Purification and properties of glutamate-phenylpyruvate aminotransferase from the ruminal protozoan Entodinium caudatum
Md. Ruhul Amin A D , Ryoji Onodera B , R. Islam Khan A , R. John Wallace C and C. Jamie Newbold CA Department of Animal Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.
B Laboratory of Animal Nutrition and Biochemistry, Miyazaki University, Miyazaki-shi 889-2192, Japan.
C Rowett Research Institute, Bucksburn, Aberdeen, AB21 9SB, UK.
D Corresponding author; email: aminmr64@hotmail.com
Australian Journal of Agricultural Research 55(9) 991-997 https://doi.org/10.1071/AR04050
Submitted: 2 March 2004 Accepted: 2 August 2004 Published: 24 September 2004
Abstract
Entodinium species are important in catabolic protein metabolism by the mixed ruminal microbial population. This study was conducted to purify, and investigate properties of one of the enzymes involved in amino acid metabolism by Entodinium caudatum, glutamate-phenylpyruvate aminotransferase (GPA; EC 2.6.1.64). GPA was purified 74-fold from a cell-free extract by ammonium sulfate precipitation and column chromatography with phenyl-superose, DEAE-Toyopearl 650M, Sephacryl S-100 HR, and Sephadex G-100. The molecular mass of GPA was estimated by SDS–PAGE to be 65.0 kDa. The optimum pH was 6.0 and it was found to be reactive over a wide range of pH from 5.0 to 10.5. Maximum activity of GPA occurred at 45°C and the activity declined at temperatures over 55°C. GPA was stable below 60°C. Aminooxyacetate and phenylhydrazine were highly inhibitory, and SDS, EDTA, and some heavy metal ions also inhibited activity. The purification and characterisation of the enzyme will help to isolate the gene and ultimately to understand the role of GPA in both anabolic and catabolic amino acid metabolism by Entodinium caudatum.
Additional keywords: rumen protozoa, transaminase, characterisation.
Acknowledgments
The authors are extremely grateful to Professor H. Ogawa, University of Tokyo, for inserting permanent rumen fistulae in goats. Md. Ruhul Amin thanks the Japanese Society for the Promotion of Science (JSPS) for the award of fellowship and financial support.
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