Recombinant sucrose phosphorylase from Leuconostoc mesenteroides: Characterization, kinetic studies of transglucosylation, and application of immobilised enzyme for production of α-d-glucose 1-phosphate

Sucrose phosphorylase catalyzes the reversible conversion of sucrose (α-d-glucopyranosyl-1,2-β-d-fructofuranoside) and phosphate into d-fructose and α-d-glucose 1-phosphate. We report on the molecular cloning and expression of the structural gene encoding sucrose phosphorylase from Leuconostoc mesenteroides (LmSPase) in Escherichia coli DH10B. The recombinant enzyme, containing an 11 amino acid-long N-terminal metal affinity fusion peptide, was overproduced 60-fold in comparison with the natural enzyme. It was purified to apparent homogeneity using copper-loaded Chelating Sepharose and obtained in 20% yield with a specific activity of 190 U mg−1. LmSPase was covalently attached onto Eupergit C with a binding efficiency of 50% and used for the continuous production of α-d-glucose 1-phosphate from sucrose and phosphate (600 mM each) in a packed-bed immobilised enzyme reactor (30 °C, pH 7.0). The reactor was operated at a stable conversion of 91% (550 mM product) and productivity of approximately 11 g l−1 h−1 for up to 600 h. A kinetic study of transglucosylation by soluble LmSPase was performed using α-d-glucose 1-phosphate as the donor substrate and various alcohols as acceptors. d- and l-arabitol were found to be good glucosyl acceptors.