JPH06197777A - Transesterification by immobilized microorganism - Google Patents

Abstract

PURPOSE:To provide a microbial transesterification process having high reaction rate and yield by using immobilized and proliferated cells of microorganism capable of performing the transesterification of esters. CONSTITUTION:A microorganism capable of catalyzing the transesterification reaction of esters is attached and immobilized to a hydrophilic immobilization carrier. The immobilized microbial phase on the carrier is made to contact with an essentially water-insoluble or scarcely soluble organic solvent containing esters and carboxylic acids or alcohols to be used for transesterification in the presence of an aqueous medium containing a nutritive source for the microorganism.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a transesterification method for esters using an immobilized microorganism.

[0002]

2. Description of the Related Art Conventionally, high value addition has been widely carried out industrially by transesterification of esters represented by glycerides. Chemically, it is common to use an inorganic catalyst such as sodium,
Poor regioselectivity, from the viewpoint of energy consumption, and from the side reactions such as oxidation reactions,
Not necessarily a good method.

On the other hand, methods utilizing a biocatalyst excellent in stereoselectivity and regioselectivity have also been widely studied. For example, technologies that utilize lipid hydrolases such as lipase and esterase and microorganisms that are the source of them as catalysts,
Transesterification of glycerides (K. Yokozeki, et al.,
Eur. J. Appl. Microbiol. Biotechnol., Vol. 14,1
(1982); RA Wisdom, et al., Biotechnol. Bioeng.,
Vol. 29, 1081 (1987)) as well as widely used in the field of optical resolution (B. Cambou & AM Klibanov,
Biotechnol. Bioeng., Vol. 26, 1449 (1984); E. Gui
be-Jampel & G. Rosseau, Tetrahedron lett. Vol. 28,
3563 (1987)).

However, the method using purified enzyme has many problems such as enzyme cost problem, separation of enzyme after reaction, management of water content in reaction solvent, and the case of using bacterial cell itself. However, there are problems such as the control of water content and the cost of separating bacterial cells after the reaction.

Therefore, there is a strong demand for the development of a novel biotransesterification method for esters, which overcomes the above-mentioned drawbacks of the chemical transesterification method for esters and which complements the deficiencies of the existing biotransesterification methods. It is rare.

[0006]

Therefore, the present inventors have conducted the transesterification reaction of esters using the above-mentioned microbial cells in an organic solvent in which a trace amount of water required for the exchange reaction can be easily controlled. As a result of intensive studies on a technique for growing microorganisms and carrying out their life activity while maintaining them, the microorganisms were inoculated on a hydrophilic immobilization carrier containing a nutrient source and water, and toxicity was exhibited to the microorganisms. When contacted with an organic solvent that does not exist, microbial cells actively grow at the interface between the organic solvent and the immobilized carrier to form a bacterial cell phase on the carrier, and the bacterial cell phase is added to the organic solvent. The inventors have found that transesterification is carried out at the interface between a carrier containing water and an organic solvent 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 catalyze the transesterification reaction of an ester is adhered and immobilized on a hydrophilic immobilization carrier, and the ester is produced in the presence of an aqueous medium containing a nutrient source for the microorganism. Provide a method for transesterification of esters, which comprises contacting a substantially water-insoluble or sparingly water-soluble organic solvent containing carboxylic acids or alcohols required for exchange with the immobilized bacterial cell phase on the carrier. To be done.

The first feature of the present invention is to grow microorganisms 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. The point is that the formed bacterial cell phase can be used as a catalyst. This allows the ester and the carboxylic acid or alcohol to be brought into contact with the bacterial cell phase in a form of being dissolved in a substantially water-insoluble or sparingly soluble organic solvent to cause the transesterification reaction of the ester, As a result, a higher reaction rate and longer-term stability of the catalyst can be obtained as compared with the conventional emulsion method, and the advantage that stirring power is virtually unnecessary is obtained.

The second feature of the present invention is that even when the esters and the carboxylic acids or alcohols which are the substrates are toxic to microorganisms, they can be added to the organic solvent at a high concentration, and the bacterial cells can be added. It is in the point of contact with the phases.
For example, aliphatic esters, carboxylic acids or alcohols having 6 to 20 carbon atoms can be brought into contact with microorganisms at a very high concentration regardless of linear or branched esters, carboxylic acids or alcohols. It is possible. Further, when the toxicity of the esters and the carboxylic acids to the microorganism is extremely weak, the concentration of the esters and the carboxylic acids is substantially 100% in the solvent, that is, the esters and the carboxylic acids can be directly used as the solvent phase. Also,
Alcohols can be added at a concentration of several times to several tens of times that of the emulsion method. Thus, as an advantage of being able to contact a high concentration of ester and carboxylic acid or alcohol to the microbial cell phase,
A high reaction rate and a high yield can be obtained, and the esters can be transesterified at a low cost.

