JPS61185193A - Production of l(+)-beta-hydroxyfatty acid - Google Patents

Abstract

PURPOSE:To produce L(+)-beta-hydroxybutyric acid and L(+)-beta-hydroxyvaleric acid, in high efficiency, by inducing a microbial strain belonging to Candida genus or Debaryomyces genus to a specific mutant strain. CONSTITUTION:A microbial strain capable of converting butyric acid to L(+)-beta- hydroxybutyric acid and valeric acid to L(+)-beta-hydroxyvaleric acid is induced to a mutant strain having low rate of metabolism or inactive to the metabolism of L(+)-beta-hydroxyfatty acid, by artificial mutagenic treatment, etc. The strain is allowed to react with a 4-5C saturated fatty acid or alpha,beta-unsaturated fatty acid or an alcohol.

Description

Detailed Description of the Invention (Industrial Field of Application) The present invention provides L(-1
Regarding the method for producing -)-β-hydroxy fatty acids, more specifically L(-1- )-A mutant strain that does not grow or grows weakly in a nutrient medium containing β-hydroxybutyric acid as the sole carbon source is reacted with saturated fatty acids having 4 or 5 carbon atoms, α, β-unsaturated fatty acids, or alcohol. , L with 4 or 5 carbon atoms to be produced (
The present invention relates to a method for producing L(t)-β-hydroxy fatty acids, which comprises collecting g)-β-hydroxy fatty acids.

(Prior art and problems) Regarding the production method of L(t)-β-hydroxybutyric acid having 4 carbon atoms, ethyl acetoacetate is asymmetrically reduced using baker's yeast to produce ethyl L(t)-β-hydroxybutyrate. There are known methods to obtain it (Mori et al.: Journal of the Chemical Society of Japan, no.
1315, 1983), it requires a large amount of yeast and is difficult to consider as an economical method.

On the other hand, regarding the production method of L(+)-β-hydroxyvaleric acid having 5 carbon atoms, a method is known in which ethyl β-ketovalerate is obtained as D(c)-β-ethyl valerate by reduction with baker's yeast. Iruga Crater et al., Helvetica Kimika Acta CG, Frater.

He1vetica Chimica Acta) 6
2, p. 2829, 1973], the configuration is reversed.

The present inventors previously discovered Candida Rugoza (Candida Rugoza)
It was discovered that L(t)-β-hydroxyvaleric acid could be produced from valeric acid by Ida rugosa) I F 0 1542 (Japanese Patent Publication No. 59-53838).
.

Ordinary microorganisms metabolize linear L(t)-β-hydroxy fatty acids very easily, resulting in extremely low yields, making them unsuitable for mass production.

Therefore, the present inventors have developed an inexpensive and efficient L(+)-
As a result of research to develop a method for producing β-hydroxy fatty acids, we mutated microorganisms belonging to the genus Satucharomycopisis, Endomyces, or Pica that have the ability to convert butyric acid to L(+)-β-hydroxybutyric acid. Improved, L(-
By inducing a mutant strain that does not grow on a nutrient medium containing 1-1-β-hydroxybutyric acid as the sole carbon source, L(+)-
He discovered that β-hydroxybutyric acid can be used to produce L(+)-β-hydroxyvaleric acid in high yield from valeric acid, 2-pentenoic acid, or n-amyl alcohol, and has already applied for a patent (Patent Application No. 59, 1983). -44753), but as a result of further investigation, we discovered a mutant strain of a microorganism belonging to the genus Candida or the genus Debaryomyces as a mutant strain having such an ability, and completed the present invention.

(Means for Solving the Problems) In carrying out the present invention, Candida paralgosa (Candida paralgosa) is used as a parent strain to be used for obtaining mutant strains.
ida pararugosa) I F OO966
゜Debaryo IJ tMyces Hansenni (Debaryo
Mycashansenales) IFo 0032, etc., but any microorganism can be used as long as it has the ability to convert butyric acid to L(t)-β-hydroxybutyric acid and valeric acid to L(t)-β-hydroxyvaleric acid. I can do it.

