M. Maestracci

Activity and regulation of an amidase (acylamide amidohydrolase, EC 3.5.1.4) with a wide substrate spectrum from a Brevibacterium sp.

The Brevibacterium R 312 strain has an amidase with a wide substrate spectrum previously named “acetamidase”. The study of its activity showed that this enzyme was able to hydrolyze a large number of amides into their corresponding organic acids. The affinity of this enzyme for the substrates varied according to the length of the carbon chain and the spatial crowding of the molecule. The comparison of the specific rates of hydrolysis showed that propionamide was the amide substrate most quickly hydrolyzed.We confirmed the inducible feature of this enzyme and noted that only acetamide and N-methylacetamide were inducers of this enzyme among the compounds tested. Thioacetamide and N-methylpropionamide, both as amide analogues, were shown to inhibit the biosynthesis of acetamidase. Similarly, the organic acids, products of the hydrolysis reaction, showed a strong repression action on the biosynthesis of the enzyme.

Acyltransferase Activity of the Wide Spectrum Amidase of Brevibacterium sp. R312

The wide spectrum amidase from Brevibacterium sp. R312, which can hydrolyse many amides to the corresponding acids, was shown to transfer the acyl groups of amides, acids and esters to hydroxylamine. The transfer rates of these reactions in cytoplasmic fractions were measured and compared. The K m and V max were determined for different substrates in the presence of hydroxylamine. The enzyme was also shown to transfer the acyl group of the amide analogue N-methylacetamide to hydroxylamide and that of acetamide to the hydroxylamine analogue methylhydroxylamine.

Nitriles as growth substrates for Brevibacterium sp. R 312 and its mutant M2

Summary The most common nitriles were tested as growth substrates for the wild type Brevibacterium sp. R 312 strain. Some (linear and branched aliphatic nitriles, β -unsaturated nitriles) are both carbon and nitrogen sources; others (ex.-unsaturated nitriles, aromatic nitriles) are not growth substrates for this strain. The causes preventing growth of the wild type on acrylonitrile were determined. A mutant strain Brevibacterium sp. M 2 was isolated. This strain can grow on the α-unsaturated nitriles tested. The results obtained indicate that these strains could be used for the biological degradation of nitriles contained in waste waters.

Bacteria in organic synthesis: γ-alkoxy-α-aminoacids from related α-aminonitriles.

The wild type strain Brevibacterium sp. R312 is able to hydrolyse water-soluble γ-alkoxyaminonitriles, under mild conditions, at pH value close to neutrality and at moderate temperature. The results reported here show that this technique is valuable for synthetic purposes.

Regulation of nitrile-hydratase synthesis in a Brevibacterium species

The regulation of nitrile-hydratase biosynthese was studied in Brevibacterium sp R 312. Enzyme biosynthesis was not influenced by any carbon and nitrogen source used in the growth medium. It was, however, repressed by amide and amide analogues. Acetamide repressed nitrile-hydratase biosynthesis and induced the wide-spectrum amidase. Therefore, it appeared reasonable to hypothesize a single repressor gene for the nitrile-hydratase/wide-spectrum amidase system.

Continuous immobilized cell reactor for amide hydrolysis

SummaryThis article deals with continuous hydrolysis of acrylamide into acrylic acid using the wild-typeBrevibacterium sp. R312 which can hydrolyze all water-soluble amides into their corresponding acids. Biotransformation has been carried out in a fluidized bed reactor specially designed to obtain good contact conditions between cells entrapped into small calcium alginate beads (2–3 mm) and low-concentration acrylamide solutions (10–40g·l−1). Different flow rates, biocatalyst loads and substrate concentrations have been investigated. Kinetic constants for the immobilized enzyme have been identified. It appears that the Michaelis constant does not change with operating conditions and remains roughly equal to the value obtained for free cells. In contrast, the maximum rate of hydrolysis is considerably decreased, as if only cells on the outskirts of beads were involved in the transformation. On the whole it is proved that corynebacteria cells could be usefully used for the bioconversion of amides in a continuous immobilized cell reactor; the higher the solid hold-up and/or the smaller the beads, the more efficient the biological transformation.

Use of nuclear magnetic resonance and gas-liquid chromatography for the study of microbial nitrile-hydratases and amidases

Abstract Some microbial strains can hydrolyse many nitriles to the corresponding organic acids by means of two enzymes: a nitrile-hydratase which hydrates the nitrile to the corresponding amide, and an amidase which hydrolyses the amide to the corresponding acid. Two methods are proposed for the assay of the enzymatic activities: proton magnetic resonance spectrometry (n.m.r.) and gas-liquid chromatography (g.l.c.). For the assay of these enzymatic activities (nitrile-hydratase and amidase), g.l.c, was better, because it was more precise and much more sensitive than the n.m.r, method. The examples of acetonitrile and acetamide are described in detail.

Development of an assay method for cyanide, α-aminonitriles and α-hydroxynitriles for the study of the biological hydrolysis of these compounds

From an initial colorimetric method for the assay of cyanide, a technique for the assay of α-aminonitriles and α-hydroxynitriles has been developed. This technique is based on the assay of cyanide resulting from the decomposition of the α-aminonitriles and α-hydroxynitriles under defined pH conditions. This assay technique proved to be sensitive and accurate, and could be used for the assay of these compounds in biological mixtures. Using this assay technique, α-aminonitrilase and α-hydroxynitrilase activity has been demonstrated for an enzyme in Brevibacterium sp.; the Km and Vmax. of the enzyme have been determined for cyanide, α-aminopropionitrile and lactonitrile.