CN113832122A - 一种7β-HSDH酶突变体及其编码基因和应用 - Google Patents

一种7β-HSDH酶突变体及其编码基因和应用 Download PDF

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CN113832122A
CN113832122A CN202111215918.4A CN202111215918A CN113832122A CN 113832122 A CN113832122 A CN 113832122A CN 202111215918 A CN202111215918 A CN 202111215918A CN 113832122 A CN113832122 A CN 113832122A
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余允东
张和平
容文西
杨卓星
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Zhongshan Bailing Biotechnology Co ltd
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Abstract

本发明公开了一种7β‑HSDH酶突变体及其编码基因和应用。所述的7β‑HSDH酶突变体的氨基酸序列与氨基酸序列如SEQ ID NO:2所示的野生型7β‑HSDH酶相比，在SEQ ID NO:2所示的氨基酸序列的第237位、第240位进行单突变或两两联合突变中的任意一种突变。所述的7β‑HSDH酶突变体可用于24‑去甲熊去氧胆酸的合成制备，将其作为生物催化剂转化底物24‑去甲‑7‑酮胆石酸生成24‑去甲熊去氧胆酸，反应后产物经HPLC验证，反应转化率>90％。本发明构建的7β‑HSDH酶突变体与野生型酶相比，催化活性显著提高，能显著降低酶的使用量，具有大规模工业应用的广泛前景。

Description

一种7β-HSDH酶突变体及其编码基因和应用
技术领域
本发明涉及生物酶工程技术领域，具体涉及一种7β-HSDH酶突变体及其编码基因和应用。
背景技术
24-去甲熊去氧胆酸(简称为norUDCA)，为熊去氧胆酸的同系物，较熊去氧胆酸侧链少一个亚甲基，具有保护肝脏、抗炎和抗纤维化活性，可改善原发性硬化性胆管炎患者的血清碱性磷酸酶水平及其他胆汁淤积的状况。最新一项IIa期随机双盲对照研究报道，大剂量的24-去甲熊去氧胆酸(norUDCA)可以显著改善非酒精性脂肪性肝病(NAFLD)患者的血清转氨酶、甘油三酯和肝脏影像学指标，且药物安全性较好[杨蕊旭、范建高.AASLD2017：24-去甲熊去氧胆酸治疗非酒精性脂肪性肝病]。
目前未有关于24-去甲熊去氧胆酸的酶法合成报道，尤其是以24-去甲-7-酮胆石酸为底物进行7位不对称还原时，现有已报道的7β-类固醇脱氢酶(7β-Hydroxysteroiddehydrogenase，7β-HSDH)的催化效率极其低下，不到其原始底物7-酮胆石酸催化活性的5％，严重限制了24-去甲熊脱氧胆酸的工业化酶法生产。
如中国专利CN109182284A公开了一种7β-羟基类固醇脱氢酶突变体、编码序列、重组基因表达载体、基因工程菌及应用，该专利公开了对来源于产气柯林斯菌的7β-羟基类固醇脱氢酶进行突变，将野生型7β-羟基类固醇脱氢酶的第175位的谷氨酸突变成天冬氨酸，得到突变体Ca7β-1；或将第175位的谷氨酸突和第197位的天冬酰胺均突变成天冬氨酸，得到突变体Ca7β-2。7β-羟基类固醇脱氢酶突变体的还原活力均得到了提高，可利用7β-羟基类固醇脱氢酶突变体催化合成熊去氧胆酸(UDCA)和牛磺熊去氧胆酸(T-DUCA)。但该专利并没有公开7β-羟基类固醇脱氢酶能作为催化剂用于合成norUDCA的相关记载。
蛋白质三维结构模拟和蛋白定向进化技术，是近年来发展起来的对原始基因序列进行人工改造、以满足工业化应用需求的高科技技术，其中蛋白定向进化技术更是获得了2018年诺贝尔化学奖。因此结合蛋白质三维结构模拟和蛋白定向进化技术，进一步寻找和开发新的适用于工业大规模生产的羟基类固醇脱氢酶是目前研究的热点。
发明内容
针对现有技术存在的不足，本发明要解决的技术问题是提供一种7β-HSDH酶突变体及其编码基因和应用，以解决现有羟基类固醇脱氢酶活性不理想，难以实现工业化生产的问题。本发明采用蛋白质三维结构模拟和蛋白定向进化技术，对来源于产气柯林斯菌(Collinsella aerofaciens)DSM 3979的7β-HSDH酶(Luo Liu,Arno Aigner,RolfD.Schmid.Appl Microbiol Biotechnol.2011,90:127-135)进行了人工定向改造，显著提高了其对24-去甲-7-酮胆石酸的催化活性，将十分有助于实现工业上减少酶量，降低生产成本。
为解决上述技术问题，本发明提供以下技术方案：
来源于Collinsella aerofaciens的野生型7β-HSDH酶的氨基酸序列如SEQ IDNO:2所示，其编码基因的核苷酸序列如SEQ ID NO:1所示。
7β-HSDH酶的编码基因核苷酸序列通过常州基宇生物技术有限公司全基因合成所得，在编码区两端分别添加NdeI和HindIII限制性内切酶位点。目的基因片段通过限制性内切酶NdeI和HindIII酶切后，与经过同样双酶切的pET21a(+)载体(Novagen公司)进行连接、转化和筛选，筛选得到的阳性质粒7β-HSDH-pET21a(+)转入BL21(DE3)宿主菌中，从而构建7β-HSDH酶的体外异源表达体系。
7β-HSDH酶的突变体的构建，是通过定向进化的技术手段得到的，即利用易错PCR、DNA重排、半理性设计及大分子建模技术模拟三维结构等定向进行技术来获得突变体。具体地，本发明通过大分子建模技术模拟三维结构来进行酶的定向进化。采用同源建模的方法来模拟7β-HSDH酶的三维结构，利用能量最低原理和分子对接技术预测出可能的与催化相关的一个或多个活性位点，然后对这些活性位点进行定点突变，从中筛选出活性有显著提高的突变体。
更为具体的过程如下：本发明通过大分子建模技术预测出可能与催化活性相关的位点，分别为N237、S240两个位点。分别对这两个位点进行定点突变，利用高压液相色谱法(HPLC)来进行突变体的筛选。更为具体的为：1、当位点237的天冬酰胺(N)突变为组氨酸(H)时，突变体的催化活性相对于野生型酶来说得到了提高；2、当位点237的天冬酰胺(N)突变为谷氨酰胺(Q)时，突变体酶活得到了提高；3、当位点240的丝氨酸(S)突变为天冬酰胺(N)时，突变体酶活相对野生型酶来说得到了提高；4、当位点240的丝氨酸(S)突变为精氨酸(R)时，突变体酶活得到了显著提高；5、当位点240的丝氨酸(S)突变为谷氨酸(E)时，突变体酶活得到了显著提高；6、当位点240的丝氨酸(S)突变为谷氨酰胺(Q)时，突变体酶活得到了显著提高。当将上述2个位点进行两两联合突变时，突变体的催化活性相对于单个突变体来说得到了更大的提高。
因此，一方面，本发明请求保护一种7β-HSDH酶突变体，其氨基酸序列与氨基酸序列如SEQ ID NO:2所示的野生型7β-HSDH酶相比，在SEQ ID NO:2所示的氨基酸序列的第237位、第240位进行单突变或两两联合突变中的任意一种突变。
具体地，所述的单突变为：
当SEQ ID NO:2所示的氨基酸序列的第237位由天冬酰胺突变为组氨酸，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:6所示；
或，当SEQ ID NO:2所示的氨基酸序列的第237位由天冬酰胺突变为谷氨酰胺，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:8所示；
或，当SEQ ID NO:2所示的氨基酸序列的第240位由丝氨酸突变为天冬酰胺，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:10所示；
或，当SEQ ID NO:2所示的氨基酸序列的第240位由丝氨酸突变为精氨酸，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:12所示；
或，当SEQ ID NO:2所示的氨基酸序列的第240位由丝氨酸突变为谷氨酸，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:14所示；
或，当SEQ ID NO:2所示的氨基酸序列的第240位由丝氨酸突变为谷氨酰胺，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:16所示。
具体地，所述的两两联合突变为：
当SEQ ID NO:2所示的氨基酸序列的第237位由天冬酰胺突变为组氨酸，第240位由丝氨酸突变为天冬酰胺，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:18所示；
或，当SEQ ID NO:2所示的氨基酸序列的第237位由天冬酰胺突变为组氨酸，第240位由丝氨酸突变为精氨酸，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:20所示；
或，当SEQ ID NO:2所示的氨基酸序列的第237位由天冬酰胺突变为组氨酸，第240位由丝氨酸突变为谷氨酸，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:22所示；
或，当SEQ ID NO:2所示的氨基酸序列的第237位由天冬酰胺突变为组氨酸，第240位由丝氨酸突变为谷氨酰胺，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:24所示；
或，当SEQ ID NO:2所示的氨基酸序列的第237位由天冬酰胺突变为谷氨酰胺，第240位由丝氨酸突变为天冬酰胺，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:26所示；
或，当SEQ ID NO:2所示的氨基酸序列的第237位由天冬酰胺突变为谷氨酰胺，第240位由丝氨酸突变为精氨酸，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:28所示；
或，当SEQ ID NO:2所示的氨基酸序列的第237位由天冬酰胺突变为谷氨酰胺，第240位由丝氨酸突变为谷氨酸，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:30所示；
或，当SEQ ID NO:2所示的氨基酸序列的第237位由天冬酰胺突变为谷氨酰胺，第240位由丝氨酸突变为谷氨酰胺，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:32所示。
另一方面，本发明还请求保护上述7β-HSDH酶突变体的编码基因。
具体地，氨基酸序列如SEQ ID NO:6所示的7β-HSDH酶突变体的编码基因的核苷酸序列如SEQ ID NO:5所示；
或，氨基酸序列如SEQ ID NO:8所示的7β-HSDH酶突变体的编码基因的核苷酸序列如SEQ ID NO:7所示；
或，氨基酸序列如SEQ ID NO:10所示的7β-HSDH酶突变体的编码基因的核苷酸序列如SEQ ID NO:9所示；
或，氨基酸序列如SEQ ID NO:12所示的7β-HSDH酶突变体的编码基因的核苷酸序列如SEQ ID NO:11所示；
或，氨基酸序列如SEQ ID NO:14所示的7β-HSDH酶突变体的编码基因的核苷酸序列如SEQ ID NO:13所示；
或，氨基酸序列如SEQ ID NO:16所示的7β-HSDH酶突变体的编码基因的核苷酸序列如SEQ ID NO:15所示；
或，氨基酸序列如SEQ ID NO:18所示的7β-HSDH酶突变体的编码基因的核苷酸序列如SEQ ID NO:17所示；
或，氨基酸序列如SEQ ID NO:20所示的7β-HSDH酶突变体的编码基因的核苷酸序列如SEQ ID NO:19所示；
或，氨基酸序列如SEQ ID NO:22所示的7β-HSDH酶突变体的编码基因的核苷酸序列如SEQ ID NO:21所示；
或，氨基酸序列如SEQ ID NO:24所示的7β-HSDH酶突变体的编码基因的核苷酸序列如SEQ ID NO:23所示；
或，氨基酸序列如SEQ ID NO:26所示的7β-HSDH酶突变体的编码基因的核苷酸序列如SEQ ID NO:25所示；
或，氨基酸序列如SEQ ID NO:28所示的7β-HSDH酶突变体的编码基因的核苷酸序列如SEQ ID NO:27所示；
或，氨基酸序列如SEQ ID NO:30所示的7β-HSDH酶突变体的编码基因的核苷酸序列如SEQ ID NO:29所示；
或，氨基酸序列如SEQ ID NO:32所示的7β-HSDH酶突变体的编码基因的核苷酸序列如SEQ ID NO:31所示。
根据现有公共知识，任何基因经操作或者改造后连入各类表达载体，转化至合适宿主细胞，经适当条件诱导均能过量表达目的蛋白。
因此，又一方面，本发明还请求保护含有上述编码基因的载体。
具体地，所述的载体可以为各种表达载体，包括但不限于pET表达载体、pCW表达载体、pUC表达载体或pPIC9k表达载体中的任意一种表达载体。
又一方面，本发明还请求保护含有上述编码基因的宿主细胞。
具体地，所述的宿主细胞可以为任一种合适的宿主细胞，包括但不限于大肠杆菌、毕赤酵母、链霉菌或枯草芽孢杆菌中的任意一种宿主细胞。
又一方面，本发明还请求保护上述7β-HSDH酶突变体、编码基因、载体、宿主细胞在制备24-去甲熊脱氧胆酸中的应用。
再一方面，本发明还提供了一种制备24-去甲熊脱氧胆酸的方法，包括以下步骤：
S1.配置反应体系，包含：1-10g/L上述的7β-HSDH酶突变体，50mM pH6.0-8.0磷酸钠缓冲液，0.2mM NADP+，10-50g/L 24-去甲-7-酮胆石酸，5-40g/L葡萄糖，0.1-10g/L葡萄糖脱氢酶；控制反应体系温度为25-40℃，pH值为6.0-8.0，进行搅拌反应；
S2.反应24h后进行HPLC检测，即得24-去甲熊去氧胆酸。
优选地，所述的方法包括以下步骤：
S1.配置反应体系，包含：1g/L上述的7β-HSDH酶突变体，50mM pH7.0磷酸钠缓冲液，0.2mM NADP+，10-50g/L 24-去甲-7-酮胆石酸，5-40g/L葡萄糖，0.5g/L葡萄糖脱氢酶；控制反应体系温度为30℃，pH值为7.0，进行搅拌反应；
S2.反应24h后进行HPLC检测，即得24-去甲熊去氧胆酸。
反应产物经HPLC检测，反应转化率＞90％。从而可证明该酶突变体可作为生物催化剂，转化底物24-去甲-7-酮胆石酸生成24-去甲熊去氧胆酸。
此外，可进行上述生物催化反应的酶包括纯酶、相应的重组菌休止细胞、粗酶液或者粗酶粉等其他存在形态。
相对于现有技术，本发明具有以下有益效果：
本发明构建的7β-HSDH酶突变体与野生型酶相比，突变体的单位酶活得到大大提高，从而可以显著加快反应速率、降低酶的使用量，降低反应时间和生产成本，可在室温、24h之内将10-50g/L的底物24-去甲-7-酮胆石酸完全转化为24-去甲熊去氧胆酸，转化率>90％。本发明构建的7β-HSDH酶突变体能扫清24-去甲熊去氧胆酸的工业化酶法生产的障碍，具有广阔的工业化应用前景。
具体实施方式
下面结合具体实施例，对本发明作进一步详细的阐述，下述实施例不用于限制本发明，仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明，实施例中未注明具体条件的实验方法，通常按照常规条件，下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例中，未注明具体条件的实验方法，通常按常规条件，如《分子克隆实验指南》(J.萨姆布鲁克，D.W.拉塞尔著，黄培堂，汪嘉玺，朱厚础等译，第三版，北京：科学出版社，2002)中所述的方法进行。
实施例一原核表达体系的构建
7β-HSDH基因片段由常州基宇生物技术有限公司合成，并重组到PUC57载体上。经限制性内切酶NdeI和HindIII(购自New England Biolabs公司，NEB)在37℃双酶切4h后，1％琼脂糖凝胶电泳分离并进行切胶回收(胶回收试剂盒购自天根生化科技(北京)有限公司)。随后与同样经过双酶切的表达载体pET21a(+)(Novagen公司)，在T4 DNA连接酶(购自Takara公司)作用下置于低温连接仪里连接过夜。连接液转化DH5a感受态细胞(购自天根生化科技(北京)有限公司)，并进行菌落PCR筛选和测序验证，从而得到阳性重组质粒7β-HSDH-pET21a(+)。
