MXPA99003403A - Method for preparing 2-hydroxy 4-methylthio butyric acid using a nitrilase - Google Patents

Abstract

The invention concerns a method for preparing 2-hydroxy 4-methylthio butyric acid and/or the ammonium salt of 2-hydroxy 4-methylthio butyric acid by enzymatic hydrolysis of 2-hydroxy 4-methythio butyronitrile characterised in that:a) in a first step a biological material having nitrilase activity is prepared, b) in a second step it is immobilised, c) in a third step, 2-hydroxy 4-methylthio butyronitrile is brought in the presence of the immobilised biological material, for obtaining the ammonium salt of 2-hydroxy 4-methylthio butyric acid, d) in a fourth step, optionally, the salt obtained in step c) is transformed into the corresponding acid, and e) in a fifth step, the product resulting from step c) or d) is concentrated.

Description

Process of preparation of 2-hydroxy 4-methylthio butyric acid by the use of a nutrilase The present invention relates to a new process for the preparation of 2-hydroxy 4- (methylthio) butanoic acid (HMTBA) and / or its ammonium salt (HMTBS). 2-Hydroxy-4-methylthio-butyric acid and its salts have been used for a long time in animal feed in the replacement of methionine. He or she has advantage over the latter to present themselves in a liquid form, which facilitates their use for food producing societies.

It has long been known to prepare 2-hydroxy 4- (methylthio) utanoic acid chemically. Thus, patents EP-142 488, EP-143 000 describing the hydrolysis of 2-hydroxy-4-methylthio-hydroxybutyronitrile (HMTBN) for a two-stage process can be cited. The first step consists in contacting the 2-hydroxy-4-methylthio butyronitrile with a strong mineral acid such as hydrochloric or sulfuric acid. In a further step, after dilution with water, the hydrolysis is completed at a higher temperature REF .: 29660. The 2-hydroxy 4- (methylthio) butanoic acid is then extracted with an organic solvent that is not very miscible with water, such as a ketone, preferably methyl isobutyl ketone, and then the solvent is removed by evaporation. This type of process, used at the industrial level, comprises at least some drawbacks. It produces a molar quantity of ammonium sulphate at least equal to the molar amount of nitrile introduced that must be eliminated, thus generating an industrial waste that goes against a policy of environmental protection. This process also requires the use of significant amounts of solvent that it is absolutely necessary to recycle.

Other processes such as those described in US Pat. Nos. 3,773,927 and 4,353,924 consist of hydrolyzing 2-hydroxy-4-methylthio-butyronitrile with hydrochloric acid and then concentrating the medium with separation of the ammonium chloride formed. The salt obtained is also difficult to remove as the previous salt and in addition the acid obtained has a strong coloration.

Also disclosed in patent EP 330 521 is a chemical hydrolysis process of methylthio hydroxypropionitrile which consists, as before, of hydrolysis in a sulfuric medium. The mixture is partially neutralized with ammonia. A two-phase separation appears. The organic phase contains the majority of 2-hydroxy-4-methylthiobutyric acid and the aqueous phase contains the majority of ammonium sulfate produced. The organic solution after the evaporation of contained water is filtered so as to recover the dissolved ammonium sulfate. The 2-hydroxy-4-methylthio butyric acid is then diluted with a little water and stabilized with a little sulfuric acid. The aqueous solution after the elimination of water makes it possible to obtain the directly marketable ammonium sulphate. This process partly solves the drawbacks of the prior art processes that refer to the use of organic solvents but does not solve at all the problems linked to the rejections of mineral salts.

Among the patents relating to salts of 2-hydoxy-4-methylthiobutyric acid, mention may be made of US Patents 2 745 745, 2 938 053 and 3 175 000 which refer to calcium and / or ammonium salts. The mixture obtained by hydrolysis of 2-hydroxy-4-methylthiobutyronitrile is treated with calcium hydroxide or carbonate. The calcium sulfate is then precipitated by releasing the ammonia which forms the ammonium salt of 2-hydroxy-4-methylthiobutyric acid. Here the problem again consists of eliminating the subsistent calcium sulfate.

A process consisting in practicing a hydrolysis and extraction with an organic solvent as in the aforementioned first document of the prior art after neutralization of the organic solution with ammonia is described again in the patent application WO 96/01808. This process, like most of the processes described above, results in the formation of at least one mole of sulfate or ammonium chloride per mole of introduced nitrile and requires the recycling of significant quantities of organic solvent.

It can not afford to obtain an ammonium salt solution of 2-hydroxy 4-methylthio butyric acid at an industrially interesting cost.

The use of a nitrilase as catalyst for the hydrolysis of a nitrile group in carboxylic group is further described in WO96 / 09403. The process described in this document, however, is not useful on an industrial scale, taking into account the insufficient activity of the microorganisms that synthesize these nitrilases.

It has now been discovered, thanks to the process of the invention, that by employing several specific steps, it would be possible to obtain 2-hydroxy-4-methylthio-butanoic acid and / or its ammonium salt enzymatically on an industrial scale, with a high yield, without using of solvents and without concomitant production of mineral salts.

A subject of the invention is a process for preparing 2-hydroxy-4-methylthio-butyric acid and / or the ammonium salt of 2-hydroxy-4-methylthio-butyric acid by enzymatic hydrolysis of 2-hydroxy-4-methylthio-butyronitrile, characterized in that: a) in a first stage a biological material having a nitrilase activity is prepared, b) in a second stage, it is immobilized, c) in a third step, 2-hydroxy-4-methylthiobutyronitrile is placed in the biological material thus immobilized, to obtain the ammonium salt of 2-hydroxy-4-methylthiobutyric acid, d) in a fourth step, optionally, the salt obtained in step c) is converted into the corresponding acid, and e) in a fifth stage, the product obtained in step c) or d) is concentrated.

The invention will be explained in detail in the following description for which the figures appended thereto will be reported: - figure 1 represents a diagram of an electrodialysis cell used in the optional stage d); Figure IA represents an electrodialysis cell with a three compartment bipolar membrane; Figure IB represents an electrodialysis cell with bipolar membrane of two behaviors.

Figure 2 represents a diagram of an installation for employing the process according to the invention; - Figure 3 represents the restriction map of plasmids pRPA-BCATl at 5.

Figure 4 depicts the sequencing strategy of the 1130 bp fragment containing the DNA sequence (referred to in Figure nitB) encoding the polypeptide having nitrilase activity according to the invention. The numbers refer to the identity of the primers used for the sequencing as well as the notations M13FWD and M13REV.

Figure 5 represents the restriction map of the plasmids pRPA-BCAT12 and pRPA-BCAT13.

Figure 6 represents the gel electrophoresis of 10% SDS-PAGE showing the expression of the DNA sequences according to the invention in strains E. coli BL21 (DE3) / pRPA-BCAT12, BL21 (DE3) / pRPA-BCAT13, BL21 (DE3) / pRPA-BCAT12 + pXL2231, BL21 (DE3) / pRPA-BCAT13 + pXL2231. Each band corresponds to an amount of soluble proteins of 10 μg and an equivalent volume of gross and insoluble fraction.

Figure 7 represents the gel electrophoresis of 10% SDS-PAGE showing the expression of the reference DNA sequences according to the invention in strains E. col i DH5o1 / pRPA-BCAT3, DH5a / pRPA-BCATβ, DH5 / pRPA-BCAT6 + pXL2035, RPA-BIOCAT76. Each band corresponds to an amount of soluble proteins of 10 μg and an equivalent volume of gross and insoluble fraction.

- Figure 8 represents the restriction map of plasmid pRPA-BCAT14.

- Figure 9 represents the e.lectrophoresis in gel % SDS-PA showing the expression of the DNA sequences according to the invention in strains P. pu ti da G2081 (pRPA-BCAT14), G2081 (pRPA-BCAT23), G2081 (pRPA-BCAT24), A. Fa ccal i s ATCC8750 (pRPA-BCAT14), A. fea cali s ATCC8750 (pRPA-BCAT23), A. ATCC8750 faccal (pRPA-BCAT24). Each track corresponds to a quantity of soluble proteins of 10 μg and an equivalent volume of crude fraction and optionally insoluble.

- Figure 10 represents the restriction map of plasmid pCGL1087.

- Figure 11 represents the restriction map of plasmid pOS48.7.

The first stage consists in preparing the biological material that have a nitrilase activity.

The biological material may consist of an enzymatic solution such as or of whole or broken cells having a nitrilase activity.

Advantageously, a microorganism that expresses a nitrilase is used.

