JPH1128097A - Microbial optical resolution of racemic primary alcohol - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microbial optical resolution process of a racemic primary alcohol by using an interfacial bioreactor. SOLUTION: In an interfacial bioreactor in which a hydrophilic immobilized carrier including a saccharide-containing liquid medium interfacially contacts with an organic solvent containing a racemic primary alcohol, a microorganism in Pichia capable of producing an alcohol-acetyl transferase is inoculated on the surface of the hydrophilic immobilized carrier and proliferated thereby enantio-selectively acetylating the racemic primary alcohol.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for the microbiological resolution of racemic primary alcohols using an interfacial bioreactor.

[0002]

2. Description of the Related Art In recent years, demand for optically active alcohols as synthetic raw materials for pharmaceuticals, agricultural chemicals, ferroelectric liquid crystal materials and the like has been increasing.
Among these optically active alcohols, various methods using lipase have been reported as enzymatic preparation methods for secondary alcohols [for example, Kakeya, H., et al., Agric, Bio].
l. Chem., 55, 1877 (1991); and Klibano
v, A. M., Biotechnol. Bioeng., 26, 1449 (1984); M
organ, B., et al., J. Org. Chem., 57, 3231 (1992)
Etc.].

However, the enzymatic optical resolution method for racemic primary alcohols generally does not have a very high enantioselectivity, and particularly for a substrate such as citronellol whose asymmetric point is far from the reaction site, almost no enantioselective method is used. Could not be expected [for example, Kawamoto, T., et al.,
Biocatalysis, 1, 137 (1987); Iwai, M., et al., Agr.
I c. Biol. Chem., 44, 2731 (1980), etc.]. Only one enantioselective resolution of racemic citronellol using porcine liver carboxylesterase has been reported [Cambou, B. et al. and Klibanov. A. M., J. Am. Che
m. Soc., 106, 2687 (1984)], it is difficult to supply this enzyme in large quantities, and this method is not suitable for mass production.

On the other hand, as an organic synthesis method of an optically active primary alcohol, an asymmetric hydrogenation method using a BINAP salt is known [Takaya, H., et al., J. Am. Am. Chem. Soc., 1
09, 1596 (1987)], but these asymmetric reagents are generally expensive, and it is desired to develop a low-cost and efficient method for preparing an optically active primary alcohol.

[0005]

DISCLOSURE OF THE INVENTION The present inventors have previously proposed, as a novel method for synthesizing acetate, acetyl coenzyme A (hereinafter referred to as "acetyl-CoA") which is a metabolite of glucose added to a carrier of an interfacial bioreactor. ) May be referred to as an acetyl donor, and the primary alcohol added to the organic solvent phase is acetylated under the action of alcohol acetyltransferase of microorganisms growing on the solid / liquid interface between the immobilized carrier and the organic solvent phase. (Hereinafter referred to as a metabolism-microbial conversion fusion system) [Oda, S., et al., Appl. Bnviron. Microbiol., 6
2, 2216 (1996)]. However, the microorganism used in this report, specifically Hansenula saturnus IFO 0809, does not have the ability to resolve racemic primary alcohols such as citronellol, and citronellol has (S)-, (R )-Both bodies undergo acetylation at a similar rate. Therefore, the metabolism-microbial conversion fusion system which the present inventors have reported so far is merely a novel non-enantioselective acetylation system, and is not directed to optical resolution.

Accordingly, the present inventors screened a microorganism capable of producing metabolism-microbial conversion fusion ability, that is, a microorganism capable of producing citronellyl acetate in a system without an acetyl donor using an interfacial bioreactor. Presence of yeast cells capable of accumulating citronellyl acetate in yeast, especially Piscia kluiberi (Pi
Chia kluyveri) IFO 1165 was found to have the ability to preferentially acetylate (S) -citronellol with high enantioselectivity, leading to the completion of the present invention.

Thus, according to the present invention, in an interfacial bioreactor in which a hydrophilic immobilized carrier containing a liquid medium containing a carbohydrate and an organic solvent containing a racemic primary alcohol are brought into interface contact, 1 on the surface of
Inoculating and growing microbial cells belonging to the genus Pichia, which have the ability to produce alcohol acetyltransferase that enantioselectively acetylates lower alcohols,
There is provided a microbiological optical resolution method for a racemic primary alcohol, which comprises enantioselectively acetylating the racemic primary alcohol.

Hereinafter, the microbiological resolution method of the present invention will be described in more detail.

