本发明涉及生物
技术领域：
，具体而言，涉及一种转氨酶突变体及其应用。
背景技术：
：手性胺广泛存在于自然界中，是很多重要生物活性分子的结构单元，是合成天然产物和手性药物的重要中间体。许多手性胺都含有一个或多个手性中心，不同手性药物的药理活性、代谢过程、代谢速率以及毒性有显著差异，通常一种对映体是有效的，而另一种对映体则是低效或无效的，甚至是有毒的。因此，如何高效立体选择性地构建含手性中心的化合物在医药研发中有着重要意义。ω－转氨酶(ω－ta)属于转移酶类，同其他的转氨酶类一样，催化一个氨基与酮基互换的过程。大部分情况下ω－转氨酶是指一类酶，只要某个酶催化的转氨反应中，反应的底物或产物中不含有α－氨基酸，就可以称该酶为ω－转氨酶。ω－转氨酶以酮类化合物为原料，通过立体选择性地转氨基作用，可以高效生产手性胺，因其底物相对廉价、产物纯度高的特点，受到研究人员越来越多的关注(greenchemistry,2017,19(2)：333-360.)。然而野生型的转氨酶多存在底物耐受性差，活性低，稳定性差等缺陷，距离工业化应用有较大的差距。技术实现要素：本发明旨在提供一种转氨酶突变体及其应用，以解决现有技术中野生型的转氨酶不适合应用于工业化生产的技术问题。为了实现上述目的，根据本发明的一个方面，提供了一种转氨酶突变体。该转氨酶突变体的氨基酸序列是由seqidno：1所示的氨基酸序列发生突变得到的氨基酸序列，突变至少包括如下突变位点之一：第19位、第41位、第43位、第72位、第76位、第92位、第107位、第125位、第132位和第226位、第292位、第295位，第308位、第332位；且第19位突变为丝氨酸，第41位突变为丝氨酸，第43位突变为天冬酰胺，第72位的甘氨酸突变为亮氨酸；第76位的亮氨酸突变为丙氨酸；第92位的赖氨酸突变为甘氨酸，第107位的亮氨酸突变为异亮氨酸；第125位的丝氨酸突变为丙氨酸；第132位的丝氨酸突变为丙氨酸；第226位的丙氨酸突变为甘氨酸；第292位的缬氨酸突变为半胱氨酸，第295位的丙氨酸突变为甘氨酸，第308位突变为丝氨酸，第332位突变为丝氨酸；或者转氨酶突变体的氨基酸序列具有发生突变的氨基酸序列中的突变位点，且与发生突变的氨基酸序列具有80％以上同源性的氨基酸序列。进一步地，转氨酶突变体的氨基酸序列为seqidno：2、seqidno：3、seqidno：4、seqidno：5、seqidno：6、seqidno：7、seqidno：8、seqidno：9、seqidno：10、seqidno：11、seqidno：12、seqidno：13、seqidno：14、seqidno：15、seqidno：16、seqidno：17、seqidno：18、seqidno：19、seqidno：20、seqidno：21、seqidno：22、seqidno：23、seqidno：24、seqidno：25、seqidno：26、seqidno：27、seqidno：28、seqidno：29或seqidno：30所示的氨基酸序列；或者转氨酶突变体的氨基酸序列为与seqidno：2、seqidno：3、seqidno：4、seqidno：5、seqidno：6、seqidno：7、seqidno：8、seqidno：9、seqidno：10、seqidno：11、seqidno：12、seqidno：13、seqidno：14、seqidno：15、seqidno：16、seqidno：17、seqidno：18、seqidno：19、seqidno：20、seqidno：21、seqidno：22、seqidno：23、seqidno：24、seqidno：25、seqidno：26、seqidno：27、seqidno：28、seqidno：29或seqidno：30所示的氨基酸序列具有95％以上同源性的氨基酸序列。根据本发明的另一个方面，提供了一种dna分子。该dna分子编码上述任一种转氨酶突变体。进一步地，dna分子的序列为seqidno：32、seqidno：33、seqidno：34、seqidno：35、seqidno：36、seqidno：37、seqidno：38、seqidno：39、seqidno：40、seqidno：41、seqidno：42、seqidno：43、seqidno：44、seqidno：45、seqidno：46、seqidno：47、seqidno：48、seqidno：49、seqidno：50、seqidno：51、seqidno：52、seqidno：53、seqidno：54、seqidno：55、seqidno：56、seqidno：57、seqidno：58、seqidno：59或seqidno：60所示序列；或者dna分子的序列为与seqidno：32、seqidno：33、seqidno：34、seqidno：35、seqidno：36、seqidno：37、seqidno：38、seqidno：39、seqidno：40、seqidno：41、seqidno：42、seqidno：43、seqidno：44、seqidno：45、seqidno：46、seqidno：47、seqidno：48、seqidno：49、seqidno：50、seqidno：51、seqidno：52、seqidno：53、seqidno：54、seqidno：55、seqidno：56、seqidno：57、seqidno：58、seqidno：59或seqidno：60具有95％以上同源性的序列。根据本发明的一个方面，提供了一种重组质粒。该重组质粒含有上述任一种dna分子。进一步地，重组质粒为pet-22b(+)、pet-22b(+)、pet-3a(+)、pet-3d(+)、pet-11a(+)、pet-12a(+)、pet-14b(+)、pet-15b(+)、pet-16b(+)、pet-17b(+)、pet-19b(+)、pet-20b(+)、pet-21a(+)、pet-23a(+)、pet-23b(+)、pet-24a(+)、pet-25b(+)、pet-26b(+)、pet-27b(+)、pet-28a(+)、pet-29a(+)、pet-30a(+)、pet-31b(+)、pet-32a(+)、pet-35b(+)、pet-38b(+)、pet-39b(+)、pet-40b(+)、pet-41a(+)、pet-41b(+)、pet-42a(+)、pet-43a(+)、pet-43b(+)、pet-44a(+)、pet-49b(+)、pqe2、pqe9、pqe30、pqe31、pqe32、pqe40、pqe70、pqe80、prset-a、prset-b、prset-c、pgex-5x-1、pgex-6p-1、pgex-6p-2、pbv220、pbv221、pbv222、ptrc99a、ptwin1、pezz18、pkk232-18、puc-18或puc-19。根据本发明的一个方面，提供了一种宿主细胞。该宿主细胞含有上述任一种重组质粒。进一步地，宿主细胞包括原核细胞、酵母或真核细胞；优选原核细胞为大肠杆菌bl21细胞或大肠杆菌dh5α感受态细胞。根据本发明的一个方面，提供了一种生产手性胺的方法。该方法包括转氨酶对酮类化合物及氨基供体进行催化转氨基反应的步骤，转氨酶为上述任一种转氨酶突变体。所述酮类化合物为其中，r1和r2各自独立地为c1～c8烷基、c5～c10环烷基、c5～c10芳基或c5～c10杂芳基，或者r1和r2与羰基上的碳共同形成c5～c10杂环基、c5～c10碳环基或c5～c10杂芳基，所述c5～c10杂环基和c5～c10杂芳基中的杂原子各自独立地选自氮、氧和硫中的至少一种，所述c5～c10芳基中的芳基、c5～c10杂芳基中的杂芳基、c5～c10碳环基中的碳环基或c5～c10杂环基中的杂环基各自独立地未被取代或被卤素、烷氧基或烷基中的至少一个基团所取代，优选地，所述酮类化合物为转氨基反应产物为更优选的，酮类化合物为转氨基反应产物为优选的，氨基供体为异丙胺。本发明的上述转氨酶突变体是在seqidno：1所示的转氨酶的基础上，通过定点突变的方法进行突变，从而改变其氨基酸序列，实现蛋白质结构和功能的改变，再通过定向筛选的方法，得到具有上述突变位点的转氨酶，本发明的转氨酶突变体具有酶活性大幅度提高的优势，其酶活相对于转氨酶母本提高了多倍，并且酶特异性也有相应提高，从而大幅度降低了手性胺工业生产中的成本。具体实施方式需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将结合实施例来详细说明本发明。来源于放线菌actinobacteriasp.的氨基转移酶(又称转氨酶)可以高选择性的催化的转化，但是其活性较低，稳定性较差，反应时添加的酶量较多。本发明的发明人通过定向进化的方法提高来源于actinobacteriasp.的氨基转移酶的活性与稳定性，降低酶的使用量。首先通过全质粒pcr的方式在来源于actinobacteriasp.的氨基转移酶上引入突变位点，对突变体进行活性和稳定性检测，挑选活性或稳定性提高的突变体。以来源于actinobacteriasp.的氨基转移酶为模版，设计了53对定点突变引物(p19s，f71y，g72y，g72l，h73t，l76a，l76v，v80t，k92g，k92d，l105i，l105m，l107i，f133m，n135t，n135s，t137s，g147f，y161s，i163c，i163v，a180l，s210i，s210v，t220l，t220a，a226c，a226g，n228g，p234s，s280l，y284l，t293s，a295g，p308s，v317i，p332s，v31y，e41s，s43n，g101s，k121v，r122d，c123m，s125a，s132a，s156n，h185t，a208r，k245v，l260k，f273v，v292c)利用定点突变手段，以pet-22b(+)为表达载体，获得带有目的基因的突变质粒。其中，定点突变：是指通过聚合酶链式反应(pcr)等方法向目的dna片段(可以是基因组，也可以是质粒)中引入所需变化(通常是表征有利方向的变化)，包括碱基的添加、删除、点突变等。定点突变能迅速、高效的提高dna所表达的目的蛋白的性状及表征，是基因研究工作中一种非常有用的手段。利用全质粒pcr引入定点突变的方法简单有效，是目前使用比较多的手段。其原理是，一对包含突变位点的引物(正、反向)，和模版质粒退火后用聚合酶“循环延伸”，所谓的循环延伸是指聚合酶按照模版延伸引物，一圈后回到引物5’端终止，再经过反复加热退火延伸的循环，这个反应区别于滚环扩增，不会形成多个串联拷贝。正反向引物的延伸产物退火后配对成为带缺刻的开环质粒。dpni酶切延伸产物，由于原来的模版质粒来源于常规大肠杆菌，是经dam甲基化修饰的，对dpni敏感而被切碎，而体外合成的带突变序列的质粒由于没有甲基化而不被切开，因此在随后的转化中得以成功转化，即可得到突变质粒的克隆。上述得将突变质粒转化至大肠杆菌细胞内，在大肠杆菌中过量表达。然后通过超声破碎细胞的方法获得粗酶。转氨酶诱导表达最佳条件：25℃，0.1mmiptg诱导过夜。通过采用软件对转氨酶的三维结构进行计算机模拟分析，g72l位于活性口袋附近，并且在蛋白二聚体界面附近，增强了蛋白二聚体的结合能力，使蛋白更稳定。v292c处于辅因子plp的结合区域，又可能增强了plp的结合能力。a226g，a295g处于底物结合口袋附近，有可能影响底物的朝向，增强催化效率。根据本发明一种典型的实施方式，提供一种转氨酶突变体。该转氨酶突变体的氨基酸序列是由seqidno：1所示的氨基酸序列发生突变得到的氨基酸序列，突变至少包括如下突变位点之一：第19位、第41位、第43位、第72位、第76位、第92位、第107位、第125位、第132位和第226位、第292位、第295位，第308位、第332位；且第19位突变为丝氨酸，第41位突变为丝氨酸，第43位突变为天冬酰胺，第72位的甘氨酸突变为亮氨酸；第76位的亮氨酸突变为丙氨酸；第92位的赖氨酸突变为甘氨酸，第107位的亮氨酸突变为异亮氨酸；第125位的丝氨酸突变为丙氨酸；第132位的丝氨酸突变为丙氨酸；第226位的丙氨酸突变为甘氨酸；第292位的缬氨酸突变为半胱氨酸，第295位的丙氨酸突变为甘氨酸，第308位突变为丝氨酸，第332位突变为丝氨酸；或者转氨酶突变体的氨基酸序列具有发生突变的氨基酸序列中的突变位点，且与发生突变的氨基酸序列具有80％以上同源性的氨基酸序列。本文使用的术语“同源性”具有本领域通常已知的含义，本领域技术人员也熟知测定不同序列间同源性的规则、标准。本发明用不同程度同源性限定的序列还必须要同时具有改进的转氨酶活性。本领域技术人员可以在本申请公开内容的教导下获得转氨酶突变体的氨基酸序列具有上述发生突变的氨基酸序列中的突变位点，且与发生突变的氨基酸序列具有80％以上同源性的氨基酸序列。优选的，转氨酶突变体的氨基酸序列为seqidno：2、seqidno：3、seqidno：4、seqidno：5、seqidno：6、seqidno：7、seqidno：8、seqidno：9、seqidno：10、seqidno：11、seqidno：12、seqidno：13、seqidno：14、seqidno：15、seqidno：16、seqidno：17、seqidno：18、seqidno：19、seqidno：20、seqidno：21、seqidno：22、seqidno：23、seqidno：24、seqidno：25、seqidno：26、seqidno：27、seqidno：28、seqidno：29或seqidno：30所示的氨基酸序列；或者转氨酶突变体的氨基酸序列为与seqidno：2、seqidno：3、seqidno：4、seqidno：5、seqidno：6、seqidno：7、seqidno：8、seqidno：9、seqidno：10、seqidno：11、seqidno：12、seqidno：13、seqidno：14、seqidno：15、seqidno：16、seqidno：17、seqidno：18、seqidno：19、seqidno：20、seqidno：21、seqidno：22、seqidno：23、seqidno：24、seqidno：25、seqidno：26、seqidno：27、seqidno：28、seqidno：29或seqidno：30所示的氨基酸序列具有95％以上同源性的氨基酸序列。本文使用的术语“同源性”具有本领域通常已知的含义，本领域技术人员也熟知测定不同序列间同源性的规则、标准。本发明用不同程度同源性限定的序列还必须要同时具有改进的转氨酶活性。