JP2006158323A - Method for producing amide compound by microorganism, microorganism used therefor, treated product of microbial cell and enzyme - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing an amide compound, with which an amide compound is efficiently produced at a higher temperature by finding a strain having highly thermostable nitrile hydratase and to obtain a microorganism used for the production method. <P>SOLUTION: The method for producing an amide compound comprises treating a nitrile compound with a culture solution, a microbial cell or a treated product of the microbial cell of a bacterium belonging to Geobacillus caldoxylosilyticus in an aqueous medium. <P>COPYRIGHT: (C)2006,JPO&amp;NCIPI

Description

  The present invention relates to a method for producing an amide compound using a culture solution, cells or treated cells of microorganisms belonging to Geobacillus caldoxylosilyticus, and microorganisms used in the method.

  In recent years, many methods for converting a nitrile compound into an amide compound using a biocatalyst such as a microorganism have been proposed. For example, a method using a microorganism belonging to the genus Bacillus, the genus Bacteridium, the genus Micrococcus, the Brevibacterium (Patent Document 1), the genus Corynebacterium, the nocardia A method using a microorganism belonging to the genus (Nocardia) (Patent Document 2), a method using a microorganism belonging to the genus Pseudomonas (Patent Document 3), a microorganism belonging to the genus Rhodococcus, and a microorganism belonging to the genus Microbacterium. Examples include a method used (Patent Document 4), a method using a microorganism belonging to the genus Fusarium (Patent Document 5), and the like. As described above, a plurality of types of microorganisms having a hydration activity for converting a nitrile compound into an amide compound are known. All of these microorganisms are mesophilic bacteria that do not grow at a high temperature of 55 ° C. or higher.

  Recently, focusing on the highly heat-resistant nitrile hydratase possessed by thermophilic bacteria that grow above 55 ° C, microorganisms with hydration activity that convert nitrile compounds to amide compounds have also been reported for thermophilic bacteria. Has been. For example, Patent Document 6 describes Bacillus smithii and Patent Document 7 describes Psuedonocardia thermophila, which uses a cell of Geobacillus caldoxylosilyticus. There is no report of converting a nitrile nitrile compound into an amide compound.

JP-B 62-21519 Shoko 56-17918 Japanese Patent Publication No.59-37951 EP188316 JP-A 64-86889 JP-A-7-255494 JP-A-8-56684

  Although attempts to use the thermostable nitrile hydratase from Bacillus smithii and Psuedonocardia thermophila have been somewhat successful, these thermophilic bacteria have upper growth limits. Was 55-60 ° C., and the enzyme also had temperature characteristics corresponding to the temperature. Therefore, the present invention produces an amide compound efficiently at a higher temperature by finding a strain having a higher heat-resistant nitrile hydratase from a strain that grows above the upper limit temperature for growth of the thermophile. It was made into the problem which should be solved to provide the microorganisms for using the manufacturing method of the amide compound which can be used, and the said manufacturing method.

  As a result of intensive studies in view of the above situation, the present inventors have found that thermophilic bacteria isolated from soil in the vicinity of a hot spring in Saitama Prefecture are efficient even in a temperature range that exceeds the temperature range of conventional thermophilic bacteria. In particular, the present inventors have found that it has a hydration activity for converting a nitrile compound into an amide compound, thereby completing the present invention. That is, the present invention allows a nitrile compound to be reacted with a culture solution, cell or treated product of a thermophilic microorganism having a hydration activity that converts a nitrile compound into an amide compound, and converts the nitrile compound into an amide compound. It is intended to provide a method for producing an amide compound, and a microorganism to be used.

That is, according to the present invention, the following inventions (1) to (7) are provided.
(1) converting a culture solution, a microbial cell or a treated product of a microorganism belonging to Geobacillus caldoxylosilyticus into a corresponding amide compound by acting on a nitrile compound in an aqueous medium, A method for producing an amide compound.
(2) The method for producing an amide compound according to (1), wherein the microorganism belonging to Geobacillus caldoxylosilyticus is Geobacillus caldoxylosilyticus M16.

(3) The manufacturing method of the amide compound as described in (1) or (2) whose reaction temperature is 20 to 75 degreeC.
(4) The manufacturing method of the amide compound as described in (1) or (2) whose reaction temperature is 55 to 75 degreeC.

