JPH09131197A - Fusion of fermentation with microbial conversion reaction - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for fusing a fermentation with a microbial conversion reaction, capable of microbially inserting the fermentation product of a saccharide such as glucose, namely a water-soluble organic acid, an alcohol or acetyl Co A, into an esterification reaction. SOLUTION: The characteristics of the method for fusing a fermentation with a microbial conversion reaction comprise sticking a microorganism having an organic acid fermentation ability, an alcohol fermentation ability or an acetyl-Co A fermentation ability and having a lipase or esterase-producing ability to a hydrophilic immobilization carrier, bringing a hydrophobic organic solvent into contact with the microorganism on the carrier in the presence of a water-soluble medium containing a saccharide as the nutritive source of the microorganism in such a concentration as not inhibiting the activities of an alcohol oxidase and an aldehyde oxidase to multiply the microorganism on the contact interface, fermentively producing the water-soluble organic acid, the alcohol or the acetyl-Co A from the fermentation raw material, and subsequently subjecting the fermentation product and a water-insoluble or slightly soluble alcohol or organic acid produced by the microbial oxidation of an alcohol dissolved in the hydrophobic organic solvent to a microbial esterification reaction.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for integrating fermentation and microbial conversion in which a water-soluble organic acid, alcohol, or acetylcoenzyme A, which is a fermentation product of sugars such as glucose, is incorporated into an esterification reaction in a microbial manner. Regarding

[0002]

2. Description of the Related Art In recent years, material production by a microbial conversion method has been attempted all over the world. As one application example of this microbial conversion method, an ester is produced by the reverse reaction of a hydrolase such as lipase or esterase. Many synthetic methods have been proposed, especially for the purpose of optical resolution [T. Sugai and H. Ohta, Agric. Biol. Chem., 55,
293 (1991); Kakeya, et al., Agric. Bio
l. Chem., 55, 1877 (1991); Sugai and
H. Ohta, Agric. Biol. Chem., 54, 3337 (19
90)]. In order to efficiently proceed the esterification reaction by this enzyme, it is necessary to reduce the amount of water present in the reaction system, and therefore the enzymatic reaction is generally carried out in an organic solvent.

On the other hand, commercially available lipases and esterases are difficult to esterify a carboxylic acid having a large steric hindrance, which is not possible to esterify a tertiary hydroxyl group, in addition to cost problems. It is difficult to esterify an α, β-unsaturated carboxylic acid, it is difficult to esterify a carboxylic acid substituted on an aromatic ring, and the optical resolution is difficult to esterify a primary hydroxyl group having relatively good symmetry. Etc. have many problems. Furthermore, when a low-molecular weight organic acid or alcohol used for esterification is added to the reaction system at a high concentration, the pH of the reaction system is lowered and lipase or esterase is deactivated, so that high-concentration ester synthesis is very difficult. [B. Cambou and A. M. Klibanov, Biotechnol.
Bioeng., 26, 1449 (1984)].

In order to overcome the above-mentioned deficiencies of such commercially available lipases or esterases, screening of microorganisms capable of producing novel lipases or esterases and microbial conversion methods using the microorganisms directly as biocatalysts are widely used. Attempts have been made, but microorganisms generally die or are unable to grow in organic solvents,
It is difficult to make the esterification reaction proceed stably.

On the other hand, an interfacial bioreactor utilizing a microorganism attached to a hydrophilic carrier containing a nutrient source and water and growing at the interface of a hydrophobic organic solvent which is substantially non-toxic to the microorganism as a biocatalyst, It can be applied to almost all microorganisms and microbial reactions, and the esterification reaction can also proceed very efficiently [S. Odaan
d H. Ohta, Biosci. Biotech. Biochem., 56, 204
1 (1992); Oda, et al., J. Ferment. Bioen
g., 78, 149 (1994), JP-A-5-34489.
No. 6]. However, in the interfacial bioreactor, it is impossible to avoid toxicity against hydrophilic poisons, and it is difficult to add low-molecular organic acids or alcohols to the reaction system at high concentrations [S. Oda and H. Ohta, Biosc
i. Biotech. Biochem., 56, 1515 (199
2)].

