构建羰基还原酶基因工程菌生物转化产l-麻黄碱

以筛选得到的Morganella morganii J-8细菌的基因组为模板，通过PCR扩增得到目的基因mdlh2。核苷酸序列测定结果表明，基因全长1046bp。以pET28a（＋）为表达载体，构建重组质粒pET28a（＋）-mldh2，并在E．coli BL21（DE3）中表达。利用表达产物进行生物转化，发现其具有催化底物1-苯基-2-甲氨基丙酮（简称MAK）产l-麻黄碱的活力。进一步考察了诱导时间和IPTG浓度对重组菌表达的羰基还原酶的影响，37℃下用0．5mmoL／L的IPTG诱导4h，重组羰基还原酶的酶活达到0．2U／mg蛋白，转化液中l-麻黄碱质量浓度达到45mg／L。

Construction and application of carbonyi reductase gene engineering strain in biosynthesis of l-ephedrine

With the genome of Morganella morganii J-8 as the plate, gene mldh was obtained by PCR amplification. Nucleotide sequencing identified the gene as 1 046 bp, with 96% similarity with that of the leucine dehydrogenase （LeuDH） gene. Using pET28a（＋） as the expressing vector, the recombinant plasmid pET28a（＋）-mldh2 was constructed and expressed in Escherichia coli BL21 （DE3）. Moreover, it was found that the expressed product could produce l-ephedrine from 1-phenyl-2-methylamine-aeetone. Additionally, the effects of IPTG concentration and the induction time on the expression of the recombinant protein were investigated. Under optimal condition （ being induced with 0.5 mmol/L IPTG for 4 h at 37 ℃ ） , the enzyme activity could reach 0. 2 U/mg protein and the concentration of l-ephedrine in transformation liquid could reach 45 mg/L.