Dominique Petre

Purification, cloning, and primary structure of a new enantiomer-selective amidase from a Rhodococcus strain: structural evidence for a conserved genetic coupling with nitrile hydratase.

A new enantiomer-selective amidase active on several 2-aryl propionamides was identified and purified from a newly isolated Rhodococcus strain. The characterized amidase is an apparent homodimer, each molecule of which has an Mr of 48,554; it has a specific activity of 16.5 mumol of S(+)-2-phenylpropionic acid formed per min per mg of enzyme from the racemic amide under our conditions. An oligonucleotide probe was deduced from limited peptide information and was used to clone the corresponding gene, named amdA. As expected, significant homologies were found between the amino acid sequences of the enantiomer-selective amidase of Rhodococcus sp., the corresponding enzyme from Brevibacterium sp. strain R312, and several known amidases, thus confirming the existence of a structural class of amidase enzymes. Genes probably coding for the two subunits of a nitrile hydratase, albeit in an inverse order, were found 39 bp downstream of amdA, suggesting that such a genetic organization might be conserved in different microorganisms. Although we failed to express an active Rhodococcus amidase in Escherichia coli, even in conditions allowing the expression of an active R312 enzyme, the high-level expression of the active recombinant enzyme could be demonstrated in Brevibacterium lactofermentum by using a pSR1-derived shuttle vector.

Aliphatic nitrilase from a soil-isolated Comamonas testosteroni sp.: gene cloning and overexpression, purification and primary structure.

An aliphatic nitrilase, active on adiponitrile and cyanovaleric acid, was identified and purified from Comamonas testosteroni sp. (Ct). Oligodeoxyribonucleotide probes were designed from limited amino acid (aa) sequence information and used to clone the corresponding gene, named nitA. High homologies were found at the aa level between Ct nitrilase and the sequences of known nitrilases. Multi-alignment of sequenced nitrilases suggests that Cys163 of Ct plays an essential role in the active site. This hypothesis is strengthened by molecular studies on nitrilases from Alcaligenes faecalis JM3, and Rhodococcus rhodochrous J1 and K22 [Kobayashi et al., Proc. Natl. Acad. Sci. USA 90 (1993) 247-251; J. Biol. Chem. 267 (1992) 20746-20751; Biochemistry 31 (1992) 9000-9007]. Large amounts of an active recombinant enzyme could be produced in Escherichia coli when nitA was overexpressed together with the E. coli groESL genes.

Enzymatic synthesis of glycamide surfactants by amidification reaction

Abstract The condensation of a secondary amine (N-methyl-glucamine) with oleic acid was selected as a reaction model to study the production of glycamide surfactants by enzymatic amidification. Reactions catalyzed by immobilized lipase from Candida antarctica were carried out in 2-methyl-2-butanol. The acido-basic conditions (through the N-methyl-glucamine / oleic acid ratio) control the chemoselectivity of the reaction allowing the synthesis of either amide, ester or amide-ester. At 90°C and with a N-methyl-glucamine / acid ratio of 1, a 100% conversion yield with 97% of amide synthesis was obtained in less than 50 hours. The process was applied to the preparation of a range of amides using various amines and acyl donors. This process is the first that describes successful amide bond synthesis from a hydroxylated secondary amine and fatty acid or fatty acid ester.

Cloning and primary structure of the wide-spectrum amidase from Brevibacterium sp. R312: high homology to the amiE product from Pseudomonas aeruginosa.

A Brevibacterium sp. R312 DNA fragment encoding the wide-spectrum amidase (EC 3.5.1.4) has been cloned and sequenced, using limited amino acid (aa) sequence information obtained from the purified enzyme. The deduced aa sequence showed more than 80% strict identity with the Pseudomonas aeruginosa aliphatic amidase, the product of the amiE gene, suggesting a horizontal transfer of the gene during evolution between Gram+ and Gram- bacteria.

