JP2663458B2 - Organic acid production method - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing an organic acid by utilizing living cells. [Problems to be Solved by the Related Art and the Invention] Conventionally, it has been known that formaldehyde can be produced from methanol using alcohol oxidase of a methanol-assimilating yeast. However, when this reaction is carried out industrially, there is a problem that alcohol oxidase is inactivated by the reaction product, formaldehyde. As a method for solving this problem, a method has been proposed in which methanol is immediately converted to formic acid in a high yield in the presence of three kinds of enzymes, alcohol oxidase, catalase and aldehyde dismutase (Japanese Patent Application Laid-Open No. 61-28891). ). The present inventors have further studied various methods capable of converting an organic acid from a monohydric alcohol to a high yield at a stretch over a long period of time. As a result, two types of specific strains are combined, and in the presence of these strains, It has been found that the intended purpose can be achieved by the reaction. Generally, when culturing is carried out in the presence of two or more strains at the same time, the growth of the other strains interferes with each other, and it is difficult to obtain a target product in high yield. Culturing with one strain and sterilizing or isolating the cells, then adding the next strain and culturing, but unexpectedly producing alcohol oxidase and catalase. The combination of the specific strain to be produced and the specific strain that produces aldehyde dismutase does not interfere with each other, and in a higher yield in a higher yield over a longer period of time than when the purified enzyme is used. The inventors have found that alcohol can be converted to an organic acid, and have completed the present invention. [Means for Solving the Problems] That is, the gist of the present invention is that Hansenula polymorpha CBS4 that produces alcohol oxidase and catalase.
Pseudomonas putida F61 strain producing 732 strain and aldehyde dismutase was suspended in the reaction solution,
A method for producing an organic acid, comprising converting a monohydric alcohol into an organic acid while passing oxygen. Hereinafter, the present invention will be described in detail. First, the strain producing alcohol oxidase and catalase used in the present invention is a methanol-assimilating yeast Hansenula polymorpha CBS4732 [The Yeasts, J. RODDER, 2nd edition (197)
0), pp. 296-299] (1983 LIST OF CULTURES, 362, CENTRAALBUREAU VOOR
SCHIMMEL CULTURES and 1983 LIST OF CULTURES, page 16, Fermentation Research Institute Foundation) The aldehyde dismutase (aldehyde disproportionating enzyme) used in the method of the present invention is obtained by adding a coenzyme. And an enzyme that simultaneously performs the redox of aldehydes without such an enzyme, and a strain that produces such an enzyme is Pseudomonas Putida F61 (Publication No. 1023). reference). The nutrients required for culturing these microorganisms are not particularly limited, and those usually used for culturing microorganisms are used. For example, as a carbon source, Hansenula polymorpha CBS4732 is methanol, Pseudomonas putida F61 is glucose, sucrose, fructose, glycerol, sorbitol, saccharide, sugar such as starch hydrolyzate, and organic acids such as acetic acid and fumaric acid. , Etc. are used. Examples of the nitrogen source include nitrates, ammonium salts, corn steep liquor, yeast extract, meat extract, yeast powder, soybean hydrolysate, cottonseed powder, polypeptone, penton, and the like. As the inorganic salt, potassium phosphate, sodium phosphate, magnesium sulfate, sodium chloride and the like can be used. The culture temperature is preferably from 20 to 40 ° C, particularly preferably from 25 to 37 ° C. The culture is usually performed aerobically for about 16 to 72 hours. In the method of the present invention, a monohydric alcohol is reacted in the presence of the above-mentioned two strains while aerating the enzyme. Examples of the monohydric alcohol include ethanol, 1-propanol, allyl alcohol, and 1-butanol. The reaction is usually carried out at pH 5.5 to 9.5, preferably pH 7 to 9, in a buffer commonly used, for example, potassium phosphate or the like. The reaction temperature is usually 10 to 45 ° C, preferably 30 to 40 ° C. The reaction time varies depending on the amount of the strain used, the concentration and type of the substrate, and the reaction temperature, but is usually selected from about 10 minutes to 100 hours, usually about 30 minutes to 48 hours. The concentration of methanol as a substrate is usually selected from 1 mM to several M. Oxygen can be present, for example, by air or oxygen blowing, but the amount is usually at least an equimolar amount of methanol. Hansenula polymorpher CBS4732 and Pseudomonas putida F61 were each pre-cultured to obtain 10 ⁵ to 10 ¹⁰ cells / ml.
At this point, main culture is performed under the above reaction conditions. According to the present invention, high activity is maintained over a long period of time as compared with the case where a purified enzyme is used, so that a monohydric alcohol serving as a substrate can be added successively, and therefore, an organic acid can be further reduced. It can be obtained in yield. The time when the monohydric alcohol is added is usually 1 hour of the culture solution.
It is preferred that the concentration of the hydric alcohol be in the range of 50 to 100 mM. The product thus obtained is prepared according to a standard method.
For example, it is isolated by ion exchange resin treatment or the like. [Effects of the Invention] According to the method of the present invention, a corresponding organic acid can be obtained more stably and in a higher yield than a monohydric alcohol for a longer period of time. EXAMPLES Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to the following Examples as long as the gist is not exceeded. Example 1 Glass cylinder with water jacket (20 × 100mm)
The mixture was stirred with a magnetic stirrer while maintaining the temperature at 30 ° C. To 20 ml of 50 mM phosphate buffer (pH 7.5), 100 mM methanol was added as a substrate, and 42 mg of Hansenula polymorpha CBS4732 and 82 mg of viable cells of Pseudomonas putida F61 strain (dry cell weight) were simultaneously added. ,
The reaction was carried out while blowing 2.5 ml · min ^{−1 of} oxygen gas. At this time, using a pH stat, titrate with 0.1 N NaOH, and
Maintained at 7.5. 100 mM methanol was completely converted to formic acid. The results are shown in FIG. Example 2 Instead of using 100 mM methanol in Example 1, 200 mM
The reaction was carried out in the same manner except that methanol was used, and a conversion of 75% was obtained. The results are shown in FIG. Comparative Example 1 A reaction was carried out in the same manner as in Example 1, except that a free enzyme was used instead of the living cells. The results are shown in FIG. Example 3 In Example 1 and Comparative Example 1, 100 mM methanol was further added, and the same reaction was repeated twice. When live cells were used, they could be reused, but free enzymes were used. In this case, the enzyme ability was inactivated and no formic acid was produced.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the change over time of formic acid production in Examples 1, 2 and Comparative Example 1.

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Claims (1)

(57) [Claims] A Hansenula polymorpha CBS4732 strain producing alcohol oxidase and catalase and a Pseudomonas putida F61 strain producing an aldehyde dismutase are suspended in a reactor, and a monohydric alcohol having 1 to 4 carbon atoms is removed from the organic acid while passing through oxygen. A process for producing an organic acid, characterized in that it is converted to an organic acid.