JPH01157374A - Organic solvent-based bioreactor system - Google Patents

Abstract

PURPOSE:To readily produce an optically active alcohol or ester, by introducing a mobile phase consisting of an a reaction substrate-containing organic solvent and a nucleophilic reagent of alcohol to a stationary phase consisting of a hydrolase and carrying out alcoholysis. CONSTITUTION:A solution of organic solvent containing at least one reactive substrate selected from an ester of a racemic alcohol, a diester of prochiral type symmetric diol and a diester of a meso type diol and a nucleophilic reagent selected from alcohols are used as a mobile phase and a water content contained in the mobile phase is adjusted to <=about 2 (w/v), especially <=about 0.5 (w/v). The reactive substrate is preferably blended with the nucleophilic reagent just before sent to a stationary phase. The prepared mobile phase is introduced to the stationary phase comprising a granular hydrolylase and alcoholysis is carried out usually at about 5-70 deg.C.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is directed to the production of optically active substances that can be used as important synthetic intermediates for pharmaceuticals, etc., by enzymatic reactions using powdered or granular enzymes in organic solvents. This invention relates to a bioreactor system that can be easily manufactured.

[Prior art]

Conventionally, methods for obtaining optically active substances include: ■ optical resolution of racemates using enzyme microorganisms, ■ optical resolution of racemates using chemical methods, ■ methods of deriving optically active substances from natural products, and ■ methods using enzymes and microorganisms. Asymmetric synthesis methods, ■Asymmetric synthesis methods using chemical methods, etc. are known, but as a method with mild reaction conditions, high selectivity, and suitable for industrialization, biocatalysts such as enzymes and microorganisms are used. In recent years, it has become common to recognize that methods (1) and (2) are the best.

As an example of the industrialization of method (2) using a bioreactor, a method has already been implemented in which a racemic form of an acylamino acid is hydrolyzed into its enantiomers by a kinetic optical resolution method using an enzyme. Production of active amino acids is taking place. This method uses an enzyme immobilized on DEAE-Sephadex using a carrier binding method.
Since the reaction system is aqueous, it has the disadvantage that hydrophobic substrates are difficult to handle (Chibata et al., Agr, Biol, Che.
m, , 33(7), 1047-1052 (196
9)). Furthermore, this method had a low ability to retain enzyme activity, and the enzyme activity of the packed bed reactor decreased to 60% in 32 days.

Regarding the method (2), industrial production of L-aspartic acid and L-malic acid is also carried out using a bioreactor using immobilized microorganisms. In this case as well, the reaction system is aqueous (Chibata et al., ApPl.

MicroMol-g 27@878 (1974)-)
In addition, this method uses an entrapping immobilization method using polyacrylamide kappa carrageenan, etc., so the immobilization operation is complicated, and the reaction with hydrophobic substrates is difficult due to the aqueous reaction. This includes the problem of

Further, as an example of research on method (2) using both water and an organic solvent, there is an example by Asada et al.

This system is a method for optically resolving racemic compounds using a hydrolase, but it requires a complicated enzyme immobilization process and requires alternate use of water and an organic solvent. This method is disadvantageous from an industrial perspective because the operation is complicated and process control is difficult.

Recently, the need for organic solvent-based bioreactors that can increase reactivity by solubilizing hydrophobic substrates has been recognized, and methods using immobilized enzymes (ionic bonding + crosslinking method) , (Matsuno et al., former 0/TEC) I
NOLOG'/, Di, 459 (1985), A method of separating hydrophobic products and enzymes using a microporous thin membrane (such as a microfiltration membrane) and causing a reaction at the interface in the pores of the thin membrane (
Yamaita et al., Oil Chemistry, 33, 683 (1984), etc. have been studied.

In the method of Matsuno et al., the enzyme is easily deactivated during immobilization, especially during crosslinking with glutaraldehyde, and the immobilization operation requires a great deal of time and effort.

In addition, the reaction rate in Yamaita et al.'s method varies significantly depending on the water content in the glycerin solution, reaching the highest value at a water content of 3-4%, and the reaction rate remains unchanged even if the water content is higher or lower than this. descend. In addition, since water is always produced in this ester synthesis reaction, it is extremely difficult to maintain a constant amount of water in the system, and it requires a complicated reactor, making it unsuitable for industrialization. On the other hand, there are two types of bioreactors for enzyme reactions: homogeneous phase bioreactors that use enzymes solubilized in aqueous solutions;
Two types of bioreactors have been used: two-phase bioreactors that use immobilized enzymes.