A third feature of the present invention is that the amount of water required for the transesterification reaction of esters can be controlled very easily. The transesterification reaction of the ester is established along with the simultaneous progress of the hydrolysis reaction of the ester and the ester synthesis reaction, and in that case, a certain amount of water content is required. However, in the conventional enzyme method or microbial conversion method, it has been difficult to control the optimum water content for the reaction, whether it is an emulsion system or a fine water system. However, according to the present invention, the water supplied to the solid / liquid interface, which is the reaction site, is continuously and constantly supplied according to the water solubility of the solvent phase. Will always be kept constant. As a result, the amount of water in the reaction system is kept constant as compared with the conventional method, so that the transesterification reaction proceeds stably.

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

An aqueous medium containing a nutrient source, that is, a 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.

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 it with 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.

There are no particular restrictions on the shape of these immobilization carriers, and they can be molded into any shape such as fibrous, film-like, granular, etc., and in the form of cloth, non-woven fabric, paper, cardboard, etc. It may be molded into.

The organic solvent in the organic liquid phase in contact with the immobilized bacterial cell phase or the organic solvent for preparing the substrate solution is preferably one which does not show substantial toxicity to the adherent microbial cells, 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, octylbenzene, etc. normal alkylbenzenes having an aliphatic chain of 5 to 15 carbon atoms; isoalkylbenzenes such as cumene; alicyclic hydrocarbons such as cyclohexane Examples thereof include ethers such as diethyl ether.

The microorganisms having the transesterification ability of the esters attached to the immobilized carrier and grown at the interface between the carrier and the organic liquid can be any microorganisms such as bacteria, yeasts, molds and actinomycetes. May be Specifically, for example, Pseudomonas genus, Chromobacterium genus, Microbacterium genus, Micrococcu
s) genus, Staphylococcus genus, Escherichia genus, Bacillus
Genus, bacterium such as Arthrobacter genus, Mycobacterium genus, yeasts such as Candida genus, Phycomyc (Phycomyc)
es), Mucor, and Penicillium
llium, Rhizopus, Tamidium
mmidium genus, Chaetostylum genus, Aspergillus genus, Geotrichum
um) and other microorganisms belonging to molds. More specifically, Pseudomonas fragii (Ps. Frag
i), Pseudomonas fluorescense (Ps. fluoresc
ence), Chromobacterium chocolate (Ch.
chocolatum), Microbacterium arborescens (Mb. arborescens), Micrococcus varians (Mc. varians), Staphylococcus aureus (S
c. aureus), Es. Coli, Bacillus subtilis subspicis niger (Ba.
subtilis subsp. niger), Candida lipolytica, Phycomyces nitens (Ph.
niteus), Mu. sufu, Penicillium roqueforti, Rh. oligosporus, Ta. elegans, Ch. fresnei, Aspergillus flavus (As. Flav
us), Geotricum candy dam (Ge. candidum)
Etc. can be mentioned.

The adhesion and immobilization of such microorganisms to the carrier is carried out, for example, by coating or spraying the microbial cell dispersion on a carrier which has previously contained an aqueous medium containing a nutrient source, or by 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 cultivate in an aqueous medium containing, but usually cultivated in contact with an organic solvent containing or not esters and carboxylic acids or alcohols as a substrate, microbial cells adhered to the carrier It can be carried out by proliferating at the interface between the organic solvent and an organic solvent to form an immobilized bacterial cell phase. 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 the bacterial cell used, and the optimum one can be selected. For example, carbon sources such as glucose and nitrogen such as urea can be selected. Sources, trace metal salts such as magnesium sulfate, and trace nutrients such as yeast extract may be used.

Culturing can be generally carried out in a culturing apparatus 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 or air required for culturing may be aerated in an organic solvent, but in general, an organic solvent has an oxygen solubility several times to several tens of times higher than that of water. Absent. Also, regarding stirring during culture,
Since the substrates such as esters, carboxylic acids, and alcohols are dissolved and present in an organic solvent, vigorous agitation such as the emulsion method is not necessary, and agitation is often not necessary at all.

The ester or carboxylic acid or alcohol as a substrate may be added from the initial stage of the above-mentioned culture, or may be added after the microorganism has grown to form the cell immobilized 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 mentioned above,
Since esters, carboxylic acids, and alcohols often exhibit a considerable toxicity to microorganisms,
Generally, higher results are achieved when the bacterial cell phase is sufficiently grown before addition.

Thus, transesterification of the ester is 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 ester and carboxylic acid or alcohol as the substrate. The reaction can be carried out.

The ester that can be used as a substrate for the transesterification reaction by the immobilized microorganism is not particularly limited as long as it undergoes transesterification by the microorganism, and various types can be used depending on the transesterification ability of the immobilized microorganism. Can be used.

