摘要
通过提高枯草芽孢杆菌来源的氨肽酶底物结合区域氨基酸的疏水性,提高氨肽酶YwaD的热稳定性。基于氨肽酶底物结合区域氨基酸特性分析,选定亲水性氨基酸残基N385为目标,通过定点突变使其突变为疏水氨基酸残基L385,获得突变体N385L。结果表明,与野生型ywaD相比,突变体N385L酶热稳定性明显提高,在80℃放置30 min,野生型YwaD完全失活,而突变体N385L仍保留20%的酶活力。另外,突变型N385L对底物氨基酰基-硝基苯胺的亲和力提高了47.4%,催化效率提高了28.5%。该研究有利于提高氨基肽酶工业应用前景。
The increase of thermostability of aminopeptidase by creating hydrophobic interactions in the substrate binding region was exploited in Bacillus subtilis 168.The hydrophilic residue N385 was targeted and substituted by hydrophobic residue L385.Based on thermostability analysis,N385L mutant was found to retain 20%while the relative activity of the wild type was reduced to 0%at the same temperature(80℃)in 30 min.Furthermore,the affinity and catalytic efficiency of the mutant N385L for the subtrate of L-Leu-pNA increased by 47.4%and 28.5%,respectively,compared to wild type.This study will inspire the production of thermostable aminopeptidase for industrial application.
作者
ULIHO Alphonse
杨套伟
苑曼曼
徐美娟
张显
MUGISHA Samson
周俊平
饶志明
ULIHO Alphonse;YANG Taowei;YUAN Manman;XU Meijuan;ZHANG Xian;MUGISHA Samson;ZHOU Junping;RAO Zhiming(School of Biotechnology,Jiangnan University,Wuxi 214122,China;Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China)
出处
《食品与生物技术学报》
CAS
CSCD
北大核心
2020年第3期9-15,共7页
Journal of Food Science and Biotechnology
基金
江苏省杰出青年科学基金项目(BK20150002)
国家自然科学基金项目(21778024)
江苏省自然科学基金项目(BK20161292)
中国博士后科学基金项目(2017M620189)
中央高校基本科研业务费专项资金项目(1012050205181860,JUSRP51708A)
江苏高校优势学科建设工程资助项目
江苏省现代发酵工业协同创新中心项目。
关键词
氨肽酶
定点突变
疏水性
热稳定性
aminopeptidase
site-directed mutagenesis
hydrophobic interactions
thermostability
