Molecular Design Of Alpha-Glucosidase Three Dimensional Structure-based And High-level Expression Of Mutant In Escherichia Coli

OBJECTIVE: To obtain sequence of α—glucosidase, SWISS-MODEL Server predicted that enzyme structure, amino acid property and characteristic, to design molecular of alpha-glucosidase,make two site-directed mutation by TaKaRa MutanBEST Kit,express the mutant gene in E.coli,and determine the mutant's enzyme activity simply.METHODS: According to the sequence of the a — glucosidase DNA in Genbank,the primers were designed with the restriction endonucleases site of EcoR Ⅰ and HindⅢ at 5'end.The genome DNA was extracted from Bacillus stearothermophilu and the sequence of DNA encoding α—glucosidase was amplified by PCR.After the insert gene and pUC18 were digested with the restriction endonucleases site of EcoR Ⅰ and HindⅢ,The gene was ligated with pUC18,transformed into the E.coli DH5α,and through α-complementary,the positive recombinant was identified and sequenced.After sequencing, the insert gene and pGEMEX-1 were digested with the restriction endonucleases site of EcoR Ⅰ and HindⅢ, transformed into the E.coli BL21 pLysS, the positive recombinant was identified and sequenced.Bioinformatics software predicted that enzyme structure,mutantion sites were searched,primers were designed, site-directed mutation was made by muantion kit at the 258 and 203. clone recombinant,expression recombinant and mutant recombinant that enzyme activity were determined.The positive recombinant was induced by IPTG for 3h, the results were observed by SDS-PAGE.RESULTS:1. A fragment about 2100bp appeared after PCR. Sequencing analysis that a— glucosidase DNA obtained by PCRwas identical to the sequence reported in Genbank.2. SWISS-MODEL Server predicted that enzyme and mutant structure, the software to evaluate it that rationalization of structures.3. site-directed mutation was made by muantion kit at the position 258 and 203.4. The expression vector transformed into ￡'.co//BL21,the recombinant was extracted and identified .Sequencing analysis indicated that the ligation was correct and could induce the protein expression.5. The level of expression recombinant enzyme production was about threefold higher than that observed for Bacillus steat-other-mophilus. Ferment-pot culture was about threefold higher than that observed for tube culture.mutant recombinant was about fourfold higher than that observed for Bacillus stearothermophilus.CONCLUSION: The a—glucosidase gene is constructed and expressed in E.coliBL21 successfully.Molecular design of alpha-glucosidase three dimensional structure-based to obtain mutant of a — glucosidase, For experiment of invert indicant .study on structure and function of a—glucosidase established base.