Xanthomonas maltophilia转化制备熊去氧胆酸及中间产物  

Transformation of ursodeoxycholic acid and intermediates by Xanthomonas maltophilia

作　　者：刘彩霞 王静[1] 王栋 吴迎超 吴庆喜 LIU Caixia;WANG Jing;WANG Dong;WU Yingchao;WU Qingxi(School of Life Sciences,Anhui University,Hefei Anhui 230601,China;Anhui Key Laboratory of Ecological Engineering and Biotechnology,Anhui Key Laboratory of Modern Biomanufacturing,Anhui University,Hefei Anhui 230601,China)

机构地区：[1]安徽大学生命科学学院,安徽合肥230601 [2]安徽大学生态工程与生物技术安徽省重点实验室,现代生物制造安徽省重点实验室,安徽合肥230601

出　　处：《阜阳师范大学学报（自然科学版）》2024年第1期54-61,共8页Journal of Fuyang Normal University:Natural Science

基　　金：国家自然科学基金(21606002);安徽省自然科学基金(2208085MB32);安徽省高校自然科学研究重大项目(2023AH040012);安徽省重点研究与开发计划项目(202004a06020021)。

摘　　要：以野生型嗜麦芽黄单胞菌(Xanthomonas maltophilia)为出发菌株,鹅去氧胆酸/β-环糊精包合物为反应底物,利用嗜麦芽黄单胞菌在液体发酵过程中产生的7α-羟基类固醇脱氢酶和7β-羟基类固醇脱氢酶,全细胞酶法催化制备熊去氧胆酸及中间产物。对嗜麦芽黄单胞菌形态鉴定,酶活测定并优化,制定产物检测方法。表明嗜麦芽黄单胞菌为杆状、单鞭毛、革兰氏阴性菌。菌株破碎后,SDS-PAGE分析表明在26-33 kDa之间存在蛋白条带,测定7α-羟基类固醇脱氢酶和7β-羟基类固醇脱氢酶酶活分别为79 U/mL、35 U/mL;优化显示,温度为35°C、pH值为9.0、添加30%甲醇时酶的活性提升;转化后UDCA得率为17.2 mg/L,7K-LCA得率为18.2 mg/L。作为一种野生型的底盘转化菌种,该研究为全细胞催化制备熊去氧胆酸及其中间产物提供了新的途径。The wild-type Xanthomonas maltophilia was chosen as the starting strain,and the inclusion complex of chenodeoxycholic acid andβ-cyclodextrin was adopted as the reaction substrate.On the basis of the 7a-hydroxysteroid dehydrogenase and 7-hydroxysteroid dehydrogenase generated during the liquid culture process of Xanthomonas maltophilia,the whole-cell systems were utilized to produce ursodeoxycholic acid and intermediates via enzymatic conversion means.The morphology of Xanthomonas maltophilia was identified,enzyme activity was determined and conditions were optimized.A standard detection method for transformation products was established using high performance liquid chromatography method(HPLC).The transformation conditions were optimized by single factor experimental design.Morphology identification showed that Xanthomonas maltophilia was a rod-shaped,monoflagellated,gram-negative bacterium.After the cell disruption of the culture,SDS-PAGE analysis indicated that there was a protein band between 26-33 kDa.The enzyme activities of 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase were of 79 U/mL and 35 U/mL,respectively.The optimization results showed that the enzyme activity increased when the temperature was 35℃,pH value was 9.0 and 30%methanol was added.The yield of UDCA and 7K-LCA were 17.2 mg/L and 18.2 mg/L respectively.As a wild-type chassis-transformed strain,this study provides a new pathway for the whole-cell catalytic production of ursodeoxycholic acid and its intermediates.

关 键 词：嗜麦芽黄单胞菌 7α-羟基类固醇脱氢酶 7β-羟基类固醇脱氢酶 鹅去氧胆酸 全细胞催化 熊去氧胆酸 

分 类 号：Q-939[生物学]