WO2003066800A2 - Method for making amides in the presence of a nitrile hydratase of microbiological origin and novel micro-organism - Google Patents

Abstract

The invention concerns a method for making amides which consists in hydrating a nitrile into a corresponding amide, in the presence of a nitrile-hydratase of microbiological origin. The invention is characterized in that said nitrile hydratase is produced by a bacterium belonging to the genus Rhodococcus pyridinovorans.

Description

PROCESS FOR THE MANUFACTURE OF AMIDES IN THE PRESENCE OF A NITRILE HYDRATASE OF MICROBIOLOGICAL ORIGIN AND NEW MICROORGANISM.

The invention firstly relates to a process for the production of amides obtained by hydration of a nitrile in the presence of a nitrile hydratase of microbiological origin. More specifically, the invention relates to a process for the manufacture of amides in the presence of a nitrile hydratase produced by a bacterium belonging to the species Rhodococcus pyridinovorans. It then relates to a method of cultivating bacteria belonging to the species Rhodococcus pyridinovorans. The invention finally relates to a specific bacterial strain belonging to the species Rhodococcus pyridinovorans.

Document FR-A-2 294 999 describes a process for the preparation of an amide by hydrolysis of the corresponding nitrile which consists in subjecting the nitrile in aqueous solution to the action of bacteria having a hydratasic nitrile activity. As a bacterium with hydratasic nitrile activity, the genus Brevibacterium, as defined in the sense of Bergey and more particularly the strain R312, is described in particular.

Document EP-188 316 describes a process for the manufacture of amides in the presence of a nitrile hydratase of microbiological origin, in particular of acrylamide, from a mononitrile containing from two to six carbon atoms. As microorganisms, bacteria belonging to the genus Rhodococcus or to the genus Microbacterium are specifically described. However, the process as described in this document remains effective only for the hydration of lower aliphatic nitriles, the activity on aromatic nitriles remaining very low (see example 2). To solve this problem, document EP-A-307 926 describes a specific strain belonging to the genus Rhodococcus, more precisely the strain

Rhodococcus rhodochrous Jl (FERM BP-1478) which, in the presence of cobalt ions, hydrates nitriles. The concentration of CoCl ₂ -6H ₂ 0 in the culture media is 10 mg / 1.

Patent EP-A-362829 presents a method for producing cells of the bacteria Rhodoccocus rhodochrous which makes it possible to obtain a high hydratasic nitrile activity by adding urea, or a derivative, to the culture medium of the bacteria. The culture of bacteria is systematically started in the presence of cobalt ion and urea, or its derivatives.

Consequently, the bacterium Rhodococcus rhodochrous J-1 described in the two aforementioned documents, by virtue of its high activity, has become, for the person skilled in the art, the reference bacterium in terms of hydratasic nitrile activity.

Document GB-A-2 290 295 also describes the use of a strain of Rhodococcus rhodochrous deposited with the Russian national collection of microorganisms under the number VKM Ac-1515D. This strain is used as a source of nitrile hydratase, for the biological manufacture of amides, in the absence of an inducer, which leads us to believe that the hydratasic nitrile activity of this bacterium is constitutive.

YOON & al. described in the document Int J Syst. Evol. Microbiol. 2000 Nov., 50 Pt, 6: 2173-80, a new bacterial strain designated "PDB9T" having given rise to the identification of a new species, namely the pyridinovorans species belonging to the genus Rhodococcus. The strain PDB9 was deposited with the Korean collection of cultures under the number KCTC 0647 BP as well as with the Korean center of culture of microorganism under the number KCCM 80005. The partial sequence of TARN 16S ribosomal is furthermore available in the GENBANK gene bank under accession number AF 173005. The document YOON indicates that Rhodococcus pyridinovorans is capable of degrading pyridine. As will be demonstrated in the examples, it turns out that the strain PDB9 does not exhibit nitrile hydratase activity.

The document Précigou et al "rapid and specifies identification of nitrile hydratase (NHase) -encoding genes in soil samples by polymerase chain reaction"

FEMS Microbiology letters 204 (2001) 155-161 indicates that out of 13 strains

Rhodococcus, tested only 2 presents the nitrile hydratase gene and therefore a hydratasic nitrile activity.

However, the Applicant has discovered that, quite surprisingly, Rhodococcus pyridinovorans exhibited a high nitrile hydratase activity, by making a microorganism advantageously usable in a process for the biological manufacture of amides.

