【摘要】 利用基因重组技术,克隆了大豆(Glycinemax(L.)Merr.)的苯丙氨酸解氨酶(phenylalanineammonialyase,PAL)全基因,构建pET-GMPAL工程表达质粒,在大肠杆菌E.coliBL21(DE3)中进行诱导表达,并利用细胞裂解液得到的粗酶液转化L-苯丙氨酸生成肉桂酸。通过8%SDS-聚丙烯酰胺凝胶电泳分析表明,获得了分子量在77.9kD的一条蛋白质特异表达带,特异蛋白质表达量达到总蛋白质含量的8%左右。通过初步发酵,获到具有PAL活性的粗酶液裂解液,粗酶的比活力达到211.7μmol/gpro·min(3529μkat/kg),高于红酵母PAL和欧芹重组PAL表达的比活力。结果表明克隆重组的大豆PAL基因通过表达质粒pET-GMPAL在E.coli中能高效地表达为有催化功能的高活力酶。更多还原

【Abstract】 Phenylalanine ammonia-lyase(PAL) gene of Glycine max L (GM) was cloned by SOE-PCR method from the genomic DNA. The full length exon of PAL expression vector pET-GMPAL was constructed and expressed in E.coli. Catalytic activity of conversion L-phenylalanine into cinnamic acid by the recombination GMPAL is achieved at high specific activity, up to 211.7μmol/g pro·min (3529μkat/kg), higher than the parsley recombinant PAL, and Rhotodotorula glutins PAL. Molecular weight of the expressed PAL protein was estimated to be 77.9kD. The amounts of PAL protein expression level was 8% of total E.coli soluble proteins by SDS-PAGE. Results indicated that soybean recombinant PAL is efficiently expressed in E.coli as fully activated functional enzyme.更多还原