GB2076820A - Process for producing acrylamide or methacrylamide utilizing micro-organisms - Google Patents

Abstract

A process for producing acrylamide or methacrylamide by enzymatic hydrolysis of acrylonitrile or methacrylonitrile in an aqueous medium by the action of micro- organisms having a nitrilase activity at temperatures as low as the freezing point of the medium to 15 DEG C and at pH 6 to 10 so as to conduct the reaction for a long period of time while maintaining a high concentration of acrylamide or methacrylamide.

Description

SPECIFICATION Process for producing acrylamide or methacrylamide utilizing microorganisms The present invention relates to an improved process for producing acrylamide or methacrylamide utilizing micro-organisms.

As a process for producing acrylamide or methacrylamide, a process of reacting acrylonitrile (AN) or methacrylonitrile (MAN) with water has been proposed using reduced copper as a catalyst. However, it has been desired to develop a novel and industrially more advantageous process since catalyts preparation and regeneration is difficult in such a process and the isolation and purification of the amide produced is onerous.

On the other hand, as a process for producing acrylamide or methacrylamide from acrylonitrile or methacrylonitrile utilizing an enzymatic reaction, an interesting process using bacteria belonging to the genus Bacillus, the genus Bacteridium in the sense of Prevot, the genus Micrococcus, the genus Brevibacterium in the sense of Bergy, has been proposed in U.S. Patent 4,001,081. This process is simply based on the discovery that the above-described bacteria hydrolyze various organic nitriles to produce the corresponding organic acid amides. In the case where acrylonitrile or methacrylonitrile (Examples 6-8 in the above-mentioned U.S. Patent) for example are used, this U.S.Patent describes that acrylamide or methacrylamide was obtained almost quantitatively when the acrylonitrile or methacrylonitrile concentration was 8 to 12% by wt, the bacterial cell concentration was 2 to 4% by wt., the pH was 7 to 9, the temperature was 250C and the reaction time was 20 to 30 minutes. It is true that acrylamide or methacrylamide can be produced at a concentration as high as 10 to 20 wt%, but the bacterial cells so rapidly lose their enzymatic activity under such conditions that it is almost impossible to use them repeatedly. In addition, the solution from which the bacterial cells are separated is very dark yellow and contains various impurities originating from the cells, and hence an onerous purifying step is necessary. Thus, the above-described process is not economically advantageously industrial applications.

A catalytic prccess for producing acrylamide or methacrylamide utilizing micro-organisms has been investigated and bacteria having an extremely high activity for hydrolyzing acrylonitrile and methacrylonitrile to produce acrylamide or methacrylamide have been discovered. Namely, the strain N771 and the strain N-774 belonging to the genus Corynebacterium, and the strain N-775 belonging to the genus Nocardia have been found in the soils around a factory producing acrylnitrile and in the waste water discharged from the factory. (Hereinafter the aforementioned bacteria will be referred to as N771, N-774 and N-775, respectively). The enzymatic nitrilase activity of these micro-organisms is surprisingly high at low temperatures.As a result of intensive investigations, a process for the hydrolysis of acrylonitrile and methacrylonitrile has been developed wherein the enzymatic activity of the bacterial cells is stably maintained at a high level for a long time, with the accumulation of produced acrylamide or methacrylamide reaching concentrations as high as 10 wt% or more, which process does not require a difficult purifying step.

According to the present invention, there is provided a process for producing acrylamide or methacrylamide which comprises subjecting acrylonitrile or methacrylonitrile in an aqueous medium to microorganisms having the ability to hydrolyze acrylonitrile or methacrylonitrile to produce acrylamide or methacrylamide, at a temperature ranging from the freezing point of the medium to 1 50C at a pH of 6 to 10.

As the microorganisms used in the present invention, any one that has the ability to hydrolyze acryionitrile or methacrylonitrile to produce acrylamide or methacrylamide may be used regardless of the taxonomic position, as well as the aforesaid strains N-771, N-774 and N-775. For example bacteria from the genus Bacillus, the genus Bacteridium, the genus Micrococcus and the genus Brevibacterium as disclosed in U.S. Patent 4,001,081 may also be used. In addition, it is also possible to use the cellular extract prepared by destroying such bacterial cells, crude enzyme preparations, etc.

