CA2153793A1 - Amidase activity polypeptides; genetic tools and host microorganisms for producing the same - Google Patents

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

WO 94/17190 ~ 15 ~ 7 9 3 PCT / FRg4 / 00080 POLYPEPTIDES WITH ~MIDASE ACTIVITY, GENETIC TOOLS AND MICRO-ORGANISMS
HOSTS ALLOWING THEM TO BE OBTAINED.

SDO M AI ~ TECHnNIOlnE:

The field of the invention is that of the enzymatic production of carboxylic derivatives (carboxylates) from compounds containing groups amides.
10The present invention relates to new polypeptides having a amidase activity, the genetic material involved in their production, as well as the micro-org ~ nicmçs containing this genetic material and exhibiting this activity.
The invention also relates to an enzymatic hydrolysis process of amides into carboxylates in which these new polypeptides are implemented 15ou micro-org ~ ni ~ m ~ s the synth ~, ti ~ nt, including, not ~ mm ~ -nt, the aforementioned micro-org ~ ni ~ my hosts.
Without this being limited, the ~mities, in which we are more interested particularly within the scope of the invention, are: 5-cyanovaleramide, ir ~ mide or adipamate of any cation, of ~ Çerence chosen from the 20list of following compounds: alkali, alkaline earth, amines, ammonium, ce dermer compound being particularly preferred. The goal is transformation enzymatic transformation of amide functions into carboxylate functions, so as to ultimately obtain 5-cyanovaleric acid, adipic acid or salts thereof, eg: 5-ammonium cyanovalerate, diammonium adipate.
25All industrial interest of ~riip~t~s, and in particular, adipate of ammonium, lies in the fact that it constitutes one of the fundamental bases for the manufacture of nylon-6,6.
As regards S-cyanovalerates, they are likely to constitute starting products of choice, for the ~Jri;l,al~tion of caprol~and~me, giving itself 30access to nylon-6.
The action of these amidases is part of a known synthetic scheme Wo 94117190 PCT/FRs4tOO080 ~1~37~3 2 diammonium adipate enzyme, from a dinitrile, namely adiponitrile.
This diagram is as follows:

,NC--R--CN

Ni/~NH\

NC--R--CONII
A~~NH~
NC--:~--COO~,+ H.NOC--R--CONH, NH~~A
H2NOC--R--COO~I+

Ni\~A/A

+4HN-OOC--R--COO~H4 NH = nitrile hydratase, lS Ni = nitrilase, A =~mid~ce, R = (CH2)n, n being an integer equal to 4 in the case of compounds adipic.

PRIOR ART:

Such a scheme remained for a long time only of interest theoretical, because we only know the general activity of Brevibacterium R 312, described not ~ mmt? nt in the following articles:
25 - ARNAUD et al., "Study of the amidase activity of some bacteria", FOLIA
MICROBIOLOGICA, 1976, 21, pages 178-185, - MAESTRACCI et al., "Use of a ~mi~ce with general activity for the bioconversion of amides into organic acids", Microbiology - food -~ 3 ~37~3 nutrition, 1986, vol. 4, pages 19-24.
According to these authors, such a ~ mitl ~ e is able to hydrolyze the ~ dip ~ mide in adipic acid (or ammonium adipate).
In fact, it was subsequently demonstrated, in particular by the Claimant, that the activity, with respect to the ~ -iir ~ mide, of this generally active amidase is extrememetlt weak, even null and this, that one uses the enzyme resulting from Brevibacterium R 312 or that derived from a recombinant microorganism comprising the expression gene for this enzyme.
This lack of general activity amidase enzymatic activity is also checks for amide substrates occurring as salts water soluble.
The industrial feasibility of the enzymatic synthesis of ~lip~tes, in particular diammonium, or adipic acid using ~ mifl ~ es could not be envisaged only from the moment when the Dem ~ nde-ress was able to highlight polypeptides expressed by Brevibacterium R 312 and Rhodococcus and characterized, not ~ mmer ~ t, by an activity ~ mi ~ e, called enantioselective, that is to say that they are capable of enantioselectively hydrolyzing racemates of amides to an acid, either S-form or R-form. This particularly concerns aryl-2 propionamides and racemic 2-aryloxy propionamides.
By isolating, purifying and characterizing these "enantioselective" amidases, it has been possible to propose a process for the enzymatic synthesis of adipate ammonium with improved yields.
This is what patent application FR 90-14 8S3 describes.
In addition to these new amidases, this patent application also describes the genetic engineering tools to produce them: DNA sequences, whether or not recombined, c~ettes of ~ cssion carrying this sequence and natural micro-organisms or recombined containing these ç ~ ettes exples ~ ion.
Although con ~ tit ~ l ~ nt a significant technical progress, these amidases "enantioselectives" suffer, however, from a handicap, already pointed out for ~ mi ~ es with general activity, which is to have a capacity for hydrolysis of amides ~, i; feels ~ t in the form of water-soluble salts, eg ammonium adipamate WO 94/17190 21 ~ 7 ~ 3 PCT / FR94 / 00080 lower than that which they have with respect to amides with little or no water solubility, such as adipamide.
However, it is well known that such a property of solubility of substrates is highly desirable, in view of improving the feasibility and profitability 5 of an industrial enzymatic synthesis.
Thus, one of the essential objectives of the present invention is to propose new enzymes with amidase activity, genetic material allowing their production and micro-organisms containing this genetic material and allowing this production, said enzymes and microorganisms being characterized, 10 at a time, by yields s ~ ti.cf ~ ic ~ ntc of production of carboxylates from amide-type substrates of various natures (mono or ~ mi(lçs) and by an activity ~ Mitl ~ ic, vis-à-vis soluble salts of amides, important and industrially interesting.
After numerous tests and research, the Applicant has succeeded 15 to achieve, among other things, this objective by isolating, purifying and characterizing new enzymes capable of hydrolyzing amides into carboxylates with high activities and into transforming, preferentially, soluble salts of amides, said enzymes being utili ~ es, either as is, or, and preferably, in the form of micro-recombinant organisms generating them.
DISCLOSURE OF THE INVENTION:

