AU669951B2 - 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

Description

POLYPEPTIDES POSSESSING A NITRILASE ACTIVITY, DNA SEQUENCE CODING FOR SAID PCLYPEPTIDES, EXPRESSION CASSETTES AND HOST MICROORGANISMS ENABLING THEM TO BE OBTAINED, AND METHOD OF CONVERTING NITRILES TO CARBOXY-LATES BY MEANS OF SAID

POLYPEPTIDES

The present invention relates to novel polypeptides having a nitrilase activity and to the genetic engineering tools for producing them, namely a DNA sequence, the expression cassettes carrying this recombinant DNA sequence, and the recombinant microorganisms (host 10 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.

15 A first particular application of the method of the invention is the enzymatic synthesis of ammonium adipate or ammonium 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:

SNC-R-C'-

NNi A NC-R-CONC2 NH IA O 2 NH nitrile hydratase Ni nitrilase A amidase R (CH2)n, 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 10 hydrolysis, there may be mentioned in particular the strains belonging to the genus Fusarium, which degrade succinonitrile and adiponitrile, although the reaction products are not indicated [Goldlust et al., Biotechnol. and Appl.

Biochem., 1989, 11, 581]; the strains belonging to the genus Pseudomonas, which degrade adiponitrile [Yanase et al., Agric. Biol. Chem., 1982, 46, 15 2925]; and the strains belonging to the genus Rhodococcus, in particular Rhodococcus rhodochrous NCIB 11 216, which hydrolyzes adiponitrile to adipic acid [Bengis-Garber et al., Appl. Microbiol. Biotechnol., 1989, 32, li], and also Rhodococcus rhodochrous K22, whose nitrilase permits the hydrolysis of adiponitrile and glutaronitrile [Yamada et al., J. Bacteriol., 1990, 172 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 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 particularly 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.

A second particular application of the invention is the enzymatic synthesis of ammonium methioninate:

CH

3 S (CH 2 2 CH COO-, NH 4 from methononitrile:H from methiononitrile:

I

CH

3 S (CHI,) CH CN

NH,

Methionine is one of the only aminoacids which is currently produced on a large industrial scale. It constitutes a sulphurized nutriment which is useful, as a growth factor, in feeding animals.

One of its main synthesis chemical routes is the hydrolysis of methionitrile to ammonium methioninate, using a strong acid. One major 10 drawback of this chemical hydrolysis is that it generates a very high coproduction of salts which are difficult to recycle and which are detrimental Sto the environment.

The Applicant takes credit for having demonstrated an advantageous enzymatic alternative to the chemical hydrolysis, said alternative solving the problem of detrimental coproduction of salts.

The present invention the; ore relates to novel polypeptides having a nitrilase activity which have been isolated from a strain of Comamonas testosteroni. More precisely, these polypeptides are prepared by extraction and purification from cultures of natural or recombinant microorganisms, the purification being effected by a series of steps consisting in preparing an enzymatic extract from the cell culture, precipitating this extract with ammonium sulfate and purifying it by different steps involving chromatography and gel filtration. These steps, which employ techniques well known to those skilled the art, are described in detail in the illustrative Examples below.

In the present description, "nitrilase activity" denotes the direct conversion of a nitrile to an ammonium carboxylate, the corresponding amide not beinga substrate for the enzyme.

The invention further relates to a DNA sequence coding for a polypeptide having a nitrilase activity. The DNA sequence coding for a polypeptide of the invention can be selected from: the DNA sequence coding for a polypeptide having a nitrilase activity, as shown in Fig. 4 and designated by SEQ ID NO 4 in the enclosed sequence listing, an analog of this sequence resulting from the degeneracy 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 polypeptide, with the aid of nucleotide probes produced from the purified polypeptide.

The inrention further relates to the expression cassettes which carry the above-defined recombinant DNA sequence together with the signals ensuring its expression. These expression cassettes can either be integrated in the genome of the host or located on an expression vector 10 such as a piasmid containing a selection means.

In particular, these expression cassettes contain transcription and translation initiation regions which contain a ribosome binding site and a promoter sequence. These regions may be homologous or heterologous with the microorganism which naturally produces the polypeptide.

The choice of these regions depends especially on the host used. In particular, when the host microorganisms are procaryotic, the heterologous promoter can be selected from strong bacterial promoters such as the tryptophan operon promoter Ptrp of E. coil, the lactose operon promoter Plac of E. coli, the phage lambda right promoter PR, the phage lambda left promoter PL, the strong promoters of Pseudomonas and S: Comamonas and the strong promoters of Corynebacteria.

More particularly, in the case of the phage lambda right promoter, the thermosensitive form PRClts may be preferred. In the case of eucaryotic microorganisms such as yeasts, the promoters can originate from glycolytic yeast genes such as the genes coding for phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GPD), lactase (LAC4) and enolase (ENO).

As far as the ribosome binding sites are concerned, the one derived from the lambda Cll gene, as well as those derived from homologous genes of Comamonas 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 translation and functional transcription of the envisaged host 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 recombinant host. The preferred markers are dominant markers, i.e. those conferring a resistance to antibiotics such as ampicillin or streptumycin, 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 Rhr dococcus, may be mentioned in particular among the host microorganisms used.

The invention further relates to the microorganisms containing the recombinant DNA sequence according to the invention, for example on a plasmid containing a selection means.

10 A recombinant microorganism containing said DNA sequence on a plasmid structure was deposited in the Collection Nationale de Cultures de Micro-organismes (Institut Pasteur, 25 rue du Docteur Roux, Paris) under no. 1-1242 on 21st July 1992. This microorganism is the strain E. coli TG1, which contains plasmid pXL2148; this microorganism is also identified by the Applicant using the reference G4207.

The invention further relates to the microorganisms capable of converting nitriles to carboxylates, 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, or else aliphatic sulphurized mononitriles such as methiononitriles.

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 discovered means for favoring the above-mentioned stabilization.

Thus any microorganism according to the invention preferably contains: at least one protein agent for assisting the folding of the polypeptides which the microorganism synthesizes, and in particular the nitrilase referred to in the present disclosure, and/or the genes coding for such an agent, this agent being present in a greater amount than that corresponding to the base level of the microorganism in question.

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 chaperone of E. coli or its homolog of eucaryotic or procaryotic origin.

The GroE chaperone of E. coli is normally present in the wildtype strains.

The genes coding for the agent are carried by the chromosome or by an extrachromosomal element (plasmid, phage). They are preferably S 10 amplified by any known and appropriate means so as to favor the synthesis of the agent in the microorganism.

