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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
  • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

• the present invention relates to the enzymatic hydrolysis of amides, in particular secondary amides.
• the invention relates first of all to a process for the enzymatic hydrolysis of substrates of the polyamide 6.6 type. leading to the two comonomers A and B of said substrates.
• the invention also relates to enzymes and / or microorganisms capable of being used in the enzymatic hydrolysis of amide functions, preferably on substrates containing at least one, such as for example polyamides (PA).
• PA polyamides
• the invention also relates to the genetic tools expressing these enzymes.
• E 3 active with respect to cyclic and linear oligomers of 6 aminohexanoic acid with DP n > 3 (PA.6).
• E 3 is described by NEGORO et al. in "Journal of Bacteriology, Dec. 1992, vol. 174, no. 24, p. 7948-7953.
• E 3 also comes from Flavobacterium sp KI 72.
• the present invention relates firstly to an enzymatic hydrolysis process and, secondly, to an enzyme.
• the process according to the invention for hydrolysis of (poly) amides characterized:
• A, B are monomeric units
• R 1 , R 3 are divalent radicals, identical or different - preferably different - and representing a (cyclo) alkylene, linear or branched, substituted or not, an arylene, an arylalkylene, the aromatic radicals being optionally polycondensates, the alkylene having a carbon number greater than or equal to 4 and preferably between 4 and 12.
• R 2 corresponds to radicals, identical or different - preferably identical - and selected from hydrogen and / or alkyl residues having, advantageously, from 1 to 6 carbons,
• X 1 OH, OM, OR 4 with M chosen from metals, preferably alkali and alkaline-earth metals and R 4 representing a linear or branched alkyl comprising from 1 to 6 carbon atoms,
• the present invention also relates to a family of enzymes of the amidase type, capable (among others) of being used in the process defined above.
• the enzyme belonging to the family according to the invention is an amidase, characterized: - ⁇ ⁇ in that it is active, in particular with respect to substrates of (poly) amide type and corresponding to the following formula:
• R 1 , R 3 are divalent radicals, identical or different - preferably different - and representing a (cyclo) alkylene, linear or branched, substituted or not, an arylene, an arylalkylene, the aromatic radicals being optionally polycondensates, the alkylene having a carbon number greater than or equal to 4 and preferably between 4 and 12,
• R 2 corresponds to radicals, identical or different - preferably identical - and selected from hydrogen and / or alkyl residues having, advantageously, from 1 to 6 carbons,
• X 1 OH, OM, OR 4 with M chosen from metals, preferably alkali and alkaline-earth metals and R 4 representing a linear or branched alkyl comprising from 1 to 6 carbon atoms,
• an enzyme is advantageously isolated having an amidase activity and comprising the peptide sequence as represented in the attached SEQ ID NO: 1, or a homologous peptide sequence having at least 80%, of preferably at least 90% and more preferably still at least 95% homology with SEQ ID NO: 1.
• this enzyme has amidase activity, in particular with respect to substrates of (poly) amide type and corresponding to the following formula (II):
• the invention firstly proceeded to the isolation of a wild strain producing the enzyme, namely: Comamonas acidovorans N 12.
• This wild strain is the result of long-term screening work. This identification was made from morphological, cultural, biochemical and antigenic characters, which could be determined by reference to official and international criteria of microbiological taxonomy.
• the Applicant's merit is not limited to the isolation of this wild strain, but also extends to the isolation of the amidase defined above.
• the microorganism Comamonas acidovorans N 12 is not the exclusive biological precursor of this amidase. It is indeed also necessary to consider all the recombinants and any wild strain having a similar enzymatic activity. Recombinant microorganisms are those having in their genome a DNA sequence coding for the amidase considered in the present invention.
• the invention also relates to an enzymatic hydrolysis process in which the abovementioned enzyme and / or the wild micro ⁇ organism referred to above and / or at least one of its recombinants mentioned above is used. -above.
• the substrates of the enzyme and / or their biological precursors are polyamides and, more precisely, oligomers whose recurrent polymerization function is a secondary amide function --CO - NH -
• These oligomers have repeating units of formula (I) and / or (H).
• the repeating units (I) are advantageously formed of two monomer units A and B, respectively dicarbonyl and diamine and linked to one another by a secondary amine function.
• a preferred example of a dimer is that formed by:
• the corresponding polyamide is PA 6.6.
• the recurring units (II) are preferably formed by noted monomeric units
• ⁇ and ® are, respectively, carboxylic and amino.
• a typical example of is a derivative of ⁇ -aminocaproic acid, a polyamide 6 (PA 6) monomer.
