Extracellular α-amylase from Thermus filiformis Ork A2: purification and biochemical characterization

Abstract

An extracellular α-amylase produced by the thermophilic bacterium Thermus filiformis Ork A2 was purified from cell-free culture supernatant by ion exchange chromatography. The molecular mass was estimated to be 60 000 Da by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was rich in both basic and hydrophobic amino acids, presenting the following NH₂-terminal amino acid sequence: Thr-Ala-Asp-Leu-Ile-Val-Lys-Ile-Asn-Phe. Amylolytic activity on soluble starch was optimal at pH 5.5–6.0 and 95°C, and the enzyme was stable in the pH range of 4.0–8.0. Calcium enhanced thermostability at temperatures above 80°C, increasing the half-life of activity to more than 8 h at 85°C, 80 min at 90°C, and 19 min at 95°C. Ethylenediaminetetraacetic acid (EDTA) inhibited amylase activity, the inhibition being reversed by the addition of calcium or strontium ions. The α-amylase was also inhibited by copper and mercuric ions, and p-chloromercuribenzoic acid, the latter being reversed in the presence of dithiothreitol. Dithiothreitol and β-mercaptoethanol activated the enzyme. The α-amylase exhibited Michaelis-Menten kinetics for starch, with a K _m of 5.0 mg·ml⁻¹ and k _cat/K _m of 5.2 × 10⁵ ml·mg⁻¹ s⁻¹. Similar values were obtained for amylose, amylopectin, and glycogen. The hydrolysis pattern was similar for maltooligosaccharides and polysaccharides, with maltose being the major hydrolysis product. Glucose and maltotriose were generated as secondary products, although glucose was produced in high levels after a 6-h digestion. To our knowledge this is the first report of the characterization of an α-amylase from a strain of the genus Thermus.