To culture the microorganism used in the present invention, ordinary culture mediums containing a carbon source (e.g., glucose, maltose, etc.), a nitrogen source (e.g., ammonium sulfate, ammonium chloride, etc.), an organic nutrient source (e.g., yeast extract, malt extract, peptone, meat extract, etc.), and an inorganic nutrient source (e.g., phosphate, magnesium, potassium, zinc, iron, manganese, etc.) are used. The culture is aerobically conducted while maintaining the pH of the culture medium at about 6 to 9 at a temperature of about 20 to 350C, preferably about 25 to 300C, for about 1 to 5 days.

The strains N-771, N-774 and N-775 to be used in the present invention were deposited at Fermentation Research Institute, Agency of Industrial Science a Technology, Ministry of International Trade and Industry, Japan, as FERM-P Nos. 4445, 4446 and 4447, respectively on March 28th, 1978.

The bacteriological characteristics of each strain are as shown below.

A: Strain N-77 1 (a) Morphology (1) Shape and size of cells: (0.5--0.8) y x (2-5) y (2) Pleomorphism of cells: At the initial stage of culture, the bacterial cells are in a long bacillary form of rods without bending, and grow with snapping and, later, break and split into a coccoid or short bacillary form.

(3) Motility: none (4) Spore: none (5) Gram staining: positive (6) Acid fastness: negative (7) Metachromatic granules: positive (b) Growth state in various culture mediums (at 300 C) (1) Nutrient agar plate culture: Circular (13 mm in diameter), with solid edges, smooth, hemispherical, opaque with luster, slightly pink.

(2) Nutrient agar slant culture: Middle growth, filament-like, surface-smooth, convex, with luster, slightly pink.

(3) Bouillon liquid culture: Vigorous growth with forming pellicle, middle-degree turbidity with growth, forming a precipitate.

(4) Bouillon gelatin stab culture: Good growth on the surface, funnet-like growth along stab, with almost no growth at the lower portion, no liquefaction of gelatin.

(5) Litmus milk: no change (c) Physiological characteristics (1) Reduction of nitrate: positive (2) Denitrification: negative (3) MR test: negative (4) VP test: negative (5) Indole production: negative (6) Hydrogen sulfide production: negative (7) Hydrolysis of starch: negative (8) Citric acid use: Kosefs culture medium: negative Christiansen's culture medium: positive (9) Use of inorganic nitrogen source: Nitrate: positive Ammonium salt: positive 10) Pigment production: negative (11) Urease: positive (12) Oxidase: negative (13) Catalase: positive (14) Hydrolysis of cellulose: negative (15) Growth range: pH 5-1 0; temp.: 5-370C (16) Oxygen relation: aerobic (17) O-F test:F (18) Heat resistance (in 10% skim milk, at 720C for 1 5 minutes: none (19) Acid and gas production from sugar Acid production Gas production L-Arabinose + D-Xylose D-Glucose + D-Mannose D-Fructose + D-Galactose Maltose Sucrose Lactose Treha lose D-Sorbitol + D-Mannitol + Inositol Glycerin + Starch Salicin B: Strain N-774 (a) Morphology (1) Shape and size of cells: (0.5-0.8) y x (2-5) u (2) Pleomorphism of cells: At the initial stage of culture, the bacterial cells are in a long bacillary form of rods without bending, and grow with snapping and, later, break and split into a coccoid or short bacillary form.

(3) Motility: none (4) Spore: none (5) Gram staining: positive (6) Acid fastness: negative (7) Metachromatic granules: positive (b) Growth state in various culture mediums (at 300C) (1) Nutrient agar plate culture: Circular (13 mm in diameter), slightly irregular, smooth with surface-drying tendency, flat, opaque, slightly pink.

(2) Nutrient agar slant culture: Middle growth, filament-like, surface-smooth, convex with drying tendency, slightly pink.

(3) Bouillon liquid culture: Vigorous growth with forming pellicle, slight turbidity, forming a precipitate with growth.

