JPS60227688A - Production of acrylic ester by microorganism producing the same - Google Patents

Abstract

PURPOSE:The utilization of a biochemical reaction of a new microorganism to produce an acrylic ester from acrylic acid and an alcohol enables safe and inexpensive production of acrylic esters. CONSTITUTION:An aqueous solution containing about 1vol% of acrylic acid and about 1vol% of an alcohol of the formula is prepared and adjusted in its pH to 5-9. The R in the formula represents a straight-chain alkyl group of 1-8 carbon atoms. Separately, a novel microorganism of Alcaligenes faecalis TH836 (FERM 7083) or Alcaligenes faecalis TK4935(FERM-7084) is cultured to provide resting cells, cell extracts or immobilized cells. One of them is added to the above-stated aqueous solution to effect biochemical reaction of acrylic acid and the alcohol at 20-40 deg.C for grood-yield production of the objective acrylic ester.

Description

DETAILED DESCRIPTION OF THE INVENTION Technical Field: The present invention relates to a method for producing acrylic esters using acrylic ester producing bacteria.

Ibara Sen: Research on ester synthesis using the 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 (achieved).

Iwai, Kolok et al., Biochemica et Bio
physica Acta.

575 (1979) p, 156-165). In this study, acetic acid, propionic acid, butyric acid, stearic acid, oleic acid, malic acid, succinic acid, etc. were considered as acid components, and primary alcohol was considered as alcohol component.

Primary diols, phenols, secondary alcohols.

All kinds of alcohols such as secondary diols, tertiary alcohols, and sugar alcohols are being considered. There are four types of lipases used, each of which originates from As
pergillus niger, Rhizopus
delemar+ Geotrichum candi
dum, Penicillium cyclopium
It is. 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 conducted an investigation using the lipase disclosed in the above report (manufactured by Amano Pharmaceutical Co., Ltd. and Seikagaku Kogyo Co., Ltd.), and confirmed that acrylic acid cannot be ester-synthesized with any alcohol. Even in other reports on conventional ester synthesis, reactions using acrylic acid as the acid component have not been reported.

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, which requires additional equipment, space, and cost. Moreover, the work environment is easily contaminated and becomes a cause of environmental pollution. This ester synthesis is carried out through a biological reaction using microorganisms.

Because it can react under mild conditions such as low temperature and low pressure and has substrate specificity, there is no risk of environmental pollution and equipment costs are low.

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 acrylic esters of the present invention involves the production of acrylic acid and the general formula ROH (
However, R is an acrylic ester produced from an alcohol represented by a straight-chain alkyl group having 1 to 8 carbon atoms, thereby achieving the above object. The alkaline earth metal fungi are Alcaligenes faecalis, especially Alcaligenes faecalis TI (
836 strain and Alcaligenes faecalis TK49
At least one of the 35 stocks. The alcohol is a linear alcohol having 1 to 8 carbon atoms.

It is methyl alcohol, ethyl alcohol, propyl alcohol, butyl alcohol, 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, and corresponding to each of the above alcohols, methyl acrylate, methyl acrylate,
Ethyl acrylate, propyl acrylate, butyl acrylate, pentyl acrylate, hexyl acrylate.

Heptyl acrylate and octyl acrylate are produced.

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 a factory that handles raw materials for the chemical industry (Ibarakifu, Toyohashi City). This new strain was developed by rBergey's based on the mycological properties described below.
Manual of Ileterminative
Bacteriolog V+ 8th editor,
Alcaligenes faecalis was identified with reference to the 1974J bacterial taxonomy and is a bacterium belonging to alkaline earth metals.
es faecalis) 78,836 shares (accession number:
FERMP-7083)
and Alcaligenes faecalis (Alcalig
enes faecalis) TK4935 (Accession number: FERM P-708
4)).

Alc used in the production of the acrylic ester of the present invention
aligenes faecalis 78836 strain and Alcal igenes faecalis TK49
Table 1 shows the mycological properties of the 35 strains.

(The following is a margin) Sawa-like ” Concave Concave Kakisen Tsukuda side peeling Saga Uki Kai Muto O○O■ ■ ■@0■■ State State State Published by ◎ ■ +++ 1.1.1111111111+ten+ ++++ + + + 111111111111++ ± + + + +10+ +++ Small [strain] [strain] 0 ■■ Top 148'' The strain used for the production of the acrylic ester of the present invention is a strain belonging to the genus Alca Ii genes. The basis for identifying this is shown below.

The present bacterial strains TH836 strain and TK4935 strain are both 1 gram negative according to the above test results, and have the characteristics that they are aerobic bacteria that proliferate by one division and do not proliferate by photosynthesis. Bacteria like this.

According to Bergey's Manua+, it is classified as part 7. The families and genera included here are as follows.

1. Family Pseudomonadaceae 2. Az
Otobacteraceae family 3, Rhyzobi
aceae family 4, Methylomonadaceae family 5, Ha
family lobacteriaceae 6, Alcalige
The properties of these 11 families and genera and the properties of the nine bacterial strains TH886 strain and TK4985 are compared below.

