JPS6317695A - Alcohol manufacturing method - Google Patents

Abstract

PURPOSE:To efficiently produce an alcohol corresponding to an aldehyde without requiring additional of a substance to be an energy source, by reacting a microorganism belonging to the genus Pseudomonas with the aldehyde. CONSTITUTION:A microorganism, e.g. Pseudomonas aeruginosa T-57814 strain (FERM-P No.8781) or Pseudomonas putida T-581020 strain (FERM-P No.8782), belonging to the genus Pseudomonas and having the ability to convert an aldehyde into an alcohol is grown in a nutrient culture medium containing one or more of an inorganic or organic ammonium compound and meat extract, peptone, etc., as a nitrogen source and, as necessary, an inorganic base, etc. In a suitable period before starting the cultivation or during the cultivation, an aldehyde expressed by the formula R.CHO (R is 2-16C n-alkyl, phenyl or phenyl-substituted alkenyl) is added and reacted at preferably 6-9 pH and 20-45 deg.C to produce the aimed alcohol.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing alcohols, and more particularly to a method for producing alcohols from aldehydes using specific microorganisms.

[Prior art 1 Problems to be solved by the invention] Conventionally,
A method for producing alcohols by allowing microorganisms to act on specific raw materials is to make the corresponding alcohols by allowing microorganisms belonging to the genus Pseudomonas to act on alkanes such as propane or aromatic hydrocarbons such as benzene in the presence of an energy source. It is known to produce similar products (Japanese Unexamined Patent Publication No. 5
9-48088).

However, using aldehydes as raw materials,
Moreover, there is still no known method for producing the corresponding alcohols without using an energy source.

[Means for solving problems]

The present inventors conducted repeated studies to develop a method for producing alcohols by allowing microorganisms to act on aldehydes without using energy sources such as glucose or sucrose, and found that The inventors have discovered that the objective can be achieved by using microorganisms, and have completed the present invention.

That is, the present invention relates to a method for producing alcohols, which is characterized by allowing a microorganism belonging to the genus Cuydomonas and having the ability to convert aldehydes into alcohols to act on aldehydes.

Microorganisms that can be used in the present invention may be microorganisms as long as they have the above-mentioned abilities, but specifically, Pseudomonas aeru 1no.
sa) strain T-57814 and Pseudomonas putida strain a) strain T-581020. All of these microorganisms were isolated by the present inventors from soil in Kimitsu District, Chiba Prefecture, using the method described below.

S shown in the table below! Dissolve the medium components in distilled water 11 and adjust the pH to 7.
, 0, was dispensed into a 5001111 capacity shake flask and sterilized under pressure at 120°C for 15 minutes. To this medium, 1 Ill of 1 g of soil suspended in 10 ml of sterilized water is added, and the culture is carried out aerobically at 30° C. for 7 days.

Using the obtained culture solution, one platinum loop was streaked on a plate medium prepared by adding 20 g of agar to the above SI medium, and the mixture was incubated at 30°C.
Culture for 7 days. The resulting colony was isolated to obtain the above microorganism.

sr medium NaxHPOa・12HzO1,5g KHzPO,0,5g M gS Oa・IHtO0,2g Fe50.・7Hz0 0.005g yeast extract 3gNH4N0h
3 g phenethyl alcohol
5 yal Pseudomonas aeruginosa T
-57814 strain and Pseudomonas putida T-58
The mycological properties of strain 1020 are as shown below.

T-57814T-581020 a) Morphological properties (1) Shape Bacillus Bacillus (2) Large scale (μ) 0.7-1. IXl
, 1-2.3 0.7-1.0X1.2-1.5(3)
Motility Yes Flagella Polar Flagella Polar Flagella (4) Sporulation None None 5) Durham staining Negative Negative (6) Growth under aerobic conditions Good Good b) Physiological properties (1) Growth Range temperature (℃) 8~48 6~40pH3
,91-9.79 3.91-9.79 (2) Production of pigment Water-soluble green-yellow Water-soluble V yellow King B medium) Pigment production Pigment production (3) Production of fluorescent pigment 10-(4) Biol cyanin Generation +
1 (King A, B medium) (5) Force ■ Generation of tinoids --(6
) Growth at 41℃ + - (7)
From sucrose -- levain
Production (8) Fulginine Degradability +
+(9) Liquefaction of Seratin +
- (10) Hydrolysis of cypress - (11) Reduction of nitrate + - [(12) Catalase + + (13) Cytoctom oxidase +
+(14)0-F test
Oxidative Oxidative (15) Carbon source availability Glucose + +Trehalose + +Meso-inodite
-- Geraniol + -β-alanine + +DL-arginine
+ +L-valine +
+(16) Other xylose decomposition + +Mannitol decomposition + +Maltose decomposition
-- Acylamidase 10 - DN-Ace production 10 - Utilization of citric acid
+ A method for classifying and identifying bacteria based on more than ten mycological properties Takeharu Haseyo, "Classification and Identification of Microorganisms", Gakkai Publishing Center, 1985, and Manual of Determinant Bacteriology (B.
ergey'3 Manual of Determinition
Inative Bacteriology), 8th edition, 1975]. As a result, both strains were negative for Durham staining, polar flagellated, and aerobic 11i.