The interfacial bioreactor used in the method of the present invention comprises a hydrophilic immobilized carrier (impregnated) containing a liquid medium containing saccharides assimilated by microbial cells, and a substrate that comes into contact with the surface of the carrier. And an organic solvent containing a racemic primary alcohol. Examples of the hydrophilic immobilization carrier to be used 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, etc. Polymer: a plate-like product of an inorganic porous material such as a foam glass plate can be used. When the carrier is repeatedly used and reused, it is preferable to use a gel-like synthetic polymer or a plate-like material of an inorganic porous material, and to impart strength, such as a strong filter plate or a foam glass plate. It is preferable to use a simple porous plate or a plate-like or rod-like material such as stainless steel as the skeleton of the carrier.

[0010] The hydrophilic immobilized carrier is impregnated with a liquid medium containing saccharides assimilated by microorganisms. The saccharide added to the liquid medium is preferably a monosaccharide or polysaccharide capable of assimilating the microorganism and metabolizing and producing acetyl coenzyme A (acetyl-CoA), for example, glucose, fructose, starch and the like. No. In addition to the carbohydrates, for example, Japanese Patent Application Laid-Open No. 5-9187
No. 8 publication may contain general medium components. The concentration of the saccharide in the liquid medium is not particularly limited, but is usually 1 to 35% by weight, particularly 10 to 10% by weight.
A range of 30% by weight is suitable.

According to the present invention, the hydrophilic immobilized carrier impregnated with the liquid medium described above is a microorganism cell belonging to the genus Pichia which has an ability to produce an alcohol acetyltransferase capable of enantioselectively acetylating a primary alcohol ( (Hereinafter referred to as a microorganism of the genus Pichia), and pre-cultured for about 12 hours to 3 days to grow a microbial membrane on the surface of the carrier.

The microorganism of the genus Pichia which can be inoculated on an immobilization carrier according to the present invention produces an alcohol transferase which assimilates saccharides, metabolizes acetyl-CoA, and enantioselectively acetylates primary alcohols. There is no particular limitation as long as it has the ability to perform, for example, P. quercuum IFO 09
49, IF of P. guilliermondii
O 10107, P. kluyveri IFO 1165 and the like can be used.
FO 1165 is suitable because it has excellent enantioselective acetylation ability of a racemic primary alcohol.

It should be noted that Piscia Kluiberg IFO 11
65 has a relatively strong citronellol oxidizing ability, but if the concentration of saccharides such as glucose in the carrier is set high, this unnecessary oxidation reaction can be suppressed. The suppression of the oxidation reaction is caused by the suppression of the production of the oxidizing enzyme alcohol dehydrogenase due to the presence of high concentration of carbohydrate [Schimpfessel, L., Biochim. Bio
Phys., Acta, 151, 317 (1968)], and the presence of a high concentration of carbohydrate is also preferable for self-production of a large amount of acetyl-CoA. Organic solvents used in the interfacial bioreactor, for example, paraffins such as decane, have about 10 times the oxygen solubility of water.
A will be aerobic and mass-produced.

Next, culturing is carried out while bringing an organic solvent containing a racemic primary alcohol into contact with the surface of the immobilized carrier having the microbial membrane, thereby culturing the microorganisms of the genus Picia. The culturing conditions at that time vary depending on the type of the microbial cells to be used, but usually, the cultivation can be carried out at a temperature of about 20 to about 30 ° C. for about 1 to about 50 days or with shaking. In addition, the inoculation method of microorganisms, reaction management, and the like are described in, for example, Patent No. 2542766, Oda, S., et al., J. Ferment.
Bioeng., 80, 559 (1995). As a result, under the catalyst of alcohol acetyltransferase produced by the microorganisms of the genus Picia, acetyl-CoA, which is metabolized by assimilating saccharides, acts on the racemic primary alcohol incorporated into the microorganisms from the organic solvent. Then, the racemic primary alcohol is enantioselectively acetylated. That is, only one of the (S) -form and the (R) -form of the racemic primary alcohol is selectively acetylated, and a mixture of an optically active primary alcohol and an optically active acetate of the primary alcohol. Is generated.

[0015] The optically active acetate ester and the residual optically active alcohol formed by the enantioselective acetylation of these primary alcohols are automatically transferred to the organic solvent phase of the interfacial bioreactor. Oda, S., et al. and Ohta, H., Biosci. Biotech. Bioche
m., 56, 1515 (1992); Oda, S., et al., J. Ferment.
Bioeng., 78, 149 (1994)], the microorganisms of the genus Pichia located at the solid / liquid interface of the carrier / organic solvent phase in the reactor can avoid the toxicity of these acetates and residual alcohols, and consequently The product accumulates in very high concentrations in the organic solvent phase.

