Purification and characterization of a new L-methioninase from solid cultures of Aspergillus flavipes

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 2+, Cu 2+, and Fe 2+, 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. © 2011 The Microbiological Society of Korea and Springer-Verlag Berlin Heidelberg.