JPH0690A - Method for oxidizing alcohols with immobilized microorganism - Google Patents

Abstract

PURPOSE:To obtain a method for biological oxidation of alcohol, having high reaction rate and yield by using immobilized multiplying microbial cells having catalytic ability of oxidation reaction of' an alcohol. CONSTITUTION:A microorganism capable of oxidizing a primary hydroxyl group and/or secondary hydroxyl group is stuck to a hydrophilic immobilizing carrier and immobilized. A substantially water-insoluble or slightly water soluble organic solvent containing an alcohol is brought into contact with an immobilized microbial cell phase on the carrier in the presence of an aqueous medium containing a nutrient source of the microorganism to oxidize the alcohol.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for oxidizing alcohols using immobilized microorganisms.

[0002]

2. Description of the Related Art Conventionally, industrially important carboxylic acids, aldehydes and ketones have been produced in large quantities by a chemical oxidation method of alcohols. In these methods, heavy metal catalysts such as potassium dichromate and potassium permanganate, hydrogen peroxide, peroxides such as organic peroxides, acids such as nitric acid, and platinum-based catalysts have been used. However, these chemical oxidation methods have many problems such as excessive progress of oxidation reaction, regioselectivity, lack of stereoselectivity, and difficulty in controlling reaction, and it is also difficult to control the progress of side reactions.

Further, in these chemical oxidation methods, problems of environmental pollution due to the use of heavy metal catalysts and acid catalysts, safety problems due to the use of peroxide catalysts, and catalyst cost problems due to the use of platinum-based catalysts. There are many problems such as.

On the other hand, in order to overcome the drawbacks of such a chemical oxidation method, a method for oxidizing alcohols using a biocatalyst having advantages such as energy saving, high selectivity and safety has been widely tried. [Hiromichi Ota, Integrated, 41
Volume, 1018 (1983); Hiromichi Ota, Yuka, 46
Vol. 726 (1989)].

However, the method for oxidizing alcohols using an oxidase is inevitably high in cost due to problems such as expensive catalysts and coenzyme requirement. Further, in the case of the method of oxidizing alcohols using a microorganism, the death of the microorganism or the reduction of the enzyme activity due to the toxicity of alcohols and oxidation products such as carboxylic acids and ketones cannot be avoided, and it has been industrialized until now. Examples have been limited to the production of very few expensive materials, such as the production of difficult-to-manufacture optically active forms and the production of regioisomers.

Further, in the oxidation of alcohols using microorganisms, when oxidizing water-insoluble or sparingly soluble alcohols, a large stirring force and aeration are indispensable in order to forcibly disperse the alcohols in water, and the production cost is high. There are problems that it is expensive and that the reaction rate is low because the raw material alcohols are in a suspended state.

Therefore, there is a strong demand for the development of a novel method for biological oxidation of alcohols which overcomes the above-mentioned drawbacks of the chemical oxidation method and which complements the defects of the existing biological oxidation method.

[0008]

Therefore, in the method of oxidizing alcohols using a microorganism, the present inventor avoids the problem of toxicity of the substrate alcohols and oxidation products such as carboxylic acids and ketones to microorganisms, As a result of diligent studies on a method for microbial oxidation of alcohols which does not require troublesome operations such as forced dispersion in water and aeration, the microorganism was inoculated on a hydrophilic immobilization carrier containing a nutrient source and water, and When contacted with an organic solvent that does not develop toxicity, microbial cells vigorously grow at the interface between the organic solvent and the immobilized carrier to form a microbial cell phase on the carrier, and this microbial cell phase exists in the organic solvent. The inventors have found that the added alcohols are oxidized at a high reaction rate and a high yield, and have completed the present invention.

Thus, according to the present invention, a microorganism having the ability to oxidize primary and / or secondary hydroxyl groups is adhered and immobilized on a hydrophilic immobilization carrier, and the immobilization is carried out in the presence of an aqueous medium containing a nutrient source for the microorganism. A method for oxidizing alcohols, which comprises contacting a substantially water-insoluble or sparingly water-soluble organic solvent containing alcohols with the immobilized bacterial cell phase on the carrier.

