本发明属于生物
技术领域：
，涉及天冬氨酸酶变体及其制备方法与应用。
背景技术：
：天冬氨酸酶(aspartase,e.c.4.3.1.1)，又名天冬氨酸解氨酶(aspartateammonia-lyase)，广泛存在于细菌、酵母和植物中。来源于大肠杆菌的天冬氨酸酶由4个相同的亚基组成，其中每个亚基由207个氨基酸残基组成，且每个亚基的分子量为52.2kda。具有活性的天冬氨酸酶均是以四聚体形式存在的，其单体无活性。从四聚体解聚成二聚体的过程是可逆的，二聚体结构的活性是四聚体活性的45％。天冬氨酸酶是一种别构酶，其最适ph值为8.0，温度为37℃。当ph>7.5时，该酶的活性主要依赖于二价金属离子和天冬氨酸；当ph<7.5时，可不需二价金属离子而催化脱氨反应。天冬氨酸酶在体内的主要功能是分解l-天冬氨酸作为碳源。以大肠杆菌为例，当培养基中含有葡萄糖时，天冬氨酸酶的表达受到抑制，在该情况下，即使培养基中l-天冬氨酸作为唯一的碳源，也不会促进天冬氨酸酶的表达。天冬氨酸酶除了在基因水平上受到调控外，酶自身也具有调控功能。当酶的表达量低时，不易形成四聚体，导致活力下降。此外，该酶受天冬氨酸激活，天冬氨酸浓度低时酶活也低。天冬氨酸酶属于富马酸酶超家族的成员，该家族包括来源于不同生物物种的天冬氨酸酶、富马酸酶、精氨琥珀酸裂解酶。天冬氨酸酶-富马酸酶甲酸的成员均不需要辅酶或辅基参与催化反应，它们可能是由一个识别富马酸的不具调节功能的四亚基酶为基础而衍生出这个家族的酶，该家族成员均保留了打开富马酸双键的这一共同功能。富马酸酶能够催化富马酸被水打开双键，精氨酸琥珀酸裂解酶能够催化富马酸被精氨酸打开双键，而天冬氨酸酶可以催化富马酸被nh4+来打开双键。正是由于天冬氨酸酶的这一特定功能，在工业中其广泛地应用于催化富马酸和氨生成l-天冬氨酸的生产中，且该反应为可逆反应。近三十年来，研究工作者在天冬氨酸酶催化富马酸和氨生成l-天冬氨酸的催化位点和反应机理进行了大量的研究工作。人们对天冬氨酸酶与底物的反应机理变得越来也清楚，这也为天冬氨酸酶的定向改造奠定了一定的理论基础。根据天冬氨酸酶能够将底物的氨基去除形成α,β双键的这一特性，人们试图改变其催化机制以合成光学纯度较高的其他种类的α氨基酸。然而，由于天冬氨酸酶的底物专一性较强，在拓宽天冬氨酸酶作用底物范围方面的研究工作进展相对缓慢。asano等人(biomol.eng(2005)22:95-101)发现对天冬氨酸酶的lys327位点进行诱变，可使其催化β-天冬酰胺脱氨，然而其催化酶活非常低。想要增加其底物作用范围可能需要对ss-loop底物结合区域进行改变，这对天冬氨酸酶的蛋白改造而言是一个巨大的挑战。与富马酸的结构类似，丙烯酸中也含有1个不饱和双键直接和羧基相连。将丙烯酸双键打开加nh4+生成β-丙氨酸，一直以来被认为是一种最简单的β-丙氨酸合成工艺。然而，目前β-丙氨酸(β-alanine)的生产仍然主其要采用传统丙烯腈加氨的化学合成方法。该方法存在副产物多、能耗大、环境污染大等问题。鉴于此，国内外的研究工作者们在丙烯酸氨化合成β-丙氨酸的研究中进行了大量的探索工作，然而至今未有报道可工业化的酶法合成工艺。据文献调研，楼坚(生物转化法生产β-丙氨酸的研究，2006)筛选得到一株含β-丙氨酸合成酶的藤黄八叠球菌。通过诱变和条件优化后，其丙烯酸转化率仍仅为1.25％，远远无法满足工业化的需求。野生型的天冬氨酸酶对富马酸有着高度的底物专一性，其催化丙烯酸加氨生成β-丙氨酸的酶活仅为0.01u/mg左右。技术实现要素：本发明的目的是对野生型天冬氨酸酶进行改造，改善其促进丙烯酸加氨生成β-丙氨酸的催化活性。本发明首先提供了如下天冬氨酸酶变体，其氨基酸序列与序列表中序列2(来源于芽孢杆菌的野生型天冬氨酸酶)相比具有96％以上同一性，并且在序列2的第187位和第326位同时存在t187i和n326c的突变，并且与序列2所示的天冬氨酸酶相比具有改善的催化丙烯酸加氨活性。其中，序列2所示的野生型天冬氨酸酶为蛋白成熟体：术语“蛋白成熟体”意指在翻译和任何翻译后修饰(如n端加工，c端截短，糖基化，磷酸化等)之后以其最终形式存在的蛋白质。换言之，蛋白成熟体指前体蛋白在切割去信号肽部分以及前肽部分(若有的话)之后遗传的部分。信号肽部分可通过本领域中已知的程序(例如signalp)来预测。序列2的第1至468位氨基酸残基是预期的成熟部分。一般而言，酶成熟部分的第一个氨基酸可通过纯化酶的n端测序来完成。这里的技术术语“同一性”是指两个氨基酸序列或两个核苷酸序列之间的相关性由参数“同一性”描述。就本发明而言，两个氨基酸序列之间的序列同一性程度使用如emboss包(emboss：theeuropeanmolecularbiologyopensoftwaresuite,等，2000，trendsgenet.16:276-277)的needle程序(优选版本3.0.0或更新版本)中执行的needleman-wunsch算法(needleman和wunsch，1970，j.mol.biol.48:443-453)来确定。使用的可选参数为缺口开放罚分为10，缺口延伸罚分为0.5，和eblosum62(blosum62的emboss版本)取代矩阵。使用标以“最长同一性(longestidentity)”(使用-nobrief选项获得)的needle输出作为百分比同一性，并如下所示计算：(相同残基*100)/(比对长度-比对中缺口总数)在本发明中，所述“96％以上同一性”可理解为96％、97％、98％或99％的序列同一性；也可以进一步理解为98.0％、98.2％、98.4％、98.6％、98.8％、99.0％、99.1％、99.2％、99.3％或99.4％，但小于100％的序列同一性。这里的“改善的催化丙烯酸加氨活性”意指在升高的温度，在升高浓度或升高ph值的底物中，在变体的工业使用或运输过程中存在的那些的条件下储存一段时间之后，显示高于亲本(序列2所示的野生型天冬氨酸酶)催化丙烯酸加氨活性的变体。本发明的天冬氨酸酶变体可与亲本天冬氨酸酶相比具有改善的催化丙烯酸加氨活性，其中改善的催化丙烯酸加氨活性作为增加的相对活性确定。在一个方面，当使用实施例中用于确定转氨活性的测定法相比较时，具有改善的催化丙烯酸加氨活性的变体的酶活是亲本酶活的至少1.5倍，例如至少2.0倍，至少5倍，至少10倍，至少15倍，至少20倍，至少25倍，至少30倍，至少35倍、至少45倍或至少55倍。进一步，所述天冬氨酸酶变体的氨基酸序列与序列表中序列2相比，还存在如下位置的突变：第20位、第75位、第89位、第156位、第164位、第204位、第226位、第258位、第285位、第321位、第324位、第381位、第389位、第406位、第426位和/或第456位；优选存在2、3、4、5、6、7、8或9处突变。更进一步，所述天冬氨酸酶变体的氨基酸序列与序列表中序列2相比，还存在下述突变：d20v、v75e、q89h、l156f、t164i、y204c、n226i、l258i、m285l、m321i、k324i、k381r、k389i、i406l、r426c和/或p456l；优选存在2、3、4、5、6、7、8或9处突变。