EXPRESSION AND CHARACTERIZATION OF α-GLUCOSIDASE FROM Weissella confusa BBK-1

Abstract

Alpha-glucosidase [EC 3.2.1.20; α-D-glucoside glucohydrolase] is an exohydrolase which catalyzes non-reducing end of substrates to release D-glucose. In addition, alpha-glucosidase also displays a transferase activity, which bring about the formation of α-glucosylated compounds such as soluble starch and glycogen. This research aims to express and characterize α-glucosidase from Weissella confusa BBK-1 (WcAG). WcAG gene was expressed in modified pET28b vector. This gene contained an open reading frame of 1,775 bps. Optimum expression condition of WcAG was cultured in LB medium containing 1% (w/v) glucose and induced with 0.4 mM IPTG at 20°C for 20 h. The recombinant WcAG containing his-tag at the C-terminus was successfully purified by His-Trap column with the specific activity of 45.29 U/ml, purification fold of 2.93 increased with 21.65% yields of total activity. WcAG had molecular mass of 124 kDa and existed as dimer in native form. For hydrolysis activity, the optimum temperature and pH were at 50 ºC and pH 6.0, respectively. Temperature and pH stability of WcAG were in range of 4 to 40 °C, and pH 6.0-8.0 in phosphate buffer, respectively. The most suitable substrate for hydrolysis activity of WcAG was maltotriose (G3) while maltose (G2) was for transglycosylation activity. For hydrolysis activity, the order of preferable substrate was maltotriose (G3), maltotetraose (G4), maltopentaose (G5), maltohexaose (G6) and maltose (G2), respectively. For kinetic study of WcAG, the apparent Km, kcat and kcat/ Km values for G3 substrate were 2.67 mM, 14.096 s-1 and 5.279 (s·mM)-1, respectively. The apparent Km, kcat and kcat/ Km values for G2 substrate were 16.22 mM, 0.738 s-1 and 0.046 (s·mM)-1, respectively. The optimal maltooligosacharide production was obtained of incubation using 200 mM of G2 with 0.4 unit/ml of WcAG at 37 ºC for 24 h. Four products were obtained from transglycosylation activity including product I, II, III and IV. Identification of all products using HPAEC-PAD revealed that product I was isomaltose, product II was panose while product III and IV still cannot be identified.