在上述实施方式中，优选转氨酶突变体的氨基酸序列与seqidno：2、seqidno：3、seqidno：4、seqidno：5、seqidno：6、seqidno：7、seqidno：8、seqidno：9、seqidno：10、seqidno：11、seqidno：12、seqidno：13、seqidno：14、seqidno：15、seqidno：16、seqidno：17、seqidno：18、seqidno：19、seqidno：20、seqidno：21、seqidno：22、seqidno：23、seqidno：24、seqidno：25、seqidno：26、seqidno：27、seqidno：28、seqidno：29或seqidno：30具有95％以上的同源性并具有或编码具有改进的转氨酶活性的氨基酸序列。本领域技术人员可以在本申请公开内容的教导下获得这样的变体序列。本发明的上述转氨酶突变体是在seqidno：1(编码dna为seqidno：31)所示的转氨酶的基础上，通过定点突变的方法进行突变，从而改变其氨基酸序列，实现蛋白质结构和功能的改变，再通过定向筛选的方法，得到具有上述突变位点的转氨酶，本发明的转氨酶突变体具有酶活性大幅度提高的优势，其酶活相对于转氨酶母本提高了6倍，并且酶特异性也有相应提高，从而大幅度降低了手性胺工业生产中的成本。根据本发明一种典型的实施方式，来源于actinobacteriasp.的转氨酶第72位由甘氨酸突变为亮氨酸；或第76位由亮氨酸突变为丙氨酸；或第107位由亮氨酸突变为异亮氨酸；或第125位由丝氨酸突变为丙氨酸；或第132位由丝氨酸突变为丙氨酸；或第76位由亮氨酸突变为丙氨酸、第125位由丝氨酸突变为丙氨酸以及第226位由丙氨酸突变为甘氨酸；或第76位由亮氨酸突变为丙氨酸、第107位由亮氨酸突变为异亮氨酸、第125位由丝氨酸突变为丙氨酸以及第226位由丙氨酸突变为甘氨酸；或第76位由亮氨酸突变为丙氨酸、第125位由丝氨酸突变为丙氨酸、第132位由丝氨酸突变为丙氨酸以及第226位由丙氨酸突变为甘氨酸；或第76位由亮氨酸突变为丙氨酸、第107位由亮氨酸突变为异亮氨酸、第125位由丝氨酸突变为丙氨酸、第132位由丝氨酸突变为丙氨酸以及第226位由丙氨酸突变为甘氨酸。根据本发明一种典型的实施方式，提供一种dna分子。该dna分子编码上述转氨酶突变体。优选的，dna分子的序列为seqidno：32、seqidno：33、seqidno：34、seqidno：35、seqidno：36、seqidno：37、seqidno：38、seqidno：39、seqidno：40、seqidno：41、seqidno：42、seqidno：43、seqidno：44、seqidno：45、seqidno：46、seqidno：47、seqidno：48、seqidno：49、seqidno：50、seqidno：51、seqidno：52、seqidno：53、seqidno：54、seqidno：55、seqidno：56、seqidno：57、seqidno：58、seqidno：59或seqidno：60所示序列；或者dna分子的序列为与seqidno：32、seqidno：33、seqidno：34、seqidno：35、seqidno：36、seqidno：37、seqidno：38、seqidno：39、seqidno：40、seqidno：41、seqidno：42、seqidno：43、seqidno：44、seqidno：45、seqidno：46、seqidno：47、seqidno：48、seqidno：49、seqidno：50、seqidno：51、seqidno：52、seqidno：53、seqidno：54、seqidno：55、seqidno：56、seqidno：57、seqidno：58、seqidno：59或seqidno：60具有95％以上同源性的序列。上述dna编码得到的转氨酶，提高了酶活性和酶的稳定性，在手性胺的工业生产中可以减少加入的酶量，降低后处理难度。本发明的上述dna分子还可以以“表达盒”的形式存在。“表达盒”是指线性或环状的核酸分子，涵盖了能够指导特定核苷酸序列在恰当宿主细胞中表达的dna和rna序列。一般而言，包括与目标核苷酸有效连接的启动子，其任选的是与终止信号和/或其他调控元件有效连接的。表达盒还可以包括核苷酸序列正确翻译所需的序列。编码区通常编码目标蛋白，但在正义或反义方向也编码目标功能rna，例如反义rna或非翻译的rna。包含目标多核苷酸序列的表达盒可以是嵌合的，意指至少一个其组分与其至少一个其他组分是异源的。表达盒还可以是天然存在的，但以用于异源表达的有效重组形成获得的。根据本发明一种典型的实施方式，提供一种重组质粒。该重组质粒含有上述任一种dna分子。上述重组质粒中的dna分子置于重组质粒的适当位置，使得上述dna分子能够正确地、顺利地复制、转录或表达。虽然本发明在限定上述dna分子时所用限定语为“含有”，但其并不意味着可以在dna序列的两端任意加入与其功能不相关的其他序列。本领域技术人员知晓，为了满足重组操作的要求，需要在dna序列的两端添加合适的限制性内切酶的酶切位点，或者额外增加启动密码子、终止密码子等，因此，如果用封闭式的表述来限定将不能真实地覆盖这些情形。本发明中所使用的术语“质粒”包括双链或单链线状或环状形式的任何质粒、粘粒、噬菌体或农杆菌二元核酸分子，优选为重组表达质粒，可以是原核表达质粒也可以是真核表达质粒，但优选原核表达质粒，在某些实施方案中，重组质粒选自pet-22b(+)、pet-22b(+)、pet-3a(+)、pet-3d(+)、pet-11a(+)、pet-12a(+)、pet-14b(+)、pet-15b(+)、pet-16b(+)、pet-17b(+)、pet-19b(+)、pet-20b(+)、pet-21a(+)、pet-23a(+)、pet-23b(+)、pet-24a(+)、pet-25b(+)、pet-26b(+)、pet-27b(+)、pet-28a(+)、pet-29a(+)、pet-30a(+)、pet-31b(+)、pet-32a(+)、pet-35b(+)、pet-38b(+)、pet-39b(+)、pet-40b(+)、pet-41a(+)、pet-41b(+)、pet-42a(+)、pet-43a(+)、pet-43b(+)、pet-44a(+)、pet-49b(+)、pqe2、pqe9、pqe30、pqe31、pqe32、pqe40、pqe70、pqe80、prset-a、prset-b、prset-c、pgex-5x-1、pgex-6p-1、pgex-6p-2、pbv220、pbv221、pbv222、ptrc99a、ptwin1、pezz18、pkk232-18、puc-18或puc-19。更优选，上述重组质粒是pet-22b(+)。根据本发明一种典型的实施方式，提供一种宿主细胞，宿主细胞含有上述任一种重组质粒。适用于本发明的宿主细胞包括但不仅限于原核细胞、酵母或真核细胞。优选原核细胞为真细菌，例如革兰氏阴性菌或革兰氏阳性菌。更优选原核细胞为大肠杆菌bl21细胞或大肠杆菌dh5α感受态细胞。根据本发明一种典型的实施方式，提供一种生产手性胺的方法。该方法包括转氨酶对酮类化合物及氨基供体进行催化转氨基反应的步骤，转氨酶为上述任一种转氨酶突变体。由于本发明的上述转氨酶突变体具有更高的酶催化活性，因而利用本发明的转氨酶突变体制备的手性胺不仅能够降低生产成本，而且所获得的手性胺ee值大于99％，de值98％。根据本发明一种典型的实施方式，所述酮类化合物为其中，r1和r2各自独立地为c1～c8烷基、c5～c10环烷基、c5～c10芳基或c5～c10杂芳基，或者r1和r2与羰基上的碳共同形成c5～c10杂环基、c5～c10碳环基或c5～c10杂芳基，所述c5～c10杂环基和c5～c10杂芳基中的杂原子各自独立地选自氮、氧和硫中的至少一种，所述c5～c10芳基中的芳基、c5～c10杂芳基中的杂芳基、c5～c10碳环基中的碳环基或c5～c10杂环基中的杂环基各自独立地未被取代或被卤素、烷氧基或烷基中的至少一个基团所取代，优选地，所述酮类化合物为转氨基反应产物为更优选的，酮类化合物为转氨基反应产物为优选的，氨基供体为异丙胺。下面将结合实施例进一步说明本发明的有益效果。本领域技术人员公知，在不背离本发明精神的情况下，可以对本发明做出许多修改，这样的修改也落入本发明的范围。且下述实验方法如无特别说明，均为常规方法，所使用的实验材料如无特别说明，均可容易地从商业公司获取。下述实施例中提提到的主原料为：主原料1：(s)-2-甲基-4-氧代哌啶-1-羧酸叔丁酯(tert-butyl(2s)-2-methyl-4-oxopiperidine-1-carboxylate)，cas790667-43-5。主原料2：(s)-3-甲基-4-氧代哌啶-1-羧酸叔丁酯((s)-tert-butyl3-methyl-4-oxopiperidine-1-carboxylate)。主原料3：n-叔丁氧羰基-3-哌啶酮(n-boc-3-piperidone)，cas98977-36-7。实施例110ml的反应瓶中，将40mg异丙胺加入1ml0.2m磷酸盐缓冲液中，调ph为7.0～7.5,加入转氨酶seqidno：1质量为120mg，加入0.4mg磷酸吡哆醛，混匀后滴入溶于0.2mldmso的40mg体系ph为7.0～7.5，35℃±3℃恒温搅拌16h。体系经gc检测转化率。按照上述步骤依次进行序列编号为seqidno：13～26的突变体转氨酶反应数据如下表1：表1序列编号突变位点16h酶用量seqidno：1无21.3％3wtseqidno：13g72l36.8％2wtseqidno：14l76a47.1％2wtseqidno：15l107i47.7％2wtseqidno：16s125a38.9％2wtseqidno：17s132a52.8％2wtseqidno：18k92g39.5％2wtseqidno：19a226g37.6％2wtseqidno：20v292c42.1％2wtseqidno：21a295g44.2％2wtseqidno：22p19s40.1％2wtseqidno：23e41s41.6％2wtseqidno：24s43n39.6％2wtseqidno：25p308s42.4％2wtseqidno：26p332s45.7％2wt其中，16h指的是反应16h，百分数指的是转化率，2wt指的是加入酶的重量是底物重量的2倍。单点突变体的转化效果较母本有所提高，但并未达到理想的效果。实施例2一般而言，单点突变的突变体性能较母本很难有较大差异，突变点的组合可以获得更优的突变体。因此通过dnashuffling的方法将突变位点进行随机重组，建立突变文库，然后进行筛选，试图拿到更优的突变体。dnashuffling是基因在分子水平上进行的有性重组。将一组同源基因用核酸酶i消化成随机片段，由这些随机片段组成文库，互为引物和模板进行pcr扩增。当一个基因拷贝片段作为另一基因拷贝的引物时，发生模板互换和基因重组。酶液制备方法：96孔板离心去掉上清培养基，每孔加入200μl酶解溶液(溶菌酶2mg/ml，多黏菌素0.5mg/ml，ph＝7.0)，37℃保温破碎3h。高通量筛选方法：250μl测活体系：终浓度0.27mg/ml，4-硝基苯乙胺盐酸盐终浓度12.24mg/ml，plp终浓度0.01mg/ml，破碎酶液加入量45μl，ph＝8.0，温度30℃。筛选得到的突变体进行摇瓶培养，然后进行放大反应。转氨酶诱导表达最佳条件：25℃，0.1mmiptg诱导过夜。将seqidno：1和制备得到的多位点突变体seqidno：2～12和27～30进行如下反应：10ml的反应瓶中，将40mg异丙胺加入1ml0.2m磷酸盐缓冲液中，调ph为7.0～7.5,加入转氨酶质量为40mg、0.4mg磷酸吡哆醛，混匀后滴入溶于0.2mldmso的40mg主原料1，体系ph为7.0～7.5，35℃±3℃恒温搅拌16h～64h。体系经gc检测转化率，突变体反应数据如下表2：表2序列编号突变位点16h64h酶用量seqidno：1无21.3％35.7％3wtseqidno：2l76a+r130a+v292c+a295g38.5％84.1％1wtseqidno：3l107i+a295g42.1％87.2％1wtseqidno：4s43n+g72l+a295g52.1％87.3％1wtseqidno：5d96n+l104i+a295g47.4％84.2％1wtseqidno：6l76a+s125a+a226g+a295g63.6％92.5％1wtseqidno：7l76a+s125a+a226g+s132a+a295g65.8％96.8％1wtseqidno：8s132a+a295g60.5％89.3％1wtseqidno：9g72l+a295g45.2％88.1％1wtseqidno：10g72l+a295g+s132a47.8％91.3％1wtseqidno：11g72l+v292c+a295g55.4％86.5％1wtseqidno：12g72l+l76a+v292c+a295g64.7％94.3％1wtseqidno：27l76a+l107i+p110q37.2％65.3％1wtseqidno：28l76a+s125a+a226g41.8％78.6％1wtseqidno：29l76a+t79s+a295g47.5％85.6％1wtseqidno：30l76a+l107i+p110q+d150g36.3％69.7％1wt实施例3取两份转氨酶seqidno：1质量都为40mg，其中一份放在4℃放置2h，另一份在50℃保温2h，然后按照如下体系进行反应。10ml的反应瓶中，将40mg异丙胺加入1ml0.2m磷酸盐缓冲液中，调ph为7.0～7.5,加入转氨酶seqidno：7质量为40mg、0.4mg磷酸吡哆醛，混匀后滴入溶于0.2mldmso的40mg主原料1，体系ph为7.0～7.5，35℃±3℃恒温搅拌16h。体系经gc检测转化率，突变体稳定性表示为50℃保温的转氨酶转化率占4℃保温的转氨酶转化率的百分比。seqidno：1～30的稳定性数据如下表3：表3实施例41000ml的反应瓶中，将4g异丙胺加入100ml0.2m磷酸盐缓冲液中，调ph为7.0～7.5,加入转氨酶seqidno：7冻干粉质量为0.4g、40mg磷酸吡哆醛，混匀后滴入溶于20mldmso的4g主原料1，体系ph为7.0～7.5，35℃±3℃恒温搅拌64h。hplc检测转化率98.2％，反应结束后，加入100ml的乙酸乙酯进行萃取3次，合并萃取有机相后加入硫酸镁进行干燥，旋转蒸发至干，得产品3.62g，收率90.2％，e.e.值99％。