Production, purification and characterization of D-aspartate oxidase from the fungus Trichoderma harzianum SKW-36

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.

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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.

Conflicts of Interest

The authors declare no conflicts of interest.

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