[Catalytic properties of aminoacylase of strain Rhodococcus armeniensis AM6.1].

Studies of substrate specificity revealed that the D-aminoacylase of Rhodococcus armeniensis AM6.1 strain exhibits absolute stereospecificity to the D-stereoisomers of N-acetyl-amino acids. The enzyme is the most active reacted with N-acetyl-D-methionine, as well as with aromatic and hydrophobic N-acetylamino acids and interacts weakly with the basic substrates. It is practically not reacted with acidic and hydrophilic N-acetyl-amino acids. Michaelis constants (K m) and maximum reaction velocities (V max) were calculated, using linear regression analysis, for the following substrates: N-acetyl-D-methionine, N-acetyl-D-alanine, N-acetyl-D-phenylalanine, N-acetyl-D-tyrosine, N-acetyl-D-valine, N-acetyl-D-oxyvaline, N-acetyl- D-leucine. Substrate inhibition of D-aminoacylase was displayed with N-acetyl-D-leucine (K s = 35.5 ± 28.3 mM) and N-acetyl-DL-tyrosine (K s = 15.8 ± 4.5 mM). Competitive inhibition of the enzyme with product­acetic acid (K i = 104.7 ± 21.7 mM, K m = 2.5 ± 0.5 mM, V max = 25.1 ± 1.5 U/mg) was observed.

A simple purification procedure of D-amino-acid oxidase from Candida guilliermondii H(see symbol)-4.

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.

Isolation, purification, and characterization of phenylpyruvate transaminating enzymes of Erwinia carotovora.

Enzymes of Erwinia carotovora that transaminate phenylpyruvate were isolated, purified, and characterized. Two aromatic aminotransferases (PAT1 and PAT2) and an aspartic aminotransferase (PAT3) were found. According to gel filtration, these enzymes have molecular weights of 76, 75, and 78 kDa. The enzymes consist of two identical subunits of molecular weights of 31.4, 31, and 36.5 kDa, respectively. The isoelectric points of PAT1, PAT2, and PAT3 were determined as 3.6, 3.9, and 4.7, respectively. The enzyme preparations considerably differ in substrate specificity. All three of the enzymes productively interacted with the following amino acids: L-aspartic acid, L-leucine (except PAT3), L-isoleucine (except PAT3), L-serine, L-methionine, L-cysteine, L-phenylalanine, L-tyrosine, and L-tryptophane. The aromatic aminotransferases display higher specificity to the aromatic amino acids and the leucine-isoleucine pair, whereas the aspartic aminotransferase displays higher specificity to L-aspartic acid and relatively low specificity to the aromatic amino acids. The aspartic aminotransferase does not use L-leucine or L-isoleucine as a substrate. PAT1, PAT2, and PAT3 show the highest activity at pH 8.9 and at 48, 53, and 58°C, respectively.