按照上述步骤依次进行序列编号为seqidno：6、7、10、12的突变体转氨酶反应数据如下表4：表4序列编号突变位点收率e.e.seqidno：6l76a+s125a+a226g+a295g89.5％99％seqidno：7l76a+s125a+a226g+s132a+a295g90.2％99％seqidno：10g72l+a295g+s132a88.9％99％seqidno：12g72l+l76a+v292c+a295g89.3％99％实施例51000ml的反应瓶中，将4g异丙胺加入100ml0.2m磷酸盐缓冲液中，调ph为7.0～7.5,加入转氨酶seqidno：7冻干粉质量为0.4g、40mg磷酸吡哆醛，混匀后滴入溶于20mldmso的4g主原料2，体系ph为7.0～7.5，35℃±3℃恒温搅拌64h。hplc检测转化率99.1％，反应结束后，加入100ml的乙酸乙酯进行萃取3次，合并萃取有机相后加入硫酸镁进行干燥，旋转蒸发至干，得产品3.75g，收率93.8％，e.e.值99％。实施例61000ml的反应瓶中，将4g异丙胺加入100ml0.2m磷酸盐缓冲液中，调ph为7.0～7.5,加入转氨酶seqidno：7冻干粉质量为0.2g、40mg磷酸吡哆醛，混匀后滴入溶于20mldmso的4g主原料3，体系ph为7.0～7.5，35℃±3℃恒温搅拌64h。hplc检测转化率99.5％，反应结束后，加入100ml的乙酸乙酯进行萃取3次，合并萃取有机相后加入硫酸镁进行干燥，旋转蒸发至干，得产品3.79g，收率94.8％，e.e.值99％。从以上的描述中，可以看出，本发明上述的实施例实现了如下技术效果：本发明的上述转氨酶突变体是在seqidno：1所示的转氨酶的基础上，通过定点突变的方法进行突变，从而改变其氨基酸序列，实现蛋白质结构和功能的改变，再通过定向筛选的方法，得到具有上述突变位点的转氨酶，本发明的转氨酶突变体具有酶活性大幅度提高的优势，并且酶特异性也有相应提高，从而大幅度降低了手性胺工业生产中的成本。以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。序列表<110>凯莱英生命科学技术（天津）有限公司<120>转氨酶突变体及其应用<130>pn74794kly<160>60<170>siposequencelisting1.0<210>1<211>341<212>prt<213>放线菌(actinobacteriasp.)<400>1metthrileserlysaspileasptyrserthrserasnleuvalser151015valalaproglyalailearggluprothrproalaglyservalile202530glntyrserasptyrgluleuaspgluserserprophealaglygly354045alaalatrpilegluglyglutyrvalproalaalaglualaargile505560serleupheaspthrglypheglyhisseraspleuthrtyrthrval65707580alahisvaltrphisglyasnilepheargleulysasphisileasp859095argvalpheaspglyalaglnlysleuargleuglnserproleuthr100105110lysalagluvalgluaspilethrlysargcysvalserleusergln115120125leuarggluserphevalasnilethrilethrargglytyrglyala130135140arglysglyglulysaspleuserlysleuthrserglniletyrile145150155160tyralaileprotyrleutrpalapheproproglugluglnilephe165170175glythrseralailevalproarghisvalargargalaglyargasn180185190thrvalaspprothrvallysasntyrglntrpglyaspleuthrala195200205alaserpheglualalysaspargglyalaargthralaileleuleu210215220aspalaaspasncysvalalagluglyproglypheasnvalvalmet225230235240vallysaspglylysleuserserproserargasnalaleuprogly245250255ilethrargleuthrvalmetglumetalaaspglumetglyileglu260265270phethrleuargaspilethrserarggluleutyrglualaaspglu275280285leuilealavalthrthralaglyglyilethrproilethrserleu290295300aspglygluproleuglyaspglythrproglyprovalthrvalala305310315320ileargaspargphetrpalametmetaspgluproserserleuval325330335glualaileglutyr340<210>2<211>341<212>prt<213>放线菌(actinobacteriasp.)<400>2metthrileserlysaspileasptyrserthrserasnleuvalser151015valalaprog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aspargphetrpalametmetaspgluproserserleuval325330335glualaileglutyr340<210>30<211>341<212>prt<213>放线菌(actinobacteriasp.)<400>30metthrileserlysaspileasptyrserthrserasnleuvalser151015valalaproglyalailearggluprothrproalaglyservalile202530glntyrserasptyrgluleuaspgluserserprophealaglygly354045alaalatrpilegluglyglutyrvalproalaalaglualaargile505560serleupheaspthrglypheglyhisseraspalathrtyrthrval65707580alahisvaltrphisglyasnilepheargleulysasphisileasp859095argvalpheaspglyalaglnlysleuargileglnserglnleuthr100105110lysalagluvalgluaspilethrlysargcysvalserleusergln115120125leuarggluserphevalasnilethrilethrargglytyrglyala130135140arglysglyglulysglyleuserlysleuthrserglniletyrile145150155160tyralaileprotyrleutrpalapheproproglugluglnilephe165170175glythrseralailevalproarghisvalargargalaglyargasn180185190thrvalaspprothrvallysasntyrglntrpglyaspleuthrala195200205alaserpheglualalysaspargglyalaargthralaileleuleu210215220aspalaaspasncysvalalagluglyproglypheasnvalvalmet225230235240vallysaspglylysleuserserproserargasnalaleuprogly245250255ilethrargleuthrvalmetglumetalaaspglumetglyileglu260265270phethrleuargaspilethrserarggluleutyrglualaaspglu275280285leuilealavalthrthralaglyglyilethrproilethrserleu290295300aspglygluproleuglyaspglythrproglyprovalthrvalala305310315320ileargaspargphetrpalametmetaspgluproserserleuval325330335glualaileglutyr340<210>31<211>1026<212>dna<213>放线菌(actinobacteriasp.)<400>31atgaccattagcaaagacattgactatagcaccagcaacctggtgagtgtggccccgggt60gcaatccgtgaacctaccccggcaggcagcgtgatccagtacagtgactacgagctggat120gaaagcagcccgtttgccggtggtgcagcctggattgaaggtgagtatgttccggcagca180gaggcccgtattagcctgtttgataccggcttcggccatagcgatctgacctacaccgtt240gcccatgtttggcacggcaacatctttcgcctgaaagaccacattgaccgcgtgtttgat300ggcgcccagaaactgcgtctgcagagcccgctgaccaaggccgaagtggaggatattacc360aaacgctgcgtgagcctgagtcagctgcgcgagagcttcgtgaacatcaccattacccgc420ggttatggcgcccgcaaaggcgagaaagatctgagcaaattaaccagccagatctacatc480tacgccatcccgtacctgtgggcctttcctccggaagagcagatcttcggtacaagtgcc540attgtgccgcgtcatgttcgtcgcgcaggccgtaataccgttgatcctaccgttaagaac600taccagtggggtgatctgaccgcagcttcttttgaagcaaaagatcgtggcgcccgcacc660gcaatcctgctggatgcagacaactgtgtggccgagggtccgggctttaacgtggtgatg720gtgaaggatggcaaactgagtagcccgagccgtaatgccctgccgggtattacacgtctg780accgtgatggagatggccgatgaaatgggcatcgaattcaccctgcgcgatatcaccagc840cgtgagttatatgaggccgacgaactgatcgccgtgaccaccgcaggtggcattaccccg900attaccagtctggatggcgaaccgctgggcgatggtacccctggtcctgtgacagtggcc960attcgcgatcgcttttgggccatgatggatgagccgagcagtctggtggaggccattgaa1020tattaa1026<210>32<211>1026<212>dna<213>放线菌(actinobacteriasp.)<400>32atgaccattagcaaagacattgactatagcaccagcaacctggtgagtgtggccccgggt60gcaatccgtgaacctaccccggcaggcagcgtgatccagtacagtgactacgagctggat120gaaagcagcccgtttgccggtggtgcagcctggattgaaggtgagtatgttccggcagca180gaggcccgtattagcctgtttgataccggcttcggccatagcgatgcgacctacaccgtt240gcccatgtttggcacggcaacatctttcgcctgaaagaccacattgaccgcgtgtttgat300ggcgcccagaaactgcgtctgcagagcccgctgaccaaggccgaagtggaggatattacc360aaacgctgcgtgagcctgagtcagctggccgagagcttcgtgaacatcaccattacccgc420ggttatggcgcccgcaaaggcgagaaagatctgagcaaattaaccagccagatctacatc480tacgccatcccgtacctgtgggcctttcctccggaagagcagatcttcggtacaagtgcc540attgtgccgcgtcatgttcgtcgcgcaggccgtaataccgttgatcctaccgttaagaac600taccagtggggtgatctgaccgcagcttcttttgaagcaaaagatcgtggcgcccgcacc660gcaatcctgctggatgcagacaactgtgtggccgagggtccgggctttaacgtggtgatg720gtgaaggatggcaaactgagtagcccgagccgtaatgccctgccgggtattacacgtctg780accgtgatggagatggccgatgaaatgggcatcgaattcaccctgcgcgatatcaccagc840cgtgagttatatgaggccgacgaactgatcgcctgcaccaccggaggtggcattaccccg900attaccagtctggatggcgaaccgctgggcgatggtacccctggtcctgtgacagtggcc960attcgcgatcgcttttgggccatgatggatgagccgagcagtctggtggaggccattgaa1020tattaa1026<210>33<211>1026<212>dna<213>放线菌(actinobacteriasp.)