Consequently, the invention relates to a process for the manufacture of amides consisting in hydrating a nitrile into a corresponding amide, in the presence of a nitrile hydratase of microbiological origin characterized in that said nitrile hydratase is produced by a bacterium belonging to the 'Rhodococcus pyridinovorans.

Even if the bacteria of the pyridinovorans species belong to the same genus Rhodococcus as the bacteria of the rhodochrous species, which exhibit a high nitrile hydratase activity, it was not however obvious that pyridinovorans was also endowed with a high nitrile hydratase activity. Indeed, to date, only the rhodochrous species has allowed industrial development for the production of amides such as acrylamide. The other major species of the genus Rhodoccocus such as erythropolis or equi exhibit only marginal activities, in particular for the production of acrylamide. In addition, the Applicant has demonstrated that the hydratase nitrile activity can be improved by applying specific culture conditions, in particular by using concentrations and / or times of addition of a carbon source, of a metal ion. and a selective inductor.

Thus and according to a first characteristic, the bacteria of the invention are cultivated in a medium containing cobalt ions (Co ²⁺ and / or Co ³⁺ ). Cobalt can be added as cobalt salts or organic compounds. It can be added all at once or continuously, at the start or during cultivation, preferably sequentially. In practice, the final cobalt concentrations (CoCl ₂ -6H ₂ O base) are between 10 and 40 mg / L and preferably between 15 and 30 mg / L.

To further improve the activity, the bacteria are cultured in the presence also of an inducer chosen from the group of amides or nitriles. Most amides (including urea and its derivatives) can be used as an inducer of nitrile-hydratase activity. These inducers can be added alone or as a mixture, in 1 step, sequentially or continuously, at the start or during the culture.

In practice, the inducer is chosen from the group comprising crotonamide, urea, propionamide, acetamide, n-butyramide, isobutyramide, n-valeramide, n-capronamide, methacrylamide and cyclohexanecarboxamide.

Certain nitrile-type inducers can also be used, such as: acetonitrile, propionitrile, isobutyronitrile.

It is preferable to use an inexpensive inducer which is not or little metabolized (urea or its derivatives). In practice, the final concentrations of urea or its derivatives are between 5 and 50 g / L, preferably greater than 10 g / L.

In other words, the Applicant has demonstrated that the nitrile hydratase activity of the bacteria of the invention is improved under standard culture conditions (CoCl ₂ -6H ₂ 0> 10 mg / 1; urea preferably> 10 g / 1 ) distinct from those used for Rhodococcus rhodochrous Jl (CoCl ₂ -6H ₂ O: 10mg / l; urea: 7.5 g / 1) [Optimum culture conditions for the production of cobalt-containing nitrile hydratase by Rhodococcus rhodochrous Jl, Appl. Microbiol. Biotechnol. (1991) 34: 783-788].

The concentration of carbon source also influences the obtaining of a satisfactory nitrile-hydratasic activity. In practice, sources of C are used at nominal concentrations varying from 5 to 50 g / L, preferably from 12 to 30 g / L, added alone or as a mixture, in 1 batch, sequentially or continuously, at the start or during cultivation. Without limitation, as a carbon source, mention will be made of D-Glucose, Pyruvate, D-Sorbitol, etc.

In practice, predetermined amounts of urea, cobalt and a carbon source are added to the basal medium. The culture is carried out at a temperature between 15 and 50 ° C, preferably around 28 ° C, at a pH between 6 and 9 lasting at least 96 hours (up to 200 hours).

In a particular embodiment, the bacterium used is the strain MW3 belonging to the species Rhodococcus pyridinovorans and deposited on January 24, 2002 at the CNCM (National Collection of Culture of Microorganisms), Institut Pasteur, 25 rue du Dr. ROUX, PARIS , France, under the number CNCM 1-2772, and the mutants of this strain. The mutants of the above strain indeed fall within the scope of the invention when they exhibit nitrile hydratase activity. In an advantageous embodiment, the microorganism can be immobilized using methods and procedures known to those skilled in the art. These methods include, for example, immobilizations by inclusion on gel (polyacrylamide, algae extracts, alginates ...) as well as immobilizations on supports by adsorption or by covalent bonds (supports of mineral origin (alumina, brick, glass , clay ...) or organic (exchange resins, collagen, cellulose ...).