A subject of the present invention is therefore new polypeptides having an amidase activity, capable of hydrolyzing amides into carboxylates and possessing 25 an enzymatic activity vis-à-vis the ~ lip ~ m ~ te ammonium greater than that which they have vis-a-vis the ip~mide.
One of these novel polypeptides was isolated from a strain of Comamonas testosteroni NI 1. Specifically, this polypeptide is prepared by extraction and purification from cultures of natural or 30 recombinants, the purification being carried out by a succession of steps consisting in prepare an enzyme extract from the cell culture, to precipitate this Wo 94/17190 PCT/FRs4/00080 s extracted with ammonium sulphate and purifying it through various stages of chromatography and gel filtration. These steps, which call upon techniques well known to those skilled in the art, are described in detail in the illustrative examples below.
By activity ~ mi ~ e, is meant, in this presentation, the hydrolysis enzymatic of an amide, such as cyano-S valeramide, ~-lip~mide and ~-lip~m~te from a cation Z, to a carboxyl, namely, respectively, cyano-S valerate of Z, adipamate Z and adipate Z. The cation Z can be any, but lechoisit, the ~ lcfe ~ this, from the following compounds: alkali, alkaline earth, amines, ammonium, the latter compound being particularly prefer.
It is to the Applicant's credit to have been able to isolate these new polypeptides and to have been able to demonstrate their use p ~ ri; lc ~ ltielle of substrates in the form of water-soluble salts, such as lip~m~te, for example ammonium, rather than nonionic, insoluble or weakly soluble substrates in water, such as lir micle.
This characteristic is verified both for enzymes in the form pure than for the natural or recombinant germs expressing these enzymes.
Among the polypeptides with activity ~ mi ~ e in accordance with the invention figure a polypeptide characterized by the peptide sequence, such as repl ~ selltée to the fig. 1 (SEQ ID: 1 of the attached sequence listing).
Another object of the invention relates to a DNA sequence encoding for a polypeptide having an amidase activity, capable of hydrolyzing amides to carboxylates, and chosen from the following sequence list:
- The DNA sequence, as shown in ~ lg. 2 (SEQ ~ D: 2 of the attached list of sequences), and coding for a polypeptide having an amidase activity, - an analog of this sequence results ~ nt of the degeneration of the genetic code7 - - a DNA sequence hybridizing with one of these sequences or a fragment of these and coding for a polypeptide having an amidase activity, The polypeptides resulting from the expression of one of the DNA sequences above are within the scope of the invention.
The wild microorganism Com~nonas testosteroni NI 1, isolated by the Wo 94/17190 PCT/FR94/00080 applicant, contains one of the above sequences in its genome.
According to the invention, it was possible to access the i ~ lentific ~ tion of these DNA sequences by the well-known method of cloning the DNA fragment genomic coding for the polypeptide whose peptide sequence is shown in FIG. 1, using nucleotide probes made from the polypeptide purified.
The invention also relates to expression cassettes which carry, with the signals ensuring its expression, one of the DNA sequences defined previously. These c~sett~s of expression can either be naturally present, 10 either integrated into the host genome or loc ~ ed on an expression vector, such pl ~ mide containing, plerelt; llce, a means of selection.
These c ~ ettes expression include, in particular, regions initiation of transcription and translation, which conti ~ nnçnt a sequence promoter and a ribosome binding site. These regions can be 15 homologous or heterologous of the micro-org ~ ni ~ me naturally producing the polypeptide.
The choice of these regions depends, not ~ mmçnt host used. In particular, when it comes to micro-org ~ ni ~ m ~ s prokaryotic hosts, the promoter heterologous can be chosen from strong bacterial promoters, such as 20 promoter of the tryptophan Ptrp operon of Escherichia coli, the promoter of the lactose operon Plac of E. coli, the right promoter of the phage lambda PR. the left promoter of phage lambda PL. the strong promoters of Pseudomonas and Comamonas, strong promoters of Corynebacteria. More particularly, in the case of the right promoter of phage lambda, the thermosensitive form PRClts is 25 preferred.
In the case of microorganisms or eukaryotes, such as yeasts, promoters may be those of yeast glycolytic genes, such as the genes encoding for phospho-glycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GPD), or even genes coding for lactase (LAC4), enolase (ENO).
Concerning the ribosome binding sites, that derived from the CII gene 7 2~S~793 of lambda, as well as those derived from Comamonas or Pseudomonas genes or those derivatives of Corynebacteria genes are used pl ~ re, elltially when the micro-host organism is prokaryotic.
A region, enabling termination of translation and 5 functional transcription of the intended host, can be positioned 3 'of the coding sequence.
Advantageously, the expression cassette can co "" ~ re; ndre, also, one or more markers making it possible to select the recombinant host. The preferred markers are domin ~ nt ~ markers, that is to say conferring a 10 resistance to antibiotics such as ampicillin, tetracycline or streptomycin or other products toxic to bacteria.
It should be noted that the amidase according to the invention already constitutes, in itself even, a selection marker for the host that contains them.
The invention also relates to the micro-gold ~ nicmes containing the 15 DNA sequence according to the invention, as well as those capable of producing at least one polypeptide according to the invention. These micro-org ~ nor ~ my co ", po, leilt or not at least one c ~ ette of expression, of the type described above.
Among these micro-org ~ ni. ~ my, we find the hosts usable for the reception of a vector of e ~ L ~, es ~ ion in accordance with the invention, mention may be made, not ~ mmçnt, of enterobacteriaceae 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 or Bacillus.
A recombinant micro-org ~ ni ~ me, containing said DNA sequence on a pl~midic structure, has been deposited in the National Collection of Cultures of 25 Microorganisms (CNCM) (Institut Pasteur, 25 rue du Docteur Roux, PARIS) under No. I 1279 on January 11, 1993. This microorganism is the E. Coli strain which contains the plasmid pXL2216 (described below). This micro-org ~ ni ~ me is also identified by the Dem ~ nderesse by the reference G 4315.
It goes without saying that the polypeptides produced by the micro-org ~ nicmes above 30 above are also included in the invention.
The latter also concerns the process of transformation of amides into ~1~3~3 8 carboxylates using a polypeptide according to the invention or a recombinant microorganism generating it. This process consists in bringing the amide into contact with transform with a recombinant polypeptide or microorganism, as defined previously. We operate, gener ~ leme ~ lt, at the te "lpi ~ -dture ambient. According to a mode S particular of re ~ ti (3n of the invention, the polypeptide or the microorganism recombinant are immobilized on or in a solid support, according to a technique conventional, such as that described, for example, in US patent US No. 4,732,851.
The process of the invention is suitable for the transformation of amides, in 10 carboxylates and, more particularly, for the transformation into carboxylates of mono and diamides of formulas:
NC--R--CONH2, H2NOC--R--CONH2 and H2NOC--R--COO~Z~
wherein R is a linear or branched alkylene or alkenylene group, containing 1 to 18 carbon atoms, R preferably being equal to--(CH2)4--and Z is chosen 15 from the following list of compounds ~ flax ~, alkaline earth, amines, ammonium, the latter compound being particularly preferred.
The process of the invention is particularly ~ p, op.ié for the synthesis enzymatic, on the one hand, of an adipate, such as ammonium adipate from of ~ ir ~ mide and especially from a ~ dir ~ m ~ t ~, such as ammonium adipamate and, 20 on the other hand, 5-cyano valerate from 5-cyano valeramide.
The following examples illustrate the characteristics and advantages of the present invention without, however, limiting its scope.

DESCRIPTION OF FIGURES
fig. 1. ~ pi; st; nte the peptide sequence of a conforming polypeptide to the invention (SEQ ~
The ~lg. 2 represents a DNA sequence according to the invention (SEQ ID: 2).
The flg. 3 represents the SDS polyacrylamide gel electrophoresis of the 30 fractions obtained at the different purification steps of Example 2.
fig. 4 represents a curve of the specific activity of the ~ mici ~ this according to Wo 94/17190 ~ 1 ~ 3 ~ 9 ~ PCT/FR94/00080 invention (micromoles of adipate x h-' x mg ~ l of protein) according to the te",p~,dtllre in C.
fig. 5, ~ feels a curve of the specific activity of amidase according to the invention (micromoles of adipate x h-' x mg ~ l of protein) as a function of pH.
S Fig. 6 represents a change in specific activity (micromoles of adipate x h-' x mg-' of protein) of the amidase according to the invention with respect to the ~ tlir ~ m ~ t ~, depending on the concentration of ~ p ~ m ~ te (millimoles x 1-').
fig. 7 represents a curve giving the ratio of the concentration in adipamate on the specific activity (liters of adipate x h-'xg~' of protein) as a function the concentration in ~ip~m~te (millimoles x 1~').
fig. 8, ~ reseilte the restriction map of the plasmid pXL2104 containing the amidase gene according to the invention.
fig. 9, ~ résellte the sequence of a fragment of 1491 bp containing the gene coding for the ~ mi ~ l ~ this of Comamonas testosteroni NI 1.
fig. 10 represents the restriction map of the plasmid pXL2216 conlellallt the gene of the ~ mi ~ e according to the invention.
fig. 11 r ~, esellte the restriction map of the plasmid pXL2144 containing the SpeI-ClaI fragment derived from pl ~ cmi ~ e pXL2104 between the XbaI and ClaI of vector pMTL22.
fig. 12, ~ r ~ feels SDS-PAGE gel electrophoresis showing expression of the DNA sequence according to the invention in the E-Coli TG 1 strain.
fig. 13, ~ p, i; se, lle the restriction map of the plasmid pXL2238 containing the gene of the ~ mi ~ e according to the invention.
The meaning of the abbreviations llti ~ é ~ s in the following description 25 is given below.
- SSC: buffer commonly used for hybridizations, it contains sodium citrate and NaCl (20XSSC = 3M NaCl-sodium citrate sodium pH 7, 0.3 M), - SDS: sodium dodecyl sulfate, - FPLC: liquid chromatography called in the language ~ngl~ e "fast protein liquid chromatography", Wo 94/17190 PCT/FRg4/00080 - SDS-PAGE: electrophoresis gel based on sodium dodecyl sulfate and polyacrylamide, - IPTG: isopropyl, BD thio galactopyranoside, - HPLC: high pt liquid chromatography; l rOl l, lance, - PS: dry weight, - X-gal.: 5 - bromo - 4 - chloro - 3 - indolyl - ~ D galactopyranoside.