<400>33atgaccattagcaaagacattgactatagcaccagcaacctggtgagtgtggccccgggt60gcaatccgtgaacctaccccggcaggcagcgtgatccagtacagtgactacgagctggat120gaaagcagcccgtttgccggtggtgcagcctggattgaaggtgagtatgttccggcagca180gaggcccgtattagcctgtttgataccggcttcggccatagcgatctgacctacaccgtt240gcccatgtttggcacggcaacatctttcgcctgaaagaccacattgaccgcgtgtttgat300ggcgcccagaaactgcgtatacagagcccgctgaccaaggccgaagtggaggatattacc360aaacgctgcgtgagcctgagtcagctgcgcgagagcttcgtgaacatcaccattacccgc420ggttatggcgcccgcaaaggcgagaaagatctgagcaaattaaccagccagatctacatc480tacgccatcccgtacctgtgggcctttcctccggaagagcagatcttcggtacaagtgcc540attgtgccgcgtcatgttcgtcgcgcaggccgtaataccgttgatcctaccgttaagaac600taccagtggggtgatctgaccgcagcttcttttgaagcaaaagatcgtggcgcccgcacc660gcaatcctgctggatgcagacaactgtgtggccgagggtccgggctttaacgtggtgatg720gtgaaggatggcaaactgagtagcccgagccgtaatgccctgccgggtattacacgtctg780accgtgatggagatggccgatgaaatgggcatcgaattcaccctgcgcgatatcaccagc840cgtgagttatatgaggccgacgaactgatcgccgtgaccaccggaggtggcattaccccg900attaccagtctggatggcgaaccgctgggcgatggtacccctggtcctgtgacagtggcc960attcgcgatcgcttttgggccatgatggatgagccgagcagtctggtggaggccattgaa1020tattaa1026<210>34<211>1026<212>dna<213>放线菌(actinobacteriasp.)<400>34atgaccattagcaaagacattgactatagcaccagcaacctggtgagtgtggccccgggt60gcaatccgtgaacctaccccggcaggcagcgtgatccagtacagtgactacgagctggat120gaaagcaacccgtttgccggtggtgcagcctggattgaaggtgagtatgttccggcagca180gaggcccgtattagcctgtttgataccggcttcctacatagcgatctgacctacaccgtt240gcccatgtttggcacggcaacatctttcgcctgaaagaccacattgaccgcgtgtttgat300ggcgcccagaaactgcgtctgcagagcccgctgaccaaggccgaagtggaggatattacc360aaacgctgcgtgagcctgagtcagctgcgcgagagcttcgtgaacatcaccattacccgc420ggttatggcgcccgcaaaggcgagaaagatctgagcaaattaaccagccagatctacatc480tacgccatcccgtacctgtgggcctttcctccggaagagcagatcttcggtacaagtgcc540attgtgccgcgtcatgttcgtcgcgcaggccgtaataccgttgatcctaccgttaagaac600taccagtggggtgatctgaccgcagcttcttttgaagcaaaagatcgtggcgcccgcacc660gcaatcctgctggatgcagacaactgtgtggccgagggtccgggctttaacgtggtgatg720gtgaaggatggcaaactgagtagcccgagccgtaatgccctgccgggtattacacgtctg780accgtgatggagatggccgatgaaatgggcatcgaattcaccctgcgcgatatcaccagc840cgtgagttatatgaggccgacgaactgatcgccgtgaccaccggaggtggcattaccccg900attaccagtctggatggcgaaccgctgggcgatggtacccctggtcctgtgacagtggcc960attcgcgatcgcttttgggccatgatggatgagccgagcagtctggtggaggccattgaa1020tattaa1026<210>35<211>1026<212>dna<213>放线菌(actinobacteriasp.)<400>35atgaccattagcaaagacattgactatagcaccagcaacctggtgagtgtggccccgggt60gcaatccgtgaacctaccccggcaggcagcgtgatccagtacagtgactacgagctggat120gaaagcagcccgtttgccggtggtgcagcctggattgaaggtgagtatgttccggcagca180gaggcccgtattagcctgtttgataccggcttcggccatagcgatctgacctacaccgtt240gcccatgtttggcacggcaacatctttcgcctgaaagaccacattaaccgcgtgtttgat300ggcgcccagatactgcgtctgcagagcccgctgaccaaggccgaagtggaggatattacc360aaacgctgcgtgagcctgagtcagctgcgcgagagcttcgtgaacatcaccattacccgc420ggttatggcgcccgcaaaggcgagaaagatctgagcaaattaaccagccagatctacatc480tacgccatcccgtacctgtgggcctttcctccggaagagcagatcttcggtacaagtgcc540attgtgccgcgtcatgttcgtcgcgcaggccgtaataccgttgatcctaccgttaagaac600taccagtggggtgatctgaccgcagcttcttttgaagcaaaagatcgtggcgcccgcacc660gcaatcctgctggatgcagacaactgtgtggccgagggtccgggctttaacgtggtgatg720gtgaaggatggcaaactgagtagcccgagccgtaatgccctgccgggtattacacgtctg780accgtgatggagatggccgatgaaatgggcatcgaattcaccctgcgcgatatcaccagc840cgtgagttatatgaggccgacgaactgatcgccgtgaccaccggaggtggcattaccccg900attaccagtctggatggcgaaccgctgggcgatggtacccctggtcctgtgacagtggcc960attcgcgatcgcttttgggccatgatggatgagccgagcagtctggtggaggccattgaa1020tattaa1026<210>36<211>1026<212>dna<213>放线菌(actinobacteriasp.)<400>36atgaccattagcaaagacattgactatagcaccagcaacctggtgagtgtggccccgggt60gcaatccgtgaacctaccccggcaggcagcgtgatccagtacagtgactacgagctggat120gaaagcagcccgtttgccggtggtgcagcctggattgaaggtgagtatgttccggcagca180gaggcccgtattagcctgtttgataccggcttcggccatagcgatgcgacctacaccgtt240gcccatgtttggcacggcaacatctttcgcctgaaagaccacattgaccgcgtgtttgat300ggcgcccagaaactgcgtctgcagagcccgctgaccaaggccgaagtggaggatattacc360aaacgctgcgtggccctgagtcagctgcgcgagagcttcgtgaacatcaccattacccgc420ggttatggcgcccgcaaaggcgagaaagatctgagcaaattaaccagccagatctacatc480tacgccatcccgtacctgtgggcctttcctccggaagagcagatcttcggtacaagtgcc540attgtgccgcgtcatgttcgtcgcgcaggccgtaataccgttgatcctaccgttaagaac600taccagtggggtgatctgaccgcagcttcttttgaagcaaaagatcgtggcgcccgcacc660gcaatcctgctggatgcggacaactgtgtggccgagggtccgggctttaacgtggtgatg720gtgaaggatggcaaactgagtagcccgagccgtaatgccctgccgggtattacacgtctg780accgtgatggagatggccgatgaaatgggcatcgaattcaccctgcgcgatatcaccagc840cgtgagttatatgaggccgacgaactgatcgccgtgaccaccggaggtggcattaccccg900attaccagtctggatggcgaaccgctgggcgatggtacccctggtcctgtgacagtggcc960attcgcgatcgcttttgggccatgatggatgagccgagcagtctggtggaggccattgaa1020tattaa1026<210>37<211>1026<212>dna<213>放线菌(actinobacteriasp.)<400>37atgaccattagcaaagacattgactatagcaccagcaacctggtgagtgtggccccgggt60gcaatccgtgaacctaccccggcaggcagcgtgatccagtacagtgactacgagctggat120gaaagcagcccgtttgccggtggtgcagcctggattgaaggtgagtatgttccggcagca180gaggcccgtattagcctgtttgataccggcttcggccatagcgatgcgacctacaccgtt240gcccatgtttggcacggcaacatctttcgcctgaaagaccacattgaccgcgtgtttgat300ggcgcccagaaactgcgtctgcagagcccgctgaccaaggccgaagtggaggatattacc360aaacgctgcgtggccctgagtcagctgcgcgaggccttcgtgaacatcaccattacccgc420ggttatggcgcccgcaaaggcgagaaagatctgagcaaattaaccagccagatctacatc480tacgccatcccgtacctgtgggcctttcctccggaagagcagatcttcggtacaagtgcc540attgtgccgcgtcatgttcgtcgcgcaggccgtaataccgttgatcctaccgttaagaac600taccagtggggtgatctgaccgcagcttcttttgaagcaaaagatcgtggcgcccgcacc660gcaatcctgctggatgcggacaactgtgtggccgagggtccgggctttaacgtggtgatg720gtgaaggatggcaaactgagtagcccgagccgtaatgccctgccgggtattacacgtctg780accgtgatggagatggccgatgaaatgggcatcgaattcaccctgcgcgatatcaccagc840cgtgagttatatgaggccgacgaactgatcgccgtgaccaccggaggtggcattaccccg900attaccagtctggatggcgaaccgctgggcgatggtacccctggtcctgtgacagtggcc960attcgcgatcgcttttgggccatgatggatgagccgagcagtctggtggaggccattgaa1020tattaa1026<210>38<211>1026<212>dna<213>放线菌(actinobacteriasp.)<400>38atgaccattagcaaagacattgactatagcaccagcaacctggtgagtgtggccccgggt60gcaatccgtgaacctaccccggcaggcagcgtgatccagtacagtgactacgagctggat120gaaagcagcccgtttgccggtggtgcagcctggattgaaggtgagtatgttccggcagca180gaggcccgtattagcctgtttgataccggcttcggccatagcgatctgacctacaccgtt240gcccatgtttggcacggcaacatctttcgcctgaaagaccacattgaccgcgtgtttgat300ggcgcccagaaactgcgtctgcagagcccgctgaccaaggccgaagtggaggatattacc360aaacgctgcgtgagcctgagtcagctgcgcgaggccttcgtgaacatcaccattacccgc420ggttatggcgcccgcaaaggcgagaaagatctgagcaaattaaccagccagatctacatc480tacgccatcccgtacctgtgggcctttcctccggaagagcagatcttcggtacaagtgcc540attgtgccgcgtcatgttcgtcgcgcaggccgtaataccgttgatcctaccgttaagaac600taccagtggggtgatctgaccgcagcttcttttgaagcaaaagatcgtggcgcccgcacc660gcaatcctgctggatgcagacaactgtgtggccgagggtccgggctttaacgtggtgatg720gtgaaggatggcaaactgagtagcccgagccgtaatgccctgccgggtattacacgtctg780accgtgatggagatggccgatgaaatgggcatcgaattcaccctgcgcgatatcaccagc840cgtgagttatatgaggccgacgaactgatcgccgtgaccaccggaggtggcattaccccg900attaccagtctggatggcgaaccgctgggcgatggtacccctggtcctgtgacagtggcc960attcgcgatcgcttttgggccatgatggatgagccgagcagtctggtggaggccattgaa1020tattaa1026<210>39<211>1026<212>dna<213>放线菌(actinobacteriasp.)