JPH0688A - Method for reducing carbonyl compound with immobilized microorganism - Google Patents

Abstract

PURPOSE:To obtain a method for biological reduction of carbonyl compound, having high reaction rate and yield by using immobilized multiplying microbial cells having catalytic ability of reducing reaction of carbonyl compound. CONSTITUTION:A microorganism capable of reducing carbonyl group and forming a secondary alcohol is stuck to a hydrophilic immobilizing carrier and immobilized. A substantially water-insoluble or slightly soluble organic solvent containing a carbonyl compound 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 reduce the carbonyl compound.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for reducing carbonyl compounds using immobilized microorganisms.

[0002]

2. Description of the Related Art Conventionally, industrially important secondary alcohols have been produced in large quantities by chemical reduction of carbonyl compounds. These chemical reduction methods include a method using a catalyst such as lithium aluminum hydride and lithium borohydride and a catalytic reduction method using a noble metal catalyst such as platinum. However, there are problems of catalyst cost, regioselectivity, and stereoselection. There are problems such as poor productivity and inability to completely maintain the safety of the production method, and the yield of the obtained product is not necessarily high.

On the other hand, particularly for the purpose of synthesizing optically active alcohols, biocatalysts having excellent regioselectivity and stereoselectivity, for example, reduction reactions of carbonyl compounds using oxidoreductases of baker's yeast and various microorganisms have been widely tried. [Fujisawa, et al., Yuka, Vol. 44, 519 (1986)], and is known to give excellent results. However, in the reduction reaction by the biocatalyst, the amount of enzyme or microbial cells used is large, and there are cost problems such as removal of the biocatalyst after the reaction and supply of coenzyme required for the biological reduction reaction.

When the carbonyl compound as a substrate and / or the alcohol as a product is insoluble or sparingly soluble in water, strong stirring force is required to forcibly disperse the substrate in water. There are problems that the reaction rate is low, it is difficult to separate the biocatalyst and the product after the reaction, and continuous production or repeated production is impossible.

When the substrate carbonyl compound and / or the product alcohol is insoluble or sparingly soluble in water, a strong stirring force is required to forcibly disperse the substrate in water. Is low, it is difficult to separate the biocatalyst and the product after the reaction, continuous production or repeated production is impossible, and the like.

Further, carbonyl compounds as substrates and alcohols as products are often toxic to enzymes and microorganisms, so that the amount of substrate added to the reaction system and the yield of products are very low. In addition, there is a problem that the long-term stability of the biocatalyst cannot be maintained.

Therefore, there is a strong demand for the development of a novel method for biological reduction of carbonyl compounds which overcomes the above-mentioned drawbacks of the chemical reduction method and which complements the deficiencies of the existing biological reduction methods.

[0008]

Therefore, the present inventor, in a method of reducing a carbonyl compound using a microorganism, avoids the toxicity of the carbonyl compound as a substrate or the reduction product to a microorganism such as secondary alcohols and forcibly disperses it in water. As a result of diligent study on the microbial reduction method of carbonyl compounds that does not require troublesome operations such as ventilation and aeration, the microorganisms were inoculated on a hydrophilic immobilization carrier containing a nutrient source and water, and the toxicity to the microorganisms was increased. When a non-expressing organic solvent is contacted, microbial cells vigorously grow at the interface between the organic solvent and the immobilized carrier to form a bacterial cell phase on the carrier, and this bacterial cell phase is added to the organic solvent. The inventors have found that the compound can be reduced with a high reaction rate and a high yield, and have completed the present invention.

[0009] Thus, according to the present invention, an aqueous medium containing a nutrient source for the microorganism, which has a carbonyl group-reducing ability and is capable of producing a secondary alcohol, is adhered and immobilized on the hydrophilic immobilization carrier. In the presence of the carbonyl compound, a substantially water-insoluble or sparingly soluble organic solvent containing the carbonyl compound is brought into contact with the immobilized bacterial cell phase on the carrier.

One feature of the present invention is that the microorganisms are 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 microorganisms. This is because the bacterial cell phase can be used as a catalyst. This makes it possible to bring the carbonyl compound into contact with the bacterial cell phase in a form in which it is dissolved in an organic solvent that is substantially insoluble or sparingly soluble in water to carry out the reduction reaction, and as a result, a high reaction rate and catalyst Long-term stability of
Advantages such as the fact that stirring power is virtually unnecessary are obtained.

