JPH0354558B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention promotes the hydration reaction of nitrile groups,
This invention relates to a method for producing amide compounds using a water-soluble enzyme protein that has a catalytic function for converting into amide groups. Examples of industrially important amide compounds include:
Acrylamide, methacrylamide, nicotinamide, etc. are known. Acrylamide is used in polymer flocculants, paper strength enhancers, fiber improvers, etc., and methacrylamide is used in paints, flocculants, adhesives, photocrosslinkable compounds, etc. Nicotinamide is also used as a fortifier, medicine, livestock feed, etc. The production method using the water-soluble enzyme protein of the present invention can be used to efficiently industrially produce these industrially important amide compounds. (Prior art) In the technology of producing an amide compound using a catalyst that hydrates a nitrile group and converts it into an amide group, a method of heat treatment using an acid or alkali as a catalyst, some nitrile compounds, for example, For acrylonitrile, methacrylonitrile, etc., heat treatment using a copper catalyst,
Methods using microbial catalysts are known. However, when it comes to converting nitrile groups into amide groups in good yields, quickly under mild conditions, and with as little catalyst as possible, methods using acid or alkali catalysts have low conversion rates; However, when the reaction temperature was raised to achieve this, there were drawbacks such as the formation of by-products. In addition, even in the method using a copper catalyst, heat treatment is required, so
By-product ammonium carboxylate is generated,
Nitrile compounds having double bonds have the disadvantage of forming polymers. On the other hand, in the method using a microbial catalyst, the hydration reaction can be carried out at room temperature, and the conversion rate is high, making it a preferable method. However, microbial catalysts have the disadvantage that they can be mixed into the reaction system as impurities. In particular, trace amounts of heavy metal ions leaked from microbial bodies often act as polymerization accelerators, and are a drawback of generating polymers when amidating nitrile compounds with double bonds. Ta. In addition, the drawback that the amide compound is further hydrated and converted into an ammonium carboxylate compound within the microorganism, and the ammonium carboxylate thus generated deactivates the catalytic function of the microorganism, cannot be ignored. (Problems to be solved by the invention) In hydration conversion of a nitrile group to an amide group,
It is desired to develop a method for producing amide compounds in a high yield, rapidly, under mild conditions, using as little catalyst as possible, and using a catalyst that does not introduce impurities into the reaction system. (Means for Solving the Problems) In order to solve these problems, the present inventors sought a catalyst for the hydration conversion of nitrile groups into amide groups in enzyme proteins, and determined that the PH of the surface charge of the protein
By applying principles such as dependence, interaction with ion exchange resins, molecular sieving effects, and electrophoretic effects, we can isolate and purify various enzyme proteins from bacteria, yeast, molds, actinomycetes, etc. The presence or absence of hydration activity ability of the group was tested. As a result, the molecular weight
We have discovered a novel enzyme protein that is composed of two different subunits of 26,500±500 and 28,500±500, and has a catalytic function to convert nitrile groups into amide groups, and have completed the present invention. This enzyme is an intracellular enzyme of the spAK-32 strain (Feikoken Bibori No. 1046) belonging to the genus Rhodocotcus.
It can be produced within the bacterial cells by growing the AK-32 bacteria in a culture solution prepared by adding an inducer to a normal nutrient medium. An example of culture conditions is as follows. Culture solution composition Meat extract 1.0% by weight Peptone 1.0 〃 Glucose 1.0 〃 NaCl 0.1 〃 Mineral aqueous solution 96.4 〃 Isobutyramide 0.5 〃 PH 7.0 〃 Mineral aqueous solution Potassium dihydrogen phosphate 0.1% by weight Potassium nitrate 0.1 〃 Ammonium sulfate 0.1 〃 Magnesium sulfate 0 .05 〃 Sulfuric acid No. Iron 0.005 〃 Manganese sulfate 0.005 〃 Distilled water 99.64 〃 Cultivated at 30℃ for 3 days An enzyme (hereinafter referred to as AKE-32 protein) that can hydrate and convert nitrile groups present in bacterial cells into amide groups After crushing by the French press method or homogenization method in the presence of glass beads, the enzyme is isolated and purified through the usual procedures of ammonium sulfate fractionation, dialysis, anion exchange resin treatment, and gel chromatography separation in the stable pH range of the enzyme. be able to.
Furthermore, by freeze-drying, it is also possible to solidify the AKE-32 protein while maintaining its activity. When using AKE-32 protein, it can be used in the form of purified enzyme, crude enzyme solution, or disrupted bacterial cell solution.
