Acceptor Specificity and Transglycosylation Reaction of Recombinant Amylomaltase on the Synthesis of Glucosyl-Polyol

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Patinya Haewpetch
Sasichai Kangsadalampai
Jarunee Kaulpiboon

Abstract

Amylomaltase (AMase) catalyzes various reactions, including an intermolecular transglycosylation reaction to produce functional glycosides, which are practically used in many industries. In this study, the p19bAMY plasmid was constructed from pET-19b vector containing Thermus sp. amylomaltase gene. This plasmid was expressed in E. coli BL21 (DE3) for producing AMase. The optimal expression was obtained when the AMase-expressing cells were cultured at 37oC 250 rpm for 24 hours with 0.8 mM IPTG. The crude AMase was subsequently purified to 9-fold by HisTrap FFTM affinity column with the specific activity of 150 units/mg and 36% yield. The relative molecular mass of the purified enzyme was 60 kDa, determined by 10% SDS-PAGE. Moreover, the acceptor specificity was investigated from transglycosylation reaction using tapioca starch as glycosyl donor and various polyols as acceptors. Among polyol acceptors, erythritol gave the highest activity, followed by maltitol and xylitol, respectively. Upon analysis of the product by TLC, the yield of synthesized erythritol glucosides (EG1-4) was 29.3% (w/w).

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วิทยาศาสตร์กายภาพ