JPS6140394B2 - - Google Patents

Description

[Detailed description of the invention]

Technical field: The present invention relates to a method for producing acrylic esters using acrylic ester producing bacteria. Prior Art: Research on performing ester synthesis by reverse reaction of esterase has been conducted for a long time. One of them is a research report on various changes in acid and alcohol components in a reaction using lipase (Tsujisaka et al.
Iwai, Okumura et al., Biochemica et Biophysica
Acta, 575 (1979) p.156−165). As acid components, acetic acid, propionic acid, butyric acid, stearic acid, oleic acid, malic acid, succinic acid, etc. were investigated, and as alcohol components, primary alcohols, primary diols, phenols, secondary alcohols, Various alcohols such as diols, tertiary alcohols, and sugar alcohols are being considered. There are four types of lipases used there, and the origin of each is Aspergillus niger,
Rhizopus delemar, Geotrichum candidum,
It is Penicillium cyclopium. It has been shown that the first two of these have the ability to synthesize esters, especially from low molecular weight acids. However, in any case, acrylic acid was not used as the acid component in this study. The present inventor has developed the lipase disclosed in the above report (manufactured by Amano Pharmaceutical Co., Ltd. and Seikagaku Corporation).
However, it was confirmed that acrylic acid cannot be ester-synthesized with any alcohol. Even in other reports on conventional ester synthesis, there are no reports of reactions using acrylic acid as the acid component. Acrylic acid esters obtained by ester synthesis of acrylic acid and alcohol have vinyl groups and are therefore extremely useful as monomers for synthesizing polymers that can be used as various industrial materials. For example, polymers obtained by copolymerizing this acrylic ester with other monomers are used as adhesives, adhesives, films, and the like. Synthesis of such acrylic esters is possible by chemical synthesis. However, chemical synthesis requires harsh reaction conditions and requires additional equipment, space, and cost. Moreover, the working environment is likely to be contaminated, causing environmental pollution. If this ester synthesis is carried out by a biological reaction using microorganisms, the reaction can be carried out under mild conditions of low temperature and low pressure, and since it has substrate specificity, there is no risk of environmental pollution and equipment costs are low. OBJECT OF THE INVENTION: An object of the present invention is to provide a method for producing acrylic esters through a biological reaction using a novel microorganism that produces acrylic esters from acrylic acid and alcohol. Another object of the present invention is to provide a method for safely and inexpensively producing acrylic esters by biological reactions. Still another object of the present invention is to provide a method for producing acrylic ester as a monomer for synthesizing useful polymers that can be used as industrial materials using acrylic ester producing bacteria. Summary of the invention: The method for producing an acrylic acid ester of the present invention involves the production of acrylic acid and an alcohol represented by the general formula ROH (where R is a linear alkyl group having 1 to 8 carbon atoms) using a bacterium belonging to the genus Alcaligenes. The above objective is achieved by producing acrylic ester from. Bacteria of the genus Alcaligenes are Alcaligenes faecalis, especially Alcaligenes faecalis.
Strain TH836 and Alcaligenes faecalis
At least one of the TK4935 strains. Alcohols are linear alcohols with 1 to 8 carbon atoms, such as methyl alcohol, ethyl alcohol, propyl alcohol, butyl alcohol,
These are pentyl alcohol, hexyl alcohol, heptyl alcohol and octyl alcohol. The acrylic ester produced by the present invention is defined by the alcohol used as a raw material,
Methyl acrylate, ethyl acrylate, propyl acrylate, butyl acrylate, pentyl acrylate, hexyl acrylate, heptyl acrylate, and octyl acrylate are produced corresponding to each of the above alcohols. The microorganism used in the production of the acrylic ester of the present invention is a new strain isolated and collected from the soil of factories that handle raw materials for the chemical industry (Ibaraki City, Toyohashi City). This new strain was developed based on the mycological properties described below.
Determinative Bacteriology, 8th editor,
Alcaligenes faecalis strain TH836 (accession number:
FERM P-7083) and Alcaligenes faecalis (FERM P-7083)
faecalis) TK4935 (Accession number:
It was named No. 7084 (FERM P-7084). Mycological properties Alcaligenes faecalis TH836 strain and Alcaligenes used in the production of the acrylic ester of the present invention
Table 1 shows the mycological properties of strain TK4935 faecalis.

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[Table] Identification of Bacterial Strain Next, the basis for identifying the bacterial strain used in the production of the acrylic ester of the present invention as belonging to the genus Alcaligenes is shown below. Both the TH836 strain and the TK4935 strain have the characteristics of being ``Gram-negative, aerobic bacteria that proliferate by division, and do not proliferate by photosynthesis'' according to the above test results. Such bacteria are classified in Division 7 according to Bergey's Manual. The families and genera included here are as follows. 1 Family Pseudomonadaceae 2 Family Azotobacteraceae 3 Family Rhyzobiaceae 4 Family Methylomonadaceae 5 Family Halobacteriaceae 6 Genus Alcaligenes 7 Genus Acetobacter 8 Genus Brucella 9 Genus Bordetella 10 Genus Francisella 11 Genus Thermus Characteristics of these 11 families and genera, and the present bacterial strains TH836 and TK4935 The properties are compared below. All of these strains are completely different from the 10 families and genera except for the genus Alcaligenes in the mycological properties shown in Table 2.

