Construction of a fusion enzyme of dextransucrase and dextranase: Application for one-step synthesis of isomalto-oligosaccharides |
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| Authors: | Young-Min Kim Mi-Young Seo Hee-Kyoung Kang Kimura Atsuo Doman Kim |
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| Affiliation: | 1. School of Biological Sciences and Technology, Chonnam National University, Gwang-Ju 500-757, South Korea;2. The Research Institute for Catalysis and The Bioindustrial Technology Center, Chonnam National University, Gwang-Ju 500-757, South Korea;3. Jeonbuk Branch Institute, Korea Research Institute of Bioscience and Biotechnology, Jeonbuk 580-185, South Korea;4. Division of Applied Bioscience, Hokkaido University, Sapporo, Japan |
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| Abstract: | The linear isomalto-oligosaccharides (IMO) with DP2–DP10 were produced by one-step process using engineered fusion enzyme (DXSR) of endo-dextranase and only α-(1–6) glucan synthesizing dextransucrase. The fusion enzyme was successfully expressed in Escherichia coli and characterized. Compared to individual enzymes, DXSR had 150% increased endo-dextranase activity and 98% decreased dextransucrase activity. The partially purified DXSR displayed molecular mass of 240 kDa as analyzed by SDS–PAGE. It showed both enzyme activities on analysis by zymogram. The thermal- and pH-stability of DXSR was around 28 °C and pH at 5.0–6.4, respectively. IMOs production by DXSR was increased by the addition of metal ions such as Fe2+, Li+, K+ and Ni2+, but the enzyme was strongly inhibited by Hg2+ and Ag+. DXSR produced linear IMO with DP2–DP10 using sucrose as a sole substrate. The molecular weight and amount of IMO could be controlled by the sucrose concentration. DXSR gave 30-fold higher production of IMO than that of an equal activity mixture of the two enzymes such as dextranase and dextransucrase. |
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