title: Cloning of a novel L-amino acid oxidase from Trichoderma harzianum ETS 323 and bioactivity analysis of overexpressed L-amino acid oxidase.
abstract: L-amino acid oxidases (L-AAOs) have been isolated from many organisms, such as snake, and are known to have antibacterial activity. To the best of the authors' knowledge, this is the first report of the cloning of cDNA encoding a novel Trichoderma harzianum ETS 323 L-amino acid oxidase (Th-L-AAO). The protein was overexpressed in Escherichia coli and purified to homogeneity. Comparisons of its deduced amino acid sequence with the sequence of other L-AAOs revealed the similarity to be between 9 and 24%. The molecular mass of the purified protein was 52 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme substrate specificity was highest for L-phenylalanine, and its optimal pH and temperature for activity were 7 and 40 °C, respectively; exogenous metal ions had no significant effect on activity. Circular dichroism spectroscopy indicated that the secondary structure of Th-L-AAO is composed of 17% α-helices, 28% β-sheets, and 55% random coils. The bacterially expressed Th-L-AAO also mediated antibacterial activity against both gram-positive and gram-negative food spoilage microorganisms. Furthermore, a three-dimensional protein structure was created to provide more information about the structural composition of Th-L-AAO, suggesting that the N-terminal sequence of Th-L-AAO may have contributed to the antibacterial activity of this protein.
authors.affiliation: Institute of Biotechnology, National Dong Hwa University, Hualien 97401, Taiwan, Republic of China.
authors.forename: Chi-Hua
authors.initials: CH
authors.lastname: Cheng
authors.forename: Chia-Ann
authors.initials: CA
authors.lastname: Yang
authors.forename: Shu-Ying
authors.initials: SY
authors.lastname: Liu
authors.forename: Chaur-Tsuen
authors.initials: CT
authors.lastname: Lo
authors.forename: Hsiou-Chen
authors.initials: HC
authors.lastname: Huang
authors.forename: Fang-Chin
authors.initials: FC
authors.lastname: Liao
authors.forename: Kou-Cheng
authors.initials: KC
authors.lastname: Peng
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D050830:L-Amino Acid Oxidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D014242:Trichoderma / Q000201:enzymology*
chemical_list: D000900:Anti-Bacterial Agents; D018076:DNA, Complementary; D050830:L-Amino Acid Oxidase