The genes coding for the agent are under the dependence of expression systems homologous or heterologous 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 S. 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 particularly for the conversion to carboxylates, on the one hand, 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, and on the other hand, of mononitriles, preferably aliphatic sulphurized mononitriles.

The method of the invention is particularly appropriate for the enzymatic synthesis of ammonium adipate from adiponitrile and ammonium methionate from methiononitrile.

The production of adipate determines a field of application of the invention in the synthesis of polyamides (NYLON@), whereas the production of methionate particularly belongs to the field of application of animal feed.

The Examples which follow afford an illustration of the characteristics and advantages of the present invention without however limiting its scope.

DESCRIPTION OF THE FIGURES Fig. 1 shows the yield of the hydrolysis of adiponitrile to cyanovalerate (curve a) and to ammonium adipate (curve b) as a function of the reaction time in hours for the strain Comamonas testosteronisp.

10 Fig. 2a and 2b show the restriction maps of plasmids pXL2075 and pXL2076.

Fig. 3 shows the restriction map of the Sstl-Sstl 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 Xbal-Sstl fragment containing the DNA sequence according to the invention is also shown in this Figure.

Fig. 4 shows the DNA sequence SEQ ID NO 4 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 TG1/pXL2027. Each lane corresponds to an amount of protein equivalent to 60 pl 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, indicating the expression of the DNA sequence according to the invention in the strains TG1/pXL2158 and TG1/pXL2158 pXL2035 (GroE).

Fig. 10 shows the restriction map of plasmid pXL2169.

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.

The abbreviations used in the remainder of the description have the following meanings:

SSC:

SDS:

FPLC:

SDS-PAGE:

IPTG:

10 EXAMPLE 1: buffer commonly used for hybridizations, containing sodium citrate and NaCI (20x SSC 3 M NaCI, 0.3 M sodium citrate, pH 7) sodium dodecylsulfate fast protein liquid chromatography gel electrophoresis based on sodium dodecylsulfate/ polyacrylamide isopropyl P-D-thiogalactopyranoside

EXAMPLES

PURIFICATION OF THE NITRILASE OF COMAMONAS

TESTOSTERONISP.

S S S S. S a o 1 PREPARATION OF THE CELLS: A strain of Comamonas testosteroni sp. was cultivated in a shake flask, at 28*C, for 15 h 30 min, in medium A having the following composition: Medium A -Glucose 5 g/l

(NH

4 2 S0 4 1 g/l Na 2

HPO

4 5.24 g/l

KHPO

4 2.77 g/l Yeast extract 5 g/I Casamino acids 1 g/l This preculture was used to inoculate a 20 I fermenter containing 15 I of medium A. The pH, temperature, air flow rate and shaking speed were set to 6.6, 28*C, 300 I/h and 350 rpm respectively. After 24 h, 84 g of wet cells were harvested. This corresponds to a content by dry weight of cells of 0.9 g/l and to an optical density at 660 nm (OD660nm) of 2.

2- DETERMINATION OF THE ENZYMATIC ACTIVITY ON ADIPONITRILE: 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 buffer, pH 7. The reaction was carried out at with shaking, and the kinetics were followed by sampling. 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 The respective rates of formation of cyanovalerate and adipate were greater than 0.45 and equal to 0.15 U/mg of dry weight of cells (1 U is equal to 1 pmol of product 0 formed per minute).

3- PURIFICATION: All the purification steps were carried out in 50 mM Tris/HCI 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 25'C in 10 mM phosphate buffer in the presence of 10 mM adiponitrile. The protein concentration 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.

Step 1: Crude extract 57 g of wet cells were taken up in 85 ml of buffer and treated with ultrasound for 30 min (VIBRACELL sonicator from Bioblock: probe 13 mm; power 7; 40% of the cycle active). The OD660nm thus dropped from 97 to 60. After centrifugation at a maximum of 48,000 g for 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 centrifuged under the same conditions and the precipitate was recovered and then dialyzed 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 KCI in the buffer 180 ml of buffer to which 0.2 M KCI had been added 270 ml of a gradient of 0.2 to 0.4 M KCI in the buffer 180 ml of buffer to which 0.4 M KCI had been added 200 ml of buffer to which 1 M KCI had been added The fraction having the nitrilase activity was eluted in a volume of 129 ml during the 0.2 M KCI stage.

The following steps are carried out on the FPLC system (Pharmacia).

10 Step 3: Get 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 at 80% saturation, followed by dialysis against the buffer. The fraction concentrated in this way (12 ml) was loaded in 2 15 batches on to the column of gel (16 x 600 mm) equilibrated with the buffer to which 0.1 M KCI 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 in a total volume of 36 ml. These fractions correspond to a molecular 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 pM CaCl 2 had been added. The column was percolated at a rate of 0.5 ml/min with the following in succession: 5 ml of equilibration buffer 15 ml of a gradient of 0 to 350 mM potassium phosphate in the equilibration 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 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 buffer containing ammonium sulfate at saturation. The column was percolated with: 6 ml of equilibration buffer 12 ml of a decreasing ammonium sulfate gradient of 15% to 0% ammonium sulfate saturation in the buffer 23 ml of buffer Some of the fraction having the nitrilase activity were eluted during the washing of the column with the equilibration buffer. These active fractions were reinjected under the same conditions. This operation S 10 was performed twice. The active fractions eluted after the gradient were pooled (volume 51 ml).

S. Step 6: Gel filtration (FPLC-Superdex 200) The 51 ml were concentrated to 3 ml by ultra-filtration on a membrane (DIAFLO PM30, AMICON). These 3 ml were loaded on to the column 15 (16 x 600 mm) equilibrated with the buffer to which 0.1 M KCI 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.

Step 7: Ion exchange column (FPLC Mono Q HR The protein solution was thawed and loaded on to the column equilibrated with the buffer containing 0.1 M KCI, at a rate of 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 KCI had been added 4.5 ml at 1 ml/min of buffer to which 0.1 M KCI had been added 15 ml at 1 ml/min of a gradient of 0.1 to 0.4 M KCI in the buffer 10 ml of buffer to whic' 0.4 M KCI had been added The active fractions were eluted between 0.15 and 0.3 M KCI. 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".

The data from each of the above purification steps are collated in Table 1 below: 0 12 TABLE 1: PURIFICATION OF THE NITRILASE OF Comamonas testosteroni sz.

