JPH0690772A - Preparation of optically active gamma- hydroxydecanoic acid - Google Patents

Abstract

PURPOSE: To obtain the subject compound in a high yield by hydrolyzing a castor oil by using a fungus and β-oxidizing the hydrolyzate since a castor oil or its hydrolyzate is nontoxic to the fungus.

CONSTITUTION: A fungus selected from Aspergillus oryzae, Candida rugosa, Geotrichum klebahnii and Yarrowia lipolytica, capable of hydrolyzing a castor oil is cultured in the presence of a castor oil so that the castor oil is hydrolyzed and the hydrolyzate is β-oxidized to give the objective compound. The culture of the fungus is preferably carried out by using a liquid medium under aerobic conditions at pH 4-7.5 at 20-30°C.

COPYRIGHT: (C)1994,JPO

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a microbiological process for producing optically active γ-decalactone.

[0002]

BACKGROUND OF THE INVENTION Considerable time and effort has been expended by microbiologists in investigating better methods for the production of optically active lactones. U.S. Pat.No. 3,076,75
No. 0 disclosed a process for the preparation of certain optically active lactones and their corresponding hydroxycarboxylic acids by the microbiological reduction of ketocarboxylic acids. The metabolism of ricinoleic acid by certain Candida species was studied by Okui and others,
They showed that γ-hydroxydecanoic acid is an intermediate in the oxidative degradation of ricinoleic acid (J. Biochem.
istry, 54,536-540,1963).

[0003]

However, due to the metabolism of γ-hydroxydecanoic acid and the toxicity of ricinoleic acid to microorganisms at the completion of fermentation, which limits the amount of substrate that can be used, only traces are found in the fermentation medium. Only an amount of γ-hydroxydecanoic acid was recovered.

[0004]

[Means for Solving the Problems] As a means for solving the above-mentioned conventional problems, the present invention is directed to Aspergillus oryzae, Candida rugosa, Geotrichum cleverny, and Yarrowia in the presence of castor oil.
Optically active γ-hydroxy which comprises culturing a microorganism capable of hydrolyzing castor oil selected from the group consisting of lipolytica, and β-oxidizing the resulting hydrolyzate to produce γ-hydroxydecanoic acid. A method for producing decanoic acid is provided. In another embodiment, the invention uses a lipase to enzymatically hydrolyze castor oil to produce an enzymatic hydrolyzate,
And a method for producing an optically active γ-hydroxydecanoic acid, which comprises culturing a microorganism capable of β-oxidizing an enzymatic hydrolyzate in the presence of the hydrolyzate to produce γ-hydroxydecanoic acid To do. In yet another embodiment, the present invention provides a method of culturing a microorganism capable of hydrolyzing castor oil and a microorganism capable of β-oxidizing a castor oil hydrolyzate in the presence of castor oil to produce γ-hydroxydecanoic acid. Provided is a method for producing an optically active γ-hydroxydecanoic acid, which comprises producing the same.

As described above, the present invention provides a fermentation process for producing optically active γ-hydroxydecanoic acid, which can be optionally converted to γ-decalactone by lactonization. According to an embodiment of the invention as applied, the fermentation step comprises culturing a microorganism capable of hydrolyzing castor oil in a suitable medium in the presence of castor oil or a castor oil hydrolyzate substrate, and To perform β-oxidation of the hydrolyzate, or to cultivate a microorganism capable of β-oxidation of the hydrolyzate of castor oil, or a microorganism capable of β-oxidation of the enzymatic hydrolyzate of castor oil. Include. The castor oil or castor oil hydrolyzate as a substrate is determined by the microorganism used during the process. Co-oxidant is added to the medium to improve the yield in the process.

