Process for producing unsaturated fatty acid or unsaturated hydrocarbon

Abstract: An unsaturated fatty acid or an unsaturated hydrocarbon is prepared from a fatty acid or a hydrocarbon with a bacterium belonging to the genus Rhodococcus and being capable of producing an unsaturated fatty acid or an unsaturated hydrocarbon in the presence of a carbohydrate or an amino acid, characterized by conducting the reaction with thiamine or an inorganic metal salt.

Description: The invention relates to a process for producing an unsaturated fatty acid or an unsaturated hydrocarbon or a derivative thereof, utilising a microorganism. Unsaturated fatty acids and derivatives thereof are widely used in, for example, perfumes, drugs, coatings, surfactants and cosmetics as well as starting materials for producing these products. Known processes for preparing such unsaturated fatty acids and derivatives thereof involve hydrolysis of animal or vegetable fats, or chemical synthesis. However the hydrolysis of a fat results in a mixture of unsaturated fatty acids having various chain lengths, whilst chemical synthesis requires a number of steps and results in a mixture of cis and trans stereoisomers. Both of these processes are disadvantageous in that the mixtures can be separated only with difficulty. On the other hand, some methods for the production of unsaturated acids are known which involve the use of microorganisms. For example, Japanese Patent Publications Nos.37096/1986 and 22199/1983 have disclosed fermentation methods for the production of γ-linolenic acid involving microorganisms of the genera Thamnidium and Mortierella , respectively. However, each of these processes is disadvantageous in that the yield is low, that the reaction is slow and that the desired unsaturated fatty acid is accumulated within the microorganism cells and thus is difficult to recover. Acyl-CoA desaturase (E.C. 1.14.99.5) is known to be one of enzymes which desaturate fatty acid derivatives. It originates from microorganisms, animals and plants. However the low activity and extremely low stability of this enzyme make it unsuitable for industrial application. Unsaturated hydrocarbons and derivatives thereof are widely used in for example, pheromones, perfumes and drugs as well as starting materials for producing these products and intermediates in the synthesis of organic compounds. Known processes for preparing such unsaturated hydrocarbons and derivatives thereof involve chemical synthesis by metathesis. However such processes are disadvantageous in that they require a number of steps and result in a mixture of cis and trans stereoisomers. U.S. Patent No. 3,629,072 discloses a process for the production of an alkene with the use of a microorganism belonging to the genus Nocardia . However this process is disadvantageous in that it requires a prolonged reaction period and gives a low yield. Accordingly although it has been urgently required to establish a process for readily producing a dialkene from a monoalkene, which is easily available on an industrial scale, no process satisfying such a requirement has been proposed so far. We have now found that a particular microorganism, which has been isolated from soil and subjected to mutation as will be shown in Referential Example 1 hereinafter, can convert a fatty acid or a hydrocarbon into the corresponding unsaturated fatty acid or hydrocarbon and that the unsaturated fatty acid or hydrocarbon thus produced is excreted from the cells and thus can be readily recovered. We have further found that the ability of said strain to producing an unsaturated fatty acid or hydrocarbon can be greatly enhanced by adding an amino acid such as L-glutamic or L-aspartic acid or a carbohydrate such as glucose to the reaction mixture comprising the strain and the substrate, i.e. a fatty acid or hydrocarbon, and further adding thiamine or an inorganic metal salt such as magnesium sulfate or manganese sulfate thereto. The invention provides a process for preparing an unsaturated fatty acid, a derivative thereof, an unsaturated hydrocarbon or a derivative thereof from a fatty acid, a derivative thereof, a hydrocarbon or a derivative thereof, respectively, utilising a bacterium belonging to the genus Rhodococcus which is capable of producing an unsaturated fatty acid or an unsaturated hydrocarbon in the presence of a carbohydrate or an amino acid, characterized by conducting the reaction in the pressure of thiamine or an inorganic metal salt, as specified in claims 1 - 7. According to a first aspect, the present invention provides a process for the production of an unsaturated fatty acid or a derivative thereof which comprises