CA2146656A1 - Nitrile-hydratase activity enzymes; genetic tools and microorganism hosts used for preparing the same; hydrolysis process applying said enzymes - Google Patents

Abstract

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

Description

21~656 ~0 95/04828 PCT/FR94/00993 El ~ A ACllV ~ MTR ~ TASE, OUIlI5 Gl ~ llQW ~: S AND ~ CRO-ORGANIS ~ D ~; H~
P1~11~TANT IEI~ ~II~ N ET PROa~ D~ .YSE I~TAl~ EN OEllVRE IF~rl~ ENZYI~

POM~TNE TEC~OllE:

The field of the invention is that of the enzymatic production of amide derivatives from compounds containing groupem ~ nt ~ nitriles.

BACKGROUND TECHNOLOGY:
The present invention relates to new enzymes having a nitrile-hydratase activity, the genetic material involved in their production, as well that the micro-org ~ nicmes con ~ enant this m ~ t ~ riel genetic and pr ~ sen ~ this activity.
The invention also relates to an enzymatic hydrolysis process lS of nitriles in amides in which one implements these new enzymes or micro-or ~: ~ ni ~ mçs the synthéti ~ nt, of which, not ~ mmPnt, the aforementioned micro-org ~ ni ~ my hosts.
Without this being limiting, nitriles, in which we are more interested particularly within the scope of the invention are: adipon ile, cyano-S
valeramide, cyano-S-valerate of any cation, of pr ~ fe, ellce selected from the list of co, l, posed following: ~ lç ~ lins ~ alkaline-earth, ~ minPs, ammonium, this the latter compound being particularly preferred. The goal is transformation enzymatic function of nitrile functions ~ mides, so as to obtain a ~ -lir ~ m ~ te, eg ammonium, convertible into diammonium adipate.
And, as everyone knows, all the industrial interest of the ~(iir~tes~ and in particular of diammonium adipate, lies in the fact that they con ~ titl ~ Pnt one fon-l ~ mPnt ~ lPs bases for the manufacture of nylon-6,6.
The action of these nitrile hydratase fits into a known pattern of enzymatic synthesis of di ~ mmonium adipate from adiponitrile. This scheme is the next:

wo gSI~ 2~ ~G ~ ~S 2 PCTIFR94/00993~

_NC--R--CN

..
NC--~--COO~I4~ H2NOC--~--CONH2 NH~~~
H2NOC--R--COO~I~+

Ni\\~~A~,/A
+ ~OOC--R--COO
NH-- nitrile hy ~ lrat ~ this, Ni = nitnl~e, A = ~mid~e, R = (CH~)n, n being an integer equal to 4 in the case of compounds adipic.
Within the framework of this scheme, the arsenal of enzymatic hydrolysis of amides and nitriles of many strains has been studied.
Y. ASANO et al. thus described in Agric. Biol. Chem., 45(5). 1165-1174. 1982, Arthrobacter. This germ has a nitrile hydratase only active on m~lononitrile and not on adiponilile.
H.YAMADA et al. refer to J. Ferment. Technol., 58, 495-500, 1980, the hydrolysis of glut ~ o ~ island by the strain Pseudomonas sp. K9, the nitrilehydratase of this hydrolyzed strain, ~ Ç ~ nLi ~] lernerlt one of the two functions CNdu ~ lin ~ you ~ while the other is only slightly hydrolyzed by the enzyme.
Patent FR 2 245 585 describes b ~ t ~ - ~ ie ~ selected nitrilase activity, preferably, among the genera ~ s, Bacteridium within the meaning of PREVOST, 21 ~ 6 ~ 5 6 , PCT/F~Rg4/00993 Micrococcus and Brevibacterium within the meaning of BERGEY. The Brevibacterium R 312 strain described in this patent, and more particularly the nitrile hydratase included in its enzymatic heritage, catalyzes the hydrolysis into amides of dinitriles of the type malononitrile, fulnaro~ile and succinonitrile. This enzyme is more active against 5 dinitriles or mono ~ itriles monoamides than vis-à-vis mononitriles monocarboxylates.
This spectrum of activity was confirmed by subsequent studies, including not ~ mm ~ -nt those of GALZY et al. in J. Gen. Microbiol, 130, 89-93, 1984.
The article by MAYAUX et al., published in J. Bacteriol, 172, 6764-6773, 1990, e.1-se; gne that Brevibacterium R 312 would be the same germ as a another coryneb ~ and ~ rie, namely: Rhodococcus N 774.
The latest patent EP o 178 106 in ~ Pi ~ ne ~ it also only Rhodococcus N 774 (or Brevibacterium R 312) has an active nitrile hydratase only on one nitrile functions of dinitriles and little ~ elr ~ l", allle vis-à-vis mononitriles carboxylated.
INGVORSEN et al describe, in CIBA Found. Symp., 140, 16-31, 1988, a strain id ~ ntifié as being a ~ odococcus sp CH5, having for because having, in its enzymatic heritage, a nitrile hydratase also hydrolyzing, plcfé ~ içllem ~ nt, only one of the CN functions of the malononitrile. This strain is even presented as not hydrolyzing acid 2-cyanoacetic acid.
We know, ~ Cg ~ l ~ m ~ nt, by patent JP 03 019 695, the strain Corynebacterium sp CS, which hydrolyzes valeronitrile to transcyano-4-cyclohexane amide acid. This nitrile hydratase also does not d~p~se the mononitrile stage monocarboxylic.
In Ann. NY Acad. Sci., 613, 142-154, 1990, YAMADA et al have describes a Pseudomonas chlororaphis involved in the hydrolysis of acrylonitrile in acrylamide.
In the mushroom family, KUWAHARA et al. (J. Ferment.
Technol., 58, 573-577, 1980) isolated Fusarium solani which proved capable of degrade succinodinitrile to succinic acid, passing through a mononitrile Wo 95/04828 2 ~ 4 6 6 ~ ~ PCT~4100993~

mono ~ mide, then a diamide and finally a monocarboxylic monoamide.
This review of the state of the art shows, on the one hand, that the collection known enzymatic NH does not offer re ~ llemçnt pelror means." ~, ~ s hydrolysis of the second nitrile function on substrates of the dinitrile type and, 5 on the other hand, that the known NH determine an enzymatic pathway of p ~ c ~ e of a ~ initrilP ~ dicarboxylic acid co, .espol dant, which involves only nitriles amides and ~ i ~ mi ~ es And we can rule that the enzymatic activities which c~ terize this path, although significant in terms of the laboldtoil~, are not not ~ticf~ ntps from an industrial perspective.
Thus, one of the essential objectives of the present invention is to propose new enzymes with nitrile hydratase activity, a genetic material allowing their production and micro-or~nicm~-s containing this genetic material, Iesdits enzymes and micro-org ~ nismçs are charact ~ ri ~ nt, at the same time, by yieldc s ~ ticf ~ i ~ ntc for producing amides from nitrile type substrates of natures 15 various (mono or linitrilPs) and by an activity ~ nitrile-hydratase, vis-à-vis the carboxylated mononitriles, specific and ~ ignifi ~ tive. Such activity is likely to provide access to an enzymatic pathway for the production of ~ -ides carboxylic from linitriles, industrialrnPnt int ~ the ~ ilte, because very pclro..nall ~ e.
After numerous tests and research, the Applicant has succeeded 20 to achieve, among other things, this objective by isolating, purifying and characterizing new enzymes:
- - capable of hydrolyzing nitriles into amides with high activities, - and having carboxylated mononitriles as the preferred substrate.
These enzymes are used, either as such, or, and preferably, under 25 the form of micro-org ~ ni ~ my recombined generating them.

