JPS58158189A - Preparation of beta-hydroxypropionic acid - Google Patents

Abstract

PURPOSE:To prepare beta-hydroxypropionic acid in high yield, by using a variant belonging to the genus Candida, having reduced dehydrogenase activity against beta-hydroxypropionic acid. CONSTITUTION:A fungus belonging to the genus Candida, capable of converting acrylic acid or propionic acid to beta-hydroxypropionic acid, is subjected to variation treatment to give a variant such as Candida rugosa IFO0750 having dehydrogenase activity against beta-hydroxypropionic acid reduced to <= half of the parent strain. This variant is introduced to Candida rugosa KT8201, which is brought into contact with acrylic acid or propionic acid or a substrate such as propyl alcohol, propionic anhydride, etc. which can be converted to propionic acid or beta-hydroxypropionic acid by the fungus.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing β-hydroxypropionic acid using microorganisms. More specifically, it belongs to the genus Cantida, has the ability to convert acrylic acid or propionic acid into β-hydroxypropionic acid, and is produced by contact reaction with a substrate capable of dehydrogenating β-hydroxypropionic acid. The present invention relates to a method for producing β-hydroxypropionic acid, which comprises collecting propionic acid.

Since β-hydroxypropionic acid is a compound having two different functional groups, it is a convenient substance as a raw material for the synthesis of medicines, agricultural chemicals, and the like. Regarding the production method of β-hydroxypropionic acid using microorganisms, using 1,3-propanediol as a raw material, acetic acid bacteria or Hansenula
Oxidation method according to the genus (K, Kerstel et al.; Biochemical Biophysical Acta Vol. 71, p. 311 19
1963, Harashi D et al.; Agricultural Rutual-Biological Chemistry, Vol. 32, p. 1'L75, 1968), or acrylic acid could be converted to β-hydroquine propionic acid by Fusaricum melisumoides (Harashi D et al., 1968); There are reports such as 1974), Journal of Fermentation Technology, Vol. 52, p. 388, but Izuh also has low crystal yield and low yield, and there are many difficulties in using it industrially. it is conceivable that.

Wood inventors are using cheap propionic acid etc. Set to +, β
- As a result of investigating the production of hydroxypropionic acid by microorganisms, it was found that if the activity of β-hydroxypropionic acid dehydrogenase fIrl of i-4tl, which has a strong ability to assimilate propionic acid, is reduced, β-hydroxypropionic acid , it was shown that it was accumulated at a yield of 11. That is, a propionic acid-assimilating microorganism was mutated to create a mutant strain with β-hydroxypropionate dehydrogenation activity less than half that of the original strain, and this mutation was subjected to a fusion reaction. Let's make it happen
It was discovered that β-hydroxypropionic acid can be produced at a high yield of '+'j. The advantage of using variable stocks is β
-Hydroxy-y''rohionic acid is highly productive.

The yeast strains of Candida L. and Ha used in the present invention include propionic acid-assimilating bacteria such as Cantida
/L/Go2za (Cand da rugosa)
IFO0750, IFO1542, IFO059
1. Candida O balapsilosis (Candida P
This also includes propionic acid-assimilating bacteria of the genus Cantida VC% isolated from nature.

The mutation is utilized to create a strain with reduced ij-hydroxypropionate dehydrogenase activity. Although either artificial mutation or natural mutation can be used for mutation, artificial mutation with high mutation efficiency is usually used. As methods for artificial mutation, X-ray irradiation, ultraviolet irradiation, T-ray treatment, and treatment with a mutagenic agent such as N-methyl-I-nitro-N-nitrosoguanidine (NTG) are used. For example, as a specific example, an example of a mutation method using NTG that the present inventors conducted to obtain a strain with low β-hydroxypropionate dehydrogenase activity is as follows. However, the method is not limited to this method, as long as a mutant strain with the desired properties can be obtained.

