JPH01273588A - Immobilized lipase - Google Patents

Abstract

PURPOSE:To exclude protons, produced by hydrolysis with a lipase and causing inhibition of products and prevent the activity of the lipase from deteriorating by the presence of anion exchange groups without binding to the lipase on the surface of a carrier bound to the lipase. CONSTITUTION:Various ion exchange resins, glass beads or silica beads containing as much ion exchange groups as possible introduced thereinto with a silane coupling agent are used as a carrier for immobilizing a lipase. The above-mentioned carrier is preferably hydrophobic. In immobilizing the lipase on the carrier, the amount of the bound lipase is regulated so that the anion exchange groups without binding to the lipase may be present on the surface of the carrier.

Description

Detailed Description of the Invention [Prior Art and its Problems] When fats and oils and esters are hydrolyzed by lipase, glycerin, alcohol, fatty acids, fatty acid ions, and protons are generated. Fatty acids and fatty acid ions are poorly soluble in water and do not inhibit the product; however, in order to eliminate inhibition, methods have been used to increase the water content in the reaction solution and dilute glycerin, alcohol, and protons ( Iwai et al., J. Ge.
n, Appl.

Micr vehicle Ro1.10.13 (1964)). However, when the water content in the reaction solution is high, it takes a lot of energy to recover the glycerin and alcohol obtained in the aqueous layer after the reaction is completed, which has been an obstacle to industrially implementing fat and oil decomposition using the enzyme method.

[Problem of invention]

It is an object of the present invention to provide an immobilized lipase that can overcome the product inhibition observed in the prior art.

[Means to solve the problem]

The present inventors have conducted extensive research on reactions using immobilized lipase. When bonding lipase, even if the reaction occurs in a reaction solution where protons generated from fatty acids are present, the protons that inhibit the product are removed from around the lipase molecule due to electrostatic repulsion of the anion exchange group. It was discovered that immobilized lipase exhibits sufficient activity, and based on this finding, the present invention was accomplished.

The origin of the lipase used in the present invention is not particularly limited, but it is preferably an enzyme that has good affinity for fats and oils, has a good reaction start-up, and can decompose fats and oils to a high decomposition rate. When decomposing fats and oils with a high melting point such as beef tallow, it is desirable to use a heat-resistant lipase that reacts at 45° C. or higher.

The lipase immobilization carrier used in the present invention is a carrier to which as many anion exchange groups as possible are bonded, and is usually a carrier using various anion exchange 4FFL fats, glass beads, or silica beads with a silane coupling agent. A hydrophobic carrier is used, such as one in which as many anion exchange groups as possible have been introduced into a metal powder, or one in which as many anion exchange groups as possible have been introduced into a metal powder. Hydrophilic vinyl polymers or cellulose in which as many anion exchange groups as possible can be introduced can also be used, but hydrophobic carriers have better affinity for the substrate and eliminate the generated hydrophilic glycerin. It is a desirable carrier because it reduces the product inhibiting effects of glycerin. Even better results can be obtained by using macroporous anion exchange resins, glass beads, silica beads, F, J. That is, the radius of a normal lipase molecule is 1 (1
Since the average pore radius is Å or more, if a porous carrier having an average pore radius larger than this is used, the lipase will also bind to the surface portion within the pores, so that the bonding area between the lipase and the anion exchange group can be widened. Moreover, when the average pore radius becomes 1000 Å or more.

Since the pore surface area to which lipase and anion exchange groups bind is reduced, it is preferable to use a carrier having an average pore radius of 10 to 1,000. A macroporous carrier can provide a wide surface area for bonding lipase and anion exchange groups, and can also prevent carrier particles from becoming finer, resulting in an immobilized enzyme column with less pressure loss.

The anion exchange group used in the present invention includes various amines,
Ammonium and its derivatives are chemical residues that dissociate in a solvent, become positively charged, and adsorb anions.

If the amount of lipase bound to the carrier used in the present invention is too small, the immobilized enzyme activity will be low and the reaction start-up by the immobilized lipase will be slow.

