海水养殖凡纳滨对虾肠道产蛋白酶菌株的筛选、鉴定

Screening and Identification of Protease-producing Bacteria from the Intestines of Maricultured Litopenaeus vannamei

摘要:

为开发海洋来源的微生物蛋白酶，本文以海水养殖凡纳滨对虾肠道为原料，利用蛋白酶水解圈法初筛得到27株产蛋白酶菌株，测定菌株的透明圈直径与菌落直径比值（即D/d）初步估计其产酶能力，通过发酵液酶活测定复筛得到一株高产蛋白酶菌株，编号为xc10，通过形态学观察、生理生化鉴定、16S rDNA基因序列分析及系统发育树分析进行菌种鉴定，并对其发酵液粗蛋白酶的酶学性质进行了初步探讨。结果表明：菌株xc10为蜡状芽孢杆菌（Bacillus cereus），蛋白酶的最适温度为50 ℃；最适pH为7.5；Fe3+、Cu2+、EDTA对该酶有抑制作用，但Zn2+对该酶有激活作用。因此，从海水养殖的凡纳滨对虾肠道中分离得到的产蛋白酶的菌株xc10为一株中性、中温蛋白酶菌，为开发利用海洋微生物蛋白酶在水产品加工的应用提供了理论基础。

Abstract:

In this study, to obtain marine-derived microbial protease, 27 protease-producing bacterial strains were isolated from the intestines of maricultured Litopenaeus vannamei using protease hydrolysis circle method. Enzyme production by different strains was preliminarily estimated by determining the ratio of transparent circle diameter to colony diameter (D/d). A high-yield protease-producing strain, named as xc10, was isolated during secondary screening by measuring protease activity of fermentation broth. It was identified as Bacillus cereus by cell morphology, physiological, and biochemical characteristics, 16S rDNA gene sequence analysis, and phylogenetic tree analysis.Enzyme properties of crude protease in the fermentation broth were investigated. The optimal temperature and the pH for protease were 50 ℃ and 7.5, respectively. Additionally, the enzyme activity was inhibited by Fe3+, Cu2+, and EDTA, while Zn2+ slightly increased the enzyme activity. Therefore, strain xc10 isolated from the intestine of maricultured Litopenaeus vannamei was confirmed as a bacterial strain producing mesophilic neutral protease. This study provides a theoretical basis for the development and application of marine-derived microbial proteases in the processing of aquatic products.