更加具体的，所述天冬氨酸酶变体的氨基酸序列为与序列表中序列2相比，存在或仅存在如下1)-385)中任一所示突变组合：1)v75e/q89h/t187i/l258i/n326c/k381r/k389i；2)d20v/q89h/l156f/t164i/t187i/y204c/l258i/m285l/n326c/k381r/r426c；3)q89h/l156f/t164i/t187i/l258i/n326c/k389i/i406l/r426c；4)l156f/t164i/t187i/l258i/k324i/n326c/k389i；5)d20v/l156f/t164i/t187i/y204c/n226i/m321i/k324i/n326c/k389i/r426c；6)l156f/t187i/y204c/k324i/n326c/k389i/i406l；7)t187i/n226i/m285l/m321i/k324i/n326c/k381r/k389i/i406l/p456l；8)d20v/q89h/l156f/t187i/m285l/n326c/k381r/k389i；9)d20v/q89h/t164i/t187i/y204c/n226i/k324i/n326c；10)l156f/t164i/t187i/l258i/n326c/i406l/r426c；11)v75e/t187i/n226i/l258i/k324i/n326c/k381r/i406l；12)v75e/t187i/l258i/m285l/n326c/i406l；13)l156f/t187i/n226i/l258i/n326c/i406l；14)d20v/l156f/t164i/t187i/n226i/m285l/m321i/k324i/n326c/i406l/r426c；15)v75e/t187i/n226i/m321i/k324i/n326c/p456l；16)v75e/q89h/l156f/t187i/n326c/k381r/r426c；17)t187i/n226i/m321i/n326c/k389i/i406l；18)v75e/l156f/t187i/m285l/m321i/k324i/n326c/k381r/p456l；19)q89h/l156f/t164i/t187i/y204c/n226i/m285l/k324i/n326c/k381r；20)t187i/n226i/m321i/n326c/i406l/r426c；21)v75e/l156f/t187i/n226i/l258i/m285l/n326c；22)v75e/q89h/l156f/t164i/t187i/n226i/n326c/k389i/i406l；23)q89h/t164i/t187i/y204c/n226i/k324i/n326c/k381r/r426c；24)d20v/q89h/l156f/t187i/y204c/m285l/k324i/n326c/k381r/k389i；25)d20v/t164i/t187i/n226i/l258i/m285l/k324i/n326c；26)v75e/q89h/t187i/y204c/l258i/m321i/n326c/k381r/k389i/i406l/r426c；27)v75e/q89h/t164i/t187i/y204c/n226i/m285l/m321i/n326c/k389i/r426c；28)d20v/v75e/t187i/n226i/l258i/m285l/n326c/r426c；29)d20v/q89h/l156f/t187i/n226i/n326c/k381r/k389i；30)d20v/v75e/l156f/t187i/n226i/m321i/k324i/n326c/r426c/p456l；31)v75e/q89h/t164i/t187i/m321i/n326c/k381r/k389i/r426c；32)d20v/v75e/t164i/t187i/y204c/m285l/m321i/n326c/k381r/r426c；33)v75e/t164i/t187i/m321i/n326c/k381r/i406l；34)v75e/l156f/t187i/y204c/m321i/n326c/k389i/r426c；35)t187i/m285l/n326c/i406l/r426c/p456l；36)d20v/q89h/t164i/t187i/m285l/n326c/k381r；37)q89h/t187i/y204c/l258i/m321i/k324i/n326c/k381r/r426c；38)v75e/q89h/t187i/y204c/l258i/m285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m321i/n326c/k381r/k389i/i406l；355)q89h/t164i/t187i/y204c/l258i/n326c/k381r；356)d20v/q89h/t187i/l258i/n326c；357)v75e/l156f/t187i/m285l/n326c/k389i；358)q89h/l156f/t187i/y204c/n226i/l258i/n326c/k389i/i406l/r426c；359)d20v/v75e/l156f/t164i/t187i/y204c/m285l/n326c/k381r/k389i；360)v75e/q89h/l156f/t164i/t187i/n226i/n326c/i406l；361)t187i/m285l/n326c/k381r/k389i/p456l；362)v75e/q89h/t164i/t187i/m321i/k324i/n326c/k381r/i406l；363)q89h/l156f/t187i/l258i/m321i/k324i/n326c；364)t187i/l258i/m285l/n326c/k389i；365)t187i/y204c/n226i/l258i/n326c/r426c；366)v75e/q89h/t164i/t187i/y204c/m285l/n326c/r426c；367)t187i/y204c/n226i/l258i/n326c；368)d20v/l156f/t164i/t187i/n226i/l258i/m285l/n326c/i406l/r426c；369)t187i/y204c/l258i/n326c/k381r；370)d20v/t187i/n326c/k381r/i406l；371)d20v/v75e/t187i/n226i/k324i/n326c/k381r/k389i/p456l；372)d20v/q89h/t187i/l258i/m321i/k324i/n326c/k389i；373)t164i/t187i/y204c/m285l/n326c/r426c；374)l156f/t187i/y204c/n326c/k381r；375)t187i/l258i/n326c/k389i/i406l/r426c；376)l156f/t187i/m285l/n326c/k389i；377)d20v/t164i/t187i/l258i/k324i/n326c；378)v75e/l156f/t164i/t187i/y204c/n226i/l258i/m285l/m321i/k324i/n326c；379)v75e/t187i/m285l/k324i/n326c/k381r/r426c；380)v75e/t187i/n226i/m285l/n326c；381)q89h/l156f/t187i/n326c/k381r；382)d20v/t187i/k324i/n326c/i406l；383)d20v/t187i/m321i/n326c/k389i；384)v75e/l156f/t187i/n326c/r426c；385)t187i/y204c/n326c/i406l/r426c。