PURIFICATION STEP Vol. Proteine ACTIVITY YIELD ml mg _PF Total Spedific Protcin Activity 1./m 0 Crude Extract 61 920 62,000 68 100 100 1 2 Q Sepharose FF 130 245 47.000 190 27 76 2.8 3 Gel Filtration 36 27 56.000 2,100 2.9 90 4 Hydroxyapatite Column 3 12 49,000 4.100 1.3 79 Phenvl Superose 51 11 11.000 1,000 1.1 18 6 Gel Filtration 9 2.7 6.300 2,300 0.3 10 34 7 Mono Q HR 5/5 2.9 1 1.200 1,200 0.01 2 18 ABBREVIATIONS: PF purification factor; U 1 pmol/h 4- DETERMINATION OF THE N-TERMINAL SEQUENCE OF THE

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, designated by SEQ ID NO 1 in the enclosed sequence listing, is as follows: Met Lys Asn Tyr Pro Thr Val Lys Val Ala Ala Val Gin Ala Ala Val Phe 5 10 Met Asn Leu Glu Ala Thr Val Asp Lys Thr 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 subject of European patent application no. 373 173.

U O

D

13 ACTIVITY OF THE PURIFIED NITRILASE: a) Influence of the pH on the activity of the nitrilase: The purified nitrilase was tested at different pH values on two substrates, adiponitrile and 5-cyanovalerate, 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 2,300 Acetate 4.0 2,900 Acetate 4,5 2,800 Adiponitrile Acetate 5.0 2,700 Phosphate 6.0 2,900 Phosphate 7.0 2,700 Phosphate 8.0 2,800 Acetate 4.0 450 Acetate 5.5 180 Phosphate 7.0 __Phosphate 8.0 6 COMMON CONDITIONS: [substrate] 10 mM; buffer 10 mM; T [nitrilase] 12 pg/ml for cyanovalerate and 3 pg/ml for adiponitrile (fraction, step U (adiponitrile) pmol of cyanovalerate formed/h, U (cyanovalerate) pmol of adipate formed/h.

b) Activity range of the purified nitrilase: The activities of the purified nitrilase were measured on adiponitrile, cyanovaleramide, 5-cyanovaleric acid, benzonitrile, propionitrile and acrylonitrile. The results are given in Table 3.

14 TABLE 3: RELATIVE ACTIVITY OF THE PURIFIED NITRILASE ON VARIOUS NITRILES SUBSTRATE RELATIVE ACTIVTY AdiDonitrile 100 28 acid 22 Acrvlonitrile 23 Prooionitrile 6 Benzonitrile 4 5 COMMON CONDITIONS: acetate buffer 10 mM, pH 4; substrate 10 mM; volume 3 ml; T 25'C; reaction time 1 or 3 h; proteins: from .:to 30 pg/ml.

EXAMPLE 2: CLONING OF THE NITRILASE OF Comamonas te ,osteroni sp.

A nucleotide probe was synthesized from the NH 2 -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.

Bacteriol., 1987, 37, 52-59) dictated a choice for the third position of the 15 codon in the case of lysines and in the case of valine. The probe is a 26 mer of degeneracy 128 (in the sequence of nucleotidic bases N replaces A, C, G or T): M K N Y P T V K V Amino acids 5' ATGAAGAATT ATCCNACNGT CAAGGT 3' Nucleotidic Bases (SEQ ID NO 3 C C G Variants The MKNYPTVKV amino acid sequence given above corresponds to the SEQ ID NO 2: in the enclosed sequence listing.

The strategy followed consisted first 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 (Sstl, Sphl, BamHI, Pstl etc.) corresponding to sites usable for cloning.

After electrophoresis on agarose gel and transfer to a nylon membrane, the various digestions were hybridized with the probe. The probe is found to have a sufficient specificity under the hybridization conditions used (hybridization buffer 5x SSC, 5x Denhardt, 0.1% SDS, 50 mM Na 3

PO

4 pH 6.5, 250 pg/ml of ssDNA; hybridization temperature 50'C; washing conditions: 1 h, 6x SSC, room temperature, and 5 min, 2x SSC, 0.1% SDS, Under these conditions, the probe made it possible to obtain important signals without ambiguity, in particular in the case of digestions with Sstl, Sphl, BamHI and Pstl. The hybridization blots show in particular the existence of a single Sstl-SstI fragment of about 4 kb. To clone this fragment, the fragments of 3.5 to 4.5 kb from an Sstl digestion of the genomic DNA were purified by preparative electrophoresis on agarose and electroelution and then ligated to plasmid pUC19 (YANISCH et al., Gene, 15 33 (1985) 103), itself digested with Sstl. After transformation in the strain (Clontech Laboratory, Palo Alto, California), 600 white clones on LB amp X-gal (SAMBROOK et al., Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, 1989) were subcultured individually, transferred to a nylon 20 membrane 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 25 greater detail (restriction mapping, partial sequencing using the probe as primer, and Southern blot). It was thus possible to show that the 5' part of the gene which hybridizes with the probe is located on an Xhol-Xbal fragment of about 150 bp orientated in the Xhol to Xbal direction. Fig. 2a and 2b show the restriction maps of these plasmids.

EXAMPLE3: SEQUENCE OF A FRAGMENT OF 1194 bp CONTAINING THE DNA CODING FOR THE POLYPEPTIDE HAVING THE NITRILASE ACTIVITY 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 of this fragment, performed by conventional methods known to those skilled in the art, is also indicated in Fig. 3. The various sequences were all obtained by the chain termination method (sequenase kit in the presence of 7-deaza-dGTP; 3 5S)dATP either on single-stranded matrices of recombinant M13 (mp 18 or 19, see YANISCH et al., op. cit.) carrying subfragments, or directly on plasmid pXL2075). Several specific primers were also synthesized for this purpose.

The DNA sequence SEQ ID NO 4 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 Comamonas (Tamaoka et al., op. cit.). An analysis of the sequence 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 corresponding to a molecular weight of 38,725 Da. The amino acid 15 sequence of this polypeptide is indicated in SEQ ID NO 4 in SEQ ID NO and in Fig. 4. This polypeptide comprises the NH 2 -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).

20 This open reading frame thus represents the 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 Klebsiella 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 homology of 34.4%, distributed over 312 amino acids, with the nitrilase of Arabidopsis specific for indole-3-acetonitrile [Bartling et al., Eur. J. Biochem., 205, 417-424, 1992].

