Site-directed Mutagenesis And Characterization Of Enzymatic Properties Of Amylosucrase

Amylosucrase(AS) from Neisseria polysaccharea is a member of the family13of theglycoside hydrolases and catalyzes the synthesis of an insoluble α-(1,4)-linked glucan polymerfrom sucrose. Unlike most amylo polysaccharide synthasis, this enzyme does not require anyexpensive activated sugar, such as ADP-or UDP-glucose. Indeed, AS only uses the energyproduced by the splitting of the linkage of sucrose. AS can synthesis amylose-like polymer,oligosaccharides or glycoconjugate according to the different receptors. Therefore, AS will be asimple, convenient, and effective glucosyl transferase for development in the industry. In thisstudy we obtain mutant strains by using the site-directed mutagenesis. We studied the activityand enzymatic properties after the tested. The results are as follows:1.Using bioinformatics technology to find the activity sites of As with3UEQ crystalstructure as a template. The hydrogen bonding connection of D294site with F191site and R292site through the Pymol software. It proves the R292site with F191site are relativelyconservative sequence by using the ClustalX2software. Therefore, this research will bechanging the R292site and F191site.2.Amylosucrase from Neisseria polysaccharea as templates to designed the mutationprimers by used the Primer5.0software and got the mutant strains of R292F、R292D、R292M、R292K and F191M. The mutant strains of purified test the enzyme properties characterization.3The specific activity of mutant strains R292F、R292M、R292D、R292K and F191M were154.84U/mg、108.39U/mg、144.84U/mg、181.06U/mg and202.38U/mg and increased by1.11、0.77、1.04、1.29and1.45times compared with the wild type, respectively. The results showedthat the optimum of pH wild type was7.0and the mutant strains of R292D、R292F、R292M、R292K and F191M were7.5、7.0、7.5、7.0and8.0.The optimum temperature of wild typewas35℃. The optimum temperature of R292D、R292F、R292M、R292K and F191M were35℃、35℃、37℃、32℃and35℃.The thermal stability shows that the half-life of wild type、R292D、R292F、R292M、R292K and F191M were20h、17h、21h、16h、21h and19h,respectively. Andthe mutant strains had well resistance to metal ions and organic solvents. The Kinetics showsthat the Vmax of R292F、R292K and F191M increased by1.19、1.44and1.45times comparedwith Wild type. The Km of R292K lower than wild type.