Construction of a fusion enzyme of dextransucrase and dextranase: Application for one-step synthesis of isomalto-oligosaccharides

The linear isomalto-oligosaccharides (IMO) with DP2-DP10 were produced by one-step process using engineered fusion enzyme (DXSR) of endo-dextranase and only alpha-(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 SIDS-PAGE. It showed both enzyme activities on analysis by zymogram. The thermal- and pH-stability of DXSR was around 28 degrees 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. (C) 2008 Elsevier Inc. All rights reserved.