supplying a fatty acid or a derivative thereof to an unsaturated fatty acid-producing bacterium belonging to the genus Rhodococcus to give desired unsaturated fatty acid or a derivative thereof, characterised in that thiamine or an inorganic metal salt is added to the reaction system. According to a second aspect, the present invention provides a process for the production of an unsaturated hydrocarbon or a derivative thereof which comprises supplying a hydrocarbon or a derivative thereof to an unsaturated hydrocarbon-producing bacterium belonging to the genus Rhodococcus to give the desired unsaturated hydrocarbon or a derivative thereof, characterized in that thiamine or an inorganic metal salt is added to the reaction system. The bacterium to be used in the invention is obtained by subjecting a strain isolated from soil of Okinawa, Japan to a mutation treatment as shown in Referential Example 1. The mycological properties of this strain are as follows. (A) Morphological properties It is a bacillus having polymorphic cells. Namely, the cells are bacillary in a young culture and coccobacillary in an aged culture. A cell is 0.5 to 0.8 µm x 1.0 to 5.0 µm in size. (B) Growth on various media (1) Sucrose-nitrate agar medium It grows poorly. Colonies are of a pale flesh color, smooth and slightly glossy. (2) Glucose-asparagine agar medium It grows poorly. Colonies are of a pale flesh color, smooth and slightly glossy. (3) Glycerol-asparagine agar medium It grows moderately well. Colonies are of a milky white color and slightly glossy. Some colonies are smooth while others are rough. (4) Starch agar medium It grows moderately well. Colonies are of a milky white color, smooth and slightly glossy. (5) Tyrosine agar medium It grows vigorously. Colonies are of a flesh color, smooth and glossy. Some colonies are slimy. (6) Nutrient agar medium It grows vigorously. Colonies are of a pale orange color, smooth and glossy. Some colonies are smooth while others are rough. (7) Yeast-malt agar medium It grows vigorously. Colonies are of an orange color, wrinkly and glossy. Some colonies are slimy. (8) Oatmeal agar medium It grows moderately well. Colonies are of a pale orange color and dimly glossy. (C) Physiological properties (i) Growth range Temperature: 15 to 37 C, preferably 25 to 35 C Medium pH value: 5 to 9.5 preferably 6 tc 8 (2) Liquefaction of gelatin (glucose-peptone-gelatin medium) Negative. (3) hydrolysis of starch (starch agar medium) Negative. (4) Coagulation and peptonization of skim-milk Both negative. (5) Melanogenesis (tyrosine medium and pepton-yeast-iron medium) Negative. (D) Utilization of carbon sourse (Pridham-Gotreeve agar medium) (1) L-Arabinose: +. (2) D-Xylose: +. (3) D-Glucose: +. (4) D-Fructose: +. (5) Sucrose: +. (6) Inositol: +. (7) L-Rhamnose: +. (8) D-Mannitol: +. (E) Chemotaxonomic properties (1) Glycolyl test Glycolyl type. (2) Menaquinone system MK-8 (H₂). Based on these mycological properties, it has been recognized that this strain belongs to the genus Rhodococcus according to Bergey's Manual of Systematic Bacteriology, vol. 2 (1986). However it is significantly different from other microorganisms belonging to the genus Rhodococcus in unsaturated fatty acid productivity. Thus it was named Rhodococcus sp. KSM-B-3M and was deposited with Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry as FERM BP-1531. on October 21, 1987. The fatty acid or derivative thereof to be used as the starting material in the process of the present invention are those represented by the following general formula (I): R-COOR₁ (I) wherein R represents a straight-chain or branched and saturated or unsaturated hydrocarbon group carrying 1 to 22 carbon atoms: and R₁ represents a hyrogen atom, a straight-chain or branched hydrocarbon group or an aromatic hydrocarbon group carrying 1 to 10 carbon atoms, an alkali metal atom or a group of the formula: wherein R₂ and R₃ represent each a hydrogen atom or a straight-chain or branched hydrocarbon group with one to five carbon atoms. In the present invention, it is preferable that R is a straight-chain hydrocarbon group with 14 to 20 carbon atoms and R₁ is a straight-chain or branched hydrocarbon group with two to four carbon atoms. According to the present invention, the above fatty acid or a derivative thereof is converted into the corresponding unsaturated fatty acid or derivative thereof. More particularly, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and tert-butyl esters of fatty acids such as n-hexadecanoic or n-tetradecanoic acid may be converted into methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and tert-butyl esters of hexadecenoic or tetradecenoic