EXAMPLE~.

EXAMPLE 1: HYDROLYSIS OF ADIPAMATE AND ADIPAMIDE BY
Comamonas testosteroni NI 1.

The Comamonas testosteroni NI 1 strain is a strain isolated from a soil sample by microbiological screening. His pelrollllances are rated on ir~m~te and ~dir~mide.

1.1. Cell preparation:
The Comamonas testosteroni NI 1 strain is cultured, in a shaken flask, at 28° C., for 19 h, in medium A, the composition of which is as follows:
- Glycerol ............................ 10.0 x 103 mgxl~
- Sodium cyano-5-valerate .........1.5 x 103 mgxl~
- K2HPO4 ............................. o~sx1o3 mgxl~' - MgSO4, 7H2O ........................ o,Sx103 mgxl~' - MnSO4, H20 ........................ 20.0 mgxl~' - FeSO4, 7H2O ........................ 20.0 mgxl~' - NaCl ............................... 10.0 mgxl~' - Yeast extract ................. o,Sx103 mgxl~' - Beef extract ................... 0.5x103 mgxl-- HCl 3N QSP pH 5 This preculture is used to inoculate a 2 liter flask filled to tenths of a wo 94/17190 ~ 3;7 9 ~ PCT~4/00080 medium A. After 22 h, 3.5 gxl ~ 'wet cells are harvested. That corresponds to an OD66,~,~ of 1.4 and a dry weight of 0.9 gxl-'.

1.2. Determination of enzymatic activity on ~-lir~mi~e and ~lir~m~te S A cell pellet containing 31 mg of dry cells is suspended in 5.5 ml of a 20 mM solution in ~-lip~m~te or ~lip~mi~e in the buffer potassium phosphate pH 7.50 mM. The reaction is carried out at 25 C under agitation and the kinetics is monitored by sampling. On each sample, are assayed by HPLC ~ tlip ~ mide, adipamate and adipate. The speeds of formation of adipate, from adipamide and adipamate, are, respectively, 8.3 and 33 Uxg~' of dry cells (1 U is equal to 1 micromole of product formed per minute) (whole cells). By comparing, we obtain the activities given in TABLE I below and measured in the same conditions, with Brevibacterium R 312 and Rhodococcus sp.
TABLE I:

~ACTIVITIES IN t~ - '01 FC DE
PRODUCE CONCERNS PER MINUTE AND
P~ GRAM OF DRY CELLS
Comamonas testosteroni A~iip~t~ 33.0 N~ k 8.3 Brevibacterium R 312 A-lipqt,. o, o A-1ip~mi~ 2.3 Rhodococcus sp A~iipqmq.~t~ 0.0 A-lip~m~

WO94/17190 ~1~ 3 ~ ~ ~ PCT~94/00080 E~ 2: P~u~CA~ONDEL'~DASEDE Comamon~ testosteroni NIl.

2.1. Culture of cells:
The Comamonas NI 1 strain is cultured in a 15 1 fermenter on the medium B whose composition is:
- Glucose ............................ 5.0 x 103 mgxl~' - Sodium cyano-5-valerate ............ 5.7 x 103 mgxl~
- Adiponitrile ............................ 1.0x103 mgxl~' - Na2HPO4, 2H2O ........................ 3.4 x 103 mgxl~' - KH2PO4 ................................ 2.7 x103 mgxl~' - MgSO4 ................................ 0.5 x 103 mgxl~' - MnSO4, H2O .............................. 20.0 mgxl~' - FeSO4, 7H2O ............................. 20.0 mgxl~' - NaCl ................................... 10.0 mgxl~' - vit~minPS
. Biotin ............................ 8 x 10-5 mgxl~' . Nicotinic acid ................... 8x 10-3 mgxl~' . Thi~mine, HCl ....................... 4 x 10-3 mgxl~' The pH, the te "lp ~ ldture, the air flow and the speed of agitation are regulated, respectively, at 6.6, 28 C, 300 l/h and 350 rpm. After 4 hours of fermentation, 15 g of adiponitrile are added. After 7:30 p.m. of culture, 188 g of cells wet, ie 40 g of dry cells are harvested. This corresponds, in the end of culture, at 2.5 g/l of dry weight cells and at an OD660m,, of 7.

