Universitas Indonesia Conferences, 1st International Conference on Advance Pharmacy and Pharmaceutical Sciences

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Characterization and transglycosylation activity of recombinant sucrose phosphorylase from Leuconostoc mesenteroides MBFWRS-3(1) expressed in E. coli
Editha Renesteen

Last modified: 2016-08-06


Sucrosephosphorylase (SPase) is an enzyme that catalyzes glucosyl transfer reaction to the amount of acceptor molecules. Various SPase molecules have been reported and their transglycosyl activities were shown differ from one to another. Glucosylation is a process that has been used widely to modify many bioactive compounds, mostly in an attempt to increase their chemical stability and to improve their characteristics. The aim of this study was to characterize the recombinant SPase (SPaseWRS-3[1]) which was cloned in our previous study, from Leuconostoc mesenteroides MBFWRS3-1 to E. coli. This study also aimed to determine its transglucosylation activity by using acid substances, i.e. benzoic acid, ascorbic acid and kojic acid, as the model. Expression was carried-out in LB medium supplemented with Tetracycline and was induced by using IPTG. The +56 kDa recombinant SPase (rec-SPase), as reported previously, was confirmed by performing SDS PAGE. Rec-SPase activity was measured by using sucrose as substrate, and the end product NADPH was measured at 340 nm, resulting 98.52% relative activity compare to reference SPase. Further, transglucosylation activity assay was conducted performing Thin Layer Chromatography by using acetonitrile+water or butanol+acetic acid+water as moving phase on a Silica gel plate. Result showed that the best glucosyl transfer reaction acitivity of recSPase as well as refSPase were obtained using benzoic acid (Rf=0.55) which were able to produce a lower Rf (0.15-0.2) substances as observed on TLC plate, while two other acid substance did not. This result is consistence to the result reported earlier on L. mesenteroides SPase.