JPH09131198A - 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 conversoin reaction comprises sticking a microorganism having an organic acid fermentation ability, an alcohol fermentation ability or an acetyl-Co A fermentation ability and having a lipase, esterase or alcohol acetyltransferase-producing ability to a hydrophilic immobilization carrier, bringing a hydrophobic organic solvent into contact with the microorganism on the carrier in the presence of an aqueous medium containing a saccharide as the nutritive source of the microorganism 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 simultaneously subjecting the fermentation product and a water-insoluble or slightly soluble alcohol or organic acid in the hydrophobic organic solvent to a microbial esterification reaction.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method of integrating fermentation and microbial conversion reaction in which a water-soluble organic acid or alcohol, which is a fermentation product of sugars such as glucose, is incorporated into an esterification reaction in a microbial manner.

[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 the lipase or esterase is deactivated, so that high-concentration ester synthesis is very difficult. [B. Cambou and A. M. Klibanov, Biotechn
ol. Bioeng., 26, 1449 (1984)].

In order to overcome the above-mentioned deficiencies of commercially available lipases or esterases, screening of microorganisms capable of producing novel lipases or esterases and microbial conversion methods using the microorganisms as they are as biocatalysts are widely used. Attempts have been made, but microorganisms generally die or cannot grow in organic solvents, and it is difficult to make the esterification reaction proceed stably.

[0005] Further, although ester formation by alcohol acetyl tranferase is known, there is no report aiming at the synthesis of useful ester since there is interest only in the production of sake aroma ester.

On the other hand, an interfacial bioreactor which uses 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 not possible 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, Bios
ci. Biotech. Biochem., 56, 1515 (199
2)].

[0007]

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. Effectively utilizing the growth ability of microorganisms and the fermentation ability of the microorganisms, while fermentatively producing water-soluble organic acids, alcohols or acetylcoenzyme A which is a precursor thereof, these water-soluble organic acids, alcohols or The inventors have found that the problem can be solved by successively incorporating acetyl coenzyme A into a microbial esterification reaction and fusing fermentation and microbial esterification reaction, and completed the present invention.

Thus, according to the present invention, the hydrophilic immobilization carrier has organic acid fermentation ability, alcohol fermentation ability or acetylcoenzyme A fermentation ability and lipase, esterase or alcohol acetyltransferase. In the presence of an aqueous medium containing a saccharide, which is a fermentation raw material which is a nutrient source for the microorganism,
A hydrophobic organic solvent containing an alcohol or an organic acid that is insoluble or sparingly soluble in water (hereinafter collectively referred to as “poorly soluble in water”) is brought into contact with the microorganism on the carrier to grow the microorganism at the contact interface, By the action of proliferating microorganisms,
While fermentation-producing a water-soluble organic acid, alcohol or acetyl coenzyme A (hereinafter, abbreviated as “acetyl-CoA”) from the fermentation raw material, the microorganism and the sparingly water-soluble alcohol or organic acid in the hydrophobic organic solvent Provided is a method for fusing fermentation and microbial conversion, which is characterized by carrying out a microbial esterification reaction between the two.

According to the present invention, the microorganisms that grow in the solid / liquid interface consisting of the carrier / hydrophobic organic solvent in the interfacial bioreactor utilize fermentation raw materials consisting of sugars such as glucose, starch and sucrose in the carrier. To produce low molecular weight water-soluble organic acids, alcohols or acetyl-CoA by fermentation. These fermented products are esterified with poorly water-soluble alcohols or organic acids in a hydrophobic organic solvent by the enzymatic action of lipase, esterase or alcohol acetyl transfruase which the microorganism has, and then, in a hydrophobic organic solvent. A large amount of esterified product is accumulated. In this case, low molecular weight water-soluble organic acids, alcohols or acetyl-Co produced by organic acid fermentation, alcohol fermentation or acetyl-CoA fermentation.
Since A is subjected to a sequential esterification reaction before reaching a concentration at which toxicity is exhibited in the carrier, it does not exhibit toxicity to microorganisms. In addition, sparingly water-soluble alcohols or organic acids in hydrophobic organic solvents can be significantly increased in addition concentration and accumulated concentration compared with the conventional technology based on the toxicity mitigation phenomenon at the solid / liquid interface, which is a great advantage of the interfacial bioreactor. It is possible. 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 microbial esterification reaction, when water-soluble organic acids are produced by fermentation, they are naturally mixed with alcohols in a hydrophobic solvent and by fermentation to produce water-soluble alcohols. When produced, it is esterified with organic acids in a hydrophobic solvent.