25 2.2 Purification:
All purification steps are carried out in buffer B (Tris-HCl 100 mM pH 7.5, 10% glycerol). A ch ~ rllne of the steps, the amidase activity of fractions is determined at pH 7.5 and at 25°C in buffer B diluted 10 times in presence of ~ -lip ~ m ~ tP 10 mM. The protein concentration of the pools is determined by the Coomassie blue method (PIERCE Protein assay kit).
The unit of activity U corresponds to 1 ~ mole of adipate produced per minute in ~ WO 94/17190 215 3 7 ~ 3 PCT / FR94 / 00080 the test conditions. The protein panel is analyzed by gel polyacrylamide-SDS (Phastsystem PHARMACIA).
The data for each of the purification steps are grouped together in the table II below and fig. 3 attached. The procedures of each step are commented on below.
- Step 0: raw extract:
66 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, p ~ nce 7, 30% of the active cycle. The DO660"", thus goes from 220 to 63. After centrifugation at 30,000 gmax for 60 min, the 125 ml of the supernatant are recovered.
- Step 1: fractionnPm ~ nt with ammonium sulphate:
The 125 ml are added with 26 g of ammonium sulphate to reach 35% saturation. After one hour, the suspension is centrifuged 30 min at 30,000 gmax. The supernatant is recovered and added with 18 g of ammonium sulfate to reach 60% saturation. After 1 hour, the suspension is centrifuged at 48,000 gmax. The precipitate is recovered then dialyzed against buffer B for about 66 hours. After dialysis, 43 ml are recovered and then analyzed (total protein l 200 mg, [prot] = 28 gxl-', total activity = 1200 U, act. spec. = 1 Uxmg~', rdt prot. = 46%, yield act. = 60%).
- Step 2: collection of ions (Q sepharose fast flow):
The dialysed fraction (43 ml) is cha, ~ ed at a rate of 130 mlxh- 'on a column (26 x 380 mm) of "Q sepharose flash flow" balanced with the buffer B at a flow rate of 200 mlxh~l. The column is percolated at a rate of 200 mlxh~' successively by the following solutions:
buffer B, QSP for return to baseline, 500 ml of a 0 to 0.4 M KCl gradient in buffer B, 200 ml of buffer B containing 0.4 M KCl, ~250 ml Buffer B supplemented with 1 M KCl.
The amidase activity is eluted in a volume of 170 ml, at an ionic strength WO 94/17190% ~ 5, ~ 7 ~ ~ 14 PCT / FR94 / 00080 between 0.05 and 250 mM in KCl. This solution is analyzed (prot.
580 mg, [prot.] = 3.4 gxl-', act. early. = 1200 U, act. spec. = 2.1 Uxmg~
of prot., rdt in prot = 47%, rdt in act. -- 100%).
- Step 3: column of hydrophobic interactions (Phenyl Sepharose):
The 168 ml are concentrated by precipitation with 65% ammonium sulphate saturation. For this, 72 g of ammonium sulphate 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 the buffer B and dialyzed against 2 l of buffer B overnight. 25ml are collected, added with 4.4 g of ammonium sulphate to reach 30%
saturation then loaded at 50 mlxh~l for 35 min on a column of "Phenyl Sepharose" (26 x 300 mm), equilibrated with buffer B
ammonium sulphate contellallt at 30% saturation. The column is washed with the same buffer at a rate of 100 mlxh ~ 'during l h. The flow is reduced to 40 mlxh ~ 'and the column is percolated with buffer B alone.
The activity is eluted after 2.8 h in a volume of 30 ml (prot. + 110 mg, [prot.] = 1.4 gxl-', act. early. = 910 U, act. spec. 8.1 Uxmg~' of prot., rdt in prot. = 20%, rdt in act. = 74%).
The following steps are carried out on the Pharmacia FPLC system.
- Step 4: filtration gel (liPLC Superdex 200):
The 80 ml are concentrated by ultrafiltration on an Amicon PM10 membrane.
then on centriprep 10. These 2.8 ml are loaded onto the gel column filtration (16 x 600 mm) balanced with buffer B supplemented with 0.1 M
KCl at a rate of 0.8 mlxmin ~ '. The activity is eluted with the above buffer above at a flow rate of 1 mlxmin ~ 'and in a volume of 9 ml (26 mg of protein, 470 U, act. spec. = 18 Uxmg~' of prot., rdt in act. = 51%, yield in prot. = 26%).
- Step 5: gel filtration (FPLC Superdex ~00):
This second gel filtration is carried out under identical conditions to the previous one, but twice. Assays are only performed on the first passage, carried out from a fraction of 3 ml of the 9 ml of the Wo 94117190;~1 ~i 3 7g 3 PCT~94/00080 15'' previous step.
- Step 6: anion exchange column (mono Q HR 5/5 Pharmacia):
On this column, equilibrated with buffer B, the proteins are not retained and are eluted directly into the dead volume. Fractions active, 16.5 ml, are analyzed (prot. = 17.1 mg, [prot.] = 1 gxl-', act.
early. = 262 U, act. spec. = lS Uxmg~' of prot., cumulative rdts for stages S and 6 = 65% for prot. and SS% for activity).
- Step 7: anion exchange column (mono Q HR 5/5 Pharmacia):
This step is performed twice, using half of the fraction previous each time and with buffer B/5. The buffer is modified by dialysis on Amicon PM10 membrane and substitution with B/S buffer. The 6 ml of retentate are loaded onto the column equilibrated with B/S buffer, at a rate of 1 mlxmin ~ '. After washing the column for 14 min, a gradient from 0 to 0.1S M in KCl is applied in 25 min. The column is lS then fed with a 1 M KCl solution in B/S buffer. Twice, 3.75 ml are collected and analyzed (prot. = 1.5 mg, [prot.] = 0.4 gxl-', act. early. = 121 U, act. spec. = 81 Uxmg~' of prot., rdt in prot. = 11%, rdt in act. = 46%.
- Step 8: anion exchange column (mono Q HR 5/5 Pharmacia):
The 3.7 ml from the previous step are diluted to 6 ml with B/S buffer, deposited on the column. The proteins are eluted under the conditions of Step 7. Active fractions are pooled and analyzed (vol. = 3 ml, protein = 1 mg, [prot.] 0.34 gxl-', act. tot. = 93 U, act.
spec. = 92 Uxmg~' of prot., rdt in prot. = 67%, rdt in act. = 77%).
- Step 9: column of hydrophobic interactions (octyl ~u~e.ose ~IR 5/5):
The 3 ml pool from the previous step is brought to 38% saturation by adding a concentrated solution of ammonium sulphate. 6ml results are loaded onto the column, equilibrated with Buffer B
added with 1.7 M of ammonium sulphate. Proteins are eluted by a decreasing gradient of ammonium sulfate from 1.7 M to 0 M in the buffer B over 1 hour at a flow rate of 0.5 mlxmin~'. The active fractions are -~ 3 7 9 3 16 combined for analysis (vol. = 1.5 ml, prot. = 0.75 mg, [prot.] = 0.5 g/l, act. early. = 53 U, act. spec. = 70 Uxmg~l of prot., rdt in prot. = 75%, rdt in act. = 56% .
- Step 10 ~ cc ~ e (column of Fast ~ p- ~ qltinf ~ HR 10 / lO Pharmacia):
S The 1.5 ml are first concentrated using the centricon PM10 until ~
a volume of 0.5 ml. This fraction is deposited on the column, balanced with 10 mM phosphate buffer pH 6.8. The protein is eluted at a rate of 4 mlxmin~l. The protein comes out in a volume of about 2 ml (prot.
early. = 360 ~Lg, [prot.] = 180 mgxl~l, act. early. = 19 U, act.
sp~c. = 53 Uxmg~l of prot., rdt in prot. = 48%, rdt in act. 36%).

TABLE II: PURIFICATION OF AMIDASE FROM Comamonas NI 1 FLIGHT. PROT. ACTIVITY YIELD
STEP PF
OF
TOTAL MG PURIFICATION SP~CIF.PROT.ACTIV.
UU/MG % %
O - Crude extract 125 '' 7002 100 0.78 100 100 I - (NH4)2SO4 35-60% 43 1,200 1,200 2.1 22 59 1.3 2 - Q ~ I.alu,c 170 580 1,200 2.1 æ 59 Z.7

3 - Phenyl s~..v,c 80 110 910 8.1 4.2 44 10

4 - Gel filtration 1~ 26 470 18 0.9'73 Z3

5 - Filtration gel

6 - Mono Q HR 5/5 16 17 26Z lS 0.6 13 19

7 - Mono Q HR 5/5 3.7 1.5 121 81 0.05 5.8 100

8 - Mono Q HR 5/5 3 1 93 9" 0.03 4.5 IZ0

9 - Octyl superose HR 5/5 1.5 0.75 53 70 0.03",5 90

10 - Desalting " 0.419 53 0.01 0.9 67 WO 94/17190 2 l ~ 3 7 9 3 PCT / FR94 / 00080 ABBR~VL~TIONS:
- PF = purification coefficient, - U = 1 mol/min.

fig. 3 attached shows the results of a polyacrylamide gel analysis SDS 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 were carried on the right of the figure (kilo Daltons). The 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 of steps 1 to 8 and well 11 to the purified extract of step 10. The band corresponding to the ~ mid ~ e according to the invention is that of approximately 40 kDa.

2.3 Characterizations:
This amidase analyzed by SDS-PAGE gel shows a main band of 43,000 Da whose N-terminal sequence is: (amino acids 1-18 of the SEQ ID: 3 in the attached sequence listing).
Met-Ile-Glu-Asn-Ile-Ile-Ala-Lys-Leu-Lys-Asn-Ile-Leu-Glu-Ser-Asn-Thr-Asn-. . .
By gel filtration, the molecular weight is 80,000 +/- 2,000 Da. Amidase is therefore a homodimeric enzyme.

2.4 Activity of the ~mi~l~se according to the te~ .ture:
A pool of fractions containing mi(l~e) was used for this study.
purification conditions, this pool contains 0.3 gxl-' of protein and has a specific activity of 26 Uxmg~l of protein. The activity of this pool was determined between 10 and 60 C.
The 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, [protein] = 7.5 mgx1~'.
The results are grouped in Fig. 4. Activity increases according to the temperature up to about 50°C. Beyond; denaturation is observed.

Wo 94/17190 PCT/FR94/00080~
~ ~ ~ 3 7 93 18 Between 10 and 40 C, the Arrhenius law is observed and allows to calculate a ~`G
reaction of 83 kJxmole ~ '.

