Fungal degradation of aromatic nitriles. Enzymology of C-N cleavage by Fusarium solani

Abstract

A strain of the fungus F. solani able to use benzonitrile as sole source of C and N was isolated by elective culture. Respiration studies indicate that the nitrile, after degradation to benzoate, is catabolized via catechol or alternatively via p-hydroxybenzoate and 3,4-dihydroxybenzoate. Cell-free extracts of benzonitrile-grown cells contain an enzyme mediating the conversion of benzonitrile into benzoate and ammonia. The nitrilase enzyme was purified by DEAE-cellulose chromatography, (NH4)SO4 precipitation and gel filtration on Sephadex G-200. The homogeneity of the purified enzyme preparation was confirmed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectric focusing on polyacrylamide gel. The enzyme showed a broad pH optimum between pH 7.8 and 9.1 and a Km with benzonitrile as substrate of 0.039 mM. The activation energy of the reaction deduced from an Arrhenius plot was 48.4 kJ/mol. The enzyme was susceptible to inhibition by thiol-specific reagents and certain heavy metal ions. Gel filtration gave a value of 620,000 for the MW of the intact enzyme. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis demonstrated that the enzyme was composed of 8 subunits of MW 76,000. Rates of enzymic attack on various substrates indicated that the nitrilase has a fairly broad specificity and that the fungus probably plays an important role in the biodegradation of certain nitrilic herbicides the environment.