米曲霉产米黑根毛霉脂肪酶高通量筛选方法的建立

Establishment of a High-Throughput Screening Method for Rhizomucor miehei Lipase Production by A.oryzae

为了快速、高效地筛选出高产米黑根毛霉脂肪酶(RML)的米曲霉菌株,利用常温常压等离子体诱变技术(ARTP)对米曲霉孢子进行诱变,优化了高通量检测RML酶活的双指示剂法,建立了24孔板米曲霉高通量培养体系。通过对诱变后的1200孔(36000~48000个孢子)进行高通量筛选,最终筛选出4株米曲霉高产突变株。经由摇瓶验证,突变株Ⅰ7-D6-7、Ⅱ4-4B-5、Ⅲ3-5C-1、Ⅴ7-6C-3酶活分别达到了265.98、240.90、253.52、293.50 U/mL,比出发菌株的酶活(176.52 U/mL)分别提高了50.68%、36.47%、43.61%、66.27%。

In order to quickly and efficiently screen out Aspergillus oryzae strains of high-yielding Rhizomucor miehei lipase(RML),the spores of Aspergillus oryzae(A.oryzae)were mutated by atmospheric room temperature plasma(ARTP)mutagenesis technology.The dual indicator method for high-throughput detection of RML enzyme activity was optimized,and a 24-well plate high-throughput culture system of A.oryzae was established.Through highthroughput screening of 1,200 wells(36,000-48,000 spores)after mutagenesis,4 high-yielding mutants,which was designatedⅠ7-D6-7,Ⅱ4-4B-5,Ⅲ3-5C-1 andⅤ7-6C-3,were finally obtained.In shake flask cultures,the enzyme activities of mutants reached 265.98,240.90,253.52 U/mL and 293.50 U/mL,which was increased by 50.68%,36.47%,43.61%and 66.27%,respectively,compared to the original strain(176.52 U/mL).