The nitrilase can be released in particular from a microorganism, in particular a microorganism of the genus Al cali genes, Rhodoco ccu s or Gordona, particularly Al caligenes fa ecali s, Rhodococcus sp. HT 29-7, Gordona terra e, preferably the strains described in WO 96/09403.

A microorganism which expresses a nitrilase activity obtained to transfer the genetic information encoding the nitrilase from a parent micro organism into a host microorganism is advantageously used. In particular, in E. coli, the co-expression of accompanying Gro ESL proteins can be used to improve the actual productions of the recombinant strain.

For this purpose, any suitable expression vector, in particular a plasmid vector, is used.

The nucleotide sequence used in this case will then also be placed under the control of signals that allow its expression in a cellular host. The cell host used can be chosen from prokaryotic systems, such as Gram-positive or Gram-negative bacteria or eukaryotes, such as yeasts, fungi or any other system. The signals that control the expression of the polypeptides are chosen according to the cell host used. For this purpose, the DNA encoding a nitrilase can be inserted into the vectors with autonomous replication within the chosen host, or the integrating vectors of the chosen host. Such vectors are prepared according to the methods commonly used by the person skilled in the art, and the resulting constructions can be introduced into an appropriate host by standard methods such as electroporation.

As a host microorganism, it can be mentioned in particular Al cali genes fa ecalis, Pseudomonas puti da, Escheri chia coli, or organisms of the genus Ba cillus, Coryneba cteri um, S trep tomyces, Sa ccha romyces, Kluyveromyces, Penicillium and Aspergillus.

A preferred vector for expression in E. coli is the plasmid pRPA-BCAT6.

The tenth stage of the process consists in immobilizing the biological material. This immobilization is advantageously carried out in the presence of a solid support and makes it possible to obtain the solid particles in which the size, shape and mechanical strength can be controlled. It also allows the simultaneous use of polyacetidine polymer and other crosslinking agents.

This process of immobilization can involve whole or permeabilized cells. It can also be applied to an enzyme solution free of cells.

The enzymes used for immobilization are the nitrilases.

The immobilization process consists of immobilizing the active biological material on a solid support, of granulometry comprising, in particular between 1 μm and 3 mm, preferably 10 μm and 2 mm, thanks to chemical agents that react with the amine functions (NH2). , NH), carboxyl (COOH), hydroxy (OH), thiol (SH) or amide (CONH2) of the biological agent and the support. These chemical agents also make it possible to insolubilize the biological material and the support in water. The mass obtained is very malleable and can also be formed in order to obtain two particles of desired shape and size. The cohesion and the hardness of these particles are obtained immediately by drying.

The biological material to be immobilized can also optionally contain an inactive biological material present at a rate of 0 to 200% by weight. This inactive biological material can be proteins (albumin, gelatin) or polysaccharides (Chitosan, k-carrageenan, alginate).

The inert support on which the biological material is deposited and the polymer can be composed of organic, or inorganic, porous or non-porous, hydrophilic or hydrophobic particles. These particles may include, but are not limited to: - ion exchange resins, - alumina, - synthetic silicas or diatoms and silica gels, - zeolites, - carbons, - water insoluble proteins. , like gluten, - polysaccharides, such as starch.

The inert support can be added at a ratio of 0.01 to 500% by weight of the biological material and preferably from 10 to 200%.

The chemical agents used to insolubilize the biological material can be polymers or bifunctional molecules that react with the functions amine (NH2, NH), carboxylic (COOH), hydroxy (OH), thiol (SH), amide (CONH2). They can be cited: - polyacetidine polymers, - polyethyleneimine polymers, - polyamide polymers, - isocyanate polymers, - alginate gels, - k-carrageenan gels, - amines such as hexamethylene diamine, - aldehydes as glutaraldehyde, - carboxylic acids such as adipic acid, and - the isocyanates.

The immobilization process may involve one or more of these chemical agents. The chemical agent is added according to a concentration comprised between 1 and 50% by weight in relation to the biological material and the support. An amount of between 5 and 30% will be preferred in order to obtain sufficiently solid particles and which preserve an important activity and which do not present too many problems of internal diffusion.

The duration of the crosslinking treatment is between 0.5 and 24 hours.

The process temperature is generally between 4 and 65 ° C. A temperature between 20 and 40 ° C will be preferred. Then the temperature used of the immobilization process can also be dependent on the stability of the biological material used.

The pH during the immobilization phase is maintained between 5 and 11. A pH between 6 and 10 is preferred with a preference against the alkaline pH. The pH is also chosen according to the resistance of the biological material which will also be determined by the person skilled in the art.

The formation of the biocatalyst should allow its use in any system, in particular in a fixed bed.

A formulation method can be extrusion. To do this, the biological material and support are crosslinked to add one or several chemical agents. After treatment, the insoluble mass is recovered by centrifugation or after flocculation and filtration. A dry matter rate of at least 10% is preferred. The dough is extruded right away. For this method, vermicellas with a diameter between 0.3 and 0.5 mm and a length comprised between 1 and 10 mm are preferably obtained. These vermicelas can be in the form of spherical. The particular obtained are dried immediately.

Another method of formulation may be film formation ("spray coating"). To do this, the biological material is mixed with one or more chemical agents. After reaction, the mixture is sprayed onto the support in the form of a thin layer. By this method, granules of average diameter between 0.1 and 2 mm are obtained.

The particles obtained can optionally then be immersed in a solution of a reducing agent such as sodium borohydride in order to reduce the imine functions formed during crosslinking.

The particles obtained are sufficiently solid and resistant to wear by being used in a fixed bed, a fluidized bed or a stirred reactor.

The third stage of the process according to the invention consists in using the immobilized biological material in one or more columns or reactors. The purpose is to be able to continuously produce the ammonium salt of 2-hydroxy-4-methylthiobutyric acid from 2-hydroxy-4-methylthiobutyronitrile.

The column or reactors are fed by a pure or diluted solution of 2-hydroxy-4-methylthiobutyronitrile or a mixture containing 2-hydroxy-4-methylthiobutyronitrile and the ammonium salt of 2-hydroxy-4-methylthiobutyric acid.

The column or reactors are preferably used at a temperature between 10 and 60 ° C and at a pH comprised between 5 and 9.

The system employed can be made up of two or several columns connected to one another in series, according to the invention with a first form of use, the feeding of the aqueous solution of 2-hydroxy-4-methylthiobutyronitrile in the upper part of the first column with simultaneous feeding of the other columns with the solution of 2-hydroxy 4-methylthiobutyronitrile in an amount limited to the solubility of this compound in the reaction mixture; This system is called stepped system. According to a second way of using the invention, one or more columns connected to one another are used in parallel in a circulation cycle. According to this installation, the 2-hydroxy-4-methylthiobutyronitrile in aqueous solution is fed continuously in the cycle, and the reaction medium is pumped continuously to preserve a constant volume in the cycle; this system system-cycle system.

The type of reactor used in this invention may be of the fixed bed, fluid bed or continuously stirred type. It is preferred to use the fixed bed type reactors because the wear problems that can be encountered with the immobilized cell particles are reduced. If the microorganism is used as such, it will be preferred to use a stirred reactor coupled to an ultrafiltration module to continuously remove the microorganism from the product of interest.

The fourth stage, which is an optional stage, consists of converting the ammonium salt of 2-hydroxy-4-methylthio butyric acid into the corresponding acid. This step can be carried out following two methods: either by electrodialysis by means of an electrodialyzer with two or three compartments, - either by heating the aqueous solution which may be followed by a liquid / liquid extraction.

Following the electrodialysis method, an electrodialyzer with two or three compartments will be used.

It is understood by "compartment", the space is already between two membranes, either in a bipolar and homopolar membrane, or between two adjacent homopolar membranes. A cell is understood as a set of a set of two or three compartments. A stack comprises between 5 and 300 cells. Electrodialysis is composed of a stack and it is well understood that it contains an anode and a cathode.

The homopolar membranes can be used within the framework of the invention by dividing into two large families, according to the mode of manufacture.

Thus, heterogeneous membranes, prepared from ion exchange resins, mixed with a binder such as polyvinyl chloride, polyethylene or other can be employed. The formed assembly can be spread over a section such as a polyester or polyacrylonitrile fabric.

It is also possible to use homogeneous membranes, obtained by introducing a functional group in an inert support, for chemical or radiochemical insertion. The most commonly used chemical method consists, in general, of functionalizing a latex of a polymer containing aromatic nuclei, such as styrene / divinylbenzene or styrene / butadiene. The latex thus functionalized can then serve to spread a stretch as for the heterogeneous membranes. The radiochemical method generally comprises the insertion, under the influence of radiation, of an aromatic compound, such as styrene, into an inert support such as a polyethylene or polytetrafluoroethylene sheet. The aromatic nucleus is functionalized immediately as in the chemical method.

