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  • C12N9/14—Hydrolases (3)
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  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes

Definitions

• the field of the invention is that of the enzymatic production of amide derivatives from compounds containing nitrile groups.
• 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.
• 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.
• an adipamate e. g. ammonium
• diammonium adipate e.g. ammonium
• NH nitrile hydratase
• Ni nitrilase
• A amidase
• R (CH Î )
• n being an integer equal to 4 in the case of adipic compounds.
• 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.
• 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.
• 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.
• These enzymes are used, either as such or, and preferably, in the form of recombinant microorganisms generating them.
• 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
• This enzyme 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.
• 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.
• 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 wild-type microorganism Comamonas testosteroni NI 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 ⁇ 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 ⁇ scherichia coli, the promoter of the lactose operon Plac of E.
• thermosensitive form P R CIts is preferred.
• 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).
• PGK phospho-glycerate kinase
• GPD glyceraldehyde-3-phosphate dehydrogenase
• ENO enolase
• 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.
• 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.
• 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.
• 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.
• 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:
• 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 .
• adipates such as ammonium adipate from a dinitrile, such as adiponitrile
• a mononitrile monoamide such as cyano-5-valeramide
• the present invention can be used for the preparation of carboxylates (adipates) useful as raw materials in industrial syntheses (polyamides).
• carboxylates adipates
• polyamides useful as raw materials in industrial syntheses
• Fig. 1a represents the peptide sequence of the ⁇ subunit of an enzyme in accordance with the invention (SEQ ID No: 1 :).
• Fig. lb represents the peptide sequence of the ⁇ 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 ⁇ 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 ⁇ 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 ⁇ and ⁇ 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 ⁇ and ⁇ and included in the strain E. coli TG1 deposited (G 4327).
• EXAMPLE 1 CHARACTERISA ⁇ ON 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.
• 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:
• 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 ⁇ ⁇ of 1.4 and a dry weight of 0.9 gxl "1 .
• 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.
• 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.
• the Comamonas Testosteroni NI 1 strain is cultured, in a stirred flask, at 28 ° C., for 23 h, in medium B:
• 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,
• 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 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.
• 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,
• 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.
• EXEMPIJS 3 CHARACTERIZATION OF THE HYDRATASE NT ⁇ ULE OF Comamonas NI 1.
• 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.
• subunit SEQ ID No: 5: in the list of sequences below
• the results of the nitrile hydratase activity measurements at different temperatures are listed in fig. 5. Between 10 and 30 ° C the ⁇ G of the reaction is 80 kJ / mole.
• 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.
• V M 35,000 moles or 5,000 kg of adipamate / h.kg of nitrile hydratase
• k ç ,, the turnover of adipamate / h.kg of nitrile hydratase
• this enzyme hydrolyzes mononitriles-monoacids and little dinitriles.
• EXAMPLE 4 CLONING OF NITRILE HYDRATASE FROM Comamonas testosteroni NI 1.
• nucleotide sequence of this first probe is designated by SEQ ID No: 7: and the corresponding amino acid sequence by SEQ ID No: 8:.
• 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:
• nucleotide sequence of this second ⁇ 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.
• 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 ⁇ g / ml ssDNA; hybridization temperature 50 ° C.
• the hybridization fingerprints show, in particular, the existence of a fragment single BglII of approximately 4 kb.
• 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.
• 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 ⁇ 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 CONIENAN ⁇ LES GENES
• 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 ⁇ 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 ⁇ 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 N ⁇ RILE HYDRATASE FROM Comamonas testosteroni NI 1 IN E. coli.
• 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).
• 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.
• 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 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.
• 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 ⁇ g / 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.
• 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 ⁇ g / ml of ampicillin and 100 ⁇ g / 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.
• MILLER JH 1972, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
• Each lane corresponds to an amount of protein equivalent to 60 ⁇ l of culture at an OD of 3 to 610 nm.
• Lane G contains the following molecular weight markers: 97.5, 66.2, 45,
• 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.

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