JPH02138975A - Production of immobilized lipase - Google Patents

Abstract

PURPOSE:To prepare a large amount of a highly active immobilized lipase by bringing a solution, regulated to >=0.1 ionic strength and containing 40-70vol.% water-miscible polar solvent added thereto into contact with an anion exchanger. CONSTITUTION:A lipase enzymic solution, regulated to <=0.1 ionic strength and containing 40-70vol.% water-miscible polar solvent added thereto is prepared. The resultant lipase enzymic solution is then passed through an anion exchanger to adsorb lipase and prepare an immobilized lipase. After adsorbing the lipase on the anion exchanger, treatment with a polyvalent reaction reagent is preferably carried out to further enhance the immobilization ratio of the lipase. An anion exchanger obtained by introducing as many anionic groups as possible into a hydrophobic substrate, such as a copolymer of polystyrene and divinylbenzene, is preferred as a carrier for the immobilized lipase.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention relates to a method for producing immobilized lipase, and more specifically, to improve the immobilization rate when preparing immobilized lipase by adsorbing lipase to an anion exchanger. The present invention relates to a method for producing immobilized lipase that is effective for mass-producing highly active immobilized lipase.

This immobilized lipase can be used to perform ester decomposition, transesterification, ester synthesis, optical resolution of racemates, etc., as well as enzyme utilization industry, oil and fat industry.
It can be widely applied to fields such as the food industry and the pharmaceutical industry.

(Prior Art) In recent years, the use of enzymes for hydrolyzing fats and oils has attracted attention, and in particular, lipase has been widely used because it is industrially easy to use and has a high ability to decompose fats and oils. However, free lipase is difficult to use repeatedly over a long period of time.
Moreover, since it cannot be used for continuous reactions using columns, it is necessary to immobilize lipase from an economical point of view in order to advance its industrial use, and the development of this technology is required.

By the way, various methods are known as general immobilization methods for lipase, such as covalent bonding, entrapping, and adsorption methods, but among them, adsorption using ion exchange resins has attracted attention as an industrially viable method. Several methods have been proposed. For example, it is a method of immobilizing so that an anion exchange group is present on the surface of the substrate, and it was found that using this immobilized lipase, high activity could be maintained even in the presence of a high concentration of fatty acids. Special Public Service 1986-1
No. 8476).

Then, a method of adsorbing mucor-derived lipase to a weakly basic anion exchange resin (Japanese Patent Application Laid-Open No. 60-989
84), a method of immobilizing polyvalent aldehyde within the macropores of a basic ion exchange resin that does not have active hydrogen (see JP-A-60-30683), (JP-A-61-15690) (see JP-A-62-155089), a method in which an enzyme is adsorbed onto a resin having an amino group and a polyether group as functional groups, and further immobilized with a polyfunctional crosslinking agent (see JP-A No. 155089/1989); Method for immobilizing lipase derived from yeast (Japanese Patent Application Laid-open No. 63-1
60583) have also been proposed.

In addition, as a method for immobilizing lipase, JP-A-62
JP-A-179388 reports an example in which a hydrophobic microporous polymer was pre-wetted with a polar solvent to significantly accelerate the rate at which lipase was immobilized. However, in this example, immobilization was performed on a hydrophobic polymer without an ion exchange group, and only the effect of improving the immobilization rate was obtained.

(Problems to be Solved by the Invention) By the way, the method according to the proposal disclosed in Japanese Patent Publication No. 1.8476/1984 has a certain effect on immobilization of lipase, but it is difficult to immobilize lipase with higher activity. It was insufficient to produce a large amount of synthetic lipase.

In order to mass produce the immobilized lipase used here, a large amount of highly purified lipase is required.

Furthermore, since the polymer substrate to which the anion exchange group is bonded is hydrophobic, it was difficult to mix it with the lipase enzyme solution.

In other words, when the immobilization rate is expressed as the amount of immobilized enzyme relative to the amount of enzyme used for immobilization, the immobilization rate worsens as the production amount of immobilized lipase increases, and also varies greatly depending on the mixing conditions. There was a problem.

The present invention focuses on such problems, and by specifically specifying the ionic strength, polar solvent concentration, etc. of the lipase enzyme solution,
Furthermore, it is an object of the present invention to increase the immobilization rate of lipase by finding a post-adsorption treatment, thereby making it possible to prepare a large amount of highly active immobilized lipase.

(Means for Solving the Problems) In order to achieve the above object, in the present invention, when preparing immobilized lipase by adsorbing lipase on an anion exchanger, the lipase enzyme solution is made to have an ionic strength of 0.1 or less, and water is Add a polar solvent miscible to the solution at a concentration of 40 to 70%.
Add volume%.

In addition, the ionic strength of the lipase enzyme solution is adjusted to 0.1 or less, and a polar solvent that is miscible with water is added to the solution to
After adding and adsorbing 70% by volume, it is treated with a polyvalent reaction reagent.