就本发明而言，使用包含于序列2中的成熟多肽来确定变体天冬氨酸酶中的相应氨基酸残基。将变体天冬氨酸酶的氨基酸序列与序列2中公开的成熟多肽进行比对，并且基于该比对，使用如执行于emboss包(emboss：欧洲分子生物学开放软件套件(theeuropeanmolecularbiologyopensoftwaresuite),赖斯(rice)等人,2000,遗传学趋势(trendsgenet).16:276-277)的尼德尔(needle)程序(优选版本5.0.0或更新版本)中所执行的尼德尔曼-翁施(needleman-wunsch)算法(尼德尔曼(needleman)和翁施(wunsch),1970,j.mol.biol.48:443-453)确定对应于序列2中公开的成熟多肽中的任何氨基酸残基的氨基酸位置编号。所使用的参数是空位开放罚分为10，空位延伸罚分为0.5，以及eblosum62(blosum62的emboss版)取代矩阵。取代：对于氨基酸取代，使用下述命名法：原始氨基酸，位置，取代氨基酸。相应地，在位置168用甘氨酸取代色氨酸命名为“trp168gly”或“w168g”。对于碱基取代，使用下述命名法：原始碱基，位置，取代碱基。相应的，在位置60处用胸腺嘧啶t取代胞嘧啶c命名为c60t。缺失：对于氨基酸缺失，使用下述命名法：“△”，原始氨基酸，位置。相应地，在位置195的甘氨酸的缺失命名为“△gly195”或“△g195”。插入：对于氨基酸插入，使用下述命名法：原始氨基酸，位置，原始氨基酸，插入氨基酸。相应地，在位置195的甘氨酸之后插入赖氨酸命名为“gly195glylys”或“g195gk”。多重改变：包含多重改变的变体由斜杠符号(“/”)分隔，例如：“arg170tyr/gly195glu”或“r170y/g195e”代表分别在位置170和195用酪氨酸和谷氨酸取代精氨酸和甘氨酸；c560t/g561t代表分别在位置560和561用胸腺嘧啶t和胸腺嘧啶t取代胞嘧啶c和鸟嘌呤g。本文中使用的用于确定氨基酸位置的命名法是基于来源于芽孢杆菌的天冬氨酸酶的氨基酸序列，其成熟多肽序列在序列表中作为序列2的第1-468位氨基酸残基给出。相应地，在本文的上下文中，编号位置的基础是序列2，以m1开始，以k468终止。为了便于所述天冬氨酸酶变体的纯化，可在所述天冬氨酸酶变体的氨基末端或羧基末端连接上如下表所示的标签。表：标签的序列标签残基序列poly-arg5-6(通常为5个)rrrrrpoly-his2-10(通常为6个)hhhhhhflag8dykddddkstrep-tagii8wshpqfekc-myc10eqkliseedl编码所述天冬氨酸酶变体的核酸分子也属于本发明的保护范围。所述核酸分子可以是dna，如cdna、基因组dna或重组dna；所述核酸分子也可以是rna，如mrna、hnrna或trna等。在本发明中，所述核酸分子为编码所述天冬氨酸酶变体的基因，所述基因的核苷酸序列与序列表中序列1相比，存在或仅存在如下1)-385)中任一所示突变组合：1)t224a/c560t/g561t/t772a/a774t/a976t/a977g/t978c/a1166t/a1167c；2)t224a/c560t/g561t/t772a/a774t/a976t/a977g/t978c/a1141c/a1142g/a1143t；3)t224a/c560t/g561t/t772a/a774t/a971t/a972t/a976t/a977g/t978c/a1166t/a1167c/c1276t/t1278c/c1366t/c1367t/a1368g；4)t224a/c560t/g561t/t772a/a774t/a853c/a976t/a977g/t978c/a1216t/t1218g；5)t224a/c560t/g561t/t772a/a774t/a853c/a976t/a977g/t978c/a1166t/a1167c；6)t224a/c560t/g561t/g963t/a1216t/t1218g/a976t/a977g/t978c/c1276t/t1278c；7)t224a/c560t/g561t/a976t/a977g/t978c/a1141c/a1142g/a1143t/a1216t/t1218g；8)t224a/c560t/g561t/a976t/a977g/t978c/a1141c/a1142g/a1143t/a1166t/a1167c/a1216t/t1218g/c1276t/t1278c；9)t224a/c560t/g561t/a971t/a972t/a976t/a977g/t978c/a1166t/a1167c；10)t224a/c560t/g561t/a853c/g963t/a971t/a972t/a976t/a977g/t978c/c1276t/t1278c；11)t224a/c560t/g561t/a853c/a971t/a972t/a976t/a977g/t978c/c1276t/t1278c；12)t224a/c560t/g561t/a853c/a971t/a972t/a976t/a977g/t978c/a1141c/a1142g/a1143t/c1276t/t1278c；13)t224a/c560t/g561t/a677t/c678t/t772a/a774t/a971t/a972t/a976t/a977g/t978c/a1141c/a1142g/a1143t/a1216t/t1218g；14)t224a/c560t/g561t/a677t/c678t/g963t/a971t/a972t/a976t/a977g/t978c/c1366t/c1367t/a1368g；15)t224a/c560t/g561t/a677t/c678t/a976t/a977g/t978c/a1141c/a1142g/a1143t；16)t224a/c560t/g561t/a677t/c678t/a853c/a976t/a977g/t978c；17)t224a/c560t/g561t/a611g/t612c/g963t/a976t/a977g/t978c；18)t224a/c560t/g561