ABSTRACT Trichoderma harzianum Rifai SKW-36 produced two kinds of D-amino acid oxidizing enzymes. One enzyme was D-aspartate oxidase acting on acidic D-amino acids such as D-aspartate and D-glutamate and another one was D-amino acid oxidase acting on neutral D-amino acid such as D-phenylalanine and D-methionine. These enzymes in the cell-free extract were separated by DEAE-Toyopearl ion-exchange column chromatography. Casamino acids, peptone, and yeast extract as carbon and nitrogen sources were effective for the production of the enzymes. No D-amino acid tested induced the production of the enzymes. Casamino acid (0.33%) as carbon and nitrogen source gave a highest specific activity of D-aspartate oxidase among media tested. D-Aspartate oxidase, which was purified by four-step column chromatography in addition to ammonium sulfate precipitation, exhibited a subunit molecular mass of 40 kDa by SDS-PAGE analysis. D-Aspartate, D-glutamate and N-methyl-D- aspartate were oxidized as substrates with the specific activities of 7.80 U/mg, 4.90 U/mg, and 4.22 U/mg, respectively. D-Asparagine, D-glutamine, D-alanine, and D-valine were slightly oxidized. No other D- amino acids tested were inert. The enzyme exhibited relatively wide substrate specificity compared to D-aspartate oxidases reported so far. The pH and temperature optima were 7.5 - 8.0 and 35°C, respectively. The enzyme was stable at pH 6.0 - 9.0. About 75% of the enzyme activity was retained even after treating the enzyme at 50°C for 10 min. The enzyme activity was inhibited not by benzoate and tartrate, but 60% and 24% by fumarate and malonate, respectively.
Cite this paper
Yano, S. , Ozaki, H. , Matsuo, S. , Ito, M. and Wakayama, M. (2012) Production, purification and characterization of D-aspartate oxidase from the fungus Trichoderma harzianum SKW-36. Advances in Bioscience and Biotechnology, 3, 7-13. doi: 10.4236/abb.2012.31002.
 Benz, F., Liersch, M., Nuesch, J. and Treichler, H.J. (1971) Methionine metabolism and Cephalosporin C synthesis in Cephalosporium acremonium D-amino acid oxidase. European Journal of Biochemistry, 20, 81-88.
Isogai, T., Ono, H., Ishitani, Y., Kojo, H., Ueda, Y. and Kohsaka, M. (1990) Structure and expression of cDNA for D-amino acid oxidase active against Cephalorporin C from Fusarium solani. Journal of Biochemistry, 108, 1063-1069.
Gabler, M. and Fischer, L. (1999) Production of a new D-amino acid oxidase from the fungus Fusarium oxysporum. Applied and Environmental Microbiology, 65, 3750-3753.
Schroder, T. and Anderson, J. R. (1996) Studies on the inactivation of the flavoprotein D-amino acid oxidase from Trigonopsis variabilis. Applied Microbiology and Biotechnology, 45, 458-464. doi:10.1007/BF00578456
Molla, G., Motteran, L., Piubelli, L., Pilone, M.S. and Pollegioni, L. (2003) Regulation of D-amino acid oxidase expression in the yeast Rhodotorula gracilis. Yeast, 20, 1061-1069. doi:10.1002/yea.1023
Yurimoto, H., Hasegawa, T., Sakai, Y. and Kato, N. (2001) Characterization and high-level production of D-amino acid oxidase in Candida boidinii. Bioscience, Biotechnology, and Biochemistry, 65, 627-633.
Fukui, K., Watanabe, F., Shibata, T. and Miyake, Y. (1987) Molecular cloning and sequence of cDNAs encoding porcine kidney D-amino acid oxidase. Biochemistry, 26, 3612-3618. doi:10.1021/bi00386a054
Tada, M., Fukui, K., Momoi, K. and Miyake, Y. (1990) Cloning and expression of a cDNA encoding mouse kidney D-amino acid oxidase. Gene, 90, 293-297.
Fukui, K. and Miyake, K. (1992) Molecular cloning and chromosomal localization of human gene encoding D-amino acid oxidase. Journal of Biological Chemistry, 267, 18631-18638.
Setoyama, C. and Miura, R. (1997) Structural and functional characterization of the human brain D-aspartate oxidase. Journal of Biochemistry, 121, 798-803.
Yamada, R., et al., (1996) Purification and properties of D-aspartate oxidase from Cryptococcus humicolus UJ1. Biocimica et Biophysica Acta, 1294, 153-158.
Wakayama, M., Nakashima, S., Sakai, K. and Moriguchi, M. (1994) Isolation, enzyme production and characterization of D-aspartate oxidase from Fusarium sacchari var. elongatum Y-105. Journal of Fermentation and Bioengineering, 78, 377-379.
Rocca, E. and Ghiretti, F. (1958) Purification and properties of D-glutamic acid oxidase from Octopus vulgaris. Lam. Archives of Biochemistry and Biophysics, 77, 336- 349. doi:10.1016/0003-9861(58)90081-X
Urich, K. (1968) D-Glutamat oxidase aus Antennendruse des Flusskrebses Orconectes limosus: Reinigung und Charakterisierung. Zeitschrift fur Naturforschung, 23b, 1508-1511.
Wakayama, M., Kitahata, S., Manoch, L., Tachiki, T., Yoshimune, K. and Moriguchi, M. (2004) Production, purification and properties of D-aminoacylase from a newly isolated Trichoderma sp. SKW-36. Process Biochemistry, 39, 1119-1124.
Iqbal, H. M. N., Ahmed, I., Zia, M.A. and Irfan, M. (2011) Purification and characterization of the kinetic parameters of cellulose produced from wheat straw by Trichoderma viride under SSF and its detergent compatibility. Advances in Bioscience and Biotechnology, 2, 149-156.
Juhasz, T., Szengyel, Z., Reczey, K., Siika-Aho, M. and Viikari, L. (2005) Characterization of cellulases and hemicellulases by Trichoderma reesei on various carbon sources. Process Biochemistry, 40, 3519-3525.
Nampoothiri, M.K., Baiju, T.V., Sandhya, C., Sabu, A., Szakacs, G. and Pandey, A. (2004) Process optimization for antifungal chitinase production by Trichoderma harzianum. Process Biochemistry, 39, 1583-1590.
Kawano, C., Katsuki, H., Yoshida, T. and Tanaka, S. (1962) A method for extraction and determination of 2, 4-dinitrophenylhydrazones of keto acids. Analytical Biochemistry, 3, 361-368.
Floch, C., Alarcon-Gutierrez, E. and Criquet, S. (2007) ABTS assay of phenol oxidase activity in soil. Journal of Microbiological Methods, 71, 319-324.
Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the folin phenol reagent. Journal of Biological Chemistry, 193, 265-275.
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685.
Sacchi, S., Lorenzi, S., Molla, G., Pilone, M.S., Rossetti, C. and Pollegioni, L. (2002) Engineering the substrate specificity of D-amino-acid oxidase. Journal of Biological Chemistry, 277, 27510-27516.