acid, respectively. The hydrocarbon or derivative thereof to be used as the starting material in the process of the present invention are those represented by the following general formula (II): R-A (II) wherein R represents a straight-chain or branched and saturated or unsaturated hydrocarbon group with 6 to 22 carbon atoms; and A represents a hydrogen atom, an aromatic hydrocarbon group, a halogen atom or a -OH, -CN, -CO-R₁ (wherein R₁ represents a straight-chain or branched hydrocarbon group or an aromatic hydrocarbon group with 1 to 10 carbon atoms), -O-R₂ (wherein R₂ represents a straight-chain or branched hydrocarbon group with one to ten carbon atoms or an aromatic hydrocarbon group), a metal atom or a group of the formula: wherein R₃ and R₄ represent each a hydrogen atom or a straight-chain or branched hydrocarbon group with one to five carbon atoms. In the present invention, it is preferable that R is a straight-chain hydrocarbon group carrying 14 to 20 carbon atoms. According to the present invention, the above hydrocarbon or a derivative thereof is converted into the corresponding unsaturated hydrocarbon or derivative thereof. More particularly, hydrocarbons and derivatives thereof such as hexadecane, octadecane, tetradecane, pentadecane, eicosane, chlorohexadecane, chlorooctadecane, hexadecanol, hexadecylbenzene, heptadecanonitrile, hexadecene, eicosene and tetradecene are converted into hexadecene, octadecene, tetradecene, eicosene, chlorohexadecene, chlorooctadecene, hexadecenol, hexadecenylbenzene, heptadecenonitrile, hexadecadiene, eicosadiene and tetradedadiene, respectively. In the present invention, cells of the abovementioned strain may be obtained by a conventional microbial culture method. The medium, pH, temperature and period for the culture may be arbitrarily selected so long as the strain can grow thereunder. It is preferred to culture this strain at 30°C for one to two days under aerobic conditions. The culture medium thus obtained may be subjected to the following reaction either as such or after collecting cells by, for example, centrifugation or filtration. 0.1 to 90%, preferably 0.2 to 0.5%,in terms of dry cells, of the culture medium or cells obtained above are suspended in water or a buffer solution (pH 6 to 8). 1 to 70%, preferably 10 to 30%, of a fatty acid or a hydrocarbon to react and 0.05 to 10%, preferably 0.5 to 2%, of an amino acid such as L-glutamic acid or L-aspartic acid or a carbohydrate such as glucose are added to the above suspension to thereby give a basic reaction mixture. Further 0.001 to 2%, preferably 0.01 to 0.3% in terms of thiamine, of a thiamine compound such as thiamine hydrochloride or 0.001 to 2% of a magnesium or manganese salt such as magnesium sulfate or manganese sulfate, preferably 0.01 to 0.3% of magnesium sulfate, are added thereto to thereby give the reaction mixture of the present invention. This reaction mixture is allowed to react at 20 to 37°C, preferably 25 to 30°C, under aerobic conditions. The addition of the thiamine or inorganic metal salt such as magnesium sulfate to the reaction mixture aims at significantly enhancing the productivity. Although it is preferable to combine these additives with an amino acid such as L-glutamic or L-aspartic acid, they may be combined with a carbohydrate. Alternately, each of these additives may be combined with an amino acid or a carbohydrate. These addtives may be added during the collection of the cells. The thiamine may be employed either in the salt-free form or as hydrochloride or sulfate. Although magnesium sulfate is preferable as the inorganic metal salt, those including sulfuric acid, magnesium or manganese such as manganese sulfate may be employed therefor. Further an extract originating from a natural material or an amino acid mixture containing amino acids, carbohydrates, thiamine and inorganic metal salt(s), for example, meat extract, yeast extract and casamino acid may be employed. When the reaction is to be conducted batchwise, the reaction period may range from one to three days. However the reaction period may be prolonged while supplying, for example, the starting fatty acid or its derivative, amino acid, vitamins and metal salt to the system. It is also possible to recover the employed cells by, for example, centrifugation or filtration and then reuse the same. This way the unsaturated hydrocarbon or fatty acid corresponding to the starting hydrocarbon or fatty acid is formed to achieve the purpose of the invention. The product of the reaction can be identified and determined in a conventional manner employed for the identification and determination of organic compounds contained