One of the features of the present invention is that the microorganism is grown at the interface between an organic solvent represented by an aliphatic hydrocarbon and a hydrophilic immobilization carrier while supplying an aqueous medium containing a nutrient source for the microorganism. The fact is that the thus-obtained bacterial cell phase can be used as a catalyst. This makes it possible to cause the oxidation reaction by contacting the bacterial cell phase with alcohols dissolved in an organic solvent that is substantially insoluble or sparingly soluble in water, resulting in a high reaction rate and catalyst. The long-term stability of is achieved and advantages such as virtually no need for stirring power are obtained. Another feature of the present invention is that the substrate alcohols can be added to an organic solvent at a very high concentration, even though they are highly toxic to microorganisms. There is a point that can contact. For example, n-octanol,
With n-decanol, only 0.5 in emulsion system.
Microbes will be killed if they exist. However, according to the invention, for example Pseudomonas putida I
FO 13696 does not die even when it contains 12% of n-octanol or 24% of n-decanol in an organic solvent, and it proliferates to form a bacterial phase. Thus, according to the present invention, it is possible to bring a high concentration of alcohols into contact with the microbial cell phase, and as a result, a high reaction rate and high yield can be obtained, and alcohols can be oxidized at low cost. can do.

Another feature of the present invention is that an organic solvent such as an aliphatic hydrocarbon generally has several times to ten times more oxygen solubility than water, so that oxygen required for oxidation of alcohols is not required. This is because there is little aeration or agitation for supply, and in some cases it is possible to oxidize alcohols at a high reaction rate and a high yield without aeration and agitation.

The present invention will be described in more detail below.

The organic solvent or the organic solvent solution of the alcohol, which is the substrate, brought into contact with the immobilized bacterial cell phase may be hereinafter referred to as an organic liquid phase.

The immobilization carrier usable in the present invention is not particularly limited as long as it is hydrophilic, and it is supplied to a microorganism existing at the interface with an organic solvent by impregnating or contacting an aqueous solution containing a nutrient source. Any material can be used as long as it can be used. Specifically, for example, natural polymers such as alginic acid, carrageenan, starch matrix, agar, and cellulose materials such as filter paper; polyvinyl alcohol, Examples include synthetic polymers such as urethane polymers, polyacrylamide, and polyacrylic acid; inorganic materials such as foam glass and silica gel.

The shape of these immobilizing carriers is not particularly limited, and they can be molded into any shape such as fibrous, membranous or granular form, and can be in the form of cloth, non-woven fabric, paper, cardboard, etc. It may be molded into.

On the other hand, the organic solvent in the organic liquid phase in contact with the immobilized bacterial cell phase or the organic solvent for preparing the organic solvent solution of the alcohol of the substrate shows substantially no toxicity to the adherent microbial cells. Preferred are, specifically, normal paraffins or liquid paraffins represented by methane hydrocarbons having 6 to 20 carbon atoms such as hexane, heptane, octane, nonane, and decane; isoparaffins such as isooctane; pentylbenzene. , Hexylbenzene,
Examples include normal alkylbenzenes having an aliphatic chain of 5 to 15 carbon atoms such as heptylbenzene and octylbenzene; isoalkylbenzenes such as quinume; alicyclic hydrocarbons such as cyclohexane; ethers such as diethyl ether. You can

Microorganisms capable of oxidizing primary and / or secondary hydroxyl groups, which are used by adhering to the above-mentioned immobilized carrier and growing at the interface between the carrier and an organic liquid, are bacteria, fungi, yeasts, and actinic radiation. Any microorganism such as fungi may be used. Specifically, for example, Gluconoba (Gluconoba)
cter), Acetobacter
r) genus, Pseudomonas
Genus, Corynebacterium
um), Arythrobacter (Arthrobacter)
r) genus, Rhodococcus genus,
Nocardia genus, Candida (Ca
genus ndida), Hansenula
Examples thereof include microorganisms belonging to the genus, the genus Aspergillus, and the like. More specifically, Gluconobacter roseus (Gl. Roseus), Gluconobacter oxidans (Gl. Oxydans),
Acetobacter liquefaction (Ac.liqu
efacins), Pseudomonas putida (Ps.p)
utida), Corynebacterium equi (Co.e)
qui), Alice Lobacter simplex (Ar.
simplex), Rhodococcus rodney (Rh.r
hodnii), Rhodococcus equi (Rh.equ)
i), Nocardia Farcinica (No. farci)
Nica), Candida utilis (Ca.util)
is), Hansenula Anomala
a), Asbergis niger (As.niger) and the like.