• polyamide-type oligomers which may be suitable for the invention, there may be mentioned:
• polyamide oligomers obtained by polycondensation of saturated aliphatic dicarboxylic acids having from 6 to 12 carbon atoms with saturated aliphatic primary diamines having from 6 to 12 carbon atoms,
• polyamino acid oligomers obtained, either by direct homopolycondensation of ⁇ -aminoalkanoic acid comprising a hydrocarbon chain having from 4 to 12 atoms carbon, either by hydrolytic opening and polymerization of lactams derived from these acids,
• the oligomers of copolyamides obtained from the starting monomers of the abovementioned polyamides possibly also consisting, in part, of aromatic acid, such as terephthalic acid and / or isophthalic acid,
• polyamides obtained by polycondensation of diacids and diamines there may be mentioned, for example:
• polyamino acids which may be suitable, mention will be made of: - polyamide 4 (polymer of 4-amino butanoic acid or of ⁇ -butyrolactam),
• Polyamide 12 (polymer of 12-amino-dodecanoic acid or of laurolactam).
• copolyamides there may be mentioned, for example: - polyamide 6.6 / 6.10 (copolymer of hexamethylenediamine, adipic acid and sebacic acid),
• the preferred substrates according to the invention are oligomers of polyamides obtained by polycondensation of diacids A and diamines B and, in particular, of PA 6.6.
• the number of monomers A and B, on the one hand, or ⁇ ⁇ ⁇ - ', on the other hand, of the substrates (I) and (H) according to the invention is advantageously between 2 and 8, preferably between 2 and 6. It should be noted that this number of monomers A and B in the molecules of oligomers or polymers will also be called DP ⁇ in the present description.
• Characteristic substrates (I) are, eg, advantageously water-soluble oligomers such as: AB, ABA and ABAB.
• this number of monomers A and B in the molecules of oligomers or polymers will also be called DP ⁇ in the present description .
• These particular substrates are part of those (I) and (H) that the enzyme of the invention and / or its biological precursors are capable of hydrolyzing, because they have at least one carboxyl end.
• the final products of hydrolysis can be monomers A and B.
• the substrates are oligomers (H) (monomers (a) (ç) - (b ⁇ )
• the final products of hydrolysis can be monomers (a ⁇ (c) (jT).
• the enzyme according to the invention can also be characterized through its activity and / or its affinity with respect to some of the above-mentioned substrates.
• a second advantageous characteristic of this amidase is to be active with respect to a substrate (I), formed by an ABA trimer and to be able to transform this trimer into a monomer A and a dimer AB, and this with a specific enzymatic activity greater than or equal to 1000 ⁇ mol of hydrolyzed substrate.h'l.mg "! of protein.
• a third advantageous characteristic of this amidase is to be active with respect to a substrate (I) formed with a type AB dimer and to be able to transform this dimer into two monomers A and B, with a specific enzymatic activity greater than 1,500 ⁇ mol of hydrolysed substrate.h "l.mg" l of protein.
• the amidase enzyme of the invention has at least one of these three non-limiting characteristics.
• the implementation of the enzyme of the invention in the enzymatic hydrolysis of amides can consist in using only the enzyme per se instead of the biological precursors (wild or recombinant microorganisms) producers of it or even a mixture of the two.
• the enzyme which is the subject of the invention is also characterized in that it is produced by microorganisms of the type Comamonas acidovorans (N 12), preferably of the type of the strain referenced and deposited in the National collection of Cultures of Microorganisms - Institut Pasteur PARIS under n ° 1 1522 on January 4, 1995, and / or by the recombinants as described supra.
• N 12 Comamonas acidovorans
• the present invention also relates to new microorganisms capable of producing the amidase enzyme according to the invention, as defined above. Due to this ability, these microorganisms have in particular the characteristics, on the one hand, of a capacity for hydrolysis of the amide functions of a polyamide compound, comprising at least one amide function and, in particular, of oligomers. More specifically, these microorganisms have a specific selectivity and hydrolysis activity, with respect to the substrates (I) and (H) defined above.
• the oligomers are, for example, the water-soluble substances mentioned above: ABAB and / or ABA and / or AB among others.
• these microorganisms can preferably be constituted by the above-mentioned Comamonas acidovorans, preferably by the strain referenced and deposited in the National Collection of Cultures of Microorganisms under the number 1 1522 on January 4, 1995, or by recombinants thereof, as described above.
• the microorganisms according to the invention are capable of allowing the hydrolysis of at least one substrate formed by a polyamide oligomer and, more precisely, by a dimer AB, dedicated to being transformed into two monomers A and B, the microorganisms being capable of ensuring hydrolysis.
• the present also relates to the genetic material under the aegis of which, it can be synthesized, via recombinant microorganisms or not.
• the present invention relates to a DNA sequence coding for an enzyme having amidase activity, characterized in that it is chosen from the list of the following sequences: ⁇ the DNA sequence, as represented by SEQ ID NO: 2 in the annexed sequence list and coding for at least one enzyme having amidase activity, ⁇ an analogue of this sequence resulting from the degeneration of the genetic code, ⁇ a DNA sequence hybridizing with the above sequence or with minus a fragment thereof and coding for an enzyme having amidase activity.