If amidase coexists, unless it is separated, the generated amide may be hydrated to ammonium carboxylate. The range of usage conditions for AKE-32 protein is as follows. PH6.0-10.0 preferably PH8.0-9.0. Ion concentration in reaction solution 0.001~
1.0mol ion/, preferably 0.01-0.1mol ion/
. An example of an ion that is preferably allowed to coexist in the reaction solution is potassium ion. Reaction temperature 0
~30°C, preferably 0-10°C. Nitrile concentration 0.1
~5% by weight. Regarding the immobilization of AKE-32 protein, any conventionally known method can be used, but under reaction conditions where the rate of diffusion of the substrate into the immobilized substance is rate-limiting, it is necessary to intentionally use an immobilization method. Good too. The reaction in which a nitrile group is converted to an amide group is an exothermic reaction, and depending on the immobilization method, the heat of reaction may accumulate within the immobilized product and deactivate the enzyme. An amide with low solubility in water, which makes it relatively easy to separate the amide compound and enzyme.
For example, in the production of methacrylamide, iso and n-butyramide, succinamide, benzamide, etc., as the amide precipitates as crystals as the reaction progresses, it is easy to separate the crude crystals and enzyme from the reaction solution by solid-liquid separation. Can be separated and recovered. The amide product thus obtained can be purified according to any suitable method to obtain the amide product. For example, minute amounts of AKE-32 protein and inorganic ions mixed into the crude crystals (under conditions of temperature and concentration that allow for crystallization during post-processing) can be removed by dissolving the crude crystals in water and then using activated carbon. It can be easily removed by treatment or ion exchange resin treatment. On the other hand, most of the remaining in the mother liquor
The AKE-32 protein can be used again in the reaction, and the amount leaked into the crude crystals may be replenished into the reaction system. The procedure for purifying the AKE-32 protein, the procedure for determining the N-terminal amino acid sequence, and the means for determining the physicochemical properties will be described in more detail. Enzyme purification procedure 1 Disruption of the cell wall of sp.AK-32 bacteria (Feikoken Bacteria No. 1046) 2 Recovery of the disrupted supernatant by centrifugation 3 Determination of the acid in the disrupted supernatant by adding an aqueous solution of streptomycin sulfate Precipitation 4 Removal of the acid by centrifugation 5 Precipitation of proteins in the supernatant by addition of saturated ammonium sulfate solution 6 Recovery of precipitated proteins by centrifugation 7 Removal of ammonium sulfate from the precipitated proteins by dialysis 8 Anion Crude purification of precipitated protein group solution after dialysis treatment by column chromatography packed with exchange resin 9 Nitrile group hydration activity test of crude protein solution 10 Concentration of nitrile hydration active protein solution after rough purification using Centricon 11 Purification of concentrated protein solution by sieve chromatography 12 Nitrile group hydration activity test of purified protein solution 13 Concentration of nitrile hydration active protein solution after purification using Centricon 14 Measuring the purity of purified protein by electrophoresis Configuring the enzyme Steps for determining the N-terminal amino acid sequence of the subunits 1. Heat treatment of purified protein solution in the coexistence of β-mercaptoethanol and sodium lauryl sulfate 2. Separation of constituent subunits by preparative electrophoresis 3. Concentration of the separated subunit solution using centricon 4. Electrophoresis Measurement of purity and molecular weight of separated subunits by electrophoresis 5 Centrifugal concentration and drying of separated subunit samples 6 Edman degradation reaction using a protein sequencer 7 Qualitative and quantitative analysis of amino acids obtained in each sequencer cycle using HPLC 8 N-terminal amino acid analysis Sequence Determination To explain the AKE-32 protein in more detail, add an equal amount of the sample buffer solution shown below to 0.25 ml of the purified enzyme solution and heat at 100° C. for 5 minutes. Sample buffer liquid composition Bromophenol blue 10 mg Glycer 1 ml 10% by weight sodium laurate aqueous solution 2 ml β-mercaptoethanol 0.5 ml Distilled water 6.5 ml Next, the heat-treated enzyme solution was poured into a stick gel packed with acrylamide gel. The two constituent subunits are separated and ingested. The conditions at that time are as follows. Electrophoresis buffer composition Tris-hydroxymethylaminomethane 6.0g Glycine 28.8g Sodium lauryl sulfate 1.0g Distilled water 1 Constant current 6mA Temperature 25℃ Flow rate 300ml/mm Transfer each of the fraction solutions in which the two subunits have been completely separated to the centricon. Contaminated glycine is removed from the electrophoresis buffer by repeating the operations of centrifugal concentration and washing with water. After centrifuging and concentrating the solutions of subunit ○ small and subunit ○ large to dryness, trifluoroacetic acid was added to dissolve the subunits, and Edman decomposition reaction was applied.
