JPH0956379A - Regeneration of immobilized lipase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To regenerate the activities of an immobilized lipase capable of providing a liquid crystal raw material or a physiologically active substance by optical resolution or asymmetric synthesis by placing the immobilized lipase having remarkably reduced activities in ester transfer reaction as compared with the initial ones in a polar solvent containing a small amount of water. SOLUTION: Chitosan is dissolved in a 3.5% aqueous solution of acetic acid and the resultant solution is then dropped into a coagulating solution such as a 7% aqueous solution of sodium hydroxide to provide a regenerated granular porous chitosan carrier having 0.1mm average grain diameter. A diglycidyl compound is then added thereto to carry out cross-linking reaction and the resultant product is further reacted with stearoyl chloride to afford a cross-linked granular regenerated porous chitosan carrier. An aqueous solution of a lipase is subsequently added to the carrier to adsorb the lipase on the carrier. The resultant carrier is then washed with water to prepare an immobilized lipase, which is then filled in a column to carry out the ester transfer reaction. When the activities thereof are reduced to those without exceeding 1/10 of the initial ones, the immobilized lipase is placed in a polar solvent (e.g. acetone) containing 1-5% water to regenerate the enzymic activities. Thereby, the immobilized lipase is regenerated.

Description

Detailed Description of the Invention

[0001]

[0001]

[0002]

[0001] The present invention relates to an immobilization with reduced activity when used as a catalyst in the synthesis and exchange reaction of an ester bond in an organic solvent where water is not present or is present in a very small amount even if present. The present invention relates to a method for regenerating and reusing lipase, which provides means for efficiently synthesizing biologically active substances such as liquid crystal raw materials, drugs, and agricultural chemicals by performing optical resolution and asymmetric synthesis using immobilized lipase. is there.

[0003]

[0002]

[0004]

2. Description of the Related Art In recent years, research has been actively conducted on the production of optically active substances using biocatalysts such as enzymes and microorganisms in organic solvents. In particular, lipase is one of the most remarkable biocatalysts because it exhibits excellent catalytic activity in many reactions involving esters in organic solutions. At this time, the lipase does not dissolve in the organic solvent and does not react effectively with the substrate.
In addition, immobilization of lipase has been actively attempted due to a demand for effective use of expensive lipase.

[0005]

It is a well-known fact that when immobilized lipase is worked in an organic solvent, the presence of water in the solvent has a great effect on the enzyme activity. However, when the amount of water in the solvent is large, the hydrolysis reaction takes precedence, and the ester transfer reaction and the synthesis reaction do not occur. As a result, the reaction of the immobilized lipase is performed in an organic solvent in which water is not present or is present in a very small amount, but water present near the lipase is taken into the organic solvent in a long-term reaction. The activity of the lipase gradually decreases. Eventually, it was inactivated and the inactivated immobilized lipase had to be discarded.

[0006]

[0004]

[0007]

DISCLOSURE OF THE INVENTION The present invention relates to an immobilized lipase having a once reduced activity when used as a catalyst in a transfer reaction of an ester bond in an organic solvent in which water is absent or in a very small amount even if water is present. And a method for reproducing and reusing them.

[0008]

[0005]

[0009]

Means for Solving the Problems The present inventors have made intensive studies and have been studying the use of immobilized lipase in repeated or continuous reactions in an organic solvent to decompose ester bonds over a long period of time. The present inventors have found that when used as a catalyst in synthesis and exchange reactions, water molecules are eliminated from the immobilized lipase and deactivated, and the present invention has been achieved. That is, the present invention relates to an immobilized lipase in which the activity of the transesterification reaction is reduced so as not to exceed one-tenth of the initial activity in a polar solvent containing 1 to 5% of water to regenerate the enzyme activity. This is the playback method.

[0010]

[0006]

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION In the immobilized lipase used in the present invention, carriers for immobilizing lipase include celite, diatomaceous earth, perlite, silica gel, activated carbon, cellulose powder, regenerated granular porous chitosan carrier, polystyrene and divinylbenzene. And a chelate resin and the like. In particular, in the case of an immobilized lipase in which a liper is immobilized on a crosslinked and regenerated granular porous chitosan carrier into which a higher fatty acid has been introduced as disclosed in JP-A-7-87974, the activity is remarkably recovered, and an extremely excellent effect is exhibited.

[0012]

The lipase to be immobilized is not particularly limited. In addition to lipase derived from pig liver and pig pancreas, Rhizopus, Aspergillus (As)
genus pergillus, genus Mucor,
The genus Geotorichum, the genus Candida, the chromobacterium (Ch)
romobacterium, Pseudomonas (P
genus pseudomonas, Penicillium (Penic)
illium), fungi, and yeast.