According to another characteristic, the substrates on which Rhodococcus pyridinovorans act can be aliphatic nitriles as well as aromatic nitriles. Mention will be made, by way of example without limiting the invention, of compounds such as acetonitrile, propionitrile, methacrylonitrile, acrylonitrile, benzonitrile, 3-cyanopyridine, 3-hydroxy isovaleronitrile, 3-hydroxypropionitrile , 2-hydroxy 4- methylthiobutyronitrile ...

It will be noted that the production of amide obtained by hydration of a nitrile in the presence of the nitrile hydratase produced by the microorganism of the present application can be carried out both by continuous and discontinuous processes (called "batch").

The invention also relates to a method for cultivating bacteria belonging to the species Rhodococcus pyridinovorans. According to a first characteristic, the culture medium contains cobalt ions Co2 + and / or Co3 + in concentrations of between 10 and 40 mg / L (base CoCl ₂ -6H ₂ O), preferably between 15 and 30 mg / L. According to an advantageous embodiment, the medium also contains urea or its derivatives in final concentrations of between 5 and 50 g / L, preferably greater than 10 g / L. According to a preferred embodiment, the medium also contains a carbon source whose nominal concentrations vary between 5 and 50 g / L, preferably between 12 and 30 g / L.

The invention also relates to the strain MW3 belonging to Rhodococcus pyridinovorans deposited on January 24, 2002 at the CNCM under the number CNCM 1-2772 and the mutants of this strain.

The invention and the advantages which result therefrom will emerge more clearly from the following exemplary embodiments.

Figure 1 is a liquid chromatography assay of acrylamide formed using M W3 as a nitrile hydratase.

Figure 2 is a liquid chromatography assay of acrylamide formed using PBD9 as nitrile hydratase.

1 / Characterization of the MW3 strain a / Culture medium

MUieu basai:

K ₂ HPO ₄ 0.5 g KH ₂ PO ₄ 0.5 g MgSO ₄ , 7H ₂ O 0.5 g Yeast extract 1.0 g Casein tryptone 7.5 g Deionized water qs 1 liter (pH 7.2) Metallic ion Co ²⁺ : 10 mg / L ( CoCl ₂ -6H ₂ O) Urea inducer: 7.5 g / L Carbon source D-Glucose monohydrate 10 g / L b / Properties of MW3

Type of colony: gram + colonies pigmented in pink Biochemical character oxidase - catalase + nitrate reductase + nitrite reductase -

Strict aerobic respiratory metabolism

c / Identification

Method used: - Amplification of the rrs gene encoding 16S rRNA

Sequencing of the rrs gene

Analysis of the 16S rRNA sequence of MW3 shows a percentage of homology equal to 100% with the 16S rRNA sequence of the strain PDB9 accessible in the GENBANK bank under the number AF173005. The homology being 100%, we conclude that MW3 belongs to Rhodococcus pyridinovorans.

2 / MW3 nitrile hydratase activity

Description of the nitrile-hydratase activity measurements carried out:

Definitions

Specific activity:. μmol of amide produced. min ^"1. mg ^{" 1} of bacterial dry weight. Relative activity: activity expressed as a percentage corresponding to the specific or total activity obtained compared to the specific or total reference activity (100%).

Total activity: μmol of amide produced. min ^'1 . g ^"1 of culture medium.

Activity test at 20 ° C

* 100 g reaction solution containing:

- 50g of lOOmM phosphate buffer

- 49g of aqueous nitrile solution respectively:. x grams of nitrile corresponding to the quantity necessary to obtain the desired final concentration. 49 - x g deionized water

* The reaction is triggered by adding 1 g of cell suspension (containing a predetermined quantity of cells centrifuged for 10 min at 10,000 g).

* Sampling of 4 mL after different incubation times and stopping the reaction with 30 μL of concentrated sulfuric acid.

* Determination by liquid phase chromatography of the amide formed.

3 / EXAMPLES

A / EXAMPLE 1

Activity test carried out under optimal standard conditions used for Rhodococcus rhodochrous Jl [Optimum culture conditions for the production of cobalt-containing nitrile hydratase by Rhodococcus rhodochrous Jl, Appl. Microbiol. Biotechnol. (1991) 34: 783-788].