The cation exchange membranes contain strong acid groups, the majority followed by sulphonate groups, or weak acid groups, followed by carboxylate groups. More rarely, the acid groups may be groups P032", HP02 ~, As032 ~, Se03".

The anion exchange membranes contain strong basic groups, most of them followed by quaternary ammonium groups, or weak basic groups, most of them followed by amines. More rarely, the basic groups may be the phosphonium quaternary groups or the sulfonium groups.

In the present process, the cationic membranes preferably contain the strong acid groups and among these preferably the sulfonate groups and the anionic membranes preferably contain strong basic groups and between these quaternary ammonium groups.

The bipolar membranes are an assembly of two membranes, one cationic, another anionic. When the membrane is subjected to a sufficient electric field, the solvation water at the interface of the membrane dissolves into H + and OH "ions, which migrate respectively towards the cathode through the cationic side and towards the anode through the anionic side. bipolar membranes, the membranes marketed by Aqualytics, Tokuyama Soda or FuMaTech can be cited as examples.

The anode of the electrodialyzer can be constituted by the materials classically used in electrodialysis, for example graphite, nickel or titanium coated with precious metals or oxides of precious metals, in particular platinum titanium. The cathode can also be constituted of materials conventionally used in electrodialysis, for example graphite, stainless steel or nickel.

The electrodialyzer is fed with the aqueous solution to be treated. It is also necessary to circulate a solution of an anolyte to the anode and a catholyte solution to the cathode. A single solution of electolite can also be used. In the present process, a single electrolyte circuit is more convenient. The role of the electrolyte solution. is to ensure a sufficient conductivity. Preferably, this conductivity will be equal to or greater than 20 millisiemens per centimeter (mS / cm), without this lower limit being considered as critical for using the process.

The electrolyte used is an ionizable compound such as a salt, an acid or a base. The electrolyte is preferably chosen from non-electroactive compounds. For example, it is preferable to use neutral salts such as sulfates, acids such as sulfuric acid, bases such as soda.

The applied current densities are generally between 0.2 and 1.5 KA / m2, and preferably between 0.4 and 1 KA / m2.

The temperature at which the process of the invention is employed is situated in a domain compatible with the stability of the membranes. It will be operated preferably at a temperature between 30 and 60 ° C.

The electrodializer can work in different ways. First, everything can work continuously, the solution to be dealt with goes through the stack continuously; several stages are then arranged in series if the treatment rate requires it. It can also work in batch, the solution to be treated is recirculated in a tank until the desired treatment rate is obtained. In short, it can work in direct passage with partial recirculation.

According to a first variant of the invention, the dissociation of the ammonium salt in 2-hydroxy-4- (methylthio) utanoic acid and in harmony can be done in an electrodialysis cell with three-compartment bipolar membranes as schematically represented in the figure IA.

A suitable electrodialysis apparatus to use the process is constituted by different compartments, delimited respectively by the cationic membranes (MC), the bipolar membranes (MB) and the anionic membranes (MA). These compartments are divided into salt compartment (S) which is impoverished from the compounds to be separated, in base behavior (B) and acid (A) where the acid and base regenerated from the salt are respectively concentrated. .

The ammonium salt is introduced into the salt compartment. Under the. action of the electric field, the ammonium ion migrates towards the cathode leaving the compartment (S) where it is, through a cation exchange membrane (cationic membrane) and combines with the OH ions "that come from the anionic phase of the bipolar membrane, in which the dissociation of water takes place under the effect of the electric field.

Simultaneously, the carboxylate ions (2-hydroxy 4- (methylthio) butanoate) migrate towards the anode leaving the compartment (S) where they are, through an anion exchange membrane (anionic membrane).

After passage in the next compartment (A), they are protonated by the contribution of H + ions that come from the cationic side of the bipolar membrane. The three adjacent compartments (B), (A), (S) form an electrodialysis cell.

In a second variant of the invention, the regeneration of 2-hydroxy-4- (methylthio) butanoic acid can be done in an electrodialysis cell with two-compartment bipolar membranes as shown in Figure IB. These two compartments are delimited respectively by the cationic membranes and the bipolar membranes. These compartments are divided into salt / acid (S / A) and base (B) compartments.

The ammonium salt is introduced into the salt / acid compartment. Under the action of the electric field, the ammonium ion migrates towards the cathode leaving the compartment (S / A) where it is, through a cation exchange membrane (cationic membrane) and combines with the OH ions "that come from the anionic side of the bipolar membrane, within which the dissociation of water takes place under the effect of the electric field.

Simultaneously, the S / A compartment is acidified by the contribution of H + ions that come from the cationic side of the bipolar membrane. The two adjacent compartments (B) and (S / A) form an electrodialysis cell.

This configuration has the advantage of a lower energy consumption and allows the desired salt / acid ratio to be varied.

In order to obtain a good functioning of the electrodialyzer, the electrical conductivity of the base compartment (as well as that of the acid compartment in the case of the configuration with three compartments) must be sufficient and can be adjusted by the addition of a supporting electrolyte, , the conductivity of the ammonia solution can be increased by the addition of an ammonium salt, such as ammonium sulfate.

In a preferred variant of the invention, 2-hydroxy-4-methylthio butanoate ammonium will be used.

In a particular embodiment realized within the framework of the present invention, a mixture of ammonium salt of 2-hydroxy-4-methylthio-butyric acid and 2-hydroxy is introduced into the salt compartment of a three compartment electrodialysis cell. 4-methylthiobutyronitrile. The salt will dissociate as before in 2-hydroxy 4-methylthiobutyrate and will migrate to the anode or will be transformed into 2-hydroxy 4-methylthio butyric acid, the ammonium ion will migrate towards the cathode or it will be transformed into harmony, in the salt compartment it will subsist water and untransformed 2-hydroxy-4-methylthiobutyronitrile which will be recycled to the hydrolysis column.

Following the heating method, the acid is recovered by displacing the balance of ammonium salt, free acid by heating the aqueous solution rich in HMTBS and removing the harmonic thus released. It can be concluded by heating under vacuum or at atmospheric pressure with or without treatment for separation. The use of C02 under pressure can be visualized in order to facilitate the displacement of equilibrium. An HMTBS and HMTBA is obtained at a ratio of 5 to 99.9% of HMTBA, and preferably 10 to 50% of HMTBA with respect to the sum of HMTBA and HMTBS. The viscosity of these solutions is between 1200 and 30 mm2.s_1 (1200 and 30 cSt) and preferably between 200 and 50 mm2.s_1 (200 and 50 cSt) at 25 ° C.

The aqueous solutions can be decomposed by extraction of the acid with the use of dissolvent products, mainly miscible or not in the water. There is a large number of possible solvents for separation. Preferred solvents are C5 to C8 ketones, in particular methyl isobutyl ketone; the ethers such as isopropyl ether; alcohols such as isopropanol; the esters; tertiary amines such as trioctylamine. In the context of the invention, the preferred solvents are: acetone, MIBC, isopropanol, isopropyl acetate, isobutyl or propyl ether or THF, trioctyl amine. The ratio of the aqueous phase and the organic phase is not critical. However, for the reasons of degree of efficiency, you should not go below a minimum ratio of 1/1 and for reasons of profitability do not go beyond a ratio of 1/3. A ratio of 1/1 to 2.0 will be preferred.

The extraction can be used in batch or continuous, regardless of the type of liquid-liquid extraction technology. For example, the cascade of decanter mixers in co- or counter-current, the centrifugal extractors, the packed or dish columns, etc. can be cited.

The aqueous solution depleted in free acid can be treated again and this, as many times as desired until the exhaustion of the ammonia is completed if desired.

The organic phase is subjected to a treatment to isolate the HMTBA. This is preferably carried out by vaporization of the solvent or by extraction with hot water. To avoid eventual deterioration of the HMTBA, the thermal stress may be kept as weak as possible for the application of a vacuum.

In these two dissociation processes of the ammonium salt, an aqueous solution of ammonia is generated. The latter can be treated in order to concentrate the ammonia. Distillation and concentration of ammonia in one or more stages with or without pressure will be preferred. A preliminary separation stage can be visualized. The concentrated solution of ammonia can be returned in the synthesis of hydrocyanic acid that comes into play in the synthesis of 2-hydroxy-4-methylthio butyronitrile.