in natural materials. For example, a given amount of the reaction mixture is extracted with an organic solvent and analyzed by, for example, gas chromatography (GC) or gas chromatography/mass spectrometry (GC/MS) to thereby identify and determine the products. The position of a double bond in an unsaturated product may be determined by, for example, treating the mixture with dimethyl disulfide and then subjected to GC/MS, i.e., methylthioetherification (cf. Shibahara et al., Yukagaku, 34 , 619 (1985)). Alternatively, the reaction mixture may be centrifuged to thereby divide it into the cells, an oily phase and an aqueous phase and then the oily phase is collected. Alternatively, the reaction mixture is extracted with an organic solvent, either as such or after removing the cells by, for example, filtration. Thus the aimed compounds may be readily collected. The compounds thus collected may be further purified or isolated by, for example, conventional column chromatography or partition extraction, if required. Brief Description of the Drawings: Fig. 1 shows the GC/FT-IR spectrum of propyl hexadecenoate produced according to the present invention. Fig. 2 shows the mass spectrum of the bismethylthioether derivative of propyl hexadecenoate produced according to the present invention. Fig. 3 shows the mass spectrum of propyl hexadecenoate produced according to the present invention. Fig. 4 shows the GC/FT-IR spectrum of cis-hexadecene produced according to the present invention. Fig. 5 shows the mass spectrum of the bismethylthioether derivative of cis-hexadecene produced according to the present invention. Fig. 6 shows the mass spectrum of cis-hexadecene produced according to the present invention. Referential Example 1 Approximately 0.5 g of a soil sample collected in Okinawa pref. was suspended in 10 ml of sterilized water. After thoroughly stirring, 0.2 ml of the suspension was inoculated into 10 ml of a liquid medium (I) of the following composition placed in a test tube (25 mm in diameter x 200 mm in height) and cultured therein at 30°C for four days under shaking. Liquid medium (I): n-hexadecane 100 g (NH₄)₂SO₄ 20 g KH₂PO₄ 20 g yeast extract 2 g MgSO₄·7H₂O 0.5 g FeSO₄·7H₂O 0.01 g MnSO₄·4∼6H₂O 0.008 g deionized water 1 ℓ pH 7 The culture medium thus obtained was appropriately diluted with sterilized water, transplanted into a common agar medium (mfd. by Eiken Kagaku K.K.) and cultured therein at 30°C for two days. The transplantation to the nutrient agar medium was repeated until formed colonies were the same with each other when observed with the naked eye and under a microscope. The strain thus isolated was suspended in a 0.1 M phosphate buffer solution (pH 7.0) and irradiated with UV light for 90 sec. Then the colonies thus obtained were transplanted into 50 ml of a medium comprising 2.5 g of glucose, 17 g of Polypeptone, 3 g of Polypeptone S, 2.5 g of monopotassium dihydrogenphosphate, 5 g of sodium chloride and 1 ℓ of deionized water contained in a 500 ml Sakaguchi flask, tradename, and subsequently cultured therein at 30°c for one day under shaking. This culture medium was centrifuged to thereby collect cells, which were then suspended in 19 ml of a 0.25 M phosphate buffer solution (pH 7.0) containing 0.5% of glucose. After adding 1 ml of hexadecane, these cells were reacted in a 500 ml Sakaguchi flask at 30°C for three days under shaking. After the completion of the reaction, the major product contained in the reaction mixture was extracted and analyzed by, for example, gas chromatography. Thus a strain producing hexadecene was obtained. This hexadecene-producing strain was appropriately diluted with sterilized water, transplanted into a nutrient agar medium and cultured therein at 30°c for two days. Then it was repeatedly transplanted into the nutrient agar medium until the colonies thus formed were the same with each other when observed with the naked eye and under a microscope. Ten of the colonies grown in the nutrient agar medium were inoculated into each a slant agar medium (II) and cultured therein at 30°C for three days. Thus it was confirmed that the strains on these ten media were the same with each other by observing them with the naked eye and under a microscope. It was further confirmed that these ten strains were the same with each other in morphological and physiological properties. Slant agar medium (II): n-hexadecane 20 g (NH₄)₂SO₄ 20 g KH₂PO₄ 2 g yeast extract 2 g MgSO₄·7H₂O 0.5 g FeSO₄·7H₂O 0.01 g MnSO₄·4∼6H₂O 0.008 g polyoxyethylene sorbitan monolaurate (average EO number: 20 mol) 0.05 g agar 20 g deionized water 1 ℓ pH 7 Then a loopful of each strain above obtained was suspended in 2 ml