Molecular cloning and biochemical characterization of a novel cold-adapted alpha-amylase with multiple extremozyme characteristics

• An α-amylase gene from Exiguobacterium sp. SH3 was cloned and expressed in Escherichia coli.
• The amylase (Amy-E) was identified as a novel cold-adapted enzyme.
• Amy-E was halotolerant with stimulated activity at high salt concentration of 1–5 M.
• The enzyme activity was also stimulated by adding Triton X-100 and Tween 20 at 50%.
• Amy-E as an extremozyme was deemed promising for applications at low temperatures.

A gene coding for an alpha-amylase (Amy-E) from Exiguobacterium sp. SH3 was successfully expressed in Escherichia coli. The enzyme was purified as a functional His-tagged protein of about 53 kDa with maximum activity at 30 °C and pH 6.5. Amy-E was also able to function well at low temperatures, retaining 41% of its maximum activity in assays conducted at 0 °C. The activation and inactivation energies of the enzyme were found to be 4.46 and 11.76 kcal mol−1, respectively. The substrate specificity of Amy-E was in the following order: soluble starch (100%), maltodextrin (87.6%); amylopectin (62.2%), wheat flour (53%), and rice flour (48.7). The Km and Vmax of soluble starch hydrolysis were found to be 2.29 mg ml−1 and 1405 U. Amy-E not only was halotolerant but also its activity was stimulated at high salt concentrations in the range of 1–5 M. The enzyme activity was also stimulated by non-ionic surfactants (Triton X-100 and Tween 20) at 20% and 50% concentrations. It was remarkably stable against sodium dodecyl sulfate (SDS), alcohols, and acetone. Amy-E as an extremozyme, seems quite promising for applications that are conducted at low temperatures and low water activity conditions.

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