[0006]

The inventors of the present invention have made extensive studies to solve various problems of the prior art as described above, and as a result, have found that the interfacial bioreactor has a great advantage in organic solvents. The water-soluble organic acids, alcohols or acetylcoenzyme A are sequentially produced by fermentatively producing water-soluble organic acids, alcohols or acetylcoenzyme A by effectively utilizing the growth ability of the microorganisms and the fermentation ability of the microorganisms. Of alcohols or organic acids that are insoluble or sparingly soluble in water (hereinafter collectively referred to as “poorly water-soluble”) into the esterification reaction and subjected to the esterification reaction by microbial reduction or microbial oxidation of alcohol and / or aldehyde compounds Fed by fermentation-microbial reduction-esterification or fermentation-microbial oxidation-esterification It can be solved by fusing, and have completed the present invention.

Thus, according to the present invention, the hydrophilic immobilization carrier has organic acid fermentation ability, alcohol fermentation ability or acetyl coenzyme A fermentation ability and lipase, esterase or alcohol acetyltransferase. Insoluble or sparingly soluble in water in the presence of a water-soluble medium that adheres microorganisms and contains a saccharide that is a nutrient source of the microorganisms and a fermentation raw material at a concentration that does not inhibit the activity of alcohol oxidase and aldehyde oxidase. The hydrophobic organic solvent containing the alcohol and / or the aldehyde compound is contacted with the microorganism on the carrier to grow the microorganism at the contact interface, and by the action of the microorganism, the water-soluble organic acid from the fermentation raw material is added. Alternatively, an alcohol is fermented and produced, and the fermentation product and a finely-diluted alcohol dissolved in a hydrophobic organic solvent are used. Fermentation and microbial conversion reaction characterized by carrying out a microbial esterification reaction between a water-insoluble or sparingly soluble alcohol or organic acid produced by oxidative oxidation and microbial oxidation or reduction of an aldehyde compound A fusion method is provided.

According to the present invention, the microorganism that grows on the solid / liquid interface consisting of the carrier / hydrophobic solvent in the interfacial bioreactor utilizes the fermentation raw material consisting of sugars such as glucose, starch and sucrose in the carrier. Low molecular weight water-soluble organic acids, alcohols or their precursors, acetylcoenzyme A and below, abbreviated as "acetyl-CoA")
To ferment and produce. These fermentation products are poorly water-soluble alcohols produced by the microbial oxidation of alcohols or the oxidative reduction of aldehyde compounds in hydrophobic organic solvents by the enzymatic action of lipases, esterases or alcohol acetyl transfruase which the microorganisms have. Are esterified with compounds or organic acids, and a large amount of esterified products are accumulated in the hydrophobic organic solvent. in this case,
Low molecular weight water-soluble organic acids, alcohols or acetyl-CoA produced by organic acid fermentation, alcohol fermentation or acetyl-CoA fermentation are sequentially esterified before reaching a concentration in the carrier that causes toxicity or feedback inhibition. Since it is used for the reaction, it does not show toxicity to microorganisms. Further, poorly water-soluble alcohols or aldehyde compounds in hydrophobic organic solvents, and also poorly water-soluble alcohols or organic acids produced by microbial conversion reaction are major advantages of interfacial bioreactors, which are toxicity mitigation at solid / liquid interface. Based on the phenomenon, the added concentration and the accumulated concentration can be remarkably increased as compared with the prior art. Furthermore, the esters produced by esterification can also be accumulated at a high concentration level far exceeding the prior art based on the toxicity mitigation phenomenon.

In the above-mentioned esterification reaction, when water-soluble alcohols are produced by fermentation, these are organic acids produced by microbial oxidation of poorly water-soluble alcohols or aldehyde compounds in a hydrophobic solvent,
Also, by fermentation, water-soluble organic acids or acetyl-Co
When A is produced, it is esterified with alcohols produced by the microbial reduction of aldehyde compounds in a hydrophobic solvent.

