Alain Arnaud

Study of the amidase signature group.

Computer methods for database search, multiple alignment and cluster analysis indicated significant homology between amino-acid sequences of 21 amidases or amidohydrolases (EC 3.5). All of them were found to be involved in the reduction of organic nitrogen compounds and ammonia production. A conserved motif was found which may be important in amide binding and in catalytic mechanisms. Homology studies between these amidases and some ureases, nitrilases and acyl-transferases or enzymes with unknown functions provided new insight into the evolution of these proteins. Dissemination of these genes seemed to be facilitated by transfer of genetic elements such as transposons and plasmids.

Purification and properties of the β-glucosidase of a yeast capable of fermenting cellobiose to ethanol: Dekkera intermedia van der walt

SummaryThe intracellular enzyme β-glucosidase of Dekkera intermedia was partially purified by ion-exchange chromatography and gel filtration. The molecular weight of this enzyme was 310,000; its optimum pH was 5.0 and optimum temperature was 55°C. This enzyme was active against different β-glucosides and was inhibited by p-chloromercurybenzoate, gluconolactone, and glucose. A “glucosyltransferase” activity appeared in the presence of ethanol. The biosynthesis of this enzyme was constitutive but repressed by glucose.

A nitrile hydratase with a wide substrate spectrum produced by a Brevibacterium sp.

SUMMARY: A mutant stain, Brevibacterium 19, defective for the enzyme nitrile hydratase but retaining all the amidase activity of the wild-type Brevibacterium R312, 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.nThe specific activities and Knm 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.

Study of the acyl transfer activity of a recombinant amidase overproduced in an Escherichia coli strain. Application for short-chain hydroxamic acid and acid hydrazide synthesis

Abstract The study of acyl transfer activity of a wide spectrum amidase from Rhodococcus sp. R312, overproduced in an Escherichia coli strain, revealed that the ‘bi-bi-ping-pong’ type reaction was efficient with only four very-short chain (C2–C3) aliphatic amides as substrates. The optimum working pH was 7.0 for all neutral amides. Very short-chain aliphatic carboxylic acids were 10–1000-fold less efficient and the corresponding optimum working pH values depended on the acid used. Very polar molecules, such as water, hydroxylamine and hydrazine, were good acyl acceptors. An [acyl donor]/[acyl acceptor] ratio lower than 0.3-0.5 had to be maintained to avoid enzyme inhibition by excess acyl donor. The different acyl-enzyme complexes generally exhibited high affinity for hydroxylamine or hydrazine (except the propionyl-enzyme complex), so that the residual hydrolysis activities were almost totally inhibited at appropriate acyl acceptor concentrations. Molar conversion yields were higher with hydrazine as acyl acceptor (e.g., 97% with acetamide as acyl donor) because of the higher Vmax values, but in all cases, interesting quantities of short-chain hydroxamic acids (2.9-6.5 g l−1) and acid hydrazides (6.4–7.8 g l−1) could be quickly obtained (10–60 min) with small amounts of enzyme (0.04-0.20 g l−1).

Biocatalyst improvement for the production of short-chain hydroxamic acids

The wide spectrum amidase gene (amiE) derived from Rhodococcus sp. R312 was overexpressed in a recombinant Escherichia coli strain. Amidase was extracted during the stationary phase by cell grinding. After partial purification with Q-Sepharose chromatography, the enzyme was immobilized on Duolite A-378 resin. A higher immobilization yield (Ri = 87%) was achieved with NaH2PO4Na2HPO4 phosphate buffer (100 mm) at pH 7. Study of the immobilized amidase demonstrated that the physicochemical properties were similar to those of the free enzyme with respect to pH and activation energy. On the other hand, the optimal working temperature was higher for the immobilized amidase and thermostability was slightly improved. n nTen g of insoluble biocatalyst produced 1 l of acetohydroxamic acid (at least 4.1 g l−1) solution after 90 min reaction time. The acetamide bioconversion rate was 55–60% (mol mol−1). After lyophilization, a 10 g powder containing 40% (wt wt−1) acetohydroxamic acid was recovered.

