联合核糖体工程与培养基优化提高解淀粉芽孢杆菌LX-6中性蛋白酶活性及其在豆粕发酵中的应用

朱怡凡; 黄心怡; 韩涛; 王骥腾; 俞晓平; 马正

doi:10.1631/jzus.B2400477

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联合核糖体工程与培养基优化提高解淀粉芽孢杆菌LX-6中性蛋白酶活性及其在豆粕发酵中的应用

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Correspondence | Updated：2025-08-25

联合核糖体工程与培养基优化提高解淀粉芽孢杆菌LX-6中性蛋白酶活性及其在豆粕发酵中的应用
Improvement of neutral protease activity of Bacillus amyloliquefaciens LX-6 by combined ribosome engineering and medium optimization and its application in soybean meal fermentation
浙江大学学报（英文版）（B辑：生物医学和生物技术） 2025年26卷第8期页码：805-812
Affiliations：

1.Key Laboratory of Microbiological Metrology, Measurement & Bio-product Quality Security, State Administration for Market Regulation, College of Life Sciences, China Jiliang University, Hangzhou 310018, China
2.Department of Aquaculture, Zhejiang Ocean University, Zhoushan 316022, China
Funds：

the Ten-thousand Talents Plan of Zhejiang Province(2022R52021);the Key Research and Development Program of Zhejiang Province(2021C04016);the Pioneer and Leading Goose R&D Program of Zhejiang(2022C04020)
DOI：10.1631/jzus.B2400477
中图分类号：
CSTR：
收稿日期：2024-09-19，

修回日期：2024-12-26，

网络出版日期：2025-07-11，

纸质出版日期：2025-08-25
Accepted：

朱怡凡,黄心怡,韩涛等.联合核糖体工程与培养基优化提高解淀粉芽孢杆菌LX-6中性蛋白酶活性及其在豆粕发酵中的应用[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2025,26(08):805-812.

Yifan ZHU, Xinyi HUANG, Tao HAN, et al. Improvement of neutral protease activity of Bacillus amyloliquefaciens LX-6 by combined ribosome engineering and medium optimization and its application in soybean meal fermentation[J]. Journal of zhejiang university-science b (biomedicine & biotechnology), 2025, 26(8): 805-812.
朱怡凡,黄心怡,韩涛等.联合核糖体工程与培养基优化提高解淀粉芽孢杆菌LX-6中性蛋白酶活性及其在豆粕发酵中的应用[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2025,26(08):805-812. DOI： 10.1631/jzus.B2400477.

Yifan ZHU, Xinyi HUANG, Tao HAN, et al. Improvement of neutral protease activity of Bacillus amyloliquefaciens LX-6 by combined ribosome engineering and medium optimization and its application in soybean meal fermentation[J]. Journal of zhejiang university-science b (biomedicine & biotechnology), 2025, 26(8): 805-812. DOI： 10.1631/jzus.B2400477.

摘要

Abstract

解淀粉芽孢杆菌（

Bacillus amyloliquefaciens

LX-6）因具有中性蛋白酶活性，在豆粕（soybean meal

SBM）发酵中具有较大应用潜力。本研究通过核糖体工程及培养基优化技术，提高解淀粉芽孢杆菌LX-6的中性蛋白酶活性。首先，利用核糖体工程技术从306个自发利福平抗性（Rif

）突变株中筛选出突变株5r-10。与野生型（WT）菌株LX-6相比，突变株的中性蛋白酶活性提高了38.45%。研究发现，突变株编码RNA聚合酶β亚基的

rpoB

基因在第469个氨基酸位点发生谷氨酸→精氨酸（glutamic acid→arginine，Glu→Arg）的突变；在形态上与WT菌株LX-6相比，突变株5r-10的细胞表面更光滑且鞭毛更少。随后，在摇瓶实验中，我们采用基于响应面方法的单因素实验和Box-Behnken设计，对菌株产中性蛋白酶培养基成分进行了优化。在最佳浓度的乳糖、酪蛋白和氯化钙（CaCl

）培养条件下，突变株5r-10的中性蛋白酶活性最高可以达到(3470.67±

23.17) U/mL，是WT菌株LX-6在原始培养基中产生的中性蛋白酶活性的1.49倍。此外，与WT菌株LX-6相比，突变株5r-10在固体发酵过程中对SBM中大分子抗原蛋白的降解更快，即与LX-6发酵相比，经突变株5r-10发酵后，SBM中大豆球蛋白和β-伴大豆球蛋白的降解率显著增加。

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