EP0665889A1 - Enzymes with a nitrile-hydratase activity, genetic tools and host micro-organisms for their production and method of hydrolysis using said enzymes - Google Patents

Abstract

Novel enzymes with a nitrile-hydratase activity, capable of catalyzing the transformation of nitriles into amides. Said enzymes are characterized by having an enzymatic activity in relation to substrates with a nitrile function and a carboxylate function greater than that relative to substrates with at least two nitrile functions. The invention also concerns a DNA sequence coding for an enzyme with a nitrile-hydratase activity capable of hydrolyzing nitriles into amides, an analogue of this sequence resulting from degenerescence of the genetic code, or a DNA sequence hybridizing with one of these sequences or a fragment of the latter and coding for an enzyme with a nitrile-hydratase activity. Finally, the invention embraces the germs capable of producing the above-mentioned enzymes.

Description

 ENZYMES λ ACΠVΓΓÉ NITRILE-HYDRATASE, GENETIC TOOLS AND HOST MICROORGANISMS

PERMITTING THEIR OBTAINING AND PROCESSING THE CYSTIC IMPLEMENTING LESMTES E ZYNŒS

TECHNICAL AREA ;

The field of the invention is that of the enzymatic production of amide derivatives from compounds containing nitrile groups.

PRIOR TECHNIQUE:

The present invention relates to new enzymes exhibiting nitrile hydratase activity, the genetic material involved in their production, as well as microorganisms containing this genetic material and exhibiting this activity.

The subject of the invention is also a process for the enzymatic hydrolysis of nitriles into amides in which these new enzymes or microorganisms synthesizing them are used, including, in particular, the above-mentioned host microorganisms.

Without being limiting, the nitriles, which are more particularly interested in the context of the invention, are: adiponitrile, cyano-5-valeramide, cyano-5-valerate of any cation, preferably chosen from the list of following compounds: alkali, alkaline earth, amines, ammonium, the latter compound being particularly preferred. The aim is the enzymatic transformation of the nitrile functions into amide functions, so as to obtain an adipamate, e. g. ammonium, convertible into diammonium adipate. And, as everyone knows, the whole industrial interest of adipates, and in particular of diammonium adipate, lies in the fact that they constitute one of the fundamental bases for the manufacture of nylon-6,6.

The action of these nitrile hydratases is part of a known scheme for the enzymatic synthesis of diammonium adipate from adiponitrile. This diagram is as follows:

NC COiNH,

NH = nitrile hydratase, Ni = nitrilase,

A = amidase, R = (CH _Î ) ,,, n being an integer equal to 4 in the case of adipic compounds.

As part of this scheme, the arsenal of enzymatic hydrolysis of amides and nitriles of many strains was studied.

Y. ASANO et al. have thus described in Agric. Biol. Chem., 45 (5). 1165-1174. 1982, Arthrobacter. This germ has a nitrile hydratase only active on malononitrile and not on adiponitrile.

H. YAMADA et al. report in J. Ferment. Technol., 58, 495-500, 1980, of the hydrolysis of glutaronitrile by the strain Pseudomonas sp. K9, the nitrile hydratase of this strain hydrolyzes, preferably one of the two CN functions of dinitrile, while the other is only slightly hydrolyzed by the enzyme.

The patent FR 2245585 describes bacteria with nitrilase activity chosen, preferably, from the genera Bacillus, Bacteridium within the meaning of PREVOST, Micrococcus and Brevibacterium as defined by BERGE Y. The strain Brevibacterium R 312 described in this patent, and more particularly the nitrile hydratase included in its enzymatic heritage, catalyzes the hydrolysis into amides of dinitriles of the malononitrile, fumaronitrile and succinonitrile type. This enzyme is more active vis-à-vis dinitriles or mononitriles monoamides than vis-à-vis mononitriles monocarboxylates.

This spectrum of activity was confirmed by subsequent studies, including in particular those of GALZY et al. in J. Gen. Microbiol, 130, 89-93, 1984.

The article by MAYAUX et al., Published in J. Bacteriol, 172, 6764-6773, 1990, teaches that Brevibacterium R 312 is the same germ as another corynebacterium, namely: Rhodococcus N 774.

Patent application EP 0178106 teaches, too, that Rhodococcus N 774 (or Brevibacterium R 312) has a nitrile hydratase active only on one of the nitrile functions of the dinitriles and ineffective with respect to the carboxylated mononitriles.

INGVORSEN et al describe, in CIBA Found. Symp., 140, 16-31, 1988, a strain identified as being a Rhodococcus sp CH5, having the characteristic of having, in its enzymatic heritage, a nitrile hydratase which also hydrolyzes, preferably, only one of the CN functions of malononitrile. This strain is even presented as not hydrolyzing 2-cyanoacetic acid.

Also known, from patent JP 03 019 695, the strain Corynebacterium sp C5, which hydrolyzes valeronitrile to transcyano-4-cyclohexane acid amide. This nitrile hydratase also does not go beyond the mononitrile monocarboxylic stage.

In Ann. N. Y. Acad. Sci., 613, 142-154, 1990, YAMADA et al have described a Pseudomonas chlororaphis involved in the hydrolysis of acrylonitrile to acrylamide.

In the family of fungi, KUWAHARA et al. (J. Ferment. Technol., 58, 573-577, 1980) have isolated Fusarium solani which has been shown to degrade succinodinitrile to succinic acid, passing through a mononitrile monoamide, then a diamide and, finally, a monocarboxylic monoamide.

This review of the state of the art shows, on the one hand, that the known enzymatic collection of NH does not offer really effective means of hydrolysis of the second nitrile function on substrates of the dinitrile type and, on the other hand on the other hand, that the known NHs determine an enzymatic pathway for the passage of a dinitrile to the corresponding dicarboxylic acid, which only involves nitride amides and diamides. And it is regrettable that the enzymatic activities which characterize this path, although significant from the laboratory point of view, are not satisfactory from an industrial perspective. Thus, one of the essential objectives of the present invention is to provide new enzymes with nitrile hydratase activity, genetic material allowing their production and microorganisms containing this genetic material, said enzymes and microorganisms being characterized, times, by satisfactory yields of production of amides from substrates of nitrile type of various natures (mono or dinitriles) and by nitrile hydratase activity, vis-à-vis carboxylated mononitriles, specific and significant. Such activity is capable of giving access to an enzymatic pathway for the production of dicarboxylic acids from dinitriles, which is industrially advantageous because it is very efficient.

After numerous tests and researches, the Applicant succeeded in achieving, among other things, this objective by isolating, purifying and characterizing new enzymes: - - capable of hydrolyzing nitriles into amides with strong activities, - and having carboxylated mononitriles as preferred substrate.

These enzymes are used, either as such or, and preferably, in the form of recombinant microorganisms generating them.

STATEMENT OF THE INVENTION;

The present invention therefore relates to new enzymes having a nitrile hydratase activity, capable of hydrolyzing nitriles into amides and having an enzymatic activity with respect to substrates having a nitrile function and a carboxylate function greater than that vis-à-vis substrates having at least two nitrile functions and that vis-à-vis substrates having at least one nitrile function and at least one other function different from the carboxylate function.

One of the very advantageous features of the enzymes according to the invention is expressed by a specific enzymatic activity (U _s ), with respect to cyano-5-valerate, expressed in moles of amide appeared xh ^"1 x kg ^{' 1} of enzyme used and under given measurement conditions, greater than or equal to 400, preferably at

1,000 and, more preferably still, to 10,000.

The above measurement conditions are: - reaction medium = 10 mM HEPES buffer,

- volume = 3 ml,

- temperature = 25 ° C,

- enzyme concentration = 16 μg / ml

One of these new enzymes was isolated from a strain of Comamonas testosteroni. More specifically, this enzyme is prepared by extraction and purification from cultures of natural or recombinant microorganisms, the purification being carried out by a succession of steps consisting in preparing an enzymatic extract from cell culture, in precipitating this extract with ammonium sulphate and to purify it by different stages of chromatography and gel filtration. These steps, which use techniques well known to those skilled in the art, are described in detail in the illustrative examples below.

