Cloning, sequencing and expression of an α-amylase gene, amyA, from the thermophilic halophile Halothermothrix orenii and purification and biochemical characterization of the recombinant enzyme a aThe GenBank accession number for the sequence reported in this paper is AF442961.

Abstract

A recombinant clone expressing an amylase was identified from an Escherichia coli generated genomic library of the thermophilic, moderately halophilic, anaerobic bacterium Halothermothrix orenii by activity screening, and the gene encoding the enzyme was designated AmyA. The amyA gene was 1545 bp long, and encoded a 515 residue protein composed of a 25 amino acid putative signal peptide and a 490 amino acid mature protein. It possessed the five consensus regions characteristic of the α-amylase family and showed the greatest homology to the Bacillus megaterium group of α-amylases. The amyA gene was expressed in E. coli as a hexahistidine-tagged enzyme and purified. The purified recombinant enzyme was optimally active at 65 SC in 5 % (w/v) NaCl at pH 75, with significant activity retained in the presence of up to 25 % (w/v) NaCl. It had a specific activity of 2232 U mg N1 and required NaCl and CaCl 2 for optimum activity and thermostability. The relatively high proportion of acidic amino acids typically observed for many enzymes from halophiles was absent in H. orenii AmyA.

Production and partial purification of α-amylase from a novel isolate Streptomyces gulbargensis

“…The apparent relative molecular mass for the -amylase by gel Wltration was very similar to that found by SDS-PAGE; hence, the enzyme seems to be a monomeric enzyme. Similar observations were made from Thermus Wliformis Ork A2 with 60 kDa [23], Thermococcus hydrothermalis, 53.6 kDa [24] and from Halothermothrix orenii, 56 kDa [25]. EVect of metal ions on amylase activity…”

Molecular cloning and sequence analysis of a novel zinc-metalloprotease gene from the Salinivibrio sp. strain AF-2004 and its extracellular expression in E. coli

“…Nowadays, there are only few reports on sequence analysis of extracellular enzymes from moderately halophilic bacteria and there is no report on proteases. The well characterized examples include two alphaamylase genes: an amyH gene from Halomonas meridiana (Coronado et al, 2000) and the other is from the thermophilic halophile, Halothermothrix orenii (Mijts and Patel, 2002).…”

Halophilic characterization of starch-binding domain from Kocuria varians α-amylase

“…Several halophilic and halo-tolerant amylolytic and pullulytic enzymes were isolated from extremely and moderately halophilic microorganisms [10][11][12][13][14] and poly-extremophilic microorganisms, e.g., thermophilic halophile [15][16][17]. We have previously reported the cloning and expression of the gene coding for ␣-amylase (KVA) from moderate halophile, K. varians, isolated from soy sauce mash [10].…”