Heterologous Expression of Cold-Adaptive Amaylase from Arthrobacter sp

In this study, the gene encoding an α-amylase from a psychrophilic Arthrobacter agilis sp. (PAMC 27388) was cloned into pET-28a(+) vector and heterologously expressed in Escherichia coli BL21 (DE3). The recombinant α-amylase with a molecular mass of 80 kDa was purified by Ni²⁺-NTA affinity chromatography and showed optimal activity at pH 3.0 and 30°C. This recombinant α-amylase were highly stable at varying temperature (30-60°C) for 24 h within the pH range of 3.0-5.0. The α-amylase activity was enhanced in the presence of FeCl₂ (1 mM), NaCl (1 mM), KCl (1 mM), β-mercaptoethanol (5 mM) and phenylmethylsulfonyl fluoride (1 mM), while CoCl₂ (1 mM), ammonium persulfate (5 mM), and urea (1%) inhibited the activity. The presence or absence of Ca²⁺ ion did not affect the enzyme activity. Thin layer chromatography (TLC) analysis showed that the cold-active α-amylase hydrolyzed starch, maltotetraose and maltotriose, and produced maltose as the major end product.