The adhesion and immobilization of such microorganisms to the carrier is carried out, for example, by coating or spraying the microbial cell dispersion liquid on a carrier containing an aqueous medium containing a nutrient source in advance, or immersing the carrier in the microbial cell culture solution. Alternatively, after the microbial cells are attached to the carrier by an appropriate method, the microbial cells are attached to the carrier by a method such as supplying an aqueous medium containing the nutrient source to the carrier, and then the carrier is previously fed with the nutrient source. It is also possible to culture in an aqueous medium containing, but usually, in the state of being contacted with an organic solvent with or without alcohols as a substrate, the adhered microbial cells at the interface between the carrier and the organic solvent. And then grow
Suitably, it is carried out by forming an immobilized bacterial cell phase on the carrier. By this culturing, the microorganisms adhere strongly to the surface of the carrier, and the immobilized bacterial cell phase hardly separates from the carrier.

The nutrient source of the microorganism that can be used in the above culture can be selected in accordance with the type of bacterial cell used, and the optimal one can be selected. For example, carbon sources such as glucose and nitrogen such as urea. Sources, trace metal salts such as magnesium sulfate, and trace nutrients such as yeast extract may be used.

The aqueous medium containing the nutrient source, that is, the culture solution, is supplied to the immobilized cells in advance if the carrier can sufficiently contain and retain the culture solution, such as agar. Alternatively, for example, the culture solution may be added to the organic liquid phase, and the microorganism-immobilized carrier may be interposed at the interface between the organic liquid phase and the culture liquid phase to be formed.

Culturing can generally be carried out in a culturing device such as a thermostat or an incubator, or the carrier is immersed in an organic solvent containing or not containing a substrate, and optionally an aqueous medium containing a nutrient source is added. It may be carried out while controlling the temperature in the reaction vessel. The optimal culture conditions such as culture temperature and culture time can be selected according to the type of microorganism used.

Oxygen required for the growth or oxidation reaction may be aerated in an organic solvent, but in general, an organic solvent has an oxygen solubility of several times to several tens of times that of water, so that it is always aerated. No need. Also, regarding the stirring during the culture, since the alcohols that are the substrate exist in the organic solvent in a dissolved state, there is no need for vigorous stirring such as the emulsion method, and there is a case where the stirring is unnecessary. Many.

The alcohol as a substrate may be added from the initial stage of the above-mentioned culture, or may be added after the microorganism has sufficiently grown to form an immobilized cell phase. Alternatively, it may be added at any time from the initial stage of culture to the formation of the immobilized bacterial cell phase. As described above, alcohols often cause toxicity to microorganisms, and therefore, generally, higher results can be achieved by adding alcohols after the bacterial cell phase has sufficiently grown.

Thus, the oxidation reaction of alcohols can be carried out by continuing the culture of the immobilized bacterial cell phase on the carrier in contact with the organic liquid phase consisting of the organic solvent solution of alcohols as the substrate. it can.

The alcohols that can be used as substrates for the alcohol oxidation reaction by the immobilized microorganisms are not particularly limited, and various alcohols can be used depending on the alcohol oxidizing ability of the immobilized microorganisms.

As alcohols that can be used as a substrate,
As the primary alcohol, methanol, ethanol, n
-Propanol, n-butanol, isobutanol, n
Lower alcohols such as amyl alcohol, isoamyl alcohol, n-octanol and n-decanol; higher alcohols such as lauryl alcohol, myristyl alcohol, cetyl alcohol, stearyl alcohol and oleyl alcohol; ethylene glycol, 1,3-propanediol, 1, Examples thereof include aliphatic polyhydric alcohols such as 4-butanediol, 1,6-hexanediol, trimethylolpropane, and pentaerythritol; aromatic alcohols such as benzyl alcohol. Further, as secondary alcohols, isopropanol, 2-butanol, 2
Examples include aliphatic alcohols such as -pentanol, 2-octanol and 2,6-dimethyl-4-peptanol; aromatic alcohols such as benzhydrol and alicyclic alcohols such as cyclohexanol.