The term “nitrile hydratase activity” denotes, in the present description, the enzymatic hydrolysis of a nitrile -mono or dinitrile, such as adiponitrile cyano-5 valeramide and cyano-5-valerate of a cation Z, to a amide, namely, in this example, respectively, cyano-5 valeramide, adipamide and adipamate of Z. The cation Z can be any, but it is preferably chosen from the following compounds: alkali, alkaline - earthy, amino, ammonium, the latter compound being particularly preferred.

It is to the credit of the Applicant to have been able to isolate these new enzymes and to have been able to demonstrate the new "mononitrile carboxylated" pathway which characterizes them. However, it should be emphasized that this new route makes it possible, as long as an amidase is associated with the enzyme of the invention, to prepare dicarboxylic acids from dinitriles passing exclusively through soluble species. It is clear that this is particularly advantageous with a view to industrial optimization of the process.

This hydrolysis characteristic of carboxylated mononitriles is verified both for enzymes in pure form and for natural or recombinant germs expressing these enzymes.

Among the enzymes with nitrile hydratase activity, in accordance with the invention, there is an enzyme characterized by two subunits a and β whose peptide sequences are shown, respectively, in FIGS. la and lb. (SEQ ID No: 1: and SEQ ID No: 2: in the sequence list below).

Another subject of the invention is a DNA sequence coding for an enzyme having nitrile hydratase activity capable of hydrolyzing nitriles to amides and chosen from the following list of sequences:

- the DNA sequence, as shown in fig. 2, (SEQ ID No: 3: in the list of sequences below) and coding for an enzyme (SEQ ID No: 4: in the list of sequences below) having a nitrile hydratase activity,

- an analogue of this sequence resulting from the degeneration of the genetic code, - a DNA sequence hybridizing with one of these sequences or a fragment thereof and coding for an enzyme having nitrile hydratase activity.

The enzymes resulting from the expression of one of the above DNA sequences are included in the scope of the invention.

The wild-type microorganism Comamonas testosteroni NI 1, isolated by the Applicant, contains one of the above sequences in its genome.

The DNA sequences coding for the enzyme were identified using nucleotic probes from the partial peptide sequences of the a and β subunits (FIGS. 1a and 1b) of the purified enzyme.

The invention also relates to the expression cassettes which carry, with the signals ensuring its expression, one of the DNA sequences defined above. These expression cassettes can either be naturally present, either integrated into the host genome or localized on an expression vector, such as a plasmid preferably containing a means of selection.

These expression cassettes include, in particular, transcription and translation initiation regions, which contain a promoter sequence and a ribosome binding site. These regions can be homologous or heterologous to the microorganism naturally producing the enzyme. The choice of these regions depends, in particular, on the host used. In particular, when it is a question of prokaryotic host microorganisms, the heterologous promoter can be chosen from strong bacterial promoters, such as the promoter of the tryptophan operon Ptrp άΕscherichia coli, the promoter of the lactose operon Plac of E. coli, the right promoter of phage lambda P _R , the left promoter of phage lambda P _L , the strong promoters of Pseudomonas and Comamonas, the strong promoters of Corynebacteria. More particularly, in the case of the right promoter of the lambda phage, the thermosensitive form P _R CIts is preferred. In the case of eukaryotic microorganisms, such as yeasts, the promoters may be those of the yeast glycolytic genes, such as the genes coding for phospho-glycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GPD), or many more genes coding for lactase (LAC4), enolase (ENO). Regarding the ribosome binding sites, that derived from the lambda CII gene, as well as those derived from Comamonas or Pseudomonas genes or those derived from Corynebacteria genes are preferably used when the host microorganism is prokaryotic.

A region, allowing termination of translation and functional transcription of the intended host, can be positioned 3 'to the coding sequence.

Advantageously, the expression cassette can also comprise one or more markers making it possible to select the recombinant host. The preferred markers are dominant markers, that is to say which confer resistance to antibiotics such as ampicillin, tetracycline or kanamycin or to other products toxic for host microorganisms. A subject of the invention is also the microorganisms containing the DNA sequence according to the invention, as well as those capable of producing at least one enzyme according to the invention. These microorganisms may or may not contain at least one expression cassette, of the type described above. Among these microorganisms, there are hosts which can be used for the reception of an expression vector in accordance with the invention, there may be mentioned, in particular, enterobacteria such as E. coli, bacteria belonging to the genera Comamonas, Pseudomonas , Streptomyces, Bacillus or coryneform bacteria, such as those belonging to the genera Corynebacterium, Brevibacterium or Rhodococcus. A recombinant microorganism, containing said DNA sequence on a plasmid structure, was deposited on July 9, 1993 under n ° I - 1330 at the National Collection of Cultures of Microorganisms (CNCM) (Institut Pasteur, 25 rue from Doctor Roux, PARIS). This microorganism is the E. Coli TG1 strain which contains the plasmid pXL2222 (described below). This microorganism is also identified by the Applicant by the reference G 4327.

It goes without saying that the enzymes produced by the microorganisms are also included in the invention.

The latter also relates to the process for converting nitriles into amides using an enzyme according to the invention or a recombinant microorganism generating it. This process consists in bringing together the nitrile to be transformed with an enzyme or a recombinant microorganism, as defined above. We

- generally operates at room temperature. According to a particular embodiment of the invention, the enzyme or the recombinant microorganism are immobilized on or in a solid support, according to a conventional technique, such as that described, for example, in American patent US 4,732,851.

The process of the invention is suitable for the transformation of nitriles into amides and, more particularly, for the transformation into amides of mono and dinitriles of formulas:

NC - R - CN, NC - R - COO ^" Z ⁺ and NC - R - CONH ₂ - R - COO ^" Z ⁺ in which R is a linear or branched alkylene or alkenylene group containing 1 to 18 carbon atoms, R being preferably equal to - (CH ₂ ) ₄ - and Z is chosen from the list of following compounds: alkali, alkaline earth, amines, ammonium, the latter compound being particularly preferred.

The process of the invention is particularly suitable for being integrated into the synthesis of adipates, such as ammonium adipate from a dinitrile, such as adiponitrile, via a mononitrile monoamide, such as cyano-5-valeramide, then either by a diamide, such as adipamide, or by a monocarboxylated mononitrile, such as cyano-5-valerate and, finally, by a monoamide monocarboxylate, such as ammonium adipamate .

POSSIBILITY OF INDUSTRIAL APPLICATION:

The present invention can be used for the preparation of carboxylates (adipates) useful as raw materials in industrial syntheses (polyamides). The following examples illustrate the characteristics and advantages of the present invention without, however, limiting its scope.

DESCRIPTION OF THE FIGURES

Fig. 1a represents the peptide sequence of the α subunit of an enzyme in accordance with the invention (SEQ ID No: 1 :).

Fig. lb represents the peptide sequence of the β subunit of the above enzyme (SEQ ID No: 2:).

Fig. 2 represents a DNA sequence according to the invention (SEQ ID No: 3:).

Fig. 3 represents the electrophoresis on an SDS polyacrylamide gel of the fractions obtained at the various purification stages of Example 2.

Fig. 4 represents a curve of the relative activity of the nitrile hydratase according to the invention as a function of the pH. Fig. 5 shows a curve of the specific activity of the nitrile hydratase according to the invention as a function of the temperature in ° C. Fig. 6 represents a variation of the specific activity (millimoles of cyano-5-valerate xh ^'1 xg ^{' 1} of protein) of the nitrile hydratase according to the invention with respect to cyano-5-valerate, depending on its cyano-5-valerate concentration (millimoles xl ^'1 ). Fig. 7 shows the restriction map of the plasmid pXL2120 containing the genes coding for the two subunits and β of the nitrile hydratase according to the invention.

Fig. 8 represents the sequence of a Ncol - Ncol - AccI fragment of 1713 bp containing the genes coding for the two subunits and β of the nitrile hydratase of Comamonas testosteroni NI 1.

