黏质沙雷氏菌发酵制备舍雷肽酶及基本酶学性质研究  

Production and Partial Characterization of Serratiopeptidase from a Serratia marcescens Strain

作　　者：梅建凤[1] 李靓[1] 唐国强 易喻[1] 应国清[1] MEI Jian-feng;LI Liang;TANG Guo-qiang;YI Yu;YING Guo-qing(College of Pharmaceutical Science,Zhejiang University of Technology,Hangzhou 310014,China;Gmax Biopharm Limited Liability Company,Hangzhou 310051,China)

机构地区：[1]浙江工业大学药学院,杭州310014 [2]杭州鸿运华宁生物医药有限公司,杭州310051

出　　处：《中国药学杂志》2020年第24期2057-2062,共6页Chinese Pharmaceutical Journal

基　　金：浙江省公益技术应用研究项目资助(GF19B060006)。

摘　　要：目的开发微生物发酵生产舍雷肽酶的工艺,并了解基本酶学特性。方法从家蚕蛹的肠道中分离产酶菌株,单因素实验和响应面实验优化发酵培养基,采用(NH4)2SO4沉淀、超滤和DEAE离子交换层析3步纯化舍雷肽酶,电泳法测定酶的相对分子质量和等电点,考察pH和温度对酶活力和稳定性的影响,并测算酶的反应动力学常数。结果分离获得一株产舍雷肽酶的黏质沙雷氏菌LL-413菌株,摇瓶发酵产酶活力达到1 126 U·mL^-1,10-L发酵罐中发酵产酶活性达到1 505 U·mL^-1;舍雷肽酶相对分子质量和等电点分别约为52×103和7.2;酶活最适pH为8.0,在pH 5.0~10.0之间稳定;酶活最适温度为40℃,在45℃以下稳定。以酪蛋白为底物时,米氏常数(Km)和最大反应速度(Vmax)分别为22.3 mg·mL^-1和1 355 mg·L·min^-1。结论利用黏质沙雷氏菌LL-413菌株生产舍雷肽酶发酵单位高,酶的蛋白水解能力强、pH和热稳定性好,研究结果为该酶的生产和应用奠定了基础。OBJECTIVE To develop a fermentation process for production of serratiopeptidase, and make clear its partial enzymatic characteristics. METHODS Enzyme-producing strains were isolated from the gut of Chinese silkworm(Bombyx mori L) pupa. The fermentation medium was optimized by single factor experiment and response surface design. A combination of(NH4)2SO4 precipitation, ultrafiltration and DEAE-ion exchange chromatography was applied to purify the enzyme. The molecular weight and isoelectric point of the enzyme were determined by electorphoresis. The effects of pH and temperature on the activity and stability of the enzyme were investigated. Kinetic constants of the purified serratiopeptidase were determined. RERULTS A serratiopeptidase-producing bacterial strain, Serratia marcescens LL-413, was isolated. The activities of the enzyme produced using optimized medium were 1 126 U·mL^-1 in the flask cultivation and 1 534 U·mL^-1 in a 10-L batch fermentor, respectively. The molecular mass and isoelectric point of the purified serratiopeptidase were about 52 k and 7.2 respectively. The optimum pH for the enzyme activity was 8.0, and it was stable between 5.0 and 10.0. The optimum temperature for the enzyme activity was 40 ℃, and it was stable below 45 ℃. Michaelis-Menten constant(Km) and maximal reaction velocity(Vmax) of the serratiopeptidase LL-413 were 22.3 mg·mL^-1 and 1 355 mg·L^-1·min^-1, respectively. CONCLUSION Fermentation of serratiopeptidase by Serratia marcescens LL-413 has a high productivity. The serratiopeptidase LL-413 has strong protein hydrolysis activity and excellent pH and thermal stability. These results are promising for future production and application of serratiopeptidase.

关 键 词：舍雷肽酶 黏质沙雷氏菌 酶的分离和纯化 酶学特性 

分 类 号：R91[医药卫生—药学] TQ925.2[轻工技术与工程—发酵工程]