• the enzymes resulting from the expression of the above DNA sequence are also included in the scope of the invention.
• Wild microorganisms eg I 1522
• recombinant microorganisms isolated by the Applicant each contain at least one expression cassette comprising the DNA sequence SEQ ID NO: 2 referred to above, and optionally, upstream thereof at least a promoter sequence and at least one ribosome binding site.
• the present invention also relates to a process for the hydrolysis of substrates, at least partly formed by those (I) and / or (H), as defined above, characterized in that it consists in implementing at least one enzyme and / or at least one of the micro ⁇ organisms as presented above. It may be advantageous, in accordance with the invention, to have several enzymes with complementary spectra.
• one of the variants of the above process can be to use at least one other type of enzyme and / or at least one of its wild and / or recombinant biological precursors.
• the substrates are, at least in part, constituted by oligomers whose DP n is less than 40, preferably 20 and, more preferably still, 12 and which are derived from polyamides at least derived from polycondensation between diacid (A) and diamine (B) monomers.
• the hydrolysis process according to this variant is characterized:
• At least one enzyme and / or at least one microorganism as defined above are provided.
• enzyme • and at least one other type of enzyme and / or at least one of its wild and / or recombinant biological precursors, said enzyme being: * an enzyme E 3 produced under the aegis of the nyl-c gene of Flavobacterium sp KI 72,
• PAM I an enzyme called PAM I, such as that defined by the peptide sequence SEQ TD NO: 3 annexed, such an enzyme can, inter alia, be produced by the microorganism referenced and deposited in the National Collection of Cultures of Microorganisms - Institut Pasteur PARIS - under n ° I
• a carbon source comprising, preferably at least one compound comprising at least one amide function, said carbon source optionally comprising a supplement chosen, advantageously, from carbohydrates, sucrose being particularly preferred,
• a compound capable of inducing the production of enzymes without being consumed by biological precursors, said compound preferably being chosen from amides.
• the process for the enzymatic hydrolysis of amides according to the invention can find numerous applications, for example in organic synthesis for the manufacture of compounds from amide compounds or for the treatment of materials containing polyamide. In particular, it could be of interest in the context of the regeneration of raw materials for polyamide polymers.
• SEQ JO NO: 3 amino acid sequence corresponding to PAM I.
• SEQ ID NO: 4 DNA coding for the enzyme PAM I.
• FIG. 1 represents the restriction map of the plasmid pXL2297 containing the gene (pam I) coding for the amidase PAM I.
• FIG. 2 represents the restriction map of the plasmid pXL2564, containing the gene (pam I) coding for PAM I.
• EXAMPLE 1 ISOLATION AND IDENTIFICATION OF THE WILD STRAIN COMAMONAS ACIDOVORANS ⁇ 12.
• MICROBIOLOGICAL SCREENING A vast microbiological screening or screening operation made it possible to select the Comamonas acidovorans N 12 strain, from various biotopes.
• microorganism was carried out using an amidasic activity test on synthetic oligomers of PA 6.6. The activity evaluation will be seen in detail below.
• the microorganism selected was then identified on the basis of its morphological, physiological and biochemical and possibly antigenic characters, in a completely traditional way, by the Bacteriology Laboratory of the iNSTITUT PASTEUR.
• the optimum culture medium is M9YE3 having the following composition:
• the cultures are carried out in Erlenmeyer flasks filled to 1/5 of their total volume. The media are incubated at 30 ° C and stirred at 150 rpm. The preculture consists of 10 ml of LB medium:
• the culture is then seeded at 1% with the optimum medium (v / v).
• the cell production is determined by a dry extract, obtained by drying overnight at 105 ° C. from a cell pellet, obtained after centrifugation (10 min at 12,000 g).
• the carbon source comprises, together with the soluble substances, a supplement chosen, advantageously, from carbohydrates, sucrose being particularly preferred.
• the activity measurement is determined at 28 ° C. in a phosphate buffer 10.5 mmol / l, at pH 7.5 and with a final volume of 1 ml. TABLE 1 below gives the results obtained.
• the activity of this enzyme is monitored by measuring the hydrolysis activity of ABAB into BAB + A.
• EDTA acid Ethylene Diamine Tetra Acetic CHAPS ⁇ 3 [(3 cholamidopropyl) -dimethylammonio] - 1 -propanesulphonate
• SDS sodium dodecyl sulphate
• Comamonas acidovorans cells are resuspended in 75 ml of 100 mM Tris-HCl pH 7.5 buffer containing 1 mM DTE, 5 mM EDTA, 100 mM KC1 and 15% v / v of glyerol.
