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Journal of Zhejiang University SCIENCE B
ISSN 1673-1581(Print), 1862-1783(Online), Monthly
2025 Vol.26 No.8 P.805-812
Improvement of neutral protease activity of Bacillus amyloliquefaciens LX-6 by combined ribosome engineering and medium optimization and its application in soybean meal fermentation
Abstract: Soybean meal (SBM) prepared by soybean crushing is the most popular protein source in the poultry and livestock industries (Cai et al., 2015) due to its economic manufacture, high protein content, and good nutritional value. Despite these benefits, SBM contains various antigen proteins such as glycinin and β-conglycinin, which account for approximately 70% of the total proteins of the SBM and reduce digestibility and damage intestinal function (Peng et al., 2018). Treating SBM with proteases (neutrase, alcalase, and trypsin) or fermentation can eliminate these antigen proteins (Contesini et al., 2018). Because of its safety and rapid growth cycle, Bacillus strains are considered ideal for the fermentation industry (Yao et al., 2021). SBM fermented by Bacillus yields products with high nutritional value and low levels of antinutritional factors (ANFs), stimulating research in this area (Yuan et al., 2017). Kumari et al. (2023) demonstrated that fermentation with Bacillus species effectively degrades antigen proteins and increases crude protein content. The degradation of antigen proteins relies on protease hydrolysis. Low protease production is the major obstacle hindering the widespread use of microbial fermentation techniques.
Key words: Bacillus amyloliquefaciens; Ribosome engineering; Fermentation optimization; Neutral protease; Soybean meal
1中国计量大学生命科学学院, 国家市场监督管理总局微生物计量检测与生物制品质量安全重点实验室, 中国杭州市, 310018
2浙江海洋大学水产养殖学院, 中国杭州市, 316022
摘要: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 r)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(CaCl2)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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DOI:
10.1631/jzus.B2400477
CLC number:
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On-line Access:
2025-08-25
Received:
2024-09-19
Revision Accepted:
2024-12-26
Crosschecked:
2025-08-25