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 degrees 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% alpha-helices, 28% beta-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.