(4) Bouillon gelatin stab culture: Good growth on the surface, funnel-like growth along stab, with almost no growth at the lower stab portion, no liquefaction of gelatin.

(5) Litmus milk: no change (c) Physiological characteristics (1) Reduction of nitrate: positive (2) Denitrification: negative ,(3) MR test: negative (4) VP test: negative (5) Indole production: negative (6) Hydrogen sulfide production: negative (7) Hydrolysis of starch: negative (8) Citric acid use: Koser's culture medium: negative Christiansen's culture medium: positive (9) Use of inorganic nitrogen source: Nitrate: positive Ammonium salt: positive (10) Pigment production: negative (11) Urease: positive (12) Oxidase: negative (13) Catalase: positive (14) Hydrolysis of cellulose: negative (15) Growth range: pH: 5-10; temp.: 1 O-400C (16) Oxygen relation: aerobic (17) O-F test:F (18) Heat resistance (in 10% skim milk, at 720C for 15 minutes): none (19) Acid and gas production from sugar Acid production Gas production L-Arabinose + D-Xylose D-Glucose + D-Mannose + D-Fructose + D-Galactose Maltose + Sucrose Lactose Trehalose + D-Sorbitol + D-Mannitol + I nositol Glycerin + Starch Salicin + - C: Strain N-775 (a) Morphology (1) Shape and size of cells: (0.6-1.0),u x (5--15) y (2) Pleomorphism of cells: At the initial stage of culture, the bacterial cells are in a long bacillary form with hypha-like appearance, and grow with branching and, later, break and split into a coccoid or short bacillary form.

(3) Motility: none (4) Spore: none (5) Gram staining: positive (6) Acid fastness: weakly positive (7) Metachromatic granules: positive (b) Growth state in various culture mediums (at 3O0C) (1) Nutrient agar plate culture: Circular (1-3 mm in diameter), irregular, smooth, in relief, opaque, slightly lustrous, slightly red.

(2) Nutrient agar slant culture: Middle growth, filament-like, surface-smooth, flat trapezoid cross section with slight luster, slightly red.

(3) Bouillon liquid culture: Vigorous growth with forming pellicle, transparent solution, slightly forming a precipitate with growth.

(4) Bouillon gelatin stab culture: Good growth on the surface, funnel-like growth along stab, with almost no growth at the lower stab portion, no liquefaction of gelatin.

(5) Litmus milk: no change (c) Physiological characteristics (1) Reduction of nitrate: positive (2) Denitrification: negative (3) MR test: negative (4) VP test: negative (5) Indole production: negative (6) Production of hydrogen sulfide: negative (7) Hydrolysis of starch: negative (8) Citric acid use: Koser's culture medium: positive Christiansen's culture medium: positive (9) Use of inorganic nitrogen source: Ammonium salt: positive Nitrate: positive (10) Pigment production: negative (11) Urease: positive (12) Oxidase: negative (13) Catalase: positive (14) Hydrolysis of cellulose: negative (15) Growth range: pH: 6-10; temp.: 10-400C (16) Oxygen relation: aerobic (17) O-Ftest:O (18) Heat resistance (in 10% skim milk, at 720C for 15 minutes): none (19) Acid and gas production from sugar: Acid production Gas production D-Arabinose + D-Xylose + D-Glucose + D-Mannose D-Fructose + D-Galactose + Maltose Sucrose + Lactose Trehalose + D-Sorbitol + D-Mannitol + I nositol Glycerin + Starch Salicin To determine taxonomic positions of the bacteria based on the above-described bacteriological characteristics according to Bergy's Manual of Determinative Bacteriology, 7th ed. (1957) and 8th ed.

