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

Year 2018, Volume: 46 Issue: 4, 481 - 494, 01.11.2018

Başak Bilen Hasan Bayraktar Seçil Önal

Abstract

-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 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

References

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