Fig. 9 shows the restriction map of the plasmid pXL2160 containing the genes coding for the two subunits α and β of the nitrile hydratase (nthA and nthB ') according to the invention, the two genes being transcribed from the promoter Plac. Fig. 10 shows the restriction map of plasmid pXL2205 containing the nthA and nthB genes. the two genes being transcribed from the Ptrp promoter.

Fig. 11 represents the SDS-PAGE gel electrophoresis showing the expression of the DNA sequence according to the invention in the E. coli TG 1 / pXL2160 and E. coli TG1 / pXL2205 strains.

Fig. 12 represents the restriction map of the plasmid pXL2222 containing all the sequences coding for the two subunits α and β and included in the strain E. coli TG1 deposited (G 4327).

The meaning of the abbreviations used in the following description is given below.

- ssc: buffer commonly used for hybridizations, it contains sodium citrate and NaCl (20XSSC = NaCl 3M-sodium citrate pH 7, 0.3 M),

- SDS: sodium dodecyl sulfate, - FPLC: liquid chromatography called in English "fast protein liquid chromatography", - SDS-PAGE: electrophoresis gel based on sodium dodecyl sulfate and polyacrylamide,

- IPTG: isopropyl β-D thio galactopyranoside,

- HPLC: high performance liquid chromatography, - PS: dry weight,

- X-gal. : 5 - bromo - 4 - chloro - 3 - indolyl - β D galactopyranoside.

BEST WAY TO IMPLEMENT THE INVENTION:

EXAMPLES

EXAMPLE 1: CHARACTERISAΉON OF THE AC IVITY OF THE STRAIN Comamonas testosteroni NI 1.

The Comamonas testosteroni NI 1 strain is a strain isolated from a soil sample by microbiological screening. Its performance is evaluated on adiponitrile, cyanovaleramide, cyanovalerate, adipamide and adipamate.

1.1. Cell preparation:

The Comamonas testosteroni NI 1 strain is cultured, in a stirred flask, at 28 ° C., for 19 h, in medium A, the composition of which is as follows:

- Glycerol

Sodium cyano-5-valerate - K ₂ HPO ₄

- MgSO ₄ , 7H ₂ O

- MnSO ₄ , H ₂ 0

- FeSO ₄ , 7H ₂ O

- NaCl - Yeast extract

- Beef extract - HC1 3N QSP pH 5

This preculture is used to inoculate a 2 liter flask filled to the tenth with medium A. After 22 h, 3.5 g × l ^'1 of cells expressed by wet weight are harvested. This corresponds to an OD _Ô ^ of 1.4 and a dry weight of 0.9 gxl ^"1 .

1.2. Determination of enzyme activity:

The reaction medium is constituted by 50 mM phosphate buffer, pH 7 and the reaction temperature is 25 ° C.

The disappearing substrates are assayed by HPLC. The specific activities U _s are given in moles of disappeared substrate xh ^'1 x kg ^"1 of dry cells. The measurements of the hydrolysis activities of adiponitrile and its hydration products by Comamonas NI 1 are listed in the table 1.

TABLE 1: SPECTRUM OF ACTIVITY OF THE COMAMONAS NI 1 STRAIN

The Comamonas NI 1 strain has an enzyme which is very active on adiponitrile. It also has an enzyme capable of hydrolyzing cyanovalerate. Since the identified hydrolysis byproducts of cyanovalerate are adipamate and adipate, it follows that Comamonas NI 1 has, in its enzymatic heritage, a nitrile hydratase and an amidase. EXAMPLE 2: PURIFICATION OF NITRILE HYDRATASE FROM Comamonas NI.

2.1 Preparation of the cells:

The Comamonas Testosteroni NI 1 strain is cultured, in a stirred flask, at 28 ° C., for 23 h, in medium B:

- Glucose 5 x 10 ³ mg / 1

- Na ₂ HPO ₄ 3.5 x 10 ³ mg / 1

- KH ₂ PO ₄ 2.8 x 10 ³ mg / 1

- MgSO ₄ , 7H ₂ O 0.5 x 10 ³ mg / 1 - MnSO ₄ , H ₂ O 20 mg / 1

- FeSO ₄ , 7H ₂ O 20 mg / 1

- NaCl 10 mg / 1

- Biotin 0.01 mg / 1

- Nicotinic acid 1 mg / 1 - Thiamine, HC1 0.5 mg / 1

- Adiponitrile 1.05 x 10 ³ g / 1

- Sodium adipate 5.7 x 10 ³ g / 1

- pH 7

This preculture is used to seed a 20 liter fermenter containing 15 liters of the same medium B. The air flow rate, the pressure and the agitation are, initially,

250 1 / h, 1.1 atm and 250 rpm. During the fermentation, the pO ₂ , the pH and the temperature are regulated, respectively, at 50% of the initial value, 6.8 and 28 ° C. After 23 h, 189 g of wet cells are harvested. This corresponds to a dry cell content of 2.3 g / l (total 35 g) and an OD ^^ of 3.8.

2.2 Purification:

All the purification steps are carried out with buffer T (HEPES / KOH 0.1 M, pH 7.2, 40 mM in butyrate) unless otherwise indicated. At each of the stages, the nitrile hydratase activity of the fractions is determined at pH 7 and at 25 ° C. in the T / 10 buffer, in the presence of 10 mM cyanovalerate. The protein concentration of the pools is determined by the blue method of Coomassie (PIERCE Protein assay kit). The protein panel is analyzed by polyacrylamide-SDS gel (Phastsystem PHARMACIA) (cf. fig. 3). In this fig. 3, lanes 1, 2, 8 and 9 each contain molecular weight standards: - phosphorylase B = 97400

- Bovine Albumin Serum = 66200

- Ovalbumin = 45,000

- Carbonic anhydrase = 31100

- Soybean trypsin inhibitor = 21500 - Lysozyme = 14400.

Lanes 3 to 7 correspond respectively to purification steps 4 to 0. The reference molecular weights are indicated in the right margin in FIG. 3.

The data for each of the purification steps are collated in Table 2. The procedures for each of the steps are discussed below.

- Step 0: Raw extract:

152 g of wet cells are taken up in 250 ml of buffer T and sonicated for 30 min ("VIBRACELL" sonicator from BIOBLOCK: 13 mm probe, power 7, 40% of the active cycle). O ₆₆₀ _ _m is reduced from 175 to 50. After centrifugation at 48,000 gmax for 60 min, the supernatant is recovered.

- Step 1: Precipitation with ammonium sulfate:

The supernatant is brought, by progressive addition of ammonium sulphate, to 15% of the saturation. After 1 h, the suspension is centrifuged for 30 min at 30,000 gmax. The supernatant is brought to 45% of the saturation. After 1 h, the suspension is centrifuged under the same conditions, the precipitate is recovered, then dialyzed against the buffer overnight.

- Step 2: Ion exchange column (Q Sepharose Fast Flow):

The dialyzed fraction is loaded at a flow rate of 150 ml / h on a column (26 x 380 mm) of "Q Sepharose Fast Flow" balanced with buffer T at a flow rate of 250 ml / h. The column is percolated at a flow rate of 250 ml / h, successively by the following solutions:. QSP of buffer T until the return to the baseline,. 500 ml of a 0 to 0.3 M KC1 gradient in buffer T,. 200 ml of buffer T supplemented with 0.3 M KC1,. 200 ml of buffer T supplemented with KC1 1 M.

The nitrile hydratase activity is eluted in a volume of 160 ml at a concentration of KC1 situated between 0.22 and 0.28 M.

- Step 3: Column of hydrophobic interactions of Phenyl Sepharose CL4B: The active fraction, brought to 15% of the ammonium sulfate saturation, is loaded at a flow rate of 150 ml / min on the column (26 x 380 mm) , equilibrated with buffer T containing ammonium sulphate at 15% of saturation. The column is percolated with:. 200 ml of balancing buffer at a flow rate of 150 ml / h,. 200 ml of buffer T containing ammonium sulphate at 10% of saturation at a flow rate of 200 ml / h,

. 200 ml of buffer T with a gradient of ammonium sulfate from 10 to

0% of the ammonium sulfate saturation at a flow rate of 200 ml / h,. wash the column with buffer T until returning to the baseline. The active fraction is eluted at an ammonium sulphate content of between 8, 1 and 6%. This active fraction is concentrated by dialysis to a volume of 12.5 ml.

