JP2001299376A - Method for producing amido compound using biocatalyst - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing an aqueous solution of an amide compound having a low foaming property. SOLUTION: Dry weight of a catalyst to be usd having a nitrile-hydratase activity is set to one three thousand and five hundredth or less to the dry weight of a produced amide compound.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing an amide compound using a catalyst having nitrile hydratase activity.

[0002]

2. Description of the Related Art In a method for synthesizing a compound using a biocatalyst having an enzymatic activity, the reaction conditions are mild, so that the reaction process can be simplified, or the purity of the reaction product due to the small amount of by-products is high. Due to such advantages, it has been used in the production of many compounds in recent years. In the production of amide compounds, since nitrile hydratase, an enzyme that converts nitrile compounds to amide compounds, has been actively studied. For example, Japanese Unexamined Patent Publication No.
129190, JP-A-54-143592, JP-A-61-162193, JP-A-2-470, JP-A-5-470
JP-A-10-103681, JP-A-11-89575 and JP-A-11-123098. At present, it is used for industrial production of acrylamide, nicotinamide and the like as an excellent reaction process from the viewpoint of operability, safety, economy and the like.

[0003]

However, an aqueous solution of an amide compound produced with a biocatalyst is liable to foam as the concentration of the amide compound in the reaction solution increases, despite the fact that a high-purity reaction solution is obtained. In the case where there is a subsequent step such as a concentration step, a distillation step, a crystallization step or a polymerization step, it may cause a trouble. Also, when the product is shipped as it is, if the product is easily foamed, the product foams during transportation and infusion, which is not preferable. Therefore, it is necessary to remove the foam-causing substance (hereinafter referred to as a foaming component) from the reaction solution. However, at the present time, a method for removing foaming components from a reaction solution is disclosed in Japanese Patent Application No. 11-254151.
In addition, the method requires special purification equipment.

[0004] In view of the above problems, an object of the present invention is to provide a method for producing an amide compound using a biocatalyst that does not require special purification.

[0005]

Means for Solving the Problems As a result of intensive studies on the above problems, the present inventors have found that the foaming component in an aqueous solution of an amide compound produced with a biocatalyst contains proteins, polysaccharides and the like derived from the biocatalyst. I found out. Furthermore, if the amount of the biocatalyst used in the reaction is reduced, the reaction rate decreases, so that the reaction requires more time and the contact time between the catalyst and the reaction solution increases, so that the extraction amount of the foaming component increases. However, it was unexpectedly found that the amount of the foaming component extracted was small, and a reaction solution having a low foaming property was obtained, which led to the present invention.

That is, the present invention relates to a method for producing an amide compound from a nitrile compound using a catalyst having nitrile hydratase activity, wherein the dry weight of the catalyst used is 3500 minutes relative to the dry weight of the amide compound produced. A method for producing an amide compound, characterized by being 1 or less. The higher the concentration of the amide compound in the reaction solution, the more the extraction of the foaming component from the biocatalyst is promoted by the solvent effect of the amide compound, and therefore the present invention is more suitable for the case where the amide compound is accumulated at a high concentration. is there. The high concentration of the amide compound in the present invention means, for example, a value of 35% by weight or more in acrylamide.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The catalyst having nitrile hydratase activity referred to in the present invention may be of any form as long as it has a nitrile hydratase enzyme. Examples of the origin of the nitrile hydratase enzyme include microorganisms, animal and plant cells, and the like, but it is preferable to use microorganism cells from the viewpoint of the enzyme expression amount per weight and ease of handling.

[0008] As the microorganism species, for example, Bacillus (Ba
genus, Bacteridium, Micrococcus and Brevibacterium (Japanese Patent Publication No. 62-21519), Corynebacterium and Nocardia (No.
cardia) (Publication No. 56-17918), Pseudomonas (Ps
eudomonas) (Japanese Patent Publication No. 59-37951), Rhodococcus (Rh
odococcus) and Microbacteri
um) genus (Japanese Patent Publication No. 4-4873), Rhodococcus rhodochrous species (Japanese Patent Publication No. 6-55148)
No.], a strain of the genus Rhodococcus [JP-B 7-40]
No. 948].