Regarding homogeneous phase bioreactors.

There are two types: stirred tank type and ultrafiltration membrane type.In particular, in those other than those that use ultrafiltration membrane, the enzyme which is the catalyst is washed away, so it is difficult to realize continuous one reaction which is advantageous for industrialization. It was difficult.

For two-phase bioreactors, immobilized enzymes are generally used, and the types include stirred tank type, fixed bed or packed bed, fluidized bed type, model, and suspended bubble column type. is also a system capable of continuous reactions.

Next, in addition to purified enzymes, the following have recently been used as catalysts for bioreactors. That is,
In the case of intracellular enzymes that are difficult to isolate, the bacterial cells can be treated with heat, organic solvents, surfactants, etc. without isolating the enzyme, leaving only the properties that cause the desired reaction. ``treated bacterial cells'', and ``dormant bacterial cells'' or ``quiescent bacterial cells'' that are viable but in a state where they do not proliferate.
, [propagated bacterial cells] etc. that proliferate in the reactor are used,
-It is now possible to perform not only step-wise reactions but also multi-step reactions.

In addition, there are methods for immobilizing enzymes to make them insoluble in water, such as carrier binding, crosslinking, and entrapping methods, but they generally require complicated processing methods, and the enzymes are Activity decreases due to deactivation. In addition, in cases such as the inclusion method, the permeability of substrates and products to the polymers surrounding the enzyme is low, resulting in decreased reactivity and inorganic carriers,
In the carrier binding method, in which enzymes are adsorbed onto the surface of a polymer, etc., the interaction between the carrier and the enzyme is weak, and the adsorbed enzyme is easily desorbed, so this method is disadvantageous in continuous reactions in an aqueous solution.

Furthermore, the use of organic solvents in addition to conventional aqueous solutions is increasing as reaction solvents, and immobilized enzymes and microorganisms are
It is relatively stable even in these organic solvents.
It recognized.

Such proposals include a method of using a powdered enzyme in the presence of an organic solvent to produce diglyceride from glycerin and fatty acids through an ester synthesis reaction (Japanese Patent Laid-Open No. 62-19090), and a method using a batch reaction to produce diglyceride. Using powdered enzymes in organic solvents.

Method for synthesizing optically active substances (Kuribach et al. (J.

Am, Chem, Soc, , 107.7072 (19
85), ).

However, although these methods allow the enzyme to be collected by filtration and used repeatedly, they are difficult to put into practical use due to the complexity of the filtration operation and the difficulty in adjusting the enzyme water dispersion during repeated use. This includes many problems.

〔the purpose〕

The present invention enables optical resolution or asymmetric synthesis to be carried out efficiently, and enzymes can be kept in a stable state for a long period of time without complicated immobilization treatment or complicated ultrafiltration or microfiltration. The purpose of the present invention is to provide an organic solvent-based bioreactor system that can be used as a catalyst in

〔composition〕

According to the present invention, a nucleophilic reagent consisting of an organic solvent and alcohol containing at least one reaction substrate shown in (a) to (c) below is used as a mobile phase, and these are immobilized with a hydrolase. An organic solvent-based bioreactor system is provided, characterized in that an organic solvent-based bioreactor system performs alcoholysis by introducing the organic solvent into a phase.

(a) Esters of racemic alcohols Cb') Diesters of prochiral symmetric diols (c)
Diester of meso-type diol Since the present invention has the above-described structure, it is possible to continuously produce optically active alcohols or optically active esters and monoesters that are alcohol derivatives, which have been difficult to produce in the past.

That is, according to the present invention, (1) optical resolution by alcoholysis of an ester of a racemic alcohol, (2) alcoholylysis of a diester of a prochiral type symmetric diol,
(2) It becomes possible to perform each reaction continuously, such as asymmetric synthesis by alcoholysis of diester of meso-type diol, using the same bioreactor system.

Typical reaction examples of the present invention are illustrated below.

