WO1994017190A1 - Polypeptides having amidase activity, genetic tools and host microorganisms for obtaining same - Google Patents

Polypeptides having amidase activity, genetic tools and host microorganisms for obtaining same Download PDF

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WO1994017190A1
WO1994017190A1 PCT/FR1994/000080 FR9400080W WO9417190A1 WO 1994017190 A1 WO1994017190 A1 WO 1994017190A1 FR 9400080 W FR9400080 W FR 9400080W WO 9417190 A1 WO9417190 A1 WO 9417190A1
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PCT/FR1994/000080
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Edith Cerbelaud
Anne-Marie Le Coq
Sophie Levy-Schil
Dominique Petre
Fabienne Soubrier
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Rhone Poulenc Chimie
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Priority to JP6516742A priority Critical patent/JPH08504596A/en
Priority to KR1019950703079A priority patent/KR960700343A/en
Priority to EP94905744A priority patent/EP0681610A1/en
Publication of WO1994017190A1 publication Critical patent/WO1994017190A1/en

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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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • 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)

Definitions

  • the field of the invention is that of the enzymatic production of carboxylic derivatives (carboxylates) from compounds containing amide groups.
  • the present invention relates to new polypeptides having amidase activity, the genetic material involved in their production, as well as the microorganisms containing this genetic material and exhibiting this activity.
  • the invention also relates to a process for the enzymatic hydrolysis of amides to carboxylates in which these new polypeptides or microorganisms are synthesized, including, in particular, the above-mentioned host micro ⁇ organisms.
  • the amides which are of particular interest in the context of the invention, are: cyano-5 valeramide, adipamide or adipamate of any cation, preferably chosen from the list of following compounds: alkaline, alkaline-earth, amino, ammonium, the latter compound being particularly preferred.
  • the aim is the enzymatic transformation of the amide functions into carboxylate functions, so as to ultimately obtain 5-cyanovaleric acid, adipic acid or salts thereof, e. g. : 5- ammonium cyanovalerate, diammonium adipate.
  • the whole industrial interest of adipates, and in particular of ammonium adipate lies in the fact that it constitutes one of the fundamental bases for the manufacture of nylon-6,6.
  • 5-cyanovalerates they are capable of constituting starting materials of choice, for the preparation of caprolactam, giving itself access to nylon-6.
  • amidases The action of these amidases is part of a known synthetic scheme enzymatic of diammonium adipate, from a dinitrile, namely adiponitrile. This diagram is as follows:
  • NH nitrile hydratase
  • Ni nitrilase
  • R (CH 2 ) n , n being an integer equal to 4 in the case of adipic compounds.
  • such an amidase is capable of hydrolyzing adipamide to adipic acid (or ammonium adipate).
  • Brevibacterium R 312 or that derived from a recombinant microorganism comprising the gene for expression of this enzyme.
  • one of the essential objectives of the present invention is to provide new enzymes with amidasic activity, genetic material allowing their production and microorganisms containing this genetic material and allowing this production, said enzymes and microorganisms being characterized. , both by satisfactory yields of production of carboxylates from substrates of the amide type of various natures (mono or diamides) and by an spreadsheet activity, with respect to the soluble salts of amides, which is significant and industrially advantageous .
  • the Applicant has succeeded in achieving, among other things, this objective by isolating, purifying and characterizing new enzymes capable of hydrolyzing amides to carboxylates with strong activities and by transforming, preferably, soluble salts of amides, said enzymes being used either as such or, and preferably, in the form of recombinant microorganisms generating them.
  • the subject of the present invention is therefore new polypeptides having an amidase activity, capable of hydrolyzing amides into carboxylates and having an enzymatic activity vis-à-vis ammonium adipamate greater than that which they have vis-à-vis -vis adipamide.
  • Comamonas testosteroni NI 1 More specifically, this polypeptide 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 the cell culture , to rush this extracted with ammonium sulfate and to purify it by various 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.
  • amidase activity is meant, in the present description, the enzymatic hydrolysis of an amide, such as cyano-5 valeramide, adipamide and adipamate of a cation Z, into a carboxyl, namely, respectively , Z-cyano-5 valerate, Z adipamate and Z adipate.
  • the cation Z can be any, but it is preferably chosen from the following compounds: alkali, alkaline earth, amino, ammonium , the latter compound being particularly preferred.
  • polypeptides with amidase activity in accordance with the invention is a polypeptide characterized by the peptide sequence, as shown in FIG. 1 (SEQ H): 1 from the attached sequence list).
  • Another subject of the invention relates to a DNA sequence coding for a polypeptide having an amidase activity, capable of hydrolyzing amides to carboxylates, and chosen from the following list of sequences:
  • polypeptides resulting from the expression of one of the above DNA sequences are included in the scope of the invention.
  • the wild micro-organism Comamonas testosteroni NI isolated by the Applicant, contains one of the above sequences in its genome.
  • 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 be either naturally present or integrated into the genome of the host 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 polypeptide.
  • the heterologous promoter can be chosen from strong bacterial promoters, such as the promoter of the tryptophan Ptrp operon from Escherichia 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.
  • the promoters can 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
  • ribosome binding sites that derived from the CII gene lambda, as well as those derived from Comamonas or Pseudomonas genes or those derived from Corynebacteria genes are preferably used when the host micro ⁇ organism 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 streptomycin or to other products toxic to bacteria.
  • amidase according to the invention already constitutes, in itself, a selection marker for the host which. contains.
  • 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 polypeptide according to the invention. These microorganisms may or may not contain at least one expression cassette, of the type described above.
  • enterobacteria such as E. coli
  • bacteria belonging to the genera Comamonas or Pseudomonas there may be mentioned, in particular, enterobacteria such as E. coli, bacteria belonging to the genera Comamonas or Pseudomonas , coryneform bacteria, such as those belonging to the genera
  • This microorganism is the E. Coli strain which contains the plasmid pXL2216 (described below).
  • This microorganism is also identified by the Applicant by the reference G 4315.
  • polypeptides produced by the above microorganisms are also included in the invention.
  • the latter also relates to the process for transforming amides into carboxylates using a polypeptide according to the invention or a recombinant microorganism generating it.
  • This method consists in bringing the amide to be transformed into contact with a polypeptide or a recombinant microorganism, as defined above.
  • the polypeptide 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 Pat. No. 4,732,851 .
  • the process of the invention is suitable for the transformation of amides, into carboxylates and, more particularly, for the transformation into carboxylates of mono and diamides of formulas:
  • 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 the enzymatic synthesis, on the one hand, of an adipate, such as ammonium adipate from adipamide and especially from an adipamate, such as ammonium adipamate and, on the other hand, cyano-5 valerate from cyano-5 valeramide.
  • an adipate such as ammonium adipate from adipamide and especially from an adipamate, such as ammonium adipamate and, on the other hand, cyano-5 valerate from cyano-5 valeramide.
  • Fig. 1 represents the peptide sequence of a polypeptide in accordance with the invention (SEQ ID: 1).
  • Fig. 2 represents a DNA sequence according to the invention (SEQ ID: 2).
  • 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 specific activity of amidase according to the invention (micromoles of adipate xh 1 x mg "1 of protein) as a function of the temperature in ° C.
  • FIG. 5 represents a curve of the specific activity of the amidase according to the invention (micromoles of adipate xh “1 x mg “ 1 of protein) as a function of the pH.
  • Fig. 6 represents a variation in the specific activity (micromoles of adipate xh "1 x mg " 1 of protein) of the amidase according to the invention with respect to adipamate, as a function of the concentration of adipamate ( millimoles xl '1 ).
  • Fig. 7 shows a curve giving the ratio of the concentration of adipamate to the specific activity (liters of adipate xh “1 xg " 1 of protein) as a function of the concentration of adipamate (millimoles xl "1 ).
  • Fig. 8 represents the restriction map of the plasmid pXL2104 containing the amidase gene according to the invention.
  • FIG. 9 represents the sequence of a 1491 bp fragment containing the gene coding for the amidase of Comamonas testosteroni NI 1.
  • FIG. 10 shows the restriction map of the plasmid pXL2216 containing the amidase gene according to the invention.
  • Fig. 11 shows the restriction map of the plasmid pXL2144 containing the Spel-ClaI fragment derived from the plasmid pXL2104 between the Xbal and ClaI sites of the vector pMTL22.
  • Fig. 12 represents the electrophoresis on SDS-PAGE gel showing the expression of the DNA sequence according to the invention in the strain E-Coli TG 1.
  • Fig. 13 shows the restriction map of the plasmid pXL2238 containing the amidase gene according to the invention.
  • IPTG isopropyl ⁇ -O thio galactopyranoside
  • HPLC high performance liquid chromatography
  • PS dry weight
  • the Comamonas testosteroni NI 1 strain is a strain isolated from a soil sample by microbiological screening. Its performance is evaluated on adipamate and adipamide.
  • 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 by medium A. After 22 h, 3.5 g ⁇ l 1 of wet cells are harvested. This corresponds to an OD 660mn of 1.4 and a dry weight of 0.9 gxl "1 .
  • a cell pellet containing 31 mg of dry cells is suspended in 5.5 ml of a 20 mM adipamate or adipamide solution in potassium phosphate buffer pH 7.50 mM. The reaction is carried out at 25 ° C. with stirring and the kinetics are followed by sampling. Adipamide, adipamate and adipate are measured on each sample by HPLC. The rates of formation of adipate, from adipamide and adipamate, are respectively 8.3 and 33 Uxg- 1 of dry cells (1 U is equal to 1 micromole of product formed per minute ) (whole cells) By comparison, the activities given in TABLE I below are obtained and measured under the same conditions, with Brevibacterium R 312 and Rhodococcus sp.
  • the Comamonas NI 1 strain is cultivated as a 15 1 fermenter on medium B, the composition of which is:
  • the pH, the temperature, the air flow and the stirring speed are regulated, respectively, at 6.6, 28 ° C, 300 l / h and 350 rpm. After 4 h of fermentation,
  • buffer B 100 mM Tris-HCl pH 7.5, 10% glycerol.
  • the amidase activity of the fractions is determined at pH 7.5 and at 25 ° C. in buffer B diluted 10 times in the presence of 10 mM adipamate.
  • the protein concentration of the pools is determined by the Coomassie blue method (PIERCE Protein assay kit).
  • the activity unit U corresponds to 1 ⁇ mole of adipate produced per minute in test conditions.
  • the protein panel is analyzed by polyacrylamide-SDS gel (Phastsystem PHARMACIA).
  • the dialyzed fraction (43 ml) is loaded at a flow rate of 130 mlxh 1 onto a column (26 x 380 mm) of "Q sepharose flast flow" balanced with buffer B at a flow rate of 200 mlxh "1.
  • the column is percolated at a rate of
  • Step 3 column of hydrophobic interactions (Phenyl Sepharose): The 168 ml are concentrated by precipitation with ammonium sulfate at 65% of saturation. For this, 72 g of ammonium sulfate are added. After one hour of stirring at 4 ° C, the suspension is centrifuged at 30,000 gmax for one hour. The pellet is resuspended in buffer B and dialyzed against 2 l of buffer B overnight.
  • the 80 ml are concentrated by ultrafiltration on an Amicon PM10 membrane and then on centriprep 10. These 2.8 ml are loaded onto the gel filtration column (16 x 600 mm) balanced with buffer B supplemented with 0.1 M KC1 at a flow rate of 0.8 mlxmin "1.
  • This second filtration gel is carried out under conditions identical to the previous one, but in two stages.
  • the assays are only carried out on the first pass, made from a fraction of 3 ml of the 9 ml of the previous step.
  • anion exchange column (mono Q HR 5/5 Pharmacia): This step is carried out in two stages, using half of the preceding fraction each time and with buffer B / 5.
  • the buffer is modified by dialysis on an Amicon PM10 membrane and substitution with B / 5 buffer.
  • the 6 ml of retentate are loaded onto the column balanced with buffer B / 5, at a flow rate of 1 ml ⁇ min 1 .
  • a gradient of 0 to 0.15 M in KC1 is applied in 25 min.
  • the column is then fed with a solution of 1 M KC1 in buffer B / 5. In two batches, 3.75 ml are collected and analyzed (prot.
  • - Step 9 column of hydrophobic interactions (octyl superose HR 5/5):
  • the 3 ml of the pool from the previous step are brought to 38% of saturation by adding a concentrated solution of ammonium sulfate.
  • the resulting 6 ml are loaded onto the column, equilibrated with buffer B supplemented with 1.7 M ammonium sulfate.
  • the proteins are eluted by a decreasing gradient of ammonium sulphate from 1.7 M to 0 M in buffer B in 1 h at a flow rate of 0.5 ml ⁇ min 1 .
  • Fig. 3 attached shows the results of a polyacrylamide gel analysis
  • the band corresponding to the amidase according to the invention is that of approximately 40 kDa.
  • the molecular weight is 80,000 +/- 2,000 Da.
  • Amidase is therefore a homodimeric enzyme.
  • a pool of fractions containing amidase was used for this study. Under the conditions of the purification, this pool contains 0.3 g ⁇ 1 ⁇ 1 of protein and has a specific activity of 26 U ⁇ mg ⁇ 1 of protein. The activity of this pool was determined between 10 and 60 ° C.
  • the results are grouped in fig. 5.
  • the optimum pH is between 8.5 and 10, which corresponds well to the behavior generally encountered for amidases.
  • EXAMPLE 3 CLONING OF THE GENE CODING FOR THE AMIDASE OF Comamonas testosteroni NI 1.
  • nucleotide probe was synthesized. It is a 20 deg 256 degenerated sea: MIENIIA (SEQ ID: 5)
  • the 20 base sequence is designated by SEQ ID: 4 while the amino acid sequence associated therewith is referenced SEQ ID: 5.
  • the strategy followed consisted, first of all, in verify the specificity of this nucleotide probe and determine 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 probe.
  • the probe has sufficient specificity under the hybridization conditions used (5x SSC, 5x Denhardt, 0.1% SDS, 50 mM NaPO 4 pH 6.5, 250 ⁇ gxml 1 ssDNA, hybridization temperature 50 ° C. washing: 1 h, 6x SSC, room temperature and 5 min in 2 x SSC, 0.1% SDS at 50 ° C). Under these conditions, the probe allowed us to obtain important and unambiguous signals. In particular, in the case of digests with Sstl, Pstl and BglLL. The hybridization fingerprints show, in particular, the existence of a single Sst1 fragment of approximately 5.5 kb.
  • EXAMPLE 4 SEQUENCE OF A 1491 bp FRAGMENT CONTAINING THE GENE ENCODING FOR THE AMIDASE OF Comamonas testosteroni NI 1.
  • the location of the 1491 bp sequence fragment containing the gene coding for the amidase of Comamonas testosteroni NI 1 is indicated under the clone insert in FIG. 9.
  • the strategy for sequencing this fragment produced according to conventional methods known to those skilled in the art, is shown in the same figure.
  • the various sequences were all obtained by the chain termination method (sequenase kit in the presence of 7-daza dGTP; ( 35 S) dATP) either on single-stranded matrices of M13 (mpl8 or m ⁇ l9 cf YANISH et al., Gene 33 (1985) 103) recombinant carrying subfragments, either directly on the plasmid pXL2104.
  • amdA the structural gene for the amidase sought. This gene will be called amdA in the remainder of this description.
  • plasmid pXL2216 was prepared by insertion of the Spel-ClaI fragment containing the amdA gene between the Xbal and ClaI sites of the vector pMTL 23 (CHAMBERS et al., Gene, 68 (1988) 139-149). This plasmid, the restriction map of which is presented in FIG. 10, was used to transform the strain of E. coli TG 1. The resulting strain called G 4315 was deposited at the National Collection of Culture of Microorganisms in Paris (Institut Pasteur, 25 rue du Do Budapest Roux) under No. 1 1279.
  • Comamonas testosteroni NI 1 a plasmid was constructed in which the amdA gene, preceded by its own ribosome binding site, is placed under the control of the promoter of the lactose operon of E. coli.
  • the plasmid pXL2144, described in FIG. 11 was obtained by insertion of the Spel-Cl ⁇ l fragment derived from the plasmid pXL2104 between the Xbal and ClaI sites of the vector pMTL 22 (CHAMBERS et al., Gene,
  • This plasmid therefore contains the promoter of the lactose operon Plac, followed by the ribosome binding site and the structural gene for amidase, as well as a gene conferring resistance to ampicillin.
  • the plasmid pXL2144 was used to transform the strain of E. coli TG 1.
  • the selection of the microorganisms is done on a picillin.
  • the expression of the amidase of Comamonas testosteroni NI 1 was visualized, after sonication of the cells, by SDS-PAGE gel, in the crude fraction or, after centrifugation, in the pellet and in the supernatant.
  • Each lane corresponds to an amount of protein equivalent to 60 ⁇ l of culture at an OD of 3 to 610 nm.
  • Fig. 12 presents the results obtained.
  • the molecular weights of the markers are indicated in kilodaltons by the arrows to the right of the gel in this figure.
  • the activity of the E. coli strain (pXL2144), whether induced or not, is measured on adipamide and adipamate and compared to that of the control strain E. coli (pMTL 22).
  • the amidase was expressed in Pseudomonas putida using the following construct: From pXL2216 described in FIG. 10, the 1.75kb SaWBgtll fragment containing the amdA gene was introduced between the SalI and Bam I sites of pDSK519 (Keen et al., 1988, Gene. 70: 191).
  • This plasmid, pXL2238, described in FIG. 13, was introduced into the strain of Pseudomonas putida G 2081.
  • G 2081 is a derivative of Pseudomonas putida 2440 (Bagdasarian and Timmis, 1981. In Hofschneid and Goebel. Topics in Microbiology and Immunology. 47 Springer Verlag. Berlin.) Made resistant to nalidixic acid and rifampicin.
  • the vector pDSK519 was used as a control plasmid.
  • strain G 2081 (pXL2238) is measured on adipamate and compared to that of the control strain G2081 (pDSK519).
  • the Pseudomonas G 2081 (pDSK519) and Pseudomonas G 2081 (pXL2238) strains were cultured in LBk medium (Tryptone 10 g / 1, NaCl 5g / l, yeast extract 5g / l, glucose 10g / l, ammonium sulfate 1 g / 1 and kanamycin 100 mg / 1). The results of the cultures are collated in Table V.
  • the Pseudomonas strain (pXL2238) expresses the amidase gene from Comamonas NI.1 and this is all the better when the cell preparation is sonic.
  • GCT ATA AAA GAT TTA ATT GAT GTT GAA GGC CAG AAA ACC ACT ATG GGT 312 Ala Ile Lys Asp Leu Ile Asp Val Glu Gly Gin Lys Thr Thr Met Gly 50 55 60
  • AGT CTA AGC CCG TCA TTA GAT CAT GTT GGA ATT ATC AGT AAA TCA TTA 696 Ser Leu Ser Pro Ser Leu Asp His Val Gly Ile Ile Ser Lys Ser Leu 175 180 185 190
  • AAG CCC CAA AAA AAC TTA GAT AAG AAG TTA ACT ATT GGC CTT CTA AAA 792 Lys Pro Gin Lys Asn Leu Asp Lys Lys Leu Thr Ile Gly Leu Leu Lys 210 215 220
  • GGT TTT TTT GAG GAA TAT ATT TGC AGC TCT GTC AAA GAA AAA TAT GAT 840 Gly Phe Phe Glu Glu Tyr Ile Cys Ser Ser Val Lys Glu Lys Tyr Asp 225 230 235

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Abstract

Novel polypeptides having amidase activity for hydrolyzing amides into carboxylates, and having a greater enzymatic activity for ammonium adipamate than for adipamide. A DNA sequence coding for a polypeptide having amidase activity for hydrolyzing amides into carboxylates, an analogue thereof resulting from genetic code degeneration, or a DNA sequence hybridizing with one of said sequences or a fragment thereof and coding for a polypeptide having amidase activity, are disclosed. Germs suitable for producing said polypeptides are also disclosed.

