Maximization of dextransucrase activity expressed in E. coli by mutation and its functional characterization.
A novel dextransucrase gene, DSRN, was obtained by ultrasoft X-ray treatment of the DSRB742 gene. The DSRN gene was further mutated via site-directed mutagenesis producing four mutants: DSRN1 (F196S), DSRN2 (Y346N), DSRN3 (K395T) and DSRN4 (P980T). Dextransucrases derived from DSRB742 and its mutants were expressed in E. coli and affinity-purified using dextran to give 80% purity. They had specific activities of 0.6-17 U/mg with Km values of 18-88 mM. DSRB742 had the lowest (0.02 s(-1) x mM(-1)) and DSRN1 had the highest (0.13 s(-1) x mM(-1)) Kcat/Km values. DSRN3 had the highest enzymatic transglycosylation efficiency with maltose (63% of theoretical), gentiobiose (39%), or salicine (40%).
Jeonnam Agricultural Research & Extension Services, Jeonnam, Naju 520-715, Korea.
Seung Hee
SH
Nam
Eun Ah
EA
Ko
Suk Sang
SS
Jang
Do Won
DW
Kim
Se Yong
SY
Kim
Dae Sung
DS
Hwang
Doman
D
Kim
D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000528:radiation effects; D024861:Genetic Enhancement / Q000379:methods*; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D013379:Substrate Specificity
D005964:Glucosyltransferases; C020025:dextransucrase