-单突变变异体Gly153AsnGly153AspGly153GlnGly153GluGly153ProGly153SerAsn154AspAsn154GlnAsn154GluAsn154Ser
Ser155AspSer155GluGly156AsnGly156AspGly156GlnGly156GluGly156ProGly156SerAsn157AspAsn157GlnAsn157GluAsn157SerSer158AspSer158GluGly159AsnGly159AspGly159GlnGly159GluGly159ProGly159SerSer160AspSer160GluThr161AsnThr161AspThr161GlnThr161GluThr161GlyThr161ProThr161SerAsn162AspAsn162GlnAsn162GluAsn162SerThr163AsnThr163AspThr163GlnThr163GluThr163GlyThr163ProThr163SerIle164AlaIle164AsnIle164AspIle164CysIle164GlnIle164GluIle164Gly
Ile164HisIle164LeuIle164MetIle164ProIle164SerIle164ThrIle164ValGly165AsnGly165AspGly165GlnGly165GluGly165ProGly165SerTyr166AlaTyr166AsnTyr166AspTyr166CysTyr166GlnTyr166GluTyr166GlyTyr166HisTyr166IleTyr166LeuTyr166MetTyr166ProTyr166SerTyr166ThrTyr166Val表15环4-双突变变异体Asn154Ser + Ser155GluGly153Glu + Ile164AlaGly159Glu + Gly165ProAsn154Glu + Gly156SerGly156Pro + Gly159AspGly153Ser + Ser160AspSer155Asp + Gly165SerIle164Gly + Gly165GluGly159Glu + Ile164AsnAsn157Gln + Asn162SerGly156Asn + Thr161GlnAsn162Glu + Tyr166GlnAsn157Glu + Gly165GlnAsn162Glu + Gly165GlnGly153Pro + Ile164Cys
Thr163Glu + Gly165AsnGly153Asp + Ile164AsnGly153Ser + Thr161AspGly156Asp + Tyr166GlnGly165Pro + Tyr1665erGly153Ser + Asn162GluGly159Glu + Tyr166LeuGly156Asn + Thr161GluAsn154Gln + Ser158GluSer160Asp + Thr161AsnSer155Glu + Gly165ProAsn154Ser + Tyr166IleGly153Asp + Thr161AsnSer158Asp + Thr161ProAsn162Glu + Thr163SerGly159Gln + Tyr166CysThr161Gly + Asn162SerGly159Asp + Tyr166AsnSer160Glu + Ile164ThrSer158Asp + Ile164LeuAsn154Ser + Asn157AspAsn154Asp + Thr161GlnGly156Ser + Ser160GluGly156Gln + Tyr166IleGly153Asn + Tyr166AlaAsn154Asp + Ile164SerSer155Asp + Thr163AsnGly156Glu + Gly159GlnGly156Ser + Asn162AspGly153Ser + Asn157GluSer160Glu + Gly165ProGly153Pro + Asn157AspGly153Gln + Ser160AspAsn154Gln + Thr163AsnGly156Ser + Gly159AspGly159Asp + Thr161GlnGly159Ser + Tyr166GluAsn157Asp + Tyr166LeuThr161Asp + Asn162GlnAsn162Gln + Gly165GluGly156Asp + Asn162GlnAsn154Gln + Ile164GlnAsn157Asp + Asn162SerAsn154Asp + Ile164AsnGly159Asp + Gly165Pro表16
环4-三突变变异体Gly153Gln + Asn154Ser + Ser155GluGly156Asp + Thr161Pro + Tyr166IleAsn157Glu + Gly159Gln + Asn162GlnAsn154Glu + Asn162Gln + Ile164LeuGly153Pro + Gly159Gln + Ile164AsnAsn154Ser + Ile164Met + Tyr166CysGly159Gln + Ser160Asp + Ile164ThrGly156Asn + Thr161Ser + Ile164CysGly156Asn + Ile164His + Tyr166AspAsn157Glu + Asn162Gln + Ile164AlaSer160Asp + Thr163Ser + Ile164GlyAsn154Glu + Thr161Pro + Ile164MetThr163Asn + Ile164Asn + Gly165ProAsn157Glu + Asn162Gln + Ile164ThrAsn162Gln + Ile164Thr + Gly165GluAsn154Gln + Gly159Asp + Tyr166ThrAsn157Gln + Thr161Gly + Thr163ProSer158Glu + Thr163Ser + Gly165SerGly153Pro + Asn157Asp + Thr163AsnGly156Pro + Ser158Asp + Gly165GlnAsn157Gln + Thr163Gly + Gly165ProSer155Glu + Gly159Ser + Tyr166ThrGly153Gln + Asn154Gln + Gly165GluGly153Gln + Asn154Ser + Gly159ProThr161Gly + Asn162Asp + Gly165AsnSer158Glu + Gly159Ser + Gly165GlnSer155Asp + Gly159Asn + Ile164SerAsn157Gln + Thr163Gly + Gly165GluGly153Ser + Gly159Ser + Asn162AspGly153Ser + Ser158Glu + Ile164ThrGly153Asp + Thr161Ser + Gly165AsnThr161Ser + Ile164Ser + Tyr166ProSer155Asp + Gly156Asn + Ile164AsnAsn154Glu + Gly159Gln + Tyr166IleGly153Gln + Gly156Gln + Thr161AspGly159Asn + Thr163Asn + Tyr166SerSer155Asp + Thr163Pro + Ile164GlyGly153Ser + Ser160Asp + Ile164ProAsn162Glu + Ile164Gln + Tyr166GlnGly156Glu + Asn157Glu + Thr161GlyGly159Asp + Ser160Glu + Ile164GlnSer155Asp + Gly156Glu + Thr163AsnSer155Glu + Gly156Asp + Gly165SerSer155Glu + Gly156Asp + Ile164MetSer160Glu + Thr161Asp + Gly165AsnAsn162Asp + Thr163Asp + Gly165Asn
Asn157Asp + Ser158Glu + Thr161GluGly159Asp + Thr161Glu + Thr163ProGly153Asp + Asn154Ser + Gly165AspGly153Glu + Asn162Gln + Gly165AspAsn154Ser + Thr161Glu + Thr163AspSer158Glu + Ser160Asp + Thr163AsnSer158Glu + Ser160Glu + Thr163GlySer158Asp + Ser160Asp + Ile164GlyAsn157Asp + Gly159Glu + Thr163GlnThr163Glu + Gly165Asp + Tyr166ProSer160Asp + Asn162Glu + Gly165AsnAsn154Glu + Gly156Asp + Asn157GluGly159Asp + Ser160Asp + Asn162GluAsn154Glu + Ser155Glu + Asn157Asp表17环4-四突变变异体Gly153Pro + Gly159Ser + Thr163Pro + Gly165AsnAsn154Gln + Asn162Glu + Ile164Cys + Gly165ProAsn154Ser + Asn157Asp + Thr161Gly + Ile164AsnGly159Glu + Thr161Gln + Thr163Asn + Gly165GlnGly159Ser + Ser160Glu + Thr163Gln + Tyr166IleGly156Ser + Ser160Glu + Thr161Asn + Gly165ProGly153Ser + Gly159Ser + Ser160Asp + Gly165SerGly153Asn + Gly159Glu + Ile164Cys + Gly165ProAsn154Ser + Gly156Glu + Thr161Pro + Tyr166HisGly153Ser + Ser160Glu + Gly165Asn + Tyr166CysAsn157Gln + Thr163Gly + Ile164His + Gly165GlnAsn154Ser + Ser155Glu + Asn162Ser + Ile164HisGly156Pro + Asn157Gln + Ser160Glu + Gly165SerGly153Gln + Ser155Asp + Thr161Gln + Tyr166GlyAsn157Ser + Thr161Gln + Thr163Asp + Ile164ThrGly153Ser + Ser160Asp + Asn162Gln + Tyr166ThrGly153Pro + Ash154Gln + Asn157Gln + Ser160GluGly156Glu + Asn162Gln + Thr163Ser + Gly165GlnGly156Asn + Asn157Glu + Thr161Gly + Thr163SerGly153Ser + Ser158Glu + Gly159Asp + Thr163GlyAsn154Gln + Ser158Asp + Gly159Asp + Thr163AsnSer158Asp + Gly159Asp + Thr163Asn + Tyr166ProGly159Asp + Ser160Glu + Thr163Ser + Gly165SerAsn154Glu + Ser155Asp + Thr163Gln + Tyr165MetGly153Glu + Asn154Asp + Ser155Glu + Gly159AsnSer158Glu + Gly159Glu + Ser160Asp + Gly165AsnSer158Asp + Gly159Asp + Ser160Asp + Asn162GlnAsn157Ser + Ser160Asp + Thr161Glu + Asn162GluGly156Asn + Ser158Asp + Ser160Glu + Thr161Glu
Gly159Glu + Thr161Asp + Thr163Asn + Tyr166ValGly156Pro + Gly159Glu + Thr161Asp + Gly165ProGly153Glu + Gly156Pro + Gly159Gln + Gly165AspGly153Glu + Asn157Gln + Gly159Gln + Gly165AspGly153Asn + Ser155Glu + Thr163Glu + Ile164GlyAsn157Glu + Ser158Asp + Ser160Asp + Thr161AsnAsn154Ser + Asn157Ser + Ser158Asp + Ser160GluSer158Glu + Gly159Gln + Ser160Glu + Thr163GlnSer158Asp + Ser160Glu + Ile164Ala + Tyr166GlnGly155Pro + Asn157Gln + Ser158Glu + Ser160AspGly153Gln + Gly156Pro + Ser158Glu + Ser160AspGly156Ser + Asn157Glu + Gly159Glu + Ile164LeuThr163Asp + Ile164Cys + Gly165Asp + Tyr166CysSer160Asp + Asn162Asp + Ile164Asn + Tyr166ThrAsn157Ser + Ser160Glu + Asn162Asp + Ile164MetSer155Glu + Asn157Asp + Asn162Ser + Gly165ProGly153Ser + Asn154Glu + Ser155Glu + Asn157GluGly153Pro + Asn154Asp + Ser155Asp + Asn157GluGly153Gln + Ser158Glu + Ser160Asp + Asn162GluAsn157Asp + Ser160Glu + Thr161Ser + Asn162GluGly156Asp + Asn157Asp + Thr161Ser + Asn162AspAsn154Gln + Asn157Asp + Ser160Glu + Tyr166SerGly153Gln + Gly156Glu + Ser158Glu + Asn162GluGly156Glu + Gly159Pro + Thr163Asp + Gly165AspSer155Glu + Thr161Ser + Thr163Ser + Gly165AspAsn154Asp + Thr163Gly + Gly165Glu + Tyr166SerSer155Asp + Asn157Glu + Gly159Glu + Gly165GlnAsn162Glu + Ile164Ala + Gly165Asp + Tyr166ThrGly156Gln + Asn162Asp + Gly165Asp + Tyr166AsnSer155Glu + Ser158Asp + Thr161Asn + Tyr166MetSer155Glu + Ser158Glu + Gly159Gln + Ile164Gly表18环5-单突变变异体Ala186AsnAla186AspAla186GlnAla186GluAla186GlyAla186HisAla186ProAla186SerAla186ThrSer187AspSer187GluPhe188Ala
Phe188AsnPhe188AspPhe188CysPhe188GlnPhe188GluPhe188GlyPhe188HisPhe188IlePhe188LeuPhe188MetPhe188ProPhe188SerPhe188ThrPhe188TyrPhe188ValSer189AspSer189GluSer190AspSer190Glu表19环5-双突变变异体Ala186Asp + Phe188GlnAla186Gly + Ser187GluAla186Gln + Ser187AspAla186His + Ser190GluSer187Asp + Phe188CysSer187Asp + Phe188ThrPhe188Gly + Ser190GluAla186Asn + Phe188SerAla186Asp + Phe188IleAla186Ser + Phe188GluAla186Thr + Phe188LeuSer187Glu + Phe188SerSer187Glu + Phe188ProSer187Asp + Phe188GlnAla186Thr + Ser190GluPhe188His + Ser190AspAla186Gln + Ser190AspPhe188Leu + Ser190GluAla186His + Ser190AspAla186Pro + Ser187GluPhe188Ile + Ser190AspAla186Asp + Phe188AsnAla186Ser + Ser187GluSer187Glu + Phe188Leu
Ala186Asn + Phe188IlePhe188Tyr + Ser190GluAla186Pro + Ser190GluAla186Thr + Phe188AspAla186His + Ser187AspAla186Gln + Phe188ThrPhe188Cys + Ser190GluSer187Glu + Phe188ThrSer187Asp + Phe188MetAla186Asn + Ser190AspPhe188Val + Ser190AspAla186Pro + Phe188SerAla186Asp + Phe188SerSer187Glu + Phe188AsnAla186Thr + Ser187AspAla186Thr + Phe188AsnAla186Gly + Phe188LeuPhe188Asn + Ser190GluAla186Thr + Ser187GluAla186Thr + Ser190AspSer187Asp + Phe188AlaAla186Asn + Ser187GluAla186Gln + Phe188TyrPhe188Ile + Ser190GluPhe188Thr + Ser190AspAla186Ser + Ser187AspPhe188Ala + Ser190AspPhe188Gln + Ser190GluAla186Gly + Ser190AspAla186Glu + Phe188ThrAla186Pro + Phe188AlaPhe188Asn + Ser190AspAla186Gly + Phe188ValPhe188Leu + Ser190AspAla186Asn + Phe188MetAla186Gln + Phe188Glu表20环5-三突变变异体Ala186Pro + Phe188Cys + Ser190GluAla186Gln + Phe188Val + Ser190GluAla186Thr + Phe188Asn + Ser190GluAla186Gly + Phe188Gln + Ser190GluAla186Pro + Phe188Gln + Ser190GluAla186Gln + Phe188Asn + Ser190AspAla186Asn + Ser187Asp + Phe188Leu
Ala186Gln + Ser187Asp + Phe188ProAla186Gln + Ser187Glu + Phe188MetAla186Pro + Phe188Val + Ser190GluAla186His + Phe188Met + Ser190AspAla186Asn + Ser187Glu + Phe188IleAla186Gln + Phe188Ala + Ser190GluAla186Pro + Phe188Thr + Ser190AspAla186Asn + Ser187Glu + Phe188ThrAla186Ser + Phe188Ala + Ser190GluAla186His + Phe188Pro + Ser190AspAla186His + Ser187Asp + Phe188LeuAla186Pro + Ser187Glu + Phe188ValAla186Asn + Ser187Asp + Phe188HisAla186Gly + Ser187Glu + Phe188LeuAla186Pro + Ser187Asp + Phe188HisAla186Gln + Phe188Ile + Ser190AspAla186Pro + Phe188Ile + Ser190AspAla186Gln + Phe188Thr + Ser190AspAla186Gln + Phe188His + Ser190GluAla186Gly + Phe188Ile + Ser190AspAla186Thr + Ser187Glu + Phe188TyrAla186His + Ser187Asp + Phe188SerAla186His + Ser187Glu + Phe188SerAla186His + Phe188Gly + Ser190GluAla186Pro + Ser187Glu + Phe188ProAla186Gln + Ser187Glu + Phe188TyrAla186Asn + Phe188Cys + Ser190AspAla186Gly + Phe188Gly + Ser190AspAla186Gln + Ser187Asp + Phe188ValAla186Pro + Ser187Glu + Phe188GlyAla186Glu + Ser187Glu + Phe188IleAla186Asp + Ser187Glu + Phe188AlaAla186Asp + Ser187Asp + Phe188ThrAla186Glu + Ser187Asp + Phe188ThrAla186Glu + Ser187Glu + Phe188TyrAla186Asp + Ser187Asp + Phe188SerAla186Asp + Ser187Glu + Phe188IleAla186Pro + Ser187Asp + Phe188AspAla186Gln + Ser187Glu + Phe188GluAla186Gln + Ser187Glu + Phe188AspAla186Glu + Ser187Glu + Phe188AspAla186Glu + Ser187Asp + Phe188GluAla186Thr + Phe188Asp + Ser190GluAla186Ser + Phe188Asp + Ser190AspAla186Gly + Phe188Asp + Ser190GluSer187Asp + Phe188Glu + Ser190AspSer187Glu + Phe188Glu + Ser190Asp
Ser187Asp + Phe188Glu + Ser190GluSer187Glu + Phe188Glu + Ser190GluAla186Glu + Phe188Glu + Ser190GluAla186Glu + Phe188Asp + Ser190AspAla186Asp + Ser187Asp + Ser190GluAla186Asp + Ser187Glu + Ser190Asp表21环5-四突变变异体Ala186Thr + Ser187Glu + Phe188Glu + Ser190AspAla186Gln + Ser187Asp + Phe188Asp + Ser190AspAla186Gly + Ser187Asp + Phe188Glu + Ser190AspAla186Thr + Ser187Asp + Phe188Glu + Ser190GluAla186His + Ser187Glu + Phe188Glu + Ser190AspAla186Gln + Ser187Glu + Phe188Glu + Ser190GluAla186Thr + Ser187Glu + Phe188Asp + Ser190AspAla186Asn + Ser187Glu + Phe188Asp + Ser190AspAla186Ser + Ser187Glu + Phe188Glu + Ser190GluAla186Asn + Ser187Asp + Phe188Asp + Ser190AspAla186Ser + Ser187Asp + Phe188Asp + Ser190GluAla186Asn + Ser187Asp + Phe188Asp + Ser190GluAla186His + Ser187Glu + Phe188Glu + Ser190GluAla186Gln + Ser187Asp + Phe188Glu + Ser190AspAla186Gly + Ser187Asp + Phe188Glu + Ser190GluAla186Pro + Ser187Asp + Phe188Asp + Ser190GluAla186Gly + Ser187Asp + Phe188Asp + Ser190GluAla186Ser + Ser187Glu + Phe188Glu + Ser190AspAla186Thr + Ser187Asp + Phe188Asp + Ser190AspAla186Gly + Ser187Glu + Phe188Asp + Ser190GluAla186Gly + Ser187Asp + Phe188Asp + Ser190AspAla186Asp + Ser187Asp + Phe188Asn + Ser190AspAla186Glu + Ser187Glu + Phe188Tyr + Ser190GluAla186Asp + Ser187Asp + Phe188Ser + Ser190GluAla186Asp + Ser187Glu + Phe188Gln + Ser190GluAla186Glu + Ser187Asp + Phe188Ala + Ser190GluAla186Glu + Ser187Glu + Phe188Gly + Ser190GluAla186Asp + Ser187Asp + Phe188Ala + Ser190AspAla186Glu + Ser187Glu + Phe188Cys + Ser190GluAla186Glu + Ser187Asp + Phe188Ile + Ser190AspAla186Glu + Ser187Glu + Phe188Ala + Ser190GluAla186Glu + Ser187Glu + Phe188His + Ser190AspAla186Asp + Ser187Asp + Phe188Leu + Ser190GluAla186Asp + Ser187Asp + Phe188Gln + Ser190GluAla186Asp + Ser187Glu + Phe188Asn + Ser190AspAla186Asp + Ser187Asp + Phe188Cys + Ser190GluAla186Asp + Ser187Asp + Phe188Thr + Ser190Asp
Ala186Glu + Ser187Glu + Phe188Leu + Ser190AspAla186Glu + Ser187Glu + Phe188Leu + Ser190GluAla186Asp + Ser187Asp + Phe188Pro + Ser190GluAla186Asp + Ser187Glu + Phe188Val + Ser190AspAla185Asp + Ser187Asp + Phe188His + Ser190GluAla186Glu + Ser187Glu + Phe188Pro + Ser190GluAla186Asp + Ser187Glu + Phe188Ser + Ser190GluAla186Glu + Ser187Glu + Phe188Val + Ser190GluAla186Asp + Ser187Glu + Phe188Gln + Ser190AspAla186Asp + Ser187Glu + Phe188Asn + Ser190GluAla186Asp + Ser187Glu + Phe188Gly + Ser190GluAla186Asp + Ser187Asp + Phe188Cys + Ser190AspAla186Asp + Ser187Glu + Phe188Tyr + Ser190GluAla186Glu + Ser187Asp + Phe188Thr + Ser190AspAla186Glu + Ser187Glu + Phe188Thr + Ser190GluAla186Glu + Ser187Glu + Phe188Met + Ser190GluAla186Asp + Ser187Asp + Phe188Gly + Ser190GluAla186Asp + Ser187Glu + Phe188His + Ser190GluAla186Asp + Ser187Glu + Phe188Met + Ser190GluAla186Glu + Ser187Asp + Phe188Ser + Ser190AspAla186Asn + Ser187Glu + Phe188Met + Ser190AspAla186Gln + Ser187Asp + Phe188His + Ser190AspAla186Asn + Ser187Glu + Phe188Ser + Ser190Glu表22环6-单突变变异体Ala199AsnAla199AspAla199GlnAla199GluAla199GlyAla199HisAla199ProAla199SerAla199ThrPro200AsnPro200AspPro200GlnPro200GluPro200GlyPro200SerGly201AsnGly201AspGly201GlnGly201GluGly201Pro
Gly201SerAla202AsnAla202AspAla202GlnAla202GlnAla202GlyAla202HisAla202ProAla202SerAla202ThrGly203AsnGly203AspGly203GlnGly203GluGly203ProGly203SerVal204AlaVal204AsnVal204AspVal204CysVal204GlnVal204GluVal204GlyVal204HisVal204MetVal204ProVal204SerVal204ThrTyr205AlaTyr205AsnTyr205AspTyr205CysTyr205GlnTyr205GluTyr205GlyTyr205HisTyr205IleTyr205LeuTyr205MetTyr205ProTyr205SerTyr205ThrTyr205ValSer206AspSer206GluThr207AsnThr207Asp
Thr207GlnThr207GluThr207GlyThr207ProThr207SerTyr208AlaTyr208AsnTyr208AspTyr208CysTyr208GlnTyr208GluTyr208GlyTyr208HisTyr208IleTyr208LeuTyr208MetTyr208ProTyr208SerTyr208ThrTyr208ValPro209AsnPro209AspPro209GlnPro209GluPro209GlyPro209SerThr210AsnThr210AspThr210GlnThr210GluThr210GlyThr210ProThr210SerAsn211AspAsn211GlnAsn211GluAsn211SerThr212AsnThr212AspThr212Glnlhr212GluThr212GlyThr212ProThr212SerTyr213AlaTyr213AsnTyr213Asp
Tyr213CysTyr213GlnTyr213GluTyr213GlyTyr213HisTyr213IleTyr213LeuTyr213MetTyr213ProTyr213SerTyr213ThrTyr213ValAla214AsnAla214AspAla214GlnAla214GluAla214GlyAla214HisAla214ProAla214SerAla214ThrThr215AsnThr215AspThr215GlnThr215GluThr215GlyThr215ProThr215SerLeu216AlaLeu216AsnLeu216AspLeu216CysLeu216GlnLeu216GluLeu216GlyLeu216HisLeu216IleLeu216MetLeu216ProLeu216SerLeu216ThrLeu216ValAsn217AspAsn217GlnAsn217GluAsn217SerGly218Asn
Gly218AspGly218GlnGly218GluGly218ProGly218SerThr219AsnThr219AspThr219GlnThr219GluThr219GlyThr219ProThr219Ser表23环6-双突变变异体Gly218Gln + Thr219GlnVal204Ala + Thr207GlnTyr213Val + Gly218ProPro209Gly + Tyr213IleThr215Ser + Thr219SerLeu216Ala + Asn217GlnPro209Asp + Thr212SerAla199Asn + Leu216IleAsn217Glu + Thr219AsnTyr208Ile + Thr215AsnAla202Gly + Pro209GluGly201Ser + Thr219GlyThr215Pro + Gly218SerTyr205Leu + Leu216HisVal204Asn + Ala214AsnAsn217Ser + Gly218ProAla202His + Tyr208ValGly201Gln + Tyr213LeuTyr213Cys + Thr219GlyPro200Asn + Thr210ProAla202Asp + Thr219AsnGly201Pro + Val204ProAla214Thr + Thr219GlnThr207Gln + Leu216AspPro200Gln + Tyr213MetVal204Ala + Thr215GluThr212Gln + Thr215AsnLeu216His + Asn217GluAla202Thr + Ala214AspVal204Pro + Thr207GlnGly203Asp + Tyr205Leu
Tyr205His + Asn211AspAsn211Gln + Thr212AsnGly203Pro + Thr219ProPro200Gly + Pro209AspThr210Asp + Gly218ProGly203Asp + Gly218SerTyr205Glu + Gly218ProThr212Gln + Thr219ProAla214Ser + Asn217GluAla202Asn + Leu216SerVal204Thr + Ala214ProPro200Asn + Asr211AspTyr205Gly + Thr219ProAla199His + Asn217SerGly201Ser + Asn211AspGly203Ser + Tyr205AlaAla202Pro + Pro209GluTyr213Pro + Gly218GluPro200Gly + Ala202GluThr210Gly + Asn211AspVal204Asn + Thr215AsnPro200Asn + Tyr208LeuAla202Pro + Ala214ThrTyr205Cys + Leu216HisLeu216Ser + Gly218GlnThr215Glu + Thr219GlnTyr205Leu + Pro209GluAla214Glu + Thr215ProGly203Glu + Gly218GlnTyr205Cys + Thr212ProLeu216Asn + Thr219AsnAla199Ser + Thr219AsnGly201Gln + Tyr213ValGly203Asn + Thr215GlnPro209Glu + Asn211GlnAla199Gly + Asn211AspAla214Glu + Leu216MetVal204Pro + Asn211AspAla202Glu + Gly218GlnTyr208Ile + Gly218GluTyr205Ser + Leu216ThrAsn211Glu + Tyr213GlyThr212Asn + Thr219AsnAsn217Asp + Thr219GlnThr207Asn + Asn217AspGly201Ser + Tyr208GlnThr210Pro + Thr212Gly
Val204Asn + Thr210GlyAla199His + Tyr208ValTyr208Gly + Tyr213ValGly201Pro + Ala214AspGly201Gln + Leu216GlyGly201Gln + Thr207AsnGly203Asn + Gly218GluTyr208Asn + Thr219SerThr207Gly + Leu216GluGly218Glu + Thr219AsnAla199Gln + Thr212GluThr212Asp + Ala214GlyGly203Asn + Ala214SerAsn211Ser + Thr212GlnPro209Asp + Tyr213ThrGly201Gln + Ala202ThrVal204Thr + Tyr208CysGly201Asn + Ala202AsnAla199His + Thr210AspVal204Ser + Thr212AspTyr208Ser + Thr219GlnTyr205Pro + Thr215AspAsn211Gln + Gly218GlnAsn217Asp + Gly218ProAla202Gln + Thr210ProTyr205Asn + Gly218GlnGly201Gln + Thr219SerAla202Asp + Thr207AsnThr212Gly + Asn217GlnThr215Asn + Thr219SerAla214Glu + Thr215GlyThr210Asn + Tyr213GlnAla199Gln + Tyr205GluTyr205His + Thr210AspGly201Asn + Gly203AspGly203Ser + Pro209AspGly201Gln + Thr210GluPro209Asn + Thr215SerThr215Gln + Thr219GlyGly201Gln + Thr215AspTyr208Gly + Tyr213ProGly201Asn + Tyr213AsnAla202Pro + Pro209AsnAla199Thr + Tyr208MetThr210Gln + Gly218AsnThr210Pro + Asn211AspAsn211Asp + Gly218Pro
Gly201Asn + Tyr205GlyAla202Gln + Tyr213GluAla199His + Ala202AspTyr205Ser + Tyr213GluGly201Gln + Pro209AspAla202Ser + Thr219GlnLeu216Met + Thr219AsnVal204Ser + Tyr205SerVal204Thr + Tyr208SerGly203Asn + Ala214ThrThr210Gly + Thr219AsnPro200Gly + Thr210ProAla214Glu + Gly218ProPro200Gly + Thr219GlnAla202Ser + Asn211AspAla199Thr + Val204GlnAla199Gln + Pro200SerPro209Ser + Leu216ThrTyr205Pro + Leu216AlaGly201Pro + Pro209GlyThr207Asn + Thr215GluPro200Ser + Tyr205AlaThr210Gln + Asn217GlnAla199Asn + Gly203GlnGly201Asn + Thr207ProGly203Asp + Tyr208AsnThr207Asn + Asn211AspTyr205Leu + Gly218AspGly203Ser + Thr210ProTyr205Cys + Asn211SerAla214Glu + Asn217SerAla199Pro + Gly201GlnGly201Asn + Tyr213MetTyr205His + Tyr208GlyGly203Glu + Thr210SerThr207Asn + Ala214SerTyr205Val + Thr219SerVal204Asn + Thr207ProThr207Ser + Asn211AspTyr205Leu + Gly218GluAsn211Ser + Thr219ProTyr205Gln + Asn211SerPro200Gly + Val204AsnAla202Glu + Tyr213HisGly201Pro + Gly218GlnAla214Asn + Asn217GlnGly201Asn + Asn211Asp
Pro209Ser + Thr212GlnGly203Gln + Val204GlnGly201Gln + Thr215ProGly201Asn + Ala202SerThr207Gln + Thr212AspVal204Gly + Thr212AspGly201Asn + Thr210AspThr210Asp + Tyr213Cys表24环6-三突变变异体Pro200Gly + Thr212Ser + Tyr213MetGly201Pro + Pro209Glu + Thr212GlyGly203Asp + Val204Met + Asn217GlnPro200Gly + Thr212Ser + Ala214AspAla199Gly + Tyr213Asp + Gly218SerGly201Asn + Thr207Ser + Thr215GluPro200Ser + Ala214Gly + Asn217GluAla199Pro + Thr207Ser + Thr210ProGly201Asn + Thr210Ser + Asn211GluGly203Asn + Thr207Asn + Asn217GluGly201Asn + Tyr205Pro + Thr207SerPro200Asn + Gly201Gln + Gly203SerPro200Gln + Asn211Glu + Tyr213AlaAla202His + Thr212Asp + Tyr213HisAla199Thr + Gly201Asn + Asn211SerPro200Ser + Gly201Gln + Pro209AsnAla202Gly + Tyr205Pro + Asn211SerGly203Gln + Tyr205Val + Thr210ProGly203Glu + Asn211Gln + Asn217SerPro200Asn + Pro209Glu + Asn211SerGly201Pro + Thr210Ser + Tyr213ThrTyr205Ile + Leu216Asp + Thr219GlyPro200Ser + Val204Met + Pro209AspTyr205Asp + Thr207Gly + Tyr208GlnPro200Ser + Thr210Asp + Thr212ProGly201Gln + Asn211Asp + Tyr213GlyTyr208Val + Thr210Gly + Leu216ValPro209Gly + Thr215Pro + Leu216AlaVal204His + Thr215Glu + Leu216CysAla199Pro + Gly203Ser + Thr210SerGly201Asn + Tyr205Met + Thr210AspPro200Gly + TyT205Cys + Thr212AsnAla199Pro + Pro200Ser + Leu216GluGly201Asn + Tyr205Asp + Tyr213llePro200Ser + Ala202Gly + Thr210Asp
Ala202Asn + Val204Ser + Gly218GluGly203Gln + Val204Cys + Thr207SerVal204Cys + Thr207Asn + Thr212AspGly201Pro + Gly203Ser + Tyr208GlyGly203Asn + Tyr208Cys + Leu216MetThr210Gln + Thr215Asp + Thr219SerPro209Asp + Tyr213Leu + Ala214ThrAla202Asp + Tyr213Pro + Thr215GlnThr215Glu + Gly218Ser + Thr219GlnPro200Gly + Val204His + Thr207SerGly203Ser + Pro209Asp + Thr219SerAla202Asn + Thr207Gln + Ala214HisThr207Ser + Ala214Glu + Leu216SerPro200Asn + Pro209Asn + Thr210ProPro209Gln + Tyr213His + Ala214ThrThr210Asn + Thr215Pro + Leu216GlnGly203Gln + Val204Ser + Thr210GluPro200Asn + Asn211Gln + Gly218SerPro200Gln + Thr207Gln + Leu216GluTyr213Pro + Gly218Glu + Thr219AsnTyr208Cys + Thr210Asp + Thr219SerPro200Gln + Tyr205His + Thr219SerPro209Asp + Thr212Gln + Thr219GlnGly201Asn + Val204Asn + Thr210AspPro200Gly + Tyr208Leu + Thr210AsnPro200Gln + Thr207Gln + Thr219GlnTyr205Val + Pro209Ser + Thr210AspGly203Pro + Val204His + Tyr213GluGly203Asn + Pro209Asn + Tyr213HisAla199Asn + Gly201Asn + Leu216AspPro209Asp + Tyr213His + Gly218SerGly201Ser + Gly203Asp + Asn211SerGly203Asn + Val204Cys + Thr212GlyGly201Pro + Tyr208Met + Leu216GluGly201Ser + Val204Gln + Leu216HisAla199Gly + Ala202Pro + Leu216SerGly203Gln + Tyr205Pro + Thr219GlyTyr205Gly + Tyr213Val + Asn217AspGly201Asn + Ala214His + Thr215GlnPro200Gly + Ala202Gln + Tyr213CysGly203Pro + Tyr208Met + Thr219ProPro200Asn + Tyr205Leu + Gly218GluPro209Gly + Thr212Glu + Tyr213ThrPro200Gln + Thr210Pro + Thr219AsnGly201Ser + Gly218Pro + Thr219AsnPro200Ser + Gly201Pro + Thr219GlyThr207Pro + Tyr213Ala + Leu216Glu