t/a611g/t612c/a976t/a977g/t978c/a1216t/t1218g；19)t224a/c560t/g561t/a611g/t612c/a853c/g963t/a976t/a977g/t978c/a1141c/a1142g/a1143t/a1216t/t1218g/c1366t/c1367t/a1368g；20)t224a/c560t/g561t/a611g/t612c/a677t/c678t/t772a/a774t/a976t/a977g/t978c/a1216t/t1218g；21)t224a/c560t/g561t/a611g/t612c/a677t/c678t/a971t/a972t/a976t/a977g/t978c/a1141c/a1142g/a1143t/a1216t/t1218g；22)t224a/c560t/g561t/a611g/t612c/a677t/c678t/a853c/a971t/a972t/a976t/a977g/t978c/a1141c/a1142g/a1143t/c1276t/t1278c；23)t224a/c491t/a492t/c560t/g561t/t772a/a774t/a976t/a977g/t978c/a1166t/a1167c；24)t224a/c491t/a492t/c560t/g561t/t772a/a774t/a976t/a977g/t978c/a1141c/a1142g/a1143t/c1276t/t1278c；25)t224a/c491t/a492t/c560t/g561t/t772a/a774t/a853c/g963t/a976t/a977g/t978c/a1141c/a1142g/a1143t/a1166t/a1167c/a1216t/t1218g/c1276t/t1278c；26)t224a/c491t/a492t/c560t/g561t/t772a/a774t/a853c/g963t/a971t/a972t/a976t/a977g/t978c/a1216t/t1218g/c1276t/t1278c；27)t224a/c491t/a492t/c560t/g561t/g963t/a976t/a977g/t978c/a1216t/t1218g；28)t224a/c491t/a492t/c560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替代地，该载体可以是这样的载体，当它被引入该宿主细胞时，被整合到基因组中并且与其中已经整合了它的一个或多个染色体一起复制。该载体优选包含一个或多个允许方便地选择转化细胞、转染细胞、转导细胞等细胞的选择性标记。选择性标记是这样一种基因，该基因的产物提供了杀生物剂抗性，或病毒抗性，或重金属抗性，或营养缺陷型的原养型等。细菌选择性标记的实例是地衣芽孢杆菌或枯草芽孢杆菌dal基因、或赋予抗生素抗性(例如氨苄青霉素、氯霉素、卡那霉素、新霉素、大观霉素、或四环素抗性)的标记。用于酵母宿主细胞的适合的标记包括但不局限于ade2、his3、leu2、lys2、met3、trp1、以及ura3。用于在丝状真菌宿主细胞中使用的选择性标记包括但不局限于amds(乙酰胺酶)、argb(鸟氨酸氨甲酰基转移酶)、bar(草丁膦乙酰转移酶)、hph(潮霉素磷酸转移酶)、niad(硝酸还原酶)、pyrg(乳清酸核苷-5'磷酸脱羧酶)、sc(硫酸腺苷基转移酶)、以及trpc(邻氨基苯甲酸合成酶)、连同其等效物。该载体优选含有允许载体整合到宿主细胞的基因组中或载体在细胞中独立于基因组自主复制的一个或多个元件。对于整合到该宿主细胞基因组中，该载体可以依靠编码该载体的多核苷酸序列或者用于通过同源或非同源重组整合到该基因组中的该载体的任何其他元件。可替代地，该载体可以包含用于指导通过同源重组而整合到宿主细胞基因组中的一个或多个染色体中的一个或多个精确位置处的另外的多核苷酸。为了增加在精确位置整合的可能性，这些整合的元件应包含足够数量的核酸，例如100至10,000个碱基对、400至10,000个碱基对、以及800至10,000个碱基对，这些碱基对与对应的靶序列具有高度的序列一致性以提高同源重组的可能性。这些整合元件可以是与宿主细胞的基因组内的靶序列同源的任何序列。此外，这些整合元件可以是非编码多核苷酸或编码多核苷酸。另一方面，该载体可以通过非同源重组整合到宿主细胞的基因组中。对于自主复制，载体可以进一步包含使用该载体能够在相应宿主细胞中自主复制的复制起点。复制起点可以是在细胞中起作用的介导自主复制的任何质粒复制子。术语“复制起点(originofreplication)”或“质粒复制子(plasmidreplicator)”意指使得质粒或载体可在体内复制的多核苷酸。细菌宿主细胞复制起点的实例是允许在大肠杆菌中复制的质粒pbr322、puc19、pacyc177以及pacyc184的复制起点，以及允许在芽孢杆菌中复制的质粒pub110、pe194、pta1060以及pamβ1的复制起点。酵母宿主细胞中复制起点的实例是2微米复制起点ars1、ars4、ars1与cen3的组合以及ars4与cen6的组合。丝状真菌宿主细胞的复制起点的实例是ama1和ans1。用于连接以上所述的元件以构建本发明的重组表达载体的步骤是本领域的普通技术人员所熟知的。在本发明中，所述重组表达载体具体为将所述基因克隆到pet21a载体后得到的重组载体。所述表达盒中含有启动所述基因转录的启动子，所述基因和转录终止序列。当然还可包含且不限于先导序列、聚腺苷酸序列、前台序列、信号肽序列。调控序列可与接头一同提供，以供导入特定限制性位点的目的，所述位点便于所述所述表达盒的连接。所述重组细胞为将所述基因导入宿主细胞后得到。这里的术语“宿主细胞”，包括各种可用于导入所述基因的转化、转染、转导等易感的细胞类型。术语“宿主细胞”涵盖亲本细胞的任何后代，其由于在复制过程中发生的突变，与亲本细胞不完全相同。宿主细胞的选择在很大程度上会取决于编码所述蛋白质的基因及其来源。所述宿主细胞可以是有用的任何细胞，例如原核细胞或真核细胞。原核宿主细胞可以是任何革兰氏阳性或革兰氏阴性细菌细胞，革兰氏阳性细菌包括但不限于：芽孢杆菌属、梭菌属、肠球菌属、土芽孢杆菌属、乳杆菌属、乳球菌属、海洋芽孢杆菌属、葡萄球菌属、链球菌属以及链霉菌属。革兰氏阴性菌包括但不限于：大肠杆菌、弯曲杆菌属、黄杆菌属、梭杆菌属、螺杆菌属、泥杆菌属、奈瑟球菌属、假单胞菌属、沙门氏菌属、以及尿原体属。所述芽孢杆菌属细胞，包括但不限于：嗜碱芽孢杆菌、解淀粉芽孢杆菌、短芽孢杆菌、球状芽孢杆菌、环状芽孢杆菌、克劳氏芽孢杆菌、凝结芽孢杆菌、坚硬芽孢杆菌、灿烂芽孢杆菌、迟缓芽孢杆菌、地衣芽孢杆菌、巨大芽孢杆菌、短小芽孢杆菌、嗜热脂肪芽孢杆菌、枯草芽孢杆菌以及苏云金芽孢杆菌。所述链球菌属细胞包括但不限于：似马链球菌、酿脓链球菌以及马链球菌兽瘟亚种细胞。所述链霉菌属细胞包括但不限于：产色链霉菌、除虫链霉菌、天蓝链霉菌、灰色链霉菌以及浅青紫链霉菌细胞。