JPH05995B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing carboxylic acids and/or alcohols. As part of our research on microbial reactions using aldehydes as substrates, the present inventors discovered a method for obtaining carboxylic acids and/or alcohols through a cross-disproportionation reaction of different aldehydes, and arrived at the present invention. That is, the gist of the present invention is that aldehydes represented by the general formula (): RCHO () (in the formula, R represents a hydrogen atom, an alkyl group, an alkenyl group, or an acyl group) and the general formula (): R' CHO () (In the formula, R' represents a hydrogen atom, an alkyl group, an alkenyl group, or an acyl group, and R and R' are different.) React with an aldehyde represented by CHO () in the presence of aldehyde dismutase. The carboxylic acids represented by the general formula (): RCOOH () (wherein R has the same meaning as in the general formula ()) and/or the general formula (): R'CH ₂ OH () (wherein, R ' has the same meaning as in general formula ()). The present invention will be explained in detail below. First, aldehyde dismutase (aldehyde dismutase) used in the method of the present invention
is an enzyme that simultaneously performs redox of aldehydes without the addition of coenzymes. For example, Pseudomonas putida F61
(FERM P-No.7165). The mycological properties of this Pseudomonas putida F61 are shown below. Primary differentiation (morphological and physiological observation when grown in Oxoid CM3 medium (30°C)) Morphology Bacillus Gram - Spore - Motility + Colony morphology Pale yellow, round, opaque, whole round,
Glossy, low concavity, diameter 0.5 mm in 24 hours Growth temperature (℃) 5 + 37 + 41 - 45 - Catalase + oxidase + O-F test 0 Identification by primary differentiation: Gram negative, decomposes sugars by oxidation. Secondary differentiation Pyocyanin - Fluorescence + DL-Arginine + Betaine + Glucose + Lactate + Acetate + Penicillin G - Streptomycin Chloramphenicol - Tetracycline Novobiocin - Polymyxin B 0/129 - Levan - Growth factor requirement - From glucose - Acid production from glycose + ONPG - Arginine dihydrogenase + Lysine decarboxylase - Ornithine decarboxylase - Reduction of nitrate to nitrite - Reduction of nitrate to nitrogen gas - Deoxyribonuclease - Gelatin-containing multistory culture (20 °C) − Plain culture containing gelatin − Casein hydrolysis − Starch hydrolysis − Egg yolk reaction − Lipase activity − NH ₃ + indole − H ₂ S − “Tween 80” hydrolysis − As a result, this strain It was identified as Monasputida. For identification, please refer to Bergey's Manual of Determinative Bacteriology.
Determinative Bacteriology), 8th edition
(1974) and Cowan et al.'s Manual for the Identification of Medical Bacteria.
Identification of Medical Bacteria)
(1974) was referred to. The nutrients necessary for culturing this microorganism are not particularly limited, and those commonly used for culturing microorganisms are used. For example, as the carbon source, carbohydrates such as glucose, sucrose, fructose, glycerol, sorbitol, molasses, and starch hydrolysates, and organic acids such as acetic acid and fumaric acid are used. Examples of nitrogen sources include nitrates, ammonium salts, cornstarch liquor, yeast extract, meat extract, yeast powder, soybean hydrolyzate, cottonseed flour, polypeptone, pentone, and the like. Potassium phosphate, sodium phosphate, magnesium sulfate, sodium chloride, etc. can be used as the inorganic salt. The culture temperature is preferably 20 to 40°C, particularly 25 to 37°C. Cultivation is usually carried out aerobically for about 16 to 72 hours. The aldehyde dismutase obtained in this way is mainly present in the cells of microorganisms, and the cells themselves collected from the culture can be used, or they can be further processed by ultrasonication, ammonium sulfate fractionation, ion exchange chromatography, etc. It can also be used after being separated and purified by known methods such as graphite and gel filtration. That is, after the cells obtained after culturing are collected by centrifugation, the cells are disrupted by mechanical means such as