Therefore, the first feature of the present invention is to utilize the growth phenomenon of a microorganism in an organic solvent, which is an advantage of the interfacial bioreactor, and to ferment the microorganism to a water-soluble organic acid, alcohol or acetyl-CoA. Is produced, and this is successively incorporated into the esterification reaction by the microorganism. In the area of traditional biotechnology,
Since the fermentation method and the microbial conversion method are completely different research fields or applied fields, and therefore the research or application in each field has been developed independently, there has been no attempt to combine the fermentation method and the microbial conversion method. However, in the present invention, since water-soluble organic acids, alcohols or acetyl-CoA are produced by fermentation and these are incorporated into the sequential esterification reaction, the fermentation and the microbial esterification reaction are fused in a preferable situation for the microorganism. be able to.

The second feature of the present invention is that the water-soluble organic acids, alcohols or acetyl-containing compounds used for esterification are used.
It is possible to suppress the expression of toxicity or feedback inhibition of CoA against microorganisms. Since water-soluble organic acids, alcohols or acetyl-CoA exhibit strong toxicity or feedback inhibition to microorganisms, when these are added to the microbial reaction system according to the conventional technique, the addition concentration must be set low. As a result, low reaction rate and low yield are forced. Further, since commercially available acetyl-CoA salt is extremely expensive, it is practically impossible to use it as an acetyl donor. However, in the present invention, water-soluble organic acids, alcohols or acetyl-CoA produced by fermentation are used.
Is subjected to a sequential esterification reaction before reaching a concentration that causes toxicity or feedback inhibition to microorganisms, and therefore does not cause a problem of toxicity or feedback inhibition expression of the water-soluble organic acids, alcohols or acetyl-CoA. Therefore, the fermentation and esterification reaction and the redox reaction can be stably continued.

A third feature of the present invention is that the sparingly water-soluble alcohols or aldehydes used for microbial reduction or oxidation reduce the added concentration and the accumulated concentration of conversion products due to the toxicity mitigation phenomenon in the interfacial bioreactor. It can be set high. The added concentration of the raw material is a very important factor for improving productivity and yield, and further for separating and purifying the product, and greatly contributes to the reduction of manufacturing cost.

The fourth feature of the present invention is that the produced water-insoluble ester is automatically transferred to the reaction solvent layer of the interfacial bioreactor, which avoids the toxicity of the ester and the esterification of the reaction equilibrium. It is in the point that shift, improvement of reaction rate, etc. can be achieved. Accumulation of high-concentration of the ester, which is the product, in the reaction solvent contributes greatly to the reduction of manufacturing cost together with the third feature.

The interfacial bioreactor which can be used in the fusion method of the invention can be known per se,
For the material, size and form of the hydrophilic immobilization carrier used and the reaction solvent that can be used, see, for example, Japanese Patent Laid-Open No.
The thing described in the Unexamined-Japanese-Patent No. 91878 can be used.

Examples of the hydrophilic immobilization carrier include natural polymers such as alginic acid, carrageenan, starch matrix, agar, and cellulose materials such as filter plates; synthesis of polyvinyl alcohol, urethane polymer, polyacrylamide, polyacrylic acid and the like. Polymer: a plate-like material of an inorganic porous material such as a foam glass plate can be used.
When the carrier is repeatedly reused, it is preferable to use a gel-like synthetic polymer or a plate-like material of an inorganic porous material. Also, in order to impart strength, a strong filter plate or a foam glass plate may be used. It is preferable to use a porous plate or a plate-shaped or rod-shaped material such as stainless steel as the skeleton of the carrier.

