摘要
本试验旨在通过定向进化技术提高角蛋白酶的热稳定性,以拓展角蛋白酶在饲料工业的适用性。试验采用易错PCR方法对地衣芽孢杆菌CP-16的角蛋白酶进行定向进化,筛选获得耐温性好的突变体,并对其进行酶学性质研究与结构功能分析。结果表明,从5000个突变体中获得3株正向角蛋白酶突变体A307V/S346T、R70G、N245K,其角蛋白酶热稳定性得到了提高。酶学性质研究发现,角蛋白酶突变体R70G热稳定性最强,在75℃热处理5 min后残留酶活性达30.65%,而野生型角蛋白酶热处理5 min后残留酶活性仅1.06%。由此可见,本研究成功获得热稳定性较好的角蛋白酶突变体,拓展了角蛋白酶的适用性,为耐热角蛋白酶开发和相关基因信息探索提供了参考。
This experiment was aimed to improve the thermostability of keratinase,and to expand the applicability of keratinase in the feed industry.The error-prone PCR method was used to carry out directed evolution of the keratinase of Bacillus licheniformis CP-16,the mutants with good temperature tolerance were screened,and the enzymatic properties and structure and function analysis were performed.The results showed that three positive keratinase mutants A307V/S346T,R70G and N245K were obtained from 5000 mutants,and the thermostability of keratinase was improved.The study of the enzymatic properties found that the keratinase mutant R70G had the strongest thermal stability,and retained 30.65%of the activity after heat treatment at 75℃for 5 min,while the wild-type keratinase only had 1.06%of the enzyme activity after heat treatment at 75℃for 5 min.This study successfully obtained the keratinase mutants with good thermostability,expand the applicability of keratinase,and provide a reference for the development of heat-resistant keratinase and the exploration of related new genetic information.
作者
傅岩
张铁鹰
孙英霞
李松育
FU Yan;ZHANG Tieying;SUN Yingxia;LI Songyu(State Key Laboratory of Animal Nutrition,Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100193,China;College of Animal Science,Shanxi Agricultural University,Taiyuan 030801,China)
出处
《动物营养学报》
CAS
CSCD
北大核心
2021年第10期5887-5894,共8页
CHINESE JOURNAL OF ANIMAL NUTRITION
基金
国家自然科学基金面上项目(31470122)
中国农业科学院科技创新工程专项经费(ASTIP-IAS08)。
关键词
角蛋白酶
定向进化
热稳定性
keratinase
directed evolution
thermostability