ultrasonication, homogenizer, and grinding with glass beads, or by chemical means such as cell wall lytic enzymes. , obtain the supernatant by centrifugation. Next, this supernatant is subjected to ammonium sulfate fractionation, ion exchange chromatography such as "DEAE-Sephacel", adsorption chromatography using hydroxyapatite etc., gel chromatography using "Sephacryl", "Sephadex" etc., phenyl-chlorceph It is purified by a combination of hydrophobic chromatography using allose, etc. The properties of this purified enzyme are as follows. Molecular weight: 2.2×10 ⁵ (gel filtration) (4 subunits of 5.5×10 ⁴ ) Isoelectric point: PH4.8 Optimum PH: 8.0 Optimum temperature: 40℃ HgCl ₂ , PCMB (p-chloromercury benzoate) , iodoacetic acid, Zn, Cu, and Fe. In the method of the present invention, aldehydes represented by general formulas () and () are reacted in the presence of the aldehyde dismutase. R and R' in general formulas () and () are
They are different from each other and are selected from a hydrogen atom and a straight or branched alkyl group, alkenyl group and acyl group, preferably a hydrogen atom, a carbon number 1
-4 alkyl group, an alkenyl group having 2 to 6 carbon atoms, and an acyl group having 2 to 6 carbon atoms. Further, it is more preferable that at least one of the two types of aldehydes represented by the general formulas () and () has a high degree of hydration in water, such as formaldehyde, acetaldehyde, or methylglyoxal. The quantitative ratio of aldehydes is
Usually, the amount is about equimolar, but when formaldehyde is used, it is preferable to use a slightly excessive amount of the other aldehyde. The reaction is usually carried out at a pH of 5.5 to 9.5, preferably at a pH of 7 to 9.
The reaction is carried out in a commonly used buffer such as potassium phosphate. Aldehyde dismutase is not limited to purified products, but also bacterial cells (immobilized bacterial cells can also be used), extracts,
Although any form of crude purified product can be used, the reaction temperature is usually 10 to 45°C, preferably 30 to 40°C. The reaction time depends on the amount of dismutase used (usually
0.01 to 10 units), 10 minutes to 100 units, depending on the concentration and type of substrate and reaction temperature.
The time is usually selected from 30 minutes to 48 hours. The concentration of aldehydes that are substrates is usually 1mM to several M.
selected from. When the reaction is completed, the corresponding carboxylic acids are produced by a cross-disproportionation reaction of the substrate aldehydes.
RCOOH and alcohols (R′CH ₂ OH (further,
Depending on the amount ratio of different aldehydes, some
RCH ₂ OH) is obtained. The reaction according to the method of the present invention is represented by the following formula. RCHO+H ₂ O+EX→RCOOH+EX−H ₂ R′CHO+EX−H ₂ →R′CH ₂ OH+EX (E is dismutase, Isolated by ion exchange resin treatment, etc. According to the method of the present invention, carboxylic acids and/or alcohols can be obtained by cross-disproportionation reaction of different aldehydes without requiring the addition of coenzymes for redox reactions. Suitable for bioreactor using a body. Hereinafter, the present invention will be explained in further detail with reference to Examples. Reference example 1 Peudomonas putida F61, 10 g of peptone, 5 g of meat extract, 1 g of K ₂ HPO ₄ , NaCl
28 flasks were inoculated with 28 medium 1 consisting of 5g of formaldehyde and 1g of formaldehyde (PH7.0),
Incubate with shaking at 30°C for 48 hours. After suspending 200 g of bacterial cells obtained from culture solution 28 in 10 mM potassium phosphate buffer (PH7.0), ultrasonication (19K
Hz, 30 minutes). Then centrifugation (16000 x g,
30 minutes) to obtain a bacterial cell extract. 25g of supernatant, water
Protamine sulfate dissolved in 500 ml is added dropwise while stirring. Stir for 30 minutes and centrifuge the solution (83,000 xg, 30 minutes). Solid ammonium sulfate is added to the obtained supernatant solution (1500 ml) at pH 7.0 to achieve 60% saturation, and the resulting precipitate is removed by centrifugation. Add ammonium sulfate to the supernatant solution until 90% saturation.
