张 红1，张卫兵1*，乔海军2*，朱建宁3，温得林4，张智良1，赵丽红1
（1.甘肃农业大学食品科学与工程学院，甘肃兰州 730070；
2.甘肃农业大学理学院，甘肃兰州 730070；
3.甘肃省药品监督管理局审评认证中心，甘肃兰州 730070；
4.甘肃天祝藏酒酒业有限公司，甘肃武威 733200）
摘要：采用酪蛋白平板对藏香型白酒大曲中高产蛋白酶菌株进行分离筛选，然后通过硫酸铵沉淀、透析和DEAESepharose FF阴离子交换层析对其产生的蛋白酶进行纯化，并研究其酶学性质。结果表明：菌株ZP-8产酶活力最高，为 197.6 U/mL，经鉴定为地衣芽孢杆菌（Bacillus licheniformis）。纯化后测得酶的分子量约为 45.43 kDa，最适pH值为8.0，在pH 7.0～11.0的范围内稳定性好，属于碱性蛋白酶。该酶最适反应温度为50 ℃，在30～50 ℃范围内稳定性较好；Mg2＋、Na＋、Ca2＋和Fe2＋对蛋白酶相对酶活力无显著影响（P＞0.05），Cu2＋和Mn2＋具有显著的激活作用（P＜0.05）。本研究对酒曲中产蛋白酶微生物的开发利用具有重要的参考价值。
关键词：藏曲；筛选鉴定；蛋白酶；分离纯化；酶学性质
中图分类号：TS262.13 文献标识码：A 文章编号：1674-506X（2022）04-0008-0006
Research on Screening of Protease-producing Strains from Tianzhu Traditional Zangqu and Enzymatic Properties
ZHANG Hong1, ZHANG Weibing1*, QIAO Haijun2*, ZHU Jianning3, WEN Delin4, ZHANG Zhiliang1, ZHAO Lihong1
（1.College of Food Science and Engineering, Gansu Agricultural University, Lanzhou Gansu 730070, China;
2.College of Science, Gansu Agricultural University, Lanzhou Gansu 730070, China;
3.Review Certification Center of Gansu Drug Administration, Lanzhou Gansu 730070, China;
4.Gansu Tianzhu Tibetan Liquor Co., Ltd., Wuwei Gansu 733200, China）
Abstract：In the experiment, the casein plate was used to isolate and screen the high-protease-producing strains from Tibetan-flavor liquor Daqu. Then, ammonium sulfate precipitation, dialysis desalting and DEAE-Sepharose FF anion exchange chromatography were used to purify the protease, and its enzymatic properties were studied. The results showed that strain ZP-8 had the highest enzyme activity of 197.6 U/mL, which was identified as Bacillus licheniformis. The molecular weight of the purified enzyme is about 45.43 kDa. The optimal pH of this enzyme is 8.0, and it is relatively stable in the pH rang of 7.0～11.0, which belongs to alkaline protease. The optimal temperature of this enzyme is 50 ℃, and it is relatively stable at 30～50 ℃; Mg2＋, Na＋, Ca2＋ and Fe2＋ have no significant effect on the relative activity of protease (P＞0.05), while Cu2＋ and Mn2＋ have remarkable activation effects (P＜0.05). This research has important reference value for the development and utilization of protease-producing microorganisms.
Keywords：Zangqu; screening and identification; protease; separation and purification; enzymatic properties
doi：10.3969/j.issn.1674-506X.2022.04-002
收稿日期：2022-01-10
基金项目：国家自然科学基金地区科学基金项目（31960486）
作者简介：张红（1996-），女，硕士。研究方向：食品微生物。
*通信作者：张卫兵（1974-），男，教授。研究方向：食品微生物；乔海军（1976-），男，副教授。研究方向：食品化学。
引用格式：张红,张卫兵,乔海军,等.天祝传统藏曲中蛋白酶产生菌的筛选与酶学特性研究[J].食品与发酵科技,2022,58(4):8-13.
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