1株枯草芽胞杆菌的鉴定及其弹性蛋白酶结构研究

鉴定弹性蛋白酶产生菌株EL32,确定该酶的基本结构。采用分子生物学、形态学及生理生化性质对菌株EL32进行鉴定;用硫酸铵沉淀、离子交换层析和分子筛层析纯化酶蛋白;借助肽指纹图谱及酶基因克隆技术研究该酶的一级结构;利用同源建模的方法研究该酶的空间结构。菌株EL32的16S rDNA与枯草芽胞杆菌16S rDNA的同源性达到99%,其菌落呈乳白色,其细胞革兰染色阳性,具有芽胞;发酵葡萄糖试验产酸不产气,明胶水解试验呈阳性,能够水解淀粉,V-P反应呈阳性,鉴定为枯草芽胞杆菌。从菌株BL32发酵液中纯化得到了弹性蛋白酶,SDS-PAGE分析显示其分子量为31 ku。用LTQ-MS测定肽指纹图谱表明该弹性蛋白酶是枯草芽胞杆菌蛋白酶subtilisin,该酶的基因和蛋白质序列与枯草芽胞杆菌蛋白酶subtilisin的同源性都高达99%。菌株EL32弹性蛋白酶的三维结构含有6个α-螺旋,7个扭曲的平行β-折叠以及2个反平行的β-折叠,His、Asp和Ser是其活性中心的关键基团。鉴定了1株产弹性蛋白酶的枯草芽胞杆菌,确定了其弹性蛋白酶是蛋白酶subtilisin,为该枯草芽胞杆菌蛋白酶的应用提供了基础。

Bacillus subtilis Strain Identification and Its Enzyme Structure of Elastase

An elastase-produeing strain EL32 was identified and confirmed the structure of the enzyme. The strain EL32 was identified adopting molecular biology, morphology, and physio-bioehemieal characterization. It was purified with ammonium sulphate precipitation, ion-exchange chromatography and molecular sieve. The enzyme was studied its primary structure with the aid of peptide fingerprint spectrum and enzymatic genetic clone; using homologous mem- brane establishment method to study its three dimensional strueture. The 16S rDNA of strain EL32 had 99% homology with Bacillus subtilis. Its colonies were milky white. Its eell was Gram positive possessing gemma; producing acid but no gas in the glucose fermentation test. Gelatin hydrolysis test positive, capable of starch hydrolysis, V-P reaction positive, and identified strain EL32 as Bacillus subtilis. The molecular weight of elastase purified from fermentation broth was 31 ku by SDS-PAGE analysis. The elastase determined by LTQ-MS peptide fingerprint speetrum indicated it was subtilisin, the gene and protein sequence of elastase shared high homology of 99% with subtilisin. The three di- mensional structure of the elastase strain EL32 consisted of six a-helices, seven-stranded parallel β-folds and two an- ti-parallel 13-folds, and His, Asp, and Set were the key groups of active center. The elastase-produeing strain EL32was identified as Bacillus subtilis, and confirmed the elastase to be subtilisin. All these results provided a foundation for the application of the subtilisin.