- Step 4: Hydrophobic interaction column on Octyl Sepharose:

The protein solution is loaded on the column (16 x 280 mm), equilibrated with buffer T containing ammonium sulfate at 15% of saturation at a flow rate of 26 ml / h. The column is then percolated at a flow rate of 47 ml / h with the equilibration buffer T. During this operation, all of the activity is eluted in the form of a peak. Only the core of the active fraction is harvested. This fraction analyzed in SDS-PAGE gel is composed of 90% of the nitrile hydratase in the form of two very close bands. TABLE 2: PURIFICATION OF NITRILE HYDRATASE FROM Comamonas NI 1.

ABBREVIATIONS: PF = purification coefficient; U = 1 mole / h.

EXEMPIJS 3: CHARACTERIZATION OF THE HYDRATASE NTΠULE OF Comamonas NI 1.

3.1 Determination of the structure:

The nitrile hydratase gel filtration gives a single peak whose elution time corresponds to 92 +/- 10 kDa. Analysis of this protein by polyacrylamide-SDS gel gives two bands of equal intensity from 26 to 25 kDa. This structure is that conventionally encountered in nitrile hydratases.

3.2 Determination of N-terminal peptide sequences:

From the purified protein, the N-terminal sequence of the two subunits was determined: subunit (SEQ ID No: 5: in the list of sequences below)

Thr-Asp-Asn-Ala-Val-Met-Glu-Gln-Arg-Val-Asp-Ala-Leu-Phe-Val-Leu-

(Thr or Arg) -Lys-Glu-Leu-Gly-Leu-Val-Thr-Asp-Gln-Thr-Val-Pro-Asp-

Tyr-Glu- (Asp or Pro) -Ala-Leu-Met-His-Asp β subunit (SEQ ID No: 6: in the list of sequences below)

Met-Asp-Gly-Met-His-Asp-Leu-Gly-Gly-Lys-Gln-Gly-Phe-Gly-Pro-Val- Ile-Lys-Thr-His-Asn-Ala-Lys-Ala-Phe-His-Glu-Glu- (Trp) - (Glu) -Val-Lys- Met-Asn-Ala-Ile

3.3 Influence of pH: The experimental conditions are as follows: ^' T ° C = 25; cyanovalerate concentration = 10 mM; nitrile hydratase concentration = 1.5 μg / 1; reaction volume = 2 ml; kinetics over 10 min; reference activity: pH 7.2, 17,000 oles / h.kg of protein; acetate buffer (3.5 to 5.5), phosphate buffer (6.5 to 8.5), Tris buffer (7 and 9), carbonate buffer (9.2 and 10.2). The conditions and results of the measurements of the nitrile hydratase activity on the cyanovalerate at different pHs are listed in fig. 4. The curve of variation of activity as a function of pH is, conventionally, bell-shaped with an optimum close to 7.5.

3.4 Influence of temperature:

The experimental conditions are as follows: cyanovalerate concentration = 10 mM; nitrile hydratase concentration = 1.5 μg / 1; reaction volume = 2 ml; kinetics over 10 min; reference activity pH 7.2, 17,000 moles / h.kg of protein; 10 mM HEPES / KOH buffer, pH 7.2. The results of the nitrile hydratase activity measurements at different temperatures are listed in fig. 5. Between 10 and 30 ° C the ΔG of the reaction is 80 kJ / mole.

3.5 Determination of kinetic constants: The substrate used is cyanovalerate. Its rate of hydrolysis to adipamate is determined at different cyanovalerate concentrations (0.5, 1, 2, 5, 10 and 20 mM) at 25 ° C and pH 7.2.

Experimental conditions: T ° C = 25, nitrile hydratase concentration = 1 μg / 1 (except 0.5 μg / 1 for a cyanovalerate concentration = 1 and 2 mM and 3.3 μg / 1 for a cyanovalerate concentration = 20 mM); reaction volume 10 ml except 5 and 3 ml for a cyanovalerate concentration = 0.5 and 20 mM, respectively; kinetics over 20 min; reference activity pH 7.2 17 moles / h. g protein; 10 mM HEPES / KOH buffer, pH 7.2. The K _M and V _M calculated for the transformation of HANES-WOOLF or LINEWEAVER-BURK are: K _M = 2.5 mM

V _M = 35,000 moles or 5,000 kg of adipamate / h.kg of nitrile hydratase According to V _M and the weight of the enzyme (92 kDa), we can calculate the turnover (k _ç ,,) of l and the rate of formation of the enzyme-substrate complex (IC _C / K _M ): _e ., = 900 s ^"1 kJK _u ≈ 36,000 l.mole- ¹ s ^{" 1}

3.6 Activity spectrum:

The activities of a purified preparation of nitrile hydratase were measured on different nitriles. The conditions and results are given in Table 3.

The spectrum of activity of this enzyme is quite special. Unlike all known nitrile hydratases, this enzyme hydrolyzes mononitriles-monoacids and little dinitriles.

TABLE 3 RELATIVE ACΠVΠÉ OF A PIMFIED PREPARATION OF NΠRILE HYDRATASE FROM COMAMONAS NI 1 ON DDTFERENTS NITRILES

COMMON CONDITIONS: 10 mM HEPES buffer, pH 7.2; reaction volume of

3 ml; T (° C) = 25; protein concentration of 16 μg / ml.

ABBREVIATIONS: T _R = reaction time: U _s = 1 mole of amide appeared / h.kg of nitrile hydratase.

EXAMPLE 4: CLONING OF NITRILE HYDRATASE FROM Comamonas testosteroni NI 1.

From the NH ₂ terminal sequences presented in Example 3, two nucleotide probes were synthesized.

For the choice of the first probe (α subunit), the use of codons was not biased. The mixture of oligonucleotides (seq. 2273) is a 23 times degenerated 23 mer containing 2 inosines. DNAVMEQR (SEQ ID No: 8:)

5 'GAYAAYGCNGTNATGGARCARMG 3' (SEQ ID No: 7:)

In the list of sequences below, the nucleotide sequence of this first probe is designated by SEQ ID No: 7: and the corresponding amino acid sequence by SEQ ID No: 8:.

For the second probe (β subunit), the high percentage of GC of the strains of

Comamonas dictated a choice for the third position of the codon in the case of alanines and in the case of lysine. The probe (seq. 2264) is a 23 sea 144 times degenerate:

H N A K A F H E (SEQ ID No: 10:)

5 'CAYAAYGCBAAGGCBTTYCAYGA 3' (SEQ ID No: 9:)

In the sequence list below, the nucleotide sequence of this second β probe is designated by SEQ ID No: 9: and the corresponding amino acid sequence by SEQ ID No: 10:.

The strategy followed consisted, first of all, in verifying the specificity of the two probes and in determining the nature of the genomic DNA fragments to be cloned. Briefly, the genomic DNA of Comamonas testosteroni NI 1 was digested with several restriction enzymes corresponding to sites which can be used for cloning. After electrophoresis on agarose gel and transfer to a nylon membrane, the various digestions were hybridized to the probes. The results show that the two probes have sufficient specificity under the hybridization conditions used (5x SSC, 5x Denhardt, 0.1% SDS, 50 mM NaPO ₄ pH 6.5, 250 μg / ml ssDNA; hybridization temperature 50 ° C. Washing conditions: 1 h, 6x SSC, room temperature and 5 min in 2x SSC, 0.1% SDS at 50 ° C). Under these conditions, the two probes allowed us to obtain specific signals. In particular in the case of digests with SstI, BglII and PstI where the hybridization signals obtained with the two probes are superimposable.

