枯草杆菌蛋白酶QK的活性与氨基酸突变位点的相关性

Correlation between the Activity of Subtilisin QK and Amino Acid Mutation Sites

目的:鉴定不同来源的枯草芽孢杆菌(Bacillus subtilis)产生的枯草杆菌蛋白酶QK(subtilisin QK)的活性与编码其序列的氨基酸位点突变的相关性。方法:以筛选得到的15株菌株的总DNA为模板,细菌16SrRNA为通用引物,进行PCR扩增,将产物测序后输入NCBI比对,确定其均为枯草芽孢杆菌属。再扩增这15株菌株的QK基因,测序后与GenBank上已提交的QK基因所对应的蛋白序列进行比对,找出氨基酸差异位点。使用纤维蛋白平板法测定菌株所产枯草杆菌蛋白酶的活性,用BCA蛋白测定试剂盒测定发酵液的总蛋白含量,并计算得到单位酶活。结果:根据突变位点的不同把15株菌株分为A、B、C、D四组。A组的突变位点为S184T,单位酶活显著最高(p<0.05);B组的突变位点为Q90K,N193S,S236T,N365S,C组的突变位点为N193S,S236T,A289V,B、C两组之间单位酶活并无显著性差异(p>0.05),但B、C两组单位酶活显著小于A组(p<0.05);D组的突变位点为Q90K,N193S,S236T,A289V,单位酶活显著低于其他三组(p<0.05)。结论:枯草杆菌蛋白酶QK的单位酶活与氨基酸突变位点之间有相关性:S184T和N365S位点变化对单位酶活性有促进作用,A289V位点变化对单位酶活性有抑制作用。据此推测,这些位点位于枯草杆菌蛋白酶QK的活性中心。

Objective:To determine whether the activity of subtilisin QK produced by Bacillus subtilis from different strains is related to the amino acid site mutation encoding its sequence.Methods:The total DNA of 15 strains was used as a template.The 16S rRNA of bacteria was used as an universal primer for PCR amplification.The products were sequenced and input into NCBI to determine that they were all B.subtilis.The QK genes of 15 strains were amplified and sequenced.The protein sequences corresponding to the submitted QK gene in GenBank were aligned to find different amino acid sites.Fibrin plate method was used to determine the activity of the subtilisin produced by these strains.BCA protein assay kit was used to determine the total protein content of the fermentation broth.The unit enzyme activity was calculated.Results:According to the mutation sites,15 strains were divided into four groups.The mutation site of group A was S184T.The unit enzyme activity of group A was the highest( p < 0.05).The mutation sites of group B were Q90K,N193S,S236T,N365S,and the mutation sites of group C were N193S,S236T,A289V.The results showed that there was no significant difference between these two groups( p > 0.05).But the enzyme activity in groups B and C was significantly lower than that of group A( p < 0.05).The mutation sites of group D were Q90K,N193S,S236T,A289V,and the unit enzyme activity was significantly lower compared with that of group A,B and C ( p < 0.05).Conclusion:There was an obvious relationship between the unit enzyme activity of subtilisin QK and amino acid mutation sites.Among these amino acid mutation sites,the sites of S184T and N365S might promote the activity of unit enzyme. And the site A289V might have an inhibitory effect.These sites might be located at the active center of subtilisin QK.