(Optical resolution) Racemic alcohol Optical activity Optically active ester Alcohol Ester of alcohol (asymmetric synthesis) X Diester of X diol Diester of monoester
Monoester [in the formula R; R': substituted or unsubstituted aliphatic hydrocarbon group or substituted or unsubstituted aromatic hydrocarbon group, n = 0-5, m 20-5
, Q = O-2, *: optically active carbon atom, X: hydrogen, halogen, '? Monosubstituted or unsubstituted aliphatic hydrocarbon group,
It is a substituted or unsubstituted aromatic hydrocarbon group, or at least one substituent containing a heteroatom such as oxygen, nitrogen, or sulfur. ] The format of the bioreactor in the present invention is a solid/formed phase system, which has conventionally been carried out using only immobilized enzymes, microorganisms, and some free microorganisms, and uses an organic solvent as the liquid. For example, it is possible to use any of the stirred tank type, fixed bed or packed bed type, fluidized bed type, model, and suspension bubble column type, but in particular, the reaction efficiency and the fixed phase of enzymes and microorganisms are From the viewpoint of retention capacity, a fixed bed or packed bed reactor is preferably used, in which enzymes and microorganisms are packed in a column and the substrate and alcohol can be passed through in an ascending or descending manner.

In addition, in this type of bioreactor, a method suitable for an actual industrial production system is to transfer the organic solvent containing the reaction substrate, which is a component of the mobile phase, from one storage tank to the other tank of the mobile phase. The component nucleophilic reagent alcohol or alcohol-containing organic solvent is introduced separately from the other storage tank, and both are mixed immediately before being supplied to the stationary phase, and then the enzyme reaction is carried out within the stationary phase. This is a system that performs alcoholysis. According to this system, since non-stereoselective alcoholysis is suppressed, it is possible to continuously produce an optically active substance having high optical purity.

The present invention will be explained in more detail below.

In the present invention, the reaction substrate used in the mobile phase is at least one of (a) an ester of a racemic alcohol, (b) a diester of a prochiral type symmetrical diol, and (C) a diester of a meso type diol.

A specific example is shown below.

That is, as racemic alcohol esters, turketanol butyrate, 2,3-dichloro-1-propatol butyrate, 3-chloro-2-acetoxy-
As esters of primary and secondary alcohols such as 1-p-toluenesulfonyloxypropane, and diesters of prochiral symmetric diols, 1,3-diacetoxy-2-
As the diester of men-type diol such as phenylpropane, cis-1,4-diacetoxy-2-cyclopentene and the like are used.

Alcohol used in the mobile phase of the present invention (Z-01()
is an alcohol having no asymmetric carbon, a racemic alcohol having an asymmetric carbon, or an optically active alcohol.

More specifically, alcohols having no asymmetric carbon atoms include straight chain or branched aliphatic alcohols having 1 to 10 carbon atoms, and especially straight chain or branched aliphatic alcohols having 3 to 8 carbon atoms. Branched chain aliphatic alcohols are preferred.

Racemic alcohols having asymmetric carbon atoms include linear or branched aliphatic alcohols having 1 to 10 carbon atoms and alcohols having an aromatic ring.

Optically active alcohols include linear or branched aliphatic alcohols having 1 to 10 carbon atoms and alcohols having an aromatic ring. When an optically active alcohol or racemic alcohol is used, the chemical structure of the alcohol must be different from the alcohol moiety of the racemic alcohol ester in the claims of the present invention.

The organic solvent used in the mobile phase of the present invention is the alcohol or other non-aqueous organic solvent shown above.

Specific examples of non-aqueous organic solvents include n-pentane,
Linear hydrocarbons such as n-hexane and n-hebutane, branched chain hydrocarbons such as isobutane, isopentane, and 2-methylpentane, alicyclic hydrocarbons such as cyclopentane and cyclohexane, methylene dichloride, and chloroform. , halogen-containing hydrocarbons such as carbon tetrachloride, dichloroethane, and trichloroethane, aromatic hydrocarbons such as benzene, toluene, and xylene, aliphatic ethers such as diethyl ether and n-dibutyl ether, and alicyclic ethers such as tetrahydrofuran and tetrahydropyran. Among them, n-hexane, toluene, diisopropylbenzene, diethyl ether, n-dibutyl ether, and carbon tetrachloride are more suitable.

The hydrolase used for the stationary phase in the present invention is:

It may be a purified product or a composition, and the form used is a powdered or granular form of hydrolytic enzyme or a dried product of bacterial cells that produce the hydrolytic enzyme (treated bacterial cells, dormant or stationary bacterial cells). I can do it.

Furthermore, the immobilization carrier 1 may be a polymer such as polystyrene, polypropylene, starch, or gluten, or an inorganic material such as activated carbon, porous glass, celite, zeolite, kaolinite, bentonite, alumina, silica gel, hydroxyapatite, calcium phosphate, or metal oxide. It is also possible to use dried immobilized enzymes obtained by supporting and immobilizing the above-mentioned hydrolytic enzymes on materials etc. by a physical adsorption method.

