Maximization of dextransucrase activity expressed in E. coli by mutation and its functional characterization |
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| Authors: | Seung Hee Nam Eun Ah Ko Suk Sang Jang Do Won Kim Se Yong Kim Dae Sung Hwang Doman Kim |
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| Affiliation: | (1) Jeonnam Agricultural Research & Extension Services, Jeonnam, Naju, 520-715, Korea;(2) School of Biological Sciences and Technology and Research Institute for Catalysis, Jeonnam National University, Gwangju, 500-757, Korea;(3) Pohang Accelerator Laboratory, Pohang, 790-784, Korea;(4) Department of Physics, Kangnung National University, Kangnung, 210-702, Korea;(5) Department of Physics, Sejong University, Gwangjing-Gu, Seoul, 143-747, Korea |
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| 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 · mM−1) and DSRN1 had the highest (0.13 s−1 · mM−1) Kcat/Km values. DSRN3 had the highest enzymatic transglycosylation efficiency with maltose (63% of theoretical), gentiobiose (39%), or salicine (40%). |
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| Keywords: | Acceptor reaction Dextransucrase Leuconostoc mesenteroides Mutation Ultrasoft X-ray |
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