LIPASE-CATALYZED CHEMOSELECTIVE N-ACYLATION OF AMINO-SUGAR DERIVATIVES IN HYDROPHOBIC SOLVENT : ACID-AMINE ION-PAIR EFFECTS

Abstract Enzymatic N-acylation of N-methyl-glucamine (1-deoxy-1-methylamino-D-glucitol) in hexane using lipase from Rhizomucor miehei (Lipozyme ® ) is described. N-methyl-glucamine was solubilized by oleic acid addition which resulted in the formation of an ion-pair between acid and amine function. This ion-pair, identified by Infra-Red spectroscopy, is essential for amide or ester synthesis. Its stability in hexane was also found to be the limiting factor of reaction yield which never exceeded 50 % of acid conversion. The chemoselectivity of the reaction between oleic acid and N-methyl-glucamine towards amide or ester synthesis was under the control of acid/amine ratio. This is the first report showing the key role of substrate ionic state when operating enzyme catalysis in non-conventional media.

Highly efficient control of iron-containing nitrile hydratases by stoichiometric amounts of nitric oxide and light.

The reaction of two iron‐containing nitrile hydratases (NHase) with NO has been studied: NHase from Rhodococcus sp. R312, which is probably similar to the photosensitive N771 NHase, and the new NHase from Comamonas testosteroni NI1 whose aminoacid sequence is quite different from those of BR312 and N771 NHases. Both enzymes are equally inactivated after addition of stoichiometric amounts of NO added as an anaerobic solution or produced in situ under physiological conditions by a rat brain NO‐synthase. Both enzymes are reactivated by photoirradiation, and two cycles of NO inactivation/photoactivation can be performed without significant loss of activity. Both iron‐containing NHases have a high affinity for NO, similar to that of methemoglobin.

Lipase-catalysed synthesis of biosurfactants by transacylation of N-methyl-glucamine and fatty-acid methyl esters

Abstract The enzymatic synthesis of a range of amide surfactants by transesterification reactions between various amines and fatty-acid methyl esters (Diester®) a low-cost natural and renewable raw material is described. The transacylation reaction catalysed by a commercial lipase from Candida antarctica (Novozym®) at 90°C, yielded 100 % conversion of fatty-acid methyl esters with 80 % amide synthesis in less than 20 hours. Yield and chemoselectivity of the reaction are under the control of the fatty-acid methyl ester/amine molar ratio.

Enzymatic amidification for the synthesis of biodegradable surfactants: Synthesis of N-acylated hydroxylated amines

Abstract Glucamide surfactants can be obtained by reacting amino–sugar derivatives with fatty acids in the presence of lipase enzymes in organic media. Reactions are catalyzed with Lipozyme in hexane or with Novozym in 2-methyl-2-butanol as solvents. In hexane, the formation of a salt complex between the fatty acid and N -methyl-glucamine allows the efficient acylation of the amine. Fatty acid conversion is limited to 50% due to salt formation. In 2-methyl-2-butanol, conversion yield of the fatty acid up to 100% can be obtained by removing the water coproduct under reduced pressure. Acido–basic conditions allow the control of the reaction chemoselectivity. This reaction can also be completed using various amines and acyl donors.

Lipase-catalysed production of N-oleoyl-taurine sodium salt innon-aqueous medium

Synthesis of N-oleoyl-taurine, a fatty amide surfactant, was achieved by condensation of taurine and oleic acid in organic media using commercial lipase preparations from Rhizomucor miehei or Candida antarctica at 55°C, yielding 25% conversion of oleic acid at 1.2 mmol.

ENZYMATIC SYNTHESIS OF SURFACTANTS VIA AMIDE BONDS

Non-ionic biodegradable surfactants can be obtained by reacting amino alditol derivatives with fatty acids in the presence of lipases in organic media. Such surfactants in contrast to ester bonds, containing an amide bond are stable under alkaline conditions. The efficient coupling of N-methyl-glucamine to oleic acid can be catalysed with Lipozyme in hexane or with Novozym in 2-methyl-2-butanol as solvents. In the first case, the acid/base ratio controls both kinetics and chemo-selectivity of the reaction. In the second case, the adoption of acid base conditions allow control of the chemo-selectivity of the reaction. This reaction can also be completed by using triglycerides or fatty acid esters as acyl donors, which opens the way to the valorization of plant oils for surfactant synthesis.