Pierre Galzy

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.

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. The specific activities and K m 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).

Substrate specificity of the lipase fromCandida parapsilosis

Substrate specificity of the acyltransferase activity of the lipase (EC 3.1.1.3) fromCandida parapsilosis CBS 604 was studied in aqueous media. The specificity toward both acid and alcohol parts of a large number of acylglycerols and aliphatic esters was investigated. This lipase showed a high activity in the presence of esters with long-chain fatty acids and particularly unsaturated fatty acids with acis-Δ9 double bond. It was observed that the activity profile depended not only on the alcohol part of the acyl ester, but also on the temperature of the reactant medium. The best lipid substrates had their melting point between −40 to +20°C, 14 to 18 carbon atoms in the acyl group and 1 to 4 carbon atoms in the alkyl group. The enzyme, defined as an acyltransferase in a previous paper, showed a high affinity for primary and secondary alcohols with a short carbon chain (1 to 5 carbon atoms) as acyl acceptors. The influence of free alcohols in the reactant medium on the hydrolysis and alcoholysis activities of the enzyme is discussed. Two phenomena seem to be involved, depending on the alcohol: competition with water for the acyltransfer reaction and lipid substrate dilution when the alcohol places at the oil/water interface.

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. Ten 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.

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.

Study of the Δ12-desaturase system ofLipomyces starkeyi

The specific activity of the microsomal Δ12-desaturase system, which transforms oleic acid into linoleic acid, was about 16 pmol/min/mg protein. However, most of the total activity was nonsedimentable even after a 200000×g centrifugation for 100 min. The study of various physicochemical parameters showed that this enzymatic complex, functioning optimally between pH 7 and 8, had low thermal stability. Ca2+, which may cause an aggregation of the microsomes, and Hg2+ completely inhibited the activity, whereas Mg2+, Mn2+, and Zn2+ were activators. The Δ12-desaturase system was relatively specific toward oleic acid, though isomers of this fatty acid also had an action, either as substrates or as competitive inhibitors, on the activity of the system. The study of the effect of the exogenous oleoyl-CoA and elaidoyl-CoA on the specific activity of the Δ12-desaturase system showed a preference toward oleoyl-CoA.

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.