JPH09275980A - Production of dried immobilized lipase carrier - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a dried immobilized lipase carrier having high ester transfer activity capable of carrying out an ester transfer reaction in high efficiency even in a very small amount of water in an organic solvent containing a substrate and high activity development ratio performance when optical resolution is carried out by preferentially subjecting one of a racemic compound of an optically active alcohol to the transfer reaction of an ester bond in an organic solvent using an enzyme. SOLUTION: A regenerated granular porous chitosan carrier is treated with an aliphatic polyalcohol glycidyl ether and then with an acid halide or an acid anhydride of a higher fatty acid in an organic solvent to give a cross-linked regenerated porous chitosan carrier, on which a lipase is adsorbed. The carrier is treated with a polyfunctional reagent to give a wet immobilized lipase carrier. The wet immobilized lipase carrier in an amount of 1 pt.wt. is shaken in 1-20 pts.wt. of an organic solvent containing 0.1-5wt.% of a surfactant and dried under reduced pressure.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD [0001] The present invention provides an excellent catalytic action when using an enzyme in an organic solvent to preferentially perform a transesterification reaction of an ester bond to one of racemic compounds of an optically active alcohol to perform optical resolution. The present invention provides a method for producing a dry immobilized lipase carrier, which exhibits excellent catalytic performance regardless of the water concentration in an organic solvent, and which can perform a transesterification reaction with high efficiency.

[0002]

2. Description of the Related Art Various synthetic reactions can be carried out by a hydrolase in an organic solvent containing only a trace amount of water. Since the enzyme reacts at room temperature and atmospheric pressure, it is possible to synthesize a thermally unstable substance, and the reaction is energy-saving and clean, and the reaction has excellent specificity and is regioselective, substrate-selective or unreactive. It has an excellent feature that a synthetic catalyst is possible, which is not available in synthetic catalysts.

However, the development of an efficient reaction method in which an enzyme has a slow reaction rate in an organic solvent has been actively studied in recent years. The use of an immobilized enzyme in an organic solvent is useful because it is highly efficient and an expensive enzyme can be repeatedly used. JP-A-64-2588 discloses that lipase is immobilized on an immobilizing carrier in the presence of an active agent selected from the group consisting of a surfactant and a polyol compound to give a water content of 300 pp.
It discloses a method for producing a modified oil and an immobilized lipase used for the same, which reforms fats and oils in a system of m or less.

According to the method disclosed in the above publication, a surfactant or a polyol compound as an activity expressing agent is dissolved in water together with lipase, and a carrier for immobilization is put into this aqueous solution to adsorb the lipase onto the carrier to immobilize the immobilized lipase. Is to be prepared. However, the immobilized lipase obtained by the method is unstable because the carrier and the lipase are not covalently immobilized and the lipase and the surfactant are only adsorbed and attached to the carrier, which is comparable to chemical synthesis. It was not suitable for continuous organic synthesis over a long period of time, such as several hundred hours, due to its stable catalytic activity.

Further, the applicant of the present invention previously disclosed in Japanese Patent Laid-Open No. 7-879.
No. 74 gazette discloses a dry immobilized lipase carrier, but this dry immobilized lipase carrier has a drawback that activation and high transesterification activity cannot be obtained unless a trace amount of water is added to the substrate solution. It was

[0006]

DISCLOSURE OF THE INVENTION The present invention involves the use of an enzyme in an organic solvent to preferentially carry out a transesterification reaction of an ester bond to one of racemic compounds of an optically active alcohol to carry out optical resolution. Provided is a method for producing a dry immobilized lipase carrier having high transesterification activity and activity expression rate, which enables highly efficient transesterification even when the organic solvent contains only a trace amount of water. It is a thing.

