CA2103616A1 - Polypeptides possessing a nitrilase activity, dna sequence coding for said polypeptides, expression cassettes and host microorganisms enabling them to be obtained, and method of converting nitriles to carboxylates by means of said polypeptides - Google Patents

Abstract

IN THE CANADIAN PATENT AND TRADEMARK OFFICE

PATENT APPLICATION

entitled: Polypeptides possessing a nitrilase activity, DNA sequence coding for said polypeptides, expression cassettes and host microorganisms enabling them to be obtained, and method of converting nitriles to carboxylates by means of said polypeptides in the names of: Dominique PETRE
Edith CERBELAUD
Sophie LEVY-SCHIL
Joël CROUZET

Assignee: RHONE POULENC CHIMIE

ABSTRACT OF THE DISCLOSURE

The present invention relates to novel polypep-tides having a nitrilase activity and to the genetic tools for producing them, namely:
- the DNA sequence coding for a polypeptide having a nitrilase activity and capable of hydrolyzing nit-riles to carboxylates, - an analog of this sequence resulting from the degene-racy of the genetic code, - a DNA sequence hybridizing with one of these sequen-ces or a fragment thereof and coding for a poly-peptide having a nitrilase activity, and - expression cassettes and microorganisms enabling them to be obtained.
Application: enzymatic conversion of nitriles to carboxylates.

Description

210 3 6 ~ ~3 POLYPEPTIDES POSSESSING A NITRILASE ACTIVITY, DNA
SE~UENCE CODING FOR SAID POLYPEPTIDES, EXPRESSION
CASS~TTES AND HOST MICROORGANISMS ENABLING THEM TO BE
OBTAINED, AND METHOD OF CONVERTING NITRILES TO CARBOXY-05 LATRS BY ~EANS OF SAID POLYPEPTIDES

The present invention relates to novel poly-peptides having a nitrilase activity and to the genetic engineering tools for producing them, namely a DNA
sequence, the expression cassettes carrying this recom-binant DNA sequence, and the recombinant microorganisms (host microorganisms) containing said DNA sequence.
The present invention further relates to an enzymatic method of converting nitriles to carboxylates by means of the polypeptides according to the invention or a host microorganism containing the DNA sequence according to the invention. One particular application of the method of the invention is the enzymatic syn-thesis of ammonium adipate or ammonium 5-cyanovalerate by the hydrolysis of adiponitrile with the aid of a polypeptide or host microorganism according to the invention.
Ammonium adipate is known to be a particularly valuable product because it can be converted to adipic acid, a product which is itself widely used for the preparation of nylon 6,6.
The enzymatic hydrolysis of dinitriles has been described by numerous authors. However, the routes by which these dinitriles are hydrolyzed to organic acids are not often referred to. The theoretical hydrolysis scheme is as follows:

- - 2103~

NC-R-Q~
Ni ~
.,~ 1`7C-R-C0NH2 ~, ~JC-R-COO- NEI4+ H~OC-R-CO~H2 N~ IOC-R-COO~

+4HN -C)OC-R-COO- NH4+

NH = nitrile hydratase Ni = nitrilase A = amidase R = (CH2)~, n being an integer equal to 4 in the case of adiponitrile.
In actual fact, it is very often observed that certain routes are preferred and that certain products are not formed or else are not hydrolyzed.
Among the microorganisms for which it has been possible to demonstrate the existence of an enzymatic activity permitting this hydrolysis, there may be men-tioned in particular the strains belonging to the genus Fusarium, which degrade succinonitrile and adiponit-rile, although th~ reaction products are not indicated [Goldlust et al., Biotechnol. and Appl. Biochem., 1989, 11, 581]; the strains belonging to the genus Pseudo-monas, which degrade adiponitrile [Yanase et al., Agric. Biol. Chem., 1982, 46, 2925]; and the strains belonging to the genus Rhodoeoeeus, in particular Rhodococeus rhodoehrous NCIB 11 216, which hydrolyzes - adiponitrile to adipic acid [Bengis-Garber et al., Appl. Microbiol. Biotechnol., 1989, 32, II], and also Rhodococeus rhodoehrous K22, whose nitrilase permits the hydrolysis of adiponitrile and glutaronitrile ;, ' ~ , . .. . . . . .
., .,, :,:

:' ' . -. ,' ~ ' 2la3~l~

[Yamada et al., J. Bacteriol., 1990, 172 (9), 4807-4815], albeit with a low activity ratio compared with that for the hydrolysis of aromatic nitriles.
Consequently, it can be seen that the enzymatic 05 hydrolysis of dinitriles is rather complex: in all cases, although the first CN group is hydrolyzed by the enzyme, the second group is not hydrolyzed at all in some cases, or else is hydrolyzed at a very low rate in other cases.
It has now been found that it is possible to hydrolyze nitriles to carboxylates, and more particu-larly dinitriles to carboxylates or dicarboxylates, totally and rapidly, by using appropriately selected enzymes either as such or, preferably, in the form of recombinant microorganisms which generate them.
The present invention therefore relates to novel polypeptides having a nitrilase activity which have been isolated from a strain of Comamonas testo-steroni. More precisely, these polypeptides are pre-pared by extraction and purification from cultures ofnatural or recombinant microorganisms, the purification being effected by a series of steps consisting in pre-paring an enzymatic extract from the cell culture, precipitating this extract with ammonium sulfate and purifying it by different steps involving chromato-graphy and gel filtration. These steps, which employ techniques well known to those skilled in the art, are described in detail in the illustrative Examples below.
In the present description, "nitrilase acti-vity" denotes the direct conversion of a nitrile to anammonium carboxylate, the corresponding amide not being - a substrate for the enzyme.
~ he invention further relates to a DNA sequence coding for a polypeptide having a nitrilase activity.
The DNA se~uence coding for a polypeptide of the - :

210361~

invention can be selected from:
- the DNA sequence coding for a polypeptide having a nitrilase activity, as shown in Fig~ 4, - an analog of this sequence resulting from the degene-05 racy of the genetic code, - or else a DNA sequence hybridizing with one of these sequences or with a fragment thereof and coding for a polypeptide having a nitrilase activity.
Such a DNA sequence can be obtained by cloning the genomic DNA fragment coding for the desired poly-peptide, with the aid of nu_leotide probes produced from the purified polypeptide.
The invention further relates to the expression cassettes which carry the above-defined recombinant DNA
sequence together with the signals ensuring its expres-sion. These expression cassettes can either be inte-grated in the genome of the host or located on an expression vector such as a plasmid containing a selec-tion means.
In particular, these expression cassettes con-tain transcription and translation initiation regions which contain a ribosome binding site and a promoter sequence. These regions may be homologous or heterolo-gous with the microorganism which naturally produces the polypeptide.
The choice of these regions depends especially on the host used. In particular, when the host micro-organisms are procaryotic, the heterologous promoter can be selected from strong bacterial promoters such as the tryptophan operon promoter Ptrp of E. coli, the lactose operon promoter Plac of E. coli, the phage - lambda right promoter P~, the phage lambda left pro-moter PT. ~ the strong promoters of Pseudomonas and comamonas and the strong promoters of Corynebacteria.
~ore particularly, in the case of the phage :
.~ . ~. , . .~ -. ~ ~ , . -:. .. : , .