E: EXPOSURE OF THE INVENTION:

The present invention therefore relates to new enzymes having a 30 nitrile hydratase activity, capable of hydrolyzing nitriles into amides and possessing an enzymatic activity vis- ~ -vis substrates having a nitrile function and a ::
~14~
~1~o 95/04828 PCT/ER94/00993 S

carboxylate function superior to that with respect to substrates having at least two nitrile functions and to that with respect to substrates having at least one nitrile function and at least one other function dirr ~ lel ~ the carboxylate function.
One of the particul ~ rit ~ s very advantageous enzymes according to the invention S is eYrrim ~ e by a specific enzymatic activity (Us) ~ vis-à-vis the cyano-5 valerate, expressed in moles of amide appeared x hl x kgl of enzyme used and under given measurement conditions, greater than or equal to 400, in preference to 1,000 and, even more pr ~ felçl-liellernent still, to 10,000.
The s~ls~ites measurement conditions are:
10 - reaction medium = 10 mM HEPES buffer, - volume = 3ml, - you ~ n ~ ule = 25 C, - enzyme concentration = 16 ~ g/ml One of these new enzymes was isolated from a strain of 15 Comamonas testosteroni. More precisely, this enzyme is l~r~ar~e by extraction and pnrific ~ tion from cultures of micro-org ~ ni ~ my natural or recombinant, the purified ~ tiQn being r ~ alized by a sl ~ cce ~ ion of stages con ~ ict ~ nt to ~ l ~ a ~ el- an extract enzymatic from the cell culture, to pl ~ cipilate this extract with sulphate ammonium and purifying it by di ~ .ei ~ your chromatography and filtration steps 20 on gel. These steps, which make use of techniques well known to those skilled in the profession, are described in detail in the illustrative examples below.
By nitrile hydratase activity, we d ~ igne, in this presentation, enzymatic hydrolysis of a nitrile-mono or dinitrile, such as adiponitrile cyano-5 valeramide and the cyano-5-valerate of a cation Z, into an amide, namely, in cet25 eYrmrle, respectively, the cyano-5 valeramide, the ~ dir ~ micle and the ~ p ~ m ~ te of Z .
The cation Z can be arbitrary, but it is chosen, pr ~ é ~ ence, among the following compounds: ~lr~lins, alkaline-earths, ~mines, ammonium, the latter colllposé being particularly preferred.
It is to the Claimant's credit to have been able to isolate these new 30 enzymes and to have been able to highlight the new "carboxylated mononitrile" pathway who r~r~rt~rize them.

Wo 95/04828 2 ~ 4 ~ ~ ~ 6 PCT/FR94tO0993~

However, it should be emphasized that this new path allows, insofar as one associates a ~ mi ~ this with the enzyme of the invention, to prepare acids dicarboxylics from dinitriles passing exclusively through esp ~ c ~ s flexible. It is clear that this is particularly advantageous in the perspective S of an optimi ~ tion in-lustri ~ lle of the process.
This characteristic of hydrolysis of carboxylated mononitriles is verified both for enzymes in pure form and for natural germs or recombinants expressing these enzymes.
Among the enzymes with nitrile hydratase activity, in accordance with the invention, 10 shows an enzyme characterized by two subunits cY and B whose sequences peptides are r ~ r ~ feels ~ Ps ~ res ~ li ~ ement, in FIGS. la and lb. (SEQ ID No: 1: and SEQ D) No: 2: in the sequence listing below).
Another object of the invention is a DNA sequence coding for a enzyme having a nitrile hydratase activity capable of hydrolyzing nitriles into 15 amides and chosen from the following list of sequences:
- The DNA sequence, such as the ~ l ~ senlée in FIG. 2, (SEQ ID No: 3: in the listing sequences below) and coding for an enzyme (SEQ ID No: 4: in the list of sequences below) having a nitrile activity hyrlr ~ t ~ this, - an analog of this sequence results ~ nt of the degeneration ~ lescence of the genetic code, 20 - a DNA sequence hybridizing with one of these sequences or a fragment of these and coding for an enzyme having a nitrile hydratase activity.
- The enzymes res ~ llt ~ nt of the e ~ - ~ lesion of one of the DNA sequences above above are included in the scope of the invention.
The wild micro-organism Comamon-st-stosteroni NI 1, isolated by the

2 ~ De-m ~ nderesse, contains one of the above sequences in its genome.
DNA sequences encoding the enzyme were identified using of nucleotic probes from the partial peptide sequences of the subunits ~ s and B (fig. la and lb) of the purified enzyme.
The invention relates, ég ~ l ~ ment, the c ~ ccettes of ~ ession which carry, 30 with the signals ensuring its e ~ .ples ~ ion, one of the DNA sequences defined prec ~ demme-nt These c ~ Ccett ~ s of e ~ r ~ ssion can be, either naturelnle-nt ~ r ~ saddles, ~ 2 ~ ~ ~ 6 ~ ~ PCT/FR94/00993 either integrated, ~ es in the genome of the host or loc ~ é ~ s on a vector of ~ l ~ iession, such a plasmid tale ~ - ~ nt, pr ~ ference, a means of selection.
These c~settes of ~A~,ession co~ lenl, not~mm~nt regions d ~ initiation of transcription and translation, which contain a sequence S promoter and a ribosome binding site. These regions can be counterparts or heterologous micro-or ~ or ~ me naturally producing the enzyme.
The choice of these regions depends, not ~ mmPnt, on the host used. In particular, when it comes to micro-or ~ nicmes prokaryotic hosts, the heterologous promoter can be chosen from strong b ~ ténçns promoters, such as the promoter of the tryplo ~ h ~ operon the Ptrp of Escherichia coli, the promoter of the operon lactose Plac of E. coli, the right promoter of phage lambda PR ~ the left promoter lambda phage PL. strong promoters of Pseudomonas and Comamonas, strong promoters of Coryn~b~ct~ries. More particularly, in the case of the promoter right of the lambda phage, the thermosPn ~ ible PRCIts form is preferred.
In the case of eukaryotic microorganisms, such as yeasts, promoters may be those of yeast glycolytic genes, such as the genes encoding for phospho-glycerate kinase (PGK), glyceraldehyde-3-phos~hale dehydrogenase (GPD), or even genes coding for lactase (LAC4), enolase (ENO).
Concerning the ribosome binding sites, that derived from the CII gene of lambda, as well as those derived from Comamonas or Pseudomonas genes or those derivatives of Coryn ~ bact ~ ries genes are used pr ~ rélç, .tiPllçmPnt when the micro-org~ni~m~ host is prokaryotic.
A region, allows a lel.~ Qn of the translation and the functional transcription of the intended host, can be positioned 3' to the cod ~ nte sequence.
AvantageusemPnt ~ c ~ sette of e ~ l.ression may also include one or more markers allows ~ nt to select the recombinant host. The ~erer~s markers are domin~nt~ markers, that is to say conferring a re ~ i ~ t ~ nce to antibiotics such as ~ mpicillin, tetracycline or kanamycin or other products toxic to host microorg ~ nisms.

Wo 95/04828 21 4 ~ ~ 5 ~ PCT/FR94/00993~

The invention also relates to the micro-org ~ nor ~ mçs containing the DNA sequence according to the invention, as well as those capable of producing at least one enzyme according to the invention. These micro-org ~ ni ~ my co.,.l) o-slow or not at least unec ~ sette of e ~ the ~ ion, of the type described above.
S Among these micro-org ~ ni ~ mPs, we find the hosts usable for the reception of a vector of e ~ ssion according to the invention, one can quote, not ~ mment, the ent ~ rob ~ ct ~ '.ries such as E. coli, the bacteria aL, pa- Lenant to the genera Comamonas, Pseudomonas, Streptomyces, Rl7cilll ~ or coryneform bacteria, such as those al ~ p ~ Lenant to the genera Corynebacterium, Brevibacterium or Rhodococcus.
A recombinant micro-or~ni~me, containing said DNA sequence on a pl ~ cmi ~ iique structure, was filed on July 9, 1993 under number I - 1330 at the CollP~ffon N~tion~le de Cultures de Micro-org~nismçs (CNCM) (Institute Pasteur, 25 rue du Doctor Roux, PARIS). This micro-org~ni~me is strain E.
Coli TG1 which contains the plasmid pXL2222 (described below). This microorganism is ég ~ lP ~ n ~ nt i (lentifi ~ by the Dem ~ nderesse by the reference G 4327.
It goes without saying that the enzymes produced by microorganisms are, ~ lPmpnt ~ included in the invention.
The latter also concerns the process of tran ~ rol ma ~ ion of nitriles in amides using an enzyme according to the invention or a micro-org ~ ni ~ recombined me génPr ~ nt This process consists in bringing together the nih island to be transformed with an enzyme or a recombinant micro-org ~ ni ~ me, as defined pr ~ demm ~ nt. We - Operates, gener ~ lemP-~ t, at room temperature. According to a particular mode of r ~ li ~ hon of the invention, the enzyme or the recombinant micro-org ~ nism are immobilized on or in a solid support, using a conventional technique, such as that described, for example, in the patent ~ mPric ~ in US 4,732,851.
The proc ~ die of the invention is suitable for the tran ~ Çol-..ation of the nihiles in amides and, more particularly, for the h ~ ro..nation in amides of mono and ~initrilP~ of formulas:
NC - R - CN, NC - R--COO~Z+ and NC--R - CONH2- R - COO~Z+
wherein R is a linear or branched alkylene or alkenylene group, containing 1 to 18 carbon atoms, R ~ both, preferably, equal ~ - (CH2)4 - and Z is chosen 5.~28 2 1 ~ 6 6 5 C PCT/FR94/00993 from the list of following compounds ~ linen ~ lino-earth, ~ mines, ammonium, the latter compound being particularly preferred.
The method of the invention is particularly adapted to be integrated to the synthesis of ~ 1ir ~ t ~ s, such as ammonium adipate from a dinitrile, such 5 than adiponitrile, via a mono-mide mononitrile, such as cyano-5-valeramide, then either with a ~ mide, such as ~ (lip ~ mide, or with a mononitrile monocarboxylated, such as cyano-5-valerate and, finally, by a monoamide monocarboxylate, such as ammonium ~ lip ~ m ~ te.

