[Characterization and molecular modification of ß-glucosidase from Citrobacter koser GXW-1].

ABSTRACT

Objective: The aim of this study was to characterize ß-glucosidase from Citrobacter koser GXW-1 isolated from soil and to improve the enzyme by molecular modification. Mehods A bacterial strain with ß-glucosidase activity was screened from the soil around Wuming sugar mill in Guangxi by esculin-ferric ammonium citrate selecting plate. The 16S rDNA of the strain was obtained and analyzed. By searching GenBank database, the genes encoding ß-glucosidase from the same genus Citrobacter were found. These sequences were aligned. Then, a gene encoding ß-glucosidase was amplified by PCR. The recombinant plasmid pQE-cbgl was constructed. The recombinant protein was purified with Ni-NTA. The enzyme properties of the recombinant protein CBGL were studied in detail. At last, the wild enzyme CBGL was reformed by error-prone PCR and site-directed random mutagenesis. Results: C. koser GXW-1 with ß-glucosidase activity was isolated from the soil. A gene encoding ß-glucosidase was cloned from the wild strain GXW-1. The properties of CBGL were identified. Its optimal pH and temperature were 6.0 and 45℃. Its Km and Vmax value were (11.280±1.073) mmol/L and (0.1704±0.0073) µmol/(mg·min), respectively. Its Ki values was (66.84±3.40) mmol/L. CBGL can hydrolyze α-pNPG, stevioside, daidzin and genistin. CBGL was modified by error-prone PCR and site directed random mutagenesis. A positive mutant W147F was obtained successfully. Its Vmax was 2.54 times that of the wild enzyme CBGL. Conclusion: CBGL not only can hydrolyze ß-glycosidic bond, but also can hydrolyze the α-glycosidic bond in α-pNPG. Furthermore, CBGL can hydrolyze stevioside, daidzin and genistin. These characteristics indicate that the ß-glucosidase CBGL has important applications in theoretical research and in industry.