Description

POLYPEPTIDES A ACTIVITE AMIDASE, OUTILS GENETIQUES ET MICRO-ORGANISMES HOTES PERMETTANT LEUR OBTENTION.POLYPEPTIDES HAVING AMIDASE ACTIVITY, GENETIC TOOLS AND HOST MICROORGANISMS FOR OBTAINING THEM.
DOMAINE TECHNIQUE :TECHNICAL AREA :
Le domaine de l'invention est celui de la production enzymatique de dérivés carboxyliques (carboxylates) à partir de composés contenant des groupements amides. La présente invention concerne de nouveaux polypeptides présentant une activité amidase, le matériel génétique impliqué dans leur production, ainsi que les micro-organismes contenant ce matériel génétique et présentant cette activité.The field of the invention is that of the enzymatic production of carboxylic derivatives (carboxylates) from compounds containing amide groups. The present invention relates to new polypeptides having amidase activity, the genetic material involved in their production, as well as the microorganisms containing this genetic material and exhibiting this activity.
L'invention a également pour objet un procédé d'hydrolyse enzymatique d' amides en carboxylates dans lequel on met en oeuvre ces nouveaux polypeptides ou des micro-organismes les synthétisant, dont, notamment, les susdits micro¬ organismes hôtes.The invention also relates to a process for the enzymatic hydrolysis of amides to carboxylates in which these new polypeptides or microorganisms are synthesized, including, in particular, the above-mentioned host micro¬ organisms.
Sans que cela ne soit limitatif, les amides, auxquels on s'intéresse plus particulièrement dans le cadre de l'invention, sont : le cyano-5 valéramide, l'adipamide ou l'adipamate d'un cation quelconque, de préférence choisi parmi la liste de composés suivants : alcalins, alcalino-terreux, aminés, ammonium, ce dernier composé étant particulièrement préféré. Le but visé est la transformation enzymatique des fonctions amides en fonctions carboxylates, de manière à obtenir in fine l'acide 5-cyanovalérique, l'acide adipique ou des sels de ceux-ci, e. g. : 5- cyanovalérate d'ammonium, adipate de diammonium. Tout l'intérêt industriel des adipates, et en particulier, de l'adipate d'ammonium, réside dans le fait qu'il constitue l'une des bases fondamentales pour la fabrication du nylon-6,6.Without being limiting, the amides, which are of particular interest in the context of the invention, are: cyano-5 valeramide, adipamide or adipamate of any cation, preferably chosen from the list of following compounds: alkaline, alkaline-earth, amino, ammonium, the latter compound being particularly preferred. The aim is the enzymatic transformation of the amide functions into carboxylate functions, so as to ultimately obtain 5-cyanovaleric acid, adipic acid or salts thereof, e. g. : 5- ammonium cyanovalerate, diammonium adipate. The whole industrial interest of adipates, and in particular of ammonium adipate, lies in the fact that it constitutes one of the fundamental bases for the manufacture of nylon-6,6.
S 'agissant des 5-cyanovalérates, ils sont susceptibles de constituer des produits de départ de choix, pour la préparation de caprolactame, donnant lui-même accès au nylon-6.As regards the 5-cyanovalerates, they are capable of constituting starting materials of choice, for the preparation of caprolactam, giving itself access to nylon-6.
L'action de ces amidases s'intègre dans un schéma connu de synthèse enzymatique d'adipate de diammonium, à partir d'un dinitrile, à savoir l'adiponitrile. Ce schéma est le suivant :The action of these amidases is part of a known synthetic scheme enzymatic of diammonium adipate, from a dinitrile, namely adiponitrile. This diagram is as follows:
Figure imgf000004_0001
Figure imgf000004_0001
NH = nitrile hydratase, Ni = nitrilase,NH = nitrile hydratase, Ni = nitrilase,
A = amidase,A = amidase,
R = (CH2)n, n étant un nombre entier égal à 4 dans le cas de composés adipiques.R = (CH 2 ) n , n being an integer equal to 4 in the case of adipic compounds.
ART ANTERIEUR ;PRIOR ART;
Un tel schéma est resté pendant longtemps uniquement d'intérêt théorique, car on ne connaissait que l' amidase à activité générale de BrevibacteriumSuch a scheme remained for a long time only of theoretical interest, because only the amidase with general activity of Brevibacterium was known.
R 312, décrite notamment dans les articles suivants : - ARNAUD et al., "Etude de l'activité amidasique de quelques bactéries", FOLIAR 312, described in particular in the following articles: - ARNAUD et al., "Study of the adidas activity of some bacteria", FOLIA
MICROBIOLOGICA, 1976, 21, pages 178-185,MICROBIOLOGICA, 1976, 21, pages 178-185,
- MAESTRACCI et al., "Utilisation d'une amidase à activité générale pour la bioconversion des amides en acides organiques" , Microbiologie - aliments - nutrition, 1986, vol. 4, pages 19-24.- MAESTRACCI et al., "Use of an amidase with general activity for the bioconversion of amides into organic acids", Microbiology - food - nutrition, 1986, vol. 4, pages 19-24.
Selon ces auteurs, une telle amidase est apte à hydrolyser l'adipamide en acide adipique (ou adipate d'ammonium).According to these authors, such an amidase is capable of hydrolyzing adipamide to adipic acid (or ammonium adipate).
En fait, il a été démontré, par la suite, notamment par la Demanderesse, que l'activité, vis-à-vis de l'adipamide, de cette amidase à activité générale est extrêmement faible, voire nulle et ce, que l'on utilise l'enzyme issue deIn fact, it has been demonstrated subsequently, in particular by the Applicant, that the activity, with respect to adipamide, of this amidase with general activity is extremely low, even zero, and this, that the we use the enzyme from
Brevibacterium R 312 ou celle issue d'un micro-organisme recombinant comprenant le gène d'expression de cette enzyme.Brevibacterium R 312 or that derived from a recombinant microorganism comprising the gene for expression of this enzyme.
Cette absence d'activité enzymatique de l' amidase à activité générale se vérifie, également, pour les substrats amides se présentant sous forme de sels solubles dans l'eau.This absence of enzymatic activity of the amidase with general activity is also verified for the amide substrates which are in the form of salts soluble in water.
La faisabilité industrielle de la synthèse enzymatique d'adipates, en particulier de diammonium, ou d'acide adipique à l'aide d'amidases n'a pu être envisagée qu'à partir du moment où la Demanderesse a pu mettre en évidence des polypeptides exprimés par Brevibacterium R 312 et Rhodococcus et caractérisés, notamment, par une activité amidase, dite énantiosélective, c'est-à-dire qu'ils sont capables d'hydrolyser énantiosélectivement des racémiques d'amides en un acide, soit de forme S, soit de forme R. Cela concerne particulièrement les aryl-2 propionamides et les aryloxy-2 propionamides racémiques. En isolant, purifiant et caractérisant ces amidases "énantiosélectives", il a été possible de proposer un procédé de synthèse enzymatique d'adipate d'ammonium présentant des rendements améliorés.The industrial feasibility of the enzymatic synthesis of adipates, in particular of diammonium, or of adipic acid using amidases could only be envisaged from the moment when the Applicant was able to demonstrate polypeptides expressed by Brevibacterium R 312 and Rhodococcus and characterized, in particular, by an amidase activity, called enantioselective activity, that is to say that they are capable of enantioselectively hydrolyzing racemides of amides into an acid, either of form S, or of form R. This particularly concerns the 2-aryl-propionamides and the 2-aryloxy-propionamides. By isolating, purifying and characterizing these "enantioselective" amidases, it has been possible to propose a process for the enzymatic synthesis of ammonium adipate having improved yields.
C'est ce que décrit la demande de brevet FR 90-14 853. Outre ces nouvelles amidases, cette demande de brevet décrit également les outils de génie génétique pour les produire : séquences d'ADN, recombinées ou non, cassettes d'expression portant cette séquence et micro-organismes naturels ou recombinés contenant ces cassettes d'expression.This is described in patent application FR 90-14 853. In addition to these new amidases, this patent application also describes the genetic engineering tools for producing them: DNA sequences, recombined or not, expression cassettes carrying this sequence and natural or recombinant microorganisms containing these expression cassettes.
Bien que constituant un progrès technique notable, ces amidasesAlthough constituting a notable technical progress, these amidases
"énantiosélectives" souffrent, néanmoins, d'un handicap, déjà signalé pour les amidases à activité générale, qui est d'avoir une capacité d'hydrolyse des amides se présentant sous forme de sels solubles dans l'eau, e. g. adipamate d'ammonium inférieure à celle qu'elles ont vis-à-vis des amides peu ou pas solubles dans l'eau, tels que l'adipamide."enantioselective" suffer, however, from a handicap, already reported for amidases with general activity, which is to have a capacity for hydrolysis of amides in the form of water-soluble salts, eg ammonium adipamate lower than that they have vis-à-vis poorly or not water-soluble amides, such as adipamide.
Or, il est bien connu qu'une telle propriété de solubilité des substrats est tout à fait souhaitable, au regard de l'amélioration de la faisabilité et de la rentabilité d'une synthèse enzymatique industrielle.However, it is well known that such a solubility property of the substrates is entirely desirable, in view of the improvement in the feasibility and the profitability of an industrial enzymatic synthesis.
Ainsi, l'un des objectifs essentiels de la présente invention est de proposer de nouvelles enzymes à activité amidasique, un matériel génétique permettant leur production et des micro-organismes contenant ce matériel génétique et permettant cette production, lesdits enzymes et micro-organismes se caractérisant, à la fois, par des rendements satisfaisants de production de carboxylates à partir de substrats de type amide de natures diverses (mono ou diamides) et par une activité amidasique, vis-à-vis des sels solubles d'amides, importante et industriellement intéressante.Thus, one of the essential objectives of the present invention is to provide new enzymes with amidasic activity, genetic material allowing their production and microorganisms containing this genetic material and allowing this production, said enzymes and microorganisms being characterized. , both by satisfactory yields of production of carboxylates from substrates of the amide type of various natures (mono or diamides) and by an adidas activity, with respect to the soluble salts of amides, which is significant and industrially advantageous .
Après de nombreux essais et recherches, la Demanderesse est parvenue à atteindre, entre autres, cet objectif en isolant, purifiant et caractérisant de nouvelles enzymes aptes à hydrolyser les amides en carboxylates avec de fortes activités et en transformant, préférentiellement, des sels solubles d'amides, lesdites enzymes étant utilisées, soit telles quelles, soit, et de préférence, sous la forme de micro¬ organismes recombinés les générant.After numerous tests and researches, the Applicant has succeeded in achieving, among other things, this objective by isolating, purifying and characterizing new enzymes capable of hydrolyzing amides to carboxylates with strong activities and by transforming, preferably, soluble salts of amides, said enzymes being used either as such or, and preferably, in the form of recombinant microorganisms generating them.
EXPOSE DE L'INVENTION :PRESENTATION OF THE INVENTION:
La présente invention a donc pour objet de nouveaux polypeptides ayant une activité amidase, capables d'hydrolyser les amides en carboxylates et possédant une activité enzymatique vis-à-vis de l'adipamate d'ammonium supérieure à celle qu'ils ont vis-à-vis de l'adipamide.The subject of the present invention is therefore new polypeptides having an amidase activity, capable of hydrolyzing amides into carboxylates and having an enzymatic activity vis-à-vis ammonium adipamate greater than that which they have vis-à-vis -vis adipamide.
L'un de ces nouveaux polypeptides a été isolé à partir d'une souche deOne of these new polypeptides was isolated from a strain of
Comamonas testosteroni NI 1. Plus précisément, ce polypeptide est préparé par extraction et purification à partir de cultures de micro-organismes naturels ou recombinants, la purification étant réalisée par une succession d'étapes consistant à préparer un extrait enzymatique à partir de la culture cellulaire, à précipiter cet extrait avec du sulfate d'ammonium et à le purifier par différentes étapes de chromatographie et filtration sur gel. Ces étapes, qui font appel à des techniques bien connues de l'homme du métier, sont décrites en détail dans les exemples illustratifs ci-après. Par activité amidase, on désigne, dans le présent exposé, l'hydrolyse enzymatique d'un amide, tel que le cyano-5 valéramide, l'adipamide et l'adipamate d'un cation Z, en un carboxyle, à savoir, respectivement, le cyano-5 valérate de Z, l'adipamate de Z et l'adipate de Z. Le cation Z peut être quelconque, mais on le choisit, de préférence, parmi les composés suivants : alcalins, alcalino-terreux, aminés, ammonium, ce dernier composé étant particulièrement préféré.Comamonas testosteroni NI 1. More specifically, this polypeptide 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 the cell culture , to rush this extracted with ammonium sulfate and to purify it by various 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. By amidase activity is meant, in the present description, the enzymatic hydrolysis of an amide, such as cyano-5 valeramide, adipamide and adipamate of a cation Z, into a carboxyl, namely, respectively , Z-cyano-5 valerate, Z adipamate and Z adipate. The cation Z can be any, but it is preferably chosen from the following compounds: alkali, alkaline earth, amino, ammonium , the latter compound being particularly preferred.
Il est du mérite de la Demanderesse d'avoir pu isoler ces nouveaux polypeptides et d'avoir pu mettre en évidence leur utilisation préférentielle de substrats sous forme de sels solubles dans l'eau, tels que l'adipamate, par exemple d'ammonium, plutôt que de substrats non ioniques, insolubles ou faiblement solubles dans l'eau, tels que l'adipamide.It is to the Applicant's credit to have been able to isolate these new polypeptides and to have been able to demonstrate their preferential use of substrates in the form of water-soluble salts, such as adipamate, for example ammonium, rather than non-ionic substrates, insoluble or poorly soluble in water, such as adipamide.
Cette caractéristique se vérifie aussi bien pour les enzymes sous forme pure que pour les germes naturels ou recombinés exprimant ces enzymes.This characteristic is true both for enzymes in pure form and for natural or recombinant germs expressing these enzymes.
Parmi les polypeptides à activité amidase conformes à l'invention figure un polypeptide se caractérisant par la séquence peptidique, telle que représentée à la fig. 1 (SEQ H) : 1 de la liste de séquences annexée).Among the polypeptides with amidase activity in accordance with the invention is a polypeptide characterized by the peptide sequence, as shown in FIG. 1 (SEQ H): 1 from the attached sequence list).
Un autre objet de l'invention est relatif à une séquence d'ADN codant pour un polypeptide ayant une activité amidase, capable d'hydrolyser les amides en carboxylates, et choisie parmi la liste de séquences suivante :Another subject of the invention relates to a DNA sequence coding for a polypeptide having an amidase activity, capable of hydrolyzing amides to carboxylates, and chosen from the following list of sequences:
- la séquence d'ADN, telle que représentée à la fig. 2 (SEQ ID : 2 de la liste de séquences annexée), et codant pour un polypeptide ayant une activité amidase,- the DNA sequence, as shown in fig. 2 (SEQ ID: 2 from the annexed sequence list), and coding for a polypeptide having amidase activity,
- un analogue de cette séquence résultant de la dégénérescence du code génétique,- an analogue of this sequence resulting from the degeneration of the genetic code,
- une séquence d'ADN hybridant avec l'une de ces séquences ou un fragment de celles-ci et codant pour un polypeptide ayant une activité amidase,a DNA sequence hybridizing with one of these sequences or a fragment thereof and coding for a polypeptide having amidase activity,
Les polypeptides résultant de l'expression de l'une des séquences d'ADN ci-dessus sont compris dans le champ de l'invention .The polypeptides resulting from the expression of one of the above DNA sequences are included in the scope of the invention.
Le micro-organisme sauvage Comamonas testosteroni NI 1 , isolé par la Demanderesse, contient l'une des séquences ci-dessus dans son génome.The wild micro-organism Comamonas testosteroni NI 1, isolated by the Applicant, contains one of the above sequences in its genome.
