Purification, recovery, and characterization of chick pea (Cicer arietinum) beta-galactosidase in single step by three phase partitioning as a rapid and easy technique


DUMAN Y., KAYA E.

PROTEIN EXPRESSION AND PURIFICATION, cilt.91, sa.2, ss.155-160, 2013 (SCI-Expanded) identifier identifier identifier

  • Yayın Türü: Makale / Tam Makale
  • Cilt numarası: 91 Sayı: 2
  • Basım Tarihi: 2013
  • Doi Numarası: 10.1016/j.pep.2013.08.003
  • Dergi Adı: PROTEIN EXPRESSION AND PURIFICATION
  • Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Sayfa Sayıları: ss.155-160
  • Kocaeli Üniversitesi Adresli: Evet

Özet

In this study chick pea beta-galactosidase was first time purified and recovered in single step by three phase partitioning (TPP). Optimal purification parameters for TPP were 60% ammonium sulfate saturation (w/v) with 1:0.5 (v/v) ratio of crude extract:t-butanol at pH 6.8, which gave 101 purification fold with 133% recovery of beta-galactosidase. SDS-PAGE analysis showed that protein has two subunits with molecular masses of 48 and 38 kDa, respectively. Characterization of enzyme showed that optimal pH of purified enzyme was 2.8 and optimal temperature was 50 degrees C. Enzyme was further characterized by the Arrhenius activation energy and Michael-Menten kinetic constants. Activation energy (E-a) was calculated by using Arrhenius equation and determined to be 15.52 kcal mol(-1). K-m value of purified enzyme was estimated for the o-nitrophenyl beta-D galactopyranoside (ONPG) substrate as 1.09 mM, while its maximum velocity, V-m was 0.90 U/mL/min at 37 degrees C. TPP improved substrate affinity of enzyme by the increased flexibility during the partitioning. TPP is simple, easy and economic technique for purification and recovery of beta-galactosidase from chick pea, and has a big potential use for industrial applications. (C) 2013 Elsevier Inc. All rights reserved.