On the other hand, when acetyl-CoA is produced by fermentation, the acetyl-CoA is esterified with an alcohol in an organic solvent as an acetyl donor.

Therefore, the first feature of the present invention is to utilize the growth phenomenon of microorganisms in an organic solvent, which is an advantage of the interfacial bioreactor, and to ferment the microorganisms with 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 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 sequentially incorporated into the esterification reaction, the fermentation and the microbial esterification reaction can be fused in a state preferable for the microorganism.

The second feature of the present invention is that water-soluble organic acids, alcohols or acetyl-containing compounds used for esterification are used.
That is, the expression of toxicity of CoA to microorganisms can be suppressed. 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 inevitable. 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, the water-soluble organic acids, alcohols or acetyl-CoA produced by fermentation are subjected to a sequential esterification reaction before reaching a concentration at which toxicity or feedback inhibition is exhibited for microorganisms, There is no problem of toxicity or feedback inhibition expression of water-soluble organic acids, alcohols or acetyl-CoA, and the fermentation and esterification reaction can be continued stably.

The third characteristic of the present invention is that the sparingly water-soluble alcohols or organic acids used for esterification are
Due to the toxicity mitigation phenomenon in the interfacial bioreactor,
The point is that the added concentration 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 since the water-insoluble ester produced is automatically transferred to the reaction solvent layer of the interfacial bioreactor, it is possible to avoid toxicity of the ester or to esterify the reaction equilibrium. It is in the point that shift, improvement of reaction rate, etc. can be achieved. Furthermore, when the poorly water-soluble alcohols or organic acids of the substrate are highly toxic, these substrates can be sequentially added at a low concentration by the fed-batch method. Accumulation of high-concentration products as esters in the reaction solvent, together with the third feature, contributes greatly to the reduction of manufacturing cost.

Further, the fifth feature of the present invention is that, when the concentration of sugars in the carrier is increased in order to improve the efficiency of fermentation production, a side reaction caused by a growing microorganism, which occurs in parallel with the esterification reaction, for example, a microorganism. The point is that the selective oxidation reaction can be effectively suppressed. Alcohol dehydrogenases that catalyze microbial oxidation reactions are generally known to be inhibited by high concentrations of sugars such as glucose [U. Lutstrof and R. Megnet, Arch. Bioch
em. Biophys., 126, 933 (1968); S. Ya
dav, et al., J. Ferment. Technol., 57, 244
(1979)]. Therefore, in the case of improving the fermentation ability with a high concentration of saccharides according to the present invention, at the same time, the side reaction, that is, the microbial oxidation reaction is suppressed, and the yield and yield of the resulting ester are significantly improved.

The interfacial bioreactor which can be used in the fusion method of the invention can be known per se,
Examples of the material, size and shape of the hydrophilic immobilization carrier used, usable reaction solvent, and the like include, for example, JP-A No. 5/1999.
The thing described in the Unexamined-Japanese-Patent No. 91878 can be used.

Examples of hydrophilic immobilization carriers 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 transparent 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;

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

On the other hand, a water / organic solvent two-phase reaction method [M. D. Hocknull and M. D. Lilly, Appl. Mi
crobiol. 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, the organic solvent or sparingly water-soluble alcohols or organic acids to be used for esterification can be used. The results obtained in the interfacial bioreactor cannot be exceeded because of the onset of toxicity.

Regarding the supply of oxygen for microbial growth, when organic acid fermentation or alcoholic fermentation is carried out by microorganisms, it is better to grow and react under anaerobic conditions without oxygen supply. Since the microorganism consumes oxygen in the reaction solvent in the process of growth, it is not always necessary to perform an operation 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, so that 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.

