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A mutant strain, Brevibacterium 19, defective for the enzyme nitrile hydratase but retaining all the amidase activity of the wild-type Brevibacterium R3 12, was isolated. This is genetic evidence in favour of the hypothesis that all the nitrile-hydratase activity of the wild-type was due to a single enzyme, the structural gene of which was lost in the mutant strain 19.The specific activities and K , of the nitrile hydratase were determined for 12 different substrates. The affinity of the enzyme increased as the number of hydrogen-bonding positions of the substrate increased, and decreased with more spatial crowding of the hydrocarbon chain. The specific activity of the enzyme for a substrate was enhanced by the nucleophilic and hydrophilic properties of the hydrocarbon side chain of that substrate. INTRODUCTIONThe wild-type strain Brevibacterium R312 (Arnaud et al., 1976a, b) was able to hydrolyse all water-soluble nitriles via the activities of its nitrile hydratase (Arnaud et al., 1977) and its amidase (Jallageas et al., 1978~). A mutant strain, Brevibacterium A4, unable to hydrolyse acetamide, was described by Jallageas et al. (1979) and Arnaud et al. (1980). Unlike the wildtype R3 12 (Amaud et al., 1976c), this mutant could no longer hydrolyse many amides such as the aliphatic amides, a-aminopropionamide, lactamide, benzamide and the D-a-aminopropionamides. This strain retained, however, the ability to hydrolyse L-a-aminoamides (Kieny-L'Homme et al., 1981). The enzyme no longer synthesized by the mutant A4 strain had previously been described and named acetamidase (Jallageas et al., 1978a).In the present work, we report the isolation of a mutant strain, Brevibacterium 19, which had lost its ability to hydrate nitriles, including cyanide, while retaining its amidase activity. Considering the industrial potential for the use of the nitrile hydratase enzyme (Bui et al., 1982), we also studied the effect of the structure of the substrate nitriles on the activity of hydratation of nitriles by the wild-type Breuibacrerium R312. The K , and V,,, of the nitrile hydratase were determined for 12 different substrates. METHODSBacterial strain. The wild-type strain used was Brecibactetium R3 12 (Arnaud et a/., 1976a, b). Media. The composition of the basal minimal medium was as previously described (Kieny-L'Homme et a/., 1981). Ammonium sulphate (5 g I-') and glucose (10 g 1-I) were added to the minimal medium. Slant cultures were maintained on YMPG-agar, the composition of which was given by Jallageas et a/. (1978~).Growth. Cultures were grown at 28 "C in conical flasks filled to 10% (v/v) RESULTS A N D DISCUSSIONIsolation and study of a nitrile hydratase defective mutant The isolation of a nitrile hydratase defective mutant was based on the following principle : in the presence of the wild-type, a halogenated nitrile would be hydrated into a halogenated amide, which would then be hydrolysed into a halogenated acid. Such compounds are toxic to some micro-organisms (Apirion, 1965;Hynes & Pateman, 1970;Clarke & Tata,...

The effect of different compounds on the enzymic action of the nitrile‐hydratase used for the bioconversion of nitriles was studied. An excess of acrylonitrile as a substrate was shown to inhibit the activity of the enzyme. This inhibition occurred only at relatively high substrate concentrations (0.2 mol/l or more). The nitrile bioconversion products (acrylamide, propionamide) and their structural analogues (acrylic acid, thioacetamide) were shown to inhibit the enzyme competitively. The most important inhibition found was that of cyanide (Ki= 0.004 mol/l), a break down product of some nitriles. By using an acetamidase‐negative mutant, amides were shown to inhibit biosynthesis of nitrile‐hydratase. An identical result was obtained with thioacetamide, a non‐substrate compound for acetamidase. This compound repressed the biosynthesis of nitrile‐hydratase by both the wild type and the acetamidase‐negative mutant to the same extent.

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