[0009] Microorganisms into which a natural or artificially improved nitrile hydratase gene has been artificially incorporated may be used. However, in order to produce an amide compound using a small amount of a microorganism having a low nitrile hydratase expression level or a microorganism having a low nitrile hydratase conversion activity from a nitrile compound to an amide compound, more reaction time is required. Therefore, it is desirable to use a microorganism that expresses nitrile hydratase as high as possible and / or a microorganism that expresses nitrile hydratase having high conversion activity.

As the form of the catalyst, microorganisms, animal and plant cells, etc., which have been washed as necessary, may be used as they are, or they may be subjected to crushing or the like, followed by inclusive methods, crosslinking methods, carrier binding methods, etc. May be fixed.
Examples of the immobilization carrier for immobilization include glass beads, silica gel, polyurethane, polyacrylamide, polyvinyl alcohol, carrageenan, agar, gelatin and the like.

The nitrile compound used in the present invention is:
There is no particular limitation as long as it is converted into the corresponding amide compound by the action of nitrile hydratase. For example, aliphatic saturated nitriles such as acetonitrile, propionitrile, succinonitrile, adiponitrile, aliphatic unsaturated nitriles such as acrylonitrile, methacrylonitrile, aromatic nitriles such as benzonitrile, phthalodinitrile and 3- Heterocyclic nitriles such as cyanopyridine and 2-cyanopyridine are exemplified. As chemical or physical properties or economic problems, typical examples suitable for production using a biocatalyst include propionitrile, acrylonitrile, methacrylonitrile,
Cyanopyridine and 2-cyanopyridine are preferable, and acrylonitrile and 3-cyanopyridine are particularly preferable.

The dry weight of the amide compound referred to in the present invention is a dry weight as a pure content obtained by subtracting impurities from the amide compound to be produced. The dry weight of the amide compound in the reaction solution can be measured by preparing a standard solution having a known amide concentration using a standard substance and using gas chromatography or liquid chromatography. In addition, the dry weight of the catalyst is the dry weight of the catalyst when an unfixed catalyst is used. In the case where an immobilized catalyst is used, it is the dry weight of the biological component after deducting the immobilized carrier.

The method for measuring the dry weight of the catalyst is as follows when measuring the amount of dry cells in the unfixed microbial cell liquid. About 1 mL of the bacterial cell fluid is spread in a weighing bottle or a Petri dish with a known constant weight and weighed (W1 (g)).
After drying at 20 ° C for 3 hours, weigh again (W2
(G)). From these values, the dry residue concentration C1
(%) Is determined by C1 = W2 / W1 × 100. Furthermore, about 10 mL of the bacterial supernatant obtained by filtering the centrifuged supernatant obtained by centrifuging about 100 mL of the bacterial cell fluid through a membrane filter (manufactured by cellulose acetate) having a pore size of 0.45 μm.
, The dry residue concentration C2 (%) of the bacterial cell supernatant is also measured in the same manner as the method for measuring the dry residue of the bacterial solution. From these values, the dry cell concentration C3 (%) in the cell liquid is calculated by the following equation. C3 = C1-C2 (however, when C2 ≦ 1; when C2> 1, the cells in the bacterial solution are washed with a 50 mM phosphate buffer (pH 7.0) and measured again.)

In the case of using a cell obtained by immobilizing cells as a catalyst, the dry cell concentration of the cell liquid before immobilization is measured, and the mixing ratio of the immobilized carrier and the cell liquid in the immobilized catalyst is measured. Calculate the dry weight of the biological component from which the immobilized carrier has been subtracted.
During immobilization, those that swell or shrink are the sum of the dry residue concentration of the immobilized carrier before immobilization and the dry residue concentration in the bacterial solution before immobilization, and the dry residue of the immobilized catalyst after immobilization. From the concentration value,
The swelling ratio or the shrinkage ratio is calculated, and the dry weight of the biological component derived from the value obtained by subtracting the immobilized carrier is calculated using the calculated value.

The smaller the dry weight of the catalyst used with respect to the dry weight of the amide compound to be produced, the lower the foamability of the reaction solution. However, it is less than 1/3500 or less that foaming property does not matter, preferably 50
If it is 1/00 or less, foamability does not matter. The lower limit of the amount of the catalyst used is not particularly limited, but in order to obtain a sufficient reaction rate, when using currently known nitrile hydratase-expressing cells, it is preferably at least 1 / 100,000.

