Bacillus tropicus来源β-D-呋喃果糖苷酶基因的克隆与表达及其酶学特性研究

Cloning and expression of theβ-D-fructofuranosidase from Bacillus tropicus and the characterization of the cloned enzyme

为获得新β-D-呋喃果糖苷酶资源并探究其酶学性质及在转化糖中的应用潜力,挖掘出Bacillus tropicus来源的β-D-呋喃果糖苷酶基因BtFFase8,成功合成后表达在大肠杆菌宿主中。通过数据库筛选β-D-呋喃果糖苷酶的基因序列,将基因克隆至载体pET-28a(+)中。将获得的重组质粒转化至大肠杆菌BL21,构建重组菌株大肠杆菌BL21/pET28a-BtFFase8。采用亲和层析纯化克隆酶,用SDS-PAGE法分析蛋白分子量,通过葡萄糖试剂盒检测酶水解产物的能力得到酶活力,用福林酚试剂法测定蛋白质含量。克隆酶BtFFase8的分子质量为57.0 kDa,对蔗糖底物的比酶活力为13.4 U/mg;BtFFase8的最适pH和最适温度分别为pH 6.5和45℃,且在pH 5.0~8.0和40℃及以下温度保持酶活力稳定,残余酶活力大于80%;BtFFase8能耐受碱性蛋白酶、酸性蛋白酶和胰蛋白酶的水解,残余酶活力大于70%。BtFFase8的发现及克隆酶优良的酶学功能为其未来应用于食品加工,特别是在转化糖的生产中奠定了良好的理论基础。

Invert sugar,a mixture of glucose and fructose,is favored for its higher sweetness than sucrose,greater resistance to crystallization and higher solubility,which is therefore widely used in the food industry.β-D-fructofuranosidase exists widely in nature and has attracted much attention and shows promise in the production of invert sugar with a variety of industrial applications.To obtain a newβ-D-fructofuranosidase resource and explore its enzymatic properties and potential applications in sugar conversion,theβ-D-fructofuranosidase gene BtFFase8 originating from Bacillus tropicus was mined,successfully synthesized and expressed in Escherichia coli.The gene sequences containing the gene encodingβ-D-fructofuranosidase were screened from NCBI GenBank search and Blast sequence comparison.The BtFFase8 gene was analyzed and homology matched using bioinformatics tools to predict the theoretical molecular mass and isoelectric point of the protein.Sequences of genes containingβ-D-fructofuranosidase were found and compared by database,and were cloned into vectors pET-28a(+).The obtained recombinant plasmid was transformed into E.coli BL21 to construct the recombinant strain E.coli BL21/pET28a-BtFFase8.Cloned enzymes BtFFase8 were purified by Ni-IDA with one-step affinity chromatography.The molecular weight size of BtFFase8 proteins was analyzed by SDS-PAGE,enzyme activity was determined by the ability of the enzyme hydrolysis products to be detected by the glucose kit,and the protein content was determined by the forintol reagent assay.Through bioinformatics and molecular biology techniques,this study successfully discovered aβ-D-fructofuranosidase gene named BtFFase8.The BtFFase8 gene encodes 491 amino acids and one stop codon,and the encoded protein possesses a theoretical molecular mass of 57.1 kDa and inferred p I of 5.56,with a signal peptide and two N-glycosylation sites.Cloned enzyme BtFFase8 was purified in one step and showed a single band in SDS-PAGE with a molecular weight of 57.0 kDa,and with a specific enzyme activity of 13.4 U/mg against the sucrose substrate.The optimal pH and temperature of BtFFase8 were pH 6.5 and 45℃,respectively.It was stable at temperatures up to 40℃and within pH range of 5.0-8.0,and the residual enzyme activity can be retained by more than 80%.In addition,BtFFase8 demonstrated resistance to various common proteases,such as alkaline proteases,acidic proteases and trypsin,more than 70%enzyme activity.The discovery of BtFFase8 and the cloning of the enzyme with excellent enzymatic properties will provide a solid theoretical foundation for its future applications in food processing,particularly in sugar conversion for production.