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    熊志岳, 许逗, 郭元昕, 田锡炜, 储炬. 米曲霉产米黑根毛霉脂肪酶高通量筛选方法的建立[J]. 华东理工大学学报(自然科学版), 2022, 48(4): 519-525. DOI: 10.14135/j.cnki.1006-3080.20210415002
    引用本文: 熊志岳, 许逗, 郭元昕, 田锡炜, 储炬. 米曲霉产米黑根毛霉脂肪酶高通量筛选方法的建立[J]. 华东理工大学学报(自然科学版), 2022, 48(4): 519-525. DOI: 10.14135/j.cnki.1006-3080.20210415002
    XIONG Zhiyue, XU Dou, GUO Yuanxin, TIAN Xiwei, CHU Ju. Establishment of a High-Throughput Screening Method for Rhizomucor miehei Lipase Production by A. oryzae[J]. Journal of East China University of Science and Technology, 2022, 48(4): 519-525. DOI: 10.14135/j.cnki.1006-3080.20210415002
    Citation: XIONG Zhiyue, XU Dou, GUO Yuanxin, TIAN Xiwei, CHU Ju. Establishment of a High-Throughput Screening Method for Rhizomucor miehei Lipase Production by A. oryzae[J]. Journal of East China University of Science and Technology, 2022, 48(4): 519-525. DOI: 10.14135/j.cnki.1006-3080.20210415002

    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

    • 摘要: 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%。

       

      Abstract: 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 high-throughput screening of 1,200 wells (36,000-48,000 spores) after mutagenesis, 4 high-yielding mutants, which was designated Ⅰ 7-D6-7, Ⅱ 4-4B-5, Ⅲ 3-5C-1 and Ⅴ 7-6C-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) .

       

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