将载体导入细菌宿主细胞可通过以下方法来实现：原生质体转化(参见，例如chang和cohen,1979,moleculargeneralgenetics168:111-115)，使用感受态细胞(参见，例如young和spizizin,1961,journalofbacteriology81:823-829，或dubnau和davidoff-abelson,1971,journalofmolecularbiology56:209-221)，电穿孔(参见，例如shigekawa和dower,1988biotechniques6:742-751)或接合(参见，例如koehler和thorne,1987,journalofbacteriology169:5771-5278)来实现。然而，可以使用本领域已知的用于将dna引入宿主细胞中的任何方法。真核宿主细胞可以是哺乳动物、昆虫、植物或真菌细胞。所述真核宿主细胞可以是真菌细胞。“真菌”如用于本文包括子囊菌门(ascomycota)、担子菌门(basidiomycota)、壶菌门(chytridiomycota)和接合菌门(zygomycota)(如由hawksworth等，于ainsworthandbisby’sdictionaryofthefungi,第八版,1995,cabinternational,universitypress,cambridge,uk中所定义)以及卵菌门(oomycota)和所有有丝分裂孢子真菌(mitosporicfungi)。所述真菌宿主细胞可以是酵母细胞。“酵母”如用于本文中包括产子囊酵母(ascosporogenousyeast)(内孢霉目(endomycetales))、产担子酵母(basidiosporogenousyeast)和属于半知菌类(fungiimperfecti)(芽孢纲(blastomycetes))的酵母。由于酵母的分类在未来可能改变，就本发明而言，应将酵母定义为如biologyandactivitiesofyeast(skinner,f.a.,passmore,s.m.和davenport,r.r.编,soc.app.bacteriol.symposiumseriesno.9,1980)中所述。所述酵母宿主细胞可以是假丝酵母属(candida)、汉逊酵母属(hansenula)、克鲁维酵母属(kluyveromyces)、毕赤酵母属(pichia)、酵母属(saccharomyces)、裂殖酵母属(schizosaccharomyces)或西洋蓍霉属(yarrowia)细胞。所述优选的酵母宿主细胞是巴斯德毕赤酵母(pichiapastoris)，甲醇毕赤酵母(pichiamethanolica)，卡尔酵母(saccharomycescarlsbergensis)、酿酒酵母(saccharomycescerevisiae)、糖化酵母(saccharomycesdiastaticus)、道格拉氏酵母(saccharomycesdouglasii)、克鲁弗酵母(saccharomyceskluyveri)、诺地酵母(saccharomycesnorbensis)、卵形酵母(saccharomycesoviformis)、乳酸克鲁维酵母(kluyveromyceslactis)细胞或解脂西洋蓍霉(yarrowialipolytica)细胞。所述真菌宿主细胞可以是丝状真菌细胞。“丝状真菌”包括真菌门(eumycota)和卵菌门的亚门的所有丝状形式。丝状真菌通常的特征在于由壳多糖(chitin)、纤维素、葡聚糖、壳聚糖(chitosan)、甘露聚糖和其它复杂多糖组成的菌丝体壁。通过菌丝延伸进行营养生长，而碳分解代谢是专性需氧的。相反，酵母例如酿酒酵母的营养生长通过单细胞菌体的出芽生殖(budding)进行，而碳分解代谢可以是发酵的。所述丝状真菌宿主细胞是枝顶孢霉属(acremonium)、曲霉属(aspergillus)、短梗霉属(aureobasidium)、烟管霉属(bjerkandera)、ceriporiopsis、鬼伞属(coprinus)、革盖菌属(coriolus)、隐球菌属(cryptococcus)、filibasidium、镰孢属(fusarium)、腐质霉属(humicola)、梨孢菌属(magnaporthe)、毛霉属(mucor)、毁丝霉属(myceliophthora)、新考玛脂霉属(neocallimastix)、脉孢菌属(neurospora)、拟青霉属(paecilomyces)、青霉属(penicillium)、平革菌属(phanerochaete)、射脉菌属(phlebia)、瘤胃壶菌属(piromyces)、侧耳属(pleurotus)、裂褶菌属(schizophyllum)、踝节菌属(talaromyces)、嗜热子囊菌属(thermoascus)、梭孢壳属(thielavia)、弯颈霉属(tolypocladium)、栓菌属(trametes)或木霉属(trichoderma)细胞。所述优选的丝状真菌宿主细胞是泡盛曲霉(aspergillusawamori)、烟曲霉(aspergillusfumigatus)、臭曲霉(aspergillusfoetidus)、日本曲霉(aspergillusjaponicus)、构巢曲霉(aspergillusnidulans)、黑曲霉(aspergillusniger)或米曲霉(aspergillusoryzae)细胞。在另一个最优选方面，丝状真菌宿主细胞是杆孢状镰孢(fusariumbactridioides)、禾谷镰孢(fusariumcerealis)、库威镰(fusariumcrookwellense)、大刀镰孢(fusariumculmorum)、禾本科镰孢(fusariumgraminearum)、禾赤镰孢(fusariumgraminum)、异孢镰孢(fusariumheterosporum)、合欢木镰孢(fusariumnegundi)、尖镰孢(fusariumoxysporum)、多枝镰孢(fusariumreticulatum)、粉红镰孢(fusariumroseum)、接骨木镰孢(fusariumsambucinum)、肤色镰孢(fusariumsarcochroum)、拟分枝孢镰孢(fusariumsporotrichioides)、硫色镰孢(fusariumsulphureum)、圆镰孢(fusariumtorulosum)、拟丝孢镰孢(fusariumtrichothecioides)或镶片镰孢(fusariumvenenatum)细胞。