[0013]

The polar solvent used in treating the immobilized lipase having reduced activity may be any organic solvent which does not deactivate lipase, such as acetone, dioxane,
Alcohol, dimethylsulfoxide, dimethylacetamide and the like can be mentioned, and acetone or dioxane is most effective.

[0014]

[0009] The concentration of water dissolved in a polar solvent for regenerating the immobilized lipase is preferably 1 to 5%. If the amount of water is less than 1%, the effect of regeneration is low, and if the concentration of water exceeds 5%, a large amount of water remains on the immobilized lipase even after the regeneration operation, and the decomposition reaction has priority over the ester transfer reaction. It is not preferable.

[0015]

The processing temperature during the regenerating process is 5 to 50 ° C., preferably 25 to 40 ° C., and the processing time is 1 to 50 ° C.
48 hours, preferably 10 to 20 hours are suitable. As a treatment method, all methods such as a method of passing a regeneration treatment solution through immobilized lipase having reduced activity, and a method of impregnation treatment can be adopted. The regeneration treatment is not advantageous for the regeneration of the activity even if it is applied to an immobilized lipase in which the activity of the transesterification reaction is reduced to more than one-tenth of the initial activity.

[0016]

[0011]

[0017]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to examples, but the present invention is not limited to these ranges.

The activity of the transesterification reaction of the immobilized lipase was measured by the following method and determined as the transesterification rate.

[0019]

(Activity measuring method) 1) A dry immobilized lipase is packed in a stainless steel column having an inner diameter of 0.4 cm and a length of 5 cm. 2) 10% R, S-α-phenylethyl alcohol and 4%
The solution is passed through a column at a temperature of 25 ° C. and a space velocity of 5 hr ⁻¹ using a hexane solution containing% vinyl acetate and 100 ppm of water as a substrate. 3) The substrate and the effluent from the stainless steel column were separated by an optical separation column (Chiral Cell O manufactured by Daicel Chemical Industries, Ltd.
Analyze in B) and measure the amount of R-α-phenylethyl alcohol in the effluent. 4) The transesterification rate is calculated by the following equation.

[0020]

[0013]

[0021]

[Equation 1]

[0022]

Example 1 1200 g of chitosan having a degree of deacetylation of 80% and an average molecular weight of 60,000 was dissolved in 1880 g of a 3.5% acetic acid aqueous solution. The aqueous solution is dropped into a coagulation solution composed of 7% sodium hydroxide, 20% ethanol, and 73% water, and the chitosan is coagulated and regenerated to a granular porous material, and is sufficiently washed with water until it becomes neutral. 1000 ml (wet) of 1 mm regenerated granular porous chitosan carrier was obtained.

[0023]

800 ml of this regenerated granular porous chitosan carrier
Then, 800 ml of water and 4.184 g of ethylene glycol diglycidyl ether were added thereto, and a crosslinking reaction was carried out at 60 ° C. for 1 hour. After the completion of the reaction, the mixture was washed with water to obtain 800 ml of a regenerated crosslinked granular porous chitosan carrier.

[0024]

The water contained in the crosslinked regenerated granular porous chitosan carrier was sufficiently removed with dimethylacetamide. 100 ml of dimethylacetamide, 4.544 g of stearoyl chloride and 1.5 ml of 100 ml of the crosslinked regenerated granular porous chitosan carrier
18 g of triethylamine was added, and the mixture was stirred at 40 ° C. and reacted for 10 hours.

[0025]

After removing the reaction residue, the residue was washed with dimethylformamide, and then dimethylacetamide was removed with pure water to obtain a regenerated cross-linked porous chitosan carrier into which stearoyl chloride had been introduced.

[0026]

10 g of a carrier in a wet state is taken by wet weight, and 10 ml of an aqueous solution in which 100 mg of lipase derived from Chromobacterium biscosme (trade name: T-01, manufactured by Asahi Kasei Kogyo Co., Ltd.) is dissolved. (pH 7.5) and 235 ml of pure water were mixed to form an enzyme solution, and a carrier was added. 37
After stirring at 1 ° C. for 1 hour to adsorb the lipase to the carrier, the carrier was separated by filtration and washed well with water. 1M-phosphate buffer (pH 7.
5), 242.5 ml of pure water, and 2.5 ml of 1% glutaraldehyde aqueous solution were mixed to prepare a fixative. The carrier after lipase adsorption was added to the fixative, and the mixture was stirred at 37 ° C. for 1 hour to convert the lipase into a carrier. Covalently immobilized. The carrier on which the lipase was immobilized was filtered off, washed well with water to obtain a wet immobilized lipase, and water contained in the immobilized lipase was sufficiently removed with acetone, dehydrated, and dried under vacuum to a moisture content of 5%. An immobilized lipase was obtained.