[0007]

DISCLOSURE OF THE INVENTION The present inventors have diligently studied and treated a regenerated granular porous chitosan carrier with a glycidyl ether of an aliphatic polyalcohol, and then treated with an acid halide or acid anhydride of a higher fatty acid in an organic solvent. Treated with a substance to give a crosslinked regenerated granular porous chitosan carrier, which is adsorbed with lipase and treated with a polyfunctional reagent to obtain a wet immobilized lipase carrier, and 1 to 1 part by weight of the wet immobilized lipase carrier is treated.
The inventors have found a method for producing a dry immobilized lipase carrier, which is shaken in an organic solvent containing 20 parts by weight of a surfactant having a concentration of 0.1 to 5% by weight, and then dried under reduced pressure. According to the method of the present invention, it is possible to obtain a dry immobilized lipase carrier that exhibits excellent catalytic performance regardless of the water concentration in an organic solvent containing a substrate and that is capable of highly efficient ester transfer reaction.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The regenerated granular porous chitosan carrier used in the present invention can be obtained by the method disclosed by the applicant in Japanese Patent Publication No. 1-16420. That is, average molecular weight 1
It is obtained by dissolving low molecular weight chitosan in the range of 20,000 to 230,000 in an acidic aqueous solution, dropping the dissolved solution into a basic solution, and solidifying and regenerating the porous chitosan.

A method for obtaining a crosslinked regenerated granular porous chitosan carrier by a crosslinking reaction of an aliphatic polyalcohol with a glycidyl ether of a regenerated granular porous chitosan carrier and a subsequent reaction with an acid halide or acid anhydride of a higher fatty acid. The applicant of the present invention is disclosed in Japanese Patent Laid-Open No. 6-23776.
According to the method disclosed in Japanese Patent No.

Examples of the glycidyl ether of the aliphatic polyalcohol used in the crosslinking reaction include polyethylene glycol diglycidyl ether such as ethylene glycol diglycidyl ether and diethylene glycol diglycidyl ether, propylene glycol diglycidyl ether and dipropylene glycol diglycidyl ether. Polypropylene glycol diglycidyl ether, glycerol polyglycidyl ether, etc. are used. The reaction of introducing the glycidyl ether of an aliphatic polyalcohol has a concentration of 0.01 to 0.5 epoxy equivalent / L and a liquid volume of regenerated granular porous. The amount is 1 to 5 times the volume of the chitosan carrier, the reaction temperature is 20 to 90 ° C., and the reaction time is 1 to 24 hours with gentle stirring. Next, the water contained in the carrier is sufficiently removed with an organic solvent, and the higher fatty acid is introduced in the organic solvent. Organic solvents include dioxane, hexane, ethanol, isopropyl alcohol, dimethylformamide, dimethylacetamide, dimethylsulfoxide,
A solvent inert to the acid halide or acid anhydride of the higher fatty acid introduced from pyridine or the like may be selected and used. Lauric anhydride, myristic anhydride, stearic anhydride are used as acid anhydrides of higher fatty acids, and lauroyl chloride, myristoyl chloride, stearoyl chloride, etc. are used as acid halides, and the reaction has a concentration of 50%. ~ 1,000 mmol / L, the amount of liquid is 1 to 5 times the volume of the carrier used, the reaction temperature is 10 to 70 ° C, the reaction time is 1 to 24 hours, and the reaction is performed with gentle stirring. It is washed with an organic solvent, and the organic solvent is replaced with pure water and removed. A deoxidizing agent such as triethylamine or pyridine may be added for the purpose of removing the fatty acid or inorganic acid generated at this time. In this way, a crosslinked regenerated granular porous chitosan carrier is obtained.

Subsequently, the crosslinked regenerated granular porous chitosan carrier is adsorbed with lipase. Examples of the lipase include Rhizopus genus, Aspergillus genus, and Mucor (Mu).
genus cor, Pseudomona
s) genus, Penicillium genus, Chromobacterium
Lipases derived from microorganisms such as genus Candida and lipases derived from animals such as pancreatic lipase are used.