Although the start-up of the reaction by chemical lipase is improved, the final decomposition rate of high fatty acid concentrations may be poor. This is because when lipase is bound, it is often bound through an anion exchange group, so the anion exchange group is blocked and there are no anion groups on the surface of the carrier to which lipase is bound. be. In order to obtain the effects of the present invention,
In general, it is necessary to use a carrier that has sufficiently bound anion exchange groups, which are low molecules, and to adjust the lipase-binding fragment so that anion exchange groups to which lipase is not bound are present on the carrier surface. .

Regarding the method of binding lipase to a carrier, since lipase is usually an acidic protein, it easily binds to an anion exchange carrier through an ionic bond. In addition, when a hydrophobic carrier having an anion exchange group is used, it is strongly bound by ionic bonds and hydrophobic bonds.

Furthermore, when those bound by ionic bonds and hydrophobic bonds are treated with geltaraldehyde or carbodiimide, the bonds become even stronger. In the case of geltaraldehyde treatment, p + (7
Above that, geltaraldehyde becomes more reactive,
pH 4.5-6.5 as lipase is inactivated
It is better to adjust the temperature to around 25°C or lower and process the process for a short time of about 10 to 20 minutes. After the reaction, excess geltaraldehyde is removed using sodium bisulfite or the like. The above operation involves pouring an appropriate amount of lipase solution onto the carrier packed in the column and adsorbing it through ionic and hydrophobic bonds. After adjusting the PL+ and temperature, pouring in an appropriately diluted gel taraldehyde solution, followed by hydrogen sulfite. This is an industrially advantageous bonding method because it is easy to prepare an immobilized enzyme column by flowing a sodium solution. If the anion exchange group is used as a functional group for binding the lipase protein as described above, it is possible that the anion exchange group will eliminate protons and weaken the effect of removing the product inhibition effect of protons. If the group is protected and the lipase is bonded via another functional group, the effects of the present invention will be further enhanced, so there is no problem with such a bonding method.

As described above, the immobilized lipase of the present invention is characterized by the presence of a large number of anion exchange groups to which lipase is not bound on the surface of the carrier. The amount of the group present on the carrier surface is usually 100% per molecule of lipase bound to the carrier.
The proportion is 0 or more, preferably 10,000 to 10,000,000. The greater the number of anion exchange groups, the more the product inhibition effect by protons can be weakened, and in general, if the carrier has 1000 or more anion groups per molecule of bound lipase, the desired effect can be achieved. Able to achieve purpose.

Regarding the pH and temperature of the reaction conditions when carrying out a lipase reaction using the immobilized lipase of the present invention, a pH and temperature near the optimum for the reaction of the immobilized lipase are used. If the desired reaction rate can be obtained even if the pH and temperature are not optimal,
There is no harm in using it. Also, if necessary, 3 gold
It is also possible to add ions, serum allamin, etc.

The immobilized lipase of the present invention is used for conventionally known lipase reactions such as those that involve product inhibition-force withdrawal by protons. Examples of such lipase reactions include decomposition reactions of lipids (glycerides and esters) and ester synthesis reactions between fatty acids and alcohols.

When using the immobilized lipase of the present invention, product interference caused by protons can be removed, so the maximum free fatty acid concentration in the reaction solution is 50% or more, especially high fat I! of 70 to 80% (by weight)! ! ! The lipase reaction can also proceed under certain concentration conditions. For example, when performing a lipid decomposition reaction using the immobilized lipase of the present invention, the free fatty acid concentration in the 5 reaction solution is at its maximum value, that is, the free fatty acid concentration in the final reaction solution is in the range of 50 to 70%. chosen to be. 5
Even if the fatty acid concentration is 0% or less, the final decomposition rate does not increase, and in order to reduce the scale of the reaction tank and to reduce the energy required to recover glycerin and alcohol obtained in the reaction double water layer, this concentration is necessary. It is more advantageous to react at the above level.

When producing fatty acids industrially, oil and water (sweet wood containing glycerin) are separated after the fat and oil decomposition process is complete.

The oil content is divided into liquid fatty acids and solid fatty acids for subsequent purification, but in addition to oleic acid, linoleic acid, and other undecomposed triglycerides may remain in the liquid fatty acids. When decomposing glycerides of about 8 or so contained in such crude fatty acids, the small amount of water contained in the crude fatty acids can be sufficient to hydrolyze them, so if the crude fats are directly reacted with the fixed lipase of the present invention, It is also possible to remove mixed triglycerides.