17 EXAMPLE 5: EXPRESSION OF THE NITRILASE IN E coli To confirm the identification of the coding frame with the purified nitrilase, the nit gene, preceded by its own ribosome binding site, was placed under 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 Xhol-Ncol fragment derived from plasmid pXL2075 between the corresponding sites of vector pMTL25 (Chambers et al., Gene, 1988, 68, 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 control strain TG1/pUC19 were cultivated for 16 h at 37*C in LB medium (Miller, 1972, Experiments in Molecular Genetics Cold Spring Harbor Laboratory, Cold Spring Harbor, containing 100 pg/ml of ampicillin, and then diluted 100-fold in the same medium and at the same temperature. When the cultures had reached an OD610 of between 0.5 and 1, IPTG was added at a final concentration of 1 mM. After 2 h of culture, 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 S. 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 25 presence of IPTG; however, this protein is essentially in insoluble form.

In Fig. 7, M represents the molecular weight marker; the molecular weights are indicated in kDa. Also, the lanes have the following meanings: TG1 pUC19 TG1 pUC19 TG pxL2087 TG1 pxL2087 IPTG IPTG Crude Fractions A D G J Residues B E H K Supernatants C F I L Starting from plasmid pXL2087, plasmid pXL2148 was prepared by insertion of the Xhol-EcoRI fragment of plasmid pXL2087, carrying the gene coding for the nitrilase, between the Sal I 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 microorganisms were selected on ampicillin. The strain E coli TG1 (pXL2148) (G4207) transformed in this way was deposited in the Collection Nationale de Cultures de Micro-organismes in Paris (Institut Pasteur, rue du Docteur Roux) under no. 1-1242 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 X 15 CII gene. To do this, an Wdel restriction site was created on the initiation codon of nit, and the Ndel/Ahall fragment of 117 bp, containing the 5' part of the nit gene, was amplified by the PCR technique starting from pXL2087.

An Ndel/Xbal fragment of 61 bp, obtained after digestion of the first fragment, was ligated to the EcoRI/Ndel fragment containing the tryptophan 20 operon promoter of E coli and the ribosome binding site of the bacteriophage X CII gene (Ptrp-RBSCII) between the EcoRI and Xbal sites of pUC19 (Yanisch et al., Gene, 33 (1985) 103) to give plasmid pXL2149.

The EcoRI/Xbal fragment of pXL2149, containing the 5' part of nit behind Ptrp-RUSCII, was ligated to the Xbal/Sall fragment of pXL2087 containing 25 the 3' part of the nit gene between the EcoRI and Sail 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 Hindlll site downstream from the superexpressed gene has been replaced by the EcoRI/Hindll multisite of M13mp18.

The final plasmid pXL2158 is therefore a derivative 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 TG1. The strain STG1/pXL2158 and the control strain TG1 containing vector pMTL22 were cultivated for 16 h at 30*C in M9 glucose medium (Miller, 1972, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, containing 100 pg/ml of ampicillin and 100 pg/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-PAGE in the crude fraction or, after centrifugation, in the residue and in the supernatant. The results are shown in Fig. 9.

TGi[pXL2158± pXL2035 TG1/pXL215 8 TG1 pMTL22 Supernatant A D G Residue B E H Total Extract C F 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/Hindlll fragment of 2.2 kb, containing the groES 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 Hindlll sites of vector pDSK519 (Keen et al., 1988, Gene, 70, 191).

Plasmid pXL2035 was introduced into the strain TG1 containing pXL2i58.

The resulting strain was cultivated under the same conditions as before, in the presence of 50 mg/I 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 nitrilase expressed from pXL2158.

The same expression system was used to produce the nitrilase in Pseudomonas putida. Thus, starting from pXL2158, the Ndel/Ncol fragment of 1256 bp and the Ncol/BamHI fragment of 535 bp were introduced between the Ndel 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) expressing a Methanobacterium ivanovii gene behind Ptrp-RBSCII.

The final plasmid pXL2169 is therefore a derivative of pKT230 containing a gene conferring kanamycin resistance and the nit gene under the control of Ptrp-RBSCII (see Fig. 10). This plasmid was introduced into the strain Pseudomonas putida G2081. G2081 is a derivative of Pseudomonas putida KT2440 (Bagdasarian and Timmis, 1981, in Hofschneid and Goebel, Topics in Microbiology and Immunology, 47, Springer Verlag, Berlin) rendered resistant to nalidixiu acid and rifampicin. Vector pDSK519 (Keen et al., 15 1988, Gene, 70, 191) was used as the control plasmid. G2081 (pXL2169) and the strain G2081 (pDSK519) were cultivated overnight at 30'C in LB medium containing 20 mg/I of kanamycin. These precultures were diluted 100-fold in the same medium. The cultures were then continued for 7 h min at the same temperature.

20 After sonication of the cells, the expression of the nitrilase of Comamonas testosteroni NI 1 was measured in 10% SDS-PAGE in the crude fraction or, after centrifugation, in the residue and in the supernatant. The results are presented in Fig. 11. Only the crude extract of the strain G2081 (pDSK519) was deposited (well For the strain G2081 (pXL2169), the 25 total extract, the sonication residue and the sonication supernatant were deposited in wells C, B and A respectively. This experiment shows that the strain of Pseudomonas putida expresses large amounts of nitrilase in soluble form.

EXAMPLE 6: ASSAY OF THE NITRILASE ACTIVITY OF RECOMBINANT

STRAINS

The Examples which follow illustrate the nitrilase activity of the recombinant strains E. coli TG1 and Pseudomonas putida G2081.

The different plasmids integrated into these strains are as follows:

I

PLASMIDS CHARACTERISTICS pXL2087 Recombinant plasmid which carries the Comamonas NI 1 nitrilase gene under the control of the promoter Pac.

pXL2158 Recombinant plasmid which carries the Comamonas 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 P.r pDSK519 Control plasmid (see page 24 line 18).

The activities of these strains, induced or non-induced, are measured on adiponitrile and 5-cyanovalerate at different pH values and are compared 15 with the negative control strains: E. coli TG1, E. coli TG1 (pXL2035) and Pseudomonas putida G2081.

6.1 PREPARATION OF THE CELLS: The cultures are carried out under the conditions described in Table 20 4. During the exponential growth phase, one of the two cultures of the recombinant strain is induced with 1 mM IPTG; after 2 h at 37'C, this culture is treated.

