定点突变对低温脂肪酶活性和热稳定性的影响  

Effects of site-directed mutations on low-temperature lipase activity and thermostability

作　　者：陈海清 于凡 王红静 张先舟[1] 亢春雨[1] 马雯[1] 檀建新[1] CHEN Haiqing;YU Fan;WANG Hongjing;ZHANG Xianzhou;KANG Chunyu;MA Wen;TAN Jianxin(Engineering Research Center of Hebei Province for Agricultural Products/College of Food Science and Technology,Hebei Agricultural University,Baoding 071001,China)

机构地区：[1]河北农业大学,食品科技学院/河北省农产品加工工程技术中心,河北保定071000

出　　处：《河北农业大学学报》2020年第2期96-102,共7页Journal of Hebei Agricultural University

基　　金：河北省重点研发计划项目(16275505D);河北省食品科学与工程学科“双一流”建设资金项目(2016SPGCA18).

摘　　要：使用在线预测软件PoPMuSiC-2.1对粘质沙雷氏菌菌株L1的脂肪酶(lipA)每个氨基酸突变后的去折叠自由能变化(ΔΔG)进行预测、筛选,用重叠延伸PCR法构建了2个脂肪酶突变体lipA-Asn86Glu、lipAAsn132Lys。经大肠杆菌表达和酶蛋白纯化后,对其酶学特性进行了表征。2个突变体和野生型的脂肪酶最适温度为30℃、最适pH值为8。与野生型相比,lipA-Asn86Glu的比酶活提高了8%,lipA-Asn132Lys的比酶活比野生型降低了19%;lipA-Asn86Glu在50℃下酶的半衰期与野生型相似,Km和Kcat值有所下降,而lipAAsn132Lys的50℃时半衰期、Km和Kcat都明显降低。上述结果为通过理性设计突变位点对酶进行分子改造,以提高酶活力或热稳定性,提供了借鉴和途径。According to the free energy change(ΔΔG) for unfolded protein, two mutations of lipA from Serratia marcescens strain L1, lipA-Asn86 Glu and lipA-Asn132 Lys, were screened using the online prediction software PoPMuSiC-2.1 and constructed using overlap extension PCR method. After expression in E. coli and purification, the lipase properties were characterized. The optimum temperature and pH of both two mutants and wild type lipase were 30 ℃ and pH8. In comparison to wild type lipase, lipase activity of lipA-Asn86 Glu was increased by 8%, but lipA-Asn132 Lys activity was 19% lower than that of the wild. LipA-Asn86 Glu had a similar half-life of the enzyme at 50℃ with the decrease of Km and Kcat values comparing to the wild type. LipA-Asn132 Lys, however, had a significantly lower Km, Kcat and half-life at 50 ℃. The above results provide a possible pathway and references for molecular modification of the lipase by rational design of mutation sites to improve enzyme activity or thermal stability.

关 键 词：脂肪酶 定点突变 重叠延伸PCR PoPMuSiC 酶活力 

分 类 号：Q786[生物学—分子生物学]