JPH0773503B2 - Biological production method of amide - Google Patents

Description

Description: BACKGROUND OF THE INVENTION The present invention relates to the hydration of nitriles in an aqueous medium by the action of a microbial-derived enzyme capable of hydrating the nitrile to form the corresponding amide. And to convert it to the corresponding amide. More specifically, the present invention is
It relates to a method for biologically producing an amide characterized by the presence of a specific surfactant in the aqueous medium.

(Prior Art) An amide is produced by hydration of a corresponding nitrile in an aqueous medium, and a method has been proposed in which this hydration is carried out by the action of an enzyme (nitrilase or nitrile hydratase) produced by a microorganism (for example, , Japanese Shogun 62-
21519, JP-B-56-17918, JP-B-59-37951, JP-A-61-162192 and JP-A-62-91189, and Japanese Patent Application No. 63-231744). The biological production method of such an amide is also practiced industrially, and is attracting attention as an advantageous production method of acrylamide.

Although many nitriles such as aliphatic and aromatic have been proposed as a substrate nitrile that can be used in the biological hydration reaction of such amides, according to the findings of the present inventors,
When a large amount of substrate that cannot be completely dissolved in water is present in the reaction system, microbial cells, substrate, products and the like are involved during the reaction to form a lump, which makes uniform stirring impossible. This phenomenon is particularly remarkable when a poorly water-soluble substrate such as an aromatic nitrile is used, and such nitrile hydration is industrially difficult to carry out.

[Outline of Invention]

(Summary) As a result of various studies aimed at solving the above problems, the present invention shows that a reaction system containing a polyoxyethylene group-containing surfactant is present in the reaction system during the nitrile hydration reaction. It has been made based on this finding that not only can it be maintained in a uniformly dispersed state, but also that the hydration reaction rate can be greatly improved.

Therefore, the method for producing an amide according to the present invention comprises the step of hydrating a nitrile in an aqueous medium by the action of a microorganism-derived enzyme capable of hydrating the nitrile to form the corresponding amide, thereby converting the nitrile into the corresponding amide. In the method for converting, a surfactant having a polyoxyethylene group is present in the aqueous medium.

(Effects) According to the present invention, the hydration reaction of the sparingly water-soluble nitrile is maintained in a uniform dispersed state by the action of the specific surfactant, and the hydration reaction can be carried out without causing troubles such as stirring. At the same time, the hydration reaction rate of nitrile can be greatly improved. It can be understood that it was totally unexpected that the improvement of the hydration reaction rate was observed in the nitrile which is hardly soluble in water and also in the nitrile which is easily soluble in water.

As described above, according to the present invention, the hydration of the aromatic nitrile can be particularly advantageously performed. 2,6 as a pesticide raw material
-Difluorobenzamide (that is, hydrate of 2,6-difluorobenzonitrile) or benzamide (that is, hydrate of benzonitrile) which is useful as a raw material for various organic industrial chemicals. It is useful.

[Specific Description of the Invention]

1. Basic Content of Biological Production of Amide The present invention is a method of hydrating a nitrile by the action of a microorganism-derived enzyme to convert this into an amide. It consists of the induction of the enzyme and the action of the resulting enzyme on the substrate nitrile.

These are known per se as a unit operation, and any mode suitable for the present invention can be adopted.

The "method of hydrating a nitrile to convert it into a corresponding amide by the action of a microorganism-derived enzyme capable of hydrating the nitrile to form the corresponding amide in an aqueous medium" which is a premise of the present invention. Includes any mode in which the action of the enzyme is purposeful. As one of such embodiments, there is a method of recovering an enzyme produced by a microorganism and using this as an enzyme preparation. However, even in the case of such a normal catalytic reaction, the present invention provides ""Biological manufacturing method".

2. Details of Hydration Reaction 1) Microorganisms Microorganisms used in the present invention include Bacillus and Bacteri (Bacteri) described in the above-mentioned publications and the like.
dium), Micrococcus, Brevibacterium, Corynebacterium
nebacterium), Nocardia, Pseudomonas, Rhodococcus,
It belongs to each genus of Arthrobacter and Microbacterium,
In particular, the nitrile hydratase enzyme produced by the microorganism of the genus Rhodococcus rhodochrous described in Japanese Patent Application No. 63-234744 (Rhodococcus rhodochrous) is particularly effective for hydration of aromatic nitriles and has excellent stability. It is more suitable to have.

A representative of this type of strain is the J-1 strain.

Details of the J-1 strain are as follows.

