Extracellular alpha-amylase from Thermus filiformis Ork A2: purification and biochemical characterization.
An extracellular alpha-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 degrees C, and the enzyme was stable in the pH range of 4.0-8.0. Calcium enhanced thermostability at temperatures above 80 degrees C, increasing the half-life of activity to more than 8 h at 85 degrees C, 80 min at 90 degrees C, and 19 min at 95 degrees C. Ethylenediaminetetraacetic acid (EDTA) inhibited amylase activity, the inhibition being reversed by the addition of calcium or strontium ions. The alpha-amylase was also inhibited by copper and mercuric ions, and p-chloromercuribenzoic acid, the latter being reversed in the presence of dithiothreitol. Dithiothreitol and beta-mercaptoethanol activated the enzyme. The alpha-amylase exhibited Michaelis-Menten kinetics for starch, with a K(m) of 5.0 mg.ml-1 and kcat/K(m) of 5.2 x 10(5) ml.mg-1s-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 alpha-amylase from a strain of the genus Thermus.
Departamento de Bioquímica, Universidade de Coimbra, Portugal. cve@imagem.ibili.uc.pt
M C
MC
Egas
M S
MS
da Costa
D A
DA
Cowan
E M
EM
Pires
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D013379:Substrate Specificity; D013816:Thermodynamics; D013824:Thermus / Q000201:enzymology*; D000516:alpha-Amylases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D010446:Peptide Fragments; D000516:alpha-Amylases