Further, the hydrophobic organic solvent which is a reaction solvent is preferably one which is not substantially toxic to the immobilized microbial cells, specifically, for example, hexane, heptane, octane and nonane. Carbon number such as decane
Normal paraffins or liquid paraffins represented by methane hydrocarbons of 20 to 20; isoparaffins such as isooctane; pentylbenzene, hexylbenzene,
Normal alkylbenzenes having 5 to 15 carbon atoms in the aliphatic chain such as heptylbenzene and octylbenzene; isoalkylbenzenes such as cumene; alicyclic hydrocarbons such as cyclohexane; aliphatic ethers such as dihexyl ether; dibutyl phthalate And the like; aliphatic esters such as ethyl decanoate; silicone oils such as polydimethylsiloxane;

The reaction solvent to be used is not limited to one kind, and may be a composite solvent system of two or more kinds in consideration of solubility of raw materials and products, toxicity to microorganisms and the like.

As a specific example of the interfacial bioreactor, for example, paraffins are used as a reaction solvent and a polyvinyl alcohol-coated filter plate is used as an immobilization carrier, which are arranged horizontally or vertically in the reaction solvent. An array packed can be used.

On the other hand, a water / organic solvent two-phase reaction method [M. D. Hocknull and M. D. Lilly, Appl. Micr
obiol. Biotechnol., 33, 148 (1990)], the fusion of the fermentation and the microbial conversion reaction in the present invention is also possible, but in that case, an organic solvent or sparingly water-soluble alcohols, aldehydes, or aldehydes to be used for esterification or Since the toxicity of organic acids is manifested, the results obtained in the interfacial bioreactor cannot be exceeded.

Regarding the supply of oxygen for the growth of microorganisms, when the microorganisms are to be subjected to organic acid fermentation or alcohol fermentation, they are rather grown under anaerobic conditions without oxygen supply.
Although it is better to carry out the reaction, in general, microorganisms consume oxygen in the reaction solvent in the process of growth, and therefore it is not always necessary to perform operations such as degassing or nitrogen substitution of the reaction solvent.
On the other hand, when acetyl-CoA is produced, it is preferable to ferment it under aerobic conditions, and therefore the interfacial bioreactor is particularly effective.

The nutrients necessary for the growth of microorganisms include water-soluble organic acids, alcohols or acetyl-CoA.
Since it is necessary to produce by fermentation, it is essential that the water-soluble organic acids, alcohols or raw materials for fermentation production of acetyl-CoA are included,
Examples of the fermentation raw material include sugars such as glucose, starch and sucrose. The nutrient source is these water-soluble organic acids, alcohols or acetyl-CoA.
In addition to a substrate for fermentative production of
It may contain the general medium components described in JP-A-8.

Sugars such as glucose as a fermentation raw material are
It is contained in an aqueous medium at a concentration that does not inhibit the activities of alcohol oxidase and aldehyde oxidase. If the sugar concentration in the medium is too high, the growth of microorganisms, enzyme activity, and fermentation activity are inhibited, but generally, the fermentation ability can be easily expressed at a concentration of about 3 to 5%. It should be noted that increasing the concentration of sugars may inhibit the microbial oxidation reaction in some cases. Specifically, if the glucose concentration exceeds 3%, oxidation inhibition occurs. May come. This phenomenon is important in that it can suppress unnecessary oxidation reactions that occur when microbial reduction is fused.
On the other hand, microbial reduction is less affected by glucose than oxidation. In addition, it is desirable that the optimum addition concentration of saccharides be individually experimentally determined.

In the present invention, examples of the organic acids produced by the organic acid fermentation of the saccharides include formic acid, acetic acid, propionic acid, lactic acid, butyric acid, amino acids and the like, and the organic acids produced by the alcohol fermentation of the saccharides. The alcohols include, for example, lower alcohols such as ethanol, propanol and butanol; diols such as butanediol. According to the present invention, these fermentation products are subjected to a microbial esterification reaction with poorly water-soluble alcohols or organic acids produced by microbial reduction or oxidation of poorly water-soluble alcohols or aldehydes.

As the sparingly water-soluble alcohols or aldehydes in the reaction solvent which can be subjected to microbial reduction or oxidation,
There is no particular limitation as long as it is soluble in a hydrophobic organic solvent which is a reaction solvent, and examples thereof include medium-chain alkanols such as 1-octanol and 1-decanol; long-chain alkanols such as 1-octadecanol; octanal. And alkanes such as decanal; terpene alcohols such as citronellol, menthol, and geraniol; terpene aldehydes such as citronellal and geranylaldehyde; aromatic alcohols such as phenylpropanol and phenylbutanol.

