초록
<P><B>Abstract</B></P><P>Sucrose synthase (SuSy) catalyzes in the presence of a pyrimidine or purine nucleoside diphosphate (NDP) the conversion of sucrose to the corresponding nucleotide‐activated derivative of glucose (NDP‐glucose). To realize the potential of SuSy for NDP‐glucose synthesis fully, a nucleoside monophosphate (NMP) should be employed in the reaction, for it is a much more cost‐effective substrate than NDP. Therefore we explored in this study the use of polyphosphate kinases (PPK) from class II and III of family 2 which catalyze in the presence of polyphosphate (polyP) the conversion of NMP into NDP. A biocatalytic cascade of PPK (from <I>Meiothermus ruber</I>) and SuSy (from <I>Acidithiobacillus caldus</I>) was established for NDP‐glucose production. The synthetic efficiency of the cascade reflected the NMP substrate specificity of the PPK, following the order of nucleoside monophosphate: adenosine (AMP)>guanosine (GMP)>cytidine (CMP)>uridine (UMP)>deoxy‐thymidine (dTMP). The efficiency was also influenced by the concentrations of magnesium (Mg<SUP>2+</SUP>) and polyphosphate (polyP) as well as by the pH. An optimized synthesis at 45 °C and pH 5.5 gave 81 mM (48 g <SMALL>L</SMALL><SUP>−1</SUP>) ADP‐glucose from 100 mM AMP and 132 mM polyP in the presence of an excess of sucrose (1 M) and 25 mM Mg<SUP>2+</SUP>. The productivity was 2.0 g <SMALL>L</SMALL><SUP>−1</SUP> h<SUP>−1</SUP> despite using an enzyme concentration of only 150 μg mL<SUP>−1</SUP>. Isolation of ADP‐glucose (∼99% purity) by anion‐exchange chromatography required prior removal of the polyP, which was achieved by fractional precipitation with ethanol. The herein developed coupling with PPK, to form the NDP substrate from NMP <I>in situ</I>, could be generally useful to advance NDP‐sugar synthesis by Leloir glycosyltransferases.</P>