Rhizobacteria with Exceptionally High Aryl Acylamidase Activity

Two Pseudomonas strains (RA2 and RB4) isolated from the rhizosphere of rice roots possessed exceptionally high aryl acylamidase activity (complete dissipation of 0.5 mM propanil [N-(3,4-dichlorophenyl)propanamide] to dichloroaniline (DCA) in broth cultures within 24 hr). These microorganisms rapidly degraded acetanilide, 2′-nitroacetanilide (2′-NAA), and propanil to the corresponding aniline product in vivo and in vitro. No aryl acylamidase activity was found on several other substrates containing substituted amide bonds: the herbicides linuron [N′-(3,4-dichlorophenyl)-N-methoxy-N-methylurea] , diuron [N′-(3,4-dichlorophenyl)-N,N-dimethylurea], chlorpropham (1-methylethyl-3-chlorophenylcarbamate), pronamide [3,5-dichloro-N-(1-dimethyl-2-propynyl)benzamide], and alachlor [2-chloro-N-(2, 6-diethylphenyl)-N-(methoxymethyl)acetamide] and one metabolite (2-chloro-2′,6′-diethylacetanilide) of alachlor. Enzyme activity from both strains exhibited Michaelis-Menten kinetics using propanil as substrate with Km values of 0.22 and 0.39 mM and apparent Vmax values of 8.1 and 13.9 μmol DCA/mg whole-cell protein/hr for RA2 and RB4, respectively. Vmax values were severalfold higher than those of similar propanil aryl acylamidases, as described in the literature. With 2′-NAA as substrate, a linear relationship between concentration and activity was observed (3 to 4 μmol product/mg protein/hr at maximum solubility of 2′-NAA), indicating that enzyme saturation was not achieved. These high levels of aryl acylamidase activities are constitutive, since neither propanil nor linuron at 500 μM was able to induce aryl acylamidase activity in these cell cultures. The insecticide carbaryl (1-naphthyl-N-methylcarbamate) inhibited enzyme activity in vivo and in vitro, but substantially only at concentrations above 100 μM, with complete inhibition at 500 μM. The enzyme activity is apparently cytosolic for both strains, and RA-2 had significant extracellular aryl acylamidase activity. These strains may facilitate rapid turnover of propanil residues in rice flood water and soils and may have potential as sources of aryl acylamidase for biotechnological applications.