Catalase (+), cytochrome oxidase (+).

Since it had a 0-F test (0), it was classified as a strain belonging to the genus Pseudomonas.

Next, the T-57814 strain utilized trehalose (medium Nidrevalose 5g, NaC 15g, Mg5Oa・7Ht
OO, 2 g, (NH4)HzPo41 g.

41g of K2HPO, 12mJ of bromothymol blue 0.2% aqueous solution, and 100,100O of distilled water are different from Pseudomonas aeruginosa described in Virgiei's manual, but since other properties are the same, the native country is Pseudomonas aeruginosa. Identified. In addition, the T-581020 strain produces fluorescent pigment (medium: King A and King B) and utilizes trehalose (medium:
Although it is different from the Pseudomonas putida described in Bargiei's manual in (same as above), the original species was identified as Pseudomonas putida because other properties were lost. Pseudomonas aeruginosa T-578
14 strains and Pseudomonas putida T-581020
The strains have been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERM P-8781 and FERM P-8782, respectively.

Microorganisms can be used in various forms, such as cells in the growth phase, cells in the resting phase, and immobilized cells (and even extraction treatment from the microorganisms). The microorganisms may be immobilized using a carrier binding method,
This can be carried out by applying conventional immobilization techniques such as cross-linking and entrapping methods. Furthermore, as the extraction process, a method can be employed in which a suspension of microbial cells is crushed using ultrasound, a French press, a high-pressure homogenizer, etc., and then a soluble extract is obtained by centrifugation or the like.

Next, the raw material aldehydes have the formula R-CHO (where R
represents an n-alkyl group, phenyl group, phenyl-substituted alkyl group, or phenyl-substituted alkenyl group having 2 to 16 carbon atoms. Specific examples of aldehydes include n-propionaldehyde, n-butyraldehyde, n-hexylaldehyde, n-octylaldehyde, n-dodecylaldehyde, palmitaldehyde, and benzaldehyde.

Examples include phenylacetaldehyde, cinnamaldehyde, and β-phenylpropionaldehyde.

When producing the desired alcohol by reacting the above-mentioned microorganisms with the above-mentioned aldehydes, if the reaction is carried out using living microorganisms, the microorganisms are inoculated into a microorganism that does not grow or multiply, and the raw material aldehyde is When using dormant cells, collect and wash the cells and suspend them in an appropriate buffer solution. The raw materials may be added to the suspension and reacted. Furthermore, by using immobilized bacterial cells, the desired alcohol can be efficiently produced through continuous reactions.

When using live bacterial cells as microorganisms, the medium may be either a natural medium or a synthetic medium, and usually contains one or more of inorganic or organic ammonium compounds, meat extract, peptone, etc. as a nitrogen source, and as necessary. Depending on the situation, one containing inorganic salts such as potassium phosphate, iron sulfate, manganese sulfate, and other nutritional substances necessary for the growth of microorganisms is used. In addition, when using a bacterial cell extract, N
It is preferable to add an electron donor such as ADH or PQQH.

Aldehydes as raw materials may be added before the start of the reaction, or may be added at an appropriate time after the start of culture, such as when using living cells. The raw materials may be added all at once, or may be divided into several portions or added sequentially.

The reaction according to the present invention may be carried out under either aerobic or anaerobic conditions, at a temperature of 5 to 55°C, preferably 20 to 45°C, and at a pH of 5 to 55°C, taking into consideration the properties of the microorganisms used.
It is sufficient to wait for a predetermined time in the range of 4 to 11, preferably 6 to 9.

The alcohols obtained according to the present invention are n-alkyl alcohol, benzyl alcohol, and phenyl-substituted alkyl alcohol, depending on the aldehyde used as the raw material.

After completion of one reaction, such as phenyl-substituted alkenyl alcohol, the resulting alcohol may be recovered and purified by conventional methods.

〔Effect of the invention〕

According to the present invention, corresponding alcohols can be efficiently produced from aldehydes using microorganisms belonging to the genus Pseudomonas. When growing bacterial cells are used as microorganisms, there is no need to add substances that serve as an energy source, such as glucose or shakerose, to the reaction system, and the reaction can be carried out under either aerobic or anaerobic conditions.

