JPS63188392A - Production of 3-chlorolactic acid - Google Patents

Abstract

PURPOSE:To obtain 3-chlorolactic acid useful as a synthetic raw material, etc., of useful compound in good yield, by reacting a bacterium cell of specific microorganism or treated product thereof with 3-chloro-1,2-propanediol. CONSTITUTION:A microorganism (e.g. Pseudomonas insueta ATCC21276) such as the genus Pseudomonas, Achromobacter, Candida, etc., having methanol- assimilating properties and ability capable of converting 3-chloro-1,2 propanediol (3CP) into 3-chlorolactic acid is aerobically cultivated in a culture medium containing a carbon source such as methanol, etc., nitrogen source, etc., such as urea, etc., at 4-8pH and 25-40 deg.C for 1-10 days. A bacterium cell separated from the resultant culture product is added to an aqueous solution having 1-100g/l concentration of 3CP and aerobically reacted at 20-70 deg.C and 5-11pH for 5-50hr to provide 3CL in the reaction liquid. Then the 3CL is recovered by solvent extraction method, ion exchange resin method, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention is directed to a compound having methanol assimilation ability and 3-chloro-1
, 3-chloro-1,2-propanediol to 3-chloro-1,2-propanediol by using bacterial cells or bacterial cell-treated products of microorganisms that have the ability to convert 2-propanediol into 3-chlorolactic acid as an enzyme source.
- A method for enzymatically producing 3-chlorolactic acid, which is characterized by producing and collecting chlorolactic acid.

3-Chlorolactic acid is a raw material for the synthesis of various useful compounds such as L-lunithine and β-chloroalanine.

Prior art Patent characterized by microorganisms of the genus Trichosporon, Geotrichum, Endomyces, Hansenula, Candida, Sporidiopolis, Rhoderomyces, and Pichia - Chloro-1,2-propanediol A method for producing 3-chlorolactic acid from [Journal of Fermentation Technology (J, F.
erment, Technol,) 64, No.
3, 251-254, (1986)).

Problems to be Solved by the Invention In the above-mentioned method for producing 3-chlorolactic acid, the reaction rate,
The reaction yield is still not satisfactory, and there is a need for the development of even better technology.

Means to Solve the Problems As a result of a new search for microorganisms that have the ability to produce 3-chlorolactic acid, the inventor discovered that some microorganisms that can assimilate methanol have the ability to produce 3-chlorolactic acid. We have discovered that there are microorganisms that have

The present invention will be explained in detail below.

The present invention has methanol assimilation ability and 3-chloro-1
, 3-chloro-1,2-propanediol is reacted with bacterial cells of a microorganism having the ability to convert 2-propanediol into 3-chlorolactic acid or a bacterial cell-treated product to produce 3-chlorolactic acid in the reaction solution. Provided is a method for producing 3-chlorolactic acid, which is characterized by accumulating and collecting 3-chlorolactic acid.

As the microorganism used in the present invention, any microorganism can be used as long as it has methanol assimilation ability and the ability to convert 3-chloro-1,2-propanediol into 3-chlorolactic acid.

For example, Pseudomonas, Achromobacter, Candida, Hyphomicrobium, Klebsiella, Methanomonas, Methylobacter, Methylobacterium, Methylococcus, Methylomonas, Methylophaga, Microcyclus, Paracoccus, Methanol-assimilating bacteria belonging to the genus Protomonas, Pichia, Arthrobacter, Nocardia, Protaminobacter, etc. can be used, and specific examples include the following strains.

Pseudomonas insueta
Pseudomonas sp.) ATCC21276 Pseudomonas sp.
CrB9133 Hyphomicrobiu/Force Lfi-(Hy
phomjcrob]utn vulgare)DS
Al1564 Klebsiera sp.
lla sp,) Nα101 FBRM P
-2498 Meta Rf S・j Chiropo 5 (Methanom
onas methylovora) ATCC213
69 Methylomonas thalassica (Methylomona
s thalassica) ^TCC33146Methyn1y'B・'2lina (Methylophaga)
marina) ATCC35842 Miku A Cyclas・
Microcyclous eburn
eus) ATCC21373 Protomonas extroque
ns) Mouth 5M1337 Pichi
a pastoris) ATCC28485 As a medium for culturing the cells of these microorganisms, a carbon source,
Any of the natural medium 9 synthetic medium can be used as long as it contains a suitable amount of nitrogen source, inorganic ions, etc. Carbon sources include alcohols such as methanol, ethanol, and 1,2-propanediol, carbohydrates such as glucose, sucrose, blackstrap molasses, starch, and starch hydrolysates, formic acid, acetic acid, lactic acid, fumaric acid, and maleic acid. Organic acids such as these are used as appropriate. As the nitrogen source, ammonia gas, aqueous ammonia, ammonium salt, urea, etc. are used. Ions such as magnesium, calcium, phosphoric acid, potassium, iron, and manganese are used as inorganic ions.