Conformément à l'invention, il a été possible d'accéder à l'identification de ces séquences d'ADN par la méthode bien connue du clonage du fragment d'ADN génomique codant pour le polypeptide dont la séquence peptidique est représentée à la fig. 1, à l'aide de sondes nucleotidiques élaborées à partir du polypeptide purifié.In accordance with the invention, it was possible to access the identification of these DNA sequences by the well-known method of cloning the genomic DNA fragment coding for the polypeptide whose peptide sequence is shown in FIG. 1, using nucleotide probes produced from the purified polypeptide.
L'invention concerne, également, les cassettes d'expression qui portent, avec les signaux assurant son expression, l'une des séquences d'ADN définie précédemment. Ces cassettes d'expression peuvent être, soit naturellement présentes, soit intégrées dans le génome de l'hôte ou localisées sur un vecteur d'expression, tel qu'un plasmide contenant, de préférence, un moyen de sélection.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 be either naturally present or integrated into the genome of the host or localized on an expression vector, such as a plasmid preferably containing a means of selection.
Ces cassettes d'expression comportent, notamment, des régions d'initiation de la transcription et de la traduction, qui contiennent une séquence promotrice et un site de fixation des ribosomes. Ces régions peuvent être homologues ou hétérologues du micro-organisme produisant naturellement le polypeptide.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 polypeptide.
Le choix de ces régions dépend, notamment, de l'hôte utilisé. En particulier, lorsqu'il s'agit de micro-organismes hôtes procaryotes, le promoteur hétérologue peut être choisi parmi les promoteurs bactériens forts, tels que le promoteur de l'opéron tryptophane Ptrp de Escherichia coli, le promoteur de l'opéron lactose Plac de E. coli, le promoteur droit du phage lambda PR, le promoteur gauche du phage lambda PL, les promoteurs forts de Pseudomonas et Comamonas, les promoteurs forts de Corynébactéries. Plus particulièrement, dans le cas du promoteur droit du phage lambda, la forme thermosensible PRCIts est préférée.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 Ptrp operon from Escherichia 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.
Dans le cas des micro-organismes eucaryotes, tels les levures, les promoteurs peuvent être ceux des gènes glycolytiques de levure, tels les gènes codant pour la phospho-glycérate kinase (PGK), la glycéraldéhyde-3-phosphate déshydrogénase (GPD), ou bien encore des gènes codant pour la lactase (LAC4), l'énolase (ENO).In the case of eukaryotic microorganisms, such as yeasts, the promoters can 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).
Concernant les sites de fixation des ribosomes, celui dérivé du gène CII de lambda, ainsi que ceux dérivés de gènes de Comamonas ou Pseudomonas ou ceux dérivés de gènes de Corynébactéries sont utilisés préférentiellement lorsque le micro¬ organisme hôte est procaryote.Regarding ribosome binding sites, that derived from the CII gene lambda, as well as those derived from Comamonas or Pseudomonas genes or those derived from Corynebacteria genes are preferably used when the host micro¬ organism is prokaryotic.
Une région, permettant une terminaison de la traduction et de la transcription fonctionnelle de l'hôte envisagé, peut être positionnée en 3' de la séquence codante.A region, allowing termination of translation and functional transcription of the intended host, can be positioned 3 'to the coding sequence.
Avantageusement, la cassette d'expression peut comprendre, également, un ou plusieurs marqueurs permettant de sélectionner l'hôte recombinant. Les marqueurs préférés sont des marqueurs dominants, c'est-à-dire conférant une résistance à des antibiotiques comme l'ampicilline, la tétracycline ou la streptomycine ou à d'autres produits toxiques pour les bactéries.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 streptomycin or to other products toxic to bacteria.
Il est à noter que l'amidase selon l'invention constitue déjà, en elle- même, un marqueur de sélection pour l'hôte qui les. contient.It should be noted that the amidase according to the invention already constitutes, in itself, a selection marker for the host which. contains.
L'invention a également pour objet les micro-organismes contenant la séquence d'ADN selon l'invention, ainsi que ceux aptes à produire au moins un polypeptide selon l'invention. Ces micro-organismes comportent ou non au moins une cassette d'expression, du type de celle décrite ci-dessus.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 polypeptide according to the invention. These microorganisms may or may not contain at least one expression cassette, of the type described above.
Parmi ces micro-organismes, on trouve les hôtes utilisables pour l'accueil d'un vecteur d'expression conforme à l'invention, on peut citer, notamment, les entérobactéries telles que E. coli, les bactéries appartenant aux genres Comamonas ou Pseudomonas, les bactéries corynéformes, telles que celles appartenant aux genresAmong 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 or Pseudomonas , coryneform bacteria, such as those belonging to the genera
Corynebacterium, Brevibacterium, Rhodococcus, Streptomyces ou Bacillus.Corynebacterium, Brevibacterium, Rhodococcus, Streptomyces or Bacillus.
Un micro-organisme recombiné, contenant ladite séquence d'ADN sur une structure plasmidique, a été déposé à la Collection Nationale de Cultures de Micro-organismes (C. N. C. M.) (Institut Pasteur, 25 rue du Docteur Roux, PARIS) sous le n° I 1279 le 11 janvier 1993. Ce micro-organisme est la souche E. Coli qui contient le plasmide pXL2216 (décrit ci-après). Ce micro-organisme est également identifié par la Demanderesse par la référence G 4315.A recombinant microorganism, containing said DNA sequence on a plasmid structure, has been deposited in the National Collection of Cultures of Microorganisms (CNCM) (Institut Pasteur, 25 rue du Docteur Roux, PARIS) under the number I 1279 January 11, 1993. This microorganism is the E. Coli strain which contains the plasmid pXL2216 (described below). This microorganism is also identified by the Applicant by the reference G 4315.
Il va de soi que les polypeptides produits par les micro-organismes ci- dessus sont, également, compris dans l'invention.It goes without saying that the polypeptides produced by the above microorganisms are also included in the invention.
Cette dernière concerne aussi le procédé de transformation des amides en carboxylates à l'aide d'un polypeptide selon l'invention ou d'un micro-organisme recombiné le générant. Ce procédé consiste à mettre en présence l'amide à transformer avec un polypeptide ou un micro-organisme recombiné, tel que défini précédemment. On opère, généralement, à la température ambiante. Selon un mode particulier de réalisation de l'invention, le polypeptide ou le micro-organisme recombiné sont immobilisés sur ou dans un support solide, selon une technique classique, comme celle décrite, par exemple, dans le brevet américain US N° 4 732 851.The latter also relates to the process for transforming amides into carboxylates using a polypeptide according to the invention or a recombinant microorganism generating it. This method consists in bringing the amide to be transformed into contact with a polypeptide or a recombinant microorganism, as defined above. We generally operate at room temperature. According to a particular embodiment of the invention, the polypeptide 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 Pat. No. 4,732,851 .
Le procédé de l'invention convient pour la transformation des amides, en carboxylates et, plus particulièrement, pour la transformation en carboxylates des mono et diamides de formules :The process of the invention is suitable for the transformation of amides, into carboxylates and, more particularly, for the transformation into carboxylates of mono and diamides of formulas:
NC - R - CONH2, H2NOC - R - CONH2 et H2NOC - R - COO~Z+ dans lesquelles R est un groupe alkylène ou alcénylène linéaire ou ramifié, contenant 1 à 18 atomes de carbone, R étant, de préférence, égal à — (CH2)4 - et Z est choisi parmi la liste de composés suivants : alcalins, alcalino- terreux, aminés, ammonium, ce dernier composé étant particulièrement préféré.NC - R - CONH 2 , H 2 NOC - R - CONH 2 and H 2 NOC - 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.
Le procédé de l'invention est particulièrement approprié pour la synthèse enzymatique, d'une part, d'un adipate, tel que l'adipate d'ammonium à partir d'adipamide et surtout à partir d'un adipamate, tel que l'adipamate d'ammonium et, d'autre part, de cyano-5 valérate à partir de cyano-5 valéramide.The process of the invention is particularly suitable for the enzymatic synthesis, on the one hand, of an adipate, such as ammonium adipate from adipamide and especially from an adipamate, such as ammonium adipamate and, on the other hand, cyano-5 valerate from cyano-5 valeramide.
Les exemples qui suivent permettent d'illustrer les caractéristiques et les avantages de la présente invention sans, toutefois, en limiter la portée.The following examples illustrate the characteristics and advantages of the present invention without, however, limiting its scope.
DESCRIPTION DES FIGURESDESCRIPTION OF THE FIGURES
La fig. 1 représente la séquence peptidique d'un polypeptide conforme à l'invention (SEQ ID : 1).Fig. 1 represents the peptide sequence of a polypeptide in accordance with the invention (SEQ ID: 1).
La fig. 2 représente une séquence d'ADN selon l'invention (SEQ ID : 2). La fig. 3 représente l'électrophorèse sur gel de polyacrylamide SDS des fractions obtenues aux différentes étapes de purification de l'exemple 2.Fig. 2 represents a DNA sequence according to the invention (SEQ ID: 2). Fig. 3 represents the electrophoresis on an SDS polyacrylamide gel of the fractions obtained at the various purification stages of Example 2.
La fig. 4 représente une courbe de l'activité spécifique de l'amidase selon l'invention (micromoles d'adipate x h 1 x mg"1 de protéine) en fonction de la température en ° C.Fig. 4 represents a curve of the specific activity of amidase according to the invention (micromoles of adipate xh 1 x mg "1 of protein) as a function of the temperature in ° C.
La fig.5 représente une courbe de l'activité spécifique de l'amidase selon l'invention (micromoles d'adipate x h"1 x mg"1 de protéine) en fonction du pH. La fig. 6 représente une variation de l'activité spécifique (micromoles d'adipate x h"1 x mg"1 de protéine) de l'amidase selon l'invention vis-à-vis de l'adipamate, en fonction de la concentration en adipamate (millimoles x l'1).FIG. 5 represents a curve of the specific activity of the amidase according to the invention (micromoles of adipate xh "1 x mg " 1 of protein) as a function of the pH. Fig. 6 represents a variation in the specific activity (micromoles of adipate xh "1 x mg " 1 of protein) of the amidase according to the invention with respect to adipamate, as a function of the concentration of adipamate ( millimoles xl '1 ).
La fig. 7 représente une courbe donnant le rapport de la concentration en adipamate sur l'activité spécifique (litres d'adipate x h"1 x g"1 de protéine) en fonction de la concentration en adipamate (millimoles x l"1).Fig. 7 shows a curve giving the ratio of the concentration of adipamate to the specific activity (liters of adipate xh "1 xg " 1 of protein) as a function of the concentration of adipamate (millimoles xl "1 ).
La fig. 8 représente la carte de restriction du plasmide pXL2104 contenant le gène de l'amidase selon l'invention.Fig. 8 represents the restriction map of the plasmid pXL2104 containing the amidase gene according to the invention.
La fig. 9 représente la séquence d'un fragment de 1491 pb contenant le gène codant pour l'amidase de Comamonas testosteroni NI 1. La fig. 10 représente la carte de restriction du plasmide pXL2216 contenant le gène de l'amidase selon l'invention.Fig. 9 represents the sequence of a 1491 bp fragment containing the gene coding for the amidase of Comamonas testosteroni NI 1. FIG. 10 shows the restriction map of the plasmid pXL2216 containing the amidase gene according to the invention.
La fig. 11 représente la carte de restriction du plasmide pXL2144 contenant le fragment Spel-Clal dérivé du plasmide pXL2104 entre les sites Xbal et Clal du vecteur pMTL22. La fig. 12 représente l'électrophorèse sur Gel SDS-PAGE montrant l'expression de la séquence d'ADN selon l'invention dans la souche E-Coli TG 1.Fig. 11 shows the restriction map of the plasmid pXL2144 containing the Spel-ClaI fragment derived from the plasmid pXL2104 between the Xbal and ClaI sites of the vector pMTL22. Fig. 12 represents the electrophoresis on SDS-PAGE gel showing the expression of the DNA sequence according to the invention in the strain E-Coli TG 1.
La fig. 13 représente la carte de restriction du plasmide pXL2238 contenant le gène de l'amidase selon l'invention.Fig. 13 shows the restriction map of the plasmid pXL2238 containing the amidase gene according to the invention.
La signification des abréviations utilisées dans la suite de la description est donnée ci-après.The meaning of the abbreviations used in the following description is given below.
- SSC : tampon couramment utilisé pour les hybridations, il contient du citrate de sodium et du NaCl (20XSSC = NaCl 3M-citrate de sodium pH 7, 0,3 M),- SSC: buffer commonly used for hybridizations, it contains sodium citrate and NaCl (20XSSC = NaCl 3M-sodium citrate pH 7, 0.3 M),
- SDS : dodécylsulfate de sodium, - FPLC : chromatographie liquide dénommée en langue anglaise "fast protein liquid chromatography", SDS-PAGE : gel d'électrophorèse à base de dodécylsulfate de sodium et de polyacrylamide,- 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-O thio galactopyranoside, CLHP : chromatographie liquide haute performance, PS : poids sec,IPTG: isopropyl β-O thio galactopyranoside, HPLC: high performance liquid chromatography, PS: dry weight,
X-gal. : 5 - bromo - 4 - chloro - 3 - indolyl - β D galactopyranoside.X-gal. : 5 - bromo - 4 - chloro - 3 - indolyl - β D galactopyranoside.
EXEMPLESEXAMPLES
EXEMPLE 1 HYDROLYSE DE L'ADIPAMATE ET DE L'ADIPAMIDE PAREXAMPLE 1 HYDROLYSIS OF ADIPAMATE AND ADIPAMIDE BY
Comamonas testosteroni NI 1.Comamonas testosteroni NI 1.
La souche Comamonas testosteroni NI 1 est une souche isolée à partir d'un échantillon de terre par criblage microbiologique. Ses performances sont évaluées sur adipamate et adipamide.The Comamonas testosteroni NI 1 strain is a strain isolated from a soil sample by microbiological screening. Its performance is evaluated on adipamate and adipamide.
1.1. Préparation des cellules :1.1. Cell preparation:
La souche Comamonas testosteroni NI 1 est cultivée, en fiole agitée, à 28° C, pendant 19 h, dans le milieu A dont la composition est la suivante :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:
Glycérol 10,0 x 103 mgxl" Glycerol 10.0 x 10 3 mgxl "
Cyano-5-valérate de sodium 1,5 x 103 mgxl' Sodium cyano-5-valerate 1.5 x 10 3 mgxl '
K2HPO4 0,5 x 103 mgxl' K 2 HPO 4 0.5 x 10 3 mgxl '
MgSO4, 7H2O 0,5 x 103 mgxrMgSO 4 , 7H 2 O 0.5 x 10 3 mgxr
MnSO4, H20 20,0 mgxl' MnSO 4 , H 2 0 20.0 mgxl '
FeSO4, 7H2O 20,0 mgxl'FeSO 4 , 7H 2 O 20.0 mgxl '
NaCl 10,0 mgxl' NaCl 10.0 mgxl '
Extrait de levure 0,5 x 103 mgxl'Yeast extract 0.5 x 10 3 mgxl '
Extrait de boeuf 0,5 x 103 mgxl' Beef extract 0.5 x 10 3 mgxl '
- HC1 3N QSP pH 5- HC1 3N QSP pH 5
Cette préculture sert à ensemencer une fiole de 2 litres remplie au dixième par le milieu A. Après 22 h, 3,5 gxl 1 de cellules humides sont récoltés. Cela correspond à une DO660mn de 1,4 et un poids sec de 0,9 gxl"1.This preculture is used to inoculate a 2 liter flask filled to the tenth by medium A. After 22 h, 3.5 g × l 1 of wet cells are harvested. This corresponds to an OD 660mn of 1.4 and a dry weight of 0.9 gxl "1 .
1.2. Détermination de l'activité enzymatique sur adipamide et adipamate :1.2. Determination of the enzymatic activity on adipamide and adipamate:
Un culot cellulaire contenant 31 mg de cellules sèches est mis en suspension dans 5,5 ml d'une solution 20 mM en adipamate ou adipamide dans le tampon phosphate de potassium pH 7,50 mM. La réaction est conduite à 25° C sous agitation et la cinétique est suivie par prélèvement. Sur chaque prélèvement, sont dosés par CLHP l'adipamide, l'adipamate et l'adipate. Les vitesses de formation de l'adipate, à partir de l'adipamide et de l'adipamate, sont, respectivement, de 8,3 et 33 Uxg"1 de cellules sèches (1 U est égale à 1 micromole de produit formé par minute) (cellules entières). Par comparaison, on obtient les activités données dans le TABLEAU I ci-dessous et mesurées dans les mêmes conditions, avec Brevibacterium R 312 et Rhodococcus sp.A cell pellet containing 31 mg of dry cells is suspended in 5.5 ml of a 20 mM adipamate or adipamide solution in potassium phosphate buffer pH 7.50 mM. The reaction is carried out at 25 ° C. with stirring and the kinetics are followed by sampling. Adipamide, adipamate and adipate are measured on each sample by HPLC. The rates of formation of adipate, from adipamide and adipamate, are respectively 8.3 and 33 Uxg- 1 of dry cells (1 U is equal to 1 micromole of product formed per minute ) (whole cells) By comparison, the activities given in TABLE I below are obtained and measured under the same conditions, with Brevibacterium R 312 and Rhodococcus sp.
