{"title":"[Characterization and molecular modification of β-glucosidase from Citrobacter koser GXW-1].","authors":"Minhua Jiang, Houmin Lin, Jinyang Yin, Zilong Wang, Hao Pang, Ribo Huang, Liqin Du","doi":"","DOIUrl":null,"url":null,"abstract":"<p><strong>Objective: </strong>The aim of this study was to characterize β-glucosidase from Citrobacter koser GXW-1 isolated from soil and to improve the enzyme by molecular modification.</p><p><strong>Mehods: </strong>A bacterial strain with β-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 β-glucosidase from the same genus Citrobacter were found. These sequences were aligned. Then, a gene encoding β-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.</p><p><strong>Results: </strong>C. koser GXW-1 with β-glucosidase activity was isolated from the soil. A gene encoding β-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.</p><p><strong>Conclusion: </strong>CBGL not only can hydrolyze β-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 β-glucosidase CBGL has important applications in theoretical research and in industry.</p>","PeriodicalId":7120,"journal":{"name":"微生物学报","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2017-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"微生物学报","FirstCategoryId":"1089","ListUrlMain":"","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0
Abstract
Objective: The aim of this study was to characterize β-glucosidase from Citrobacter koser GXW-1 isolated from soil and to improve the enzyme by molecular modification.
Mehods: A bacterial strain with β-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 β-glucosidase from the same genus Citrobacter were found. These sequences were aligned. Then, a gene encoding β-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 β-glucosidase activity was isolated from the soil. A gene encoding β-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 β-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 β-glucosidase CBGL has important applications in theoretical research and in industry.