The selection of a suitable microorganism for the process is largely dependent on the embodiment of the invention applied, the desired product yield, and the resistance to fatty acid toxicity found in the castor oil hydrolyzate. To be done. The microorganism in the present invention is a bacterium, yeast, or filamentous fungus. Desirable microorganisms to be used for the hydrolysis of the castor oil substrate and the β-oxidation of the resulting hydrolyzate are Aspergillus oryzae, Candida rugosa, Geotrichum cleverny, or Yarrowia lipolytica (formerly Saccharomycopsis.
Known as Lipolitica and until recently known as Candida lipolytica), a particularly desirable one is Yarrowia lipolytica. The preferred microorganisms used only for the β-acid of the castor oil hydrolyzate are Hansenula saturnus, Candida guilliermondi, Candida albicans, Candida crusei, Candida paracursei, Candida pseudotropicals, Candida stella toidea, Candida tropicalis, Aspergillus oryzae. , Candida rugosa,
Geotrichum cleverney, or Yarrowia lipolytica, especially preferred is Candida guilermondi. Hansenula is the preferred microorganism used in the combination of lipase and castor oil.
Saturnus, Candida Guilliermondi, Candida albicans, Candida Crusei, Candida Paracrusei, Candida Pseudotropicalis, Candida Stella Toidea, Candida tropicalis, Aspergillus oryzae, Candida rugosa, Geotrichum
Cleverney, or Yarrowia lipolytica, especially preferred is Candida guilliermondi. Generally, any type of lipase enzyme
Used to hydrolyze castor oil, including that caused by fungi or yeast.

In lipases used with microorganisms in the process of the invention, the formation of the enzymatic hydrolyzate is regulated by limiting the amount of lipase used in the process. This will avoid the toxicity due to the presence of excessive amounts of hydrolyzate. The appropriate amount of lipase required is conveniently found by experimentation,
And it will depend on the lipase, the microorganism used and the medium conditions. Most preferably, the hydrolysis with lipase is performed simultaneously with the fermentation in the same reaction vessel. However, the hydrolysis could also be carried out prior to fermentation if the appropriate amount was taken to avoid the toxic effects of the hydrolyzate.
When castor oil is used in the present invention, toxicity concerns are eliminated because triglyceride is non-toxic to living organisms. In addition, the use of castor oil and castor oil hydrolysates as substrates provides a co-oxidant for processes with increased efficiency due to the presence of other fatty acids due to the hydrolysis of castor oil.

The form in which the microorganism is used is not critical. They can be used, for example, as a medium (suspension) containing cells and a matching nutrient solution, or in the form of cells suspended in a buffer solution. The cells or enzyme extract are then fixed to a suitable solid support used to carry out the transfer. Media suspensions are prepared by inoculating the appropriate medium with the microorganisms. Suitable media include those containing carbon sources, nitrogen sources, inorganic salts, and growth factors. Suitable carbon sources include, for example, glucose, galactose, L-sorbose, maltose, sucrose, cellobiose, trehalose, L.
-Arabinose, L-rhamnose, ethanol, glycerol, L-erythritol, D-mannitol, lactose, melibiose, raffinose, melerytose,
There are starch, D-xylose, D-sorbitol, α-methyl-D-glucoside, lactic acid, citric acid, and succinic acid. Suitable nitrogen sources include, for example, peptone, meat extract, yeast extract, corn steep liquor, and nitrogen containing casein, urea, amino acids, or inorganic compounds such as nitrates, nitrites, and inorganic ammonium salts. Suitable inorganic salts include, for example, phosphate, magnesium, potassium, calcium, sodium. The nutrients in the medium are optionally supplemented with, for example, one or more trace vitamins of the vitamin B group and / or one or more trace minerals such as Fe, Mo, Cu, Mn, B. . However, the process can also be carried out in a vitamin-free medium, for example vitamins or trace minerals may not be needed if a small amount of yeast extract is added to the medium.

Cultivation of the microorganism is preferably carried out under aerobic conditions as a fixed medium or a submerged medium (eg, shaking medium, fermenter). A pH range of 3.5 to about 8.0 and preferably about 4.0 to about 7.5 is suitable. The pH is adjusted by the addition of inorganic or organic bases such as sodium hydroxide, potassium hydroxide, calcium hydroxide, calcium carbonate, by ion exchange resins or by the addition of buffers such as phosphates or phthalates. Sent. Culture temperature is about 15 ℃
It is suitable to maintain the temperature between 30 and 33 ° C, preferably in the range of about 20 to 30 ° C.