Here, the lipase in the present invention is an enzyme produced by microorganisms and higher animals and plants, and is a biopolymer that acts on ester bonds to catalyze ester decomposition, transesterification, or ester synthesis.

It also includes substances called esterases and phospholipases as long as they have the above-mentioned effects.

In particular, when decomposing fats and oils with a high melting point such as beef tallow, lipase derived from Pseudomonas fluorescens is highly heat resistant, has good affinity with lipids, starts a reaction quickly, and decomposes lipids to a high rate. Most preferred.

The carrier for the immobilized lipase of the present invention is a hydrophobic substrate such as a copolymer of polystyrene and divinylbenzene, a copolymer of acrylate and divinylbenzene, a phenolic resin, an acrylic resin, or an epoxy resin. An anion exchanger into which an ion exchange group has been introduced is used. Since the anion exchanger increases the lipase binding area, a macroporous carrier is desirable. In this case, the pore diameter of the macroporous carrier is 10 to 10% of the lipase molecule used.
A thickness of 102 to 104 angstroms, which is 100 times larger, is used. Specific examples of such carriers include DOWEX MWA-1 (manufactured by Dow Chemical Company) and DOWEX 6.
6 (manufactured by Dow Chemical Company), and Diaion wn-30 (manufactured by Mitsubishi Chemical Industries, Ltd.).

The polar solvent used may be any polar solvent that is miscible with water and does not deactivate the enzyme, specifically ethanol,
Examples include isopropyl alcohol.

To produce immobilized lipase, first add 40 to 70% by volume of a water-miscible polar solvent to a lipase enzyme solution that has been desalted to an ionic strength of 0.1 or less, remove impure proteins by centrifugation, etc. When brought into contact with the carrier, lipase, which is generally an acidic protein, forms an ionic bond with the anion exchanger, and lipase protein, which is rich in hydrophobic amino acids, forms a strong hydrophobic bond with the hydrophobic carrier. Thereafter, by washing the unimmobilized lipase, immobilized lipase can be easily obtained.

In addition, in order to immobilize more firmly, treatment is performed with a polyvalent reaction reagent such as glutaraldehyde. However, since polyvalent reaction reagents are undesirable if mixed into foods, etc., after the reaction is complete, unreacted polyvalent reaction reagents are completely removed using a reducing agent, etc., and then thoroughly washed with water.

Furthermore, the amount of immobilization by this lipase is such that there are 1000 or more anion exchange groups per molecule of lipase. In reality, lipase is adsorbed onto a carrier into which as many ion exchange groups as possible are introduced. As the amount of adsorbed lipase increases, the amount of unadsorbed lipase gradually increases, but when the amount exceeds a certain level, it suddenly increases. If the amount of lipase adsorption is kept below that amount, the above objective can be reliably achieved.

(Function) In the present invention, the ionic strength is lowered to 0.1 or less, and lipase is adsorbed to the carrier by the ionic bonding action between the carrier and lipase protein. When the ionic strength is set to 0.1 or more, the ionic bond between the carrier and lipase protein is weakened.
The amount of lipase adsorbed also decreases, making it impossible to obtain highly active immobilized lipase.

Therefore, a highly active immobilized lipase can be obtained by setting it to 0.1 or less.

Furthermore, in the present invention, by adding a polar solvent of 40 to 70% by volume to the lipase enzyme solution, hydrophobic polystyrene, etc., which is the base of the anion exchanger, can be easily mixed and adsorbed with the lipase enzyme solution, and the lipase protein and the hydrophobic base can be easily mixed and adsorbed. The immobilization rate is significantly improved despite the decrease in hydrophobic binding. Moreover, when 40 to 70% by volume of a polar solvent is added, contaminant proteins other than lipase can be easily removed. If immobilization is performed using an enzyme solution from which contaminant proteins have been removed, the amount of contaminant proteins adsorbed to the immobilization carrier will decrease, so the amount of lipase protein adsorbed will increase, and highly active gluebase will be obtained. In addition, if the polar solvent is less than 40%, it is difficult to remove contaminant proteins, and if the polar solvent is more than 70%, the lipase protein concentration decreases due to the increase in the volume of the polar solvent, and a large amount of lipase protein is immobilized. It becomes difficult to adsorb onto a carrier, making it unsuitable for practical use.

(Example) Examples of the present invention will be further described below.

In addition, the lipase activity in the examples was determined by the modified method of Nord et al.
(1962)), and the amount of enzyme that liberated 1 microequivalent of acid per minute at 60°C and P) 17.0 was defined as 1 unit.

In addition, the immobilization rate in the table is based on the lipase solution used before immobilization and after immobilization.
No. 95) was used, which was obtained by desalting and concentrating the culture supernatant.