Ho (slant for f (Candida Rugoza IF0 0
750) 1 platinum loop of glucose 40g1 (NH4)
2HPO413g, KH2PO47g, Mg family) 4
・7H200,8g, ZnSO47H2060m+f
, FeSO4・7H2090q, CuSO4・5H20
5q, MnSO4.4 H2010'f % Na
C/! o, 1 g % piotin lq1 thiamine 2~, water 11! -, 500 tons of S culture medium consisting of 1 set of pH 7.2 was placed in a volume flask and inoculated.
Culture was carried out with shaking at 30°C for 120 hours. The culture solution 1.5
After washing ml with 0.5M phosphate buffer (pH 7,0),
0.5 cells/nttSuspended in 3 liters of NTG solution and heated at 4°C.
It was left for 60 minutes. Afterwards, it was washed 3 times with ji'ii buffer solution, and solid plate C medium (glucose 2
0g1 yeast extract 5g, meat extract 10g, peptone lug, agar 20g1 water 11X pH 7.0) was coated on 41 to extrude colonies. A replica of this Colony-8 medium was made into P medium of pH 7.0 to which 10 g of propionic acid and 20 g of agar were added instead of glucose. A strain (bacterium that cannot assimilate propionic acid) that grows poorly on this P medium was selected. The propionic acid non-assimilating bacteria obtained in this manner were subjected to a culture reaction under the conditions shown in Example IVc, and a strain accumulating a high β-hydroxypropionic acid yield was selected. All of the mutant strains selected in this way have significantly lower β-hydroxypropionate dehydrogenase activity than the parent strain, and can all be used in the present invention. The above enzyme activity was reported by Haseyo, one of the inventors, at Agricultural Biological Laboratory.
Gummistry 3u (Agricultural Bi
45, p. 2899 (1981) 1" using β-hydroxypropionic acid instead of β-hydroxyisobutyric acid as a substrate under the same conditions as the β-hydroxyisoacetate dehydrogenase activity measurement reported in It can be implemented if necessary.

An example of a mutant strain obtained by the above method for carrying out the present invention is Cantida rugosa KT8201 strain. As shown in Table 1, there is almost no difference in the bacteriological properties of this mutant strain from the parent strain, but there is a difference in propionic acid assimilation ability, β-
Significant differences are observed in hydroxypropionate dehydrogenase activity and β-hydroxyisobutyrate dehydrogenase activity. 01L It is not an essential condition that the mutant strain of the present invention has low activity of β-hydroxyisoacetate dehydrogenase.

Table 1: Application form for microorganism storage kettle has been deposited with the Institute of Microbial Industry at the Institute of Medical Sciences as receipt number 110.

L of β-hydroxypropionic acid in the present invention, for example, the above microorganisms in a medium containing a substrate selected from propyl alcohol, propional □dehyde, etc., a carbon source, a nitrogen source, an apolar salt, and a suitable nutrient source. aerobically cultivating the microorganisms, or using the culture solution obtained by culturing the microorganisms in an appropriate medium in advance, or collecting only the microbial cells and suspending them in a solution containing suitable hot plate salt, etc. There is a method of adding a substrate such as an acid, carrying out an aerobic contact reaction, and producing one product of β-hydroxypropionic acid. In addition, in order to accumulate a large amount of β-hydroxypropionic acid, it is necessary to use a suitable material such as glucose, glycerose, ethanol, or acetic acid, in order to supply the energy source that the microorganism uses during the culture or contact reaction with the microorganism. Preferably, an available source of carbonic acid is added.

The medium used in the present invention contains glucose, glycerin, carbon sources, ammonia, urea, ammonium sulfate, peptone, inorganic salts necessary for the growth of organic nitrogen-containing compounds such as casamino acids, and vitamins such as biotin and thiamine. If necessary, various nutrient sources commonly used for culturing microorganisms can be appropriately mixed and used in the pond.

The culture is carried out aerobically by inoculating the bacteria into a sterilized medium and maintaining the pH at 6 to 9 at a temperature of 20 to 45° C. for 1 to 10 days using aerobic agitation culture, shaking culture, etc.

At the initial stage of culture, bacterial cells grow, and then β-hydroxypropionic acid is produced. During the production of β-hydroxypropionic acid, if the substrate such as propionic acid and the above-mentioned energy sources such as glucose are further supplied, it can be efficiently produced.
A large amount of β-hydroxypropionic acid can be accumulated. Depending on the purpose, stationary cells of the above-mentioned microorganisms or cells immobilized using various polymers or the like may also be used.

To recover human β-hydroxypropionic acid from a culture or a static bacterial cell reaction solution, methods commonly used for recovering human β-hydroxycarboxylic acid can be used. For example, the β-hydroxypropionic acid-containing solution after bacterial cell removal is
β-hydroxypropionic acid can be obtained by condensing, adjusting the pH to 2 with sulfuric acid, extracting with ether, ethyl acetate, etc., and removing the solvent.

The present invention will be specifically explained below using examples, but the present invention is not limited only to the examples.

Example 1 Glucose 40g, (NH4)2HPO413g.

KH2PO47g, MgSO4,・7H200,8g
, ZnSO4・7H2060q, FeSO4・7H
2090119, CuSO4”5H205, If, Mn
A medium consisting of 11 g of SO4.4H, 10.1 g of NaC, 5 g of yeast extract, 20 g (11 g) of propionic acid was adjusted to pH 7.2 with caustic soda, and 30 tx
Candida rugosa IFO0750゜ and its mutant KT8
201 strain was inoculated and cultured with shaking for 3°.