As a method for converting microorganisms into L(t)-β-hydroxy fatty acids by interacting with a substrate, microorganisms are cultured in a nutrient medium, and microorganisms are separated from the culture solution or culture solution and the microorganisms are suspended. There are methods such as preparing a suspension and reacting it with a substrate, or culturing microorganisms in a medium to which a substrate is added, and then reacting the microorganisms with the substrate. The isolated bacterial cells can also be used in a state where they are immobilized with a water-insoluble polymer or the like.

The productivity of L(t)-β-hydroxy fatty acids is improved by supplying an energy source that can be used by the microorganisms during the contact reaction between the microorganisms and the substrate. In this case, preferred energy sources include glucose, glycerol, and the like.

Since ordinary microorganisms have a fast metabolic rate of L(-1-)-β-hydroxy fatty acids, the amount of L-lees-β-hydroxy fatty acids accumulated is extremely small, making it difficult to produce them economically. Therefore, in order to efficiently accumulate a large amount, L(t)−β
-Use a mutant strain that has a slow metabolic rate of hydroxy fatty acids or does not metabolize them, in other words, a mutant strain that does not grow or grows weakly in a nutrient medium containing L(t)-β-hydroxy fatty acids as the sole carbon source. It is advantageous to do so.

Artificial mutation or natural mutation is used to obtain such mutant strains, but artificial mutation is usually used for efficient mutation. Artificial mutation methods include X-ray irradiation, ultraviolet irradiation,
Treatment with r-rays, treatment with mutagenic agents such as N-methyl-N-nitro-N'-nitrosoguanidine (NTG) are used. As a specific example, the present inventors
An example of the NTG mutation method used to obtain a mutant strain that does not metabolize -β-hydroxy fatty acids is as follows:
The next one is auction. However, the method is not limited to this method as long as the desired mutant strain can be obtained.

Preservation slant (Candida paralgoza IF0
0966) to 1 platinum loop of glucose 40? , (NH4
)2HPO4131P, KH2PO47y, MgSO4
・7H200, 8y, ZnSO44H2060fttg
, FeSO4'7H2090fng, CuSO4・5H
2051+19. MnSO4・4H201011v,
NaC10,1y, biotin p also thiamine--HCl2
) n, water 1/, pH 7,2 S medium 30-
was inoculated into a 500-volume flask and heated at 30°C.

The cells were incubated for 20 hours. 1.51 nl of the culture solution was
．． After washing with 5M phosphate buffer (pH 7,0), 0.5f
Suspended in 3rnl of nq/1nlNTG solution, 4°C, 60
Leave it for a minute. Thereafter, the solid plate medium C (glucose 202, glucose 202,
Yeast extract 5y, meat extract 1 (1, peptone 10y
, agar 20y, water 11%, pH 7.0) and colonies were allowed to appear. This colony was added to S medium containing 10 y of butyric acid and 20 y of agar instead of glucose at pH 7.0.
The cells were replicated onto S medium and cultured at 30°C for 2 days. Bacteria that grew poorly on this S medium (non-butyric acid assimilation) were selected.

The non-assimilated butyric acid flavor obtained in this way was planted on S medium, and
℃ for 48 hours, and the production of L(t)-β-hydroxybutyric acid was determined by gas chromatography [Haseyo et al.; Journal of Fermentation Technology (J
, Ferm, Tech, ) volume 59, page 257,
1981] and selected L(t)-β-hydroxybutyric acid producing strains. The mutant strain selected in this way is L (t)
-Growth on a nutrient medium containing β-hydroxybutyric acid as the sole carbon source (for example, a medium in which L(t)-β-hydroxybutyric acid is added instead of glucose in S medium) is significantly reduced and the present invention is not suitable. Can be used. Mutations of microorganisms of other genera can be carried out using similar techniques, and as an example of a mutant strain obtained by the above method for carrying out the present invention, Candida pararugosa (Candida pararugosa) is used.
KT 84015, Debaryomyces hansenji K
There are T 84016 etc. There was almost no difference in the properties of this mutant strain from that of the parent strain), but as shown in Table 1, there was a significant difference in the ability to assimilate L(t)-β-hydroxy fatty acids.

