食品科学 ›› 2025, Vol. 46 ›› Issue (15): 120-117.doi: 10.7506/spkx1002-6630-20250224-111

• 生物工程 • 上一篇    下一篇

ARTP-UV复合诱变结合含盐平板选育产耐盐高活性蛋白酶的米曲霉

孙思城,李浩然,苏国万,吴惠贞,林伟锋   

  1. (1.华南理工大学食品科学与工程学院,广东 广州 510640;2.鹤山市东古调味食品有限公司,广东 江门 529738)
  • 出版日期:2025-08-15 发布日期:2025-07-22
  • 基金资助:
    国家自然科学基金面上项目(32172329)

Combined Mutagenesis by Atmospheric and Room Temperature Plasma and Ultraviolet and Screening on Salt-Containing Plates of Aspergillus oryzae for High Salt Tolerance and Activity of Protease

SUN Sicheng, LI Haoran, SU Guowan, WU Huizhen, LIN Weifeng   

  1. (1. College of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China; 2. Heshan Donggu Flavoring Food Co. Ltd., Jiangmen 529738, China)
  • Online:2025-08-15 Published:2025-07-22

摘要: 对从高盐稀态酱醪中筛选得到的米曲霉(Aspergillus oryzae)K9进行常压室温等离子体(atmospheric and room temperature plasma,ARTP)复合紫外诱变,并利用高盐含量平板进行驯化培养和筛选,旨在得到产耐盐高活性蛋白酶的菌株。首先对K9进行ARTP诱变,筛选得到优势菌株S-G27,其中性蛋白酶活性较K9提高35.82%;接着以S-G27为出发菌株进行紫外诱变,筛选得到优势菌株G-U2,其中性蛋白酶活性较K9提高56.81%;G-U2所产蛋白酶的耐盐性进一步提高,在15% NaCl溶液中,G-U2中性蛋白酶活性残留率为K9的1.94 倍;G-U2稳定性良好,第7代时菌株的中性蛋白酶活性残留率为92%;和出发菌株K9相比,G-U2的菌丝更短、孢子更多、孢子体积更大且表面纹饰更多;诱变未引起黄曲霉毒素相关基因的表达,符合生产安全要求。在模拟发酵酱油实验中,发酵终点G-U2中性蛋白酶活性、酸性蛋白酶活性、氨基酸态氮质量浓度分别较K9高出120.19%、52.29%、18.37%。

关键词: 米曲霉;常压室温等离子体诱变;紫外线诱变;耐盐蛋白酶

Abstract: This study aimed to obtain strains producing high levels of salt-tolerant protease from Aspergillus oryzae K9, isolated from high-salt liquid-state moromi, by combined mutagenesis induced by atmospheric and room temperature plasma (ARTP) and ultraviolet (UV) followed by domestication and screening using high-salt plates. The dominant mutant S-G27, whose neutral protease activity was increased by 35.82% compared with K9, was obtained after ARTP treatment of K9. The dominant mutant G-U2, whose neutral protease activity was increased by 56.81% compared with K9, was obtained after subsequent UV treatment of S-G27. In 15% NaCl solution, the residual activity of neutral protease from G-U2 was 1.94 times as high as that of K9. G-U2 exhibited good stability, and 92% of the initial enzyme activity remained after the seventh passage. Compared with the starting strain K9, G-U2 showed shorter mycelia, more spores, larger spore volume, and more surface ornaments. Mutagenesis did not induce the expression of aflatoxin-related genes, meeting production safety requirements. In simulated soy sauce fermentation experiments, the neutral protease activity, acid protease activity, and amino nitrogen content of G-U2 at the end of fermentation were 120.19%, 52.29%, and 18.37% higher than those of K9, respectively.

Key words: Aspergillus oryzae; atmospheric and room temperature plasma mutagenesis; ultraviolet mutagenesis; salt tolerant protease

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