Esters that can be used as a substrate include
For example, ethyl acetate, butyl acetate, amyl acetate, acetic acid-2
-Ethylhexyl, cyclohexyl acetate, benzyl acetate, ethyl butyrate, butyl butyrate, ethyl heptanoate, butyl heptanoate, ethyl octanoate, butyl octanoate,
Ethyl decanoate, butyl decanoate, ethyl laurate,
Ethyl stearate, ethyl oleate, ethyl benzoate, benzyl benzoate, dioctyl adipate, diethyl oxalate, diethyl phthalate, dioctyl phthalate,
Examples thereof include triolein glyceride (triolein), glycerin ester of long chain fatty acid such as olive oil, cetyl palmitate, long chain alcohol ester of long chain fatty acid such as beeswax.

The concentration of the ester in the organic solvent is not particularly limited and can be determined according to the toxicity to the bacterial cells. Since esters are often insoluble in water, it is possible to add them in an organic solvent at a very high concentration to bring them into contact with the bacterial cell phase, especially medium-chain carboxylic acids such as octanoic acid and decanoic acid. In the case of ethyl ester, butyl ester, glycerin ester of long-chain fatty acid such as triolein, olive oil, etc., because of their low toxicity, those esters are 100% concentration, that is, the esters themselves are organic liquid phase. Can be brought into contact with the bacterial cell phase.

On the other hand, the carboxylic acids required for transesterification can be supplied from the aqueous phase on the side of the hydrophilic carrier when they are water-soluble, but only when they do not show toxicity to the bacterial cells. Ordinarily, it is added to the organic liquid phase and subjected to transesterification reaction.

Examples of the carboxylic acids that can be used as a substrate include lower fatty acids such as acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, heptanoic acid, octanoic acid and decanoic acid, lauric acid, myristic acid, palmitic acid and stearic acid. Higher fatty acids such as oleic acid, linoleic acid and linolenic acid, and aromatic monocarboxylic acids such as benzoic acid, methylbenzoic acid and salicylic acid.

Further, when the transesterification reaction is carried out on the alcohol portion, if the alcohols required for the reaction are water-soluble, they can be supplied from the aqueous phase on the side of the hydrophilic carrier, but they are toxic to the bacterial cells. It is usually added to the organic liquid phase and subjected to a transesterification reaction only when it does not develop.

The alcohols that can be used as the substrate include, for example, methanol, ethanol, propanol,
Lower alcohols such as butanol, amyl alcohol, octanol and decanol, lauryl alcohol, myristyl alcohol, higher alcohols such as cetyl alcohol, stearyl alcohol and oleyl alcohol, aromatic alcohols such as benzyl alcohol, and alicyclic alcohols such as cyclohexanol. Can be mentioned.

The concentration of the carboxylic acid or alcohol in the organic solvent is not particularly limited and can be determined depending on the toxicity to the microbial cells. When the carboxylic acids or alcohols are substantially insoluble or sparingly soluble in water, they do not migrate to the aqueous phase, that is, the hydrophilic immobilization carrier side, so that the toxicity to the bacterial cells can be greatly reduced. For example, medium-chain alkanols such as octanol and decanol are 10 to many microbial cells.
It can be contacted at a concentration of 20%.

On the other hand, when the carboxylic acids or alcohols are water-soluble, they can be supplied from the aqueous phase side as long as they do not exhibit toxicity to the cells. For example, if a resistant strain is selected, ethanol or propanol can be added to water at a concentration of 10%. Also, when adding carboxylic acids to water, the pH will drop, so neutralize or
Alternatively, it is necessary to add the carboxylic acids in salt form.

According to the method of the present invention described above, the exchange reaction of the esters such as aliphatic and aromatic can be carried out extremely efficiently by using the growing bacterial cell phase of the immobilized microorganism. At that time, if there is a possibility that side reactions such as β-oxidation of carboxylic acid based on the growth cell may occur, addition of an appropriate metabolic or conversion inhibitor may inhibit it, or such side reactions may occur. A metabolic deficient strain that has been bred and improved so as not to occur can be used.

According to the method of the present invention, a trace amount of water required for transesterification of esters is supplied from the hydrophilic carrier side, and the amount of water at the solid / liquid interface, which is the reaction site, is kept constant. A steady transesterification reaction proceeds. Thus, since the ester of the product is constantly accumulated on the organic liquid phase side, the exchange ester and the exchanged carboxylic acid or the exchanged alcohol accumulated in the organic liquid phase are recovered and the ester and carboxylic acid of the substrate are recovered. By performing a method such as supplementing acids or alcohols, it is possible to dramatically increase the frequency of contact between the immobilized cell phase and the substrate, and it is possible to significantly improve the reaction rate, yield, and yield. It becomes possible and continuous operation becomes possible.

