Transglycosylation activity and characterization of recombinant sucrose phosphorylase from Leuconostoc Mesenteroides MBFWRS-3(1) expressed in Escherichia Coli

Objective: Sucrose phosphorylase (SPase) is an enzyme that catalyzes the transfer of glucosyl to various acceptor molecules. Distinct types of SPases have been reported, and their transglycosylase activities have been shown to differ. In general, glycosylation is a process that is used to modify bioactive compounds. As such, glycosylation can increase the chemical stability of compounds and improve their characteristics such as reduce strong smell and sour taste. We previously cloned recombinant SPase (SPaseWRS-3[1]) from Leuconostoc mesenteroides MBFWRS-3[1] in Escherichia coli. In the current study, we aimed to characterize SPaseWRS-3 and determine its transglycosylation activity using benzoic acid (BA), ascorbic acid, and kojic acid (KA). Methods: Expression analyses were conducted in lysogeny broth (LB) medium supplemented with tetracycline and expression was induced using isopropyl-p-d-thiogalactopyranoside. The characteristics of the 56 kDa recombinant SPase (rec-SPase) were confirmed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Rec-SPase activity was determined spectrophotometrically using sucrose as the substrate and NADPH as the end-product at 340 nm. Transglycosylation activity was evaluated using thin-layer chromatography (TLC) on silica gel plates. Results: Our results demonstrated that the rec-SPase had an activity of 98.52% relative to the reference SPase (ref-SPase). BA and KA were determined to undergo glucosyl transfer by rec-SPase using ref-SPase, as observed with TLC. Our findings are consistent with those reported previously for the SPase isolated from L. mesenteroides. Conclusion: Recombinant SPase activity is comparable to reference SPase activity. Our study could be the initial study to deeply observe SPase activity in other substrates as well.