<400>39atgaccattagcaaagacattgactatagcaccagcaacctggtgagtgtggccccgggt60gcaatccgtgaacctaccccggcaggcagcgtgatccagtacagtgactacgagctggat120gaaagcagcccgtttgccggtggtgcagcctggattgaaggtgagtatgttccggcagca180gaggcccgtattagcctgtttgataccggcttcctacatagcgatctgacctacaccgtt240gcccatgtttggcacggcaacatctttcgcctgaaagaccacattgaccgcgtgtttgat300ggcgcccagaaactgcgtctgcagagcccgctgaccaaggccgaagtggaggatattacc360aaacgctgcgtgagcctgagtcagctgcgcgagagcttcgtgaacatcaccattacccgc420ggttatggcgcccgcaaaggcgagaaagatctgagcaaattaaccagccagatctacatc480tacgccatcccgtacctgtgggcctttcctccggaagagcagatcttcggtacaagtgcc540attgtgccgcgtcatgttcgtcgcgcaggccgtaataccgttgatcctaccgttaagaac600taccagtggggtgatctgaccgcagcttcttttgaagcaaaagatcgtggcgcccgcacc660gcaatcctgctggatgcagacaactgtgtggccgagggtccgggctttaacgtggtgatg720gtgaaggatggcaaactgagtagcccgagccgtaatgccctgccgggtattacacgtctg780accgtgatggagatggccgatgaaatgggcatcgaattcaccctgcgcgatatcaccagc840cgtgagttatatgaggccgacgaactgatcgccgtgaccaccggaggtggcattaccccg900attaccagtctggatggcgaaccgctgggcgatggtacccctggtcctgtgacagtggcc960attcgcgatcgcttttgggccatgatggatgagccgagcagtctggtggaggccattgaa1020tattaa1026<210>40<211>1026<212>dna<213>放线菌(actinobacteriasp.)<400>40atgaccattagcaaagacattgactatagcaccagcaacctggtgagtgtggccccgggt60gcaatccgtgaacctaccccggcaggcagcgtgatccagtacagtgactacgagctggat120gaaagcagcccgtttgccggtggtgcagcctggattgaaggtgagtatgttccggcagca180gaggcccgtattagcctgtttgataccggcttcctacatagcgatctgacctacaccgtt240gcccatgtttggcacggcaacatctttcgcctgaaagaccacattgaccgcgtgtttgat300ggcgcccagaaactgcgtctgcagagcccgctgaccaaggccgaagtggaggatattacc360aaacgctgcgtgagcctgagtcagctgcgcgaggccttcgtgaacatcaccattacccgc420ggttatggcgcccgcaaaggcgagaaagatctgagcaaattaaccagccagatctacatc480tacgccatcccgtacctgtgggcctttcctccggaagagcagatcttcggtacaagtgcc540attgtgccgcgtcatgttcgtcgcgcaggccgtaataccgttgatcctaccgttaagaac600taccagtggggtgatctgaccgcagcttcttttgaagcaaaagatcgtggcgcccgcacc660gcaatcctgctggatgcagacaactgtgtggccgagggtccgggctttaacgtggtgatg720gtgaaggatggcaaactgagtagcccgagccgtaatgccctgccgggtattacacgtctg780accgtgatggagatggccgatgaaatgggcatcgaattcaccctgcgcgatatcaccagc840cgtgagttatatgaggccgacgaactgatcgccgtgaccaccggaggtggcattaccccg900attaccagtctggatggcgaaccgctgggcgatggtacccctggtcctgtgacagtggcc960attcgcgatcgcttttgggccatgatggatgagccgagcagtctggtggaggccattgaa1020tattaa1026<210>41<211>1026<212>dna<213>放线菌(actinobacteriasp.)<400>41atgaccattagcaaagacattgactatagcaccagcaacctggtgagtgtggccccgggt60gcaatccgtgaacctaccccggcaggcagcgtgatccagtacagtgactacgagctggat120gaaagcagcccgtttgccggtggtgcagcctggattgaaggtgagtatgttccggcagca180gaggcccgtattagcctgtttgataccggcttcctacatagcgatctgacctacaccgtt240gcccatgtttggcacggcaacatctttcgcctgaaagaccacattgaccgcgtgtttgat300ggcgcccagaaactgcgtctgcagagcccgctgaccaaggccgaagtggaggatattacc360aaacgctgcgtgagcctgagtcagctgcgcgagagcttcgtgaacatcaccattacccgc420ggttatggcgcccgcaaaggcgagaaagatctgagcaaattaaccagccagatctacatc480tacgccatcccgtacctgtgggcctttcctccggaagagcagatcttcggtacaagtgcc540attgtgccgcgtcatgttcgtcgcgcaggccgtaataccgttgatcctaccgttaagaac600taccagtggggtgatctgaccgcagcttcttttgaagcaaaagatcgtggcgcccgcacc660gcaatcctgctggatgcagacaactgtgtggccgagggtccgggctttaacgtggtgatg720gtgaaggatggcaaactgagtagcccgagccgtaatgccctgccgggtattacacgtctg780accgtgatggagatggccgatgaaatgggcatcgaattcaccctgcgcgatatcaccagc840cgtgagttatatgaggccgacgaactgatcgcctgcaccaccggaggtggcattaccccg900attaccagtctggatggcgaaccgctgggcgatggtacccctggtcctgtgacagtggcc960attcgcgatcgcttttgggccatgatggatgagccgagcagtctggtggaggccattgaa1020tattaa1026<210>42<211>1026<212>dna<213>放线菌(actinobacteriasp.)<400>42atgaccattagcaaagacattgactatagcaccagcaacctggtgagtgtggccccgggt60gcaatccgtgaacctaccccggcaggcagcgtgatccagtacagtgactacgagctggat120gaaagcagcccgtttgccggtggtgcagcctggattgaaggtgagtatgttccggcagca180gaggcccgtattagcctgtttgataccggcttcctacatagcgatgcgacctacaccgtt240gcccatgtttggcacggcaacatctttcgcctgaaagaccacattgaccgcgtgtttgat300ggcgcccagaaactgcgtctgcagagcccgctgaccaaggccgaagtggaggatattacc360aaacgctgcgtgagcctgagtcagctgcgcgagagcttcgtgaacatcaccattacccgc420ggttatggcgcccgcaaaggcgagaaagatctgagcaaattaaccagccagatctacatc480tacgccatcccgtacctgtgggcctttcctccggaagagcagatcttcggtacaagtgcc540attgtgccgcgtcatgttcgtcgcgcaggccgtaataccgttgatcctaccgttaagaac600taccagtggggtgatctgaccgcagcttcttttgaagcaaaagatcgtggcgcccgcacc660gcaatcctgctggatgcagacaactgtgtggccgagggtccgggctttaacgtggtgatg720gtgaaggatggcaaactgagtagcccgagccgtaatgccctgccgggtattacacgtctg780accgtgatggagatggccgatgaaatgggcatcgaattcaccctgcgcgatatcaccagc840cgtgagttatatgaggccgacgaactgatcgcctgcaccaccggaggtggcattaccccg900attaccagtctggatggcgaaccgctgggcgatggtacccctggtcctgtgacagtggcc960attcgcgatcgcttttgggccatgatggatgagccgagcagtctggtggaggccattgaa1020tattaa1026<210>43<211>1026<212>dna<213>放线菌(actinobacteriasp.)<400>43atgaccattagcaaagacattgactatagcaccagcaacctggtgagtgtggccccgggt60gcaatccgtgaacctaccccggcaggcagcgtgatccagtacagtgactacgagctggat120gaaagcagcccgtttgccggtggtgcagcctggattgaaggtgagtatgttccggcagca180gaggcccgtattagcctgtttgataccggcttcctacatagcgatctgacctacaccgtt240gcccatgtttggcacggcaacatctttcgcctgaaagaccacattgaccgcgtgtttgat300ggcgcccagaaactgcgtctgcagagcccgctgaccaaggccgaagtggaggatattacc360aaacgctgcgtgagcctgagtcagctgcgcgagagcttcgtgaacatcaccattacccgc420ggttatggcgcccgcaaaggcgagaaagatctgagcaaattaaccagccagatctacatc480tacgccatcccgtacctgtgggcctttcctccggaagagcagatcttcggtacaagtgcc540attgtgccgcgtcatgttcgtcgcgcaggccgtaataccgttgatcctaccgttaagaac600taccagtggggtgatctgaccgcagcttcttttgaagcaaaagatcgtggcgcccgcacc660gcaatcctgctggatgcagacaactgtgtggccgagggtccgggctttaacgtggtgatg720gtgaaggatggcaaactgagtagcccgagccgtaatgccctgccgggtattacacgtctg780accgtgatggagatggccgatgaaatgggcatcgaattcaccctgcgcgatatcaccagc840cgtgagttatatgaggccgacgaactgatcgccgtgaccaccgcaggtggcattaccccg900attaccagtctggatggcgaaccgctgggcgatggtacccctggtcctgtgacagtggcc960attcgcgatcgcttttgggccatgatggatgagccgagcagtctggtggaggccattgaa1020tattaa1026<210>44<211>1026<212>dna<213>放线菌(actinobacteriasp.)