〔Example〕

Next, the present invention will be explained in detail with reference to examples.

Example 1 Pseudomonas aeruginosa strain T-57814 (FE
RMP-8781) was cultured on a peptone and meat extract agar slant medium at 30°C for 24 hours. 50m7 medium with the composition shown in Table 1! After the mixture was dispensed into a 500 ml Sakaro flask and autoclaved at 120°C for 15 minutes, one platinum loop of the seed culture was inoculated thereto and cultured with shaking at 30°C. 12 hours after the start of culture, cinnamic alcohol was added to 0.
It was added at a concentration of 1% and cultured for 24 hours.

Collect the culture solution from 20 flasks to make 11, and add this to 5.
Cells obtained by centrifugation for 10 minutes at 11.0 OO It got cloudy.

Table 1 Meat extract 5g Peptone 15g NaC 15g Kz HP 045g Distilled water 11 (adjusted to pH 7 using IN HCl, IN NaOH) 1st L-Table Nag HPO47.1g KH2PO45.25g 7 Distilled water 1l (IN HCl, IN Adjust to p) 17 using NaOH) Add 50ml of the above bacterial suspension to 500++j! The mixture was placed in a Yosaka Lough flask, kept under nitrogen gas under anaerobic conditions, and 300 μm of cinnamaldehyde was sequentially added at a rate of 30 μm every 30 minutes, followed by a shaking reaction at 30° C. for 6 hours. After the reaction solution was sterilized, it was analyzed by gas chromatography, and it was confirmed that 120 mg of cinnamic alcohol had been produced.

Examples 2 to 5 The same procedures as in Example 1 were carried out except that the reaction raw materials and the amounts added were changed to those shown in Table 3. The results are shown in Table 3.

Meji L-Table 2 Benzfludehy F (300)
Benzyl alcohol (85)3
Phenyl 7 Setofludehyde F (200) Phenethyl alcohol (60) 4 β-phenylpropiosi β-phenylaldehyde (200)
Property tools (62)5
n-Butyraldahi F (200) Butanol (58) Examples 6 to 10 Pseudomonas aeruginosa T-5 in Example 1
Pseudomonas putida T-58 instead of 781'4 strain
The test was carried out in the same manner except that 1020 strain (FERM P-8782) was used and the raw materials and amounts added were changed to those shown in Table 4. The results are shown in Table 4.

6 Cinnamon fulldehyde F (300)
Cinnamon alcohol (115) 7 Pensaldehyde (200) Benzyl alcohol
1 (58) 8 Phenylacetaldehyde (200) Phenethyl alcohol (57)
(Continued) Practicing Doctor: Haratsukiji (Vli Engineering) Makoto JL Garden U 糺i Ball)
9 β-phenylpropion
β-phenyl 71L Techt (200)
BuC Panol (48)10
n-propylflutehyde (300) n-
Bupanol (92) Example 11 Culture fluids from 160 flasks obtained by repeating the same method as in Example 1 were collected, and bacterial cells were obtained in the same manner as in Example 1. This bacterial body was divided into 8 equal parts, and various buffers (pH
4: 0. IM citrate buffer, pits, 6+7
, 8゜ζ Mountain 9: 0.1M phosphate buffer, pH 10, 11M CAP
After washing with S buffer), 6 bacterial cells were added to the same buffer.
It was suspended so that it might be g/l.

50mf of each bacterial cell suspension was placed in a 5001111 volume Sakaro flask, 150μ of cinnamaldehyde was added thereto, the mixture was made anaerobic with nitrogen, and a shaking reaction was carried out at 30°C for 2 hours. The results are shown in Table 5.

Star-1-head reaction p)! 4 5 6 7 8 9
10 11 Cinnamon alcohol (■) 20 28 32 4
2 58 46 3 + Example 12 A bacterial suspension (pH 7) obtained by repeating the same method as Example 1
) 50 n+j! Add cinnamaldehyde to the flask containing 5
0 ■ was added, and the shaking reaction was carried out aerobically for 10 minutes.

After the reaction solution was sterilized, it was analyzed by gas chromatography, and it was confirmed that 1.2+ng of cinnamic alcohol had been produced.

Claims (3)

[Claims] (1) A method for producing alcohols, which comprises allowing a microorganism belonging to the genus Pseudomonas and having the ability to convert aldehydes into alcohols to act on aldehydes. (2) A patent claim in which the aldehyde is represented by the formula R.CHO (where R represents an n-alkyl group having 2 to 16 carbon atoms, a phenyl group, a phenyl-substituted alkyl group, or a phenyl-substituted alkenyl group) The method described in item 1. (3) The method according to claim 1, wherein the microorganism is any one of cells in a growth phase, cells in a resting phase, immobilized cells, and a processed cell extract.