黑曲霉多聚半乳糖醛酸酶AnPGA耐热性和酶活性的定向改造  

Directional modification of heat resistance and enzyme activity ofpolygalacturonase AnPGA from Aspergillus niger

作　　者：颜佳超 曹瑜 杨丹妮 吴勇 徐辉 乔代蓉 曹毅 YAN Jiachao;CAO Yu;YANG Danni;WU Yong;XU Hui;QIAO Dairong;CAO Yi(Microbiology and Metabolic Engineering of Key Laboratory of Sichuan Province,College of Life Sciences,Sichuan University,Chengdu 610065,China;Laboratory and Equipment Management Office,Sichuan University,Chengdu 610065,China)

机构地区：[1]四川大学生命科学学院微生物与代谢工程四川省重点实验室,成都610065 [2]四川大学实验室及设备管理处,成都610065

出　　处：《应用与环境生物学报》2021年第5期1304-1310,共7页Chinese Journal of Applied and Environmental Biology

基　　金：四川省科技厅项目(2020YFH0076);成都市科技局项目(2020-GH02-00037-HZ)资助。

摘　　要：果胶是一种蕴藏丰富的生物质资源,多聚半乳糖醛酸酶(polygalacturonase,PG)是降解果胶的关键酶,在食品领域也有很广泛的应用,其酶活性和耐热性是制约果胶及其自身利用的关键.采用PoPMuSiC工具结合分子动力学模拟对Aspergillus niger SC323来源的多聚半乳糖醛酸酶AnPGA进行改进,预测影响解折叠自由能显著的位点结合柔性位点的策略,旨在提高其耐热性或酶活性.通过重叠PCR进行定点突变,并构建突变体的表达载体,电转化Pichia pastoris GS115后,进行诱导表达,成功筛选到酶活力和热稳定性均提高的突变体D73P,其最大反应速度是野生型的2.72倍,在65℃下的半衰期t_(1/2)比野生型增加了7 min,在65℃下处理30 min后的残余酶活性为野生型的1.86倍.本研究利用PoPMuSiC结合分子动力学模拟的设计策略提高了AnPGA的酶活力和耐热性,为工业应用提供了新的酶资源,也为其他工业酶的定向改造提供了参考.Pectin is a rich source of biomass.Polygalacturonase is the key enzyme for degrading pectin and is widely used in the food industry.However,its enzyme activity and heat resistance are key factors that restrict its utilization as well as that of pectin.In this study,polygalacturonase AnPGA from Aspergillus niger SC323 was modified to improve either its heat resistance or enzyme activity.PoPMuSiC was used to predict the amino acid sites that significantly affected the unfolding free energy,and a simulation of molecular dynamics was used to predict flexible amino acid sites.These strategies were combined to design the mutation sites for AnPGA.Sitedirected mutagenesis was carried out by overlapping PCR,expression vectors were constructed for mutants,and the mutant Pichia pastoris GS115 expression strains were constructed.After all mutants were expressed,the mutant D73P which had improved enzyme activity and thermal stability was successfully screened.The Vmax of D73P was 2.72 times higher than that of the wild type,and the half-life(t_(1/2))of D73P was extended by 7 min at 65℃.Furthermore,the D73P showed more residual activity than the wild type(1.86 times greater)after 30 min of treatment at 65℃.This study was the first to use a PoPMuSiC design strategy combined with a simulation of molecular dynamics to improve the activity and heat resistance of AnPGA.The results provided new enzyme resources for industrial applications and a reference for the directional modification of other industrial enzymes.

关 键 词：多聚半乳糖醛酸酶 定向改造 定点突变 热稳定性 酶活性 

分 类 号：TS201.2[轻工技术与工程—食品科学]