If the concentration of saccharides such as glucose as a fermentation raw material in the aqueous medium is too high, it inhibits the growth of microorganisms, the enzyme activity and the fermentation ability.
A concentration of about wt% is appropriate, but by raising the concentration of saccharides to about 3 wt% or more, undesirable side reactions such as oxidative decomposition reaction of poorly water-soluble alcohols or organic acids in the reaction solvent are significantly increased. In many cases, it is suppressed, and as a result, the yield of ester is significantly improved.
It is desirable to experimentally determine the optimum addition concentration of saccharides individually.

In the present invention, examples of the organic acids produced by the organic acid fermentation of the above-mentioned saccharides include formic acid, acetic acid, propionic acid, lactic acid, butyric acid, amino acids and the like, and also produced by the alcohol fermentation of the above-mentioned 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 and acetyl-CoA are subjected to a microbial esterification reaction with poorly water-soluble alcohols or organic acids as described below.

The sparingly water-soluble alcohols or organic acids in the reaction solvent used for the esterification reaction are not particularly limited as long as they can be dissolved in the hydrophobic organic solvent which is the reaction solvent, and for example, 1- Medium-chain alkanols such as octanol and 1-decanol; long-chain alkanols such as 1-octadecanol; citronellol and menthol,
Terpene alcohols such as geraniol; aromatic alcohols such as phenylpropanol and phenylbutanol; alkanoic acids such as octanoic acid and decanoic acid; 2
-Aromatic acids such as hydroxy-4-phenylbutanoic acid; Terpenic acids such as citronellic acid and geranilic acid;
And the like. The addition amount of these sparingly water-soluble alcohols or organic acids can be generally set to a high concentration based on the toxicity mitigation phenomenon in the interfacial bioreactor, but is also influenced by 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 their toxicity is largely avoided even by an interfacial bioreactor. Depending on the microbial species, it may be possible to add only a concentration of 1 to 5%. In such a case, the raw materials terpell alcohol and alkanols are added by the sequential addition method (fed-batch method). It can be solved by doing.

Even in the case of organic acids or alcohols having relatively high water solubility, the ester of the present invention can be obtained by controlling the addition amount to the reaction solvent to a low level and applying the stepwise addition method. It can be used as a substrate for chemical reaction.

The microbial species that can be used in the present invention have the ability to produce water-soluble organic acids, alcohols or acetyl-CoA by fermentation of sugars (organic acid fermentation ability, alcohol fermentation ability or acetyl-CoA fermentation ability). And is capable of producing lipase, esterase or alcohol acetyl tranferase, there is no particular limitation, and for example, Propionibacterium (Propiobacterium spp.
nibacterium), Acetobacter, Lactobacillus, Pseudomonas, Bacillus, etc .; Issatchenkia, Hansenula, Candida. , Saccharomyces, Kluyveromyces (Klu
yberomyces), yeasts such as Pichia; Rhizopus, Aspergillu
s), Penicillium and other filamentous fungi. L. brevis), Pseudomonas fluorescens, H. saturnus, H. anomala
a), Candida utilis, Isatienchia tericola, B. subtili subsp. niger
ger), Saccharomyces cerevisiae
e), P. heedii, P. quercuum, R. delemar
mar), Aspergillus terreus (A. terreus), Penicillium notaum (P. notaum) and the like.
In particular, microorganisms capable of producing lipase and esterase excellent in stereoselectivity, for example, when using microorganisms such as Pseudomonas sp., Candida sp., Aspergillus sp.
Depending on the type of alcohols or organic acids in the reaction solvent, the microbial esterification reaction proceeds stereoselectively, and thus can 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 a hydrophobic water-soluble alcohol or organic acid is dissolved depending on the nature of the microorganisms. The 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 esters.

[0032]

EXAMPLES 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.

Example 1 In a glass petri dish having a diameter of 21 cm, 5 g of peptone, 3 g of yeast extract, 3 g of malt extract, and 1 magnesium sulfate.
g, glucose 40 g, agar 20 g and distilled water 1 lit
200 ml of agar plate (pH 6.0)
Dispense into the H.saturnas (H. satur)
nus) IFO 0809 2 ml of a daily culture solution was inoculated with a conradi stick. After static culture overnight, fermented with 70% of 2% citronellol / decane solution for 5 days-
A conversion test was conducted. As a result of the gas chromatography analysis of the decane layer, the accumulation of citronellyl acetate in the decane layer was confirmed from the first day after the start of the reaction, and the accumulated concentration reached 18 g / l on the fifth day.