The reaction vessels used in the present invention may be of a batch type in which the immobilized lipase and the reaction solution are mixed together, or of a continuous type in which the immobilized lipase is packed in a column. Equivalent lipase can be easily recovered using both batch and column methods.
This method has the advantage that it is not necessary to remove the lipase protein during the product purification step, and the recovered enzyme can be used repeatedly. The column type has further advantages such as less contact with air during the reaction, so the unsaturated fatty acids are not oxidized by the air, and because it can be continuous, it can be easily incorporated into the currently used high-pressure continuous cracking process.

〔Effect of the invention〕

By using the immobilized lipase of the present invention, it has become possible to decompose fats and oils and esters to a high decomposition rate with extremely low water content in the reaction solution. This not only reduces the energy required to recover the glycerin and alcohol obtained after the reaction by distillation, but also reduces the size of the reaction tank when decomposing large amounts of fats and oils, and has other effects on the industrial fats and oils decomposition process. big. Moreover, the use of an immobilized lipase column has the effect of enabling continuous reaction and preventing air oxidation of unsaturated fatty acids. Moreover, it is an excellent method for industrial enzymatic decomposition of fats and oils, as it is easy to prepare and regenerate the immobilized enzyme column.

〔Example〕

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

Example 1 1 g DOWEX HuA-1 (total exchange capacity 1, 2 m
Dow, a macroporous type weakly basic anion exchange resin whose exchange group is a tertiary amine with a matrix in which polystyrene chains are crosslinked with divinylbenzene.
Chemical Co., Ltd.) was washed with distilled water and 1/15M pine quill vain buffer pH 5.0, 50AL12 lipase solution (1450 units) and 1-component pine quill vain buffer were added, and the mixture was shaken at 8°C overnight. . Next, 1 mQ of pine irvain buffer solution and 80 μQ of 2-color gel tar aldehyde solution were added, and the mixture was shaken at 8° C. for 10 minutes to bind to DOWEX 1nA-1. Finally, add 0.2 parts of 20 parts of sodium bisulfite and shake at 8°C for 10 minutes.
After removing excess gel taraldehyde, it was thoroughly washed with water and a buffer solution to obtain DOWEX MWA-1 immobilized lipase. The amount of lipase used here is an amount sufficient to express the activity of the immobilized lipase, and an amount that provides a high final decomposition rate.

1g of DOWEX WGR (total exchange capacity 1, 6meq
/ mQ, a gel-type weakly basic anion exchange resin containing a condensate of shrimp chlorohydrin and ammonia and using polyamine as an exchange group (manufactured by Dow Chemical Company) was treated in the same manner as above to obtain a large amount of inorganic ions. We obtained lipase immobilized on a gel-type anion exchange resin synthesized to have many extremely fine pores that allow it to pass through.

Revachit CNP80 (total exchange capacity 4.7meq/
1 g of mfl, a macroporous weakly acidic cation exchange resin with acrylic as the base and carboxylic acid as the exchange group (manufactured by Bayer AG), ■-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide・Add 50 μm of p-toluenemethosulfonic acid (hereinafter abbreviated as CMC) and 10 μm of water, continue the reaction at room temperature while keeping the pH at 4 to 5 with 6N HCQ, and when the pH stabilizes, remove excess CMC. Wash thoroughly.

Next, add 2 mQ of 1/15M pine irvain buffer pH 4.
, 50 μQ of lipase solution (1450 units) was added, and the mixture was allowed to react overnight in a cold room to obtain Revatit CNP80-immobilized lipase.

The lipase activity was determined using 3.14 g of tributylene, 35-mer two-stone polyvinyl alcohol (Kurashiki Popal 117) solution, 40 ml of 0.1 M phosphate buffer (pH 7), 12 ml of
Mix 1 g of tributyrin emulsion 9-1 enzyme suspension prepared by mixing water from step 2 and sonicating it for 10 minutes,
The reaction was carried out at 0°C for 20 minutes with shaking. The reaction was stopped by adding a 1:1 mixture of methanol and acetone, and the resulting fatty acid was added dropwise with 0.05N NaOH to measure the activity.

