Cloning and expression of the β-D-fructofuranosidase from Bacillus tropicus and the characterization of the cloned enzyme
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 indus-try.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 new β-D-fructofuranosi-dase resource and explore its enzymatic properties and potential applications in sugar conversion,the β-D-fructofuranosidase gene BtFFase8 originating from Bacillus tropicus was mined,successfully synthesized and expressed in Escherichia coli.The gene sequences containing the gene encoding β-D-fructofuranosi-dase 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 iso-electric point of the protein.Sequences of genes containing β-D-fructofuranosidase were found and com-pared 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 a β-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 in-ferred pI of 5.56,with a signal peptide and two N-glycosylation sites.Cloned enzyme BtFFase8 was puri-fied 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,BtF-Fase8 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.
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