TABLEAU I :TABLE I:
ACTIVITÉS EN MICROMOLES DEMICROMOLES ACTIVITIES FROM
SOUCHES SUBSTRATS PRODUIT FORMÉ PAR MINUTE ETSUBSTRATE STRAINS PRODUCT FORMED PER MINUTE AND
PAR GRAMME DE CELLULES SÈCHESBY RANGE OF DRY CELLS
Comamonas testosteroni Adipamate 33,0 NI 1Comamonas testosteroni Adipamate 33.0 NI 1
Adipamide 8,3Adipamide 8.3
Brevibacterium R 312 Adipamate 0,0Brevibacterium R 312 Adipamate 0.0
Adipamide 2,3Adipamide 2,3
Rhodococcus sp Adipamate 0,0Rhodococcus sp Adipamate 0.0
Adipamide 35,0 EXEMPLE 2 : PURIFICATION DE L'AMIDASE DE Comamonas testosteroni NI 1.Adipamide 35.0 EXAMPLE 2 PURIFICATION OF THE AMIDASE OF Comamonas testosteroni NI 1.
2.1. Culture des cellules :2.1. Culture of cells:
La souche Comamonas NI 1 est cultivée en fermenteur de 15 1 sur le milieu B dont la composition est :The Comamonas NI 1 strain is cultivated as a 15 1 fermenter on medium B, the composition of which is:
Glucose 5,0 x 103 mgxl" Glucose 5.0 x 10 3 mgxl "
Cyano-5-valérate de sodium 5,7 x 103 mgxl" Sodium cyano-5-valerate 5.7 x 10 3 mgxl "
Adiponitrile 1,0 x 103 mgxl" Adiponitrile 1.0 x 10 3 mgxl "
- Na2HPO4, 2H2O 3,4 x 103 mgxl" - KH2PO4 2,7 x 103 mgxl" - Na 2 HPO 4 , 2H 2 O 3.4 x 10 3 mgxl " - KH 2 PO 4 2.7 x 10 3 mgxl "
- MgSO4 0,5 x 103 mgxï- MgSO 4 0.5 x 10 3 mgxï
- MnSO4, H2O 20,0 mgxl" - MnSO 4 , H 2 O 20.0 mgxl "
- FeSO4, 7H2O 20,0 mgxl" - FeSO 4 , 7H 2 O 20.0 mgxl "
- NaCl 10,0 mgxl" - Vitamines :- NaCl 10.0 mgxl " - Vitamins:
. Biotine 8 x 10"5 mgxl" . Biotin 8 x 10 "5 mgxl "
. Acide nicotinique 8 x 10"3 mgxl" . Nicotinic acid 8 x 10 "3 mgxl "
. Thiamine, HC1 4 x 10"3 mgxl" . Thiamine, HC1 4 x 10 "3 mgxl "
Le pH, la température, le débit d'air et la vitesse d'agitation sont régulés, respectivement, à 6,6, 28° C, 300 1/h et 350 tpm. Après 4 h de fermentation,The pH, the temperature, the air flow and the stirring speed are regulated, respectively, at 6.6, 28 ° C, 300 l / h and 350 rpm. After 4 h of fermentation,
15 g d'adiponitrile sont ajoutés. Après 19 h 30 de culture, 188 g de cellules humides, soit 40 g de cellules sèches sont récoltées. Cela correspond, en fin de culture, à 2,5 g/1 de cellules poids sec et à une DO^onm de 7.15 g of adiponitrile are added. After 19 h 30 of culture, 188 g of wet cells, ie 40 g of dry cells are harvested. This corresponds, at the end of the culture, to 2.5 g / l of dry weight cells and to an OD 50 nm of 7.
2.2 Purification :2.2 Purification:
Toutes les étapes de la purification sont réalisées dans le tampon B (Tris-HCl 100 mM pH 7,5, glycérol 10 %). A chacune des étapes, l'activité amidase des fractions est déterminée à pH 7,5 et à 25° C dans le tampon B dilué 10 fois en présence d'adipamate 10 mM. La concentration en protéine des pools est déterminée par la méthode au bleu de Coomassie (kit PIERCE Protein assay).All the purification steps are carried out in buffer B (100 mM Tris-HCl pH 7.5, 10% glycerol). At each of the stages, the amidase activity of the fractions is determined at pH 7.5 and at 25 ° C. in buffer B diluted 10 times in the presence of 10 mM adipamate. The protein concentration of the pools is determined by the Coomassie blue method (PIERCE Protein assay kit).
L'unité d'activité U correspond à 1 μmole d'adipate produite par minute dans les conditions du test. Le pannel de protéines est analysé par gel de polyacrylamide-SDS (Phastsystem PHARMACIA).The activity unit U corresponds to 1 μmole of adipate produced per minute in test conditions. The protein panel is analyzed by polyacrylamide-SDS gel (Phastsystem PHARMACIA).
Les données de chacune des étapes de la purification sont regroupées dans le tableau II ci-après et la fig. 3 annexée. Les procédures de chacune des étapes sont commentées ci-après.The data for each of the purification steps are collated in Table II below and FIG. 3 attached. The procedures for each step are discussed below.
- Etape 0 : extrait brut :- Step 0: raw extract:
66 g de cellules humides sont reprises dans 100 ml de tampon B et soniquées pendant 28 min en temps effectif (sonicateur VIBRACELL de BIOBLOCK : sonde 13 mm, puissance 7, 30 % du cycle actif. La DO660nm passe ainsi de 220 à 63. Après centrifugation à 30 000 gmax pendant66 g of wet cells are taken up in 100 ml of buffer B and sonicated for 28 min in effective time (VIBRACELL sonicator from BIOBLOCK: 13 mm probe, power 7, 30% of the active cycle. The OD 660nm thus goes from 220 to 63. After centrifugation at 30,000 gmax for
60 min, les 125 ml du surnageant sont récupérés.60 min, the 125 ml of the supernatant are recovered.
- Etape 1 : fractionnement au sulfate d'ammonium :- Step 1: fractionation with ammonium sulfate:
Les 125 ml sont additionnés de 26 g de sulfate d'ammonium pour atteindre 35 % de la saturation. Après une heure, la suspension est centrifugée 30 min à 30 000 gmax. Le surnageant est récupéré et additionné de 18 g de sulfate d'ammonium pour atteindre 60 % de la saturation. Après 1 h, la suspension est centrifugée à 48 000 gmax. Le précipité est récupéré puis dialyse contre le tampon B pendant 66 heures environ. Après dialyse, 43 ml sont récupérés puis analysés (protéine totale 1 200 mg, [prot] = 28 gxl"1, activité totale = 1 200 U, act. spéc. = 1 Uxmg 1, rdt prot. = 46 % , rdt act. = 60 %).The 125 ml are added with 26 g of ammonium sulfate to reach 35% of the saturation. After one hour, the suspension is centrifuged for 30 min at 30,000 gmax. The supernatant is recovered and added with 18 g of ammonium sulphate to reach 60% of the saturation. After 1 h, the suspension is centrifuged at 48,000 gmax. The precipitate is recovered and then dialyzed against buffer B for approximately 66 hours. After dialysis, 43 ml are recovered and then analyzed (total protein 1,200 mg, [prot] = 28 gxl " 1 , total activity = 1,200 U, specific act = 1 Uxmg 1 , rdt prot. = 46%, rdt act . = 60%).
- Etape 2 : colonne échangeuse d'ions (Q sepharose fast flow) :- Step 2: ion exchange column (Q sepharose fast flow):
La fraction dialysée (43 ml) est chargée à un débit de 130 mlxh 1 sur une colonne (26 x 380 mm) de "Q sepharose flast flow" équilibrée avec le tampon B à un débit de 200 mlxh"1. La colonne est percolée à un débit deThe dialyzed fraction (43 ml) is loaded at a flow rate of 130 mlxh 1 onto a column (26 x 380 mm) of "Q sepharose flast flow" balanced with buffer B at a flow rate of 200 mlxh "1. The column is percolated at a rate of
200 mlxh 1 successivement par les solutions suivantes :200 mlxh 1 successively by the following solutions:
< tampon B, QSP pour retour à la ligne de base,<buffer B, QSP for return to the baseline,
< 500 ml d'un gradient de KC1 0 à 0,4 M dans le tampon B,<500 ml of a 0 to 0.4 M KC1 gradient in buffer B,
< 200 ml de tampon B contenant 0,4 M de KC1, < 250 ml de tampon B additionné de 1 M de KC1.<200 ml of buffer B containing 0.4 M of KC1, <250 ml of buffer B supplemented with 1 M of KC1.
L'activité amidase est éluée dans un volume de 170 ml, à une force ionique comprise entre 0,05 et 250 mM en KC1. Cette solution est analysée (prot. 580 mg, [prot.] = 3,4 gxl 1, act. tôt. = 1 200 U, act. spéc. = 2, 1 Uxmg"1 de prot., rdt en prot = 47 %, rdt en act. = 100 %).Amidase activity is eluted in a volume of 170 ml, at an ionic strength between 0.05 and 250 mM in KC1. This solution is analyzed (prot. 580 mg, [prot.] = 3.4 gxl 1 , early act. = 1200 U, specific act = 2, 1 Uxmg "1 prot., Rdt in prot = 47 %, rdt in act. = 100%).
- Etape 3 : colonne d'interactions hydrophobes (Phenyl Sepharose) : Les 168 ml sont concentrés par précipitation au sulfate d'ammonium à 65 % de la saturation. Pour cela, 72 g de sulfate d'ammonium sont additionnés. Après une heure d'agitation à 4° C, la suspension est centrifugée à 30 000 gmax pendant une heure. Le culot est remis en suspension dans le tampon B et dialyse contre 2 1 de tampon B pendant une nuit. 25 ml sont recueillis, additionnés de 4,4 g de sulfate d'ammonium pour atteindre 30 % de la saturation puis chargés à 50 mlxh"1 pendant 35 min sur une colonne de "Phenyl Sepharose" (26 x 300 mm), équilibrée avec du tampon B contenant le sulfate d'ammonium à 30 % de la saturation. La colonne est lavée avec le même tampon à un débit de 100 mlxh"1 pendant 1 h. Le débit est réduit à 40 mlxh"1 et la colonne est percolée avec du tampon B seul.- Step 3: column of hydrophobic interactions (Phenyl Sepharose): The 168 ml are concentrated by precipitation with ammonium sulfate at 65% of saturation. For this, 72 g of ammonium sulfate are added. After one hour of stirring at 4 ° C, the suspension is centrifuged at 30,000 gmax for one hour. The pellet is resuspended in buffer B and dialyzed against 2 l of buffer B overnight. 25 ml are collected, added with 4.4 g of ammonium sulphate to reach 30% of saturation and then loaded at 50 mlxh "1 for 35 min on a" Phenyl Sepharose "column (26 x 300 mm), balanced with buffer B containing ammonium sulphate at 30% saturation. The column is washed with the same buffer at a flow rate of 100 ml × h −1 for 1 h. The flow rate is reduced to 40 ml × h −1 and the column is percolated with buffer B alone.
L'activité est éluée après 2,8 h dans un volume de 30 ml (prot. + 110 mg, [prot.] = 1,4 gxl"1, act. tôt. = 910 U, act. spéc. 8, 1 Uxmg 1 de prot. , rdt en prot. ≈ 20 %, rdt en act. ≈ 74 %). Les étapes suivantes sont réalisées sur le système FPLC Pharmacia. - Etape 4 : gel filtration (FPLC Superdex 200) :The activity is eluted after 2.8 h in a volume of 30 ml (prot. + 110 mg, [prot.] = 1.4 gxl "1 , early act. = 910 U, specific act 8, 1 Uxmg 1 of prot., Rdt in prot. At 20%, rdt in active. At 74%). The following steps are carried out on the FPLC Pharmacia system. - Step 4: gel filtration (FPLC Superdex 200):
Les 80 ml sont concentrés par ultrafiltration sur membrane Amicon PM10 puis sur centriprep 10. Ces 2,8 ml sont chargés sur la colonne de gel filtration (16 x 600 mm) équilibrée avec le tampon B additionné de 0, 1 M de KC1 à un débit de 0,8 mlxmin"1. L'activité est éluée avec le tampon ci- dessus à un débit de 1 mlxmin"1 et dans un volume de 9 ml (26 mg de protéine, 470 U, act. spéc. = 18 Uxmg"1 de prot., rdt en act. = 51 % , rdt en prot. ≈ 26 %).The 80 ml are concentrated by ultrafiltration on an Amicon PM10 membrane and then on centriprep 10. These 2.8 ml are loaded onto the gel filtration column (16 x 600 mm) balanced with buffer B supplemented with 0.1 M KC1 at a flow rate of 0.8 mlxmin "1. The activity is eluted with the above buffer at a flow rate of 1 mlxmin " 1 and in a volume of 9 ml (26 mg of protein, 470 U, specific act = 18 Uxmg "1 of prot., Rdt in act. = 51%, rdt in prot. At 26%).
- Etape 5 : gel filtration (FPLC Superdex 200) :- Step 5: filtration gel (FPLC Superdex 200):
Cette deuxième gel filtration est effectuée dans des conditions identiques à la précédente, mais en deux fois. Les dosages ne sont effectués que sur le premier passage, réalisé à partir d'une fraction de 3 ml des 9 ml de la précédente étape.This second filtration gel is carried out under conditions identical to the previous one, but in two stages. The assays are only carried out on the first pass, made from a fraction of 3 ml of the 9 ml of the previous step.
- Etape 6 : colonne échangeuse d'anions (mono Q HR 5/5 Pharmacia) :- Step 6: anion exchange column (mono Q HR 5/5 Pharmacia):
Sur cette colonne, équilibrée avec le tampon B, les protéines ne. sont pas retenues et sont éluées directement dans le volume mort. Les fractions actives, 16,5 ml, sont analysées (prot. = 17, 1 mg, [prot.] = 1 gxl"1, act. tôt. = 262 U, act. spéc. ≈ 15 Uxmg"1 de prot., rdts cumulés pour les étapes 5 et 6 = 65 % pour les prot. et 55 % pour l'activité).On this column, balanced with buffer B, the proteins do not. are not retained and are eluted directly in the dead volume. The active fractions, 16.5 ml, are analyzed (prot. = 17, 1 mg, [prot.] = 1 gxl "1 , early act. = 262 U, specific act. U 15 Uxmg " 1 of prot. , cumulative rdts for steps 5 and 6 = 65% for the prots and 55% for the activity).
- Etape 7 : colonne échangeuse d'anions (mono Q HR 5/5 Pharmacia) : Cette étape est réalisée en deux fois, en utilisant la moitié de la fraction précédente à chaque fois et avec le tampon B/5. Le tampon est modifié par dialyse sur membrane Amicon PM10 et substitution par du tampon B/5. Les 6 ml de rétentat sont chargés sur la colonne équilibrée avec le tampon B/5, à un débit de 1 mlxmin 1. Après lavage de la colonne pendant 14 min, un gradient de 0 à 0,15 M en KC1 est appliqué en 25 min. La colonne est ensuite alimentée avec une solution de KC1 1 M dans le tampon B/5. En deux fois, 3,75 ml sont recueillis et analysés (prot. = 1,5 mg, [prot.] = 0,4 gxl"1, act. tôt. = 121 U, act. spéc. = 81 Uxmg"1 de prot., rdt en prot. = 11 % , rdt en act. = 46 % .- Step 7: anion exchange column (mono Q HR 5/5 Pharmacia): This step is carried out in two stages, using half of the preceding fraction each time and with buffer B / 5. The buffer is modified by dialysis on an Amicon PM10 membrane and substitution with B / 5 buffer. The 6 ml of retentate are loaded onto the column balanced with buffer B / 5, at a flow rate of 1 ml × min 1 . After washing the column for 14 min, a gradient of 0 to 0.15 M in KC1 is applied in 25 min. The column is then fed with a solution of 1 M KC1 in buffer B / 5. In two batches, 3.75 ml are collected and analyzed (prot. = 1.5 mg, [prot.] = 0.4 gxl "1 , early act. = 121 U, specific act = 81 Uxmg " 1 prot., prot. rdt = 11%, active rdt. = 46%.
- Etape 8 : colonne échangeuse d'anions (mono Q HR 5/5 Pharmacia) : Les 3,7 ml de l'étape précédente sont dilués à 6 ml avec le tampon B/5, déposés sur la colonne. Les protéines sont éluées dans les conditions de l'étape 7. Les fractions actives sont réunies et analysées (vol. = 3 ml, protéines = 1 mg, [prot.] 0,34 gxl"1, act. tôt. = 93 U, act. spéc. = 92 Uxmg"1 de prot., rdt en prot. = 67 %, rdt en act. = 77 %). - Etape 9 : colonne d'interactions hydrophobes (octyl superose HR 5/5) :- Step 8: anion exchange column (mono Q HR 5/5 Pharmacia): The 3.7 ml of the previous step are diluted to 6 ml with buffer B / 5, deposited on the column. The proteins are eluted under the conditions of step 7. The active fractions are combined and analyzed (vol. = 3 ml, proteins = 1 mg, [prot.] 0.34 gxl "1 , early act. = 93 U , specific act = 92 Uxmg "1 of prot., rdt in prot. = 67%, rdt in act. = 77%). - Step 9: column of hydrophobic interactions (octyl superose HR 5/5):
Les 3 ml du pool de l'étape précédente sont amenés à 38 % de la saturation par addition d'une solution concentrée en sulfate d'ammonium. Les 6 ml résultants sont chargés sur la colonne, équilibrée avec le tampon B additionné de 1,7 M de sulfate d'ammonium. Les protéines sont éluées par un gradient décroissant de sulfate d'ammonium de 1,7 M à 0 M dans le tampon B en 1 h à un débit de 0,5 mlxmin 1. Les fractions actives sont réunies pour analyse (vol. = 1,5 ml, prot. = 0,75 mg, [prot.] = 0,5 g/1, act. tôt. = 53 U, act. spéc. = 70 Uxmg"1 de prot. , rdt en prot. = 75 %, rdt en act. = 56 % .The 3 ml of the pool from the previous step are brought to 38% of saturation by adding a concentrated solution of ammonium sulfate. The resulting 6 ml are loaded onto the column, equilibrated with buffer B supplemented with 1.7 M ammonium sulfate. The proteins are eluted by a decreasing gradient of ammonium sulphate from 1.7 M to 0 M in buffer B in 1 h at a flow rate of 0.5 ml × min 1 . The active fractions are assembled for analysis (vol. = 1.5 ml, prot. = 0.75 mg, [prot.] = 0.5 g / 1, early act. = 53 U, specific act = 70 Uxmg "1 of prot., prot. rd = 75%, active rd = 56%.
Etape 10 : dessalage (colonne de Fast desalting HR 10/1O Pharmacia) : Les 1,5 ml sont tout d'abord concentrés à l'aide du centricon PM 10 jusqu'à un volume de 0,5 ml. Cette fraction est déposée sur la colonne, équilibrée avec le tampon phosphate 10 mM pH 6,8. La protéine est éluée à un débit de 4 mlxmin"1. La protéine sort dans un volume de 2 ml environ (prot. tôt. = 360 μg, [prot.] = 180 mgxl"1, act. tôt. = 19 U, act. spéc. = 53 Uxmg 1 de prot., rdt en prot. = 48 %, rdt en act. 36 %).Step 10: desalting (column of Fast desalting HR 10 / 1O Pharmacia): The 1.5 ml are first concentrated using the PM 10 centricon to a volume of 0.5 ml. This fraction is deposited on the column, equilibrated with the 10 mM phosphate buffer pH 6.8. The protein is eluted at a flow rate of 4 ml × min "1. The protein leaves in a volume of approximately 2 ml (early prot. = 360 μg, [prot.] = 180 mg × l " 1 , early active. = 19 U, specific act = 53 Uxmg 1 of prot., rdt in prot. = 48%, rdt in act. 36%).
TABLEAU H PURIFICATION DE L'AMIDASE DE Comamonas NI 1TABLE H PURIFICATION OF THE AMIDASE OF Comamonas NI 1
VOL. PROT. ACTIVITE RENDEMENTFLIGHT. PROT. YIELD ACTIVITY
ETAPE PFSTEP PF
DEOF