将阳性重组质粒7β-HSDH-pET21a(+)转化表达宿主菌BL21(DE3)(购自天根生化科技(北京)有限公司)，得到原核表达菌株7β-HSDH-pET21a(+)/BL21(DE3)，作为后续定向进化和发酵的原代菌株。
用于NADPH再生的葡萄糖脱氢酶(GDH，来源于B.subtilis)由常州基宇生物技术有限公司合成，后续重组表达质粒的构建同7β-HSDH-pET21a(+)质粒的构建，转入BL21(DE3)中后得到表达菌株。
实施例二酶的摇瓶发酵制备酶冻干粉
上述构建的表达菌株7β-HSDH-pET21a(+)/BL21(DE3)，在加有终浓度为100μg/mL氨苄青霉素的5mL LB液体培养基【10g/L胰蛋白胨(OXIOD)，5g/L酵母粉(OXIOD)，10g/L氯化钠(国药试剂)】中于37℃、200rpm振荡培养过夜后，按1％(V/V)比例接种于含有终浓度为100μg/mL氨苄青霉素的400mL LB液体培养基中，于37℃、200rpm振荡培养。待OD600在0.8-1.0之间时，加入终浓度为0.1mM的诱导剂IPTG(异丙基-β-D-硫代半乳糖苷，IPTG)，并在30℃诱导过夜。菌体在4℃、8000rpm条件下离心收集，然后悬浮于50mM pH7.0磷酸钠缓冲液中，超声破碎(200W，3s/5s，20min)，于4℃、12000rpm离心20min，取上清进行冷冻干燥，即得酶冻干粉。
实施例三突变体的构建和筛选
突变体的构建：采用大分子建模技术预测出可能有益的突变位点为N237、S240两个位点，分别对这两个位点进行定点突变(N237K、N237H、N237Q、S240N、S240R、S240E、S240Q)。随后以7β-HSDH-pET28a(+)重组质粒为模板，使用合成的相应引物，第一次PCR扩增突变DNA片段，然后以PCR将所得片段作为模板，第二次PCR扩增全长得到7β-HSDH的突变基因。(具体突变操作参照stratagene公司的
Figure BDA0003310795400000071
Site-Directed Mutagenesis Kit操作说明)。
其中：
N237K位点突变(第237位点天冬酰胺突变为赖氨酸)
正向引物(SEQ ID NO:33)：5'TCGCCGGTCAACGTAAAAAAGATAGCGTCC 3'，
反向引物(SEQ ID NO:34)：5'GGACGCTATCTTTTTTACGTTGACCGGCGA 3'；
N237H位点突变(第237位点天冬酰胺突变为组氨酸)
正向引物(SEQ ID NO:35)：5'TCGCCGGTCAACGTCATAAAGATAGCGTCCAT 3'，
反向引物(SEQ ID NO:36)：5'ATGGACGCTATCTTTATGACGTTGACCGGCGA 3'；
N237Q位点突变(第237位点天冬酰胺突变为谷氨酰胺)
正向引物(SEQ ID NO:37)：5'TCGCCGGTCAACGTCAGAAAGATAGCGTCC 3'，
反向引物(SEQ ID NO:38)：5'GGACGCTATCTTTCTGACGTTGACCGGCGA 3'；
S240N位点突变(第240位点丝氨酸突变为天冬酰胺)
正向引物(SEQ ID NO:39)：5'CGGTCAACGTAATAAAGATAATGTCCATGACTGG 3'，
反向引物(SEQ ID NO:40)：5'CCAGTCATGGACATTATCTTTATTACGTTGACCG 3'；
S240R位点突变(第240位点丝氨酸突变为精氨酸)
正向引物(SEQ ID NO:41)：5'CGTAATAAAGATCGAGTCCATGACTGG 3'，
反向引物(SEQ ID NO:42)：5'CCAGTCATGGACTCGATCTTTATTACG 3'；
S240E位点突变(第240位点丝氨酸突变为谷氨酸)
正向引物(SEQ ID NO:43)：5'CGTAATAAAGATGAAGTCCATGACTGG 3'，
反向引物(SEQ ID NO:44)：5'CCAGTCATGGACTTCATCTTTATTACG 3'；
S240Q位点突变(第240位点丝氨酸突变为谷氨酰胺)
正向引物(SEQ ID NO:45)：5'CGTAATAAAGATCAGGTCCATGACTGG 3'，
反向引物(SEQ ID NO:46)：5'CCAGTCATGGACCTGATCTTTATTACG 3'。
突变体培养：将上述突变得到的质粒转化BL21(DE3)宿主菌后，涂布于含100μg/mL氨苄青霉素的LB固体培养基上，37℃倒置培养过夜，随后从平板上挑取单克隆置于含有100μg/mL氨苄青霉素的5mL LB液体培养基中进行培养。过夜培养的菌液再按1％(V/V)比例接种于含有100μg/mL氨苄青霉素的100mL LB液体培养基中，于37℃、200rpm振荡培养4h后加入终浓度为0.1mM的IPTG进行诱导，于30℃培养过夜。于4℃、8000rpm离心10min收集菌体，用50mM pH7.0磷酸钠缓冲液悬浮后超声破碎(200W，3s/5s，30min)，于4℃、12000rpm离心20min，取上清进行单位酶活测定。
突变体的活性筛选：底物浓度2g/L(DMSO配置)，NADPH 0.2mM，加入适量上述制备的上清液，用50mM pH7.0磷酸钠缓冲液补充体积至3mL，室温反应，实时检测NADPH在340nm处吸光值的变化。根据NADPH的消耗量和降低速率来计算突变体的单位酶活(U/mg)。1U定义为1min内消耗1μmol NADPH所需的酶量。
试验结果如下表1所示。
表1野生型与不同突变体的酶活
氨基酸编号 野生型/突变体名称 单位酶活(U/mg) 提高倍数
SEQ ID NO:2 野生型7β-HSDH 0.35 ---
SEQ ID NO:4 N237K 0.13 ---
SEQ ID NO:6 N237H 0.68 1.94
SEQ ID NO:8 N237Q 0.54 1.54
SEQ ID NO:10 S240N 1.00 2.86
SEQ ID NO:12 S240R 1.11 3.17
SEQ ID NO:14 S240E 0.54 1.54
SEQ ID NO:16 S240Q 0.95 2.71
以上结果显示，突变体酶活得到显著提高的克隆中含有的突变位点如下：位点237的天冬酰胺(N)突变为组氨酸(H)；位点237的天冬酰胺(N)突变为谷氨酰胺(Q)；位点240的丝氨酸(S)突变为天冬酰胺(N)；位点240的丝氨酸(S)突变为精氨酸(R)；位点240的丝氨酸(S)突变为谷氨酸(E)；位点240的丝氨酸(S)突变为谷氨酰胺(Q)。
其中，当SEQ ID NO:2所示的氨基酸序列的第237位由天冬酰胺突变为组氨酸，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:6所示，相应地，其编码基因的核苷酸序列如SEQ ID NO:5所示。
当SEQ ID NO:2所示的氨基酸序列的第237位由天冬酰胺突变为谷氨酰胺，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:8所示，相应地，其编码基因的核苷酸序列如SEQ ID NO:7所示。
当SEQ ID NO:2所示的氨基酸序列的第240位由丝氨酸突变为天冬酰胺，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:10所示，相应地，其编码基因的核苷酸序列如SEQ ID NO:9所示。
当SEQ ID NO:2所示的氨基酸序列的第240位由丝氨酸突变为精氨酸，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:12所示，相应地，其编码基因的核苷酸序列如SEQID NO:11所示。
当SEQ ID NO:2所示的氨基酸序列的第240位由丝氨酸突变为谷氨酸，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:14所示，相应地，其编码基因的核苷酸序列如SEQID NO:13所示。
当SEQ ID NO:2所示的氨基酸序列的第240位由丝氨酸突变为谷氨酰胺，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:16所示，相应地，其编码基因的核苷酸序列如SEQ ID NO:15所示。
实施例四突变位点的两两联合突变
将上述筛选到的活性得到显著提高的突变体位点，进行两两联合突变，活性筛选方法和酶活定义方法同上。试验结果如下表2所示。
表2野生型与不同突变体的酶活
氨基酸编号 野生型/突变体名称 单位酶活(U/mg) 提高倍数
SEQ ID NO:2 野生型7β-HSDH 0.35 ---
SEQ ID NO:18 N237H/S240N 2.61 7.46
SEQ ID NO:20 N237H/S240R 5.07 14.48
SEQ ID NO:22 N237H/S240E 1.51 4.31
SEQ ID NO:24 N237H/S240Q 2.70 7.71
SEQ ID NO:26 N237Q/S240N 1.58 4.51
SEQ ID NO:28 N237Q/S240R 2.31 6.60
SEQ ID NO:30 N237Q/S240E 1.13 3.23
SEQ ID NO:32 N237Q/S240Q 1.68 4.80
其中，当SEQ ID NO:2所示的氨基酸序列的第237位由天冬酰胺突变为组氨酸，第240位由丝氨酸突变为天冬酰胺，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:18所示，相应地，其编码基因的核苷酸序列如SEQ ID NO:17所示。
当SEQ ID NO:2所示的氨基酸序列的第237位由天冬酰胺突变为组氨酸，第240位由丝氨酸突变为精氨酸，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:20所示，相应地，其编码基因的核苷酸序列如SEQ ID NO:19所示。
当SEQ ID NO:2所示的氨基酸序列的第237位由天冬酰胺突变为组氨酸，第240位由丝氨酸突变为谷氨酸，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:22所示，相应地，其编码基因的核苷酸序列如SEQ ID NO:21所示。
当SEQ ID NO:2所示的氨基酸序列的第237位由天冬酰胺突变为组氨酸，第240位由丝氨酸突变为谷氨酰胺，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:24所示，相应地，其编码基因的核苷酸序列如SEQ ID NO:23所示。
当SEQ ID NO:2所示的氨基酸序列的第237位由天冬酰胺突变为谷氨酰胺，第240位由丝氨酸突变为天冬酰胺，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:26所示，相应地，其编码基因的核苷酸序列如SEQ ID NO:25所示。
当SEQ ID NO:2所示的氨基酸序列的第237位由天冬酰胺突变为谷氨酰胺，第240位由丝氨酸突变为精氨酸，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:28所示，相应地，其编码基因的核苷酸序列如SEQ ID NO:27所示。
当SEQ ID NO:2所示的氨基酸序列的第237位由天冬酰胺突变为谷氨酰胺，第240位由丝氨酸突变为谷氨酸，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:30所示，相应地，其编码基因的核苷酸序列如SEQ ID NO:29所示。
当SEQ ID NO:2所示的氨基酸序列的第237位由天冬酰胺突变为谷氨酰胺，第240位由丝氨酸突变为谷氨酰胺，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:32所示，相应地，其编码基因的核苷酸序列如SEQ ID NO:31所示。
实施例五突变体的生物催化
将1.6g底物24-去甲-7-酮胆石酸溶解于32mL乙酸正丁酯中，待底物完全溶解后，依次加入45mL 50mM pH7.0磷酸钠缓冲液、1.15g葡萄糖、0.2mM NADP二钠盐、1g/L酶突变体和0.5g/L GDH酶(葡萄糖脱氢酶)粉，在30℃恒温机械搅拌器下搅拌反应。反应期间使用2M氢氧化钠溶液实时维持pH值在7.0。反应24h后进行HPLC检测，不同突变体的底物转化率和产物生成率见下表3。
表3野生型与不同突变体的底物转化率和产物生成率
Figure BDA0003310795400000101
Figure BDA0003310795400000111
最后应当说明的是，以上内容仅用以说明本发明的技术方案，而非对本发明保护范围的限制，本领域的普通技术人员对本发明的技术方案进行的简单修改或者等同替换，均不脱离本发明技术方案的实质和范围。
序列表
<110> 中山百灵生物技术股份有限公司
<120> 一种7β-HSDH酶突变体及其编码基因和应用
<160> 46
<170> SIPOSequenceListing 1.0
<210> 1
<211> 792
<212> DNA
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 1
atgaatctgc gtgaaaaata cggtgaatgg ggtctgatcc tgggtgctac ggaaggtgtc 60
ggtaaagcgt tctgtgaaaa aatcgccgcg ggcggtatga acgtggttat ggtcggccgt 120
cgcgaagaaa aactgaatgt gctggcaggc gaaattcgtg aaacctatgg tgttgaaacg 180
aaagtcgtgc gtgctgattt ttcccagccg ggtgcagcag aaaccgtctt cgcagctacg 240
gaaggcctgg acatgggttt tatgtcttac gtggcctgcc tgcatagttt cggtaaaatt 300
caagataccc cgtgggaaaa acacgaagca atgatcaacg tgaatgttgt cacgtttctg 360
aaatgtttcc atcactatat gcgtatcttt gcggcccagg atcgcggtgc cgtgattaac 420
gttagctcta tgaccggcat cagttcctca ccgtggaatg gtcaatacgg cgcgggtaaa 480
gccttcattc tgaaaatgac cgaagcagtt gcttgcgaat gtgaaggcac gggtgtggac 540
gttgaagtca tcaccctggg taccacgctg acgccgtcgc tgctgagcaa cctgccgggc 600
ggtccgcagg gtgaagcagt gatgaaaatt gctctgaccc cggaagaatg cgttgatgaa 660
gcgtttgaaa aactgggcaa agaactgtca gttatcgccg gtcaacgtaa taaagatagc 720
gtccatgact ggaaagcaaa ccacaccgaa gacgaataca tccgctacat gggctcattt 780
taccgtgact aa 792
<210> 2
<211> 263
<212> PRT
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 2
Met Asn Leu Arg Glu Lys Tyr Gly Glu Trp Gly Leu Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ala Gly Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Lys Leu Asn Val Leu
35 40 45
Ala Gly Glu Ile Arg Glu Thr Tyr Gly Val Glu Thr Lys Val Val Arg
50 55 60
Ala Asp Phe Ser Gln Pro Gly Ala Ala Glu Thr Val Phe Ala Ala Thr
65 70 75 80