Monohydroxamic acid biosynthesis

Abstract Hydroxamic acids (HA), with the general formula R–CO–NHOH, are chelating agents which may be used in a number of interesting applications, such as medicine and waste water treatment. In this paper, we describe the enzymatic synthesis of HA with various chain lengths (from C2 to C18), using three microbial enzymes. For short- and middle-chain HA synthesis, using amidases from Rhodococcus sp. R312, the optimal working pH was found to be pH 7 or 8, depending on the amide substrate used. Different Michaelis–Menten constants were also determined. For fatty HA synthesis, using lipase from Candida parapsilosis, the optimal working conditions were determined to be pH 6, 1 M hydroxylamine and 40°C. Because of the favorable bioconversion yields achieved, the enzymatic synthesis of HA with the appropriate biocatalysts appears to be an interesting alternative to chemical synthesis.

Transcriptional analysis of the nitrile‐degrading operon from Rhodococcus sp. ACV2 and high level production of recombinant amidase with an Escherichia coli– T7 expression system

Northern blotting analysis with RNA probes derived from amidase and nitrile hydratase genes from Rhodococcus sp. ACV2 revealed that both genes are part of the same operon. RNase protection mapping and sequence analysis indicated that the operon is probably under the control of a sigma 70‐like promoter located upstream from the amidase gene. Plasmids were constructed with the cloned genes under tac and lac promoter control. Expression of amdA was demonstrated in Escherichia coli. In another construction, the amdA gene was inserted under the control of the bacteriophage T7 promoter. Large amounts of recombinant amidase (at least 20% of total proteins) in a soluble and active form were obtained with the E. coli– T7 expression system by lowering the growth temperature to 29u2003°C, without IPTG induction. The ratio of amidase activity of strain ACV2 to E. coli was approximatelyu20031:3. Purification of the recombinant amidase was carried out in one chromatographic step, giving an enzyme preparation that could be used directly in a biotechnological process.

Spectrophotometric assay of aliphatic monohydroxamic acids and α-, β-, and γ-aminohydroxamic acids in aqueous medium

Abstract Chelating properties of hydroxamic acids with iron(III) were used for the quantitative determination of several monohydroxamic acids. Maximum absorption was obtained at a wavelength of ca. 500 nm for all iron(III)/monohydroxamic acid complexes tested. Determination of the molar extinction coefficient (ϵ M ) for each complex simplified the monohydroxamic acid assay since a mean ϵ M value could be determined for each family of iron(III)/monohydroxamic acid complexes. These results confirmed the influence of the NH 2 group in the coordination of α-, β-, and γ-aminohydroxamic acids with iron(III). Finally, this study showed the high sensitivity of the spectrophometric hydroxamic acid assay, with a minimum required concentration of 2.5 × 10 −5 to 5.0 × 10 −5 mol l −1 as a function of the monohydroxamic acid concerned. The method was validated under biological conditions for Spectrophotometric quantification of an enzyme-catalyzed acyl transfer reaction resulting in hydroxamic acid production.

Application of high-performance liquid chromatography to the study of the biological transformation of adiponitrile by nitrile hydratase and amidase.

A procedure for the assay of nitrile hydratase and amidase activities by high-performance liquid chromatography is described. The method can be used to assay the intermediate compounds resulting from the hydrolysis of adiponitrile to adipic acid, and to determine the kinetics of the hydrolysis of these compounds using whole cells and enzyme extracts. The precision of the method makes it suitable for the determination of the enzymic parameters.

Sizing of the Rhodococcus sp. R312 genome by pulsed-field gel electrophoresis. Localization of genes involved in nitrile degradation.

The two restriction enzymesAsnI andDraI were found to produce DNA fragment sizes that could be used for mapping theRhodococcus sp. R312 (formerlyBrevibacterium sp. R312) genome by pulsed-field gel electrophoresis.AsnI produced 24 fragments (4 to 727 kb) andDraI yielded 15 fragments (8.5 to 2400 kb). The fragment lengths in each digest were summed, indicating that the size of the chromosome ranged from 6.31 to 6.56 Mb, with a mean of 6.44 Mb. In addition, the wide-spectrum amidase gene (amiE) and the operon containing the enantiomer-selective amidase gene (amdA) and the nitrile hydratase structural gene (nthA, nthB) were localized on theAsnI andDraI fragments.