Identification of catalytic acidic residues of levan fructotransferase from Microbacterium sp. AL-210

摘要: β-Fructofuranosidases, a family 32 of glycoside hydrolases (GH32), share three conserved domains including the W(L/M)(C/N)DP(Q/N), FRDPK, and ECP(D/G) motifs. The functional role acidic residues within levan fructotransferase, one β-fructofuranosidases, from Microbacterium sp. AL-210 was studied by site-directed mutagenesis. Each mutant overexpressed in E. coli BL21(DE3) purified using Hi-Trap chelating affinity chromatography fast performance liquid chromatography. Substitution Asp-63 Ala, Asp-195 Asn, Glu-245 Ala Asp decreased enzyme activity approximately 100-fold compared to wild-type enzyme. This result indicates that Asp-63, Asp-195, play major catalysis. Since are present position inulinase, levanase, levanfructotransferase, invertase, they likely have common as nucleophile, transition state stabilizer, general acid β-fructofuranosidases.