(1) Origin and Deposit The J-1 strain was collected by the present inventors from soil in Sakyo Ward, Kyoto City and deposited on September 18, 1987 at the Institute for Microbial Technology, Institute of Industrial Science and Technology. Obtained the accession number of FERM BP-1478.

(2) Bacteriological properties (a) Morphology (1) Cell shape and size 0.9 to 1.0 μ × 3 to 10 μ (2) Presence or absence of polymorphism in cells It develops without snapping and with snapping, and later ruptures into a short rod-like shape.

(3) Motility None (4) Presence or absence of spores None (5) Gram stainability positive (6) Anti-acid negative (7) Metachromatic bodies are observed (b) Growth state in each medium (30 ° C) (1) Meat broth agar plate culture 1mm diameter (48 hours) round, irregular, smooth and dry on the surface, flat, opaque, pale orange pink (2) Slope culture on broth agar, filamentous, smooth on the surface, slightly bumpy and dry, Light orange pink (3) Liquid culture of broth A pellicle is formed and grows vigorously. Medium turbidity and precipitation occur as it grows.

(4) Meat broth gelatin stab culture It grows well on the surface and grows like a funnel along the stab, but hardly grows in the lower layer. Liquefaction of gelatin is not observed.

(5) Litmus milk unchanged (c) Physiological properties (1) Nitrate reduction positive (2) Denitrification reaction negative (3) MR test negative (4) VP test negative (5) Indole formation positive (6) Sulfide Generation of hydrogen Positive (7) Hydrolysis of starch Negative (8) Utilization of citric acid Coser's medium: Negative Christensen's medium: Positive (9) Inorganic nitrogen source utilization Nitrate: Positive Ammonium salt: Positive (10) Dye formation Negative (11) Urease positive (12) Oxidase negative (13) Catalase positive (14) Cellulose hydrolysis negative (15) Growth range pH: 5-10 Temperature: 10-41 ° C (16) Attitude toward oxygen Aerobic ( 17) Tyrosine degradation positive (18) Adenine degradation positive (19) Phosphatase positive (20) Tween80 hydrolysis positive (21) OF test 0 (weak) (22) Heat resistance (10% skimmed (23) in water, 15 minutes) None (23) Production of acid and acid from sugars Production of gas Production of gas L-arabinose-D-xylose-D-glucose + -D-mannose-D-fructose +- Maltose + -sucrose + -lactose-trehalose-D-sorbit + -D-mannitol + -glycerin +-(24) Growth as a single carbon source Inositol-maltose + D-mannite + rhamnose-D- Sorbit + m-hydroxy Benzoic acid + Sodium adipate + Sodium benzoate + Sodium citrate + Sodium lactate + Testtetron + L-tyrosine + Glycerol (1%) (+/-) Trehalose (+) p-Hydroxy + Benzoic acid (1%) (W / V) (+) Weak but positive.

(25) Fatty acid and cell wall analysis Includes unsaturated, saturated linear fatty acid, and tuberculostearic acid. Mycolic acid TLC gives a single spot.

Based on the above bacterial characteristics, Bergy's Bacterial Classification Document (Bergy's M
When classified based on the anual of Systematic Bacteriology (1986), the J-1 strain is an aerobic, gram-positive, weak acid-fast, catalase-positive, endospore-free bacillus that does not set flagella. In addition, in the early stage of development, it appears to be a Nocardia-type bacterium by exhibiting long-rod-like fungal shape, showing growth accompanied by branching (Branching), and later rupturing into short-rod fungi. .

Analysis of fatty acid composition includes unsaturated, saturated straight chain fatty acids including tuberculostearic acid. Mycolic acid TLC is distinguished from Mycobacterium because it gives a single spot showing the same Rf as the standard strain Rodococcus rhodochrous (IFO 3338). In addition, from the composition (carbon number) of mycolic acid
Distinguished from the genus cardia. From the examination of other biochemical properties, this bacterium is recognized as Rhodococcus rhodochous.

2) Substrate / Nitrile The nitrile serving as a substrate for the enzyme produced by the microorganism as described above is an aromatic or aliphatic mononitrile or dinitrile, especially mononitrile.

Aromatic nitrites, especially those having 4 to 10 carbon atoms forming an aromatic ring, are the ones who enjoy the features of the present invention most. Some specific examples of the aromatic nitrile are shown below, and compounds represented by the following general formulas [I] to [VI] can be mentioned.

For example, 4-, 3-, and 2-cyanopyridine.

