Comparative study on amylosucrases derived from Deinococcus species and catalytic characterization and use of amylosucrase derived from Deinococcus wulumuqiensis

Abstract

Abstract Amylosucrase (ASase; EC 2.4.1.4), a versatile enzyme, exhibits three characteristic activities: hydrolysis, isomerization, and transglycosylation. In this study, a novel ASase derived from Deinococcus wulumuquiensis (DWAS) was identified and expressed in Escherichia coli. The optimal reaction temperature and pH for the sucrose hydrolysis activity of DWAS were determined to be 45 °C and 9.0, respectively. DWAS displays relatively high thermostability compared with other ASases, as demonstrated by half-life of 96.7 and 4.7 min at 50 °C and 55 °C, respectively. DWAS fused with 6×His was successfully purified to apparent homogeneity with a molecular mass of approximately 72 kDa by Ni-NTA affinity chromatography and confirmed by SDS-PAGE. DWAS transglycosylation activity can be used to modify isovitexin, a representative flavone C-glucoside contained in buckwheat sprouts to increase its limited bioavailability, which is due to its low absorption rate and unstable structure in the human body. Using isovitexin as a substrate, the major transglycosylation product of DWAS was found to be isovitexin monoglucoside. The comparison of transglycosylation reaction products of DWAS with those of other ASases derived from Deinococcus species revealed that the low sequence homology of loop 8 in ASases may affect the acceptor specificity of ASases and result in a distinctive acceptor specificity of DWAS.