The process according to the invention is conveniently carried out by adding castor oil or castor oil hydrolyzate as a substrate to the medium medium at the beginning of the culture as the sole carbon source. Alternatively, the substrate may be combined with other carbon sources such as dextrose and added during or when the culture is complete. The amount, level, or concentration of the substrate in the medium is set variously. For example, about 0.3 for hydrolyzed castor oil.
% To about 5% of the medium is initially formed or added during the course of the fermentation, while virtually any level of castor oil is used.

The reaction time varies depending on the composition of the medium and the substrate concentration. Generally, shake flask cultures take about 2 hours to about 2 depending on the composition of the microorganism and the medium.
It takes 40 hours. However, if a fermentor is used, the fermentation time will be reduced to about 100 hours or less. The fermentation may be carried out with microbial cells isolated from the culture or with an enzyme extract isolated from the cells by means known per se. In this case, the fermentation may conveniently be carried out in an aqueous solution, for example a buffer solution, saline, fresh nutrient solution or water. The isolated cells or enzyme extract are fixed on a solid support and the desired transfer is carried out in the absence of live microorganisms. The transfer of the substrate is carried out by a microbial mutant. Such mutants are readily prepared by methods well known in the art such as exposing cells to UV or X-rays or conventional mutagens such as acridine orange. Can be obtained.

The substrate is generally added directly to the medium.
A surfactant or dispersant such as Tween 80 (polyoxyethylene sorbitan monostearate) is also added to the aqueous suspension of the substrate. Conventional defoamers such as silicone oil (eg UCON), polyalkylene glycol derivatives, corn oil, or soybean oil can be used to control foaming. Substrate transfer can be performed by GLC, TLC, HPLC,
It can be monitored using standard analytical techniques such as IR and NMR. If a rapid disappearance of substrate is observed, more substrate is added to maximize the transfer capacity of the microorganism. The culture is generally terminated when all of the substrate has disappeared from the medium.

After the fermentation process is completed, the γ-hydroxydecanoic acid is lactonized in the medium or isolated and then purified by conventional techniques including solvent extraction and distillation. When in situ lactonization is desired, the pH of the medium is adjusted to between about 1 and about 5, preferably between about 1 and about 3, by the addition of a suitable acid such as hydrochloric acid, and The resulting mixture is approximately 50 ° C
To about 100 ° C., preferably about 70 ° C. to about 100 ° C., for about 10 minutes, and the heating converts γ-hydroxydecanoic acid into γ-decalactone. The γ-decalactone is then recovered and purified by a usual method. If the γ-hydroxydecanoic acid is recovered, it will be lactonized by known methods (eg IL Finar, Organic Chemistry, 6th ed., Vo.
l. 1 p 469 (1973)).

[0014]

In the present invention, castor oil or a hydrolyzate of castor oil is used as a substrate for microorganisms, and the caster oil or a hydrolyzate of castor oil is non-toxic to microorganisms. Further, other fatty acids are present in the hydrolyzate of the castor oil, which improves the efficiency of the process.

[0015]

EXAMPLES The following examples serve to illustrate preferred embodiments of the invention in practice but are not meant to limit the scope of the invention. Unless stated otherwise, weight is in grams, temperature is in degrees Celsius, and pressure is
It is mmHg. Example 1 100 ml of 2% beef extract and 0.02% T
The flask containing ween 80 was autoclaved at 120 ° C. for 20 minutes. 1 ml of medium for the medium
A 10 ⁷ cell count of Yarrowia lipolytica (Saccharomycopsis lipolytica) was inoculated and 10 g of castor oil was added. The medium was cultivated on a rotary shaker (200 rpm) at 26 ° C. for 1 week.
The pH of the medium was sometimes adjusted from 6.5 to 7.0. At the end of the fermentation period the pH of the medium was adjusted to 1.5 by the addition of mineral acid and the mixture was heated at 100 ° C for 10 minutes. After cooling, the organic product was extracted with hexane, the hexane was evaporated and the residue was distilled, and the residue was distilled to give 0.61 g of γ-decalactone with 90% GLC purity.