Another feature of the present invention is that although the carbonyl compound as a substrate shows a strong toxicity to microorganisms, it can be added to an organic solvent at a very high concentration, and There is a point that can contact. For example, 2
In the case of octane and 2-decanone, microorganisms cannot grow because only 0.5% is present in the emulsion type. However, in accordance with the present invention, for example, C. petitida IFO 13696 is not killed by the inclusion of 2-octanone at a concentration of 12% in the organic solvent and 2-decanone at a concentration of 24%. Does not die, but proliferates to form a bacterial cell phase. According to the invention,
In this way, a high concentration of carbonyl compound can be contacted with the microbial cell phase, and as a result, a high reaction rate and high yield can be achieved, and advantages such as reduction of the carbonyl compound at low cost can be obtained. To be

The present invention will be described in more detail below.

The organic solvent or the organic solvent solution of the carbonyl compound, which is a substrate, which is 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 immobilization carriers is not particularly limited, and they can be formed into any shape such as fibrous, film-like, and granular shapes, 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 carbonyl compound of the substrate does not substantially show toxicity to adherent microbial cells. Preferred is, 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, heptylbenzene, Examples thereof include normal alkylbenzenes having an aliphatic chain of 5 to 15 carbon atoms such as octylbenzene; isoalkylbenzenes such as quinume; alicyclic hydrocarbons such as cyclohexane; and ethers such as diethyl ether.

The microorganism having the ability to reduce a carbonyl group, which is attached to the immobilized carrier and is grown at the interface between the carrier and an organic liquid, can be any microorganism such as bacteria, fungi, yeasts and actinomycetes. May be Specifically, for example, Bacillus genus, Xanthonas (Xantho
monas genus, Lactobacillus genus, Leuconostoc genus, Streptococcus (Stre
Bacteria such as genus ptococcus, genus Pseudomonas, genus Pichia, genus Candida,
The genus Rhodotorula, Saccha lomyces
genus romyces, Schizosacchromyce
s), Kloeckera, Cryptococcus
(Cryptococcus) genus, Depodascus (Dipodascus) genus,
Yeasts such as the genus Loderderomyces, the genus Geotrichum, and the glioeosporiu (Gloeosporiu)
m) genus, Aureobasidium genus, Aspergillus genus, Penicilliu
genus m), genus Sporotrichum, vulbraria
(Curuvlaria) and other microorganisms belonging to molds and the like. More specifically, Bacillus thuringiensis, Xanthomonas oryzae (X.
a. Oryzae), Lactobacillus brevis,
Leuconostok Dextranicum
m), Streptococcus faecalis (St. faecali
s), Picia tericola (Pi. tericola), Candida albicans (Ca. albicans), Rhodotorula rubra (Rh. ru)
bra), Sa. cerevisia
e), Kl. corticis, Cryptococcus magerans, Cr. macerans, Di. uninucleatus, Lo. elongisporus, Ge. Aureobasidium pullulans (Au. Pullulans), Aspergillus ocraceus (As. Ochraceus), Penicillium
Decumbens, Sporotricham Exil, Cu. Lunata
Etc. can be mentioned.

The adhesion and immobilization of such microorganisms on the carrier can be carried out, for example, by applying or spraying a bacterial cell suspension on a carrier containing an aqueous medium containing a nutrient source in advance, or immersing the carrier in a bacterial cell culture solution. Or, after attaching the microbial cells to the carrier by an appropriate method, after attaching the microbial cells on the carrier by a method such as supplying an aqueous medium containing a nutrient source to the carrier, the carrier is pre-nutrified. Although it can be cultured in an aqueous medium containing a source, it is usually cultured in a state of being contacted with an organic solvent containing or not containing a carbonyl compound as a substrate,
Suitably, the adhered microbial cells are grown at the interface between the carrier, the organic solvent and the organic solvent to form an immobilized microbial 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.

As the nutrient source of the microorganism that can be used in the above-mentioned culture, an optimum one can be selected according to the type of the bacterial cell used, for example, a carbon source such as glucose,
A general source consisting of a nitrogen source such as urea, a trace metal salt such as magnesium sulfate, and a trace nutrient source 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. And / or for example, by adding a culture solution to the above organic liquid phase and interposing a microorganism-immobilized carrier 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.