In the fifth stage, the aqueous solution of HMTBS and / or free acid is concentrated. This is preferably done by evaporating the water.

The object of the invention is an installation for using the process according to the invention. Such installation is illustrated schematically in Figure 2 and comprises the conduits 1,2 intended to introduce respectively the cyanhydrin of the AMTP and the water in the reactor 3. the reactor 3 is a fixed bed with recirculation that is filled with the immobilized strains or enzymes . A part of the solution is removed from the reactor 3 and sent to a final reactor 4. The final reactor 4 is a fixed-bed type filled with the immobilized strains or enzymes. A solution concentrated in HMTBS is quickly recovered at the outlet of reactor 4 via line 5.

This solution concentrated in HMTBS can undergo two totally independent treatments according to the type of final product desired.

If it is desired to recover a product containing a high proportion of HMTBS, the solution of line 5 is sent to an evaporator 6 which allows concentrating the product and a concentrated solution composed mainly of HMTBS is recovered in line 8 and contains a small amount of HMTBS. free acid. The excess water as well as a fraction of ammonia are evacuated through the conduit 7.

If it is desired to recover a product containing a high proportion of free acid, the concentrated HMTBS solution of line 5 is sent to a bipolar electrodialysis system 9. In this apparatus, the ammonia is more or less separated from the acid by electrodialysis. The ammonium depleted mixture is recovered in conduit 11 and then concentrated in evaporator 12 to obtain one. Concentrated solution composed mainly of free acid and containing little or no HMTBS. The solution rich in ammonia is removed through line 10. The ammonia is recovered by a separation 15.

This ammonia effluent can be concentrated by distillation 16, the water is recovered by 18. The concentrated ammonia solution 17 can be returned to the synthesis of HCN.

The following examples illustrate the invention.

EXAMPLES Example 1: Nter purification and sequencing of nitrilase The nitrilase was purified in four stages. The summary of the purification is given in table 1.

The cells of Al cali genes fa ecal i s were cultured 24 hours, at 30 ° C, in a minimal medium in the presence of benzonitrile (0.5 g / 1). After centrifugation of the culture, the package was -resuspended in a TG buffer (25 mM Tris-HCl, 10% glycerol (w / v), pH 7.5). The cell suspension was treated with ultrasound and then centrifuged in order to obtain the crude extract. The crude extract was then treated with ammonium sulfate up to 30% saturation. The precipitate obtained was resuspended in the TG buffer and then dialysed against 2 liters of the same buffer overnight. The obtained solution was then deposited on an Q Sepharose Fast Flow HR 26/10 anion exchange column previously equilibrated with the TG buffer. The activity was then eluted with a gradient of 0 to 1 M NaCl. The active fractions were then deposited on an Mono Q HR 5/5 anion exchange column previously equilibrated with TG buffer. The nitrilase was eluted with the aid of a gradient of 0 to 1 M NaCl. To end, the fractions containing activity were combined, the concentration of ammonium sulphate was then brought to 1 M. This solution was then deposited in a column of hydrophobic interactions Phenyl Superóse HR 5/5 previously equilibrated with TG buffer added (NH4) 2S04 1 M. The activity was then eluted by a gradient of 1 M to 0 M ammonium sulfate.

Table 1: Summary of purification of nitrilase Operating conditions: [nitrile] = 50 mM; 100 mM phosphate buffer pH 7.0; 30 ° C.

The molecular weights of the protein were determined by gel filtration. It is approximately 260 kDa. On SDS-PAGE gel, a single band of 43 kDa (95% purity) is observed. It is by consequence probably a protein of structure 6 with which it weighs 43kDa.

Example 2: Cloning of Nitrilasa from Al Cali genes fa ecali s ATCC8750 The terminal NH2 sequence presented in the example 1 presents a total identity with the sequence of the N-terminal extremity of the nitrilase of A. Facials JM3 (Kobayashi et al., 1993, Proc. Na ti Acad.Sci. USA 90: 247-251), while the N-terminal extremities of bacterial nitrilases have 35 to 57% identity in 14 waste. The inventors have hypothesized that the nitrilase of the invention purified from strain ATCC8750 would be similar to that described by Kobayashi et al. (above). The cloning strategy then consisted in amplifying, by PCR reaction in genomic DNA of strain ATCC8750, the gene of this nitrilase, with the help of two nucleotide probes determined from the sequence given by Kobayashi et al. (above).

The two probes were synthesized, one that could anneal to the 5 'part of the sequence given by Kobayashi et al. (above) and the other with part 3 ': Part 5 '(PCRAF1): CCGGGAATTCATATGCAGACAAGAAAAATCGTCC Part 3' (PCRAF2): TCCTTCTGCGTCCCCGATCCCGCAT The PCRAF1 primer contains 12 nucleotides at 5 'that allow introduction upstream of the initiation codon ATG restriction sites for enzymes .EcoRI and Ndel. The PCRAF2 primer allows the introduction of the restriction site of the BamHI enzyme. The genomic DNA of strain A. Faccal ATCC8750 was extracted according to the CTAB protocol described by Ausubel et al. (Current Protocols in Molecular Biology, John Willey & Sons Inc.

Ed, 2.4.1-2.4.5) and 100 ng were used for each PCR reaction. In order to represent in the amplification specificity, different concentrations of MgCl2 were tested as indicated in Ausubel et al. (above) in a total sample volume of lOOμl and with 2.5 units of Taq DNA polymerase (Perkin Elmer). Two additional reactions were performed with 1.5 mM MgCl2 and 5% DMSO or 5% formamide. A Perkin Elmer 9600 thermal cycler was programmed with the following chaining: 5 min at 90 ° C, 30 cycles (30 sec at 95 ° C, 30 sec at 55 ° C, 45 sec at 72 ° C) and 4 min at 72 ° C . The different amplification products were analyzed in 0.8% agarose gel. A majority band, in which the size corresponds to the expected size of 1.15 kb, was amplified in all reactions but more specifically with 1.5 mM MgCl2 and 5% DMSO. The products of the reaction under these conditions were maintained for the continuation. The sample was treated with protein K, precipitated with ethanol after a phenol-chloroform extraction. The background was resuspended, incubated for 2 h at 37 ° C with 40 units of EcoRl enzyme and 40 units of BamEl enzyme. After migration in 0.7% agarose gel, the 1.15 kb band was cut out and extracted to be cloned into the pBSK vector "(Stratagene, La Jolla, USA), open EcoRI -BamEl by classical methods, five independent clones, called pRPA-BCATl at 5, were analyzed by enzymatic digestion of the plasmid DNA with Ndel, EcoRl, BamE l, BspMI and Bg / I l enzymes (Figure 3) The profiles obtained correspond to the theoretical restriction profiles of a plasmid obtained by this method with the sequence described by Kobayashi et al. (above) Strain XL1 Blue (pRPA-BCAT3) was deposited in the CBS under the number CBS 998-96.

Example 3: Sequencing of a 1130 bp fragment containing the DNA encoding the polypeptide having nitrilase activity.

The insertion cloned in the plasmid pRPA.BCAT3 was sequenced by the company Génome Express S.A. (Grenoble, France) from a DNA preparation made in the laboratory (Kit Wizzard Midi-prep, Promega). The sequencing strategy of this fragment, carried out according to the classical methods known to the person skilled in the art, is indicated in figure 4. The ten internal nucleotide primers (identified by numbers 623 to 629 and 681 to 682 in Figure 4) were synthesized according to the sequence of the nitrilase of A. Faccal s JM3 (Kobayashi et al., cited above). This set was completed by the universal primers "Reverse" and "M13 Forward". Each region was ligated at least once in each strand of DNA.

The DNA sequence obtained has two differences with respect to the published sequence: one in the assumed structure serves as a transcription terminator and the other in the nitrilase gene, called nitB, leads to the substitution Asn279-- > Asp These two regions were then sequenced in the laboratory in the pRPA-BCATl, 2,4,5 plasmids with two specific primers 710 and 682 (see Figure 4). The change C- > T in position 1412 (according to the Kobayashi numbering, above) in the terminator was found in all the clones. It is a mutation that differentiates the two strains of A. fa ecal i s. The change A- > G at position 1138. is not present in the plasmid pRPA-BCAT3. It is then a mutation introduced when PCR by Taq DNA polymerase.

Example 4: Expression of nitrilase in E. col i BL21 (DE3).