ML MGML MG
PURIFICATION TOTALE 1 SPÉCIF. PROT. j ACTIV. U ! U/MG % j %TOTAL PURIFICATION 1 SPECIFIC. PROT. j ACTIV. U! U / MG% j%
0 - Extrait brut 125 2 700 2 100 j 0,78 100 100 10 - Raw extract 125 2 700 2 100 d 0.78 100 100 1
1 - (NH4)2S04 35-60 % 43 1 200 1 200 j 2,1 22 j 59 1 ,31 - (NH 4 ) 2 S0 4 35-60% 43 1,200 1,200 d 2.1 22 d 59 1, 3
2 - Q sepharose 170 580 1 200 j 2,1 22 | 59 2,72 - Q sepharose 170 580 1 200 d 2.1 22 | 59 2.7
3 - Phenyl sepharose 80 110 910 j 8,1 4,2 | 44 103 - Phenyl sepharose 80 110 910 d 8.1 4.2 | 44 10
4 - Gel filtration 12 26 470 j 18 0,9 j 23 234 - Gel filtration 12 26 470 d 18 0.9 d 23 23
5 - Gel filtration - - - j - j -5 - Gel filtration - - - d - d -
6 - Mono Q HR 5/5 16 17 262 ; 15 0,6 j 13 196 - Mono Q HR 5/5 16 17 262; 15 0.6 d 13 19
7 - Mono Q HR 5/5 3,7 1 ,5 121 j 81 0,05 j 5,8 1007 - Mono Q HR 5/5 3.7 1.5 121 d 81 0.05 d 5.8 100
8 - Mono Q HR 5/5 3 1 93 j 92 0,03 j 4,5 1208 - Mono Q HR 5/5 3 1 93 d 92 0.03 d 4.5 120
9 - Octyl superose HR 5/5 1 ,5 0,75 53 j 70 0,03 j 2,5 909 - Octyl superose HR 5/5 1, 5 0.75 53 d 70 0.03 d 2.5 90
10 - Dessalage 2 0,4 19 | 53 0,01 j 0,9 67 ABRÉVIATIONS :10 - Desalting 2 0.4 19 | 53 0.01 d 0.9 67 ABBREVIATIONS:
- PF = coefficient de purification,- PF = purification coefficient,
- U = 1 mole/ min.- U = 1 mole / min.
La fig. 3 annexée montre les résultats d'une analyse en gel de polyacrylamideFig. 3 attached shows the results of a polyacrylamide gel analysis
SDS des fractions obtenues aux différentes étapes de la purification. Les puits sont numérotés de 1 à 12, de gauche à droite, les poids moléculaires du marqueur ont été portés sur la droite de la figure (kilo Daltons). Les puits 1 et 12 contiennent des protéines standard de poids moléculaire connus, le puits 2 l'extrait brut et les puits 3 à 10 correspondent aux extraits purifiés des étapesSDS of the fractions obtained at the various stages of purification. The wells are numbered from 1 to 12, from left to right, the molecular weights of the marker have been shown on the right of the figure (kilo Daltons). Wells 1 and 12 contain standard proteins of known molecular weight, well 2 the crude extract and wells 3 to 10 correspond to the purified extracts from steps
1 à 8 et le puits l i a l'extrait purifié de l'étape 10. La bande correspondant à l'amidase selon l'invention est celle d'environ 40 kDa.1 to 8 and well 1 i has the purified extract from step 10. The band corresponding to the amidase according to the invention is that of approximately 40 kDa.
2.3 Caractérisations : Cette amidase analysée par gel SDS-PAGE présente une bande principale de2.3 Characterizations: This amidase analyzed by SDS-PAGE gel has a main band of
43 000 Da dont la séquence N-terminale est : (acides aminés 1-18 de la SEQ BD : 3 dans la liste de séquences annexée). Met-De-Glu- Asn-Ile-Ile- Ala-Lys-Leu-Lys- Asn-Ile-.Leu-Glu-Ser- Asn-Thr- Asn- ...43,000 Da whose N-terminal sequence is: (amino acids 1-18 of SEQ BD: 3 in the annexed sequence list). Met-De-Glu- Asn-Ile-Ile- Ala-Lys-Leu-Lys- Asn-Ile-.Leu-Glu-Ser- Asn-Thr- Asn- ...
Par gel filtration, le poids moléculaire est de 80 000 +/- 2 000 Da. L'amidase est donc une enzyme homodimérique.By gel filtration, the molecular weight is 80,000 +/- 2,000 Da. Amidase is therefore a homodimeric enzyme.
2.4 Activité de l'amidase en fonction de la température :2.4 Amidase activity as a function of temperature:
Un pool de fractions contenant l'amidase a été utilisé pour cette étude. Dans les conditions de la purification, ce pool contient 0,3 gxl"1 de protéine et possède une activité spécifique de 26 Uxmg"1 de protéine. L'activité de ce pool a été déterminée entre 10 et 60° C.A pool of fractions containing amidase was used for this study. Under the conditions of the purification, this pool contains 0.3 g × 1 −1 of protein and has a specific activity of 26 U × mg −1 of protein. The activity of this pool was determined between 10 and 60 ° C.
Les conditions de cette étude sont les suivantes : [adipamate] = 10 mM, tampon tris-HCl 10 mM, pH 7,5. Volume réactionnel 2 ml, temps de réactionThe conditions of this study are as follows: [adipamate] = 10 mM, 10 mM tris-HCl buffer, pH 7.5. Reaction volume 2 ml, reaction time
15 min, [protéine] = 7,5 mgxl 1. Les résultats sont regroupés dans la fig. 4. L'activité augmente en fonction de la température jusqu'à 50° C environ. Au-delà; une dénaturation est observée. Entre 10 et 40° C, la loi d'Arrhénius est observée et permet de calculer un ΔG de réaction de 83 kJxmole"1.15 min, [protein] = 7.5 mgxl 1 . The results are grouped in fig. 4. The activity increases as a function of the temperature up to approximately 50 ° C. Beyond; denaturation is observed. Between 10 and 40 ° C, Arrhenius' law is observed and makes it possible to calculate a reaction ΔG of 83 kJxmole "1 .
2.5 Activité en fonction du pH : A partir de la même préparation enzymatique que celle utilisée précédemment, l'activité est mesurée à différents pH. Les conditions de cette étude sont les suivantes : [adipamate] = 10 mM, tampon carbonate 10 mM, pH 10, tris-HCl 10 mM, pH 9, 8.5, 7.5, phosphate 10 mM, pH 6.5, acétate 10 mM, pH 3.5, 4.5, 5.5, T = 25° C, volume reactionnel 2 ml, temps de réaction 15 min, [protéine] = 7,5 mgxl"1.2.5 Activity as a function of pH: From the same enzymatic preparation as that used previously, the activity is measured at different pHs. The conditions of this study are as follows: [adipamate] = 10 mM, carbonate buffer 10 mM, pH 10, tris-HCl 10 mM, pH 9, 8.5, 7.5, phosphate 10 mM, pH 6.5, acetate 10 mM, pH 3.5 , 4.5, 5.5, T = 25 ° C, reaction volume 2 ml, reaction time 15 min, [protein] = 7.5 mgxl "1 .
Les résultats sont regroupés dans la fig. 5. Le pH optimum se situe entre 8,5 et 10, ce qui correspond bien au comportement généralement rencontré pour les amidases.The results are grouped in fig. 5. The optimum pH is between 8.5 and 10, which corresponds well to the behavior generally encountered for amidases.
2.6 Détermination du Km de l'amidase :2.6 Determination of the Km of amidase:
Le substrat utilisé est l'adipamate. Sa vitesse d'hydrolyse en adipate est déterminée à différentes concentrations en adipamate (0,2, 1, 2, 5 et 10 mM). Les conditions de cette étude sont les suivantes : T = 25° C, tampon Tris-HCl 10 mM, pH 7,5, extrait enzymatique 100 μl (cf. paragraphe 2.5), volume reactionnel 4 ml, sauf 10 ml pour 0,2 et 1 mM en adipamate, temps de réaction cinétique sur 2 h.The substrate used is adipamate. Its rate of hydrolysis to adipate is determined at different adipamate concentrations (0.2, 1, 2, 5 and 10 mM). The conditions of this study are as follows: T = 25 ° C, 10 mM Tris-HCl buffer, pH 7.5, enzyme extract 100 μl (see paragraph 2.5), reaction volume 4 ml, except 10 ml for 0.2 and 1 mM adipamate, kinetic reaction time over 2 h.
Comme montré à la fig. 6, la courbe obtenue en traçant la vitesse en fonction de la concentration en substrat est conforme à une cinétique de Michaelis- Menten. En conséquence, la transformation de Hanes S/V = f(S), représentée à la fig. 7 donne une constante de Michaelis de 0,25 mM.As shown in fig. 6, the curve obtained by plotting the speed as a function of the substrate concentration is in accordance with Michaelis-Menten kinetics. Consequently, the transformation of Hanes S / V = f (S), shown in fig. 7 gives a Michaelis constant of 0.25 mM.
EXEMPLE 3 : CLONAGE DU GÈNE CODANT POUR L'AMIDASE DE Comamonas testosteroni NI 1.EXAMPLE 3: CLONING OF THE GENE CODING FOR THE AMIDASE OF Comamonas testosteroni NI 1.
A partir de la séquence N-terminale présentée dans l'exemple 2.3, une sonde nucléotidique a été synthétisée. Il s'agit d'un 20 mer 256 fois dégénéré : M I E N I I A (SEQ ID : 5)From the N-terminal sequence presented in Example 2.3, a nucleotide probe was synthesized. It is a 20 deg 256 degenerated sea: MIENIIA (SEQ ID: 5)
5 ' ATG ATT GAA AAT ATT ATT GC 3' (SEQ ID : 4) C G C C C A A A5 'ATG ATT GAA AAT ATT ATT GC 3' (SEQ ID: 4) C G C C C A A A
Dans la liste de séquences annexée, la séquence de 20 bases est désignée par SEQ ID : 4 tandis que la séquence d'aminoacides y associée est quant à elle référencée SEQ ID : 5. La stratégie suivie a consisté, tout d'abord, à vérifier la spécificité de cette sonde nucléotidique et à déterminer la nature des fragments d'ADN génomique à cloner. Brièvement, l'ADN génomique de Comamonas testosteroni NI 1 a été digéré par plusieurs enzymes de restriction correspondant à des sites utilisables pour le clonage. Après électrophorèse sur gel d'agarose et transfert sur membrane de nylon, les diverses digestions ont été hybridées à la sonde. La sonde a une spécificité suffisante dans les conditions d'hybridation utilisées (5x SSC, 5x Denhardt, 0,1 % SDS, 50 mM NaPO4 pH 6,5, 250 μgxml 1 ssDNA, température d'hybridation 50° C. Conditions de lavage : 1 h, 6x SSC, température ambiante et 5 min en 2 x SSC, 0, 1 % SDS à 50° C). Dans ces conditions, la sonde nous a permis d'obtenir des signaux importants et sans ambiguité. En particulier, dans le cas des digestions par Sstl, Pstl et BglLL. Les empreintes d'hybridation montrent, en particulier, l'existence d'un fragment unique Sstl d'environ 5,5 kb. Afin de cloner ce fragment, les fragments de 5 à 6 kb d'une digestion Sstl de l'ADN génomique ont été purifiés par électrophorèse préparative sur agarose et électroélution, puis ligaturés au plasmide pUC 19, lui-même digéré par Sstl. Après transformation dans la souche DH5α, 500 clones blancs sur LB ampicilline X-gal ont été repiqués individuellement, transférés sur membrane de nylon, puis analysés par hybridation avec la sonde ayant servi pour hybrider le Southern blot et ce dans les mêmes conditions de stringence. Deux clones ont ainsi été repérés comme hybridant très fortement avec la sonde. Ces deux clones se sont avérés contenir un plasmide ayant inséré le même fragment d'environ 5,5 kb dans la même orientation. L'un de ces deux plasmides (pXL2104) a été analysé plus en détail (cartographie de restriction, séquençage partiel en utilisant la sonde comme amorce et Southern blot). Il a ainsi pu être montré que la partie 5' du gène, qui hybride avec la sonde, est localisée sur un fragment de Pstl/Spel de 420 pb environ et que le gène est orienté dans le sens Spel vers Pstl. La fig. 8 présente une carte de restriction de ce plasmide.In the annexed sequence list, the 20 base sequence is designated by SEQ ID: 4 while the amino acid sequence associated therewith is referenced SEQ ID: 5. The strategy followed consisted, first of all, in verify the specificity of this nucleotide probe and determine 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 probe. The probe has sufficient specificity under the hybridization conditions used (5x SSC, 5x Denhardt, 0.1% SDS, 50 mM NaPO 4 pH 6.5, 250 μgxml 1 ssDNA, hybridization temperature 50 ° C. washing: 1 h, 6x SSC, room temperature and 5 min in 2 x SSC, 0.1% SDS at 50 ° C). Under these conditions, the probe allowed us to obtain important and unambiguous signals. In particular, in the case of digests with Sstl, Pstl and BglLL. The hybridization fingerprints show, in particular, the existence of a single Sst1 fragment of approximately 5.5 kb. In order to clone this fragment, the 5 to 6 kb fragments of an Sst1 digestion of the genomic DNA were purified by preparative electrophoresis on agarose and electroelution, then ligated to the plasmid pUC 19, itself digested with Sst1. After transformation into the DH5α strain, 500 white clones on LB ampicillin X-gal were individually subcultured, transferred to a nylon membrane, then analyzed by hybridization with the probe used to hybridize the Southern blot, under the same stringency conditions. Two clones were thus identified as very strongly hybridizing with the probe. These two clones were found to contain a plasmid having inserted the same fragment of approximately 5.5 kb in the same orientation. One of these two plasmids (pXL2104) was further analyzed detail (restriction mapping, partial sequencing using the probe as primer and Southern blot). It has thus been possible to show that the 5 ′ part of the gene, which hybridizes with the probe, is located on a Pstl / Spel fragment of approximately 420 bp and that the gene is oriented in the Spel towards Pstl direction. Fig. 8 shows a restriction map of this plasmid.
EXEMPLE 4 : SÉQUENCE D'UN FRAGMENT DE 1491 pb CONTENANT LE GÈNE CODANT POUR L'AMIDASE DE Comamonas testosteroni NI 1.EXAMPLE 4 SEQUENCE OF A 1491 bp FRAGMENT CONTAINING THE GENE ENCODING FOR THE AMIDASE OF Comamonas testosteroni NI 1.
La localisation du fragment de 1491 pb séquence, contenant le gène codant pour l'amidase de Comamonas testosteroni NI 1, est indiquée sous l'insert clone sur la fig. 9. La stratégie de séquençage de ce fragment, réalisé selon des méthodes classiques connues par l'homme de métier, est représentée sur la même figure. Les diverses séquences ont toutes été obtenues par la méthode de terminaison de chaînes (kit sequenase en présence de 7-déaza dGTP ; (35S)dATP) soit sur des matrices simple brin de M13 (mpl8 ou mρl9 cf YANISH et al., Gène 33 (1985) 103) recombinant portant des sous fragments, soit directement sur le plasmide pXL2104. Plusieurs amorces spécifiques ont également été synthétisées dans ce but. La séquence est présentée sur la fig. 2. et sur SEQ BD : 2 de la liste de séquences annexée. Le GC % moyen de la séquence obtenue est de 33,6 % , ce qui est inférieur au GC % de 61,5 % décrit chez d'autres souches de Comamonas (TAMAOKA et al. , Int. J. Syst. Bacteriol, 1987, 37, 52-59). Une analyse de la séquence obtenue a permis de caractériser une phase ouverte de 1254 pb codant pour un polypeptide de 418 résidus correspondant à un poids moléculaire de 46 148 Da. Ce polypeptide comprend la séquence NH2 terminale utilisée pour synthétiser la sonde.The location of the 1491 bp sequence fragment containing the gene coding for the amidase of Comamonas testosteroni NI 1 is indicated under the clone insert in FIG. 9. The strategy for sequencing this fragment, produced according to conventional methods known to those skilled in the art, is shown in the same figure. The various sequences were all obtained by the chain termination method (sequenase kit in the presence of 7-daza dGTP; ( 35 S) dATP) either on single-stranded matrices of M13 (mpl8 or mρl9 cf YANISH et al., Gene 33 (1985) 103) recombinant carrying subfragments, either directly on the plasmid pXL2104. Several specific primers have also been synthesized for this purpose. The sequence is shown in fig. 2. and on SEQ BD: 2 from the attached sequence list. The average GC% of the sequence obtained is 33.6%, which is lower than 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 an open phase of 1254 bp coding for a polypeptide of 418 residues corresponding to a molecular weight of 46,148 Da. This polypeptide comprises the terminal NH 2 sequence used to synthesize the probe.
Ainsi cette phase ouverte représente le gène structural de l'amidase recherchée. Ce gène sera dénommé amdA dans la suite du présent exposé.Thus this open phase represents the structural gene for the amidase sought. This gene will be called amdA in the remainder of this description.
A partir du plasmide pXL2104, le plasmide pXL2216 a été préparé par insertion du fragment Spel-Clal contenant le gène amdA entre les sites Xbal et Clal du vecteur pMTL 23 (CHAMBERS et Al. , Gène, 68 (1988) 139-149). Ce plasmide, dont la carte de restriction est présentée sur la figure 10, a été utilisé pour transformer la souche de E. coli TG 1. La souche résultante appelée G 4315 a été déposée à la Collection Nationale de Culture de Micro-organismes à Paris (Institut Pasteur, 25 rue du Docteur Roux) sous le n° 1 1279.From plasmid pXL2104, plasmid pXL2216 was prepared by insertion of the Spel-ClaI fragment containing the amdA gene between the Xbal and ClaI sites of the vector pMTL 23 (CHAMBERS et al., Gene, 68 (1988) 139-149). This plasmid, the restriction map of which is presented in FIG. 10, was used to transform the strain of E. coli TG 1. The resulting strain called G 4315 was deposited at the National Collection of Culture of Microorganisms in Paris (Institut Pasteur, 25 rue du Docteur Roux) under No. 1 1279.
EXEMPLE 5 : EXPRESSION DE L'AMIDASE DE Comamonas Testosteroni NI 1EXAMPLE 5 EXPRESSION OF THE AMIDASE OF Comamonas Testosteroni NI 1
DANS E. coli.IN E. coli.
Afin de confirmer l'identité entre le gène amdA et le gène codant pour l'amidase deIn order to confirm the identity between the amdA gene and the gene coding for amidase
Comamonas testosteroni NI 1 , un plasmide a été construit dans lequel le gène amdA, précédé de son propre site de fixation des ribosomes, est placé sous le contrôle du promoteur de l'opéron lactose de E. coli. Ainsi, le plasmide pXL2144, décrit sur la fig. 11, a été obtenu par insertion du fragment Spel-Clάl dérivé du plasmide pXL2104 entre les sites Xbal et Clal du vecteur pMTL 22 (CHAMBERS et al., Gène,Comamonas testosteroni NI 1, a plasmid was constructed in which the amdA gene, preceded by its own ribosome binding site, is placed under the control of the promoter of the lactose operon of E. coli. Thus, the plasmid pXL2144, described in FIG. 11, was obtained by insertion of the Spel-Clάl fragment derived from the plasmid pXL2104 between the Xbal and ClaI sites of the vector pMTL 22 (CHAMBERS et al., Gene,