The pH was maintained at 0 with NaOH. After completion of the culture, the produced β-hydroxypropionic acid was analyzed by gas chromatography (for Hase, etc.; Journal of Fermentation Technology).
Fermentation Technology
) Vol. 59, p. 203, 1981), the results shown in Table 2 were obtained.

Table 2 Culture solution 11 obtained by culturing a large amount of the mutant strain under the above conditions was collected,
Centrifugation N1. After removing the bacterial cells, remove the upper samurai under reduced pressure for 200 minutes
The mixture was concentrated to a pH of 2.5 m/m and adjusted to pH 2.5 with sulfuric acid. Add this to 500ml of ethyl acetate! Extracted three times.

After dehydration with Glauber's salt, the solvent was removed under reduced pressure and the oily yellow-brown substance was
I got 5g. This was prepared using a silica gel column (Wakogel C-200, 14X 30 cm) with benzene.
) Loaded with VC, extracted with benzene:acetone (3:l), collected fractions containing β-hydroxypropionic acid, and removed the solvent under reduced pressure to obtain 14.3 g of β-hydroxypropionic acid as a colorless oil. .

Measurement of the NMR spectrum, IR spectrum, elemental analysis, refractive index, etc. of the wood material matched the literature values for β-hydroxypropionic acid, and the product was identified as β-hydroxypropionic acid.

Example 2 Medium 3 obtained by removing propionic acid from the medium shown in Example 1
After sterilizing 0txt in a 500 ml flask, the mutant strain KT8201 was inoculated and cultured with shaking at 30°C for 24 hours. To each of the culture solutions were added 600 # of acrylic acid, 6 oowg of propyl alcohol, and 150 caps of propionaldehyde, 900 q+ of glucose was added, and the pH was adjusted to 7.0 with caustic soda. Then, it was cultured for 3 days. The pH is 7. When the mixture was OK, 1.2 g of sugar glucose was added. After the culture was completed, the produced β-hydroxypropionic acid was analyzed by gas chromatography, and the results shown in Table 3 were obtained.

Table 3 Example 3 Mutant strain KT8201 was inoculated into the medium used in Example 1 (50 g of propionic acid was added) and pre-cultured at 30°C for 4 days while maintaining the pH at 7.0. 28,54.76 hours after the start of culture, 1 g of glucose as an energy source! or glycerol 'g'-4 or 28j3
Ethanol every 6,54,62.76 hours 0.3. H
1, and 0.3 ml of acetic acid was added. After culturing, β
The results shown in Table 4 were obtained when the amount of -hydroxypropionic acid produced was measured.

Patent applicant Kanebuchi Chemical Industry Co., Ltd. Agent Patent Attorney Makoto Asano -

Claims (9)

[Claims] (1) A mutant strain of a microorganism belonging to the genus Candida that has the ability to convert acrylic acid or propionic acid to β-hydroxypropionic acid and has a decreased β-hydroxypropionic acid dehydrogenase activity, Alternatively, a method for producing β-hydroxypropionic acid, which comprises carrying out a contact reaction with the microorganism and a substrate that can be converted into propionic acid or β-hydroxypropionic acid, and collecting the produced β-hydroxypropionic acid. (2) The production method according to claim 1, wherein the microorganism is a mutant strain or a microorganism derived from Candida rugosa. (3) The production method according to claim 1 or 2, wherein the β-hydroxypropionate dehydrogenase activity of the mutant strain is less than half that of the parent strain. (4) The substrate that the microorganism can convert into propionic acid or β-hydroxypropionic acid is propyl alcohol,
The manufacturing method according to claim 1, which is propionaldehyde, propionamide, and propionic anhydride. (5) A method according to claim 1, wherein microorganisms are cultured in a nutrient medium, and propionic acid, acrylic acid, or a substrate that can be converted by the microorganisms into propionic acid or β-hydroxypropionic acid is applied to the obtained culture solution. Manufacturing method. (6) By culturing microorganisms in a medium supplemented with propionic acid, acrylic acid, or a substrate that the microorganisms can convert into propionic acid or β-hydroxypropionic acid, Alternatively, the production method according to claim 1, in which the microorganism acts on a substrate that can be converted into propionic acid. (7) Cultivate microorganisms in a nutrient medium, isolate microbial cells from the obtained culture medium, prepare a bacterial cell suspension, and use propionic acid, acrylic acid, or - The manufacturing method according to claim 1, wherein the method acts on a substrate that can be converted into hydroxypropionic acid. (8) Claim 1 which supplies an energy source that can be used by the microorganism during a contact reaction between the microorganism and propionic acid or acrylic acid or a substrate that the microorganism can convert into propionic acid or β-hydroxypropionic acid. Manufacturing method described in section. (9) The production method according to claim 8, wherein the energy source that can be used by the microorganisms is glucose, chrycerol, ethanol, or acetic acid.