When oxygen is required for culturing, it may be aerated with an organic solvent, but in general, an organic solvent has oxygen solubility of several times to several tens of times that of water, so that it is always aerated. do not have to. When oxygen interferes with the culture or reaction, the dissolved oxygen in the organic solvent may be replaced with carbon dioxide, nitrogen, or the like. Since the solubility of these substitution gases in the organic solvent is generally several times higher than that of water, substitution can be performed efficiently. On the other hand, as for the stirring during culture, the carbonyl compound that is the substrate is dissolved and present in the organic solvent, so that vigorous stirring such as the emulsion method is not necessary, and the stirring is often unnecessary. .

The carbonyl compound as a substrate may be added from the initial stage of the above culture, or may be added after the microorganism has sufficiently grown to form an immobilized bacterial 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, since carbonyl compounds often exhibit toxicity to microorganisms, generally, higher results are achieved by adding them after the bacterial cell phase has sufficiently grown.

Thus, the reduction reaction of the carbonyl compound 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 the carbonyl compound as the substrate. it can.

The carbonyl compound that can be used as a substrate for the reduction reaction of the carbonyl compound by the immobilized microorganism is not particularly limited, and various compounds can be used depending on the reducing ability of the carbonyl group of the immobilized microorganism.

Examples of the carbonyl compound that can serve as a substrate include acetone, methyl ethyl ketone, methyl n-propyl ketone, methyl isopropyl ketone, methyl isobutyl ketone, diethyl ketone, 2-hexanone, 3-hexanone, 2-octanone, 2-decanone. Alkanones such as; ketoesters such as ethyl 3-ketobutanoate, ethyl 4-chloro-3-ketobutanoate; diketones such as acetylacetone, acetonylacetone, 2,4-octanedione; acetophenone, fosenzophenone, benzyl, 2- Aromatic carbonyl compounds such as acetoxy-1-phenylethanone; cyclohexanone, 2-
Examples thereof include alicyclic carbonyl compounds such as methylcyclohexanone.

The concentration of the carbonyl compound in the organic solvent is not particularly limited and can be determined depending on the toxicity to the microbial cells. When the carbonyl compound is substantially water-insoluble or sparingly soluble, it does not migrate to the water phase, that is, the hydrophilic immobilization carrier side, and thus the toxicity to the bacterial cells can be greatly reduced. For example, in the case of a medium-chain aliphatic carbonyl compound such as 2-octanone or 2-decanone, yeasts such as Satsucaromyces, Roderomyces, Picia and Candida have a concentration in the organic liquid phase of 3 to 5%. However, it can grow. On the other hand, in the case of water-soluble carbonyl compounds such as acetone and methyl ethyl ketone, these may migrate to the water phase side and develop toxicity to the bacterial cells, so the concentration should be adjusted according to the resistance of the strain. I need to decide.

According to the above-mentioned method of the present invention, the reduction reaction of an aliphatic, aromatic, alicyclic carbonyl compound, etc. can be carried out extremely efficiently by using the proliferating cells of the immobilized microorganism. . At that time, if a side reaction is likely to occur, a metabolic deficient strain that has been bred and improved by blocking it by the addition of an appropriate metabolism or conversion inhibitor or preventing such side reaction from occurring is selected. Can be used.

According to the method of the present invention, the carbonyl compound of the substrate is supplied from the organic liquid phase side, and the carbonyl group-reducing compound of the product, that is, the secondary alcohol is accumulated on the organic liquid phase side. Therefore, the frequency of contact between the immobilized cell phase and the substrate can be dramatically increased by collecting the product accumulated in the organic liquid phase and supplementing the substrate with a carbonyl compound. The reaction rate, yield, and yield can be significantly improved, and continuous operation is also possible.

Thus, the method of the present invention can be applied to industrially important secondary alcohols in the fields of, for example, industrial chemicals, pharmaceuticals, cosmetics, fragrances, detergents, surfactants, fiber treating agents, oils and fats, dyes, paints and printing materials. By applying it to manufacturing processes of the same class, it is possible to obtain various industrially advantageous advantages such as reduction of production cost, energy saving and labor saving of processes.