: Activité spécifique relative (%) * : sur la base des résultats du brevet EP 362829 (AS = 497 ; ex 2, tab 1)Culture conditions: CoCl -6H ₂ O: 10 mg / 1; urea: 7.5 g / 1; glucose: 10 g / L; substrate: acrylonitrile 50 mM

: Relative specific activity (%) *: on the basis of the results of patent EP 362829 (AS = 497; ex 2, tab 1)

It is noted that, under standard conditions described as optimal for J1, MW3 exhibits a moderate hydratasic nitrile activity.

B / EXAMPLE 2: Variation of the culture conditions of MW3 as a function of the concentrations and / or the times of addition of the cobalt.

Culture conditions: CoCl ₂ -6H ₂ 0: variable; urea: 7.5 g / 1; glucose: 10 g / L; substrate: acrylonitrile 50 mM

: Activité spécifique relative (%) En multipliant par 2 la concentration en cobalt et en fractionnant son ajout, l'activité de M 3 peut de façon surprenante être multipliée par 4.

: Relative specific activity (%) By multiplying by 2 the concentration of cobalt and by fractioning its addition, the activity of M 3 can surprisingly be multiplied by 4.

C / EXAMPLE 3: Variation in the culture conditions of MW3 as a function of the concentrations and / or the times of addition of the inducer (urea).

Culture conditions: CoCl ₂ -6H ₂ O: 10 mg / 1; urea: variable; glucose: 10 g / L; substrate: acrylonitrile 50 mM

¹ : Relative specific activity (%) Surprisingly, when the addition of urea only occurs after the start of cultivation of MW3 (at least 12 hours) and the quantity used is increased, the hydrile nitrile activity of the bacteria is over multiplied.

D / EXAMPLE 4: Variation of the culture conditions of MW3 as a function of the concentrations and / or the times of addition of the carbon source (glucose).

Culture conditions: CoCl ₂ -6H ₂ O: 10mg / l; urea: 7.5 g / 1; glucose: variable; substrate: acrylonitrile 50 mM

: Total relative activity (%)

Surprisingly, when the amount of carbon source is increased and its addition is fractionated after the start of cultivation of MW3, the hydratasic nitrile activity of the bacteria is amplified. E / EXAMPLE 5: Variation of the substrate (nitrile) used during the activity test. Culture conditions: CoCl ₂ -6H ₂ 0: 10mg / l; urea: 7.5 g / 1; glucose: 10 g / L; substrate: variable

; Activité spécifique relative (%)

; Relative specific activity (%)

MW3 exhibits nitrile hydratase activity on a wide variety of aliphatic, aromatic and unsaturated substrates.

F / EXAMPLE 6: Specific activity measurements (substrate: acrylonitrile 377 mM).

Test l Test 2 Test 3

CoCl2-6H20 20 mg / L 20 mg / L 20 mg / L

Addition time T = 0 T = 0 and 24h T = 0

Urea 2 x 7.5 g / L 2 x 7.5 g / L 2 x 7.5 g / L

Addition time T = 0 and 24h T = 24 and 48h T = 0 and 24h

Glucose 2 x 10 g / L 16 g / L 2 x 10 g / L

Addition time T = 0 and 24h T = 0 T = 0 and 24h

Cultivation time 168h 168h 120h

Concentration of 9 g / L 8.1 g / L 6.2 g / L cells

Amide formation

Specific Activity 340 220 250

Total Activity 3,060 1,760 1,550 Note :

When the addition of urea is carried out entirely at T = 0, the total activity obtained was clearly lower than those observed for the tests presented in the table above (less than 1000).

G / EXAMPLE 7: immobilization

140 g of a MW-3 cell suspension (containing 15% w / w of dry cells in 50 mM phosphate buffer pH 7.0) obtained by fermentation on a nutrient medium as described by Example 6 (test 1) , is immobilized in a polyacrylamide gel according to a process well known to those skilled in the art. The gel thus obtained is ground in a mill in order to obtain after sieving particles of homogeneous size between 500 and 1500 μm. These particles are washed in the pH 7.0 phosphate buffer and then stored in this same buffer.

H / EXAMPLE 8: bioconversion test No. 1

120 g of drained gel obtained according to Example 7 and having a total activity of 15,000 U are taken. 2.895 kg of deionized water containing 148 ppm of sodium acrylate, pH 8.0 are added. The mixture is maintained, with stirring at 200 rpm, at 12 ° C for the first 12 hours and then at 20 ° C for the following 12 hours. 2624 ml of acrylonitrile are added at a constant flow rate of 164 ml / h for 16 h, the pH being regulated by adding 50 mM sodium hydroxide. After a total duration of 24 h of bioconversion, a final solution is obtained having a concentration of 56.4% of acrylamide and with an acrylonitrile residue <50 ppm (verification carried out by High Pressure Liquid Chromatography). The product is separated from the catalyst by filtration over 0.2 μm.