of a sterilized 10% aqueous solution of glycerol contained in a cryopreservation vial and cryopreserved at -80°C. Three months thereafter, it was rapidly thawed and a loopful of the reserved suspension was regenerated in an agar medium. Then it was confirmed that the morphological and physiological properties thereof on various media showed no change under the same conditions as those employed above. The cryopreservation and thawing were repeated five times at intervals of one month. Then the strains showed no change in morphological and physiological properties on various media. The morphological and physiological properties of this strain on various media are as described above. (Examples) To further illustrate the present invention, the following Examples will be given. Example 1 50 ml of a medium comprising 2.5 g of glucose, 17 g of Polypeptone (mfd. by Daigo Eiyo K.K.), 3 g of Polypeptone S (mfd. by Daigo Eiyo K.K.), 2.5 g of monopotassium dihydrogenphosphate, 5 g of sodium chloride and 1 ℓ of deionized water was introduced into a 500-ml Sakaguchi flask and inoculated with Rhodococcus sp. KSM-B-3M strain. This strain was cultured in the medium at 30°C for one day under shaking. Then 1 ml of the culture medium was transplanted into 100 ml of a medium of the same composition as the one described above in a 500-ml Sakaguchi flask and cultured therein at 30°C for one day under shaking. The culture medium was centrifuged at 5,000 x g to thereby give 2 g of cells. 1 g of these cells were suspended in 20 ml of a 0.25 M phosphate buffer solution (pH 7.0) containing 1% of monosodium glutamate, 0.1% of thiamine hydrochloride and 0.1% of magnesium sulfate. Then 4 ml of n-propyl palmitate (n-propyl hexadecanoate) was added thereto and the resulting mixture was allowed to react in a 500-ml Sakaguchi flask at 26°C for two days under shaking. After the completion of the reaction, the major product contained in the reaction mixture was extracted with ethyl acetate and analyzed by GC, GC/MS or gas chromatography/infrared spectrocopy (GC/FT-IR) (cf. Fig. 1). Thus it was confirmed that the major product was propyl cis-6-hexadecenoate. The position of the double bond was determined by converting the product into a dimethyl disulfide derivative and analyzing the same with mass spectroscopy (cf. Fig. 2). Fig. 3 shows the mass spectrum of the major product. 1 ml of the reaction mixture was extracted with 3 ml of ethyl acetate and the content of the propyl cis-6-hexadecenoate therein was determined by gas chromatography. As a result, the productivity of this strain of the above compound was found to be 15 g/l·2 days. Example 2 The procedure of Example 1 was repeated except that the propyl hexadecanoate was replaced with methyl hexadecanoate, ethyl hexadecanoate, isopropyl hexadecanoate, isobutyl hexadecanoate and n-butyl hexadecanoate. The unsaturated fatty acid ester in each reaction mixture was identified and determined in the same manner as the one described in Example 1. Table 1 shows the results. Substrate Major product Production g/l·2 days methyl hexadecanoate methyl cis-9-hexadecenoate 0.5 ethyl hexadecanoate ethyl cis-6-hexadecenoate 8.5 iso-propyl hexadecanoate iso-propyl cis-6-hexadecenoate 28.0 iso-butyl hexadecanoate iso-butyl cis-6-hexadecenoate 10.4 n-butyl hexadecanoate n-butyl cis-4-hexadecenoate 5.4 Example 3 The procedure of Example 1 was repeated except that the propyl hexadecanoate was replaced with sodium hexadecanoate, isopropyl tetradecanoate and ethyl tetradecanoate. The unsaturated fatty acid or its ester in each reaction mixture was identified and determined in the same manner as the one described in Example 1. Table 2 shows the results. Substrate Major product Production g/l·2 days sodium hexadecanoate hexadecenoic acid 0.2 iso-propyl tetradecanoate iso-propyl tetradecenoate 10.5 ethyl tetradecanoate ethyl tetradecenoate 2.1 Comparative Example 1 50 ml of a medium comprising 2.5 g of glucose, 17 g of Polypeptone, 3 g of Polypeptone S, 2.5 g of monopotassium dihydrogenphosphate, 5 g of sodium chloride and 1 ℓ of deionized water was introduced into a 500-ml Sakaguchi flask and inoculated with Rhodococcus sp. KSM-B-3M strain. This strain was cultured in the medium at 30°C for one day under shaking. Then 1 ml of the culture medium was transplanted into 100 ml of a medium of the same composition as the one described above in a 500-ml Sakaguchi flask and cultured therein at 30°C for one day under shaking. The culture medium was centrifuged at 5,000 x g to thereby give 2 g of cells. 