Molecular Cloning and Characterization of a Novelα-Amylase from Antarctic Sea Ice BacteriumPseudoalteromonassp. M175 and Its Primary Application in Detergent

Abstract

A novel cold-adapted and salt-tolerant α-amylase gene (amy175) from Antarctic sea ice bacterium Pseudoalteromonas sp. M175 was successfully cloned and expressed. The open reading frame (ORF) of amy175 had 1722 bp encoding a protein of 573 amino acids residues. Multiple alignments indicated Amy175 had seven highly conserved sequences and the putative catalytic triad (Asp244, Glu286, and Asp372). It was the first identified member of GH13_36 subfamily which contained QPDLN in the CSR V. The recombinant enzyme (Amy175) was purified to homogeneity with a molecular mass of about 62 kDa on SDS-PAGE. It had a mixed enzyme specificity of α-amylase and α-glucosidase. Amy175 displayed highest activity at pH 8.0 and 25°C and exhibited extreme salt-resistance with the maximum activity at 1 M NaCl. Amy175 was strongly stimulated by Mg2+, Ni2+, K+, 1 mM Ca2+, 1 mM Ba2+, 1 mM Pb2+, 1 mM sodium dodecyl sulphate (SDS), and 10% dimethyl sulfoxide (DMSO) but was significantly inhibited by Cu2+, Mn2+, Hg2+, 10 mM β-mercaptoethanol (β-ME), and 10% Tween 80. Amy175 demonstrated excellent resistance towards all the tested commercial detergents, and wash performance analysis displayed that the addition of Amy175 improved the stain removal efficiency. This study demonstrated that Amy175 would be proposed as a novel α-amylase source for industrial application in the future.

Microbial Alpha-Amylase Production: Progress, Challenges and Perspectives

“…M175 has been isolated from Antarctic sea ice, which is a common source of cold-stable alpha-amylase. 27 The microorganisms produce cold-active amylases that have a flexible polypeptide chain to make an easier accommodation of substrates at the low-temperature condition. Also, the enzymes contain lipid composition to maintain greater membrane fluidity.…”

Recapitulation of stability diversity of microbial α-amylases

“…When the wild-type α-amylase wtAmy175 from Pseudoalteromonas sp. M175 was expressed and studied, the optimum temperature and pH for enzyme activity were found as 30 °C and 7.5, respectively [82]. The enzyme also showed high activity and remarkable stability in 0-10 mM sodium dodecyl sulphate suggesting its capability as an outstanding candidate in detergent and textile industries [83].…”

“…In 2018, a novel α-amylase-producing strain Pseudoalteromonas sp. M175 (KU726544) was identified from Antarctic ice cover by Wang et al [82] and the α-amylase gene amy175 isolated from this microbe was cloned and expressed in E. coli. By analysing the characteristics of the expressed enzyme, it was concluded that the α-amylase gene amy175 could be used as a novel α-amylase source for industrial application with highest activity at 25 °C and pH 8.0, exhibiting the extreme salt-resistance [82].…”

“…M175 (KU726544) was identified from Antarctic ice cover by Wang et al [82] and the α-amylase gene amy175 isolated from this microbe was cloned and expressed in E. coli. By analysing the characteristics of the expressed enzyme, it was concluded that the α-amylase gene amy175 could be used as a novel α-amylase source for industrial application with highest activity at 25 °C and pH 8.0, exhibiting the extreme salt-resistance [82]. The stain removal efficiency of various tested commercial detergents got improved with the incorporation of Amy175, and thus it can be considered as a novel α-amylase source for industrial application.…”

Enzymes from Marine Polar Regions and Their Biotechnological Applications

“…A cold-adapted α-amylases from the Antarctic sea-ice bacterium Pseudoalteromonas sp. M175 exhibited resistance towards all the tested commercial detergents and was shown to improve their stain removal efficiency [93]. Heat-labile α-amylases displaying high activity at low temperature isolated from bacteria of Antarctic seawater were structurally studied [96,97].…”