TABLE 4: CULTURE OF THE STRAINS S. MiCROORGANISMS MEDIUM OD660nm DW (g/)1 1 E. coliTG1 a 3.1 0.90 2 E. coi (pXL2087) b 3.2 0.90 3 E. coli (pXL2087) c 2.5 0.90 4- E. coli (pXL2035) d 2.1 0.90 E. coli(pXL2158) b 3.1 0.80 6 E. coli(pXL2035, 2158) e 4.2 1.30 7 P. putida (pXL1289) d 2.1 0.98 8 P. putida (pXL2169) d 2.3 0.98 -L _1 IIU ABBREVIATIONS: a LB medium; b LB medium 100 pg/ml of Amp; c medium b addition of 1 mM IPTG to OD6onm 1; d LB medium mg/I of kanamycin; e M9 medium 100 mg/I of ampicillin 50 mg/I 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; T 37*C.

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 6.2 SPECIFIC ACTIVITY MEASUREMENTS: The conditions of the specific activity measurements and the results 15 are collated in Table TABLE 5: DETERMINATION OF THE ACTIVITIES OF THE CONTROL STRAINS FOR THE RECOMBINANT STRAINS

S

MICROORGANISM OPERATING CONDITIONS Activty Nature IPTG State Substrate [DW: Volume pH Ua Ub (ml) 1 E. coliTG1 W CVA 15.5 1 5.2 0 W CVA 15.5 1 7.0 0 W AdN 15.5 1 5.2 0 W AdN 15.5 1 7.0 0 2 E. coiTG1 W CVA 1.4 1 4.0 28 pXL2087 W CVA 1.4 1 5.2 27 W CVA 1.4 2 7.0 8 S CVA 1.4 1 5.2 S CVA 1.4 2 7.0 8 MICROORGANISM OPERATING CONDITIONS -civt Nature IPTG State Substrate [O0M Volume pH Ua Ub W AdN 0.3 1 4.2 159 W AdN 1.4 1 4.3 38 W MdN 1.4 2 6.2 18 W MdN 1.4 2 7.0 11 S AdIN 1.4 2 6.2 17 S AdIN 1.4 2 7.0 3 -E.cooiTG1 W CVA 1.2 2 4.0 pXL2087 W CVA 1.2 2 5.2 14 W CVA 1.2 2 7.0 3.4 S CVA 1.0 1 5.2 13 S CVA 1.0 1 7.0 3.2 W MdN 0.3 1 4.2 N 1.2 2 4.3 16 W MN 1.2 2 6.2 11 W MN 1.2 2 7.0 3.4 S AdN 1.0 1 6.2 3 S AdN 1.0 1 7.0 4 4 -E.coofiTGl W AdN 0.06 1 7.0 0 0 pXL2035 coli TG1 W MN 0.24 1 7.0 270 1.25 1 7.0 8.3 6 coli TGl W AdN 0.06 1 7.0 1500 pXL2035, 2158 CVA 0.2 1 7.0 7 putida W MN 0.3 1 7.0 0 G2081 pDSK5i19 8 P. put ida j W CVA 0.25 1 7.0 130 G 2081 pXL2169

I

COMMON CONDITIONS: [substrate] 50 mM; T 25C; buffer 50 mM; kinetics over 90 min for 1 to 3 and over 120 min for 4 to 8.

ABBREVIATIONS: W whole cells; S sonicated cells; Ua pmols of cyanovalerate produced/h; Ub pmols of adipate produced/h; AdN adiponitrile; CVA 5-cyanovalerate. DW dry weight.

EXAMPLE 7: SYNTHESIS OF AMMONIUM ADIPATE BY THE BATCH HYDROLYSIS OF ADIPONITRILE WITH E. coli (pXL2087) IN

SUSPENSION

S

120 pj or 1068 pmol 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 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 pl 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: MEAN ACTIVITIES OF THE E. coil (pXL2087) CELLS DURING THE HYDROLYSIS OF ADIPONITRILE r ar, o REACTION TIME SPECIFIC ACTIVITY pmol of adipate/hx rrio dry cells 0.5 16 11 11 7.7 EXAMPLE 8: SYNTHESIS OF AMMONIUM ADIPATE BY THE HYDROLYSIS 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.

I,

The resulting biocatalyst was then used in a fixed bed column for the hydrolysis of adiponitrile to ammonium adipate.

8.1- FIXING OF E. coli (pXL2087) TO RESIN: The cells were fixed according to the following protocol: Ig (wet weight) of E. coli (pXL2087) with a solids content of 22%, Ig of POLYCUP polyazetidine, Ig of DUOLITE A 171 resin.

POLYCUP is a solution of polyamide resin modified with epichlorohydrin, and DUOLITE A 171 is a highly basic anion exchanger resin of type 1, with macroporous structure and high porosity.

The gram of cells was suspended in the polyazetidine solution. After homogenization, the resin was poured into the cell suspension. The whole was stirred with a spatula and then left to dry for 18h, open to the air, under a hood. 4mL or 1.3g of biocatalyst were thus collected. The activities of the immobolized and free cells were 15 determined at 25 0 C and pH 7 on a 50mM solution of adiponitrile. They are respectively 30 and 110mol of 2-cyanovalerate/h/mg of cells DW, from which a fixing yield of 26% is deduced.

8.2- HYDROLYSIS OF ADIPONITRILE IN A FIXED BED REACTOR: The half-line is determined in a continuously fed fixed bed reactor under the 20 conditions indicated below: T 28 0 C; catalyst 0.5g or 2mL or 85mg of cells (dry weight); [adiponitrile] :phosphate buffer 50mM, pH 7; flow rate 3.7 ±0.1ml/h; column: diameter 1cm, height 3cm.

SThe inital activity of the cells was 1.5p.mol of adipate/h/mg of cells (dry weight).

66% of the initial activity is preserved after 32 days or 770h.

EXAMPLE 9 SYNTHESIS OF AMMONIUM METHIONINATE BY HYDROLYSIS OF METHIONITRILE, WITH RECOMBINANT STRAINS IN SUSPENSION The recombinant strains used in this example are those from E. coli TG1, integrating the plasmids PXL2158 and PXL2035 described above and containing the nitrilase gene of Comamonas testosteroni.

[N:\LIBM X)00606:AN1

-I

Tests 9 to 13 show the nitrilase activity at 280C, of these recombinant strains with respect to the methiononitrile, according to various operating conditions. The strain culture is performed as explained in example 6.1.

The following Table 7 summarizes the conditions and the results of tests 9 tol13.