1 g of these cells were suspended in 20 ml of a 0.25 M phosphate buffer solution (pH 7.0) containing 1.0% of glucose. Then 4 ml of n-propyl hexadecanoate was added thereto and the resulting mixture was allowed to react in a 500-ml Sakaguchi flask at 26°C for two days under shaking. 1 ml of the reaction mixture was extracted vith 3 ml of ethyl acetate and the propyl hexadecenoate thus formed was identified and determined in the same manner as the one described in Example 1. As a result, the productivity of this strain of the above compound was found to be 4.0 g/l·2 days. Comparative Example 2 The procedure of Comparative Example 1 was repeated except that the 0.25 M phosphate buffer solution containing 1.0% of glucose was replaced with a 0.25 M phosphate buffer solution containing 1.0% of monosodium L-glutamate. The isopropyl hexadecenoate thus formed in the reaction mixture was identified and determined in the same manner as the one described in Example 1. As a result, the productivity of this strain of the above compound was found to be 5.2 g/l·2 days. Example 4 The procedure of Comparative Example 1 was repeated except that the 0.25 M phosphate buffer solution containing 1.0% of glucose was replaced with: (1) a 0.25 M phosphate buffer solution containing 1.0% of glucose and 0.1% of thiamine hydrochloride: (2) a 0.25 M phosphate buffer solution containing 1.0% of monosodium L-glutamate and 0.1% of magnesium sulfate: (3) a 0.25 M phosphate buffer solution containing 1.0% of monosodium L-glutamate and 0.1% of thiamine hydrochloride: (4) a 0.25 M phosphate buffer solution containing 1.0% of monosodium L-glutamate, 0.1% of thiamine hydrochloride and 0.1% of magnesium sulfate: (5) a 0.25 M phosphate buffer solution containing 1.0% of monosodium L-aspartate, 0.1% of thiamine hydrochloride and 0.1% of magnesium sulfate: (6) a 0.25 M phosphate buffer solution containing 1.0% of glycine and 0.1% of thiamine hydrochloride: 0.1% of magnesium sulfate: (7) a 0.25 M phosphate buffer solution containing 1.0% of L-tyrosine, 0.1% of thiamine hydrochloride and 0.1% of magnesium sulfate: (8) a 0.25 M phosphate buffer solution containing 1.0% of L-threonine, 0.1% of thiamine hydrochloride and 0.1% of magnesium sulfate: (9) a 0.25 M phosphate buffer solution containing 1.0% of L-proline, 0.1% of thiamine hydrochloride and 0.1% of magnesium sulfate: and (10) a 0.25 M phosphate buffer solution containing 1.0% of monosodium L-glutamate and 0.1% of manganese sulfate. The unsaturated fatty acid ester in each reaction mixture was identified and determined in the same manner as the one described in Example 1. Table 3 shows the results. Example 5 50 ml of a medium comprising 2.5 g of glucose, 17 g of Polypeptone (mfd. by Daigo Eiyo K.K.), 3 g of Polypeptone S (mfd. by Daigo Eiyo K.K.), 2.5 g of monopotassium dihydrogenphosphate, 5 g of sodium chloride and 1 ℓ of deionized water was introduced into a 500-ml Sakaguchi flask and inoculated with Rhodococcus sp. KSM-B-3M strain. This strain was cultured in the medium at 30°C for one day under shaking. Then 1 ml of the culture medium was transplanted into 100 ml of a medium of the same composition as the one described above in a 500-ml Sakaguchi flask and cultured therein at 30°C for one day under shaking. The culture medium was centrifuged at 5,000 x g to thereby give 2 g of cells. 1 g of these cells were suspended in 20 ml of a 0.25 M phosphate buffer solution (pH 7.0) containing 1% of monosodium glutamate, 0.1% of thiamine hydrochloride and 0.1% of magnesium sulfate. Then 5 ml of n-hexadecane was added thereto and the resulting mixture was allowed to react in a 500-ml Sakaguchi flask at 30°C for two days under shaking. After the completion of the reaction, the major product contained in the reaction mixture was extracted with ethyl acetate and analyzed by GC, GC/MS or gas chromatography/infrared spectrocopy (CG/FT-IR) (cf. Fig. 4). Thus it was confirmed that the major product was cis-7-hexadecene. The position of the double bond was determined by converting the product into a dimethyl disulfide derivative and analyzing th same with mass spectroscopy (cf. Fig. 5). Fig. 6 shows the mass spectrum of the major product. 