JPH0669378B2 - Method for converting water-insoluble or almost water-insoluble organic substrate using microorganisms - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for converting an organic substrate by a microorganism.

More specifically, when a water-insoluble or almost water-insoluble ketone or aldehyde represented by the following formula [I] is reduced by using a microorganism, a vegetable oil containing triglyceride as a main component is added to obtain a higher conversion rate. It relates to a method for advantageously reducing ketones or aldehydes.

In the formula, R ₁ and R ₂ are each a hydrogen atom or a carbon atom of 1 to
15 represents an alkyl group, an alkoxy group, an allyl group or an allyloxy group.

2. Description of the Related Art and Problems When converting organic compounds, the use of microorganisms or enzymes is advantageous in terms of energy because the reaction can be carried out at room temperature and atmospheric pressure. Further, the microorganism or enzyme has the ability to recognize isomers such as optical isomers and geometrical isomers, and only more useful isomers can be produced.

With enzymes, expensive coenzymes must be added if coenzymes are required for the enzymatic reaction. When the enzyme is isolated, the enzyme is often unstable.

On the other hand, when a microorganism is used, the microorganism has the ability to produce or regenerate a coenzyme, and it is not necessary to add an expensive coenzyme. In addition, when used as a microorganism, the enzyme is often more stable than when isolated. In such a case, it is advantageous to use a microorganism.

When using microorganisms, water is required for culture or survival.
However, many organic compounds are insoluble in water or almost insoluble in water. There are also water-insoluble or almost water-insoluble organic compounds that are solid at room temperature and atmospheric pressure.

Even if an attempt is made to convert these water-insoluble or almost water-insoluble organic compounds into a microorganism culture solution or a microorganism suspension by using microorganisms, the contact area between the microorganisms and the substrate is small and the incorporation is poor. The conversion efficiency was poorer than that of sexual substrates.

When converting an organic substrate, when the substrate is a solid at room temperature and atmospheric pressure and is water-insoluble or almost water-insoluble, a method of dissolving the substrate in an organic solvent and adding it has been attempted.

Further, it has been attempted to improve the conversion rate of an oily substrate at room temperature and atmospheric pressure by further dissolving it in an organic solvent to increase the contact area with microorganisms. Further, it has also been attempted to add a surfactant or the like to the reaction solution to form an emaldiene to improve the conversion rate.

However, the cell membrane and cell wall of microorganisms are made of phospholipids, proteins, etc., and the cell membrane and cell wall change due to organic solvents, and enzymes and coenzymes are often denatured by organic solvents, and conversion activity disappears or decreases. To do Further, when a surfactant is used, the conversion activity is reduced, or it becomes difficult to take out the product due to emulsification.

Under the circumstances, the present inventors have conducted research to establish a more advantageous method for reducing water-insoluble or almost water-insoluble ketones and aldehydes, and as a result, It has been found that a higher conversion rate can be obtained by adding triglyceride, and various studies have been conducted on this to complete the present invention.

The triglyceride added in the method of the present invention may be either a natural product or a synthetic product as long as it is a liquid at normal temperature and pressure, and is not particularly limited.

A vegetable oil containing triglyceride extracted and separated from nature as a main component is preferable because it is easily available. Specific examples of such a vegetable oil containing triglyceride as a main component include soybean oil, olive oil, rice bran oil, rapeseed oil, castor oil, safflower oil, coconut oil, corn oil, cottonseed oil,
Examples include peanut oil and sesame oil. Of the above triglycerides, soybean oil and olive oil are more preferable.

As a method of addition, sterilized triglyceride and the substrate may be added separately when the substrate is added, or sterilized triglyceride and the substrate may be mixed and added.

Further, the substrate may be mixed and added to a mixed solvent of sterilized triglyceride and a small amount of an organic solvent such as acetone.

As the addition conditions, the substrate is 0.1 g to 5.0 per 100 ml of the culture solution.
g, triglyceride 0.5 to 30 g is preferable. It is preferable to increase the amount of triglyceride by increasing the base mass.