Glu Gly Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Leu His Ser
85 90 95
Phe Gly Lys Ile Gln Asp Thr Pro Trp Glu Lys His Glu Ala Met Ile
100 105 110
Asn Val Asn Val Val Thr Phe Leu Lys Cys Phe His His Tyr Met Arg
115 120 125
Ile Phe Ala Ala Gln Asp Arg Gly Ala Val Ile Asn Val Ser Ser Met
130 135 140
Thr Gly Ile Ser Ser Ser Pro Trp Asn Gly Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ala Phe Ile Leu Lys Met Thr Glu Ala Val Ala Cys Glu Cys Glu Gly
165 170 175
Thr Gly Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Leu Thr Pro
180 185 190
Ser Leu Leu Ser Asn Leu Pro Gly Gly Pro Gln Gly Glu Ala Val Met
195 200 205
Lys Ile Ala Leu Thr Pro Glu Glu Cys Val Asp Glu Ala Phe Glu Lys
210 215 220
Leu Gly Lys Glu Leu Ser Val Ile Ala Gly Gln Arg Asn Lys Asp Ser
225 230 235 240
Val His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Tyr Ile Arg Tyr
245 250 255
Met Gly Ser Phe Tyr Arg Asp
260
<210> 3
<211> 792
<212> DNA
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 3
atgaatctgc gtgaaaaata cggtgaatgg ggtctgatcc tgggtgctac ggaaggtgtc 60
ggtaaagcgt tctgtgaaaa aatcgccgcg ggcggtatga acgtggttat ggtcggccgt 120
cgcgaagaaa aactgaatgt gctggcaggc gaaattcgtg aaacctatgg tgttgaaacg 180
aaagtcgtgc gtgctgattt ttcccagccg ggtgcagcag aaaccgtctt cgcagctacg 240
gaaggcctgg acatgggttt tatgtcttac gtggcctgcc tgcatagttt cggtaaaatt 300
caagataccc cgtgggaaaa acacgaagca atgatcaacg tgaatgttgt cacgtttctg 360
aaatgtttcc atcactatat gcgtatcttt gcggcccagg atcgcggtgc cgtgattaac 420
gttagctcta tgaccggcat cagttcctca ccgtggaatg gtcaatacgg cgcgggtaaa 480
gccttcattc tgaaaatgac cgaagcagtt gcttgcgaat gtgaaggcac gggtgtggac 540
gttgaagtca tcaccctggg taccacgctg acgccgtcgc tgctgagcaa cctgccgggc 600
ggtccgcagg gtgaagcagt gatgaaaatt gctctgaccc cggaagaatg cgttgatgaa 660
gcgtttgaaa aactgggcaa agaactgtca gttatcgccg gtcaacgtaa aaaagatagc 720
gtccatgact ggaaagcaaa ccacaccgaa gacgaataca tccgctacat gggctcattt 780
taccgtgact aa 792
<210> 4
<211> 263
<212> PRT
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 4
Met Asn Leu Arg Glu Lys Tyr Gly Glu Trp Gly Leu Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ala Gly Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Lys Leu Asn Val Leu
35 40 45
Ala Gly Glu Ile Arg Glu Thr Tyr Gly Val Glu Thr Lys Val Val Arg
50 55 60
Ala Asp Phe Ser Gln Pro Gly Ala Ala Glu Thr Val Phe Ala Ala Thr
65 70 75 80
Glu Gly Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Leu His Ser
85 90 95
Phe Gly Lys Ile Gln Asp Thr Pro Trp Glu Lys His Glu Ala Met Ile
100 105 110
Asn Val Asn Val Val Thr Phe Leu Lys Cys Phe His His Tyr Met Arg
115 120 125
Ile Phe Ala Ala Gln Asp Arg Gly Ala Val Ile Asn Val Ser Ser Met
130 135 140
Thr Gly Ile Ser Ser Ser Pro Trp Asn Gly Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ala Phe Ile Leu Lys Met Thr Glu Ala Val Ala Cys Glu Cys Glu Gly
165 170 175
Thr Gly Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Leu Thr Pro
180 185 190
Ser Leu Leu Ser Asn Leu Pro Gly Gly Pro Gln Gly Glu Ala Val Met
195 200 205
Lys Ile Ala Leu Thr Pro Glu Glu Cys Val Asp Glu Ala Phe Glu Lys
210 215 220
Leu Gly Lys Glu Leu Ser Val Ile Ala Gly Gln Arg Lys Lys Asp Ser
225 230 235 240
Val His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Tyr Ile Arg Tyr
245 250 255
Met Gly Ser Phe Tyr Arg Asp
260
<210> 5
<211> 792
<212> DNA
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 5
atgaatctgc gtgaaaaata cggtgaatgg ggtctgatcc tgggtgctac ggaaggtgtc 60
ggtaaagcgt tctgtgaaaa aatcgccgcg ggcggtatga acgtggttat ggtcggccgt 120
cgcgaagaaa aactgaatgt gctggcaggc gaaattcgtg aaacctatgg tgttgaaacg 180
aaagtcgtgc gtgctgattt ttcccagccg ggtgcagcag aaaccgtctt cgcagctacg 240
gaaggcctgg acatgggttt tatgtcttac gtggcctgcc tgcatagttt cggtaaaatt 300
caagataccc cgtgggaaaa acacgaagca atgatcaacg tgaatgttgt cacgtttctg 360
aaatgtttcc atcactatat gcgtatcttt gcggcccagg atcgcggtgc cgtgattaac 420
gttagctcta tgaccggcat cagttcctca ccgtggaatg gtcaatacgg cgcgggtaaa 480
gccttcattc tgaaaatgac cgaagcagtt gcttgcgaat gtgaaggcac gggtgtggac 540
gttgaagtca tcaccctggg taccacgctg acgccgtcgc tgctgagcaa cctgccgggc 600
ggtccgcagg gtgaagcagt gatgaaaatt gctctgaccc cggaagaatg cgttgatgaa 660
gcgtttgaaa aactgggcaa agaactgtca gttatcgccg gtcaacgtca taaagatagc 720
gtccatgact ggaaagcaaa ccacaccgaa gacgaataca tccgctacat gggctcattt 780
taccgtgact aa 792
<210> 6
<211> 263
<212> PRT
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 6
Met Asn Leu Arg Glu Lys Tyr Gly Glu Trp Gly Leu Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ala Gly Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Lys Leu Asn Val Leu
35 40 45
Ala Gly Glu Ile Arg Glu Thr Tyr Gly Val Glu Thr Lys Val Val Arg
50 55 60
Ala Asp Phe Ser Gln Pro Gly Ala Ala Glu Thr Val Phe Ala Ala Thr
65 70 75 80
Glu Gly Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Leu His Ser
85 90 95
Phe Gly Lys Ile Gln Asp Thr Pro Trp Glu Lys His Glu Ala Met Ile
100 105 110
Asn Val Asn Val Val Thr Phe Leu Lys Cys Phe His His Tyr Met Arg
115 120 125
Ile Phe Ala Ala Gln Asp Arg Gly Ala Val Ile Asn Val Ser Ser Met
130 135 140
Thr Gly Ile Ser Ser Ser Pro Trp Asn Gly Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ala Phe Ile Leu Lys Met Thr Glu Ala Val Ala Cys Glu Cys Glu Gly
165 170 175
Thr Gly Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Leu Thr Pro
180 185 190
Ser Leu Leu Ser Asn Leu Pro Gly Gly Pro Gln Gly Glu Ala Val Met
195 200 205
Lys Ile Ala Leu Thr Pro Glu Glu Cys Val Asp Glu Ala Phe Glu Lys
210 215 220
Leu Gly Lys Glu Leu Ser Val Ile Ala Gly Gln Arg His Lys Asp Ser
225 230 235 240
Val His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Tyr Ile Arg Tyr
245 250 255
Met Gly Ser Phe Tyr Arg Asp
260
<210> 7
<211> 792
<212> DNA
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 7
atgaatctgc gtgaaaaata cggtgaatgg ggtctgatcc tgggtgctac ggaaggtgtc 60
ggtaaagcgt tctgtgaaaa aatcgccgcg ggcggtatga acgtggttat ggtcggccgt 120
cgcgaagaaa aactgaatgt gctggcaggc gaaattcgtg aaacctatgg tgttgaaacg 180
aaagtcgtgc gtgctgattt ttcccagccg ggtgcagcag aaaccgtctt cgcagctacg 240
gaaggcctgg acatgggttt tatgtcttac gtggcctgcc tgcatagttt cggtaaaatt 300
caagataccc cgtgggaaaa acacgaagca atgatcaacg tgaatgttgt cacgtttctg 360
aaatgtttcc atcactatat gcgtatcttt gcggcccagg atcgcggtgc cgtgattaac 420
gttagctcta tgaccggcat cagttcctca ccgtggaatg gtcaatacgg cgcgggtaaa 480
gccttcattc tgaaaatgac cgaagcagtt gcttgcgaat gtgaaggcac gggtgtggac 540
gttgaagtca tcaccctggg taccacgctg acgccgtcgc tgctgagcaa cctgccgggc 600
ggtccgcagg gtgaagcagt gatgaaaatt gctctgaccc cggaagaatg cgttgatgaa 660
gcgtttgaaa aactgggcaa agaactgtca gttatcgccg gtcaacgtca gaaagatagc 720
gtccatgact ggaaagcaaa ccacaccgaa gacgaataca tccgctacat gggctcattt 780
taccgtgact aa 792
<210> 8
<211> 263
<212> PRT
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 8
Met Asn Leu Arg Glu Lys Tyr Gly Glu Trp Gly Leu Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ala Gly Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Lys Leu Asn Val Leu
35 40 45
Ala Gly Glu Ile Arg Glu Thr Tyr Gly Val Glu Thr Lys Val Val Arg
50 55 60
Ala Asp Phe Ser Gln Pro Gly Ala Ala Glu Thr Val Phe Ala Ala Thr
65 70 75 80
Glu Gly Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Leu His Ser
85 90 95
Phe Gly Lys Ile Gln Asp Thr Pro Trp Glu Lys His Glu Ala Met Ile
100 105 110