POSSIBILITY OF ~PPl, ICATlON INDUSTRIAL:

The pr ~ invention can be implemented for the ~ le ~ a ~ ation of carboxylates (~-7ir~tes) useful as raw materials in syntheses industrial (polyamides).
The following examples illustrate the characteristics and advantages of the present invention without, however, limiting its scope.

DESCRIPTION OF FIGURES

The ~Ig. the re ~ r ~ feels the peptide sequence of the subunit ~ of a enzyme confo., lle to the invention (SEQ ID No: 1:).
fig. lb .epl ~ feels the peptide sequence of the subunit, ~ of the aforesaid enzyme (SEQ ID No: 2: ).
fig. 2 r ~. ~ feels a DNA sequence according to the invention (SEQ ID No :3: ).
fig. 3 represents the SDS polyacrylamide gel electrophoresis of the - fractions obtained at the different stages of purification of 1"PYPmP1~P 2.
fig. 4. ~ r ~ feels a curve of the relative activity of the nitrile hydratase according to the invention as a function of the pH.
fig. 5 represents a curve of the specific activity of the nitrile hydratase according to the invention depending on the te, llpél ~ L ~ Ire in C.

WO 95/04828 21 4 6 ~ ~ 6 PCT / FR ~ 94/00993 fig. 6 rcpr ~ feels a variation of the specific activity (millimoles of cyano-5-valerate x h- 'xg ~' protein) of the nitrile hydratase according to the invention vis-à-against cyano-5-valerate, depending on its cyano-5-valerate concentration (millimoles x 1~
S Fig. 7 the ~ resel ~ you the restriction map of the pl ~ cmide pXL2120 contains the genes encoding the two cr and B subunits of nitrile hydratase according to the invention.
fig. 8 re ~ r ~ se-lte the sequence of an NcoI - NcoI - AccI fragment of 1713 bp containing the genes encoding the two nitrile a and ~ subunits Comamonas testosteroni NI 1 hydratase.
The ~lg. 9 the ~ serves the restriction map of the pl ~ cmide pXL2160 contains the genes encoding the two subunits ~ and ~ of nitrile hydratase (nthA and nthB! according to the invention, the two genes being transcribed from the promoter Place.
fig. 10 ~r~sel~te the restriction map of pl~crnide pXL2205 col-ten~nt the nth~ and nthB genes, both genes being t*anscrits ~ from Ptrp promoter.
fig. 11 lc~l~sel~le SDS-PAGE gel electrophoresis showing the e,. ~ the ~ ion of the DNA sequence according to the invention in E. coli strains TG1/pXL2160 and E. coli TG1/pXL2205.
fig. 12 repf ~ feels the pl ~ mide restriction map pXL2222 cQnt ~ nt all of the sequences coding for the two a and ~ B subunits and included in the deposited E. coli TG1 strain (G 4327).
The significance of the abbreviations used in the following description Lion is given below.
- sSc: court buffer 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: chr~---liquid dlography denominated in ~npl~i~e language "fast protein liquid chromatography", ~O 95/04828 2 14 6 6 5 6 PCT/FRg4/00993 - SDS-PAGE: electrophoresis gel based on sodium dodecyl sulfate and polyacrylamide, - IPTG: isopropyl ~BD thio galactopyranoside, - HPLC: high pelrollllallce liquid chromatography, S - PS: dry weight, - X-gal.: S-bromo-4-chloro-3-indolyl-~D galactopyranoside.

~F~n~T~F~uRE HOW TO ACHIEVE THE ~nENTION:

EXAMPLES

EXAMPLE 1: CHARACTERIZATION OF A~11V11~ FROM COMARNONAS SOUCEE
testosterone NI 1.

The Comamonas t~t~steroni NI 1 strain is a strain isolated from a ~ h ~ ntillon of soil by microbiological screening. His ~lrollllances are rated on adiponillile, cyanovaleramide, cyanovalerate, ~tlir~mide and ~-lip~m~te 20 1.1. Preparation Dec c~ c:
The Comamonas t ~, stosteroni NI 1 strain is cultured, in a shaken flask, at 28 C, - for 19 h, in medium A, the collposis of which is as follows:
- Glycerol ........................ 10.0x103 mgxl-- Sodium cyano-S-valerate ......1.5 x 103 mgxl~
K2HPO4 - - - - - - - - - 2 x 103 mgxl-- MgSO4, 7H2O ...................... o,sx1o3 mgxl~' - MnSO4, H20 ....................... 20.0 mgxl~
- FeSO4, 7H2O ............................ 20.0 mgxl~
- NaCl ................................ 10.0 mgxl-l - Yeast extract ................. 0.Sx103 mgxl-- Beef extract .............. o,sx1o3 mgxl-WO 95/04828 ~; PCT/FR94/0099:~

- ~Cl 3N QSP pH 5 This preculture is used to inoculate a 2 liter flask filled to tenths of a medium A. After 22 h, 3.5 gxl-' of cells expressed in wet weight are harvested. This corresponds to an OD660nm of 1.4 and a dry weight of 0.9 gxl~
S
1.2. Det~,ation of the activity~ e~y~ ue:
The reaction medium consists of 50 mM phosphate buffer, pH 7 and the reaction temperature is 25°C.
The disappearing substrates are assayed by HPLC. Us specific activities are given in moles of substrate disappeared x hl x kg' of dry cells.
Measurements of the hydrolysis activities of adiponitrile and its products of hydration by Comamonas NI 1 are listed in Table 1.

TABLE 1: SPECTRUM OF A~;11V11~; DE LA SOUC~3 Comamonas NI 1 CONMRLONS OP~RATOD~ES: .
NA~ ACTIVITY
:. T ISubstrate~{oellulc¦ vs~ , SP~C~QUE
~mM) (g/l) ~ml~ ~U
S0 3.4 22 8.0 Cyanoval~ramide 58 13.0 1.0 1.1 Cyanovalcratc 37 13.0 1.0 2.2 A~ip~ e 20 5.6 S.5 0.5 Adipamatc 20 5.6 S,5 ~,o The Comamon ~ s NI 1 strain has a very active enzyme on adi ~ onillile. She also has an enzyme capable of hydrolyzing cyanoval ~ spleen. Being since the identified hydrolysis by-products of cyanovalerate are ip~m~t~ and the ~ip~te, it follows that Comamonas NI 1 possesses, in its enzymatic heritage, a nitrile-hydratase and an amidase.

~4~&
~0 95/04828 PCT~R94/00993 EXAMPLE 2 ~ R ~ CATION OF NlTRILE HYDR ~ TASE OF Comamonas NI.

2.1 Cell preparation:
The Comamonas Testosteroni NI 1 strain is cultured, in a shaken flask, at 28C, for 23 h, in medium B:
- Glucose ............................ 5 x 103 mg/l - Na2HPO4 ............................ 3.5 x 103 mg/l - KH2PO4 --------------------- 2.8 x 103 mg/l - MgSO4, 7H2O ........................ 0.5 x 103 mg/l - MnSO4, H2O ........................ 20 mg/l - FeSO4.7H2O ........................ 20 mg/l - NaCl ............................... 10 mg/l - Biotin ............................ 0.01 mg/l - Nic-tinic acid .................. 1 mg/l - Thi~mine, HCl ...................... 0.5 mg/l - Adi~oni~ile .................... 1.05 x103 g/l - Sodium adipate .............. 5.7 x 103 g/l pH 7 This preculture is used to en ~ elnencP ~ r a ferm ~ nte-lr of 201 containing 15 1 of same medium B. The air flow, the pressure and the agitation are, initiated ~ l ~ ment, de250 l / h, 1.1 atm and 250 rpm. During fermentation, PO2, pH and ~,.,~ ldl-lre are regulated, ~ ement, at 50% of the initial value, 6.8 and 28 C. After 23 h, 189 g of wet cells are collected. This col.esl)olld at a dry cPllules content of 2.3 g/l (total 35 g) and at a DOooo ~ of 3.8.