The substrate of the present invention has the formula [In the formula, R ₁ and R ₂ are each a hydrogen atom or a carbon atom 1
To 15 are alkyl, alkoxy, allyl or allyloxy groups. ] Water-insoluble or almost water-insoluble ketones or aldehydes represented by

The microorganisms used in the present invention are not particularly limited, but include Rhodotorula genus, Schizosaccharomyces genus, Chluyveromyces genus (Kluyveromyces) genus, Hansenula genus, Vicia (Pichia) genus, Torulopsis genus, Sporobolomyces genus, Arthrobacter genus, Saccharomyces
ces), Rhodococcus genus, Pseudomonas genus, Micrococcu
s) genus, Bacillus genus, Brevibacterium genus, Aspergillu
s) genus, Mucor genus, Kloecker
a) Genus, Endomycopsis, Candida, Rhodosp
genus oridium, Saccharomycopsi
s) genus, Lipomyces genus, Rhiz
genus opus, genus Penicillium, genus Fusarium, genus Gibberella, genus Gliocladium, genus Aureobasidium, bisclamis
Genus, Phytophtora, Cylindrocarpon, Schvanniomyces
hwanniomyces, Debaryomayce
s) genus, Cryptococcus genus, Torula genus, Citromyces genus, Aerobacter genus, Serratia
Genus, Achromobacter genus, Flavobacterium genus, Agrobacterium genus, Corynebacterium
terium), Staphylococcus
Genus, Acinetobacter genus, Xanthomonas genus, Mycobacterium (Mycob
Acterium), Streptomyces
Genus, Endomyces, Trametes
genus metes, genus Pycnoporus, genus Gloeophyllum, Metschinik
Microorganisms belonging to the genus owia, the genus Alcaligenes and the genus Escherichia are preferred.

Representative strain names belonging to each of these genera are exemplified, but the microorganism of the present invention is not limited to these examples.

(1) Rhodotorula rubraIFO-0901 (2) Diso Saccharomyces pombe Shizosaccharomyc
es pombe IFO-0346 (3) Kluyveromyces lactis
lactis IFO-1090 (4) Hansenula anomala IFO-0707 (5) Pichia farinosa IFO-0193 (6) Torulopsis candida IFO-0880 (7) Sporoboromyces salmonicolor Sporobolom
yces salmonicolor IFO-0374 (8) Arthrobacter simplex
simplex IFO-12069 (9) Saccharomyces cerevisiae (Baker's yeast) IFO-2044 (10) Rhodococcus eryt
hropolis IFO-12320 (11) Pseudomonas putida IFO-12996 (12) Micrococcus luteus IFO-8066 (13) Bacillus licheniformis Bacillus licheniform
is IFO-12197 (14) Brevibaca ammoniagenes Brevibac
terium ammoniagenes IFO-12072 (15) Aspergillus niger ATCC-9642 (16) Mucor ramannianus ATCC-9628 (17) Kloeckera corticis IFO-0868 (18) Endomycopsis fibrigella Endomycopsis
fibuligera IFO-1665 (19) Candida lipolytica
NRRL-Y-6795 (20) Rhodospo Rhodospo
ridium diobovatum IFO-0688 (21) Saccharomycopsis lipolytica Saccharomy
copsis lipolytica ATCC-34088 (22) Lipomyces starkeyi Lipomyces starkeyi IFO-0678 (23) Rhizopus oryzae IFO-5440 (24) Penicillium chrysogenum Penicillium chryso
genum IFO-4626 (25) Fusarium culmorum IFO-5902 (26) Gibberella fujikuroi IFO-6356 (27) Gliocladium deliquescence Glocladium del
iquescens IFO−6790 (28) Aureobasidiu
m pullulans IFO-4465 (29) Byssochlamys fulva IFO−7901 (30) Phytophthora i
nfestans IFO-9173 (31) Cylindro Cylindro
carpon destructans IFO-5998 (32) Schwanniomyces oxydentalis Schwan
niomyces occidentalis IFO-0371 (33) Debaryomyces caste
llii IFO-1359 (34) Cryptcoccu neoformance Cryptcoccu
s neoformans ATCC-11239 (35) Torula herbarum IFO-9500 (36) Citeromyces ma
tritensis IFO-0954 (37) Aerobacter cloacae IAM-1221 (38) Serratia marcescens IFO-3046 (39) Achromobacter species Achromobacter
sp. ATCC-21910 (40) Flavobac
terium arborescens IFO-3750 (41) Agrobactus tumefaciens
erium tumefaciens IFO-13264 (42) Corynebacter
ium glutamicum ATCC-13287 (43) Staphylococcus Staphylococcus
aureus IFO-3060 (44) Acinetob, Acinetobacter calcoaceticus
acter calcoaceticus IFO-13006 (45) Xanthomonas
compestris IFO-13551 (46) Mycobacteria smegmatis Mycobacter
ium smegmatis IFO-3082 (47) Streptomyces griseus subsp.griseus IFO-3355 (48) Endomyces ovetensis
sis IFO-1201 (49) Trametes orientali
s IFO−6483 (50) Pycnoporus coccineus IFO−9768 (51) Gleophyllum trabeum
um IFO-6430 (52) Metschnikowia pulche
rrima IFO-0863 (53) Alcaligenes faec
alis IFO-13111 (54) Escherichia coli IFO-3302 All of these strains are American Type Culture Collec
tion (ATCC), Fermentation Research Institute (IFO) in Osaka,
Institute for Applied Microbiology (IAM) or Agricultura
● It is stored in the Research Service Culture Collection (NRRL) and can be obtained from these storages.