在另一个最优选的方面，丝状真菌宿主细胞是黑刺烟管菌(bjerkanderaadusta)、ceriporiopsisaneirina、ceriporiopsisaneirina、ceriporiopsiscaregiea、ceriporiopsisgilvescens、ceriporiopsispannocinta、ceriporiopsisrivulosa、ceriporiopsissubrufa或ceriporiopsissubvermispora、灰盖鬼伞(coprinuscinereus)、毛革盖菌(coriolushirsutus)、特异腐质霉(humicolainsolens)、疏棉状腐质霉(humicolalanuginosa)、米黑毛霉(mucormiehei)、嗜热毁丝霉(myceliophthorathermophila)、粗糙脉菌(neurosporacrassa)、产紫青霉(penicilliumpurpurogenum)、黄孢平革菌(phanerochaetechrysosporium)、辐射射脉菌(phlebiaradiata)、刺芹侧耳(pleurotuseryngii)、土生梭孢霉(thielaviaterrestris)、trametesvillosa、杂色栓菌(trametesversicolor)、哈茨木霉(trichodermaharzianum)、康宁木霉(trichodermakoningii)、长枝木霉(trichodermalongibrachiatum)、里氏木霉(trichodermareesei)或绿色木霉(trichodermaviride)菌株细胞。可以将真菌细胞通过涉及原生质体形成、原生质体转化和细胞壁重建的方法以本身公知的方式转化。用于转化曲霉属和木霉属宿主细胞的方法可以参考ep238023和约尔顿(yelton)等人，1984，美国科学院杂志(proc.nat.acad.sci.usa)81:1200-1204中的介绍。用于转化镰孢属菌种的方法可以参考马拉迪尔(malardier)等人,1989,gene78:120-156和wo96/00787所述的内容。用于转化酵母宿主细胞的方法可以参考以下文献：贝克尔(becker)和古伦特(guarente),于艾本尔森j.n.(abelson,j.n.)和赛蒙m.i.(simon,m.i.)编著,酵母遗传学与分子生物学指南(guidetoyeastgeneticsandmolecularbiology)，酶学方法(methodsinenzymology)，第194卷,第182-187页，学术出版社有限公司(academicpress,inc.)，纽约(newyork)；埃托(ito)等人,1983,细菌学杂质(journalofbacteriology)153:163；和辛伦hinnen等人,1978,美国国家科学院院刊(proc.nat.acad.sci.usa)75:1920。在本发明中，所述重组细胞具体为含有所述基因的大肠杆菌。所述大肠杆菌具体为大肠杆菌bl21。本发明还保护所述天冬氨酸酶变体的生产方法，包括培养含有所述核酸分子的宿主细胞，并从细胞培养物中回收所述天冬氨酸酶变体的步骤。在本发明天冬氨酸酶变体的产生方法中，可使用本领域熟知的方法在适合产生所述变体的营养培养基中培养所述宿主细胞。例如，可以通过在合适培养基中且在允许表达和/或分离所述天冬氨酸酶变体的条件下进行的摇瓶培养，和实验室或工业发酵罐中的小规模或大规模发酵(包括连续、分批、分批补料或固态发酵)来培养所述宿主细胞。使用本领域已知的方法在合适的营养培养基中进行培养，所述营养培养基包含碳源，氮源和无机盐。合适的培养基能够从商业供应商获得，或可以根据公开的组成制备(例如，在美国典型培养物保藏中心的目录中)。如果天冬氨酸酶变体被所述宿主细胞分泌到营养培养基中，该变体能够从所述培养基中直接回收。如果天冬氨酸酶变体不能够被所述宿主细胞分泌到营养培养基中，则该变体能够从细胞裂解物中(lysate)回收。可使用本领域已知的对这些变体特异的方法来检测该变体。这些检测方法包括但不限于：特异性抗体的使用、酶作用产物的形成或酶作用底物的消失。例如，可以使用酶测定来确定该变体的活性。可使用本领域已知的方法回收所述天冬氨酸酶变体。例如，该变体可以通过常规方法从营养培养基中回收，所述常规方法包括但不限于：离心、过滤、提取、喷雾干燥、蒸发或沉淀。可通过多种本领域已知的方法纯化所述天冬氨酸酶变体，所述方法包括但不限于：色谱法(例如，离子色谱交换、亲和色谱、疏水作用色谱、色谱聚焦、以及尺寸排阻色谱)、电泳法(例如，制备型(preparative)等电聚焦)、差示溶解度(例如，硫酸铵沉淀)、sds-page或萃取(参见，例如，蛋白质纯化(proteinpurification)，詹森(j.-c.janson)和莱登(larsryden)编著，vch出版社(vchpublishers)，newyork,1989)。所述天冬氨酸酶变体或所述核酸分子或所述重组载体或所述表达盒或所述重组细胞在制备具有催化丙烯酸加氨活性的产品中的应用也属于本发明的保护范围。所述天冬氨酸酶变体或所述核酸分子或所述重组载体或所述表达盒或所述重组细胞在生产目的产物中的应用也属于本发明的保护范围；所述目的产物为如下中的任一种或多种：β-丙氨酸，β-丙氨酸盐、β-丙氨酸的多聚体。本发明还提供了一种生产目的产物的方法，包括如下步骤：使用丙烯酸或丙烯酸盐和含氨材料在所述天冬氨酸酶变体的催化下进行反应，得到所述目的产物；所述目的产物为如下中的任一种或多种：β-丙氨酸，β-丙氨酸盐、β-丙氨酸的多聚体。在本发明一个实施例中，所述含氨材料具体为氨水。在所述反应的体系中，所述丙烯酸或丙烯酸盐20-400mm(具体如400mm)，ph值为7.0-9.5(如7.5、8.0-8.5、9.0)，反应温度为30-55℃(如35℃、37℃、40-45℃、50℃)，反应时间为0.5-5h(如1h、2h、3h、4h)。实验证明，本发明的天冬氨酸酶变体与野生型亲本天冬氨酸酶相比具有改善的催化丙烯酸加氨活性，且热稳定性和ph谱更优，在丙烯酸加氨反应中可以大大提高丙烯酸的转化率。具体实施方式下述实施例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。实施例1、天冬氨酸酶突变体的制备及性能鉴定本实施例共涉及300种天冬氨酸酶突变体，均是在来源于芽孢杆菌的如序列2所示的野生型天冬氨酸酶蛋白成熟体(对应的编码基因如序列表中序列1所示)的基础进行突变后得到的。300种天冬氨酸酶突变体在蛋白和基因水平的突变位点具体如表1中所示。一、克隆使用本领域已知的方法构建含有编码所述天冬氨酸酶及其变体基因的质粒，并且将所得重组质粒转化至合适的宿主细胞中。在本实施例中，使用的载体质粒具体是pet21a。以含有天冬氨酸酶及其变体编码基因的基因片段为模板，以tatggctagcatgactggtatgaataccgatgttcgtattg和gctagttattgctcagcggttttcttccagcaattcccg为引物进行pcr扩增相应的核苷酸基因片段。之后，将所述pcr扩增得到的基因片段和pet21a按照1:1的摩尔比例混合，通过gibsonassembly的方法(参见：戢志呈等，应用gibsonassembly方法构建植物表达载体，华南农业大学学报，2014,35(5)：112-116)构建含有天冬氨酸酶或其变体编码基因的质粒。以大肠杆菌bl21(de3)作为宿主细胞，将含有所述天冬氨酸酶编码基因的重组质粒导入到该宿主细胞中。二、表达使用自诱导培养基培养含有步骤一构建的重组质粒的宿主细胞。