2.~ Activity as a function of pH:
From the same enzymatic preparation as that used previously, the activity is measured at different pH. The conditions of this study are following: [~r~ip~m~te] = 10 mM, 10 mM carbonate buffer, pH 10, tris-HCl 10 mM, pH 9, 8.5, 7.5, 10 mM phosphate, pH 6.5, 10 mM acetate, pH 3.5, 4.5, 5.5, T = 25 C, reaction volume 2 ml, reaction time 15 min, [protein] = 7.5 mgxl~l.
The results are grouped in the ~Ig. ~. The optimum pH is between 8.5 and 10, which corresponds well to the behavior generally encountered for amidases.

2.6 Determination of the Km of the ~mitlqce:
The substrate used is the ~ -lir ~ m ~ you. Its rate of hydrolysis to adipate is determined at different concentrations in ~ ip ~ m ~ tt ~ (0.2, 1, 2, 5 and 10 mM).
The conditions of this study are as follows: T = 25 C, Tris-HCl buffer 10 mM, pH 7.5, enzymatic extract 100 ~1 (see paragraph 2.5), volume reaction 4 ml, except 10 ml for 0.2 and 1 mM in ~ rlir ~ m ~ t ~., time of kinetic reaction over 2 h.
As shown in fig. 6, the curve obtained by plotting the speed as a function of the substrate concentration conforms to a Michaelis-Lie. As a result, the Hanes transformation S/V = f(S), represented to the ~Ig. 7 gives a Michaelis constant of 0.25 mM.

EXAMPLE 3: CLONING OF G1~NE ENCODING COMAMONAS AMIDASE
testosterone NI 1.

30 From the N-terminal sequence ~I~; sentée in Example 2.3, a probe nucleotide was synthesized. This is a 20 mer 256 times degenerate:

Wo 94/17190 PCT/FR94/00080 7~3 19 , ~, .

MIENIA (SEQ~:5) S' ATG ATT GAA AAT ATT ATT GC 3' (SEQ ID: 4) CGCCC
AAA
In the ~annexed sequence listing, the 20 base sequence is ~ignee by SEQ ID: 4 while the amino acid sequence associated therewith is referenced SEQ ID: 5.
The strategy followed consisted, first of all, in verifying the specificity of this probe 10 nucleotide and to determine the nature of the genomic DNA fragments to be cloned.
Briefly, the genomic DNA of Comamonas testosteroni NI 1 was digested with several co ~ ondant restriction enzymes at sites usable for cloning.
After electrophoresis on agarose gel and transfer onto a nylon membrane, the various digests were hybridized to the probe. The probe has sufficient specificity 15 under the hybridization conditions used (5x SSC, 5x Denhardt, 0.1% SDS, 50 mM NaPO4 pH 6.5, 250 µgxml ~~ ssDNA, hybridization temperature 50C.
Wash conditions: 1 h, 6x SSC, room temperature and 5 min in 2 x SSC, 0.1% SDS at 50C).
Under these conditions, the probe enabled us to obtain important signals without 20 ambiguity. In particular, in the case of digestions by SstI, PstI and BglII. The hybridization fingerprints show, in particular, the existence of a single fragment SstI of about 5.5 kb. In order to clone this fragment, the 5 to 6 kb fragments of a SstI digestion of genomic DNA were purified by preparative electrophoresis on agarose and electroelution, then ligated to plasmid pUC 19, itself digested 25 per SstI. After transformation into the DH5a ~ strain, 500 white clones on LBampicillin X-gal were subcultured individually, transferred to a membrane of nylon, then analyzed by hybridization with the probe used to hybridize the Southern blot under the same stringency conditions. Two clones have thus were identified as hybridizing very strongly with the probe. These two clones 30 found to contain a plasmid having inserted the same approximately 5.5 kb fragment into the same direction. One of these two pl ~ mides (pXL2104) was analyzed further Wo 94/17190 PCT/FR94/00080 ~37~3 detail (restriction cal~ogldyhie, partial sequencing using the probe as primer and Southern blot). It was thus able to be shown that the 5' part of the gene, which hybrid with the probe, is located on a PstI/SpeI fragment of approximately 420 bp and that the gene is oriented in the direction SpeI towards PstI. fig. 8 presents a map restriction of this pl ~ mide.

EXAMPLE 4: Sl~:QUENOE OF A 1491 bp FRAGMENT CONTAINING THE G~NE
CODING FOR AMIDASE FROM Comamonas testosteroni NI 1.

The location of the sequenced 1491 bp fragment, containing the gene coding for amidase from Comamonas testosteroni NI 1, is indicated under the cloned insert on the fig. 9. The sequencing strategy for this fragment, carried out using methods known to those skilled in the art, is r ~, i; se ~ e in the same figure. The various sequences were all obtained by the chain termination method.
(sequenase kit in the presence of 7-deaza dGTP; ~5S)dATP) or on matrices single strand of M13 (mpl8 or mpl9 cf YANIS ~ et al., Gene 33 (1985) 103) recombinant carrying sub-fragments, either dir ~; ~", ent on the plasmid pXL2104.
Several specific primers have ég ~ lt m-have been synthesized for this purpose.
The sequence is shown in Fig. 2. and on SEQ ID: 2 from the sequence listing ~ne~ee. The average GC% of the sequence obtained is 33.6%, which is lower to the GC% of 61.5% described in other strains of Comamonas (TAMAOKA et al., Int. J.Syst. Bacteriol, 1987, 37, 52-59). An analysis of the obtained sequence has made it possible to characterize an open frame of 1254 bp coding for a polypeptide of 418 colle~yol-ant residues at a molecular weight of 46,148 Da. This polypeptide co---ylends the terminal NEI2 sequence used to synthesize the probe.
Thus this open phase ~ cyresellte the structural gene of the desired amidase. This gene will be referred to as amdA in the remainder of this presentation.
From the pl ~ mi ~ the pXL2104, the pl ~ micle pXL2216 was prepared by inserting the SpeI-ClaI fragment containing the amdA gene between the XbaI and ClaI sites of the vector pMTL 23 (CHAMBERS et al., Gene, 68 (1988) 139-149). This plasmid, whose map restriction is shown in Figure 10, was used to transform the strain WO 94/17190 2 1 ~ 3 7 ~ 3 PCT / ~ 94/00080 of E. coli TG 1. The resulting strain called G 4315 has been deposited in the Collection National Culture of Microorganisms in Paris (Institut Pasteur, 25 rue du Doctor Roux) under number I 1279.

5 EXAMPLE 5: EXPRESSION OF AMIDASE FROM Comamonas Testosteroni NI 1 IN E. coli.

In order to confirm the identity between the am~4 gene and the gene encoding the amidase of Comamonas testosteroni NI 1, a plasmid was constructed in which the amdA gene, 10 preceded by its own ribosome binding site, is placed under the control of the promoter of the lactose operon of E. coli. Thus, the pl ~ mide pXL2144, described on the fig. 11, was obtained by insertion of the SpeI-ClaI fragment derived from the plasmid pXL2104 between the XbaI and ClaI sites of the vector pMTL 22 (CHAMR ~ R ~ et al., Gene, 68 (1988) 139-149).
15 This plasmid therefore contains the promoter of the lactose operon Plac, followed by the site of binding of the ribosomes and the structural gene of the ~ mi ~ e, as well as a gene conferring the resistance to ampicillin.
The plasmid pXL2144 was used to transform the strain of E. coli TG 1. The selection of micro-org ~ ni ~ me ~ is done on ampicillin.
20 The e ~ p, ession of the ~ mid ~ e of Comamonas testosteroni NI 1 was vicu ~ ed, 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 a quantity of protein equivalent to 60 ~ l of culture at an OD of 3 to 610 nm.
25 The f~lg. 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 contents of the di~erent tracks are shown in the following table.