Ala199Ser + Thr207Asn + Thr212AspThr207Gln + Tyr208Ile + Thr210AspTyr213Ser + Thr215Gln + Thr219ProPro200Gly + Tyr208Ile + Ala214SerGly201Asn + Pro209Ser + Thr215AspPro200Ser + Pro209Asp + Tyr213MetTyr208Gly + Thr210Glu + Thr215SerAla202Ser + Ala214Asp + Thr219SerVal204Pro + Pro209Asn + Thr219AsnThr207Asn + Pro209Asn + Gly218SerPro200Gly + Leu216Cys + Gly218SerPro209Gly + Leu216Val + Gly218GluAsn211Gln + Tyr213Thr + Thr215SerTyr213Val + Thr215Glu + Gly218SerTyr213Pro + Gly218Gln + Thr219SerGly201Pro + Ala202Thr + Val204CysAla202Ser + Tyr208Leu + Pro209AspTyr208Thr + Thr210Asn + Ala214ThrTyr205Pro + Pro209Ser + Thr215GlnTyr205Cys + Tyr208Gly + Leu216GluAla199Gly + Gly203Asp + Pro209AsnVal204Gln + Thr207Gly + Gly218GlnAla202His + Gly218Asp + Thr219GlnGly203Asp + Ash211Ser + Leu216MetPro200Ser + Val204Cys + Thr215GluThr207Asn + Leu216Pro + Asn217GluAla199His + Tyr205His + Thr212ProAla199Gln + Gly203Asp + Thr219GlyAla199His + Pro200Gln + Asn217AspVal204Ser + Tyr213Leu + Thr219GlnVal204Ser + Pro209Ser + Leu216GluAla202Gly + Pro209Asn + Thr219AsnThr215Gln + Gly218Asn + Thr219GlnAla199Pro + Ala214Ser + Leu216ValGly203Asn + Thr215Asn + Gly218GluThr207Pro + Tyr208Ala + Thr219AsnAla199Ser + Thr210Ser + Gly218GlnPro209Asn + Thr210Ser + Asn211SerGly201Asn + Val204Cys + Tyr213ValGly201Ser + Val204Ser + Pro209AsnGly201Pro + Thr210Ser + Asn211SerTyr205Gln + Tyr208His + Thr210AspTyr208Ile + Thr215Ser + Gly218GluThr212Glu + Ala214His + Thr215GlnGly201Asn + Val204Gly + Tyr205GlnAla199Thr + Pro209Glu + Thr210SerPro209Gly + Thr210Asn + Gly218Glu
Gly203Pro + Pro209Asn + Thr210AsnGly203Glu + Tyr213Pro + Leu216ThrAla199Pro + Thr212Glu + Ala214ThrThr207Gln + Pro209Asn + Thr219SerAla199Thr + Tyr205Gln + Thr219ProVal204Met + Ala214Gly + Asn217GluGly201Gln + Val204Pro + Thr210GluTyr208Leu + Thr212Asp + Gly218AsnGly201Gln + Tyr205His + Thr210GluGly201Pro + Ala214Gly + Thr219SerGly203Asp + Tyr205Ala + Tyr208SerThr212Pro + Asn217Ser + Gly218AspTyr205Asp + Thr207Gly + Tyr208LeuGly201Pro + Ala202Glu + Leu216SerAla202His + Tyr208Pro + Thr215AspTyr208Thr + Thr212Glu + Thr219SerVal204Met + Pro209Gln + Leu216AspThr207Pro + Thr210Asn + Tyr213GluGly201Ser + Val204Gln + Asn211AspGly203Gln + Tyr205Asn + Tyr213SerAla199Thr + Thr210Asn + Tyr213GluTyr208Ile + Pro209Ser + Thr212ProTyr205Leu + Tyr208Thr + Ala214GluPro200Gln + Val204Gln + Ala214GluGly203Pro + Asn211Asp + Asn217GlnGly201Asn + Gly218Gln + Thr219ProAla199Gln + Gly203Gln + Ala214AspAla202Thr + Tyr205Cys + Tyr213IleGly201Gln + Val204Cys + Asn211GluTyr208Thr + Pro209Gly + Thr215AspThr210Glu + Asn211Gln + Tyr213LeuVal204Cys + Tyr208Pro + Thr219ProAla202Asp + Gly203Asn + Gly218AsnAla199Asn + Val204Gly + Tyr205ProGly203Pro + Val204Met + Tyr208HisPro200Gly + Tyr213Pro + Leu216CysVal204Ala + Ala214His + Asn217GluAla202Gln + Thr207Asn + Ala214GlyGly203Ser + Val204Gly + Tyr213ThrAsn211Asp + Leu216Cys + Thr219AsnPro200Asn + Thr212Gly + Gly218AsnTyr205His + Tyr208Leu + Thr215GlnGly203Ser + Tyr205His + Thr219SerVal204Asn + Thr212Pro + Thr215AspThr212Ser + Thr215Ser + Leu216AlaTyr208Leu + Thr212Glu + Leu216MetThr207Asn + Tyr208Leu + Leu216Asp
Thr210Gly + Thr212Pro + Asn217AspAsn211Ser + Ala214Asn + Leu216GluThr207Ser + Tyr208Thr + Ala214HisAla202Asp + Val204Ser + Tyr208Thr表25环6-四突变变异体Pro200Ser + Thr207Ser + Tyr213Cys + Gly218GlnAla202Gln + Tyr208Cys + Thr210Pro + Tyr213AsnGly203Asp + Tyr205Ile + Thr215Pro + Thr219SerGly201Gln + Gly203Gln + Val204Asn + Thr207AsnPro200Ser + Gly201Ser + Ala202Ser + Pro209GluGly201Ser + Thr207Pro + Thr215Asp + Thr219ProAla202Gln + Asn211Gln + Tyr213Ser + Thr219AsnGly201Pro + Tyr213Ala + Ala214Pro + Thr215SerAla199Gly + Pro200Gly + Ala202Glu + Gly218ProTyr205Thr + Tyr208Met + Thr210Ser + Asn217GlnGly201Ser + Ala214Ser + Leu216Gly + Gly218AsnPro200Gln + Gly203Gln + Asn211Glu + Thr215ProPro200Ser + Tyr208Leu + Asn211Ser + Gly218AspAla199Asn + Pro209Asn + Thr210Ser + Tyr213MetAla199His + Gly203Ser + Pro209Asp + Thr212GlnTyr205Gly + Thr212Gly + Thr215Asn + Asn217GlnAla199Gln + Tyr208Thr + Thr210Asn + Tyr213GlnGly203Ser + Pro209Asp + Asn211Ser + Ala214ThrPro209Asn + Thr215Ser + Gly218Asp + Thr219GlyAla199Pro + Thr207Gln + Thr210Glu + Thr215ProAla202Asn + Val204Gly + Thr207Gly + Thr212AspVal204Thr + Tyr208Leu + Asn211Ser + Asn217AspPro200Gly + Tyr205Val + Leu216Asp + Thr219GlyPro200Gly + Tyr208Ser + Ala214Ser + Thr219SerAla199Thr + Gly203Glu + Thr207Asn + Tyr213CysPro200Gly + Tyr213Thr + Asn217Gln + Thr219ProGly203Asn + Val204Thr + Thr207Asn + Thr210SerAla202Gly + Thr207Gln + Pro209Gln + Thr210AspAla199His + Gly201Gln + Tyr205Asp + Thr219GlnThr207Ser + Asn211Asp + Tyr213Thr + Leu216SerAla199Ser + Thr210Glu + Asn211Gln + Leu216IleGly201Ser + Gly203Ser + Thr212Pro + Ala214GluPro200Gly + Gly201Gln + Gly203Gln + Leu216HisAla199Pro + Gly201Gln + Gly203Pro + Thr212GluAla199His + Gly201Asn + Val204Thr + Tyr208SerVal204Thr + Tyr208Ile + Ala214Glu + Thr219ProAla199Ser + Tyr205Pro + Thr212Ser + Thr219GlnVal204Met + Leu216Cys + Asn217Ser + Thr219GlyGly201Ser + Tyr205His + Pro209Gln + Thr219Ser
Pro200Gln + Pro209Asn + Asn211Asp + Thr219GlyVal204Met + Thr210Gly + Thr212Asp + Ala214SerPro200Gly + Thr207Asn + Asn211Glu + Leu216HisTyr208Ala + Thr210Asp + Thr215Gln + Thr219GlyGly203Asn + Thr207Gln + Pro209Asp + Thr215GlyAla199Ser + Pro209Glu + Leu216His + Thr219ProGly203Asp + Tyr205His + Tyr213Val + Ala214ThrPro200Asn + Thr212Asn + Leu216His + Gly218ProAla199Ser + Pro209Gln + Thr215Gln + Asn217GluGly203Asp + Thr207Pro + Thr212Gly + Ala214GlnGly201Pro + Gly203Gln + Thr207Pro + Ala214AspVal204Gln + Ala214Asp + Leu216Thr + Gly218SerThr207Asn + Tyr208Pro + Pro209Asp + Tyr213AsnPro209Asp + Asn211Gln + Thr215Gln + Gly218ProVal204Gln + Asn211Ser + Asn217Gln + Gly218ProGly201Gln + Ala202His + Asn217Asp + Gly218GlnVal204Thr + Tyr208Leu + Ala214Pro + Gly218AspGly201Ser + Thr207Pro + Thr212Ser + Thr215ProAla202Gly + Val204Gly + Asn211Glu + Leu216GlyAla199Ser + Gly201Gln + Ala214Ser + Asn217SerAla202Gln + Thr207Pro + Pro209Glu + Tyr213ProAla199Thr + Ala202Ser + Val204Ala + Thr207GlnAla199His + Thr207Ser + Pro209Gln + Ala214GluAla202His + Gly203Asp + Val204Pro + Gly218ProAla202Asn + Gly203Glu + Val204Gln + Thr210GlnPro200Asn + Val204Gly + Tyr205Gln + Thr207SerAla202Glu + Gly203Pro + Thr207Gly + Thr219GlyGly201Asn + Tyr208Val + Gly218Glu + Thr219AsnPro200Ser + Gly201Gln + Tyr205Asp + Thr219GlyPro200Ser + Thr207Gly + Ala214Pro + Thr215AspVal204Ala + Thr207Gln + Asn211Asp + Thr215ProAla202Thr + Val204Asn + Thr207Ser + Pro209AsnAla202Asn + Val204Thr + Thr207Gly + Pro209SerAla199Pro + Thr210Gln + Tyr213Cys + Ala214ThrAla199Pro + Pro200Gln + Ala214Glu + Thr215AsnPro200Ser + Val204Gln + Thr207Ser + Leu216ThrAla199Ser + Thr207Asn + Leu216Gln + Gly218GlnGly201Ser + Tyr208Ala + Ala214Ser + Leu216GlnVal204Thr + Thr207Ser + Thr215Asp + Asn217SerGly201Asn + Thr207Pro + Tyr213Met + Asn217AspVal204Ala + Asn211Gln + Thr215Glu + Gly218SerPro200Gly + Pro209Ser + Thr212Gly + Asn217GluAla199Pro + Tyr205Ile + Thr210Gly + Thr219GlyPro200Gly + Tyr205Ala + Thr212Pro + Tyr213GluGly203Ser + Thr212Ser + Leu216Ala + Asn217AspGly201Pro + Gly203Gln + Pro209Asp + Thr215GlyAla202His + Gly203Ser + Tyr208Cys + Gly218Asn
Ala199Pro + Ala202Gln + Val204Gly + Thr212GlyAla199Asn + Pro200Gly + Gly201Gln + Val204GlnGly201Asn + Gly203Pro + Pro209Ser + Thr215AspGly201Asn + Thr210Ser + Tyr213Asp + Leu216GlnGly203Gln + Val204His + Gly218Gln + Thr219GlyGly201Asn + Tyr213Val + Ala214Glu + Thr219GlyAla202Glu + Thr207Ser + Thr215Ser + Thr219SerGly203Glu + Thr212Asn + Asn217Gln + Thr219SerGly203Ser + Thr210Asn + Thr212Glu + Leu216MetVal204Ala + Asn211Asp + Tyr213Met + Leu216IleThr207Pro + Tyr208Asn + Tyr213Pro + Leu216GlnGly201Gln + Gly203Glu + Tyr213Met + Ala214ThrPro200Gln + Thr212Ser + Tyr213Pro + Thr215AsnPro200Asn + Val204Asn + Asn211Glu + Tyr213GlnPro209Ser + Asn211Gln + Asn217Asp + Thr219AsnPro200Gly + Gly203Asn + Tyr205Val + Tyr208HisAla202Glu + Gly203Pro + Thr212Ser + Gly218AsnAla202Gln + Val204Gly + Pro209Asp + Thr215AsnTyr208Ala + Asn211Glu + Thr215Gln + Thr219SerTyT208Gln + Tyr213Ala + Leu216Thr + Asn217AspAla199Thr + Pro200Gln + Gly201Asn + Asn211AspAla199Thr + Pro200Ser + Gly201Asn + Tyr208GlyVal204Ser + Thr207Gly + Thr212Asp + Gly218GlnPro200Asn + Ala214His + Thr215Gly + Thr219AsnThr207Ser + Tyr208Ser + Leu216Asn + Gly218AspPro200Gly + Gly203Glu + Thr212Gly + Gly218AsnPro200Ser + Gly201Ser + Tyr208Gln + Thr219AsnPro200Asn + Gly201Pro + Asn211Glu + Asn217GlnVal204Met + Thr207Asn + Asn217Gln + Thr219GlyPro200Gly + Gly201Pro + Thr210Glu + Leu216ThrPro200Asn + Gly203Pro + Val204Gln + Thr219GlnAla202Asp + Pro209Asn + Tyr213Cys + Leu216AlaPro200Gln + Gly201Ser + Ala202Glu + Tyr205GlyThr207Pro + Tyr208Asn + Asn211Ser + Tyr213SerAla202Ser + Val204Gly + Thr207Asn + Tyr213GlnGly201Ser + Gly203Gln + Thr212Ser + Leu216AspAla199Thr + Tyr205Met + Tyr208Val + Ala214ThrGly201Gln + Ala202Thr + Val204Cys + Tyr205LeuPro200Gln + Tyr205Cys + Thr210Glu + Thr212AsnPro200Gln + Tyr213Pro + Asn217Glu + Gly218AsnTyr208Pro + Tyr213His + Ala214Glu + Asn217SerPro200Ser + Ala202Gly + Tyr208Val + Thr212GlnPro200Asn + Tyr205Gly + Pro209Gln + Asn211SerTyr205Gly + Asn211Gln + Thr215Gly + Thr219GlyGly201Asn + Gly203Asn + Thr212Asp + Ala214GlyVal204Pro + Pro209Gly + Thr210Glu + Leu216ProTyr205Pro + Tyr213Ala + Gly218Asn + Thr219Pro
Ala199Gln + Val204His + Asn211Asp + Thr215GlyPro209Gln + Thr215Gln + Asn217Asp + Thr219AsnGly201Asn + Asn211Glu + Tyr213Ile + Asn217GlnGly201Ser + Thr207Asn + Tyr213Glu + Ala214GlyAla199Ser + Thr210Asn + Thr215Gln + Gly218ProVal204Asn + Tyr205Thr + Tyr208Met + Thr215ProVal204Gly + Tyr205Val + Thr210Asp + Thr215GlnAla202Ser + Val204Ala + Leu216Ser + Asn217AspAsn211Asp + Thr212Ser + Ala214Asn + Thr215GlyAla199His + Tyr208Leu + Asn217Gln + Thr219AsnAla202Pro + Tyr208Cys + Leu216Ala + Thr219AsnAla199Gly + Pro200Gly + Gly201Asn + Ala202AspPro200Ser + Ala202Gln + Thr210Gln + Leu216AspGly201Gln + Gly203Glu + Val204Gly + Leu216AsnVal204Pro + Tyr208Thr + Pro209Ser + Asn211GluTyr208Thr + Thr215Ser + Leu216Glu + Gly218GlnGly201Asn + Tyr205Ser + Tyr213Asp + Gly218ProAla202Gln + Gly203Gln + Tyr205Ile + Asn217GluThr212Ser + Tyr213Glu + Thr215Ser + Leu216CysGly203Glu + Thr212Pro + Tyr213Leu + Ala214SerPro200Ser + Gly201Gln + Ala202Asp + Thr210AsnVal204Gln + Thr210Asp + Tyr213Ser + Ala214ThrAla199Pro + Gly201Ser + Val204Gly + Asn217GlnPro200Asn + Ala202Ser + Thr207Gln + Tyr208MetPro200Gln + Gly203Pro + Ala214Glu + Gly218ProPro200Ser + Thr207Gly + Gly218Asn + Thr219GlyGly203Ser + Thr207Gln + Thr210Gln + Asn217SerGly201Ser + Thr210Glu + Asn211Glu + Thr219AsnThr207Gly + Thr210Asp + Asn211Asp + Gly218ProPro200Asn + Thr210Glu + Asn211Glu + Thr219ProVal204Ser + Thr210Asp + Asn211Glu + Thr219AsnAla199Ser + Tyr205Thr + Ala214Asp + Thr215AspPro200Asn + Thr215Asp + Leu216Asp + Thr219SerTyr208Asn + Tyr213His + Thr215Asp + Leu216AspGly201Ser + Asn211Glu + Thr212Asp + Ala214HisVal204Thr + Asn211Glu + Thr212Asp + Ala214ProPro200Gly + Thr212Gly + Asn217Glu + Gly218AspThr207Gln + Ala214Gly + Asn217Glu + Gly218GluThr207Asn + Pro209Gly + Asn217Asp + Gly218GluTyr213Thr + Leu216Val + Asn217Glu + Gly218GluAla199Thr + Ala202Asp + Gly203Glu + Gly218ProVal204His + Tyr205Asp + Thr212Gly + Thr215GluPro200Ser + TyT205Glu + Thr215Glu + Leu216CysAla202Glu + Gly203Asp + Asn217Glu + Thr21SProPro209Glu + Asn211Asp + Leu216His + Thr219ProThr207Pro + Tyr208Ser + Pro209Glu + Asn211AspVal204Thr + Pro209Asp + Asn211Asp + Thr212Pro
表26环6-五取代变异体Pro200Asn + Ala202Asn + Val204Gly + Asn211Asp + Thr219GlyPro200Gly + Gly201Pro + Pro209Glu + Asn211Ser + Thr212GlyPro200Ser + Asn211Gln + Ala214Gly + Asn217Glu + Thr219AsnGly201Asn + Gly203Ser + Thr207Ser + Tyr213Pro + Thr215GlyPro200Ser + Tyr205Pro + Tyr208Leu + Asn211Ser + Gly218AspPro200Gly + Pro209Gly + Thr212Asp + Thr215Asn + Leu216ThrGly201Gln + Ala202Pro + Thr212Asn + Ala214Gln + Thr215GluTyr205Pro + Tyr208Ser + Thr215Asp + Asn217Ser + Thr219GlnPro200Gly + Thr207Gly + Thr212Gln + Ala214Asn + Thr215AsnTyr205Ile + Thr207Pro + Tyr208Ala + Ala214Gln + Leu216AsnGly203Pro + Val204His + Thr207Gln + Tyr213Glu + Thr219SerTyr205Met + Thr207Gly + Pro209Glu + Thr215Ser + Thr219AsnPro200Gly + Thr207Gln + Asn211Asp + Thr215Pro + Gly218SerAla202His + Gly203Pro + Asn211Glu + Tyr213Gly + Thr219GlnAla199Gly + Tyr208Val + Thr210Glu + Tyr213Leu + Thr219GlnAla199His + Pro200Gln + Val204Cys + Thr215Asn + Asn217AspAla199Gly + Pro200Gln + Tyr205Ile + Thr210Gly + Thr215AspTyr208Met + Pro209Asn + Thr210Ser + Asn211Ser + Ala214GluPro200Asn + Gly201Gln + Pro209Asn + Leu216Ala + Gly218GluGly203Gln + Pro209Gly + Thr210Asn + Asn211Ser + Tyr213ValGly201Pro + Thr207Asn + Thr210Gln + Tyr213His + Ala214ThrPro200Gln + Gly201Ser + Val204Cys + Thr210Asn + Asn217GluAla199Pro + Gly203Gln + Tyr205Val + Asn211Asp + Thr215AsnAla202Asn + Val204Ser + Asn211Ser + Tyr213Asn + Leu216AspThr210Glu + Thr212Ser + Ala214Gly + Thr215Gln + Leu216IleAla199Asn + Ala202Asn + Val204Gly + Tyr205Met + Thr215AsnThr210Gln + Thr212Pro + Tyr213Thr + Leu216Gln + Asn217GlnAla199Gln + Pro200Gly + Ala202Pro + Thr212Gln + Thr215SerAla202Gln + Thr207Asn + Thr210Gln + Asn211Glu + Ala214GlyGly203Gln + Thr207Asn + Pro209Gly + Tyr213Asn + Gly218AspVal204Cys + Tyr205Thr + Thr207Asn + Leu216Thr + Thr219GlnGly201Gln + Val204His + Tyr208Ser + Thr212Ser + Thr215GlyPro200Gln + Ala202Asp + Tyr208Gly + Thr212Ser + Leu216ThrAla199Ser + Gly201Ser + Asn211Asp + Ala214Thr + Thr219GlyAla202Gln + Val204Ser + Tyr208Leu + Pro209Asp + Tyr213GlnAla199Ser + Tyr205Ala + Thr210Glu + Thr212Gly + Leu216AlaAla199His + Ala202Glu + Thr210Gly + Asn211Gln + Tyr213ProAla199Ser + Gly203Glu + Tyr213Met + Leu216Ala + Gly218SerGly201Gln + Gly203Ser + Thr207Asn + Gly218Pro + Thr219ProAla199Gly + Pro209Asp + Thr212Ser + Leu216Met + Gly218SerPro200Ser + Tyr205Ala + Ash211Ser + Thr212Glu + Thr215ProPro209Glu + Thr212Gly + Ala214Thr + Leu216Asn + Thr219ProGly203Asn + Tyr205Gln + Pro209Asn + Gly218Asn + Thr219Ser
Gly201Asn + Ala202Gln + Gly203Pro + Thr210Gly + Asn217SerPro200Ser + Gly201Pro + Tyr208Val + Thr210Gly + Thr212AsnAla199His + Gly201Gln + Tyr205Glu + Leu216Met + Gly218AsnTyr205Ser + Asn211Gln + Thr212Asp + Thr215Ser + Thr219GlyAla199Thr + Gly201Gln + Ala202Thr + Gly203Glu + Tyr213ProAla199Gln + Val204Asn + Thr207Asn + Tyr208Gly + Gly218SerAla199His + Gly203Pro + Thr207Gln + Asn211Asp + Ala214AsnAla202Ser + Gly203Pro + Tyr205Ser + Thr210Glu + Leu216AsnThr207Gly + Tyr208Gly + Thr212Asp + Leu216Pro + Thr219AsnAla202Asp + Val204Tnr + Tyr208Ile + Thr212Asn + Gly218SerGly203Gln + Val204Ala + Tyr205Pro + Asn211Gln + Thr215GlnPro200Ser + Gly203Pro + Thr212Pro + Leu216Met + Gly218GluAla199Gly + Gly201Gln + Thr207Ser + Leu216Cys + Asn217GluAla199Thr + Tyr205Met + Tyr208Val + Ala214Thr + Leu216IleAla202Asp + Tyr205Pro + Thr207Ser + Tyr208Thr + Thr210ProAla199Gln + Pro200Gln + Thr207Asn + Leu216Val + Gly218SerGly201Ser + Tyr208Met + Asn211Asp + Thr212Ser + Thr219GlyGly203Ser + Thr207Pro + Asn211Ser + Asn217Asp + Gly218GlnGly201Pro + Gly203Asp + Tyr208Asn + Thr212Ser + Thr215AsnAla199Pro + Pro200Ser + Tyr205Asp + Thr212Pro + Leu216ValTyr208Ala + Tyr213Ile + Ala214Gly + Thr215Pro + Asn217GlnAla199Ser + Pro200Gly + Gly203Asp + Leu216His + Asn217SerGly201Pro + Gly203Ser + Val204Asn + Leu216Ala + Asn217AspVal204Gln + Tyr208His + Asn211Asp + Tyr213His + Leu216ThrPro200Gly + Gly201Asn + Val204Gln + Tyr208Cys + Thr210ProAla202Asn + Tyr205Gln + Pro209Glu + Thr210Gly + Gly218AsnPro200Gly + Thr207Pro + Tyr208Met + Leu216Val + Asn217GluGly201Asn + Tyr205Gln + Thr212Asp + Thr215Pro + Asn217GlnGly203Glu + Tyr208Ile + Asn211Gln + Thr215Ser + Thr219GlyGly201Asn + Ala202Ser + Gly203Gln + Asn211Gln + Ala214AsnPro200Gly + Val204Asn + Pro209Gln + Thr210Pro + Thr219SerTyr208Leu + Pro209Asn + Thr210Gln + Thr215Asn + Gly218AsnAla202Ser + Val204Cys + Thr210Asn + Ala214Glu + Thr215GlyPro200Asn + Gly203Gln + Tyr208Ser + Pro209Gln + Gly218ProAla202Thr + Gly203Asp + Tyr205Leu + Thr212Gly + Tyr213GlnGly203Gln + Val204Pro + Thr210Asp + Gly218Gln + Thr219GlyTyr208His + Thr210Ser + Asn211Asp + Leu216Asn + Asn217GlnGly201Gln + Tyr205His + Thr210Glu + Tyr213lle + Leu216AlaPro200Asn + Gly201Ser + Ala202Glu + Gly203Gln + Thr219SerAla199Gln + Gly201Asn + Ala202Asp + Thr212Asn + Tyr213ThrAla199Pro + Val204Ser + Tyr213His + Asn217Asp + Gly218GlnPro200Asn + Gly201Ser + Ala202Pro + Thr207Ser + Ala214ThrAla199Asn + Gly201Pro + Gly203Asp + Tyr213Cys + Gly218GlnGly201Gln + Gly203Asn + Val204Thr + Thr210Ser + Thr212GlnGly201Asn + Ala202Pro + Thr210Gln + Asn211Gln + Thr215GlnPro200Asn + Gly203Glu + Tyr205Val + Thr207Pro + Tyr208SerAla199Gln + Gly201Ser + Ala202Pro + Thr207Pro + Ash217Glu
Pro200Gln + Tyr205Met + Thr212Pro + Leu216Glu + Thr219SerAla199Pro + Pro200Gln + Ala202Gly + Thr207Gln + Asn217GluTyr208Thr + Asn211Ser + Tyr213Gln + Leu216Gly + Thr219ProGly203Ser + Val204Thr + Tyr208Ala + Tyr213Pro + Thr219AsnGly201Asn + Gly203Pro + Val204Ala + Asn211Ser + Thr219GlyGly203Glu + Thr207Ser + Tyr208Ser + Thr215Asn + Leu216CysGly201Asn + Tyr208Leu + Asn211Glu + Leu216Gly + Asn217SerGly201Pro + Tyr208Val + Thr210Pro + Leu216Gln + Gly218AsnPro200Asn + Ala202Thr + Thr207Ser + Thr212Ser + Ala214AspGly203Glu + Val204Asn + Thr210Pro + Ala214His + Gly218AsnGly201Ser + Val204Met + Ala214Gly + Leu216Ser + Thr219SerPro200Gly + Tyr208Val + Tyr213His + Leu216Ala + Asn217GluThr212Glu + Tyr213Asn + Ala214Thr + Leu216Gln + Gly218ProGly203Pro + Val204Asn + Tyr208Cys + Asn217Glu + Thr219SerPro200Asn + Gly201Pro + Val204Thr + Leu216Asp + Asn217GlnTyr205Met + Tyr208Asn + Pro209Asn + Tyr213Ile + Ala214HisGly201Gln + Gly203Pro + Tyr205Cys + Thr210Gln +Asn217AspAla202Asn + Gly203Asp + Thr207Gly + Asn211Ser + Thr219ProPro200Gly + Gly201Ser + Val204Cys + Thr207Gly + Gly218AspPro200Ser + Gly203Pro + Tyr208Pro + Thr210Asn + Asn211GluPro200Gly + Gly201Gln + Tyr205Leu + Pro209Asn + Ala214HisTyr205Ser + Thr207Gln + Tyr208Ser + Thr212Ser + Thr219AsnAla202Gly + Asn211Asp + Thr212Gln + Ala214Thr + Leu216HisAla202Pro + Val204Ala + Pro209Gln + Asn211Ser + Thr212GluAla202Gly + Thr207Pro + Pro209Gly + Asn217Ser + Gly218AspGly201Gln + Pro209Asn + Thr215Ser + Leu216Thr + Thr219GlyVal204Thr + Tyr208Val + Thr215Asn + Asn217Glu + Gly218AsnTyr205Gln + Tyr213Pro + Thr215Ser + Leu216Gly + Gly218GlnGly203Asn + Thr207Ser + Thr210Asn + Asn211Asp + Leu216MetTyr208His + Pro209Ser + Leu216Gly + Asn217Glu + Gly218GlnThr207Ser + Pro209Gly + Asn211Glu + Thr215Ser + Leu216IleAla199Gly + Ala202Glu + Thr207Pro + Tyr213Val + Ala214GlyAla199His + Pro200Gly + Gly201Ser + Gly218Asp + Thr219ProAla202Glu + Tyr208Ala + Thr212Ser + Tyr213His + Thr219AsnAla199Thr + Val204Thr + Thr207Asn + Thr210Asn + Tyr213GluTyr208Gln + Pro209Gly + Ala214Asn + Thr215Glu + Gly218AsnTyr208Thr + Tyr213Val + Thr215Asp + Leu216Val + Gly218GlnThr207Pro + Pro209Ser + Thr210Glu + Asn217Ser + Thr219ProPro200Ser + Gly201Gln + Tyr205Ile + Pro209Gly + Thr219GlnVal204Met + Thr207Pro + Tyr208Gln + Pro209Asn + Thr215AspVal204Ala + Pro209Asn + Thr210Gly + Tyr213Asp + Ala214ProPro200Ser + Tyr208His + Tyr213Glu + Leu216Cys + Thr219SerGly201Gln + Ala202Asp + Tyr205Ala + Leu216Thr + Asn217GlnAla199Gly + Gly201Asn + Val204Gly + Ala214Asn + Thr219ProPro200Asn + Ala202Asn + Gly203Gln + Tyr208Asn + Asn217AspVal204Gly + Tyr205Asp + Asn211Gln + Tyr213Met + Ala214GlyGly201Asn + Thr207Gln + Pro209Gln + Ala214Asp + Gly218Pro