Example 2 Gel of 10% polyvinyl alcohol (ENTV-50
0, filter plate (6 × 13c) covered with Kansai Paint
The internal cavity b of m) was replaced with a liquid medium having the same composition as in Example 1, and Pichia heedii IF was added thereto.
Thirteen plate-shaped carriers were inoculated with a 1-day culture solution of O 10019 and cultured for 1 day, and then filled in a stainless steel tank having an internal capacity of 3 liters in a vertical arrangement using a stainless frame.

3% geraniol / undecane solution
A magnet placed on the bottom of the reactor by dispensing 1 liter
The mixture was reacted for 5 days under stirring. Undecane layer gas chromatograph
As a result of the analysis by matography,
Significant accumulation of geranyl acetate was confirmed from the first day after the reaction started.
The accumulated concentration reached 26 g / l on the 5th day. Example 3 A glass petri dish having a diameter of 21 cm and the same set as in Example 1
Adult agar plate medium was dispensed. Isachenkia
Of Issatchnkia terricola IFO0933
2 ml of the 1-day culture solution was used to inoculate using a conradi stick. 1
After overnight static culture, add 70 ml of 2% decanoic acid / decane solution.
Fermentation-conversion test was carried out for 5 days by layering. Deccan layer moth
As a result of analysis by chromatography, in the decane layer
Accumulation of ethyl decanoate was confirmed from the first day after the reaction started.
The accumulated concentration reached 9 g / l on the 5th day.

Example 4 An agar plate medium having the same composition as in Example 1 was dispensed into a glass dish having a diameter of 21 cm. Bacillus subtilis su
bsp. niger) IFO 3108 2 ml daily culture was inoculated with a conradi stick. After overnight static culture, 1%
A fermentation-conversion test was conducted for 5 days by overlaying 70 ml of the menthol-decane solution. As a result of the gas chromatography analysis of the decane layer, accumulation of ethyl decanoate in the decane layer was confirmed from the first day of the reaction start, and the accumulated concentration reached 6 g / l on the fifth day.

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Claims (6)

[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, In the presence of an aqueous medium containing a saccharide as a nutrient source of a microorganism and a fermentation raw material, a hydrophobic organic solvent containing an alcohol or an organic acid insoluble or sparingly soluble in water is brought into contact with the microorganism on the carrier. The fermentation product and water in the hydrophobic organic solvent are grown while fermenting the water-soluble organic acid, alcohol, or acetylcoenzyme A from the fermentation raw material by the action of the growth microorganisms to grow the microorganism at the contact interface. Characterized by carrying out a microbial esterification reaction with an insoluble or sparingly soluble alcohol or organic acid Method of fusion fermentation and microbial conversion. 2. The microorganism is Propionibacterium shermanii, Acetobactor aceti, Lactobacillus brevis, Pseudomonas fluorescen.
s), Hansenula saturnu
s), Hansenula anomala, Pichiaheedii, Pichiaquercuum, Candid utilis.
a utilis), Issatchenkia
terricola), Bacillus subtilis subspice niger (Bacillus subtilissubsp. niger) Saccharomyces cerevisiae (Saccharomyces cerevisiae),
The fusion method according to claim 1, which is selected from Rhizopus delemar, Aspergillus terreus, and Penicillium notaum. 3. The fusion method according to claim 1, wherein the saccharide as a fermentation raw material is selected from glucose, starch and sucrose. 4. The hydrophobic organic solvent is normal paraffin,
The fusion method according to claim 1, which is selected from liquid paraffin, isoparaffin, normal alkylbenzene, isoalkylbenzene alicyclic hydrocarbon, and aliphatic ethers. 5. The fusion method according to claim 1, wherein the water-insoluble or sparingly soluble alcohol is selected from medium-chain or long-chain alkanols, terpene alcohols and aromatic alcohols. 6. The fusion method according to claim 1, wherein the water-insoluble or sparingly soluble organic acid is selected from alkanoic acids, aromatic acids and terpenic acids.