TABLE 7: MICROORGANISM OPERATING CONDITIONS Initial specific activity pimoVh mg Nature State Substrate LOW] Volume pH of OW (mM) (ml) 9 coi W MetCN 17.2 5 7 8 PXL2158 (100) coli W MetCN 1.4 5 7 77 PXL2158 2035 (50) 11 coi W MetCN 1.4 5 7 62 PXL2158 2035 (50) 12- E. coi W MetCN 1.4 5 7 73 -PXL2158 2035. (100) 13 coi W MetCN 1.4 5 7 61 PXL2 158 2035 (200) r 0

S

S.

ABBREVIATIONS: W methiononitrile.

whole cells; DW dry weight of cells; MetON COMMENTS ON TABLE 7: Tests 9 and 10 show that the recombinant strain E coi PXL2158 possesses a good nitrilase activity but that it is recombinant strain E. coi combining PXL2P158 and PXL-2035, which is the most interesting strain as regards performances, thanks to an activity which is almost 10 times greater: about 80 pmol of MEtCN/hxmg of DW.

Tests 11 to 13 show that the initial quantity of MetON substrate has only a small influence on the activities obtained.

27 EXAMPLE 10: SYNTHESIS OF AMMONIUM METHIONINATE BY HYDROLYSIS OF METHIONONITRILE, WITH E. coil PXL 2158 2035 CELLS FIXED ON RESIN The E. coli PXL 2158 2035 free cells are fixed on resin according to the technique described in US patent 4 732 851, so as to form a biocatalyst.

10.1 FIXING YIELD AND ACTIVITY OF THE BIOCATALYST: The results of the initial activities of the biocatalyst and of the free cells are given in Table 8 below.

TABLE 8: SPECIFIC INITIAL ACTIVITIES OF HYDROLYSIS OF THE METHIONONITRILE OF THE FREE AND FIXED CELLS OF E.

15 coli PXL 2158 +2035 TEST No. Dry biocatalyst Dry calls Specific Initial Activity pmol/hxmg pmolihx mg i:__DB DC 14 10.3 1.4 3 22 1.3 63 CONDITIONS: volume 5 ml; potassium phosphate buffer 100 mM pH7, [MetCN] 50 mM; T 28'C; kinetics over 3 h; DB dry biocatalyst; DC dry cells.

COMMENTS: the activity of the biocatalyst is 3 pmol/hxmg of dry biocatalyst at 28"C and the fixing yield is With the free or fixed cells, 5% to 6% of amides are produced at the end of the hydrolysis.

e 10.2 INFLUENCE OF THE INITIAL METHIONONITRILE CONCENTRATION: Two methiononitrile batches were tested: purified methiononitrile in the form of a sulfate and free methiononitrile.

The results are summarized in Table 9 below.

TABLE 9: INFLUENCE OF THE INITIAL METHIONONITRILE CONCENTRATION ON THE SPECIFIC INITIAL ACTIVITY OF E. coil PXL 2158 2035 TEST [MetCN Dry Specific Initial activity Amide No. biocatalyst formed* .:::sufat: i ::::iiFree pmo::. i hxmg pm: :ol/hxmg ii:ii::. mM i: mM DB B DC 16 50 10.3 3.0 22.0 5.9 17 100 10.0 3.4 25.1 18 200 10.5 4.2 30.5 8.3 19 300 10.2 4.2 30.5 10.6 105 9.9 2.7 20.0 9.6 21 210 10.0 2.9 20.8 12 22 315 9.7 2.4 17.7 16 CONDITIONS: volume 5 ml; potassium phosphate buffer 100 mM pH7; T 28*C; kinetics over 3 h; percent with respect to the formed acid; DB dry biocatalyst; DC dry cells.

COMMENTS: the specific initial activity slightly increases up to 200 mM of methiononitrile. The percent of formed amide increases with the initial nitrile concentration. The purified methiononitrile makes it possible to obtain a better activity and a low percent of amide.

o 29 SEQUENCE LISTING INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 27 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: Met Lys Asn Tyr Pro Thr Val Lys Val Ala Ala Val Gin Ala Ala Val 1 5 10 Phe Met Asn Leu Glu Ala Thr Val Asp Lys Thr 20 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid TOPOLOGY: linear S (ii) MOLECULE TYPE: peptide (vii) IMMEDIATE SOURCE: CLONE: deduced sequence of probe of SEQ ID NO:3 oeeee: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Lys Asn Tyr Pro Thr Val Lys Val 1 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vii) IMMEDIATE SOURCE: CLONE: probe (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ATGAAGAAYT AYCCNACNGT SAAGGT 26 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 1194 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO o (iv) ANTI-SENSE: NO (vii) IMMEDIATE SOURCE: CLONE: seq. coding for the polypeptide having nitrilase activity (ix) FEATURE: NAME/KEY: CDS LOCATION: 87..1148 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CTCGAGACAA AATTGGGACA GTCGCCCCCT ATCTGCAAAA TGGAACCTCC TTTGCACATC TATAAAATT TTTGAGGAAG ACAGCA ATG AAA AAT TAT CCT ACA GTC AAG GTA 113 Met Lys Asn Tyr Pro Thr Val Lys Val 1 GCA GCA GTG CAA GCT GCT CCT GTA TTT ATG AAT CTA GAG GCA ACA GTA 161 Ala Ala Val Gln Ala Ala Pro Val Phe Met Asn Leu Glu Ala Thr Val 15 20

C--

GAT

Asp AAA ACT TOT AAO 'ITA ATA GCA OAA OCA OCA TCT ATG Lys Thr Cys Lys Leu Ile Ala Olu Ala Ala Ser Met GGC GCC AAO Gly Ala Lys 140 209 OTT ATC GGC TTC CCA Val Ile Oly Phe Pro GAA OCA TTT Glu Ala Phe CCC GOC TAT Pro Oly Tyr TOO ACA TCA Trp Thr Ser AAT ATO GAC TTC ACT Asn Met Asp Phe Thr OGA ATG ATG TOO Oly Met Met Trp CCA TAT TOO TT Pro Tyr Trp Ile 0CC OTC CTT TTC Ala Val Leu Phe CAA ATT AOT OAT Oln Ile Ser Asp 257 305 AAO AAT Lys Asn 0CC ATT OAA ATC Ala Ile iu Ile AOC AAA OAA OTT Ser Lys Olu Val

