Molecular Cloning and Expression of Alpha Amylase Gene from Thermophilic Bacillus subtilis in Escherichia coli

KIRAN F., Simsek T., OSMANAĞAOĞLU Ö.

RESEARCH JOURNAL OF BIOTECHNOLOGY, vol.9, no.7, pp.1-6, 2014 (SCI-Expanded) identifier identifier

  • Publication Type: Article / Article
  • Volume: 9 Issue: 7
  • Publication Date: 2014
  • Journal Name: RESEARCH JOURNAL OF BIOTECHNOLOGY
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Page Numbers: pp.1-6
  • Keywords: Bacillus subtilis, Thermostable Enzyme, alpha-amylase, Gene Cloning, BIOCHEMICAL-CHARACTERIZATION, GROWTH, OPTIMIZATION, PURIFICATION, ENZYME
  • Ankara University Affiliated: Yes

Abstract

The aim of this experiment was to isolate thermostable alpha-amylase producing bacteria and to clone the gene (AmyE) into Escherichia coli DH alpha for obtaining a low-cost source of a thermostable enzyme. Fourteen potential strains of a-amylase producer were isolated from water samples of different natural thermal springs in Turkey. A strain exhibiting the highest alpha-amylase activity (1.8 U/L) was selected for the molecular cloning experiments and was identified as Bacillus subtilis by 16S rDNA sequence analysis. The gene encoding an extracellular thermostable. amylase was amplified by Polymerase Chain Reaction (PCR). Gel purified PCR product and the vector pBR322 partially double digested with restriction enzymes and then in vitro ligations were performed.