The amount of acid released in 1 minute under the above conditions was defined as 1 unit.

The lipase preparation is Pseudomonas fluor
escesbiotype I-Ha 1021 (
The lipase produced by Kaikouken Kyoiku No. 5495) was subjected to ammonium sulfate salting out, dialysis, degreasing with chloroform, etc., and then freeze-dried.

In a 100 mQ Erlenmeyer flask, prepare reaction solutions of various beef tallow concentrations with beef tallow and 0.1N phosphate buffer P)17.0.
The immobilized lipase, which had been well removed from moisture, was added and reacted for 160 to 200 hours at 60°C with shaking 178 times per minute. The decomposition rate (final decomposition rate) was determined from the ratio of the oxidation value and saponification value. . The results are shown in Table 1.

Table 1 → (The table shows the final decomposition rate of fats and oils at high concentrations of raw beef tallow for those with anion exchange groups such as tertiary amines and polyamines on the surface of the carrier to which lipase is bound (Dowex N
It can be seen that those using WA-1 or DOWEX WGR as an immobilizing carrier) are better than those containing a carboxylic acid as a cation exchange group (using Revatit CNP80 as an immobilizing carrier). In addition, lipase immobilized on a macroporous anion exchange resin, which has an anion exchange group on the surface of the carrier and where the lipase binds extends into the pores, has a particularly good decomposition rate at high substrate concentrations. It shows.

The number of anion exchange groups per molecule of lipase bound to the immobilized carrier lipase is determined based on the molecular weight of the lipase (120,000), the activity unit of the immobilized lipase, and the total exchange capacity of the immobilization carrier. When calculated, DOWEX NWA-
In the case of No. 1, there were 11X10' or more, and in the case of DOWEX υGH, there were 9X10' or more. On the other hand, lipase protein with a molecular weight of 120,000 has over 800 amino acids, of which about 80, or 10%, are acidic amino acids. Since most of it is neutralized with basic amino acids, the number of residues on the lipase molecule that can be dissociated on the protein molecule and bonded to the anion exchange group is less than half (40 or less).

Example 2 The final decomposition rates of various macroporous anion exchange resins were compared using immobilized lipase in the same manner as shown for DOWEX MVA-1 in Example 1.

The results are shown in Table 2.

Table 2 Table 2 shows that when immobilized lipase with an appropriate amount of lipase bound to various anion exchangers of Macro Polar Stamp and anion exchange type chelate resin (Diaion CR20) is used, lipase is activated. Since an anion exchange group exists on the surface of the bonded carrier, proton product interference is removed, indicating that the decomposition rate of fats and oils is good.

Note that lipase 1 bound to the carrier surface of immobilized lipase
When the number of anion exchange groups per molecule is calculated in the same manner as in Example 1, in the case of Amberlite IRA93, it is 12X
105, 6.5 for Amberlight IRA904
X 10', 2 for Diaion WA21 (7). O
X to5, 2 for Diaion HPA25. O
X 10', 1.5 X for Diaion CR20
10', 19X for Duolite A-4], 05
In the case of Duolite A-7, it was 23 x 105.

Example 3 DOWEX MWA-1 prepared in the same manner as Example 1
The immobilized lipase was thoroughly dried, reaction solutions with various concentrations of beef tallow were prepared with beef tallow and 0.1 M phosphate buffer, and lipase reactions were performed to compare the final decomposition rates.

The results are shown in Table 3. The results of soluble lipase used during immobilization are also shown as a control.

Note that when 80% beef tallow is reacted with immobilized lipase, the free fatty acid concentration after the reaction becomes 71%. Furthermore, when 94% beef tallow is reacted with immobilized lipase, the free fatty acid concentration after the reaction becomes 79%.

Table 3 Table 3 shows that when using soluble lipase, the final decomposition rate is significantly reduced at high fatty acid concentrations, whereas when using immobilized lipase prepared in such a way that a large number of anion exchange groups exist on the surface of the carrier to which lipase is attached, We show that a high decomposition rate can be obtained using chemical lipase even at high fatty acid concentrations.

Claims (1)

[Claims] (1) An immobilized lipase characterized in that an anion exchange group to which lipase does not bind is present on the surface of a carrier to which lipase is bound.