S PARTIAL PURIFICATION AND BIOCHEMICAL CHARACTERIZATION OF -GLUCOSIDASE FROM CORN BY THREE-PHASE PARTITIONING

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Başak Bilen Hasan Bayraktar Secıl Onal

Abstract

α-Glucosidase was first purified from corn by three-phase partitioning(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% (w/v) ammonium sulfate saturation with 1.0:0.75 (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 electrophoresis(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 (KM and Vmax) were calculated from Lineweaver-Burk plot as 0.64 mM and 0.45 U, respectively. With 20 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.

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How to Cite
BILEN, Başak; BAYRAKTAR, Hasan; ONAL, Secıl. S PARTIAL PURIFICATION AND BIOCHEMICAL CHARACTERIZATION OF -GLUCOSIDASE FROM CORN BY THREE-PHASE PARTITIONING. Hacettepe Journal of Biology and Chemistry, [S.l.], v. 46, n. 4, p. 481-494, jan. 2019. ISSN 1303-5002. Available at: <http://hjbc.hacettepe.edu.tr/index.php/hjbc/article/view/90>. Date accessed: 16 jan. 2019.
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