All of the present bacterial strains are completely different from the 10 families and genera except for the genus Al ca I igenes in the mycological properties shown in Table 2.

Table 2 (18) On the other hand, all nine strains are as shown in Table 3.

It shares many characteristics with the genus Alcaligenes.

Table 3: 1, aerobic 2, gram negative 3, non-fermentative 4, motile, flagella (1-8) 5, size 0.5-1.2 μm by 0.5-2.
6 μm6, usually exists alone7, does not produce pigment8, ogisidase: positive9, does not decompose agar10, does not decompose casein and gelatin -11, suitable growth temperature 20-37°C12, good at pH 7.0 From the above results, the strains T836 and TK4935 used in the production of the acrylic ester of the present invention are A
It is identified as a bacterium belonging to the genus lcaligenes. Next, to determine the species of this strain, we used four known strains of this genus.
Table 4 shows the results compared to the species.

Table 4 0 Favorite→4 strains are all Alcaligenes f
The only difference from S. aecalis is that it lacks the ability to assimilate propionic acid and fermentation, and all other characteristics are the same. Therefore, nine strains were classified as Alcaligenes fa.
Alcaligenes f.
It was identified as P. aecalis. , the culture conditions do not need to be exceptional as a medium, meat extract.

Organic nutritional sources such as yeast ffl extract, malt extract, peptone, fumo-yoU)) dicate, magnesium, sodium.

A conventional medium containing appropriate inorganic nutrients such as potassium, manganese, iron, etc. may be used.

As the carbon source, 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.

Culture temperature is 20°C to 85°C, preferably 25°C to 3
It is 0°C. Culture pH is 5-8, preferably 6°5-7
．． Step 5: Culture is carried out aerobically for 1 to 3 days with stirring or shaking.

Reaction Composition The ester reaction of the present invention is carried out by culturing the present M strain under such culture conditions and using the resting bacterial cells, bacterial cell extract (crude enzyme solution), purified enzyme, immobilized bacterial cells, and immobilized enzyme. Appropriately select and use at least one of the states. The basic composition of the reaction system necessary for the synthesis of the acrylic ester of the present invention is
Shown in the table. Note that in one synthesis, an aqueous medium is generally used as the medium.

Table 5 Phosphate buffer (pH 8,0) 0.2M acrylic acid 1% (v/v) Various alcohol water Bacterial cells or cell extracts The amounts of cells and extracts added are as follows: Concentration is 6600m (7) Absorbance (OD660) f O.
1 or more, usually in the range of 10-20. The bacterial cell extract is obtained by treating the bacterial cells with an ultrasonic crusher or a French press, and has an absorbance (A280) of 0.2801 m 17).
1 or more, usually in the range of 0.5 to 20.0.

The reaction pH may be in the range of 5-9. Preferably 6~
Adjusted to a range of 8.

The acid component is acrylic acid. Methacrylic acid cannot be used. The usable concentration of acrylic acid is 0.1% final concentration.
(/v) to 5% (v/v), preferably 0.5%
(/v) to 2% (Y'v). The use of acrylic acid lowers the pH of the reaction system composition, and as the acrylic acid is consumed, the pH increases. In order to minimize such pH fluctuations so that the pH of the reaction system is always around 7, a 0.2M phosphate buffer is used as the buffer in this basic composition.

Instead of the phosphate buffer, bicarbonate ζ carbonate Na buffer, Tris-HCl buffer, etc. can also be used. Buffer concentration is also 0. It is not limited to 2M.
It is appropriately selected depending on the amount of acrylic acid used, reaction rate, etc.

The amount of alcohol used is per mole of acrylic acid.

Usually, it is about 0.1 to 10 moles, but it is preferable to use an excess amount, per mole of acrylic acid.

Preferably, 1 to 5 mol is used. However, if too much alcohol is used, there is a risk of enzyme deactivation.
Usually, the final concentration is 0.1% ('/v) to 10% (text VL
It is preferably used in a range of 0.5% (/v) to 2% (/v).

The reaction temperature may normally 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 the product can be detected if the reaction time is about 10 minutes or longer.

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 homogenized by means such as stirring.

Generally, after the reaction is complete, the bacterial cells are removed from the system by centrifugation. The supernatant liquid is directly subjected to gas chromatography (2R) or liquid chromatography to detect and quantify the produced substances, or the supernatant liquid is further heat-treated as necessary (for example, at 100'C for 1 minute). ) to remove contaminating proteins, and then apply the gas chromatograph or liquid chromatograph to detect and quantify the produced substances.

The produced substances were confirmed as follows.

■． As long as acrylic acid, alcohol, and enzyme were present at the same time, a product peak was seen (in liquid chromatography), and the peak increased 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 (Hitachi tank) that the produced substance had the same molecular weight as the known substance.