.

2~03~1~

lambda right promoter, the thermosensitive form P~CIts may be preferred. In the case of eucaryotic micro-organisms such as yeasts, the promoters can originate from glycolytic yeast genes such as the genes coding 05 for phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GPD), lactase (LAC4) and enolase (ENO).
As far as the ribosome binding sites are con-cerned, the one derived from the lambda CII gene, as well as those derived from homologous genes of Coma-monas or Pseudomonas or those derived from genes of Corynebacteria, are used preferentially when the host microorganism is procaryotic.
A region permitting a termination of the trans-lation and functional transcription of the envisagedhost can be positioned at the 3' end of the coding sequence. The expression cassette also comprises one or more markers making it possible to select the recom-binant host. The preferred markers are dominant mar-kers, i.e. those conferring a resistance to antibioticssuch as ampicillin or streptomycin, or to other toxic products.
Enterobacteria such as E. coli, bacteria belonging to the genera comamonas or Pseudomonas, and corynebacteria such as those belonging to the genera Corynebacterium, Brevibacterium or Rhodococcus, may be mentioned in particular among the host microorganisms used.
The invention further relates to the micro-organisms containing the recombinant DNA sequenceaccording to the invention, for example on a plasmid - containing a selection means.
A recombinant microorganism containing said DNA
sequence on a plasmid structure was deposited in the Collection Nationale de Cultures de Micro-organismes 2~3fi~

(C.N.C.M.) (Institut Pasteur, 25 rue du Docteur Roux, Paris) under no. I-1242 on 21st July 1992. This microorganism is the strain E. coli TGl, which contains plasmid pXL2148; this microorganism is also identified 05 by the Applicant using the reference G4207.
The invention further relates to the micro-organisms capable of converting nitriles to carboxy-lates, and more particularly aliphatic dinitriles of the formula NC-R-CN, in which R is a linear or branched alkylene group having from 1 to 10 carbon atoms, to carboxylates.
Beyond acquiring their structures I and II
during their synthesis by the microorganisms according to the invention, it is important that the polypeptides in question stabilize in their structures III and IV so as to possess an optimal nitrilase activity.
The Applicant takes credit for having dis-covered means for favoring the above-mentioned stabili-zation.
20Thus any microorganism according to the inven-tion preferably contains:
- at least one protein agent for assisting the folding of the polypeptides which the microorganism syn-thesizes, and in particular the nitrilase referred to 25in the present disclosure, - and/or the genes coding for such an agent, this agent being present in a greater amount than that corres-ponding to the base level of the microorganism in question.
30 In terms of the present invention, base level is understood as meaning the maximum level which can be - attained by the corresponding wild-type microorganism in question.
Advantageously, this agent is the GroE chape-rone of E. coli or its homolog of eucaryotic or pro-~ . -: ' . ' ..

- 21036~

caryotic origin.
The GroE chaperone of E. coli is normally pre-sent in the wild-type strains.
The genes coding for the agent are carried by 05 the chromosome or by an extrachromosomal element (plas-mid, phage). They are preferably amplified by any known and appropriate means so as to favor the synthe-sis of the agent in the microorganism.
The genes coding for the agent are under the dependence of expression systems homologous or hetero-logous with their host microorganism.
The invention further relates to the method of converting nitriles to carboxylates with the aid of a polypeptide according to the invention or a recombinant microorganism which generates it. This method consists in bringing the nitrile to be converted into contact with a polypeptide or recombinant microorganism as defined above. The process is generally carried out at room temperature. In one particular embodiment of the invention, the polypeptide or recombinant microorganism is immobilized on or in a solid support.
The method of the invention is suitable for the conversion of nitriles to carboxylates and more parti-cularly for the conversion of dinitriles of the formula NC-R-CN, in which R is a linear or branched alkylene group containing 1 to 10 carbon atoms, to carboxylates.
The method of the invention is particularly appropriate for the enzymatic synthesis of ammonium adipate from adiponitrile.
30The Examples which follow afford an illustra-tion of the characteristics and advantages of the pre-- sent invention without however limiting its scope.

.
- . :
, ' .- . .

21~3G~ ~i DESCRIPTION OF THE FIGURES

Fig. 1 shows the yield (%) of the hy~rolysis of adiponitrile to cyanovalerate (curve a) and to ammonium 05 adipate (curve b) as a function of the reaction time in hours for the strain Comamonas testosteroni sp.
Fig. 2a and 2b show the restriction maps of plasmids pXL2075 and pXL2076.
Fig. 3 shows the restriction map of the SstI-SstI fragment of 4.1 kb containing the DNA sequence(called "nitrilase gene" in the Figure) coding for the polypeptide having the nitrilase activity according to the invention, said fragment being present in plasmids pXL2075 and pXL2076. The strategy for producing the XbaI-SstI fragment containing the DNA sequence accor-ding to the invention is also shown in this Figure.
Fig. 4 shows the DNA sequence according to the invention with its deduced amino acid sequence.
Fig. 5 shows the restriction map of plasmid pXL2087.
Fig. 6 shows the restriction map of plasmid pXL2148.
Fig. 7 shows the SDS-PAGE, 10% SDS, indicating the expression of the DNA sequence according to the invention in the strain E. coli TGl/pXL2027. Each lane corresponds to an amount of protein equivalent to 60 ~l of culture at an optical density of 3 at 610 nm.
Fig. 8 shows the restriction map of plasmid pXL2158.
Fig. 9 shows the SDS-PAGE, 12.5% SDS, indica-ting the expression of the DNA sequence according to ~ the invention in the strains TGl/pXL2158 and TGl/
pXL2158 + pXL2035 (GroE).
Fig. lO shows the restriction map of plasmid pXL2169.