WO 94tl7190 PCT / FRg4 / 00080~
~537g3 22 TG1 + TG1 + TG1 + TG1 +
p~144 pXL2144 pMTL22pMTL22 + lrrG + IPrC
Sul.la~ ADGJ
BEHK Base CFIL gross fraction It appears from fig. 12 than the cell extracts comprising the plasmid pXL2144 express the ~ mi ~ e according to the invention, essentially in soluble form a band at about 45 kDa is visible for the ACDF tracks, whereas this one does not appear not for extracts G to L.
10 In addition, the co ", p ~ udison of the band at about 45 kDa of AC extracts and DF extracts shows that the cells cultured in the presence of ITPG have a level d't;~,cssion higher.

EXAMPLE 6: AC11VrF- AMIDASE C-EZ Escherichia coli.

The activity of the E. coli strain (pXL2144), induced or not, is measured on adipamide and adipamate and co ", by ~ e to that of the control strain E. coli (pMTL 22).

20 6.1 Cell preparation:
The cultures are carried out under the conditions described in TABLE III below. During the exponential phase of growth, one of the two cultures of the recombinant strain is induced by 1 mM IPTG, after further 2 h at 37 C this culture is treated.5 WO 94/17190 215 3 7 9 3 PCT~94/00080 TABLE m STRAIN CULTURE

PS
MICRO-ORG~NI$~ES MEDIUM 660 nm g/I

E. coli (PMTL22) B 2.2 0.84 E. coli (pXL2144) B 1.8 0.75 E. coli (pXL2144) C 2.3 1.10 ABBR~VL~TIONS:
- B = LB medium + Amp 100 ~gxml~', - C = medium B + addition of 1 mM IPTG at 0.8 OD
COMMON CONDITIONS:
Inoculation to 1/100th from a preculture aged 17 h, time of culture 5.5 h, T = 37 C.
15 6.2 Measures of specific activities:
The conditions for measuring specific activities and the results are Listed in TABLE IV below.
The common conditions for these measurements are: T = 25 C, 50 mM phosphate buffer pH 7, kinetics over 90 min.

WO 94/17190 S 3 7 ~ 3 PCT / FR94 / 00080 TABLE IV: MEASUREMENTS OF AMIDAS ACr/VrrES OF STRAINS
RECOMBINANTS THAT EXPRESS COMAMONAS AMIDASE
NI 1.

SUBSTRATE ACTIVITY
SPECIFIC SOUCHHS
Nature [cel.]Induced Et. t Nature [S]~mole/min/g g/l +I mMde PS
E. coli (pXL2144) 6.2 + E A-1ipnm~ 15 14 6.1+ST A~iipnm: ip12 15 6.1 + SC Ariipnm: ~f. 12 0 6.1 + SS A-iipnmi~3 12 14 6.2 - EA~lip mi~ 11 0 0.3 + E ~ . 50700 0.25 + ST A-iipnmA~,, 502,000 2.5 0 + SCA ~ 5 09 7 0.25 + SS A~iip-mn~R 501,900 0.69 - E Arlirqmntt~50130 1.4 - ST A~^50500 2.8 - SC Affiliation~.50 46 1.4 - SS A~'. ~50433 E. coli (pMTL22) 10 E ~ jpnmA~ 25 0 14 EJ~fiipnrr: ilo.13 0 ABBREVIATIONS:
- ~ = whole cells, - ST = crushed cell, - SC = insoluble fraction of the ground cell, - SS = soluble fraction of the ground cell.

WO 94/17190 3 PCT/FRg4/00080 EXAMPLE 8: EXPRESSION AND A~:11V11~; AMIDASE IN Pseudomonas putida The ~mid~e was expressed in Pseudomonas putida using the construct following S: From pXL2216 described in FIG. 10, the Sa~/BglII fragment of 1.75kb containing the amdA gene was introduced between the SalI and BamHI sites of pDSK519 (Keen et al., 1988, Gene. 70:191). This pl ~ cmide, pXL2238, described on Figure 13 was introduced into the strain of Pseudomonas putida G 2081. G 2081 is a derivative of Pseu ~ lomonas p ~ tida 2440 (Bagdasarian and Timmis, 1981. In 10 Hofschneid and Goebel. Topics in Microbiology and Immunology. 47. Springers Verlag. Berlin.) rendered re~i~t~nt~ to nalidixic acid and rifampicin. Levector pDSK519 was used as pl ~ mide control.
The activity of strain G 2081 (pXL2238) is measured on ~(lip~m~t~ and compared to that of the control strain G2081 (pDSK519).
Pseudomonas G 2081 (pDSK519) and Pseudomonas G 2081 (pXL2238) strains were cultured in LBk medium (Tryptone 10 g/l, NaCl 5g/l, yeast extract 5g/l, glucose 10 g/l, ammonium sulphate 1 g/l and kanamycin 100 mg/l). The results of cultures are listed in Table V.
0 TABLE V: CULTURE OF STRAINS ESTIMATED FOR THEIR ACTIVITY
AMIDASE

Microorganism DO660~ Dry Weight (g/l) Pseudomonas (pDSK519) 2.3 0.9 25 Pseudomonas (pXL2238) 1.8 0.5 The activities of these strains were determined on ~ tlip ~ m ~ te and adipamide. The conditions and results are listed in Table VI.

WO 94/17190 PCT / FR94 / 00080 ~
~3~3 26 TABLE VI: REACTIONAL CONDITIONS AND RESULTS OF MEASUREMENTS OF
L~A~; 11V11~ AMIDASE OF THE RECOMMENDED STRAIN RES
PSEUDOMONAS (PDSK519) AND PSEUDOMONAS (PXI2238) Con~ ion~ r~ictinnnPll~oc Strain duration volume Substrate Activity ((U/kg of CS) nature (g/l) state (h) (ml) na~re (mM) Comamonas 5.6 E 4 5,sæ~lir~m ~.o 20 0.5 testosteroni Nll ~ I;p-,.. t~ 20 2.0 P. putida 2 E 3 4 r', ~ 20 o (pDSK519) P. Putida 2 E 1.5 2 r ' .~ 18 0 (pXL223 8) E r ~ t' 45 3, 8 S ~ip~ 50 5, 1 Common conditions: SO mM pll 7 phosphate buffer; ~C25-28;
Abbreviations: E whole cells, S cell homogenate; U mole of amide hydrolyzed/h; CS dry cells;

The Pseudomonas strain (pXL2238) expresses the Comamonas mi~e gene NEITHER. 1 and all the better as the prep ~ r ~ tinn cell ~ ire is sonicated.
These experiments show that the ~mi~e is expressed and active in Pseudomonas putida.

WO 94/17190 215 3 7 9 3 PCT / FRg4 / 00080 27 . ~

EXAMPLE 9: COMPARISON OF SPECIFIC ACTIVITIES ON
ADIPAMIDE AND ADIPAMATE FROM EXPERIMENTAL STRAINS
AMIDASE ACCORDING TO ART A~R (FR 90-14 853) AND
CONCERNS EXPRESSING AN AMIDASE ACCORDING TO THE 1NVENTION.

