水貂源高产蛋白酶枯草芽孢杆菌的分离筛选及酶学性质

Isolation,identification,and enzymatic characterization of Bacillus subtilis from mink

【背景】开发、筛选优良益生菌菌种是当下畜牧业的研究热点,益生菌的潜在功能也被广为挖掘。【目的】分离、筛选具有良好耐受性且高产胞外蛋白酶的菌株,研究其生物学特性和酶学性质,为后续微生物蛋白酶的制备和微生态制剂的开发提供菌种资源。【方法】采集健康水貂新鲜粪便,配制酪蛋白培养基初筛和优化Folin-酚法复筛,对筛选菌进行生物学特性研究,获得产蛋白酶能力较强、耐受性较优的菌株,并进行常规鉴定和分子生物学鉴定,最后对蛋白酶酶学性质进行研究。【结果】筛选得到一株高产蛋白酶、耐受性较优的芽孢杆菌,经鉴定为枯草芽孢杆菌(Bacillus subtilis),编号为3。在初始发酵培养基条件下,酶学性质研究结果表明,该蛋白酶的最适反应温度为70℃,最适反应pH值为9.0,最佳金属离子激活剂为K^(+),Cu^(2+)和Fe^(2+)对酶活力有明显的抑制作用,在20%浓度的有机溶剂作用时蛋白酶未变性失活。【结论】从水貂粪便中分离获得一株具有良好的生物学特性、酶学性质和碱性蛋白酶活性的枯草芽孢杆菌,为该菌株在实际生产应用中提供了基础保障。

[Background]The development and screening of elite probiotics is the research hotspot of animal husbandry,and the potential function of probiotics has been extensively explored.[Objective]To isolate and screen the strains with good tolerance and high yield of extracellular protease and study their biological and enzymatic properties,so as to provide strain resources for the preparation of microbial protease and the development of microecological preparations.[Methods]Fresh feces samples were collected from healthy minks,from which the target strains were preliminarily screened with casein-containing medium and re-screened with optimized Folin method.The biological characteristics of the strains screened out were studied.The strains with high protease yield and good tolerance were obtained and identified by routine and molecular methods.Finally,the enzymatic properties of the protease were studied.[Results]A Bacillus strain with high protease yield and good tolerance was obtained and identified as B.subtilis(number 3).In the initial fermentation medium,the protease showed that the optimal reaction performance at 70℃and pH 9.0,with the activity improved by K^(+)and inhibited by Cu^(2+)and Fe^(2+).In addition,20%organic solvent did not deactivate the protease.[Conclusion]A B.subtilis strain with good biological characteristics,enzymatic characterization,and alkaline protease activity was isolated from mink feces.The findings of this study provide a basis for the production and application of this strain.