The hybridization fingerprints show, in particular, the existence of a fragment single BglII of approximately 4 kb. In order to clone this fragment, the 2.5 to 4.5 kb fragments of a BglII digestion of genomic DNA were purified by preparative electrophoresis on agarose and electroelution, then ligated to the plasmid pUC19, itself digested with BamHI. After transformation into the DH5α strain, 800 white clones on LB amp X-gal were individually subcultured, transferred to a nylon membrane, then analyzed by hybridization with the seq 2264 probe under the same stringency conditions as during the hybridization of the " Southern blot ". A clone was thus identified as specifically hybridizing with the probe. This clone was found to contain a plasmid (pXL2120), having inserted a fragment of approximately 4.2 kb, also hybridizing with the seq probe. 2273. This plasmid was analyzed in more detail (restriction mapping, partial sequencing using the probe seq. 2264 as primer and "Southern blot"). It has thus been possible to show that the 5 ′ part of the gene coding for the β subunit, which hybridizes with the seq probe. 2264, is located on a fragment of Sphl-Kpnl of around 280 bp, oriented in the direction SphI towards Kpnl. Fig. 7 shows a restriction map of this plasmid.

EXAMPLE 5: SEQUENCE DTJN FRAGMENT OF 1713 PB CONIENANΓ LES GENES

CODING FOR THE TWO SUBTIUNITIES OF NTIRILE HYDRATASE

DE Comamonas testosteroni NI 1.

The localization of the NcoI-NcoI-AccI fragment of 1713 bp sequence, containing the

• genes coding for the two subunits of the nitrile hydratase of Comamonas testosteroni NI 1, is indicated under the clone insert in fig. 8. The sequencing strategy for this fragment, produced according to the conventional methods known to those skilled in the art, is indicated in the same figure. The various sequences were all obtained by the chain termination method (sequenase kit in the presence of 7-deaza dGTP; ( ³⁵ S) dATP), either on single-stranded matrices of recombinant M13 carrying subfragments, or directly on the plasmid pXL2120. Several specific primers have also been synthesized for this purpose. The sequence is shown in fig. 2. The average GC% of the sequence obtained is 56.6%, which is close to the GC% of 61.5% described in other strains of Comamonas (TAMAOKA et al., Int. J; Syst. Bacteriol., 1987, 37, 52-59). An analysis of the sequence obtained made it possible to characterize a first open phase of 645 bp potentially translated into a protein of 215 amino acids. The NH ₂ terminal sequence of the a subunit, used to synthesize the seq probe. 2273, is contained in this protein and makes it possible to locate the initiation codon of the gene coding for the α subunit, called nthA in the rest of the description. This gene contains 621 bp, code for a protein of 207 residues corresponding to a molecular weight of 22,967 Da. A second open phase of 621 bp is potentially translated into a polypeptide of 207 residues. This polypeptide present in position NH ₂ terminal, the terminal NH ₂ sequence used to synthesize the probe seq. 2264, which makes it possible to identify the second phase open to the structural gene for the β subunit of the nitrile hydratase, called nthB in the rest of the description. This protein has a theoretical molecular weight of 22,653 Da;

EXAMPLE 6: EXPRESSION OF NΠRILE HYDRATASE FROM Comamonas testosteroni NI 1 IN E. coli.

In order to confirm the identity between the nthA and nthB genes and the genes coding for the two subunits of the nitrile hydratase of Comamonas testosteroni NI 1, the expression plasmids pXL2160 and pXL2205, presented in FIGS. 9 and 10, were built in several stages.

First, the 779 bp Ncol / BglI fragment (containing the 282 bp located upstream of the nthA gene and the first 496 bp of nthA) and the 789 bp BglI / AhalI fragment (containing the 3 ′ part of nthA and all the nthB gene), fragments isolated from pXL2120, were cloned between the Ncol and CJal sites of pMTL22 (CHAMBERS et al., 1988, Gene, 68, 139-149).

The resulting plasmid, pXL2160, therefore carries a gene conferring resistance to ampicillin and the nthA and nthB genes under the control of their own ribosome binding sites, the two genes being transcribed from the promoter of the lactose operon of E. coli. pXL2160 carries, moreover, in the cloning multisite, downstream of the fragment of Comamonas testosteroni, a unique Xhol site. In parallel, an Ndel restriction site was created on the initiation codon of nthA and the 180 bp Ndel / HindIII fragment containing the 5 ′ part of the nthA gene was amplified by the PCR technique. This fragment was ligated to the HindIII / XhoI fragment of pXL2160 between the NdeI / Xhol sites of pMTL22, to result in the plasmid pXL2203.

The Ndel / Xhol fragment of pXL2203, carrying the nthA and nthB genes, was introduced behind the EcoRI / Ndel fragment containing the promoter of the tryptophan operon from E. coli and the binding site of the ribosomes of the Cili gene of the bacteriophage λ ( Ptrp-RBSCII), between the EcrjRI and SalI sites of the plasmid pXL534 (LATTA et al., 1990, DNA Cell. Biol., 9, 129-137).

The final plasmid pXL2205 therefore carries a gene conferring resistance to ampicillin, the nthA gene under control of the RBSCII ribosome binding site and the nthB gene under control of its own ribosome binding site, the two genes being transcribed from from the promoter Ptrp. The expression of the nitrile hydratase was visualized in the E. coli TG 1 strain containing the plasmids pXL2160 and pXL2205.

For this purpose, the strain TG l / pXL2160, as well as the control strain TGl / pUC19, were cultivated for 16 h in LB medium at 37 ° C. (MILLER, JH 1972, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) containing 100 μg / ml ampicillin, then diluted 100 times in the same medium and at the same temperature. When the cultures reached an OD ₆₁₀ of between 0.5 and 1, IPTG, at the final concentration of 1 mM, was added. After 2 h of culture, the bacteria were collected. In addition, the TG1 / pXL2205 strain, as well as the control strain TG1 containing the vector pUC19, were cultivated for 16 h in M9 glucose medium at 30 ° C. (MILLER, JH 1972, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), containing 100 μg / ml of ampicillin and 100 μg / ml of tryptophan, these cultures were diluted 100 times in the same medium in the absence of tryptophan and cultured for 6 h at the same temperature. The expression of the nitrile hydratase of Comamonas testosteroni NI 1 was visualized, after sonication of the cells, in polyacrylamide-SDS gel, in the crude fraction or after centrifugation in the pellet and in the supernatant. The results are shown in fig. 11.

Each lane corresponds to an amount of protein equivalent to 60 μl of culture at an OD of 3 to 610 nm.

Lane G contains the following molecular weight markers: 97.5, 66.2, 45,

31, 21.5 and 14.4 kDa.

The content of the other tracks is shown in the following table.

It appears from fig. 7 that the cell extracts, containing the plasmid pXL2205 (cf. lanes D, E, F), strongly express the two subunits of the nitrile hydratase (indicated and β next to the gel), essentially in soluble form. These two subunits are also expressed in cell extracts containing the plasmid pXL2160 (cf. lanes H, I, J), after induction with IPTG. The expression is then weaker and, for the most part, in the insoluble fraction. ^~~ Furthermore, plasmin pXL2222, containing all the sequences coding for the two subunits of nitrile hydratase and presented in FIG. 12, was transformed into the E. coli TG 1 strain. The resulting strain, called G4327, was deposited at the National Collection of Culture of Microorganisms in PARIS (INSTITUT PASTEUR, 25, rue du Docteur Roux), under the no. 11,330. Plasmid PXL2222 groups together two fragments extracted from pXL2160, the 934 bp Nrul / Kpnl fragment containing the nthA gene and the 5 'part of nthB cloned in front of the 370 bp Kpnl / Stul fragment containing the 3' part of nthB. These two fragments are inserted between the Nael and EcoRV sites of pMTL22 (CHAMBERS et al., 1988, Gene, 68, 139-149). The EcoRV site used is that located downstream of lacZ 'and not that in the multisite. EXAMPLE 7: DETERMINATION OF THE ACTIVITY OF THE RECOMBINANT STRAINS.

Different recombinant strains, in which the genes coding for nitrile hydratase have been cloned, are tested for the hydrolysis of CVA and / or DNA. The cultures of the strains are listed in Table 4.

TABLE 4: CULTURE OF STRAINS

Middle M9:

. Glucose. . . .