(5) Geobacillus caldoxylosilyticus M16 (reception number: FERM AP-20315) or a variant thereof.
(6) A culture solution, a microbial cell, or a treated microbial cell of a microorganism belonging to Geobacillus caldoxylosilyticus, which is used for the method for producing an amide compound according to (1).
(7) An enzyme agent for producing an amide compound from a nitrile compound, which contains, as an active ingredient, a culture solution, cells or treated cells of microorganisms belonging to Geobacillus caldoxylosilyticus.

According to the present invention, a reaction at a higher temperature is possible as compared with the case where a thermophilic bacterium having a conventionally known upper limit temperature of growth up to 60 ° C. is used, thereby enabling efficient production of an amide compound. Become.

  Hereinafter, the present invention will be described in more detail. The microorganism used in the present invention may be any microorganism as long as it belongs to Geobacillus caldoxylosilyticus and has a hydration activity for converting a nitrile compound into an amide compound. In addition, as these microorganisms, microorganisms stored by various microorganism storage institutions may be used, or strains isolated from the natural world by a so-called screening technique may be used. Specifically, in the present invention, screening was performed by the following method. First, take a small amount of soil collected at various places, put it in a test tube containing water or physiological saline, and incubate in a shaking incubator at 70 ° C. for 2 to 14 days. . A portion of this culture solution is taken and put into a general-purpose microbial growth medium such as glucose, polypeptone, yeast extract, malt extract, etc. as a main component, and cultured for 1 day to 7 days. Incubate to the extent. Thermophilic microorganisms can be separated by spreading a part of the resulting culture solution on the agar plate medium containing the aforementioned microorganism growth medium components and further culturing at 70 ° C. to form colonies. The microorganism thus obtained is used for a suitable period of time, for example, about a period of time using, for example, a test tube or flask containing a liquid medium obtained by adding a nitrile compound such as n-valeronitrile or an amide compound such as crotonamide to the medium components. It is grown by culturing at a culture temperature of 70 ° C. for about 12 hours to 7 days. The culture solution or cells are added to an aqueous solution of a nitrile compound such as propionitrile or acrylonitrile, and reacted at a reaction temperature of, for example, 20 ° C. for about 10 to 60 minutes, and then an amide compound is formed in the reaction solution. By examining the presence or absence, thermophilic microorganisms having a hydration activity for converting a nitrile compound into an amide compound can be easily separated. For detection of the amide compound, for example, an analysis method by liquid chromatography as described later can be used. Further, the produced compound was separated by liquid chromatography and then identified by gas chromatography / mass spectrometry (GC / MS), infrared absorption spectrum (IR) and nuclear magnetic resonance spectrum (NMR). The strain obtained this time is a thermophilic microorganism having a high hydration activity with respect to the nitrile group of the nitrile compound isolated from nature by the present inventors. As a result of identification of the strain, Geobacillus geobacillus (Geobacillus It was proved to be caldoxylosilyticus) and deposited at the Patent Organism Depository Center of the National Institute of Advanced Industrial Science and Technology (Central 1-1, Tsukuba City, Ibaraki Prefecture) under the receipt number: FERM AP-20315 Sunday: December 7, 2004). Further, even if various literatures are examined, there is no description about the hydration activity for converting a nitrile compound into an amide compound in a microorganism belonging to Geobacillus caldoxylosilyticus. In this respect, Geobacillus caldoxylosilyticus M16 (reception number: FERM AP-20315) is recognized as a new strain. In addition, mutants, cell fusion strains and genetically modified strains derived from mutants of the strains, that is, Geobacillus caldoxylosilyticus M16 (reception number: FERM AP-20315) are also used in the method of the present invention. It can be used. As a mutant of Geobacillus caldoxylosilyticus M16, as long as it is a microorganism having a hydration activity that converts a nitrile compound into an amide compound, it is derived from the above-mentioned Geobacillus caldoxylosilyticus M16. Any variant can be used. Specific examples include natural mutations, chemical mutants, and mutants that have undergone artificial mutations such as ultraviolet rays.

  The bacteriological properties of the above-mentioned Geobacillus caldoxylosilyticus M16 strain are as follows.