In order to confirm the identification of the DNA sequence cloned with the nitrilase gene I purified, the nitB gene was placed under the control of the promoter of the F 10 phage T7 (PT7) gene according to the form of operation described below: the Ndel -BamUl insertions of 1.13-kb of the pPRA-BCAT3 and pRPA plasmids -BCAT4 were cloned into vector pXL2432 to give respectively the vectors pRPA-BCAT12 and pRPA-BCAT 13 described in figure 5. The vector origin pXL2432 is a hybrid between the plasmid pET9 region (Studier et al., 1990, Methods In Enzymol 185, 60-89), included between the Accl and EcoRl sites, which encompasses the origin of replication (ORÍ) and the selection marker carries resistance to kanamycin (kan), and the region between the sites coRI and Accl from the PETlla plasmid (Novagen Inc., Madison Wl, USA) which encompasses the expression cassette, the lacl repressor gene and the ROP copy number regulator gene.

The cultures were brought to the induction condition according to the following form of operation: Strain BL21 (DE3) (Novagen Inc., Madison Wl, USA) containing the plasmid pRPA-BCAT12, strain BL21 (DE3) containing plasmid pRPA-BCAT13 as well as strain BL21 (DE3) containing plasmid pXL2432 were cultured 16 h in LB medium at 37 ° C (Miller, 1972, Experiments in Molecular Genetics - Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) containing 50 μg / ml kanamycin, and then diluted to the 100th in the same medium and at the same temperature. When the cultures reached an OD 600 between 0.5 and 1, IPTG was added to the final concentration of 1 mM. After 6 hours of culture, the bacteria were collected. A similar mode of operation was adopted to culture strain BL21 (DE3) which contained plasmids pRPA-BCAT12 and pXL2231, strain BL21 (DE3) which contained plasmids pRPA-BCAT13 and pXL2231, as well as strain BL21 (DE3) which contained the plasmids pXL2432 and pXL2231 by adding tetracycline to the culture medium at a rate of 12 μg / ml medium. Plasmid pXL2231, which is derived from vector pXLl635 (Application FR 90/05185 from 04/24/1990), belongs to the IncP incompatibility group and is then compatible with plasmids pRPA-BCAT12 and 13 which possess the origin of replication of the plasmid ColEl. Its selection marker is resistance to tetracycline and carries a jBcoRI-iTindlII fragment of 2.2 kb containing the GroES and GroEL genes that code for the molecular companions of E. col i (Fayet et al., 1986, Mol. Gen. Genet, 202: 435-445).

The expression of the nitrilase was analyzed in gel at % of SDS-PA in the crude fraction after sonication of the cells, and after centrifugation in the background and in the supernatant. The results are presented in Figure 6 and show a high level of expression of the nitrilase (NitB) in cell extracts of which one of the plasmids contains at least the nitB insert; however, this protein is essentially in insoluble form although the presence of plasmid pXL2231 allows to increase the amount of nitrilic acid polypeptide in the soluble fraction.

In Figure 6, M represents the molecular weight marker; these are indicated in kDa. On the other hand, the bands have the following meanings: - A, D, G represent the gross fractions respectively of BL21 (DE3) + p / RPA-BCAT12 / RPA-BCAT13 / XL2432; - B, E, H represent the supernatants obtained respectively from the same strains; - C, F, I represent the funds obtained respectively from the same strains; - J, N, Q represent the crude fractions obtained respectively from BL21 (DE3) + pXL2231 + p / RPA-BCAT12 / RPA-BCAT13 / XL2432: - K, O, R represent the supernatants obtained respectively from these strains; -L, P, S represent the funds obtained respectively from these strains.

Strain BL21 (DE3) / pRPA-BCAT12 + pXL2231 was named RPA-BIOCAT126. Strain BL21 (DE3) / pRPA-BCAT13 + PXL2231 was named RPA-BIOCAT127. The nitrilásicas activities of cultures of RPA-BIOCAT126, RPA-BIOCAT127, and RPA-BIOCAT66 that correspond to BL21 (DE3) / pXL2432 were determined as follows: the cultures were carried out according to the protocol described before modifying the volume of culture (50 ml ) and the final concentration of IPTG (0.1 mM). The cultures were centrifuged and the cell packs were put back in 10 ml of potassium phosphate buffer lOOmM pH7. Hydrolysis of HMTBN was performed with 500μl of this suspension added to 500μl of 200mM HMTBN pH7 solution. A kinetics of hydrolysis was obtained by mixing lOOμl of this mixture in 900 μl of 0.1 N phosphoric acid at regular intervals for 1 to 4 hours. The amounts of HMTBA produced were analyzed by HPLC as described in the application WO 96/09403. The results are grouped in table 2.

Table 2: Activities of the strains RPA-BIOCAT66, 126 and 127 ABBREVIATIONS Km: Canamycin 50μg / ml; Te: tetracycline 12 μg / ml; h: hours; U: kg of HMTBA formed per hour and per kg of dry weight.

In order to improve the solubilization of the expressed nitrilic polypeptide, the plasmid pRPA-BCAT37 was constructed as follows. Plasmid pXL2391 was obtained by ligating the 5.9 kb £ coRI-PvuII fragment from plasmid pDSK519 (Keen et al., 1988, Gene 70: 191-197), treated with Klenow polymerase, with the 2056 bp HindlII fragment extracted from the plasmid pHP45OSp (Prentki et Krisch, 1984, Gene 29: 303-313) treated with the Mung Bean Nuclease. The plasmid pXL2391 was then digested by Sma l et Sacl and the insert 2.3 kb carrying the GroESL operon, extracted from the plasmid pXL2231 digested by ffindlll, treated with Klenow and digested by Sacl, where it was introduced. The plasmid pRPA-BCAT37 is consequently a derivative of the plasmid RSF1010 with a stronger copy number than the plasmid pXL2231, compatible with plasmids carrying the ColEl origin and carrying a streptomycin resistance marker. This plasmid was introduced into strain BL21 (DE3) / pRPA-BCAT12 to give the strain RPA-BIOCAT171. The activity of a culture was carried out according to a form of operation similar to that described above, but replacing tetracycline with streptomycin with 100 μg / ml, was determined and reported in table 3.

Table 3: Activity of strain RPA-BI0CAT171 ABBREVIATIONS Km: Canamycin 50μg / ml; Sm: Streptomycin lOOμg / ml; h: hours; U: kg of HMTBA formed per hour and per kg of dry weight.

Example 5: Nitrylase expression of E. DH5alfa coli.

Plasmid pRPA-BCAT6 was constructed by cloning pBCAT3 (see Figure 3) the 0.6 kb S cal-Nde I fragment from pXL2158 containing the Ptrp promoter and the RBScII ribosome binding site (Levy-Schill et al., 1995, Gene 161: 15-20). Expression was carried out with the strain DH5alpha containing the plasmid pRPA-BCAT6 and / or the plasmid pXL2035 (Levy-Schill et al., Cited above) with the following form of operation: the pre-culture was incubated 16 h at 37 ° C in M9 medium glucose (Miller, JH 1972. Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) containing 0.4% casamino acids, 100 μg / ml tryptophan, 100 μg / ml carbenicillin. For strains containing pXL2035, kanamycin was added at a rate of 50 mg / ml. Expression was made after dilution of 1/100 of the culture saturated in an identical medium but without tryptophan and incubation 8 to 16 h at 37 ° C.

The SDS-PAGE analysis of the extracts of different strains was carried out as described in the previous example and is presented in figure 7.

In this figure, M represents the molecular weight marker; those that are identical in kDa. On the other hand, the bands have the following meanings: L, I, F, C represent the crude fractions obtained respectively from the strains DH5alpha + pRPA-BCAT / 3, / 6, / 6 + pXL2035, RPA-BIOCAT76; - K, H, E, B represent the supernatants obtained respectively from these same strains; J, G, D, A represent the funds obtained respectively from these same strains.

Plasmid pRPA-BCAT6 makes it possible to obtain a weak accumulation of a 43 kDa polypeptide, which is mostly insoluble. Coexpression of GroE from plasmid pXL2035 reduces overexpression, but after three successive transplants of the DH5alpha strain (pRPA-BCAT6 + pXL2035), the selected RPA-BIOCAT76 strain returns to an initial level of expression of the nitrilic acid polypeptide, which It is almost completely soluble. The doses of crop activities carried out according to the forms of operation described above and in the previous example are presented in table 4.

Table 4: Activity of strains DHdalfa (pRPA-BCAT3), DHdalfa (pRPA-BCAT6), DHdalfa (pRPA-BCAT6 + pXL2035), RPA-BIOCAT76 ABBREVIATIONS Km: Canamycin 50μg / ml; Cb: Carbenicillin 100 μg / ml; h: hours; U: kg of HMTBA formed per hour and per kg of dry weight.