OCT

Ala OCA MAA AAO AAT Ala Lys Lys Asn OTT TAC OTT C Val Tyr Val Cys TCT OTA TCA GAO Ser Val Ser Glu OAT MAT 0CC TCO Asp Asn Ala Ser CTA TAT Leu Tyr 110 TTO AG CAA Leu Thr Oln TOO TIT OAC CCC Trp Phe Asp Pro AAT GOT Asn Cly 120 AAT TTO ATT 0CC AAO Asn Leu Ile Oly Lys 125 CAC AGO AAA TTC His Arg Lys Phe 130 AAO CCC ACT ACT Lys Pro Thr Ser ACT CAA AGA Ser Oiu Arg- 135 71T AAA ACA Phe Lys Thr OCT OTAi TOO Ala Val Trp 140 OCA OAT 000 OAT Oly Asp 0iy Asp AOC ATO OCT CCC Ser Met Ala Pro p497 5145 593 GAG TAT Olu Tyr 155 000 AAT CTT 000 Oly Asn Leu Oly ATT C C ATO le Ala Ala Met 175 CTC CAC TCC TOO Leu Cmn Cys Trp CAT OCT CTC CCA His Ala Leu Pro

AAC

Asn CCC TCA Gly Ser TTO AAC CAA Leu Asn Olu 180 CAC OTA CAT OTT OCT Cmn Val His Val Ala 185 TCC TOG CCA 0CC TTC OTC CCT AAA GC Ser Trp Pro Ala Phe Val Pro Lys Cly 190

OCA

Ala 195 OTA TCA TCC AGA Val Ser Ser Arg

I

OTA TCA 689 Val Ser 200 TCC AGC GTC TGT GCG TCT ACT AAT GCG ATG CAT CAG ATC ATT AGT CAG Ser Ser Val Ala Ser Thr Asn Met His Gin Ile Ile Ser Gin 215 ACC AAT CTC Thr Asn Leu TTT TAC GCG ATC AGC AAT CAG GTA Phe Tyr Ala Ile Ser Asn Gin Val 220 225 TAT GTA ATT ATG Tyr Val Ile Met GTT GGC Val Gly 235 CAA GAC ATG ATT Gin Asp Met Ile GAC ATG ATT GGG AAA Asp Met Ile Gly Lys 240 TCT GGA AAC ACA GCG Ser Gly Asn Thr Ala 260 GAT GAA TIT TCC AAA Asp Glu Phe Ser Lys 245 ATT ATT TCT AAC ACC Ile Ile Ser Asn Thr 265 TTT CTA CCG CTT Phe Leu Pro Leu

GGT

Gly 255 785 833 881 929 977 1025 oo o GGT GAG ATT TTG Gly Glu Ile Leu TCA ATT CCA CAA Ser Ile Pro Gin GAC GCG GAG GGA ATT Asp Ala Glu Gly Ile 275 TAT GGA AAG TGG TTA Tyr Gly Lys Trp Leu 295 GCT GTT Ala Val 280 CTG GAT Leu Asp GCA GAG ATT Ala Glu lle CCC GCC GGT Pro Ala Gly 300

GAC

Asp 285 CTT AAC CAA ATA Leu Asn Gin Ile CAT TAC TCT ACT CCC His Tyr Ser Thr Pro 305 GGC TTC TTA AGT TTG ACA TTT GAT Gly Phe Leu Ser Leu Thr Phe Asp 310 r a a c CAG TCT Gin Ser 315 GAA CAT GTA CCC Glu His Val Pro AAA AAA ATA GGT Lys Lys Ile Gly GAG CAG ACA AAC CAT Glu Gin Thr Asn His 325 ATG GAT ATG CTA ACG Met Asp Met Leu Thr 345 1073

TTC

Phe 330 ATC TCT TAT GAA GAC TTA CAT GAA GAT lie Ser Tyr Glu Asp Leu His Glu Asp 335 1121 ATT CCG Ile Pro CCG AGG CGC GTA GCC ACA GCG TGATCGCCGC CTCTCGGGGC Pro Arg Arg Val Ala Thr Ala 1168 350 GTTCGGTTGC TGATAGCCAT CGCCTT INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 354 amino acids

I

TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Lys Asn Tyr Pro Thr Val Lys Val Ala Ala Val Gin Ala Ala Pro o 1 Val I Ala Phe Thr Ser Tyr Thr Lys Gly 145 Leu Ser Lys Asn ?he Glu Ile Gly Lys Val Gin Phe 130 Ser Gin Leu Gly Ala Met Asn Ala Ala Pro Gly Met Met Glu Va3 Cys Va] Leu Tri 115 Lys Prc Met Al Cys Tri Asn Gl 18 Ala Va 195 SMet Hi

P

a p u 0 1 Leu Ser Tyr Trp Gin Ser Phe Thr Pro Glu 165 Gin Gln Ser Gin Glu Met Pro Ala 70 Gin Val Asp Ser Val 150 His Val Ser Ile Ala Thr Gly Ala I 40 Tyr Trp 55 Val Leu Ile Ser Ser Glu Pro Asn 120 Ser Glu 135 Phe Lys Ala Leu His Val Arg Val 200 Ile Ser 215 Thr Asn Val 25 Lys Iie Phe Asp Lys 105 Gly Arg Thr Pro Ala 185 Ser Glr Lei 10 Asp I Val Trp Lys Ala 90 Asp Asn Ala Glu Leu 170 Ser SSer SPhe i Val Lys lle rhr Asn 75 Ala Asn Leu Val Tyr 155 Asn Trp Set Tyr Gl Thr C Gly E Ser Ala Lys Ala Ile Trp 140 Gly lie SPro Val r Ala 220 y Gln ,ys Phe Asn Ile Lys Ser Gly 125 Gly Asn Ala Ala Cys 205 Ile As Lys L Pro C Met Glu Asn Leu 110 Lys Asp Leu SAla Phe 190 Ala SSer p Met ,eu 3lu Asp lie Gly Tyr His Gly Gly Met 175 Val Ser Asr Il1 Ile Ala Phe Pro Val Leu Arg Asp Gly 160 Gly SPro SThr 1 Gin e Asp 210 Val Tyr Val Ile Met Sei 225 230 235 240 Met lie Gly Lys Glu Phe Ser Lys Asn 250 Phe Leu Pro Leu Gly Ser 255 34 Gly Asn Thr Ala Ile lie Ser Asn Thr Gly Giu Ile Leu Ala Ser Ile 260 -265 270 Pro Gin Asp Ala Glu Gly Ile *Aia Val Ala Giu Ile Asp Leu Asn Gln 275 280 285 Ile Ile Tyr Gly Lys Trp Leu Leu Asp Pro Ala Gly His Tyr Ser Thr 290 295 300 Pro Gly Phe Leu Ser. Leu Thr Phe Asp Gin Ser Giu His Val Pro Val 305 310 315 1320 Lys Lys le Gly Glu Gin Thr Asn His Phe Ile Ser Tyr Giu Asp Leu 325 330 335 His Giu Asp Lys Met Asp Met Leu Thr Ile Pro Pro Arg Arg Val Ala 340 3)45 350 Thr Ala

