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 Meiothermus ruber) and SuSy (from Acidithiobacillus caldus) 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 (Mg2+) and polyphosphate (polyP) as well as by the pH. An optimized synthesis at 45 °C and pH 5.5 gave 81 mM (48 g L−1) ADP-glucose from 100 mM AMP and 132 mM polyP in the presence of an excess of sucrose (1 M) and 25 mM Mg2+. The productivity was 2.0 g L−1 h−1 despite using an enzyme concentration of only 150 μg mL−1. 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 in situ, could be generally useful to advance NDP-sugar synthesis by Leloir glycosyltransferases.
Fields of Expertise
- Human- & Biotechnology