1 / EXAMPLE 9: bioconversion test n ° 2

20 g of MW-3 immobilized and drained as described in Example 7 are added to 811 g of deionized water containing 148 ppm of sodium acrylate, pH 8.0. The temperature of the mixture being maintained at 20 ° C. with stirring of 200 rpm. 189 ml of methacrylonitrile are added at a constant rate for 24 h. After a total duration of 30 h of bioconversion, 19.1% of methacrylamide containing less than 100 ppm of methacrylonitrile are obtained. The product is separated from the catalyst by filtration through 0.2 μm, then crystallized with a yield of 46%.

J / EXAMPLE 10: bioconversion test n ° 3

3 g of a suspension of free cells of MW-3 (310 mg of dry cells in 0.85% NaCl) are added to 687 g of deionized water containing 148 ppm of sodium acrylate, pH 8.0. This mixture being maintained at 20 ° C. with stirring at 200 rpm, 389 ml of acrylonitrile are added at a constant rate during 16 h. After a total duration of 24 h bioconversion, a 42% solution of acrylamide containing less than 50 ppm of residual acrylonitrile is obtained. The cells are separated from the final product by filtration through 0.2 μm.

4 / Absence of PBD9 nitrile hydratase activity

In this example, the nitrile hydratase activity of the strain PBD9 described by Yoon is compared with MW3, under the following standard conditions at 20 ° C:

100g reaction solution containing:

- 50g of 100 mM phosphate buffer

- 49g of aqueous nitrile solution respectively:

- x grams of nitrile corresponding to the quantity necessary to obtain the desired final concentration

- 49-x grams of deionized water

The reaction is started by adding 1 g of cell suspension (containing a predetermined quantity of cells centrifuged for 10 min at 10,000 g) 4 mL sample after 3 min incubation and reaction stopped with 30 μL of concentrated sulfuric acid.

The amide formed is assayed by liquid phase chromatography.

The results appear in Figures 1 and 2. As these 2 figures clearly show, there is no formation of acrylamide using PBD9 (fig 2) unlike M 3 (fig 1).

Claims

1 / Process for the manufacture of amides consisting in hydrating a nitrile into a corresponding amide, in the presence of a nitrile hydratase of microbiological origin, characterized in that said nitrile hydratase is produced by a bacterium belonging to the species Rhodococcus pyridinovorans . 2 / A method according to claim 1, characterized in that the bacteria are cultured in the presence of cobalt ions. 3 / A method according to claim 2, characterized in that the final concentrations of cobalt (base CoCl ₂ -6H2θ) are between 10 and 40 mg / L, preferably between 15 and 30 mg / L. 4 / A method according to the preceding claim, characterized in that the bacteria are grown in the presence of urea or its derivatives. 5 / A method according to claim 4, characterized in that the final concentrations of urea or its derivatives are between 5 and 50 g / L, preferably greater than 10 g / L. 61 Method according to one of the preceding claims, characterized in that bacteria are cultivated in the presence of a carbon source whose nominal concentrations vary between 5 and 50 g / L, preferably between 12 and 30 g / L. Method according to one of the preceding claims, characterized in that the bacterium used is the strain MW3 deposited on January 24, 2002 at the CNCM under the number CNCM 1-2772 and the mutants of this strain. 8 / A method of cultivating bacteria belonging to the Rhodococcus pyridinovorans species, characterized in that the medium contains cobalt ions in concentrations of between 10 and 40 mg / L (base C0CI ₂ .6H ₂ O), preferably between 15 and 30 mg / L. 9 / A method according to claim 8, characterized in that the medium contains urea or its derivatives in final concentrations between 5 and 50 g / L, preferably greater than 10 g / L. 10 / Method according to one of claims 8 or 9, characterized in that the medium contains a carbon source whose nominal concentrations vary between 5 and 50 g / L, preferably between 12 and 30 g / L. 11 / MW3 strain belonging to Rhodococcus pyridinovorans deposited on January 24, 2002 at the CNCM under the number CNCM 1-2772 and the mutants of this strain.