Abstract:

Abstract:Objective To clone the  glucosidase gene from Thermotoga neapolitana (DSM 4359) and express it in  Escherichia coli,and to study the characteristics of the recombinant enzyme. Methods Based on the glucosidase gene  DNA of Thermotoga neapolitana (DSM 4359),the primers were designed.The glucosidase gene was obtained by PCR method,and the  glucosidase gene was inserted into the expression vector pET-28a(+)and transformed into E.coli BL21(DE3).The glucosidase gene was induced by IPTG and   expressed in a high efficiency in vitro.The optimum reaction temperature,pH value,and the specificity of substrate of the   recombinant  enzyme were observed.Results 825 bp DNA fragment was obtained by PCR amplification,which encoded 274 amino the acid residues,and the  glucosidase gene was inserted into the expression vector pET-28a(+).The recombinant  glucosidase was successfully transferred into  E.coli BL21(DE3) and highly expressed in vitro.The recombinant protein was obtained and had a relative molecular mass of 63 000  indicated by non-denaturing polyacrylamide gel electrophoresis analysis.The optimum reaction temperature of glucosidase was 75℃,and the activity  maintained 60% with the temperature in the range of 70℃-85℃.And the optimum pH value was 5.0,and the activity  maintained 70% with the pH value in the  range of 3.0-6.0.The preferred substrate was trehalose,followed by  gentiobiose,and the  activities  of the other  disaccharides  were  lower or zero.Conclusion Glucosidase gene from Thermotoga neapolitana is successfully cloned and highly expressed in E.coli BL21 (DE3 ).The glucosidase can specifically catalyze the hydrolyzed hydrolysis of α,α-1,1-glucosidic bond.