The concentration of alcohols in the organic solvent is not particularly limited and can be determined depending on the toxicity to the bacterial cells. In particular, when the alcohols to be subjected to the oxidation reaction are substantially water-insoluble or sparingly soluble, they do not migrate to the aqueous phase, that is, the hydrophilic immobilization carrier side, so that the toxicity to the cells can be greatly reduced. For example, in the case of n-octanol, Pseudomonas putida (Ps. Putida) and Pseudomonas frazi (Ps.
s. Fragium) and other Pseudomonas bacteria can grow even at a concentration of 12% in the organic liquid phase. In addition, A. duodecadis (Ar. duodeca
bacterium such as Bacillus subtilis, bacterium such as Bacillus subtilis, Acetobacter aceti (A)
c. aceti) and Gluconobacter oxidans (Gl. oxydans) can grow even at a concentration of 3% in the organic liquid phase. N- which is less toxic
In the case of decanol, many bacteria withstand concentrations above 24% and many yeasts above 6%.

On the other hand, even if the alcohols used for the oxidation reaction are water-soluble and exhibit strong toxicity to microorganisms in water, the toxicity of the alcohols may be alleviated by the method of the present invention. . For example, n-butanol has a solubility of about 7% in water and is toxic to microorganisms in water at a low concentration of only 0.2%. According to the method of the invention, a bacterium of the genus Bacillus, a genus of Arthrobacter, a genus of Corynebacterium, a genus of Microbacterium, or Saccharomyces.
When contacted with a yeast of the genus ces) or the like, it is possible to grow even when the butanol concentration is 3% or more.

According to the above-mentioned method of the present invention, the oxidation reaction of primary and / or secondary alcohols such as aliphatic, aromatic and alicyclic compounds is extremely efficient by using the microbial cells of the immobilized microorganism. You can In that case, if a side reaction may occur, block it by adding an appropriate metabolic or conversion inhibitor or use a breeding-improved metabolism-deficient strain so that such side reaction does not occur. You can

According to the method of the present invention, most of the oxygen required for the oxidation reaction is supplied from the organic liquid phase side, and the product alcohol oxides (aldehydes, ketones, carboxylic acids) are accumulated in the organic liquid phase. . Therefore, the frequency of contact between the immobilized bacterial cell phase and the substrate can be dramatically increased by recovering the product accumulated in the organic liquid phase and supplementing the alcohol of the substrate. The reaction rate, yield, and yield can be significantly improved, and continuous operation is also possible.

Thus, the method of the present invention is applied to various industrially important aldehydes in the fields of, for example, industrial chemicals, pharmaceuticals, cosmetics, fragrances, detergents, surfactants, fiber treatment agents, oils and fats, dyes, paint printing materials and the like. By applying it to the production process of ketones and carboxylic acids, various industrially advantageous advantages such as reduction of production cost, energy saving and labor saving of the process can be obtained.

[0032]

EXAMPLES The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples. Parts and% are based on weight.

Example 1 An agar plate consisting of 1.0% polypeptone, 0.2% yeast extract, 0.1% magnesium sulfate, and 1.5% agar was prepared in a glass petri dish (surface area: 38.5 cm ² ) and a table was prepared. 200 μl of the suspension of various microorganisms in 1 was smeared using a conradi stick. After drying, the cells were cultured at 30 ° C. for 2 days to form a bacterial cell phase, 10 ml of 10% 2-octanol in n-hexadecane was overlaid, and static culture was performed at 30 ° C. for 7 days. After culturing, 1 μl of the solvent phase was analyzed by gas chromatography, and the concentration of 2-octanone produced was measured. The results are shown in Table 1.

[0034]

[Table 1]

Example 2 100 ml of the same medium as in Example 1 was poured into a glass container having an inner volume of 480 ml which can be tightly closed to prepare an agar plate (surface area 43 cm ² ). 100 μl of each microbial suspension shown in Table 2 was smeared on this using a conradi stick, and the mixture was kept at 30 ° C. for 2 hours.
Cultured for a day. After that, 10 ml of a 10% solution of 2-octanol in ethyl caprylate was overlaid, and 120 r was added at 30 ° C.
The cells were cultured for 2 days under pm reciprocal shaking. After culturing, culture phase 1μ
1 was analyzed by gas chromatography, and the concentration of 2-octanone produced was measured. The results are shown in Table 2.