Applicable to Applied Biosystems' gas phase protein sequencer. HPLC was used to identify PTA (phenylthiohydantoin) amino acids, and the
The peak position and peak height of PTH-amino acid are compared with those of standard PTH-amino acid for qualitative and quantitative determination. The N-terminal amino acid sequences of the two subunits are determined from the amino acids that are sequentially analyzed. Other methods for solving physical and chemical properties Optimum PH After dissolving a certain amount of nitrile compound in a certain amount of phosphate buffer solution of various pH values from 3 to 11, a certain amount of AKE-32 protein is dissolved. is added, and the time course of the nitrile hydration reaction is followed by gas chromatography. Compare the hydration reaction rates of each, and select the pH of the phosphate buffer solution where the reaction proceeds most rapidly as the optimal pH. PH Stability Two samples are prepared by adding a certain amount of AKE-32 protein to each of a certain amount of phosphate buffer solution with a pH value of 3 to 11. To one sample, a certain amount of nitrile compound was added immediately, and to the other sample, after standing for 24 hours at 20 °C, a certain amount of nitrile compound was added, and for each sample, the nitrile hydration The reaction is followed over time by gas chromatography. The PH range of the phosphate buffer solution where the reaction rate decreases least before and after standing is defined as the stable range. Temperature stability A certain amount of AKE− in a phosphate buffer solution of PH8.5
Two samples with 32 protein added were prepared, and a certain amount of nitrile compound was added to one sample immediately, and a certain amount of nitrile compound was added to the other sample after it was left at 30℃ for 16 hours. The nitrile hydration reaction of each sample is then monitored over time by gas chromatography.
Compare the degree of deactivation of the reaction rate before and after standing. Inhibitor Add a certain amount of AKE- to a phosphate buffer solution at pH 8.5.
After adding a substance that can serve as an inhibitor to the 32-protein mixture, a certain amount of a nitrile compound is added, and the hydration reaction of the nitrile is monitored over time by gas chromatography. Compare whether the reaction rate decreases with or without an inhibitor. Infrared absorption spectrum After freeze-drying the purified enzyme protein, use an infrared absorption spectrometer to determine the absorption band wavelength using the KBr tablet method. (Effects of the Invention) According to the present invention, when hydration converting a nitrile group into an amide group, the hydration reaction can be rapidly promoted with an extremely small amount of catalyst and even at temperatures below room temperature. , the conversion rate to amide groups is extremely high (selectivity: 100%), and an industrially satisfactory method for producing amide is provided that does not generate impurities or by-products in the reaction system. (Example) Example 1 100 parts of a solution prepared by adding solid sodium hydroxide to a 0.05 mol/aqueous potassium dihydrogen phosphate solution and adjusting the pH to 8.5 were taken and cooled to 0.5°C using ice water. Add 0.01% by weight of AKE-32 protein to this solution, then add methacrylonitrile.
17 parts were added, and the hydration reaction of the nitrile group was carried out while stirring at 0.5°C. At this time, a part of methacrylonitrile was dissolved and the rest was not dissolved and separated into two phases, but the reaction was continued regardless. Two hours after the start of the reaction, methacrylonitrile was no longer present in the reaction system, and methacrylamide crystals were observed.
After the crystals were separated from the mother liquor by suction, the mother liquor adhering to the surface of the crystals was quickly washed away using cold water at 0°C. The methacrylamide crystals obtained here could be used as raw materials for producing various polymers. Example 2 Take 50 parts of a solution prepared by adding phosphoric acid to 0.035 mol/potassium monohydrogen phosphate and adjusting the pH to 8.0,
Cooled to 2°C. AKE-32 protein was added to this solution in an amount equivalent to 0.02% by weight, and then 8 parts of methacrylonitrile was added, and a hydration reaction of the nitrile group was carried out with stirring at 2°C. Two hours after the start of the reaction, methacrylonitrile, which had already separated into two phases, disappeared, and methacrylamide crystals were precipitated. An additional 50 parts of distilled water was added to the reaction system to dissolve the precipitated methacrylamide, and then the methacrylamide was quantified and the presence or absence of by-products was analyzed using gas chromatography. As a result, more than 99.9% of the added methacrylonitrile was converted to methacrylamide, and no detected peaks derived from by-products were observed. Furthermore, after the reaction was completed, the reaction solution was allowed to stand at room temperature for an additional 24 hours, and then the presence or absence of an organic acid was examined using liquid chromatography analysis, but no production of methacrylic acid was observed. Examples 3 to 14 50 parts of a 0.02 mol/potassium hydrogen carbonate aqueous solution was taken and cooled to 0°C. To this solution was added AKE-32 protein in an amount equivalent to 0.02% by weight, and then 1 part of each type of nitrile was added, and a hydration reaction of the nitrile group was carried out with stirring at 0°C. The conversion rate up to 30 minutes after the start of the reaction was monitored using gas chromatography, and the conversion rate of various nitrile compounds to amide compounds was compared with that of methacrylonitrile. Table 1 shows the results.