These show that the coexpression of GroE allows to improve the expression of the nitrilase and that a selection of classical strain for successive transplants also contributes to the improvement of the activity of the recombinants.

Example 6: Expression of the nitrilase in Pseudomonas putida and Alcaligenes faecalis.

The expression system described in Example 5 was used to produce the nitrilase in Pseudomonas putida and A. faecalis. The 1.14 kb Ndel-Xbal fragment of pRPA-BCAT6 containing the nitB gene was introduced into the Ndel-Xbal sites of vector pXL1289. The vector pXL1289 is a derivative of pKT230 (Bagdasarian et al., 1981, Gene 15: 237-247) that carries the origin of replication (oriV) multi-host in Gram-negative bacteria and that contain a fragment íJcoRI-Ndel of 120 bp carrier of the Ptrp promoter and the RBScII of pXL534 (Latta et al., 1990, DNA and Cell Biology, 9: 129-137), an Ndel-Xbal insert coding for the cobA gene (Crouzet et al., 1990, J. Bacteriol '172: 5968-5979) and a 500 bp Xbal-BamEI insert containing the TrrnB terminator of pXL534 (Latta et al., 1990, DNA and Cell Biology, 9: 129-137). The plasmid obtained pRPA-BCAT14 is by sequence a derivative of pKT230 containing a gene that confers resistance to kanamycin (Kan) and the nitB gene under the control of Ptrp: RBScII (figure 8).

While the introduction of a plasmid into the strain of E. col i DH5alfa, a particular clone was selected: it would express a nitrilase of which the molecular weight in SDS-PA gel is 44 kDa and instead of 43 kDa. The plasmid housed by this clone has the same restriction profile as pRPA-BCAT14 and was designated pRPA-BCAT24. Finally, the plasmid pRPA-BCAT23 was constructed according to the same form of operation as pRPA-BCAT14 with the following modification: the 136 bp Stul-Bsml fragment of pRPA-BCAT6 was replaced by the Stul-Bsml fragment of pRPA- BCAT4. Plasmid pRPA-BCAT23 thus expresses a NitB nitrilase with an Asn residue at position 279.

The plasmids pRPA-BCATl4, 23 and 24 were introduced by electroporation into the strain Pseudomonas s pu ti da G2081. Strain G2081 is derived from strain KT2440 (Bagdasarian and Timmis, 1981, in Hofschneid and Goebel, Topics in Microbiology and Immunology, 47 Springer Verlag, Berlin) by selection of spontaneous resistance to nalidixic acid and rifampicin. The vector pKT230 was used as a control plasmid.

Plasmids pRPA-BCAT14, pRPA-BCAT23, pRPA-BCAT24 and pKT230 were then extracted from strains of P. puti da to be introduced by electroporation into strain A. Faccal s ATCC8750 (= RPA-BI0CAT1).

Strains G2081 (pRPA-BCAT14), G2081 (pRPA-BCAT23), G2081 (pRPA-BCAT24), G2081 (pKT230) and RPA-BIOCAT1 (pRPA-BCAT14), RPA-BIOCAT1 (pRPA-BCAT23), RPA-BIOCAT1 ( pRPA-BCAT24), RPA-BIOCAT1 (pKT230) were grown overnight at 30 ° C in LB medium containing 50 mg / ml kanamycin. These precultures were diluted 1/100 in an M9 medium containing 50 mg / ml kanamycin and incubated 20 h at 30 ° C.

After sonication of the cells, the expression of the nitrilase was measured in a 10% gel of SDS-PA in the crude fraction after sonication of the cells, and after centrifugation in the package and in the supernatant. The results are presented in figure 9. For strains RPA-BIOCAT1 (PKT230) and G2081 (pKT230), only the crude extracts were deposited (bands A and I respectively). In this figure, M represents the molecular weight marker; which are identical in kDa. On the other hand, the bands have the following meanings: -B, D, F represent the crude fractions obtained respectively from strains RPA-BI0CAT1 (pRPA-BCAT14), RPA-BI0CAT1 (pRPA-BCAT23), RPA-BI0CAT1 (pRPA-BCAT24); - C, E, G represent the supernatants obtained respectively from the same strains; - H represents the package obtained from RPA-BIOCAT1 (pRPA-BCAT24); - J, L, O represent the crude fractions obtained respectively from strains G2081 (pRPA-BCAT14), G2081 (pRPA-BCAT23), G2081 (pRPA-BCAT24); - K, N, P represent the supernatants obtained respectively from the same strains; - Q represents the package obtained from G2081 (pRPA-BCAT24).

This experiment shows that the strains of C. pu ti da express significant amounts of three soluble nitrilic acid polypeptides and that only the 44 kDa nitrilic acid polypeptide is overexpressed by the A strain. fa ecali s. The activity doses of these cultures, carried out according to the protocol described in example 4, show that the strains G2081 (pRPA-BCAT23), G2081 (pRPA-BCAT24), and RPA-BI0CAT1 (pRPA-BCAT24) have a nitrilastic activity in HMTBN.

Example 7: Expression of nitrilase in Coryneba cteri um gl u tami cum.

The expression of the nitrilase was carried out in the strain CGL1010 (= ATCC 14752) with the help of the promoter P cspB (Peyret et al., 1993, Molecular Microbiol., 9: 97-109). A 530 bp fragment containing the PcspB promoter was amplified from the plasmid pCGL815 (Peyret et al., Cited above) with the help of the following primers KS1 and KS2: KS1: 5 * -ACGCGTCGACCAGATCGTCAAGTTGTGG-3 'KS2: 5'-CATAGAGGCGAAGGCTCCTTG-3' After digestion with Sal I, the amplified 530 bp fragment was cloned into the pBSK plasmid (Stratagene, La Jolla USA) opened by Sal I and EcoKV to give the plasmid pCGLl084. A £ coNI / Nde I adapter was manufactured by hybridizing the following two oligonucleotides KS8 and KS9: KS8: 5'-TCAAGGAGCCTTCGCCTCA-3 'KS9: 5' -TATGAGGCGAAGGCTCCTTG-3 ' The nitrilase gene was extracted from the pRPA-BCAT6 plasmid in the form of a 1.1 kb Ndel-Xba l fragment. It was introduced in pCGL1084. open with EcoNl and Xba l using the EcoNl / Ndel adapter described above to give the plasmid pCGL1086. The Sal 1 -BamE I fragment from pCGL1086 containing PcspB: i or tB was then cloned into the pCGL482 vector (Peyret et al., Cited above) at the Sal I and BamHI sites that led to the plasmid pCGL1087. Plasmid pCGL1087 (FIG. 10) is thus a pBIl-based vector carrier (Santamaría et al., 1984, J. Gen. Microbiol. 130: 2237-2246) containing the origin of replication of pACYC 184 recognized in E. coli, a gene that confers resistance to chloramphenicol (Cm) and fusion P cspB '. : nor tB.

Plasmids pCGL1087 and pCGL 482 were introduced by electroporation into CGL1010 as described by Bonnamy et al. , 1990 (FEMS Microbiol, Lett 66: 263-270). After 20 hours of culture at 30 ° C in 3.7% Brain Heart Infusion (Difco Laboratories, Detroit, USA) added with 5 mg / ml chloramphenicol, the doses of nitrilase activities were performed in the culture samples as described in Example 4. The results show that the strain CGL1010 (pCGLl087) possess a nitrilase activity that the strain CGL1010 (pCGL1087) possesses a nitrilase activity while the strain CGL1010 (pCGL482) does not.

Example 8: Expression of nitrilase in Streptomyces lividans.

The expression of the nitrilase was carried out in the strain of S. li vi dans TK24 (Hopwood et al., 1985, Genetic Manipulation of Streptomyces, A Laboratory Manual, The John Innes Foundation, Norwich) with the help of the plasmid pIJ6021 (Takano et al. al., 1995, Gene 166: 133-137) using the P tipA promoter (Holmes et al., 1993; EMBO J. 12: 3183-3191).