Example 1 Desalted and concentrated culture supernatant of Shutomonas fluorescens biotype (hereinafter abbreviated as pfC)
Lipase was immobilized on DOWEX-66 using For immobilization, various concentrations of ethanol were added to pf-c.
After centrifugation at 000 PPM for 20 minutes, 40 g of the supernatant was taken, added to 10 g of Dowex-66, and fixed at 8° C. overnight. After immobilization, gluculaldehyde (
Add 6g of 25% N, stir at 8°C for 10 minutes, then add 20%
After adding 2 g of sodium bisulfite and further stirring at 8°C for 10 minutes to remove excess glutaraldehyde, the mixture was thoroughly washed with water to obtain immobilized lipase.

As shown, by adding 40% or more ethanol, the immobilization rate was 90% or more.

Table 1 Immobilization treatment results - Effect of ethanol concentration Example 2 5.5% of ethanol was added to pf-c that had been sufficiently desalted.
After adding 0% and centrifuging the supernatant, salt was added at various concentrations to prepare enzyme solutions with an ionic strength of 0 to 0.5. This enzyme solution was immobilized on Dowex-66 according to the previous method.

The fat and oil decomposition ability was measured using the obtained immobilized lipase.

That is, 1 g of immobilized lipase, 1 g of olive oil, and 1 g of water were added and reacted at 60''C for 48 hours, and then the oil/fat hydrolysis rate was measured.

The fat and oil hydrolysis rate was determined from the equation of fat and oil hydrolysis rate (%) - (oxidation/saponification value) x 100.

As shown in Table 2, the immobilization rate was 95% or more when using an enzyme solution with an ionic strength of 0 to 0.1.
An immobilized lipase with high lipolytic activity was obtained.

Table 2 Immobilization treatment results - ionic strength Example 3 PF-C: (1) water, (2) methanol, (3) ethanol.

(4) isopropyl alcohol, (5) ace1-one 5
After adding 0% and centrifuging at 5000 PPM for 20 minutes, the supernatant was used to immobilize the enzyme on DOWEX-66. 3rd result
Shown in the table.

Table 3 Immobilization treatment results - About solvent treatment Example 4 Add equal volume of ethanol to pf-c and mix the mixture at 400O
Centrifuged for 20 minutes at RPM. 800g of supernatant liquid (
330 units/g) was added with 200 g of Dowex-66, followed by infiltration treatment at 8°C overnight for immobilization.

After fixation, add 32g of glutaraldehyde (25%) at 8°
After washing with C for 10 minutes to remove excess glutaraldehyde, the mixture was thoroughly washed with water to obtain immobilized lipase.

The immobilization rate was 82.7%, and the fat and oil decomposition ability was 92.5%.

This example shows that even if a large amount of immobilized lipase is prepared, highly active lipase can be obtained.

(Effects of the Invention) As described above, in preparing immobilized lipase by adsorbing lipase to an anion exchanger, the present invention lowers the ionic strength of the lipase enzyme solution to 0.1 or less and reduces the polar solvent to 40-70%. This is a method of adding % by volume, and the ionic strength is 0.
By lowering the amount to 1 or less, lipase is adsorbed to the carrier by the ionic bonding action between the carrier and lipase protein, and a sufficient amount of lipase can be secured.Additionally, by adding a predetermined amount of polar solvent, the anion exchanger substrate can be The hydrophobic resin becomes easier to mix and adsorb with the lipase enzyme solution,
Although the hydrophobic bond between the lipase protein and the hydrophobic substrate is reduced, the immobilization rate can be significantly improved.

Moreover, by adding a polar solvent, contaminant proteins other than lipase can be easily removed, the amount of contaminant protein adsorbed is reduced, and lipase with higher activity can be obtained, and the method of the present invention has a higher immobilization rate than conventional methods. It has the remarkable effect of being able to prepare a large quantity of highly active immobilized lipase.

In particular, when treated with a polyvalent reaction reagent as described in claim 2, more solid immobilization can be achieved.

In addition, the immobilized lipase obtained above is stable and can maintain high activity even when used continuously for a long time, which is extremely advantageous in practice.

Claims (1)

[Claims] 1. In preparing immobilized lipase by adsorbing lipase on an anion exchanger, the ionic strength of the lipase enzyme solution is reduced to 0.1 or less, and a water-miscible polar solvent is added to the solution. A method for producing immobilized lipase, which comprises adding 40 to 70% by volume of immobilized lipase. 2. When preparing immobilized lipase by adsorbing lipase on an anion exchanger, the ionic strength of the lipase enzyme solution is 0.1 or less, and the amount of a water-miscible polar solvent is 40 to 70% by volume based on the solution. A method for producing immobilized lipase, which comprises further adsorbing the lipase and then treating it with a polyvalent reaction reagent. 3. The method for producing immobilized lipase according to claim 1 or 2, wherein a macroporous hydrophobic resin is used as the substrate of the anion exchanger. 4. The method for producing immobilized lipase according to claim 1, claim 2, or claim 3, wherein the lipase adsorption amount is 1/1000 or less of the anion exchange capacity.