.

~1~351 ~

Fig. 11 shows the SDS-PAGE, 10~ SDS, indicating the expression of the DNA sequence according to the invention in the strain Pseudomonas putida G2081-pXL2169.
05 The abbreviations used in the remainder of the description have the following meanings:
SSC: buffer commonly used for hybridizations, containing sodium citrate and NaCl (20x SSC =

3 M NaCl, 0.3 M sodium citrate, pH 7) 10 SDS: sodium dodecylsulfate FPLC: fast protein liquid chromatography SDS-PAGE: sodium dodecylsulfate/polyacrylamide gel electrophoresis IPTG: isopropyl ~-D-thiogalactopyranoside EXAMPLES

EXAMPL~ 1: PURIFICATION OF THE NITRILASE OF Comamonas testosteroni sp.
1 - PREPARATION OF THE CELLS:
A strain of Comamonas testosteroni sp. was cultivated in a shake flask, at 28C, for 15 h 30 min, in medium A having the following composition:
25 Medium A
- Glucose 5 g/l - (NH~)2SO~ 1 g/l - Na~HPO~ 5.24 g/l - KHPO~ 2.77 g/l - Yeast extract 5 g/l - Casamino acids 1 g/l This preculture was used to inoculate a 20 l fermenter containing 15 l of medium A. The pH, tem-perature, air flow rate and shaking speed were set to 6.6, 28C, 300 l/h and 350 rpm respectively. After - 2~3~

24 h, 84 g of wet cells were harvested. This corras-ponds to a content by dry weight of cells of 0.9 g/l and to an optical density at 660 nm (OD660nm) of 2.

NITRILE:
A cellular residue containing 13.1 mg of dry weight of cells was suspended in 2 ml of a 52.3 mM
solution of adiponitrile in 50 mM potassium phosphate lQ buffer, pH 7. The reaction was carried out at 25 C, with shaking, and the kinetics were followed by sam-pling. 5-Cyanovaleramide, adipamide, 5-cyanovalerate, adipamate and adipate were determined on each sample by high performance liquid chromatography (HPLC). The results are collated in Fig. 1, which shows the curves of the yield (on the ordinate) of cyanovalerate (curve a) and ammonium adipate (curve b). The respective rates of formation of cyanovalerate and adipate were greater than 0.4S and e~ual to 0.15 U/mg of dry weight of cells (1 U is equal to 1 ~mol of product formed per minute).

3 - PURIFICATION: -All the purification steps were carried out in 50 mM Tris/HCl buffer, pH 7.5, 1 mM dithioerythritol (DTE), unless indicated otherwise. At each step, the nitrilase activity of the fractions was determined at pH 7 and at 25CC in 10 mM phosphate buffer in the presence of 10 mM adiponitrile. The protein concen-tration of the pools was determined by the Coomassie blue method (PIERCE Protein assay kit). The proteins were analyzed by SDS-PAGE (Phastsystem, PHARMACIA).
The procedures of each step are discussed below.

2 ~ 3'~

Step 1: Crude extract 57 g of wet cells were taken up in 85 ml of buffer and treated with ultrasound for 30 min (VIBRA-CELL sonicator from Bioblock: probe 13 mm; power 7; 40%
05 of the cycle active). The OD660nm thus dropped from 97 to 60. After centrifugation at a maximum of 48,000 g for 60 min, the supernatant was recovered.
This supernatant was brought to 15% saturation by the gradual addition of ammonium sulfate. After 1 h, the suspension was centrifuged for 30 min at a maximum of 30,000 g. The supernatant was brought to 50% saturation. After 1 h, the suspension was centri-fuged under the same conditions and the precipitate was recovered and then dialy2ed against the buffer for two days.
Step 2: Ion exchange column (Q Sepharose Fast Flow) The dialyzed fraction was loaded at a rate of 125 ml/h on to a column (26 x 380 mm) of "Q Sepharose Fast Flow" equilibrated with the buffer at a rate of 250 ml/h. The column was percolated at a rate of 250 ml/h by the following solutions in succession:
- 166 ml of buffer - 180 ml of a gradient of 0 to 0.2 M KCl in the buffer 25 - 180 ml of buffer to which 0.2 M KCl had been added - 270 ml of a gradient of 0.2 to 0.4 M KCl in the buffer - 180 ml of buffer to which 0.4 M KCl had been added - 200 ml of buffer to which 1 M KCl had been added 3~ The fraction having the nitrilase activity was eluted in a volume of 129 ml during the 0.2 M KCl - stage.
The following steps are carried out on the FPLC
system (Pharmacia).

step 3: Gel filtration (FPLC Superdex 200) The previously obtained fraction having the nitrilase activity (129 ml) was concentrated to 12 ml by precipitation of the proteins with ammonium sulfate 05 at 80~ saturation, followed by dialysis against the buffer. The fraction concentrated in this way (12 ml) was loaded in 2 batches on to the column of gel (16 x 600 mm) equilibrated with the buffer to which 0.1 M KCl had been added, at a rate of 0.8 ml/min. The fractions having the nitrilase activity were eluted with the above buffer at a rate of 1 ml/min and i-n a total volume of 36 ml. These fractions correspond to a mole-cular weight of 280 kDa.
Step 4: Column of hydroxyapatite (BIO-RAD HPHT;
7~8 x 100 mm) The fractions obtained above were concentrated to 8 ml by ultrafiltration (DIAFLO PM39 membrane, AMICON). The concentrated solution was injected on to the column of hydroxyapatite equilibrated with the buffer to which 10 ~M CaCl2 had been added. The column was percolated at a rate of 0.5 ml/min with the fol-lowing in succession:
- 5 ml of equilibration buffer - 15 ml of a gradient of 0 to 350 mM potassium phos-phate in the eguilibration buffer - 10 ml of the equilibration buffer to which 350 mM
potassium phosphate had been added The fractions having the nitrilase activity were eluted between 62 and 135 mM potassium phosphate in a volume of 3 ml.
Step 5: Hydrophobic interaction column (FPLC-Phenyl Superose HR 5/5) The active fractions obtained above, brought to 15% saturation with ammonium sulfate/ were loaded at a rate of 0.5 ml/min on to the column equilibrated with 2:~36 ~