2.2 Purification All the steps of the purification are r ~ li ~ s with the buffer T (HEPES / KOH
0.1 M, pH 7.2, 40 mM butyrate) unless otherwise indicated contlail ~. At each of stages, the nitrile hydratase activity of the fractions is d ~ l ~ llninée at pH 7 and at 25° C. in T/10 buffer, in the presence of 10 mM cyanovalerate. The protein concentration of the pools is determined by the blue method of wo 95/04828 214 ~ 6 ~ ~ 14 PCT~Rg4/00993~

Coomassie (PIERCE Protein assay kit). The protein panel is analyzed by polyacrylamide-SDS gel (Phastsystem PHARMACIA) (cf. fig. 3).
In this fig. 3, tracks 1, 2, 8 and 9 contain each of the standards of molecular weights:
S-phosphorylase B = 97400 - Bovine Serum Albumin = 66200 - Ovalbumin = 45000 - Carbonic anhydrase = 31100 - Soy Trypsin Inhibitor = 21500 - Lysozyme = 14400.
Tracks 3 to 7 glue ~ o, ldent respectively to purification steps 4 to 0.
The reference molecular weights are indicated in the right margin on Ia fig- 3-The data of ch ~ cnn ~ of the purification steps are regio--~) ed in the Table 2. The procedures for each of the steps are commented on below.
- Step 0: Raw extract:
152 g of wet cells are taken up in 250 ml of T buffer and sonicated for 30 min (~VIBRACELLr sonicator from BIOBLOCK; 13 mm probe, power 7, 40% of the active cycle). The D660nm thus goes from 175 to 50. After centrifugation at 48,000 gmax for 60 min, the supernatant is recovered.
- Step 1: Pre ipjt ~ tion with sulphate of ~ mmon; um:
The supernatant is brought, by adding p, ~ -essi ~ e of sulphate ammonium, 15% of the ~ t ~ tiQn. After 1 h, the suspension is centrifuged for 30 min at 30,000 gmax. The supernatant is brought to 45% of the saturation. After 1 h, the suspension is centrifuged in the same conditions, the precipitate is r ~ cuy ~ then dialyzed against the buffer for a night.
- Step 2: Ion exchange column (Q Sepharose Fast ~low):
The dialysed fraction is ch ~ e at a flow rate of 150 mVh on a column (26 x 380 mm) of "Q Sepha-ose Fast Flow" ~equilibrated with T buffer at a flow rate of 250 mlJh. The column is percol ~ e ~ a flow rate of 250 ml / h, ~1~6~6 ~0 95/04828 PCT/FR94/00993 successively by the following solutions:
. QSP of buffer T until return to baseline, 500 ml of a 0 to 0.3 M gradient in KCl in buffer T, 200 ml of buffer T supplemented with 0.3 M KCl, . 200 ml of buffer T supplemented with 1 M KCl.
The nitrile hydratase activity is elected in a volume of 160 ml at a KCl concentration between 0.22 and 0.28 ~.
- Step 3: Phenyl Sepharose CL4B hydrophobic interaction column:
The active fraction, ~m~n~e at 15% ammonium sulphate saturation, is loaded at a flow rate of 150 ml/min onto the column (26 x 380 mm), equilibrated with buffer T containing 15% ammonium sulphate saturation. The column is percolated with:
200 ml of equilibration buffer at a flow rate of 150 ml/h, . 200 ml buffer T containing 10% ammonium sulphate saturation at a flow rate of 200 ml/h, 200 ml of buffer T with a gradient of ammonium sulphate from 10 to O% of the saturation in sulphate of ~ mmonium at a flow rate of 200 ml/h, washing of the column with buffer T until the return to the base line.
The active fraction is eluted at an ammonium sulphate content of between 8.1 and 6%. This active fraction is concentrated by dialysis to a volume of 12.5ml.
- Step 4: Hydrophobic interaction column on Octyl Sel)har~se:
The protein solution is cha ~ ged on the column (16 x 280 mm), balanced with buffer T containing ammonium sulphate at 15% saturation at a rate of 26 ml/h. The column is then percolated at a rate of 47 ml/h with the balancing buffer T. During this operation, all of the activity is eluted as a peak. Only the core of the active fraction is harvested. This fraction analyzed in SDS-PAGE gel is composed of 90%
nitrile hydratase in the form of two very close bands.

WO 95/04828 2 14 ~ 6 5 6 PCT / FR94 / 00993 TABLE 2: PUR~CATION OF N1TR~E~DR~TASE FROM Comamonas NI 1.

AcrtvlT~ ~F~t~
:. ::,.': '~',- '''-,:.'.'''' r~PE DE iURlP3C~TJoN Vo~P~6~ PF
m~~rng3. - 'burp. Spe~ - Pro~ ~ct.
m~(U/kd: t%) (%3 o - gross output 3926 750 65095 100 100 1 - SO,5NH,)~ 35-45% 2024 040 710176 60 109 1.7 2 - MonoQ Seph-roseFF 160384 7151 860 5.6 110 20

3 - Phenyl SephArose CL4B 12 38540 14,200 0.6 83,150

4-OCTYLSeph~roseCL4B 29 14 25217000 0.2 39 180 ABR~ TIONS: PF = purification coefficient; U = 1 mole/h.

EXAMPLE 3: CHARACTERISTICS OF N1TRILE DRATASE FROM Comamonas NI 1.

15 3.1 Determination of the structure:
Gel filtration of nitrile hydratase gives a single peak whose time elution collespond to 92 ~ / - 10 l ~ Da. The analysis of this protein by gel of polyacrylamide-SDS gives two bands of equal int ~ n ~ it ~ - 26 to 25 kDa.
This structure is that conventionally encountered in nitriles hy ~ t ~ es.
3.2 Determining s~ue,.cæc pepti~ uecN-te..~ .qlPc:
From the purified protein, the N-terminal sequence of the two subunits has been determined:
- cY subunit (SEQ ID No: ~: in the sequence listing below) Thr-Asp-Asn-Ala-Val-Met-Glu-Gln-Arg-Val-As~Ala-Leu-Phe-Val-Leu-(Thr or Arg)-Lys-Glu-Leu-Gly-Leu-Val-Thr-Asp-Gln-Thr-Val-Pro-Asp-Tyr-Glu-(Asp or Pro)-Ala-Leu-Met-His-Asp - subunit ,~ (SEQ~) No: 6: in the sequence listing below) Met-Asp-Gly-Met-His-Asp-Leu-Gly-Gly-Lys-Gln-Gly-Phe-Gly-Pro-Val -~O 95/04828 21 4 6 6 5 6 PCT/El~94/0o993 Ile-Lys-Thr-His-Asn-Ala-Lys-Ala-Phe-His-Glu-Glu-(Trp)-(Glu)-Val-Lys-Met-Asn-Ala-Ile 3.3 Influence of pH:
S The eXFé ~ merlt ~ conditions are as follows: r C = 25; concentration in cyanovalerate=10 mM; nitrile hydratase concentration=1.5 ~ g/l;
reaction volume = 2 ml; kinetics over 10 min; reference activity: pH
7.2, 17,000 moles/h.kg protein; acetate buffer (3.5 to 5.5), buffer phosphate (6.5 to 8.5), Tris buffer (7 and 9), carbonate buffer (9.2 and 10.2).
The conditions and results of measurements of the activity of nitrile hydratase on cyanovalerate at dirr~re.~ts pH are listed in fig. 4.
The curve of variation of activity as a function of pH is, classically, in bell with an optimum close to 7.5.

15 3.4 Tnfl~Pn~e de la t~ .dlun~:
The experimental conditions are as follows: concentration in cyanovalerate = 10 mM; nitrile hydratase concentration=1.5 ~ g/l;
re~tiorlnel volume--2 ml; kinetics over 10 min; pH reference activity 7.2, 17,000 moles/h.kg protein; 10 mM HnEPESAKOH buffer, pH 7.2.
The results of measurements of nitrile hydratase activity at different the ~ d ~ ures are re ~ e.Loliés in fig. 5.
Between 10 and 30 C the /`G of the reaction is 80 kJ/mole.