The reaction method for producing the amide compound may be any of a fixed bed, a moving bed, a fluidized bed, a stirring tank and the like, and may be a batch reaction or a continuous reaction. The reaction mode is selected depending on the physical properties of the reaction substrate, reaction solution, target compound, etc., the production scale, and the like, and the reaction apparatus is designed. Further, it is preferable to carry out the reaction while controlling the reaction conditions such as the reaction temperature and the reaction pH under optimum conditions so that the amide can be produced in a smaller scale or in a shorter time.

[0017]

Embodiment (Embodiment 1) Preparation of biocatalyst Rhodococcus rhodoc with nitrile hydratase activity
hrous J1 (FERM BP-1478), glucose 2%, urea 1
%, Peptone 0.5%, yeast extract 0.3%, cobalt chloride 0.05% (all by weight) (pH 7.
The cells were cultured aerobically at 30 ° C according to 0). 50 mM
The cells were washed with a phosphate buffer (pH 7.0) to obtain a cell suspension (15% by weight of dried cells). On the other hand, acrylamide, methylenebisacrylamide and 2-dimethylaminopropyl methacrylamide were 30,1,4, respectively.
The monomer mixed aqueous solution was adjusted so as to be% by weight.

Subsequently, a cell suspension, an aqueous monomer solution, 10
Wt% N, N, N ', N'-tetramethylethylenediamine aqueous solution, 10 wt% aqueous ammonium persulfate solution, 5,
After mixing at 2, 0.1 and 0.1 L / hr for polymerization, the mixture was cut into particles of about 0.5 mm square to obtain immobilized bacterial cell particles. The immobilized bacterial cell particles were washed with a 0.1% aqueous solution of sodium acrylate (adjusted to pH 7) to obtain an immobilized bacterial cell catalyst. (Dry cell weight was 10% in this catalyst)

2. Reaction of acrylonitrile to acrylamide by immobilized bacterial cell catalyst.
3510 g of a 2 g / L aqueous solution of sodium acrylate was added, and 3 g of the above-mentioned immobilized cell catalyst (dry cells 0.3
g). This is pH 7.0, temperature 10 ° C
, And two flat stirring blades having a blade length of 120 mm and a blade width of 20 mm were stirred at 80 rpm. This was continuously fed so that the acrylonitrile concentration was always 2% by weight, and the accumulation reaction was carried out until the concentration of acrylamide became 40% (2000 g of acrylamide produced). From this solution, the immobilized bacterial cell catalyst was separated by a wire mesh having a mesh size of 300 μm to obtain a reaction solution. (Dry weight of bacterial cells used / dry weight of generated acrylamide = 1/6700) The obtained acrylamide aqueous solution had little foaming.

3. Concentration 4 L of the obtained reaction solution is placed in a separable flask having an internal volume of 10 L, and the concentration of acrylamide becomes 50% in a warm bath at 45 mmHg while introducing air at 150 mL / min in order to prevent polymerization of acrylamide. Concentrated. As a result, about 4 L of transparent acrylamide 50
% Aqueous solution was obtained.

Comparative Example 1 A 25% acrylamide reaction solution was obtained in the same manner as in Example 1 using 25 g of the immobilized cell catalyst (corresponding to 2.5 g of dried cells) (dry weight of cells used / dry weight of acrylamide produced). = 1/800). Although it was an aqueous solution of acrylamide which easily foamed, when the concentration was attempted to be increased to 50% in the same manner as in Example 1, the reaction solution foamed violently, and it became difficult to continue the concentration operation to 50%.

[0022]

According to the present invention, the production of an amide compound using a biocatalyst such as cells, immobilized cells or immobilized enzymes can simplify the reaction process or increase the purity of the reaction product due to less by-products. Although it is very effective because of its advantages, it has a drawback that the reaction solution easily foams. According to the present invention, an aqueous solution of an amide compound having a low foaming property can be produced.

Claims (4)

[Claims] 1. A method for producing an amide compound from a nitrile compound using a catalyst having nitrile hydratase activity, wherein the dry weight of the catalyst used is 1/3500 or less of the dry weight of the produced amide compound. A method for producing an amide compound, comprising: 2. The method according to claim 1, wherein the nitrile compound is acrylonitrile,
2. A methacrylonitrile or cyanopyridine.
A method for producing the amide compound as described above. 3. The method for producing an amide compound according to claim 1, wherein the nitrile compound is acrylonitrile. 4. The method for producing an amide compound according to claim 3, wherein the concentration of the produced amide compound in the reaction solution is 35% or more.