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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。
In order to achieve high-efficiency bio-oxidation of cholesterol, a DNA fragment encoding Chlamydia simplexerr oxidase was cloned into plasmid pTY2 by genetic engineering to construct the expression vector pTY2-5332. The vector uses the 16S rDNA site in the genome of Arthrobacter simplex as an integration point to increase the copy number of the cholesterol oxidase in the genome and achieve the overexpression of the simple Arthrobacter cholerae oxidase. Experimental analysis of the growth of recombinant bacteria showed that the cholesterol oxidase inserted into the 16S rDNA site did not affect the growth of A. simplex. Recombinant bacteria could completely convert 2g / L cholesterol into 4-cholesten-3one at 20h, shortened the transformation time of original bacteria by 4h and improved the conversion efficiency of cholesterol. After optimization of the transformation conditions, the recombinant strain was further cultured for 16h with 2% inoculum size, then the amount of cholesterol was 2g / L when it was sieved through 120 mesh, and 2% (volume ratio) dimethylformamide was added as promoting Solvent; cholesterol can be completely converted to 4-cholesten-3-one after 18h, which is 1.11 times before optimization.