JPH0458313B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a method for producing formic acid. (Prior Art) It has been known that formaldehyde can be produced from methanol by alcohol oxidase of methanol-assimilating yeast. (Biotechnolo.Bioeng., 20, 333-348 (1978)). CH ₃ OH + O ₂ = HCHO + H ₂ O ₂ (reaction by alcohol oxidase) H ₂ O ₂ H ₂ O + 1/2O ₂ (reaction by catalase) ₃ +12 ₂ = + ₂ (total) However, when performing this reaction industrially,
The problem is that alcohol oxidase is inactivated by its reaction product, formaldehyde, and it is necessary to continuously trap the resulting target product, formaldehyde, out of the reaction system, or to channel it into a valuable substance that does not inhibit the yeast reaction. be. The present inventors conducted various studies on methods of producing formic acid at a higher yield than methanol without adding any coenzyme. Among these, the present invention was achieved by paying attention to the fact that formaldehyde dismutase reacts with formaldehyde as a substrate (Agric.Biol.Chem., 47(1), 39-46, 1983). That is, the gist of the present invention is that when producing formic acid from methanol, methanol is converted to formic acid by reacting in the presence of oxygen and in the coexistence of alcohol oxidase, catalase, and formaldehyde dismutase. There is a method for producing formic acid. (Structure of the Invention) The present invention will be described in detail below. The reaction formula for the process of producing formic acid from methanol in the present invention is as follows. CH ₃ OH + O ₂ → HCHO + H ₂ O ₂ (reaction by alcohol oxidase) H ₂ O ₂ → H ₂ O + 1/2O ₂ (reaction by catalase) HCHO + 1/2H ₂ O → 1/2CH ₃ OH + 1/2HCOOH (reaction by formaldehyde dismutase) reaction) 12 ₃ +12 ₂ →12+12 ₂
In other words, if formaldehyde dismutase, which is considered to be a coenzyme-intrinsic enzyme, is used, it acts on formaldehyde, which is the substrate, without adding any coenzyme, and therefore converts methanol to formic acid almost 100%. It can be converted. First, alcohol oxidase and catalase used in the present invention are usually obtained from yeast, for example, from the methanol-assimilating yeast Hansenula Polymorpha CBS4732. The mycological properties of Hansenula polymorpha CBS4732 are, for example,
Yeasts) J.LODDER 2nd edition (1970) No. 296-299
It is written on the page. In addition, representative examples of other yeasts include Kloeckera sp, Candida boidinii (both European Journal of Biochemistry, 64 ,
341-350, 1976). Furthermore, aldehyde dismutase (aldehyde dismutase) used in the method of the present invention
is oxygen that simultaneously performs the redox of aldehydes without the addition of coenzymes, and is obtained, for example, from Pseudomonas Putida F61 (Feikoken Jokyo No. 1023). The mycological properties of DaF61 are shown below. Primary identification (morphological and physiological observation when grown in oxoid CM3 medium (30°C)) Morphology Bacillus Gram - Spore - Motility + Colony morphology Pale yellow; circular, opaque, full circle,
Glossy, low concavity, diameter approximately 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 - Firefly + DL-Arginine + Betaine + Glucose + Lactate + Acetate + Penicillin G - Streptomycin +++ Chloramphenicol - Tetracycline ++ Novopyocin - Polymyxin B ++ 0/129 - Levan - Growth factor requirement − Gas production from glucose − Acid production from glucose + ONPG − Arginine dihydrogenase + Lysine decarboxylase − Ornithine decarboxylase − Reduction of nitrate to nitrite − Reduction of nitrate to nitrogen gas − Deoxyribonuclease − Gelatin-containing stratum Culture (20℃) − Gelatin-containing flat culture − Casein hydrolysis − Starch hydrolysis − Egg yolk reaction − Lipase activity NH ₃ + indole − H ₂ S − “Tween 80” hydrolysis − As a result of the above, this strain It was identified as Pseudomonas putida. For fixing, please refer to (Bergey's) Manual of (Determinative) Bacteriology, 8th edition (1974) and ()
Manual by Cowan et al.
for (the) 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, yeast extract, meat extract, yeast powder, soybean hydrolyzate, cottonseed flour, polypeptone, penton, 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, gel chromatography using "Sephacryl", "Sephadex", etc., phenyl-chromatography, etc. It is purified by a combination of hydrophobic chromatography using luciferose, 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-chloromer kyuri-
benzoate), iodoacetic acid, Zn, Cu, and Fe. In the method of the present invention, methanol is reacted in the coexistence of the alcohol oxidase, catalase, and formaldehyde dismutase and in the presence of oxygen. The reaction is usually carried out at a pH of 5.5 to 9.5, preferably a pH of 7 to 9.
The reaction is carried out in a commonly used buffer such as potassium phosphate. The above-mentioned enzymes in the present invention are not limited to purified products, but can be used in the form of bacterial cells (immobilized bacterial cells can also be used), extracts, or crudely purified products, but the reaction temperature is usually 10 to 45°C, preferably is 30-40℃. The reaction time varies depending on the amount of enzyme such as dismitase used (usually about 0.01 to 10 units), the concentration and type of substrate, and the reaction temperature,
Selected from minutes to 100 hours, usually 30 minutes to 48 hours. The concentration of methanol as a substrate is usually 1mM.
~Selected from several M. Oxygen can be present, for example, by air or oxygen blowing methods, but
The amount used is usually an equimolar amount or more of methanol. The product thus obtained is isolated according to conventional methods, for example by treatment with an ion exchange resin. (Effects of the Invention) According to the method of the present invention, formic acid can be obtained at once from methanol in high yield without requiring the addition of coenzymes and without being inhibited by the intermediate product formaldehyde. (Example) Hereinafter, the present invention will be explained in further detail with reference to Examples. In the Examples, FDM means formaldehyde dismutase, AOD means alcohol oxidase, and CAT means catalase. Example 1 (Used strain) Methanol-assimilating yeast, Hansenula
PolymorphaDL1 was cultured in medium A (Table 1) at 30℃ for 48 hours with shaking, and after collection and washing, it was frozen and stored (-15
℃). FDM-producing bacteria, Pseudomonas putida
F61 was cultured with shaking in medium B (Table 2) at 30°C for 72 hours, and stored frozen in the same manner as above.