• the suspension is then subjected to a discontinuous treatment (10% treatment, 90% rest) by ultrasound in melting ice for 70 min.
• this pellet which also contains polyamidase II activity which is subsequently treated. It will be noted that it is possible after desalting the 90 ml of supernatant in the same buffer without glycerol and new ultracentrifugation to purify and to reconcentrate in a new very fine pellet the polyamidase II activity apparently soluble during the first centrifugation. In fact, by decreasing the density of the buffer (by eliminating the glycerol), the fall of very fine particles is accelerated.
• the supernatant (S2) is then collected and subjected to a discontinuous treatment (10% treatment, 90% rest) with ultrasound in melting ice for 20 min. Then, centrifuged again for 2 h at 50,000 g and the supernatant (S3) is collected.
• Tris-HCl pH 8 containing 1 mM DTE, 8 mM CHAPS, 1.5 M ammonium sulphate and 15% v / v of glycerol. These 10 ml are then chromatographed twice on a Phenylsuperose HR10 / 10 column (Pharmacia) balanced in the 25 mM Tris-HCl pH 8 buffer. containing 2 mM DTE, 8 mM CHAPS, IM ammonium sulphate and 15% v / v glycerol. The elution is done by a linear gradient over 70 min from 1 to OM ammonium sulfate with a flow rate of 1.25 ml / min.
• the polyamidase activity is eluted at about 0.8 M ammonium sulfate.
• the active fractions are combined and concentrated to 400 ⁇ l on Centriprep 10 (Amicon).
• Step 3 The previous 400 ⁇ l is chromatographed twice on a column
• TSK G3000 SW (Supelco) balanced in 100 mM Tris-HCl pH 7.5 buffer containing 2 mM DTE, 8 mM CHAPS, 150 mM NaCl and 15% v / v glyerol and eluted at 0.5 ml / min.
• the activity is found from exclusion to a molecular weight of around 30 kDa.
• sequence SEQ H) NO: 1 was obtained using standard nucleotide sequencing techniques.
• EXAMPLE 4 ENZYMATIC HYDROLYSIS OF OLIGOMERS FORMED BY MONOMERS A AND B CONNECTED TO EACH OTHER BY SECONDARY AMIDE LINES USING A MIXTURE OF ENZYMES ACCORDING TO THE INVENTION AND HYDROLASE PAM I.
• the present example it is not the pure enzymes that are used, but their biological precursors, namely the Comamonas acidovorans NI 2 strain according to the invention and a strain harboring the pam I gene (SEQ ID NO: 4 attached) and producing the enzyme PAM I (SEQ D3 NO: 3 attached), said strain being constructed according to the protocol given below.
• This construction aims to obtain the pam I gene (coding for the enzyme PAM I) which is preceded by the binding site of the ribosomes of the cil gene on page ⁇ and which is expressed from the promoter of the tryptophan érE operon. Coli.
• an Ndei restriction site was created at the initiation codon of pam I by the PCR technique using as matrix the plasmid pXL2297 (FIG. 1) hosted by the strain I 1495 deposited at the CNCM on 29 November 1994.
• the amplified Hindlll-Apal fragment was cloned into pUC29 (Benes et al. (1993), Gene 130: 151-152) digested with Hindlll] and Apal and the pam I gene was reconstituted by introducing the Apal fragment of Apal to the Apal site.
• the plasmid pXL2564 (described in FIG. 2), is a derivative of pBR322 (Sucliffe (1978), Nucleic Acid. Res. 5: 2721) containing a gene conferring resistance to ampicillin and the pam I gene under control of the Pt ⁇ -RBScll expression cassette.
• the plasmid pXL2564 was introduced into the strain of E. Coli TG1, the selection of microorganisms being done on LB ampicillin.
• a clone containing pXL2564 (called strain PAM I) was subcultured twice on agar plates and cultured at 37 ° C. in M9 glucose medium containing 100 ⁇ g / ml of ampicillin according to the procedure described in patent FR 2 694 571.
• the Comamonas acidovorans NI 2 strain was cultivated for 19 hours under the same conditions as those defined in paragraph 2.1 above.
• oligomers of average DP n 8 are resuspended in 300 ml of water. 200 ml of water containing the cells (ie 3.1 g in total) are added. The whole is stirred for 18 hours under a stream of nitrogen.
• the solution is centrifuged and the supernatant (400 ml) is recovered. A dry weight of ⁇ 4 g / l is measured on the supernatant. A fraction is given for analysis of the oligomers of adipic acid and HMD.
• the molar yield of adipic acid monomer formed is 66%, while that of HMD is 58%.
• the molar yield of monomers and oligomers of Dp of less than 4, expressed in moles of monomers, is 72%.
• CAAATTGCAT CGAGCCTATT CAGCACAACT CCCGAAAACC GGGCGGCAAC TTTCCGGAAT 180

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