Pro200Gly + Tyr208Ser + Asn211Asp + Tyr213Ser + Thr219GlyAla202Thr + Val204Pro + Thr210Ser + Asn211Asp + Leu216ThrGly203Glu + Val204Met + Tyr205His + Asn211Gln + Thr219GlnGly201Gln + Ala202Gly + Val204Ser + Thr207Gln + Asn211GluGly203Asn + Tyr208Ala + Pro209Asp + Thr212Asn + Thr215GlnAla199Gln + Pro200Asn + Tyr205Met + Asn211Asp + Thr219GlnVal204Gln + Tyr208Val + Thr210Pro + Asn211Gln + Leu216AspPro200Asn + Val204Pro + Tyr205Gly + Thr210Pro + Ala214GlyGly201Asn + Tyr205Met + Tyr213Cys + Leu216Ile + Thr219AsnAla199Gly + Gly201Asn + Gly203Gln + Val204Thr + Pro209GluAla199His + Ala202Gly + Pro209Asp + Thr210Asp + Gly218SerGly201Gln + Tyr208Ser + Pro209Asp + Thr210Glu + Gly218SerAla199Asn + Pro200Ser + Thr212Asn + Ala214Asp + Thr215GluAla199His + Pro200Ser + Thr215Gly + Asn217Asp + Gly218GluTyr205Thr + Thr207Ser + Asn217Glu + Gly218Asp + Thr219GlyAla202Thr + Tyr205Glu + Tyr208Thr + Thr215Glu + Leu216GlnTyr205Asp + Thr212Gly + Thr215Glu + Asn217Ser + Gly218SerPro200Gly + Ala202Glu + Val204Ala + Tyr208Leu + Asn217AspPro200Gln + Ala202Asp + Tyr205Gly + Tyr213Ile + Asn217AspPro209Asp + Asn211Asp + Tyr213Thr + Leu216Cys + Thr219AsnAla202Asn + Val204His + Pro209Glu + Asn211Glu + Thr219AsnPro200Ser + Ala202Asp + Val204Gln + Leu216Glu + Asn217GluAla199Gly + Pro200Gln + Ala202Gln + Thr210Glu + Thr212AspGly203Asp + Pro209Gly + Tyr213His + Asn217Asp + Gly218SerGly203Asp + Val204Thr + Tyr208Ala + Tyr213Gln + Thr215AspTyr205Pro + Thr207Gly + Pro209Glu + Thr212Asp + Gly218SerAla199His + Ala202Glu + Gly203Gln + Asn211Gln + Gly218AspAla199Asn + Ala202Glu + Thr207Gln + Thr212Gln + Gly218AspAla202Asp + Gly203Asn + Tyr213Val + Leu216Ile + Gly218GluPro200Gly + Ala202Thr + Tyr208Met + Ala214Glu + Leu216AspGly203Glu + Thr207Gln + Thr212Ser + Ala214Glu + Thr215GluVal204His + Tyr205Asp + Tyr208Met + Tyr213Glu + Asn217GlnTyr205Gly + Tyr213Ile + Leu216Glu + Gly218Glu + Thr219GlyVal204Ser + Asn211Gln + Tyr213Thr + Thr215Glu + Asn217GluTyr208Leu + Thr210Gly + Thr212Gln + Tyr213Glu + Thr215GluAla202Glu + Tyr205Glu + Thr210Gly + Asn211Gln + Leu216AspPro200Asn + Thr210Ser + Thr212Asp + Tyr213Glu + Thr215AspAla199Thr + Thr207Ser + Thr210Asp + Thr212Asn + Tyr213AspAla202Glu + Val204Pro + Tyr205Thr + Tyr208His + Thr215GluGly203Glu + Tyr208Ile + Thr210Gln + Tyr213Gly + Gly218AspTyr208Gln + Pro209Glu + Tyr213Thr + Ala214Glu + Thr219GlnTyr205Asp + Thr210Ser + Thr215Gln + Asn217Asp + Gly218GluGly201Asn + Tyr205Glu + Tyr208Leu + Asn217Asp + Gly218Glu表27环6-六取代变异体Pro200Gly + Gly203Asp + Thr210Gln + Thr212Asn + Ala214His +Leu216AsnPro200Gly + Thr207Asn + Thr210Gln + Thr212Pro + Thr215Asp +Gly218ProPro200Gln + Gly203Ser + Thr207Asn + Tyr208Ile + Pro209Ser +Thr210GluGly203Pro + Tyr205Gly + Tyr208Gly + Asn211Glu + Tyr213Pro +Gly218GlnAla199His + Gly201Gln + Gly203Gln + Thr212Pro + Leu216Ile +Gly218AspVal204Met + Tyr205Thr + Thr207Ser + Tyr213His + Ala214Glu +Thr219AsnAla199Thr + Gly201Ser + Gly203Asp + Thr207Pro + Thr210Gly +Thr212ProPro200Gly + Tyr208Ile + Asn211Glu + Tyr213Ser + Ala214Ser +Thr215GlnAla199Thr + Gly201Gln + Pro209Gln + Thr212Asn + Tyr213His +Thr219GlyAla199Gly + Pro200Gly + Gly201Pro + Val204Cys + Thr207Gly +Thr215GlnAla199Gly + Pro200Ser + Thr207Pro + Leu216Ala + Asn217Glu +Gly218AsnPro200Gln + Val204Met + Asn211Gln + Ala214Gln + Thr215Gln +Leu216AspAla199Ser + Gly203Asn + Thr207Gln + Pro209Asp + Thr215Gly +Leu216HisGly201Pro + Tyr208Ile + Thr210Glu + Ala214Gly + Leu216Val +Thr219SerAla199Gln + Ala202Asn + Pro209Asn + Tyr213Asn + Thr215Gln +Gly218SerPro200Gln + Gly203Pro + Val204Gly + Thr212Pro + Ala214Pro +Thr215GluVal204Met + Tyr208Asn + Tyr213Asp + Ala214His + Thr215Gln +Leu216ThrAla199Gly + Pro200Asn + Ala202Thr + Val204Ser + Gly218Glu +Thr219ProAla199Ser + Gly201Pro + Gly203Asn + Thr207Ser + Thr212Gly +Thr219GlnAla199His + Tyr205Asn + Tyr208Ala + Asn211Gln + Tyr213Asn +Asn217AspTyr205Asp + Thr207Gly + Pro209Asn + Thr210Pro + Tyr213Ser +Leu216SerGly201Gln + Ala202His + Thr207Gly + Pro209Glu + Thr210Ser +Ala214GlyPro200Asn + Val204Gln + Tyr205Cys + Thr207Gln + Thr212Gln +Thr219SerPro200Ser + Tyr205Asp + Thr210Ser + Thr215Gln + Leu216Asn +Asn217GlnAla202His + Tyr205Thr + Tyr208His + Thr212Asp + Tyr213Ile +Gly218GlnGly201Asn + Pro209Glu + Asn211Gln + Thr212Ser + Tyr213Gln +Thr219GlnAla199Pro + Pro200Asn + Tyr205Val + Thr210Glu + Leu216Gly +Gly218AsnAla199Gln + Pro200Gln + Val204Thr + Tyr208Thr + Leu216Met +Asn217AspVal204Met + Thr210Ser + Thr212Asn + Tyr213Cys + Thr215Asn +Thr219ProAla199Gln + Gly201Pro + Ala202His + Thr207Asn + Leu216Val +Gly218SerAla199Thr + Gly203Ser + Tyr205Val + Tyr208Val + Thr210Pro +Ala214GluAla199Asn + Pro200Gln + Tyr205Met + Tyr208Cys + Thr212Glu +Ala214GlnAla202Ser + Thr207Gln + Thr210Glu + Asn211Gln + Tyr213Ile +Leu216AlaAla199Gly + Pro200Gly + Thr207Pro + Tyr213Ser + Leu216Asn +Asn217SerGly201Ser + Ala202Gly + Tyr205Asn + Pro209Gly + Thr210Asn +Asn211GlnGly201Ser + Ala202Gly + Gly203Asp + Thr207Asn + Asn211Gln +Thr215GlyVal204Gln + Tyr205His + Thr207Gln + Tyr208His + Asn211Asp 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Thr210Gln + Asn211Gln +Ala214AspGly203Asn + Pro209Ser + Thr210Glu + Ala214Pro + Gly218Asn +Thr219AsnPro200Ser + Gly201Pro + Ala202Thr + Ala214Gly + Leu216Asp +Thr219SerAla202Ser + Tyr205His + Thr212Gln + Thr215Glu + Asn217Gln +Thr219SerAla202Gly + Thr207Asn + Pro209Gly + Thr210Pro + Tyr213Met +Thr215GlyPro200Gln + Val204Ser + Thr207Ser + Tyr208Cys + Thr212Asn +Gly218GlnAla199Pro + Pro200Gly + Ala202Thr + Thr207Gly + Thr212Glu +Thr219SerAla199His + Gly203Gln + Tyr205Gly + Thr207Ser + Thr210Asn +Thr212SerGly203Glu + Val204Asn + Tyr205Pro + Thr210Pro + Ala214His +Gly218AsnPro200Gln + Val204Ala + Tyr208Cys + Ala214Gln + Leu216Val +Asn217GluGly201Gln + Ala202Asp + Tyr205Gly + Thr210Gly + Leu216Val +Gly218SerPro200Asn + Gly201Ser + Thr207Gln + Thr212Asp + Tyr213Gly +Thr215ProAla202Asn + Gly203Asp + Val204Cys + Thr207Gly + Asn211Ser +Thr219ProAla199Ser + Gly201Ser + Tyr205Ser + Thr207Asn + Tyr213Val +Leu216AlaPro200Gln + Tyr205Ser + Thr207Gln + Tyr208Ser + Thr212Ser +Thr219AsnPro200Asn + Ala202Gly + Asn211Asp + Thr212Gln + Ala214Thr +Leu216HisGly201Pro + Gly203Glu + Val204Met + Pro209Gly + Asn217Ser +Thr219SerPro200Gly + Gly201Pro + Tyr208Asn + Thr212Ser + Tyr213Leu +Ala214AsnAla199Ser + Gly201Pro + Gly203Asn + Asn211Asp + Leu216Cys +Thr219AsnGly203Pro + Val204Gly + Thr207Gln + Leu216Gly + Asn217Ser +Gly218AsnVal204Gln + Thr207Gln + Thr210Gly + Asn211Glu + Ala214Gly +Thr219ProAla202His + Tyr208Asn + Thr210Asn + Leu216Asn + Gly218Pro +Thr219GlyGly201Pro + Gly203Asp + Tyr205Met + Thr207Ser + Tyr213Gln +Asn217GlnGly201Ser + Ala202Ser + Thr207Asn + Tyr208Gly + Asn211Ser +Leu216ProAla202Gln + Gly203Asn + Val204Met + Tyr208Ser + Thr212Gln +Leu216AspAla199Asn + Ala202Thr + Pro209Glu + Thr210Pro + Asn217Ser +Thr219GlyGly203Asn + Tyr208Ser + Asn211Glu + Ala214His + Leu216Met +Gly218ProAla199Ser + Pro200Gln + Val204Asn + Tyr208Asn + Thr215Asp +Thr219GlnGly201Asn + Thr207Asn + Tyr208Gln + Pro209Asn + Asn211Asp +Leu216CysPro200Ser + Gly203Asn + Thr207Gln + Thr212Gln + Asn217Asp +Gly218AsnVal204His + Tyr205Ala + Thr210Asn + Thr215Pro + Leu216Val +Asn217GluAla199Pro + Gly201Asn + Ala202Asp + Val204Thr + Thr210Gly +Thr219SerPro200Asn + Ala202Gly + Gly203Glu + Thr207Gln + Tyr208His +Asn211GlnPro200Gly + Ala202Asn + Gly203Asn + Thr212Asp + Ala214Ser +Leu216ProAla199Gly + Gly201Pro + Thr207Gly + Pro209Asp + Thr215Asn +Gly218SerGly203Asn + Tyr205Thr + Asn211Asp + Ala214Gly + Gly218Asn +Thr219GlyGly201Pro + Val204His + Tyr208Cys + Thr215Glu + Leu216Asn +Gly218ProVal204Ala + Pro209Asn + Thr210Asp + Tyr213Leu + Leu216Ser +Thr219GlyAla202Asp + Tyr205Val + Thr207Asn + Thr210Asn + Tyr213Pro +Ala214AsnGly201Ser + Thr207Gly + Thr212Pro + Thr215Ser + Leu216Asn +Thr219GlyAla199His + Gly203Gln + Tyr205Gln + Tyr208His + Ala214Gln +Thr215AsnVal204Asn + Tyr205Ile + Pro209Glu + Thr210Ser + Tyr213Val +Leu216SerAla199Ser + Pro200Ser + Gly201Asn + Val204Thr + Thr207Pro +Gly218GluAla199Gly + Pro200Gly + Pro209Gln + Thr215Asp + Leu216Cys +Thr219GlyGly201Ser + Gly203Ser + Tyr205Cys + Thr207Gln + Thr212Gly +Ala214ThrPro200Asn + Ala202Gln + Pro209Gly + Thr210Asp + Thr215Pro +Gly218AsnTyr205Pro + Thr207Gln + Thr210Glu + Tyr213Cys + Leu216Pro +Thr219SerGly201Gln + Tyr205Val + Tyr208Val + Thr210Gln + Thr215Glu +Gly218AsnAla199Ser + Tyr208Asn + Asn211Gln + Leu216Ile + Asn217Glu +Gly218ProPro200Asn + Gly201Pro + Val204Thr + Tyr205His + Thr215Glu +Leu216ThrGly203Pro + Val204His + Prn209Gly + Thr210Glu + Thr215Pro +Gly218AsnTyr205Leu + Asn211Gln + Ala214Gln + Leu216Gly + Asn217Asp +Gly218AsnAla199Ser + Gly201Gln + Gly203Pro + Tyr208Thr + Thr215Gly +Thr219AsnPro209Glu + Thr210Ser + Thr212Ser + Tyr213Pro + Ala214Thr +Thr215ProPro200Ser + Ala202Pro + Gly203Asn + Thr207Pro + Thr210Asp +Tyr213ValGly201Asn + Thr207Gly + Pro209Ser + Tyr213Met + Leu216Ile +Gly218AspGly201Pro + Val204His + Thr212Gly + Leu216Asn + Asn217Ser +Thr219GlyThr212Gln + Ala214Glu + Thr215Ser + Asn217Ser + Gly218Ser +Thr219AsnGly203Ser + Tyr205Met + Tyr208Thr + Thr210Asn + Ala214Glu +Leu216IleGly201Gln + Tyr208Gln + Thr210Gly + Thr212Gln + Asn217Ser +Thr219AsnGly201Gln + Tyr205Cys + Thr212Pro + Tyr213Met + Leu216Asn +Gly218GlnAla202Gln + Tyr205Val + Pro209Gln + Thr210Asp + Asn211Ser +Tnr215GlnGly201Pro + Val204Pro + Thr207Asn + Asn211Gln + Asn217Glu +Thr219AsnAla199Ser + Tyr213Ser + Ala214Thr + Leu216Ser + Asn217Ser +Gly218GluPro200Gln + Gly201Ser + Val204Ala + Thr207Gln + Thr210Asn +Ala214GluTyr205Gln + Tyr208Cys + Thr212Gln + Leu216Ser + Gly218Gln +Thr219SerPro200Gly + Gly201Pro + Gly203Pro + Thr207Gln + Thr210Gly +Thr219ProAla199Gln + Pro200Gly + Val204Ser + Thr215Glu + Asn217Ser +Thr219ProAla199Ser + Pro200Gln + Gly201Pro + Val204Pro + Thr210Pro +Thr219GlnAla202Gln + Thr207Gln + Thr210Glu + Ala214Ser + Thr215Pro +Gly218GlnPro200Gln + Ala202Glu + Thr207Pro + Pro209Asn + Thr212Gln +Leu216ValGly201Asn + Asn211Glu + Tyr213Cys + Ala214Thr + Thr215Ser +Leu216HisAla199His + Ala202Gly + Tyr205Ala + Thr207Gly + Asn211Asp +Ala214GlnGly203Ser + Thr207Asn + Pro209Glu + Thr210Asp + Asn217Gln +Thr219GlyGly201Gln + Val204Met + Thr210Asp + Asn211Asp + Leu216Gln +Gly218SerGly203Pro + Tyr205Ile + Tyr208Gln + Asn211Asp + Thr212Glu +Gly218GlnGly203Ser + Tyr205Val + Thr207Asn + Leu216Glu + Asn217Asp +Thr219SerVal204Ala + Tyr205Pro + Pro209Ser + Leu216Ala + Asn217Asp +Gly218GluPro200Asn + Gly201Ser + Tyr208Thr + Thr215Gln + Asn217Asp +Gly218AspAla202Asn + Tyr208Met + Thr210Gln + Asn211Gln + Asn217Asp +Gly218AspAla199Gln + Ala202Asp + Gly203Glu + Thr207Gln + Tyr208Asn +Asn211GlnAla202Asp + Gly203Asp + Thr207Ser + Tyr208Cys + Thr210Asn +Thr215ProAla202Glu + Gly203Asp + Thr207Gly + Tyr208Leu + Leu216Val +Thr219GlnPro200Ser + Gly201Asn + Pro209Asp + Thr210Asp + Asn211Asp +Leu216SerAla199Gln + Val204Gln + Tyr205Asp + Tyr208Leu + Thr210Gln +Thr215GluPro200Gln + Gly203Gln + Tyr205Glu + Thr212Gly + Thr215Glu +Thr219ProPro200Asn + Thr207Ser + Thr210Asp + Asn211Asp + Thr212Glu +Leu216MetAla202Asp + Gly203Asp + Val204Ser + Thr210Ser + Asn217Asp +Gly218ProVal204Ser + Thr207Pro + Tyr213His + Thr215Glu + Leu216Asp +Asn217GluVal204Gly + Asn211Ser + Ala214Gln + Thr215Glu + Leu216Glu +Asn217GluTyr208Ile + Tyr213Ser + Ala214His + Leu216Glu + Asn217Asp +Gly218AspGly203Asp + Thr207Ser + Ala214His + Thr215Asp + Leu216Asp +Thr219SerAla199Ser + Gly201Asn + Thr207Ser + Pro209Glu + Asn211Asp +Thr219SerGly203Glu + Tyr205Glu + Thr210Gly + Tyr213Asn + Ala214Gln +Thr215AspAla199His + Gly203Asp + Val204His + Thr212Ser + Asn217Asp +Gly218ProTyr205Glu + Thr210Gly + Asn211Ser + Ala214Asp + Thr215Ser +Asn217GlnGly201Asn + Tyr205Asp + Asn211Gln + Thr212Ser + Ala214Glu +Gly218SerAla199Asn + Gly201Asn + Gly203Asp + Tyr208Thr + Thr215Asp +Leu216ThrPro200Gly + Tyr208Pro + Pro209Glu + Thr212Asp + Gly218Ser +Thr219SerPro200Asn + Val204Gly + Thr207Asn + Pro209Asp + Thr212Glu +Tyr213AlaAla199Pro + Gly203Asp + Tyr205Glu + Pro209Ser + Ala214His +Thr215AsnPro200Gly + Gly203Glu + Thr207Ser + Thr215Gln + Asn217Asp +Gly218AspAla202Glu + Gly203Glu + Tyr208Gln + Tyr213Thr + Ala214Ser +Gly218AspPro200Gln + Gly203Asp + Tyr213Asn + Ala214Asp + Thr215Asp +Leu216IleAla202Ser + Gly203Ser + Tyr205Glu + Pro209Gly + Thr215Asp +Asn217GluAla202Gln + Thr207Pro + Thr212Asp + Tyr213Pro + Ala214Glu +Thr219GlyPro200Ser + Val204Pro + Thr212Asp + Tyr213Ala + Ala214Asp +Thr219AsnAla199Ser + Ala202Asp + Val204Pro + Pro209Ser + Thr215Asn +Leu216AspAla199Gly + Ala202Asn + Val204Asn + Tyr213Asp + Ala214Glu +Leu216GluAla199Thr + Gly203Gln + Thr210Gln + Thr212Gln + Tyr213Glu +Thr215GluPro200Gln + Gly201Pro + Pro209Asp + Tyr213Glu + Thr215Pro +Gly218AsnVal204Asn + Tyr205Val + Pro209Asp + Thr212Pro + Tyr213Asp +Ala214GlyTyr205Leu + Asn211Glu + Thr212Glu + Tyr213Leu + Ala214Glu +Thr215GlyAla199Ser + Ala202Glu + Tyr213Pro + Ala214Gly + Thr215Asp +Gly218ProPro200Gly + Gly203Asp + Val204His + Thr210Asn + Asn211Gln +Gly218AspGly201Ser + Ala202Gly + Gly203Asp + Ala214Asn + Gly218Glu +Thr219GlyAla199Gln + Pro200Ser + Ala202Ser + Thr210Asp + Asn211Glu +Ala214GluAla202Glu + Thr207Ser + Asn211Gln + Ala214Asp + Leu216Glu +Gly218SerAla199Gln + Pro200Ser + Tyr205Asp + Pro209Ser + Thr215Gly +Asn217GluAla199Ser + Tyr205Asp + Tyr213Met + Ala214Thr + Asn217Asp +Thr219AsnPro200Gln + Ala202Asn + Gly203Gln + Tyr213Glu + Leu216Glu +Thr219AsnAla199Pro + Ala202Gln + Gly203Asn + Thr212Glu + Thr215Asp +Leu216AspAla199Gln + Gly201Gln + Val204Thr + Asn211Asp + Ala214Asp +Thr215AspGly203Gln + Val204Met + Asn211Glu + Thr212Ser + Ala214Asp +Thr219GlyThr207Gly + Tyr208Val + Thr210Gly + Thr215Glu + Leu216Asn +Gly218AspPro200Gly + Val204Asn + Thr207Gly + Thr210Glu + Tyr213Val +Ala214AspGly203Glu + Thr207Asn + Thr210Gly + Thr212Asp + Thr215Asp +Leu216GlnGly203Gln + Val204Cys + Pro209Asn + Thr210Ser + Thr212Asp +Thr215AspGly203Asn + Val204Gly + Tyr205Asp + Ala214Glu + Thr215Pro +Gly218Asp表28环6-七取代突变变异体Pro200Asn + Ala202Gln + Tyr208Cys + Thr210Pro + Tyr213Asn +Ala214Asn + Thr219SerGly203Pro + Val204Gln + Tyr205Gly + Tyr208Gly + Asn211Glu +Tyr213Pro + Gly218GlnGly201Asn + Gly203Gln + Val204Cys + Thr207Ser + Thr212Asp +Thr215Pro + Thr219GlyGly203Gln + Val204Gln + Tyr205Pro + Thr212Gly + Tyr213Val +Asn217Asp + Thr219GlyGly201Gln + Ala202Gln + Val204Pro + Asn211Asp + Tyr213Ile +Leu216Gly + Gly218ProPro200Gly + Ala202Gln + Val204Pro + Pro209Asn + Tyr213Ala +Asn217Asp + Thr219ProAla199Thr + Gly201Ser + Val204Ser + Thr210Gly + Asn211Asp +Gly218Ser + Thr219SerGly201Gln + Tyr208Val + Pro209Gln + Asn211Ser + Thr215Pro +Asn217Gln + Thr219GlnAla199His + Pro200Gly + Pro209Asn + Thr212Gln + Tyr213Ile +Leu216Gln + Thr219GlyPro200Gln + Thr210Pro + Asn211Ser + Thr212Gln + Leu216Gln +Asn217Asp + Thr219ProPro200Ser + Val204His + Tyr205Ala + Tyr208Ser + Asn211Gln +Ala214His + Thr215AsnAla199Ser + Pro200Ser + Gly201Pro + Tyr205Met + Thr207Pro +Thr210Asp + Thr212SerGly201Gln + Val204Ser + Tyr205Asn + Tyr213Ala + Ala214Glu +Thr215Gln + Leu216ThrAla199Gln + Val204Thr + Thr207Ser + Tyr208Asn + Pro209Asn +Thr212Gln + Gly218ProPro200Asn + Gly203Pro + Thr207Pro + Thr210Gln + Tyr213His +Leu216Ile + Gly218AspAla202Gly + Gly203Gln + Val204Gly + Tyr205Ser + Thr212Gln +Asn217Ser + Thr219GlyPro209Gly + Thr210Gln + Asn211Asp + Tyr213Gln + Ala214Ser +Leu216Asn + Asn217SerAla199His + Val204Pro + Tyr205Glu + Pro209Asn + Tyr213Ala +Leu216Thr + Gly218AsnAla199Gln + Pro200Asn + Ala202Thr + Gly203Pro + Val204Gln +Thr212Ser + Tyr213GlnGly201Asn + Ala202Asn + Gly203Asp + Tyr208Thr + Thr212Gln +Tyr213Thr + Gly218AsnAla199Thr + Gly203Asp + Val204Thr + Thr207Asn + Thr210Asn +Asn211Ser + Tyr213ValPro200Gly + Thr207Pro + Tyr208Met + Pro209Glu + Tyr213His +Thr215Pro + Leu216GlnPro200Ser + Gly201Asn + Ala202Asp + Tyr205Cys + Tyr208Ala +Tyr213His + Thr219AsnAla199Pro + Val204Met + Thr207Gln + Tyr208Val + Asn211Glu +Ala214Ser + Thr219SerGly201Asn + Tyr205Leu + Tyr208Pro + Thr210Pro + Thr212Asn +Tyr213Leu + Ala214AsnPro200Ser + Gly201Pro + Gly203Pro + Val204Asn + Thr207Asn +Ala214Asp + Leu216HisPro200Gly + Gly201Gln + Val204Gln + Thr207Gln + Tyr213His +Ala214Ser + Gly218ProPro200Gln + Val204His + Pro209Ser + Asn211Ser + Thr212Glu 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Ala202Pro + Val204Met + Tyr205Ile + Tyr208Ile +Asn217Asp + Thr219ProAla199Thr + Gly203Asn + Tyr208Leu + Pro209Gly + Tyr213Ile +Ala214Glu + Leu216GlyGly201Gln + Val204Ala + Tyr208Gly + Thr210Gln + Thr215Gln +Asn217Gln + Gly218AsnPro200Gln + Gly201Asn + Val204Ser + Tyr205Glu + Tyr213Ser +Ala214Gln + Asn217GlnPro200Ser + Gly203Asn + Thr207Gly + Thr210Pro + Tyr213His +Ala214Thr + Thr215ProAla199Asn + Tyr208Ile + Pro209Asp + Thr212Gln + Tyr213Ser +Ala214His + Asn217GlnAla199Asn + Pro200Gln + Ala202Pro + Val204Cys + Tyr208His +Thr215Gln + Thr219AsnAla199Ser + Ala202His + Val204Pro + Thr210Asn + Thr212Pro +Gly218Asp + Thr219AsnGly201Pro + Tyr205Glu + Thr210Gly + Asn211Ser + Tyr213Gln +Ala214Thr + Gly218GlnAla199Pro + Gly203Gln + Thr207Asn + Asn211Asp + Ala214Gln +Thr215Gly + Thr219GlyAla199Gln + Pro200Asn + Thr212Ser + Tyr213Ser + Leu216Ile +Asn217Asp + Thr219ProPro209Asn + Thr212Gln + Ala214Ser + Leu216Gly + Asn217Asp +Gly218Asn + Thr219AsnGly201Ser + Gly203Pro + Thr207Ser + Thr212Ser + Ala214His +Asn217Asp + Gly218GlnAla199Ser + Pro200Gln + Gly201Asn + Tyr205Asn + Ala214Ser +Asn217Asp + Gly218ProAla199His + Ala202Gly + Gly203Glu + Tyr208Gln + Pro209Gln +Leu216Asn + Thr219SerAla199Pro + Thr207Ser + Tyr208Cys + Asn211Gln + Tyr213Gly +Leu216Thr + Thr219GlyPro200Gly + Ala202Thr + Tyr205Cys + Thr212Glu + Leu216Thr +Asn217Ser + Gly218ProAla199Thr + Gly203Asn + Tyr208Ser + Pro209Gly + Asn211Ser +Tyr213Pro + Gly218ProAla199Gly + Tyr205Gln + Pro209Asn + Asn211Glu + Ala214His +Thr215Gln + Thr219AsnAla199Asn + Pro200Asn + Gly203Pro + Tyr208Ile + Pro209Gly +Tyr213Asp + Ala214HisGly203Asn + Val204Thr + Asn211Gln + Thr212Pro + Thr215Asp +Leu216His + Thr219GlnAla199Gln + Gly201Ser + Val204Pro + Tyr205Asn + Pro209Asp +Thr210Gln + Thr219AsnPro200Asn + Gly201Ser + Val204Ser + Tyr205Asp + Thr210Asn +Thr212Pro + Asn217SerPro200Asn + Gly201Asn + Tyr205Asp + Tyr208Pro + Thr215Pro +Asn217Gln + Thr219GlyAla199Gln + Gly201Pro + Tyr205Ile + Tyr208Asn + Thr215Gly +Gly218Asp + Thr219ProAla199Pro + Gly201Gln + Tyr205Asn + Thr207Ser + Thr210Gly +Tyr213Met + Ala214SerPro200Asn + Ala202Glu + Val204Thr + Thr207Asn + Tyr208His +Tyr213Gln + Asn217GlnGly201Ser + Ala202Asp + Val204Pro + Thr207Gly + Pro209Gln +Tyr213Pro + Gly218SerGly201Asn + Ala202Asn + Tyr205Cys + Thr210Gly + Tyr213Gln +Ala214Pro + Thr219GlyPro200Asn + Gly203Ser + Thr207Gly + Thr210Pro + Thr215Asp +Leu216Ser + Gly218SerGly201Gln + Ala202Glu + Tyr208Gly + Thr212Pro + Ala214His +Leu216Thr + Thr219GlnAla199Asn + Pro200Gln + Ala202Asn + Tyr205Pro + Thr207Pro +Tyr208Pro + Pro209AspGly201Pro + Ala202Glu + Pro209Ser + Thr212Ser + Thr215Pro +Leu216Cys + Gly218AsnAla202Thr + Gly203Asp + Thr207Gln + Tyr208Cys + Ala214Pro +Leu216Gln + Asn217SerGly201Ser + Ala202Asn + Gly203Pro + Tyr205Met + Asn211Ser +Leu216Glu + Gly218GlnAla199Gly + Tyr205Gly + Asn211Ser + Thr212Ser + Ala214Gln +Asn217Glu + Thr219AsnGly203Gln + Tyr205Gln + Tyr208Met + Pro209Glu + Thr210Glu +Thr215Pro + Thr219GlnPro200Gly + Tyr205Ile + Thr207Gln + Tyr208Pro + Pro209Asp +Thr210Glu + Leu216ProAla199Pro + Pro200Gly + Gly203Gln + Tyr208Asn + Thr210Asp +Asn211Glu + Leu216ProThr207Asn + Tyr208Gln + Pro209Gly + Ala214Asn + Thr215Glu +Leu216Asp + Gly218AsnAla199Thr + Pro200Gln + Thr210Ser + Ala214Gly + Thr215Asp +Leu216Glu + Gly218GlnAla202Gln + Gly203Ser + Val204Cys + Tyr213Ile + Ala214Pro +Leu216Asp + Asn217AspPro200Gly + Gly203Pro + Tyr205Asp + Tyr208Asn + Thr212Gly +Thr215Asp + Gly218ProAla202Glu + Gly203Glu + Val204Cys + Tyr205Pro + Asn217Glu +Gly218Gln + Thr219ProPro200Gly + Ala202Glu + Val204Cys + Thr207Asn + Leu216Ile +Asn217Glu + Gly218GluAla199Pro + Gly201Pro + Thr207Gln + Tyr208Leu + Thr212Glu +Tyr213Asp + Ala214AspAla199Gly + Gly201Pro + Ala202Pro + Tyr208Ala + Pro209Glu +Asn211Asp + Thr212GlyThr207Pro + Pro209Glu + Asn211Glu + Tyr213Met + Ala214Ser +Thr215Gln + Leu216CysGly201Gln + Gly203Glu + Thr207Gln + Tyr208Met + Thr215Asn +Asn217Asp + Thr219ProGly201Asn + Ala202His + Gly203Pro + Tyr205Asp + Thr212Gln +Ala214Glu + Thr219SerVal204Ser + Thr207Pro + Pro209Glu + Thr212Asp + Tyr213Ser +Leu216Gln + Thr219ProAla199Thr + Thr207Pro + Pro209Glu + Thr212Glu + Tyr213Thr +Leu216Ser + Asn217SerPro200Asn + Tyr205His + Pro209Asp + Thr212Asp + Tyr213Ala +Ala214Pro + Thr219GlyAla199Pro + Pro200Gly + Gly201Ser + Val204His + Pro209Glu +Thr212Asp + Thr219GlnAla199Pro + Ala202Gln + Gly203Asp + Thr207Gly + Tyr208Gln +Leu216Glu + Gly218ProGly203Glu + Val204Ser + Tyr208Val + Tyr213Pro + Ala214Asp +Thr215Glu + Leu216AspGly201Gln + Gly203Glu + Val204Gln + Thr207Ser + Thr215Asp +Leu216Ala + Asn217GluAla202Asn + Val204Gly + Asn211Ser + Ala214Asp + Thr215Glu +Leu216Glu + Asn217GluAla199His + Pro200Ser + Gly201Gln + Ala202Glu + Leu216Ile +Gly218Asp + Thr219GlyAla202Gly + Gly203Glu + Val204Gln + Thr207Gly + Thr215Gln +Asn217Glu + Gly218AspAla199Asn + Gly203Glu + Thr210Gly + Ala214Asn + Thr215Pro +Asn217Glu + Gly218AspAla202Pro + Gly203Asn + Tyr205Glu + Asn211Gln + Thr215Gln +Leu216Glu + Thr219AsnPro200Gly + Val204Thr + Thr207Ser + Tyr208Cys + Thr210Asp +Asn211Glu + Tyr213GluAla202Glu + Gly203Pro + Val204Thr + Thr212Ser + Tyr213Val +Thr215Glu + Asn217AspPro200Gln + Val204Met + Pro209Gly + Thr210Asp + Thr212Asp +Tyr213Asp + Ala214AspAla199His + Pro200Gln + Ala202Gly + Val204Cys + Thr215Glu +Leu216Cys + Asn217AspVal204Gly + Tyr208Leu + Pro209Glu + Thr210Asn + Tyr213Glu +Ala214Asp + Thr215SerPro200Gly + Gly201Gln + Ala202Ser + Pro209Gly + Thr212Glu +Ala214Asp + Leu216AlaGly201Asn + Tyr205His + Tyr208Ser + Tyr213Asp + Ala214Asp +Thr215Ser + Leu216AspGly203Asn + Tyr208Gln + Asn211Gln + Tyr213Asp + Thr215Asp +Asn217Gln + Thr219GlnAla202Ser + Thr207Pro + Tyr208Cys + Tyr213Asp + Ala214Ser +Thr215Asp + Leu216CysAla199Thr + Gly201Pro + Tyr205Ser + Thr207Asn + Tyr208Ile +Asn211Glu + Tyr213GluAla199Pro + Ala202Ser + Gly203Pro + Tyr208Gly + Asn211Asp +Tyr213Glu + Thr215GlnAla199Gln + Pro200Asn + Gly203Asp + Tyr205Glu + Tyr208Asn +Tyr213Met + Ala214AspGly201Asn + Thr207Pro + Thr210Asp + Asn211Glu + Thr212Glu +Ala214Glu + Leu216ProPro200Gln + Gly201Asn + Gly203Asn + Thr207Asn + Asn211Asp +Thr212Glu + Ala214GluThr210Gly + Asn211Asp + Tyr213Asp + Ala214Asp + Asn217Ser +Gly218Pro + Thr219AsnAla199Asn + Gly201Ser + Tyr208Ala + Thr210Glu + Tyr213Glu +Ala214Pro + Leu216HisAla202Asn + Gly203Asp + Tyr205Gln + Thr212Ser + Thr215Glu +Gly218Asp + Thr219ProPro200Gly + Gly203Glu + Thr207Gln + Ala214Gln + Thr215Asp +Leu216Asn + Gly218GluAla199Ser + Gly201Ser + Ala202Glu + Gly203Gln + Thr207Ser +Thr215Asp + Thr219SerAla202Asp + Tyr205Thr + Thr207Asn + Tyr208Ala + Thr215Glu +Leu216Gln + Gly218AsnPro200Asn + Gly203Asp + Val204Pro + Tyr208Cys + Leu216Cys +Asn217Ser + Gly218GluAla199Asn + Pro200Ser + Gly201Asn + Gly203Glu + Val204Gly +Asn211Gln + Gly218GluAla199Thr + Gly201Gln + Ala202Pro + Thr210Asp + Asn211Glu +Ala214Asp + Thr219AsnAla199Gly + Pro200Ser + Tyr205Glu + Thr210Pro + Asn211Asp +Tyr213Asp + Thr215GluPro200Ser + Ala202Glu + Thr207Gln + Asn211Ser + Ala214Glu +Asn217Glu + Gly218AsnAla202Ser + Val204His + Thr207Ser + Thr212Asp + Tyr213Asp +Thr215Asn + Leu216AspGly203Pro + Tyr205Pro + Pro209Asp + Asn211Asp + Ala214Glu +Thr215Asp + Thr219GlyThr207Ser + Pro209Ser + Thr212Glu + Tyr213Glu + Tnr215Glu +Leu216Gln + Asn217AspAla199Ser + Pro200Gly + Gly201Ser + Thr212Gly + Ala214Glu +Asn217Glu + Gly218ProGly201Gln + Tyr208Thr + Ala214Asp + Thr215Pro + Leu216Asn +Asn217Glu + Gly218ProAla199Ser + Tyr205Glu + Thr207Ser + Thr210Asn + Tyr213Glu +Ala214Gln + Asn217AspGly203Glu + Pro209Asp + Thr210Asp + Asn211Glu + Ala214Thr +Thr215Gln + Thr219AsnAla202Pro + Gly203Asp + Tyr205Cys + Thr212Asp + Thr215Pro +Leu216Glu + Asn217AspAla199Asn + Gly201Pro + Ala202Glu + Gly203Glu + Tyr205Glu +Thr207Gly + Thr212AspAla202Asn + Thr207Pro + Asn211Glu + Thr212Asp + Ala214Ser +Thr215Asp + Leu216ValGly201Gln + Ala202Glu + Val204Ser + Tyr205Asp + Tyr213Glu +Thr215Asn + Leu216ThrPro200Gly + Gly203Asn + Tyr205Glu + Thr212Glu + Tyr213Val +Ala214Asn + Leu216GluAla202His + Thr207Gln + Pro209Gln + Thr210Gln + Thr215Glu +Asn217Gln + Gly218GluAla199Gly + Gly201Gln + Pro209Gly + Thr212Gly + Thr215Glu +Asn217Ser + Gly218GluGly203Asp + Val204Met + Thr212Asp + Tyr213Val + Ala214Asp +Asn217Glu + Thr219ProAla202Asp + Gly203Asn + Tyr208Met + Thr212Asp + Asn217Glu +Gly218Glu + Thr219GlyPro200Gln + Val204Asn + Thr207Gly + Thr210Glu + Asn211Gln +Tyr213Val + Ala214AspGly203Gln + Tyr205lle + Tyr208His + Thr210Asp + Ala214Asp +Thr215Pro + Leu216ValGly203Gln + Tyr208Ala + Pro209Gln + Thr210Glu + Asn211Gln +Ala214Asp + Leu216CysGly201Gln + Gly203Glu + Val204Ser + Pro209Asn + Asn211Glu +Tyr213Asp + Thr215GluTyr205Asp + Thr207Pro + Pro209Ser + Thr210Glu + Ala214Glu +Asn217Ser + Thr219ProAla199Asn + Gly201Ser + Ala202Thr + Gly203Asp + Thr210Pro +Ala214Asp +Gly218GluGly203Glu + Thr207Ser + Tyr208Thr + Pro209Gln + Thr212Glu +Ala214Ser + Thr215GluAla199Pro + Gly201Ser + Val204Gln + Tyr205Asp + Thr212Asp +Leu216Ala + Thr219ProPro200Gln + Tyr208Ile + Pro209Glu + Ala214Glu + Leu216Asp +Asn217Ser + Gly218GluAla199Thr + Ala202Glu + Gly203Glu + Thr210Asp + Ala214Ser +Gly218Glu + Thr219GlnAla199Thr + Pro200Ser + Ala202Asp + Gly203Asp + Thr210Gly +Thr212Glu + Gly218GluAla199Pro + Ala202Glu + Tyr208Leu + Thr210Asp + Ala214Ser +Leu216Glu + Gly218AspGly201Asn + Tyr205Ala + Thr207Pro + Thr210Asp + Asn211Asp +Ala214Asp + Asn217GluAla199Ser + Gly203Gln + Tyr205Gln + Pro209Glu + Thr212Glu +Tyr213Asn + Leu216AspAla199Gln + Pro200Asn + Tyr205Glu + Tyr208Leu + Pro209Glu +Thr210Asp + Tyr213GlyPro200Gly + Gly203Glu + Pro209Glu + Thr212Ser + Tyr213Pro +Thr215Asp + Asn217GlnGly201Pro + Tyr205Val + Thr207Ser + Pro209Asp + Ala214Glu +Asn217Glu + Thr219GlnPro200Asn + Ala202Thr + Pro209Gly + Asn211Glu + Thr212Glu +Ala214Asn + Leu216AspGly201Ser + Thr207Ser + Thr212Asp + Ala214Pro + Leu216Asp +Asn217Asp + Gly218ProTyr205Asp + Thr207Asn + Pro209Asp + Asn211Glu + Thr212Pro +Gly218Ser + Thr219ProPro200Gly + Ala202Asn + Gly203Asn + Thr210Ser + Thr212Glu +Tyr213Asp + Asn217GluGly203Pro + Pro209Ser + Thr212Asn + Tyr213Glu + Asn217Glu +Gly218Asp + Thr219GlnAla202Ser + Tyr205Ala + Pro209Glu + Thr210Asp + Leu216Ser +Asn217Asp + Gly218GlnVal204Met + Pro209Asp + Thr210Glu + Ala214Gln + Leu216Gln +Gly218Asp + Thr219ProAla202Glu + Val204Met + Thr207Ser + Pro209Glu + Thr210Glu +Thr212Ser + Gly218ProPro200Ser + Ala202His + Thr210Asp + Asn211Glu + Leu216Gly +Gly218Asp + Thr219GlyPro200Gly + Gly201Asn + Ala202Ser + Thr210Asp + Asn211Glu +Ala214His + Thr215AspPro200Gln + Pro209Gly + Thr210Glu + Asn211Asp + Thr212Gln +Tyr213Met + Leu216AspPro200Gly + Gly203Glu + Tyr208Thr + Thr210Asp + Asn211Asp +Ala214Thr + Leu216AsnGly203Ser + Val204Cys + Thr207Asn + Pro209Ser + Thr210Glu +Thr215Glu + Leu216GluAla199Asn + Pro200Asn + Gly201Pro + Gly203Asn + Asn211Asp +Thr215Asp + Leu216AspAla202Thr + Tyr208Met + Pro209Asn + Asn211Glu + Thr212Glu +Ala214Gly + Gly218GluGly203Gln + Val204Cys + Tyr205Gly + Tyr208Pro + Asn211Glu +Thr212Asp + Gly218GluPro200Gln + Ala202Asp + Tyr205Met + Thr212Asp + Tyr213Glu +Gly218Gln + Thr219GlnAla199Gln + Gly203Pro + Pro209Asn + Thr210Gln + Thr212Asp +Asn217Asp + Gly218AspGly201Asn + Tyr208Asn + Pro209Ser + Thr212Glu + Tyr213Pro +Asn217Asp + Gly218AspAla199Gln + Gly201 Pro + Tyr205Ser + Asn211Glu + Ala214Asn +Asn217Glu + Gly218Glu表29多环双突变变异体Leu 95Gly + Gly203ProThr 58Gln + Asn211GluAsp 59Glu + Leu216GlnTyr103Cys + Thr212GlyGly156Glu + Ala202GlnGly126Gln + Thr215ProSer187Glu + Ala214GlyAsn 96Gln + Ile106AlaGly101Asp + Tyr205ProAsn 61Ser + Gly165GlnGly 64Gln + Thr132GlyAsp 59Glu + Tyr205AsnSer100Glu + Tyr205HisAla128Ser + Thr161GlnSer158Asp + Gly203SerAsn 61Asp + Asn162SerThr161Glu + Pro209GlnGly 62Asp + Pro209AsnIle106Leu + Asn211AspTyr103Glu + Gly203ProTyr166Pro + Thr215GluIle106Leu + Pro209GluThr163Pro + Ala202ProThr132Gly + Leu216SerSer104Glu + Phe188Val
Tyr166Asp + Ala186ThrSer160Glu + Thr215SerGly 62Gln + Gly165AspLeu 95Gln + Ala128GluSer 97Glu + Asn211SerLeu 95Asn + Phe188LeuAsn157Gln + Thr212SerLeu125Gln + Gly159AspAsn211Asp + Tyr213AlaSer158Asp + Tyr205CysSer160Glu + Asn162GlnSer187Asp + Tyr213ProSer158Asp + Leu216GlnMet198Ser + Ala202AspGly 99Asn + Ala214GluThr 58Asp + Gly130GlnGly156Glu + Tyr208ProGly105Pro + Leu216IleSer187Asp + Tyr205GlnThr215Ser + Gly218AspGly 60Asn + Ala128GluThr 65Gln + Leu125AsnGly101Gln + Gly218AspAsn211Ser + Thr212AspThr212Gln + Thr215GlyGly203Glu + Tyr205GlyThr132Pro + Thr215SerTyr103Asp + Gly165GlnThr 65Pro + Gly203GluThr 58Glu + Gly 60ProGly153Pro + Leu216ValAsn 96Gln + Pro209GluGly 99Asn + Gly153AsnAla128Asn + Ala202HisGly105Glu + Gly130SerThr212Gly + Gly218GluPhe188Val + Tyr205SerAsn217Glu + Thr219GlyLeu 95Gln + Thr215AspVal 94Gln + Thr212GluAsn 61Ser + Gly203AspSer129Glu + Asn154GlnGly 62Glu + Thr219AsnSer129Glu + Asn157GlnTyr103Ile + Thr215ProIle164Thr + Thr215SerGly101Gln + Tyr208Gln
Asn 61Gln + Thr212AspThr212Asp + Leu216IleAsn211Glu + Leu216HisGly153Glu + Thr163GlnGly203Asp + Thr215AsnAsn 61Ser + Thr132AsnSer158Glu + Gly203ProLeu 95Asn + Thr215AsnThr 58Ser + Ser100AspIle164Met + Tyr205AlaSer158Glu + Tyr205MetPhe188Leu + Thr212GluGly130Pro + Ser190GluGly101Asp + Gly126SerAsn 61Ser + Ala186SerAsn162Gln + Phe188SerThr 58Gly + Thr 65AspSer129Asp + Thr210ProSer129Glu + Gly153AsnAsp 59Glu + Tyr213ThrAsn154Glu + Leu216GlnSer 97Asp + Ala199ProGly203Asn + Gly218AspThr 65Ser + Gly126GlnThr215Gln + Leu216AlaAsn 96Glu + Tyr205HisThr 65Ser + Gly156AspAla128Ser + Thr163GlnThr215Asp + Leu216ValSer129Glu + Leu216AlaAsn211Asp + Tyr213LeuVal 94Ser + Phe188ValSer158Glu + Thr212GlnGly 99Pro + Asn154GlnGly203Asn + Thr215GlnAla202Ser + Tyr205GluTyr205Val + Asn217GluVal 94Gly + Thr215AspSer104Glu + Thr215GlnGly201Pro + Tyr205GluAsn 96Ser + Gly127GluTyr205Thr + Ala214ThrGly 62Ser + Thr215GlyThr 65Ser + Asn211GluGly153Pro + Gly203SerAla128Thr + Leu216GluAsn 96Glu + Leu216Gln
Thr215Asn + Leu216AlaAsp 59Glu + Ala202ProVal 94Thr + Leu216MetSer 97Glu + Thr212GlnGly101Asp + Ala202AsnSer129Asp + Ala202GlyTyr103Ala + Thr132SerLeu 95Met + Leu216AspSer100Asp + Gly101ProSer100Asp + Thr219ProVal 94Asn + Thr215ProTyr103Asn + Ala128AspGly201Asn + Tyr205AspThr 58Glu + Ile106CysThr 65Glu + Tyr103ProVal 94Met + Ser 98GluGly203Glu + Thr210ProPro209Glu + Gly218SerThr161Pro + Gly203SerSer 97Glu + Leu125ProAla128Asp + Ala199HisIle106Gly + Ala214ProAsp 59Glu + Tyr205IleAsn154Asp + Tyr208HisThr210Asp + Leu216ValThr215Asp + Thr219GlnGly 60Glu + Leu125SerSer 97Asp + Gly153AsnTyr166Pro + Tyr205GlySer129Glu + Gly203GlnGly153Gln + Pro209GlnGly203Gln + Thr215GlnAsn 61Glu + Thr212SerThr163Asp + Tyr205HisAsn154Glu + Tyr166ThrAsn 96Ser + Gly105AspPhe188Gly + Asn211SerGly153Glu + Gly218AsnSer100Asp + Thr212ProGly126Pro + Gly156AspLeu125Ser + Thr215GluAsp 59Glu + Thr 65GlyGly156Pro + Gly159AspThr 58Asp + Ala202SerLeu 95Met + Gly 99SerAsn 61Gln + Tyr205GluGly126Asn + Gly159Pro
Leu 95Thr + Gly218AsnThr 65Gln + Leu216AlaGly130Pro + Gly203GluGly 64Gln + Ser 98GluGly126Asn + Gly127GlnThr132Asn + Gly153AsnThr 58Pro + Gly105GluGly130Asp + Ala214HisGly126Glu + Tyr205ProTyr103His + Ser129AspSer 98Asp + Pro209SerAsp 59Glu + Tyr205SerGly 60Gln + Thr215AsnPro209Asn + Asn211AspThr132Asp + Ala202AsnGly218Ser + Thr219GlyThr 58Asp + Gly203GlnIle106Asp + Ala202ThrThr161Asn + Ser190AspSer129Glu + Thr215GlnThr212Asn + Leu216AspAsn 96Gln + Ser158GluGly203Pro + Thr215AspVal 94Asp + Tyr205AsnAla128Pro + Thr215AspGly201Gln + Leu216ProThr161Pro + Leu216CysGly126Asp + Tyr205MetSer 97Asp + Thr212ProThr210Asn + Leu216HisAla186Asn + Tyr205ProSer131Asp + Phe188ThrThr132Ser + Leu216AsnAsn 61Asp + Gly130ProLeu 95Asn + Thr132GlySer100Asp + Thr215AsnTyr166Thr + Asn211GlnThr207Pro + Leu216ThrIle106Ala + Gly165AspTyr205Asn + Thr215AsnAsn 96Gln + Gly218AsnSer160Asp + Phe188GlyThr210Gly + Thr212SerGly 64Ser + Asn 96GlnGly101Asp + Asn154GlnGly 64Asn + Asn157GluAsn157Glu + Gly203Gln
Tyr213Asn + Thr215SerGly101Asn + Ala186AsnSer160Glu + Leu216IleTyr103His + Ser187GluAsn 96Ser + Ala214AsnGly 62Asn + Ser 98AspIle106Ser + Asn157GluThr 65Glu + Thr212GlyGly 62Asn + Thr215ProGly127Ser + Thr210AspGly156Ser + Asn217GluSer 98Asp + Tyr103ThrGly127Asn + Gly165ProGly153Glu + Leu216MetSer129Asp + Gly165ProAsn 61Asp + Thr163GlnGly126Asn + Tyr205MetAsn157Gln + Thr212GlnSer190Glu + Thr215GlySer129Glu + Gly165GlnThr 65Glu + Gly101ProVal 94Gln + Gly165GluTyr103Ile + Serl90GluAsn217Gln + Gly218SerSer 98Glu + Gly 99AsnSer102Asp + Thr212SerThr212Ser + Thr215AspGly130Glu + Pro209AsnAla128Pro + Gly165ProAsn217Gln + Gly218AsnLeu125His + Thr163SerThr212Asp + Tyr213AlaThr 65Pro + Thr210SerGly105Pro + Ala202GlnVal 94Thr + Ser158GluThr132Gly + Tyr205CysGly105Gln + Ala214HisGly127Gln + Tyr205CysGly127Glu + Gly130SerPhe188Tyr + Asn211SerGly 64Asn + Phe188AspThr212Ser + Leu216AspMet198His + Gly203ProTyr208Val + Thr210AspGly165Glu + Tyr205MetAsn 96Gln + Thr132GluSer 98Glu + Gly101Pro
Leu125Cys + Thr215AspLeu125Cys + Gly126SerSer190Asp + Thr215AsnGly 99Gln + Gly153AspGly 60Asp + Thr210SerSer160Asp + Phe188LeuVal204Gln + Thr215GluAsn157Glu + Gly203SerTyr213Glu + Leu216CysAsn 61Gln + Leu216AspLeu125Gly + Ser155AspAla186Pro + Val204GlnIle106Met + Ser160AspLeu125Ile + Leu216SerGly 60Gln + Gly203AsnGly203Asn + Pro209GluGly 60Ser + Gly 62AspAsn154Glu + Tyr213GlnAsn154Gln + Gly203AspAsn154Glu + Thr219GlnThr212Ser + Leu216HisTyr205His + Thr215AspGly 60Asp + Gly126ProGly203Glu + Leu216AlaGly156Ser + Gly203SerSer155Asp + Gly165GlnAla202Gly + Thr212AspAsn157Gln + Tyr213SerGly 99Ser + Gly130SerGly203Ser + Thr219AspGly130Ser + Ala214AsnThr210Gln + Thr212AspAsn211Gln + Thr212GluGly153Asn + Thr212AspGly153Asp + Tyr205SerSer100Glu + Tyr205ThrGly126Asn + Ash154GluSer190Asp + Tyr205ValSer102Glu + Tyr166ThrAsn157Ser + Pro209GlyAsp 59Glu + Tyr205LeuSer131Glu + Thr132AsnGly127Asp + Leu216ThrThr210Asn + Asn217GlnGly203Ser + Gly218AspIle106Gln + Gly203AspThr161Asp + Tyr205Pro
Gly105Pro + Thr212AspTyr205Thr + Leu216CysAsn 96Ser + Ser158GluAsn 61Glu + Tyr205AlaTyr205Ser + Thr212GluThr163Gln + Phe188GluTyr103Ala + Thr212AspGly101Asp + Ala202HisSer155Glu + Gly218AsnVal 94Thr + Thr163SerSer102Glu + Asn217GlnGly159Ser + Ala202GluAsn154Ser + Tyr166AlaAsp 59Glu + Phe188GlyThr163Gln + Gly218SerGly153Asn + Tyr205ValSer129Glu + Leu216ProGly126Ser + Thr212GluGly203Asp + Leu216AsnGly126Glu + Thr212ProVal 94Pro + Ala186GluGly165Gln + Thr215GluSer129Asp + Gly153AsnSer 98Glu + Thr215SerGly203Ser + Leu216ProGly105Asp + Leu216HisIle106Met + Thr210AspLeu216Asn + Asn217GluGly127Gln + Leu216GluVal 94Glu + Ala202GlyThr132Asn + Leu216HisAsp 59Glu + Ala202AsnLeu 95Ile + Tyr205ProThr 65Gln + Thr215ProSer104Glu + Thr215AsnSer160Glu + Gly203ProGly203Asp + Leu216ProAla128Glu + Tyr213MetSer 98Asp + Leu216CysSer158Asp + Thr215GlnVal 94Asp + Thr215AsnGly127Asp + Asn154SerGly126Ser + Gly156GlnLeu125Gly + Thr212AspThr210Glu + Leu216AsnAsn154Ser + Ala202AsnAsn 96Gln + Gly126Asn
Gly 64Ser + Gly127SerAsn211Ser + Thr212GlySer160Asp + Gly218GlnTyr205Ala + Thr210GluThr212Asp + Leu216SerSer158Asp + Thr215Ser表33多环三突变变异体Leu 95Gly + Gly203Pro + Asn211GluThr 58Gln + Asp 59Glu + Leu216GlnTyr103Cys + Ala202Gln + Thr212GlyGly126Gln + Gly156Glu + Thr215ProAsn 96Gln + Ile106Ala + Tyr205ProAsn 61Ser + Gly101Asp + Gly165GlnGly 64Gln + Thr132Gly + Gly153GluAla128Ser + Ser158Asp + Gly203SerTyr103Glu + Ile106Leu + Gly203ProThr163Pro + Ala202Pro + Leu216SerSer104Glu + Thr132Gly + Phe188ValLeu 95Asn + Phe188Leu + Asn211SerAsn162Gln + Ser187Asp + Tyr213ProThr 58Asp + Gly130Gln + Tyr208ProGly105Pro + Gly156Glu + Leu216IleGly 60Asn + Thr215Ser + Gly218AspThr 65Gln + Leu125Asn + Ala128GluAsn211Ser + Thr212Asp + Gly218GlnGly203Glu + Thr212Gln + Thr215GlyThr132Pro + Tyr205Gly + Thr215SerGly 60Pro + Thr 65Pro + Gly203GluAsn 96Gln + Gly203Asp + Thr215GlyGly 99Asn + Gly153Asn + Pro209GluGly105Glu + Ala128Asn + Ala202HisTyr205Ser + Thr212Gly + Gly218GluGly 99Gln + Asn217Glu + Thr219GlyLeu 95Gln + Ala202Thr + Thr215AspSer129Glu + Asn157Gln + Thr219AsnAsp 59Glu + Tyr103Ile + Thr215ProGly 99Asp + Ile164Thr + Thr215SerAsn 61Gln + Tyr208Gln + Thr212AspGly153Glu + Thr163Gln + Thr215AsnAsn 61Ser + Thr132Asn + Gly203ProLeu 95Asn + Ser158Glu + Thr215AsnThr 58Ser + Ser100Asp + Tyr205AlaSer158Glu + Phe188Leu + Tyr205MetAsn 61Ser + Ala186Ser + Phe188Ser
Asn154Glu + Tyr213Thr + Leu216GlnSer 97Asp + Ala199Pro + Gly203AsnThr 65Ser + Gly126Gln + Gly218AspTyr205His + Thr215Gln + Leu216AlaAla128Ser + Thr163Gln + Thr215AspVal 94Ser + Ser100Asp + Phe188ValGly 99Pro + Asn154Gln + Gly203AsnAsn 96Ser + Thr163Asp + Ala214ThrTyr166Asp + Tyr205Thr + Ala214ThrGly 62Ser + Thr 65Ser + Thr215GlyGly153Pro + Gly203Ser + Thr219GluAsp 59Glu + Ala202Pro + Leu216AlaVal 94Thr + Thr212Gln + Leu216MetSer129Asp + Thr132Ser + Ala202GlyLeu 95Met + Tyr103Ala + Leu216AspSer104Asp + Gly203Asn + Thr215AsnThr 65Glu + Tyr103Prc + Ile106CysVal 94Met + Ser 98Glu + Thr210ProSer 97Glu + Thr161Pro + Gly203SerLeu125Pro + Ala128Asp + Ala199HisAsp 59Glu + Tyr205Ile + Ala214ProSer129Glu + Tyr166Pro + Gly203GlnGly203Gln + Pro209Gln + Thr215GlnAsn 96Ser + Asn154Glu + Tyr166ThrGly105Asp + Phe188Gly + Asn211SerThr 58Asp + Gly156Pro + Ala202SerLeu 95Met + Gly 99Ser + Ser131GluGly126Asn + Gly159Pro + Tyr205GluLeu 95Thr + Leu216Ala + Gly218AsnGly105Glu + Gly130Pro + Leu216ThrThr 58Pro + Thr132Asn + Gly153AsnGly 60Gln + Pro209Asn + Thr215AsnThr132Asp + Ala202Asn + Thr219GlyThr 58Asp + Gly203Gln + Gly218SerSer129Glu + Thr212Asn + Thr215GlnGly203Pro + Tyr205Asn + Thr215AspAla128Pro + Thr215Asp + Leu216ProThr161Pro + Gly201Gln + Leu216CysGly126Asp + Tyr205Met + Thr212ProSer 97Asp + Thr210Asn + Leu216HisAla186Asn + Phe188Thr + Tyr205ProAsn 61Asp + Thr132Ser + Leu216AsnLeu 95Asn + Thr132Gly + Thr215AspIle106Asp + Tyr166Thr + Asn211GlnIle106Ala + Thr207Pro + Leu216ThrTyr205Asn + Thr215Asn + Gly218AsnAsn 96Gln + Ser160Asp + Phe188Gly
Gly 64Ser + Gly101Asp + Asn154GlnThr 58Asn + Gly 64Asn + Asn157GluGly101Asn + Ala186Asn + Thr215SerGly 62Asn + Asn 96Ser + Ala214AsnGly130Asp + Phe188Asn + Gly218ProGly 99Ser + Ala128Glu + Leu216GlyVal 94Ala + Pro209Ser + Thr215AspLeu 95Thr + Gly126Gln + Leu216GlyAsp 59Glu + Gly159Ser + Tyr205ProAla199Gly + Ala202Gln + Gly203GlnSer158Glu + Thr161Gly + Tyr205AsnThr 58Ser + Ser190Glu + Tyr205ProLeu 95Asn + Tyr205Ala + Pro209GluThr 58Asn + Gly153Pro + Thr210ProAla128Gly + Gly203Asn + Tyr205ProVal 94Cys + Asn157Gln + Tyr205GlnSer129Glu + Tyr205Asn + Thr215AsnThr 65Gly + Gly127Asn + Thr212GlnGly 60Asn + Tyr205Thr + Asn217GlnGly126Pro + Ala214Pro + Thr215GlyThr163Gly + Ala202Pro + Thr212ProThr 58Pro + Gly130Ser + Ser160AspGly153Ser + Asn154Glu + Thr212GlyLeu125Gly + Tyr205Thr + Thr210GluTyr103Val + Thr132Gly + Pro200GlnSer100Glu + Gly127Asn + Leu216AlaSer160Glu + Ala186Ser + Tyr205SerGly159Ser + Thr163Glu + Ile164HisGly101Asp + Tyr166Cys + Gly203ProThr 58Asn + Asn 61Glu + Ala214SerGly127Glu + Phe188Met + Ala202GlyAsn162Glu + Gly203Ser + Gly218AsnTyr166Asn + Thr210Glu + Ala214HisIle106Asp + Thr212Ser + Thr215GlnAsn162Glu + Ala202Ser + Thr215AsnTyr103Pro + Ser187Glu + Tyr205ProVal204Thr + Thr212Ser + Thr215GlnSer100Asp + Gly101Asn + Leu216HisLeu 95Cys + Gly203Ser + Leu216ThrAsn 96Ser + Gly153Glu + Tyr205ProIle106Pro + Ala202Ser + Thr212ProSer102Asp + Gly203Ser + Thr215SerLeu 95Cys + Ser104Glu + Phe188CysAsp 59Glu + Ala214Gly + Thr215GlyLeu125Cys + Thr212Gly + Thr215SerLeu125Val + Ala202His + Thr215ProGly101Asp + Gly127Asn + Gly203Gln
Thr 58Pro + Gly105Asn + Gly203GluTyr166Cys + Gly203Ser + Tyr205AspGly126Gln + Ile164Val + Leu216HisGly153Ser + Thr215Pro + Leu216GluSer160Asp + Gly218Gln + Thr219AsnAsn 61Glu + Asn157Ser + Ala214SerGly 62Ser + Ala128Ser + Asn154AspIle106Gly + Tyr205Val + Thr215SerSer160Glu + Asn211Gln + Thr215ProThr 65Gly + Ser100Glu + Thr215ProThr 65Ser + Ser104Glu + Gly203ProAsn 61Ser + Ile106Ser + Thr219ProSer102Asp + Gly153Pro + Leu216HisThr 65Glu + Gly126Gln + Gly203SerSer187Glu + Phe188Ile + Leu216ThrThr132Gln + Gly165Asp + Thr212SerSer129Asp + Asn211Ser + Leu216ThrLeu125Ile + Asn154Ser + Leu216MetThr 65Pro + Gly101Pro + Thr212AspThr 65Gln + Leu125Asn + Leu216MetPro209Gly + Ala214Glu + Thr215ProLeu125Val + Tyr205Ser + Leu216HisLeu 95Ash + Gly156Asp + Thr212GlnLeu125Pro + Thr210Asp + Thr215SerGly105Asn + Thr161Asn + Tyr205ValGly126Ser + Ser160Glu + Gly218AsnVal 94Thr + Thr207Gly + Thr212GluTyr208Gln + Thr212Gln + Gly218AspIle106Asn + Gly159Asn + Phe188AspTyr205Met + Thr212Gln + Tyr213ThrSer131Asp + Thr132Pro + Tyr213AsnGly105Ser + Ala128Thr + Thr215ProLeu 95His + Asn 96Asp + Thr215GlyGly 60Ser + Gly156Asp + Pro209AsnAsn162Asp + Thr163Asn + Ala199GlyTyr103Glu + Leu125His + Thr215SerGly127Glu + Asn157Gln + Tyr205LeuAsp 59Glu + Gly105Pro + Ala186AsnAla128Asp + Tyr213His + Leu216HisThr 65Pro + Ile106His + Ser131GluGly126Gln + Thr161Asp + Ala202SerGly 99Gln + Gly126Asp + Thr212GlyThr 58Asp + Gly159Ser + Thr212SerAsp 59Glu + Ala186Gly + Gly203GlnGly 62Glu + Tyr205Ala + Leu216GlnAsn154Gln + Tyr205Ser + Tyr213MetLeu 95Ser + Ser158Glu + Thr212Gly
Tyr166Asn + Tyr205Glu + Leu216AsnAsp 59Glu + Ala128Asn + Gly153ProGly 99Asp + Gly159Asn + Asn211SerGly165Pro + Thr215Gln + Leu216ThrGly165Asn + Thr215Asn + Leu216AspGly 60Asp + Gly159Asn + Thr212GlnThr 58Glu + Thr163Gly + Thr210GlnGly159Ser + Ala186Gly + Ala199ThrGly203Glu + Asn211Gln + Tyr213GlnGly 99Ser + Asn162Asp + Thr215GlnThr 65Gly + Ser190Glu + Thr219SerVal 94Met + Asn162Ser + Ser190GluAsn 61Gln + Leu 95Ser + Tyr205AspGly126Glu + Gly201Pro + Gly203SerThr 58Gly + Thr212Pro + Gly218GluSer104Asp + Gly105Ser + Gly159ProThr 58Gln + Thr163Gly + Tyr205ProGly 60Ser + Thr 65Pro + Thr163GlyLeu125Met + Ser131Glu + Thr210GlyPhe188Gln + Gly201Ser + Ala202GlyGly203Pro + Leu216Glu + Asn217GlnGly 62Asn + Gly126Pro + Thr210AsnGly 60Asn + Ser104Asp + Gly127GlnSer155Glu + Thr163Pro + Gly203ProLeu 95Met + Thr132Asp + Asn211SerTyr103Ile + Gly159Asp + Ala202GlyGly101Gln + Tyr166Pro + Asn211GlnAsn157Ser + Ala202Ser + Thr215GlyThr132Pro + Ser155Glu + Ala214HisThr132Ser + Thr212Asp + Leu216GlyGly126Asn + Thr212Asp + Gly218GlnTyr205Cys + Thr212Pro + Leu216MetAsn 96Gln + Thr163Gln + Leu216GluAla202Ser + Tyr205Asn + Gly218AspLeu 95Asp + Gly126Asn + Ala202SerSer100Glu + Ile106Gln + Ala202GlnThr 58Ser + Ser100Glu + Ile164LeuGly101Ser + Phe188Asp + Tyr213GlySer104Glu + Gly156Gln + Thr212GlyLeu125Val + Gly203Asn + Tyr205ThrThr163Gln + Thr215Asn + Asn217GluGly 99Pro + Gly165Glu + Ala214GlnMet198Ser + Gly203Glu + Tyr213ValAsn 96Ser + Ser 97Asp + Thr212SerAla202Glu + Gly203Ser + Tyr205GlnGly 64Asn + Asn154Gln + Asn157GluThr 65Ser + Gly130Pro + Tyr205Asp
Gly126Gln + Thr161Gln + Pro209SerAsp 59Glu + Phe188Leu + Leu216AlaSer131Asp + Thr212Pro + Thr215GlnVal204Pro + Gly218Asn + Thr219GluThr 58Ser + Gly203Glu + Thr212ProGly 62Gln + Tyr205Gly + Thr212GluThr 65Gly + Ser129Asp + Met198GlyLeu 95Cys + Asn162Ser + Ala202ThrLeu125Asp + Tyr205Thr + Leu216ThrAsn157Asp + Tyr205Ser + Leu216ProGly156Asn + Gly165Asn + Thr215GluAla128Ser + Thr163Glu + Ala199SerVal 94Ser + Ser187Glu + Thr215GlnVal 94Pro + Thr215Gly + Asn217GluVal 94Asn + Gly130Glu + Gly203SerGly127Gln + Ile164Cys + Thr210GlnSer187Glu + Thr212Gln + Thr215AsnAsn 96Gln + Gly126Gln + Gly203AspGly165Asp + Thr210Ser + Tyr213IleThr 58Gln + Gly153Ser + Asn217GlnGly203Asp + Tyr205Met + Thr210SerAla128Pro + Thr132Glu + Thr215ProSer104Glu + Phe188Ser + Thr212GlySer104Glu + Asn157Ser + Ala214SerIle106Cys + Tyr205Glu + Thr215AsnGly156Gln + Tyr205Pro + Thr219AspAsn157Glu + Tyr213Thr + Leu216IleThr 58Ser + Asn162Ser + Ser190AspGly 62Ser + Thr 65Asp + Tyr205IleLeu125Pro + Gly203Asp + Thr212SerThr 58Gln + Ser 98Asp + Thr212ProSer 97Asp + Leu125Val + Tyr213IleIle106Leu + Phe188Glu + Thr212AsnThr 58Pro + Gly153Pro + Asn162GlnSer190Asp + Thr210Gln + Leu216AlaSer100Glu + Thr215Gly + Leu216MetGly127Glu + Ala128Gln + Thr212ProVal 94Pro + Gly127Ser + Thr215GluThr161Asp + Ala199Asn + Tyr213GlnThr132Pro + Tyr166Leu + Thr212ProGly203Gln + Thr215Asn + Leu216MetGly127Pro + Gly203Asp + Tyr205IleThr163Ser + Tyr205Ala + Pro209GlnVal 94Cys + Thr215Asn + Gly218ProGly126Glu + Gly165Pro + Ala202ThrThr 58Glu + Tyr103Asn + Pro200GlnPhe188Thr + Ala202Asn + Asn217Ser
Gly 60Asp + Leu 95Thr + Tyr205IleSer102Glu + Ala202Ser + Leu216GlnGly 99Asn + Ser102Asp + Gly218GlnIle106Ala + Thr132Pro + Asn157AspGly203Ser + Tyr205Gln + Thr212GluSer 97Asp + Ile106His + Asn211GlnSer100Glu + Gly201Asn + Tyr205HisThr212Gln + Ala214Glu + Thr215GluAsn 61Glu + Gly 62Glu + Gly203ProAsn211Glu + Thr212Asp + Leu216IleGly 99Glu + Ser100Asp + Gly156ProAla202Asp + Gly203Glu + Tyr205ProSer 97Asp + Ser 98Glu + Thr163GlnAla128Thr + Tyr205Asp + Thr215AspSer102Asp + Gly105Asp + Leu216GlyGly127Glu + Gly165Glu + Tyr205ThrGly203Asp + Tyr205Ala + Asn217GluAsp 59Glu + Asn 61Glu + Gly130SerGly 60Glu + Ser 97Glu + Gly 99AsnTyr166Ala + Gly203Glu + Leu216AspSer158Asp + Ser160Glu + Tyr205CysSer129Asp + Tyr166Glu + Thr215GlnSer160Asp + Asn162Glu + Thr210GlySer129Asp + Ser131Asp + Gly218AsnAsn 61Gln + Leu 95Asp + Ser 97AspGly 62Ser + Leu216Asp + Gly218GluAsp 59Glu + Gly165Asn + Pro209GluLeu 95Glu + Gly105Glu + Gly203SerAsn157Glu + Asn162Asp + Thr215GlnTyr103His + Asn162Glu + Ser190AspSer158Asp + Asn162Glu + Thr163ProGly 62Glu + Ser 97Glu + Gly153GlnGly 99Gln + Asn154Asp + Ser187GluSer155Asp + Gly156Asp + Thr219AspGly1535er + Asn154Asp + Gly165AspAsp 59Glu + Asn 96Glu + Tyr205SerAsp 59Glu + Asn 96Asp + Leu216IleSer104Asp + Ser131Asp + Thr210GlySer 97Glu + Gly101Asp + Ala202AsnAsn 96Asp + Ser102Glu + Ile106GlyAsn 61Asp + Leu 95Glu + Val204GlnGly156Asp + Thr161Asp + Gly203SerSer100Asp + Tyr103Asp + Gly126AsnThr 65Asp + Tyr205Asp + Thr215AspThr 58Asp + Ser 97Asp + Thr132GlyThr215Pro + Leu216Asp + Thr219GluSer100Glu + Gly105Glu + Asn162Gln
