通过失活Sec途径阻遏蛋白和胞外蛋白酶提高地衣芽孢杆菌产碱性蛋白酶的能力<sup>*</sup>

doi:10.13523/j.cb.2307018

中国生物工程杂志

2024, Vol. 44

Issue (2/3): 39-47 DOI: 10.13523/j.cb.2307018

研究报告

通过失活Sec途径阻遏蛋白和胞外蛋白酶提高地衣芽孢杆菌产碱性蛋白酶的能力^*

郝漫,惠威,邵岚莹,史超硕,路福平,张会图^**(

)

天津科技大学生物工程学院应用微生物与酶工程实验室天津 300457

Improving the Ability of Bacillus licheniformis to Produce Alkaline Protease by Inactivating Sec Pathway Repressor Protein and Extracellular Proteases

HAO Man,HUI Wei,SHAO Lanying,SHI Chaoshuo,LU Fuping,ZHANG Huitu^**(

)

Laboratory of Applied Microbiology and Enzyme Engineering, College of Biotechnology, Tianjin University of Science &Technology, Tianjin 300457, China

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摘要：

为了探究Sec分泌途径对地衣芽孢杆菌(Bacillus licheniformis) 碱性蛋白酶产量的影响,对地衣芽孢杆菌 TCCC11470 (BL Δupp Δeps Δpgs)中的分子伴侣阻遏蛋白基因hrcA和基因组中3个Sec途径分泌的胞外蛋白酶基因epr、bpr和vpr进行叠加敲除。通过对比分析基因缺失前后的碱性蛋白酶酶活力发现,敲除菌株 TCCC11470ΔhrcA和TCCC11470ΔhrcAΔeprΔbprΔvpr在 42 h 的碱性蛋白酶酶活力分别达到 18 521.2 U/mL和20 048.5 U/mL,分别高出对照菌株 BLΔuppΔepsΔpgs (14 478.6 U/mL) 27.9%和38.5%。这一结果指出,通过改进Sec分泌通路可以显著提升碱性蛋白酶的催化效能,为构建优化的工业酶生产宿主提供了新思路和研究方向。

关键词： 地衣芽孢杆菌; Sec分泌途径; 碱性蛋白酶; HrcA; 胞外蛋白酶

Abstract:

In order to investigate the influence of the Sec secretion pathway on alkaline protease production in Bacillus licheniformis, the molecular chaperone-blocking protein gene hrcA and the three extracellular protease genes epr, bpr, and vpr in the genome of B. licheniformis TCCC11470 (BLΔuppΔepsΔpgs) were sequentially deleted. By comparing the alkaline protease activities before and after gene deletion, it was found that the knockout strains TCCC11470ΔhrcA and TCCC11470ΔhrcAΔeprΔbprΔvpr reached alkaline protease activities of 18 521.2 U/mL and 20 048.5 U/mL after 42 hours, respectively, which were 27.9% and 38.5% higher than the control strain BLΔuppΔepsΔpgs (14 478.6 U/mL), respectively. These results indicate that optimizing the Sec secretion pathway can effectively enhance the enzymatic activity of alkaline protease, providing new insights and research strategies for the construction of industrial enzyme production hosts.

Key words: Bacillus licheniformis Sec secretion pathway Alkaline protease HrcA Extracellular protease

收稿日期: 2023-07-13 出版日期: 2024-04-03

ZTFLH:

Q814

基金资助: *绿色生物制造国家重点研发专项(2021YFC2100400)

通讯作者: **电子信箱:hzhang@tust.edu.cn

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引用本文:

郝漫, 惠威, 邵岚莹, 史超硕, 路福平, 张会图. 通过失活Sec途径阻遏蛋白和胞外蛋白酶提高地衣芽孢杆菌产碱性蛋白酶的能力^*[J]. 中国生物工程杂志, 2024, 44(2/3): 39-47.

HAO Man, HUI Wei, SHAO Lanying, SHI Chaoshuo, LU Fuping, ZHANG Huitu. Improving the Ability of Bacillus licheniformis to Produce Alkaline Protease by Inactivating Sec Pathway Repressor Protein and Extracellular Proteases. China Biotechnology, 2024, 44(2/3): 39-47.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2307018 或 https://manu60.magtech.com.cn/biotech/CN/Y2024/V44/I2/3/39

图1 地衣芽孢杆菌碱性蛋白酶表达和分泌过程

表1 研究中使用的菌株和质粒

表2 研究中使用的引物

图2 基因敲除菌株的构建 A:基因敲除菌株构建流程 B:基因敲除菌株验证。 M:DNA标尺;WT:对照菌株;MT:基因敲除菌株

图3 hrcA敲除后groEL和dnaK基因转录水平的变化

图4 BLΔUEPΔhrcA的生物量与AprE合成能力分析

图5 删除hrcA对宿主aprE表达水平的影响

图6 胞外蛋白酶缺失对AprE表达的影响 A:胞外蛋白酶缺失对菌体AprE产量与转录水平的影响 B:敲除菌株蛋白质电泳,泳道1~8分别为BLΔUEP、CK、ΔE、ΔB、ΔV、ΔEB、ΔEBV 和 ΔAprE菌株

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