Purification and characterization of 5-oxo-L-prolinase from Paecilomyces varioti F-1, an ATP-dependent hydrolase active with L-2-oxothiazolidine-4-carboxylic acid.

Abstract

An enzyme cleaving L-2-oxothiazolidine-4-carboxylic acid to L-cysteine was purified 75-fold with 8% recovery to near homogeneity from crude extracts of Paecilomyces varioti F-1, which had been isolated as a fungus able to assimilate L-2-oxothiazolidine-4-carboxylic acid. The molecular mass was estimated to be 260 kDa by gel filtration. The purified preparation migrated as a single band of molecular mass 140 kDa upon SDS-PAGE. The maximum activity was observed at a range of pH 7.0-8.0 and at 50 degrees C. The enzyme activity was completely inhibited by SH-blocking reagents such as AgNO(3), p-chloromercuribenzoic acid, N-ethylmaleimide, and N-bromosuccinimide. The enzyme required ATP, Mg(2+), and KCl for the cleavage of L-2-oxothiazolidine-4-carboxylic acid. The enzyme also cleaved 5-oxo-L-proline to L-glutamic acid and is considered to be 5-oxo-L-prolinase.