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

[0035]

EXAMPLES The present invention will 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 For the bacteria shown in Table 1, polypeptone 1.0%,
An agar medium (pH 7.0) consisting of yeast extract 0.2%, magnesium sulfate 0.1%, and agar 1.5%, peptone 0.5%, yeast extract 0.3%, and malt extract 0 for molds. An agar medium (pH 6.0) containing 0.3% of glucose, 1.0% of glucose and 1.5% of agar was dispensed into a glass petri dish to prepare an agar plate (surface area: 38.5 cm ² ). Table 1
100 μl of the suspensions of various standard strains shown in (1) were smeared with a Conradi stick, dried, and then overlaid with 10 ml of ethyl heptanoate containing 1% n-decanol, followed by static culture at 30 ° C. for 5 days. After culturing, 1 μl of the solvent phase was analyzed by gas chromatography to quantify the decyl heptanoate formed. The results are shown in Table 1.

[0037]

[Table 1]

Example 2 In the same manner as in Example 1, 100 ml of an agar plate was prepared in a glass container having an internal capacity of 480 ml and capable of being sealed (surface area 43 cm ² ).
100 μl of suspensions of various standard strains shown in Table 2 were smeared using a conradi stick. After standing at 30 ° C. for 2 days to form an immobilized bacterial cell phase, n-containing 2% n-butanol was added.
Overlay with 10 ml of ethyl octoate, 30 ℃, 100rpm
The cells were cultured under shaking for 5 days. After culturing, 1 μl of the solvent phase was analyzed by gas chromatography to quantify the amount of n-butyl octoate produced. The results are shown in Table 2.

[0039]

[Table 2]

Example 3 150 ml of agar plate prepared in the same manner as in Example 2 was used.
100 μl of each standard strain suspension shown in 1 was smeared using a conradi stick, and statically cultured at 30 ° C. for 1 day to form an immobilized bacterial cell phase. Then, 10 ml of an n-decanoic acid solution containing 2% n-butyl heptanoate was overlaid, and the temperature was 30 ° C and 100 rp.
It was cultured for 3 days under shaking at m 2. After culturing, 1 μl of the solvent phase was analyzed by gas chromatography to measure the concentration of the produced n-butyl decanoate. The results are shown in Table 3.

[0041]

[Table 3]

Example 4 100 ml of agar plate prepared in the same manner as in Example 2 was added to Table 4.
100 μl of each standard strain suspension shown in 1) was smeared with a Conraji rod and incubated at 30 ° C. for 1 day to form an immobilized bacterial phase, and then 20 ml of a triolein solution containing 3% n-butanol was added. Were overlaid and cultured at 30 ° C. under shaking at 100 rpm for 5 days. After the completion of the culture, 10 μl of the solvent phase was analyzed using an Iatroscan to quantify the transesterification product. The results are shown in Table 4.

[0043]

[Table 4]

Example 5 A silica gel thin layer chromatography plate was cut into 5 cm pieces, and the plate was immersed in a culture solution of Pseudomonas fradi IFO 12049 having the same composition as in Example 2 for 1 day.
Then, the plate slice was put into 50 ml of a triolein solution containing 3% n-butanol, and statically cultured at 30 ° C. for 7 days. After culturing, 10 μl of the solvent layer was analyzed by an iatroscan to quantify the transesterification product produced. As a result, accumulation of 1.2 g / l of mono-exchanger and 16.7 g / l of di-exchanger was confirmed.

Comparative Example 50 ml of a liquid medium (pH 7.0) consisting of 1.0% polypeptone, 0.2% yeast extract, 0.1% magnesium sulfate and 0.02% span-80 was used for Bacillus subtilis.
5 ml of a 1-day culture solution of Subsposis Niger IFO 3108 was inoculated and cultured at 30 ° C. under shaking at 150 rpm. After the culturing, ethyl heptanoate and n-decanol were added at 1% and 0.3%, respectively, and the cells were cultivated with shaking under the same conditions for 5 days. After culturing, the mixture was extracted 3 times with diethyl ether, dried,
After dilution, the produced decyl heptanoate was quantified using gas chromatography. As a result, accumulation of 0.9 g / l decyl heptanoate was confirmed.

Claims (1)

[Claims] 1. A microorganism having the ability to catalyze the transesterification reaction of an ester is adhered and immobilized on a hydrophilic immobilization carrier, and the ester and the carboxyl required for the exchange are present in the presence of an aqueous medium containing a nutrient source for the microorganism. A method for transesterification of esters, which comprises contacting a substantially water-insoluble or sparingly water-soluble organic solvent containing acids or alcohols with the immobilized bacterial cell phase on the carrier.