buffer containing ammonium sulfate at 15% saturation.
The column was percolated with:
- 6 ml of equilibration buffer - 12 ml of a decreasing ammonium sulfate gradient of 05 15% to 0~ saturation in the buffer - 23 ml of buffer Some of the fractions having the nitrilase activity were eluted during the washing of the column with the equilibration buffer. These active fractions were reinjected under the sama conditions. This opera-tion was performed twice. The active fractions eluted after the gradient were pooled (volume 51 ml~.
Step 6: Gel filtration (FPLC-Superdex 200) The 51 ml were concentrated to 3 ml by ultra-filtration on a membrane (DIAFLO PM30, AMICON). These3 ml were loaded on to the column (16 x 600 mm) e~uili-brated with the buffer to which 0.1 M KCl had been added. The 9 ml containing the activity were eluted at a position corresponding to a molecular weight of 280 kDa. This solution was brought to 36% with glycerol and then frozen for 15 days.
St2p 7^ Ion exchange column (FPLC Mono Q HR
5/5) The protein solution was thawed and loaded on to the column equilibrated with the buffer containing 0.1 M KCl, at a rate of 0.5 ml/min. The column was percolated with the following in succession:
- 15 ml at 0.5 ml/min of buffer to which 0.1 M KCl had been added - 4.5 ml at 1 ml/min of buffer to which 0.1 M KCl had been added - 15 ml at 1 ml/min of a gradient of 0.1 to 0.4 M KCl in the buffer - 10 ml of buffer to which 0.4 M KCl had been added The active fractions were eluted between 0.15 :, .

2 ~ J ~

and 0.3 M KCl. These fractions are homogeneous~ SDS-PAGE analysis reveals two bands very close to 38 and 39 kDa. The fractions thus obtained will hereafter be called "purified nitrilase".
05 The data from each of the above purification steps are collated in Table 1 below:

TABLE 1: PURIFICATION OF THE NITRILASE OF Comamonas testosteroni sp.

PURIFICATION Vol. Protein ACTIVITY YIELD PF
STEP ml mg Total Specific Protein Activity _ 0 - Crude 61 92062,000 68 100 100 extract _ _ 2 - Q Sepharose13024547,000 190 27 76 2 8 3 - Gel 36 2756,000 2100 2.9 90 30 filtration _ 4 - Hydroxyapa- 3 1249,000 4100 1. 3 79 60 tite column 5 - Phenyl 51 1111,000 1000 1.1 18 15 Superose _ 6 - Gel 9 2.76, 300 2300 0. 3 10 34 filtration _ HR 5/5 2.9 11, 2DO 1200 0 01 2 18 ABBREVIATIONS: PF ~ purification factor; U ~ 1 ~mol/h NITRILASE:
Taking the purified protein, the N-terminal ~ sequence of 27 amino acids was determined by Edman automatic sequential degradation using an "Applied Biosystems Model 470 A" apparatus. This sequence is as follows:

~3~

Met Lys Asn Tyr Pro Thr Val Lys Val Ala Ala ~al Gln Ala Ala Val Phe Met Asn Leu Glu Ala Thr Val Asp Lys Thr ~5 A search of sequence libraries made it possible to find a 53% identity with the nitrilase of Klebsiella pneumoniae active on bromoxynil, which forms the sub-ject of European patent application no. 373 173.
5 - ACTIVITY OF THE PURIFIED NITRILASE:
a) - Influence of the pH on the activity of the nitri-lase:
The purified nitrilase was tested at different pH values on two substrates, adiponitrile and 5-cyano-valerate, under the conditions indicated in Table 2 below.

TABLE 2: ACTIVITY OF THE PURIFIED NITRILASE ON ADIPONITRILE AND

CYANOVAlERATE AS A FUNCTION OF THE pH

SUBSTRATE ~ BUFFER SPECIFIC ACTIVITY

U/mg of protein Nature pH

Acetate 3.0 2300 Acetate 4.0 2900 Acetate 4.5 2800 Adiponitrile Acetate 5.0 2700 Phosphate 6.0 2900 Phosphate 7.0 2700 Phosphate 8.0 2800 Acetate 4.0 450 5-Cyanovalerate Acetate 5.5 180 Phosphate 7.0 30 Phosphate 8.0 COMMON CONDITIONS: [substrate] e 10 ~M; buffer 10 mM; T 25 C;

~ 1 9 ~

[nitrilase] - 12 ~g/ml for cyanovalerate and 3 ~g/ml for adiponitrile (fraction, step 6);
U (adiponitrile) ~ ~mol of cyanovalerat~ .
formed/h, U (cyanovalerate) /lmol of adipate formed/h 05 b) - Activity range of the purified nitrilase:
The activities of the purified nitrilase were measured on adiponitrile, 5-cyanovaleramide, 5-cyano-valeric acid, benzonitrile, propionitrile and acrylo-nitrile. The results are given in Table 3.

TABLE 3: RELATIVE ACTIVITY OF THE PURIFIED ~ITRILASE ON VARIOUS
NITRILES

SUBSTRATERELATIVE ACTIVITY (%) Adiponitrile100 5-Cyanovaleramide 28 5-Cyanovaleric acid 22 Acrylonitrile _ Propionitrile Benzonitrile 4 COMNON CONDITIONS: acetate buffer 10 mM, pH 4; substrate 10 mM;
volume - 3 ml; T - 25 C; reaction time - 1 or 3 h; proteins: from 5 to 30 ~g/ml EX ~ PLE 2: CLONING OF ~ ; NITRILAS~S OF comamonas testosteroni sp.