3.5 Determination of kinetic conct~nt~C:
The sul ~ sL ~ dt used is cyanoval ~ rate. Its rate of hydrolysis in ~flip~m~t~ is determined at different concentrations of cyanovalerate (0.5, 1, 2, 5, 10 and 20 mM) at 25 C and pH 7.2.
Experimental conditions: rc = 25, nitrile hydratase concentration =
1 ~ g / l (except 0.5 ~ g / l for a concentration of cyanovalérdte = 1 and 2 mM and 3.3 ~ gtl for a cyanovalerate concentration = 20 mM); volume reaction 10 ml except 5 and 3 ml for a cyanovalerate concentration =

WO g5 / 04828 ~ G6S ~ 18 PCT / FRg4 / 00993 0.5 and 20 mM, respectively, kinetics over 20 min; pH reference activity 7.2 17 moles/hg of protein; 10 mM HEPES/KOH buffer, pH 7.2.
The KM and VM calculated for the transformation of HANES-WOOLF OR of LINEWEAVER BURK are from:
KM = 2.5mM
MV = 35,000 moles or 5,000 kg of ~dip~m~t~/h.kg of nitrile hydratase According to VM and the weight of the enzyme (92 kDa), one can calculate the turnover ~,) of the enzyme as well as the rate of formation of the enzyme-complex substrate (k~~/KM) kC,t = 900 S-k~,/KM = 36,000 l.mole~l s-3.6 Spectrum of activity:
The activities of a purified addition of nitrile hydratase were measured on dirré ~ G ~ ts nitriles. The conditions and results are given in Table 3.
The spectrum of activity of this enzyme is quite particular. Unlike all known nitrile hydratases, this enzyme hydrolyzes mononitriles-monoats and few linitrils 21466~6 ~O 95/04828 - PCT/FR94/00993 TABLE 3 ACm~}~nvE D~E PR~AnON H~; D~ L~ Nr~E
~nDRATASE OF Gomamonas NI 1 ON D~ NITR~ES

~ONS oi~ o~ : ~A~
S:::: . N~TURE suss~ -: . ---:. .: .
:-. :... .. . ~ - :::~ ~ ~l. :: . ~ ~ S~ Re~tiv rmM~ .. rn~ J ~ ~.:. a.~~.:.. .
Cyano-5-valerate 20 10 18,000 100 t.. ~ ;the 9,S 15 290 1.7 A~ lur~ ;lc 9.4 15 2S0 1.4 168 0.g 0AA~20 30 180 0.8 Ir 10 60 87 0.5 ~1 ' ~ ' 10 10 84 0,S

COMMON CONDITIONS: Tarpon HEPES 10 mM, pH 7.2; reaction volume of 3ml; T(C)=25; protein concentration of 16 ~ g / ml.
ABR~VIATIONS: TR = reaction time: Us = 1 mole of amide appeared/h.kg of nitrile hydratase.

EXAMPLE 4: CLONING THE N1TR~E ~DR~TASE OF Comamonas test~steroni NI 1.

From the NH2 terminal pr ~ sequences in Example 3, two probes nucleotides were synth ~ ti ~ es.
For the choice of the first probe (subunit a), the codon usage was not biased. The mixture of oligonucleotides (seq. 2273) is a 23 sea 32 times degenerate contained ~ lt 2 inosines.

wo 95/04828 21~ 6 ~ 5 & PCT~4/00993~

DNAVMEQR (SEQlDNo:8:) S' GAYAAYGCNGTNATGGARCARMG 3' (SEQ ID No: 7: )

5 In the sequence listing below, the nucleotide sequence of this first probe a is designated SEQ ID No: 7: and the corresponding amino acid sequence by SEQ n) No: 8: .
For the second probe (subunit O, the high GC percentage of Comamonas strains dictated a choice for the third position of the codon in the case of ~ l ~ ninPs and in the case of Iysine. The probe (seq. 2264) is a 23 sea 144 times degenerate:
HNAKAFHE (SEQ II) No: 10: ) S' CAYAAYGCBAAGGCBITYCAYGA 3' (SEQID No: 9: ) In the sequence listing below, the nucleotide sequence of this second probe ,B is ~ign~ by SEQ ID No: 9: and the corresponding ~minoacid sequence by SEQ n~ No: 10: .
The strategy followed con ~ ist ~, first of all, to verify the specificity of the two probes and to determine the nature of the genomic DNA fragments to be cloned. Briefly, the genomic DNA of Comamonas testosteroni NI 1 was digested by several restriction enzymes co, lesl) ol dant to sites usable for cloning. After electrophoresis on agarose gel and transfer on nylon membrane, the various digests were hybridized to the probes. The results show that the two probes have sufficient specificity in the hybridization conditions used (Sx SSC, 5x Denhardt, 0.1% SDS, 50 mM NaPO4 pH 6.5, 250 μg/ml ssDNA; le~ dLule hybridization 50 C. Washing conditions: 1 h, 6x SSC, lempéldl ~ lre ambient etS min in 2x SSC, 0.1% SDS at 50 C). Under these conditions, the two probes allowed to obtain s ~ ific signals. Not ~ mm ~ nt in the case of digestions by ~ I, ~ II and ~ I where the hybridization signals obtained with the two probes are stackable.
The hybridization samples show, in particular, the existence of a fragment ~ 21~ 6 6 5 6 PCT/FR94/00993 unique ~III of about 4 kb. In order to clone this fragment, the fragments from 2.5 to 4.5 kb of a ~ glII digestion of genomic DNA were purified by electrophoresis ~ ali ~ e on agarose and electroelution, then ligated to the plasmid pUCl9, him even digested by R~m~T. After transfol,-,alion in the DH5cx strain, 800 clones 5 blanks on LB amp X-gal were subcultured individually, transferred to membrane of nylon, then analyzed by hybridization with the probe seq 2264 in the same snngence conditions than during the hybridization of the "Southern blot". A clone has thus been identified as hybridizing specifically with the probe. This clone turned out coll ~ ne pl ~ cmide (pXL2120), having inserted a fragment of approximately 4.2 kb, 10 also hybridizing with the probe seq. 2273. This plasmid was further analyzed (c~logl~hie restriction, partial sequencing using probe seq. 2264 as primer and "Southern blot"). It was thus able to be shown that the 5' part of the gene coding for the ,B subunit, which hybridizes with the probe seq. 2264, is located on a ~ mpnt of ~ LI-KpnI of approximately 280 bp, oriented in the direction SphI towards ~ I.
15 Fig. 7 p,is a restriction map of this plasmid.

EXAMPLE 5: S~QUENCE OF A l;RkGM~T OF 17L3 PB OO~I~T LE:SG~ES

OF Comamonas testosteroni NI 1.
The loc ~ li ~ tion of the NcoI-NcoI-AccI fragment of 1713 bp sequenced, containing the genes encoding the two Comamonas nitrile hydratase subunits estosteroni NI 1, is indicated under the cloned insert in fig. 8. The strategy of seql ~ enç ~ ge of this fr ~ gment, carried out according to the classic methods known by 25 skilled in the art, is indicated in the same figure. The various sequences have all were obtained by the method of tel-",. ~ on of chain (kit sequenase in presence of 7-dea-a dGTP; (35S)dATP), or on single-stranded matrices of M13 recombinant carrying subfragments, either di.eclel..ent on the plasmid pXL2120.
Several specific primers have also been synthesized for this purpose.
30 The sequence is shown on f~lg. 2. The average GC% of the obtained sequence is of 56.6%, which is close to the GC% of 61.5% described in other strains of -21~ 6 6 5 6 22 PCT/FR94/00993~

Gomamonas (TAMAOKA et al., Int. J; Syst. R~teriol., 1987, 37, 52-59). A
analysis of the sequence obtained made it possible to car ~ ct ~ .ricer a first open phase of 645 bp pot ~ ntiPllempnt translated into a protein of 215 acids ~ minéC La terminal NH2 sequence of the subunit ~, used for synth ~ tic ~ r the probe seq.
2273, is contained in this protein and makes it possible to locate the initiation codon of the gene coding for the cY subunit, called nthA in the remainder of the description. This gene colllpolle 621 bp, codes for a protein of 207 residues co, ~ ~ ndant to a weight molecular weight of 22,967 Da. A second open phase of 621 bp is potentiçll ~ ment translated into a polypeptide of 207 residues. This polypeptide has in the terminal NH2 position, the terminal NH2 sequence used to synthesize the probe seq. 2264, which allows idçntifi ~ r of the ~ lxi ~ me phase open to the gene of structure of the ~ subunit of nitrile hydratase, called nthB in the sequel to the presentation. This protein has a theoretical molecular weight of 22,653 Da;

EXAMPLE 6: EXPRESSION OF THE NITRILE HYDRATASE OF Coma~nonas tetesteroni NI 1 IN E. coli.

In order to confirm the identity between the nthA and nthB genes and the genes encoding the two subunits of the nitrile hydratase of Comamonas tes~osleroni NI 1, the pl ~ cmides of e, ~; ssion pXL2160 and pXL2205, presented on the ~ lg. 9 and 10, have been collsLI.li~ in several steps.
First, the 779 bp NcoI/~_I fragment (conlh ~ ing the 282 bp loç ~ licées upstream of the nthA gene and the first 496 bp of nthA) and the ~g_I / AhaII fragment of 789 bp (containing part 3 'n ~ and all the nthB gene), fragments isolated from of pXL2120, were cloned between the NcoI and ~! ~ I sites of pMTL22 (CHAMBERS and al., 1988, Gene, 68, 139-149).
The resulting plasmid ~ nt7 pXL2160, therefore carries a gene conferring the r ~ cict ~ nce to ~ mpicillin and the nthA and nthB genes under the control of their own sites of binding of the ribosomes, the two genes being transcribed from the promoter of the lactose operon of E. coli. pXL2160 carries, moreover, in the multisite of cloning, downstream of the Gomamonas testosteroni fragment, a unique XhoI site.