(A) Morphological properties Culture conditions: TM medium Medium composition: 8 g peptone (Bact Peptone) per 1000 ml
MgCl ₂ 0.15 g
Bact Yeast Extract 4 g
NaCl 2 g
CaCl ₂ 0.18 g
Gellan gum 15g
Culture temperature: 70 ° C

1. Cell shape and size: Aspergillus oryzae Size: 0.8 × 2.0-3.0μm
2. Presence or absence of cell polymorphism: −
3. Existence of mobility: +
Flagellar state: Peripheral hair4. Presence of spores: +
Spore site:

(B) Culture properties Culture conditions: TM medium (described above) 70 ° C
1. Color: Transparent to light cream color Gloss: +
3. Pigment production: −
4). Presence or absence of surface growth:-
5. Presence or absence of medium turbidity: +

Culture conditions: gelatin puncture culture 70 ° C
1. Growth state: +
2. Gelatin liquefaction: +

Culture conditions: Litmus milk 70 ° C
1. Coagulation:-
2. Liquefaction:-

(C) Physiological properties Gram staining: indefinite 2. Reduction of nitrate:-
3. Denitrification reaction:-
4). MR test:-
5. VP test:-
6). Generation of hydrogen sulfide:-
7). Hydrolysis of starch:-
8). Use of citric acid
Koser ： −
Christensen ： −
9. Use of inorganic nitrogen source Nitrate:-
Ammonium salt: +
10. Dye generation11. Urease activity:-
12 Oxidase: +
13. Colorase: +
14 Growth temperature pH: 5.5-8.0
Temperature: 50 ° C to 75 ° C
15. Attitude toward oxygen: facultative anaerobic 16. OF test:-/-

(D) Acid production / gas production from sugar L-arabinose:-/-
2. D-xylose: +/-
3. D-glucose: +/-
4). D-Mannose: +/-
5. D-fructose: +/-
6). D-galactose:-/-
7). Maltose: +/-
8). Circus: +/-
9. Lactose:-/-
10. Trehalose: +/-
11. D-sorbitol:-/-
12 D-mannitol: +/-
13. Inositol:-/-
14 Glycerin:-/-

(E) Other properties β-galactosidase:-
2. Arginine dihydrase activity:-
3. Lysine decarboxylase activity:-
4). Tryptophan deaminase activity:-
5. Gelatinase activity: +

  The microorganism used in the method of the present invention can be cultured in accordance with a general microorganism culture method. Solid culture or liquid culture [test tube shaking culture, reciprocating shaking culture, rotary shaking culture, jar fermenter Culture, culture tank, etc.] are all possible. Culture is usually performed under anaerobic conditions. Also, when using a jar fermenter, aseptic air is not introduced. Although culture | cultivation temperature can be suitably changed in the range in which microorganisms grow, it is the range of 50 to 75 degreeC, for example. The pH of the medium is, for example, 5.5 to 8.0. In particular, in the case of Geobacillus caldoxylosilyticus M16 microorganism, a medium pH of about 6 to about 8 at a culture temperature in the range of about 60 ° C. to about 72 ° C., preferably about 65 ° C. to about 70 ° C. It is. The culture time varies depending on various conditions, but is usually about 1 day to about 7 days.

  For culture of the microorganism used in the method of the present invention, various media appropriately containing a carbon source, a nitrogen source, organic or inorganic salts, etc., used for normal culture in general bacteria can be used. Examples of the carbon source include glucose, glycerol, dextrin, sucrose, organic acid, animal and vegetable oils, and sugar-tightness. Examples of the nitrogen source include meat extract, peptone, yeast extract (yeast extract), malt extract, soybean flour, cottonseed flour, dry yeast, casamino acid, sodium nitrate, urea and other organic or inorganic nitrogen sources. Organic or inorganic salts include potassium, sodium, magnesium, iron, manganese, cobalt, zinc and other chlorides, sulfates, acetates, carbonates and phosphates, specifically potassium chloride, sodium chloride, sulfuric acid Magnesium, ferrous sulfate, manganese sulfate, cobalt chloride, zinc sulfate, copper sulfate, sodium acetate, calcium carbonate, sodium carbonate, 1 potassium hydrogen phosphate, 2 potassium hydrogen phosphate, 1 sodium hydrogen phosphate, 2 hydrogen phosphate Sodium etc. can be mentioned. In order to increase the hydration activity of the microorganisms used in the method of the present invention with respect to the nitrile group, it is preferable to add nitrile compounds such as n-valeronitrile and crotononitrile and amide compounds such as crotonamide to the medium. Examples of the added amount include about 10 mg to about 1 g per 100 ml of the medium.