A nucleotide probe was synthesized from the NH2-terminal sequence presented in Example 1; the high - percentage of GC in the strains of comamonas described in the literature (Tamaoka et al., Int. J. Syst. Bac-teriol., 1987, 37, 52-59) dictated a choice for the third position of the codon in the case of lysines and - : , 2:~36~

in the case of valine. The probe is a 26 mer of degeneracy 128 (N replaces A, C, G or T):

M K N Y P T V K V
05 5' ATGAAGAATT ATCCNACNGT CAAGGT 3' C C G

The strategy followed consisted firs~ of all in verifying the specificity of this nucleotide probe and determining the nature of the genomic DNA fragments to be cloned. Briefly, the genomic DNA of Comamonas testosteroni sp. was digested with several restriction enzymes (SstI, SphI, BamHI, PstI etc. ) corresponding to sites usable for cloning.
After electrophoresis on agarose gel and trans-fer to a nylon membrane, the various digestions were hybridized with the probe~ The probe is found to have a sufficient specificity under the hybridization condi-tions used (hybridization buffer = 5x SSC, 5x Denhardt, 0.1% SDS, 50 mM Na~P0~, pH 6.5, 250 ~g/ml of ssDNA;
hybridization temperature 50C; washing conditions:
1 h, 6x SSC, room temperature, and 5 min, 2x SSC, 0.1 SDS, 50 C).
Under these conditions, the probe made it pos-sible to obtain important signals without ambiguity, in particular in the case of digestions with SstI, SphI, BamHI and PstI. The hybridization blots show in par-ticular the existence of a single SstI-SstI fragment of about 4 kb. To clone this fragment, the fragments of 3.5 to 4.5 kb from an SstI digestion of the genomic DNA
were purified by preparative electrophoresis on agarose - and electroelution and then ligated to plasmid pUC19 (YANISCH et al., Gene, 33 (1985~ 103), itself digested with SstI. After transformation in the strain DH5 (Clontech Laboratory, Palo Alto, California), 600 white clones on LB amp X-gal (SAMBROOK et al., Molecular ('loning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, N.Y., 1989) were subcultured individually, transferred 05 to a nylon men~rane and then analyzed by hybridization with the probe used to hybridize the Southern blot, under the same conditions of stringency. Six clones were thus identified as hybridizing very strongly with the probe. Two clones which had inserted the same fragment of about 4.1 kb in both orientations (pXL2075 [Fig. 2a] and pXL2076 [Fig. 2b]) were analyzed in greater detail (restriction mapping, partial sequencing using the probe as primer, and Southern blot). It was thus possible to show that the ~' part of the gene which hybridizes with the probe is located on an XhoI-XbaI fragment of about 150 bp orientated in the XhoI to XbaI direction. Fig. 2a and 2b show the restriction maps of these plasmids.

EXAMPLE 3: SEQUENCE OF A FRAGMENT OF 1194 bp CONTAINING
TH$ DNA CODING FOR THE POLYPEPTID~ HAVING
THE NITRITA~E A~TIVITY

The location, on the cloned insert, of the fragment of 1194 bp containing the sequenced nitrilase gene is indicated in Fig. 3. The strategy for the sequencing o~ this fragment, performed by conventional methods known to those skilled in the art, is also indicated in Pig. 3. The various sequences were all obtained by the chain termination method (sequenase kit in the presence of 7-deaza-dGTP; (35S)dATP either on - single-stranded matrices of recombinant M13 (mp 18 or 19, see YANISCH et al., op~ cit. ) carrying subfrag-ments, or directly on plasmid pXL2075). Several spe-cific primers were also synthesized for this purpose.

2.~

The DNA sequence according to the invention is shown in Fig. 4. The average G+C content of the sequence obtained is 45.7%, which is lower than the G~C
content of 61.5% described for other strains of Coma-05 monas (Tamaoka et al ., op. cit.). An analysis of thesequence obtained made it possible to characterize an open reading frame of 1064 bp, hereafter called the nit gene, coding for a polypeptide of 354 residues corres-ponding to a molecular weight of 38,725 Da. The amino acid sequence of this polypeptide is indicated in Fig. 4. This polypeptide comprises the NH2-terminal sequence used to synthesize the probe, as well as three internal sequences determined on tryptic fragments of the purified nitrilase (these internal sequences are underlined in Fig. 4).
This open reading frame thus represents tne DNA
sequence according to the invention.

EXAMPLE 4: HOMOLOGY WITH OTHER PROTEINS, IDENTIFICATION
OF HOMOLOGOUS SEQUENCE

The DNA sequence according to the invention was compared with all the sequences in the NBRF protein library; only one significant homology was found with the nitrilase of ~lebsiella ozaenae specific for the herbicide bromoxynil (Stalker et al., J. Biol. Chem., 1988, 263, 6310-6314). The two nitrilases exhibit a strict homology of 34.9% distributed over 320 amino acids. Furthermore, this protein exhibits a strict ~0 homology of 34.4%, distri~uted over 312 amino acids, with the nitrilase of Arabidopsis specific for indole-- 3-acetonitrile [Bartling et al., Eur. J. Biochem., 205, 417-424, 1~92].

2 ~

EXAMPLE 5: EXPRESSION OF THE ~ITRILA~E IN E. coli To confirm the identification of the coding frame with the purified nitrilase, the n~ gene, pre-os ceded by its own ribosome binding site, was placedunder the control of the lactose operon promoter of E.
coli in accordance with the procedure described below:
Plasmid pXL2087, described in Fig. 5, was obtained by insertion of the XhoI-NcoI fragment derived from plasmid pXL2075 between the corresponding sites of vector pMTL25 (Chambers et al., Gene, 1988, 6~, 139-149). This plasmid therefore contains the lactose operon promoter Plac, followed by the ribosome binding site and the structural nitrilase gene, as well as a gene conferring ampicillin resistance.
The expression of the nitrilase was visualized in the strain E. coli TG1 containing plasmid pXL2087.
For this purpose, the strain TG1/pXL2087 and the con-trol strain TG1/pUC19 were cultivated for 16 h at 37 C
in LB medium (Miller, J.H., 1972, Experiments in Mole-cular Genetics Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) containing 100 ~g/ml of ampi-cillin, and then diluted 100-fold in the same medium and at the same temperature. When the cultures had 25 reached an OD6l0 f between 0.5 and 1, IPTG was added at a final concentration of 1 mM. After 2 h of cul-ture, the bacteria were collected.
After sonication of the cells, the expression of the nitrilase was measured by SDS-PAGE in the crude fraction or, after centrifugation, in the residue and the supernatant. The results are presented in Fig. 7 - and show a high level of expression of the nitrilase in the extracts of cells cultivated in the presence of IPTG; however, this protein is essentially in insoluble form.

