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

Abstract

1. A strain of the fungus Fusarium solani able to use benzonitrile as sole source of carbon and nitrogen was isolated by elective culture. 2. Respiration studies indicate that the nitrile, after degradation to benzoate, is catabolized via catechol or alternatively via p-hydroxybenzoate and 3,4-dihydroxybenzoate. 3. Cell-free extracts of benzonitrile-grown cells contain an enzyme mediating the conversion of benzonitrile into benzoate and ammonia. 4. The nitrilase enzyme was purified by DEAE-cellulose chromatography, (NH(4))(2)SO(4) 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. 5. The enzyme showed a broad pH optimum between pH7.8 and 9.1 and a K(m) with benzonitrile as substrate of 0.039mm. The activation energy of the reaction deduced from an Arrhenius plot was 48.4kJ/mol. 6. The enzyme was susceptible to inhibition by thiol-specific reagents and certain heavy metal ions. 7. Gel filtration gave a value of 620000 for the molecular weight of the intact enzyme. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis demonstrated that the enzyme was composed of eight subunits of mol.wt. 76000. 8. 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 in the environment.

Cloning and expression in Escherichia coli of a Klebsiella ozaenae plasmid-borne gene encoding a nitrilase specific for the herbicide bromoxynil

“…Cloning of the gene locus (bxn) encoding the bromoxynilspecific nitrilase will facilitate comparison at the gene level of this enzyme to nitrilases identified in other organisms (4,17,18). The bxn gene is also being tailored for expression in plants to confer a bromoxynil-resistant phenotype to bromoxynil-sensitive crops.…”

“…A number of soil microbes that are capable of utilizing benzonitrile or 4-hydroxybenzonitrile as a sole source of carbon, nitrogen, or both have been identified (4,17,18). These organisms contain an enzyme, termed nitrilase, that converts benzonitrile and related compounds to their corresponding benzoic acid derivatives, releasing ammonia as a by-product (4,17,18). These nitrilases, however, cannot catalyze the conversion of bromoxynil.…”

“…Although bromoxynil and ioxynil are rapidly metabolized in soil, little is known about the microbial mechanism for such breakdown other than the fact that the cyano moiety of these compounds is subject to initial hydrolysis. A number of soil microbes that are capable of utilizing benzonitrile or 4-hydroxybenzonitrile as a sole source of carbon, nitrogen, or both have been identified (4,17,18). These organisms contain an enzyme, termed nitrilase, that converts benzonitrile and related compounds to their corresponding benzoic acid derivatives, releasing ammonia as a by-product (4,17,18).…”

Nitrilase from Pseudomonas fluorescens EBC191: cloning and heterologous expression of the gene and biochemical characterization of the recombinant enzyme

“…Also the nitrilases from Rhodococcus rhodochrous NCIMB 11215, R. rhodochrous NCIMB 11216, Fusarium species and Arthrobacter sp. strain J1 were rapidly inactivated during storage (Bandyopadhyay et al, 1986;Harper, 1976Harper, , 1977Harper, , 1985Goldlust & Bohak 1989). Several investigators have added reducing agents such as DTT to the enzyme preparations in order to prevent oxidation of the thiol residues which are supposed to be involved in the hydrolysing reaction (Kobayashi et al, 1992;Nagasawa et al, 1990).…”

Metabolism of Bismuth Subsalicylate and Intracellular Accumulation of Bismuth by Fusarium sp. Strain BI

“…Cain et al reported a Fusarium oxysporum strain that did not grow on salicylate but grew well on catechol (9). Catechol and gentisate are intermediates in other catabolic pathways that do not include salicylate (18), and catechol 1,2-dioxygenase activity and gentisate 1,2-dioxygenase activity have been detected in fusaria grown on other aromatic substrates (6). Growth on other aromatic compounds.…”