The conditions of the measurements and the results are given in TABLE VIII below.

vm TABLE:

STRAINS SULS rRAT ACnVITY
SPi~ClFlQUE
NatUre [Cel.] IndUit State NatUre tS] ~mole/
(mM)min/g Brevibacteria (R 312) 0.06 + EA~;r~m;~ 20 2.3 0.3 + ST A~;r~m;~1~ 20 10 0.3 + ST Adipamatc20 4.2 E. coli (PXL1751) 0.3 + E Adipamide 20 75 ART
0.3 + E Adipamate 20 16 ~I~UhUK
0.3 + ST Adipamate20 16 Rho~co.. ~ ~ sp 0.76 + E~1ir:~mi~ 20 35 0.76 + ST A~;r~m;~1~ 20 62 0.3'' + ST Adipamate 20 55 C. testosteroni sp 5.6 + EA~;P~ 20 8.3 5.6 + E Adipamate 20 33 INV~ON
E. coli (PXL2144) 6.2 + EA~i;r~m;~ 5 14 6.1 + ST .A~1;r~m;~Jr 12 15 0.3 + E Adipamate 50 700 0.25 + ST Adipamate 50 2000 WO 94/17190 ~ 1 ~ 3 7 9 3 PCT ~ 94/00080 COMMON CONDITIONS:
T=25C
- 50 mM phosphate buffer pH 7.
ABBREVIATIONS:
- E = whole cells, - ST = crushed cell.
The pl ~ mi (the pXL1751 of the recombinant strain E. Coli is described in detail in patent application FR 90-14 853.
TABLE VII clearly highlights the greater specificity of the ~ mi ~ e according to the invention vis-à-vis the ~ (lip ~ m ~ te that vis-à-vis the adipamide, contrary to what is observed for known amidases. This observation is worth both for an amidase e ~ les ~ ion by natural germs and by recombinant germs.

WO 94/17190 PCT~FR94/00080 SEQUENCE LIST

(l) GENERAL INFORMATION:

(i) APPLICANT:
(A) NAME: RHONE-POULENC CHIMIE
(B) STREET: 25, Quai Paul Doumer (C) CITY: Courbevoie .
(E) COUNTRY: France (E) ZIP CODE: 92400 (G) TELEPHONE: 47 68 12 34 (H) FAX: 47 68 16 56 (ii) TITLE OF THE INVENTION: Polypeptides with amidase activity, tools genetics and micro-org~ni~m~s hosts allowing their obtaining, hydrolysis process using said polypeptides (iii) NUMBER OF SEQUENCES: 5 (iv) COMPUTER READABLE FORM:
(A) SUP'PORT TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #10, Version #1.25 (EPO) (vi) PRIOR APPLICATION DATA:
(A) DEPOSIT NUMBER: FR 93 01062 (B) FILING DATE: 27-JAN-1993 (2) INFORMATION FOR SEQ ID NO: l:

(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 418 amino acids (B) TYPE: amino acid (D) CONFIGURATION: lin~P;re (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ Ip NO: l:

REPLACEMENT LIGHT (RULE 26) WO 94/17190~~5~~93` PCT~R94100080 Met Ile Glu Asn Ile Ile Ala Lys Leu Lys Asn Ile Leu Glu Ser Asn 1 5 or 15 Thr Asn Ser Phe Ile Ser Leu Asp Leu Val Asp Val Ser Lys Gln Leu Lys Lys Gln Asp Ser Asn Gln Pro Leu Asn Gly Val Thr Val Ala Ile Lys Asp Leu Ile Asp Val Glu Gly Gln Lys Thr Thr Met Gly Ser Ala Gln Tyr Gln Asn Asn Island Ala Arg Ser Asp Ala Asp Val Val Arg Arg Leu Arg Thr Ala Gly Ala Val Ile Phe Gly Lys Thr Asn Thr His Glu 95he Ala Tyr Gly Ser Thr Gly Asp Lys Ser Phe Phe Gly Pro Val Leu 100 105 llo Asn Pro Asn Asn Glu Asn His Ile Thr Gly Gly Ser Ser Ser Gly Ser Ala Ala Ala Val Ala Ala Asp Leu Cys Asp Val Ala Ile Gly Thr Asp Thr Ala Ala Ser Val Arg Leu Pro Ala Ala Leu Cys Gly Val Ile Gly Leu Lys Pro Thr Tyr Asn Ser Ile Lys Arg Asp Gly Val Phe Ser Leu er Pro Ser Leu Asp His Val Gly Ile Ile Ser Lys Ser Leu Asp Leu 180 18~ l9o Ile Glu Lys Thr Phe Asn Ser Thr Lys Thr Ser Ile Asp Gln Lys Pro Gln Lys Asn Leu Asp Lys Lys Leu Thr Ile Gly Leu Leu Lys Gly Phe Phe Glu Glu Tyr Ile Cys Ser Ser Val Lys Glu Lys Tyr Asp Glu Thr Ile Glu Leu Leu Arg Ser Asn Gly Cys Glu Ile Lys Glu Ile Asn Ile ys Glu Ala Phe Asp Island Tyr Thr Asn Ser Gln Island Val Leu Lys Tire Glu Ala Tyr Lys Ile His Glu Glu Ser Leu Lys Leu Asn Tyr Pro Phe Asp Pro Glu Val Lys Gly Arg Ile Leu Arg Gly Glu Asn Ile Thr Lys ~ SUBSTITUTE SHEET (RULE 26) .3 WO 94/17190 'PCT ~ FR94100080 Asp Glu Tyr Asp Ile Ala Lys Lys Tyr Gln Glu Asn Ala Arg Ile Ile Phe Asp Thr Ala Leu Lys Asp Val His Val Leu Ala Ser Leu Thr Ser Gly Ile Leu Pro Pro Lys Ile Phe Glu Arg Gln Thr Lys Val Asn Glu Asn Thr Val Glu Thr Phe Phe Leu Leu Thr Arg Leu Thr Ala Pro Ile Asn Phe Thr Gly His Pro Ala Ile Ser Tyr Pro Ile Gly Lys Ile Asn Asn Leu Pro Val Gly Ile Gln Phe Ile Ser Asp Phe His Asn Glu Glu Ile Leu Phe Gln Ala Cys Asn Leu Leu Gln Lys Ser Phe Leu Ala Lys Gly Asn (2) INFORMATION FOR SEQ ID NO: 2:

(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 1491 base pairs (B) TYPE: nucleic acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: Genomic DNA) (ix) ADDITIONAL FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 127..1380 (xi) SEQUENCE DESCRIPTION: SEa ID NO: 2:

TCCGCAATTC ATGATTGAAA GATGTGAGTT TCCATGAGAT CAGAGACCGA GM m AGAC 60 Met Ile Glu Asn Ile Ile Ala Lys Leu Lys Asn Ile Leu Glu 1 5 lo Ser Asn Thr Asn Ser Phe Ile Ser Leu Asp Leu Val Asp Val Ser Lys SUBSTITUTE SHEET (RULE 26) 7~

Gln Leu Lys Lys Gln Asp Ser Asn Gln Pro Leu Asn Gly Val Thr Val Ala Ile Lys Asp Leu Ile Asp Val Glu Gly Gln Lys Thr Thr Met Gly Ser Ala Gln Tyr Gln Asn Asn Ile Ala Arg Ser Asp Ala Asp Val Val Arg Arg Leu Arg Thr Ala Gly Ala Val Ile Phe Gly Lys Thr Asn Thr 80 85gb His Glu Phe Ala Tyr Gly Ser Thr Gly Asp Lys Ser Phe Phe Gly Pro Val Leu Asn Pro Asn Asn Glu Asn His Ile Thr Gly Gly Ser Ser Ser Gly Ser Ala Ala Ala Val Ala Ala Asp Leu Cys Asp Val Ala Ile Gly Thr Asp Thr Ala Ala Ser Val Arg Leu Pro Ala Ala Leu Cys Gly Val Ile Gly Leu Lys Pro Thr Tyr Asn Ser Ile Lys Arg Asp Gly Val Phe Ser Leu Ser Pro Ser Leu Asp His Val Gly Ile Ile Ser Lys Ser Leu Asp Leu Ile Glu Lys Thr Phe Asn Ser Thr Lys Thr Ser Ile Asp Gln SUBSTITUTE SHEET (RULE 26) WO 94/17190~`~'`
PCT/E~94/00080 Lys Pro Gln Lys Asn Leu Asp Lys Lys Leu Thr Ile Gly Leu Leu Lys GGT m 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 Glu Thr Ile Glu Leu Leu Arg Ser Asn Gly Cys Glu Ile Lys Glu Ile Asn Ile Lys Glu Ala Phe Asp Ile Tyr Thr Asn Ser Gln Ile Val Leu Lys Tyr Glu Ala Tyr Lys Ile His Glu Glu Ser Leu Lys Leu Asn Tyr CCA I~C GAC CCT GM GTT AAG GGG CGA ATA TTA AGG GGT GM AAT ATA 1032 Pro Phe Asp Pro Glu Val Lys Gly Arg Ile Leu Arg Gly Glu Asn Ile Thr Lys Asp Glu Tyr Asp Ile Ala Lys Lys Tyr Gln Glu Asn Ala Arg ATC ATA m 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 Thr Ser Gly Ile Leu Pro Pro Lys Ile Phe Glu Arg Gln Thr Lys Val Asn Glu Asn Thr Val Glu Thr Phe Phe Leu Leu Thr Arg Leu Thr Ala Pro Ile Asn Phe Thr Gly His Pro Ala Ile Ser Tyr Pro Ile Gly Lys SUBSTITUTE SHEET (RULE 26) -WO 94/17190 7 9 ~ PCT ~ R94 / 00080 Ile Asn Asn Leu Pro Val Gly Ile Gln Phe Ile Ser Asp Phe His Asn Glu Glu Ile Leu Phe Gln Ala Cys Asn Leu Leu Gln Lys Ser Phe Leu Ala Lys Gly Asn TTCCA m CC T 1491 (2) INFORMATION FOR SEQ ID NO: 3:

