一株高效氧化胆固醇的简单节杆菌构建与转化条件优化  

Construction of Artificial Arthrobacter simplex for Oxidizing Cholesterol,and Optimization of Bioconversion Condition

作　　者：张璟[1] 张玉富[1] 秦慧民[1] 毛淑红[1] 路福平[1] 

机构地区：[1]天津科技大学生物工程学院工业发酵微生物教育部重点实验室,天津300457

出　　处：《中国生物工程杂志》2016年第11期70-75,共6页China Biotechnology

基　　金：国家高技术研究发展计划资助项目(2011AA02A211-06)

摘　　要：为实现胆固醇的高效生物氧化,利用基因工程手段将编码简单节杆菌胆固醇氧化酶的DNA片段克隆到质粒pTY2中,构建pTY2-5332插入表达载体。该载体以简单节杆菌基因组中的16S rDNA位点为整合点,提高了胆固醇氧化酶在基因组中的拷贝数,实现了简单节杆菌胆固醇氧化酶的过表达。重组菌的生长实验分析表明,插入到16S rDNA位点的胆固醇氧化酶没有影响简单节杆菌的生长。重组菌可在20h将2g/L的胆固醇完全转化为4-胆甾烯-3酮,比原始菌的转化时间缩短了4h,提高了胆固醇的转化效率。经过转化条件优化确定了该重组菌以2%的接种量后继续培养16h,然后胆固醇经120目过筛后投料量为2g/L,并添加2%(体积比)二甲基甲酰胺作为促溶剂;胆固醇添加18h后可完全转化为4-胆甾烯-3-酮,是优化前的1.11倍。To improve the ability of conversion cholesterol to 4-cholesten-3-one,the cholesterol oxidase gene from the Arthrobacter simplex was cloned and the integrative expression vector p TY2-5332 was constructed.The recombinant plasmid p TY2-5332 was transformed into Arthrobacter simplex by eletroporation and inserted into the 16 S r DNA site to increase the copy number of the cholesterol oxidase in genome. Result of the growth data indicates that a small amount of the 16 S r DNA site disrupted has little effect on the growth of the Arthrobacter simplex. The recombinant strain can convert cholesterol completely to 4-cholesten-3-one in 20 hours when the cholesterol concentration is 2g / L,reduce 4 hours compared with the wild-type Arthrobacter simplex,and are capable of increasing cholesterol conversion efficiency. The optimal conversion condition of cholesterol was 2%inoculum size and continuous culture of 16 h,then 2g / L substrate passed through a 120 mesh screen and 2%DMF were added to the medium. Under this contions,the cholesterol converted into 4-cholesten-3-one in 18 hours completely,which was 1. 11 times higher than the recombinant strain.

关 键 词：简单节杆菌 胆固醇氧化酶 胆固醇 4-胆甾烯-3-酮 16S RDNA 

分 类 号：Q78[生物学—分子生物学]