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

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。

The Mutation Breeding of a Lipase-producing Strain by Atmospheric and Room Temperature Plasma and Its High Throughput Screening

Benefiting from various substrates, regioselectivity and stereoselectivity, lipases have been widely used in a multitude of fields, especially structure lipids synthesis and oil modification. Trichosporon sp. is a kind of lipase-producing strain, but relatively low yield limits its application in industry. To obtain high-producing mutants, the atmospheric and room temperature plasma (ARTP) was applied to this 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-nitrophenyl palmitate (p-NPP) method. With this approach, 60 mutants were successfully screened. The mutation rate and positive one were 51.7% and 28.3% respectively, when lipase activity was regarded as the screening factor. According to the results of shake-flask fermentation for 96 h, lipase yields of mutant A13 and mutant A5 were respectively 2.64 and 1.54 times higher than that of the wild strain. And both of them showed good genetic stability. Further study revealed that mutant A13 could reach the plateau of lipase output 24 h ahead of the wild strain, which is the most important advantage to this mutant.