Claims (14)

1. 2- Recombinant DNA sequence according to claim 1, characterized in that it contains the following nucleotide sequence: (SEQ ID NO: Z:LUJV,A. C44%t C4 Z V C AAA ==49A AC~a;AG C7.; -A"-C A~~r.4tUr%%.C7 C4. A4.* -G '4 =ZZ--A=G ;CC7 Z C Z A -Z .U4 C A .LAC.A4 UA k; A.T-
2. 7--ACAAGA C=:AG3 -AaAAa.~A C AA 9 GAA-== A f4A ;CA- I C2 A7ACT: =A.A7--4A4 Ur AAA A ZA'. .PC AACC 3 Polypeptides resulting from the expression of a DNA sequence according to one of claims 1 or 2 and possessing a nitrilase activity. 4 Polypeptide according to claim 3, characterized in that it comprises the sequence designated by SEQ ID NO :5 :in the enclosed sequence listing and given hereinunder. M4e: Lys Asn 7ar h. Va. L- Vaj Va Val ?he Me: G u Cl G~ C-I Se: a vel Me: 7-e 7:1 ZIn. Se:- a Val au' :1 C- Ala t2;-s *'0 I, As~Me: .eu ?he Se: :'sr Cl: z.sz Ser As=. Gly e>C e 2~ 2 7 Pro G. Asp Ala Glu Gly 11e Ala Val Ala Glu Ile Asp Leu As- Gi- 275 2B0 285 e Ty Gly Ly:s Tr-p Leu Leu Asp Pro -Ala Gly :is Ty- Se: T"' 290 295 300 Pro Gly Phe Leu Ser Leu Tnr ?he Asp Gin Ser Glu E±s Val ?Pr Va 305 310o -5 320 Lys Lys :e Gly Gl GGin Thr Asr s ?he Ie Ser Cu s= Leu 325 330 335 H-s G21u As; Lys Asp Me: L A-g Ar -g Val- .a T-h A 5 Microorganism containing the DNA sequence (SEQ ID NO 4 according to claims 1 or 2. 15 6 Microorganism containing the DNA sequence (SEQ ID NO 4 according to claims 1 or 2 on a plasmid containing a selection means. 7 Microorganism consisting of the strain E. coli TG1 containing plasmid pXL2148, said strain having the reference G4207 and being deposited in the Collection Nationale de Cultures de Micro- organismes under No. 1-1242. 8 Microorganism containing an expression cassette consisting of the DNA sequence (SEQ ID NO 4 according to claim 1 or 2 under the dependence of signals ensuring the expression of this sequence in the host microorganism. 25 9 Microorganism according to claim 8, characterized in that it comprises, upstream from the DNA sequence (SEQ ID NO 4 a ribosome binding site and a promoter sequence homologous or heterologous with the polypeptide produced. Microorganism according to claim 9, characterized in that 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 Corynebacterium, Comamonas or Pseudomonas. 11 Microorganism according to claim 9, characterized in that the ribosome binding site can be the one derived from the phage lambda CII gene or those derived from genes of E. coil, Comamonas, Pseudomonas or Corynebacterium.
3. 12- Microorganism according to claims 8 to 11, characterized in that the expression cassette is carried by a plasmid containing a selection means.
4. 13- Microorganism according to claim 12, characterized in that the selection means is a marker conferring antibiotic resistance.
5. 14- Microorganism according to claims 5 to 13, characterized in that it is selected from the strains of E. coli, Comamonas, Corynebacterium, Brevibacterium, Rhodococcus and Pseudomonas.
6. 15- Microorganism according to any one of claims 5 to 14, characterized: in that it contains at least one protein agent for assisting the folding of the polypeptides which the microorganism 15 synthesizes, and in particular the polypeptides according to claim 3 or claim 4, and/or the genes coding for such an agent, in that this agent is present in a greater amount than that corresponding to the base level of the microorganism in question.
7. 16- Microorganism according to claim 15, characterized in that the agent is the GroE chaperone of E. coli or its homolog of eucaryotic or procaryotic origin.
8. 17- Microorganism according to claim 15 or claim 16, characterized in that the genes coding for the agent are carried by the chromosome or by an extrachromosomal element (plasmid, phage) and in that said genes are amplified.
9. 18- Microorganism according to claim 17, characterized in that the genes coding for the agent are under the dependence of expression systems homologous or heterologous with said microorganism.
10. 19- Enzymatic method of converting nitriles, characterized in that it consists in bringing the nitriles into contact with a polypeptide having a nitrilase activity, according to any one of claims 3 or 4, or a host microorganism according to any one of claims 5 to 19. u Method according to claim 19, characterised in that the nitrile is a dinitrile of the formula NC-R-CN in which R is an alkylene group having from 1 to 10 carbon atoms.
11. 21. Method according to one of claims 19 and 20, characterised in that the nitrile is adiponitrile.
12. 22. Method according to claim 19, characterised in that the nitrile is a mononitrile preferably belonging to the class of aliphatic sulphurised mononitriles.
13. 23. Method according to claim 22, characterised in that the nitrile is the methiononitiile.
14. 24. DNA sequence coding for a polypeptide having a nitrilase activity and capable of hydrolysing nitriles to carboxylates substantially as hereinbefore described with reference to any one of the Examples. Enzymatic method of converting nitriles substantially as hereinbefore described with reference to any one of the Examples. Dated 12 January, 1994 15 Rhone Poulenc Nutrition Animale Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON ooo ce [N:\LIBW101408:GSA 1 of 1 I ABSTRACT 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 S" CARBOXYLATES BY MEANS OF SAID POLYPEPTIDES The present invention relates to novel polypeptides 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 nitriles to carboxylates, an analog of this sequence resulting from the degeneracy of the genetic code, a DNA sequence hybridizing with one of these sequences or a fragment thereof and coding for a polypeptide having a nitrilase activity, expression cassettes and microorganisms enabling them to be obtained. Application: enzymatic conversion of nitriles to carboxylates.