The crosslinked regenerated granular porous chitosan carrier is immersed in the above-mentioned aqueous solution containing lipase and stirred to adsorb the lipase onto the carrier. At this time, the temperature of the aqueous solution of lipase is in a range that does not cause inactivation of lipase, for example, 0 to 60.
℃, preferably 5 to 40 ℃ is good. Further, the pH of the aqueous solution of lipase may be in the range where enzyme denaturation does not occur, and p
H3-9 are preferred.

Then, in order to improve the durability of the lipase, a treatment for covalently bonding the enzyme and the carrier with a polyfunctional reagent is performed. Examples of polyfunctional reagents include glyoxal, glutaraldehyde, malonaldehyde, succinyl aldehyde, bissulphosuccinimidyl suberate, dimethyl suberimilate, ethylene glycol bissulphosuccinimidyl succinate, dicyclohexylcarbodiimide, and hexamethylene diisocyanate. For example, 1.2 to 2 times mol may be used per 1 mol of the lipase molecule. Thus, a wet immobilized lipase is obtained.

In the treatment with these polyfunctional reagents, the crosslinked regenerated particulate chitosan carrier may be reacted in advance prior to the adsorption of lipase. The obtained wet immobilized lipase is dipped in an organic solvent to sufficiently replace and remove the contained water with the organic solvent.

At this time, the organic solvent used is acetone, ethanol, methanol, tetrahydrofuran,
Although a polar solvent such as dioxane is used, acetone is preferable because it does not deactivate the enzyme. Then, the wet immobilized lipase carrier is immersed in the same organic solvent as the above-mentioned organic solvent in which the following surfactant is dissolved and shaken.

At this time, the surfactant used is
Examples thereof include acidic, neutral and basic surfactants, but weakly acidic, neutral and weakly basic surfactants that do not deactivate lipase are preferable. Specifically, nonaethylene glycol octylphenol ether, polyoxyethylene lauryl ether, polyoxyethylene sorbitan monolaurate, nonaethylene glycol octyl phenyl ether, polyethylene glycol, t-octyl phenyl ether, polyoxyethylene sorbitan monolaurate, sorbitan Examples thereof include monolaurate, heptaethylene glycol ether, polyethylene glycol hexadecyl ether, a block copolymer of ethylene and ethylene glycol, 1-S-octyl-β-D-thioglucopyranoside, and octyl-β-glucoside.

However, when the surfactant is in a liquid state, when the wet-immobilized lipase carrier is dried under reduced pressure, the contraction of the carrier is remarkable, and in order to prevent the contraction, the solid surfactant at room temperature is added to the above-mentioned organic compound. It is preferably dissolved in a solvent and used, and 1-S-octyl-β-D-thioglucopyranoside and octyl-β-glucoside are preferable.

The concentration of the surfactant in the organic solvent is 0.1-0.1%.
It is 5% by weight, preferably 0.2 to 5% by weight. 0.1
If it is less than 5% by weight, the effect is low, and if it exceeds 5% by weight, it is difficult to dissolve the surfactant in the organic solvent. The amount of the organic solvent containing the surfactant is 1 to 20 parts by weight, preferably 2 to 10 parts by weight based on 1 part by weight of the wet fixed lipase carrier.
If the amount is less than 1 part by weight, it is difficult to shake because the amount of the liquid is small, and even if the amount is 20 parts by weight or more, no noticeable improvement in effect is observed.

The wet immobilized lipase carrier is immersed and shaken in an organic solvent containing a surfactant for 15 minutes to 2 hours, preferably 30 minutes to 2 hours. If it is less than 10 minutes, the surfactant does not sufficiently diffuse into the inside of the carrier, and if it exceeds 2 hours, there is no effect on the diffusion of the surfactant into the inside of the carrier and the deactivation of lipase becomes remarkable, which is not preferable. The treatment temperature is 5 to 40 ° C, preferably 5 to 30 ° C. 5 ℃
If less than 40, the diffusion of the surfactant into the carrier is poor, and
When the temperature exceeds ℃, heat inactivation of lipase becomes remarkable.