Of the above genera, Rhodotorula, Shizosaccharomyces, Kluyveromces, and Hansenul
a) Genus, Torulopsis genus, Sporobolomyces genus, Sacchamyces
More preferred are microorganisms belonging to the genus romyces, the genus Rhodococcus, and the genus Saccharomycopsis.

In the method of the present invention, the culturing of the microorganism used is usually accomplished by liquid culture according to a conventional method. Moreover, you may perform solid culture as needed.

The culture conditions of the bacterium vary slightly depending on the strain, but generally speaking, glucose, fructose, sucrose, maltose, sugars such as soluble starch as a carbon source, yeast extract, peptone, ammonium sulfate, ammonium chloride, NZ amine as a nitrogen source (Type A), potassium nitrate, urea, amino acid, casamino acid, corn steep liquor,
Inorganic salts such as bran, polypeptone, rice bran, magnesium sulfate, sodium chloride, calcium carbonate, potassium monohydrogen phosphate, potassium dihydrogen phosphate, manganese sulfate, zinc sulfate, copper sulfate, ferrous sulfate, sodium molybdate, etc., As other nutrients, a medium containing malt extract, meat extract, pharma media, etc. is used,
It is not limited to this. This medium is inoculated with the strain and cultured aerobically or anaerobically. Culture temperature is 10-70
C. is suitable, 40 to 65.degree. C. is preferable for culturing thermophilic bacteria, and 20 to 50.degree. C. is preferable for culturing mesophilic bacteria.

Usually, the reaction is carried out by appropriately growing the bacterium by culturing for 12 to 72 hours and then adding the substrate, but the substrate may be added from the beginning or may be added in several times. Or 12 fungi
Alternatively, the cells may be grown by culturing for 96 hours to 96 hours, then removed by centrifugation or filtration, the obtained cells are added to the newly prepared reaction solution, and a substrate is further added. An example of such a reaction system is a system consisting of 2.0 mol or less of a phosphate buffer or / and 20% or less of a sugar such as glucose / sucrose,
Water alone may be used, or a small amount of malt extract, magnesium sulfate, etc. may be added. Also, immobilized cells can be used.

When adding the substrate, sterilized triglyceride is added at the same time, the substrate is mixed with sterilized triglyceride, or the substrate is mixed with sterilized triglyceride and an organic solvent such as acetone. .

As the conditions for carrying out the reaction, a reaction temperature of 10 to 70 ° C. is suitable, 40 to 65 ° C. when thermophilic bacteria are used, and 20 to 50 ° C. when mesophilic bacteria are used. Reaction time is usually 12 to 170
It's time. The pH of the culture medium during the reaction is slightly different depending on the cells, and is preferably pH 7 to 11 for alkalophilic bacteria and pH 5 to 9 for non-alkalophilic bacteria.

Next, after carrying out the asymmetric reduction reaction in this manner, the product is recovered. In this recovery, solvent extraction, recrystallization, column chromatography, etc. can be appropriately adopted.