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[Table] On the other hand, as shown in Table 3, all of the present strains have many properties in common with the genus Alcaligenes.

[Table] From the above results, the bacterial strains TH836 and TK4935 used for the production of the acrylic ester of the present invention
The strain is identified as a bacterium belonging to the genus Alcaligenes. Next, Table 4 shows the results of comparison with four known species of this genus to determine the species of this strain.

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[Table] This strain differs from Alcaligenes faecalis only in that it lacks the ability to assimilate propionic acid and butyric fermentation, and all other characteristics are consistent with this strain. Therefore, this strain was identified as Alcaligenes faecalis as it is very closely related to Alcaligenes faecalis. Culture conditions The medium does not need to be special, and organic nutritional sources such as meat extract, yeast extract, malt extract, and peptone, and inorganic nutritional sources such as phosphate, magnesium, sodium, potassium, manganese, and iron may be used as appropriate. Any ordinary medium containing the above ingredients may be used. As carbon sources, various amino acids that served as substrates in the carbon source assimilation test, organic acids of TCA cycle members, etc. are used. As a nitrogen source,
Inorganic nitrogen such as nitrates, nitrites and ammonium salts and organic nitrogen such as amino groups are used.
The culture temperature is 20°C to 35°C, preferably 25°C to 30°C. The culture pH is 5 to 8, preferably 6.5 to 7.5, and 1
Culture is carried out aerobically for 3 days to 3 days with stirring or shaking. Reaction composition The ester reaction of the present invention is carried out by culturing the present bacterial strain under such culture conditions and preparing the resting bacterial cells, bacterial cell extract (crude enzyme solution), purified enzyme, immobilized bacterial cells, and the state of the immobilized enzyme. Appropriately select and use at least one of them. The basic composition of the reaction system required for the synthesis of the acrylic ester of the present invention is shown in Table 5.
Incidentally, in the synthesis, an aqueous medium is generally used as the medium.

[Table] The amount of bacterial cells and extract to be added is such that the final concentration as a bacterial cell suspension is 0.1 or more in terms of absorbance at 660 nm (OD660), usually in the range of 1.0 to 20. The bacterial cell extract is obtained by treating bacterial cells with an ultrasonic crusher or a French press, and is used when the absorbance at 280 nm (A280) is 0.1 or more, usually in the range of 0.5 to 20.0. The reaction pH may be in the range of 5 to 9. Preferably it is adjusted to a range of 6 to 8. The acid component is acrylic acid. Methacrylic acid cannot be used. The usable concentration of acrylic acid is a final concentration of 0.1% (v/v) to 5% (v/v), preferably in the range of 0.5% (v/v) to 2% (v/v). To be elected. The use of acrylic acid lowers the PH of the reaction system composition, and as acrylic acid is consumed, the PH increases. Such a PH
In order to minimize fluctuations and ensure that the pH of the reaction system is always around 7, this basic composition uses a buffer as a buffer.
A 0.2M phosphate buffer is used. In place of the phosphate buffer, bicarbonate-sodium carbonate buffer, Tris-hydrochloric acid buffer, etc. can also be used. The buffer concentration is not limited to 0.2M, but is appropriately selected depending on the amount of acrylic acid used, reaction rate, etc. The amount of alcohol used is usually about 0.1 to 10 moles per mole of acrylic acid, but it is preferable to use an excess molar amount, and preferably 1 to 5 moles per mole of acrylic acid. but,
If too much alcohol is used, there is a risk of enzyme deactivation, so the final concentration is usually 0.1% (v/v) or more.
It is used in a range of 10% (v/v), preferably 0.5% (v/v) to 2% (v/v). The reaction temperature may generally be within the range of 20°C to 40°C. Preferably it is 25°C to 35°C. The reaction time varies somewhat depending on the amount of substrate added, but is approximately 10
Detection of the product is possible if it is longer than 1 minute. The reaction is preferably carried out in a homogeneous system. When the alcohol is butanol, if the amount used is large, it becomes difficult to mix with water, so it is preferable to stir the system to form a uniform emulsion. For the same reason, alcohols having 5 or more carbon atoms are preferably made into a homogeneous system by means such as stirring. Generally, after the reaction is completed, bacterial cells are removed from the system by centrifugation. The supernatant can be directly subjected to gas chromatography or liquid chromatography to detect and quantify the produced substances, or if necessary, the supernatant can be further heat-treated (for example, at 100°C for 1 minute) to remove contaminating proteins. Then, the product is detected and quantified using a gas chromatograph or liquid chromatograph. Confirmation of produced substances The produced substances were confirmed as follows. 1 As long as acrylic acid, alcohol and enzyme are present at the same time, a product peak is seen (in liquid chromatography) and the peak increases with time. 2 Showed the same retention time as known substances by gas chromatography and/or liquid chromatography. (The known substance was obtained through chemical synthesis.) 3 It was confirmed by the CI method using GC-Mass (manufactured by Hitachi) that the produced substance had the same molecular weight as the known substance. Conventional distillation or solvent extraction is used to collect the produced acrylic ester. Examples: Next, the present invention will be specifically described with reference to Examples. Example 1 (Preparation of bacterial cell suspension) Meat extract 10g, polypeptone 10g, NaCl 5g,
1 part distilled water, and gravy liquid medium with a composition of PH7.0
100ml was prepared. Pour this into a 500ml Sakaguchi flask and sterilize it, then add Alcaligenes faecalis TH836 to it.
The strain was inoculated. This was cultured with shaking at 30°C for 24 hours. This culture solution was centrifuged (10,000 rpm,
10 min.), and the precipitated bacterial cells were washed once with 0.05M phosphate buffer of pH 7.0. The washed cells were diluted with the same buffer to give an OD660 of 10.0 to obtain a cell suspension. (Reaction system) A reaction was carried out at 30° C. for 1 hour using the following reaction composition.