Example 2 The same method and materials as described in Example 1 were used except that 0.05 g of decanoic acid was added daily. 0.69 g of γ-decalactone having a GLC purity of 92% was obtained.

Example 3 A method and material similar to that described in Example 1 was used, except that Candida cyril mondi was used and 3 g of castor oil hydrolyzate was added. 34% yield of desired product γ-
Decalactone was obtained.

Example 4 The desired product γ-by using the same method and materials as described in Example 1 except that lipase is added with the castor oil.
Decalactone was obtained.

[Embodiment 5] For example, C.I. Albicans,
C. Crusay, C.I. Paracleuse, C.I. Pseudotropicalis, C.I. Stella Toidea, C.I. The desired product γ-decalactone was obtained by using the same methods and materials as described in Examples 1, 2 and 3 except that other Candida species such as T. picalis were used.

Example 6 The desired product γ-by using the same method and materials as described in Example 1, except that Aspergillus oryzae was used as the microorganism and 3 g of castor oil was added. Decalactone (0.86 g / l) was obtained.

[Example 7] Geotrichum as a microorganism
The desired product γ-decalactone (0.2 g / l) was obtained by using the same procedure and materials as described in Example 1, except that Cleverney was used and 3 g of castor oil was added. Was given.

Example 8 Candida Guilliermondi was used as the microorganism and 100 mg of lipase (Steapsin, Nutritional Bi) for each 100 ml of medium.
The desired product γ-decalactone was obtained by using the same method and materials as described in Example 1, except that ochem Corp.) was added.

Although the present invention has been described above, it will be apparent that the same can be varied in various ways. Such modifications should not be construed as departing from the spirit and scope of the present invention, and all such modifications are intended to be within the scope of the following claims.

[0024]

Therefore, in the present invention, optically active γ-hydroxydecanoic acid is produced in high yield.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication (C12P 7/42 C12R 1:69) (C12P 7/42 C12R 1:72) (C12P 7/42 (C12R 1: 725) (C12P 7/42 C12R 1:74) (C12P 17/08 C12R 1:85) (C12P 17/08 C12R 1:72) (C12P 17/08 C12R 1: 725) (C12P 17/08 (C12R 1:74) (C12P 17/08 C12R 1:69) (C12P 17/08 C12R 1: 645)

Claims (6)

[Claims] 1. Culturing a microorganism capable of hydrolyzing a caster oil selected from the group consisting of Aspergillus oryzae, Candida rugosa, Geotrichum cleverny, and Yarrowia lipolytica in the presence of castor oil, Method for producing optically active γ-hydroxydecanoic acid, which comprises β-oxidizing a decomposed product to produce γ-hydroxydecanoic acid 2. The method according to claim 1, wherein cooxidant is added or present during the culture of said microorganism. 3. In the presence of a hydrolyzate of castor oil, Hansenula Saturnus, Candida Guilliermondi, Candida albicans, Candida krusei, Candida paracrusei, Candida pseudotropicalis, Candida stellatoidea, Candida tropicalis, Aspergillus oryzae, An optically active γ consisting of culturing a microorganism capable of β-oxidizing a castor oil hydrolyzate selected from the group consisting of Candida rugosa, Geotrichum cleverney, and Yarrowia lipolytica to produce γ-hydroxydecanoic acid.
-Method for producing hydroxydecanoic acid 4. The method according to claim 3, wherein the hydrolyzate of castor oil is an enzymatic hydrolyzate enzymatically hydrolyzed with lipase. 5. The lipase enzyme is derived from a microorganism, pancreas, fungus, or yeast.
Method described in 6. The method according to claim 4, wherein cooxidant is added or present during the culture of said microorganism.