Ala186Glu + Ala202Glu + Thr215AspAsn 61Asp + Ser100Glu + Asn162SerSer104Asp + Thr132Glu + Tyr205MetGly 99Asn + Thr210Asp + Ala214GluAsn157Asp + Ala186Glu + Thr212SerGly 62Asp + Pro209Asn + Asn211AspGly105Pro + Gly153Glu + Asn162AspAla186Ser + Thr212Asp + Thr215AspGly 99Glu + Ser102Asp + Leu125GlyGly 62Ser + Gly 99Glu + Ser102AspGly126Glu + Ser155Glu + Tyr205ProSer158Glu + Phe188Asp + Ala202SerGly 60Glu + Leu 95Glu + Ile106LeuAla128Asp + Asn154Asp + Thr215GlnAla128Asp + Asn162Glu + Asn217SerAsn157Gln + Ser187Glu + Asn217AspGly 62Asp + Ser 98Asp + Thr212GluGly159Glu + Ser160Asp + Phe188AspGly126Asp + Thr210Glu + Asn211GluSer104Asp + Gly105Asp + Ala128GluAsp 59Glu + Tyr205Glu + Thr215GluGly 60Asp + Tyr205Glu + Thr215AspAsn 61Glu + Pro209Asp + Thr215GluAsn 61Asp + Ser 97Glu + Tyr205AspSer102Asp + Gly105Glu + Pro209AspSer158Asp + Gly203Asp + Thr215GluAsn157Asp + Gly203Asp + Thr215AspGly127Glu + Ser129Glu + Thr215GluAsn 96Glu + Ser100Asp + Thr212GluSer190Glu + Gly203Glu + Tyr205GluSer129Asp + Phe188Glu + Ser190AspSer100Asp + Ser104Glu + Thr212ProSer129Glu + Gly153Glu + Asn157GlnSer 97Glu + Thr212Glu + Ala214GluSer100Asp + Ser102Glu + Ser160AspAsp 59Glu + Ser 97Glu + Asn157AspSer 97Glu + Asn157Glu + Ser160GluGly101Asn + Leu125Glu + Ser129GluGly101Glu + Gly130Glu + Thr161SerAsn154Glu + Ser158Glu + Gly203GlnVal 94Asp + Ser187Asp + Ser190Glu表34多环四突变变异体Thr 58Gln + Leu 95Gly + Gly203Pro + Asn211GluAsp 59Glu + Tyr103Cys + Thr212Gly + Leu216Gln
Gly126Gln + Gly156Glu + Ala202Gln + Thr215ProAsn 615er + Gly101Asp + Gly165Gln + Tyr205ProSer104Glu + Thr132Gly + Phe188Val + Leu216SerGly127Glu + Thr212Asn + Thr215Ser + Leu216GlnLeu125Thr + Thr210Gln + Thr212Asp + Thr215SerThr132Pro + Gly203Glu + Tyr205Gly + Thr215GlyAsn 96Gln + Gly153Pro + Pro209Glu + Leu216ValGly 99Asn + Ala128Asn + Gly153Asn + Ala202HisPhe188Val + Tyr205Ser + Thr212Gly + Gly218GluTyr103Ile + Ser129Glu + Asn157Gln + Thr215ProLeu 95Asn + Ser158Glu + Gly203Pro + Thr215AsnThr 58Ser + Ser100Asp + Ile164Met + Tyr205AlaThr 65Ser + Gly126Gln + Gly203Asn + Gly218AspAsn 96Glu + Tyr205His + Thr215Gln + Leu216AlaThr 65Ser + Ala128Ser + Gly156Asp + Thr163GlnAsn154Gln + Asn157Glu + Gly203Asn + Thr215GlnGly 62Ser + Tyr205Thr + Ala214Thr + Thr215GlyAsp 59Glu + Asn 61Gln + Ala202Pro + Leu216AlaLeu 95Met + Tyr103Ala + Thr132Ser + Tyr205AspSer 97Glu + Leu125Pro + Thr161Pro + Gly203SerAsp 59Glu + Ile106Gly + Tyr205Ile + Ala214ProSer129Glu + Tyr166Pro + Gly203Gln + Tyr205GlyAsn 61Glu + Gly203Gln + Thr212Ser + Thr215GlnGly153Glu + Phe188Gly + Asn211Ser + Gly218AsnTyr103Val + Gly126Asn + Pro209Asn + Leu216AspAla128Ser + Ser190Glu + Tyr213Ala + Leu216ValGly127Ser + Asn154Ser + Tyr205Leu + Thr212GlnAla128Glu + Asn154Gln + Asn157Gln + Leu216HisSer155Asp + Gly156Ser + Gly203Ser + Leu216AlaGly 99Ser + Gly130Ser + Asn157Gln + Tyr213SerGly130Ser + Gly203Ser + Ala214Asn + Thr219AspAsp 59Glu + Asn157Ser + Tyr166Thr + Pro209GlyGly203Ser + Thr210Asn + Leu216Thr + Asn217GlnTyr103Ala + Thr163Gln + Ala202His + Thr212AspGly 62Glu + Asn154Ser + Tyr166Ala + Phe188GlyAsp 59Glu + Thr163Gln + Tyr205Val + Gly218SerVal 94Pro + Gly153Asn + Gly165Gln + Thr215GluSer 98Glu + Asn154Gln + Gly203Ser + Leu216ProIle106Glu + Thr132Asn + Ala202Gly + Leu216HisAsp 59Glu + Leu 95Ile + Ala202Asn + Tyr205ProThr 65Gln + Asn 96Gln + Ser104Glu + Thr215ProLeu125Gly + Gly126Ser + Asn154Ser + Gly156GlnAsn 96Gln + Gly126Asn + Gly127Ser + Ala202AsnGly 64Ser + Ser160Asp + Asn211Ser + Thr212GlyGly101Pro + Ser158Asp + Thr215Ser + Leu216SerVal 94Glu + Ala202Ser + Tyr205Ser + Thr212GlyGly153Ser + Asn154Glu + Thr161Asn + Thr212Gly
Tyr103Val + Leu125Gly + Thr132Gly + Pro200GlnGly127Pro + Phe188Met + Ala202Gly + Thr215GluAsn162Glu + Gly203Ser + Ala214His + Gly218AsnAsn 96Glu + Gly 99Pro + Thr132Asn + Thr212ProGly101Asn + Tyr205Gly + Tyr213Met + Thr215AspLeu125Pro + Gly126Asn + Ala202Gly + Gly203AspGly 99Pro + Gly127Glu + Tyr205His + Thr212GlnTyr103Pro + Tyr166Ile + Gly203Ser + Thr207GlyThr 58Gly + Gly 62Ser + Gly105Glu + Ala128SerIle106Gly + Tyr205Val + Thr215Ser + Leu216AspAsn 61Ser + Ile106Ser + Ser190Asp + Thr219ProThr 65Glu + Gly126Gln + Gly203Ser + Leu216HisSer187Glu + Phe188Ile + Thr212Ser + Leu216ThrLeu125Ile + Asn154Ser + Asn211Ser + Leu216MetThr 65Gln + Leu125Asn + Pro209Gly + Ala214GluLeu125Val + Tyr205Ser + Thr215Pro + Leu216HisLeu 95Asn + Leu125Pro + Gly156Asp + Thr212GlnGly126Ser + Ser160Glu + Thr161Asn + Gly218AsnLeu 95Gln + Phe188Ile + Ala202His + Thr212AsnSer104Glu + Thr212Gln + Tyr213Thr + Thr215ProAla128Thr + Ser131Asp + Thr132Pro + Tyr213AsnLeu 95His + Ser129Glu + Thr215Gly + Leu216AsnGly105Pro + Ala128Asp + Tyr213His + Leu216HisGly159Ser + Tyr205Ala + Thr212Gly + Leu216AlaGly 62Glu + Asn154Gln + Tyr205Ser + Leu216GlnLeu 95Ser + Ser158Glu + Val204Gln + Thr212GlySer 98Asp + Leu216Ile + Asn217Ser + Gly218AsnLeu125Gly + Ala128Gln + Gly203Glu + Pro209GlnGly105Asn + Tyr166Val + Ala202Asp + Thr215ProSer 97Asp + Ala202Gln + Pro209Gln + Thr215GlyLeu 95Thr + Tyr205Ile + Leu216Val + Thr219SerGly 99Asn + Ser102Asp + Tyr205Ser + Gly218GlnThr 65Pro + Gly165Asn + Ala186Pro + Gly201SerAsn154Asp + Gly203Gln + Tyr205Asn + Thr212AsnIle106His + Ala186Gly + Thr212Asn + Leu216AspGly126Glu + Gly127Gln + Ala128Asn + Ala202AsnLeu 95Ser + Ala128Gly + Thr161Gln + Asn217GluAsp 59Glu + Gly101Pro + Ala199Gly + Gly203SerAsn 61Asp + Val204Thr + Tyr205Cys + Thr215SerGly101Asn + Asn162Asp + Phe188Ala + Tyr205HisAla186Thr + Gly203Asp + Thr215Pro + Asn217SerTyr103Gln + Phe188Met + Thr210Glu + Thr219ProGly 62Asp + Gly126Ser + Leu216Pro + Thr219AsnGly 60Gln + Tyr205Pro + Thr212Ser + Thr215AspIle106Val + Ser187Glu + Thr212Pro + Thr215SerGly159Asp + Asn211Ser + Ala214Gln + Thr219ProAsn 96Ser + Ser160Asp + Ala214Gln + Leu216Gly
Ser104Asp + Gly203Ser + Tyr205Ser + Thr215ProGly153Gln + Phe188Tyr + Thr215Glu + Leu216IleSer129Asp + Thr132Pro + Thr212Gln + Leu216ThrAla128Gln + Ala186Asn + Thr215Asp + Gly218AsnAsn 96Asp + Ala202Pro + Tyr205Gln + Pro209SerThr132Asp + Gly165Pro + Thr210Pro + Leu216CysGly105Pro + Gly130Ser + Thr161Glu + Thr215ProIle106Cys + Asn157Gln + Tyr205His + Thr212GlnLeu 95Gly + Ser100Glu + Gly101Pro + Gly126SerGly 60Ser + Gly105Asp + Thr132Pro + Tyr205ThrThr132Ser + Ile164Val + Gly203Glu + Gly218AsnGly203Gln + Thr212Pro + Thr215Asn + Leu216ProAsn 61Ser + Val 94Gln + Thr132Asp + Val204GlyGly201Ser + Thr212Gly + Thr215Pro + Leu216ProGly153Pro + Ser160Glu + Gly165Gln + Gly218SerTyr166Ile + Tyr213Ser + Ala214Glu + Gly218AsnThr132Ser + Thr163Asp + Tyr166Val + Ala199HisThr132Gln + Asn157Ser + Tyr208Gly + Thr215AspGly203Glu + Tyr205Ile + Tyr208Pro + Thr215SerGly127Ser + Gly165Ser + Tyr213Ile + Leu216HisGly156Glu + Pro209Asn + Asn211Ser + Thr212SerGly165Gln + Phe188Leu + Ala202His + Thr212GluSer102Asp + Gly130Asn + Asn162Ser + Leu216ThrVal 94Cys + Gly156Ser + Tyr205Ala + Leu216AlaGly 99Pro + Tyr103Glu + Phe188His + Leu216CysTyr103Gln + Gly126Gln + Gly156Pro + Tyr166AsnLeu125Pro + Gly153Glu + Asn162Ser + Thr215SerGly 64Ser + Gly 99Asn + Gly203Asp + Thr210GlnGly 99Asn + Ala202Pro + Thr212Asn + Gly218SerAla128Ser + Gly203Gln + Pro209Glu + Gly218SerThr 58Ser + Tyr103Gly + Leu125Gly + Tyr205GlyAsn154Glu + Thr161Ser + Asn162Gln + Gly203GlnGly101Pro + Asn154Ser + Leu216His + Asn217GluThr163Asp + Ala186Ser + Tyr205His + Leu216ProGly159Glu + Gly203Ser + Thr210Gly + Leu216HisLeu125Ser + Thr163Glu + Ala202Pro + Thr210GlnGly153Pro + Ser155Glu + Thr210Asn + Leu216CysGly101Asp + Gly126Gln + Tyr166Ile + Gly203ProSer158Asp + Gly201Asn + Va1204Asn + Pro209SerTyr166Pro + Tyr205Pro + Thr215Asp + Leu216AlaGly 64Asn + Ser160Asp + Tyr205Leu + Thr215GlyGly101Ser + Thr132Gln + Gly159Pro + Thr161SerTyr103Val + Asn154Gln + Gly159Ser + Asn211AspGly127Glu + Asn157Ser + Thr161Asn + Thr212GlyAsn 61Asp + Ile164Asn + Pro209Asn + Leu216ValSer158Asp + Asn162Ser + Tyr205Asn + Thr212GlyLeu125Val + Ala202Thr + Pro209Asn + Thr219Pro
Gly 60Asp + Asn154Gln + Gly203Pro + Tyr205LeuLeu125Gln + Asn162Ser + Gly203Asp + Thr215GlySer102Asp + Tyr103Ile + Tyr205Met + Thr212GlnGly 99Gln + Ala214Gln + Leu216Ala + Gly218GluGly105Asn + Gly156Gln + Gly203Pro + Gly218AspIle106Ser + Thr212Asn + Leu216Glu + Gly218GlnAsn 96Asp + Tyr166Thr + Gly203Ser + Gly218ProSer155Asp + Gly203Gln + Tyr213Thr + Thr215GlyGly101Ser + Thr212Gly + Thr215Asn + Leu216HisThr 65Ser + Leu 95Asn + Thr132Ser + Asn162AspLeu 95Met + Thr132Asn + Gly203Asp + Thr215GlyAsn154Ser + Gly203Glu + Thr212Gly + Leu216AlaGly 60Ser + Ser131Glu + Thr210Ser + Asn217GlnGly 62Asn + Asn162Gln + Gly203Gln + Thr215SerGly 99Ser + Tyr103Ala + Thr210Gln + Thr219SerAsn162Gln + Gly203Glu + Thr215Gln + Thr219GlyAsn162Gln + Gly165Asn + Leu216Ile + Gly218SerThr 65Asn + Gly 99Gln + Thr212Ser + Leu216AspPhe188Val + Ala214Asp + Thr215Asp + Leu216GlnGly 99Glu + Ser100Asp + Gly156Pro + Tyr205GlnVal 94Asn + Tyr103Asp + Ser104Asp + Thr215ProGly 64Gln + Asn154Asp + Ser155Glu + Leu216SerAsn154Glu + Ser155Glu + Gly156Gln + Thr215ProGly153Pro + Asn157Ser + Tyr205Asp + Thr215AspTyr103Leu + Phe188Met + Tyr205Glu + Thr215GluTyr205Ala + Thr210Glu + Thr212Asp + Gly218GlnAla202His + Tyr205Val + Thr210Glu + Thr212AspGly203Asp + Tyr205His + Thr212Ser + Thr215AspGly127Glu + Ser129Asp + Gly153Asn + Thr210ProAla128Asn + Tyr166Ala + Gly203Asp + Leu216AspSer 98Glu + Ser100Glu + Gly203Ser + Asn217GlnAsn 61Asp + Gly 62Pro + Asn 96Asp + Ser 97GluGly159Ser + Thr161Glu + Thr163Glu + Ile164HisSgr160Glu + Asn162Glu + Asn211Gln + Thr215ProAsn 61Asp + Gly 99Asp + Thr161Gly + Thr212ProIle106Val + Ala186Asp + Ala202Glu + Thr215GlnAsp 59Glu + Gly 64Asn + Thr 65Glu + Tyr213SerVal 94Pro + Thr215Asp + Leu216His + Asn217AspAsp 59Glu + Gly127Asn + Ala186Pro + Pro209AspTyr103Glu + Ser131Glu + Tyr213His + Thr215SerSer129Asp + Thr132Gln + Gly165Asp + Leu216ThrTyr205Ile + Thr212Glu + Ala214Glu + Thr219GlnThr 58Gly + Thr212Gln + Tyr213Asp + Thr215AspAsp 59Glu + Gly 62Gln + Ser 97Glu + Tyr205GlyPhe188Asp + Tyr208Gln + Thr212Gln + Gly218AspAla186Asp + Ala202Asp + Gly203Glu + Tyr205ProAsn154Asp + Ser155Asp + Gly165Ser + Ser187Asp
Ser 97Asp + Ser100Glu + Gly201Asn + Tyr205HisSer 97Asp + Ser100Asp + Gly165Ser + Tyr205ThrGly105Ser + Asn157Glu + Ser160Glu + Thr215ProGly156Asp + Thr161Asp + Ser190Asp + Gly203SerThr 65Gly + Ser100Glu + Leu125Glu + Thr215ProGly 62Glu + Ser 97Glu + Gly153Gln + Pro209GlnGly 62Glu + Asn157Gln + Gly203Gln + Thr210GluAsn154Asp + Gly165Glu + Thr212Gly + Gly218ProAsn162Asp + Gly165Asp + Thr212Gln + Leu216CysSer100Glu + Gly126Glu + Ala186Gln + Tyr205AlaAsn154Ser + Ser158Asp + Ser190Glu + Thr215GlnSer129Asp + Thr132Asp + Gly203Gln + Thr215GlnAsn 96Ser + Thr163Asp + Tyr166Asp + Ala214ThrGly 60Glu + Ser 97Glu + Tyr166Asn + Thr210GluIle106Gln + Asn154Glu + Gly203Asp + Gly218AspSer100Glu + Gly105Glu + Asn162Gln + Phe188SerAla186Glu + Ala202Glu + Thr215Asp + Asn217GlnAsp 59Glu + Gly 99Asp + Ile164Thr + Thr215SerSer129Glu + Asn154Glu + Asn162Ser + Gly165AspSer104Glu + Thr132Glu + Tyr205Met + Thr215ProGly 62Asp + Ile106Leu + Pro209Asn + Asn211AspAsp 59Glu + Ser 98Asp + Gly156Asn + Gly218AsnAsn 96Asp + Ser100Asp + Ser104Glu + Thr132AsnAla128Asp + Asn154Asp + Tyr213Met + Thr215GlnSer131Glu + Gly153Asp + Gly165Asp + Tyr205GlyGly101Asp + Gly105Asp + Gly126Asn + Ser131GluSer129Asp + Asn157Glu + Thr163Glu + Thr212ProGly 99Asn + Ala128Asp + Asn162Glu + Asn217SerSer 98Asp + Ser100Asp + Ser104Glu + Thr212ProAsn 96Gln + Ile106Ala + Gly156Glu + Ser160AspGly203Asp + Pro209Glu + Thr215Asp + Leu216ProAsn 96Asp + Ser 97Glu + Ser104Asp + Ala214SerGly159Asn + Gly165Glu + Leu216Cys + Thr219AspGly153Asp + Ser187Asp + Gly203Asn + Leu216SerThr 65Glu + Tyr103Pro + Thr215Asp + Asn217AspGly 62Asp + Ala202Glu + Thr215Asp + Leu216SerThr 65Asp + Gly101Gln + Gly130Ser + Tyr205AspAsn 61Ser + Thr 65Asp + Thr132Asn + Tyr205AspGly 99Pro + Ser187Glu + Gly203Glu + Thr215AspThr 65Ser + Gly126Asp + Thr210Glu + Asn211GluAsn 96Asp + Thr161Asp + Asn162Glu + Ala202AsnGly101Glu + Ile106Met + Ala214Asp + Thr215GluAsn 61Asp + Ala186Asp + Ser187Glu + Leu216ProSer104Asp + Tyr205Val + Thr215Asp + Leu216AspLeu 95Asp + Ala202His + Thr215Glu + Leu216GluSer129Asp + Gly130Asp + Thr210Gln + Thr212AspIle106Glu + Asn162Ser + Asn211Asp + Thr212Glu
Thr 58Asp + Asp 59Glu + Ile106Glu + Phe188SerSer104Asp + Gly105Asp + Ala128Glu + Thr215GlyGly 99Asp + Ser100Glu + Thr161Glu + Pro209GlnGly 99Asp + Ser100Glu + Ser190Asp + Tyr205HisAsp 59Glu + Ala202Asp + Gly203Glu + Leu216CysAsp 59Glu + Gly 60Glu + Ala128His + Thr215AspGly126Asn + Asn154Glu + Ser155Asp + Gly203AspSer 97Glu + Ser 98Asp + Ser131Glu + Asn162SerSer 97Glu + Ser 98Glu + Ser104Asp + Asn217GlnGly156Ser + Ser158Glu + Tyr205Asp + Thr215AspAsn 61Glu + Ser 97Glu + Ala128His + Tyr205AspGly 64Asn + Thr 65Glu + Phe188Tyr + Thr215GluTyr103Glu + Ile106Glu + Gly203Pro + Gly218GluGly 99Glu + Ser102Asp + Leu125Gly + Ser131GluSer102Glu + Asn154Glu + Ser190Asp + Tyr205ValGly127Glu + Gly165Glu + Tyr205Thr + Thr215GluVal 94Glu + Gly105Asp + Gly165Pro + Thr212GluAsn 96Glu + Ser 98Asp + Tyr103Leu + Ser155AspAsp 59Glu + Asn 61Glu + Gly105Glu + Gly130SerAsp 59Glu + Asn 61Asp + Thr 65Gly + Tyr205GluLeu 95Gln + Tyr103Asp + Gly203Asp + Thr215AspAsn157Asp + Gly203Asp + Thr215Asp + Thr219GlnSer 98Glu + Gly203Glu + Thr215Glu + Gly218SerSer131Asp + Asn154Gln + Gly203Asp + Thr215GluGly101Asn + Ile106Glu + Gly159Asp + Thr161AspGly127Glu + Ser129Glu + Gly130Ser + Thr215GluGly153Gln + Ser155Asp + Pro209Glu + Thr212AspGly203Glu + Thr210Asp + Leu216Asp + Gly218ProVal 94Ser + Ser 98Glu + Ser100Asp + Phe188AspAsn 96Asp + Ser100Asp + Gly153Ser + Asn162GluGly 62Glu + Leu 95Glu + Ile106Leu + Thr212GluSer160Asp + Asn162Asp + Ala202Glu + Thr215GlnAsn 61Glu + Ser 98Asp + Gly126Glu + Leu216AsnSer100Asp + Ser104Asp + Gly105Asn + Tyr205CysAsn 61Asp + Gly 99Glu + Tyr103Thr + Ser131AspVal 94Glu + Asn157Asp + Thr163Glu + Tyr205AlaGly127Asp + Ser131Glu + Thr132Asn + Ser190AspGly 60Glu + Gly156Ser + Asn211Asp + Thr212GlnGly126Asp + Gly156Glu + Ser158Glu + Tyr205MetAsn157Asp + Tyr205Val + Thr215Asp + Asn217GluAsp 59Glu + Gly203Glu + Tyr205Asn + Pro209AspGly105Glu + Ser131Asp + Gly156Asn + Leu216ProAsp 59Glu + Ser 97Glu + Asn157Asp + Phe188HisSer 97Asp + Ser100Asp + Ala202Pro + Thr212GluVal 94Gly + Ser104Glu + Asn157Asp + Ser160AspAsn 61Glu + Asn162Glu + Ser190Asp + Thr215GlyAsn 81Asp + Asn 96Glu + Gly127Ser + Gly130Asp
Thr 65Glu + Gly165Glu + Phe188Val + Ser190GluTyr103Pro + Asn154Glu + Ser158Glu + Tyr205ProSer131Glu + Gly156Glu + Tyr166Asp + Ala186ThrAsn 61Glu + Thr 65Asp + Tyr103Pro + Ser131AspGly130Glu + Asn154Asp + Ser187Asp + Tyr208HisSer131Glu + Thr163Pro + Gly203Glu + Gly218GluAsp 59Glu + Asn162Asp + Thr163Gln + Thr210GluLeu125Asp + Gly165Asp + Gly203Asp + Tyr213GlnGly101Asp + Asn162Asp + Gly165Glu + Leu216AsnSer155Glu + Ser158Asp + Tyr205Glu + Leu216ProGly 60Asp + Tyr205Pro + Thr212Asp + Thr215GluSer160Glu + Thr163Asp + Tyr205His + Thr215AspThr 58Pro + Asn 61Glu + Asn154Glu + Asn157AspAsp 59Glu + Gly 99Ser + Gly153Asn + Thr212AspSer 97Glu + Gly101Asp + Thr132Glu + Ala202AsnThr 58Glu + Asn 61Asp + Ala186Gly + Ser187AspThr 58Glu + Asn 61Glu + Ile106Cys + Thr132AspSer158Glu + Gly203Glu + Asn211Gln + Ala214AspSer129Asp + Gly203Glu + Ala214Glu + Thr215SerSer104Asp + Ser155Glu + Asn157Gln + Asn162GluThr 65Glu + Ser160Glu + Tyr205Cys + Thr210AspAsn 61Asp + Leu 95Glu + Tyr205Glu + Leu216AsnSer100Glu + Gly127Glu + Thr215Asp + Leu216ProSer102Glu + Gly127Glu + Ala128Pro + Asn162GluAsn 96Asp + Gly105Glu + Asn162Asp + Thr212GlnSer 98Glu + Gly127Asp + Ser131Glu + Ala202SerAsp 59Glu + Leu 95Asp + Gly159Asp + Tyr205ValThr 65Glu + Ser187Glu + Thr215Gln + Gly218GluGly156Glu + Gly159Glu + Tyr205Met + Thr215GluGly 64Gln + Gly130Glu + Thr132Gly + Gly153GluAsp 59Glu + Gly 99Glu + Ser129Glu + Tyr205AsnGly126Glu + Ala186Glu + Gly203Asp + Thr212ProThr 58Asp + Tyr103Val + Ser104Glu + Thr132AspSer104Glu + Thr132Glu + Ala202Glu + Asn217GlnSer 98Glu + Thr132Gly + Ser187Glu + Thr219GluGly 62Asp + Ser100Asp + Gly105Asn + Tyr205ValSer 97Asp + Ala186Asn + Thr212Glu + Thr215AspSer187Asp + Ala202Gly + Thr212Glu + Thr215GluAsp 59Glu + Ser 98Glu + Gly153Ser + Asn154GluGly126Glu + Thr132Asp + Ser155Asp + Ala202GlyAsp 59Glu + Gly 62Pro + Ser158Glu + Phe188GluSer104Asp + Gly130Asp + Tyr205Pro + Thr212GluThr 65Pro + Ser102Asp + Gly126Asp + Ala202AspGly 62Asp + Ser129Glu + Thr215Asp + Leu216ValAla128Asp + Asn162Asp + Ala202Ser + Tyr205GluSer 97Glu + Tnr132Gly + Gly153Asp + Ser187AspSer102Asp + Leu125Asp + Gly156Asp + Gly218Asn
Asn 61Gln + Ser160Glu + Ser190Asp + Gly203GluAsn 96Asp + Tyr166Asp + Ser190Glu + Leu216CysThr 65Asp + Asn162Glu + Tyr205Asp + Thr215ProThr 65Asp + Ser 97Glu + Gly105Glu + Leu216MetVal 94Asp + Ser 98Asp + Ser158Asp + Thr215GlnThr 65Glu + Gly101Asp + Thr215Glu + Gly218AsnThr 58Asp + Gly156Ser + Asn211Asp + Thr215GluGly 99Gln + Gly126Gln + Asn154Asp + Thr161AspGly156Asn + Ala186Asp + Thr212Glu + Leu216AspSer 97Asp + Ile106Gln + Leu125Asp + Thr215SerSer100Glu + Ser104Glu + Tyr166Ala + Ser187AspSer 98Asp + Ser102Glu + Thr215Glu + Leu216GlnSer129Asp + Thr132Gln + Ser155Glu + Ser160AspSer 97Glu + Thr163Asp + Ser187Glu + Thr219SerLeu125Cys + Ser129Glu + Ser155Glu + Thr215GlyAsn 61Asp + Gly126Glu + Ala214Asp + Thr219GlnThr 58Asn + Gly 64Pro + Thr 65Glu + Val 94AspTyr205Leu + Tyr213Asp + Thr215Gly + Asn217AspAsp 59Glu + Ala202Gln + Thr212Asp + Asn217AspAsp 59Glu + Ser187Asp + Pro209Asn + Thr212AspThr 58Asn + Leu 95Glu + Phe188Asp + Gly203AspSer187Glu + Tyr205Ile + Thr212Glu + Leu216GluSer129Glu + Gly156Gln + Thr212Asp + Leu216AspGly101Glu + Ser129Glu + Asn157Glu + Tyr205ThrGly 60Glu + Leu125Ser + Gly153Glu + Ala202AspAsn 61Glu + Phe188Glu + Tyr205Ala + Thr212GluAsn 61Glu + Gly153Asn + Gly159Asp + Thr212AspThr 65Gly + Tyr205Glu + Asn211Glu + Leu216CysThr 65Glu + Asn 96Glu + Gly156Asp + Leu216Cys表35多环五突变变异体Tyr103Cys + Gly156Glu + Ala202Gln + Thr212Gly + Leu216GlnAsn 61Ser + Gly101Asp + Thr132Gly + Gly165Gln + Tyr205ProGly 60Asn + Asn162Gln + Thr212Pro + Tyr213Gln + Asn217AspGly 60Ser + Leu 95His + Ser100Glu + Gly156Pro + Thr215GlyLeu 95Ile + Thr163Gln + Tyr205Glu + Tyr213Ala + Thr219AsnGly159Glu + Phe188Tyr + Gly203Pro + Tyr205Gly + Ala214ThrGly126Pro + Gly127Gln + Tyr208Asn + Thr212Gln + Leu216AlaLeu 95Gly + Asn162Ser + Gly165Glu + Pro209Gly + Thr210GlyThr 58Ser + Leu 95Asn + Ser100Asp + Tyr205Ala + Thr215AsnAsn 61Ser + Ser100Glu + Asn162Gln + Phe188Ser + Gly203SerTyr103Ser + Thr161Pro + Phe188Gln + Gly201Gln + Leu215ProSer 97Asp + Gly 99Pro + Tyr205Pro + Thr210Asn + Leu216HisLeu 95Met + Ser187Asp + Tyr205Val + Thr212Gly + Leu216AsnIle106Asp + Tyr166Thr + Thr207Pro + Asn211Gln + Leu216Thr
Asn 96Gln + Ser160Asp + Phe188Gly + Thr215Asn + Gly218AsnThr 58Pro + Ser 98Asp + Gly130Pro + Tyr205Val + Tyr208ProVal 94Ala + Gly 99Ser + Ala128Glu + Phe188Asn + Leu216GlyLeu 95Thr + Gly126Gln + Pro209Ser + Thr215Asp + Leu216GlySer 98Glu + Asn154Gln + Gly203Ser + Thr215Ser + Leu216ProThr 58Asp + Leu125Pro + Ala202Thr + Gly203Pro + Tyr205ValAla128Asp + Gly153Gln + Ala186His + Ala199Gln + Gly203AsnAsp 59Glu + Gly159Gln + Ala186His + Leu216Ser + Gly218SerAla186His + Phe188Gln + Ser190Glu + Pro200Ser + Tyr205ValVal 94Gly + Ser 98Asp + Tyr166Pro + Ala202Gly + Tyr205HisAsn 61Gln + Tyr103Met + Asn157Asp + Thr161Ser + Gly203AsnThr 65Gln + Gly203Gln + Pro209Ser + Thr215Gln + Asn217AspGly126Ser + Ser160Asp + Thr163Gln + Gly203Ser + Tyr205GlyTyr103Gln + Gly156Pro + Tyr166Asn + Phe188Tyr + Thr215GluVal 94Met + Gly 99Asn + Thr212Asn + Tyr213Gly + Thr215GluAla128Ser + Ala202Pro + Gly203Gln + Pro209Glu + Gly218SerThr 58Ser + Tyr103Gly + Leu125Gly + Tyr205Gly + Gly218SerLeu125Pro + Asn154Glu + Thr161Ser + Asn162Gln + Gly203GlnGly 60Asp + Gly 62Pro + Asn 96Ser + Ala186Gly + Thr210GlnAsn 61Gln + Thr 65Ser + Leu 95Met + Tyr103Gly + Ser104AspAsn 61Ser + Gly155Gln + Gly156Gln + Ala202Gly + Asn211GlnSer102Asp + Gly126Gln + Ala202Asn + Tyr205Leu + Thr212GlyGly 60Asn + Ser104Asp + Gly105Asn + Thr163Ser + Tyr205AlaVal 94Asn + Gly101Asn + Leu125Asn + Gly130Asp + Leu216CysGly159Pro + Gly165Pro + Ala186Thr + Gly201Pro + Leu216SerLeu 95Cys + Gly130Gln + Thr212Ser + Ala214Asn + Leu216ValGly 60Glu + Thr161Gly + Gly203Gln + Tyr205Ile + Thr212GlyGly153Ser + Gly159Asn + Ala186Pro + Gly203Ser + Thr219AsnAsp 59Glu + Gly 62Gln + Leu125Ala + Thr215Asn + Asn217GlnVal 94Gly + Leu125Ser + Gly203Glu + Thr215Gln + Leu216AsnAla186Gln + Ser187Glu + Gly201Gln + Ala202Pro + Gly203SerAsn157Asp + Gly203Gln + Thr212Pro + Ala214Gln + Leu216AlaThr 65Ser + Ser 98Asp + Thr161Ser + Gly203Ser + Tyr205CysGly 99Asp + Gly165Ser + Met198Cys + Pro209Gly + Thr215GlyGly153Pro + Tyr208Ile + Thr212Ser + Ala214His + Gly218GluSer 98Asp + Ile106Ala + Tyr166Leu + Ala202Gln + Ala214ThrGly 99Gln + Ser129Glu + Thr161Asn + Phe188Cys + Tyr205ThrGly 60Asn + Gly159Gln + Ser160Glu + Thr163Pro + Ala186GlyAsn 96Asp + Gly 99Pro + Val204Ala + Tyr205Val + Asn217GlnGly 60Ser + Phe188Ile + Thr212Asn + Thr215Ser + Leu216AspAla128Gly + Thr132Gln + Gly165Gln + Met198Thr + Gly203GluAsn 96Asp + Asn157Ser + Gly201Asn + Thr212Gly + Thr215GlyGly 99Asp + Phe188Val + Gly203Asn + Thr207Asn + Leu216ProAla128Pro + Thr132Pro + Thr163Pro + Phe188Thr + Thr212GluVal 94His + Gly105Glu + Ala186Pro + Thr210Gln + Thr212GlyGly101Pro + Thr132Asp + Asn162Ser + Thr212Gln + Leu216ProGly 60Asn + Tyr103Asn + Gly105Gln + Ile106Ala + Leu216Gln