68 (1988) 139-149). Ce plasmide contient donc le promoteur de l'opéron lactose Plac, suivi du site de fixation des ribosomes et du gène structural de l'amidase, ainsi qu'un gène conférant la résistance à l'ampicilline.68 (1988) 139-149). This plasmid therefore contains the promoter of the lactose operon Plac, followed by the ribosome binding site and the structural gene for amidase, as well as a gene conferring resistance to ampicillin.
Le plasmide pXL2144 a été utilisé pour transformer la souche de E. coli TG 1. La sélection des micro-organismes se fait sur a picilline. L'expression de l'amidase de Comamonas testosteroni NI 1 a été visualisée, après sonication des cellules, par gel SDS-PAGE, dans la fraction brute ou, après centrifugation, dans le culot et dans le surnageant.The plasmid pXL2144 was used to transform the strain of E. coli TG 1. The selection of the microorganisms is done on a picillin. The expression of the amidase of Comamonas testosteroni NI 1 was visualized, after sonication of the cells, by SDS-PAGE gel, in the crude fraction or, after centrifugation, in the pellet and in the supernatant.
Chaque piste correspond à une quantité de protéine équivalente à 60 μl de culture à une DO de 3 à 610 nm. La fig. 12 présente les résultats obtenus. Les poids moléculaires des marqueurs sont indiqués en kilodaltons par les flèches à droite du gel sur cette figure.Each lane corresponds to an amount of protein equivalent to 60 μl of culture at an OD of 3 to 610 nm. Fig. 12 presents the results obtained. The molecular weights of the markers are indicated in kilodaltons by the arrows to the right of the gel in this figure.
Le contenu des différentes pistes est indiqué sur le tableau suivant. TGl + TGl + TG1 + TG1 + pXL2144 PXL2144 pMTL22 pMTL22 + IPTG + IPTGThe content of the different tracks is shown in the following table. TGl + TGl + TG1 + TG1 + pXL2144 PXL2144 pMTL22 pMTL22 + IPTG + IPTG
Surnageant A D G JSupernatant A D G J
Culot B E H KBase B E H K
Fraction brute C F I LGross fraction C F I L
Il ressort de la fig. 12 que les extraits de cellules comprenant le plasmide pXL2144 expriment l'amidase selon l'invention, essentiellement sous forme soluble une bande à environ 45 kDa est visible pour les pistes A. CD. F, alors que celle-ci n'apparait pas pour les extraits G à L.It appears from fig. 12 that the cell extracts comprising the plasmid pXL2144 express the amidase according to the invention, essentially in soluble form a band at approximately 45 kDa is visible for the A. CD lanes. F, whereas this one does not appear for extracts G to L.
De plus, la comparaison de la bande à 45 kDa environ des extraits A.C et des extraits D.F montre que les cellules cultivées en présence d'ITPG ont un niveau d'expression plus élevé.In addition, comparison of the band at around 45 kDa of the A.C extracts and the D.F extracts shows that the cells cultured in the presence of ITPG have a higher level of expression.
EXEMPLE 6 : ACTIVITÉ DE L'AMIDASE CHEZ Escherichia coli.EXAMPLE 6 ACTIVITY OF AMIDASE IN Escherichia coli.
L'activité de la souche E. coli (pXL2144), induite ou non, est mesurée sur adipamide et adipamate et comparée à celle de la souche témoin E. coli (pMTL 22).The activity of the E. coli strain (pXL2144), whether induced or not, is measured on adipamide and adipamate and compared to that of the control strain E. coli (pMTL 22).
6.1 Préparation des cellules :6.1 Cell preparation:
Les cultures sont réalisées dans les conditions décrites dans le TABLEAU HI ci- dessous. Au cours de la phase exponentielle de croissance, une des deux cultures de la souche recombinante est induite par l'IPTG 1 mM, après encore 2 h à 37° C cette culture est traitée. TABLEAU : CULTURE DES SOUCHESThe cultures are carried out under the conditions described in TABLE HI below. During the exponential growth phase, one of the two cultures of the recombinant strain is induced by 1 mM IPTG, after another 2 h at 37 ° C. this culture is treated. TABLE: CULTURE OF STRAINS
P. S.P. S.
MICRO-ORGANISMES MILIEU DO 660 nm g/iMICROORGANISMS MEDIUM DO 660 nm g / i
E. coli (PMTL22) B 2,2 0,84E. coli (PMTL22) B 2.2 0.84
E. coli (pXL2144) B 1,8 0,75E. coli (pXL2144) B 1.8 0.75
E. coli (pXL2144) C 2,3 1,10E. coli (pXL2144) C 2.3 1.10
ABRÉVIATIONS :ABBREVIATIONS:
- B = milieu LB + Amp 100 μgxml"1,- B = medium LB + Amp 100 μgxml " 1 ,
- C = milieu B + ajout de IPTG 1 mM à 0,8 de DOe^. CONDITIONS COMMUNES :- C = medium B + addition of 1 mM IPTG to 0.8 DOe ^. COMMON CONDITIONS:
Ensemencement au l/100ème à partir d'une préculture âgée de 17 h, temps de culture 5,5 h, T = 37° C. 6.2 Mesures des activités spécifiques :Seeding in l / 100th from a preculture aged 17 h, culture time 5.5 h, T = 37 ° C. 6.2 Measures of specific activities:
Les conditions de mesure des activités spécifiques et les résultats sont répertoriés dans le TABLEAU IV ci dessous.The conditions for measuring specific activities and the results are listed in TABLE IV below.
Les conditions communes de ces mesures sont les suivantes : T = 25° C, tampon phosphate 50 mM pH 7, cinétique sur 90 min. The common conditions for these measurements are as follows: T = 25 ° C, phosphate buffer 50 mM pH 7, kinetics over 90 min.
TABLEAU TV : MESURES DES ACTIVITÉS AMIDASIQUES DES SOUCHES RECOMBINANTES QUI EXPRIMENT L'AMIDASE DE Comamonas NI 1.TV TABLE: MEASUREMENTS OF THE AMIDASIC ACTIVITIES OF THE RECOMBINANT STRAINS THAT EXPRESS THE AMIDASE OF Comamonas NI 1.
SUBSTRAT ACTIVITÉSUBSTRATE ACTIVITY
SOUCHES SPÉCIFIQUESPECIFIC STRAINS
Nature [cel.] Induit Etat Nature [S] μmole/min/g g/i +/- mM de PSNature [cel.] Induced Nature State [S] μmole / min / g g / i +/- mM of PS
E. coli (pXL2144) 6,2 + E Adipamide 15 14E. coli (pXL2144) 6.2 + E Adipamide 15 14
6,1 + ST Adipamide 12 156.1 + ST Adipamide 12 15
6, 1 + se Adipamide 12 06, 1 + se Adipamide 12 0
6,1 + SS Adipamide 12 146.1 + SS Adipamide 12 14
6,2 - E Adipamide 11 06.2 - E Adipamide 11 0
0,3 + E Adipamate 50 7000.3 + E Adipamate 50 700
0,25 + ST Adipamate 50 2 0000.25 + ST Adipamate 50 2,000
2,50 + se Adipamate 50 972.50 + se Adipamate 50 97
0,25 + ss Adipamate 50 1 9000.25 + ss Adipamate 50 1 900
0,69 - E Adipamate 50 1300.69 - E Adipamate 50 130
1,4 - ST Adipamate 50 5001.4 - ST Adipamate 50 500
2,8 - se Adipamate 50 462,8 - se Adipamate 50 46
1,4 - ss Adipamate 50 4331.4 - ss Adipamate 50 433
E. coli (pMTL22) 10 E Adipamate 25 0E. coli (pMTL22) 10 E Adipamate 25 0
14 E Adipamide 13 014 E Adipamide 13 0
ABRÉVIATIONS :ABBREVIATIONS:
- E = cellules entières,- E = whole cells,
- ST = broyât cellulaire,- ST = cell ground material,
- SC = fraction insoluble du broyât cellulaire,- SC = insoluble fraction of the ground cell,
- SS = fraction soluble du broyât cellulaire. EXEMPLE 8 : EXPRESSION ET ACTΓVΠΈ DE L'AMIDASE DANS Pseudomonas putida- SS = soluble fraction of the ground cell. EXAMPLE 8 EXPRESSION AND ACTΓVΠΈ OF AMIDASE IN Pseudomonas putida
L'amidase a été exprimée chez Pseudomonas putida à l'aide de la construction suivante : A partir de pXL2216 décrit sur la figure 10, le fragment SaWBgtll de 1.75kb contenant le gène amdA a été introduit entre les sites Sali et Bam l de pDSK519 (Keen et al., 1988, Gène. 70 : 191). Ce plasmide, pXL2238, décrit sur la figure 13 a été introduit dans la souche de Pseudomonas putida G 2081. G 2081 est une dérivée de Pseudomonas putida 2440 (Bagdasarian and Timmis, 1981. In Hofschneid and Goebel. Topics in Microbiology and Immunology. 47. Springer Verlag. Berlin.) rendue résistante à l'acide nalidixique et à la rifampicine. Le vecteur pDSK519 a été utilisé comme plasmide témoin.The amidase was expressed in Pseudomonas putida using the following construct: From pXL2216 described in FIG. 10, the 1.75kb SaWBgtll fragment containing the amdA gene was introduced between the SalI and Bam I sites of pDSK519 (Keen et al., 1988, Gene. 70: 191). This plasmid, pXL2238, described in FIG. 13, was introduced into the strain of Pseudomonas putida G 2081. G 2081 is a derivative of Pseudomonas putida 2440 (Bagdasarian and Timmis, 1981. In Hofschneid and Goebel. Topics in Microbiology and Immunology. 47 Springer Verlag. Berlin.) Made resistant to nalidixic acid and rifampicin. The vector pDSK519 was used as a control plasmid.
L'activité de la souche G 2081 (pXL2238) est mesurée sur adipamate et comparée à celle de la souche témoin G2081 (pDSK519). Les souches Pseudomonas G 2081 (pDSK519) et Pseudomonas G 2081 (pXL2238) ont été cultivées en milieu LBk (Tryptone 10 g/1, NaCl 5g/l, extrait de levure 5g/l, glucose 10g/l, sulfate d'ammonium 1 g/1 et kanamycine 100 mg/1). Les résultats des cultures sont regroupés dans le tableau V.The activity of strain G 2081 (pXL2238) is measured on adipamate and compared to that of the control strain G2081 (pDSK519). The Pseudomonas G 2081 (pDSK519) and Pseudomonas G 2081 (pXL2238) strains were cultured in LBk medium (Tryptone 10 g / 1, NaCl 5g / l, yeast extract 5g / l, glucose 10g / l, ammonium sulfate 1 g / 1 and kanamycin 100 mg / 1). The results of the cultures are collated in Table V.
TABLEAU V : CULTURE DES SOUCHES ESTIMEES POUR LEUR ACTIVITE AMIDASETABLE V: CULTURE OF ESTIMATED STRAINS FOR THEIR AMIDASE ACTIVITY
Micro-organisme OOβήOnn, Poids secMicroorganism OO βή Onn, Dry weight
(g/D(l / R
Pseudomonas (pDSK519) 2,3 0,9Pseudomonas (pDSK519) 2.3 0.9
Pseudomonas (pXL2238) 1,8 0,5Pseudomonas (pXL2238) 1.8 0.5
Les activités de ces souches ont été déterminées sur adipamate et adipamide. Les conditions et résultats sont répertoriés dans le tableau VI. TABLEAU VI CONDITIONS REACTÏONNELLES ET RESULTATS DES MESURES DE L'ACTTVΓΓÉ AMIDASE DES SOUCHES RECOMBINANTES PSEUDOMONAS (PDSK519) ET PSEUDOMONAS (PXL2238)The activities of these strains were determined on adipamate and adipamide. The conditions and results are listed in Table VI. TABLE VI REACTIONAL CONDITIONS AND RESULTS OF MEASUREMENTS OF THE ACIDTVΓΓÉ AMIDASE OF THE RECOMBINANT STRAINS PSEUDOMONAS (PDSK519) AND PSEUDOMONAS (PXL2238)
Conditions réactionnellesReaction conditions
Souches durée volume Substrat Activité ((U/kg de CS)Strains duration volume Substrate Activity ((U / kg of CS)
nature (g/1) état (h) (ml) nature (mM)nature (g / 1) state (h) (ml) nature (mM)
Comamonas 5,6 E 4 5,5 adipamide 20 0,5 testosteroni Nil adipamate 20 2,0Comamonas 5.6 E 4 5.5 adipamide 20 0.5 testosteroni Nile adipamate 20 2.0
P. putida 2 E 3 4 adipamide 20 0 (pDSK519)P. putida 2 E 3 4 adipamide 20 0 (pDSK519)
2 adipamate 50 02 adipamate 50 0
P. Putida 2 E 1,5 2 adipamide 18 0 (pXL2238)P. Putida 2 E 1.5 2 adipamide 18 0 (pXL2238)
E adipamate 45 3,8E adipamate 45 3.8
S adipamate 50 5, 1S adipamate 50 5, 1
Conditions communes : tampon phosphate 50 mM pli 7 ; T C 25-28 ; Abréviations : E cellules entières, S broyât cellulaire ; U mole d 'amide hydrolysée/h ; CS cellules sèches ;Common conditions: phosphate buffer 50 mM fold 7; T C 25-28; Abbreviations: E whole cells, S ground cell; U mole of hydrolyzed amide / h; CS dry cells;
La souche Pseudomonas (pXL2238) exprime le gène de l'amidase de Comamonas NI.1 et cela d'autant mieux que la préparation cellulaire est soniquee.The Pseudomonas strain (pXL2238) expresses the amidase gene from Comamonas NI.1 and this is all the better when the cell preparation is sonic.
Ces expériences montrent que l'amidase est exprimée et active chez Pseudomonas putida. EXEMPLE 9 COMPARATIF DES ACTIVITÉS SPÉCIFIQUES SUR ADIPAMIDE ET ADIPAMATE DE SOUCHES EXPRIMANT UNE AMIDASE SELON L'ART ANTÉRIEUR (FR 90-14 853) ET SOUCHES EXPRIMANT UNE AMIDASE SELON L'INVENTION.These experiments show that amidase is expressed and active in Pseudomonas putida. COMPARATIVE EXAMPLE 9 ACTIVITIES SPECIFIC TO ADIPAMIDE AND ADIPAMATE OF STRAINS EXPRESSING AN AMIDASE ACCORDING TO THE PRIOR ART (FR 90-14 853) AND STRAINS EXPRESSING AN AMIDASE ACCORDING TO THE INVENTION.
Les conditions des mesures et les résultats sont donnés dans le TABLEAU VIII ci- dessous.The conditions of the measurements and the results are given in TABLE VIII below.
TABLEAU VfflVffl TABLE
SOUCHES SUBSTRAT ACTIVITÉSUBSTRATE ACTIVITY STRAINS
SPÉCIFIQUESPECIFIC
Nature [Cel.] Induit Etat Nature [S] (μmole/Nature [Cel.] Induced Nature State [S] (μmole /
+/- (mM) min/g (g/1)+/- (mM) min / g (g / 1)
Brevibacterium (R 312) 0,06 + E Adipamide 20 2,3Brevibacterium (R 312) 0.06 + E Adipamide 20 2.3
0,3 + ST Adipamide 20 100.3 + ST Adipamide 20 10
0,3 + ST Adipamate 20 4,20.3 + ST Adipamate 20 4.2
E. coli (pXL1751) 0,3 + E Adipamide 20 75E. coli (pXL1751) 0.3 + E Adipamide 20 75
ART 0,3 + E Adipamate 20 16 ANTÉRIEUR 0,3 + ST Adipamate 20 16ART 0.3 + E Adipamate 20 16 PREVIOUS 0.3 + ST Adipamate 20 16
Rhodococcus sp 0,76 + E Adipamide 20 35 0,76 + ST Adipamide 20 62 0,32 + ST Adipamate 20 55Rhodococcus sp 0.76 + E Adipamide 20 35 0.76 + ST Adipamide 20 62 0.32 + ST Adipamate 20 55
C. testosteroni sp 5,6 + E Adipamide 20 8,3 5,6 + E Adipamate 20 33C. testosteroni sp 5.6 + E Adipamide 20 8.3 5.6 + E Adipamate 20 33
INVENTIONINVENTION
E. coli (pXL2144) 6,2 + E Adipamide 15 14 6,1 + ST Adipamide 12 15 0,3 + E Adipamate 50 700 0,25 + ST Adipamate 50 2 000 CONDITIONS COMMUNES :E. coli (pXL2144) 6.2 + E Adipamide 15 14 6.1 + ST Adipamide 12 15 0.3 + E Adipamate 50 700 0.25 + ST Adipamate 50 2 000 COMMON CONDITIONS:
- T = 25° C- T = 25 ° C
- Tampon phosphate 50 mM pH 7. ABRÉVIATIONS : - E = cellules entières,- 50 mM phosphate buffer pH 7. ABBREVIATIONS: - E = whole cells,
- ST ≈ broyât cellulaire.- ST ≈ ground cell.
Le plasmide pXL1751 de la souche recombinée E. Coli est décrit en détail dans la demande de brevet FR 90-14 853.The plasmid pXL1751 of the recombinant E. Coli strain is described in detail in patent application FR 90-14 853.
Le TABLEAU Vu met clairement en évidence la plus grande spécificité de l'amidase suivant l'invention vis-à-vis de l'adipamate que vis-à-vis de l'adipamide, à l'inverse de ce que l'on observe pour les amidases connues. Cette observation vaut aussi bien pour une expression amidasique par les germes naturels que par les germes recombinés. The TABLE seen clearly highlights the greater specificity of the amidase according to the invention with respect to adipamate than with respect to adipamide, contrary to what is observed. for known amidases. This observation is just as valid for an adidas expression by natural germs as by recombinant germs.
LISTE DE SEQUENCESLIST OF SEQUENCES
(1) INFORMATION GENERALE:(1) GENERAL INFORMATION:
(i) DEPOSANT:(i) DEPOSITOR:
(A) NOM: RHONE-POULENC CHIMIE(A) NAME: RHONE-POULENC CHEMISTRY
(B) RUE: 25, Quai Paul Doumer(B) STREET: 25, Quai Paul Doumer
(C) VILLE: Courbevoie(C) CITY: Courbevoie
(E) PAYS: France(E) COUNTRY: France
(F) CODE POSTAL: 92400(F) POSTAL CODE: 92400
(G) TELEPHONE: 47 68 12 34 (H) TELECOPIE: 47 68 16 56(G) TELEPHONE: 47 68 12 34 (H) FAX: 47 68 16 56
(ii) TITRE DE L' INVENTION: Polypeptides à activité amidase, outils génétiques et micro-organismes hôtes permettant leur obtention, procédé d'hydrolyse mettant en oeuvre lesdits polypeptides(ii) TITLE OF THE INVENTION: Amidase activity polypeptides, genetic tools and host microorganisms for obtaining them, hydrolysis process using said polypeptides
(iii) NOMBRE DE SEQUENCES: 5(iii) NUMBER OF SEQUENCES: 5
(iv) FORME LISIBLE PAR ORDINATEUR:(iv) COMPUTER-READABLE FORM:
(A) TYPE DE SUPPORT: Floppy disk(A) TYPE OF SUPPORT: Floppy disk
(B) ORDINATEUR: IBM PC compatible(B) COMPUTER: IBM PC compatible
(C) SYSTEME D' EXPLOITATION: PC-DOS/MS-DOS(C) OPERATING SYSTEM: PC-DOS / MS-DOS
(D) LOGICIEL: Patentin Release #1.0, Version #1.25 (OEB)(D) SOFTWARE: Patentin Release # 1.0, Version # 1.25 (EPO)
(vi) DONNEES DE LA DEMANDE ANTERIEURE:(vi) DATA FROM THE PREVIOUS APPLICATION:
(A) NUMERO DE DEPOT: FR 93 01062(A) DEPOSIT NUMBER: FR 93 01 062
(B) DATE DE DEPOT: 27-JAN-1993(B) DEPOSIT DATE: 27-JAN-1993
(2) INFORMATION POUR LA SEQ ID NO: 1:(2) INFORMATION FOR SEQ ID NO: 1:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 4l8 acides aminés(A) LENGTH: 4l8 amino acids
(B) TYPE: acide aminé(B) TYPE: amino acid
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: protéine(ii) TYPE OF MOLECULE: protein
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 1: Met Ile Glu Asn Ile Ile Ala Lys Leu Lys Asn Ile Leu Glu Ser Asn(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: Met Ile Glu Asn Ile Ala Lys Leu Lys Asn Ile Leu Glu Ser Asn
1 • 5 10 151 • 5 10 15
Thr Asn Ser Phe Ile Ser Leu Asp Leu Val Asp Val Ser Lys Gin LeuThr Asn Ser Phe Ile Ser Leu Asp Leu Val Asp Val Ser Lys Gin Leu
20 25 3020 25 30
Lys Lys Gin Asp Ser Asn Gin Pro Leu Asn Gly Val Thr Val Ala IleLys Lys Gin Asp Ser Asn Gin Pro Leu Asn Gly Val Thr Val Ala Ile
35 40 4535 40 45
Lys Asp Leu Ile Asp Val Glu Gly Gin Lys Thr Thr Met Gly Ser AlaLys Asp Leu Ile Asp Val Glu Gly Gin Lys Thr Thr Met Gly Ser Ala
50 55 6050 55 60
Gin Tyr Gin Asn Asn Ile Ala Arg Ser Asp Ala Asp Val Val Arg Arg 65 70 75 80Gin Tyr Gin Asn Asn Ile Ala Arg Ser Asp Ala Asp Val Val Arg Arg 65 70 75 80
Leu Arg Thr Ala Gly Ala Val Ile Phe Gly Lys Thr Asn Thr His GluLeu Arg Thr Ala Gly Ala Val Ile Phe Gly Lys Thr Asn Thr His Glu