Table 1 In MS medium, 1 g of each compound was added as a single carbon source and cultured (30°C, 4 days).

Book: Good growth - 2 Poor growth These mutant strains have been deposited with the following numbers at the Institute of Microbial Technology, Agency of Industrial Science and Technology, as shown in Table 2.

Table 2 The microorganism culture medium used in the present invention contains carbon sources such as glucose and glycerol, ammonia, and ammonium sulfate.

Nitrogen sources such as inorganic and organic nitrogen-containing compounds such as peptone and casamino acids, inorganic salts necessary for growth such as potassium phosphate and magnesium sulfate, vitamins such as biotin, and others used for normal microbial culture as necessary. Various nutrient sources can be appropriately mixed and used. For culturing, bacteria are inoculated into a sterilized medium, and culture is carried out aerobically, such as with aeration and shaking, for 1 to 10 days while maintaining the pH at 6 to 9 at a temperature of 20 to 45°C. There is bacterial cell production in the early stage of culture, and then L(t)-
Production of β-hydroxy fatty acids takes place. Furthermore, by supplying glucose, glycerol, or the like as an energy source during the production of L(t)-β-hydroxy fatty acids, L(t)-β-hydroxy fatty acids can be produced more efficiently. Even if the substrate is added to the medium from the early stage of culture,
It may be added after the bacterial cells have grown.

L(t)-β- produced from culture solution or bacterial cell reaction solution
Hydroxy fatty acids can be recovered by means commonly used for recovering β-hydroxy fatty acids. For example, after removing the bacterial cells, concentrating the L(-1-)-β-hydroxy fatty acid-containing liquid and adjusting the pH to 2.5 or less with sulfuric acid, etc.
L(t)-β-hydroxy fatty acid can be obtained by extracting this with ether, ethyl acetate, etc., removing the solvent, and then distilling it under reduced pressure. Since L(t)-β-hydroxy fatty acid is not a very stable substance, the L(-1-1-β-hydroxy fatty acid obtained by the above solvent extraction etc.) is dissolved in alcohol such as methanol or ethanol, and then extracted with sulfuric acid etc. It can be easily converted into an alkyl ester by heating in the presence of a catalyst.

If this product is distilled, high yield and high purity L(t)-β-
Hydroxy fatty acid esters can be obtained, and these esters are relatively stable.

(Effects of the Invention) According to the present invention, optically active L(t)-β-hydroxybutyric acid and L(+)-β-hydroxyvaleric acid can be industrially advantageously produced, and useful synthetic intermediates for pharmaceuticals etc. can be supplied. It became so.

(Examples) The present invention will be specifically described below with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Glucose 40y, yeast extract 5y.

(NH4)2HPO413y, KH2PO479, Mg
SO4・7H200, 8f, ZnSO4・7H2060
1n9. FeSO4・7H2090y, CuSO4・5
H2054, MnSO4'4820 1011tf,
NaCl O,ly, butyric acid 10m/or valeric acid 101n! A medium consisting of a composition of (per 11) NaOH
Adjust the pH to 7.2, put this 31 in a 51 capacity mini jar fermentor, sterilize it, and add Candida pararugosa IFO09.
66 Ihasono mutant strain KT 84015, mata is Debaryomyces hansenii (Debaryomyces hansenii) I
FO0032 or its mutant strain KT 84016 was planted and cultured at 30° C. for 4 days with aeration of 1 vvm and stirring at 500 rpm.

During cultivation, the pH was maintained at 7.0, and glucose was added at 60% daily.
y were added. After the completion of the culture, the produced L(+)-β-
As a result of measuring hydroxybutyric acid or L(t)-β-hydroxyvaleric acid, accumulation as shown in Table 3 was observed.

Table 3 XL(t)-β-HBA: L(t)-β-hydroxybutyric acid L(t)-β-HVA: L(t)-β-hydroxyvaleric acid. Even in stocks, L(
No accumulation of +)-β-hydroxybutyric acid or L←)-β-hydroxyvaleric acid was observed.