Asn Val Asn Val Val Thr Phe Leu Lys Cys Phe His His Tyr Met Arg
115 120 125
Ile Phe Ala Ala Gln Asp Arg Gly Ala Val Ile Asn Val Ser Ser Met
130 135 140
Thr Gly Ile Ser Ser Ser Pro Trp Asn Gly Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ala Phe Ile Leu Lys Met Thr Glu Ala Val Ala Cys Glu Cys Glu Gly
165 170 175
Thr Gly Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Leu Thr Pro
180 185 190
Ser Leu Leu Ser Asn Leu Pro Gly Gly Pro Gln Gly Glu Ala Val Met
195 200 205
Lys Ile Ala Leu Thr Pro Glu Glu Cys Val Asp Glu Ala Phe Glu Lys
210 215 220
Leu Gly Lys Glu Leu Ser Val Ile Ala Gly Gln Arg Gln Lys Asp Ser
225 230 235 240
Val His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Tyr Ile Arg Tyr
245 250 255
Met Gly Ser Phe Tyr Arg Asp
260
<210> 9
<211> 792
<212> DNA
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 9
atgaatctgc gtgaaaaata cggtgaatgg ggtctgatcc tgggtgctac ggaaggtgtc 60
ggtaaagcgt tctgtgaaaa aatcgccgcg ggcggtatga acgtggttat ggtcggccgt 120
cgcgaagaaa aactgaatgt gctggcaggc gaaattcgtg aaacctatgg tgttgaaacg 180
aaagtcgtgc gtgctgattt ttcccagccg ggtgcagcag aaaccgtctt cgcagctacg 240
gaaggcctgg acatgggttt tatgtcttac gtggcctgcc tgcatagttt cggtaaaatt 300
caagataccc cgtgggaaaa acacgaagca atgatcaacg tgaatgttgt cacgtttctg 360
aaatgtttcc atcactatat gcgtatcttt gcggcccagg atcgcggtgc cgtgattaac 420
gttagctcta tgaccggcat cagttcctca ccgtggaatg gtcaatacgg cgcgggtaaa 480
gccttcattc tgaaaatgac cgaagcagtt gcttgcgaat gtgaaggcac gggtgtggac 540
gttgaagtca tcaccctggg taccacgctg acgccgtcgc tgctgagcaa cctgccgggc 600
ggtccgcagg gtgaagcagt gatgaaaatt gctctgaccc cggaagaatg cgttgatgaa 660
gcgtttgaaa aactgggcaa agaactgtca gttatcgccg gtcaacgtaa taaagataat 720
gtccatgact ggaaagcaaa ccacaccgaa gacgaataca tccgctacat gggctcattt 780
taccgtgact aa 792
<210> 10
<211> 263
<212> PRT
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 10
Met Asn Leu Arg Glu Lys Tyr Gly Glu Trp Gly Leu Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ala Gly Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Lys Leu Asn Val Leu
35 40 45
Ala Gly Glu Ile Arg Glu Thr Tyr Gly Val Glu Thr Lys Val Val Arg
50 55 60
Ala Asp Phe Ser Gln Pro Gly Ala Ala Glu Thr Val Phe Ala Ala Thr
65 70 75 80
Glu Gly Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Leu His Ser
85 90 95
Phe Gly Lys Ile Gln Asp Thr Pro Trp Glu Lys His Glu Ala Met Ile
100 105 110
Asn Val Asn Val Val Thr Phe Leu Lys Cys Phe His His Tyr Met Arg
115 120 125
Ile Phe Ala Ala Gln Asp Arg Gly Ala Val Ile Asn Val Ser Ser Met
130 135 140
Thr Gly Ile Ser Ser Ser Pro Trp Asn Gly Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ala Phe Ile Leu Lys Met Thr Glu Ala Val Ala Cys Glu Cys Glu Gly
165 170 175
Thr Gly Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Leu Thr Pro
180 185 190
Ser Leu Leu Ser Asn Leu Pro Gly Gly Pro Gln Gly Glu Ala Val Met
195 200 205
Lys Ile Ala Leu Thr Pro Glu Glu Cys Val Asp Glu Ala Phe Glu Lys
210 215 220
Leu Gly Lys Glu Leu Ser Val Ile Ala Gly Gln Arg Asn Lys Asp Asn
225 230 235 240
Val His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Tyr Ile Arg Tyr
245 250 255
Met Gly Ser Phe Tyr Arg Asp
260
<210> 11
<211> 792
<212> DNA
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 11
atgaatctgc gtgaaaaata cggtgaatgg ggtctgatcc tgggtgctac ggaaggtgtc 60
ggtaaagcgt tctgtgaaaa aatcgccgcg ggcggtatga acgtggttat ggtcggccgt 120
cgcgaagaaa aactgaatgt gctggcaggc gaaattcgtg aaacctatgg tgttgaaacg 180
aaagtcgtgc gtgctgattt ttcccagccg ggtgcagcag aaaccgtctt cgcagctacg 240
gaaggcctgg acatgggttt tatgtcttac gtggcctgcc tgcatagttt cggtaaaatt 300
caagataccc cgtgggaaaa acacgaagca atgatcaacg tgaatgttgt cacgtttctg 360
aaatgtttcc atcactatat gcgtatcttt gcggcccagg atcgcggtgc cgtgattaac 420
gttagctcta tgaccggcat cagttcctca ccgtggaatg gtcaatacgg cgcgggtaaa 480
gccttcattc tgaaaatgac cgaagcagtt gcttgcgaat gtgaaggcac gggtgtggac 540
gttgaagtca tcaccctggg taccacgctg acgccgtcgc tgctgagcaa cctgccgggc 600
ggtccgcagg gtgaagcagt gatgaaaatt gctctgaccc cggaagaatg cgttgatgaa 660
gcgtttgaaa aactgggcaa agaactgtca gttatcgccg gtcaacgtaa taaagatcga 720
gtccatgact ggaaagcaaa ccacaccgaa gacgaataca tccgctacat gggctcattt 780
taccgtgact aa 792
<210> 12
<211> 263
<212> PRT
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 12
Met Asn Leu Arg Glu Lys Tyr Gly Glu Trp Gly Leu Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ala Gly Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Lys Leu Asn Val Leu
35 40 45
Ala Gly Glu Ile Arg Glu Thr Tyr Gly Val Glu Thr Lys Val Val Arg
50 55 60
Ala Asp Phe Ser Gln Pro Gly Ala Ala Glu Thr Val Phe Ala Ala Thr
65 70 75 80
Glu Gly Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Leu His Ser
85 90 95
Phe Gly Lys Ile Gln Asp Thr Pro Trp Glu Lys His Glu Ala Met Ile
100 105 110
Asn Val Asn Val Val Thr Phe Leu Lys Cys Phe His His Tyr Met Arg
115 120 125
Ile Phe Ala Ala Gln Asp Arg Gly Ala Val Ile Asn Val Ser Ser Met
130 135 140
Thr Gly Ile Ser Ser Ser Pro Trp Asn Gly Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ala Phe Ile Leu Lys Met Thr Glu Ala Val Ala Cys Glu Cys Glu Gly
165 170 175
Thr Gly Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Leu Thr Pro
180 185 190
Ser Leu Leu Ser Asn Leu Pro Gly Gly Pro Gln Gly Glu Ala Val Met
195 200 205
Lys Ile Ala Leu Thr Pro Glu Glu Cys Val Asp Glu Ala Phe Glu Lys
210 215 220
Leu Gly Lys Glu Leu Ser Val Ile Ala Gly Gln Arg Asn Lys Asp Arg
225 230 235 240
Val His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Tyr Ile Arg Tyr
245 250 255
Met Gly Ser Phe Tyr Arg Asp
260
<210> 13
<211> 792
<212> DNA
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 13
atgaatctgc gtgaaaaata cggtgaatgg ggtctgatcc tgggtgctac ggaaggtgtc 60
ggtaaagcgt tctgtgaaaa aatcgccgcg ggcggtatga acgtggttat ggtcggccgt 120
cgcgaagaaa aactgaatgt gctggcaggc gaaattcgtg aaacctatgg tgttgaaacg 180
aaagtcgtgc gtgctgattt ttcccagccg ggtgcagcag aaaccgtctt cgcagctacg 240
gaaggcctgg acatgggttt tatgtcttac gtggcctgcc tgcatagttt cggtaaaatt 300
caagataccc cgtgggaaaa acacgaagca atgatcaacg tgaatgttgt cacgtttctg 360
aaatgtttcc atcactatat gcgtatcttt gcggcccagg atcgcggtgc cgtgattaac 420
gttagctcta tgaccggcat cagttcctca ccgtggaatg gtcaatacgg cgcgggtaaa 480
gccttcattc tgaaaatgac cgaagcagtt gcttgcgaat gtgaaggcac gggtgtggac 540
gttgaagtca tcaccctggg taccacgctg acgccgtcgc tgctgagcaa cctgccgggc 600
ggtccgcagg gtgaagcagt gatgaaaatt gctctgaccc cggaagaatg cgttgatgaa 660
gcgtttgaaa aactgggcaa agaactgtca gttatcgccg gtcaacgtaa taaagatgaa 720
gtccatgact ggaaagcaaa ccacaccgaa gacgaataca tccgctacat gggctcattt 780
taccgtgact aa 792
<210> 14
<211> 263
<212> PRT
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 14
Met Asn Leu Arg Glu Lys Tyr Gly Glu Trp Gly Leu Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ala Gly Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Lys Leu Asn Val Leu
35 40 45
Ala Gly Glu Ile Arg Glu Thr Tyr Gly Val Glu Thr Lys Val Val Arg
50 55 60
Ala Asp Phe Ser Gln Pro Gly Ala Ala Glu Thr Val Phe Ala Ala Thr
65 70 75 80
Glu Gly Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Leu His Ser
85 90 95
Phe Gly Lys Ile Gln Asp Thr Pro Trp Glu Lys His Glu Ala Met Ile
100 105 110
Asn Val Asn Val Val Thr Phe Leu Lys Cys Phe His His Tyr Met Arg
115 120 125
Ile Phe Ala Ala Gln Asp Arg Gly Ala Val Ile Asn Val Ser Ser Met
130 135 140