1 ml of the reaction mixture was extracted vith 3 ml of ethyl acetate and the content of the cis-7-hexadecene therein was determined by gas chromatography. As a result, the productivity of this strain of the above compound was found to be 18 g/l·2 days. Example 6 The procedure of Example 5 was repeated except that the n-hexadecane was replaced with n-dodecane, n-tetradecane, n-octadecane, n-eicosane, 1-chlorotetradecane, 1-chlorohexadecane, 1-chlorooctadecane and 1-chloroeicosane. The unsaturated hydrocarbon or its derivative in each reaction mixture was identified and determined in the same manner as the one described in Example 5. Table 4 shows the results. Substrate Major product Production g/l·2 days n-dodecane cis-3-dodecene 2.1 n-tetradecane cis-5-tetradecene 8.3 n-octadecane cis-9-octadecene 16.2 n-eicosane cis-11-eicosene 10.6 1-chloro-tetradecane 1-chloro-cis-5-tetradecene 7.2 1-chloro-hexadecane 1-chloro-cis-7-hexadecene 25.6 1-chloro-octadecane 1-chloro-cis-9-octadecene 22.5 1-chloro-eicosane 1-chloro-cis-11-eicosene 15.2 Example 7 The procedure of Example 5 was repeated except that the n-hexadecane was replaced with n-hexadecanol, n-hexadecylbenzene and n-heptadecanonitrile. The unsaturated hydrocarbon in each reaction mixture was identified and determined in the same manner as the one described in Example 5. Table 5 shows the results. Substrate Major product Production g/l·2 days 1-hexadecanol hexadecenol 0.4 n-hexadecylbenzene hexadecenyl benzene 0.4 n-heptadecanonitorile heptadecenonitorile 6.0 Example 8 The procedure of Example 5 was repeated except that the n-hexadecane was replaced with 1-hexadecene, 1-octadecene, 1-eicosene and 1-tetradecene. The unsaturated hydrocarbon in each reaction mixture was identified and determined in the same manner as the one described in Example 5. Table 6 shows results. Substrate Major product Production g/l·2 days 1-hexadecene hexadeca-di-ene 22.7 1-octadecene octadeca-di-ene 20.1 1-eicosene eicosa-di-ene 19.3 1-tetradecene tetradeca-di-ene 7.2 Comparative Example 3 50 ml of a medium comprising 2.5 g of glucose, 17 g of Polypeptone, 3 g of Polypeptone S, 2.5 g of monopotassium dihydrogenphosphate, 5 g of sodium chloride and 1 ℓ of deionized water was introduced into a 500-ml Sakaguchi flask and inoculated with Rhodococcus sp. KSM-B-3M strain. This strain was cultured in the medium at 30°C for one day under shaking. Then 1 ml of the culture medium was transplanted into 100 ml of a medium of the same composition as the one described above in a 500-ml Sakaguchi flask and cultured therein at 30°C for one day under shaking. The culture medium was centrifuged at 5,000 x g to thereby give 2 g of cells. 1 g of these cells were suspended in 20 ml of a 0.25 M phosphate buffer solution (pH 7.0) containing 1.0% of glucose. Then 4 ml of 1-chlorohexadecane was added thereto and the resulting mixture was allowed to react in a 500-ml Sakaguchi flask at 30°C for two days under shaking. 1 ml of the reaction mixture was extracted with 3 ml of ethyl acetate and the 1-chlorohexadecene thus formed was identified and determined in the same manner as the one described in Example 1. As a result, the productivity of this strain of the above compound was found to be 5.5 g/l·2 days. Comparative Example 4 The procedure of Comparative Example 3 was repeated except that the 0.25 M phosphate buffer solution containing 1.0% of glucose was replaced with a 0.25 M phosphate buffer solution containing 1.0% of monosodium L-glutamate. The 1-chlorohexadecene thus formed in the reaction mixture was identified and determined in the same manner as the one described in Example 3. As a result, the productivity of this strain of the above compound was found to be 6.2 g/l·2 days. Example 9 The procedure of Comparative Example 3 was repeated except that the 0.25 M phosphate buffer solution containing 1.0% of glucose was replaced with: (1) a 0.25 M phosphate buffer solution containing 1.0% of monosodium L-glutamate and 0.1% of magnesium sulfate; (2) a 0.25 M phosphate buffer solution containing 1.0% of glucose and 0.1% of thiamine hydrochloride; (3) a 0.25 M phosphate buffer solution containing 1.0% of monosodium L-glutamate and 0.1% of thiamine hydrochloride: (4) a 0.25 M phosphate buffer solution containing 1.0% of monosodium L-glutamate, 0.1% of thiamine hydrochloride and 0.1% of magnesium sulfate: (5) a 0.25 M phosphate buffer solution containing 1.0% of monosodium L-aspartate, 0.1% of thiamine hydrochloride and 0.1% of magnesium sulfate: (6) a 0.25 M phosphate buffer solution containing 1.0% of glycine, 0.1% thiamine hydrochloride and 0.1% of magnesium sulfate: (7) a 0.25 M phosphate buffer solution containing 1.0% of L-tyrosine, 0.1% of thiamine hydrochloride and 0.1% of magnesium sulfate: (8) a 0.25 M phosphate buffer solution containing 1.0% of L-threonine, 0.1% of thiamine hydrochloride and 0.1% of magnesium sulfate: (9) a 0.25 M phosphate buffer solution containing 1.0% of L-proline, 0.1% of thiamine hydrochloride and 0.1% of magnesium sulfate: and (10) a 0.25 M phosphate buffer solution containing 1.0% of monosodium L-glutamate and 0.1% of manganese sulfate. The 1-chlorohexadecene in each reaction mixture was identified and determined in the same manner as the one described in Example 5. Table 7 shows results.

