尖孢镰刀菌M1耐热蛋白酶纯化及酶学性质  

Purification and characterization of the thermostable protease from Fusarium oxysporim M1

作　　者：王世伟 王卿惠 向文胜[2] 莫继先 李婷婷 翟丽萍 王韬 WANG Shiwei;WANG Qinghui;XIANG Wensheng;MO Jixian;LI Tingting;ZHAI Liping;WANG Tao(Key Laboratory of Resistance Gene Engineering and Preservation of Biodiversity in Cold Areas,College of Life Sciences and Agriculture and Forestry,Qiqihar University,Qiqihar,Heilongjiang 161005,China;School of Life Science,Northeast Agricultural University,Harbin,Heilongjiang 150030,China)

机构地区：[1]齐齐哈尔大学生命科学与农林学院抗性基因工程与寒地生物多样性保护重点实验室,黑龙江齐齐哈尔161005 [2]东北农业大学生命科学学院,黑龙江哈尔滨150030

出　　处：《微生物学通报》2021年第5期1604-1615,共12页Microbiology China

基　　金：2018年度黑龙江省省属本科高校基本科研业务费科研项目(135309482)。

摘　　要：【背景】前期工作中,从北大仓白酒大曲分离到一株真菌,经形态学和分子生物学方法,将其鉴定为尖孢镰刀菌(Fusarium oxysporim)M1,研究发现该菌能产中性蛋白酶。中性蛋白酶是应用于工业化生产的重要酶制剂。由于其作用条件温和、催化速率较高,被广泛应用于食品、医药、皮革、饲料、化工和废弃物处理行业。【目的】为了使该菌蛋白酶应用于相关工业生产,需要对该蛋白酶进行纯化和酶学特性研究。【方法】采用硫酸铵分级分离、疏水和离子交换层析对该菌蛋白酶进行纯化,通过SDS-PAGE测定酶的纯度和分子量,并研究其热稳定性和酸碱适应性。【结果】经各步层析,蛋白酶纯化倍数达26.1,得率为7.9%;经测定纯酶的分子量为62 kD;该酶最适温度为40℃,最适pH为7.0,属于中性蛋白酶;该酶对酸较敏感,对碱有较强的耐受性;耐热性较强,但酶活性不受乙二胺四乙酸二钠盐抑制。【结论】由于该中性蛋白酶具有较好的耐热性,因此,可作为工业生产上潜在的生物催化剂。[Background] A fungus producing a neutral protease was isolated from Daqu of Beidacang liquor and identified as Fusarium oxysporim M1 by morphology and molecular biology in our preliminary work. Neutral proteases are important enzyme preparations which have been widely used in industrial production, such as food, medicine, leather, feed, chemical and waste treatment owing to its mild reaction conditions and high catalytic rate. [Objective] In order to apply the neutral protease from Fusarium oxysporim M1 to related industrial production, it is very necessary to purify the protease and study its enzymatic characteristics. [Methods] The protease was purified through ammonium sulfate fractionation, hydrophobic and ion exchange chromatography in this paper, and then its molecular weight was determined by SDS-PAGE electrophoresis. At same time, the thermal stability and acid-base adaptability of the enzyme were also studied. [Results] The protease purification fold and yield were 26.1 and 7.9% by different chromatography steps. The molecular weight of the purified enzyme was 62 kD. The optimum temperature and pH value were 40 ℃ and 7.0 respectively. The protease belongs to a neutral protease which is sensitive to acid but strong tolerance to alkali. However, it is interesting that the enzyme have strong heat resistance, but is not inhibited by EDTA. [Conclusion] Therefore, the protease from Fusarium oxysporim M1 can be used as a biological catalyst used in different industrial applications in the future.

关 键 词：镰刀菌属 耐热蛋白酶 纯化 特征 

分 类 号：TQ925[轻工技术与工程—发酵工程] TQ426.97