Then, excess surfactant and excess organic solvent adhering to the carrier are removed using a glass filter or the like,
The dried immobilized lipase carrier is obtained by drying under reduced pressure.

[0021]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to examples, but the present invention is not limited to these ranges.
Incidentally, the measurement method of the immobilization rate of lipase, which was measured in the example,
The method for measuring the transesterification expression activity of free lipase, the method for measuring the transesterification expression activity of the dry immobilized lipase carrier and the method for measuring the activity expression rate were performed as follows.

1. Method for measuring immobilization rate of lipase 20 g of olive oil (manufactured by Kanto Kagaku Co., Ltd.) 20 was added to ADEKA TOL SO-120 (trade name, manufactured by Asahi Denka Kogyo Co., Ltd.)
g and 60 ml of pure water to prepare an emulsion substrate. To 25 ml of the substrate, 10 ml of pure water is added and preheated at 37 ° C for 10 minutes. 0.1 ml of the enzyme aqueous solution used for the immobilization operation is added to the substrate and reacted at 37 ° C. for 5 minutes. Add 80 ml of ethanol containing 50% of acetone and stir to stop the enzyme reaction. The fatty acid released by the enzymatic reaction is titrated with 50 mM NaOH to determine the titer. As a blank, in the same operation as above, by the operation of, 0.1 ml of pure water is added instead of the enzyme aqueous solution, and the titer is determined. The emulsion lipid decomposition expression activity value A (u / ml aqueous solution) of the lipase aqueous solution before the immobilization operation is determined by the following formula.

[Equation 1] The activity B (u / ml aqueous solution) of emulsion lipid decomposition expression of the filtrate after the immobilization operation is measured in the same manner as above. The immobilization rate C is obtained by the following equation from A and B obtained by and.

[Equation 2]

2. Transesterification activity of free lipase
Measurement method A hexane solution containing 2.5% α-D, L-phenylethyl alcohol and 2% vinyl acetate monomer is prepared, and pure water is appropriately added to obtain a substrate solution containing a predetermined concentration of water. A dry immobilized lipase carrier having a hydrolytic activity of 5 u was weighed, added to 2 ml of the substrate solution, and added at 25 ° C for 3 hours.
Stir for a period of time to carry out a transesterification reaction. After completion of the reaction, the immobilized lipase carrier is quickly removed by filtration, and the reaction solution is cooled to -50 ° C to stop the reaction. The composition of the reaction solution is measured by high performance liquid chromatography using an optical resolution column, Chiralcel OB (trade name, manufactured by Daicel Chemical Industries, Ltd.) to determine the decrease amount (μmol) of α-L-phenylethyl alcohol. The transesterification expression activity value of free lipase is calculated as 1 u when acylating 1 μmol of α-L-phenylethyl alcohol per minute at 25 ° C., and calculated from the following formula.

(Equation 3)

[0024] 3. Ester conversion of dry immobilized lipase carrier
Method for measuring transfection activity Weigh a dry immobilized lipase carrier with a weight equivalent to 10 u in terms of lipolysis expression activity and add to 2 ml of a substrate solution containing the same water as the substrate solution described in the method for measuring transesterification activity of free lipase. The mixture is stirred at 25 ° C. for 3 hours to carry out a transesterification reaction. After completion of the reaction, the immobilized lipase carrier is rapidly removed by filtration to stop the reaction. The composition of the reaction solution is measured by high performance liquid chromatography using an optical resolution column, Chiralcel OB (trade name, manufactured by Daicel Chemical Industries, Ltd.) to determine the decrease amount (μmol) of α-L-phenylethyl alcohol. The transesterification expression activity value of the dry immobilized lipase carrier is determined by the following formula when 1 μmol of α-L-phenylethyl alcohol is acylated per minute at 25 ° C. for 1 u.

(Equation 4)

[0025] 4. Method of measuring activity expression rate The above measuring method 2. And 3. Transesterification activity value F (u / mg) of free lipase and transesterification activity value G (u / mg) of immobilized lipase carrier determined by the method shown in
From the above, the expression rate (%) of the ester transfer expression activity was calculated by the following formula.