21~3~

In Fig. 7, M represents the molecular weight marker; the molecular weights are indicated in kDa.
Also, the lanes have the following meanings:

05 _ TGl + pUCl9 TGl + pUC19 TGl + TGl +

+ IPTG pXL2087 pXL2087 +

Crude fractions A D _ J

Residues B E K

Supernatants C F I L

_ _ ,. ~
Starting from plasmid pXL2087, plasmid pXL2148 was prepared by insertion of the XhoI-EcoRI fragment of plasmid pXL2087, carrying the gene coding for the nitrilase, between the SalI and EcoRI sites of pBR322 ~SUTCLIFFE, Nucleic Acid Res., 5 (1978) 2721-2730].
This plasmid pXL2148, whose restriction map is shown in Fig. 6, was also used to transform the strain E. coli TG1 by the calcium chloride method. The micro-organisms were selected on ampicillin. The strain E.
coli TG1 (pXL~048) (G4207) transformed in this way was deposited in the Collection Nationale de Cultures de Micro-organismes in Paris (Institut Pasteur, 25 rue du Docteur Roux) under no. I-1242 on 21st July 1992.
Other expression systems were used to produce the nitrilase in a recombinant microorganism.
First of all, the nit gene was expressed in E.
coli behind the tryptophan operon promoter of E. coli under the dependence of the RBS of the phage ~ CII
gene. To do this, an NdeI restriction site was created on the initiation codon of nit, and the NdeI/~h~II
fragment of 117 bp, containing the 5' part of the nit gene, was amplified by the PCR technique starting from pXL2087. An NdeI/XbaI fragment of 61 bp, obtained ~ 1 0 ~ f,r after digestion of the first fragment, was ligated to the EcoRI/NdeI fragment containing the tryptophan operon promoter of E. coli and the ribosome binding site of the bacteriophage ~ CII gene (Ptrp-RBSCII) 05 between the EcoRI and XbaI sites of pUC19 ~Yanisch et al., Gene, 33 (1985) 103) to give plasmid pXL2149. The ~oRI/XbaI fragment of pXL2149, containing the 5' part of nit behind Ptrp-RBSCII, was ligated to the XbaI/
SalI fragment of pXL2087 containing the 3' part of the nit gene between the EcoRI and SalI sites of pXL642 (Mayaux, unpublished results): pXL642 is a derivative of pXL534 (Latta et al., 1990, DNA Cell Biol., 9, 129) in which the superexpressed gene codes for a tissue inhibitor of metalloproteases and in which the HindIII
site downstream from the superexpressed gene has been replaced by the EcoRI/HindIII multisite of M13mpl8.
The final plasmid pXL2158 is therefore a deri-vative of pBR322 (Sutcliffe, Nucleic Acid Res., 5 (1978) 2721) containing a gene conferring ampicillin resistance and the nit gene under the control of Ptrp-RBSCII. The restriction map of this plasmid pXL2158 is shown in Fig. 8.
Plasmid pXL2158 was used to transform the strain E. coli TGl. The strain TGl/pXL2158 and the control strain TG1 containing vector pMTL22 were culti-vated for 16 h at 30C in M9 glucose medium tMiller, J.H., 1972, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) containing 100 ~g/ml of ampicillin and 100 ~g/ml of tryptophan. These cultures were diluted 100-fold in the same medium, but without tryptophan, and cultivated for 6 hours at the same temperature.
After sonication of the cells, the expression of the nitrilase of Comamonas testosteroni NI 1 was measured in 12.5% SDS/polyacrylamide gel in the crude 2 1 ~ ~3 ~ e ~-~

fraction or, after centrifugation, in the residue and l_he supernatant. The results are shown in Fig. 9.

05 TGl/pXL2158 TGl/pXL2158 TGl + pMTL22 Supernatant D
Residue E H

Total extract C

This gel shows that pXL2158 induces a strong expression of the nitrilase, predominantly in insoluble form.
The efficacy of the GroE chaperone was then tested (Hemmingsen et al., 1988, Nature, 333, 330) in order to assist the correct folding of the nitrilase.
For this purpose, plasmid pXL2035 was constructed in the following manner: The EcoRI/HindIII fraqment of 2.2 kb, containing the aroES and groEL genes coding for the two subunits of GroE, was extracted from plasmid pOF39 (Fayet et al., 1986, Mol. Gen. Genet., 202, 435) and introduced between the EcoRI and HindIII sites of vector pDSK519 (Keen et al., lg88, Gene, 70, 191).
Plasmid pXL2035 was introduced into the strain TG1 containing pXL2158. The resulting strain was cul-tivated under the same conditions as before, in the presence of 50 mg/l of kanamycin; the expression results are visualized in Fig. 9. It is found that the superexpression of GroE (only the GroEL subunit is visible on the gel) solubilizes the bulk of the nitri-lase expressed from pXL2158.
The same expression system was used to produce the nitrilase in Pseudomonas putid~. Thus, starting from pXL2158, the NdeI/NcoI fragment of 1256 bp and the . . :, .
, 21 B ~ t;

NcoI/BamHI fragment of 535 bp were introduced between t:he NdeI and BamHI sites of pXL1841. pXL1841 (Blanche et al., 1991, J~ Bacteriol., 173, 4637) is a derivative of pKT230 (Bagdasarian et al., 1981, Gene, 15, 237) 05 expressing a Methanobacterium ivanovii gene behind Ptrp-RBSCII.
The final plasmid pXL2169 is therefore a deri-vative of pKT230 containing a gene conferring kanamycin resistance and the nit gene under the control of Ptrp-RBSCII ( cf. Fig. 10). This plasmid was introduced intothe strain Pseudomonas putida G2081. G2081 is a deri-vative of Pseudomonas putida KT2440 (~agdasarian and Timmis, 1981, in Hofschneid and Goebel, Topics in Microbiology and Immunology, 47, Springer Verlag, Berlin) rendered resistant to nalidixic acid and rifampicin. Vector pDSK519 (Keen et al., 198~, Gene, 70, 191) was used as the control plasmid. G2081 (pXL2169) and the strain G2081 (pDSK519) were cultiva-ted overnight at 30C in LB medium containing 20 mg/l of kanamycin. These precultures were diluted 100-fold in the same medium. The cultures were then continued for 7 h 30 min at the same temperature. After sonica-tion of the cells, the expression of the nitrilase of Comamonas testosteroni NI 1 was measured in 10% SDS/
polyacrylamide gel in the crude fraction or, after centrifugation, in the residue and the supernatant.
~he results are presented in Fig. 11. Only the crude extract of the strain G2081 (pDSK519) was deposited (well D); for the strain G2081 (pXL2169), the total extract, the sonication residue and the sonication supernatant were deposited in wells C, B and A respec-tively. This experiment shows that the strain of Pseudomonas putida expresses large amounts of nitrilase in soluble form.