Thr Gly Ile Ser Ser Ser Pro Trp Asn Gly Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ala Phe Ile Leu Lys Met Thr Glu Ala Val Ala Cys Glu Cys Glu Gly
165 170 175
Thr Gly Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Leu Thr Pro
180 185 190
Ser Leu Leu Ser Asn Leu Pro Gly Gly Pro Gln Gly Glu Ala Val Met
195 200 205
Lys Ile Ala Leu Thr Pro Glu Glu Cys Val Asp Glu Ala Phe Glu Lys
210 215 220
Leu Gly Lys Glu Leu Ser Val Ile Ala Gly Gln Arg Asn Lys Asp Glu
225 230 235 240
Val His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Tyr Ile Arg Tyr
245 250 255
Met Gly Ser Phe Tyr Arg Asp
260
<210> 15
<211> 792
<212> DNA
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 15
atgaatctgc gtgaaaaata cggtgaatgg ggtctgatcc tgggtgctac ggaaggtgtc 60
ggtaaagcgt tctgtgaaaa aatcgccgcg ggcggtatga acgtggttat ggtcggccgt 120
cgcgaagaaa aactgaatgt gctggcaggc gaaattcgtg aaacctatgg tgttgaaacg 180
aaagtcgtgc gtgctgattt ttcccagccg ggtgcagcag aaaccgtctt cgcagctacg 240
gaaggcctgg acatgggttt tatgtcttac gtggcctgcc tgcatagttt cggtaaaatt 300
caagataccc cgtgggaaaa acacgaagca atgatcaacg tgaatgttgt cacgtttctg 360
aaatgtttcc atcactatat gcgtatcttt gcggcccagg atcgcggtgc cgtgattaac 420
gttagctcta tgaccggcat cagttcctca ccgtggaatg gtcaatacgg cgcgggtaaa 480
gccttcattc tgaaaatgac cgaagcagtt gcttgcgaat gtgaaggcac gggtgtggac 540
gttgaagtca tcaccctggg taccacgctg acgccgtcgc tgctgagcaa cctgccgggc 600
ggtccgcagg gtgaagcagt gatgaaaatt gctctgaccc cggaagaatg cgttgatgaa 660
gcgtttgaaa aactgggcaa agaactgtca gttatcgccg gtcaacgtaa taaagatcag 720
gtccatgact ggaaagcaaa ccacaccgaa gacgaataca tccgctacat gggctcattt 780
taccgtgact aa 792
<210> 16
<211> 263
<212> PRT
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 16
Met Asn Leu Arg Glu Lys Tyr Gly Glu Trp Gly Leu Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ala Gly Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Lys Leu Asn Val Leu
35 40 45
Ala Gly Glu Ile Arg Glu Thr Tyr Gly Val Glu Thr Lys Val Val Arg
50 55 60
Ala Asp Phe Ser Gln Pro Gly Ala Ala Glu Thr Val Phe Ala Ala Thr
65 70 75 80
Glu Gly Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Leu His Ser
85 90 95
Phe Gly Lys Ile Gln Asp Thr Pro Trp Glu Lys His Glu Ala Met Ile
100 105 110
Asn Val Asn Val Val Thr Phe Leu Lys Cys Phe His His Tyr Met Arg
115 120 125
Ile Phe Ala Ala Gln Asp Arg Gly Ala Val Ile Asn Val Ser Ser Met
130 135 140
Thr Gly Ile Ser Ser Ser Pro Trp Asn Gly Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ala Phe Ile Leu Lys Met Thr Glu Ala Val Ala Cys Glu Cys Glu Gly
165 170 175
Thr Gly Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Leu Thr Pro
180 185 190
Ser Leu Leu Ser Asn Leu Pro Gly Gly Pro Gln Gly Glu Ala Val Met
195 200 205
Lys Ile Ala Leu Thr Pro Glu Glu Cys Val Asp Glu Ala Phe Glu Lys
210 215 220
Leu Gly Lys Glu Leu Ser Val Ile Ala Gly Gln Arg Asn Lys Asp Gln
225 230 235 240
Val His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Tyr Ile Arg Tyr
245 250 255
Met Gly Ser Phe Tyr Arg Asp
260
<210> 17
<211> 792
<212> DNA
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 17
atgaatctgc gtgaaaaata cggtgaatgg ggtctgatcc tgggtgctac ggaaggtgtc 60
ggtaaagcgt tctgtgaaaa aatcgccgcg ggcggtatga acgtggttat ggtcggccgt 120
cgcgaagaaa aactgaatgt gctggcaggc gaaattcgtg aaacctatgg tgttgaaacg 180
aaagtcgtgc gtgctgattt ttcccagccg ggtgcagcag aaaccgtctt cgcagctacg 240
gaaggcctgg acatgggttt tatgtcttac gtggcctgcc tgcatagttt cggtaaaatt 300
caagataccc cgtgggaaaa acacgaagca atgatcaacg tgaatgttgt cacgtttctg 360
aaatgtttcc atcactatat gcgtatcttt gcggcccagg atcgcggtgc cgtgattaac 420
gttagctcta tgaccggcat cagttcctca ccgtggaatg gtcaatacgg cgcgggtaaa 480
gccttcattc tgaaaatgac cgaagcagtt gcttgcgaat gtgaaggcac gggtgtggac 540
gttgaagtca tcaccctggg taccacgctg acgccgtcgc tgctgagcaa cctgccgggc 600
ggtccgcagg gtgaagcagt gatgaaaatt gctctgaccc cggaagaatg cgttgatgaa 660
gcgtttgaaa aactgggcaa agaactgtca gttatcgccg gtcaacgtca taaagataat 720
gtccatgact ggaaagcaaa ccacaccgaa gacgaataca tccgctacat gggctcattt 780
taccgtgact aa 792
<210> 18
<211> 263
<212> PRT
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 18
Met Asn Leu Arg Glu Lys Tyr Gly Glu Trp Gly Leu Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ala Gly Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Lys Leu Asn Val Leu
35 40 45
Ala Gly Glu Ile Arg Glu Thr Tyr Gly Val Glu Thr Lys Val Val Arg
50 55 60
Ala Asp Phe Ser Gln Pro Gly Ala Ala Glu Thr Val Phe Ala Ala Thr
65 70 75 80
Glu Gly Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Leu His Ser
85 90 95
Phe Gly Lys Ile Gln Asp Thr Pro Trp Glu Lys His Glu Ala Met Ile
100 105 110
Asn Val Asn Val Val Thr Phe Leu Lys Cys Phe His His Tyr Met Arg
115 120 125
Ile Phe Ala Ala Gln Asp Arg Gly Ala Val Ile Asn Val Ser Ser Met
130 135 140
Thr Gly Ile Ser Ser Ser Pro Trp Asn Gly Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ala Phe Ile Leu Lys Met Thr Glu Ala Val Ala Cys Glu Cys Glu Gly
165 170 175
Thr Gly Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Leu Thr Pro
180 185 190
Ser Leu Leu Ser Asn Leu Pro Gly Gly Pro Gln Gly Glu Ala Val Met
195 200 205
Lys Ile Ala Leu Thr Pro Glu Glu Cys Val Asp Glu Ala Phe Glu Lys
210 215 220
Leu Gly Lys Glu Leu Ser Val Ile Ala Gly Gln Arg His Lys Asp Asn
225 230 235 240
Val His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Tyr Ile Arg Tyr
245 250 255
Met Gly Ser Phe Tyr Arg Asp
260
<210> 19
<211> 792
<212> DNA
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 19
atgaatctgc gtgaaaaata cggtgaatgg ggtctgatcc tgggtgctac ggaaggtgtc 60
ggtaaagcgt tctgtgaaaa aatcgccgcg ggcggtatga acgtggttat ggtcggccgt 120
cgcgaagaaa aactgaatgt gctggcaggc gaaattcgtg aaacctatgg tgttgaaacg 180
aaagtcgtgc gtgctgattt ttcccagccg ggtgcagcag aaaccgtctt cgcagctacg 240
gaaggcctgg acatgggttt tatgtcttac gtggcctgcc tgcatagttt cggtaaaatt 300
caagataccc cgtgggaaaa acacgaagca atgatcaacg tgaatgttgt cacgtttctg 360
aaatgtttcc atcactatat gcgtatcttt gcggcccagg atcgcggtgc cgtgattaac 420
gttagctcta tgaccggcat cagttcctca ccgtggaatg gtcaatacgg cgcgggtaaa 480
gccttcattc tgaaaatgac cgaagcagtt gcttgcgaat gtgaaggcac gggtgtggac 540
gttgaagtca tcaccctggg taccacgctg acgccgtcgc tgctgagcaa cctgccgggc 600
ggtccgcagg gtgaagcagt gatgaaaatt gctctgaccc cggaagaatg cgttgatgaa 660
gcgtttgaaa aactgggcaa agaactgtca gttatcgccg gtcaacgtca taaagatcga 720
gtccatgact ggaaagcaaa ccacaccgaa gacgaataca tccgctacat gggctcattt 780
taccgtgact aa 792
<210> 20
<211> 263
<212> PRT
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 20
Met Asn Leu Arg Glu Lys Tyr Gly Glu Trp Gly Leu Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ala Gly Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Lys Leu Asn Val Leu
35 40 45
Ala Gly Glu Ile Arg Glu Thr Tyr Gly Val Glu Thr Lys Val Val Arg
50 55 60
Ala Asp Phe Ser Gln Pro Gly Ala Ala Glu Thr Val Phe Ala Ala Thr
65 70 75 80
Glu Gly Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Leu His Ser
85 90 95
Phe Gly Lys Ile Gln Asp Thr Pro Trp Glu Lys His Glu Ala Met Ile
100 105 110
Asn Val Asn Val Val Thr Phe Leu Lys Cys Phe His His Tyr Met Arg
115 120 125
Ile Phe Ala Ala Gln Asp Arg Gly Ala Val Ile Asn Val Ser Ser Met
130 135 140
Thr Gly Ile Ser Ser Ser Pro Trp Asn Gly Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ala Phe Ile Leu Lys Met Thr Glu Ala Val Ala Cys Glu Cys Glu Gly
165 170 175