<400>44atgaccattagcaaagacattgactatagcaccagcaacctggtgagtgtggccccgggt60gcaatccgtgaacctaccccggcaggcagcgtgatccagtacagtgactacgagctggat120gaaagcagcccgtttgccggtggtgcagcctggattgaaggtgagtatgttccggcagca180gaggcccgtattagcctgtttgataccggcttcggccatagcgatgcgacctacaccgtt240gcccatgtttggcacggcaacatctttcgcctgaaagaccacattgaccgcgtgtttgat300ggcgcccagaaactgcgtctgcagagcccgctgaccaaggccgaagtggaggatattacc360aaacgctgcgtgagcctgagtcagctgcgcgagagcttcgtgaacatcaccattacccgc420ggttatggcgcccgcaaaggcgagaaagatctgagcaaattaaccagccagatctacatc480tacgccatcccgtacctgtgggcctttcctccggaagagcagatcttcggtacaagtgcc540attgtgccgcgtcatgttcgtcgcgcaggccgtaataccgttgatcctaccgttaagaac600taccagtggggtgatctgaccgcagcttcttttgaagcaaaagatcgtggcgcccgcacc660gcaatcctgctggatgcagacaactgtgtggccgagggtccgggctttaacgtggtgatg720gtgaaggatggcaaactgagtagcccgagccgtaatgccctgccgggtattacacgtctg780accgtgatggagatggccgatgaaatgggcatcgaattcaccctgcgcgatatcaccagc840cgtgagttatatgaggccgacgaactgatcgccgtgaccaccgcaggtggcattaccccg900attaccagtctggatggcgaaccgctgggcgatggtacccctggtcctgtgacagtggcc960attcgcgatcgcttttgggccatgatggatgagccgagcagtctggtggaggccattgaa1020tattaa1026<210>45<211>1026<212>dna<213>放线菌(actinobacteriasp.)<400>45atgaccattagcaaagacattgactatagcaccagcaacctggtgagtgtggccccgggt60gcaatccgtgaacctaccccggcaggcagcgtgatccagtacagtgactacgagctggat120gaaagcagcccgtttgccggtggtgcagcctggattgaaggtgagtatgttccggcagca180gaggcccgtattagcctgtttgataccggcttcggccatagcgatctgacctacaccgtt240gcccatgtttggcacggcaacatctttcgcctgaaagaccacattgaccgcgtgtttgat300ggcgcccagaaactgcgtatacagagcccgctgaccaaggccgaagtggaggatattacc360aaacgctgcgtgagcctgagtcagctgcgcgagagcttcgtgaacatcaccattacccgc420ggttatggcgcccgcaaaggcgagaaagatctgagcaaattaaccagccagatctacatc480tacgccatcccgtacctgtgggcctttcctccggaagagcagatcttcggtacaagtgcc540attgtgccgcgtcatgttcgtcgcgcaggccgtaataccgttgatcctaccgttaagaac600taccagtggggtgatctgaccgcagcttcttttgaagcaaaagatcgtggcgcccgcacc660gcaatcctgctggatgcagacaactgtgtggccgagggtccgggctttaacgtggtgatg720gtgaaggatggcaaactgagtagcccgagccgtaatgccctgccgggtattacacgtctg780accgtgatggagatggccgatgaaatgggcatcgaattcaccctgcgcgatatcaccagc840cgtgagttatatgaggccgacgaactgatcgccgtgaccaccgcaggtggcattaccccg900attaccagtctggatggcgaaccgctgggcgatggtacccctggtcctgtgacagtggcc960attcgcgatcgcttttgggccatgatggatgagccgagcagtctggtggaggccattgaa1020tattaa1026<210>46<211>1026<212>dna<213>放线菌(actinobacteriasp.)<400>46atgaccattagcaaagacattgactatagcaccagcaacctggtgagtgtggccccgggt60gcaatccgtgaacctaccccggcaggcagcgtgatccagtacagtgactacgagctggat120gaaagcagcccgtttgccggtggtgcagcctggattgaaggtgagtatgttccggcagca180gaggcccgtattagcctgtttgataccggcttcggccatagcgatctgacctacaccgtt240gcccatgtttggcacggcaacatctttcgcctgggagaccacattgaccgcgtgtttgat300ggcgcccagaaactgcgtctgcagagcccgctgaccaaggccgaagtggaggatattacc360aaacgctgcgtgagcctgagtcagctgcgcgagagcttcgtgaacatcaccattacccgc420ggttatggcgcccgcaaaggcgagaaagatctgagcaaattaaccagccagatctacatc480tacgccatcccgtacctgtgggcctttcctccggaagagcagatcttcggtacaagtgcc540attgtgccgcgtcatgttcgtcgcgcaggccgtaataccgttgatcctaccgttaagaac600taccagtggggtgatctgaccgcagcttcttttgaagcaaaagatcgtggcgcccgcacc660gcaatcctgctggatgcagacaactgtgtggccgagggtccgggctttaacgtggtgatg720gtgaaggatggcaaactgagtagcccgagccgtaatgccctgccgggtattacacgtctg780accgtgatggagatggccgatgaaatgggcatcgaattcaccctgcgcgatatcaccagc840cgtgagttatatgaggccgacgaactgatcgccgtgaccaccgcaggtggcattaccccg900attaccagtctggatggcgaaccgctgggcgatggtacccctggtcctgtgacagtggcc960attcgcgatcgcttttgggccatgatggatgagccgagcagtctggtggaggccattgaa1020tattaa1026<210>47<211>1026<212>dna<213>放线菌(actinobacteriasp.)<400>47atgaccattagcaaagacattgactatagcaccagcaacctggtgagtgtggccccgggt60gcaatccgtgaacctaccccggcaggcagcgtgatccagtacagtgactacgagctggat120gaaagcagcccgtttgccggtggtgcagcctggattgaaggtgagtatgttccggcagca180gaggcccgtattagcctgtttgataccggcttcggccatagcgatctgacctacaccgtt240gcccatgtttggcacggcaacatctttcgcctgaaagaccacattgaccgcgtgtttgat300ggcgcccagaaactgcgtctgcagagcccgctgaccaaggccgaagtggaggatattacc360aaacgctgcgtggccctgagtcagctgcgcgagagcttcgtgaacatcaccattacccgc420ggttatggcgcccgcaaaggcgagaaagatctgagcaaattaaccagccagatctacatc480tacgccatcccgtacctgtgggcctttcctccggaagagcagatcttcggtacaagtgcc540attgtgccgcgtcatgttcgtcgcgcaggccgtaataccgttgatcctaccgttaagaac600taccagtggggtgatctgaccgcagcttcttttgaagcaaaagatcgtggcgcccgcacc660gcaatcctgctggatgcagacaactgtgtggccgagggtccgggctttaacgtggtgatg720gtgaaggatggcaaactgagtagcccgagccgtaatgccctgccgggtattacacgtctg780accgtgatggagatggccgatgaaatgggcatcgaattcaccctgcgcgatatcaccagc840cgtgagttatatgaggccgacgaactgatcgccgtgaccaccgcaggtggcattaccccg900attaccagtctggatggcgaaccgctgggcgatggtacccctggtcctgtgacagtggcc960attcgcgatcgcttttgggccatgatggatgagccgagcagtctggtggaggccattgaa1020tattaa1026<210>48<211>1026<212>dna<213>放线菌(actinobacteriasp.)<400>48atgaccattagcaaagacattgactatagcaccagcaacctggtgagtgtggccccgggt60gcaatccgtgaacctaccccggcaggcagcgtgatccagtacagtgactacgagctggat120gaaagcagcccgtttgccggtggtgcagcctggattgaaggtgagtatgttccggcagca180gaggcccgtattagcctgtttgataccggcttcggccatagcgatctgacctacaccgtt240gcccatgtttggcacggcaacatctttcgcctgaaagaccacattgaccgcgtgtttgat300ggcgcccagaaactgcgtctgcagagcccgctgaccaaggccgaagtggaggatattacc360aaacgctgcgtgagcctgagtcagctgcgcgaggccttcgtgaacatcaccattacccgc420ggttatggcgcccgcaaaggcgagaaagatctgagcaaattaaccagccagatctacatc480tacgccatcccgtacctgtgggcctttcctccggaagagcagatcttcggtacaagtgcc540attgtgccgcgtcatgttcgtcgcgcaggccgtaataccgttgatcctaccgttaagaac600taccagtggggtgatctgaccgcagcttcttttgaagcaaaagatcgtggcgcccgcacc660gcaatcctgctggatgcagacaactgtgtggccgagggtccgggctttaacgtggtgatg720gtgaaggatggcaaactgagtagcccgagccgtaatgccctgccgggtattacacgtctg780accgtgatggagatggccgatgaaatgggcatcgaattcaccctgcgcgatatcaccagc840cgtgagttatatgaggccgacgaactgatcgccgtgaccaccgcaggtggcattaccccg900attaccagtctggatggcgaaccgctgggcgatggtacccctggtcctgtgacagtggcc960attcgcgatcgcttttgggccatgatggatgagccgagcagtctggtggaggccattgaa1020tattaa1026<210>49<211>1026<212>dna<213>放线菌(actinobacteriasp.)<400>49atgaccattagcaaagacattgactatagcaccagcaacctggtgagtgtggccccgggt60gcaatccgtgaacctaccccggcaggcagcgtgatccagtacagtgactacgagctggat120gaaagcagcccgtttgccggtggtgcagcctggattgaaggtgagtatgttccggcagca180gaggcccgtattagcctgtttgataccggcttcggccatagcgatctgacctacaccgtt240gcccatgtttggcacggcaacatctttcgcctgaaagaccacattgaccgcgtgtttgat300ggcgcccagaaactgcgtctgcagagcccgctgaccaaggccgaagtggaggatattacc360aaacgctgcgtgagcctgagtcagctgcgcgagagcttcgtgaacatcaccattacccgc420ggttatggcgcccgcaaaggcgagaaagatctgagcaaattaaccagccagatctacatc480tacgccatcccgtacctgtgggcctttcctccggaagagcagatcttcggtacaagtgcc540attgtgccgcgtcatgttcgtcgcgcaggccgtaataccgttgatcctaccgttaagaac600taccagtggggtgatctgaccgcagcttcttttgaagcaaaagatcgtggcgcccgcacc660gcaatcctgctggatggagacaactgtgtggccgagggtccgggctttaacgtggtgatg720gtgaaggatggcaaactgagtagcccgagccgtaatgccctgccgggtattacacgtctg780accgtgatggagatggccgatgaaatgggcatcgaattcaccctgcgcgatatcaccagc840cgtgagttatatgaggccgacgaactgatcgccgtgaccaccgcaggtggcattaccccg900attaccagtctggatggcgaaccgctgggcgatggtacccctggtcctgtgacagtggcc960attcgcgatcgcttttgggccatgatggatgagccgagcagtctggtggaggccattgaa1020tattaa1026<210>50<211>1026<212>dna<213>放线菌(actinobacteriasp.)