Gly126Pro + Gly203Pro + Tyr205Val + Thr215Asn + Leu216SerThr 58Glu + Thr161Gly + Tyr205Asn + Tyr208Leu + Leu216ThrGly101Asn + Gly159Pro + Asn162Ser + Ala202Gly + Tyr205GlnTyr103Ile + Gly105Pro + Ala202Gln + Thr210Gln + Leu216ProGly 60Asp + Gly153Ser + Thr163Gly + Thr212Pro + Leu216MetGly101Pro + Ser104Glu + Gly105Pro + Gly153Ser + Tyr205ThrAsn162Asp + Phe188His + Ala199Gly + Thr212Pro + Leu216ProAla186Gly + Ala202Pro + Tyr205Met + Thr210Gln + Leu216ValGly 62Ser + Tyr103Met + Asn154Glu + Thr212Gly + Leu216MetThr 65Ser + Gly101Pro + Tyr103Leu + Thr161Asp + Thr219SerGly105Ser + Ile106His + Ala186Thr + Met198Pro + Thr212ProThr163Gly + Gly203Asp + Thr212Asn + Leu216His + Asn217GlnGly126Ser + Thr161Gln + Gly203Pro + Thr210Pro + Leu216ValThr 65Gln + Ser131Asp + Ala186Gly + Thr215Ser + Leu216HisThr132Gln + Asn162Ser + Gly165Asp + Tyr205Gly + Tyr208CysGly127Gln + Gly153Glu + Gly203Pro + Thr215Gly + Thr219GlyIle106Met + Leu125Cys + Gly126Ser + Ser131Glu + Gly156AsnSer 97Glu + Thr132Gly + Pro200Gly + Tyr205Ala + Asn211SerThr 65Asp + Tyr166Gln + Gly203Asn + Thr210Ser + Thr212GlnGly 99Ser + Tyr166Ser + Ser187Glu + Tyr205Gln + Leu216SerGly126Ser + Gly130Asn + Thr161Asp + Phe188Val + Thr207GlnLeu 95Pro + Pro200Ser + Tyr205Leu + Thr210Glu + Leu216CysTyr103Glu + Gly127Gln + Tyr166Thr + Ala202His + Thr210SerThr 58Gly + Ala202Pro + Pro209Gln + Thr212Ser + Ala214GluGly 62Gln + Tyr103Thr + Thr163Gly + Ser187Asp + Gly203GlnGly101Gln + Leu125Glu + Thr132Ser + Gly203Ser + Thr212GlnAsn 96Ser + Ala128His + Ala186Gln + Tyr205His + Thr215SerGly101Ser + Gly165Asp + Thr210Ser + Thr212Gly + Thr215GlyGly156Gln + Ala202Gly + Ala214His + Thr215Pro + Leu216ThrVal 94Cys + Gly 99Gln + Gly126Asn + Gly159Pro + Thr219ProLeu 95Pro + Gly105Pro + Thr161Asp + Gly203Pro + Leu216AsnGly 60Glu + Asn 61Gln + Thr 65Gln + Thr210Gly + Leu216GlnLeu125Gly + Ser187Asp + Phe188Gln + Ala202Asn + Thr212AsnPhe188Ile + Gly203Glu + Thr210Gly + Thr212Gln + Leu216ProAsp 59Glu + Leu 95Ile + Ile106Leu + Gly203Gln + Tyr213IleGly 99Gln + Ala186Pro + Tyr205Gly + Thr215Glu + Gly218GlnSer 98Glu + Thr161Pro + Ala202Gln + Tyr205Leu + Thr207GlnSer100Glu + Gly153Asn + Thr163Gln + Thr212Pro + Leu216GlnLeu 95Ser + Ile106Asp + Tyr205Val + Leu216Pro + Thr219AsnTyr166Val + Ser190Asp + Thr207Gly + Thr212Ser + Leu216AsnAsp 59Glu + Gly105Gln + Thr132Gln + Ala186Ser + Tyr205IleGly 60Pro + Tyr205Ile + Asn211Gln + Leu216Ile + Asn217GlnGly 99Asn + Leu125T r + Asn157Gln + Asn162Glu + Thr212GlnIle106Ala + Asn162Glu + Tyr166His + Asn211Ser + Thr215GlnAsn154Asp + Phe188Gly + Gly203Pro + Tyr205Ala + Pro209SerLeu 95Asn + Gly105Glu + Tyr166Ala + Tyr205Leu + Thr212SerVal 94Ala + Ser131Glu + Thr132Glu + Phe188His + Thr212Gly
Gly 64Gln + Gly159Asp + Ser160Glu + Thr215Pro + Leu216AlaThr 58Glu + Asp 59Glu + Pro209Gln + Thr212Asn + Thr215GlnAsn 96Asp + Ser 97Glu + Tyr205Thr + Ala214Thr + Leu216SerAsp 59Glu + Gly 60Glu + Gly159Asn + Thr210Pro + Gly218AsnAsp 59Glu + Gly 60Asp + Leu 95Ser + Gly203Pro + Tyr205SerTyr103Ile + Asn154Ser + Tyr205Glu + Ala214His + Thr215AspGly105Asn + Gly127Gln + Ala202Glu + Gly203Ser + Asn217GluAsn 61Asp + Thr 65Gln + Ser 97Asp + Tyr205Ala + Leu216SerAsn 61Glu + Ser 97Glu + Gly105Gln + Asn162Ser + Thr212SerAsn 61Asp + Leu 95Ser + Ser 97Glu + Val204Gln + Thr212GlyTyr103His + Asn154Glu + Phe188Ile + Ser190Glu + Thr212GlnVal 94Pro + Asn 96Asp + Ser 98Glu + Tyr103Pro + Thr212SerVal 94Met + Ser158Asp + Thr161Glu + Gly203Ser + Gly218SerGly203Asp + Tyr213Pro + Thr215Asn + Asn217Asp + Gly218AspSer160Glu + Asn162Glu + Tyr205Val + Asn211Gln + Thr215ProAsn 61Asp + Ser 98Glu + Tyr103Cys + Gly203Ser + Tyr213AlaGly101Asn + Tyr103Pro + Ile106Asn + Asn157Glu + Thr161AspGly130Asn + Ala186Glu + Ala202Glu + Thr215Pro + Thr219AsnGly126Asp + Ala128Glu + Thr132Ser + Met198Cys + Gly218SerVal 94Pro + Leu125Gly + Thr215Asp + Leu216His + Asn217AspGly 62Ser + Ile106Ala + Ala128Thr + Thr215Asp + Asn217GluGly105Pro + Ser129Glu + Thr132Asn + Gly165Glu + Asn211GlnAsp 59Glu + Asn 96Asp + Ser 97Glu + Phe188His + Thr210ProAsn 61Gln + Gly153Glu + Thr163Asp + Gly203Gln + Leu216SerAsp 59Glu + Thr 65Gly + Leu 95Gly + Ser 97Asp + Tyr166GlyAsn157Asp + Ser160Asp + Asn162Glu + Thr210Gly + Thr212SerSer 97Glu + Ser100Asp + Gly101Ser + Thr207Gln + Thr212AsnGly 64Ser + Gly203Ser + Thr212Asp + Ala214Asp + Thr215AspGly 60Asn + Thr132Asn + Ser155Glu + Gly156Glu + Asn162AspGly 99Ser + Tyr103Ile + Gly130Gln + Phe188Glu + Thr219GluGly156Asp + Thr161Asp + Ser190Asp + Gly203Ser + Ala214GlnGly101Asp + Ser104Asp + Gly127Ser + Thr210Gln + Thr212GlnGly130Gln + Ser155Asp + Tyr213Met + Gly218Asp + Thr219GluGly 99Ser + Asn154Ser + Gly203Asp + Leu216Asn + Gly218AspSer102Glu + Gly130Asp + Ser131Asp + Ala202His + Thr210AsnGly153Pro + Gly159Asp + Asn162Asp + Ala186Gln + Ala202GlnAsn 96Asp + Ser100Glu + Gly105Asp + Val204Thr + Thr215ProAsn162Asp + Gly165Asp + Thr212Gln + Thr215Ser + Leu216CysSer155Asp + Gly165Ser + Ser187Glu + Tyr205His + Thr212GlySer100Glu + Gly126Glu + Ala186Pro + Tyr205Asn + Tyr213GlnGly 62Pro + Ser104Glu + Ser131Asp + Thr212Gln + Tyr213ThrGly203Asp + Tyr205Asn + Asn211Gln + Thr212Gly + Ala214AspIle106Cys + Asn154Glu + Ala186Glu + Gly203Ser + Leu216AlaThr 58Pro + Asn 61Asp + Val 94Glu + Asn 96Asp + Gly165ProLeu 95Glu + Ser 97Glu + Ser102G1u + Thr161Ser + Tyr205LeuThr 58Gly + Ala128Pro + Gly156Asp + Thr161Asp + Gly203AsnGly 60Ser + Gly126Pro + Thr161Glu + Ser190Glu + Thr212Gly
Gly 60Ser + Ser102Glu + Gly105Asp + Ser131Glu + Gly203AsnAsn154Gln + Ala186Ser + Tyr213Glu + Leu216Glu + Thr219ProGly101Asp + Ile106Cys + Gly126Asp + Met198Gln + Thr219ProLeu 95Gln + Asn154Asp + Ser190Asp + Thr212Gly + Asn217GluThr 58Gln + Asn 61Glu + Ser100Asp + Tyr205Ala + Thr215GlyAsn 61Glu + Gly 99Gln + Ser100Glu + Tyr103Met + Leu125SerAla128Ser + Gly130Ser + Gly159Asp + Thr163Glu + Tyr20SCysAsp 59Glu + Gly 99Asp + Tyr103Ile + Ile164Thr + Thr215SerLeu 95Ile + Ser102Asp + Ser131Asp + Ile164Val + Leu216IleAsn 96Gln + Thr212Pro + Ala214Asp + Leu216Glu + Thr219GluIle106Glu + Ser131Asp + Ala202Gly + Tyr205Asn + Thr215SerIle106Glu + Gly130Ser + Ser131Asp + Phe188Ala + Tyr205ProThr 65Gln + Asn154Asp + Thr215Glu + Leu216His + Asn217GluAsn 61Gln + Gly 99Gln + Asn154Glu + Ser158Asp + Ser187GluGly101Ser + Gly126Asn + Thr212Glu + Thr215Asp + Leu216GlnAsp 59Glu + Thr 65Pro + Ser 98Asp + Tyr103Pro + Leu125IleGly153Ser + Ser155Asp + Gly165Asp + Ser187Asp + Asn217SerSer100Asp + Ser102Glu + Gly130Glu + Gly156Pro + Tyr205IleGly105Ser + Gly153Glu + Thr215Asn + Leu216Asp + Asn217GluThr 58Asp + Asn 96Glu + Ala186Gly + Gly203Pro + Thr219GlyAsn154Asp + Ser160Asp + Gly165Ser + Ser190Asp + Ala202SerAsp 59Glu + Ser 98Glu + Tyr103Asn + Thr132Ser + Pro209GluAla128Asp + Asn154Asp + Tyr166Ala + Pro200Gly + Tyr205MetGly101Gln + Asn157Gln + Gly203Asp + Thr215Gly + Thr219AspAsn 61Gln + Gly126Asp + Asn162Glu + Ser190Asp + Thr215GlyThr 58Gln + Asp 59Glu + Leu 95Gly + Gly203Pro + Asn211GluIle106Asn + Leu125Val + Ser129Glu + Thr163Asp + Ala214GlyGly126Gln + Gly127Asp + Ala128Ser + Ser155Asp + Ser158GluSer190Glu + Ala202Glu + Thr212Asn + Thr215Gly + Asn217AspSer155Glu + Gly156Glu + Ala202Asp + Thr215Gly + Thr219GlnSer160Glu + Ser190Asp + Gly203Gln + Tyr205Asn + Thr212AsnGly156Asn + Tyr166Ala + Gly203Glu + Thr212Glu + Leu216AspGly 99Gln + Ser102Glu + Gly126Asp + Asn154Glu + Tyr205AsnThr 65Pro + Gly127Glu + Ser131Glu + Ser155Asp + Tyr205CysAsn 96Gln + Thr161Gly + Ser190Glu + Thr210Glu + Asn211AspGly130Asn + Ser187Glu + Pro209Gln + Ala214Glu + Thr215GluGly101Glu + Ile106Met + Thr210Ser + Ala214Asp + Thr215GluSer104Asp + Thr210Gly + Thr212Gln + Ala214Glu + Thr215GluAsn 96Gln + Ser158Asp + Gly159Asp + Thr163Asn + Leu216GluAsn 96Gln + Ser158Glu + Gly203Pro + Thr215Asp + Leu216AspGly127Asn + Gly130Gln + Ser160Glu + Thr215Asp + Leu216AspGly 60Asn + Asn 61Glu + Gly 62Glu + Asn157Asp + Tyr205SerGly105Asn + Ala128Glu + Ser129Glu + Gly153Pro + Ala202AspThr 58Asp + Tyr103Met + Gly153Ser + Asn157Glu + Ser158GluGly 64Ser + Gly 99Asn + Ser129Asp + Gly130Asp + Asn157AspAsn154Gln + Ala202Asp + Asn211Glu + Thr212Asp + Leu216IleGly101Asp + Ser102Glu + Gly159Glu + Ile164Cys + Asn217Gln
Gly 62Pro + Gly101Asp + Ser102Glu + Ser190Glu + Thr215AsnThr 58Glu + Asp 59Glu + Gly130Glu + Ala214Gln + Leu216ValAsn 61Ser + Ser102Glu + Ser160Glu + Thr161Asp + Leu216ValSer102Asp + Ser160Glu + Thr161Asp + Tyr205Ser + Asn211GlnGly 62Pro + Ser104Glu + Gly105Asp + Leu125Met + Ala214AspVal 94Cys + Gly105Asp + Asn162Glu + Thr163Glu + Leu216IleSer 97Asp + Ser 98Asp + Ala202Asp + Tyr205His + Leu216ThrGly 62Glu + Asn 96Glu + Gly105Asp + Tyr166Ser + Pro209GlyVal 94Asp + Ala128Asp + Thr132Asn + Gly165Asp + Thr212SerSer102Glu + Gly105Glu + Asn154Asp + Gly156Asn + Leu216GlyTyr103Glu + Gly130Gln + Ile164Leu + Thr210Glu + Thr212AspAsp 59Glu + Asn157Ser + Tyr166Met + Gly203Asp + Asn217AspGly 60Gln + Gly130Asn + Ser155Glu + Thr215Glu + Asn217AspVal 94Thr + Gly 99Asp + Gly153Pro + Tyr205Glu + Ala214GluGly101Asn + Ser131Asp + Asn154Gln + Gly203Asp + Thr2l5GluGly101Pro + Gly127Pro + Asn157Glu + Gly203Asp + Thr215AspThr 65Gln + Gly153Glu + Gly165Glu + Gly203Asp + Leu216CysSer 98Asp + Ser100Glu + Ala128Gly + Gly159Asp + Gly165ProSer 98Asp + Ser100Asp + Ser187Asp + Gly203Asn + Thr212SerSer 98Glu + Ser100Glu + Ser155Asp + Asn162Ser + Thr210AsnAsn 96Asp + Ser100Asp + Gly127Asn + Tyr205Cys + Thr219AspAsn 96Asp + Ser100Glu + Gly159Asp + Tyr205Gln + Ala214ProLeu 95Gln + Gly101Pro + Ser102Asp + Gly153Asp + Tyr166GluAsn 96Ser + Gly159Asp + Ser190Asp + Tyr205Ile + Ala214GlyThr 65Glu + Ser 97Glu + Gly101Glu + Ala186Pro + Pro200GlyGly 99Asn + Gly126Asp + Ser158Glu + Ser160Glu + Tyr205GlnGly 62Glu + Leu 95Glu + Ile106Leu + Thr212Glu + Ala214SerAsn 61Ser + Gly 99Pro + Ser104Asp + Gly203Asp + Tyr205AspGly101Ser + Tyr103Ala + Gly203Gln + Tyr205Asp + Gly218AspSer160Asp + Asn162Asp + Phe188Pro + Thr212Glu + Leu216SerIle106Ser + Ser160Glu + Asn162Glu + Gly203Asp + Leu216ValVal 94Asp + Gly126Asp + Gly130Pro + Thr212Ser + Leu216AsnThr 65Pro + Ser155Asp + Asn157Asp + Ala186Thr + Leu216AspSer100Glu + Gly105Pro + Ala128Gly + Asn157Asp + Thr163GluGly126Glu + Ala186Asp + Ala202Asp + Gly203Gln + Leu216AsnThr132Gly + Ser160Asp + Ala186Glu + Ala202Asp + Tyr205GlnTyr103Pro + Ser129Glu + Ala186Glu + Ala202Glu + Thr215AsnLeu125Pro + Ser129Asp + Gly153Glu + Asn162Ser + Leu216IleAsp 59Glu + Thr 65Glu + Gly105Ser + Ser131Asp + Tyr213SerGly126Asp + Gly156Glu + Ser158Glu + Ile164Met + Tyr205MetAsn 96Glu + Tyr103Gly + Leu125Glu + Asn154Glu + Thr163GlnThr 58Glu + Ala128Ser + Gly156Asp + Thr163Asp + Thr210GlnGly 99Asp + Leu125Thr + Gly156Glu + Ser158Asp + Gly203ProGly101Glu + Gly156Asp + Ser158Asp + Ala202Gln + Pro209AsnSer131Glu + Gly156Asp + Ser158Glu + Phe188Leu + Gly203AsnGly 62Ser + Ser129Glu + Asn157Gln + Gly165Asp + Thr215GluSer 97Asp + Ser129Glu + Gly165Asp + Thr215Asn + Asn217Ser
Ala186Asp + Tyr205Cys + Thr212Asp + Ala214Asp + Thr215SerVal 94His + Ser158Glu + Thr212Asp + Ala214Glu + Thr215GlnLeu125Glu + Gly159Pro + Ala186His + Thr212Asp + Ala214GluAsn 61Asp + Tyr103Gln + Gly105Gln + Leu125Asp + Pro209AspGly 99Gln + Gly159Ser + Pro209Asp + Thr215Glu + Leu216AlaSer100Asp + Ser102Asp + Gly105Asn + Gly203Asp + Asn211GlnSer 97Glu + Ser100Glu + Gly165Glu + Phe188Val + Leu216AsnAsn 96Ser + Leu125Asp + Ser129Glu + Tyr205Gln + Thr215ProAsn 96Glu + Asn157Asp + Gly165Gln + Ser187Asp + Tyr205ValLeu 95Asp + Ser102Asp + Gly203Glu + Val204Ser + Leu216SerGly 62Glu + Thr 65Gln + Asn157Asp + Asn162Asp + Thr215GlnSer131Glu + Gly165Asp + Leu216Cys + Asn217Gln + Thr219AspVal 94Asp + Ser187Asp + Ser190Glu + Tyr205Asn + Thr215ProAsp 59Glu + Asn 96Gln + Ser187Glu + Ser190Asp + Thr215AsnSer158Asp + Asn162Asp + Gly203Ser + Leu216Glu + Asn217SerGly126Pro + Ser158Asp + Asn162Asp + Thr212Asp + Leu216ThrSer102Asp + Ser158Asp + Thr163Asp + Met198Gly + Thr207ProGly 60Glu + Leu125Val + Ser155Glu + Gly165Glu + Val204AsnSer131Glu + Asn154Glu + Ser187Glu + Tyr213Val + Asn217GlnGly 62Asp + Ala128His + Asn154Glu + Ser187Glu + Gly203SerAsn 61Gln + Gly 62Pro + Gly105Asp + Gly203Glu + Gly218AspSer102Asp + Gly203Glu + Ala214Gly + Thr215Pro + Gly218AspAsp 59Glu + Asn157Asp + Thr163Pro + Pro209Gly + Thr210AspLeu125Glu + Asn162Ser + Gly165Glu + Thr212Asp + Thr215GlnIle106Asn + Ser155Glu + Tyr166Val + Ser187Asp + Thr212AspGly105Pro + Ser155Glu + Ser187Asp + Tyr205Pro + Thr215AspAsn 96Glu + Ser155Glu + Ser158Asp + Thr161Pro + Thr215ProThr 58Asp + Thr 65Asp + Tyr103Val + Thr132Asp + Phe188GlyAsp 59Glu + Asn 96Glu + Ser190Glu + Gly203Ser + Ala214SerSer155Glu + Ile164Ser + Ala202Asp + Tyr205Asp + Leu216ProTyr103Ala + Ser104Asp + Ser131Asp + Ser160Asp + Tyr213LeuThr 58Glu + Asn 61Asp + Ala186Gly + Ser187Asp + Tyr205AlaAsn 96Glu + Ser102Glu + Gly153Asp + Gly203Pro + Asn217GlnAsn 96Glu + Ser102Asp + Gly105Pro + Asn162Glu + Tyr213ValSer129Asp + Gly153Asn + Gly203Glu + Ala214Glu + Thr215GlnSer100Glu + Gly127Glu + Gly203Glu + Tyr205Ala + Tyr213ThrSer158Asp + Ser190Asp + Gly203Asp + Thr212Pro + Thr215GlyLeu 95Ile + Thr132Gly + Ser187Asp + Tyr213Asp + Leu216GluGly 60Glu + Gly 62Pro + Ala128Glu + Ser155Glu + Ala202SerThr 58Asp + Ser 97Asp + Gly153Ser + Gly159Glu + Ala214ThrAsn 61Asp + Ser100Glu + Gly126Asn + Ser129Asp + Thr219SerAsp 59Glu + Leu 95Asp + Phe188Glu + Pro209Ser + Gly218SerTyr103Glu + Ser187Glu + Thr212Ser + Thr215Gln + Gly218AspSer104Glu + Ser190Asp + Tyr205Asn + Tyr208Val + Gly218AspAla128Asp + Ser131Glu + Thr161Gln + Tyr205Glu + Leu216MetSer102Asp + Ala128Glu + Ile164Gly + Gly203Asn + Thr212SerGly 62Asp + Gly105Glu + Tyr208Ala + Thr210Ser + Thr212Asp
Ser 97Glu + Ser102Glu + Ile106Ser + Asn157Ser + Leu216GlyGly 62Asn + Leu 95Met + Ser 97Asp + Ser102Asp + Asn217SerSer102Glu + Leu125Cys + Ser131Asp + Ala202Ser + Asn217GluSer102Asp + Ser131Glu + Thr163Asp + Thr207Gly + Thr215AsnSer 97Asp + Ile106Thr + Ala186Asp + Gly203Asp + Thr215GlyThr 58Asp + Gly127Asp + Ala128Thr + Ser155Asp + Gly203SerVal 94Gly + Gly127Asp + Ser155Asp + Gly156Gln + Tyr205GluIle106Glu + Gly126Asp + Gly127Asn + Gly156Glu + Ala202AsnSer 97Asp + Gly203Pro + Asn211Ser + Thr215Asp + Gly218GluGly 99Asp + Ser104Asp + Ile106Val + Thr132Asp + Thr215AsnGly 62Glu + Ser100Glu + Gly127Pro + Tyr205Ile + Thr212GlnGly 99Glu + Gly126Asp + Ala186Glu + Gly203Ser + Asn217SerThr132Asp + Tyr205Glu + Pro209Gln + Tnr212Glu + Asn217SerSer 97Asp + Gly127Gln + Ala186Asn + Thr212Glu + Thr215AspSer102Asp + Asn154Gln + Thr212Asp + Thr215Asp + Leu216GlyLeu125Gln + Ser129Asp + Asn162Gln + Thr212Glu + Thr215AspAsp 59Glu + Ser 98Glu + Leu125Gly + Ala128Asn + Ser129AspAsp 59Glu + Ser 98Asp + Gly156Asn + Ala202Asp + Gly218AsnAsn 61Ser + Asn157Glu + Gly203Asp + Tyr205His + Thr219AspIle106Asp + Gly130Asp + Tyr166Ile + Ala186Ser + Thr212AspAsp 59Glu + Gly156Asp + Thr161Asn + Leu216Ala + Gly218AspThr 58Asp + Asn 96Glu + Ser131Asp + Phe188Val + Thr215GlyLeu 95Val + Ser155Glu + Tyr166Ala + Ala186Glu + Thr210AspSer 98Asp + Leu125Cys + Ala128Asp + Thr161Gly + Asn162GluAsn 96Gln + Ile106Ala + Gly156Glu + Ser160Asp + Thr210GluAsp 59Glu + Tyr166Glu + Tyr205Asn + Thr210Gly + Asn211GluAsp 59Glu + Gly153Glu + Pro209Ser + Asn211Asp + Thr212ProAsp 59Glu + Tyr103Gly + Gly153Glu + Ser187Glu + Asn211GlnAsp 59Glu + Thr163Ser + Ser187Glu + Val204Thr + Asn217GluSer104Glu + Ile106Ala + Gly156Asp + Gly165Asp + Tyr205AsnGly126Glu + Ser131Asp + Asn154Ser + Tyr205Ser + Thr215AspAsp 59Glu + Gly101Gln + Thr212Glu + Ala214Pro + Leu216AspAsn154Ser + Ile164Cys + Thr212Asn + Ala214Asp + Gly218GluThr 65Glu + Ile106Asp + Gly127Asn + Thr215Asp + Leu216HisGly 60Gln + Gly 99Asp + Gly127Glu + Tyr205Glu + Thr215AsnThr 58Gln + Gly 60Glu + Ile106Val + Tyr205Glu + Asn211GluGly130Pro + Gly159Glu + Phe188Leu + Ser190Glu + Thr212GluAsn 61Glu + Gly127Asp + Ser190Glu + Tyr205Ala + Leu216SerSer187Glu + Pro209Asp + Thr212Asn + Ala214Ser + Thr215AspSer155Glu + Tyr166Glu + Gly203Glu + Val204Pro + Thr215SerSer 98Asp + Ser102Glu + Ala202Ser + Thr215Glu + Leu216GlnVal 94Asn + Gly101Asn + Leu125Glu + Ser129Glu + Thr212AspSer129Asp + Ser155Asp + Ala202His + Gly203Asn + Asn211SerAsn 96Asp + Tyr103His + Gly105Asn + Ala202Glu + Ala214GluLeu 95His + Gly 99Gln + Ser190Glu + Ala202Asp + Thr210AsnGly101Asp + Ala128Glu + Thr132Glu + Gly156Asn + Tyr166IleGly105Asp + Leu125Asp + Ala202Pro + Gly203Glu + Leu216Gln
Gly105Asp + Leu125Glu + Gly203Glu + Thr212Gly + Leu216CysGly 62Glu + Val 94Ala + Ser129Asp + Tyr205Asp + Thr212SerAsn 61Asp + G1y101Glu + Tyr205Glu + Thr215Pro + Leu216HisSer104Asp + Leu125Glu + Ser190Glu + Asn211Gln + Leu216AsnGly105Asn + Gly127Asp + Ala128Gly + Ser158Asp + Thr219GluSer 97Asp + Ser155Glu + Ser160Asp + Asn211Gln + Thr215SerAsp 59Glu + Gly 64Gln + Gly130Glu + Gly153Glu + Tyr205AsnAsn157Asp + Tyr205Leu + Thr212Glu + Thr215Gly + Leu216GluThr 58Glu + Gly101Glu + Gly105Pro + Gly130Ser + Tyr166AspSer 97Glu + Gly159Asp + Tyr166Ala + Ser187Glu + Leu216ValGly101Asp + Ser131Glu + Thr212Gln + Thr215Asp + Leu216IleGly153Asn + Asn157Asp + Tyr213Leu + Asn217Ser + Thr219AspGly153Asp + Ser158Glu + Tyr205Gly + Leu216Val + Gly218SerGly 99Ser + Ser187Asp + Gly203Glu + Pro209Asp + Asn211GlnLeu 95Ala + Ser102Asp + Ser187Glu + Gly203Asp + Thr219GlnGly 62Asp + Ala128Thr + Ser187Glu + Gly203Glu + Asn211SerGly101Asp + Ala128Thr + Asn154Gln + Phe188Asp + Leu216Glu表36多环六突变变异体Tyr103Cys + Gly126Gln + Gly156Glu + Ala202Gln + Thr212Gly +Thr215ProAsn 61Gln + Gly101Asp + Ile106Ala + Asn162Ser + Gly165Gln +Tyr205ProGly126Pro + Ala128His + Ser187Glu + Gly203Ser + Tyr208Asn +Leu216AlaThr 65Ser + Gly126Gln + Gly203Asn + Thr215Gln + Leu216Ala +Gly218AspIle106Ala + Gly165Asp + Tyr166Thr + Thr207Pro + Asn211Gln +Leu216ThrSer158Glu + Thr161Gly + Ala199Gly + Ala202Gln + Gly203Gln +Tyr205AsnThr 58Asn + Leu 95Asn + Gly153Pro + Tyr205Ala + Pro209Glu +Thr210ProGly126Glu + Ala128Gly + Asn157Gln + Gly165Asn + Gly203Asn +Tyr205ProGly 64Ser +Asn 96Gln + Gly126Asn + Gly127Ser + Asn211Ser +Thr212GlyGly126Asn + Ser158Asp + Gly203Pro + Tyr205Met + Thr215Gly +Thr219GlnLeu125Val + Gly156Asp + Tyr205Ser + Thr212Gln + Thr215Pro +Leu216HisGly105Asn + Gly126Ser + Ser160Glu + Thr161Asn + Tyr205Val +Gly218AsnGly105Ser + Ala128Thr + Thr132Pro + Ser160Glu + Tyr213Asn +Thr215ProAsp 59Glu + Gly105Pro + Asn157Gln + Ala186Asn + Tyr213His +Leu216HisGly 99Gln + Gly126Gln + Asn154Asp + Gly159Ser + Tyr205Ala +Thr212GlyThr 58Gln + Ser104Asp + Gly105Ser + Gly159Pro + Thr163Gly +Tyr205ProLeu125Met + Ser131Glu + Phe188Gln + Gly201Ser + Ala202Gly +Thr210GlyGly 60Asn + Gly 62Asn + Ser104Asp + Gly126Pro + Gly127Gln +Thr210AsnGly101Gln + Asn157Ser + Tyr166Pro + Ala202Ser + Asn211Gln +Thr215GlySer 97Aslp + Gly130Pro + Thr163Pro + Val204Thr + Tyr205Cys +Ala214ProTyr103Ala + Ala128His + Gly153Gln + Asn154Ser + Ser187Glu +Gly203SerAla128His + Tyr166Pro + Thr207Ser + Thr212Pro + Thr215Pro +Gly218GluAsn157Gln + Ser187Asp + Phe188Ala + Gly203Pro + Tyr205Ser +Thr212SerVal 94Pro + Tyr103Gly + Gly105Gln + Gly126Ser + Ala214Asp +Thr215GlyLeu 95Asn + Gly203Pro + Tyr205Met + Thr212Asn + Thr215Gln +Leu216HisLeu 95His + Tyr166Cys + Ala202Gly + Thr207Gln + Pro209Gly +Leu216HisGly 62Ser + Asn 96Gln + Thr132Gln + Gly159Ser + Gly165Pro +Thr219GluThr132Gln + Asn157Ser + Tyr166Val + Ala199His + Tyr208Gly +Thr215AspGly127Ser + Gly165Ser + Gly203Glu + Tyr205Ile + Tyr208Pro +Thr215SerGly156Glu + Pro209Asn + Asn211Ser + Thr212Ser + Tyr213Ile +Leu216HisTyr103Gln + Gly126Gln + Ala128Gln + Gly156Pro + Tyr166Asn +Ala202ThrThr 58Ser + Tyr103Gly + Leu125Gly + Thr161Ser + Asn162Gln +Tyr205GlyGly 60Ser + Gly101Gln + Gly127Asp + Gly203Pro + Pro209Gly +Thr215GlnGly105Gln + Leu125Asn + Ser187Glu + Pro200Gly + Ala214Ser +Leu216ThrAsn 61Glu + Leu 95Ile + Asn 96Ser + Tyr166Asn + Thr210Ser +Gly218SerGly 60Asn + Ser104Asp + Gly105Asn + Thr161Ser + Thr163Ser +Tyr205AlaVal 94Gly + Asn162Asp + Gly165Asn + Asn211Gln + Leu216Ile +Gly218SerSer 97Glu + Tyr103Met + Gly153Pro + Pro200Ser + Gly203Ser +Leu216MetThr 65Gln + Tyr103Leu + Ala128Asp + Ile164His + Tyr205Thr +Leu216SerGly127Glu + Phe188Gln + Gly203Gln + Pro209Asn + Leu216Met +Gly218ProGly 62Glu + Gly156Asn + Asn162Gln + Gly201Ser + Thr210Pro +Thr212AsnGly 60Pro + Tyr103Asn + Gly127Asn + Thr161Gly + Thr215Asp +Gly218GlnGly 60Asn +Gly159Gln + Ser160Glu + Thr163Pro + Ala186Gly +Pro209GlnAla202Thr + Gly203Ser + Tyr205Ala + Pro209Glu + Thr210Gln +Thr215GlnVal 94Asn + Ile106Asp + Gly126Pro + Tyr205Cys + Thr215Ser +Leu216CysVal 94Gln + Gly105Pro + Gly159Asp + Ala202Ser + Thr210Ser +Thr215SerAsn 61Ser + Asn154Asp + Ala186Thr + Pro209Ser + Tyr213Ile +Ala214GlnThr 58Pro + Gly127Ser + Gly159Ser + Ala186Glu + Thr215Pro +Thr219AsnAla128Gly + Ser129Glu + Thr132Gln + Gly165Gln + Met198Thr +Thr219GlnVal 94Gln + Asn157Asp + Tyr166Cys + Tyr205Ser + Thr207Pro +Thr215AsnAsn 96Gln + Thr132Glu + Ile164Ser + Thr210Pro + Thr212Gln +Leu216MetGly101Asn + Tyr103Ile + Gly159Pro + Asn162Ser + Ala202Gly +Thr210GlnGly 60Asn + Val 94Gln + Ser 97Glu + Gly203Gln + Tyr205Pro +Leu216MetTyr103Gln + Gly130Pro + Ser155Glu + Gly159Asn + Tyr205Pro +Thr212SerAsn157Gln + Gly203Asp + Pro209Ser + Thr215Ser + Leu216Cys +Thr219SerGly101Pro + Ile106Val + Gly126Glu + Ala128His + Asn157Ser +Thr219ProGly 64Pro + Gly203Ser + Tyr205Pro + Pro209Asp + Thr210Asp +Ala214GlnThr 65Gln + Gly203Pro + Pro209Gln + Thr212Pro + Gly218Glu +Thr219AspAsn 61Ser + Ser104Asp + Gly105Asp + Asn162Gln + Ala186Ser +Phe188SerTyr103Asp + Ser104Asp + Gly153Ser + Asn162Ser + Ala202Gly +Tyr208GlnAsn154Asp + Ser155Glu + Asn162Ser + Gly203Asn + Pro209Gly +Leu216AsnSer 97Glu + Ser 98Glu + Asn157Gln + Ala202Asn + Tyr205Asn +Leu216MetThr 65Gln + Gly 99Ser + Thr132Ser + Asn154Asp + Ser155Asp +Ser190AspAsn 96Asp + Gly 99Asp + Gly165Pro + Gly203Asn + Thr215Gln +Leu216ThrAsp 59Glu + Asn 61Asp + Val 94Asn + Gly101Ser + Asn217Gln +Thr219SerGly101Asn + Ile106Gln + Gly130Gln + Gly159Asp + Thr161Asp +Phe188SerSer 98Glu + Gly 99Ser + Ser100Glu + Tyr103Ala + Gly203Ser +Asn217GlnThr 65Gly + Val 94Met + Asn157Asp + Ser190Glu + Leu216Ser +Thr219SerGly130Asn + Ser160Glu + Asn162Glu + Ala186Asn + Gly203Pro +Tyr205GlnAsn 61Glu + Leu 95Pro + Ser 98Glu + Leu125Cys + Phe188Cys +Gly203ProSer129Asp + Ser131Asp + Gly203Pro + Tyr205Asn + Thr212Ser +Thr215GlnGly101Asp + Tyr103Asp + Leu125Thr + Thr161Asn + Thr212Gly +Leu216AsnGly105Pro + Ser129Glu + Thr132Asn + Gly165Glu + Ala202Asn +Asn211GlnAla186Pro + Phe188Met + Ala202Glu + Gly203Ser + Tyr213Val +Leu216GluIle106His + Gly130Ser + Tyr205Ser + Thr210Gly + Asn211Asp +Tyr213AspThr 65Asn + Gly203Gln + Pro209Gln + Thr212Asp + Tyr213Glu +Thr215AspGly 62Asn + Asn 96Ser + Tyr103His + Asn162Glu + Ser190Asp +Ala214AsnIle106Asn + Gly156Glu + Gly159Asn + Phe188Asp + Tyr208Gln +Gly218AspGly 60Pro + Gly 62Gln + Ala202Asp + Thr215Gly + Leu216Thr +Thr219GluGly153Asp + Gly156Pro + Ser187Glu + Phe188Asp + Gly203Gln +Ala214HisAla186Glu + Ala202Glu + Tyr205Ala + Tyr213Val + Thr215Asp +Asn217GluAsp 59Glu + Asn157Gln + Ala186Thr + Gly203Gln + Pro209Glu +Thr212AspAsp 59Glu +Asn 96Glu + Gly 99Glu + Gly101Glu + Tyr103His +Tyr205SerGly159Ser + Ser160Glu + Thr163Glu + Ile164His + Ala186Ser +Tyr205SerThr 58Asp + Gly 62Asp + Thr132Ser + Asn154Gln + Gly203Pro +Tyr205HisAsn154Ser + Ser158Asp + Ser190Glu + Thr212Gly + Thr215Gln +Gly218ProThr 58Asp +Asn 96Ser + Ser 97Asp + Ile106Pro + Thr132Gly +Thr212ProSer155Asp + Ser160Asp + Asn162Glu + Phe188Gly + Thr210Gly +Thr212SerAsp 59Glu + Gly 99Asp + Ser100Asp + Tyr103Ile + Ile164Thr +Thr215SerGly127Gln + Ser187Glu + Gly203Gln + Thr212Gln + Thr215Asn +Gly218GluGly 60Glu + Ser 97Glu + Gly 99Asn + Tyr166Asn + Thr210Glu +Ala214HisLeu 95Ser + Tyr103Met + Leu125Glu + Gly165Glu + Ser190Glu +Gly218ProAsn 96Asp + Ser 97Glu + Ser100Asp + Ser104Glu + Thr132Asn +Leu216HisLeu 95Ile + Ser104Glu + Ala128Glu + Ser129Glu + Ser131Asp +Asn157GlnAsp 59Glu + Asn 61Asp + Ser100Glu + Gly203Asn + Tyr205Ile +Thr212GlnThr 58Asn + Thr132Ser + Asn157Ser + Thr163Glu + Phe188Glu +Tyr205ProSer129Glu + Asn154Glu + Gly156Asn + Asn162Ser + Gly165Asp +Leu216CysGly 60Glu + Gly153Asn + Gly159Ser + Tyr205Asp + Ala214Asp +Thr215GluVal 94Glu + Ser102Asp + Gly105Asp + Ala128Asp + Thr215Gly +Leu216GlyGly126Asp + Gly156Glu + Ile164Met + Phe188Leu + Ser190Glu +Tyr205AlaGly 62Gln + Ser155Asp + Ser190Asp + Leu216Glu + Asn217Ser +Thr219GluVal 94Cys + Gly203Asp + Val204Asn + Tyr205Ile + Thr212Glu +Thr215AspAsn 61Ser + Val 94Gln + Leu 95Gln + Gly203Asp + Thr212Glu +Thr215AspAsn 61Gln + Gly 99Gln + Gly153Gln + Asn154Glu + Ser158Asp +Ser187GluGly 99Glu + Gly101Gln + Gly126Asp + Phe188Met + Tyr205Ser +Thr212ProGly101Asn + Tyr205Gly + Thr212Asp + Tyr213Met + Thr215Asp +Teu216IleGly130Pro + Asn154Glu + Ser160Glu + Ser187Asp + Ser190Glu +Tyr205LeuLeu125Pro + Asn154Glu + Thr161Ser + Ala202Glu + Gly203Gln +Leu216GluGly101Asp + Ile106Ala + Gly153Asp + Ser190Asp + Leu216Asn +Thr219AspGly 60Gln + Tyr103Gly + Tyr205Leu + Thr212Glu + Thr215Glu +Asn217AspGly 62Gln + Asn154Asp + Ser155Asp + Ser160Asp + Gly165Glu +Tyr205MetGly 62Glu + Asn154Gln + Tyr205Ala + Tyr213Met + Thr215Asp +Leu216GlnAsn154Ser + Asn157Asp + Ala202Asp + Gly203Glu + Tyr205Asp +Thr219GlyLeu 95Gln + Gly126Glu + Asn154Ser + Gly159Asn + Ser190Asp +Gly218AspAsn 61Ser + Thr 65Asp + Thr132Asn + Gly203Asp + Tyr205Asp +Thr215AsnAsn 61Glu + Leu 95Asp + Ser 97Glu + Thr132Asp + Thr161Pro +Thr163SerGly126Ser + Ser131Glu + Gly203Glu + Tyr205Ala + Leu216Asp +Gly218GluGly 60Glu + Ser100Asp + Gly101Asp + Ile106His + Gly130Gln +Ala214HisGly 62Pro + Gly101Asp + Ser102Glu + Gly126Glu + Ser190Glu +Thr215AsnAsn 61Asp + Gly 99Asp + Ser100Glu + Thr161Glu + Pro209Gln +Ala214AsnThr 58Gln + Asp 59Glu + Leu 95Gly + Gly203Pro + Asn211Glu +Leu216GlnAsn 96Gln + Gly156Asp + Gly165Glu + Tyr205Asn + Thr215Asn +Gly218AsnGly 60Pro + Ser102Glu + Tyr103Glu + Thr163Glu + Tyr166Asp +Gly203SerGly127Gln + Gly153Gln + Asn157Glu + Ser160Glu + Thr163Glu +Asn217GluAsn 61Gln + Ser102Asp + Gly126Asp + Ser155Glu + Ser190Asp +Ala202SerLeu125Pro + Gly156Asp + Phe188Glu + Tyr205Cys + Thr212Gly +Leu216AspThr 58Gly + Asn 61Glu + Gly126Ser + Thr212Pro + Leu216Asp +Gly218GluVal 94Ser + Gly156Asp + Thr161Ser + Thr212Gly + Thr215Asp +Leu216GluVal 94Gly + Gly101Glu + Gly159Asp + Ser160Asp + Ala186Pro +Leu216ProAsn 61Asp + Gly 62Glu + Leu125Gln + Asn154Asp + Gly159Asn +Thr212GlnAsn 96Gln + Gly101Asp + Ser102Glu + Gly159Glu + Ile164Cys +Asn217GlnThr 58Asp + Asp 59Glu + Ser190Glu + Gly203Gln + Tyr205Cys +Pro209SerGly 62Pro + Gly105Glu + Ile106Glu + Asn154Asp + Leu216Gln +Thr219AsnVal 94Ser + Ala128Asn + Ala202Glu + Gly203Asp + Tyr205Cys +Thr212AspAsn 61Glu + Tyr103Glu + Ser104Glu + Gly203Asn + Thr212Asn +Thr215ProThr 65Ser + Ser 97Asp + Ser 98Glu + Ser104Glu + Thr163Gln +Gly203ProGly126Asp + Ala128Pro + Gly156Asp + Ser160Glu + Thr161Asp +Gly203AsnThr 65Gly + Ser100Glu + Gly101Asn + Leu125Glu + Ser129Glu +Thr215ProAsn 61Glu + Gly159Ser + Ala186Gly + Pro209Asp + Asn211Gln +Thr215GluVal 94Ser + Ser100Glu + Gly101Gln + Gly126Asp + Ser131Asp +Gly203SerAsn 61Asp + Thr 65Gln + Ser 97Asp + Gly203Glu + Tyr205Ala +Leu216ValGly156Ser + Ser158Glu + Ala186Ser + Tyr205Asp + Thr212Asp +Thr215AspAsp 59Glu + Gly 60Glu + Tyr205Ala + Tyr213Glu + Thr215Glu +Leu216CysThr132Ser + Thr163Asp + Ser190Glu + Tyr205Gly + Pro209Asp +Thr215AsnGly 62Glu +Asn 96Glu + Ser 98Glu + Ser155Asp + Tyr166Ala +Leu216ValSer160Asp + Tyr205Ala + Thr210Glu + Thr212Asp + Leu216Ser +Gly218Glnlhr 65Pro + Leu 95Glu + Gly 99Glu + Ile106His + Leu125Gly +Ser131GluGly 60Glu + Thr 65Gly + Gly 99Ser + Gly203Glu + Tyr213Gln +Thr215GluGly 62Asp + Asn 96Gln + Gly101Glu + Leu125Cys + Gly127Ser +Pro209AspSer 98Asp + Ser100Asp + Ala202Gln + Thr210Glu + Thr212Gly +Thr215AsnSer 98Glu + Ser100Asp + Ala128Gln + Gly130Gln + Thr210Gln +Asn217AspLeu 95Pro + Gly130Asn + Ser155Glu + Phe188Glu + Tyr205Asn +Thr215GluAsp 59Glu + Val 94Glu + Ser 98Asp + Ser104Glu + Gly126Ser +Tyr213GlySer100Asp + Ser102Asp + Gly127Asn + Tyr205Asp + Thr215Asp +Leu216HisGly127Asp + Ala186Gly + Ser190Glu + Tyr205Val + Thr212Gln +Leu216ThrLeu 95Glu + Ser 97Glu + Ser102Glu + Gly159Asp + Tyr205Leu +Leu216AlaGly 60Glu +Asn 61Glu + Leu125Asp + Gly165Glu + Phe188His +Gly203AsnSer102Glu + Ser104Asp + Ala202Thr + Gly203Ser + Thr212Glu +Ala214GluGly 62Glu + Leu 95Glu + Ile106Leu + Tyr166Ile + Gly203Ser + Thr212GluAsn154Ser + Ala202Asn + Gly203Asp + Tyr205Glu + Thr210Glu +Leu216AsnAsn 61Glu + Ser 98Asp + Gly 99Ser + Gly101Glu + Asn154Asp +Gly203GlnSer160Asp + Asn162Asp + Ala202Glu + Tyr208Thr + Asn211Ser +Thr215GlnGly101Asp + Ser160Asp + Asn162Glu + Thr163Ser + Tyr205Asn +Thr212GlnVal 94Asn + Ser160Glu + Asn162Glu + Asn211Gln + Thr212Asp +Thr215ProThr 58Asn + Ser 97Glu + Ser155Asp + Asn157Asp + Thr212Ser +Leu216CysSer104Glu + Leu125Thr + Ala186Thr + Tyr205Glu + Pro209Asn +Leu216AspAsp 59Glu + Gly 64Gln + Ser 97Asp + Ser100Asp + Ser160Asp +Tyr213ValGly101Pro + Gly130Asp + Asn154Ser + Thr215Glu + Leu216His +Asn217GluAla186Asp + Gly203Asn + Tyr205Cys + Thr212Asp + Ala214Asp +Thr215SerGly 99Asp + Ile106Gly + Ala128Asn + Asn154Gln + Thr212Asp +Ala214GluThr 65Gln + Asn 96Ser + Ser100Asp + Gly126Pro + Ala202Asp +Leu216AspThr 65Ser + Ser129Asp + Gly153Asn + Tyr205Leu + Tyr213Asp +Thr215AspAsn 61Ser + Ser104Asp + Ile106Glu + Ala128Asp + Thr163Asp +Thr212GlnGly105Glu + Ser131Asp + Gly156Asn + Tyr205Ala + Thr215Pro +Leu216ProGly 62Asp + Ser100Asp + Ala128Asn + Tyr166Ala + Gly203Asp +Leu216AspGly 60Asn + Asn 61Glu + Gly 62Asp + Ser187Glu + Ala202Glu +Tyr205SerSer 97Glu + Ser100Asp + Leu125Asp + Gly203Pro + Thr207Gln +Thr215AspAsn 96Ser + Ser 98Asp + Ser102Glu + Tyr205Gln + Thr212Gln +Thr215GlyGly 64Ser + Ser 98Glu + Ser100Glu + Ser155Asp + Ser187Asp +Ala199GlnSer104Asp + Leu125His + Ser129Asp + Ser131Asp + Ala202Glu +Gly218AsnSer155Glu + Thr163Glu + Ala202Glu + Tyr205Glu + Thr207Gly +Gly218SerAsn 61Ser + Thr132Gln + Ser155Glu + Gly159Asp + Thr212Pro +Thr215GlnSer100Glu + Gly127Glu + Gly130Asn + Gly203Asp + Thr215Asp +Leu216ThrAsn162Asp + Phe188His + Ser190Asp + Pro209Glu + Thr212Pro +Leu216ProSer129Glu + Gly130Asp + Gly153Asn + Asn162Ser + Asn211Glu +Ala214GluGly 60Asp + Thr 65Asn + Ser 98Asp + Gly159Asn + Leu216Asp +Gly218GluGly 62Pro + Ser131Glu + Gly165Glu + Tyr205Ser + Leu216Thr +Thr219AspGly 62Glu + Asn 96Asp + Ser100Asp + Thr132Glu + Thr215Pro +Gly218ProThr 58Asn + Gly105Gln + Ser187Asp + Ser190Glu + Thr212Glu +Leu216ProGly127Glu + Ser129Glu + Gly153Ser + Asn154Glu + Thr212Gly +Thr215GluThr 65Pro + Ser102Asp + Ser104Glu + Gly126Asp + Asn162Ser +Ala202AspThr 58Asn + Gly 64Pro + Thr 65Glu + Val 94Asp + Tyr205Asp +Thr212AspSer102Glu + Ser104Asp + Gly203Pro + Thr207Pro + Thr212Asp +Thr215AspGly 62Asp + Ser104Glu + Gly159Gln + Thr210As1 + Thr212Pro +Leu216ProGly105Gln + Gly153Ser + Gly156Asp + Ala186Glu + Ala214Glu +Thr215ProThr 58Gln + Leu 95Pro + Ser155Glu + Gly165Glu + Gly203Pro +Leu216GluGly 99Glu + Tyr103Pro + Asn154Glu + Ser158Glu + Ser187Glu +Tyr205ProAsn 96Asp + Ser 97Asp + Leu125Val + Tyr166Asp + Ser190Glu +Leu216CysLeu 95Thr + Asn154Asp + Ser187Asp + Phe188Asn + Thr212Asp +Thr215GlySer131Glu + Asn154Glu + Ser187Glu + Tyr205Gln + Tyr213Val +Asn217GlnGly 62Asp + Ser129Glu + Gly203Asp + Tyr213Leu + Thr215Asp +Leu216ValGly101Asp + Ala128Asp + Ser155Asp + Ser187Asp + Thr207Gly +Thr212GlnSer131Asp + Asn162Ser + Gly203Asp + Thr212Ser + Thr215Asn +Gly218GluGly101Gln + Gly130Pro + Gly159Glu + Ser190Glu + Asn211Glu +Thr212GluTyr103Asn + Ile106Asp + Tyr166Ala + Ala186Glu + Leu216Pro +Thr219GluAsn 96Glu + Ile106Glu + Ala128Ser + Ser158Asp + Ser160Asp +Ala214AsnThr 58Asp + Val 94His + Ser155Glu + Ser158Asp + Gly159Asn +Tyr213ProAsp 59Glu + Val 94Pro + Asn 96Asp + Tyr103Pro + Gly159Glu +Leu216ValSer 97Asp + Asn154Glu + Asn157Ser + Thr163Glu + Ala202Ser +Asn217GluLeu 95Gly + Gly127Asp + Ser131Glu + Asn154Ser + Thr161Asn +Asn162AspGly 60Asp + Leu125Gly + Gly126Ser + Gly156Gln + Thr212Asp +Thr215GluSer100Glu + Ala128Glu + Gly165Pro + Phe188Tyr + Gly203Ser +Tyr205GlyLeu 95Gly + Asn157Asp + Asn162Ser + Gly165Glu + Pro209Gly +Thr210GlySer104Glu + Ser131Glu + Thr132Gly + Gly156Glu + Phe188Val +Leu216SerGly 99Glu + Ser100Asp + Ala128Glu + Pro209Gly + Thr212Asn +Ala214GluThr 58Asn + Asn 96Glu + Ser102Asp + Tyr103Met + Ser155Asp +Gly156GlnLeu 95His + Gly126Glu + Gly165Gln + Ser190Glu + Tyr213Asp +Thr215AspGly105Glu + Leu125Cys + Ser129Glu + Gly165Glu + Thr212Ser +Thr219GluSer 98Asp + Ser102Asp + Gly105Asp + Phe188Leu + Tyr205Glu +Thr212ProLeu 95Val + Gly 99Asp + Tyr103Val + Gly105Asp + Tyr205Asp +Thr215AspGly159Glu + Gly165Asp + Thr215Ser + Leu216Cys + Asn217Glu +Thr219AspTyr103Ile + Ser129Asp + Gly159Asp + Phe188Glu + Ser190Asp +Ala202GlyAsp 59Glu + Asn 96Asp + Gly105Glu + Asn162Asp + Thr163Gln +Thr212GlnGly 99Pro + Gly101Glu + Ser104Glu + Gly153Glu + Asn162Asp +Thr215GlnAsn 61Glu + Gly126Asp + Gly159Gln + Asn162Glu + Ser190Asp +Thr215GlyGly 99Asp + Ser158Asp + Asn162Asp + Thr163Asn + Ser187Glu +Ala199GlyAsn154Glu + Ser155Glu + Gly156Gln + Thr210Asp + Leu216Asp +Gly218ProGly126Asn + Ser129Asp + Asn162Glu + Ala186Ser + Ala202Glu +Asn217AspSer100Asp + Gly101Asp + Ala128Thr + Asn154Gln + Phe188Asp +Leu216GluAsn 61Ser + Ile106Ser + Ser158Glu + Ser190Asp + Tyr205Asp +Thr219ProThr 58Ser + Val 94Ala + Ser 98Glu + Ser158Glu + Ser190Glu +Tyr205ProSer100Glu + Gly127Glu + Asn154Glu + Gly203Glu + Tyr205Ala +Tyr213ThrGly101Glu + Leu125Gly + Ser155Asp + Tyr205Val + Leu216Pro +Thr219AspSer 97Asp + Asn157Gln + Gly165Ser + Tyr205Ser + Leu216Asp +Thr219AspSer 97Glu + Ser 98Asp + Gly105Glu + Gly165Gln + Ser190Glu +Thr215SerGly 60Gln + Thr 65Gln + Val 94Asp + Leu125Glu + Ser131Asp +Ala202GlyTyr166Glu + Ser187Asp + Ser190Glu + Gly203Pro + Tyr205Gly +Leu216AspThr 58Pro + Val 94Glu + Ser 97Asp + Gly165Glu + Tyr205Ser +Tyr213HisGly 62Asp + Val 94Ala + Ser129Glu + Gly203Gln + Tyr205Val +Thr212GluTyr103Asn + Gly130Glu + Ser131Asp + Ser155Glu + Phe188Met +Thr212AspSer155Asp + Gly203Asn + Pro209Asp + Thr212Asp + Thr215Ser +Asn217GluAsn 96Glu + Ser160Glu + Asn162Ser + Asn211Gln + Ala214Glu +Thr215AspAsn 96Glu + Ser158Asp + Gly159Asp + Thr163Asn + Thr210Pro +Leu216GluThr 58Asn + Ser129Asp + Gly130Glu + Gly156Glu + Thr161Ser +Tyr205AspGly153Gln + Ser158Asp + Ala202Asp + Asn211Glu + Thr212Asp +Leu216HisSer104Glu + Leu125Cys + Gly156Pro + Ala202Glu + Gly203Asp +Pro209GluThr 65Asp + Ser100Glu + Gly105Asp + Gly127Ser + Gly203Ser +Tyr208MetSer 97Asp + Ser 98Asp + Ser158Asp + Thr212Gln + Thr215Pro +Leu216GluGly 62Gln + Ser 97Glu + Ser 98Asp + Gly126Glu + Gly130Pro +Thr212GluGly 60Asp + Asn157Glu + Tyr205Glu + Tyr213Thr + Thr215Asp +Leu216IleGly 62Glu + Ser102Asp + Gly127Glu + Ser155Asp + Tyr213Pro +Ala214GlnThr 58Pro + Asn 96Asp + Gly127Glu + Ser155Glu + Asn162Ser +Thr212SerGly 62Gln + Ser100Glu + Gly130Glu + Asn154Gln + Gly203Glu +Asn217GluAsp 59Glu + Gly 62Pro + Phe188Glu + Ala202Ser + Tyr205Glu +Pro209AspAsn 61Glu + Ser 97Glu + Ala128His + Asn162Gln + Tyr166Glu +Tyr205AspIle106Pro + Gly126Pro + Gly153Glu + Ser158Glu + Gly203Glu +Thr215GluSer102Glu + Tyr103Pro + Gly105Ser + Gly130Glu + Ser158Glu +Ala199ProAsp 59Glu + Asn 61Ser + Ser158Asp + Thr161Asn + Ser190Glu +Asn211AspAsn 61Gln + Ser104Asp + Gly127Glu + Asn157Glu + Asn162Glu +Tyr205AlaVal 94Asp + Gly105Glu + Gly165Asp + Ala186Pro + Ala202Asp +Gly203GlnAsn 61Glu + Val 94Gln + Ser102Asp + Leu125Asp + Gly159Gln +Gly165SerIle106Asp + Ser131Glu + Asn162Glu + Ala202Ser + Thr212Ser +Thr215AsnIle106Asp + Ser131Asp + Gly159Asp + Gly203Pro + Leu216Ala +Thr219GlnAsn154Gln + Gly159Asp + Ser190Asp + Ala214Glu + Leu216Pro +Gly218AspAsn 61Asp + Ser158Asp + Gly203Asp + Thr212Gln + Ala214Gly +Thr215GluGly 62Asp + Thr 65Glu + Leu 95Met + Ser104Asp + Thr132Gln +Asn162AspAsn 96Asp + Ser 98Asp + Gly127Asn + Ser129Asp + Tyr205Asn +Thr215AspSer 97Asp + Gly126Pro + Thr161Ser + Ala186Glu + Phe188Glu +Thr212GluGly 60Asn + Ser 98Glu + Gly159Asn + Gly203Glu + Thr212Asp +Asn217GluSer131Asp + Ser190Asp + Gly203Asp + Tyr205His + Thr215Asp +Leu216GlnAsn 61Glu + Gly105Gln + Ser131Asp + Met198Gly + Pro209Gly +Thr210GluThr132Pro + Gly153Asp + Asn162Glu + Gly203Asn + Pro209Gln +Leu216GluSer100Glu + Gly105Pro + Asn157Asp + Gly203Glu + Asn211Ser +Leu216GluThr 58Asn + Ser 98Glu + Gly156Glu + Gly203Asp + Ala214Gln +Leu216AspAsp 59Glu + Gly 99Gln + Ser100Asp + Gly101Pro + Ser102Glu +Gly203GluSer 98Asp + Gly126Gln + Thr161Asn + Ala202Asn + Thr212Glu +Thr215GluSer160Asp + Thr161Asn + Thr212Glu + Thr215Glu + Leu216Val +Thr219SerSer 98Glu + Ser100Asp + Ser160Asp + Phe188Asp + Val204Pro +Asn211GlnLeu 95Gly + Ser 98Glu + Ser100Glu + Gly101Asn + Gly203Asp +Thr210GluAsn 96Glu + Ser100Asp + Asn157Asp + Thr163Pro + Ala202Pro +Thr212GluAsp 59Glu + Gly 99Asp + Ser104Glu + Gly127Glu + Gly203Asn +Tyr205GlyGly 62Glu + Thr132Gln + Gly156Glu + Thr163Asp + Ala186Pro +Ala202AspSer 98Asp + Ser158Glu + Ser160Asp + Phe188Ile + Pro209Asn +Gly218GluThr 58Asn + Gly 62Glu + Gly 64Asn + Asn157Glu + Ser190Glu +Gly203GluAsn 61Asp + Gly 99Pro + Ser104Asp + Gly203Asp + Tyr205Asp +Thr212ProGly 60Asp + Ile106Pro + Gly127Glu + Ala202Asp + Leu216Ile +Gly218AspAsn 61Asp + Ser129Glu + Ser131Asp + Thr161Asn + Ala214Gln +Thr215GluAsn 61Asp + Ser 98Asp + Ile106Ser + Ser131Asp + Ser160Glu +Ile164HisSer100Glu + Ser160Glu + Tyr205Asp + Thr210Pro + Thr212Ser +Leu216GluLeu 95Pro + Gly105Asp + Thr161Glu + Tyr205Glu + Leu216Asp +Thr219GlySer 98Asp + Ser102Glu + Leu125Glu + Ala128Pro + Tyr213Gly +Leu216AspLeu 95Val + Ser155Glu + Tyr166Ala + Ala186Glu + Thr210Asp +Leu216MetThr 58Asn + Gly 99Glu + Gly105Glu + Gly127Glu + Ser190Asp +Tyr205LeuAsp 59Glu + Gly 99Glu + Leu125Ser + Thr212Glu + Leu216Asp +Gly218SerThr 58Gln + Gly130Glu + Thr132Glu + Gly203Asn + Thr212Glu +Asn217AspSer 97Glu + Gly127Gln + Ser129Asp + Gly156Asp + Ser158Glu +Asn211SerSer102Glu + Gly126Glu + Gly153Ser + Ser190Glu + Ala202Thr +Asn211AspTyr103Glu + Leu125His + Ser131Glu + Gly203Asp + Asn211Asp +Thr215SerSer129Asp + Gly130Gln + Gly165Asp + Gly203Asp + Thr212Glu +Leu216ValGly127Asn + Ser131Asp + Gly159Glu + Tyr205Gln + Thr212Asp +Ala214GluSer 97Glu + Ile106Glu + Tyr166Gly + Ala202Asp + Thr215Ser +Leu216AspLeu 95Glu + Leu125His + Gly126Asp + Tyr205Gln + Thr215Asp +Thr219AsnAsp 59Glu + Ser 97Glu + Ala128Asn + Thr132Glu + Asn157Asp +Tyr205GlySer 98Asp + Ser102Asp + Ala128His + Ser187Glu + Thr215Pro +Gly218AspThr 58Pro + Gly130Ser + Gly156Asp + Ser160Asp + Thr212Ser +Thr215GluThr 58Ser + Gly 60Glu + Thr 65Asp + Ser100Glu + Gly159Glu +Leu216HisSer 97Glu + Ser100Glu + Asn154Glu + Gly203Pro + Thr212Asn +Thr215GluSer 97Asp + Ser100Asp + Ile106Val + Ser155Asp + Gly203Pro +Thr215AspSer 97Glu + Asn157Glu + Ser160Asp + Thr210Gln + Thr212Asn +Asn217AspAsp 59Glu + Leu 95Cys + Ala128Asn + Thr132Gly + Gly153Asp +Ser187AspThr 65Asp + Leu125Asp + Ser190Glu + Gly203Asn + Asn211Asp +Thr219SerAsp 59Glu + Asn 96Asp + Leu125Cys + Thr215Asp + Leu216Ile +Thr219AspSer 98Glu + Gly105Asp + Leu125His + Asn162Asp + Ser190Glu +Ala202SerGly 60Ser + Thr 65Glu + Ser158Glu + Thr163Gly + Thr215Pro +Leu216AspVal 94Glu + Asn 96Ser + Gly101Pro + Ser102Asp + Ser155Glu +Thr212AspSer100Glu + Gly127Asn + Ser129Asp + Thr132Glu + Asn162Glu +Leu216AlaSer 98Glu + Ser102Asp + Ala202His + Thr212Pro + Thr215Asp +Gly218AspAsn 61Asp + Asn 96Asp + Gly165Glu + Tyr166Ser + Gly203Asp +Asn211GlnAsn 61Asp + Asn 96Glu + Gly127Ser + Gly130Asp + Gly203Glu +Thr212ProSer 97Glu + Gly101Asp + Ser129Glu + Ser158Glu + Thr161Ser +Ala214GlnGly 62Glu + Ser 97Asp + Ser129Asp + Ala199Pro + Leu216Gln +Gly218AspSer129Glu + Asn157Gln + Ser187Asp + Ser190Glu + Gly203Asn +Tyr205AspGly156Asn + Asn162Gln + Ser187Glu + Gly203Glu + Thr215Gln +Thr219GlyAla128Ser + Ser187Glu + Gly203Glu + Tyr205Leu + Thr210Ser +Leu216ThrThr 58Gly + Thr 65Asp + Ser100Glu + Gly105Glu + Ala128Asp +Leu216ThrAsp 59Glu + Thr 65Gly + Gly156Asp + Tyr205Glu + Leu216Cys +Asn217AspThr 58Pro + Asn 96Asp + Ser160Asp + Ser190Asp + Ala202Asn +Thr212AsnSer160Glu + Ser190Asp + Tyr205Asp + Thr207Pro + Thr212Gln +Leu216MetAsn 61Asp + Leu 95Glu + Tyr166Asn + Tyr205Glu + Asn211Glu +Leu216AsnSer 98Glu + Ser158Glu + Thr163Glu + Tyr166Leu + Gly203Glu +Tyr205ThrAsp 59Glu + Ser104Glu + Ser158Asp + Thr163Glu + Phe188His +Thr212ProSer158Asp + Thr163Glu + Ala202Asp + Tyr205Cys + Thr212Asp +Leu216ProTyr166Glu + Ser190Glu + Met198Ser + Gly203Asn + Asn211Asp +Tyr213ProAsn 61Ser + Gly 64Ser + Thr 65Asp + Ser129Glu + Tyr205Glu +Tyr213GlyAla128Pro + Thr132Glu + Gly153Ser + Asn154Asp + Gly165Asp +Gly203GluAsp 59Glu + Thr132Glu + Thr163Pro + Tyr166Ile + Thr210Asp +Leu216AspThr 58Asp + Asn 96Gln + Ser104Asp + Gly126Gln + Gly203Asp +Pro209GluGly 64Asn + Thr 65Glu + Gly126Glu + Tyr166Ala + Phe188Tyr +Thr215GluAsp 59Glu + Asn 96Asp + Gly105Asn + Gly127Asp + Ser158Glu +Thr215SerSer104Asp + Ser131Asp + Gly159Glu + Phe188Asp + Thr210Gly +Thr212GlnVal 94Met + Ser104Asp + Gly126Ser + Ser131Glu + Thr210Glu +Thr215AspThr 58Asp + Ser 97Asp + Gly105Asp + Ser187Glu + Met198Thr +Tyr205ValVal 94Asp + Gly 99Asp + Gly126Asn + Thr163Glu + Gly203Glu +Leu216CysThr 58Glu + Gly 62Glu + Gly105Glu + Gly156Glu + Phe188Thr +Thr212GlyThr 58Gln + Gly159Asp + Ser190Glu + Gly203Asp + Tyr205Ile +Gly218SerThr 58Pro + Ser131Glu + Tyr166Thr + Tyr205Asp + Thr2155er +Gly218AspGly 60Asp + Thr161Asp + Gly165Glu + Pro209Gln + Thr215Gly +Thr219GlyAsp 59Glu + Ser102Asp + Thr161Asp + Asn162Gln + Ser190Glu +Ala202SerLeu 95Cys + Ser100Glu + Ser104Glu + Phe188Cys + Tyr205Met +Leu216AspSer100Asp + Ser104Asp + Gly105Asn + Tyr205Cys + Tyr208Asn +Pro209GluGly 64Ser + Thr 65Gln + Ser100Glu + Ser104Glu + Ser160Asp +Thr207AsnThr 65Gly + Gly 99Asp + Ala128Glu + Ser155Glu + Gly203Gln +Thr215AspGly 99Asp + Ala128Asp + Ser155Asp + Ala202Glu + Thr210Asn +Leu216ValThr 58Asp + Ser 97Asp + Thr132Asp + Thr215Gln + Leu216Asn +Gly218GluThr 58Asp + Ser 97Glu + Gly127Asp + Tyr166Ala + Ala186Ser +Asn217GluThr 65Asp + Thr132Glu + Asn154Asp + Thr212Glu + Leu216Gly +Asn217SerGly 99Gln + Ser131Glu + Thr161Asn + Tyr205Thr + Pro209Asp +Thr215GluTyr103His + Ser131Glu + Gly156Asp + Gly159Glu + Thr212Glu +Leu216MetThr 58Gln + Ser 98Asp + Ser102Glu + Ala202His + Thr212Pro +Thr215AspGly127Glu + Asn162Glu + Phe188Met + Ala202Gly + Gly203Ser +Gly218AsnII洗涤组合物在本发明的另一实施方案中，有效量的一种或多种酶变异体包括在用于洗涤各种需要除去蛋白质污垢的表面的组合物中。这种洗涤组合物包括用于洗涤硬表面的非限制形式洗涤组合物(例如，液体或颗粒)；用于洗涤织物的非限制形式的洗涤组合物(颗粒、液体或条状制剂)；餐具洗涤组合物(非限制形式)；非限制形式的口腔用洗涤组合物(例如，洁牙剂、牙膏和漱口剂制剂)；非限制形式的假牙洗涤组合物(例如，液体、片剂)和非限制形式的接触透镜洗涤组合物(例如，液体、片剂)。
除了本文上述的卡氏枯草杆菌蛋白酶变异体之外，洗涤组合物还包括与蛋白酶相容的一种或多种洗涤组合物材料。本文使用的术语“洗涤组合物材料”指的是选择用于所需的洗涤组合物的具体类型和产物形式(例如，液体、颗粒、条状、喷雾剂、棒状、膏状、凝胶)的任何液体、固体或气态材料，该材料还应与组合物中使用的卡氏枯草杆菌蛋白酶变异体相容。通过考虑所洗涤的表面材料、在使用过程中用于洗涤条件下所需的组合物的形式(例如通过洗涤剂使用)很容易地具体选择洗涤组合物材料。本文中使用的术语“相容”指的是洗涤组合物材料不降低卡氏枯草杆菌蛋白酶变异体的蛋白水解活性至在正常使用场合根据需要蛋白酶失效的程度。具体的洗涤组合物材料在下文详细举例说明。
本文中使用的“有效量的酶变异体”指的是为获得在具体洗涤组合物中所需的酶活性而必需的酶变异体的量。这种有效量易于被本领域的技术人员确定，其基于许多因素，例如所用的具体的酶变异体、洗涤应用、洗涤组合物的具体组成，和是否需要液体或无水(例如颗粒、条状)组合物等等。洗涤组合物优选含有约0.0001％-约10％一种或多种本发明的酶变异体，更优选约0.001％-约1％，还优选约0.01％-约0.1％。其中使用酶变异体的各种洗涤组合物的若干实例在下文中进一步地讨论。本文使用的所有份数、百分数和比率均为按重量计，除非另有说明。
本文中使用的“非织物洗涤组合物”包括硬表面洗涤组合物、餐具洗涤组合物、口腔洗涤组合物、假牙洗涤组合物和接触透镜洗涤组合物。A.用于硬表面、餐具和织物的洗涤组合物本发明的酶变异体可用于其中需要高泡和良好不溶底物去除的各种洗涤组合物中。因此，酶变异体可与各种常规成分一起使用以提供全配制的硬表面洗涤剂。餐具洗涤组合物、织物洗涤组合物等等。这种组合物可以是液体、颗粒、条状形式等等。这种组合物可配制成含有多达按表面活性剂重量计30％-60％的现代“浓缩”洗涤剂。
本发明的洗涤组合物可选择性地，并优选地含有各种阴离子、非离子、两性离子等等的表面活性剂。这些表面活性剂通常以组合物的约5％-约35％的含量存在。
用于本发明的表面活性剂的非限制性实例包括常规的C11-C18烷基苯磺酸盐和伯和无规烷基硫酸盐、式为CH3(CH2)x(CHOSO3c-M+)CH3和CH3(CH2)y(CHOSO3-M+)CH2CH3的C10-C18仲(2，3)烷基硫酸盐，其中x和(y+1)是至少约7，优选至少约9的整数，M是水溶性阳离子，尤其是钠、C10-C18烷基烷氧基硫酸盐(尤其是EO1-5乙氧基硫酸盐)、C10-C18烷基烷氧基羧酸盐(尤其是EO1-5乙氧基羧酸盐)、C10-C18烷基聚糖苷和它们相应的硫酸化聚糖苷，和C12-C18α-磺化脂肪酸酯、C12-C18烷基和烷基苯酚烷氧基化物(尤其是乙氧基化物和混合的乙氧基化物/丙氧基化物)、C12-C18甜菜碱和磺基甜菜碱(“sultaines”)、C10-C18胺氧化物等等。本发明优选烷基烷氧基硫酸盐(AES)和烷基烷氧基羧酸盐(AEC)。(根据配制者的需要，使用这种表面活性剂与上述的胺氧化物和/或甜菜碱或磺基甜菜碱表面活性剂的混合物也是优选的)。其它常规使用的表面活性剂在标准文献中列出。尤其有用的表面活性剂包括在1993年3月16日颁布的Connor等人的US5194639中的C10-C18N-甲基葡糖酰胺，列为本文参考文献。
用于洗涤组合物中的各种各样的其它成分可包括在本发明的组合物中，其包括其它活性成分、载体、增溶剂、加工助剂、染料或颜料、用于液体配方的溶剂等等。如果需要附加地增加泡沫，在组合物中，通常以约1％-约10％的含量加入发泡剂，例如C10-C16烷醇酰胺。C10-C14单乙醇和二乙醇酰胺说明了这种发泡剂的典型种类。与上述的高泡辅助表面活性剂，例如胺氧化物、甜菜碱和磺基甜菜碱一起使用这类发泡剂也是有利的。如果需要，溶解的镁盐，例如氯化镁、硫酸镁等等可以通常约0.1％-约2％的含量加入以提供附加的起泡。
本发明的液体洗涤组合物可含有作为载体的水和其它溶剂。低分子量的伯或仲醇，例如甲醇、乙醇、丙醇和异丙醇是适宜的。单羟基醇对于表面活性剂的增溶是优选的，但多元醇，例如含有约2-约6个碳原子和约2-约6个羟基的多元醇(例如，1，3-丙二醇、乙二醇、甘油和1，2-丙二醇)也可以使用。组合物可含有约5％-约90％，典型地约10％-约50％的这种载体。
本发明的洗涤组合物将优选配制成在使用过程以含水洗涤操作，洗涤水将具有约6.8-约11.0的pH，最终的产物因此通常在该范围内配制。控制pH在所需要的范围内的技术包括使用缓冲剂、碱、酸等等，是本领域技术人员已知的。
在配制本发明的硬表面洗涤组合物和织物洗涤组合物时，配制者可以按重量计约5％-约50％的含量使用各种助剂。典型的助剂包括1-10微米沸石、多羧酸盐，例如柠檬酸盐和氧联二琥珀酸盐、层状硅酸盐、磷酸盐等等。其它常规的助剂在标准配方中列出。
同样，配制者可在组合物中，通常以按重量计约0.001％-1％的含量使用各种其它的酶，例如纤维素酶、脂酶、淀粉酶和蛋白酶。在洗衣洗涤剂领域中已知各种洗涤和织物护理酶。
各种漂白化合物，例如过碳酸盐、过硼酸盐等等可通常以按重量计约1％-约15％的含量用于该组合物中。如果需要，该组合物还可以含有漂白活性剂，例如四乙酰基乙二胺、壬酰氧基苯磺酸盐等等，它们也是现有技术中已知的。使用含量通常为按重量计约1％-约10％。
各种污垢解脱剂，尤其是阴离子低聚酯类型、各种螯合剂，尤其是氨基膦酸盐和乙二胺二琥珀酸盐、各种粘土去污剂，尤其是乙氧基化四亚乙五胺、各种分散剂，尤其是聚丙烯酸酯和聚天冬氨酸酯、各种增白剂，尤其是阴离子增白剂、各种泡沫抑制剂，尤其硅烷和仲醇、各种织物柔软剂，尤其是绿土等等部可以按重量计约1％-约35％的含量用于组合物。在标准配方和公开的所得中有此常规物质的详细描述。
