相关研究 RELATED RESEARCH

通过定向进化技术提高角蛋白酶的热稳定性研究

  • 傅岩 ,
  • 张铁鹰 ,
  • 孙英霞 ,
  • 李松育
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  • 1. 中国农业科学院北京畜牧兽医研究所, 动物营养学国家重点实验室, 北京 100193;
    2. 山西农业大学动物科学学院, 太原 030801
傅岩(1996-),女,重庆人,硕士研究生,研究方向为酶与抗营养因子。E-mail:564136274@qq.com

收稿日期: 2021-03-30

  网络出版日期: 2021-10-16

基金资助

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)

Enhancing Thermostability of Keratinase by Directed Evolution Technology

  • FU Yan ,
  • ZHANG Tieying ,
  • SUN Yingxia ,
  • LI Songyu
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  • 1. State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
    2. College of Animal Science, Shanxi Agricultural University, Taiyuan 030801, China

Received date: 2021-03-30

  Online published: 2021-10-16

Supported by

 

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5 000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。

本文引用格式

傅岩 , 张铁鹰 , 孙英霞 , 李松育 . 通过定向进化技术提高角蛋白酶的热稳定性研究[J]. 动物营养学报, 2021 , 33(10) : 5887 -5894 . DOI: 10.3969/j.issn.1006-267x.2021.10.047

Abstract

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 5 000 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.

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