自诱导培养基的组成如下：蛋白胨10g/l，酵母粉5g/l，磷酸氢二钠3.55g/l，磷酸二氢钾3.4g/l，氯化铵2.68g/l，硫酸钠0.71g/l，七水硫酸镁0.493g/l，六水氯化铁0.27g/l，100％甘油20ml/l，葡萄糖0.5g/l，乳糖2g/l，氨苄青霉素钠50mg/l。将含有重组质粒的宿主细胞接种于自诱导培养基中，按照分批发酵的方式进行发酵。在30℃和200rpm条件下振荡培养20小时。三、细胞收集可采用以下三种方法：①离心法：将细胞培养液在4000g转速下，离心10分钟，收集细胞；②中空纤维膜过滤：将细胞培养液用0.22微米中空纤维膜，收集细胞；③陶瓷膜过滤：用50kda陶瓷膜过滤，收集细胞。实验室条件下优选离心法收集细胞(本实施例采用该方法)，比较酶活。四、表达蛋白的纯化用超纯水重悬细胞至od550值为200。采用超声破碎的方法破碎细胞，超声破碎条件如下：工作1秒，间歇两秒，180w，共破碎20-25分钟。也可采用高压均质破碎的方法裂解细胞，高压均质破碎的条件：50hz，800bar，破碎2遍。首先，采用高速离心的方法去除细胞碎片和大分子杂质，离心条件如下：取10-50ml细胞破碎液，12000rpm，4℃，离心20-40min。之后，采用超滤离心的方法对去除细胞碎片和大分子杂质的清液进行浓缩，所选用的超滤离心管为装有ultracel-50超滤膜的amiconultra-15(或50)ml离心超滤管，离心条件：12000rpm，4℃，离心20-40min。加入1-5ml4℃纯水重悬超滤管所截留的天冬氨酸酶蛋白。五、酶蛋白浓度的测定将酶蛋白重悬液稀释100-500倍后，采用bradford法对其浓度进行测定。六、催化丙烯酸加氨活性的测定天冬氨酸酶变体所作用底物的配制：丙烯酸或丙烯酸盐20-400mm，用氨水调ph值至7.0-9.5。天冬氨酸酶变体酶活测定的操作步骤如下：取一定量的发酵所得细胞或纯化的酶蛋白，加入底物后，在一定温度下反应反应0.5-5h。采用高效液相色谱检测β-丙氨酸，使用外标法确定反应前后β-丙氨酸量的变化。液相色谱检测的条件如下：色谱柱eclipsexdb-c18，流动相ph2.6磷酸二氢钾：乙腈＝95:5(体积比)，流速1.0ml/min，检测波长210nm。ph2.6的磷酸二氢钾(kh2po4)的配制：取1.44gkh2po4溶于1l超纯水，用0.6-0.7ml85％磷酸调节ph至2.6。催化丙烯酸加氨酶活的定义：1分钟内反应生成1mm的β-丙氨酸定义为1u。单位细胞催化丙烯酸加氨酶活力的定义：1.0od550细胞浓度下1分钟内反应生成1mm的β-丙氨酸定义为1u/od550。单位蛋白催化丙烯酸加氨酶活力的定义：1.0mg天冬氨酸酶蛋白在1分钟内反应生成1mm的β-丙氨酸定义为1u/mg。转化率的计算：转化率％＝a/b×100％，其中：a—β-丙氨酸的生成摩尔浓度mm；b—丙烯酸的初始摩尔浓度，单位mol/l单位细胞酶活的计算：单位细胞酶活＝β-丙氨酸生成摩尔浓度/反应时间/细胞浓度，其中：反应时间，单位min；细胞浓度为反应体系中的od550值。单位蛋白酶活的计算：单位蛋白酶活＝β-丙氨酸生成摩尔浓度/反应时间/蛋白浓度，其中：反应时间，单位min；蛋白浓度为反应体系中的粗酶蛋白浓度，单位g/l。七、相对酶活力将25mg野生型天冬氨酸酶或其变体的粗酶蛋白与1.0ml400mmph8.0的底物混合，在40℃搅拌反应1.0h后，测定其催化丙烯酸加氨酶活性。相对酶活力，即天冬氨酸酶变体酶活力与野生型天冬氨酸酶酶活力的比值。天冬氨酸酶变体的相对酶活力结果见表1。表1天冬氨酸酶变体蛋白和基因突变位点以及催化丙烯酸加氨酶相对活性测定结果注：蛋白取代的编号是从序列2所示氨基酸序列的n端为起始的；基因取代的编号是从序列1所示核苷酸序列的5’端为起始的。结果表明，与野生型天冬氨酸酶相比，所得突变体的酶活提高5-45倍。八、ph谱的测定通过调整底物中氨水的加量，分别配制不同ph值(7.0、7.5、8.0、8.5、9.0、9.5)浓度为400mm的底物。将25mg野生型天冬氨酸酶或其变体的粗酶蛋白与不同ph底物混合后，在40℃条件下搅拌反应1.0h，测定催化丙烯酸加氨酶活力。为了测定相对活性，将在ph8.0测量的活性设定为100％，结果见表2。表2天冬氨酸酶变体的ph谱测定结果结果表明，与野生型天冬氨酸酶相比，所得变体的ph谱明显拓宽，某些变体催化底物反应的最适ph值增加至8.5-9.0左右。九、温度谱的测定将25mg野生型天冬氨酸酶或其变体的粗酶蛋白与1.0mlph8.0、400mm底物混合，分别在不同温度下(30℃、35℃、40℃、45℃、50℃、55℃)搅拌反应1.0h，测定其催化丙烯酸加氨酶活力。为了测定相对活性，将在40℃测量的活性设定为100％，结果见表3。表3天冬氨酸酶变体的温度谱测定结果结果表明，与野生型天冬氨酸酶相比，所得变体的温度谱明显拓宽，某些变体催化底物的最适温度增加至45℃-50℃。十、热稳定性的测定将25mg野生型天冬氨酸酶或其变体的粗酶蛋白在40℃温育4小时后，与1.0mlph8.0、400mm底物混合，在40℃条件下搅拌反应1.0h，测定其催化丙烯酸加氨酶活力，其中实验组为一个或多个(若干个)的天冬氨酸酶变体，对照组为野生型天冬氨酸酶。结果见表4。表4天冬氨酸酶变体热稳定性的测定结果变体酶活保留率wt21.40％ahb00139.03％ahb03260.64％ahb06356.16％ahb08550.88％ahb11658.08％ahb14844.40％ahb22952.24％ahb24457.52％ahb29451.36％ahb32754.80％ahb35764.08％ahb37360.64％结果表明，与野生型天冬氨酸酶相比，所得变体的热稳定性明显增加。40℃温育4小时后，酶活保留率提高约2-3倍。十一、丙烯酸转化率的测定将25mg野生型天冬氨酸酶或其变体的粗酶蛋白与1.0mlph8.5的底物(浓度400mm)混合，在45℃条件下搅拌反应0-5h，液相检测丙烯酸残留情况，计算转化率。结果见表5。表5丙烯酸转化率的测定如表5所示：反应5h后，野生型天冬氨酸酶底物转化率仅为1.34％，ahb001变体的底物转化率达到68.33％，其他所示变体的底物转化率均达到84％以上。本文描述和要求保护的本发明并不局限于本文公开的具体实施方案内，因为以上实施方案旨在作为对本发明的某个具体方面说明，并且旨在将任何等同的实施方案包含于本发明的范围内。<110>秦皇岛华恒生物工程有限公司<120>天冬氨酸酶变体及其制备方法与应用<130>gncln171338<160>2<170>patentinversion3.5<210>1<211>1404<212>dna<213>芽孢杆菌（bacillussp.）