A novel pH and thermo-tolerant halophilic alpha-amylase from moderate halophile Nesterenkonia sp. strain F: gene analysis, molecular cloning, heterologous expression and biochemical characterization

Abstract

A novel pH and thermo-tolerate halophilic alpha-amylase from moderately halophilic bacterium, Nesterenkonia sp.strain F was cloned and expressed in Escherichia coli. 16S rRNA sequence of the strain shared 99.46% similarities with closely related type species. Also, the genome sequence shared ANI values below 92% and dDDH values below 52% with the closely related type species. Consequently, it is proposed that strain F represents a novel species. The AmyF gene was 1390 bp long and encodes an alpha-amylase of 463 amino acid residues with pI of 4.62. The deduced AmyF shared very low sequence similarity (< 24%) with functionally characterized recombinant halophilic alpha-amylases. The recombinant alpha-amylase was successfully purified from Ni-NTA columns with a molecular mass of about 52 KDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was active over a wide range of temperature (25-75 °C) and pH (4-9) with optimum activity at 45 °C and 7.5, respectively. Also, although it was active over a various concentrations of NaCl and KCl (0-4 M), increasing activity of the enzyme was observed with increasing concentration of these salts. Low concentrations of Ca²⁺ ion had no activating effect, but high concentrations of the ion (40-200 mM) enhanced activity of AmyF. The enzyme activity was increased by increasing concentrations of Mg²⁺, Zn²⁺, Hg²⁺ and Fe³⁺. However, it was inhibited only at very high concentrations of these metal ions. Cu²⁺ did not decrease the amylase activity and the highest activity was observed at 100 mM of the ion. These properties indicate wide potential applications of this recombinant enzyme in starch processing industries. This is the first isolation, cloning and characterization of a gene encoding alpha-amylase from Nesternkonia genus.

Keywords: 3D structure prediction; Biochemical characterization; Cloning; Gene analysis; Halophilic α-amylase; Nesterenkonia sp. strain F.