Enzymes immobilized by such physical adsorption methods (a type of carrier binding method), which are easy to immobilize and have little possibility of enzyme deactivation during the immobilization process, are generally However, this method was not effective in aqueous reactions because it was weaker than other ionic bonding methods or covalent bonding methods, and the enzyme easily detached from the carrier. Therefore, detachment of the enzyme from the carrier is suppressed, so it can be used preferably.

Specific examples of the hydrolase used in the present invention include lipases such as porcine pancreatic lipase, yeast lipase derived from the genus Candida, bacterial lipase derived from the genus Aspergillus, Mucor, and Pseudomonas, and lipases derived from porcine liver. Examples include esterase, or proteolytic enzymes such as trypsin, chymotrypsin, and subtilisin.

In the present invention, the water content of the reaction system is extremely low,
Although it is necessary to carry out the reaction in a substantially nonaqueous system, the presence of a trace amount of water in the enzyme is necessary, so the water content in the reaction substrate, alcohol, and organic solvent that is the mobile phase is 2. % (It/V) or less, and furthermore, 0.5% (
W/V) or less is desirable. In addition, the water content of the powdered or granular enzyme that is the stationary phase is 0.1 to 10% (S/V).
and 0.5 to brocade (W/V) is desirable.

The moisture content of each substance mentioned above can be determined by various drying methods, e.g.
For liquids, it is adjusted by using a suitable desiccant, and for solids, it is dried in a vacuum desiccator.

One of the features of the present invention is that, by keeping the water content of the reaction system extremely low, non-stereoselective reaction substrates can be used without enzymes, which can cause a decrease in optical purity due to carboxylic acids produced by ester hydrolysis. At the same time, in a water/organic solvent coexistence system, an unstable enzyme can be used in a continuous reaction system in a highly active state for a long time.

In the case of optical resolution, the molar ratio of the reaction substrate and the alcohol as a nucleophilic reagent used in the present invention is 0.5 or more: substrate:alcohol=l:0.5 or more in the case of asymmetric synthesis.
There is no particular restriction as long as the ratio of alcohol is 1:1 or more.

In addition, in the present invention, the solution of the reaction substrate and alcohol is preferably mixed just before the enzyme column, which is the stationary phase, and supplied into the column, but the flow rate and the amount of enzyme packed in the column are , the reactivity of each reaction substrate determines its optimal rate and amount. Furthermore, there is no particular restriction on the reaction temperature as long as it is lower than the boiling point of the substance with the lowest boiling point among the reaction substrate, alcohol, and organic solvent, but a temperature of about 5 to 70°C is usually used. .

〔effect〕

Since the present invention has the above-mentioned configuration, an unstable enzyme can be stably present in a highly active state for a long period of time in a continuous reaction system without using a complicated ultrafiltration membrane or microfiltration membrane device. It can be said to be a bioreactor system with high production efficiency.

Furthermore, since it is possible to suppress the non-enzymatic decomposition reaction of the non-stereoselective reaction substrate, which causes a decrease in optical purity, it becomes possible to efficiently obtain an optically active substance having high optical purity.

Therefore, according to the present invention, pharmaceutical synthesis intermediates such as (R)-turketal, (R), (S)-epichlorohydrin, etc., which have conventionally been produced from natural optically active substances through a long synthetic route, can be efficiently synthesized. It can be manufactured as follows.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 A tapered glass column with an inner diameter of 40 mm and a length of 300 mm was packed with commercially available lipase ``Amano PJ'' (manufactured by Jinko Pharmaceutical Co., Ltd., 220 g). After piping this column to a pump, it was placed in a 35°C hot water bath. to keep the column temperature constant.

A methylene dichloride solution containing racemic turketal butyrate at a concentration of 50+++M (Tank A) and a methylene dichloride solution containing n-butyl alcohol at a concentration of 50mM (Tank B) were prepared at a flow rate of 0.5-min. Inject into column. The solutions in tanks A and 8 are only mixed via the pump just before entering the column. Undecomposed (S) was extracted from the methylene dichloride solution coming out of the column.
-Tulketal butyrate was obtained with an optical purity of 85%.

Further, the alcoholysis rate at this time was 60%, and the optical purity and decomposition rate were maintained as they were even after 30 days of continuous operation.

