产脂肪酶菌株的常压室温等离子体诱变及高通量筛选方法的建立

Mutagenesis of Atmospheric and Room Temperature Plasma for Lipase-Producing Strain and Establishment of High Throughput Screening Method

脂肪酶由于可催化的反应种类和底物类别多,且具有位置选择性和异构体选择性等特点而常用于结构酯的合成和油脂改性等领域。丝孢酵母(Trichosporon sp.)是一种产脂肪酶菌种,但生产能力偏低是限制该菌实现工业化生产的重要因素。本研究利用常压室温等离子体(ARTP)对丝孢酵母野生菌进行诱变处理,并建立了96孔板培养结合对硝基苯酚棕榈酸酯(P-NPP)法测定酶活力的高通量筛选方法,实现了60个突变菌株的初筛。以酶活力为筛选指标时,突变率和正突变率分别为51.7%和28.3%。8株初筛菌株的摇瓶发酵结果显示,A13和A5的产酶提高最显著,培养96 h后分别比野生菌增加2.64倍和1.54倍,且2个突变菌株的遗传稳定性良好。对比研究发现,突变菌株A13相较野生菌的最大优势在于提前24 h便能达到最高产酶量。

Because of various reaction types, substrates, regioselectivity, isomer selectivity and other features, lipases have been widely used in a multitude of fields, especially structure lipids synthesis and oil modification. Tri- chosporon sp. is a kind of lipase - producing strain, but relatively low yield limits its industrial production. The at- mospheric and room temperature plasma （ARTP） were used to conduct mutagenic treatment on trichosporon wild strain and a high throughput screening method using 96 - well plates was set up ; in this protocol, enzyme activity of fermented broth was measured by p - nitrophenylpalmitate （p - NPP） method and preliminary screening of 60 mutant strains were finished. The mutation rate and positive mutation rate were 51.7% and 28.3% respectively when en- zyme activity was regarded as the screening index. According to the results of shake - flask fermentation of 8 prelimi- nary screening strains, enzyme yields of A13 and mutant A5 were prominent which were respectively 2.64 and 1.54 times higher than that of the wild strain after 96 h cultivation. And both of them showed good genetic stability. Com- pared with wild strain mutant A13 reached the plateau of yield of enzyme 24 h ahead of time, which is the most im- portant advantage to this mutant.