Three-phase partitioning as a rapid and efficient method for purification of invertase from tomato


Özer B., Akardere E., Celem E. B. , Onal S.

BIOCHEMICAL ENGINEERING JOURNAL, vol.50, pp.110-115, 2010 (Journal Indexed in SCI) identifier identifier

  • Publication Type: Article / Article
  • Volume: 50
  • Publication Date: 2010
  • Doi Number: 10.1016/j.bej.2010.04.002
  • Title of Journal : BIOCHEMICAL ENGINEERING JOURNAL
  • Page Numbers: pp.110-115
  • Keywords: Bioseparation, t-Butanol, Invertase, Protein purification, Three-phase partitioning, Tomato, ACID INVERTASE, AGROINDUSTRIAL RESIDUES, SOLUBLE INVERTASE, CARBON-SOURCES, PROTEINS, FRUIT, FRUCTOFURANOSIDASE, LOCALIZATION, PEROXIDASE

Abstract

Three-phase partitioning (TPP), a technique used in protein purification, was used to purify invertase from tomato (Lycopersicon esculentum). The method consists of simultaneous addition of ammonium sulfate and t-butanol to the crude enzyme extract in order to obtain the three phases. Different parameters (ammonium sulfate saturation, crude extract to t-butanol ratio and pH) essential for the extraction and purification of invertase were optimized to get highest purity fold and yield. It was seen that, 50% (w/v) ammonium sulfate saturation with 1:1 (v/v) ratio of crude extract to t-butanol at pH 4.5 gave 8.6-fold purification with 190% activity recovery of invertase in a single step. Finally, the purified enzyme was also characterized and the general biochemical properties were determined. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of enzyme showed considerable purification and its molecular weight was nearly found to be as 20 kDa. This work shows that, TPP is a simple, quick and economical technique for purification of invertases. (C) 2010 Elsevier B.V. All rights reserved.