The precipitate collected by centrifugation is dissolved in 200 ml of 10 mM potassium phosphate buffer (PH7.0). Solid NaCl (4M) is added to the enzyme solution (220ml) and the mixture is introduced into a "Phenyl-Sepharose" column (4 x 50cm) (equilibrated with 10mM potassium phosphate buffer containing 4M NaCl). After washing the column with buffer, elution is performed with a gradient of decreasing NaCl concentration and increasing ethylene glycol concentration (final concentration 0, 50%; total volume 6) (flow rate: 19 cm/hour). Collect the active fraction and add it to 10mM potassium phosphate buffer (PH7.0)
Dialyze with The dialyzed solution was concentrated to 100ml by ultrafiltration and applied to a “DEAE-Sefacel” column (2.5 x 45cm) using 10mM potassium phosphate buffer (PH7.0).
to be introduced. The column is washed with Equilibration Buffer 2.5 and then with Buffer 2 containing 100mM NaCl. Enzyme increases NaCl (100→300mM,
It is eluted by the linear gradient method (flow rate 10 cm/hour) according to 2.5). The active ingredient is collected and dialyzed against 10mM potassium phosphate buffer. 50 ml of the enzyme solution is introduced into a hydroxyapatite column (2.5 x 21 cm) (pre-equilibrated with the above buffer). Potassium phosphate buffer concentration 10, 50, 100
and increase the concentration to 200 mM and elute stepwise (flow rate: 8 cm/hour). Enzyme activity appears in the fraction eluted with 200mM potassium phosphate buffer (PH7.0). Collect these fractions
Dialyze against 10mM potassium phosphate buffer (PH7.0),
Concentration was performed by ultrafiltration to obtain 18 ml (3470 units) of purified enzyme. This purified enzyme solution is stored at 5°C. (Measurement of activity) Aldehyde dismutase activity was measured at 20mM
aldehyde, 100mM KCl and enzyme (final volume 10
ml) using a PH stat. The reaction was carried out at 30 °C with stirring under _N2 atmosphere, and acid production was maintained at pH 7 with 10 mM NaOH.
Titrate. One unit of activity is defined as the enzyme activity that produces 1 μmol of acid per minute. Example 1 Two types of aldehydes shown in Table 1 were reacted for 20 minutes at 30°C in the presence of 5 units/3 ml of dismutase obtained in Reference Example 1 and 300 μmol/3 ml of KCl (PH8.0). The amount of acid was 10mM
The titration of NaOH was measured, and the amount of alcohol produced was determined by gas chromatography.

[Table] Example 2 Using 10mM of HCHO and 20mM of R'CHO shown in Table 2 as aldehyde (RCHO), Reference Example 1
0.5 units/ml of dismutase obtained in
The reaction was carried out at 30°C for 30 minutes in the presence of (PH
8.0). The amount of alcohol produced is shown in Table 2 (formic acid was successfully used in an amount approximately equimolar to the amount of alcohol).

[Table] Example 3 In Reference Example 1, the microbial cells obtained from the culture solution were centrifuged and the microbial cells obtained were washed with a 0.9% NaCl solution and dried by a conventional method to obtain dry microbial cells. Using 1 mg/ml of these bacterial cells, a reaction was carried out in 0.05 M potassium phosphate buffer using 10 mM formaldehyde and 10 mM acetaldehyde as substrates to obtain 9.5 mM formic acid and 9.5 mM methanol.

Claims (1)

[Claims] 1 Aldehydes represented by the general formula (): RCHO () (in the formula, R represents a hydrogen atom, an alkyl group, an alkenyl group, or an acyl group) and the general formula (): R′CHO () (In the formula, R' represents a hydrogen atom, an alkyl group, an alkenyl group, or an acyl group, and R and R' are different.) are reacted in the presence of aldehyde dismutase. and/or carboxylic acids represented by the general formula (): RCOOH () (wherein R has the same meaning as in the general formula ()) and/or the general formula (): R'CH ₂ OH () (wherein R' is the same as in the general formula ()) A method for producing carboxylic acids and/or alcohols, characterized by obtaining alcohols represented by the formula ().