在洗涤组合物中也可使用酶稳定剂。酶稳定剂包括丙二醇(优选约1％-约10％)、甲酸钠(优选约0.1％-约1％)和甲酸钙(优选约0.1％-约1％)。1.硬表面洗涤组合物本文使用的“硬表面洗涤组合物”指的是用于洗涤硬表面，例如地板、墙壁、浴室瓷砖等等的液体和颗粒洗涤组合物。本发明的硬表面洗涤组合物含有有效量的一种或多种本发明的酶变异体，按组合物活性酶重量计优选为约0.001％-约10％，更优选约0.01％-约5％，还优选约0.05％-约1％。除了含有一种或多种酶变异体之外，硬表面洗涤组合物通常含有表面活性剂和水溶性螯合剂。然而，在某些特殊的产物，例如喷雾玻璃清洁剂中，有时是不使用表面活性剂的，因为它们会产生残留在玻璃表面的膜状/条纹残余物。
当存在时，表面活性剂组分可含有少至组合物的0.1％，但通常组合物将含有约0.25％-约10％，更优选约1％-约5％的表面活性剂。
组合物通常含有约0.5％-约50％的洗涤剂助剂，优选为约1％-约10％。
pH值优选在约8-12的范围内。如果需要进行调节，可使用常规的pH调节剂，例如氢氧化钠、碳酸钠或盐酸。
在组合物中可包括溶剂。有用的溶剂包括，但不限制于，乙二醇醚，例如二乙二醇单己基醚、二乙二醇单丁基醚、乙二醇单丁基醚、乙二醇单己基醚、丙二醇单丁基醚、二丙二醇单丁基醚，和二醇，例如2，2，4-三甲基-1，3-戊二醇和2-乙基-1，3-己二醇。当使用时，溶剂通常以约0.5％-约15％，优选约3％-约11％的含量存在。
此外，当组合物以“完全强度”应用于表面后表面不进行漂洗时，在本发明的组合物中可使用高挥发性溶剂，例如异丙醇或乙醇，以有助于组合物从表面快速蒸发。当使用时，挥发性溶剂通常以约2％-约12％的含量存在于组合物中。
本发明的硬表面洗涤组合物的实施方案通过如下实施例说明。
实施例7-12液体硬表面洗涤组合物实施 例组分 7 8 9 101112Thr58Asn0.05 0.50 0.02 0.03 0.10 0.03Thr58Gln+Asn61Glu - - - - 0.20 0.02Na2DIDA*EDTA**- - 2.90 2.90 - -柠檬酸钠- - - - 2.90 2.90C12烷基苯磺酸钠1.95 - 1.95 - 1.95 -C12烷基硫酸钠 - 2.20 - 2.20 -2.20C12(乙氧基)***硫酸钠 - 2.20 - 2.20 -2.20C12二甲基胺氧化物 - 0.50 - 0.50 -0.50枯烯磺酸钠 1.30 - 1.30 - 1.30 -己基卡必醇***6.30 6.30 6.30 6.30 6.30 6.30水****平衡至100％*N-二乙二醇-N，N-亚氨基二乙酸二钠**乙二胺二乙酸四钠***二乙二醇单己基醚****所有配方调节到pH7
在实施例7-10中，用表3-36中所列的卡氏枯草杆菌蛋白酶变异体代替Thr58Asn，得到基本上类似的结果。
在实施例11-12中，用表3-36中所列的卡氏枯草杆菌蛋白酶变异体的任何组合代替Thr58Asn和Thr58Gln+Asn6lGlu，得到基本上类似的结果。
实施例13-18用于清洁硬表面和除去家庭霉变的喷雾组合物实施例组分1314151617 18Val94Ala 0.50 0.05 0.60 0.30 0.20 0.30Leu125Asn+Gly126Pro+Ser129Glu - - - - 0.30 0.10辛基硫酸钠 2.00 2.00 2.00 2.00 2.00 2.00十二烷基硫酸钠 4.00 4.00 4.00 4.00 4.00 4.00氢氧化钠 0.80 0.80 0.80 0.80 0.80 0.80硅酸盐(钠) 0.04 0.04 0.04 0.04 0.04 0.04香料 0.35 0.35 0.35 0.35 0.35 0.35水平衡至100％产物pH为约7。
在实施例13-16中，用表3-36中所列的卡氏枯草杆菌蛋白酶变异体代替Val94Ala，得到基本上类似的结果。
在实施例17-18中，用表3-36中所列的卡氏枯草杆菌蛋白酶变异体的任何组合代替Val94Ala和Leu125Asn+Gly126Pro+Ser129Glu，得到基本上类似的结果。2.餐具洗涤组合物在本发明的另一实施方案中，餐具洗涤组合物含有一种或多种本发明的酶变异体。本文使用的“餐具洗涤组合物”指的是用于洗涤餐具的组合物的所有形式，其包括，但不限制于，颗粒和液体形式。本发明的餐具洗涤组合物的实施方案由如下实施例说明。
实施例19-24餐具洗涤组合物实施例组分 19 2021222324Gly153Asn0.05 0.50 0.02 0.40 0.10 0.03Ala186asp+Phe188Gln - - - -0.40 0.02C12-C14N-甲基葡糖酰胺 0.90 0.90 0.90 0.90 0.90 0.90C12乙氧基(1)硫酸盐 12.00 12.00 12.00 12.00 12.00 12.002-甲基十一烷酸 4.50 4.50 4.50 4.50 4.50 4.50C12乙氧基(2)羧酸盐 4.50 4.50 4.50 4.50 4.50 4.50C12醇乙氧基化物(4) 3.00 3.00 3.00 3.00 3.00 3.00C12胺氧化物 3.00 3.00 3.00 3.00 3.00 3.00枯烯磺酸钠 2.00 2.00 2.00 2.00 2.00 2.00乙醇 4.00 4.00 4.00 4.00 4.00 4.00Mg++(作为氯化镁)0.20 0.20 0.20 0.20 0.20 0.20Ca++(作为氯化钙)0.40 0.40 0.40 0.40 0.40 0.40水 平衡至100％产物pH调节至7。
在实施例19-22中，用表3-36中所列的卡氏枯草杆菌蛋白酶变异体代替Gly153Asn，得到基本上类似的结果。
在实施例23-24中，用表3-36中所列的卡氏枯草杆菌蛋白酶变异体的任何组合代替Gly153Asn和Ala186asp+Phe188Gln，得到基本上类似的结果。3.织物洗涤组合物在本发明的另一实施方案中，织物洗涤组合物含有一种或多种本发明的酶变异体。本文使用的“织物洗涤组合物”指的是用于洗涤织物的组合物的所有形式，其包括，但不限制于，颗粒、液体和条状形式。优选的织物洗涤组合物是液体形式。a.颗粒织物洗涤组合物本发明的颗粒织物洗涤组合物含有有效量的一种或多种本发明的酶变异体，按组合物活性酶重量计优选为约0.001％-约10％，更优选约0.005％-约5％，还优选约0.01％-约1％。除了含有一种或多种酶变异体之外，颗粒织物洗涤组合物通常含有至少一种表面活性剂、一种或多种助剂和，在某些情况下，漂白剂。
本发明的颗粒织物洗涤组合物的实施方案由如下实施例说明。
实施例25-28颗粒织物洗涤组合物实施例组分 25262728Pro200Asn 0.10 0.20 0.03 0.05Gly203Asp+Tyr205Ala+Tyr208Ser- - 0.02 0.05C13直链烷基苯磺酸盐22.00 22.00 22.00 22.00磷酸盐(作为三聚磷酸钠) 23.00 23.00 23.00 23.00碳酸钠 23.00 23.00 23.00 23.00硅酸钠 14.00 14.00 14.00 14.00沸石8.20 8.20 8.20 8.20螯合剂(二亚乙三胺五乙酸)0.40 0.40 0.40 0.40硫酸钠 5.50 5.50 5.50 5.50水平衡至100％在实施例25-26中，用表3-36中所列的卡氏枯草杆菌蛋白酶变异体代替Pro200Asn，得到基本上类似的结果。
在实施例27-28中，用表3-36中所列的卡氏枯草杆菌蛋白酶变异体的任何组合代替Pro200Asn和Gly203Asp+Tyr205Ala+Tyr208Ser，得到基本上类似的结果。
实施例29-32颗粒织物洗涤组合物实施例组分29 30 3132Pro200Asn+Ala202Asn+Vla204 0.10 0.20 0.03 0.05Gly+Asn211Asp+Thr219GlyLeu95Gly+Gly203Pro - -0.02 0.05C12烷基苯磺酸盐12.00 12.00 12.00 12.00沸石A(1-10微米) 26.00 26.00 26.00 26.002-丁基辛酸 4.00 4.00 4.00 4.00C12-C14仲(2，3)烷基硫酸钠 5.00 5.00 5.00 5.00柠檬酸钠 5.00 5.00 5.00 5.00荧光增白剂 0.10 0.10 0.10 0.10硫酸钠 17.00 17.00 17.00 17.00水和少量组分平衡至100％在实施例29-30中，用表3-36中所列的卡氏枯草杆菌蛋白酶变异体代替Pro200Asn+Ala202Asn+Vla204Gly+Asn211Asp+Thr219Gly，得到基本上类似的结果。
在实施例31-32中，用表3-36中所列的卡氏枯草杆菌蛋白酶变异体的任何组合代替Pro200Asn+Ala202Asn+Vla204Gly+Asn211Asp+Thr219Gly和Leu95Gly+Gly203Pro，得到基本上类似的结果。
实施例33-36颗粒织物洗涤组合物实施例组分33343536Thr58Gln+Asp59Glu+Leu216Gln 0.10 0.20 0.03 0.05Pro200Asn+Ala202Gln+Tyr208Cys+Thr210 -- 0.02 0.05Pro+Tyr213Asn+Ala214Asn+Thr219SerC13直链烷基苯磺酸盐22.00 22.00 22.00 22.00磷酸盐(作为三聚磷酸钠) 23.00 23.00 23.00 23.00碳酸钠 23.00 23.00 23.00 23.00硅酸钠 14.00 14.00 14.00 14.00沸石8.20 8.20 8.20 8.20螯合剂(二亚乙三胺五乙酸)0.40 0.40 0.40 0.40硫酸钠 5.50 5.50 5.50 5.50水 平衡至100％在实施例33-34中，用表3-36中所列的卡氏枯草杆菌蛋白酶变异体代替Thr58Gln+Asp59Glu+Leu216Gln，得到基本上类似的结果。
在实施例35-36中，用表3-36中所列的卡氏枯草杆菌蛋白酶变异体的任何组合代替Thr58Gln+Asp59Glu+Leu216Gln和Pro200Asn+Ala202Gln+Tyr208Cys+Thr210Pro+Tyr213Asn+Ala214Asn+Thr219Ser，得到基本上类似的结果。
实施例37-40颗粒织物洗涤组合物实施例组分37383940Asp59Glu+Gly60Pro+ 0.10 0.20 0.03 0.05Gly62Asn+Gly64GlLeu125Ala - - 0.02 0.05C12烷基苯磺酸盐 12.00 12.00 12.00 12.00沸石(1-10微米) 26.00 26.00 26.00 26.002-丁基辛酸 4.00 4.00 4.00 4.00C12-C14仲(2，3)烷基硫酸钠5.00 5.00 5.00 5.00柠檬酸钠 5.00 5.00 5.00 5.00荧光增白剂 0.10 0.10 0.10 0.10硫酸钠 17.00 17.00 17.00 17.00水和少量组分 平衡至100％在实施例37-38中，用表3-36中所列的卡氏枯草杆菌蛋白酶变异体代替Asp59Glu+Gly60Pro+Gly62Asn+Gly64Gl，得到基本上类似的结果。
在实施例39-40中，用表3-36中所列的卡氏枯草杆菌蛋白酶变异体的任何组合代替Asp59Glu+Gly60Pro+Gly62Asn+G1y64Gl和Leu125Ala，得到基本上类似的结果。
实施例41-42颗粒织物洗涤组合物实施例组分 4142直链烷基苯磺酸盐 11.4 10.70动物脂烷基硫酸盐 1.80 2.40C14-15烷基硫酸盐 3.003.10C14-15醇7倍乙氧基化4.004.00动物脂醇11倍乙氧基化 1.801.80分散剂 0.070.1硅氧烷液体 0.800.80柠檬酸三钠 14.00 15.00柠檬酸 3.002.50沸石 32.50 32.10马来酸丙烯酸共聚物 5.005.00二亚乙三胺五亚甲基膦酸 1.000.20Gly127Glu+Asn162Glu+Phe218Met+ 0.300.30Ala202Gly+Gly203Ser+Gly218Asn脂酶 0.360.40淀粉酶 0.300.30硅酸钠 2.002.50硫酸钠 3.505.20聚乙烯吡咯烷酮 0.300.50过硼酸盐 0.5 1苯酚磺酸盐 0.1 0.2过氧化酶 0.1 0.1少量组分 至100 至100实施例43-44颗粒织物洗涤组合物实施例组分4344直链C12烷基苯磺酸钠6.5 8.0硫酸钠 15.0 18.0沸石A 26.0 22.0次氮基三乙酸钠 5.0 5.0聚乙烯吡咯烷酮 0.5 0.7四乙酰基乙二胺 3.0 3.0硼酸 4.0 -过硼酸盐 0.5 1苯酚磺酸盐 0.1 0.2Gly218Gln+Thr219Gln0.4 0.4填料(例如，硅酸盐；碳酸盐；香料；水) 至100至100实施例45压型颗粒织物洗涤组合物组分 重量％烷基硫酸盐 8.0烷基乙氧基硫酸盐2.0C25和C45醇3和7倍乙氧基化的混合物 6.0多羟基脂肪酸酰胺2.5沸石17.0层状硅酸盐/柠檬酸盐 16.0碳酸盐 7.0马来酸丙烯酸共聚物 5.0污垢解脱聚合物 0.4羧甲基纤维素0.4聚(4-乙烯基吡啶)-N-氧化物 0.1乙烯基咪唑和乙烯基吡咯烷酮共聚物0.1PEG2000 0.2Pro209Gln 0.5脂酶0.2纤维素酶0.2四乙酰基乙二胺 6.0过碳酸盐22.0乙二胺二琥珀酸 0.3泡沫抑制剂 3.54，4’-双(2-吗啉基-4-苯胺基-s-三嗪-6-基氨基)芪-2，2’- 0.25二磺酸二钠4，4’-双(2-磺基苯乙烯基)联苯二钠 0.05水、香料和少量组分 至100
实施例46颗粒织物洗涤组合物组分 重量％直链烷基苯磺酸盐 7.6C16-C18烷基硫酸盐 1.3C14-15醇7倍乙氧基化物4.0椰子烷基二甲基羟基乙基铵氯化物 1.4分散剂 0.07硅氧烷液体 0.8柠檬酸三钠 5.0沸石4A 15.0马来酸丙烯酸共聚物 4.0二亚乙三胺五亚甲基膦酸 0.4过硼酸盐 15.0四乙酰基乙二胺 5.0绿土 10.0聚(环氧乙烷)(MW300000) 0.3Thr58Pro+Gly64Ser+Thr65Gly 0.4脂酶 0.2淀粉酶 0.3纤维素酶 0.2硅酸钠 3.0碳酸钠 10.0羧甲基纤维素 0.2增白剂 0.2水、香料和少量组分 至100
实施例47颗粒织物洗涤组合物组分 重量％直链烷基苯磺酸盐 6.92动物脂烷基硫酸盐 2.05C14-15醇7倍乙氧基化物4.4C12-15烷基乙氧基硫酸盐-3倍乙氧基化 0.16沸石 20.2柠檬酸盐 5.5碳酸盐 15.4硅酸盐 3.4马来酸丙烯酸共聚物 4.0羧甲基纤维素酶 0.31污垢解脱聚合物 0.30Val94Gln+Ser100Glu 0.2脂酶 0.36纤维素酶 0.13过硼酸盐四水合物 11.64过硼酸盐单水合物 8.7四乙酰基乙二胺 5.0二亚乙三胺五甲基膦酸 0.38硫酸镁 0.40增白剂 0.19香料、硅氧烷、泡沫抑制剂 0.85少量组分 至100b.液体织物洗涤组合物本发明的液体织物洗涤组合物含有有效量的一种或多种本发明的酶变异体，按组合物活性酶重量计优选为约0.005％-约5％，更优选为约0.01％-约1％。该液体织物洗涤组合物通常还含有一种阴离子表面活性剂、脂肪酸、水溶性洗涤剂助剂和水。
本发明的液体织物洗涤组合物的实施方案由如下实施例说明。
实施例48-52液体织物洗涤组合物实施例组分 48 49 505152Leu125Ala+Gly126Glu 0.05 0.03 0.30 0.03 0.10Gly153Glu+Ile164Ala - - - 0.01 0.20C12-C14烷基硫酸钠 20.00 20.00 20.00 20.00 20.002-丁基辛酸 5.00 5.00 5.00 5.00 5.00柠檬酸钠 1.00 1.00 1.00 1.00 1.00C10醇乙氧基化物(3) 13.00 13.00 13.00 13.00 13.00单乙醇胺 2.50 2.50 2.50 2.50 2.50水/丙二醇/乙醇(100∶1∶1)平衡至100％在实施例48-50中，用表3-36所列的卡氏枯草杆菌蛋白酶变异体代替Leu125Ala+Gly126Glu，得到基本上类似的结果。
在实施例51-52中，用表3-36中所列的卡氏枯草杆菌蛋白酶变异体的任何组合代替Leu125Ala+Gly126Glu和Gly153Glu+Ile164Ala，得到基本上类似的结果。
实施例53-57液体织物洗涤组合物实施例组分 53 54 55 56 57Phe188Ser0.05 0.03 0.30 0.03 0.10Ala186Gln+Ser187Glu - - - 0.01 0.20+Phe188AspC12-C14烷基硫酸钠 20.00 20.00 20.00 20.00 20.002-丁基辛酸 5.00 5.00 5.00 5.00 5.00柠檬酸钠 1.00 1.00 1.00 1.00 1.00C10醇乙氧基化物(3) 13.00 13.00 13.00 13.00 13.00单乙醇胺 2.50 2.50 2.50 2.50 2.50水/丙二醇/乙醇(100∶1∶1) 平衡至100％在实施例53-55中，用表3-36中所列的卡氏枯草杆菌蛋白酶变异体代替Phe188Ser，得到基本上类似的结果。
在实施例56-57中，用表3-36中所列的卡氏枯草杆菌蛋白酶变异体的任何组合代替Phe188Ser和Ala186Gln+Ser187Glu+Phe188Asp，得到基本上类似的结果。
实施例58-59颗粒织物洗涤组合物实施例组分 5859C12-14烯基琥珀酸 3.0 8.0柠檬酸单水合物 10.0 15.0C12-15烷基硫酸钠 8.0 8.0C12-15醇2倍乙氧基化硫酸钠 -3.0C12-15醇7倍乙氧基化物 -8.0C12-15醇5倍乙氧基化物 8.0 -二亚乙三胺五(亚甲基膦酸) 0.2 -油酸 1.8 -乙醇 4.0 4.0丙二醇 2.0 2.0Thr58Ser+Ser100Asp+Ile164Met+Tyr205Ala 0.2 0.2聚乙烯吡咯烷酮 1.0 2.0泡沫抑制剂 0.15 0.15氢氧化钠 至pH 7.5过硼酸盐 0.5 1苯酚磺酸盐 0.1 0.2过氧化酶 0.4 0.1水和少量组分 至10 0份在实施例58和59中，用表3-36中所列的卡氏枯草杆菌蛋白酶变异体代替Thr58Ser+Ser100Asp+Ile164Met+Tyr205Ala，得到基本上类似的结果。
实施例60-62液体织物洗涤组合物实施例组分 60 61 62柠檬酸 7.103.003.00脂肪酸 2.00 - 2.00乙醇1.933.203.20硼酸2.223.503.50单乙醇胺0.711.091.091，2-丙二醇 7.898.008.00枯烯磺酸钠 1.803.003.00甲酸钠 0.080.080.08氢氧化钠6.703.803.80含硅消泡剂 1.161.181.18Asn61Glu0.0145 - -Tyr103Cys+Gly156Glu+Ala202Gln - 0.0145 -+Thr212Gly+Leu216GlnGly105Asn+Gly126Ser+Ser160Glu -- 0.0145+Thr161Asn+Tyr205Val+Gly218Asn脂酶0.200 0.200 0.200纤维素酶- 7.507.50污垢解脱聚合物 0.290.150.15消泡剂 0.060.085 0.085增白剂360.095- -增白剂3 - 0.050.05C12烷基苯磺酸 9.86 - -C12-15烷基多乙氧基化(2.5)硫酸盐 13.80 18.00 18.00C12葡糖酰胺- 5.005.00C12-13烷基聚乙氧基化物(9) 2.00 2.002.00水、香料和少量组分 平衡至 100％c.条状织物洗涤组合物本发明的适合于手洗脏织物的条状织物洗涤组合物含有有效量的一种或多种本发明的酶变异体，按组合物活性酶重量计优选为约0.001％-约10％，更优选为约0.01％-约1％。
本发明的条状织物洗涤组合物的实施方案由如下实施例说明。
实施例63-66条状织物洗涤组合物实施例组分 63646566Thr207Glu 0.3 - 0.1 0.02Leu125Ile+Ser131Glu- - 0.4 0.03C12-C16烷基硫酸钠20.0 20.0 20.0 20.00C12-C14N-甲基葡糖酰胺5.0 5.0 5.0 5.00C11-C13烷基苯磺酸钠 10.0 10.0 10.0 10.00碳酸钠 25.0 25.0 25.0 25.00焦磷酸钠 7.0 7.0 7.0 7.00三聚磷酸钠 7.0 7.0 7.0 7.00沸石A(0.1-10μ)5.0 5.0 5.0 5.00羧甲基纤维素 0.2 0.2 0.2 0.20聚丙烯酸酯(MW1400) 0.2 0.2 0.2 0.20椰子烷基单乙醇胺 5.0 5.0 5.0 5.00增白剂、香料 0.2 0.2 0.2 0.20硫酸钙 1.0 1.0 1.0 1.00硫酸镁 1.0 1.0 1.0 1.00水 4.0 4.0 4.0 4.00填料*平衡至 100％*可以选自常规材料，例如碳酸钙、滑石、粘土、硅酸盐等等。
在实施例63-64中，用表3-36中所列的卡氏枯草杆菌蛋白酶变异体代替Thr207Glu，得到基本上类似的结果。
在实施例65-66中，用表3-36中所列的卡氏枯草杆菌蛋白酶变异体的任何组合代替Thr207Glu和Leu125Ile+Ser131Glu，得到基本上类似的结果。
实施例67-70条状织物洗涤组合物实施例组分 67 68 6970Pr0200Ser+Ala214Gly+Asn217Glu 0.3 -0.1 0.02Gly101Asp+Ala128Asp+Ser155Asp+- 0.3 0.4 0.03Ser187Asp+Thr207Gly+Thr212GlnC12-C16烷基硫酸钠 20.020.0 20.0 20.0C12-C14N-甲基葡糖酰胺 5.0 5.0 5.05.00C11-C13烷基苯磺酸钠 10.010.0 10.0 10.00碳酸钠25.025.0 25.0 25.00焦磷酸钠 7.0 7.0 7.07.00三聚磷酸钠7.0 7.0 7.07.00沸石A(0.1-10μ) 5.0 5.0 5.05.00羧甲基纤维素 0.2 0.2 0.20.20聚丙烯酸酯(MW1400)0.2 0.2 0.20.20椰子烷基单乙醇胺 5.0 5.0 5.05.00增白剂、香料 0.2 0.2 0.20.20硫酸钙1.0 1.0 1.01.00硫酸镁1.0 1.0 1.01.00水4.0 4.0 4.04.00填料*平衡至100％*可以选自常规材料，例如碳酸钙、滑石、粘土、硅酸盐等等。
在实施例67中，用表3-36中所列的卡氏枯草杆菌蛋白酶变异体代替Pro200Ser+Ala214Gly+Asn217Glu，得到基本上类似的结果。
在实施例68中，用表3-36中所列的卡氏枯草杆菌蛋白酶变异体代替Pro200Ser+Ala214Gly+Asn217Glu和Gly101Asp+Ala128Asp+Ser155Asp+Ser187Asp+Thr207Gly+Thr212Gln，得到基本上类似的结果。
在实施例69-70中，用表3-36中所列的卡氏枯草杆菌蛋白酶变异体的任何组合代替Va1203Glu和Gly100Glu+Ilel07Ser，得到基本上类似的结果。B.其它洗涤组合物除了上述硬表面洗涤、餐具洗涤和织物洗涤组合物之外，本发明的一种或多种酶变异体可加入各种需要水解不溶解底物的其它洗涤组合物中。该附加的洗涤组合物包括，但不限制于，口腔洗涤组合物、假牙洗涤组合物和接触透镜洗涤组合物。1.口腔洗涤组合物在本发明的另一实施方案中，在组合物中包含一种或多种可药用量的本发明的酶变异体以从牙齿或假牙中除去蛋白质污垢。本文使用的“口腔洗涤组合物”指的是洁齿粉、牙膏、牙凝胶、牙粉、漱口剂、口腔喷雾剂、口腔凝胶、咀嚼胶、锭剂、香囊、片剂、生物凝胶、预防膏、假牙处理溶液等等。按组合物的重量计，洗涤组合物优选含有约0.0001-约20％，更优选约0.001％-约10％，还优选约0.01％-约5％的一种或多种本发明的酶变异体和可药用的载体。本文使用的“可药用的”指的是该术语描述的药物、药剂或惰性成分适合于与人体和低级动物的组织接触，而没有不适宜的毒性、不适性、不稳定性、刺激性、过敏反应等等，与有益效果/风险比率相当。
口腔洗涤组合物的可药用的口腔洗涤载体组分按组合物重量计通常占约50％-约99.99％，优选约65％-约99.99％，更优选约65％-约99％。
可包括在本发明的口腔洗涤组合物中的可药用的载体组分和选择性的组分是本领域技术人员已知的。用于口腔洗涤组合物的各种组合物类型、载体组分和选择性组分在1992年3月17日颁布的Seibel的US5096700；1991年7月2日颁布的Sampathkumar的US5028414和1991年7月2日颁布的Benedict、Bush和Sunberg的US5028415中，列为本文参考文献。
本发明的口腔洗涤组合物的实施方案在如下实施例中说明。
实施例71-74洁牙粉组合物实施例组分7172 73 74Thy212Gln 2.000 3.500 1.500 2.000山梨糖醇(70％水溶液) 35.000 35.000 35.000 35.000PEG-6*1.000 1.000 1.000 1.000氧化硅牙用磨料**20.000 20.000 20.000 20.000氟化钠0.243 0.243 0.243 0.243二氧化钛 0.500 0.500 0.500 0.500糖精钠0.286 0.286 0.286 0.286烷基硫酸钠(27.9％水溶液) 4.000 4.000 4.000 4.000香料 1.040 1.040 1.040 1.040羧乙烯基聚合物***0.300 0.300 0.300 0.300Carrageenan****0.800 0.800 0.800 0.800水平衡至 100％*PEG-6＝分子量600的聚乙二醇。**由J.M.Huber提供的沉淀的氧化硅Zeodent119***由B.F.Goodrich Chemical Company提供的Carbopol****由Hercules Chemical Company提供的lota Carrageenan。
在实施例71-74中，用表3-36中所列的卡氏枯草杆菌蛋白酶变异体代替Thy212Gln，得到基本上类似的结果。
实施例75-78漱口剂组合物实施例组分 75767778Val204Asn+Thr215Asn 3.00 7.50 1.00 5.00SDA40醇 8.00 8.00 8.00 8.00香料0.08 0.08 0.08 0.08乳化剂 0.08 0.08 0.08 0.08氟化钠 0.05 0.05 0.05 0.05甘油 10.0010.0010.0010.00增甜剂 0.02 0.02 0.02 0.02苯甲酸 0.05 0.05 0.05 0.05氢氧化钠 0.20 0.20 0.20 0.20染料 0.04 0.04 0.04 0.04水 平衡至100％在实施例75-78中，用表3-36中所列的卡氏枯草杆菌蛋白酶变异体代替Val204Asn+Thr215Asn，得到基本上类似的结果。
实施例79-82锭剂组合物实施例组分79808182Thr65Asp+Thr132Glu+Asn154Asp+ 0.01 0.03 0.10 0.02Thr212Glu+Leu216Gly+Asn217Ser山梨糖醇17.50 17.50 17.50 17.50甘露糖醇17.50 17.50 17.50 17.50淀粉13.60 13.60 13.60 13.60增甜剂 1.20 1.20 1.20 1.20香料11.70 11.70 11.70 11.70色素0.10 0.10 0.10 0.10玉米糖浆 平衡至100％在实施例79-82中，用表3-36中所列的卡氏枯草杆菌蛋白酶变异体代替Thr65Asp+Thr132Glu+Asn154Asp+Thr212Glu+Leu216Gly+Asn217Ser，得到基本上类似的结果。
实施例83-86口香糖组合物实施例组分83 848586Tyr103Ala 0.03 0.02 0.10 0.05山梨糖醇结晶 38.44 38.40 38.40 38.40Palija-T胶基料*20.0020.0020.00 20.00山梨糖醇(70％水溶液) 22.0022.0022.00 22.00甘露糖醇 10.0010.0010.00 10.00甘油 7.56 7.56 7.56 7.56香料 1.00 1.00 1.00 1.00*L A.Dreyfus Company提供。
在实施例83-86中，用表3-36中所列的卡氏枯草杆菌蛋白酶变异体代替Tyr103A1a，得到基本上类似的结果。2.假牙洗涤组合物在本发明的另一实施方案中，用于在口腔外洗涤假牙的假牙洗涤组合物含有一种或多种本发明的酶变异体。该假牙洗涤组合物含有有效量的一种或多种酶变异体，优选按组合物重量计，约0.0001-约50％，更优选约0.001％-约35％，还优选约0.01％-约20％的一种或多种本发明的酶变异体，和假牙洗涤载体。各种假牙洗涤组合物配方，如泡腾片等等是现有技术中已知的(参见例如Young的US5055305，列为本文参考文献)，通常适合于加入一种或多种酶变异体以便从假牙除去蛋白质污垢。
本发明的假牙洗涤组合物的实施方案由如下实施例说明。
实施例87-90双层泡腾假牙洗涤片实施例组分87888990酸性层Leu216Pro 1.0 1.5 0.01 0.05酒石酸 24.0 24.0 24.00 24.00碳酸钠 4.0 4.0 4.00 4.00氨基磺酸10.0 10.0 10.00 10.00PEG200004.0 4.0 4.00 4.00碳酸氢钠24.5 24.5 24.50 24.50过硫酸钾15.0 15.0 15.00 15.00酸性焦磷酸钠7.0 7.0 7.00 7.00热解氧化硅 2.0 2.0 2.00 2.00TAED*7.07.07.007.00蓖麻酰基磺基琥珀酸盐0.50.50.500.50香料1.01.01.001.00碱性层过硼酸钠单水合物32.0 32.0 32.00 32.00碳酸氢钠19.0 19.0 19.00 19.00EDTA3.03.03.003.00三聚磷酸钠 12.0 12.0 12.00 12.00PEG200002.02.02.002.0过硫酸钾26.0 26.0 26.00 26.00碳酸钠 2.02.02.002.00热解氧化硅 2.02.02.002.00染料/香料 2.02.02.002.00*四乙酰基乙二胺在实施例87-90中，用表3-36中所列的卡氏枯草杆菌蛋白酶变异体代替Leu216Pro，得到基本上类似的结果。2.接触透镜洗涤组合物在本发明的另一实施方案中，接触透镜洗涤组合物含有一种或多种本发明的酶变异体。该接触透镜洗涤组合物含有有效量的一种或多种酶变异体，优选按组合物重量计，约0.01-约50％，更优选约0.01％-约20％，还优选约1％-约5％的一种或多种本发明的酶变异体，和接触透镜洗涤载体。各种接触透镜洗涤组合物配方，例如片剂、液体等等在现有技术中是已知的(参见例如1989年9月5日授权的Davies，Meaken和Rees，US4863627；1988年5月24日再次授权的Huth，Lam和Kirai，US Re32672；有1986年9月2日授权的Schafer，US4609493；1987年9月1日授权的Ogunbiyi和Smith，US4690793；1986年9月30日授权的Ogunbiyi，Riedhammer和Smiht，US4614549；和1981年8月25日颁布的Ogata，US4285738；均列为本文参考文献)，通常适合于加入本发明的一种或多种酶变异体以便从接触透镜除去蛋白质污垢。
本发明的接触透镜洗涤组合物的实施方案由如下实施例说明。
实施例91-94酶解接触透镜洗涤溶液实施例组分 91929394Ala199Thr+Val204Gln 0.01 0.5 0.1 2.0葡萄糖 50.00 50.0 50.0 50.0非离子表面活性剂(聚氧乙烯- 2.00 2.0 2.0 2.0聚氧丙烯共聚物)阴离子表面活性剂(聚氧乙烯- 1.00 1.0 1.0 1.0烷基苯基醚硫酸酯钠)氯化钠 1.00 1.0 1.0 1.0硼砂0.30 0.3 0.3 0.3水 平衡至100％在实施例91-94中，用表3-36中所列的卡氏枯草杆菌蛋白酶变异体代替Ala199Thr+Val204Gln，得到基本上类似的结果。
尽管描述了本发明的具体实施方案，但显然本领域的技术人员可作出本发明的各种改变和改进而不会违背本发明的精神和范围。在所附的权利要求中，本发明的范围覆盖了所有这些改进。
序列表(1)一般信息(i)申请人BRODE，PHILIPF。
BARNETT，BOBBYL。
RUBINGH，DONNN。
(ii)发明项目SUBTILISIN CARLSBERG VARIANTS HANINGDECREASED ASDORPTION AND INCREASEDHYDROLYSIS(iii)序列数1(iv)通信地址(A)收信人THE PROCTER & GAMBLE COMPANY(B)街11810 East Miami River Road(C)城市Ross(D)州OH(E)国家USA(F)邮区45061(v)计算机可读形式(A)介质类型软盘(B)计算机IBM PC兼容(C)操作系统PC-DOS/MS-DOS(D)软件Patentln Release#1.0，Version#1.03(vi)当前申请资料(A)申请号(B)申请日(C)分类(viii)代理人/事务所信息(A)姓名CORSTANJE，BRAHMJ。
(B)登记号34，804(C)参考/存档号5602(ix)电讯信息(A)电话(513)627-2858(B)电传(513)627-0260(2)SEQ ID NO1的信息(i)序列特征(A)长度274氨基酸(B)类型氨基酸(C)链型单(D)拓扑学线性(xi)序列描述SEQID NO1(xi) SEQUENCE DESCRIPTIONSEQ ID NO1Ala Gln Thr Val Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val15 10 15Gln Ala Gln Gly Phe Lys Gly Ala Asn Val Lys Val Ala Val Leu Asp20 25 30Thr Gly Ile Gln Ala Ser His Pro Asp Leu Asn Val Val Gly Gly Ala35 40 45Ser Phe Val Ala Gly Glu Ala Tyr Asn Thr Asp Gly Asn Gly His Gly5055 60Thr His Val Ala Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val65 70 7580Leu Gly Val Ala Pro Ser Val Ser Leu Tyr Ala Val Lys Val Leu Asn8590 95Ser Ser Gly Ser Gly Ser Tyr Ser Gly Ile Val Ser Gly Ile Glu Trp100 105 110Ala Thr Thr Asn Gly Met Asp Val Ile Asn Met Ser Leu Gly Gly Ala115120 125Ser Gly Ser Thr Ala Met Lys Gln Ala Val Asp Asn Ala Tyr Ala Arg130 135140Gly Val Val Val Val Ala Ala Ala Gly Asn Ser Gly Asn Ser Gly Ser145 150 155 160Thr Asn Thr Ile Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val165 170175Gly Ala Val Asp Ser Asn Ser Asn Arg Ala Ser Phe Ser Ser Val Gly180 185 190Ala Glu Leu Glu Val Met Ala Pro Gly Ala Gly Val Tyr Ser Thr Tyr195200 205Pro Thr Asn Thr Tyr Ala Thr Leu Asn Gly Thr Ser Met Ala Ser Pro210215220His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Leu225230 235240Ser Ala Ser Gln Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Tyr Leu245250 255Gly Ser Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala Ala260 265 270Ala Gln
权利要求
1.具有卡氏枯草杆菌蛋白酶野生型氨基酸序列的修饰氨基酸序列的卡氏枯草杆菌蛋白酶变异体，野生型氨基酸序列包括第一环区域、第二环区域、第三环区域、第四环区域、第五环区域和第六环区域；其中修饰的氨基酸序列在一个或多个环区域内的一个或多个位点上含有取代基；其中A.当取代发生在第一环区域中时，取代发生在位点58、59、60、61、62、64或65的一个或多个上；其中a.当取代发生在位点58时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser；b.当取代发生在位点59时，取代氨基酸是Glu；c.当取代发生在位点60时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser；d.当取代发生在位点61时，取代氨基酸是Asp、Gln、Glu或Ser；e.当取代发生在位点62时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser；f.当取代发生在位点64时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser；和g.当取代发生在位点65时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser；B.当取代发生在第二环区域中时，取代发生在位点94、95、96、97、98、99、100、101、102、103、104、105或106的一个或多个上；其中a.当取代发生在位点94时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Met、Pro、Ser或Thr；b.当取代发生在位点95时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Met、Pro、Ser、Thr或Val；c.当取代发生在位点96时，取代氨基酸是Asp、Gln、Glu、Ser；d.当取代发生在位点97时，取代氨基酸是Asp或Glu；e.当取代发生在位点98时，取代氨基酸是Asp或Glu；f.当取代发生在位点99时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser；g.当取代发生在位点100时，取代氨基酸是Asp或Glu；h.当取代发生在位点101时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser；i.当取代发生在位点102时，取代氨基酸是Asp或Glu；j.当取代发生在位点103时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr或Val；k.当取代发生在位点104时，取代氨基酸是Asp或Glu；l.当取代发生在位点105时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser；和m.当取代发生在位点106时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Leu、Met、Pro、Ser、Thr或Val；C.当取代发生在第三环区域中时，取代发生位点125、126、127、128、129、130、131或132的一个或多个上；其中a.当取代发生在位点125时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Met、Pro、Ser、Thr或Val；b.当取代发生在位点126时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser；c.当取代发生在位点127时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser；d.当取代发生在位点128时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、His、Pro、Ser或Thr；e.当取代发生在位点129时，取代氨基酸是Asp或Glu；f.当取代发生在位点130时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser；g.当取代发生在位点131时，取代氨基酸是Asp或Glu；和h.当取代发生在位点132时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser；D.当取代发生在第四环区域中时，取代发生在位点153、154、155、156、157、158、159、160、161、162、163、164、165或166的一个或多个上；其中a.当取代发生在位点153时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser；b.当取代发生在位点154时，取代氨基酸是Asp、Gln、Glu或Ser；c.当取代发生在位点155时，取代氨基酸是Asp或Glu；d.当取代发生在位点156时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser；e.当取代发生在位点157时，取代氨基酸是Asp、Gln、Glu、Ser；f.当取代发生在位点158时，取代氨基酸是Asp或Glu；g.当取代发生在位点159时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser；h.当取代发生在位点160时，取代氨基酸是Asp或Glu；i.当取代发生在位点161时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser；j.当取代发生在位点162时，取代氨基酸是Asp、Gln、Glu或Ser；k.当取代发生在位点163时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser；l.当取代发生在位点164时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Met、Pro、Ser、Thr或Val；m.当取代发生在位点165时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser；和n.当取代发生在位点166时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr或Val；和E.当取代发生在第五环区域中时，取代发生在位点186、187、188、189或190一个或多个上；其中a.当取代发生在位点186时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、His、Pro、Ser或Thr；b.当取代发生在位点187时，取代氨基酸是Asp或Glu；c.当取代发生在位点188时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr、Tyr或Val；d.当取代发生在位点189时，取代氨基酸是Asp或Glu；e.当取代发生在位点190时，取代氨基酸是Asn或Glu；和F.当取代发生在第六环区域中时，取代发生在位点199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218或219的一个或多个上；其中a.当取代发生在位点199时，取代氨基酸是Asn、Asp、gln、Glu、Gly、His、Pro、Ser或Thr；b.当取代发生在位点200时，取代氨基酸是Asn、Asp、Gln、Glu、Gly或Ser；c.当取代发生在位点201时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser；d.当取代发生在位点202时，取代氨基酸是Asp、Asp、Gln、Glu、Gly、His、Pro、Ser或Thr；e.当取代发生在位点203时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser；f.当取代发生在位点204时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Met、Pro、Ser或Thr；g.当取代发生在位点205时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr或Val；h.当取代发生在位点206时，取代氨基酸是Asp或Glu；i.当取代发生在位点207时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser；j.当取代发生在位点208时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr或Val；k.当取代发生在位点209时，取代氨基酸是Asn、Asp、Gln、Glu、Gly或Ser；l.当取代发生在位点210时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser；m.当取代发生在位点211时，取代氨基酸是Asp、Gln、Glu或Ser；n.当取代发生在位点212时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser；o.当取代发生在位点213时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr或Val；p.当取代发生在位点214时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、His、Pro、Ser或Thr；q.当取代发生在位点215时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser；r.当取代发生在位点216时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Met、Pro、Ser、Thr或Val；s.当取代发生在位点217时，取代氨基酸是Asp、Gln、Glu或Ser；t.当取代发生在位点218时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser；和u.当取代发生在位点219时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser；从而，与野生型卡氏枯草杆菌蛋白酶相比较，卡氏枯草杆菌蛋白酶变异体对不溶解底物具有降低的吸附作用和增加的水解作用。
2.权利要求1的卡氏枯草杆菌蛋白酶变异体，其中一个或多个取代发生在第一环区域。
3.权利要求1的卡氏枯草杆菌蛋白酶变异体，其中一个或多个取代发生在第二环区域。
4.权利要求1的卡氏枯草杆菌蛋白酶变异体，其中一个或多个取代发生在第三环区域。
5.权利要求1的卡氏枯草杆菌蛋白酶变异体，其中一个或多个取代发生在第四环区域。
6.权利要求1的卡氏枯草杆菌蛋白酶变异体，其中一个或多个取代发生在第五环区域。
7.权利要求1的卡氏枯草杆菌蛋白酶变异体，其中一个或多个取代发生在第六环区域。
8.一种选自硬表面洗涤组合物、餐具洗涤组合物、口腔洗涤组合物、假牙洗涤组合物、接触透镜洗涤组合物和织物洗涤组合物的洗涤组合物，其特征在于其中洗涤组合物含有权利要求1-7的任何一项的卡氏枯草杆菌蛋白酶变异体和洗涤组合物载体；洗涤组合物优选是硬表面洗涤组合物或洗涤组合物是织物洗涤组合物；组合物优选含有按组合物重量计至少约5％的表面活性剂和至少约5％助剂；组合物还优选含有选自溶剂、缓冲剂、酶、污垢释放剂、粘土去污剂、分散剂、增白剂、泡沫抑制剂、织物柔软剂、增泡剂、酶稳定剂、漂白剂、染料、香料和它们的混合物的洗涤组合物材料。
9.编码权利要求1-7的任何一项的卡氏枯草杆菌蛋白酶变异体的突变体卡氏枯草杆菌蛋白酶基因。
全文摘要
本发明涉及具有野生型卡氏枯草杆菌蛋白酶氨基酸序列的修饰氨基酸序列的卡氏枯草杆菌蛋白酶变异体,野生型氨基酸序列包括第一环区域、第二环区域、第三环区域、第四环区域、第五环区域和第六环区域;其中,修饰的氨基酸序列在一个或多个环区域中的特定标记的位点上含有与野生型卡氏枯草杆菌蛋白酶不同的氨基酸(即取代),从而与野生型卡氏枯草杆菌蛋白酶相比,卡氏枯草杆菌蛋白酶变异体具有对不溶解底物降低的吸附作用和增加的水解作用。本发明还涉及编码这种卡氏枯草杆菌蛋白酶变异体的基因。本发明还涉及含有这种卡氏枯草杆菌蛋白酶变异体的用于洗涤各种表面的组合物。
文档编号C11D3/00GK1183119SQ96193612
公开日1998年5月27日 申请日期1996年2月14日 优先权日1995年3月8日
发明者P·F·布罗德三世, B·L·巴尔内特, D·N·鲁宾 申请人:普罗格特-甘布尔公司