(Here, R ¹ and R ² are H, CH ₃ , OH, and OCH, respectively.
₃ , Cl, F, CN, NH ₂ or NO ₂ . ) For example, benzonitrile, o-, m- and p-chlorobenzonitrile, o-, m- and p-fluorobenzonitrile, o- and m-nitrobenzonitrile, p-
Aminobenzonitrile, o-, m- and p-tolunitrile, 4-cyanophenol, anisonitrile, phthalonitrile, isophthalonitrile, terephthalonitrile,
They are 2,6-dichlorobenzonitrile, 2,4-dichlorobenzonitrile and 2,6-difluorobenzonitrile.

For example, α- and β-naphthonitrile.

(Where X is S or O) For example, 2-thiophenecarbonitrile and 2-furonitrile.

It is ril and 2-furonitrile.

For example, 5-cyanoindole, 3-indoleacetonitrile.

That is, it is cyanopyrazine.

Another group of nitriles of interest in the present invention are aliphatic nitriles. Suitable are mono- or dinitriles having 2 to 6 carbon atoms, especially mononitrile. Acrylonitrile is a typical example of the usefulness of the produced amide, and the productivity is good.

Needless to say, the amides corresponding to these nitriles are those in which the CN group has been converted into a CONH ₂ group. In addition,
In the case of dinitriles, the conversion of at least one of the CN groups to CONH ₂ shall be considered as the corresponding amide.

3) Surfactant The surfactant used in the present invention has a polyoxyethylene group, for example, an alkyl ether type,
Examples thereof include alkylaryl ether type, alkyl ester type, sorbitan monoalkyl ester type, polyoxyethylene alkylamine and the like. Specifically, polyoxyethylene stearyl ether (n = 14) [Emulgen 320P (Kao Corporation)] as the alkyl ether type, and polyoxyethylene octyl phenyl ether (n = 10) as the alkyl allyl ether type. [Triton X100 (Wako Pure Chemical Industries, Ltd.), sorbitan monoalkyl ester type sorbitan polyoxyethylene monooleate (n = 20) [Tween 80
(Hanui Chemical Co., Ltd.), polyoxyethylene dodecylamine (n
= 5) [Amiet 105 (Kao Corporation)] and the like.

The amount of these surfactants used varies depending on the type of substrate and tends to increase as the concentration of the substrate in the reaction system increases, but it is usually in the range of 0.1 to 5% by weight relative to the reaction system.

4) Culture / Production of Enzyme The culture of the microorganism used in the present invention is not limited as long as it is purposeful, and can be performed according to the methods described in the above-mentioned publications.

5) Hydration of Nitrile The "method of hydrating nitrile by the action of a microorganism-derived enzyme to convert it into a corresponding amide", which is a prerequisite for the present invention, is a purposeful method for the action of the enzyme. It has been described above that it includes various aspects.

One of such embodiments is to allow the substrate nitrile to be present in the culture system of the microorganism to form an amide in the medium.

Another one of the modes in which the enzyme acts is to add a substrate nitrile to the culture medium in which the enzyme is accumulated to cause a hydration reaction. As a modified example of this embodiment, there may be mentioned a method of using a culture solution in which cells are crushed.

Yet another embodiment of the action of the enzyme is a method of separating the bacterial cells accumulating the enzyme from the culture solution, preferably supporting this on a suitable carrier and "immobilizing", and contacting with the substrate. Is. It can be said that this method, particularly this preferred embodiment, is suitable for industrial implementation, alongside the second embodiment or more than the second embodiment. This technique is well known, including the type of carrier and the method of loading the microorganisms, and also the use of immobilized microorganisms as so-called bioreactors.

Another one of the modes of making the enzyme act is to obtain an enzyme preparation,
This enzyme is a non-biological method of hydrating nitriles. It goes without saying that the hydration reaction is carried out under pH and temperature conditions in which enzyme activity is not lost, and these conditions are generally the same as those in the above-mentioned biological method. As described above, the aspect in which the microorganism does not exist at the time of enzyme action is treated as the "biological production method" in the present invention.

The substrate concentration in the culture solution or hydration reaction solution varies depending on the type of the substrate, but is usually 0.5 to 15 mol / liter, the reaction temperature is usually in the range of 0 to 30 ° C, and the optimum pH is 7 to
The range is 9. For adjusting the pH, it is desirable to use a buffering agent such as potassium phosphate buffer, Tris / HCl buffer, HEPES / KOH buffer, sodium borate buffer.

3. Experimental Example In the following experimental example, one unit (U) of enzyme activity is defined as the amount of enzyme that produces the corresponding amide from each substrate at a rate of 1 μmol / min under each reaction condition. is there.