214~656 ~O 95/04828, PCT/E~R94/00993 At the same time, a ~5LI restriction site was created on the initiation codon of ~h~
and the 180 bp ~ HindIII fragment containing the 5' part of the nthA gene was ~ mplified by the PCR technique. This fragment was ligated to the fr ~ gment ~ TTTlxhoI
of pXL2160 between the ~deI/~hoI sites of pMTL22, to end up with the plasmid pXL2203.
The NdeI / ~ oI fragment of pXL2203, carrying the nthA and nthB genes, was h ~ llod ~ it behind the fr ~ ment ~ RI / NdeI tale ~ t the promoter of the trvptophan operon of E. coli and the ribosome binding site of the bacteriophage CII gene ~ (Ptrp-RBSCII), between the EcoRI and SalI sites of the plasmid pXL534 (LArrA et al., 1990, DNA Cell. Biol., 9, 129-137).
The final plasmid pXL2205 therefore carries a gene conferring resistance to ampic-illin, the nthA gene under control of the RBSCII ribosome binding site and the nthB gene under control of its own ribosome binding site, both genes being tr ~ n ~ crit ~ from the Ptrp promoter.
The e ~ p.cssion of nitrile hydratase was visu ~ e in the strain E. coli TG 1 containing pl ~ smids pXL2160 and pXL2205.
For this purpose, the strain TG1/pXL2160, as well as the control strain TG1/pUC19, were cultured for 16 h in LB medium at 37 C (MILLER, JH 1972, Experiments in Molecular Genetics~ Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) containing lOO ~ g / ml of ampicillin, then diluted to lOOth in the same medium and at the same te-~-péld~re. When the cultures have reached an OD6l0 between 0.5 and 1, IPTG, to the final concentration of 1 mM, was added. After 2 hours of culture, the bacteria were collected ~ .s.
In addition, the TG1/pXL2205 strain, as well as the control strain TG1 containing the pUC19 vector, were cultured for 16 h in M9 glucose medium at 30 C (MILLER, JH1972, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), containing 100 g/ml ampicillin and 100 ug/ml tryptophan, these cultures were diluted 100 times in the same medium in the absence of tryptophan and cultured for 6 h at the same te", ~ alul ~.
The ~; ~, ession of the nitrile hydratase of (: ~ omamonas t ~ .stosteroni NI 1 was vi ~ ed, after sonic ~ tion of this ~ them, in polyacrylamide-SDS gel, in the crude fraction WO 95/04828 2 ~ 4 ~ 6 ~ ~ PCT / FRg4 / 00993 ~

or after centrifugation in the pellet and in the supernatant. The results are p ~ se, ltés on the f ~ lg. 11.
Each track COll ~ s ~ ond to a quantity of proteins equivalent to 60 ~ 1 of culture at an OD of 3 to 610nm.
Lane G contains the following molecular weight markers: 97.5, 66.2, 45, 31, 21.5 and 14.4 kDa.
The contents of the other tracks are shown in the following table.

.
:~..TGlJpUCl9. TG1/pXLæOSrG1/pXL2160 TG11pUC19 . ~ . + IPrG + II'TG
" , :
0 S~ t ADHK
BEIL base Gross fraction CF ~ M

It appears from fig. 7 than the cell extracts, containing the plasmid pXL2205 (cf.
15 lanes D, E, F), strongly express both nitrile hydratase subunits (indicated ~ and ~ B next to the gel), essçnt ~ nt in soluble form. These two subunits are also expressed in cell extracts containing the plasmid pXL2160 (see tracks H, I, J), after in ~ uctiQn with IPTG. The e~ression is then more weak and, for the most part, in the insoluble fraction.
20 `~Furthermore, the pl ~ mine pXL2222, contellal t all the sequences coding for the two subunits of nitrile hydratase and shown in fig. 12, has been transformed in the E. coli TG 1 strain. The resulting strain, called G4327, was deposited at the Golle~tion Nationale de Culture de Micro-or~ni~nles à PARIS (n~lsTrrurpAsTEuR~
25, rue du Docteur Roux), under n 11 330. The plasmid PXL2222 groups together two fra$~ment~ extracted from pXL2160, the 934 bp NruI/~LnI fragment containing the gene nthA and the 5' part of nthB cloned in front of the 370 bp KpnI/$tuI fragment contained the 3' part of nthB. These two fr~gm~nt~ are inserted between the NaeI and E~coRV sites of pMTL22 (CHAMBERS et al., 1988, Gene, 68, 139-149). The EcoRV site used is the one located downstream of lacZ' and not the one in the multisite.

~ 214~65~
0951~ ~ .A - PCT~4/00993 EXAMPLE 7: DETERMINATION OF THE A~;11V11~ OF THE CONCERNS
RECOMBINAN RES.

Dirré.~"~your recombinant strains, in which the genes coding for nitrile 5 hydratase were ~ clon ~ s, are tested for the hydrolysis of CVA and / or DNA. The cultures of the strains are lC ~ lk, -ied in Table 4.

TABLE 4: CULTURE OF CULTURES
.

0 ~ u~: M~ieu DO~0~ ~i~en~ul~:
~:dry~JI3 E.co~ ~XL2205) M9 1.4 0.4 E. co~ ~XL2160) LB~ 1.7 0.S
E. co~ LB 3.1 0.9 - Middle M9: .
. Glucose ....................... 4 xl~ mg/l . Na2HPO4.2H2O ................. 7 x 103 mg/l KH2PO4 - - .................3 x 103 mg/l . NaCl ........................ 0.5 xl~ mg/l . NH4Cl ........................ 1 x1~ mg/l .~mino~cides .................. 4 x 103 mg/l .hi~mine,HCl .................. 10 mg/l . MgSO4 ........................ 1mM
. CaCl2, 2H2O .................. 1 mM
.~mrjc.illine .................. 0.1 xl~ mg/l - Middle LB~:
. middle LB
.~TG ........................... 1mM
. Ampicillin .................. 0.1 x103 mg/l - Middle LB:
. Tryptone ................... 10 x 10 3 mg/l . Yeast extract .......... 5 x 10 3 mg/l . NaCl ....................... 5 x 10 3 mg/l COMMON CONDITIONS: inoculation at 1/100th from a preculture 5 p.m.; T° C = 37, except M9 at 30° C.
The activities of the recombinant strains and the host strain are listed in table 5.

TABLE 5: DETERMINATION OF THE NITRILE HYDRATASE ACTIVITIES OF E.
coli (pXL2205) AND E. coli (pXL2160).
CONDITIONS OPERATIONS ACTIVITY
MICROORGANISMS (moles/h.kg cel. PS) Substrate Cellulose(g/l) E.coli CVA 16 0 E. coli (pXL2160) DNA 1.8 0 CV 0.9 10 E. coli (pXL2205) CVA 0.6 105 ABBREVIATIONS: CVA = cyanovalerant; AdN = adiponitrile.
COMMON CONDITIONS: T (°C) = 28; 50 mM phosphate buffer, H 7;
[S]=50mM; kinetics over 2 h.