  Examples of the nitrile compound used in the present invention include aliphatic nitrile compounds such as acetonitrile, n-propionitrile, n-butyronitrile, isobutyronitrile, n-valeronitrile, n-hexanenitrile, and 2-chloropropionitrile. Nitrile compounds containing halogen atoms such as, aliphatic nitrile compounds containing unsaturated bonds such as acrylonitrile, crotononitrile, methacrylonitrile, hydroxynitrile compounds such as lactonitrile, mandelonitrile, 2-phenylglycinonitrile, etc. Examples thereof include aromatic nitrile compounds such as aminonitrile compounds, benzonitrile and cyanopyridine, dinitrile compounds such as malononitrile, succinonitrile and adiponitrile, and trinitrile compounds.

  In the method of the present invention, a nitrile compound is hydrated and converted to a corresponding amide compound by allowing a culture solution of a microorganism, a microbial cell, or a treated microbial cell to act on the nitrile compound in an aqueous medium. As used herein, “hydrating a nitrile compound to convert it to the corresponding amide compound” means that water molecules are added to the nitrile compound, such as acetonitrile for n-propionitrile, n-propioamide for n-propionitrile, and acrylamide for acrylonitrile. The amide compound.

  In the present invention, a culture solution, microbial cells or processed microbial cells of a microorganism are allowed to act on a nitrile compound in an aqueous medium. The phrase “acting on a nitrile compound in an aqueous medium” as used herein refers to adding the above-described nitrile compound to an aqueous solution such as water or a phosphate buffer. For example, in addition to an aqueous solution of a nitrile compound such as acrylonitrile, for example, it has the ability to be converted to an amide compound by reacting at a reaction temperature of 20 ° C. for 5 to 60 minutes.

  The method of the present invention can be performed, for example, as follows. The culture solution of the microorganisms cultivated by the above-mentioned method, the microbial cells or the processed microbial cells are suspended in an aqueous aqueous solution such as water or a buffer solution such as a phosphate buffer solution, and this is added to the nitrile compound and reacted. Here, the treated microbial cells are crushed cells or enzymes crushed by usual treatment methods such as ultrasonic waves, homogenizers, and French presses, or microbial cells, crushed cells, enzymes, etc. It is insolubilized by immobilization methods such as a carrier binding method that binds to a carrier by covalent bond, ionic bond, adsorption, etc., or an entrapment method confined in a polymer network structure, and processed into a state that can be easily separated. is there. As reaction conditions, the density | concentration of the microbial cell to be used or microbial cell crushed material is 0.01 to 20 weight%, for example, Preferably, 0.1 to 10 weight% can be mentioned. In addition, the concentration of the enzyme and the immobilized product varies depending on the degree of purification or the immobilization method, but for example, it is prepared so that hydration activity for nitrile groups equivalent to that of the above-mentioned bacterial cells or disrupted cells exists. It is preferable to do. Although the culture solution can be used as it is by adding a nitrile compound, it is preferably diluted so that there is a hydration activity for the nitrile group equivalent to that of the above-mentioned cells or cell disruptions. Or it is preferable to prepare by concentration etc.

  The reaction temperature at the time of carrying out the method of the present invention is not particularly limited as long as the conversion reaction from the nitrile compound to the amide compound proceeds, but generally 5 ° C to 80 ° C, preferably 10 ° C to 75 ° C, more Preferably 20 ° C to 75 ° C, more preferably 30 ° C to 75 ° C, more preferably 40 ° C to 75 ° C, more preferably 50 ° C to 75 ° C, more preferably 55 ° C to 75 ° C, and particularly preferably 60 to 75 ° C. It can be in the range of ° C.

  Moreover, reaction pH is 5-10, for example, Preferably it is 6-9. The reaction time is, for example, 5 minutes to 72 hours. If the reaction pH is maintained within the above range, the microorganism used in the method of the present invention can also produce and accumulate the amide compound at a high concentration. Recovery of the amide compound from the reaction solution can be performed by any generally known method. For example, after removing bacterial cells and the like from the reaction solution by centrifugation or the like, impurities and the like are removed by treatment with activated carbon, ion exchange resin, or the like. Then, the target amide compound can be obtained by recrystallizing a crystal precipitated by concentration under reduced pressure or concentration by distillation using an organic solvent such as methanol.