For example, if the product is crystalline, the reaction solution is ether,
The product can be obtained by extracting the product with an organic solvent such as ethyl acetate, toluene, n-hexane and the like, concentrating the organic layer and then cooling the product to crystallize it. When the product is oily, the product can be obtained by similarly extracting and concentrating and then fractionally distilling.

As described in detail above, according to the method of the present invention, the product can be obtained at a higher conversion rate than in the case of converting only the substrate into the reaction solution to convert the microorganism, which is extremely advantageous industrially.

Next, the present invention will be described in more detail with reference to Examples.
The present invention is not limited to this.

Example 1 A test tube having a diameter of 25 mm was placed in a liquid medium [water 1 to glucose 10.0;
g, yeast extract 3.0 g, peptone 5.0 g, malt extract 1.0 g was dissolved and adjusted to pH 6.0] 10 ml and then sterilized,
Tolulopsis candida IFO-0380 was inoculated with 1 platinum loop from a slant medium and aerobically shake-cultured at 28 ° C for 24.0 hours. Then, in a 500 ml Erlenmeyer flask, liquid medium [water 1 to glucose 40.0 g, polypeptone 10.0 g, yeast extract 5.0
g, magnesium sulfate 3.0 g, potassium dihydrogen phosphate 5.0
g, manganese sulfate 5.0 mg, zinc sulfate 20.0 mg, copper sulfate 2.5 m
g, ferrous sulfate 20.0 mg, sodium molybdate 25 μg
1 ml of the above culture solution was inoculated into a sterilized medium containing 100 ml, and after culturing for 24.0 hours, 0.5 g of phenoxy-2-propanone as a substrate was added to 2 ml of acetone.
Phenoxy-2-propanone dissolved in and added to
0.5 g of soybean oil 1 g, 3 g, 5 g, 10 g of clarified bacteria, dissolved in a mixed solvent of soybean oil and acetone, each containing 2 ml of acetone, and 4.0 g of glucose dissolved in 10 ml of water and sterilized Was added. After aerobically shaking at 30 ° C. for 96.0 hours, the reaction was extracted with diethyl ether. The extract was subjected to gas chromatography (column: SiliconDC-QF-1 2.1
Table 1 shows the results of analysis using a m, glass column (column temperature: 120 ° C).

From these tables, it can be seen that soybean oil is effective in improving the conversion rate.

Example 2 Cultivation, addition of a substrate, reaction, and extraction were performed in the same manner as in Example 1 except that Saccharomycopsis lipolytica ATCC-34088 was used. Further, the extract was analyzed in the same manner as in Example 1 to measure the conversion rate.

The results are shown in Table 2.

Example 3 Rhodotorula rubra IFO-0901 was used, the same culture and addition of substrate as in Example 1 except that preculture was carried out for 48 hours.
The reaction was carried out and the reaction product was extracted with diethyl ether. The extract was analyzed in the same manner as in Example 1 to measure the conversion rate. The results are shown in Table 3.

Example 4 After culturing, adding a substrate and carrying out a reaction in the same manner as in Example 1 except that 1- (4-phenoxy) propan-2-one was used as a substrate, the reaction product was extracted with toluene.
The extract was subjected to gas chromatography (column: SiliconDC-
QF-1 2.1 m, glass column, column temperature: 200 ° C) was used for analysis.

The results are shown in Table 4.

Examples 5 to 12 and Reference Examples 1 to 9 In the same manner as in Example 1 except that the amounts of phenoxy-2-propanone as a cultured microorganism and a substrate, the amount of soybean oil, and the amount of acetone were as shown in Table 5. Culture, addition of substrate, and reaction were performed. Then the reaction product was extracted with diethyl ether,
The extract was analyzed in the same manner as in Example 1 to measure the conversion rate. For comparison, the same reaction and analysis were performed when soybean oil was not added. (Reference Examples 1-9) Examples 13 to 17 and Reference Examples 10 to 12 Using Mizosaccharomyces pombe IFO-0346, the preculture time was set to 48 hours, and 1- (4-phenoxyphenoxy) propan-2-one was used as a substrate. Incubation and reaction were carried out in the same manner as in Example 1 except that the amounts of the substrate, the amount of fats and oils, and the amount of acetone were set as shown in Table 6. Thereafter, the reaction product was extracted with toluene, and the extract was analyzed in the same manner as in Example 4 to measure the conversion rate. For comparison, the same reaction and analysis were performed in the case where oils and fats were not added. (Reference Examples 10 to 12) The results are shown in Table 6.