(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 418 amino acids (B) TYPE: amino acid (D) CONFIGURATION: ]; born~; D

(ii) TYPE OF MOLECULE: protein (xi) SE W ENCE DESCRIPTION: SEQ ID NO: 3:

Met Ile Glu Asn Ile Ile Ala Lys Leu Lys Asn Ile Leu Glu Ser Asn Thr Asn Ser Phe Ile Ser Leu Asp Leu Val Asp Val Ser Lys Gln Leu Lys Lys Gln Asp Ser Asn Gln Pro Leu Asn Gly Val Thr Val Ala Ile Lys Asp Leu Ile Asp Val Glu Gly Gln Lys Thr Thr Met Gly Ser Ala Gln Tyr Gln Asn Asn Island Ala Arg Ser Asp Ala Asp Val Val Arg Arg Leu Arg Thr Ala Gly Ala Val Ile Phe Gly Lys Thr Asn Thr His Glu Phe Ala Tyr Gly Ser Thr Gly Asp Lys Ser Phe Phe Gly Pro Val Leu Asn Pro Asn Asn Glu Asn His Ile Thr Gly Gly Ser Ser Ser Gly Ser SUBSTITUTE ILLE (RULE 26) 21~37~3 WO 94117190 ~ PCT ~ R94 / 00080 Ala Ala Ala Val Ala Ala Asp Leu Cys Asp Val Ala Ile Gly Thr Asp Thr Ala Ala Ser Val Arg Leu Pro Ala Ala Leu Cys Gly Val Ile Gly Leu Lys Pro Thr Tyr Asn Ser Ile Lys Arg Asp Gly Val Phe Ser Leu er Pro Ser Leu Asp His Val Gly Ile Ile Ser Lys Ser Leu Asp Leu Ile Glu Lys Thr Phe Asn Ser Thr Lys Thr Ser Ile Asp Gln Lys Pro Gln Lys Asn Leu Asp Lys Lys Leu Thr Ile Gly Leu Leu Lys Gly Phe Phe Glu Glu Tyr Ile Cys Ser Ser Val Lys Glu Lys Tyr Asp Glu Thr Ile Glu Leu Leu Arg Ser Asn Gly Cys Glu Ile Lys Glu Ile Asn Ile ys Glu Ala Phe Asp Island Tyr Thr Asn Ser Gln Island Val Leu Lys Tire Glu Ala Tyr Lys Ile His Glu Glu Ser Leu Lys Leu Asn Tyr Pro Phe Asp Pro Glu Val Lys Gly Arg Ile Leu Arg Gly Glu Asn Ile Thr Lys Asp Glu Tyr Asp Ile Ala Lys Lys Tyr Gln Glu Asn Ala Arg Ile Ile Phe Asp Thr Ala Leu Lys Asp Val His Val Leu Ala Ser Leu Thr Ser Gly Ile Leu Pro Pro Lys Ile Phe Glu Arg Gln Thr Lys Val Asn Glu Asn Thr Val Glu Thr Phe Phe Leu Leu Thr Arg Leu Thr Ala Pro Ile Asn Phe Thr Gly His Pro Ala Ile Ser Tyr Pro Ile Gly Lys Ile Asn Asn Leu Pro Val Gly Ile Gln Phe Ile Ser Asp Phe His Asn Glu Glu Le Leu Phe Gln Ala Cys Asn Leu Leu Gln Lys Ser Phe Leu Ala Lys Ly Asn ~ SUBSTITUTE SHEET (RULE 26) ~lS37~3 WO 94/17190, 36 PCTn~R94/00080 (2) INFORMATION FOR SEQ ID NO: 4:

(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETIC: NO

(iii) ANTI-SENSE: NO

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

(2) INFORMATION FOR SEQ ID NO: 5:

(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 7 amino acids (B) TYPE: amino acid (D) CONFIGURATION: 1; neA; D

(ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Met Ile Glu Asn Ile Ile Ala REPLACEMENT SHEET (RULE 26)

Claims (15)

CLAIMS: 1 - Polypeptides having an amidase activity, capable of hydrolyzing amides to carboxylates, characterized in that they possess enzymatic activity against of ammonium adipamate greater than that which they have vis-à-vis adipamide. 2 - Polypeptide according to claim 1, characterized by the sequence peptide SEQ ID: 1 as shown in FIG. 1. 3 - DNA sequence coding for a polypeptide having an activity amidase, capable of hydrolyzing amides into carboxylates, characterized in that it is chosen from the list of sequences following:
- the DNA sequence SEQ ID: 2, as shown in FIG. 2 and encoding a polypeptide having an amidase activity, - an analog of this sequence resulting from the degeneration of the genetic code, - a DNA sequence hybridizing with one of these sequences or a fragment thereof and encoding a polypeptide having a amidase activity. 4 - Polypeptides resulting from the expression of a DNA sequence according to claim 3 and having amidase activity. 5 - Microorganism consisting of the E. coli strain containing the plasmid pXL2216, strain referenced G 4315 and deposited in the National Collection of Cultures of Microorganisms under No. I 1279 on January 11, 1993. 6 - Microorganism capable of producing at least one polypeptide according to the claim 4. 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 binding of ribosomes. 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: E. coli tryptophan Ptrp operon promoter, promoter of the lactose operon Plac of E. coli, right promoter of the phage lambda PR, promoter left of phage lambda PL, strong promoters of Pseudomonas, Comamonas or corynebacteria. 9 - Microorganism according to claim 7, characterized in that it belongs to the prokaryotic family and in that the ribosome binding site is selected from that derived from the CII gene of lambda, those derived from genes of Comarmonas or Pseudomonas, or those derived from corynebacteria genes. 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 yeast glycolytic genes, such as the genes encoding phospho-glycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, or those of lactase or enolase. 11- Microorganism according to one of claims 6 to 10, characterized in that the expression cassette is carried by a plasmid. 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, Streptomyces, Bacillus. 13 - Polypeptides, characterized in that they are produced by micro-organisms according to any one of claims 6 to 12. 14 - Process for the enzymatic hydrolysis of amides into carboxylates, characterized in that it consists in placing the mono or di-amides corresponding to one of following general formulas:
H2NOC-R-CONH2, H2NOC-R-COO-Z+, NC-R-CONH2 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 following list of compounds: alkaline, alkaline-earth, amines, 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 one any of claims 6 to 12. 15 - Process according to claim 14, characterized in that R=-(CH2)4-.