枯草杆菌蛋白酶QK的活性与氨基酸突变位点的相关性
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摘要
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16S rRNA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.
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.
引文
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[9]雒焕贞,宋焕禄,陈存社,等.市售纳豆产品中菌株的分离、鉴定及纳豆激酶活力比较[J].食品工业科技,2017(1):141-146.
[10]刘卫今,蒋勇,完迪迪.简单高效的感受态细胞制备和质粒转化体系的建立[J].安徽农业科学,2008,36(7):2745-2746.
[11]Astrup T,Sten Müllertz.The fibrin plate method for estimating fibrinolytic activity[J].Archives of Biochemistry and Biophysics,1952,40(2):346-351.
[12]陈锡雄,阮俊锋,高文,等.传统纳豆中纳豆菌分离筛选的初步研究[J].宁德师专学报:自然科学版,2008(2):134-136.
[2]惠明,窦丽娜,田青,等.枯草芽孢杆菌的应用研究进展[J].安徽农业科学,2008(27):11623-11624.
[3]Ko J H,Yan J P,Zhu L,et al.Identification of two novel fibrinolytic enzymes from Bacillus subtilis QK02[J].Comp Biochem Physiol C Toxicol Pharmacol,2004,137(1):65-74.
[4]牛树壹.枯草杆菌蛋白酶的分离纯化、酶学性质及体外溶栓性质[D].青岛:中国海洋大学,2014.
[5]曾祥燕,赵良忠.纳豆芽孢益生杆菌的筛选鉴定及耐受性、抑菌能力的研究[J].食品工业科技,2015(1):160-165.
[6]Weng M,Deng X,Bao W,et al.Improving the activity of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation[J].Biochem Biophys Res Commun,2015,465(3):580-586.
[7]刘中美,何孝天,崔文璟,等.通过定点突变增强纳豆激酶的热稳定性[J].现代食品科技,2015(2):37-41.
[8]马明,杜金华,于玲,等.高纳豆激酶酶活枯草芽孢杆菌的筛选及菌种鉴定[J].中国食物与营养,2006(8):29-32.
[9]雒焕贞,宋焕禄,陈存社,等.市售纳豆产品中菌株的分离、鉴定及纳豆激酶活力比较[J].食品工业科技,2017(1):141-146.
[10]刘卫今,蒋勇,完迪迪.简单高效的感受态细胞制备和质粒转化体系的建立[J].安徽农业科学,2008,36(7):2745-2746.
[11]Astrup T,Sten Müllertz.The fibrin plate method for estimating fibrinolytic activity[J].Archives of Biochemistry and Biophysics,1952,40(2):346-351.
[12]陈锡雄,阮俊锋,高文,等.传统纳豆中纳豆菌分离筛选的初步研究[J].宁德师专学报:自然科学版,2008(2):134-136.