Purification and characterization of a new L-methioninase from solid cultures of <Emphasis Type="Italic">Aspergillus flavipes</Emphasis> |
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Authors: | Ashraf S A El-Sayed |
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Institution: | (1) Department of Botany and Microbiology, Faculty of Science, Zagazig University, Zagazig, Egypt;(2) Present address: Division of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Sakyo-ku Kyoto, 606-8585, Japan; |
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Abstract: | L-Methioninase was purified to electrophoretic homogeneity from cultures of Aspergillus flavipes using anion-exchange and gel filtration chromatography by 12.1 fold compared to the crude enzyme preparation. The purified
enzyme had a molecular mass of 47 kDa under denaturing conditions and an isoelectric point of 5.8 with no structural glycosyl
residues. The enzyme had optimum activity at pH 7.8 and pH stability from 6.8–8.0 at 35°C. The enzyme appeared to be catalytically
stable below 40°C. The enzyme activity was strongly inhibited by DL-propargylglycine, hydroxylamine, PMSF, 2-mercaptoethanol,
Hg+, Cu2+, and Fe2+, with slight inhibition by Triton X-100. A flavipes L-methioninase has a higher catalytic affinity towards L-methionine (Km, 6.5 mM and Kcat, 14.1 S−1) followed by a relative demethiolating activity to L-homo-cysteine (Km, 12 mM and Kcat, 9.3 S−1). The enzyme has two absorption maxima at 280 and 420 nm, typical of other PLP-enzymes. Apo-L-methioninase has the ability
to reconstitute its structural catalytic state completely upon addition of 0.15 mM PLP. L-Methioninase has neither an appreciable
effect on liver function, platelet aggregation, nor hemolysis of human blood. The purified L-methioninase from solid cultures
of A. flavipes displayed unique biochemical and catalytic properties over the currently applied Pseudomonad enzyme. |
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