高产硝酸盐还原酶Staphylococcus simulans ZSJ6的复合诱变选育及其酶学性质研究  

Investigation on the Complex Mutagenesis Selection of High-yield Nitrate Reductase Strain Staphylococcus simulans ZSJ6 and Its Enzymatic Properties

作　　者：赵赛赛 张小丹 贾晓妍 陶大炜 刘可玉 宁喜斌[1,2,3] ZHAO Sai-sai;ZHANG Xiao-dan;JIA Xiao-yan;TAO Da-wei;LIU Ke-yu;NING Xi-bin(College of Food Science&Technology,Shanghai Ocean University,Shanghai 201306;Laboratory of Quality&Safety Risk Assessment for Aquatic Products on Storage and Preservation(Shanghai),Ministry of Agriculture and Rural Affairs,Shanghai 201306;National R&D Branch Center for Freshwater Aquatic Products Processing Technology(Shanghai),Shanghai 201306)

机构地区：[1]上海海洋大学食品学院,上海201306 [2]农业农村部水产品贮藏保鲜质量安全风险评估实验室(上海),上海201306 [3]国家淡水水产品加工技术研发中心(上海),上海201306

出　　处：《生物技术通报》2023年第4期103-113,共11页Biotechnology Bulletin

摘　　要：硝酸盐还原酶可以将天然肉制品中的硝酸盐还原为亚硝酸盐,避免了直接添加亚硝酸盐导致其含量骤增,降低产生亚硝胺的概率,对天然肉制品的品质和安全非常重要。以腊肠中筛选出的模仿葡萄球菌(Staphylococcus simulans D14)为出发菌株,探究了单一诱变条件包括紫外、微波、氯化锂诱变,以及紫外-微波-氯化锂联合诱变的两种复合诱变方式对菌株的不同影响,以期获得硝酸盐还原酶高产菌株,并通过摇瓶发酵初步探究其酶学性质,以期为天然肉制品的发酵剂提供菌株选择。确定了紫外、微波和氯化锂诱变的最佳条件:紫外照射60 s,微波辐照80 s,氯化锂浓度1.5%。筛选出诱变菌株Staphylococcus simulans ZSJ6,菌株酶活力可达603.29 U/mg蛋白,是出发菌株酶活力的3.59倍,且突变菌株连续传代8次后,其酶活力并无显著变化(P>0.05),表明其遗传稳定性较好。酶学性质结果显示,其最适作用温度为30℃,最适作用pH为7.5,且稳定性良好,在pH 7.5下孵育2 h,酶活力仍保留90%以上。Mg^(2+)、Ca^(2+)、K^+均能促进酶活力,其中Ca^(2+)对酶活力的促进作用最大,相对于空白组酶活力提升了1.63倍。Cu^(2+)、Fe^(2+)、Hg^(2+)、Mn^(2+)均能抑制酶活,其中Cu^(2+)和Hg^(2+)对酶活力的抑制作用最大,酶活力均被抑制到30%以下。研究结果为天然肉制品中硝酸盐的转化,以及生产初期使用亚硝酸盐带来的亚硝酸盐浓度过高等问题提供了解决思路,具有研究价值和应用潜力。Nitrate reductase can reduce the nitrate in natural meat products to nitrite,avoiding a sudden increase in its content due to direct addition of nitrite and reducing the chance of producing nitrosamines,which is very important for the quality and safety of natural meat products.Staphylococcus simulans D14 screened from sausage as the starting strain,and the different effects of single mutagenesis condition including UV,microwave,and lithium chloride mutagenesis,and two compound mutagenesis methods of UV and microwave combined with lithium chloride on the strains were investigated to obtain high-yield strains of nitrate reductase,and their enzymatic properties were initially explored by shake flask fermentation in order to provide strain selection for fermenters of natural meat products.The optimal conditions for UV,microwave and lithium chloride mutagenesis were determined:UV irradiation for 60 s,microwave irradiation for 80 s and lithium chloride concentration of 1.5%.The mutagenic strain S.simulans ZSJ6 was screened,and the enzyme activity of the strain reached 603.29 U/mg protein,which was 3.59 times higher that of the starting strain,and there was no significant change in the enzyme activity of the mutagenic strain after 8 consecutive passages(P>0.05),indicating that its genetic stability was good.From the results of enzymatic properties,its optimal action temperature was 30℃,the optimal action pH was 7.5,and its stability was excellent,and the enzyme activity retained more than 90%after incubation at pH 7.5 for 2 h.Mg^(2+),Ca^(2+),and K^(+)all promoted enzyme activity,of which Ca2+had the largest role in promoting the enzyme activity,which was 1.63 times higher than that blank group.Cu^(2+),Fe^(2+),Hg^(2+)and Mn^(2+)suppressed enzyme activity,among which Cu^(2+)and Hg^(2+)had the maximum inhibitory action on the enzyme activity,and the enzyme activity was all inhibited to less than 30%.The above results provide a solution for the transformation of nitrate in natural meat products and the problem of high ni

关 键 词：模仿葡萄球菌 硝酸盐还原酶 天然肉制品 复合诱变 酶学性质 亚硝酸盐 硝酸盐 

分 类 号：TS201.25[轻工技术与工程—食品科学]