(1974), the strains N-771 and N-774 fall under the aerobic, Gram-positive, non-acid fastness and catalase-positive bacillary category forming no endo-spores and no flagella. From the fact that the bacteria are in a long bacillary form at the initial stage of growth, not showing filament-like appearance but showing snapping growth without branching and that the bacteria break and split into a coccoid or short bacillary form, it is clear that they fall under the category of Coryneform bacteria.In addition, comparison with the Coryneform bacteria described in the Bergy's Manual precludes the bacteria of the present invention from belonging to: (1) the genus Cellulomonas, because they do not have cellulosedecomposing ability, (2) the genusArthrobacter, because Gram-staining is not variable, (3) the genus Microbacterium, because they do not have heat resistance in 10% skim milk at 720C for 1 5 minutes, and (4) the genus Kurthia because they do not have flagella. Accordingly, it is concluded the bacteria of the present invention belong to the genus Corynebacterium.

The strain N-775 falls under the aerobic, Gram-positive, weakly acid fastness and catalasepositive bacillary category forming no endospores and no flagella. From the fact that the bacteria are in a long bacillary form at the initial stage of growth, showing hypha-like appearance, and grow with branching to break and split later into a coccoid or short bacillary form, they are considered to belong to the genus Nocardia.

In practising the process of the present invention, all that is required is to select a microorganism having the ability to hydrolyze acrylonitrile or methacrylonitrile or one of the above-described microorganisms, culture it for 2 to 3 days in the aforesaid manner, collect the bacterial cells from the culture solution by centrifugation, suspend the cells in water or physiological saline and subject acrylonitrile or methacrylonitrile to the action of the cells.

That is, the reaction may usually be conducted in an aqueous suspension containing about 1 to 10 dry wt% of bacterial cells and 0.5 to 10 wt% of acrylonitrile or methacrylonitrile at a temperature ranging from about the freezing point to 1 50C, at a pH of about 6 to 10, preferably about 7 to 9, for about 0.5 to 10 hours. Additionally, upon reaction, it is preferable to subsequently add acrylonitrile or methacrylonitrile as their concentration in the system falls while limiting the concentration of acrylonitrile or methacrylonitrile in the system to a level of not higher than 2 wt%, since they possess a strong toxicity and would inhibit the enzymatic reaction.Generally slightly higher concentrations of acrylonitrile and methacrylonitrile are possible with the batch process than with the continuous process described below because it is possible to stir the reaction system and a homogeneous system can be obtained.

During the reaction, the pH is preferably controlled to be in the range of about 7 to 9 by consecutively adding a caustic alkali, ammonia or the like or by previously adding a buffer solution to the system. pH values outside the above range would lead to further hydrolysis of the produced and accumulated acrylamide or methacrylamide to form by-products or would lead to reduction in stability of the cell enzyme. Thus, acrylamide or methacrylamide can be produced and accumulated with almost 100% conversion.

It is particularly noted that the accumulated concentration of produced acrylamide or methacrylamide attainable and the life of cell enzyme activity are remarkaly improved by conducting the reaction at a temperature as low as the freezing point of the medium to 1 50C, which is based on the following knowledge which has so far been unexpected.

That is, it has been found that: (1) the nitrilase as the hydrolase of the present invention produced and accumulated in the aforesaid bacterial cells has an extremely higher activity than generally well known hydrolases by 10 to 50 times and, therefore, the reaction can be conducted at an economical reaction rate even at temperatures as low as 1 50C or lower, (2) the enzyme of the present invention has relatively low heat resistance and it could be inactivated in an extremely short time at temperatures usually employed for ordinary enzymatic reactions (25 to 300 C), and hence its effect is not fully exhibited when various chemical treatments are conducted to assure its stability, unless the reaction is conducted at low temperatures, (3) since the enzyme of the present invention has a relatively high activity and can react at low temperature, it is possible to remarkably reduce the enzymatic activity inhibition of acrylonitrile or methacrylonitrile and acryiamide or methacrylamide, and as a result attain concentrations of accumulated acrylamide or methacrylamide as high as 10 to 30 wt% while stably maintaining the enzymatic activity for a long period of time.

These microorganisms may be used as intact cells but, from the standpoint of repeated use, continuous operation and purification, immobilized cells, in particular, immobilized cells entrapped by a polyacrylamide and related polymer gels, are preferred.