. Na ₂ HPO ₄ , 2H ₂ O

. K_H. PO ₄ . . . .

. NaCl

. NI ^ Cl

. Casamino acids

. Thiamine, HC1

. MgSO ₄ . . . .

. CaCl ₂ , 2H ₂ O.

. Ampicillin Middle LB ^AI :

. middle LB

. IPTG 1 mM

. Ampicillin. 0.1 x 10 ³ mg / 1 - LB medium:

. Tryptone 10 x 10 ³ mg / 1

. Yeast extract 5 x 10 ³ mg / 1

. NaCl 5 x 10 ³ mg

COMMON CONDITIONS: seeding in l / 100th from a preculture aged 17 h; T ° C = 37, except M ₉ at 30 ° C.

The activities of the recombinant strains and of the host strain are listed in Table 5.

TABLE 5: DETERMINATION OF NITRILE HYDRATASE ACTIVITIES OF E. coli (pXL2205) AND E. coli (pXL2160).

_ ABBREVIATIONS: CVA = cyanovalerate; AdN = adiponitrile. COMMON CONDITIONS: T (° C) = 28; 50 mM phosphate buffer, H 7; [S] = 50 mM; kinetics over 2 h. LIST OF SEQUENCES

(1) GENERAL INFORMATION:

(i) DEPOSITOR:

(A) NAME: RHONE-POULENC CHEMISTRY

(B) STREET: 25, Quai Paul Doumer

(C) CITY: Courbevoie

(E) COUNTRY: France

(F) POSTAL CODE: 92400

(G) TELEPHONE: 7 68 12 3 (H) FAX: 7 68 1656

(ii) TITLE OF THE INVENTION: Nitrile-hydratase enzymes, genetic tools and host microorganisms for obtaining them and hydrolysis process using said enzymes

(iii) NUMBER OF SEQUENCES: 10

(iv) COMPUTER-READABLE FORM:

(A) TYPE OF SUPPORT: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patentln Release # 1.0, Version # 1.25 (EPO)

(vi) DATA FROM THE PREVIOUS APPLICATION:

(A) DEPOSIT NUMBER: FR 93 09990

(B) DEPOSIT DATE: 10-AUG-1993

(2) INFORMATION FOR SEQ ID NO: 1:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 207 amino acids

(B) TYPE: amino acid

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: protein

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:

Met Thr Asp Asn Ala Val Met Glu Gin Arg Val Asp Ala Leu Phe Val 1 10 15

Leu Thr Lys Glu Leu Gly Leu Val Thr Asp Gin Thr Val Pro Asp Tyr 20 25 30 Glu Asp Ala Leu Met His Asp Trp Leu Pro Gin Asn Gly Ala Lys Leu

35 40 45

Val Ala Lys Ala Trp Thr Asp Pro Val Phe Lys Ala Gin Leu Leu Ser

50 55 60

Glu Gly Val Ala Ala Ser Glu Ser Leu Gly Phe Ser Phe Pro Lys His 65 70 75 80

His Lys His Phe Val Val Leu Glu Asn Thr Pro Glu Leu His Asn Val

85 90 95

Ile Cys Cys Ser Leu Cys Ser Cys Thr Ala Phe Thr Ile Gly Met

100 105 110

Ala Pro Asp Trp Tyr Lys Glu Leu Glu Tyr Arg Ala Arg Ile Val Arg

115 120 125

Gin Ala Arg Thr Val Leu Lys Glu Ile Gly Leu Asp Leu Pro Glu Ser

130 135 140

Ile Asp Ile Arg Val Trp Asp Thr Thr Ala Asp Thr Arg Tyr Met Val 145 150 155 160

Leu Pro Leu Arg Pro Gin Gly Thr Glu Asp Trp Ser Glu Ala Gin Leu

165 170 175

Ala Thr Leu Ile Thr Gin Asp Cys Leu Ile Gly Val Ser Arg Leu Glu

180 185 190

Ala Pro Phe Ala Ala Leu Pro Ala Pro Ala Val Ala Leu Gly Ala 195 200 205

(2) INFORMATION FOR SEQ ID N0: 2:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 207 amino acids

(B) TYPE: amino acid

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: protein

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:

Met Asp Gly Met His Asp Leu Gly Gly Lys Gin Gly Phe Gly Pro Val

1 5 10 15

Ile Lys Thr His Asn Ala Lys Ala Phe His Glu Glu Trp Glu Val Lys

20 25 30

Met Asn Ala Ile Ser Gly Ala Leu Val Ser Lys Gly Ile Tyr Asn Met

35 KB 45

Asp Glu Tyr Arg His Gly Ile Glu Arg Met Glu Pro Arg His Tyr Leu

50 55 60

Thr Ala Ser Tyr Phe Glu Arg Val Phe Thr Thr Ala Val Thr Leu Cys 65 70 75 80

Ile Glu Lys Gly Val Phe Thr Ala Ala Glu Leu Glu Ala Lys Leu Gly 85 90 95 Thr Ser Val Pro Leu Ser Leu Pro Ser Ser Pro Gly Arg Gin ^' Pro Ala

100 105 no

Lys Gly Pro Glu Gly Gly Phe Lys Leu Gly Gin Arg Val His Val Lys

115 120 125

Asn Glu Phe Val Pro Gly His Thr Arg Phe Pro Ala Tyr Ile Arg Gly

130 135 10

Lys Ala Gly Val Val Val Gly Ile Ser Pro Ala Tyr Pro Tyr Pro Asp 145 150 155 - 160

Ala Ala Ala His Gly Glu Tyr Gly Phe Ser Glu Pro Thr Tyr Asp Val

165 170 175

Cys Phe Lys Ser Lys Asp Leu Trp Pro Asp Gly Cys Glu Ala Ala Asp

180 185 190

Val His Val Gly Val Phe Gin Ser Tyr Leu Leu Ser Ala Glu Glu 195 200 205

(2) INFORMATION FOR SEQ ID N0: 3:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 1713 base pairs

(B) TYPE: nucleic acid

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: DNA (genomics)

(iii) HYPOTHETIC: NO

(iii) ANTI-SENSE: NO

(ix) ADDITIONAL FEATURE:

(A) NAME / KEY: CDS

(B) LOCATION: join (283- -903. 90 -.1 9)

(ix) ADDITIONAL FEATURE:

(A) NAME / KEY: intron

(B) LOCATION: 904..908

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3:

CCATGGAGAA GATGGGGGAG TGGGGCGTGA GCGAAAAGCT GCTGAATCGT GCACCCAAGG 60

TGAGTCGGCT CGCCAACATC CAACTCGCCC GCTCAGTTGA CACCGAGATG GCTAAGAGCG 120

ACTTCGACTG GCGCTCGGTG AAGGTCAAGG TTGCCTGAGC CCCGTCTGCC TGACTCCATC 180

CCTGTTGCAG GCGCGTGTAG CACAGAGATA CCCCCTTATC ACACAGACTC AGCGCAAGCT 240

GGTCGCAATC TGCATCAAAT GTCGCGAATA GGAGAAACCC AG ATG ACT GAT AAC 294

Met Thr Asp Asn 1 GCC GTA ATG GAA CAA CGC GTG GAC GCA CTC TTT GTG CTC ACC AAA GAG 342 Ala Val Met Glu Gin Arg Val Asp Ala Leu Phe Val Leu Thr Lys Glu 5 10 15 20

CTT GGT TTG GTA ACT GAC CAA ACC GTG CCC GAC TAC GAA GAC GCC CTC 30 Leu Gly Leu Val Thr Asp Gin Thr Val Pro Asp Tyr Glu Asp Ala Leu 25 30 35

ATG CAC GAC TGG CTG CCG CAA AAT GGT GCC AAG CTG GTG GCC AAA GCT 38 Met His Asp Trp Leu Pro Gin Asn Gly Ala Lys Leu Val Ala Lys Ala 40 45 50

TGG ACC GAT CCA GTC TTC AAA GCT CAA CTG CTC AGT GAA GGT GTG GCA 486 Trp Thr Asp Pro Val Phe Lys Ala Gin Leu Leu Ser Glu Gly Val Ala 55 60 65