The pUC19 plasmid (Yanisch-Perron et al., 1985, Gene 33: 103-119) was modified by removing the 140 bp Tfi I fragment by Tfi I digestion, Klenow treatment and ligation of the vector thereon. The obtained plasmid was opened with EcoRl and BamEl in order to clone the 1.15 kb EcoRI-BamEl fragment of pRPA-BCAT3 containing the tB gene. The pOS48 plasmid thus obtained possesses a unique site Tfi I located between the tB gene and the BamE l site. The site was used to insert a 2.3 kb Ohyg fragment extracted from the plasmid pHP45Ohyg (Blondelet-Rouault et al., Gene, presented) after digestion with BajpHI and Klenow treatment. The Ohyg fragment contains the hygromycin phosphotransferase (hyg) gene from Streptomyces hygroscopius (Zalacain et al., 1986, Nucí Acids Res., 14: 1565-1581) and confers S. li vi dans a resistance to hygromycin. Plasmid pOS48.5 thus obtained was used to isolate a 3.45 kb Ndel-BamE I fragment containing the nitrilase gene followed by the hyg gene, which was cloned into the plasmid pIJ6021 (Takano et al., Cited above) opened by Ndel and BamE l. Plasmid pIJ6021 was not replicated more than in Streptomyces, the ligation mixture was transformed into S. li vi dans TK24 by classical methods (Hopwood et al., Cited above) selecting clones resistant to 200 mg / 1 hygromycin (Boehringer Manheim ). The plasmid DNAs of these clones were extracted by classical methods (Hopwood et al., Cited above) and their restriction profiles correspond well to the expected construction that was named pOS48.7 (Figure 11).

Two clones of S. li vidans containing pOS48.7 as well as a clone containing the plasmid pIJ6021 were cultured in 50 ml of TSB medium (Tryptic Soy Broth, Difco) at 30 ° C for 72 h with a selection of kanamycin 5 mg / 1, and then Thiostrepton was added at the concentration of 5 mg / 1 and the culture was continued for 18 h more according to the conditions described (Hopwood et al., cited above). The culture was harvested and the activity dose, performed under the conditions described in example 4, shows that strain S. l i vi dans TK24 (pOS48.7) expresses a nitrilase activity contrary to strain TK24 (pIJ6021).

Example 9: Hydrolysis of HMTBN with other nitrilases The primary sequence of the nitrilase of Comamonas tes tos teroni sp. , described in Levy-schill et al. , 1995 (above) presents 31% identity with the primary sequence of the nitrilase described in this invention. The recombinant strain of E. coli TG1 (pXL2158, pXL20.35), which expresses the nitrilase of C. Teroni tes, was cultivated under the conditions described by Lévy-Schill et al. , (above) and a cell pack was incubated in the 100 mM phosphate buffer at pH 7 with 50 mM HMTBN, at 30 ° C. The activity measured was 2.3 kg / h.kg CS. As the nitrilase of the invention, the nitrilase of C. testos teroni is capable of hydrolyzing the HMTBN.

In addition, the donors presented in Example 4 with the plasmids pBCAT12 and pBCAT13 show that the Asn279- > Asp279 in the nitrilase of A. The ATCC8750 allows you to preserve the activity in HMTBN.

Example 10: Support contribution g of dried cells were added to 20 g of water adjusted to pH 7.0. The amount of support (CELITE 545, Prolabo, France) indicated was added immediately and after perfect homogenization, the suspension was reticulated by the addition of 15% glutaraldehyde based on the total dry mass followed by 10% polyethyleneamine (SEDIPUR, BASF, Germany) . The suspension was stirred 1 hour at room temperature.

The suspension was then flocculated by the addition of 0.001% of an anionic flocculant, Superfloc AlOO. The crosslinked mass was recovered by filtration.

This mass was then reticulated by the addition of 10% polyacetidine (KYMENE 557, Hercules, USA) based on the total dry mass. Then the wet mass was extruded again through a 0.5 mm diameter hole and dried.

The activity of the particles obtained was then determined following a procedure described in WO 96/09403.

The addition of support for the cells markedly improves the activity.

The experience was repeated replacing the CELITE with wheat gluten (Roquette, France) particularly soluble in water or gelatin (SBI, France) totally soluble in water.

'The cell suspension does not flocculate and is not filterable This example shows that gluten could be substituted with CELITE. On the contrary, if the addition is of gelatin (totally soluble), the formation of catalyst is impossible by the technique described above (extrusion).

Example 11 g of Escheri chia col i BIOCAT171 from example 4 prepared by aerobic culture in an LB medium were mixed with 10 g of CLARCEL 78 (CECA, France) in 500 g of 100 mM phosphate buffer pH 7.0. After homogenization, 6 g of 25% glutaraldehyde were added and the suspension was stirred 15 minutes at room temperature. 2 g of polyethyleneamine were then added as well as 6 g of 25% glutaraldehyde. Everything was stirred for one hour at room temperature. The whole was flocculated by the addition of 5 ml of a 0.2% SUPERFLOC AlOO solution (CYTEC, France). The mass was filtered and the paste thus obtained was mixed with 16 g of 12.5% polyacetidine (KYMENE 557, Hercules) and then extruded through a 0.5 mm diameter hole. The vermicelas obtained were dried at 35 ° C in an oven and then immersed for 30 minutes in a bath of 1% NaBH 4 prepared in a buffer of 50 mM borate pH 10.0. The biocatalyst was then washed with distilled water. The catalyst was stored at 5 ° C at room temperature in a 500 mM phosphate buffer pH 8.0.

A thermostatic column 3 cm in internal diameter and 45 cm in height was filled with 100 g of biocatalyst. This column met a pump via a recirculation cycle. The total volume of the reactor is 430 ml. The cycle is filled with an ammonium salt solution of 25% hydroxymethyl butyric acid. The solution was fed at the top of the column to the base at a rate of 20 1 / hr. The water that circulates in the double envelope of the column and in the heat exchanger allows to maintain the temperature at 35 ° C. The demineralized water was added to the cycle at a flow rate of 80 g / h. The 2-hydroxy 4-methylthiobutyronitrile was added at 20 g / h. The excess volume of reaction medium is evacuated through the base of the column in such a way that the volume of the cycle remains constant. Thus. obtains a continuous flow of 95% nitrile transformation. In addition, the concentration of the aqueous solution of ammonium salt of the 2-hydroxy-4-methylthio butyric acid obtained at the outlet of the reactor is 25%.

Example 12 The electrodialyzer used is constituted by a stack of 9 cells of 2 dm2 active surface, each composed of 2 compartments designated as follows: - salt / acid compartment: limited on the cathode side by a Neosepta CMB cation exchange membrane from Tokuyama Soda, and on the anode side by the cationic side of a bipolar membrane Aqualytics - base compartment: limited on the anode side by the cationic membrane, and on the cathode side by the anionic face of the bipolar membrane.

The anode is made of platinum titanium. The cathode is made of stainless steel.

The electrolyte is constituted by an aqueous solution of sodium sulphate which has a conductivity of 100 mS / cm at 40 ° C. The circulation flow at the electrodes is 2x100 1 / h. The volume is 5 1.

The "base" compartment is initially filled with 5 liters of a 1% solution of ammonium sulfate.

The "salt / acid" compartment is initially filled with 5 liters of a 1.44 mol / l solution of the ammonium salt of 2-hydroxy-4- (methylthio) butanoic acid.

The recirculation flow of the solutions is initially set at 130 1 / h for the salt / acid compartment and at 190 1 / h for the base compartment.

The electrodialysis is carried out discontinuously (recirculation operation), at an average temperature of 40 ° C. The intensity was imposed at 9 A, with a current density of 0.45 kA / m2.

After 155 minutes of operation, the conductivity of the salt / acid compartment increased from 59 to 7.8 mS / cm.

The salt / acid compartment contains 1.35 mol / l of 2-hydroxy-4- (meth ilthio) butanoic acid, 100% in acid form.

The faradic yield is estimated at 71% and the energy consumption of 0.53 kWh per kg of acid formed.

Example 13 The electrolyser used is constituted by a stack of 8 cells of identical configuration to that of example 12.

The "base" compartment is initially filled with 5.27 liters of a 1% solution of ammonium sulfate.

The "salt / acid" compartment is initially filled with 4.85 liters of a 1.39 mol / l solution of the ammonium salt of 2-hydroxy-4- (methylthio) butanoic acid.

The recirculation flow of solutions is initially set at 60 1 / h for the salt / acid compartment and at 150 1 / h for the base compartment.

The electrolysis is carried out discontinuously (operation in recirculation), at an average temperature of 40 ° C. The intensity was imposed at 9 A, with a current density of 0.45 kA / m2.

After 172 minutes of operation, the conductivity of the salt / acid compartment increased from 59 to 9.5 mS / cm.

The final volume of salt / acid compartment is 4.67 liters.

The composition is as follows: 2-hydroxy-4- (methylthio) butanoic acid: 1.24 mol / 1 ammonium 2-hydroxy-4- (methylthio) butanoate: 0.148 mol / 1 The transformation rate is 86%. The final product is 89% in acid form.