Example 1 Prepared with or without addition of a predetermined amount of 2,6-difluorobenzonitrile 40 mM and the surfactants shown in Table 1.
To 5 ml of 0.1 M phosphate buffer (pH 7), 185 μg of an enzyme (nitrile hydratase) purified and isolated from Rhodococcus rhodochrous J-1 was added, and the reaction was carried out at 20 ° C. for 30 minutes with stirring. Enzyme activity was quantified by high performance liquid chromatography (HPLC) for the formed amides, and the activity was shown as the mutual activity when the activity in the case where no surfactant was added was 100. The reaction was carried out in the state where most of the substrate was insoluble, but in the systems to which the surfactant was added, the substrate was uniformly dispersed and a good stirring state was obtained.

The results are shown in Table-1.

The conditions for culturing the J-1 bacterium and for the purification and isolation of the enzyme are as follows.

(1) Culture Basic medium (in 1) Glucose 10 g 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 Peptone 7.5 g Distilled water balance (pH 7.2) CoCl ₂ 7.5 g / liter of urea was added to the above basic medium containing 10 ml / liter, and 4 ml of a pre-culture liquid (using the above basic medium) of Rhodococcus rhodochrous J-1 strain (FERM BP-1478) was added to 60 ml of this medium. Shaking culture was performed at 28 ° C. for 96 hours.

(2) Preparation of cells for reaction Cells were collected by centrifugation, washed with a 0.85% NaCl solution, centrifuged again, and then suspended in a 0.85% NaCl solution to obtain a wet cell solution for reaction.

(3) Preparation of enzyme for reaction The cells cultured by the above method were collected by centrifugation and then homogenized to prepare a cell-free extract. The purified enzyme was prepared by performing the ammonium sulfate fractionation and various chromatographies shown in the table below. The obtained enzyme was used as a reaction enzyme.

Example 2 Rod Coccus rhodochrous J-obtained in the same manner as in Example 1 in 5 ml of 0.1 M phosphate buffer (pH 7) containing various substrates shown in Table 2 at a predetermined concentration using the surfactant Amate 105. Add 0.1 to 4 mg (dry weight) of one cell and stir
The reaction was performed at 20 ° C for 20 to 30 minutes, and the relative activity was measured in the same manner as in Example 1.

Table 2 shows the surfactant concentration when the maximum activity was obtained and the relative activity at that time.

In addition, benzonitrile (10 mM), 3-cyanopyridine,
For acrylonitrile and propionitrile, these were dissolved, and most of the other substrates were insoluble, but even in this case, the system in which the surfactant was added showed uniform dispersion of the substrates and good results. A stirred state was obtained.

Activity without addition of surfactant Example 3 0.85 mg (dry weight) of Rhodococcus rhodochrous J-1 cells obtained in the same manner as in Example 1 was added to 0.1 M phosphate buffer (pH 7) with or without addition of amate 105 in a predetermined amount. To 4.5 ml, and to this suspension was added 0.5 g of benzonitrile and maintained at 20 ° C. with stirring. The progress of the reaction was examined by HPLC analysis of benzonitrile and benzamide formed, and the reaction was continued until the benzonitrile was completely consumed.

In this reaction, most of the substrate was reacted in an insoluble state, but in the system to which the surfactant was added, the substrate was uniformly dispersed and a good stirring state was obtained. On the other hand, in the system to which the surfactant was added, the cells, the substrate and the product were entangled with each other to form a lump and the stirring could not be performed well.

The results are shown in Table-3.

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Claims (5)

[Claims] 1. A process for hydrating a nitrile to convert it into a corresponding amide by the action of a microorganism-derived enzyme having the ability to hydrate the nitrile to produce the corresponding amide in an aqueous medium. A method for producing an amide biologically characterized by the presence of a surfactant having a polyoxyethylene group in an aqueous medium. 2. The method for biologically producing an amide according to claim 1, wherein the microorganism is a J-1 strain (FERM BP-1478) of Rhodococcus sp. 3. A nitrile has 4 carbon atoms forming an aromatic ring.
The method for biologically producing an amide according to any one of claims 1 to 2, wherein the aromatic nitrile is from 10 to 10. 4. An aromatic nitrile is represented by the following general formula [I]:
The method for biologically producing an amide according to claim 3, which is any of the compounds represented by [VI]. (Here, R ¹ and R ² are H, CH ₃ , OH, OC, respectively.
H ₃ , Cl, F, CN, NH ₂ or NO ₂ ) (Where X is S or O) 5. The method for biologically producing an amide according to claim 1, wherein the nitrile is an aliphatic nitrile having 2 to 6 carbon atoms.