2 ~

EXAMPLE 6: ASSAY OF THE NITRIIl~SE ACTIVITY OF RECO~-The Examples which follow illustrate the nitri-05 lase activity of the recombinant strains E. coli TGland Pseudomonas putida G2081.
The different plasmids integrated into these strains are as follows:

pXL2087 Recombinant plasmid which carries the Coma-monas NI l nitrilase gene under the control of the promoter P,~c.
pXL2158 Recombinant plasmid which carries the Coma-monas NI 1 nitrilase gene under the control of the tryptophan promoter.
pXL2035 Recombinant plasmid which carries the genes coding for GroE and S.
pXL2169 Broad host range plasmid with an insertion, carrying the Comamonas NI 1 nitrilase gene under the control of Ptrp-pDSK519 Control plasmid (cf. page 24 line 18).

The activities of these strains, induced or non-induced, are measured on adiponitrile and 5-cyano-valerate at different pH values and are compared with the negative control strains: E. coli TG1, E. coli TG1 (pXL2035) and Pseudomonas putida G2081.

1 - PREPARATION OF THE CELLS:
The cultures are carried out under the condi-tions described in Table 4. During the exponential growth phase, one of the two cultures of the recombi-nant strain is induced with 1 mM IPTG: after 2 h at 37 C, this culture is treated.

~ 3 ~ ~l $-~

TABLE 4: CULTURE OF THE STRAINS

MICROORGANISM MEDIUM D66~nm DW ~g/l ) 1 - E. col i TGl a 3.1 0.90 2 - E . col i (pXL2087) b 3.2 0.90 __ 3 - E . col i (pXL2087) c 2.5 0.90 4 - E. coli (pXL2035) d 2.1 0.90 5 - E . col i (pXL2148)(l) b 3.1 0.80 6 - E . col i (pXL2035, 2158)(~) e 4.2 1.30 7 - P. putida (pXL1289) d 2.1 0.98 8 - P. putida (pXL2169) 2.3 0.98 ABBREVIATIONS: 8: LB medium; b: LB medium + 100 ~g/ml of Amp;
c: medium b + addition of 1 mM IPTG to OD66onm =
1; d: LB medium + 50 mg/l of kanamycin; e: M9 medium + 100 mg/l of ampicillin + 50 mg/l of kanamycin; DW: dry weight COMMON CONDITIONS: 1 to 3: Inoculation in a ratio of 1/100 with a 16-hour-old preculture; culture time 5.75 h;

4 to 8: Inoculation in a ratio of 1/100 with a 17-hour-old preculture at 37 C with the addition of tryptophan; culture time in 15 1 fermenter: 23 h for E. coli and 7.5 h for P. putida; T 30 C
2 - SPECIFIC ACTIVITY MEAS ~ EMENTS:
The conditions of the specific activity meas-urements and the results are collated in Table 5.

'.
, .

2~3~

l'ABLE 5: DETER~INATION OF THE ACTIVITIES OF THE CONTROL STRAINS
FOR THE RECOMBINANT STRAINS

MICROORGANISM OPERATING CONDITIONS
Activity 05Nature IPTG State Substrate [DW] Volume pH U
(g/l) (ml) mg of DW
_ 1 - E. coli W CVA15.5 1 5.2 O
TGl W CVA15.5 1 7 0 O
W AdN15.5 1 5.2 O
_ _ W AdN15.5 1 7 0 O
2 - E. coli + W CVA1.4 1 4.0 28 TGl ~ W CVA1.4 1 5.2 27 pXL2087 + W CVA1.4 2 7.0 8 + S CVA1.4 1 5.2 25 + S CVA1.4 2 7.0 + W AdN0.3 1 4.2 159(a) + W AdN1.4 1 4.3 38 + W AdN1.4 2 6.2 18 + W AdN1.4 2 7.0 11 + S AdN1.4 2 6.2 17 + S AdN1.4 2 7.0 10 . _ 3 - E . col i W CVA1.2 2 4.0 10 20 TGl W CVA1.2 2 5.2 14 pXL2087 W CVA1.2 2 7.0 3.4 _ S CVA1.0 1 5.2 13 _ S CVA1.0 1 7.0 3.2 _ W AdN0.3 1 4.2 75(a) _ W AdN1.2 2 4.3 16 W AdN1.2 2 6.2 11 _ W AdN1.2 2 7.0 3.4 _ S AdN1.0 1 6.2 3 _ S AdN1.0 1 7.0 4 - E. coli _ W AdN0.06 1 7.0 Ua - O
TGl Ub - not pXL2035 _ _ determined 5 - E. coli _ W AdN0.24 1 7.0Ua - 270 TGl Ub - -pXL2168 CVA1.25 1 7.0 Ua - -Ub - 8.3 _ _ . __ _ .
, - . '-: ' ' ~- .'' ,. ' . .

2 1 ~ r ~ 2~3 _ _ _ _ MICROORGANISM OPERATING CONDITIONS
_- Activity Nature IPTG State Substrate [DW] Volume pH U
(g/l) (ml) ~ mg of DW

05 TGl W AdN 0.06 1 7.0 Ua = 1500 pXL2035, . _ _ 2158 CVA O.2 1 7.0 UUb = 70 7 - P. putida U AdN 0.3 1 7.0 Ua - 0 pDSK519 Ub ~ -_ _ 8 - P. putida W CVA 0.25 1 7.0 Ua ~ 130 G2081 Ub = -pXL2169 _ _ 15 CO~MON CONDITIONS: [substrate] - 50 mM; T 25 C; bufer 50 mM; kinetics over ~0 min f~r 1 to 3 and over 120 min for 4 to 8 ABBREVIATIONS: W: whole cells; S: sonicated cells; U: 1 ~mol of adipate produced/h, except (a) 1 ~mol of 5-cyano-valerate produ~ed/h; Ua - ~mol of cyanovalerate pro-duced/h/mg of cells DW; Ub - ~mol of adipate produced/
h/mg of dry cells; AdN: adiponitrile; CVA: 5-cyano-valerate; D~: dry weight E~ PLE 7: SYN~EESIS OF A~D~ONI ~ ~ IPATE BY IqIE ~ TCH
~YDROLYSIS OF ~ IPONIIqRILE WI ~ E. coli (pXL2087~ IN SUSPENSION

120 ~l or 1068 ~mol of adiponitrile were added at 25 C and with magnetic stirring, at the reaction times 0, 1, 2, 3, 5, 6 and 7 h, to an initial volume of 5 ml of 50 mM phosphate buffer, pH 7, containing the strain E. coli ( pXL2087) at an initial concentration of 21 g/l. The reaction was monitored analytically by taking 100 ~l samples of the reaction volume every hour. The hydrolysis was found to proceed without a notable loss of kinetics.
The mean activities calculated over 30 min after addition of the adiponitrile are collated in Table 6 below.