<400>1atgaataccgatgttcgtattgagaaagactttttaggagaaaaggagattccgaaagac60gcttattatggcgtacaaacaattcgggcaacggaaaattttccaattacaggttatcgt120attcatccagaattaattaaatcactagggattgtaaaaaaatcagccgcattagcaaac180atggaagttggcttactcgataaagaagttgggcaatatatcgtaaaagctgctgacgaa240gtgattgaaggaaaatggaatgatcaatttattgttgacccaattcaaggcggggcagga300acttccattaatatgaatgcaaatgaagtgattgctaaccgcgcattagaattaatggga360gaggaaaaaggaaactattcaaaaattagtccaaactcccatgtaaatatgtctcaatca420acaaacgatgctttccctactgcaacgcatattgctgtgttaagtttattaaatcaatta480attgaaactacaaaatacatgcaacaagaattcatgaaaaaagcagatgaattcgctggc540gttattaaaatgggaagaacgcacttgcaagacgctgttcctattttattaggacaagag600tttgaagcatatgctcgtgtaattgcccgcgatattgaacgtattgccaatacgagaaac660aatttatacgacatcaacatgggtgcaacagcagtcggcactggcttaaatgcagatcct720gaatatataagcatcgtaacagaacatttagcaaaattcagcggacatccattaagaagt780gcacaacatttagtggacgcaactcaaaatacagactgctatacagaagtttcttctgca840ttaaaagtttgcatgatcaacatgtctaaaattgccaatgatttacgcttaatggcatct900ggaccacgcgcaggcttatcagaaatcgttcttcctgctcgacaacctggatcttctatc960atgcctggtaaagtgaatcctgttatgccagaagtgatgaaccaagtggcattccaagtg1020ttcggtaatgatttaacaattacatctgcttctgaagcaggccaatttgaattaaatgtg1080atggaacctgtgttattcttcaatttaattcaatcgatttcgattatgactaatgtcttt1140aaatcctttacagaaaactgcttaaaaggtattaaggcaaatgaagaacgcatgaaagaa1200tatgttgagaaaagcattggaatcattactgcaattaacccacatgtaggctatgaaaca1260gctgcaaaattagcacgtgaagcatatcttacaggggaatccatccgtgaactttgcatt1320aagtatggcgtattaacagaagaacagttaaatgaaatcttaaatccatatgaaatgaca1380catccgggaattgctggaagaaaa1404<210>3<211>468<212>prt<213>芽孢杆菌（bacillussp.）<400>2metasnthraspvalargileglulysasppheleuglyglulysglu151015ileprolysaspalatyrtyrglyvalglnthrileargalathrglu202530asnpheproilethrglytyrargilehisprogluleuilelysser354045leuglyilevallyslysseralaalaleualaasnmetgluvalgly505560leuleuasplysgluvalglyglntyrilevallysalaalaaspglu65707580valilegluglylystrpasnaspglnpheilevalaspproilegln859095glyglyalaglythrserileasnmetasnalaasngluvalileala100105110asnargalaleugluleumetglygluglulysglyasntyrserlys115120125ileserproasnserhisvalasnmetserglnserthrasnaspala130135140pheprothralathrhisilealavalleuserleuleuasnglnleu145150155160ilegluthrthrlystyrmetglnglngluphemetlyslysalaasp165170175gluphealaglyvalilelysmetglyargthrhisleuglnaspala180185190valproileleuleuglyglnglupheglualatyralaargvalile195200205alaargaspilegluargilealaasnthrargasnasnleutyrasp210215220ileasnmetglyalathralavalglythrglyleuasnalaasppro225230235240glutyrileserilevalthrgluhisleualalyspheserglyhis245250255proleuargseralaglnhisleuvalaspalathrglnasnthrasp260265270cystyrthrgluvalserseralaleulysvalcysmetileasnmet275280285serlysilealaasnaspleuargleumetalaserglyproargala290295300glyleusergluilevalleuproalaargglnproglyserserile305310315320metproglylysvalasnprovalmetprogluvalmetasnglnval325330335alapheglnvalpheglyasnaspleuthrilethrseralaserglu340345350alaglyglnphegluleuasnvalmetgluprovalleuphepheasn355360365leuileglnserileserilemetthrasnvalphelysserphethr370375380gluasncysleulysglyilelysalaasnglugluargmetlysglu385390395400tyrvalglulysserileglyileilethralaileasnprohisval405410415glytyrgluthralaalalysleualaargglualatyrleuthrgly420425430gluserilearggluleucysilelystyrglyvalleuthrgluglu435440445glnleuasngluileleuasnprotyrglumetthrhisproglyile450455460alaglyarglys465当前第1页12