Cultivation is carried out under aerobic conditions, pH 4-8, temperature 25-40°C.
Do this for 1 to 10 days, keeping it within this range. The obtained microbial cells can be used for the reaction as they are, or the processed products obtained by subjecting the microbial cells to various treatments may be used for the reaction.

Examples of bacterial cell-treated products include mechanically ground bacterial cells, ultrasonic-treated products, freeze-dried products, solvent-treated products, enzyme-treated products, dry-processed products, surfactant-treated products, and bacterial cell protein components. For example, microorganisms, microbial cells, and immobilized products of the processed microbial cells can be used.

The reaction is carried out by aerobically reacting the bacterial cells obtained as described above or the treated product thereof in an aqueous solution containing 3-chloro-1,2-propanediol.

3-chloro-1,2-propanediol used in the reaction is added in a concentration range of 1 to 100 g/β.

The operating conditions include a temperature of 20 to 70°C, preferably 20°C.
~45°C, pH 5-11, preferably 7-8, 5-5
The reaction is carried out for 0 hours.

In this way, 3-chlorolactic acid is produced in the reaction solution.

As a method for recovering 3-chlorolactic acid from the reaction solution, a solvent extraction method, an ion exchange resin method, a precipitation method, etc. are used.

Examples are shown below.

Example 1 Far Eastern powdered bouillon 2%, yeast extract 0.5%, agar 1.
Pseudomonas sp. NCl3913 was added to a slanted agar medium prepared by heat sterilizing a medium with a composition of 5% at 120°C for 20 minutes, then solidifying it by adding 1% methanol that had been sterilized with a membrane filter (pore size 0.45μ).
3 was plated and cultured for 48 hours at 28°C, and the resulting bacterial cells were used as a seed culture.

The obtained bacterial cells were transferred to a liquid medium 3001111 containing 3 Q+nl, which was obtained by removing agar from the medium with the above composition.
Inoculated into a Erlenmeyer flask and incubated at 28°C for 24 hours at 21O
Shaking culture was carried out under shaking conditions of rpm. 60m of this culture solution
1 was inoculated into a 2β Erlenmeyer flask with protrusions containing 503 ml of a growth medium with the following composition, and 1% methanol sterilized in the same manner as above was added to the flask at 21 Orpm.

Shaking culture was carried out at 28°C, and 1% methanol was further added at 24 hours of culture, and shaking culture was carried out for a total of 72 hours. The composition of the growth medium is as follows.

(NH4)2SO40.4%, KH2P port, 0.1
%, K, HPo, 0.7%, Fe50<4H
2010mg/ml, unso4・4H208mg/
L Thiamine・1Ici'1mg#! , biotin lh
g/j! , NaCβ0. old%, casamino acid 0.05%,
After completion of the pH 7.2 culture, the bacterial cells were centrifugally washed once using physiological saline to collect the bacterial cells, and the obtained bacterial cells were stored frozen at -20°C. After thawing the cells, add 10 ml of the reaction solution (3-
The suspension was suspended in 0.1 M phosphorus buffer, pH 7.0, containing 10 mg/ml of chloro-1,2-propanediol, and reacted for 72 hours under shaking conditions at 28° C. and 21 Orpm.

After extracting the generated 3-chlorolactic acid with ethyl acetate, Hitachi 1
As a result of quantitative analysis using 63 gas chromatography, it was found that 2.1 mg of 3-chlorolactic acid was produced per 1 ml of the reaction solution. As a control, the quantitative value of 3-chlorolactic acid in a reaction solution carried out in the same manner without the addition of 3-chloro-1,2-propanediol was 0.01 mg or less per 1 ml of the reaction solution.

Effects of the Invention According to the present invention, 3-chloro-1,2-propanediol can be enzymatically converted using microorganisms to produce 3-chlorolactic acid in good yield.

Claims (2)

[Claims] (1) Has methanol assimilation ability and 3-chloro-1,2
- 3-chloro-1,2-propanediol is reacted with the cells of a microorganism having the ability to convert propanediol into 3-chlorolactic acid or a bacterial cell-treated product, and 3-chlorolactic acid is added to the reaction solution.
- A method for producing 3-chlorolactic acid, which comprises producing and accumulating chlorolactic acid and collecting it. (2) The microorganism is of the genus Pseudomonas, Achromobacter, Candida, Hyphomicrobium, Klebsiella, Methanomonas, Methylobacter, Methylobacterium, Methylococcus, Methylomonas,
Claim 1, characterized in that the invention belongs to a genus selected from the genus Methylophaga, Microcyclus, Paracoccus, Protomonas, Pichia, Arthrobacter, Nocardia, and Protaminobacter. manufacturing method.