Faaric performance is 74%. The energy consumption is estimated at 0.58 kWh per kg of acid formed.

Example 14 A device analogous to that of example 13 is used.

The "base" compartment is initially filled with 5.46 liters of a 1% solution of ammonium sulfate.

The "salt / acid" compartment is initially filled with 5.23 liters of a 1.24 mol / 1 solution of the ammonium salt of 2-hydroxy-4- (methylthio) butanoic acid.

The recirculation flow of the solutions is initially set at 90 1 / h for the salt / acid compartment and at 150 1 / h for the base compartment.

The electrodialysis is carried out discontinuously (recirculation operation), at an average temperature of 40 ° C. The intensity was imposed at 14 A, with a current density of 0.7 kA / m2.

After 105 minutes of operation, the conductivity of the salt / acid compartment increased from 57.6 to 9.8 mS / cm.

The final composition of the salt / acid compartment is as follows: 2-hydroxy-4- (methylthio) butanoic acid: 1.28 mol / 1 2-hydroxy-4- (methylthio) butanoate ammonium: 0. 14 mol / 1 The product is thus 90% in acid form.

Example 15 A device analogous to that of example 13 is used.

The test is used with the content of the base compartment obtained from the output of example 14.

The "salt / acid" compartment is initially filled with 5.2 liters of a 1.44 mol / l solution of the ammonium salt of 2-hydroxy-4- (methylthio) butanoic acid.

The recirculation flow of the solutions is initially set at 50 1 / h for the salt / acid compartment and at 150 1 / h for the base compartment.

The electrodialysis is carried out discontinuously (operation in recirculation), at an average temperature of 42 ° C. The intensity was imposed at 19 A, with a current density of 0.95 kA / m2.

After 53 minutes of operation, the salt / acid compartment conductivity went from 59 to 27 mS / cm.

The final volume of the salt / acid compartment is 4.85 liters.

The composition is as follows: 2-hydroxy-4- (methylthio) butanoic acid: 0.87 mol / 1 ammonium 2-hydroxy-4- (methylthio) butanoate: 0.56 mol / 1 The transformation rate is 56%. The final product is 61% in acid form.

The faaric efficiency is 83%. The energy consumption is estimated at 0.7 kWh per. kg of acid formed.

Example 16 200.1 g of HMTBS solution are concentrated in batch to approximately 1.5 M in a rotary evaporator. The temperature of the bath is 45 +/- 5 ° C. The pressure is regulated in the vicinity of 2.5.103 Pa (25 mbar). The temperature in the kettle evolves from 25 ° C at the start of the distillation to 40 ° C at the end. At the end of 3 hours, 41.9 g of a yellow viscous medium of which the characteristics are the following are recovered: After the title adjustment (dilution with H20), a product is obtained in which the characteristics are the following: By potentiometry we have the following mass distribution: In HPLC, the ratio of the monomer or dimers / surfaces (monomer + dimers) monomer / (monomer + dimers) 100 dimers / (monomer + dimers) (*) 0 is measured (*) no dimers are detected Example 17 200.0 g of HMTBS solution is concentrated in batch to approximately 1.5 M of a rotary evaporator. The bath temperature is 121 +/- 5 ° C. The pressure is regulated around 9.5.104 Pa. The temperature in the kettle evolves from 100 ° C at the beginning of the distillation to 113 ° C at the end. At the end of 3 hours, 41.5 g of a brown viscous medium are recovered in which the characteristics are the following: After the title adjustment (dilution with H20), a product is obtained in which the characteristics are the following: By potentiometry, we have the following mass distribution: In HPLC, the ratio of the monomer or dimers / surfaces (dimers + monomer) is measured monomer / (monomer + dimers [95.0 dimers / (monomer + dimers) 5.0 Example 18 To 100.0 g of the medium described in Example No. 17, 40.0 g of H20 are added. The medium is extracted with 75.0 g of isopropyl ether. After phase separation, 25.6 g of HMTBA are recovered in the organic phase after evaporation, in which the characteristics are the following: Example 19: aging of the solutions described in the previous examples The solutions of examples 16 and 17 did not evolve in their appearance in dimers after 40 days of storage.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, the content of the following is claimed as property.

Claims (23)

1. Preparation process of 2-hydroxy-4-methylthio-butyric acid and / or of the ammonium salt of 2-hydroxy-4-methylthio-butyric acid by enzymatic hydrolysis of 2-hydroxy-4-methylthio-butyronitrile, characterized in that: a) in a first stage a biological material having a nitrilase activity is prepared, b) in a second stage, it is immobilized, c) in a third step, 2-hydroxy-4-methylthiobutyronitrile is put in presence with the biological material thus immobilized, to obtain the ammonium salt of 2-hydroxy-4-methylthiobutyric acid, d) in a fourth step, optionally, the salt obtained in step c) is converted into the corresponding acid, and e) in a fifth stage, the product obtained in step c) or d) is concentrated.

2. Process according to claim 1, characterized in that the nitrilase activity is obtained from a nitrilase of Al cali genes, preferably faecali s.

3. Process according to claim 1 or claim 2, characterized in that the genetic information encoding a nitrilase that is expressed in a host microorganism is used.

4. Process according to any of claims 1 to 3, characterized in that the host microorganism is chosen from Escheri chi a coli or a member of the genus Ba ci llus, Coryneba cteri um, Streptomyces, Sa ccharomyces, Kl uyveromyces, Peni cilli um and Aspergill us.

5. Process according to claim 1, characterized in that the nitrilase activity is obtained from the nitrilase encoded by the gene cloned in the plasmid pRPA-BCAT3, deposited in the CBS under the number CBS 998-96.

6. Process according to any of claims 1 to 3, characterized in that the nitrilase is co-expressed with an accompanying protein.

7. Process according to claim 5, characterized in that the accompanying protein is GroESL in E. coli

8. Process according to any of the preceding claims, characterized in that the biological material of step a) is immobilized on a solid support.

9. Process according to claim 8, characterized in that the solid support has a granulometry comprised between 1 μm and 3 mm, preferably 10 μm and 2 mm, and because it is added at a ratio of 0.01 to 500%, preferably 10 to 200% in weight of the biological material.

10. Process according to claim 9, characterized in that the solid support is chosen from: - ion exchange resins, - alumina, - synthetic silicas or diatoms and silica gels, - zeolites, - carbons, - proteins partially soluble in water, and - polysaccharides.

11. Process according to claim 10, characterized in that the solid support is gluten.

12. Process according to claim 8, characterized in that one or more chemical agents are used to crosslink and / or insolubilize the biological material and the support.

13. Process according to claim 12, characterized in that the chemical agents are chosen from: - polyacetidine polymers, - polyethyleneimine polymers, - polymers of polyamides, - polymers of isocyanates, - gels of alginate, - gels of k-carrageenan, - amines, - aldehydes, - carboxylic acids , and - the isocyanates.

14. Process according to any of claims 10 to 13, characterized in that the biological material and the support are mixed in the presence of chemical agents to obtain a paste that is extruded and then dried.

15. Process according to any of claims 10 to 13, characterized in that the biological material and the chemical agents are mixed and then deposited in the support in the form of a thin layer.

16. Process according to any of the preceding claims, characterized in that a mixture of HMTBS and HMTBA containing at least 60% of HMTBA, and preferably at least 80% of HMTBA, the complement that is of HMTBS, is obtained by electrolytic dissociation of the corresponding ammonium salt.

17. Process according to claim 16, characterized in that the dissociation is carried out in an electrodialyzer of bipolar membranes with three compartments.

18. Process according to claim 16, characterized in that the dissociation is carried out in an electrodialyzer of two-compartment bipolar membranes.

19. Process according to any of claims 1 to 15, characterized in that a mixture of HMTBS and HMTBA is obtained by heating an ammonium salt solution.

20. Process according to claims 16 to 19, characterized in that the ammonia released is distilled, concentrated and recycled in the synthesis of HCN.

21. Aqueous solution obtained according to claims 16 to 19 constituted of a mixture of HMTBA and HMTBS in which the weight ratio HMTBA / (HMTBA + HMTBS) is from 5 to 99.9% and preferably from 10 to 50%.

22. Installation for employing the process according to the invention, characterized in that it comprises > - one or several reactors packed with immobilized enzymes having a nitrilase activity, or immobilized host microorganisms, which express an enzyme of nitrilase activity, - optionally, one or several means of electrodialysis, - means of concentration of the final product.

23. Plasmid pRPA / BCAT3, deposited in the CBS under the number CBS 998/96.