title: A simple purification procedure of D-amino-acid oxidase from Candida guilliermondii H(see symbol)-4.
abstract: D-amino-acid oxidase (EC 1.4.3.3) was purified about 1480-fold from the yeast Candida guilliermondii H(see symbol)-4 using chromatofocusing method. The purification procedure gave an enzyme preparation which is greater than 90% homogenous on SDS-polyacrylamide gels with a specific activity of 11.54 U/mg at 30 degrees C with D-proline as substrate with the yield of total activity 9.3%. The molecular weights of subunit and native enzyme were determined to be 38.4 and 78.6 kDa by SDS-polyacrylamide gel electrophoresis and gel-filtration, respectively, suggesting that the native enzyme exists as a homodimer. A single molecular form with an isoelectric point of 6.85 was detected in analytical isoelectrofocusing. The optimum pH and temperature were 8.0 and 33 degrees C. An enzyme shows stability in the pH range from 7.4 to 9.0 and at the temperature no higher than 38 degrees C. Activation energy for D-amino-acid oxidase reaction was calculated to be 60 kJ/mol at 30 degrees C. The strict D-isomer specificity of the enzyme is confirmed, since no reaction could be detected with L-amino acids, and a large number of D-amino acids could be substrates for this enzyme. K(m) and V(max) values were determined for D-proline and D-alanine, which, among 22 tested, were the best substrates of the enzyme. D-amino-acid oxidase from the yeast C. guilliermondii is a flavoprotein oxidase in which the prosthetic group is tightly, but not covalently, bound FAD. The enzyme is completely inhibited by sodium benzoate, SH-oxidizing agents, but not by sodium azide, toluene or chloroform.
authors.affiliation: Department of Biochemistry, Yerevan State University, 0025 Yerevan, Armenia. grigor87@mail.ru
authors.forename: G K
authors.initials: GK
authors.lastname: Gevorgyan
authors.forename: M A
authors.initials: MA
authors.lastname: Davtyan
authors.forename: A A
authors.initials: AA
authors.lastname: Hambardzumyan
mesh_terms: D002175:Candida / Q000201:enzymology*; D002850:Chromatography, Gel; D003605:D-Amino-Acid Oxidase / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D003605:D-Amino-Acid Oxidase