Thr Gly Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Leu Thr Pro
180 185 190
Ser Leu Leu Ser Asn Leu Pro Gly Gly Pro Gln Gly Glu Ala Val Met
195 200 205
Lys Ile Ala Leu Thr Pro Glu Glu Cys Val Asp Glu Ala Phe Glu Lys
210 215 220
Leu Gly Lys Glu Leu Ser Val Ile Ala Gly Gln Arg His Lys Asp Arg
225 230 235 240
Val His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Tyr Ile Arg Tyr
245 250 255
Met Gly Ser Phe Tyr Arg Asp
260
<210> 21
<211> 792
<212> DNA
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 21
atgaatctgc gtgaaaaata cggtgaatgg ggtctgatcc tgggtgctac ggaaggtgtc 60
ggtaaagcgt tctgtgaaaa aatcgccgcg ggcggtatga acgtggttat ggtcggccgt 120
cgcgaagaaa aactgaatgt gctggcaggc gaaattcgtg aaacctatgg tgttgaaacg 180
aaagtcgtgc gtgctgattt ttcccagccg ggtgcagcag aaaccgtctt cgcagctacg 240
gaaggcctgg acatgggttt tatgtcttac gtggcctgcc tgcatagttt cggtaaaatt 300
caagataccc cgtgggaaaa acacgaagca atgatcaacg tgaatgttgt cacgtttctg 360
aaatgtttcc atcactatat gcgtatcttt gcggcccagg atcgcggtgc cgtgattaac 420
gttagctcta tgaccggcat cagttcctca ccgtggaatg gtcaatacgg cgcgggtaaa 480
gccttcattc tgaaaatgac cgaagcagtt gcttgcgaat gtgaaggcac gggtgtggac 540
gttgaagtca tcaccctggg taccacgctg acgccgtcgc tgctgagcaa cctgccgggc 600
ggtccgcagg gtgaagcagt gatgaaaatt gctctgaccc cggaagaatg cgttgatgaa 660
gcgtttgaaa aactgggcaa agaactgtca gttatcgccg gtcaacgtca taaagatgaa 720
gtccatgact ggaaagcaaa ccacaccgaa gacgaataca tccgctacat gggctcattt 780
taccgtgact aa 792
<210> 22
<211> 263
<212> PRT
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 22
Met Asn Leu Arg Glu Lys Tyr Gly Glu Trp Gly Leu Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ala Gly Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Lys Leu Asn Val Leu
35 40 45
Ala Gly Glu Ile Arg Glu Thr Tyr Gly Val Glu Thr Lys Val Val Arg
50 55 60
Ala Asp Phe Ser Gln Pro Gly Ala Ala Glu Thr Val Phe Ala Ala Thr
65 70 75 80
Glu Gly Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Leu His Ser
85 90 95
Phe Gly Lys Ile Gln Asp Thr Pro Trp Glu Lys His Glu Ala Met Ile
100 105 110
Asn Val Asn Val Val Thr Phe Leu Lys Cys Phe His His Tyr Met Arg
115 120 125
Ile Phe Ala Ala Gln Asp Arg Gly Ala Val Ile Asn Val Ser Ser Met
130 135 140
Thr Gly Ile Ser Ser Ser Pro Trp Asn Gly Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ala Phe Ile Leu Lys Met Thr Glu Ala Val Ala Cys Glu Cys Glu Gly
165 170 175
Thr Gly Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Leu Thr Pro
180 185 190
Ser Leu Leu Ser Asn Leu Pro Gly Gly Pro Gln Gly Glu Ala Val Met
195 200 205
Lys Ile Ala Leu Thr Pro Glu Glu Cys Val Asp Glu Ala Phe Glu Lys
210 215 220
Leu Gly Lys Glu Leu Ser Val Ile Ala Gly Gln Arg His Lys Asp Glu
225 230 235 240
Val His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Tyr Ile Arg Tyr
245 250 255
Met Gly Ser Phe Tyr Arg Asp
260
<210> 23
<211> 792
<212> DNA
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 23
atgaatctgc gtgaaaaata cggtgaatgg ggtctgatcc tgggtgctac ggaaggtgtc 60
ggtaaagcgt tctgtgaaaa aatcgccgcg ggcggtatga acgtggttat ggtcggccgt 120
cgcgaagaaa aactgaatgt gctggcaggc gaaattcgtg aaacctatgg tgttgaaacg 180
aaagtcgtgc gtgctgattt ttcccagccg ggtgcagcag aaaccgtctt cgcagctacg 240
gaaggcctgg acatgggttt tatgtcttac gtggcctgcc tgcatagttt cggtaaaatt 300
caagataccc cgtgggaaaa acacgaagca atgatcaacg tgaatgttgt cacgtttctg 360
aaatgtttcc atcactatat gcgtatcttt gcggcccagg atcgcggtgc cgtgattaac 420
gttagctcta tgaccggcat cagttcctca ccgtggaatg gtcaatacgg cgcgggtaaa 480
gccttcattc tgaaaatgac cgaagcagtt gcttgcgaat gtgaaggcac gggtgtggac 540
gttgaagtca tcaccctggg taccacgctg acgccgtcgc tgctgagcaa cctgccgggc 600
ggtccgcagg gtgaagcagt gatgaaaatt gctctgaccc cggaagaatg cgttgatgaa 660
gcgtttgaaa aactgggcaa agaactgtca gttatcgccg gtcaacgtca taaagatcag 720
gtccatgact ggaaagcaaa ccacaccgaa gacgaataca tccgctacat gggctcattt 780
taccgtgact aa 792
<210> 24
<211> 263
<212> PRT
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 24
Met Asn Leu Arg Glu Lys Tyr Gly Glu Trp Gly Leu Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ala Gly Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Lys Leu Asn Val Leu
35 40 45
Ala Gly Glu Ile Arg Glu Thr Tyr Gly Val Glu Thr Lys Val Val Arg
50 55 60
Ala Asp Phe Ser Gln Pro Gly Ala Ala Glu Thr Val Phe Ala Ala Thr
65 70 75 80
Glu Gly Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Leu His Ser
85 90 95
Phe Gly Lys Ile Gln Asp Thr Pro Trp Glu Lys His Glu Ala Met Ile
100 105 110
Asn Val Asn Val Val Thr Phe Leu Lys Cys Phe His His Tyr Met Arg
115 120 125
Ile Phe Ala Ala Gln Asp Arg Gly Ala Val Ile Asn Val Ser Ser Met
130 135 140
Thr Gly Ile Ser Ser Ser Pro Trp Asn Gly Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ala Phe Ile Leu Lys Met Thr Glu Ala Val Ala Cys Glu Cys Glu Gly
165 170 175
Thr Gly Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Leu Thr Pro
180 185 190
Ser Leu Leu Ser Asn Leu Pro Gly Gly Pro Gln Gly Glu Ala Val Met
195 200 205
Lys Ile Ala Leu Thr Pro Glu Glu Cys Val Asp Glu Ala Phe Glu Lys
210 215 220
Leu Gly Lys Glu Leu Ser Val Ile Ala Gly Gln Arg His Lys Asp Gln
225 230 235 240
Val His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Tyr Ile Arg Tyr
245 250 255
Met Gly Ser Phe Tyr Arg Asp
260
<210> 25
<211> 792
<212> DNA
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 25
atgaatctgc gtgaaaaata cggtgaatgg ggtctgatcc tgggtgctac ggaaggtgtc 60
ggtaaagcgt tctgtgaaaa aatcgccgcg ggcggtatga acgtggttat ggtcggccgt 120
cgcgaagaaa aactgaatgt gctggcaggc gaaattcgtg aaacctatgg tgttgaaacg 180
aaagtcgtgc gtgctgattt ttcccagccg ggtgcagcag aaaccgtctt cgcagctacg 240
gaaggcctgg acatgggttt tatgtcttac gtggcctgcc tgcatagttt cggtaaaatt 300
caagataccc cgtgggaaaa acacgaagca atgatcaacg tgaatgttgt cacgtttctg 360
aaatgtttcc atcactatat gcgtatcttt gcggcccagg atcgcggtgc cgtgattaac 420
gttagctcta tgaccggcat cagttcctca ccgtggaatg gtcaatacgg cgcgggtaaa 480
gccttcattc tgaaaatgac cgaagcagtt gcttgcgaat gtgaaggcac gggtgtggac 540
gttgaagtca tcaccctggg taccacgctg acgccgtcgc tgctgagcaa cctgccgggc 600
ggtccgcagg gtgaagcagt gatgaaaatt gctctgaccc cggaagaatg cgttgatgaa 660
gcgtttgaaa aactgggcaa agaactgtca gttatcgccg gtcaacgtca gaaagataat 720
gtccatgact ggaaagcaaa ccacaccgaa gacgaataca tccgctacat gggctcattt 780
taccgtgact aa 792
<210> 26
<211> 263
<212> PRT
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 26
Met Asn Leu Arg Glu Lys Tyr Gly Glu Trp Gly Leu Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ala Gly Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Lys Leu Asn Val Leu
35 40 45
Ala Gly Glu Ile Arg Glu Thr Tyr Gly Val Glu Thr Lys Val Val Arg
50 55 60
Ala Asp Phe Ser Gln Pro Gly Ala Ala Glu Thr Val Phe Ala Ala Thr
65 70 75 80
Glu Gly Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Leu His Ser
85 90 95
Phe Gly Lys Ile Gln Asp Thr Pro Trp Glu Lys His Glu Ala Met Ile
100 105 110
Asn Val Asn Val Val Thr Phe Leu Lys Cys Phe His His Tyr Met Arg
115 120 125
Ile Phe Ala Ala Gln Asp Arg Gly Ala Val Ile Asn Val Ser Ser Met
130 135 140
Thr Gly Ile Ser Ser Ser Pro Trp Asn Gly Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ala Phe Ile Leu Lys Met Thr Glu Ala Val Ala Cys Glu Cys Glu Gly
165 170 175
Thr Gly Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Leu Thr Pro
180 185 190
Ser Leu Leu Ser Asn Leu Pro Gly Gly Pro Gln Gly Glu Ala Val Met