The addition amount of these sparingly water-soluble alcohols or aldehydes can be generally set to a high concentration based on the toxicity mitigation phenomenon in the interfacial bioreactor.
It also depends on the microbial species and the polarity of the raw material. For example,
Terpene alcohols such as citronellol and medium-chain alkanols such as 1-octanol particularly damage water-soluble alcohols, organic acids, and acetyl-CoA production pathways, and thus avoid their toxicity even with an interfacial bioreactor. Depending on the microbial species, it may be possible to add only a concentration of 1 to 4%, but in such a case, the raw materials terper alcohol and alkanols are added by the sequential addition method (fed-batch method). By doing so, it can be easily solved.

Whether to employ microbial reduction or oxidation in the present invention can be determined according to the water-soluble organic acid, alcohol or acetyl-CoA produced by fermentation. Specifically, when a water-soluble organic acid or acetyl-CoA is accumulated by fermentation, it is preferable to produce an alcohol from an aldehyde compound by microbial reduction, while when a water-soluble alcohol is produced by fermentation, It is preferable to produce a poorly water-soluble organic acid from a poorly water-soluble alcohol by microbial oxidation. Microbial oxidation of aldehyde to organic acid is also possible, but in that case, the reduction reaction may proceed at a faster rate.

The microorganism species usable in the present invention include the ability to produce water-soluble organic acids, alcohols or acetyl-CoA by fermentation of sugars, the ability to reduce poorly water-soluble aldehydes dissolved in the reaction solvent of an interfacial bioreactor, or alcohol. There is no particular limitation as long as it has the ability to oxidize aldehydes and aldehydes and the ability to produce lipase, esterase or alcohol acetyl tranferase. For example, Propionibacteriu (Propionibacteriu)
m), genus Acetobacter, genus Lactobacillus, genus Pseudomonas
as), Bacillus and other bacteria; Issatchenkia, Hansenul
a), yeasts such as Candida, Pichia, Saccharomyces, Kluyberomyces; Rhizopus (R)
hizopus), Aspergillus (Aspergillus), Penicillium (Penicillium) and other filamentous fungi.
More specifically, for example, Propionibacterium shermanii (P. shemanii), Acetobacter aceti (A. aceti), Lactobacillus brevis (L. brevi)
s), Pseudomonas fluorescens (P. fluoresc
ens), Hansenula Saturnus,
H. anomala, Candida utilis, Isatienchia terricola, B. subtilis subsp. Niger, Saccharomyces cerevisiae. cerevisiae), R. delemar, Aspergillus terreus, Penicillium notaum (P. notau)
m), Picia Heedii, P. quercuum, etc. In particular, when a microorganism capable of producing lipase or esterase having excellent stereoselectivity, for example, Pseudomonas sp., Candida sp., Aspergillus sp., Penicillium sp., Etc. is used, the type of alcohols or organic acids in the reaction solvent In some cases, the esterification reaction proceeds in a stereoselective manner, so that it can also be used as an optical resolution method.

The adhesion of these microorganisms to the above-mentioned hydrophilic immobilization carrier is carried out, for example, by allowing the hydrophilic immobilization carrier to be impregnated with an aqueous medium (medium) containing a nutrient source containing the above-mentioned saccharide as an essential component. It can be carried out by inoculating a desired microorganism and culturing for about 0 to 5 days under optimal conditions of the microorganism.

If necessary, the hydrophilic immobilization carrier to which the microorganisms are adhered is supplemented with an aqueous medium containing a nutrient source, and sparingly water-soluble alcohols and / or aldehyde compounds are dissolved depending on the properties of the microorganisms. The hydrophobic organic solvent is added and the culture is continued until the ester is sufficiently accumulated. The culturing temperature at that time can be a temperature preferable for the microorganism to be used, for example, an optimum temperature between about 20 and about 40 ° C.