(Equation 5)

Example 1 3.5% of 1,200 g of chitosan having a deacetylation degree of 80% and an average molecular weight of 60,000
It was dissolved in 18,800 g of an acetic acid aqueous solution. The aqueous solution is
It drops into a coagulation solution consisting of% sodium hydroxide, 20% ethanol, 73% water, coagulates and regenerates chitosan into a granular porous material, thoroughly rinses with water until it becomes neutral, and has an average particle size of 0.1 mm.
Regenerated granular porous chitosan carrier 10,000ml (wet)
I got

500 ml of water and 5.36 g of ethylene glycol diglycidyl ether (epoxy equivalent 87.13) were added to 500 ml of each of the regenerated granular porous chitosan carriers and reacted at 60 ° C. for 1 hour.
500 ml of the regenerated granular porous chitosan carrier was obtained.

The water contained in the regenerated granular porous chitosan carrier was thoroughly removed with dimethylacetamide. Dimethylacetamide 50 was added to 500 ml of the regenerated granular porous chitosan carrier.
0 ml, 7.620 g stearoyl chloride and 2.530 g
Of triethylamine was added.

This was stirred at 25 ° C. and reacted for 18 hours. After removing the reaction residual liquid, it was washed with dimethylacetamide, and then dimethylacetamide was removed with pure water to obtain a crosslinked regenerated granular porous chitosan carrier in a wet state.

10 g of this wet carrier was collected under wet weight, and a lipase AP derived from bacteria (Asahi Kasei Co., Ltd.) 1
10 ml of 0 mg / ml aqueous solution, 1M phosphate buffer (pH
5 ml of 7.5) and 235 ml of pure water were mixed to prepare an enzyme solution, a crosslinked regenerated granular porous chitosan carrier was put therein, and the mixture was stirred at 37 ° C. for 1 hour to adsorb lipase, filtered and washed well with water.

1M-phosphate buffer solution (pH 7.5) was added to 5 m.
l, 242.5 ml of pure water and 2.5 ml of 1% glutaraldehyde aqueous solution were mixed to prepare a fixing solution, and a carrier having lipase adsorbed therein was put in this solution and stirred at 37 ° C. for 1 hour,
The carrier was filtered off and washed thoroughly with water to obtain 10 g of a wet immobilized lipase carrier.

Next, the water contained in the carrier was removed by displacement with acetone. Octyl-β-glucoside was used as a surfactant, and its concentration (% by weight) was 0.05%, 0.1%.
Prepare 10 g of each solution dissolved in acetone to have a concentration of 0.5%, 0.5%, 1%, 5%, and 7%, and immerse 1 g of the carrier obtained by the above-mentioned method in each, at 25 ° C. Shake for 1 hour. After removing the excess organic solvent containing a surfactant with a glass filter, it was dried under reduced pressure at 25 ° C. for 12 hours to obtain 0.29 g of dry immobilized lipase carriers (Sample Nos. A to f), respectively. As a comparative example, a carrier obtained by simply drying the wet immobilized lipase under a reduced pressure without treating it with an acetone solution of a surfactant was used as a sample N.
o. g. Sample No. For a to g, the expression activity and the activity expression rate, which are the transesterification expression activity, were measured, and the results are shown in Table 1.

[0033]

[Table 1]

As is clear from the results of Table 1, the sample No. obtained by the method of the present invention. The dry immobilized lipase carriers of a to af were treated with an organic solvent containing a surfactant, and the untreated sample No. Compared with g, it shows much better transesterification expression activity, and the surfactant concentration is preferably 0.1 to 5% by weight, and even if the concentration is 7% by weight, the effect is unchanged at 5% by weight. .