As above-mentioned, an amide compound can be manufactured from a nitrile compound by using the culture solution, microbial cell, or microbial cell processed material of the microorganisms which belong to Geobacillus caldoxylosilyticus (Geobacillus caldoxylosilyticus). That is, the culture solution, fungus body, or treated product of a microorganism belonging to Geobacillus caldoxylosilyticus can be used as an enzyme agent for producing an amide compound from a nitrile compound. An enzyme agent for producing an amide compound from a nitrile compound, which contains, as an active ingredient, a culture broth of a microorganism belonging to Geobacillus caldoxylosilyticus, a fungus body or a treated product of the fungus body is also within the scope of the present invention. Is.
Examples of the present invention are shown below, but the present invention is not limited to these examples.

Example 1 (bacteria cell isolation)
Take a small amount (about 1g) of soil collected in Nagao Park, Kitakyushu City, Kitakyushu City, Fukuoka Prefecture, and place it in a test tube containing 5ml of physiological saline and shake it in a shaking incubator at 70 ° C for 3 days. Cultured at last. A part (0.5 ml) of this culture solution is taken and added to a medium (pH 7.0) consisting of 1.0% by weight of glucose, 0.5% by weight of polypeptone and 0.3% by weight of yeast extract for 2 days. Incubated with reciprocal shaking at ℃. A part (0.1 ml) of the culture solution thus obtained was spread on an agar plate medium containing the above-mentioned medium components and cultured at 70 ° C. for further 2 days to form colonies, thereby separating thermophilic microorganisms. The separated cells are inoculated into a liquid medium in which 0.1% by weight of n-valeronitrile is added to a medium having the same composition as described above, and then cultured at 70 ° C. for 24 hours to obtain a culture solution as a specimen. Obtained. 1 ml of the culture was added to 9 ml of a 1.1% by weight acrylonitrile solution (0.05M-phosphate buffer pH 7.7), and the reaction was started at a reaction temperature of 20 ° C. After 10 minutes, the reaction was stopped by adding 1 ml of 1N hydrochloric acid. A part of the reaction solution was analyzed by liquid chromatography, and a strain having hydration activity with respect to the nitrile group was selected by examining the presence or absence of acrylamide formation. In this way, Geobacillus caldoxylosilyticus M16 was obtained as a thermophilic microorganism having hydration activity for converting a nitrilo compound into an amide compound.

(Liquid chromatography analysis conditions)
HPLC: Hitachi D-7000 series column; Inertsil ODS-3 (manufactured by GL Sciences)
Length: 200mm
Column temperature: 35 ° C
Flow rate: 1 ml / min
Sample injection volume: 10 μl
Solution: 0.1 wt% phosphoric acid aqueous solution

Example 2 (Growth upper limit temperature of cell culture)
An attempt was made to compare Geobacillus caldoxylosilyticus M16 obtained in Example 1 with Bacillus smithii described in JP-A-7-255494.・ We were unable to order Smithy (Bacillus smithii). Therefore, after separating cells from soil by the method described in JP-A-7-255494, cells identified as Bacillus smithii by 16S rRNA homology search were used. These two types of cells were each applied to an agar plate medium containing the medium components used in Example 1, cultured at a plurality of different temperatures, and the growth state of the cells was examined. The results are shown in Table 1. Geobacillus caldoxylosilyticus M16 (invention strain) was a thermophilic microorganism having a higher growth upper limit temperature than Bacillus smithii (control strain). Moreover, the growth temperature range of Bacillus smithii (control strain) was the same as that described in Japanese Patent Application Publication No. 7-255494.