The ester accumulated at a high concentration in the reaction solvent is recovered according to a general separation and purification method, for example, a step of concentrating by distilling off the reaction solvent by distillation or an adsorbent such as silica gel or alumina. It can be carried out by a method of adsorbing and separating the produced esters.

[0031]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples, and various applications are possible.

Example 1 In a glass dish having a diameter of 21 cm, 5 g of peptone, 3 g of malt extract, 1 g of magnesium sulfate and 40 g of glucose.
200 ml of an agar plate medium (pH 6.0) consisting of 20 g of agar, 20 g of agar and 1 liter of distilled water was dispensed, and Hansenula Saturnus IFO 0809 was added thereto.
2 ml of the 1-day culture solution of was inoculated with a conradi stick.
After overnight static culture, 2% citronellal / decane solution 7
Fermentation-conversion test under shaking culture was carried out by layering 0 ml for 5 days. As a result of gas chromatographic analysis of the decane layer, microbial esterification of citronellol produced by microbial reduction of citronellal and acetic acid produced by acetic acid fermentation was observed, and the accumulation of citronellyl acetate in the decane layer was 1 Confirmed from day one. As a result of the 5-day fermentation-conversion test, the accumulated concentration of citronellyl acetate in the decane layer reached 19 g / l.

Example 2 Gel of 10% polyvinyl alcohol (ENTV-50
0, filter plate (6 × 13c) covered with Kansai Paint
The internal pores of m) were replaced with a liquid medium having the same composition as in Example 1, and P. quercuum was added to the liquid medium.
Thirteen plate-shaped carriers were inoculated with the 1-day culture solution of IFO 0949 and cultured for 1 day, and then filled in a stainless steel tank with an internal volume of 3 l in a vertical arrangement using a stainless frame.

1 l of a 3% geranyl aldehyde / undecane solution was dispensed into this and reacted under aeration for 5 days while stirring with an agitator installed at the bottom of the reactor. As a result of gas chromatography analysis of the undecane layer, microbial esterification of geraniol, which is a geranylaldehyde microbial reduction product, and acetic acid produced by acetic acid fermentation was observed, and the accumulated concentration of geranyl acetate in the undecane layer was 5 days. Reached 28 g / l.

Example 3 An agar plate medium having the same composition as in Example 1 was dispensed into a glass dish having a diameter of 21 cm. Isachenkia
2 ml of a 1-day culture of Issatchnkia terricola IFO0933 was inoculated using a conradi stick. 1
After static culture at night, 70 ml of 3% decanol / decane solution
Were subjected to a fermentation-conversion test for 5 days. As a result of gas chromatography analysis of the decane layer, accumulation of ethyl decanoate, which is an esterification product of decanoic acid, which is a microbial oxidation product of decanol, and ethanol produced by ethanol fermentation was confirmed, and the accumulated concentration in 10 days was 10
g / l was reached.

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Claims (1)

[Claims] 1. A hydrophilic immobilization carrier on which a microorganism having an organic acid fermenting ability, an alcohol fermenting ability, or an acetyl coenzyme A fermenting ability and capable of producing a lipase, an esterase, or an alcohol acetyl transferase is attached, Alcohols and / or aldehydes that are insoluble or sparingly soluble in water in the presence of a water-soluble medium containing a saccharide that is a nutrient source for microorganisms and a fermentation raw material at a concentration that does not inhibit the activity of alcohol oxidase and aldehyde oxidase. A hydrophobic organic solvent containing a compound is brought into contact with the microorganism on the carrier to grow the microorganism at the contact interface, and by the action of the growing microorganism, a water-soluble organic acid, alcohol or acetylcoenzyme A from the fermentation raw material. Fermentation production of alcohol, and a slight amount of alcohol produced by dissolving the fermentation product and a hydrophobic organic solvent Fermentation and microbial conversion reaction characterized by carrying out microbial esterification reaction between water-insoluble or sparingly soluble alcohol or organic acid produced by physical oxidation and microbial oxidation or reduction of aldehyde compound Fusion method.