GCG TCT GAA AGC CTT GGC TTT AGT TTT CCC AAA CAC CAC AAG CAC TTC 534 Ala Ser Glu Ser Leu Gly Phe Ser Phe Pro Lys His His Lys His Phe 70 75 80

GTG GTG CTG GAG AAC ACC CCC GAG TTG CAT AAC GTG ATT TGC TGT TCA 582 Val Val Leu Glu Asn Thr Pro Glu Leu His Asn Val Ile Cys Cys Ser 85 90 95 100

CTG TGT TCG TGT ACG GCA TTC ACC ATC ATT GGC ATG GCG CCC GAT TGG 630 Leu Cys Ser Cys Thr Ala Phe Thr Ile Ile Gly Met Ala Pro Asp Trp 105 no 115

TAC AAA GAG CTT GAG TAC CGC GCC CGC ATT GTG CGT CAG GCA CGT ACC 678 Tyr Lys Glu Leu Glu Tyr Arg Ala Arg Ile Val Arg Gin Ala Arg Thr 120 125 130

GTG CTG AAG GAA ATA GGC CTG GAT CTG CCA GAG TCC ATC GAT ATC CGC 726 Val Leu Lys Glu Ile Gly Leu Asp Leu Pro Glu Ser Ile Asp Ile Arg 135 1 ^ 0 145

GTG TGG GAC ACC ACA GCA GAC ACC CGC TAC ATG GTG CTG CCC CTA CGC 774 Val Trp Asp Thr Thr Ala Asp Thr Arg Tyr Met Val Leu Pro Leu Arg 150 155 160

CCC CAA GGC ACC GAG GAC TGG AGT GAA GCT CAA CTT GCC ACA CTC ATC 822 Pro Gin Gly Thr Glu Asp Trp Ser Glu Ala Gin Leu Ala Thr Leu Ile 165 170 175 180 ACG CAA GAC TGT CTG ATT GGC GTG AGC CGC TTG GAA GCA CCG TTC GCT 870 Thr Gin Asp Cys Leu Ile Gly Val Ser Arg Leu Glu Ala Pro Phe Ala 185 1 0 195

GCA CTG CCA GCA CCC GCT GTT GCT TTA GGA GCC TGACC ATG GAC GGC 917 Ala Leu Pro Ala Pro Ala Val Ala Leu Gly Ala Met Asp Gly 200 205 210

ATG CAC GAT TTG GGG GGC AAG CAA GGC TTT GGC CCC GTA ATC AAA ACG 965 Met His Asp Leu Gly Gly Lys Gin Gly Phe Gly Pro Val Ile Lys Thr 215 220 225

CAC AAC GCT AAG GCC TTC CAC GAA GAG TGG GAG GTG AAG ATG AAC GCT 1013 His Asn Ala Lys Ala Phe His Glu Glu Trp Glu Val Lys Met Asn Ala 230 235 240

ATC AGC GGT GCG CTG GTT AGC AAG GGC ATC TAC AAC ATG GAC GAG TAT 106l Ile Ser Gly Ala Leu Val Ser Lys Gly Ile Tyr Asn Met Asp Glu Tyr 245 250 255

CGG CAC GGA ATT GAG CGC ATG GAG CCG CGC CAT TAC CTG ACT GCC TCG 1109 Arg His Gly Ile Glu Arg Met Glu Pro Arg His Tyr Leu Thr Ala Ser 260 265 270

TAT TTC GAG CGC GTG TTC ACC ACA GCT GTG ACC TTG TGT ATT GAA AAA 1157 Tyr Phe Glu Arg Val Phe Thr Thr Ala Val Thr Leu Cys Ile Glu Lys 275 280 285 290

GGC GTT TTC ACC GCC GCA GAG TTG GAG GCC AAG CTG GGT ACC TCG GTG 1205 Gly Val Phe Thr Ala Ala Glu Leu Glu Ala Lys Leu Gly Thr Ser Val 295 300 305

CCC TTG TCT CTG CCC AGC TCA CCC GGT CGC CAA CCT GCC AAA GGC CCT 1253 Pro Leu Ser Leu Pro Ser Ser Pro Gly Arg Gin Pro Ala Lys Gly Pro 310 315 320

GAG GGT GGC TTC AAG CTG GGT CAG AGA GTG CAT GTG AAG AAC GAG TTT 1301 Glu Gly Gly Phe Lys Leu Gly Gin Arg Val His Val Lys Asn Glu Phe 325 330 335

GTA CCC GGC CAC ACA CGT TTT CCG GCC TAC ATC CGT GGC AAA GCA GGT 1349 Val Pro Gly His Thr Arg Phe Pro Ala Tyr Ile Arg Gly Lys Ala Gly 340 345 350 GTT GTA GTG GGT ATC TCG CCT GCC TAC CCC TAT CCA GAC GCG GCG GCG 1397 Val Val Val Gly Ile Ser Pro Ala Tyr Pro Tyr Pro Asp Ala Ala Ala 355 360 365 370

CAC GGC GAG TAC GGC TTT TCG GAA CCG ACG TAT GAC GTG TGC TTC AAG 14 His Gly Glu Tyr Gly Phe Ser Glu Pro Thr Tyr Asp Val Cys Phe Lys 375 380 385

TCG AAA GAC CTG TGG CCA GAC GGC TGC GAA GCC GCG GAT GTA CAC GTT 1493 Ser Lys Asp Leu Trp Pro Asp Gly Cys Glu Ala Ala Asp Val His Val 390 395 00

GGC GTA TTC CAA AGT TAC CTG TTG TCC GCA GAG GAG TGATGTCATG 153

Gly Val Phe Gin Ser Tyr Leu Leu Ser Ala Glu Glu 405 410

AGCCATGCTT CTGCCTTGCT CGATTCCGAC GCCGTTGACG CTGCTGCGTT CAGCAGCGTA 1599 ACGGAAATTC TCCACTGGTA TGGCTCTGGA AAGCGCACAC CTTCGACGGT GGTTGAAGTG 1659 TATTTGCAGC GCCTTGAAAA GCTACTGAG

(2) INFORMATION FOR SEQ ID NO: 4:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 4l4 amino acids

(B) TYPE: amino acid

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: protein

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4:

Met Thr Asp Asn Ala Val Met Glu Gin Arg Val Asp Ala Leu Phe Val

1 5 10 15

Leu Thr Lys Glu Leu Gly Leu Val Thr Asp Gin Thr Val Pro Asp Tyr

20 25 30

Glu Asp Ala Leu Met His Asp Trp Leu Pro Gin Asn Gly Ala Lys Leu

35 40 5

Val Ala Lys Ala Trp Thr Asp Pro Val Phe Lys Ala Gin Leu Leu Ser

50 55 60

Glu Gly Val Ala Ala Ser Glu Ser Leu Gly Phe Ser Phe Pro Lys His 65 70 75 80

His Lys His Phe Val Val Leu Glu Asn Thr Pro Glu Leu His Asn Val

85 90 95

Ile Cys Cys Ser Leu Cys Ser Cys Thr Ala Phe Thr Ile Gly Met 100 105 110 Ala Pro Asp Trp Tyr Lys Glu Leu Glu Tyr Arg Ala Arg Ile Val Arg