TABLE 6: MEA~ ACTIVITIES OF THE E. coli (pXL.2087) C~LLS DURI~G THE
HYDROLYSIS OF ADIPONITRILE

REACTION TIME SPECIFIC ACTIVITY
(h) ~mol of adipate h x m~; of cells 0.5 16 2.5 15 5.5 11 6.5 11 7.7 15 EXAMPLE 8: SYNTHESIS OF AMMONIUM ADIPATE BY THE HYDRO-LYSIS OF ADIPONITRILE IN A FIXED BED
REACTOR WITH E. coli (pXL2087) IMMOBILIZED
ON RESIN
The E. coli (pXL2087) cells were first fixed by the technique described in US patent 4 732 851.
The resulting biocatalyst was then used in a fixed bed column for the hydrolysis of adiponitrile to Z5 ammonium adipate.

1 -- FIXING OF E. coli (pXL2087) TO RESIN:
The cells were fixed according to the following protocol:
- 1 g (wet weight) of E. coli (pXL2087) with a solids content of 22%
- 1 g of POLYCUP polyazetidine - 1 g of DUOLITE A 171 resin The gram of cells was suspended in the poly-35 azetidine solution. After homogenization, the resin - ~ ~
, " , ' .
' : . ' . ~

.: :

2 1 ~ 3 ~i ~ r;3 was poured into the cell suspension. The whole was stirred with a spatula and then left to dry for 18 h, c,pen to the air, under a hood. 4 ml or 1.3 g of bio-c:atalyst were thus collected.
05 The activities of the immobilized and free cells were determined at 25C and pH 7 on a 50 mM
solution of adiponitrile. They are respectively 30 and llO ymol of 2-cyanovalerate/h/mg of cells DW, from which a fixing yield of 26% is deduced.
2 - HYDROLYSIS OF ADIPONITRILE IN A FIXED BED REACTOR:
The half-life is determined in a continuously fed fixed bed reactor under the conditions indicated below:
T 28 C; catalyst 0.5 g or 2 ml or 85 mg of cells (dry weight); [adiponitrile] 50 mM; phosphate buffer 50 mM, pH 7; flow rate 3.7 + 0.1 ml/h; column: diameter 1 cm, height 3 cm.
The initial activity of the cells was 1.5 ~mol of adipate/h/mg of cells tdry weight). 66% of the initial activity is preserved after 32 days or 770 h.

Claims (21)

1. A DNA sequence coding for a polypeptide having a nitrilase activity and capable of hydrolyzing nit-riles to carboxylates, which is selected from:
- the DNA sequence coding for a polypeptide having a nitrilase activity, as shown in Fig. 4, - an analog of this sequence resulting from the degene-racy of the genetic code, and - a DNA sequence hybridizing with one of these sequen-ces or a fragment thereof and coding for a poly-peptide having a nitrilase activity.

2. A recombinant DNA sequence according to claim 1 which contains the following nucleotide sequence:

3. A polypeptide resulting from the expression of a DNA sequence according to one of claims 1 or 2 and possessing a nitrilase activity.

4. A polypeptide according to claim 3 which com-prises the following sequence:

5. A microorganism containing the DNA sequence according to claims 1 or 2.

6. A microorganism containing the DNA sequence according to claims 1 or 2 on a plasmid containing a selection means.

7. A microorganism consisting of the strain E.
coli TG1 containing plasmid pXL2148, said strain having the reference G4207 and being deposited in the Collec-tion Nationale de Cultures de Micro-organismes under no. I-1242.

8. A microorganism containing an expression cas-sette consisting of the DNA sequence according to claim 1 or 2 under the dependence of signals ensuring the expression of this sequence in the host microorganism.

9. A microorganism according to claim 8 which com-prises, upstream from the DNA sequence, a ribosome binding site and a promoter sequence homologous or heterologous with the polypeptide produced.

10. A microorganism according to claim 9 wherein the promoter can be the tryptophan operon promoter Ptrp of E. coli, the lactose operon promoter Plac of E.
coli, the phage lambda right promoter PR, the phage lambda left promoter PL or strong promoters of Coryne-bacterium, Comamonas or Pseudomonas.

11. A microorganism according to claim 9 wherein the ribosome binding site can be the one derived from the phage lambda CII gene or those derived from genes of E. coli, Comamonas, Pseudomonas or Corynebacterium.

12. A microorganism according to claims 8 to 11 wherein the expression cassette is carried by a plasmid containing a selection means.

13. A microorganism according to claim 12 wherein the selection means is a marker conferring antibiotic resistance.

14. A microorganism according to claims 5 to 13 which is selected from the strains of E. coli, Coma-monas, Corynebacterium, Brevibacterium, Rhodococcus and Pseudomonas.

15. A microorganism according to any one of claims 5 to 14 - which contains at least one protein agent for assis-ting the folding of the polypeptides which the microorganism synthesizes, and in particular the polypeptides according to claim 3 or claim 4, and/or the genes coding for such an agent, - and wherein this agent is present in a greater amount than that corresponding to the base level of the microorganism in question.

16. A microorganism according to claim 15 wherein the agent is the GroE chaperone of E. coli or its homolog of eucaryotic or procaryotic origin.

17. A microorganism according to claim 15 or claim 16 wherein the genes coding for the agent are carried by the chromosome or by an extrachromosomal element (plasmid, phage) and wherein said genes are amplified.

18. A microorganism according to claim 17 wherein the genes coding for the agent are under the dependence of expression systems homologous or heterologous with said microorganism.

19. An enzymatic method of converting nitriles, which consists in bringing the nitriles into contact with a polypeptide having a nitrilase activity, accor ding to either one of claims 3 or 4, or a host micro-organism according to any one of claims 5 to 19.

20. A method according to claim 19 wherein the nitrile is a dinitrile of the formula NC-R-CN, in which is an alkylene group having from 1 to 10 carbon atoms.

21. A method according to one of claims 19 and 20 wherein the nitrile is adiponitrile.