After sterilizing the culture solution obtained under the above culture conditions, remove the supernatant solution under reduced pressure for 5 minutes.
The solution was concentrated to 0.01 ft and then adjusted to pH 2.0 with sulfuric acid. This was extracted three times with ethyl acetate, and after removing the solvent, each L(t)-β-hydroxy fatty acid was obtained in the form of a yellow oil.

This was purified by vacuum distillation to obtain a colorless oily L(t)-β-hydroxy fatty acid as shown in Table 4. These are N
According to MR, gas chromatography, and measurement of specific rotation after conversion to methyl ester, L(t)-β-hydroxybutyric acid can be obtained from butyric acid, and L(+)-β-hydroxyvaleric acid can be obtained from valeric acid. was confirmed.

Margin table below -4 Example 2 Medium 31 obtained by removing fatty acids from the medium shown in Example 1 was used for 5! After sterilization by placing in a mini jar incubator, inoculate with Candida parargosa KT 84015 or Debaryomyces hansenji KT 84016,
Culture was carried out at 30° C. for 24 hours with aeration of 1 vvm and stirring of 500 rpm.

Add 15% of crotonic acid, n-butyl alcohol, 2-pentenoic acid, or n-amyl alcohol to this culture wave.
Glucose was added at a rate of 60y each day while maintaining the pH at 7.0, and the reaction was continued for an additional 3 days. After the reaction, the L(+)-β-hydroxy fatty acids produced were measured by gas chromatography, and the results showed that each L
Accumulation of (g)-β-hydroxy fatty acids was observed.

Table-5 Example 3 Same as Example 1, Candida paralgoza KT
84015 or Debaryomyces hansenii K
After culturing T 84016 and starting the culture, 24.48.
72 hours glucose 60f or glycerol 60
y were added and cultured for 96 hours while maintaining the pH at 7.0. The amount of L(+)-β-hydroxy fatty acid produced in the culture solution after completion of the culture was as shown in Table 6.

Table-6

Claims (8)

[Claims] (1) L(+)-β-hydroxybutyric acid derived from microorganisms that belong to the genus Candida and Debaryomyces and have the ability to convert butyric acid to L(+)-β-hydroxybutyric acid. A mutant strain that does not grow or grows poorly on a nutrient medium using a single carbon source is treated with saturated fatty acids having 4 or 5 carbon atoms, α,β-unsaturated fatty acids, or alcohol to produce 4 or 5 carbon atoms. L(+)-β characterized by collecting L(+)-β-hydroxy fatty acids of
- A method for producing hydroxy fatty acids. (2) The microorganism is Candida paralgosa (Can
dida pararugosa) or Debaryomyces hansenii
3. The production method according to claim 1, which is a mutant strain derived from C. senii). (3) The saturated fatty acid used as a substrate is butyric acid or valeric acid, the α,β-unsaturated fatty acid is crotonic acid or 2-pentenoic acid, and the alcohol is n-butyl alcohol or n-amyl alcohol, each corresponding to the target product. L(+)-β-hydroxybutyric acid, or L(+
)-β-hydroxyvaleric acid. (4) Claims 1, 2 or 3 in which microorganisms are cultured in a nutrient medium and a substrate is applied to the obtained culture solution.
Manufacturing method described in section. (5) The production method according to claim 1, 2, or 3, wherein the method is cultured in a medium to which a substrate is added, and the substrate and microorganisms are allowed to interact with each other. (6) A microorganism is cultured in a nutrient medium, the microorganism cells are separated from the obtained culture solution to prepare a cell suspension, and a substrate is applied to the cell suspension. The manufacturing method described in paragraph 3. (7) The production method according to any one of claims 1 to 6, wherein an energy source that can be used by the microorganism is supplied when the microorganism and the substrate are allowed to interact with each other. (8) The production method according to claim 7, wherein the energy source that can be used by the microorganism is glucose or glycerol.