<400>50atgaccattagcaaagacattgactatagcaccagcaacctggtgagtgtggccccgggt60gcaatccgtgaacctaccccggcaggcagcgtgatccagtacagtgactacgagctggat120gaaagcagcccgtttgccggtggtgcagcctggattgaaggtgagtatgttccggcagca180gaggcccgtattagcctgtttgataccggcttcggccatagcgatctgacctacaccgtt240gcccatgtttggcacggcaacatctttcgcctgaaagaccacattgaccgcgtgtttgat300ggcgcccagaaactgcgtctgcagagcccgctgaccaaggccgaagtggaggatattacc360aaacgctgcgtgagcctgagtcagctgcgcgagagcttcgtgaacatcaccattacccgc420ggttatggcgcccgcaaaggcgagaaagatctgagcaaattaaccagccagatctacatc480tacgccatcccgtacctgtgggcctttcctccggaagagcagatcttcggtacaagtgcc540attgtgccgcgtcatgttcgtcgcgcaggccgtaataccgttgatcctaccgttaagaac600taccagtggggtgatctgaccgcagcttcttttgaagcaaaagatcgtggcgcccgcacc660gcaatcctgctggatgcagacaactgtgtggccgagggtccgggctttaacgtggtgatg720gtgaaggatggcaaactgagtagcccgagccgtaatgccctgccgggtattacacgtctg780accgtgatggagatggccgatgaaatgggcatcgaattcaccctgcgcgatatcaccagc840cgtgagttatatgaggccgacgaactgatcgcctgcaccaccgcaggtggcattaccccg900attaccagtctggatggcgaaccgctgggcgatggtacccctggtcctgtgacagtggcc960attcgcgatcgcttttgggccatgatggatgagccgagcagtctggtggaggccattgaa1020tattaa1026<210>51<211>1026<212>dna<213>放线菌(actinobacteriasp.)<400>51atgaccattagcaaagacattgactatagcaccagcaacctggtgagtgtggccccgggt60gcaatccgtgaacctaccccggcaggcagcgtgatccagtacagtgactacgagctggat120gaaagcagcccgtttgccggtggtgcagcctggattgaaggtgagtatgttccggcagca180gaggcccgtattagcctgtttgataccggcttcggccatagcgatctgacctacaccgtt240gcccatgtttggcacggcaacatctttcgcctgaaagaccacattgaccgcgtgtttgat300ggcgcccagaaactgcgtctgcagagcccgctgaccaaggccgaagtggaggatattacc360aaacgctgcgtgagcctgagtcagctgcgcgagagcttcgtgaacatcaccattacccgc420ggttatggcgcccgcaaaggcgagaaagatctgagcaaattaaccagccagatctacatc480tacgccatcccgtacctgtgggcctttcctccggaagagcagatcttcggtacaagtgcc540attgtgccgcgtcatgttcgtcgcgcaggccgtaataccgttgatcctaccgttaagaac600taccagtggggtgatctgaccgcagcttcttttgaagcaaaagatcgtggcgcccgcacc660gcaatcctgctggatgcagacaactgtgtggccgagggtccgggctttaacgtggtgatg720gtgaaggatggcaaactgagtagcccgagccgtaatgccctgccgggtattacacgtctg780accgtgatggagatggccgatgaaatgggcatcgaattcaccctgcgcgatatcaccagc840cgtgagttatatgaggccgacgaactgatcgccgtgaccaccggaggtggcattaccccg900attaccagtctggatggcgaaccgctgggcgatggtacccctggtcctgtgacagtggcc960attcgcgatcgcttttgggccatgatggatgagccgagcagtctggtggaggccattgaa1020tattaa1026<210>52<211>1026<212>dna<213>放线菌(actinobacteriasp.)<400>52atgaccattagcaaagacattgactatagcaccagcaacctggtgagtgtggcctcgggt60gcaatccgtgaacctaccccggcaggcagcgtgatccagtacagtgactacgagctggat120gaaagcagcccgtttgccggtggtgcagcctggattgaaggtgagtatgttccggcagca180gaggcccgtattagcctgtttgataccggcttcggccatagcgatctgacctacaccgtt240gcccatgtttggcacggcaacatctttcgcctgaaagaccacattgaccgcgtgtttgat300ggcgcccagaaactgcgtctgcagagcccgctgaccaaggccgaagtggaggatattacc360aaacgctgcgtgagcctgagtcagctgcgcgagagcttcgtgaacatcaccattacccgc420ggttatggcgcccgcaaaggcgagaaagatctgagcaaattaaccagccagatctacatc480tacgccatcccgtacctgtgggcctttcctccggaagagcagatcttcggtacaagtgcc540attgtgccgcgtcatgttcgtcgcgcaggccgtaataccgttgatcctaccgttaagaac600taccagtggggtgatctgaccgcagcttcttttgaagcaaaagatcgtggcgcccgcacc660gcaatcctgctggatgcagacaactgtgtggccgagggtccgggctttaacgtggtgatg720gtgaaggatggcaaactgagtagcccgagccgtaatgccctgccgggtattacacgtctg780accgtgatggagatggccgatgaaatgggcatcgaattcaccctgcgcgatatcaccagc840cgtgagttatatgaggccgacgaactgatcgccgtgaccaccgcaggtggcattaccccg900attaccagtctggatggcgaaccgctgggcgatggtacccctggtcctgtgacagtggcc960attcgcgatcgcttttgggccatgatggatgagccgagcagtctggtggaggccattgaa1020tattaa1026<210>53<211>1026<212>dna<213>放线菌(actinobacteriasp.)<400>53atgaccattagcaaagacattgactatagcaccagcaacctggtgagtgtggccccgggt60gcaatccgtgaacctaccccggcaggcagcgtgatccagtacagtgactacgagctggat120tcaagcagcccgtttgccggtggtgcagcctggattgaaggtgagtatgttccggcagca180gaggcccgtattagcctgtttgataccggcttcggccatagcgatctgacctacaccgtt240gcccatgtttggcacggcaacatctttcgcctgaaagaccacattgaccgcgtgtttgat300ggcgcccagaaactgcgtctgcagagcccgctgaccaaggccgaagtggaggatattacc360aaacgctgcgtgagcctgagtcagctgcgcgagagcttcgtgaacatcaccattacccgc420ggttatggcgcccgcaaaggcgagaaagatctgagcaaattaaccagccagatctacatc480tacgccatcccgtacctgtgggcctttcctccggaagagcagatcttcggtacaagtgcc540attgtgccgcgtcatgttcgtcgcgcaggccgtaataccgttgatcctaccgttaagaac600taccagtggggtgatctgaccgcagcttcttttgaagcaaaagatcgtggcgcccgcacc660gcaatcctgctggatgcagacaactgtgtggccgagggtccgggctttaacgtggtgatg720gtgaaggatggcaaactgagtagcccgagccgtaatgccctgccgggtattacacgtctg780accgtgatggagatggccgatgaaatgggcatcgaattcaccctgcgcgatatcaccagc840cgtgagttatatgaggccgacgaactgatcgccgtgaccaccgcaggtggcattaccccg900attaccagtctggatggcgaaccgctgggcgatggtacccctggtcctgtgacagtggcc960attcgcgatcgcttttgggccatgatggatgagccgagcagtctggtggaggccattgaa1020tattaa1026<210>54<211>1026<212>dna<213>放线菌(actinobacteriasp.)<400>54atgaccattagcaaagacattgactatagcaccagcaacctggtgagtgtggccccgggt60gcaatccgtgaacctaccccggcaggcagcgtgatccagtacagtgactacgagctggat120gaaagcaacccgtttgccggtggtgcagcctggattgaaggtgagtatgttccggcagca180gaggcccgtattagcctgtttgataccggcttcggccatagcgatctgacctacaccgtt240gcccatgtttggcacggcaacatctttcgcctgaaagaccacattgaccgcgtgtttgat300ggcgcccagaaactgcgtctgcagagcccgctgaccaaggccgaagtggaggatattacc360aaacgctgcgtgagcctgagtcagctgcgcgagagcttcgtgaacatcaccattacccgc420ggttatggcgcccgcaaaggcgagaaagatctgagcaaattaaccagccagatctacatc480tacgccatcccgtacctgtgggcctttcctccggaagagcagatcttcggtacaagtgcc540attgtgccgcgtcatgttcgtcgcgcaggccgtaataccgttgatcctaccgttaagaac600taccagtggggtgatctgaccgcagcttcttttgaagcaaaagatcgtggcgcccgcacc660gcaatcctgctggatgcagacaactgtgtggccgagggtccgggctttaacgtggtgatg720gtgaaggatggcaaactgagtagcccgagccgtaatgccctgccgggtattacacgtctg780accgtgatggagatggccgatgaaatgggcatcgaattcaccctgcgcgatatcaccagc840cgtgagttatatgaggccgacgaactgatcgccgtgaccaccgcaggtggcattaccccg900attaccagtctggatggcgaaccgctgggcgatggtacccctggtcctgtgacagtggcc960attcgcgatcgcttttgggccatgatggatgagccgagcagtctggtggaggccattgaa1020tattaa1026<210>55<211>935<212>dna<213>放线菌(actinobacteriasp.)