195 200 205
Lys Ile Ala Leu Thr Pro Glu Glu Cys Val Asp Glu Ala Phe Glu Lys
210 215 220
Leu Gly Lys Glu Leu Ser Val Ile Ala Gly Gln Arg Gln Lys Asp Asn
225 230 235 240
Val His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Tyr Ile Arg Tyr
245 250 255
Met Gly Ser Phe Tyr Arg Asp
260
<210> 27
<211> 792
<212> DNA
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 27
atgaatctgc gtgaaaaata cggtgaatgg ggtctgatcc tgggtgctac ggaaggtgtc 60
ggtaaagcgt tctgtgaaaa aatcgccgcg ggcggtatga acgtggttat ggtcggccgt 120
cgcgaagaaa aactgaatgt gctggcaggc gaaattcgtg aaacctatgg tgttgaaacg 180
aaagtcgtgc gtgctgattt ttcccagccg ggtgcagcag aaaccgtctt cgcagctacg 240
gaaggcctgg acatgggttt tatgtcttac gtggcctgcc tgcatagttt cggtaaaatt 300
caagataccc cgtgggaaaa acacgaagca atgatcaacg tgaatgttgt cacgtttctg 360
aaatgtttcc atcactatat gcgtatcttt gcggcccagg atcgcggtgc cgtgattaac 420
gttagctcta tgaccggcat cagttcctca ccgtggaatg gtcaatacgg cgcgggtaaa 480
gccttcattc tgaaaatgac cgaagcagtt gcttgcgaat gtgaaggcac gggtgtggac 540
gttgaagtca tcaccctggg taccacgctg acgccgtcgc tgctgagcaa cctgccgggc 600
ggtccgcagg gtgaagcagt gatgaaaatt gctctgaccc cggaagaatg cgttgatgaa 660
gcgtttgaaa aactgggcaa agaactgtca gttatcgccg gtcaacgtca gaaagatcga 720
gtccatgact ggaaagcaaa ccacaccgaa gacgaataca tccgctacat gggctcattt 780
taccgtgact aa 792
<210> 28
<211> 263
<212> PRT
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 28
Met Asn Leu Arg Glu Lys Tyr Gly Glu Trp Gly Leu Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ala Gly Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Lys Leu Asn Val Leu
35 40 45
Ala Gly Glu Ile Arg Glu Thr Tyr Gly Val Glu Thr Lys Val Val Arg
50 55 60
Ala Asp Phe Ser Gln Pro Gly Ala Ala Glu Thr Val Phe Ala Ala Thr
65 70 75 80
Glu Gly Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Leu His Ser
85 90 95
Phe Gly Lys Ile Gln Asp Thr Pro Trp Glu Lys His Glu Ala Met Ile
100 105 110
Asn Val Asn Val Val Thr Phe Leu Lys Cys Phe His His Tyr Met Arg
115 120 125
Ile Phe Ala Ala Gln Asp Arg Gly Ala Val Ile Asn Val Ser Ser Met
130 135 140
Thr Gly Ile Ser Ser Ser Pro Trp Asn Gly Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ala Phe Ile Leu Lys Met Thr Glu Ala Val Ala Cys Glu Cys Glu Gly
165 170 175
Thr Gly Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Leu Thr Pro
180 185 190
Ser Leu Leu Ser Asn Leu Pro Gly Gly Pro Gln Gly Glu Ala Val Met
195 200 205
Lys Ile Ala Leu Thr Pro Glu Glu Cys Val Asp Glu Ala Phe Glu Lys
210 215 220
Leu Gly Lys Glu Leu Ser Val Ile Ala Gly Gln Arg Gln Lys Asp Arg
225 230 235 240
Val His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Tyr Ile Arg Tyr
245 250 255
Met Gly Ser Phe Tyr Arg Asp
260
<210> 29
<211> 792
<212> DNA
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 29
atgaatctgc gtgaaaaata cggtgaatgg ggtctgatcc tgggtgctac ggaaggtgtc 60
ggtaaagcgt tctgtgaaaa aatcgccgcg ggcggtatga acgtggttat ggtcggccgt 120
cgcgaagaaa aactgaatgt gctggcaggc gaaattcgtg aaacctatgg tgttgaaacg 180
aaagtcgtgc gtgctgattt ttcccagccg ggtgcagcag aaaccgtctt cgcagctacg 240
gaaggcctgg acatgggttt tatgtcttac gtggcctgcc tgcatagttt cggtaaaatt 300
caagataccc cgtgggaaaa acacgaagca atgatcaacg tgaatgttgt cacgtttctg 360
aaatgtttcc atcactatat gcgtatcttt gcggcccagg atcgcggtgc cgtgattaac 420
gttagctcta tgaccggcat cagttcctca ccgtggaatg gtcaatacgg cgcgggtaaa 480
gccttcattc tgaaaatgac cgaagcagtt gcttgcgaat gtgaaggcac gggtgtggac 540
gttgaagtca tcaccctggg taccacgctg acgccgtcgc tgctgagcaa cctgccgggc 600
ggtccgcagg gtgaagcagt gatgaaaatt gctctgaccc cggaagaatg cgttgatgaa 660
gcgtttgaaa aactgggcaa agaactgtca gttatcgccg gtcaacgtca gaaagatgaa 720
gtccatgact ggaaagcaaa ccacaccgaa gacgaataca tccgctacat gggctcattt 780
taccgtgact aa 792
<210> 30
<211> 263
<212> PRT
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 30
Met Asn Leu Arg Glu Lys Tyr Gly Glu Trp Gly Leu Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ala Gly Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Lys Leu Asn Val Leu
35 40 45
Ala Gly Glu Ile Arg Glu Thr Tyr Gly Val Glu Thr Lys Val Val Arg
50 55 60
Ala Asp Phe Ser Gln Pro Gly Ala Ala Glu Thr Val Phe Ala Ala Thr
65 70 75 80
Glu Gly Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Leu His Ser
85 90 95
Phe Gly Lys Ile Gln Asp Thr Pro Trp Glu Lys His Glu Ala Met Ile
100 105 110
Asn Val Asn Val Val Thr Phe Leu Lys Cys Phe His His Tyr Met Arg
115 120 125
Ile Phe Ala Ala Gln Asp Arg Gly Ala Val Ile Asn Val Ser Ser Met
130 135 140
Thr Gly Ile Ser Ser Ser Pro Trp Asn Gly Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ala Phe Ile Leu Lys Met Thr Glu Ala Val Ala Cys Glu Cys Glu Gly
165 170 175
Thr Gly Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Leu Thr Pro
180 185 190
Ser Leu Leu Ser Asn Leu Pro Gly Gly Pro Gln Gly Glu Ala Val Met
195 200 205
Lys Ile Ala Leu Thr Pro Glu Glu Cys Val Asp Glu Ala Phe Glu Lys
210 215 220
Leu Gly Lys Glu Leu Ser Val Ile Ala Gly Gln Arg Gln Lys Asp Glu
225 230 235 240
Val His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Tyr Ile Arg Tyr
245 250 255
Met Gly Ser Phe Tyr Arg Asp
260
<210> 31
<211> 792
<212> DNA
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 31
atgaatctgc gtgaaaaata cggtgaatgg ggtctgatcc tgggtgctac ggaaggtgtc 60
ggtaaagcgt tctgtgaaaa aatcgccgcg ggcggtatga acgtggttat ggtcggccgt 120
cgcgaagaaa aactgaatgt gctggcaggc gaaattcgtg aaacctatgg tgttgaaacg 180
aaagtcgtgc gtgctgattt ttcccagccg ggtgcagcag aaaccgtctt cgcagctacg 240
gaaggcctgg acatgggttt tatgtcttac gtggcctgcc tgcatagttt cggtaaaatt 300
caagataccc cgtgggaaaa acacgaagca atgatcaacg tgaatgttgt cacgtttctg 360
aaatgtttcc atcactatat gcgtatcttt gcggcccagg atcgcggtgc cgtgattaac 420
gttagctcta tgaccggcat cagttcctca ccgtggaatg gtcaatacgg cgcgggtaaa 480
gccttcattc tgaaaatgac cgaagcagtt gcttgcgaat gtgaaggcac gggtgtggac 540
gttgaagtca tcaccctggg taccacgctg acgccgtcgc tgctgagcaa cctgccgggc 600
ggtccgcagg gtgaagcagt gatgaaaatt gctctgaccc cggaagaatg cgttgatgaa 660
gcgtttgaaa aactgggcaa agaactgtca gttatcgccg gtcaacgtca gaaagatcag 720
gtccatgact ggaaagcaaa ccacaccgaa gacgaataca tccgctacat gggctcattt 780
taccgtgact aa 792
<210> 32
<211> 263
<212> PRT
<213> 产气柯林斯菌(Collinsella aerofaciens)
<400> 32
Met Asn Leu Arg Glu Lys Tyr Gly Glu Trp Gly Leu Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ala Gly Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Lys Leu Asn Val Leu
35 40 45
Ala Gly Glu Ile Arg Glu Thr Tyr Gly Val Glu Thr Lys Val Val Arg
50 55 60
Ala Asp Phe Ser Gln Pro Gly Ala Ala Glu Thr Val Phe Ala Ala Thr
65 70 75 80
Glu Gly Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Leu His Ser
85 90 95
Phe Gly Lys Ile Gln Asp Thr Pro Trp Glu Lys His Glu Ala Met Ile
100 105 110
Asn Val Asn Val Val Thr Phe Leu Lys Cys Phe His His Tyr Met Arg
115 120 125
Ile Phe Ala Ala Gln Asp Arg Gly Ala Val Ile Asn Val Ser Ser Met
130 135 140
Thr Gly Ile Ser Ser Ser Pro Trp Asn Gly Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ala Phe Ile Leu Lys Met Thr Glu Ala Val Ala Cys Glu Cys Glu Gly
165 170 175
Thr Gly Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Leu Thr Pro
180 185 190
Ser Leu Leu Ser Asn Leu Pro Gly Gly Pro Gln Gly Glu Ala Val Met
195 200 205
Lys Ile Ala Leu Thr Pro Glu Glu Cys Val Asp Glu Ala Phe Glu Lys
210 215 220
Leu Gly Lys Glu Leu Ser Val Ile Ala Gly Gln Arg Gln Lys Asp Gln