If the racemic primary alcohol to be subjected to optical resolution is extremely toxic, the toxicity can be avoided as much as possible by feeding (adding fed-batch) this alcohol.

Acetyl coenzyme A by metabolism
Fusion of production and acetylation catalyzed by alcohol acetyltransferase can be carried out by a method other than the interfacial bioreactor method, for example, an aqueous-organic solvent two-phase reaction method [Hocknull,
MD, and Lilly, MD, Appl. Microbiol. Biotechno
l, 33, 148 (1990)], but the two-phase reaction method does not exhibit sufficient effect of avoiding toxicity of the substrate alcohol, and contains a liquid-liquid interface or a biocatalyst. The permeation and diffusion of the substrate or product in the aqueous phase is rate-limiting and cannot outperform the results achieved using an interfacial bioreactor.

The separation of one optically active component from the mixture of the resulting optically active acetate of the primary alcohol and the optically active primary alcohol can be carried out, for example, by a separation operation such as chromatography or the like. Microorganisms that selectively oxidize only alcohol without acting on the mixture can be used to induce a mixture of acetate and carboxylic acid, followed by extraction or distillation.

For example, by treating racemic citronellol as a racemic primary alcohol according to the method of the present invention, a quantitative mixture of (S) -citronellyl acetate and (R) -citronellol can be obtained. The mixture can be separated by column chromatography. For example, the mixture can be separated from a microorganism having no hydrolytic capacity of citronellyl acetate but capable of oxidizing alcohol (for example, Pichia angsta) (Pichia angusta), Isachienkia orientalis (Issatchenkia orientalis), etc., and oxidatively induces only (R) -citronellol to (R) -citronellic acid. (C) -citronellyl acetate can be easily separated from ())-citronellic acid, and (S) -citronellol can be quantitatively recovered by hydrolyzing the separated (S) -citronellyl acetate. .

The racemic primary alcohols that can be subjected to the optical resolution of the present invention include, in addition to the above-mentioned racemic citronellol, racemic 2-phenyl-1-propanol, 2-phenyl-1-butanol, 3-phenyl -1
-Highly toxic aromatic alcohols such as butanol.

On the other hand, as the organic solvent for dissolving these racemic primary alcohols, those which are substantially non-toxic to hydrophobic immobilized microbial cells are preferable.
Specifically, for example, 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 Normal alkylbenzenes having 5 to 15 carbon atoms in the aliphatic chain, such as octylbenzene and octylbenzene; isoalkylbenzenes such as cumene; alicyclic hydrocarbons such as cyclohexane; aliphatic ethers such as dihexyl ethers; Examples thereof include aromatic esters such as phthalate; aliphatic esters such as ethyl decanoate; and silicone oils such as polydimethylsiloxane.

Thus, as a specific example of the interfacial bioreactor, for example, a paraffin is used as a reaction solvent, and a filter plate coated with polyvinyl alcohol is used as an immobilization carrier, and these are arranged in a horizontal or vertical direction in the reaction solvent. An array can be mentioned.

[0023]

In general, enzymatic optical resolution of a primary alcohol with lipase or esterase is very difficult, and particularly, an asymmetric point (chiral center) such as citronellol is separated from a reaction point (CH ₂ OH). Enzymatic optical resolution of primary alcohols was virtually impossible [Kawamot
o, T., et al., Biocatalysis, 1, 137 (1987)].

The present invention uses an interfacial bioreactor which exerts an effect on the microbial exchange reaction of a water-insoluble substrate, and is an esterification enzyme, alcohol acetyl, which has never been applied to useful ester synthesis or optical resolution. This is the first application of transferase to optical resolution.

According to the present invention, acetyl-CoA (extremely expensive) required for the enzyme reaction can be produced in-house by using the growing microbial cells, so that the raw material cost can be significantly reduced. is there.

[0026]

EXAMPLES Hereinafter, the optical resolution of racemic primary alcohols by the metabolism-microbial conversion fusion method using the interfacial bioreactor of the present invention will be described more specifically with reference to examples.