In conventional immobilized cells prepared by entrapping cells with polyacrylamide and related polymer, the level of enzymatic activity in the immobilized cells is 30 to 60% of the activity of intact cells in most cases. On the other hand, the cells of the present invention can be immobilized at the activity level of almost 100%, because the microorganisms are acrylamide-producing bacteria and are stable against highly concentrated acrylamide, and because immobilizing can be conducted at 15"C or less.

The cell immobilization can be conducted by suspending the aforesaid microorganisms in a suitable aqueous medium (e.g., water, a physiological saline, a buffer solution, etc.) containing an acryiamide series monomer and a cross linking agent, adding a suitable polymerization initiator and a polymerization accelerator to the suspension, and conducting polymerization and gellation at about 0 to 300 C, preferably 0 to 15 C, at a pH of about 5 to 10, preferably about 6 to 8. The content of microorganisms in the polymerization reaction solution depends upon the kind and the form of the microorganisms used, but it is usually about 0.1 to 50 wt%, preferably about 1 to 20 dry wt%.

The acrylamide series monomers used to immobilize the cells in the present invention include, for example, acrylamide, methacrylamide, etc. and, if necessary, ethylenically unsaturated monomers copolymerizable with them may be used in combination. The concentration of such monomers in the reaction should at least be at a level high enough to form gels as a result of the polymerization, and is usually about 2 to 30 wt%, preferably about 5 to 20 wt%, based on the reaction solution.

The cross-linking agents include N,N'-methylenebisacrylamide, 1 ,3-di-(acrylamidomethyl)-2- imidazolidone, etc. As the polymerization initiator and the polymerization accelerator, those which least inhibit the activity of microorganisms are selected. Usually, potassium persulfate, ammonium persulfate, etc. are used as the initiator, and dimethylaminopropionitrile, triethanoiamine, etc. are used as the accelerator, each in an amount of about 0.01 to 10 wt%.

Thus, there can be obtained polymer gels containing bacterial cells, i.e., immobilized cells.

The reaction of the present invention may be conducted either in a batchwise manner or in a continuous manner.

Additionally, a more concentrated acrylamide or methacrylamide aqueous solution or crystals of acrylamide or methacrylamide can be obtained from the thus obtained acrylamide or methacrylamide aqueous solution of the present invention using conventional techniques. For example, the aforesaid reaction solution is treated, if necessary, with active carbon, ion-exchange resin, etc., and then concentrated under reduced pressure to obtain a more concentrated acrylamide or methacrylamide aqueous solution or crystals thereof.

The present invention will now be described in more detail by the following examples of preferred embodiments of the present invention which, however, should not be construed as limiting the present invention. Additionally, all parts and percents in the following examples are by weight. The reaction products such as acrylamide and methacrylamide, unreacted materials such as acrylonitrile and methacrylonitrile, and by-products like methacrylic acid and acrylic acid were determined by means of gas chromatography.

EXAMPLE 1 8 parts of the washed cells of the strain N-771 (water content: 75%) prepared by aerobic culture using a culture medium (pH: 7.2) containing 1% glucose, 0.5% peptone, 0.3% yeast extract and 0.3% malt extract was mixed with 92 parts water, and acrylonitrile was intermittently added dropwise at a rate of 2 parts per hour while controlling the pH of the solution at 8.0 by properly adding a 0.5 N KOH aqueous solution under stirring, at various reaction temperatures ranging from about OOC to 300C as shown in Table 1. The reaction was continued until unreacted acrylonitrile was detected and, at that stage, the reaction was stopped and the cells were removed by centrifugation to obtain a clear solution.

The content of acrylamide was determined with respect to each solution to compare the concentrations of accumulated acrylamide at respective reaction temperatures. Thus, the results shown in Table 1 were obtained. It is seen from the results, that the enzymatic activity of the cells became stable and the concentration of produced and accumulated acrylamide was greatly increased when the reaction was conducted at temperatures of not higher than 1 50C.

TABLE 1 Run No.

