α-Amylase from the thermophilic bacterial strain Anoxybacillus vranjensis ST4 (AVA) was cloned into the pMALc5HisEk expression vector and successfully expressed and purified from the Escherichia coli ER2523 host strain. AVA belongs to the GH13_5 subfamily of glycoside hydrolases and has 7 conserved sequence regions (CSRs) distributed in three distinct domains (A, B, C). In addition, there is a starch binding domain (SBD) from the CBM20 family of carbohydrate binding modules (CBMs). AVA is a monomer of 66 kDa that achieves maximum activity at 60-80 °C and is active and stable over a wide pH range (4.0-9.0). AVA retained 50 % of its activity after 31 h of incubation at 60 °C and was resistant to a large number of denaturing agents. It hydrolyzed starch granules very efficiently, releasing maltose, maltotriose and maltopentaose as the main products. The hydrolysis rates of raw corn, wheat, horseradish, and potato starch, at a concentration of 10 %, were 87.8, 85.9, 93.0, and 58 %, respectively, at pH 8.5 over a 3 h period. This study showed that the high level of expression as well as the properties of this highly stable and versatile enzyme show all the prerequisites for successful application in industry.
Keywords: Anoxybacillus vranjensis; Starch; pMALc5HisEk expression vector; α-Amylase.
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