一种新型糖苷水解酶的异源表达及酶学性质研究  

Heterologous expression and enzymatic properties of a novel glycoside hydrolase

作　　者：徐龙威 雍婕[1] 周海燕[1] XU Longwei;YONG Jie;ZHOU Haiyan(College of Bioscience and Biotechnology,Hunan Agricultural University,Changsha,Hunan 410128,China)

机构地区：[1]湖南农业大学生物科学技术学院,湖南长沙410128

出　　处：《湖南农业大学学报（自然科学版）》2022年第6期650-658,共9页Journal of Hunan Agricultural University(Natural Sciences)

基　　金：湖南农业大学“双一流”建设工程(SYL201802002)。

摘　　要：为得到MtGH6最优的表达宿主,构建了质粒pET21a-MtGH6、pBES-MtGH6、pPICZαA-MtGH6,以大肠埃希菌BL21、枯草芽孢杆菌RIK1285及毕赤酵母GS115为工程菌,对MtGH6进行异源表达及分离纯化,并对表达MtGH6的3个宿主菌的生长状况、产酶量、纯化回收率、酶活力等参数进行比较分析。结果表明:大肠埃希菌BL21及枯草芽孢杆菌RIK1285在生长稳定后,OD值维持在1.5,而毕赤酵母GS115生长稳定后,OD值维持在2.0;毕赤酵母GS115生产的MtGH6为77mg/L,纯化回收率为15.40%,产酶量和纯化回收率均高于大肠埃希菌BL21、枯草芽孢杆菌RIK1285生产的MtGH6;由毕赤酵母GS115表达的MtGH6的酶活力为15.60U/mg,由大肠埃希菌BL21及枯草芽孢杆菌RIK1285表达的MtGH6分别为7.45、10.06U/mg,说明毕赤酵母GS115是MtGH6的最优表达宿主;对其分泌的MtGH6的酶学性质展开研究,结果表明在降解微晶纤维素的反应中,该酶最适pH为8.0,最适温度为60℃,添加0.5 mmol/L Mn2+可使其活性提高,表明MtGH6在生物燃料生产中可能具有良好的应用前景。In order to obtain the optimal expression host of MtGH6, we used plasmid pET21a-MtGH6,pBES-MTGH6 and pPICZαA-MtGH6 to express the MtGH6 by use of EScherichia coli BL21、Bacillus subtilis RIK1285 and Pichia pastoris GS115. The growth status, enzyme production, purification efficiency and enzyme activity of the three host bacteria expressing MtGH6 were compared and analyzed. The results showed that the OD value of Escherichia coli BL21and Bacillus subtilis RIK1285 remained at 1.5 after stable growth, while that of Pichia pastoris GS115 remained at 2.0after stable growth. MtGH6 produced by Pichia pastoris GS115 was 77 mg/L, and the purification recovery was 15.40%.The enzyme production and purification recovery were higher than those produced by Escherichia coli BL21 and Bacillus subtilis RIK1285. The enzyme activity of MtGH6 expressed by Pichia pastoris GS115 was 15.60 U/mg, and the activity of MtGH6 expressed by Escherichia coli BL21 and Bacillus subtilis RIK1285 was 7.45 U/mg and 10.06 U/mg,respectively, indicating that Pichia Pastoris GS115 was the optimal host for MtGH6 expression. The enzyme properties of MtGH6 secreted by Pichia Pastoris GS115 were studied. The results showed that the optimum pH and temperature of this enzyme were 8.0 and 60 ℃, and the addition of 0.5 mmol/L Mn2+ could increase its activity, indicating that MTGH6had a good application prospect in the production of biofuels.

关 键 词：糖苷水解酶 异源表达 宿主菌 酶学性质 

分 类 号：Q556.2[生物学—生物化学]