6-1 6-2 6-3 6-4 6-5 6-6 Reaction Temperature (OC) -3 to 0 5 10 1 5 20 30 Reaction Time Before Acrylonitrile was Detected (hrs.) 1 6 1 6 14 12 5 4 Acrylamide in the Reaction Solution (%) 31.8 31.0 28.1 25.0 10.7 9.3 EXAMPLE 2 13.5 parts of the washed cells of the strain N-775 (water content: 78%) obtained by culturing in the same manner as in Example 6 were mixed with 86.5 parts water, and acrylonitrile was intermittently added dropwise at a rate of 2 parts per hour while controlling the pH of the solution at 8.0 by properly adding a 0.5 N potassium hydroxide aqueous solution under stirring, at various reaction temperatures ranging from about OOC to about 30 C as shown in Table 2. Subsequently, the reaction was continued in the same manner as in Example 6 until unreacted acrylonitrile was detected. The concentration of acrylamide was determined with respect to each solution to obtain the results in Table 2. It is seen in this example, too, that the produced and accumulated acrylamide concentration greatly increased in the experiments wherein the reaction was conducted at temperature of not higher than 15"C.

TABLE 2 Run No.

7-1 7-2 7-3 7-4 7-5 7-6 Reaction Temperature (OC) -3 to 0 5 10 1 5 20 30 Reaction Time Before Acrylonitrile was Detected (hrs.) 14 13 11 10 5 4 Acrylamide in the Reaction Solution (%) 28.2 27.5 23.1 21.0 11.5 9.5 EXAMPLE 3 13.5 parts of the washed cells of the strain CBS 717.73 (the strain described in the Examples of USP 4,001,081) obtained by culturing in the same manner as in Example 6 (water content: 78%) was mixed with 86.5 parts water, and acrylonitrile was intermittently added dropwise at a rate of 2 parts per hour while controlling the pH of the soltuion at 8.0 by properly adding a 0.5 N potassium hydroxide aqueous solution under stirring, at various reaction temperatures ranging from about OOC to 300C as shown in Table 3. Subsequently, the reaction was continued in the same manner as in Example 6 until unreacted acrylonitrile was detected. The acrylamide concentration of each solution was determined.

The results obtained are shown in Table 3. It is seen in this example, too, that the produced and accumulated acrylamide concentration greatly increased in the experiments wherein the reaction was .conducted at temperatures of not higher than 1 50C.

TABLE 3 Run No.

8-1 8-2 8-3 8-4 8-5 8-6 Reaction Temperature (OC) -3 to 0 5 10 1 5 20 30 Reaction Time Before Acrylonitrile was Detected (hrs.) 13 13 3 11 10 5 4 Acrylamide in the Reaction Solution (%) 27.5 26.2 22.9 21.3 10.2 9.5 EXAMPLE 4 4 parts of the washed cells of the strain N-771 obtained in the same manner as in Example 6, 0.45 parts of acrylamide, 0.05 part of N,N'-methylenebisacrylamide and 4 parts of physiological saline were mixed to prepare a uniform suspension. To this suspension were added 0.5 part of a 5% dimethylaminopropionitrile aqueous solution and 1 part of a 2.5% potassium persulfate aqueous solution, and the system was maintained at 1 00C for 30 minutes to polymerize.The thus obtained massive, cell-containing gels were crushed into small particles and washed with physiological saline to obtain 10 parts of immobilized cells. To 20 parts of the immobilized cells was added 72 parts of a 0.05 M phosphate buffer (pH: 8.0), and acrylonitrile was dropwise added thereto at a rate of 2 parts per hour to react for 4 hours under stirring. A clear solution obtained by separating and removing the bacterial cells from the reaction product contained 10.6% acrylamide and almost no by-products like acrylic acid and unreacted acrylonitrile were detected. Thus the reaction proceeded almost quantitatively to completion.

The separated immobilized cells were repeatedly used to conduct the same reaction. On the other hand, similar experiments were conducted at 300C for comparison. The results obtained are shown in Table 4.