Claims: A process for preparing: (i) an unsaturated fatty acid or a derivative thereof from a fatty acid or derivative thereof represented by the general formula (I): R-COOR₁ (I) wherein R represents a straight-chain or branched and saturated or unsaturated hydrocarbon group with 1 to 22 carbon atoms: and R₁ represents a hydrogen atom, a straight-chain or branched hydrocarbon group or an aromatic hydrocarbon group with 1 to 10 carbon atoms, an alkali metal atom or a group of the formula: wherein each of R₂ and R₃ represents a hydrogen atom or a straight-chain or branched hydrocarbon group with one to five carbon atoms, or (ii) an unsaturated hydrocarbon or a derivative thereof from a hydrocarbon or derivative thereof represented by the general formula (II): R-A (II) wherein R represents a straight-chain or branched saturated or unsaturated hydrocarbon group with 6 to 22 carbon atoms; and A represents a hydrogen atom, an aromatic hydrocarbon group, a halogen atom or a -OH, -CN, -CO-R₁ (wherein R₁ represents a straight-chain or branched hydrocarbon group or an aromatic hydrocarbon group with 1 to 10 carbon atoms), or -O-R₂ (wherein R₂ represents a straight-chain or branched hydrocarbon group with one to ten carbon atoms or an aromatic hydrocarbon group), or a metal atom or a group of the formula: wherein each of R₃ and R₄ represents a hydrogen atom or a straight-chain or branched hydrocarbon group with one to five carbon atoms, wherein the process is carried out in the presence of Rhodococcus sp. KSM-B-3M strain (FERM-BP-1531), characterised by conducting the reaction by suspending 0.1 to 90% of dry cells in water or a buffer solution at a pH of 6 to 8, adding 1 to 70% of the fatty acid or derivative thereof or the hydrocarbon or derivative thereof and 0.05 to 10% of an amino acid or carbohydrate to give a basic reaction mixture, adding 0.001 to 2% or thiamine or a thiamine compound or a magnesium or manganese salt, and allowing the resulting mixture to react at 20 to 37°C under aerobic conditions. A process as claimed in claim 1, in which said carbohydrate is selected from glucose, sorbitol, arabinose, xylose, fructose, inositol, rhamnose and mannitol. A process as claimed in claim 1, in which said amino acid is selected from L-glutamic acid, L-aspartic acid, glycine, L-threonine, L-tyrosine, L-isoleucine and L-proline. A process as claimed in claim 1, in which said magnesium or manganese salt is a sulphate. A process as claimed in claim 1, in which said hydrocarbon or derivative thereof is selected from n-dodecane, n-heptadecane, n-hexadecane, n-octadecane, n-tetradecane, n-pentadecane, n-eicosane, chloroheptadecane, chlorohexadecane, chlorooctadecane, hexadecylalcohol, hexadecylbenzene, heptadecanonitrile, hexadecene, eiscosene, octadecene and tetradecene. A process as claimed in claim 1, in which said fatty acid or derivative thereof is selected from n-heptadecanoic acid, n-hexadecanoic acid, n-pentadecanoic acid and n-tetradecanoic acid as well as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tertbutyl, n-heptyl and n-hexyl esters thereof. A process according to claim 1, wherein the Rhodococcus sp.KSM-B-3M strain is that deposited at the Fermentation Research Institute, Japan, under the accession number FERM-BP-1531.

Inventors: ITOH SUSUMU, KOIKE KENZO, TAKAIWA MIKIO, ITOH, SUSUMU, KOIKE, KENZO, TAKAIWA, MIKIO
Applicants: AGENCY IND SCIENCE TECHN, THE AGENCY OF INDUSTRIAL SCIENCE AND TECHNOLOGY
Application year: 1988
Main classification: C12P7
IPC codes: C12P7, C12P13, C12P5