85 90 9585 90 95
Phe Ala Tyr Gly Ser Thr Gly Asp Lys Ser Phe Phe Gly Pro Val LeuPhe Ala Tyr Gly Ser Thr Gly Asp Lys Ser Phe Phe Gly Pro Val Leu
100 105 no100 105 no
Asn Pro Asn Asn Glu Asn His Ile Thr Gly Gly Ser Ser Ser Gly SerAsn Pro Asn Asn Glu Asn His Ile Thr Gly Gly Ser Ser Ser Gly Ser
115 120 125115 120 125
Ala Ala Ala Val Ala Ala Asp Leu Cys Asp Val Ala Ile Gly Thr AspAla Ala Ala Val Ala Ala Asp Leu Cys Asp Val Ala Ile Gly Thr Asp
130 135 140130 135 140
Thr Ala Ala Ser Val Arg Leu Pro Ala Ala Leu Cys Gly Val Ile Gly 145 150 155 160Thr Ala Ala Ser Val Arg Leu Pro Ala Ala Leu Cys Gly Val Ile Gly 145 150 155 160
Leu Lys Pro Thr Tyr Asn Ser Ile Lys Arg Asp Gly Val Phe Ser LeuLeu Lys Pro Thr Tyr Asn Ser Ile Lys Arg Asp Gly Val Phe Ser Leu
165 170 175165 170 175
Ser Pro Ser Leu Asp His Val Gly Ile Ile Ser Lys Ser Leu Asp LeuSer Pro Ser Leu Asp His Val Gly Ile Ile Ser Lys Ser Leu Asp Leu
180 185 190180 185 190
Ile Glu Lys Thr Phe Asn Ser Thr Lys Thr Ser Ile Asp Gin Lys ProIle Glu Lys Thr Phe Asn Ser Thr Lys Thr Ser Ile Asp Gin Lys Pro
195 200 205195 200 205
Gin Lys Asn Leu Asp Lys Lys Leu Thr Ile Gly Leu Leu Lys Gly PheGin Lys Asn Leu Asp Lys Lys Leu Thr Ile Gly Leu Leu Lys Gly Phe
210 215 220210 215 220
Phe Glu Glu Tyr Ile Cys Ser Ser Val Lys Glu Lys Tyr Asp Glu Thr 225 230 235 240Phe Glu Glu Tyr Ile Cys Ser Ser Val Lys Glu Lys Tyr Asp Glu Thr 225 230 235 240
Ile Glu Leu Leu Arg Ser Asn Gly Cys Glu Ile Lys Glu Ile Asn IleIle Glu Leu Leu Arg Ser Asn Gly Cys Glu Ile Lys Glu Ile Asn Ile
245 250 255245 250 255
Lys Glu Ala Phe Asp Ile Tyr Thr Asn Ser Gin Ile Val Leu Lys TyrLys Glu Ala Phe Asp Ile Tyr Thr Asn Ser Gin Ile Val Leu Lys Tyr
260 265 270260 265 270
Glu Ala Tyr Lys Ile His Glu Glu Ser Leu Lys Leu Asn Tyr Pro PheGlu Ala Tyr Lys Ile His Glu Glu Ser Leu Lys Leu Asn Tyr Pro Phe
275 280 285275 280 285
Asp Pro Glu Val Lys Gly Arg Ile Leu Arg Gly Glu Asn Ile Thr Lys 290 295 300 Asp Glu Tyr Asp Ile Ala Lys Lys Tyr Gin Glu Asn Ala Arg Ile Ile 305 310 315 320Asp Pro Glu Val Lys Gly Arg Ile Leu Arg Gly Glu Asn Ile Thr Lys 290 295 300 Asp Glu Tyr Asp Asp Ala Lys Lys Tyr Gin Glu Asn Ala Arg Ile Ile 305 310 315 320
Phe Asp Thr Ala Leu Lys Asp Val His Val Leu Ala Ser Leu Thr SerPhe Asp Thr Ala Leu Lys Asp Val His Val Leu Ala Ser Leu Thr Ser
325 330 335325 330 335
Gly Ile Leu Pro Pro Lys Ile Phe Glu Arg Gin Thr Lys Val Asn GluGly Ile Leu Pro Pro Lys Ile Phe Glu Arg Gin Thr Lys Val Asn Glu
340 3+5 350340 3 + 5 350
Asn Thr Val Glu Thr Phe Phe Leu Leu Thr Arg Leu Thr Ala Pro IleAsn Thr Val Glu Thr Phe Phe Leu Leu Thr Arg Leu Thr Ala Pro Ile
355 360 365355 360 365
Asn Phe Thr Gly His Pro Ala Ile Ser Tyr Pro Ile Gly Lys Ile AsnAsn Phe Thr Gly His Pro Ala Ile Ser Tyr Pro Ile Gly Lys Ile Asn
370 375 380370 375 380
Asn Leu Pro Val Gly Ile Gin Phe Ile Ser Asp Phe His Asn Glu Glu 385 390 395 400Asn Leu Pro Val Gly Ile Gin Phe Ile Ser Asp Phe His Asn Glu Glu 385 390 395 400
Ile Leu Phe Gin Ala Cys Asn Leu Leu Gin Lys Ser Phe Leu Ala LysIle Leu Phe Gin Ala Cys Asn Leu Leu Gin Lys Ser Phe Leu Ala Lys
405 410 415405 410 415
Gly AsnGly Asn
(2) INFORMATION POUR LA SEQ ID N0: 2:(2) INFORMATION FOR SEQ ID N0: 2:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 1-491 paires de bases(A) LENGTH: 1-491 base pairs
(B) TYPE: acide nucléique(B) TYPE: nucleic acid
(C) NOMBRE DE BRINS: simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: ADN (génomique)(ii) TYPE OF MOLECULE: DNA (genomics)
(ix) CARACTERISTIQUE ADDITIONELLE:(ix) ADDITIONAL FEATURE:
(A) NOM/CLE: CDS(A) NAME / KEY: CDS
(B) EMPLACEMENT: 127..1380(B) LOCATION: 127..1380
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 2:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:
TCCGCAATTC ATGATTGAAA GATGTGAGTT TCCATGAGAT CAGAGACCGA GAATTTAGAC 60TCCGCAATTC ATGATTGAAA GATGTGAGTT TCCATGAGAT CAGAGACCGA GAATTTAGAC 60
ATTCATCGAA AGTGAAAATA ATAACTAGTT TTATATATTA GTTATTGAAA TTTAGTGGAT 120ATTCATCGAA AGTGAAAATA ATAACTAGTT TTATATATTA GTTATTGAAA TTTAGTGGAT 120
AAGAAA ATG ATT GAA AAT ATT ATT GCG AAA TTA AAA AAT ATT CTA GAG 168 Met Ile Glu Asn Ile Ile Ala Lys Leu Lys Asn Ile Leu GluAAGAAA ATG ATT GAA AAT ATT ATT GCG AAA TTA AAA AAT ATT CTA GAG 168 Met Ile Glu Asn Ile Ala Lys Leu Lys Asn Ile Leu Glu
1 5 101 5 10
TCG AAC ACC AAT TCG TTT ATA AGT TTA GAT TTA GTT GAT GTC TCA AAA 216 Ser Asn Thr Asn Ser Phe Ile Ser Leu Asp Leu Val Asp Val Ser Lys 15 20 25 30 CAA TTA AAA AAA CAA GAT AGC AAT CAA CCA CTA AAT GGA GTA ACA GTT 264 Gin Leu Lys Lys Gin Asp Ser Asn Gin Pro Leu Asn Gly Val Thr Val 35 40TCG AAC ACC AAT TCG TTT ATA AGT TTA GAT TTA GTT GAT GTC TCA AAA 216 Ser Asn Thr Asn Ser Phe Ile Ser Leu Asp Leu Val Asp Val Ser Lys 15 20 25 30 CAA TTA AAA AAA CAA GAT AGC AAT CAA CCA CTA AAT GGA GTA ACA GTT 264 Gin Leu Lys Lys Gin Asp Ser Asn Gin Pro Leu Asn Gly Val Thr Val 35 40
GCT ATA AAA GAT TTA ATT GAT GTT GAA GGC CAG AAA ACC ACT ATG GGT 312 Ala Ile Lys Asp Leu Ile Asp Val Glu Gly Gin Lys Thr Thr Met Gly 50 55 60GCT ATA AAA GAT TTA ATT GAT GTT GAA GGC CAG AAA ACC ACT ATG GGT 312 Ala Ile Lys Asp Leu Ile Asp Val Glu Gly Gin Lys Thr Thr Met Gly 50 55 60
TCT GCT CAA TAT CAG AAT AAT ATA GCA AGA AGT GAT GCC GAT GTT GTG 360 Ser Ala Gin Tyr Gin Asn Asn Ile Ala Arg Ser Asp Ala Asp Val Val 65 70 75TCT GCT CAA TAT CAG AAT AAT ATA GCA AGA AGT GAT GCC GAT GTT GTG 360 Ser Ala Gin Tyr Gin Asn Asn Ile Ala Arg Ser Asp Ala Asp Val Val 65 70 75
CGC AGG CTC AGA ACA GCA GGT GCT GTG ATA TTT GGA AAA ACT AAT ACA 408 Arg Arg Leu Arg Thr Ala Gly Ala Val Ile Phe Gly Lys Thr Asn Thr 80 85 90CGC AGG CTC AGA ACA GCA GGT GCT GTG ATA TTT GGA AAA ACT AAT ACA 408 Arg Arg Leu Arg Thr Ala Gly Ala Val Ile Phe Gly Lys Thr Asn Thr 80 85 90
CAC GAG TTT GCT TAT GGA TCT ACT GGG GAT AAA TCA TTT TTT GGA CCA 456 His Glu Phe Ala Tyr Gly Ser Thr Gly Asp Lys Ser Phe Phe Gly ProCAC GAG TTT GCT TAT GGA TCT ACT GGG GAT AAA TCA TTT TTT GGA CCA 456 His Glu Phe Ala Tyr Gly Ser Thr Gly Asp Lys Ser Phe Phe Gly Pro
95 100 105 11095 100 105 110
GTT CTA AAT CCA AAC AAT GAA AAC CAT ATT ACT GGT GGG TCT AGC AGT 50 Val Leu Asn Pro Asn Asn Glu Asn His Ile Thr Gly Gly Ser Ser Ser 115 120 125GTT CTA AAT CCA AAC AAT GAA AAC CAT ATT ACT GGT GGG TCT AGC AGT 50 Val Leu Asn Pro Asn Asn Glu Asn His Ile Thr Gly Gly Ser Ser Ser 115 120 125
GGA TCA GCT GCA GCT GTC GCA GCC GAC TTG TGC GAT GTT GCA ATA GGT 552 Gly Ser Ala Ala Ala Val Ala Ala Asp Leu Cys Asp Val Ala Ile Gly 130 135 140GGA TCA GCT GCA GCT GTC GCA GCC GAC TTG TGC GAT GTT GCA ATA GGT 552 Gly Ser Ala Ala Ala Val Ala Ala Asp Leu Cys Asp Val Ala Ile Gly 130 135 140
ACT GAT ACG GCT GCT TCT GTA AGA CTA CCT GCA GCA CTA TGT GGT GTT 600 Thr Asp Thr Ala Ala Ser Val Arg Leu Pro Ala Ala Leu Cys Gly Val 145 150 155ACT GAT ACG GCT GCT TCT GTA AGA CTA CCT GCA GCA CTA TGT GGT GTT 600 Thr Asp Thr Ala Ala Ser Val Arg Leu Pro Ala Ala Leu Cys Gly Val 145 150 155
ATT GGA TTA AAG CCA ACT TAC AAC TCC ATT AAA AGA GAT GGA GTT TTT 648 Ile Gly Leu Lys Pro Thr Tyr Asn Ser Ile Lys Arg Asp Gly Val Phe 160 165 170ATT GGA TTA AAG CCA ACT TAC AAC TCC ATT AAA AGA GAT GGA GTT TTT 648 Ile Gly Leu Lys Pro Thr Tyr Asn Ser Ile Lys Arg Asp Gly Val Phe 160 165 170
AGT CTA AGC CCG TCA TTA GAT CAT GTT GGA ATT ATC AGT AAA TCA TTA 696 Ser Leu Ser Pro Ser Leu Asp His Val Gly Ile Ile Ser Lys Ser Leu 175 180 185 190AGT CTA AGC CCG TCA TTA GAT CAT GTT GGA ATT ATC AGT AAA TCA TTA 696 Ser Leu Ser Pro Ser Leu Asp His Val Gly Ile Ile Ser Lys Ser Leu 175 180 185 190
GAT TTA ATT GAA AAA ACC TTT AAC TCC ACT AAA ACA TCA ATC GAT CAA 744 Asp Leu Ile Glu Lys Thr Phe Asn Ser Thr Lys Thr Ser Ile Asp Gin 195 200 205 80GAT TTA ATT GAA AAA ACC TTT AAC TCC ACT AAA ACA TCA ATC GAT CAA 744 Asp Leu Ile Glu Lys Thr Phe Asn Ser Thr Lys Thr Ser Ile Asp Gin 195 200 205 80
3333
AAG CCC CAA AAA AAC TTA GAT AAG AAG TTA ACT ATT GGC CTT CTA AAA 792 Lys Pro Gin Lys Asn Leu Asp Lys Lys Leu Thr Ile Gly Leu Leu Lys 210 215 220AAG CCC CAA AAA AAC TTA GAT AAG AAG TTA ACT ATT GGC CTT CTA AAA 792 Lys Pro Gin Lys Asn Leu Asp Lys Lys Leu Thr Ile Gly Leu Leu Lys 210 215 220
GGT TTT TTT GAG GAA TAT ATT TGC AGC TCT GTC AAA GAA AAA TAT GAT 840 Gly Phe Phe Glu Glu Tyr Ile Cys Ser Ser Val Lys Glu Lys Tyr Asp 225 230 235GGT TTT TTT GAG GAA TAT ATT TGC AGC TCT GTC AAA GAA AAA TAT GAT 840 Gly Phe Phe Glu Glu Tyr Ile Cys Ser Ser Val Lys Glu Lys Tyr Asp 225 230 235
GAA ACT ATA GAA TTA TTA AGA AGT AAC GGT TGT GAA ATC AAA GAA ATT 888 Glu Thr Ile Glu Leu Leu Arg Ser Asn Gly Cys Glu Ile Lys Glu Ile 240 245 250GAA ACT ATA GAA TTA TTA AGA AGT AAC GGT TGT GAA ATC AAA GAA ATT 888 Glu Thr Ile Glu Leu Leu Arg Ser Asn Gly Cys Glu Ile Lys Glu Ile 240 245 250
AAT ATT AAA GAA GCA TTT GAT ATT TAC ACA AAT TCG CAA ATA GTT CTT 93 Asn Ile Lys Glu Ala Phe Asp Ile Tyr Thr Asn Ser Gin Ile Val Leu 255 260 265 270AAT ATT AAA GAA GCA TTT GAT ATT TAC ACA AAT TCG CAA ATA GTT CTT 93 Asn Ile Lys Glu Ala Phe Asp Ile Tyr Thr Asn Ser Gin Ile Val Leu 255 260 265 270
AAA TAC GAA GCT TAT AAA ATA CAT GAA GAA TCT TTG AAA TTA AAT TAT 984 Lys Tyr Glu Ala Tyr Lys Ile His Glu Glu Ser Leu Lys Leu Asn Tyr 275 280 285AAA TAC GAA GCT TAT AAA ATA CAT GAA GAA TCT TTG AAA TTA AAT TAT 984 Lys Tyr Glu Ala Tyr Lys Ile His Glu Glu Ser Leu Lys Leu Asn Tyr 275 280 285
CCA TTC GAC CCT GAA GTT AAG GGG CGA ATA TTA AGG GGT GAA AAT ATA 1032 Pro Phe Asp Pro Glu Val Lys Gly Arg Ile Leu Arg Gly Glu Asn Ile 290 295 300CCA TTC GAC CCT GAA GTT AAG GGG CGA ATA TTA AGG GGT GAA AAT ATA 1032 Pro Phe Asp Pro Glu Val Lys Gly Arg Ile Leu Arg Gly Glu Asn Ile 290 295 300
ACC AAA GAC GAA TAT GAT ATC GCA AAA AAA TAT CAA GAA AAT GCA AGA 1080 Thr Lys Asp Glu Tyr Asp Ile Ala Lys Lys Tyr Gin Glu Asn Ala Arg 305 310 315ACC AAA GAC GAA TAT GAT ATC GCA AAA AAA TAT CAA GAA AAT GCA AGA 1080 Thr Lys Asp Glu Tyr Asp Ile Ala Lys Lys Tyr Gin Glu Asn Ala Arg 305 310 315
ATC ATA TTT GAC ACA GCT TTA AAA GAT GTT CAT GTA TTA GCA TCC TTA 1128 Ile Ile Phe Asp Thr Ala Leu Lys Asp Val His Val Leu Ala Ser Leu 320 325 330ATC ATA TTT GAC ACA GCT TTA AAA GAT GTT CAT GTA TTA GCA TCC TTA 1128 Ile Ile Phe Asp Thr Ala Leu Lys Asp Val His Val Leu Ala Ser Leu 320 325 330
ACA AGC GGA ATA CTA CCG CCA AAA ATC TTT GAA CGT CAA ACA AAG GTT 1176 Thr Ser Gly Ile Leu Pro Pro Lys Ile Phe Glu Arg Gin Thr Lys Val 335 340 35 350ACA AGC GGA ATA CTA CCG CCA AAA ATC TTT GAA CGT CAA ACA AAG GTT 1176 Thr Ser Gly Ile Leu Pro Pro Lys Ile Phe Glu Arg Gin Thr Lys Val 335 340 35 350
AAT GAA AAT ACA GTG GAA ACA TTC TTT TTA TTG ACA AGA CTA ACA GCT 1224 Asn Glu Asn Thr Val Glu Thr Phe Phe Leu Leu Thr Arg Leu Thr Ala 355 360 365AAT GAA AAT ACA GTG GAA ACA TTC TTT TTA TTG ACA AGA CTA ACA GCT 1224 Asn Glu Asn Thr Val Glu Thr Phe Phe Leu Leu Thr Arg Leu Thr Ala 355 360 365
CCA ATA AAT TTT ACT GGT CAC CCT GCT ATC TCA TAT CCG ATT GGA AAA 1272 Pro Ile Asn Phe Thr Gly His Pro Ala Ile Ser Tyr Pro Ile Gly Lys 370 375 380 ATA AAT AAC TTG CCC GTA GGC ATC CAG TTT ATT TCT GAT TTT CAC AAT 1320 Ile Asn Asn Leu Pro Val Gly Ile Gin Phe Ile Ser Asp Phe His Asn 385 390 395CCA ATA AAT TTT ACT GGT CAC CCT GCT ATC TCA TAT CCG ATT GGA AAA 1272 Pro Ile Asn Phe Thr Gly His Pro Ala Ile Ser Tyr Pro Ile Gly Lys 370 375 380 ATA AAT AAC TTG CCC GTA GGC ATC CAG TTT ATT TCT GAT TTT CAC AAT 1320 Ile Asn Asn Leu Pro Val Gly Ile Gin Phe Ile Ser Asp Phe His Asn 385 390 395
GAG GAA ATT CTT TTC CAG GCG TGT AAT CTT CTA CAG AAA TCA TTT TTA 1368 Glu Glu Ile Leu Phe Gin Ala Cys Asn Leu Leu Gin Lys Ser Phe Leu 400 405 410GAG GAA ATT CTT TTC CAG GCG TGT AAT CTT CTA CAG AAA TCA TTT TTA 1368 Glu Glu Ile Leu Phe Gin Ala Cys Asn Leu Leu Gin Lys Ser Phe Leu 400 405 410
GCG AAA GGT AAT TAAATGAAAT TCAAGATCAA AACCACTTTG TGTGCGTCAT 1420GCG AAA GGT AAT TAAATGAAAT TCAAGATCAA AACCACTTTG TGTGCGTCAT 1420
Ala Lys Gly AsnAla Lys Gly Asn
415415
TGTTATGCTT ATCTGGCTTG GCTCAAGCCG CATATCCCTC TAAGAATATT ACCCTTGTGG 1480 TTCCATTTCC T 1491TGTTATGCTT ATCTGGCTTG GCTCAAGCCG CATATCCCTC TAAGAATATT ACCCTTGTGG 1480 TTCCATTTCC T 1491
(2) INFORMATION POUR LA SEQ ID NO: 3=(2) INFORMATION FOR SEQ ID NO: 3 =
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 4l8 acides aminés(A) LENGTH: 4l8 amino acids
(B) TYPE: acide aminé(B) TYPE: amino acid
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: protéine(ii) TYPE OF MOLECULE: protein
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 3:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3:
Met Ile Glu Asn Ile Ile Ala Lys Leu Lys Asn Ile Leu Glu Ser AsnMet Ile Glu Asn Ile Ala Lys Leu Lys Asn Ile Leu Glu Ser Asn
1 5 10 151 5 10 15
Thr Asn Ser Phe Ile Ser Leu Asp Leu Val Asp Val Ser Lys Gin LeuThr Asn Ser Phe Ile Ser Leu Asp Leu Val Asp Val Ser Lys Gin Leu
20 25 3020 25 30
Lys Lys Gin Asp Ser Asn Gin Pro Leu Asn Gly Val Thr Val Ala IleLys Lys Gin Asp Ser Asn Gin Pro Leu Asn Gly Val Thr Val Ala Ile
35 40 4535 40 45
Lys Asp Leu Ile Asp Val Glu Gly Gin Lys Thr Thr Met Gly Ser AlaLys Asp Leu Ile Asp Val Glu Gly Gin Lys Thr Thr Met Gly Ser Ala
50 55 6050 55 60
Gin Tyr Gin Asn Asn Ile Ala Arg Ser Asp Ala Asp Val Val Arg Arg 65 70 75 80Gin Tyr Gin Asn Asn Ile Ala Arg Ser Asp Ala Asp Val Val Arg Arg 65 70 75 80
Leu Arg Thr Ala Gly Ala Val Ile Phe Gly Lys Thr Asn Thr His GluLeu Arg Thr Ala Gly Ala Val Ile Phe Gly Lys Thr Asn Thr His Glu
85 90 9585 90 95
Phe Ala Tyr Gly Ser Thr Gly Asp Lys Ser Phe Phe Gly Pro Val LeuPhe Ala Tyr Gly Ser Thr Gly Asp Lys Ser Phe Phe Gly Pro Val Leu
100 105 no100 105 no
Asn Pro Asn Asn Glu Asn His Ile Thr Gly Gly Ser Ser Ser Gly Ser 115 120 125 Ala Ala Ala Val Ala Ala Asp Leu Cys Asp Val Ala Ile Gly Thr AspAsn Pro Asn Asn Glu Asn His Ile Thr Gly Gly Ser Ser Ser Gly Ser 115 120 125 Ala Ala Ala Val Ala Ala Asp Leu Cys Asp Val Ala Ile Gly Thr Asp
130 135 140130 135 140
Thr Ala Ala Ser Val Arg Leu Pro Ala Ala Leu Cys Gly Val Ile Gly 145 150 155 160Thr Ala Ala Ser Val Arg Leu Pro Ala Ala Leu Cys Gly Val Ile Gly 145 150 155 160
Leu Lys Pro Thr Tyr Asn Ser Ile Lys Arg Asp Gly Val Phe Ser LeuLeu Lys Pro Thr Tyr Asn Ser Ile Lys Arg Asp Gly Val Phe Ser Leu
165 170 175165 170 175
Ser Pro Ser Leu Asp His Val Gly Ile Ile Ser Lys Ser Leu Asp LeuSer Pro Ser Leu Asp His Val Gly Ile Ile Ser Lys Ser Leu Asp Leu
180 185 190180 185 190
Ile Glu Lys Thr Phe Asn Ser Thr Lys Thr Ser Ile Asp Gin Lys ProIle Glu Lys Thr Phe Asn Ser Thr Lys Thr Ser Ile Asp Gin Lys Pro
195 200 205195 200 205
Gin Lys Asn Leu Asp Lys Lys Leu Thr Ile Gly Leu Leu Lys Gly PheGin Lys Asn Leu Asp Lys Lys Leu Thr Ile Gly Leu Leu Lys Gly Phe
210 215 220210 215 220
Phe Glu Glu Tyr Ile Cys Ser Ser Val Lys Glu Lys Tyr Asp Glu Thr 225 230 235 240Phe Glu Glu Tyr Ile Cys Ser Ser Val Lys Glu Lys Tyr Asp Glu Thr 225 230 235 240
Ile Glu Leu Leu Arg Ser Asn Gly Cys Glu Ile Lys Glu Ile Asn IleIle Glu Leu Leu Arg Ser Asn Gly Cys Glu Ile Lys Glu Ile Asn Ile
245 250 255245 250 255
Lys Glu Ala Phe Asp Ile Tyr Thr Asn Ser Gin Ile Val Leu Lys TyrLys Glu Ala Phe Asp Ile Tyr Thr Asn Ser Gin Ile Val Leu Lys Tyr
260 265 270260 265 270
Glu Ala Tyr Lys Ile His Glu Glu Ser Leu Lys Leu Asn Tyr Pro PheGlu Ala Tyr Lys Ile His Glu Glu Ser Leu Lys Leu Asn Tyr Pro Phe
275 280 285275 280 285
Asp Pro Glu Val Lys Gly Arg Ile Leu Arg Gly Glu Asn Ile Thr LysAsp Pro Glu Val Lys Gly Arg Ile Leu Arg Gly Glu Asn Ile Thr Lys
290 295 300290 295 300
Asp Glu Tyr Asp Ile Ala Lys Lys Tyr Gin Glu Asn Ala Arg Ile Ile 305 310 315 320Asp Glu Tyr Asp Ile Ala Lys Lys Tyr Gin Glu Asn Ala Arg Ile Ile 305 310 315 320
Phe Asp Thr Ala Leu Lys Asp Val His Val Leu Ala Ser Leu Thr SerPhe Asp Thr Ala Leu Lys Asp Val His Val Leu Ala Ser Leu Thr Ser
325 330 335325 330 335
Gly Ile Leu Pro Pro Lys Ile Phe Glu Arg Gin Thr Lys Val Asn GluGly Ile Leu Pro Pro Lys Ile Phe Glu Arg Gin Thr Lys Val Asn Glu
340 345 350340 345 350
Asn Thr Val Glu Thr Phe Phe Leu Leu Thr Arg Leu Thr Ala Pro IleAsn Thr Val Glu Thr Phe Phe Leu Leu Thr Arg Leu Thr Ala Pro Ile
355 360 365355 360 365
Asn Phe Thr Gly His Pro Ala Ile Ser Tyr Pro Ile Gly Lys Ile AsnAsn Phe Thr Gly His Pro Ala Ile Ser Tyr Pro Ile Gly Lys Ile Asn
370 375 380370 375 380
Asn Leu Pro Val Gly Ile Gin Phe Ile Ser Asp Phe His Asn Glu Glu 385 390 395 400Asn Leu Pro Val Gly Ile Gin Phe Ile Ser Asp Phe His Asn Glu Glu 385 390 395 400
Ile Leu Phe Gin Ala Cys Asn Leu Leu Gin Lys Ser Phe Leu Ala LysIle Leu Phe Gin Ala Cys Asn Leu Leu Gin Lys Ser Phe Leu Ala Lys
405 410 415405 410 415
Gly Asn (2) INFORMATION POUR LA SEQ ID NO: 4:Gly Asn (2) INFORMATION FOR SEQ ID NO: 4:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 20 paires de bases(A) LENGTH: 20 base pairs
(B) TYPE: acide nucléique(B) TYPE: nucleic acid
(C) NOMBRE DE BRINS: simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: ADN (génomique)(ii) TYPE OF MOLECULE: DNA (genomics)
(iii) HYPOTHETIQUE: NON(iii) HYPOTHETIC: NO
(iii) ANTI-SENS: NON(iii) ANTI-SENSE: NO
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 4:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4:
ATGATHGARA AYATHATHGC 20ATGATHGARA AYATHATHGC 20
(2) INFORMATION POUR LA SEQ ID NO: 5:(2) INFORMATION FOR SEQ ID NO: 5:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 7 acides aminés(A) LENGTH: 7 amino acids
(B) TYPE: acide aminé(B) TYPE: amino acid
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: peptide(ii) TYPE OF MOLECULE: peptide
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 5:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5:
Met Ile Glu Asn Ile Ile Ala 1 5 Met Ile Glu Asn Ile Ala Island 1 5