[0027]

The dried immobilized lipase thus obtained was packed into five stainless steel columns, respectively, and the transesterification reaction described in the method for measuring the activity was carried out. The activity of the transesterification reaction was measured. %, And the transesterification rate after 800 hours was 25%.

[0028]

Next, regeneration treatment was carried out by flowing acetone containing water shown in Table 1 into each of the five columns at 25 ° C. for 18 hours.

[0029]

[0021]

[0030]

[Table 1]

[0031]

The transesterification reaction was carried out again as described above, and the activity of the transesterification reaction was measured.

[0032]

[0023]

[0033]

[Table 2]

[0034]

As is clear from the results, the immobilized lipase could be effectively regenerated by flowing an acetone solution containing 1 to 5% water.

[0035]

Example 2 A dry immobilized lipase having a moisture content of 5% was obtained in the same manner as in Example 1, and a stainless steel column 5 was obtained.
Filled the book. The transesterification reaction was performed in the same manner as in Example 1, and the transesterification activity was measured. The initial transesterification rate and the transesterification rate after a predetermined time were as shown in Table 3.

[0036]

[0026]

[0037]

[Table 3]

[0038]

Then, acetone containing 3% water was flowed through each of the five columns at 25 ° C. for 18 hours to perform a regeneration treatment.

The transesterification reaction was carried out again for each of them, and the transesterification activity was measured. The transesterification rate was as shown in Table 4.

[0040]

[0028]

[0041]

[Table 4]

[0042]

Using the regenerated immobilized lipase, the same transesterification reaction was further carried out, and the transesterification activity was measured.
It was as follows.

[0043]

[0030]

[0044]

[Table 5]

[0045]

Next, acetone containing 3% water was flowed through each of the five columns at 25 ° C. for 18 hours to perform regeneration treatment.

The transesterification was carried out again and the transesterification activity was measured. The transesterification rate was as shown in Table 6.

[0047]

[0032]

[0048]

[Table 6]

[0049]

As is clear from the results, Experiment No. 6
The immobilized lipase can be effectively regenerated by flowing an acetone solution containing water through the immobilized lipase in which the activity of the transesterification reaction does not exceed one-tenth of the initial activity as shown in ８8. However, when the transesterification activity exceeds one-tenth of the initial activity as in Experiment Nos. 9 and 10, the transesterification activity is not sufficiently restored even after regeneration and regeneration.

[0050]

Example 3 A dry immobilized lipase having a water content of 5% was obtained in the same manner as in Example 1, and the same amount was packed in two stainless steel columns having an inner diameter of 0.4 cm and a length of 5 cm. The transesterification reaction was carried out in the same manner as in Example 1, and the activity of the transesterification reaction was measured.
%, And the transesterification rate after 800 hours was 15%.

[0051]

Next, dioxane containing 2% of water was passed through one column, and dioxane containing 5% of water was passed through the other column at 25 ° C. for 18 hours for regeneration.

The transesterification reaction was carried out again, and the transesterification activity was measured. The transesterification rate was 48% for dioxane containing 2% water and 52% for dioxane containing 5% water. And the immobilized lipase was effectively regenerated.

[0053]

Example 4 Nippon Rensui Co., Ltd. as a carrier
Made by DIAION HP21 and Dow Chemical Co., Ltd.
And 10 g of wet weight using Dowex MWA-1 and a lipase derived from Chromobacterium biscom (manufactured by Asahi Kasei Kogyo Co., Ltd.) in the same manner as in Example 1.
A dried and immobilized lipase on which T-01) was immobilized was obtained.

[0054]

Using the obtained dried immobilized lipase, a transesterification reaction was carried out in the same manner as in Example 1, and the activity was measured. The initial transesterification rate of Diaion HP21 was 8%, and after 800 hours. Has a transesterification rate of 2
%, The initial transesterification rate of Dowex MWA-1 was 8%, and the transesterification rate after 800 hours was 1.5%.

[0055]

Next, acetone containing 5% water was passed through each column at 25 ° C. for 18 hours for regeneration. The transesterification reaction was conducted again in the same manner and the activity of the transesterification reaction was measured.
5%, and 4.5% for Dowex MWA-1, indicating that the immobilized lipase had lower activity than the initial stage, but was effectively regenerated as such.

[0056]

[0039]

[0057]

The immobilized lipase whose activity has been reduced to not more than one-tenth of the initial activity when used as a catalyst in the transfer reaction of an ester bond in an organic solvent contains 1 to 5% of water. When the enzyme activity is regenerated in a polar solvent, the immobilized lipase can be efficiently regenerated.

Claims (1)

[Claims] 1. The activity of the transesterification reaction is one of the initial activities.
Immobilized lipase reduced to no more than 1/0
A method for regenerating immobilized lipase, wherein the enzyme activity is regenerated by placing it in a polar solvent containing up to 5% water.