Example 3 (comparison of nitrile hydratase activity at each temperature)
0.2% by weight of glycerol, 0.2% by weight of trisodium citrate dihydrate, 0.1% by weight of potassium dihydrogen phosphate, 0.1% by weight of dipotassium hydrogen phosphate, 0.1% by weight of polypeptone, Yeast extract 0.1% by weight, sodium chloride 0.1% by weight, n-valeronitrile 0.1% by weight, magnesium sulfate heptahydrate 0.02% by weight, iron (II) sulfate heptahydrate 0.003 Geobacillus caldoxylosilyticus M16 previously cultivated in a 500 ml Erlenmeyer flask containing 100 ml of a sterilized medium (pH 7.0) containing wt% cobalt chloride hexahydrate 0.0002% 1 ml of the culture solution was inoculated. This was cultured with shaking at 200 stroke / min for 1 day at 70 ° C. to obtain a cell culture broth. The cells were collected from 300 ml of the cell culture solution of Geobacillus caldoxylosilyticus M16 by centrifugation (10000 × g, 15 minutes) and washed with 0.05 M phosphate buffer (pH 7.5). Then, it was suspended in 50 ml of the same buffer. The cell suspension prepared in this manner was measured for hydration activity for converting a nitrile compound into an amide compound at the reaction temperatures listed in Table 2. Hydration activity was determined as follows. First, the unit (unit) of enzyme activity is defined as 1 unit (hereinafter referred to as U) for converting 1 μmol of acrylonitrile into acrylamide per minute, and the cell suspension is 5 U / ml at 10 ° C. The suspension was adjusted (reaction time was 10 minutes). Using this bacterial cell suspension, hydration activity was similarly determined under the conditions of 30 ° C., 40 ° C., 50 ° C., 60 ° C., 70 ° C., and 75 ° C. Next, Bacillus smithii was similarly determined for hydration activity and used as a control group. For ease of comparison, the value of Bacillus smithii at each temperature is set to 100, and the relative hydration activity of Geobacillus caldoxylosilyticus M16 is shown in the table. 2. The relative hydration activity of Geobacillus caldoxylosilyticus M16 was shown to increase with increasing temperature from 30 ° C to 75 ° C.

Example 4 (Comparison of thermal stability of nitrile hydratase)
In order to examine the thermal stability of the activity, a 25 U / mL cell suspension at 10 ° C. was prepared, subjected to a heat treatment at a predetermined temperature for a predetermined time, and the residual activity was measured. In addition, the hydration activity which converts a nitrile compound into an amide compound was measured by the following method. 1 mL of the cell suspension was added to 9 mL of 1.1% acrylonitrile solution (0.05 M potassium phosphate buffer, pH 7.5), and the reaction was started at a reaction temperature of 10 ° C. After 10 minutes, the reaction was stopped by adding 1 mL of 1N hydrochloric acid. In each strain, the activity (residual activity) after the storage treatment relative to the activity before the storage treatment is calculated, and the converted activity with the calculated residual activity of Bacillus smithy as the standard (100) is shown in the table.

Example 5 (Reaction to various nitrile compounds)
The hydration characteristics for converting various nitrile compounds shown in Table 4 to the corresponding amide compounds were examined. 1 mL of the cell suspension was added to 9 mL of a 1.1% nitrile solution (0.05 M potassium phosphate buffer, pH 7.5), and the reaction was started at a reaction temperature of 30 ° C. After 10 minutes, the reaction was stopped by adding 1 mL of 1N hydrochloric acid. As a result, all had the ability about the hydration activity to convert a nitrile compound into a corresponding amide compound.

According to the present invention, an amide compound can be synthesized from a nitrile compound under higher temperature conditions than in the conventional case. According to the present invention, an efficient amide compound can be produced.

Claims (7)

An amide compound comprising the action of a culture solution, cell or treated product of a microorganism belonging to Geobacillus caldoxylosilyticus in an aqueous medium to convert it to a corresponding amide compound in an aqueous medium. Production method. The manufacturing method of the amide compound of Claim 1 whose microorganisms which belong to Geobacillus caldoxylosilyticus (Geobacillus caldoxylosilyticus) are Geobacillus caldoxylosilyticus (Geobacillus caldoxylosilyticus) M16. The manufacturing method of the amide compound of Claim 1 or 2 whose reaction temperature is 20 to 75 degreeC. The manufacturing method of the amide compound of Claim 1 or 2 whose reaction temperature is 55 to 75 degreeC. Geobacillus caldoxylosilyticus M16 (reception number: FERM AP-20315) or a variant thereof. A culture solution, cell or treated cell of a microorganism belonging to Geobacillus caldoxylosilyticus, which is used for the method for producing an amide compound according to claim 1. An enzyme preparation for producing an amide compound from a nitrile compound, which contains a culture solution, cells or treated cells of microorganisms belonging to Geobacillus caldoxylosilyticus as an active ingredient.