[Table] Methyl alcohol, ethyl alcohol, propyl alcohol, butyl alcohol, pentyl alcohol, hexyl alcohol, and octyl alcohol were used as alcohol components. (Analysis) After the reaction was completed, the bacterial cells were removed by centrifugation, and the supernatant was analyzed by gas chromatography or liquid chromatography to quantify the yield of the product. (Results) The results are shown in Table 6.

[Table] Example 2 Alcaligenes prepared in the same manner as Example 1
20 ml of bacterial cell suspension of faecalis TH836 strain was placed in a 100 ml glass beaker, and the bacterial cells were crushed for 10 minutes using an ultrasonic crusher manufactured by Kaiyo Denki Co., Ltd. while cooling with ice-cold water. The resulting crushed product was centrifuged at 15,000 rpm for 20 minutes, and the resulting supernatant was used as a crude enzyme solution. Except for using this crude enzyme solution instead of the bacterial cell suspension.
An ester reaction was carried out using the same reaction system as in Example 1. The results are shown in Table 7.

[Table] Example 3 (Preparation of crude enzyme solution) In the broth liquid medium shown in Example 1
Alcaligenes faecalis TK4935 was cultured at 30°C for 24 hours. Centrifuge the culture solution (10000 rpm, 10 min.
) and the collected bacteria in a phosphate buffer with a pH of 7.0.
Washed once with 0.05M. The washed cells were diluted with the same buffer to give an OD660 of 10.0 to prepare a cell suspension. 100ml of this bacterial suspension
The microbial cells were placed in a ml glass beaker and crushed for 10 minutes using an ultrasonic crusher manufactured by Kaiyo Denki Co., Ltd. while cooling with ice-cold water. The obtained crushed material was crushed at 15000 rpm.
The mixture was centrifuged for 20 minutes, and the resulting supernatant was used as a crude enzyme solution. (Reaction system) A reaction was carried out at 30° C. for 2 hours using the following reaction composition.

[Table] Furthermore, in place of the above ethyl alcohol, use butyl alcohol, hexyl alcohol, or octyl alcohol at a final concentration of 1% (v/v) each.
It was used so that We also conducted reactions using methacrylic acid instead of acrylic acid for various alcohols. The reaction was stopped by immersion in 100°C water bath for 1 minute. The precipitated protein is
It was removed by centrifugation at 5000 rpm for 10 minutes, and the supernatant was analyzed by liquid chromatography to quantify the yield of the produced ester. (Results) The results are shown in Table 8.

[Table] Effects of the invention: In the method for producing acrylic ester of the present invention,
Strains Alcaligenes faecalis TH836 and TK4935
Using this method, acrylic acid ester can be produced and accumulated in a reaction system containing acrylic acid and alcohol. Enzymes necessary for esterification are produced in proportion to cell growth, regardless of the type of medium. Since the acrylic acid ester product has a vinyl group, it is extremely useful as a monomer for synthesizing polymers that can be used as industrial materials. By copolymerizing this with other known monomers, it can be used for many more useful applications. According to the present invention, since the ester reaction is carried out biologically, it has the advantage of being extremely safe and inexpensive compared to chemical synthesis.

Claims (1)

[Claims] 1. Acrylic acid and the general formula ROH (where R has 1 to 8 carbon atoms) are produced by a bacterium belonging to the genus Alcaligenes.
A method for producing acrylic esters using microorganisms, in which acrylic esters are produced from alcohols represented by straight-chain alkyl groups). 2 The Alcaligenes bacterium is Alcaligenes
The manufacturing method according to claim 1, which is faecalis. 3. The production method according to claim 2, wherein the Alcaligenes faecalis is at least one of Alcaligenes faecalis strain TH836 and Alcaligenes faecalis TK4935 strain.