Example 1 5.0 g of peptone, 3.0 g of yeast extract as nutrients,
Malt extract 3.0 g, magnesium sulfate heptahydrate 7.0
g, 200 g of glucose, and 850 liters of distilled water were prepared in a nutrient agar plate (agar 20.0 g, pH 6.0) in a glass dish of 21 cm in diameter at a volume of 500 ml / dish. Piscia Kluiberg on this agar plate
3 ml of the IFO 1165 suspension was uniformly inoculated and pre-cultured for 1 day to grow a microbial membrane. Thereafter, 50 ml of a 2% decane solution of racemic citronellol was overlaid, and cultured with shaking at a temperature of 30 ° C. and a shaking number of 40 strokes / min. The decane phase was sampled daily, and the concentrations of citronellol and the produced citronellyl acetate in the decane phase were determined by gas chromatography. The decrease in citronellol and the accumulation of citronellyl acetate were observed from the first day of the reaction, and after 5 days of reaction, the remaining concentration of the former was 10.7 mg / ml and the latter was the accumulated concentration of 11.2 mg / ml.
It became. At this time, the decane phase was recovered, decane was removed to half the amount by distillation, and then citronellyl acetate and citronellol were separated and recovered by column chromatography using a silica gel column. For the latter,
After inducing to citronellic acid by Jones oxidation,
After modification with (R) -1- (naphthylethyl) amine, the optical purity was measured using liquid chromatography. In addition, for the former, after adding ethanol and alkali-hydrolyzing to give citronellol, optical purity was measured as an amine derivative by the same method. As a result, the absolute configuration of the produced citronellyl acetate was (S)
And its enantiomeric excess is 94% ee. (Enantio
meric excess). The absolute configuration of the remaining citronellol is (R), and its enantiomeric excess is 9%.
2% ee. Met.

Example 2 In Example 1, the difference between Piscia Kluiberg IFO 11
Piscia Guillermonde IFO 10 in place of 65
Optical resolution of citronellol was performed in the same manner except that 107 was used. The residual concentration of citronellol is 9.8 g.
/ Ml and the accumulated concentration of citronellyl acetate is 11.2 g /
ml.

Next, as a result of measuring the optical purity, the absolute configuration of the produced citronellyl acetate was (R), and the enantiomeric excess was 69% ee. Met. The absolute configuration of the residual citronellol is (S), and its enantiomeric excess is 66% ee. Met.

Comparative Example 1 In the same manner as in Example 1, Hansenula saturnas was used.
Acetylation of racemic citronellol with IFO 0809 was performed. As a result, on the 5th day from the start of the reaction, racemic citronellol alcohol was almost quantitatively converted to citronellyl acetate, and no enantioselectivity was observed in the acetylation.

Example 3 In the same manner as in Example 1, acetylation of racemic 3-phenyl-1-butanol was attempted. Substrate concentration is 1%,
It was added in the form of a decane solution. The reaction proceeded smoothly from the first day of the reaction, and on the fourth day from the start of the reaction, the concentration of the formed acetate and the concentration of the residual alcohol became almost equal (the former was 5.3).
mg / ml, the latter being 5.5 mg / ml). At this time, the decane phase was recovered, half of the decane was distilled off, and then the acetate ester and the residual alcohol were separated by column chromatography. The optical purity of each of these was measured using liquid chromatography equipped with an optical resolution column, and the light application was measured using a photometer. As a result, the produced acetic ester showed (+), and 93.2% ee. Of enantiomeric excess. On the other hand, the residual alcohol is (-)
And 96.6% ee. Of enantiomeric excess.

Claims (5)

[Claims] 1. An interfacial bioreactor in which a hydrophilic immobilized carrier containing a liquid medium containing a saccharide and an organic solvent containing a racemic primary alcohol are in interfacial contact, the surface of the hydrophilic immobilized carrier is attached 1. Inoculating and growing microbial cells belonging to the genus Pichia having the ability to produce an alcohol acetyltransferase capable of enantioselectively acetylating primary alcohols, and enantioselectively acetylating the racemic primary alcohols A method for microbiological resolution of a racemic primary alcohol. 2. The optical resolution method according to claim 1, wherein the racemic primary alcohol is selected from an aromatic alcohol, an aliphatic alcohol, a heterocyclic alcohol and a terpene alcohol. 3. The optical resolution method according to claim 1, wherein the racemic primary alcohol is substantially insoluble or hardly soluble in water. 4. The microbial cell belonging to the genus Pichia is
Pichia quercuum IFO 094
9. Pichia guilliermond
ii) IFO 10107 and Pichia Kluiberg (Pich
ia kluyveri) selected from IFO 1165
Optical resolution method as described. 5. The method according to claim 1, wherein the saccharide is selected from glucose, fructose and starch.