Examples 18 to 21 and Reference Examples 13 to 17 Cultivation, addition of substrate, reaction as in Example 1 except that acetophenone was used as a substrate and the amounts of cultured microorganisms and soybean oil and the amount of acetone were as shown in Table 7. Was done. Thereafter, the reaction product was extracted with toluene, and similarly analyzed by gas chromatography to measure the conversion rate. For comparison, the same analysis was performed in the case where soybean oil was not added (Reference Examples 13 to 17). Examples 22 to 23 Reference Examples 18 to 20 Using 3-nonanone as a substrate, the amount of cultured microorganisms and soybean oil, and the amount of acetone were the same as in Example 1 except that the amounts were the same as in Example 1, culture, addition of substrate, The reaction was carried out. Thereafter, the reaction product was extracted with diethyl ether, and similarly analyzed by gas chromatography to measure the conversion rate. For comparison, the same analysis was performed without adding soybean oil. (Reference examples 18 to 20)

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hiroshi Kishida 4-2-1 Takashi Takarazuka-shi, Hyogo Prefecture Sumitomo Kagaku Kogyo Co., Ltd. (72) Inventor Hideo Hirohara 4-2-1 Takashi Takarazuka-shi, Hyogo Prefecture Sumitomo Chemical Co., Ltd.

Claims (4)

[Claims] 1. When a water-insoluble or almost water-insoluble ketone or aldehyde represented by the following formula [I] is reduced by using a microorganism, a vegetable oil containing triglyceride as a main component is added to a culture solution. Characteristic method for microbial reduction of water-insoluble or almost water-insoluble ketone or aldehyde In the formula, R ₁ and R ₂ are each a hydrogen atom or a carbon atom of 1 to
15 represents an alkyl group, an alkoxy group, an allyl group or an allyloxy group. 2. A vegetable oil containing triglyceride as a main component is soybean oil, olive oil, rice bran oil, rapeseed oil, castor oil, sawarawa oil, coconut oil, corn oil, cottonseed oil, peanut oil or sesame oil. The method described. 3. The microorganism is a genus Rhodotorula,
Genus Shizosaccharomyces, Kluyveromyces, Hansenula
genus senula, genus Pichia, Torupsis
lopsis), Sporobolomyces
Genus, Arthrobacter genus, Saccharomyces genus, Rhodococc
us) genus, Pseudomonas genus, Micrococcus genus, Bacillus genus,
Brevibacterium genus, Aspergillus genus, Mucor genus, Kloeckera genus, Endomycosis
genus opsis, genus Candida, genus Rhodosporidium, genus Saccharomycopsis, lipomyces
Genus, Rhizopus, Penicillium
genus, Fusarium genus, Gibberella
rella) genus, Gliocladium genus, Aureobasidium genus, Byssochlamys genus, Phytophthora
Genus, Cylindrocarpon genus, Schwanniomyces genus, Debaryomyces genus, Cryptococcs
us), Torula, Citromyces
yces), aerobacter (Aerobacter), serratia (Serratia), achromobacter (Achromobacter)
Genus, Flavobacteriu genus, Agrobacterium genus, Corynebacterium genus, Staphylococcus (Staph)
ylococcus), Acinetobacter
Genus, Xanthomonas genus, Mycobacterium genus, Streptomyces (Str
eptomyces), Endomyces, Trametes, Pycnoporus
Genus, genus Gloeophyllum, genus Metschinikowia, Alcaligene
The method according to claim 1, which is a microorganism belonging to the genus s) or the genus Escherichia. 4. The microorganism is a genus Rhodotorula,
Genus Shizosaccharomyces, Kluyveromyces, Hansenula
A microorganism belonging to the genus senula, the genus Torulopsis, the genus Sporobolomyces, the genus Saccharomyces, the genus Rhodoccus, or the genus Saccharomycopsis. The method according to item 2 or item 2.