[Table] In addition, by passing the aqueous solutions of various amides obtained in Examples 3 to 14 above through a cation exchange resin packed column and an anion exchange resin packed column in that order, potassium ions that were present in the system were removed. It was also possible to remove hydrogen carbonate ions. The purity of the various amide aqueous solutions thus obtained was 99.9% or more in terms of the amide compound. Comparative Example 1 Each of the acid and base aqueous solutions in Table 2 below was
Add 1% by weight of various nitrile compounds and heat at 30°C.
Although the mixture was left stirring for 24 hours, no amide compound was observed to be formed.

[Table] (Note) In the table, an x mark indicates that the corresponding amide compound was not observed.
Comparative Example 2 Sodium borohydride was added to a 1.0 mol/aqueous copper chloride solution to create an activated copper catalyst. After adding 20g of this copper catalyst to 50ml of distilled water,
Add methacrylonitrile equivalent to 2% by weight,
Although the mixture was left for 1 hour while stirring at ℃, no formation of methacrylamide was observed. However, when this catalyst was heated to 100°C in a glass autoclave and contacted with the reaction solution for about 1 hour while stirring, it was found that in addition to methacrylonitrile, methacrylamide and methacrylic acid were present in the system. This was revealed by gas chromatography analysis. However, a white polymer was produced in the autoclave. Comparative Example 3 AK-32 bacteria was added to 50 ml of 0.035 mol/phosphate buffer solution adjusted to pH 8.0 at 2°C in terms of dry weight.
After adding 10mg, add 1g of propionitrile,
As a result of monitoring the hydration reaction of the nitrile group every 5 minutes with gas chromatography analysis while stirring, it was found that 20
In minutes, only 10% of the propionitrile added was converted to propionamide. Furthermore, after the reaction solution was left at room temperature for 24 hours, the presence or absence of carboxylic acid production was examined by liquid chromatography analysis, and the presence of propionic acid was observed.

Claims (1)

[Scope of Claims] 1. Derived from a bacterium of the genus Rhodococcus, which has the ability to catalyze the conversion of nitrile groups into amide groups, and which has the ability to catalyze the conversion of acrylonitrile and methacrylonitrile to the corresponding amide compounds. A method for producing an amide compound, which is characterized by producing a corresponding amide compound from a nitrile compound using a water-soluble enzyme protein composed of two heterogeneous subunits with excellent speed as a catalyst. 2. The method according to claim 1, wherein the nitrile compound has 6 or less carbon atoms. 3. The method according to claim 2, wherein the nitrile compound is an unsaturated nitrile, and the nitrile group is bonded to an unsaturated carbon. 4. The method according to claim 1, wherein the nitrile compound is methacrylonitrile. 5. The method according to claim 1, wherein the nitrile compound is acrylonitrile. 6. The method according to any one of claims 1 to 5, wherein the water-soluble enzyme protein catalyst composed of two heterogeneous subunits has the following physical and chemical properties. Molecular weight Subunit ○Large molecular weight 28500±500 (SDS electrophoresis measurement) Subunit ○Small molecular weight 26500±500 (SDS electrophoresis measurement) N-terminal amino acid sequence Subunit ○Large; 1 Met -Asp-Gly-Val-5 His -Asp-Leu −Ala−Gly−10 Lys −Gln−Gly−Phe−Gly −15 Pro −Val−Asp−His−Thr−20 Ile −Asn −Glu−Tyr−Glu−25 Lys −Gly−Gln−Pro −Val−30 Pro - subunit ○ small; 1 Ser -Lue-Met-Ile-5 Asp -Arg-Glu -His-Asp-10 Gln -Gly-Val-Thr-His -15 Ala -Pro-Gly-Val-Pro-20 Glu -Gln -Ala-Pro-Ala-25 Gly -Asp-Arg-Ala - Enzyme action Acts on the nitrile group of a nitrile compound and acts as a catalyst for the reaction of hydration converting the nitrile group into an amide group. Optimum PH The hydration effect of the nitrile group is optimal around PH8.5. PH Stability PH8.0 to PH9.0 is a stable region, and as it deviates from this region, stability decreases. PH
3.0 immediately loses hydration activity. Temperature stability At pH 8.5, more than 95% of the hydration activity of the nitrile group remains even after being left at 30°C for 16 hours. Significantly inhibited by inhibitors Ag ⁺ and Hg ²⁺ . It is also inhibited by ammonium methacrylate. Infrared absorption spectrum The absorption bands in the infrared absorption spectrum by KBr tablet method are 3420, 3330, 2960, 2930, 1650,
1540，1450，1400，1350，1300，1240，1190，
1170, 1110, 1030, 930, 880, 850, 750, 550
and 510 cm ^-1 .