[Table] Place the above stick 1 into a 2 volume Sakaguchi flask.

[Table] Table 2 Composition of medium B Peptone 10g Meat extract 5 NaCl 5 K ₂ HPO 1 Glyose 10 Deionized water 1000ml PH 7.0 Place the above medium 1 into a 2 volume Sakaguchi flask. (Oxygen purification) () AOD and CAT: A cell-free extract obtained by ultrasonic disruption of H. polymorpha DL1 cells was precipitated with ammonium sulfate (80% saturation), dialyzed, and applied to a DEAE-Sefacel column. . CAT
was eluted with 0.1M phosphate buffer, and AOD was eluted with 0.1M phosphate buffer + 0.2M NaCl. Each active fraction was collected, precipitated with ammonium sulfate (80% saturation), dialyzed, and stored in a refrigerator. In this state, AOD is safe for about 2 weeks, but for longer periods, mix the enzyme solution with an equal amount of glycerol at -15°C.
It needs to be saved below. () FDM: A cell-free extract of P.putida F61 is treated with protamine and then fractionated with acetone.
Purified by Sephacel column chromatography. The activity of each enzyme preparation used is as shown in Table 3. Here, the amount of enzyme that changes 1 μmol of substrate per minute was defined as 1 unit (U). Table 3 Specific activity of partially purified enzyme Enzyme ratio correction (μmol/mm・mg protein) 7.58 CAT 6600 FDM 90.9 (Reaction conditions) Reaction solution composition is 0.05M phosphate buffer (PH7.5), 0.1
~1M methanol, 5.7U/ml AOD, 200U/ml
It contained CAT, 50U/ml FDM, and the total volume was 5.0ml. The reaction was carried out at 30°C using a PH stat.
This was done while maintaining the pH at 7.5 with 0.1M NaOH. Also,
Pure oxygen was blown into the reaction solution using a cylinder. The results are shown in Table 4.

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Claims (1)

[Claims] 1. A method for producing formic acid from methanol, which comprises converting methanol into formic acid by reacting it in the coexistence of alcohol oxidase, catalase, and formaldehyde dismutase and in the presence of oxygen. .