降解亚硝酸盐乳杆菌的筛选鉴定及其NiRs酶学性质

Screening and Identification of Nitrite-Degrading Lactobacillus and Characterization of Its Nitrite Reductase

控制食品中的亚硝酸盐含量是食品工业的热点话题之一,因此该研究以乳酸菌为研究对象,从中筛选出亚硝酸盐降解能力最强的菌株进行鉴定,随后分离纯化目的菌株的亚硝酸还原酶(nitrite reductase,NiRs),并研究其酶学性质。结果表明,亚硝酸盐降解率在90%以上的32株乳酸菌中,亚硝酸盐降解率最高的菌株36(目的菌株)的亚硝酸盐降解率为(96.36±0.37)%,为革兰氏阳性杆状菌,其糖类发酵试验结果与植物乳杆菌(Lactobacillus plantarum)一致;其16S rDNA基因序列长度为1464 bp,在NCBI中登录号为MN611150,与L.plantarum JCM1149同源性达100%,确定菌株36为植物乳杆菌。菌株36的NiRs比酶活为99.35 U/mg,回收率为15.5%,亚基相对分子质量约为47.8 kD;该酶最适温度为50℃,最适pH值为6.5,K_(m)值为7.79 mmol/L,V_(max)为5.84 mg/(L·min);当金属离子添加浓度在1 mmol/L~10 mmol/L,Fe^(2+)、Fe^(3+)和Ca^(2+)对NiRs酶活力有显著激活作用(P<0.05),1 mmol/L和10 mmol/L的Na^(+)以及10 mmol/L Ba^(2+)也有显著激活作用(P<0.05);而1 mmol/L和5 mmol/L的Ba^(2+)和K^(+)对NiRs酶活力有显著抑制作用(P<0.05),但乙二胺四乙酸(ethylenediaminetetraacetic acid,EDTA)对酶活无显著影响。

Controlling the nitrite content in food is one of the hot topics in the food industry.Therefore,this study aims to screen out and identify the lactic acid bacterial strain with the strongest nitrite-degrading ability.Then,the nitrite reductase(NiR)of the target strain was isolated and purified,and its enzymatic properties were studied.The results showed that the nitrite degradation rate was above 90% in 32 strains of lactic acid bacteria and was the highest[(96.36±0.37)%]in strain 36(target strain).Strain 36 was a Gram-positive rod-shaped bacterium,with the sugar fermentation test results consistent with those of Lactobacillus plantarum.The 16S rDNA sequence(NCBI accession No.MN611150)of this strain was 1464 bp and had the homology of 100% with that of L.plantarum JCM1149.Therefore,strain 36 was identified as L.plantarum.The NiR of strain 36 showed the activity of 99.35 U/mg,the recovery of 15.5%,and the molecular weight of 47.8 kD.The optimal temperature,optimal pH,K_(m),and V_(max)of this enzyme were 50℃,6.5,7.79 mmol/L and 5.84 mg/(L·min),respectively.When added at the concentration within 1 mmol/L~10 mmol/L,Fe^(2+),Fe^(3+)and Ca^(2+)significantly activated the NiR(P<0.05),and 1 mmol/Land 10 mmol/L Na^(+)and 10 mmol/L Ba^(2+)also had significant activation effects(P<0.05).However,1 mmol/L and 5 mmol/L Ba^(2+)and K^(+)significantly inhibited the activity of NiR(P<0.05),and ethylenediaminetetraacetic acid(EDTA)had no significant effect on the enzyme activity.