115 120 125

Gin Ala Arg Thr Val Leu Lys Glu Ile Gly Leu Asp Leu Pro Glu Ser

130 135 140

Ile Asp Ile Arg Val Trp Asp Thr Thr Ala Asp Thr Arg Tyr Met Val 145 150 155 160

Leu Pro Leu Arg Pro Gin Gly Thr Glu Asp Trp Ser Glu Ala Gin Leu

165 70 175

Ala Thr Leu Ile Thr Gin Asp Cys Leu Ile Gly Val Ser Arg Leu Glu

180 185 190

Ala Pro Phe Ala Ala Leu Pro Ala Pro Ala Val Ala Leu Gly Ala Met

195 200 205

Asp Gly Met His Asp Leu Gly Gly Lys Gin Gly Phe Gly Pro Val Ile

210 215 220

Lys Thr His Asn Ala Lys Ala Phe His Glu Glu Trp Glu Val Lys Met 225 230 235 240

Asn Ala Ile Ser Gly Ala Leu Val Ser Lys Gly Ile Tyr Asn Met Asp

245 250 255

Glu Tyr Arg His Gly Ile Glu Arg Met Glu Pro Arg His Tyr Leu Thr

260 265 270

Ala Ser Tyr Phe Glu Arg Val Phe Thr Thr Ala Val Thr Leu Cys Ile

275 280 285

Glu Lys Gly Val Phe Thr Ala Ala Glu Leu Glu Ala Lys Leu Gly Thr

290 295 300

Ser Val Pro Leu Ser Leu Pro Ser Ser Pro Gly Arg Gin Pro Ala Lys 305 310 315 ^• 320

Gly Pro Glu Gly Gly Phe Lys Leu Gly Gin Arg Val His Val Lys Asn

325 330 335

Glu Phe Val Pro Gly His Thr Arg Phe Pro Ala Tyr Ile Arg Gly Lys

340 345 350

Ala Gly Val Val Val Gly Ile Ser Pro Ala Tyr Pro Tyr Pro Asp Ala

355 360 365

Ala Ala His Gly Glu Tyr Gly Phe Ser Glu Pro Thr Tyr Asp Val Cys

370 375 380

Phe Lys Ser Lys Asp Leu Trp Pro Asp Gly Cys Glu Ala Ala Asp Val 385 390 395 400

His Val Gly Val Phe Gin Ser Tyr Leu Leu Ser Ala Glu Glu 405 410

(2) INFORMATION FOR SEQ ID NO: 5 '

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 38 amino acids

(B) TYPE: amino acid

(D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: peptide

(ix) ADDITIONAL FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 17

(D) OTHER INFORMATION: / note≈ "Xaa = Thr or Arg"

(ix) ADDITIONAL FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 33

(D) OTHER INFORMATION: / note≈ "Xaa ≈ Àsp or Pro"

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5:

Thr Asp Asn Ala Val Met Glu Gin Arg Val Asp Ala Leu Phe Val Leu

1 5 10 15

Xaa Lys Glu Leu Gly Leu Val Thr Asp Gin Thr Val Pro Asp Tyr Glu

20 25 30

Xaa Ala Leu Met His Asp 35

(2) INFORMATION FOR SEQ ID NO: 6:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 36 amino acids

(B) TYPE: amino acid

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: peptide

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6:

Met Asp Gly Met His Asp Leu Gly Gly Lys Gin Gly Phe Gly Pro Val

1 10 15

Ile Lys Thr His Asn Ala Lys Ala Phe His Glu Glu Trp Glu Val Lys

20 25 30

Met Asn Ala Ile 35

(2) INFORMATION FOR SEQ ID NO: 7:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomics)

(iii) HYPOTHETIC: NO

(iii) ANTI-SENSE: NO

(ix) ADDITIONAL FEATURE:

(A) NAME / KEY: modified_base

(B) LOCATION: 9

(D) OTHER INFORMATION: / note≈ "N ≈ inosine"

(ix) ADDITIONAL FEATURE:

(A) NAME / KEY: modified_base

(B) LOCATION: 12

(D) OTHER INFORMATION: / note≈ "N = inosine"

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7:

GAYAAYGCNG TNATGGARCA RMG 23

(2) INFORMATION FOR SEQ ID NO: 8:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 8 amino acids.

(B) TYPE: amino acid

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: peptide

(vii) IMMEDIATE SOURCE:

(B) CLONE: sequence deduced from the probe SEQ ID NO: 7

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8:

Asp Asn Ala Val Met Glu Gin Arg 1 5

(2) INFORMATION FOR SEQ ID NO: 9:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomics)

(iii) HYPOTHETIC: NO

(iii) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9:

CAYAAYGCBA AGGCBTTYCA YGA 23

(2) INFORMATION FOR SEQ ID NO: 10:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: peptide

(vii) IMMEDIATE SOURCE:

(B) CLONE: sequence deduced from the probe SEQ ID NO: 9

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10:

His Asn Ala Lys Ala Phe His Glu 1 5

Claims

CLAIMS:

1 - Enzymes having a nitrile hydratase activity, capable of catalyzing the transformation of nitriles into amides, characterized in that they have an enzymatic activity with respect to substrates having a nitrile function and a carboxylate function, greater than that vis with respect to substrates having at least two nitrile functions.

2 - Enzymes according to claim 1, characterized by a specific enzymatic activity (U _s ), with respect to cyano-5-valerate, expressed in moles of amide appeared x h ' ¹ x kg ^"1 of enzyme put in work and under given measurement conditions, greater than or equal to 400, preferably 1,000 and, more preferably still, 10,000.

3 - Enzyme according to claim 1 or claim 2, characterized in that it consists of two subunits,, β, whose peptide sequences are as shown in fig. la and lb (SEQ ID No: 1: and: 2 :) 4 - DNA sequence coding for an enzyme having nitrile hydratase activity, capable of hydrolyzing nitriles to amides, characterized in that it is chosen from the list of the following sequences:

- the DNA sequence, as shown in fig. 2 (SEQ ID No: 3:) and coding for an enzyme (SEQ ID No: 4:) having a nitrile hydratase activity,

- an analogue of this sequence resulting from the degeneration of the genetic code,

- a DNA sequence hybridizing with one of these sequences or a fragment thereof and coding for an enzyme having nitrile hydratase activity.

5 - Enzymes resulting from the expression of a DNA sequence according to claim 4 and having a nitrile hydratase activity.

6 - Microorganism constituted by the E. coli strain containing the plasmid pXL2222, strain referenced and deposited in the National Collection of Cultures of

Microorganisms under n ° I - 1 330 on July 9, 1993. 7 - Microorganism capable of producing at least one enzyme according to any one of claims 1 to 3 and 5.

8 - Microorganism according to claim 7, characterized in that it contains at least one expression cassette comprising the DNA sequence according to claim 4 and, upstream thereof, a promoter sequence and a site of ribosome fixation.

9 - Microorganism according to claim 8, characterized in that it belongs to the family of prokaryotes and in that the promoter is chosen from the following promoters: promoter of the tryptophan operon Ptrp from E. coli, promoter of l lactose operon Plac from E. coli, right promoter of phage lambda P _R , left promoter of phage lambda P, strong promoters of Pseudomonas, Comamonas or corynebacteria.

10 - Microorganism according to claim 8, characterized in that it belongs to the prokaryotic family and in that the ribosome binding site is chosen from that derived from the C11 gene from lambda, those derived from Comamonas genes or Pseudomonas, or those derived from corynebacteria genes.

11 - Microorganism according to claim 8, characterized in that it belongs to the family of eukaryotes, such as yeasts and in that the promoters are chosen from those of the yeast glycolytic genes, such as the genes coding for phospho -glycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, or those of lactase or enolase.

12 - Microorganism according to one of claims 7 to 11, characterized in that the expression cassette is carried by a plasmid.

13 - Microorganism according to claim 12, characterized in that it is selected from the following list of strains:

E. coli, Comamonas, Pseudomonas, Corynebacterium, Brevibacterium, Rhodococcus, Streptomyces, Bacillus.

14 - Enzymes, characterized in that they are produced by the micro¬ organisms according to any one of claims 7 to 13. 15 - Process for the enzymatic hydrolysis of nitriles into amides, characterized in that it consists: - putting the mono or di-nitriles corresponding to one of the following general formulas: NC - R - CN, NC - R - COO ^" Z ⁺ , NC - R - CONH ₂ in which R is an alkylene or alkenylene group, linear or branched, having from 1 to 18 carbon atoms and Z is chosen from the list of following compounds: alkaline, alkaline-earth, amino, ammonium, the latter compound being particularly preferred, in the presence of enzymes according to any one of claims 2, 3 and 14, or in the presence of a microorganism according to any one of claims 7 to 13. 16 - Method according to claim 15, characterized in that R = - (CH ^ -.