JP3154646B2 - Microbial production of glycolic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing glycolic acid from glycolonitrile by using a microorganism. Glycolic acid is an important substance as a raw material for pharmaceutical intermediates, cosmetics and industrial detergents.

[0002]

2. Description of the Related Art In recent years, high purity glycolic acid containing almost no impurities has been demanded for use as a raw material for medicines and cosmetics. The industrial production of glycolic acid is based on formaldehyde, carbon monoxide and water as raw materials,
Although performed under high pressure, a high-level purification step is required to remove impurities from the glycolic acid obtained here. On the other hand, as a microbiological production method of glycolic acid, a method using a microorganism belonging to the genus Corynebacterium is known (see JP-A-61-56086).

[0003]

In the method using this microorganism, a product of high purity can be obtained with almost no formation of by-products, but the microorganism used has a low nitrile hydrolytic activity and the product has a low activity. Due to the low accumulated concentration, it has not reached a level that can be implemented industrially. Therefore, an object of the present invention is to efficiently produce high-purity glycolic acid.

[0004] The present inventor has sought to solve the above problems.
As a result of intensive studies on the production of glycolic acid by microbiological hydrolysis of glycolonitrile, Rhodococcus
The present inventors have found that a microorganism belonging to the genus (Rhodococcus) or the genus Gordona has a high ability to hydrolyze glycolonitrile to glycolic acid, and completed the present invention.

That is, the present invention relates to a method for producing glycolic acid from glycolonitrile by the action of a microbial hydrolase, wherein the microorganism used belongs to the genus Rhodococcus or the genus Gordona. A microbiological production method of glycolic acid.

[0006]

DETAILED DESCRIPTION OF THE INVENTION The microorganism used in the present invention is a genus Rhodococcus or Gordona.
Belongs to the genus, specifically, Rhodococcus rhodochrous ATCC 33025 strain,
Rhodococcus sp. SK92 strain (F
ERM BP-3324) and its mutant Rhodococcus erythropolis SK92-B1 strain
(FERM P-14853) and Gordona te
rrae) MA-1 strain (FERM BP-4535) and the like.

[0007] Among these microorganisms, the ATCC 33025 strain can be easily obtained from the American Type Culture Collection (ATCC). The SK92 strain, SK92-B1 strain and MA-1 strain have been deposited with the National Institute of Advanced Industrial Science and Technology,
The mycological properties of strain -1 are described in JP-A-3-280889 and JP-A-6-237789, respectively. Note that SK
The 92 strain was identified as Rhodococcus in JP-A-3-280889, but further from the following detailed mycological properties,
Rhodococcus erythropolis.

Strain SK92 Test items Test results Adenine decomposition + tyrosine decomposition + urea decomposition + assimilation Inoseal + maltose-mannitol + rhamnose-sorbitol + sodium m-hydroxybenzoate-sodium benzoate + sodium citrate + sodium lactate + testosterone + Acetamide + sodium pyruvate + 0.02% growth in the presence of sodium azide + growth at 10 ° C + growth at 40 ° C-growth in the presence of 0.001% crystal violet-growth in the presence of 0.3% phenylethanol- Growth in the presence of 5% NaCl + Growth in the presence of 7% NaCl +

For culturing the microorganism of the present invention, a usual medium in which these microorganisms can grow can be used. For example, a medium to which glucose, sucrose, glycerol or the like as a carbon source, peptone, meat extract, yeast extract, amino acid, other various inorganic salts, organic salts, vitamins and the like as a nitrogen source as appropriate are used. At this time, in a microorganism in which glycolonitrile hydrolase is inductively produced,
It is effective to add, for example, a nitrile compound, an amide compound, a lactam compound or the like to the medium as the enzyme inducer.

The culture is carried out under aerobic conditions at pH 5 to 10
The temperature may be controlled at 15 to 40 ° C. for 1 to 7 days in a range suitable for each microorganism.

The hydrolysis reaction is carried out by culturing the microorganisms as described above, and culturing the culture, the cells separated from the culture, or the treated cells (crushed cells, extracted enzymes, etc.) or the cells immobilized by a conventional method. The body or the enzyme may be suspended in water, a buffer, a physiological saline, or the like, and glycolonitrile may be allowed to coexist.

The concentration of bacterial cells and the concentration of glycolonitrile in the reaction solution are not particularly limited, but usually 0.1 to 20% by weight of glycolonitrile in an aqueous solution containing 0.01 to 10% by weight of microbial cells. Control within the range. Usually, the reaction temperature ranges from 1 to 50 ° C and the pH ranges from 5 to 10. The reaction time varies depending on the substrate concentration, the cell concentration, and other conditions, but the reaction may usually be performed for about 10 minutes to 1 week. Regarding the reactor type, either a batch type or a continuous type is possible.