[0031]

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 Peptone 0.5%, yeast extract 0.3%, malt extract 0.1%
An agar plate consisting of 3%, glucose 1.0% and agar 1.5% was prepared in a glass petri dish (surface area 38.5 cm ² ), and 100 μl of suspensions of various microorganisms shown in Table 1 were coated with a conradi stick. It spattered. After drying, 10% 2-decanone n-
Hexadecane solution (10 ml) was overlaid and statically cultured at 30 ° C. for 7 days. After culturing, 1 μl of the solvent phase was analyzed by gas chromatography to measure the concentration of the produced 2-octyl acid n-butyl ester. The results are shown in Table 1.

[0033]

[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 sealed, to prepare an agar plate (surface area 43 cm ² ). Various microbial suspensions 1 shown in Table 2
00 μl was smeared with a conradi stick and cultured at 30 ° C. for 2 days to form a bacterial cell phase. Then 10% 2-
Overlay 10 ml of n-hexadecane solution of octane,
Culturing was carried out for 7 days under shaking at 100 ° C at 100 ° C. After culturing, 1 μl of the solvent phase was analyzed by gas chromatography,
The produced 2-octanol concentration was measured. The results are shown in Table 2.

[0034]

[Table 2] Example 3 A glass container (internal volume: 500 ml) vertically packed with a 3 mm-thick filter plate (manufactured by Toyo Advantech) was prepared (surface area: 800 cm ² ). To this, 200 ml of a liquid medium consisting of 0.5% peptone, 0.3% yeast extract, 0.3% malt extract, 1.0% glucose and 0.080% span-80 was added and sterilized under high pressure steam. After cooling, inject 50 ml of Roderomyces elongisporus daily culture solution into it,
The cells were cultured for 2 days under aeration of 0.2 vvm and stirring at 300 rpm. After culturing, 10% 2-decanone n-paraffin solution was injected while removing the culture solution, and 50 ml of the culture solution, solvent phase 1
50 ml was cultured at 30 ° C. under stirring at 300 rpm for 7 days without aeration. During the culture period, 1 μl of the solvent phase was analyzed by gas chromatography to measure the concentration of 2-decanol produced. The results are shown in Table 3.

[0035]

[Table 3] Example 4 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 Lactobacillus. Brevis IAM 1082 daily culture solution 10
0 μl was inoculated with a conradi rod and 10 ml of n-paraffin in which dissolved oxygen was replaced with nitrogen was overlaid. After stationary culture at 30 ° C. for 2 days to form a bacterial cell phase, 200 μl of 4-methylcyclohexanone was added and dissolved, and stationary culture was carried out for 7 days. After the culturing, 1 μl was collected from the solvent phase, and the concentration of produced 2-methylcyclohexanol was measured using gas chromatography. As a result, accumulation of 1.2 g of 2-methylcyclohexanol was confirmed.

Comparative Example 1 Peptone 0.5%, yeast extract 0.3%, malt extract 0.1%.
A liquid medium consisting of 3%, glucose 1.0%, and span-80 0.02% was inoculated with Roderomyces elongisporus, and cultured at 30 ° C. for 1 day with shaking. 2-Octanone was added to this so as to be 0.2, 0.4, 0.6, 0.8, and 1.0%, and shake culture was carried out for 7 days. After culturing, the mixture was extracted three times with diethyl ether, dried and diluted, and the concentration of produced 2-octanol was measured by gas chromatography. As a result, the produced 2-octanol concentration was 0.1 g / l or less at any concentration.

Comparative Example 2 1 ml of a 1-day culture of Lactobacillus brevis IAM 1082 was added to 20 ml of the medium of Example 1 which had been replaced with nitrogen by adding 0.02% of Span-80 and nitrogen substitution, and grown for 1 day. Let Then, 2-methylcyclohexanone was added to the medium so that the concentration was 0.2, 0.4, 0.6, 0.8, and 1.0%, and the mixture was cultured with shaking at 150 rpm for 7 days. After culturing, the mixture was extracted three times with diethyl ether, dried and diluted, and the concentration of produced 2-methylcyclohexanol was measured using gas chromatography. As a result, the produced 2-methylcyclohexanol concentration was 0.1 g / l or less at any addition concentration.

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Claims (1)

[Claims] 1. A carbonyl compound in the presence of an aqueous medium containing a nutrient source for said microorganism, wherein a microorganism capable of reducing a carbonyl group and capable of producing a secondary alcohol is adhered and immobilized on a hydrophilic immobilization carrier. A method for reducing a carbonyl compound, which comprises contacting a substantially water-insoluble or sparingly water-soluble organic solvent containing the above with the immobilized bacterial cell phase on the carrier.