To collect the produced acrylic ester I26) Conventional distillation or solvent extraction methods are used.

Implementation example 2 Next, the present invention will be specifically described by way of examples.

Example 1 (Preparation of bacterial cell suspension) Meat extract 10g, polypeptone lQg, NaC
15g, distilled water 11. Then, 100 ml of a meat juice liquid medium having a composition of pH 7.0 was prepared. After sterilizing this in a 500m1 volume slope flask, add Alcaligene to it.
S faecalis TH886 strain was inoculated. This was cultured with shaking at 30°C for 24 hours. This culture solution was centrifuged (10,00 Orpm, lQmin,) l.
The precipitated bacterial cells were washed with pH 7.0, 0.0N acid buffer.
Washed once with 5M. The washed cells were diluted with the same buffer to 0D660 = 10°0 to obtain a cell suspension.

(reaction system) The reaction was carried out at 30°C for 1 hour using the following reaction composition.

Phosphate buffer IM (pHs, o) Q, 2ml
Acrylic acid 10% aqueous solution (v/v) 0.1ml Various alcohol 10% aqueous solution ('''v), 04ml 1 bacterial cell suspension 0D660 = 10 0.6ml Alcohol components include methyl alcohol, ethyl alcohol, propyl alcohol, butyl Alcohol, pentyl alcohol, hexyl alcohol, and octyl alcohol were used.

(Analysis) After the reaction was completed, the microbial cells were removed by centrifugation, and the supernatant was analyzed by gas chromatography or liquid chromatography to quantify the yield of the product.

(result) The results are shown in Table 6.

Example 2 Alcaligenes prepared in the same manner as Example 1
faecal 1sTI (Bacterial suspension of 836 strains 20
ml was placed in a 100 ml glass beaker, and while cooling with ice-cold water, the bacterial cells were crushed for 10 minutes using an ultrasonic crusher manufactured by Kaiyo Denki ■. 15. OOQr
The mixture was centrifuged at pm for 20 minutes, and the resulting supernatant was used as a crude enzyme solution. Except that this crude enzyme solution was used 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.

The following margin is Example 3 (Preparation of crude enzyme solution) Al ca I i in the broth liquid medium shown in Example 1
genesfaecalis TK4935 at 30°C
The cells were cultured for 24 hours.

Centrifuge the culture solution (100,00 rpm, 10 m1n
, ) L. The collected bacterial cells were washed once with phosphate buffer 005M, pH 7.0. Wash the washed bacterial cells with the same buffer.

A bacterial cell suspension was prepared by diluting it to 0D660=10.0. 20 ml of this bacterial cell suspension was placed in a 100 ml glass beaker, and while cooling with ice-cold water, the bacterial cells were crushed for 10 minutes using an ultrasonic crusher manufactured by Kaiyo Denki ■. The resulting crushed product was centrifuged at 15.00 Or pm for 20 minutes, and the resulting supernatant was used as a crude enzyme solution.

(reaction system) The reaction was carried out at 30°C for 2 hours using the following reaction composition.

Reaction composition in the margin below Addition amount Phosphate buffer ■M (pt-rB, 0) 0.2ml
Acrylic acid 10% aqueous solution (1'v) 0.1 ml
Ethyl alcohol/l/20% aqueous solution (%)' 0.1
ml crude enzyme solution A280 = 14.0 0.6 ml Furthermore, in place of the above ethyl alcohol, butyl alcohol
)v, hexyl alcohol or octyl alcohol were used at a final concentration of 1% (%), respectively.

Also, against various types of alcohol.

Reactions were also conducted using methacrylic acid instead of acrylic acid. The reaction was stopped by immersion in 100°C water bath for 1 minute. The precipitated protein is 1 at 5000 rpm.
It was removed by centrifugation for 0 minutes, and the supernatant was analyzed by liquid chromatography to quantify the yield of the produced ester.

(result) The results are shown in Table 8.

Table 8 Effect of the invention: In the method for producing acrylic ester of the present invention.

Strain Alcaligenes faecalis TH
836 and TK4935 can be used to produce and accumulate acrylic acid esters in a reaction system containing acrylic acid and alcohol. The enzymes required for esterification are
It is produced in proportion to bacterial 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 physically, it has the advantage of being extremely safe and inexpensive compared to chemical synthesis.

that's all Agent: Patent Attorney Shusaku Yamamoto 5

Claims (1)

[Claims] 1. Acrylic acid ester produced from acrylic acid and an alcohol represented by the general formula ROH (where R is a straight-chain alkyl group having 1 to 8 carbon atoms) by a bacterium belonging to alkaline earth metals. to produce. A method for producing acrylic esters using microorganisms. 2. The manufacturing method according to claim 1, wherein the alkaline earth metal fungus is Alcaligenes faecalis. 3. The Alcaligenes faecalis strain is Alcaligenes faecalis strain TH836 and Alcaligenes faecalis strain TH836.
The production method according to claim 2, wherein at least one of the P. faecalis TK4935 strains is used.