生物学杂志

生物学杂志 ›› 2021, Vol. 38 ›› Issue (1): 41-.doi: 10.3969/j.issn.2095-1736.2021.01.041

• 研究报告 • 上一篇    下一篇

基于前导肽和成熟区共进化提高米黑霉脂肪酶热稳定性研究

  

  1. 山西中医药大学 基础医学院， 晋中 030600
  • 出版日期:2021-02-18 发布日期:2021-02-22
  • 通讯作者: 李睿，硕士，副教授，研究方向为食品工程、生物制药，E-mail：46116288@qq.com
  • 作者简介:王珏，硕士，讲师，研究方向为分子生物学、基因工程和蛋白定向进化，E-mail:wj.happysmile@163.com
  • 基金资助:
    山西中医药大学科技创新能力培育计划（ 2018PY-20）；山西省自然基金青年项目（ 201801D221278）

Enhancing the thermostability of Rhizomucor miehei lipase through coevolution of the propeptide and the mature region#br#

  1. Department of Basic Medicine, Shanxi University of Chinese Medicine, Jinzhong 030600, China
  • Online:2021-02-18 Published:2021-02-22

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摘要： 研究结合理性设计和定向进化技术，通过2轮易错PCR、2次定点突变和4轮定点饱和突变提高米黑霉脂肪酶(Rhizomucor miehei Lipase，RML)的热稳定性，共得到9株理想的脂肪酶突变体，在经70 ℃热处理2 h后，均能保留对甘油三酯的水解活性。以M9（D48V/V67A/S168P/S240A/S311C/D313H）的热稳定性最佳，热处理后保留活性可达(1 316±31)U/mg，这一数据是野生型RML常温水解活性的5.2倍（野生型RML经热处理后完全失活）。经测序分析，突变位点D48V和V67A位于目的基因前导肽上，S168P、S240A、S311C和D313H位于目的基因成熟区，且对前导肽上的两个突变点进行饱和突变后，突变体的热稳定性会发生改变，说明前导肽的突变会影响RML全基因的热稳定性，因此，RML热稳定性得以提高是源于其前导肽和成熟区的共进化作用。

关键词: 前导肽, 基因突变, 米黑霉脂肪酶, 热稳定性

Abstract: The present study enhanced the Rhizomucor miehei lipase (RML) thermostability through the rational design and the directed evolution, including 2 rounds of error-prone PCR, 2 rounds of site-directed mutagenesis and 4 rounds of saturation mutagenesis totally. Nine enzyme mutants were obtained and all of these mutants could maintain the activities after being the heat treatment at 70 ℃ for 2 h. The activity of the mutant M9(D48V/V67A/S168P/S240A/S311C/D313H) could reach (1 316±31)U/mg after the heat treatment, which was 5.2-fold of wild type at 37 ℃ (the wild type RML has no activity after the heat treatment). With sequencing analysis, the mutant sites D48V and V67A were located on the propeptide of the gene, and the mutant sites of S168P, S240A, S311C and D313H were located on the mature region. After saturation mutation of the two mutants on the propeptide, the thermostability of the mutant changed, which indicated that the mutation of the propeptide could affect the thermostability of the whole RML gene.

Key words: propeptide, gene mutation, Rhizomucor miehei lipase, thermostability

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