<< Example 2 >> 10 g in the same manner as in Example 1.
To obtain a wet immobilized lipase carrier. Next, the water contained in the carrier was replaced and removed with acetone. Using 1-S-octyl-β-D-thioglucopyranoside as a surfactant, 0.5 g, 1 g, 2 g, 20 g, 50 g of a solution dissolved in acetone to a concentration of 1% by weight was prepared. , 1 g of the obtained wet immobilized lipase carrier was dipped, shaken at 25 ° C for 1 hour, the organic solvent containing excess surfactant was removed by a glass filter, and dried under reduced pressure at 25 ° C for 12 hours, Dry immobilized lipase carrier with a dry weight of 0.29 g, sample no. hl was obtained. The expression activity and the activity expression rate, which are these transesterification expression activities, were measured in a substrate solution containing 150 ppm of water, and the results are shown in Table 2.

[0036]

[Table 2]

As is clear from Table 2, 1-S was used instead of the octyl-β-glucoside used in Example 1 as the surfactant.
The dry immobilized lipase carrier exerts excellent performance even if it is replaced with octyl-β-D-thioglucopyranoside, and 1 to 50 parts by weight of the surfactant is added to 1 part by weight of the wet immobilized lipase carrier. It is apparent that 1 to 20 parts by weight is suitable because it exhibits preferable performance when treated in an organic solution containing it, and the result does not improve at 20 parts by weight or more.

<< Example 3 >> 10 g in the same manner as in Example 1
To obtain a wet immobilized lipase carrier. Next, the water contained in the carrier was replaced and removed with acetone. As a surfactant, 20 g of a solution of octyl-β-glucoside dissolved in acetone to have a concentration of 1% by weight was prepared, 10 g of a wet immobilized lipase carrier was immersed in the solution, and the mixture was shaken at 25 ° C for 1 hour to obtain an excess. After removing the organic solvent containing the surfactant with a glass filter, it was dried under reduced pressure at 25 ° C. for 12 hours to obtain 2.9 g of a dry immobilized lipase carrier. 10 mg each of the obtained dry immobilized lipase carrier was added to the substrate solution having a water concentration of 150 ppm,
In addition to 280 ppm, 620 ppm and 1180 ppm of the substrate solution, the expression activity and the activity expression rate, which are the transesterification expression activity, were measured, and the results are shown in Table 3. As a comparison, for the dry immobilized lipase carrier obtained without treatment with an organic solvent containing a surfactant, the expression activity and the activity expression rate were similarly measured in a substrate solution in which the water concentration was the same,
The results are shown in Table 3.

[0039]

[Table 3]

As is clear from the results shown in Table 3, even when the substrate solution contains a very small amount of water, the dry immobilized lipase carrier treated in a solution in which the surfactant is dissolved in acetone solution is It shows excellent transesterification expression activity and activity expression rate.

[0041]

The regenerated granular porous chitosan carrier is treated with a glycidyl ether of an aliphatic polyalcohol and then crosslinked regenerated granular porous chitosan carrier treated with an acid halide or an acid anhydride of a higher fatty acid is adsorbed with lipase, To obtain a wet immobilized lipase carrier treated with a polyfunctional reagent,
The dry immobilized lipase carrier obtained by shaking this in an organic solvent containing a surfactant and drying under reduced pressure does not depend on the water concentration in the organic solvent containing a substrate because it is treated with a surfactant. It has an excellent catalytic performance and an effect that it can be subjected to a transesterification reaction with high efficiency.

Claims (1)

[Claims] 1. A regenerated granular porous chitosan carrier is treated with a glycidyl ether of an aliphatic polyalcohol and then with an acid halide or acid anhydride of a higher fatty acid in an organic solvent to obtain a crosslinked regenerated granular porous chitosan carrier, Lipase is adsorbed on this and treated with a polyfunctional reagent to obtain a wet fixed lipase carrier, and a surfactant having a concentration of 1 to 20 parts by weight per 0.1 part by weight of the wet fixed lipase carrier is 0.1 to 5% by weight. A method for producing a dry-immobilized lipase carrier, which comprises: shaking in an organic solvent containing; and then drying under reduced pressure.