Partial Purification and Biochemical Characterization of α-Glucosidase from Corn by Three-Phase Partitioning

Authors : Başak BİLEN, Hasan BAYRAKTAR, Seçil ÖNAL

Pages : 481-494

View : 21 | Download : 9

Publication Date : 2018-11-01

Article Type : Research Paper

Abstract :-Glucosidase was first purified from corn by three-phase partitioninginsert ignore into journalissuearticles values(TPP); and then characterized. Ammonium sulfate and t-butanol were used in order to obtain three phases. Effect of different process parameters such as; ammonium sulfate concentration, enzyme to t-butanol ratio and pH required for efficient purification of the corn α-glucosidase was studied to get highest purification fold and activity recovery. Optimum purification parameters of the TPP system were determined as 50% insert ignore into journalissuearticles values(w/v); ammonium sulfate saturation with 1.0:0.75 insert ignore into journalissuearticles values(v/v); ratio of crude extract: t-butanol at pH 4.5. Under optimized conditions α-glucosidase was purified with 3.8 purification fold and 60% activity recovery. The molecular weight of α-glucosidase was determined approximately as 29 kDa by using sodium dodecyl sulfate–polyacrylamide gel electrophoresisinsert ignore into journalissuearticles values(SDS–PAGE);. Characterization studies showed that, optimum pH and temperature of α-glucosidase were pH 4.5 and 55°C, respectively. The purified enzyme was found to be very stable at a temperature range of 25-60°C and a pH range of 3.5-5.5. Kinetic constants insert ignore into journalissuearticles values(KM and Vmax); were calculated from Lineweaver-Burk plot as 0.64 mM and 0.45 U, respectively. With 10 mM of CaCl2 concentration α-glucosidase activity was significantly increased to 130%. TPP is a useful strategy to concentrate and purify α-glucosidase for its applications.
Keywords : Three phase partitioning TPP, α glucosidase, corn, purification