<400>55tgatccagtacagtgactacgagctggatgaaagcagcccgtttgccggtggtgcagcct60ggattgaaggtgagtatgttccggcagcagaggcccgtattagcctgtttgataccggct120tcggccatagcgatctgacctacaccgttgcccatgtttggcacggcaacatctttcgcc180tgaaagaccacattgaccgcgtgtttgatggcgcccagaaactgcgtctgcagagcccgc240tgaccaaggccgaagtggaggatattaccaaacgctgcgtgagcctgagtcagctgcgcg300agagcttcgtgaacatcaccattacccgcggttatggcgcccgcaaaggcgagaaagatc360tgagcaaattaaccagccagatctacatctacgccatcccgtacctgtgggcctttcctc420cggaagagcagatcttcggtacaagtgccattgtgccgcgtcatgttcgtcgcgcaggcc480gtaataccgttgatcctaccgttaagaactaccagtggggtgatctgaccgcagcttctt540ttgaagcaaaagatcgtggcgcccgcaccgcaatcctgctggatgcagacaactgtgtgg600ccgagggtccgggctttaacgtggtgatggtgaaggatggcaaactgagtagcccgagcc660gtaatgccctgccgggtattacacgtctgaccgtgatggagatggccgatgaaatgggca720tcgaattcaccctgcgcgatatcaccagccgtgagttatatgaggccgacgaactgatcg780ccgtgaccaccgcaggtggcattaccccgattaccagtctggatggcgaatcgctgggcg840atggtacccctggtcctgtgacagtggccattcgcgatcgcttttgggccatgatggatg900agccgagcagtctggtggaggccattgaatattaa935<210>56<211>1026<212>dna<213>放线菌(actinobacteriasp.)<400>56atgaccattagcaaagacattgactatagcaccagcaacctggtgagtgtggccccgggt60gcaatccgtgaacctaccccggcaggcagcgtgatccagtacagtgactacgagctggat120gaaagcagcccgtttgccggtggtgcagcctggattgaaggtgagtatgttccggcagca180gaggcccgtattagcctgtttgataccggcttcggccatagcgatctgacctacaccgtt240gcccatgtttggcacggcaacatctttcgcctgaaagaccacattgaccgcgtgtttgat300ggcgcccagaaactgcgtctgcagagcccgctgaccaaggccgaagtggaggatattacc360aaacgctgcgtgagcctgagtcagctgcgcgagagcttcgtgaacatcaccattacccgc420ggttatggcgcccgcaaaggcgagaaagatctgagcaaattaaccagccagatctacatc480tacgccatcccgtacctgtgggcctttcctccggaagagcagatcttcggtacaagtgcc540attgtgccgcgtcatgttcgtcgcgcaggccgtaataccgttgatcctaccgttaagaac600taccagtggggtgatctgaccgcagcttcttttgaagcaaaagatcgtggcgcccgcacc660gcaatcctgctggatgcagacaactgtgtggccgagggtccgggctttaacgtggtgatg720gtgaaggatggcaaactgagtagcccgagccgtaatgccctgccgggtattacacgtctg780accgtgatggagatggccgatgaaatgggcatcgaattcaccctgcgcgatatcaccagc840cgtgagttatatgaggccgacgaactgatcgccgtgaccaccgcaggtggcattaccccg900attaccagtctggatggcgaaccgctgggcgatggtacccctggtcctgtgacagtggcc960attcgcgatcgcttttgggccatgatggatgagtcgagcagtctggtggaggccattgaa1020tattaa1026<210>57<211>1026<212>dna<213>放线菌(actinobacteriasp.)<400>57atgaccattagcaaagacattgactatagcaccagcaacctggtgagtgtggccccgggt60gcaatccgtgaacctaccccggcaggcagcgtgatccagtacagtgactacgagctggat120gaaagcagcccgtttgccggtggtgcagcctggattgaaggtgagtatgttccggcagca180gaggcccgtattagcctgtttgataccggcttcggccatagcgatgcgacctacaccgtt240gcccatgtttggcacggcaacatctttcgcctgaaagaccacattgaccgcgtgtttgat300ggcgcccagaaactgcgtatacagagccagctgaccaaggccgaagtggaggatattacc360aaacgctgcgtgagcctgagtcagctgcgcgagagcttcgtgaacatcaccattacccgc420ggttatggcgcccgcaaaggcgagaaagatctgagcaaattaaccagccagatctacatc480tacgccatcccgtacctgtgggcctttcctccggaagagcagatcttcggtacaagtgcc540attgtgccgcgtcatgttcgtcgcgcaggccgtaataccgttgatcctaccgttaagaac600taccagtggggtgatctgaccgcagcttcttttgaagcaaaagatcgtggcgcccgcacc660gcaatcctgctggatgcagacaactgtgtggccgagggtccgggctttaacgtggtgatg720gtgaaggatggcaaactgagtagcccgagccgtaatgccctgccgggtattacacgtctg780accgtgatggagatggccgatgaaatgggcatcgaattcaccctgcgcgatatcaccagc840cgtgagttatatgaggccgacgaactgatcgccgtgaccaccgcaggtggcattaccccg900attaccagtctggatggcgaaccgctgggcgatggtacccctggtcctgtgacagtggcc960attcgcgatcgcttttgggccatgatggatgagccgagcagtctggtggaggccattgaa1020tattaa1026<210>58<211>1026<212>dna<213>放线菌(actinobacteriasp.)<400>58atgaccattagcaaagacattgactatagcaccagcaacctggtgagtgtggccccgggt60gcaatccgtgaacctaccccggcaggcagcgtgatccagtacagtgactacgagctggat120gaaagcagcccgtttgccggtggtgcagcctggattgaaggtgagtatgttccggcagca180gaggcccgtattagcctgtttgataccggcttcggccatagcgatgcgacctacaccgtt240gcccatgtttggcacggcaacatctttcgcctgaaagaccacattgaccgcgtgtttgat300ggcgcccagaaactgcgtctgcagagcccgctgaccaaggccgaagtggaggatattacc360aaacgctgcgtggccctgagtcagctgcgcgagagcttcgtgaacatcaccattacccgc420ggttatggcgcccgcaaaggcgagaaagatctgagcaaattaaccagccagatctacatc480tacgccatcccgtacctgtgggcctttcctccggaagagcagatcttcggtacaagtgcc540attgtgccgcgtcatgttcgtcgcgcaggccgtaataccgttgatcctaccgttaagaac600taccagtggggtgatctgaccgcagcttcttttgaagcaaaagatcgtggcgcccgcacc660gcaatcctgctggatggagacaactgtgtggccgagggtccgggctttaacgtggtgatg720gtgaaggatggcaaactgagtagcccgagccgtaatgccctgccgggtattacacgtctg780accgtgatggagatggccgatgaaatgggcatcgaattcaccctgcgcgatatcaccagc840cgtgagttatatgaggccgacgaactgatcgccgtgaccaccgcaggtggcattaccccg900attaccagtctggatggcgaaccgctgggcgatggtacccctggtcctgtgacagtggcc960attcgcgatcgcttttgggccatgatggatgagccgagcagtctggtggaggccattgaa1020tattaa1026<210>59<211>1026<212>dna<213>放线菌(actinobacteriasp.)<400>59atgaccattagcaaagacattgactatagcaccagcaacctggtgagtgtggccccgggt60gcaatccgtgaacctaccccggcaggcagcgtgatccagtacagtgactacgagctggat120gaaagcagcccgtttgccggtggtgcagcctggattgaaggtgagtatgttccggcagca180gaggcccgtattagcctgtttgataccggcttcggccatagcgatgcgacctacagcgtt240gcccatgtttggcacggcaacatctttcgcctgaaagaccacattgaccgcgtgtttgat300ggcgcccagaaactgcgtctgcagagcccgctgaccaaggccgaagtggaggatattacc360aaacgctgcgtgagcctgagtcagctgcgcgagagcttcgtgaacatcaccattacccgc420ggttatggcgcccgcaaaggcgagaaagatctgagcaaattaaccagccagatctacatc480tacgccatcccgtacctgtgggcctttcctccggaagagcagatcttcggtacaagtgcc540attgtgccgcgtcatgttcgtcgcgcaggccgtaataccgttgatcctaccgttaagaac600taccagtggggtgatctgaccgcagcttcttttgaagcaaaagatcgtggcgcccgcacc660gcaatcctgctggatgcagacaactgtgtggccgagggtccgggctttaacgtggtgatg720gtgaaggatggcaaactgagtagcccgagccgtaatgccctgccgggtattacacgtctg780accgtgatggagatggccgatgaaatgggcatcgaattcaccctgcgcgatatcaccagc840cgtgagttatatgaggccgacgaactgatcgccgtgaccaccggaggtggcattaccccg900attaccagtctggatggcgaaccgctgggcgatggtacccctggtcctgtgacagtggcc960attcgcgatcgcttttgggccatgatggatgagccgagcagtctggtggaggccattgaa1020tattaa1026<210>60<211>1026<212>dna<213>放线菌(actinobacteriasp.)<400>60atgaccattagcaaagacattgactatagcaccagcaacctggtgagtgtggccccgggt60gcaatccgtgaacctaccccggcaggcagcgtgatccagtacagtgactacgagctggat120gaaagcagcccgtttgccggtggtgcagcctggattgaaggtgagtatgttccggcagca180gaggcccgtattagcctgtttgataccggcttcggccatagcgatgcgacctacaccgtt240gcccatgtttggcacggcaacatctttcgcctgaaagaccacattgaccgcgtgtttgat300ggcgcccagaaactgcgtatacagagccagctgaccaaggccgaagtggaggatattacc360aaacgctgcgtgagcctgagtcagctgcgcgagagcttcgtgaacatcaccattacccgc420ggttatggcgcccgcaaaggcgagaaaggtctgagcaaattaaccagccagatctacatc480tacgccatcccgtacctgtgggcctttcctccggaagagcagatcttcggtacaagtgcc540attgtgccgcgtcatgttcgtcgcgcaggccgtaataccgttgatcctaccgttaagaac600taccagtggggtgatctgaccgcagcttcttttgaagcaaaagatcgtggcgcccgcacc660gcaatcctgctggatgcagacaactgtgtggccgagggtccgggctttaacgtggtgatg720gtgaaggatggcaaactgagtagcccgagccgtaatgccctgccgggtattacacgtctg780accgtgatggagatggccgatgaaatgggcatcgaattcaccctgcgcgatatcaccagc840cgtgagttatatgaggccgacgaactgatcgccgtgaccaccgcaggtggcattaccccg900attaccagtctggatggcgaaccgctgggcgatggtacccctggtcctgtgacagtggcc960attcgcgatcgcttttgggccatgatggatgagccgagcagtctggtggaggccattgaa1020tattaa1026当前第1页12