Thus, glycolonitrile can be converted to glycolic acid and ammonia with a molar yield of almost 100% and can be produced and accumulated as a high-concentration aqueous solution of ammonium glycolate.

[0014]

EXAMPLES The present invention will be described below in detail with reference to examples, but the present invention is not limited to these examples.

Example 1 Medium comprising 1.5% glucose, 0.75% sodium glutamate, 0.1% monopotassium phosphate, 0.2% dipotassium phosphate, 0.05% magnesium sulfate heptahydrate, 0.1% yeast extract and 0.5% ε-caprolactam (pH 7.2) 100ml to 500
The mixture was placed in a ml Erlenmeyer flask and autoclaved at 120 ° C. for 20 minutes. This medium contains Rhodococcus rhodochrous ATC
The C33025 strain was inoculated and cultured with shaking at 30 ° C. for 65 hours. The cells were collected by centrifugation from the culture solution, washed with 50 mM potassium phosphate buffer (pH 7.0), and the cells were washed with the same amount of 50 mM potassium phosphate buffer (pH 7.0) as the culture solution. Suspended in water. Next, 0.2 ml of the above cell suspension was added to 0.8 ml of 50 mM potassium phosphate buffer (pH 7.0) containing 1% by weight of glycolonitrile, and reacted at 30 ° C. for 15 hours. 1N hydrochloric acid
After stopping the reaction by adding 0.2 ml, the bacteria were removed by centrifugation,
The produced glycolic acid was quantified by HPLC (using Wakobeads T-132-E column manufactured by Wako Pure Chemical Industries, Ltd.). As a result, all glycolonitrile is converted to glycolic acid,
No formation of glycolamide was observed.

Example 2 Glycerol 2%, monopotassium phosphate 0.68%, dipotassium phosphate 0.71%, sodium sulfate 0.28%, magnesium chloride 0.04%, calcium chloride 0.004%, manganese sulfate
Medium consisting of 0.003%, iron chloride 0.0006%, zinc sulfate 0.0003%, yeast extract 0.3% and benzyl cyanide 0.05%
(pH 7.5) 100 ml was put into a 500 ml Erlenmeyer flask, 120 ° C,
Autoclaved for 20 minutes. This medium was inoculated with Gordona terae MA-1 strain and cultured with shaking at 30 ° C. for 72 hours. The cells were collected from this culture by centrifugation, and
When the reaction was carried out for 48 hours in the same manner as described above, all of the glycolonitrile was converted to glycolic acid, and no formation of glycolamide was observed.

Example 3 1500 ml of a medium (pH 7.2) consisting of sucrose 2%, polypeptone 0.5%, monopotassium phosphate 0.1%, dipotassium phosphate 0.1%, magnesium sulfate heptahydrate 0.05% and yeast extract 0.3% was used. In a 2000ml mini jar, add 12
The solution was autoclaved at 0 ° C for 20 minutes. This was inoculated with Rhodococcus erythropolis SK92-B1 strain, and
The cells were cultured with aeration and stirring for hours. The cells were collected by centrifugation from the culture solution, and a cell suspension was prepared in the same manner as in Example 1. Next, the cell suspension was added to 30 ml of 50 mM potassium phosphate buffer (pH 7.0) containing 1% by weight of glycolonitrile.
1 ml (containing 40 mg of bacterial cells as a dry weight per ml) was added, and the reaction was carried out at 20 ° C., and the production rate of glycolic acid was measured. As a result, 0.53 μm per minute per 1 mg dry cells
Molar glycolic acid was formed.

The cells cultured in the same manner as in Example 3
Wash twice with an aqueous solution of 0.4% by weight ammonium glycolate (pH 7.0)
H 7.0). This suspension (as dry weight per ml)
20 ml was placed in a 50 ml Erlenmeyer flask and stirred in a constant temperature bath at 20 ° C. % Glycolonitrile aqueous solution was supplied. The pH of the reaction solution was controlled by adding aqueous ammonia so as to be 7.0. When the reaction was carried out for 24 hours, 48.2% by weight of ammonium glycolate was accumulated, and the yield from glycolonitrile was almost 100%.

[0019]

According to the present invention, the reaction activity and the accumulated concentration of glycolic acid are extremely high, and the selectivity to glycolic acid is almost 100%. An industrial recipe may be provided.

Claims (1)

(57) [Claims] 1. A method for producing glycolic acid from glycolonitrile by the action of a microbial hydrolase, wherein the microorganism used is Rhodococcus.
A microbiological process for producing glycolic acid, wherein the process belongs to the genus or Gordona.