[0036]

[Table 2]

Example 3 An agar plate (surface area 43c, prepared in the same manner as in Example 2)
m ² ), 100 μl of a suspension of Rhodococcus equi IFO 3730 was smeared and cultured at 30 ° C. for 2 days. To this, 10 ml of a 2% solution of 2-methylcyclohexanol in n-hexadecane was overlaid and cultured for 4 days under reciprocal shaking at 100 rpm. After the culture, 1 μl of the culture phase was analyzed by gas chromatography to measure the concentration of produced 2-methylcyclohexanone. As a result, accumulation of 9.8 g / l of 2-methylcyclohexanone was confirmed.

Example 4 10 g of calcium alginate beads was added to 200 ml of a 1-day culture solution of Gluconobacter oxidans IFO 3189 and left standing for 5 minutes to attach microbial cells to the surface of the beads. Then pull up the beads from the culture,
After removing excess water, normal paraffin 150 ml
The mixture was placed in a glass container having a glass content of 500 ml containing and was cultured for 1 day to form an immobilized bacterial cell phase on the bead surface. Then, n-decanol was adjusted to 10%, and acrylic acid, which is a β-oxidation blocker, was added to 0.005%.
Add, aeration of 0.2 vvm, 5 under stirring 20 rpm
Cultured for one day. The beads were strongly aggregated and were not dispersed in the solvent phase. After the culturing, 1 μl of the culture phase was analyzed by gas chromatography to measure the concentration of produced n-decanoic acid.
As a result, accumulation of 9.5 g / l n-decanoic acid was confirmed.

Example 5 A fold-fold filter paper having a surface area of 1600 ° C. (20 × 40 cm ² ) was placed vertically in a reaction tank, 200 ml of a daily culture solution of Candida utilis IFO 0396 was poured, and the mixture was allowed to stand for 5 minutes. The cells were attached to the filter paper. Next, the culture solution was removed, 20 ml of a medium containing no cells was placed in the bottom of the reaction tank, and 2-ethyl- was added to the n-paraffin layer, which was the solvent phase.
1,3-hexanediol was added at the 10% level.
The culture was carried out for 7 days under stirring at 30 ° C. and 200 rpm under aeration of 0.2 vvm. After culturing, 1 μl of the solvent phase was analyzed by gas chromatography to quantify the produced 2-ethyl-3-hydroxyhexanoic acid. As a result, 6.7 g / l of 2-ethyl-3-hydroxyhexanoic acid was accumulated on the solvent phase side.

Comparative Example 1 2 ml of a 1-day culture of Rhodococcus equi IFO 3730 was inoculated into 20 ml of a medium containing 1% polypeptone, 0.2% yeast extract, 0.1% magnesium sulfate and 0.01% Span-80. Then, after culturing at 30 ° C. for 1 day, 2-
Octanol (82, 164, 246, 328 mg) was added, and the mixture was shake-cultured at 120 rpm for 3 days. After culturing, the mixture was extracted three times with diethyl ether, dried and diluted, and the concentration of produced 2-octanone was measured by gas chromatography. As a result, in each case, only 0.1 g / or less of 2-octanone was accumulated.

Comparative Example 2 2 ml of a 1-day culture of Rhodococcus equi IFO 3730 was inoculated into 20 ml of the same medium as in Comparative Example 1, and 30
The cells were cultured at 0 ° C for 1 day. Then, 2-methylcyclohexanol 94, 187, 281, 374 mg was added, and 30
Culturing was carried out at 120 ° C. and reciprocal shaking for 3 days. After culturing, the mixture was extracted 3 times with diethyl ether, dried and diluted, and the concentration of the produced 2-methylcyclohexanone was measured by gas chromatography. As a result, it was confirmed that only 0.1 g / l of 2-methylcyclohexanone was accumulated in each case.

Continuation of front page (51) Int.Cl. ⁵ Identification code Office reference number FI Technical display area C12R 1: 365) (C12P 7/26 C12R 1:01) (C12P 7/26 C12R 1:06) (C12P 7 / 26 C12R 1:40) (C12P 7/40 C12R 1: 365) (C12P 7/40 C12R 1:01) (C12P 7/40 C12R 1:06) (C12P 7/40 C12R 1:40)

Claims (1)

[Claims] 1. A hydrophilic immobilization carrier having a first grade and / or a second grade.
A microorganism having the ability to oxidize a primary hydroxyl group is adhered and immobilized, and in the presence of an aqueous medium containing a nutrient source for the microorganism, a substantially water-insoluble or sparingly soluble organic solvent containing alcohols is immobilized on the carrier. A method for oxidizing alcohols, which comprises contacting with a bacterial cell phase.