Maximization of dextransucrase activity expressed in E. coli by mutation and its functional characterization
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Updated: Jul 13, 2026
Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
Published on: March 16, 2011
Seung Hee Nam1, Eun Ah Ko, Suk Sang Jang
1Jeonnam Agricultural Research & Extension Services, Jeonnam, Naju 520-715, Korea.
Abstract:
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%).
