Cloning, expression and characterization of highly thermo- and pH-stable maltogenic amylase from a thermophilic bacterium Geobacillus caldoxylosilyticus TK4


Kolcuoglu Y., ÇOLAK A., FAİZ Ö., BELDÜZ A. O.

PROCESS BIOCHEMISTRY, vol.45, no.6, pp.821-828, 2010 (SCI-Expanded) identifier identifier

  • Publication Type: Article / Article
  • Volume: 45 Issue: 6
  • Publication Date: 2010
  • Doi Number: 10.1016/j.procbio.2010.02.001
  • Journal Name: PROCESS BIOCHEMISTRY
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Page Numbers: pp.821-828
  • Keywords: Characterization, Cloning, Geobacillus caldoxylosilyticus TK4, Maltogenic amylase, Thermophilic, ALPHA-AMYLASE, ENZYMATIC CHARACTERIZATION, TRANSGLYCOSYLATION, CYCLOMALTODEXTRINASE, SPECIFICITY, ACARBOSE, ENZYMES, GENE
  • Karadeniz Technical University Affiliated: Yes

Abstract

Maltogenic amylases (MAases), a subclass of cyclodextrin (CD)-hydrolyzing enzymes, belong to glycoside hydrolase family 13. A gene corresponding to MA in Geobacillus caldoxylosilyticus TK4 (GcaTK4MA) was cloned Into pET28a(+) vector and expressed in Escherichia coli with 6xHis-tag at the N-terminus. Herein, we report on the biochemical properties of a new thermo- and pH-stable MA. GcaTK4MA has similar properties those of other MAases in terms of the primary structure, preference for CD over starch and having an extra domain at its N- and C-terminals. The recombinant protein was purified efficiently by using one-step nickel affinity chromatography. The purified enzyme exhibited optimal activity for beta-CD hydrolysis at 50 degrees C and pH 7.0. When the enzyme was separately incubated at 4 degrees C and 50 degrees C in the buffer solutions (pH 3.0-9.0) up to 7 days, it was seen that the enzyme had the higher stability at 50 degrees C than 4 degrees C. The enzyme retained about 80% of its original activity when it was incubated at 50 degrees C for 7 days. The enzyme activity was significantly inhibited by SDS and EDTA at the final concentration of 1%. These results suggest that this is the first reported MA having an extremely pH- and thermal stabilities. (C) 2010 Elsevier Ltd. All rights reserved.