Example 2 A flange-type glass column with an inner diameter of 23 mm and a length of 250 mm was packed with commercially available lipase "Amano PJ (manufactured by Ohno Pharmaceutical) (enzyme amount: too much). After piping this column to a pump, the column was placed in a constant temperature bath at 22 ° C. Keep the temperature constant.

25 m
Toluene solutions (tank B) dissolved at a concentration of M are each injected into the column at a flow rate of 0.25-min. Tanks A and R
The solutions are mixed only just before entering the column via the pump. Undecomposed (R)-3-chloro-2-acetoxy- was extracted from the toluene solution coming out of the column.
1-p-Toluenesulfonyloxypropane and (S)-
3-chloro-1-p-toluenesulfonyloxy-2-
Each propatool was obtained with an optical purity of 99% or more and a yield of 50%. The optical purity and decomposition rate were maintained even after 30 days of continuous operation.

Example 3 Commercially available lipase [Amano CESJ (Ohno Pharmaceutical i
) (100 g of enzyme fik), and after piping the column to a pump, the column temperature is kept constant in a constant temperature bath at 22°C.

A benzene solution (tank A) containing racemic 3-chloro-2-acetoxy-1-p-toluenesulfonyloxypropane dissolved at a concentration of 20mM (tank A) and 25m
Benzene solutions (tank B) dissolved at a concentration of M are each injected into the column at a flow rate of 0.25-min. The solutions in tanks A and 8 are only mixed via the pump just before entering the column. Undecomposed (R)-3-chloro-2-acetoxy-1-p-toluenesulfonyloxypropane and (S) are extracted from the benzene solution coming out of the column.
-3-chloro-1-p-toluenesulfonyloxy-2
- Optical purity of each propatool is 99% or more, yield 50%
The optical purity and decomposition rate obtained in 1 were maintained as they were even after 30 days of continuous operation.

Example 4 Using porcine pancreatic lipase (manufactured by Sigma) as the lipase and racemic 2,3-dichloro-1-propatur butyrate as the substrate, under the same conditions as in Example 1,
An alcoholysis reaction is carried out to produce undecomposed (S)-2,3
-dichloro-1-propatur butyrate and (R)
-2,3-dichloropropatool was obtained.

Example 5 Alcoholysis was carried out in the same manner as in Example 2 using Amano P (manufactured by Ohno Pharmaceutical Co., Ltd.) as the lipase and 1,3-diacetoxy-2-phenylpropane as the substrate to produce optically active 3-acetoxy -2-phenyl-propatool was obtained.

Example 6 Alcoholysis was carried out in the same manner as in Example 2 using Amano P (manufactured by Ohno Pharmaceutical Co., Ltd.) as the lipase and cis-1,4-diacetoxy-2-cyclopentene as the substrate to obtain optically active 4- Acetoxy-2-cyclobentenol was obtained.

Comparative Example 1 Substrate (R,5)-3-chloro-2-acetoxy-1-ρ- was added to 100% 0.1M phosphate buffer (pH 7,0).
A toluenesulfonylysis reaction was performed. This reaction solution 200
- was extracted with methylene chloride, dried and concentrated under reduced pressure, and separated by silica gel column chromatography, (R)-3-chloro-2-acetoxy-1-ρ-toluenesulfonyloxypropane 8.5 g, (S)- -chloro-1-p-
Toluenesulfonyloxy-2-propatol 7.4g
I got it. When the relative luminosity of each group was measured (a ID”
-9,2° (C=5.0, Meoll), (a)o”
-2,2' (C: 5.0. Meoll).

or.

When the optical purity was measured by +1 PLC, all showed values of 99% or more.

However, water droplets have the disadvantage that the enzyme is dissolved in water, making it impossible to carry out the reaction continuously, and the reaction must be carried out while neutralizing the free carboxylic acid produced.

Claims (2)

[Claims] (1) Using a nucleophilic reagent consisting of an organic solvent and alcohol containing at least one reaction substrate shown in (a) to (c) below as a mobile phase, these are introduced into a stationary phase consisting of a hydrolase. An organic solvent-based bioreactor system characterized by performing alcoholysis by. (a) Esters of racemic alcohols (b) Diesters of prochiral symmetrical diols (c) Diesters of meso diols (2) The organic solvent-based bioreactor system according to claim 1, wherein the organic solvent containing the reaction substrate and the nucleophilic reagent consisting of alcohol are mixed immediately before being supplied to the stationary phase.