~ g5/~ 214 6 6 5 6 PCT~R94/00993 SEQUENCE LIST

(1) 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: Enzymes with nitrile-hydratase activity, tools genetics and microorganisms: where hosts allow their production and hydrolysis process using said enzymes (iii) NUMBER OF SEQUENCES: 10 (iv) T~T~IR!~F~ FORM BY COMPUTER:
(A) MEDIA TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release ~1..0, Version #1.2~ (EPO) (vi) PRIOR APPLICATION DATA:
(A) DEPOSIT NUMBER: FR 93 09990 (B) FILING DATE: 10-AUG-1993 (2) INFORMATION FOR SEQ ID NO: 1:

(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 207 amino acids (B) TYPE: amino acid (D) CONFIGURATION: l in~i re (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Met Thr Asp Asn Ala Val Met Glu Gln Arg Val Asp Ala Leu Phe Val Leu Thr Lys Glu Leu Gly Leu Val Thr Asp Gln Thr Val Pro Asp Tyr WO 95/04S28 2 i ~ 6 ~ 5 ~ PCT ~ R94 / 00993 ~
^28 Glu Asp Ala Leu Met His Asp Trp Leu Pro Gln Asn Gly Ala Lys Leu Val Ala Lys Ala Trp Thr Asp Pro Val Phe Lys Ala Gln Leu Leu Ser ~5 60 Glu Gly Val Ala Ala Ser Glu Ser Leu Gly Phe Ser Phe Pro Lys His His Lys His Phe Val Val Leu Glu Asn Thr Pro Glu Leu His Asn Val Island Cys Cys Ser Leu Cys Ser Cys Thr Ala Phe Thr Island Island Gly Met Ala Pro Asp Trp Tyr Lys Glu Leu Glu Tyr Arg Ala Arg Ile Val Arg Gln Ala Arg Thr Val Leu Lys Glu Ile Gly Leu Asp Leu Pro Glu Ser Island Asp Island Arg Val Trp Asp Thr Thr Ala Asp Thr Arg Tyr Met Val Leu Pro Leu Arg Pro Gln Gly Thr Glu Asp Trp Ser Glu Ala Gln Leu Ala Thr Leu Ile Thr Gln Asp Cys Leu Ile Gly Val Ser Arg Leu Glu Ala Pro Phe Ala Ala Leu Pro Ala Pro Ala Val Ala Leu Gly Ala (2) INFORMATION FOR SEQ ID NO: 2:
(i) CHARACTERISTICS OF THE S~:~u~c~:
(A) LENGTH: 207 amino acids (B) TYPE: amino acid (D) CONFIGURATION: 1;n~ re (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Asp Gly Met His Asp Leu Gly Gly Lys Gln Gly Phe Gly Pro Val Ile Lys Thr His Asn Ala Lys Ala Phe His Glu Glu Trp Glu Val Lys Met Asn Ala Ile Ser Gly Ala Leu Val Ser Lys Gly Ile Tyr Asn Met Asp Glu Tyr Arg His Gly Ile Glu Arg Met Glu Pro Arg His Tyr Leu Thr Ala Ser Tyr Phe Glu Arg Val Phe Thr Thr Ala Val Thr Leu Cys Ile Glu Lys Gly Val Phe Thr Ala Ala Glu Leu Glu Ala Lys Leu Gly ~ 95/04828 214 6 6 5 6 PCTA~R94/00993 Thr Ser Val Pro Leu Ser Leu Pro Ser Ser Pro Gly Arg Gln Pro Ala Lys Gly Pro Glu Gly Gly Phe Lys Leu Gly Gln Arg Val His Val Lys Asn Glu Phe Val Pro Gly His Thr Arg Phe Pro Ala Tyr Ile Arg Gly - Lys Ala Gly Val Val Val Gly Ile Ser Pro Ala Tyr Pro Tyr Pro Asp Ala Ala Ala His Gly Glu Tyr Gly Phe Ser Glu Pro Thr Tyr Asp Val Cys Phe Lys Ser Lys Asp Leu Trp Pro Asp Gly Cys Glu Ala Ala Asp Val His Val Gly Val Phe Gln Ser Tyr Leu Leu Ser Ala Glu Glu (2) INFORMATION FOR SEQ ID NO: 3:

(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 1713 base pairs (8) TYPE: nucleic acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: l;n~;re (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETIC: NO
(iii) ANTI-SENSE: NO

(ix) ADDITIONAL FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: join(283..903, 909..1~29) (ix) ADDITIONAL FEATURE:
(A) NAME/KEY: intron (B) LOCATION: 904..908 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
CCATGGAGAA GAl~GGGGAG lGGGGC~-lGA GCGAAAAGCT GCTGAATCGT GCACCCAAGG 60 TGA~ GG~l CGCCAACATC CAACTCGCCC GCTCAGTTGA CACCGAGATG GCTAAGAGCG 120 ACTTCGACTG GCG~lCG~lG AAGGTCAAGG~CClGAGC CCC~-l~lGCC TGACTCCATC 180 C~l~llGCAG GCGCGTGTAG CACAGAGATA CCCCCTTATC ACACAGACTC AGCGCAAGCT 240 Met Thr Asp Asn 214~6~
WO 95/04828 PCT / FR94 / 0099:~

GCC GTA ATG GM CM CGC GTG GAC GCA CTC m GTG CTC ACC MM GAG 342 Ala Val Met Glu Gln Arg Val Asp Ala Leu Phe Val Leu Thr Lys Glu 5 lo 15 20 Leu Gly Leu Val Thr Asp Gln Thr Val Pro Asp Tyr Glu Asp Ala Leu Met His Asp Trp Leu Pro Gln Asn Gly Ala Lys Leu Val Ala Lys Ala Trp Thr Asp Pro Val Phe Lys Ala Gln Leu Leu Ser Glu Gly Val Ala Ala Ser Glu Ser Leu Gly Phe Ser Phe Pro Lys His His Lys His Phe Val Val Leu Glu Asn Thr Pro Glu Leu His Asn Val Ile Cys Cys Ser 85 90 95 loo Leu Cys Ser Cys Thr Ala Phe Thr Ile Ile Gly Met Ala Pro Asp Trp Tyr Lys Glu Leu Glu Tyr Arg Ala Arg Ile Val Arg Gln Ala Arg Thr Val Leu Lys Glu Ile Gly Leu Asp Leu Pro Glu Ser Ile Asp Ile Arg Val Trp Asp `Thr Thr Ala Asp Thr Arg Tyr Met Val Leu Pro Leu Arg Pro Gln Gly Thr Glu Asp Trp Ser Glu Ala Gln Leu Ala Thr Leu Ile 21~6656 0 95/04828 PCT~R94/00993 hr Gln Asp Cys Leu Ile Gly Val Ser Arg Leu Glu Ala Pro Phe Ala Ala Leu Pro Ala Pro Ala Val Ala Leu Gly Ala Met Asp Gly Met His Asp Leu Gly Gly Lys Gln Gly Phe Gly Pro Val Ile Lys Thr His Asn Ala Lys Ala Phe His Glu Glu Trp Glu Val Lys Met Asn Ala Ile Ser Gly Ala Leu Val Ser Lys Gly Ile Tyr Asn Met Asp Glu Tyr Arg His Gly Ile Glu Arg Met Glu Pro Arg His Tyr Leu Thr Ala Ser Tyr Phe Glu Arg Val Phe Thr mr Ala Val Thr Leu Cys Ile Glu Lys Gly Val Phe Thr Ala Ala Glu Leu Glu Ala Lys Leu Gly Thr Ser Val Pro Leu Ser Leu Pro Ser Ser Pro Gly Arg Gln Pro Ala Lys Gly Pro Glu Gly Gly Phe Lys Leu Gly Gln Arg Val His Val Lys Asn Glu Phe Val Pro Gly His Thr Arg Phe Pro Ala Tire Ile Arg Gly Lys Ala Gly WO 95/04828 21 ~ 6 6 5 ~ PCT ~ R94 / 00993 ~

Val Val Val Gly Ile Ser Pro Ala Tyr Pro Tyr Pro Asp Ala Ala Ala His Gly Glu Tyr Gly Phe Ser Glu Pro Thr Tyr Asp Val Cys Phe Lys Ser Lys Asp Leu Trp Pro Asp Gly Cys Glu Ala Ala Asp Val His Val Gly Val Phe Gln Ser Tyr Leu Leu Ser Ala Glu Glu AGCCATGCTT CTGCCTTGCT CGATTCCGAC GCC~l-lGACG w G~lGC~ l CAGCAGCGTA 1599 ACGGAAATTC TCCACTGGTA lGG~l~lGGA AAGCGCACAC CTTCGACGGT GGTTGAAGTG 1659 (2) INFORMATION FOR SEQ ID NO: 4:

(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 414 amino acids (B) TYPE: amino acid (D) CONFIGURATION: n~ire (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Met Thr Asp Asn Ala Val Met Glu Gln Arg Val Asp Ala Leu Phe Val Leu Thr Lys Glu Leu Gly Leu Val Thr Asp Gln mr Val Pro Asp Tyr Glu Asp Ala Leu Met His Asp Trp Leu Pro Gln Asn Gly Ala Lys Leu Val Ala Lys Ala Trp Thr Asp Pro Val Phe Lys Ala Gln Leu Leu Ser Glu Gly Val Ala Ala Ser Glu Ser Leu Gly Phe Ser Phe Pro Lys His His Lys His Phe Val Val Leu Glu Asn mr Pro Glu Leu His Asn Val Island Cys Cys Ser Leu Cys Ser Cys Thr Ala Phe Thr Island Island Gly Met 21~6656 g~ PCT~R94/00993 Ala Pro Asp Trp Tyr Lys Glu Leu Glu Tyr Arg Ala Arg Ile Val Arg Gln Ala Arg Thr Val Leu Lys Glu Ile Gly Leu Asp Leu Pro Glu Ser Island Asp Island Arg Val Trp Asp Thr Thr Ala Asp Thr Arg Tyr Met Val 145 150 155 160eu Pro Leu Arg Pro Gln Gly Thr Glu Asp Trp Ser Glu Ala Gln Leu 165 170 175la Thr Leu Ile Thr Gln Asp Cys Leu Ile Gly Val Ser Arg Leu Glu Ala Pro Phe Ala Ala Leu Pro Ala Pro Ala Val Ala Leu Gly Ala Met Asp Gly Met His Asp Leu Gly Gly Lys Gln Gly Phe Gly Pro Val Ile Lys Thr His Asn Ala Lys Ala Phe His Glu Glu Trp Glu Val Lys Met 225 230 235 240sn Ala Ile Ser Gly Ala Leu Val Ser Lys Gly Ile Tyr Asn Met Asp 245 250 255lu Tyr Arg His Gly Ile Glu Arg Met Glu Pro Arg His Tyr Leu Thr Ala Ser Tyr Phe Glu Arg Val Phe Thr Thr Ala Val Thr Leu Cys Ile Glu Lys Gly Val Phe Thr Ala Ala Glu Leu Glu Ala Lys Leu Gly Thr Ser Val Pro Leu Ser Leu Pro Ser Ser Pro Gly Arg Gln Pro Ala Lys' 305 310 315 320ly Pro Glu Gly Gly Phe Lys Leu Gly Gln Arg Val His Val Lys Asn 325 330 335lu Phe Val Pro Gly His Thr Arg Phe Pro Ala Tyr Ile Arg Gly Lys Ala Gly Val Val Val Gly Ile Ser Pro Ala Tyr Pro Tyr Pro Asp Ala Ala Ala His Gly Glu Tyr Gly Phe Ser Glu Pro Thr Tyr Asp Val Cys Phe Lys Ser Lys Asp Leu Trp Pro Asp Gly Cys Glu Ala Ala Asp Val His Val Gly Val Phe Gln Ser Tyr Leu Leu Ser Ala Glu Glu (2) INFORMATION FOR SEQ ID NO: 5:

(i) CHARACTERISTICS OF THE S~Qu~
(A) LENGTH: 38 amino acids tB) TYPE: amino acid (D) CONFIGURATION: lin~P;re WO 95/04828 2 ~ ~ 6 ~ 5 6 PCT~4/00993~

(ii) TYPE OF MOLECULE: peptide (ix) ADDITIONAL FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 17 (D) OTHER INFORMATION: /note= "Xaa ~ Thr or Arg"

(ix) CM ADDITIONAL ACTERISTIC:
(A) NAME/KEY: Modified-site (B) LOCATION: 33 (D) OTHER INFORMATION EXTS: /note= nXa8 5 Asp or Pro"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Thr Asp Asn Ala Val Met Glu Gln Arg Val Asp Ala Leu Phe Val Leu Xaa Lys Glu Leu Gly Leu Val Thr Asp Gln Thr Val Pro Asp Tyr Glu Xaa Ala Leu Met His Asp (2) INFORMATION FOR SEQ ID NO: 6:

(i) CHARACTERISTICS OF THE S~:~U~NC~:
(A) LENGTH: 36 amino acids (B) TYPE: amino acid (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: peptide (xi) S~4U~ DESCRIPTION: SEQ ID NO: 6:

Met Asp Gly Met His Asp Leu Gly Gly Lys Gln Gly Phe Gly Pro Val Ile Lys Thr His Asn Ala Lys Ala Phe His Glu Glu Trp Glu Val Lys Met Asn Ala Ile (2) INFORMATION FOR SEQ ID NO: 7:

(i) CA~AC'l~KISTI W ES OF THE SEQUENCE:
(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: l; n~i re ~ 0 95/~ 2 1 4 6 6 5 6 PCT~R94/00993 (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETIC: NO

(iii) ANTI-SENSE: NO
(ix) ADDITIONAL FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION: 9 (D) OTHER INFORMATION: /note= "N=inos;ne"

(ix) ADDITIONAL FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION: 12 (D) OTHER INFORMATION~M~Nl~: /note~ nN = inosine"

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

(2) INFORMATION FOR SEQ ID NO: 8:

(i) CHARACTERISTICS OF THE S~Q~k~
(A) LENGTH: 8 amino acids (B) TYPE: amino acid~
(D) CONFIGURATION: l;n~P;ne (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE:
(B) CLONE: sequence deduced from probe SEQ ID NO: 7 (xi) DESCRIPTION OF S~u~CE: SEQ ID NO: 8:

Asp Asn Ala Val Met Glu Gln Arg (2) INFORMATION FOR SEQ ID NO: 9:

(i) CHARACTERISTICS OF THE S~Qu~N~:
(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: lin~;re WO 95/0482B 2 ~ 4 ~ ~ ~ 6 36 PCTA~R94/0099 (ii) TYPE OF MOLECULE: DNA (genomic) (iii) liY~til~lh-llQuE: NO

(iii) ANTI-SENSE: NO
(xi) SE W ENCE DESCRIPTION: SEQ ID NO: 9 CAYAAYGCBA AG~ lrCA YGA 23 (2) INFORMATION FOR SEQ ID NO: 10:

(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) CONFIGURATION: lin~;re (ii) TYPE OF MOLECULE: peptide (vii~ IMMEDIATE SOURCE:
(B) CLONE: sequence deduced from the probe SEQ ID NO: 9 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

His Asn Ala Lys Ala Phe ~is Glu

Claims (16)

CLAIMS: 1 - Enzymes with nitrile-hydratase activity, capable of catalyzing the transformation of nitriles into amides, characterized in that they possess enzymatic activity against vis substrates having a nitrile function and a carboxylate function, greater than that with respect to substrates having at least two nitrile functions. 2 - Enzymes according to claim 1, characterized by an activity specific enzyme (Us), with respect to cyano-5-valerate, expressed in moles of amide appeared x h-1 x kg-1 of enzyme used and under conditions of measurement given, greater than or equal to 400, preferably 1000 and, more more preferably still, at 10,000. 3 - Enzyme according to claim 1 or claim 2, characterized in that it consists of two subunits, .alpha., .beta., the sequences of which peptides are as shown in FIGS. 1a and 1b (SEQ ID No: 1: and: 2:) 4 - DNA sequence coding for an enzyme having nitrile activity-hydratase, capable of hydrolyzing nitriles into amides, characterized in that it is chosen from the list of sequences following:
- the DNA sequence, as shown in FIG. 2 (SEQ ID
No: 3:) and encoding an enzyme (SEQ n) No: 4:) having a nitrile-hydratase 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 an enzyme having a nitrile hydratase activity. 5 - Enzymes resulting from the expression of a DNA sequence according to the claim 4 and having nitrile hydratase activity. 6 - Micro-organism consisting of the E. coli strain containing the plasmid pXL2222, strain referenced and deposited in the National Collection of Cultures of Micro-organisms under number I - 1 330 on July 9, 1993. 7 - Micro-organism capable of producing at least one enzyme according to one any of claims 1 to 3 and 5. 8 - Microorganism according to claim 7, characterized in that it contains at least one expression cassette comprising the DNA sequence according to claim 4 and, upstream thereof, a promoter sequence and a site of binding of ribosomes. 9 - Microorganism according to claim 8, 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. 10 - Microorganism according to claim 8, 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 Comamonas or Pseudomonas, or those derived from corynebacteria genes. 11- Microorganism according to claim 8, characterized in that it belongs to the family of eukaryotes, such as yeasts and in that the promoters are chosen from those of yeast glycolytic genes, such as the genes encoding phospho-glycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, or those of lactase or enolase. 12 - Microorganism according to one of claims 7 to 11, characterized in that the expression cassette is carried by a plasmid. 13 - Microorganism according to claim 12, characterized in that it is selected from the following list of strains:
E.coli, Comamonas, Pseudomonas, Corynebacterium, Brevibacterium, Rhodococcus, Streptomyces, Bacillus, 14 - Enzymes, characterized in that they are produced by micro-organisms according to any one of claims 7 to 13. 15 - Process for the enzymatic hydrolysis of nitriles to amides, characterized in that it consists of:

- to put the mono or di-nitriles corresponding to one of the following general formulas:
NC-R-CN, NC-R-COO-Z+, NC-R-CONH2 in which R is an alkylene or alkenylene group, linear or branched, having 1 to 18 carbon atoms and Z is selected from the following list of compounds: alkaline, alkaline-earth, amines, ammonium, the latter compound being particularly preferred, in the presence of enzymes according to any one of claims 2, 3 and 14, or in the presence of a microorganism according to one any of claims 7 to 13. 16 - Process according to claim 15, characterized in that R = - (CH2)4 -.