降解植物甾醇为甾体9α-OH-AD菌株的筛选与鉴定  

Screening and identification of a strain degrading phytosterols to 9α-hydroxyandrost-4-ene-3,17-dione

作　　者：宋宇迪 董玉秀 马新宇 张晓娟 陈志蔚 蔡兆培 李会 史劲松 许正宏 

机构地区：[1]江南大学药学院,无锡214122 [2]大丰市佳丰油脂有限责任公司,盐城224100 [3]江南大学粮食发酵工艺与技术国家工程实验室,无锡214122

出　　处：《应用与环境生物学报》2017年第6期1022-1027,共6页Chinese Journal of Applied and Environmental Biology

基　　金：江苏省大学生创新创业训练计划项目(2016102950 47X);江苏高校品牌专业建设工程资助项目(PPZ Y2015B146);国家863计划项目(2011AA02A211)资助~~

摘　　要：9α-羟基雄甾-4-烯-3,17-二酮(9α-OH-AD)是重要的甾体药物中间体,多用于生产糖皮质激素类药物.通过微生物法转化植物甾醇生成9α-OH-AD,将为工业化生产提供极大便利.从榨油厂、油菜花田等多处土壤中筛选出1株高效转化植物甾醇为9α-OH-AD的菌株,根据16S r DNA序列比对并结合菌株的形态特征、生理生化特征分析,鉴定其为分枝杆菌属,并将其命名为Mycobacterium sp.LY-1.该菌在往复式摇床中培养,当培养条件为温度30℃、转速120 r/min、转化时间7 d、底物添加浓度为5 g/L时,9α-OH-AD产物得率达到16.2%.为解决底物植物甾醇在水中溶解性较差的问题,考察了助溶剂(吐温80、β-环糊精)对植物甾醇转化产甾药中间体9α-OH-AD效率的影响.经过筛选,最终确定最适助溶剂为0.1%的吐温80.在此条件下,当底物投料浓度为15 g/L时,9α-OH-AD的浓度达到3.9 g/L,产物摩尔得率提高至35.1%.本研究表明菌株LY-1转化效率好,可为后续的代谢工程改造提供便利并为后期用于工业化生产奠定基础.9α-Hydroxyandrost-4-ene-3,17-dione （9α-OH-AD） is an important precursor in the manufacture of a number of modern glucocorticoid drugs. It may provide great convenience via the microbial conversion of phytosterols to 9α-OH-AD for industrial production. Here, a strain that can transform phytosterols to 9α-OH-AD was isolated from the soils of an oil mill, rape field, and other sources. Based on the sequence analysis of 16S rDNA and a series of morphological and biochemical characteristics, it was identified as Mycobacterium sp. and named as Mycobacterium sp. LY-1. Strain LY-1 was then cultivated in a rotary shaker at 30 ℃ and 120 r/min. After transformation for 7 days, the molar yield of 9α-OH-AD reached 16.2% with the addition of substrate at 5 g/L. To solve the low-solubility problem of the substrate phytosterols in water, the effects of a cosolvent （Tween 80 and β-cyclodextrin） on the biotransformation efficacy of phytosterols to drug intermediate 9α-OH-AD were investigated. Through the screening, the optimal feeding method was addition of 0.1% Tween 80. In this condition, the concentration of 9α-OH-AD was 3.9 g/L, and the molar yield of 9α-OH-AD reached 35.1% with the addition of substrate at 15 g/L. These results suggest that strain LY-1 has good transformation efficacy for a potential industrial application, and it laid the foundation for later industrial production.

关 键 词：植物甾醇 分枝杆菌 微生物转化 9α-OH-AD 吐温80 

分 类 号：Q939.9[生物学—微生物学]