Claims

REVENDICATIONS :CLAIMS:
1 - Polypeptides ayant une activité amidase, capables d'hydrolyser les amides en carboxylates, caractérisés en ce qu'ils possèdent une activité enzymatique vis-à-vis de l'adipamate d'ammonium supérieure à celle qu'ils ont vis-à-vis de l'adipamide.1 - Polypeptides having an amidase activity, capable of hydrolyzing amides into carboxylates, characterized in that they have an enzymatic activity vis-à-vis ammonium adipamate greater than that which they have vis-à-vis adipamide screw.
2 - Polypeptide selon la revendication 1, caractérisé par la séquence peptidique SEQ ID : 1 telle que représentée à la fig. 1.2 - Polypeptide according to claim 1, characterized by the peptide sequence SEQ ID: 1 as shown in FIG. 1.
3 - Séquence d'ADN codant pour un polypeptide ayant une activité amidase, capable d'hydrolyser les amides en carboxylates, caractérisée en ce qu'elle est choisie parmi la liste des séquences suivantes :3 - DNA sequence coding for a polypeptide having an amidase activity, capable of hydrolyzing amides to carboxylates, characterized in that it is chosen from the list of the following sequences:
- la séquence d'ADN SEQ ID : 2, telle que représentée à la fig. 2 et codant pour un polypeptide ayant une activité amidase,- the DNA sequence SEQ ID: 2, as shown in fig. 2 and coding for a polypeptide having an amidase activity,
- un analogue de cette séquence résultant de la dégénérescence du code génétique,- an analogue of this sequence resulting from the degeneration of the genetic code,
- une séquence d'ADN hybridant avec l'une de ces séquences ou un fragment de celles-ci et codant pour un polypeptide ayant une activité amidase.- a DNA sequence hybridizing with one of these sequences or a fragment thereof and coding for a polypeptide having amidase activity.
4 - Polypeptides résultant de l'expression d'une séquence d'ADN selon la revendication 3 et ayant une activité amidase.4 - Polypeptides resulting from the expression of a DNA sequence according to claim 3 and having an amidase activity.
5 - Micro-organisme constitué par la souche E. coli contenant le plasmide pXL2216, souche référencée G 4315 et déposée dans la Collection Nationale de Cultures de Micro-organismes sous le n° I 1279 le 11 janvier 1993.5 - Microorganism constituted by the E. coli strain containing the plasmid pXL2216, strain referenced G 4315 and deposited in the National Collection of Cultures of Microorganisms under the n ° I 1279 on January 11, 1993.
6 - Micro-organisme apte à produire au moins un polypeptide selon la revendication 4.6 - Microorganism capable of producing at least one polypeptide according to claim 4.
7 - Micro-organisme selon la revendication 6, caractérisé en ce qu'il contient au moins une cassette d'expression comprenant la séquence d'ADN selon la revendication 3 et, en amont de celle-ci, une séquence promotrice et un site de fixation des ribosomes. 8 - Micro-organisme selon la revendication 7, caractérisé en ce qu'il appartient à la famille des procaryotes et en ce que le promoteur est choisi parmi les promoteurs suivants : promoteur de l'opéron tryptophane Ptrp de E. coli, promoteur de l'opéron lactose Plac de E. coli, promoteur droit du phage lambda PR, promoteur gauche du phage lambda PL, promoteurs forts de Pseudomonas, Comamonas ou de corynébactéries. 9 - Micro-organisme selon la revendication 7, caractérisé en ce qu'il appartient à la famille des procaryotes et en ce que le site de fixation des ribosomes est choisi parmi celui dérivé du gène Cil de lambda, ceux dérivés de gènes de Comamonas ou Pseudomonas, ou ceux dérivés de gènes de corynébactéries.7 - Microorganism according to claim 6, characterized in that it contains at least one expression cassette comprising the DNA sequence according to claim 3 and, upstream thereof, a promoter sequence and a site of ribosome fixation. 8 - Microorganism according to claim 7, characterized in that it belongs to the family of prokaryotes and in that the promoter is chosen from following promoters: promoter of the tryptophan Ptrp operon from E. coli, promoter of the lactose operon Plac from E. coli, right promoter of phage lambda P R , left promoter of phage lambda P L , strong promoters of Pseudomonas, Comamonas or of corynebacteria. 9 - Microorganism according to claim 7, characterized in that it belongs to the family of prokaryotes 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.
10 - Micro-organisme selon la revendication 7, caractérisé en ce qu'il appartient à la famille des eucaryotes, tels que les levures et en ce que les promoteurs sont choisis parmi ceux des gènes glycolytiques de levure, tels les gènes codant pour la phospho-glycérate kinase, le glycéraldéhyde-3-phosphate déshydrogénase, ou ceux de la lactase ou l'énolase.10 - Microorganism according to claim 7, 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.
11- Micro-organisme selon l'une des revendications 6 à 10, caractérisé en ce que la cassette d'expression est portée par un plasmide.11- Microorganism according to one of claims 6 to 10, characterized in that the expression cassette is carried by a plasmid.
12 - Micro-organisme selon la revendication 11, caractérisé en ce qu'il est sélectionné parmi la liste suivante de souches :12 - Microorganism according to claim 11, characterized in that it is selected from the following list of strains:
E. coli, Comamonas, Pseudomonas, Corynebacterium, Brevibacterium, Rhodococcus, Streptqmyces, Bacillus. 13 - Polypeptides, caractérisés en ce qu'il sont produits par les micro¬ organismes selon l'une quelconque des revendications 6 à 12.E. coli, Comamonas, Pseudomonas, Corynebacterium, Brevibacterium, Rhodococcus, Streptqmyces, Bacillus. 13 - Polypeptides, characterized in that they are produced by the micro¬ organisms according to any one of claims 6 to 12.
14 - Procédé d'hydrolyse enzymatique d'amides en carboxylates, caractérisé en ce qu'il consiste à mettre les mono ou di-amides répondant à l'une des formules générales suivantes : Ε NOC - R - CONJH2, H,NOC - R - COO"Z+, NC - R - CONH, dans lesquelles R est un groupe alkylène ou alcénylène, linéaire ou ramifié, ayant de 1 à 18 atomes de carbone et Z est choisi parmi la liste de composés suivants : alcalins, alcalino-terreux, aminés, ammonium, ce dernier composé étant particulièrement préféré, en présence d'un polypeptide selon l'une quelconque des revendications 2, 4 et 13 ou en présence d'un micro-organisme selon l'une quelconque des revendications 6 à 12. 15 - Procédé selon la revendication 14, caractérisé en ce que R = - (CΑJ4 -. 14 - Process for the enzymatic hydrolysis of amides into carboxylates, characterized in that it consists in putting the mono or di-amides corresponding to one of the following general formulas: Ε NOC - R - CONJH 2 , H, NOC - R - COO " Z + , NC - R - CONH, 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: alkali, alkaline- earthy, amino, ammonium, the latter compound being particularly preferred, in the presence of a polypeptide according to any one of claims 2, 4 and 13 or in the presence of a microorganism according to any one of claims 6 to 12 . 15 - Process according to claim 14, characterized in that R = - (CΑJ 4 -.
PCT/FR1994/000080 1993-01-27 1994-01-21 Polypeptides having amidase activity, genetic tools and host microorganisms for obtaining same WO1994017190A1 (en)

Priority Applications (3)

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JP6516742A JPH08504596A (en) 1993-01-27 1994-01-21 Polypeptides having amidase activity, genetic tools and host microorganisms for producing them, and hydrolysis process using the polypeptides
KR1019950703079A KR960700343A (en) 1993-01-27 1994-01-21 POLYPEPTIDES WITH AMIDASE ACTIVITY, GENETIC TOOLS AND HOST MICROORGANISMS FOR THEIR PREPARATION, AND HYDROLYSIS PROCESS USING SAID POLYPEPTIDES
EP94905744A EP0681610A1 (en) 1993-01-27 1994-01-21 Polypeptides having amidase activity, genetic tools and host microorganisms for obtaining same

Applications Claiming Priority (2)

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FR9301062A FR2700777B1 (en) 1993-01-27 1993-01-27 Amidase activity polypeptides, genetic tools and host microorganisms for obtaining them, hydrolysis process using said polypeptides.
FR93/01062 1993-01-27

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998044094A2 (en) * 1997-03-28 1998-10-08 Institut Pasteur Helicobacter aliphatic amidase polypeptides, dna sequences encoding those polypeptides and uses thereof
US5866379A (en) * 1997-01-28 1999-02-02 Novus International Enzymatic conversion of α-hydroxynitriles to the corresponding .alpha.

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2736928B1 (en) * 1995-07-18 1997-10-17 Rhone Poulenc Fibres & Polymer ENZYMES HAVING AMIDASE ACTIVITY MICRO-ORGANISMS LIKELY TO PRODUCE SUCH ENZYMES AND METHOD FOR HYDROLYSIS OF AMIDES USING THE SAME

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0488916A1 (en) * 1990-11-28 1992-06-03 Rhone-Poulenc Fiber And Resin Intermediates Process for enzymatic preparation of ammonium adipate

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0488916A1 (en) * 1990-11-28 1992-06-03 Rhone-Poulenc Fiber And Resin Intermediates Process for enzymatic preparation of ammonium adipate

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5866379A (en) * 1997-01-28 1999-02-02 Novus International Enzymatic conversion of α-hydroxynitriles to the corresponding .alpha.
WO1998044094A2 (en) * 1997-03-28 1998-10-08 Institut Pasteur Helicobacter aliphatic amidase polypeptides, dna sequences encoding those polypeptides and uses thereof
WO1998044094A3 (en) * 1997-03-28 1999-01-07 Pasteur Institut Helicobacter aliphatic amidase polypeptides, dna sequences encoding those polypeptides and uses thereof
US6248551B1 (en) 1997-03-28 2001-06-19 Institut Pasteur Helicobacter aliphatic amidase AmiE polypeptides, and DNA sequences encoding those polypeptides

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KR960700343A (en) 1996-01-19
FR2700777B1 (en) 1995-04-14

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