TABLE 4 Number of times of Yield of acrylamide (%) repeatedly using the microorganisms 1 50C 300C 1 100 100 2 100 100 3 100 85 4 100 5 5 100 0 6 100 0 7 100 0 EXAMPLE 5 40 g of the immobilized cells of the strain N-771 obtained in the same manner as in Example 9 were filled in a jacketed column (3 cm in inside diameter and 25 cm in length), and a 4% acrylonitrile aqueous solution or 2.6% methacrylonitrile aqueous solution was continuously fed via the top of the column at a rate of 100 ml/hr at 1 00C and at 250C (for comparison) to react the times shown in Table 5. Ratios of produced acrylamide or methacrylamide at respective reaction stages were determined to obtain the results shown in Table 5.

TABLE 5 Yield of acrylamide Yield of methacrylamide Reaction (%) (%) Time (hr) 100C 250C 100C 250C 10 100 100 100 100 20 100 32 100 100 30 100 0 100 100 40 100 0 100 4 50 100 0 100 0 100 100 0 100 0 150 100 0 100 0 200 100 0 100 0 250 100 0 100 0 300 100 0 100 0 EXAMPLE 6 Table 6 given below comparatively shows the activity of intact cells and of immobilized cells with respect to various microorganisms having an acrylamide-producing ability.

Preparation ofimmobilized cells: 4 parts of intact cells (water content: 75%), 0.45 part of acrylamide, 0.05 part of N,N'methylenebisacrylamide, and 4 parts of physiological saline were mixed to prepare a uniform suspension. To this suspension were added 0.5 part of a 5% dimethylaminopropionitrile aqueous solution and 1 part of a 2.5% potassium persulfate aqueous solution, and the system was maintained as 10 to 1 50C for 30 minutes to polymerize. Subsequently, the thus obtained cell-containing gels were crushed and washed with physiological saline to obtain 10 parts of the immobilized cells.

Measurement of the acrylamide-producing stability: 0.8 part of the intact cells or 2 parts of the immobilized cells were diluted with a 0.05 M phosphate buffer (pH: 8.0) to make 100 parts. Then, 1 part of each of the thus diluted solution was mixed with 1 part of a 0.05 M phosphate buffer (pH: 8.0) containing 2% acrylonitrile and, after reacting at 1 O0C for 30 minutes under stirring, acrylamide produced in the reaction solution was determined to calculate the acrylamide-producing ability of each of intact cells and immobilized cells.

TABLE 6 Acrylamide-producing ability* Microorganism Intact cells Immobilized cells Strain N-771, 10.7 10.8 genus Corynebacterium (FERM-P No. 4445) Strain N-774, 6.0 5.8 genus Corynebacterium (FERM-P No. 4446) Strain N-775, 2.6 2.5 genus Nocardia (FERM-P No. 4447) *Amount of acrylamide (g) produced by reacting for 1 hour per 1 g of dry bacterial cells.

Claims (10)

1. A process for producing acrylamide or methacrylamide which comprises subjecting acrylonitrile or methacrylonitrile in an aqueous medium to the action of micro-organisms having the ability to hydrolyze acrylonitrile or methacrylonitrile, at a temperature of from the freezing point of the medium to l50CatapHof6to 10.

2. A process as claimed in claim 1, wherein said micro-organisms are selected from bacteria belonging to the genus Corynebacterium, the genus Nocardia, the genus Bacillus, the genus Bacteridium in the sense of Prevot, the genus Micrococcus and the genus Brevibacterium in the sense of Bergey.

3. A process as claimed in claim 2, wherein said bacteria are from the genus Corynebacterium or the genus Nocardia.

4. A process as claimed in claim 2, wherein said bacteria are of the genus Corynebacterium.

5. A process as claimed in claim 2, wherein said bacteria are of the genus Nocardia.

6. A process as claimed in any preceding claim, wherein said bacteria are immobilized with a polymer gel.

7. A process as claimed in claim 6, wherein said immobilized bacteria are entrapped with a polyacrylamide and related polymer gel.

8. A process as claimed in claim 1 , wherein said bacteria are the strains N-771 or N-774 belonging to the genus Corynebacterium or the strain N-775 belonging to the genus Nocardia.

9. A process as claimed in claim 1, substantially as hereinbefore described in any one of Examples 1 to 6.

10. Acrylamide or methacrylamide when produced by the process as claimed in any preceding claim.