225 230 235 240
Val His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Tyr Ile Arg Tyr
245 250 255
Met Gly Ser Phe Tyr Arg Asp
260
<210> 33
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
tcgccggtca acgtaaaaaa gatagcgtcc 30
<210> 34
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
ggacgctatc ttttttacgt tgaccggcga 30
<210> 35
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
tcgccggtca acgtcataaa gatagcgtcc at 32
<210> 36
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
atggacgcta tctttatgac gttgaccggc ga 32
<210> 37
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
tcgccggtca acgtcagaaa gatagcgtcc 30
<210> 38
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
ggacgctatc tttctgacgt tgaccggcga 30
<210> 39
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
cggtcaacgt aataaagata atgtccatga ctgg 34
<210> 40
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
ccagtcatgg acattatctt tattacgttg accg 34
<210> 41
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
cgtaataaag atcgagtcca tgactgg 27
<210> 42
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
ccagtcatgg actcgatctt tattacg 27
<210> 43
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
cgtaataaag atgaagtcca tgactgg 27
<210> 44
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
ccagtcatgg acttcatctt tattacg 27
<210> 45
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
cgtaataaag atcaggtcca tgactgg 27
<210> 46
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
ccagtcatgg acctgatctt tattacg 27

Claims (10)

1.一种7β-HSDH酶突变体，其特征在于，所述的7β-HSDH酶突变体的氨基酸序列与氨基酸序列如SEQ ID NO:2所示的野生型7β-HSDH酶相比，在SEQ ID NO:2所示的氨基酸序列的第237位、第240位进行单突变或两两联合突变中的任意一种突变。
2.根据权利要求1所述的7β-HSDH酶突变体，其特征在于，所述的单突变为：
当SEQ ID NO:2所示的氨基酸序列的第237位由天冬酰胺突变为组氨酸，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:6所示；
或，当SEQ ID NO:2所示的氨基酸序列的第237位由天冬酰胺突变为谷氨酰胺，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:8所示；
或，当SEQ ID NO:2所示的氨基酸序列的第240位由丝氨酸突变为天冬酰胺，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:10所示；
或，当SEQ ID NO:2所示的氨基酸序列的第240位由丝氨酸突变为精氨酸，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:12所示；
或，当SEQ ID NO:2所示的氨基酸序列的第240位由丝氨酸突变为谷氨酸，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:14所示；
或，当SEQ ID NO:2所示的氨基酸序列的第240位由丝氨酸突变为谷氨酰胺，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:16所示。
3.根据权利要求1所述的7β-HSDH酶突变体，其特征在于，所述的两两联合突变为：
当SEQ ID NO:2所示的氨基酸序列的第237位由天冬酰胺突变为组氨酸，第240位由丝氨酸突变为天冬酰胺，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:18所示；
或，当SEQ ID NO:2所示的氨基酸序列的第237位由天冬酰胺突变为组氨酸，第240位由丝氨酸突变为精氨酸，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:20所示；
或，当SEQ ID NO:2所示的氨基酸序列的第237位由天冬酰胺突变为组氨酸，第240位由丝氨酸突变为谷氨酸，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:22所示；
或，当SEQ ID NO:2所示的氨基酸序列的第237位由天冬酰胺突变为组氨酸，第240位由丝氨酸突变为谷氨酰胺，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:24所示；
或，当SEQ ID NO:2所示的氨基酸序列的第237位由天冬酰胺突变为谷氨酰胺，第240位由丝氨酸突变为天冬酰胺，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:26所示；
或，当SEQ ID NO:2所示的氨基酸序列的第237位由天冬酰胺突变为谷氨酰胺，第240位由丝氨酸突变为精氨酸，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:28所示；
或，当SEQ ID NO:2所示的氨基酸序列的第237位由天冬酰胺突变为谷氨酰胺，第240位由丝氨酸突变为谷氨酸，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:30所示；或，当SEQ ID NO:2所示的氨基酸序列的第237位由天冬酰胺突变为谷氨酰胺，第240位由丝氨酸突变为谷氨酰胺，所述的7β-HSDH酶突变体的氨基酸序列如SEQ ID NO:32所示。
4.权利要求2或3所述的7β-HSDH酶突变体的编码基因，其特征在于，氨基酸序列如SEQID NO:6所示的7β-HSDH酶突变体的编码基因的核苷酸序列如SEQ ID NO:5所示；
或，氨基酸序列如SEQ ID NO:8所示的7β-HSDH酶突变体的编码基因的核苷酸序列如SEQ ID NO:7所示；
或，氨基酸序列如SEQ ID NO:10所示的7β-HSDH酶突变体的编码基因的核苷酸序列如SEQ ID NO:9所示；
或，氨基酸序列如SEQ ID NO:12所示的7β-HSDH酶突变体的编码基因的核苷酸序列如SEQ ID NO:11所示；
或，氨基酸序列如SEQ ID NO:14所示的7β-HSDH酶突变体的编码基因的核苷酸序列如SEQ ID NO:13所示；
或，氨基酸序列如SEQ ID NO:16所示的7β-HSDH酶突变体的编码基因的核苷酸序列如SEQ ID NO:15所示；
或，氨基酸序列如SEQ ID NO:18所示的7β-HSDH酶突变体的编码基因的核苷酸序列如SEQ ID NO:17所示；
或，氨基酸序列如SEQ ID NO:20所示的7β-HSDH酶突变体的编码基因的核苷酸序列如SEQ ID NO:19所示；
或，氨基酸序列如SEQ ID NO:22所示的7β-HSDH酶突变体的编码基因的核苷酸序列如SEQ ID NO:21所示；
或，氨基酸序列如SEQ ID NO:24所示的7β-HSDH酶突变体的编码基因的核苷酸序列如SEQ ID NO:23所示；
或，氨基酸序列如SEQ ID NO:26所示的7β-HSDH酶突变体的编码基因的核苷酸序列如SEQ ID NO:25所示；
或，氨基酸序列如SEQ ID NO:28所示的7β-HSDH酶突变体的编码基因的核苷酸序列如SEQ ID NO:27所示；
或，氨基酸序列如SEQ ID NO:30所示的7β-HSDH酶突变体的编码基因的核苷酸序列如SEQ ID NO:29所示；
或，氨基酸序列如SEQ ID NO:32所示的7β-HSDH酶突变体的编码基因的核苷酸序列如SEQ ID NO:31所示。
5.含有权利要求4所述的编码基因的载体。
6.根据权利要求5所述的载体，其特征在于，所述的载体为pET表达载体、pCW表达载体、pUC表达载体或pPIC9k表达载体。
7.含有权利要求4所述的编码基因的宿主细胞，其特征在于，所述的宿主细胞为大肠杆菌、毕赤酵母、链霉菌或枯草芽孢杆菌。
8.权利要求1-3任一项所述的7β-HSDH酶突变体、权利要求4所述的编码基因、权利要求5或6所述的载体、权利要求7所述的宿主细胞在制备24-去甲熊脱氧胆酸中的应用。
9.一种制备24-去甲熊脱氧胆酸的方法，其特征在于，所述的方法包括以下步骤：
S1.配置反应体系，包含：1-10g/L权利要求1-3任一项所述的7β-HSDH酶突变体，50mMpH6.0-8.0磷酸钠缓冲液，0.2mM NADP+，10-50g/L 24-去甲-7-酮胆石酸，5-40g/L葡萄糖，0.1-10g/L葡萄糖脱氢酶；控制反应体系温度为25-40℃，pH值为6.0-8.0，进行搅拌反应；
S2.反应24h后进行HPLC检测，即得24-去甲熊去氧胆酸。
10.根据权利要求9所述的方法，其特征在于，所述的方法包括以下步骤：
S1.配置反应体系，包含：1g/L权利要求1-3任一项所述的7β-HSDH酶突变体，50mMpH7.0磷酸钠缓冲液，0.2mM NADP+，10-50g/L 24-去甲-7-酮胆石酸，5-40g/L葡萄糖，0.5g/L葡萄糖脱氢酶；控制反应体系温度为30℃，pH值为7.0，进行搅拌反应；
S2.反应24h后进行HPLC检测，即得24-去甲熊去氧胆酸。
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114276401A (zh) * 2021-12-27 2022-04-05 中山百灵生物技术股份有限公司 一种24-去甲熊去氧胆酸的合成方法
CN114480319A (zh) * 2022-01-27 2022-05-13 南京桦冠生物技术有限公司 一种单胺氧化酶突变体及其应用
CN114752572A (zh) * 2022-02-18 2022-07-15 深圳希吉亚生物技术有限公司 甲酸脱氢酶突变体及其应用
CN114854707A (zh) * 2022-06-14 2022-08-05 苏州百福安酶技术有限公司 一种7β-羟基甾体脱氢酶突变体

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108546691A (zh) * 2018-05-09 2018-09-18 华东理工大学 7β-羟基甾醇脱氢酶突变体及其在制备熊脱氧胆酸中的应用
US20200407766A1 (en) * 2016-06-20 2020-12-31 Pharmazell Gmbh Coupled, Self-Sufficient Biotransformation of Chenodeoxcholic Acid to Ursodeoxycholic Acid and Novel Enzyme Mutants Applicable in said Process
CN113388592A (zh) * 2021-06-30 2021-09-14 中山百灵生物技术股份有限公司 一种7β-HSDH酶突变体及其编码基因和应用
CN113462665A (zh) * 2021-06-30 2021-10-01 中山百灵生物技术股份有限公司 一种7α-HSDH酶突变体及其编码基因和应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200407766A1 (en) * 2016-06-20 2020-12-31 Pharmazell Gmbh Coupled, Self-Sufficient Biotransformation of Chenodeoxcholic Acid to Ursodeoxycholic Acid and Novel Enzyme Mutants Applicable in said Process
CN108546691A (zh) * 2018-05-09 2018-09-18 华东理工大学 7β-羟基甾醇脱氢酶突变体及其在制备熊脱氧胆酸中的应用
CN113388592A (zh) * 2021-06-30 2021-09-14 中山百灵生物技术股份有限公司 一种7β-HSDH酶突变体及其编码基因和应用
CN113462665A (zh) * 2021-06-30 2021-10-01 中山百灵生物技术股份有限公司 一种7α-HSDH酶突变体及其编码基因和应用

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
MING-MIN ZHENG ET AL.: "Engineering 7β-Hydroxysteroid Dehydrogenase for Enhanced Ursodeoxycholic Acid Production by Multiobjective Directed Evolution" *
SIMONE SAVINO ET AL.: "Structural and biochemical insights into 7b-hydroxysteroid dehydrogenase stereoselectivity" *
ZHI-NENG YOU ET AL.: "Switching Cofactor Dependence of 7β-Hydroxysteroid Dehydrogenase for Cost-Effective Production of Ursodeoxycholic Acid" *
董新星等: "3β和17β羟基类固醇脱氢酶的研究进展" *
贺俊斌等: "多酶催化串联策略在复杂天然产物合成中的应用" *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114276401A (zh) * 2021-12-27 2022-04-05 中山百灵生物技术股份有限公司 一种24-去甲熊去氧胆酸的合成方法
CN114480319A (zh) * 2022-01-27 2022-05-13 南京桦冠生物技术有限公司 一种单胺氧化酶突变体及其应用
CN114752572A (zh) * 2022-02-18 2022-07-15 深圳希吉亚生物技术有限公司 甲酸脱氢酶突变体及其应用
CN114752572B (zh) * 2022-02-18 2023-07-18 深圳希吉亚生物技术有限公司 甲酸脱氢酶突变体及其应用
CN114854707A (zh) * 2022-06-14 2022-08-05 苏州百福安酶技术有限公司 一种7β-羟基甾体脱氢酶突变体
CN114854707B (zh) * 2022-06-14 2023-09-12 苏州百福安酶技术有限公司 一种7β-羟基甾体脱氢酶突变体

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