BMMYA'平板-Fast blue RR顶层琼脂法高效筛选高酶活脂肪酶

BMMYA' plate -Fast blue RR top agar——an efficient plate screening method for lipase with high enzyme activity

摘建立了一种高效筛选高酶活或高产脂肪酶菌株的平板方法。该方法以华根霉(Rhizopus chinensis CCTCCM201021)脂肪酶基因proRCL在毕赤酵母中构建的基因突变文库为筛选对象,利用BMMYA'平板-FastblueRR顶层琼脂法对其中高酶活或高产的脂肪酶突变株进行筛选,将待筛菌株接种至含有2%甲醇的BMMYA'平板上,30℃生长并诱导4～5 d后,平板经脂肪酶致死温度65℃处理1 h,冰浴、室温平衡后,向平板中倾入Fast blue RR顶层琼脂。2 min内周围显示出明显的黑褐色的菌株为高酶活或高产突变株。该方法简便,快速,高效而且准确,筛选阳性率可达到90%。

An efficient plate screening method for lipase with high enzyme activity was established. This novel method was used as a powerful tool to screen a mutant library for strains with high enzyme activity or high yield of enzyme. Taking lipase gene proRCL from Rhizopus chinensis CCTCCM201021 as the template, the mutant pool was constructed in Pichia pastoris GSll5 in earlier experiments. Candidate strains were inoculated into BMMYA＇ plates containing 2 % （V/V） methanol, grew and induced for 4- 5 d at 30 ℃, afterward undergone heat-treatment at enzyme letlhal temperature for 1 h, and then, cooled at ice and room temperature. Fast blue RR top agar was then poured into plates and positive strains demonstrated apparent dark brown around within 2 rain. This method was very convenient, fast, efficient and accurate and with more than 90 % screening credibility.