JPH01285188A - Lipase-immobilized polyacrylic acid-based material and utilization thereof - Google Patents

Abstract

PURPOSE:To improve washing effect by immobilizing lipase on a carrier composed of water-insoluble polyacrylic acid or derivative of said polyacrylic acid, forming polyacrylic acid-based material and adding to various system used as a detergent. CONSTITUTION:A carrier composed of water-insoluble polyacrylic acid or derivative of said polyacrylic acid is bonded and immobilized with lipase by applying a physical bonding method, adsorption method, ionic bonding method or chemical bonding method, etc., to form a lipase-immobilized polyacrylic acid-based material. An immobilizing by a chemical bonding is preferable for the case of utilizing as drugs in which purity of material is especially required and as the definite means of the immobilizing, polyacrylic acid is made to an active ester and reacted with lipase. Water-soluble carbodiimide is especially preferable as an esterifying agent of said case.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field] The present invention relates to a lipase-immobilized polyacrylic acid material useful in the pharmaceutical field, oleochemical field, or cleaning chemistry field.

[Background technology]

Lipase is a type of ester degrading enzyme that decomposes fats and oils into glycerin and fatty acids, which are constituent components of the chemical structure, and is classified as EC3,1,1,3. Lipase has an extremely wide distribution in the living world, and is found in plants, molds, bacteria, milk, animal tissues, etc.
It is known that the pancreas contains particularly large amounts of it. However, lipase belongs to a relatively unstable group of enzymes, and when lipase isolated from a biological system is dissolved in water, it is deactivated at a relatively rapid rate.

For this reason, isolated lipase is usually dried and then stored at low temperatures.

For the above reasons, although lipase is expected to have a wide range of applications, particularly in the field of oil and fat chemistry, it is currently only used in limited fields such as digestive aids and the production of cheese flavors. Given this current situation, if stable immobilized lipase can be obtained and used repeatedly, in other words, if lipase can be used as a so-called bioreactor, a wide range of applications can be expected. It should be.

Many methods have already been proposed for immobilizing enzymes on carriers while retaining enzymatic activity, and in some cases this goal has been achieved. Even if immobilization is possible, identical results may not be obtained for different enzymes. For example, diethylaminoethyl cellulose is well known as a general immobilization carrier for enzymes, but lipase adsorbed to this carrier does not express lipase activity.

[Disclosure of the invention]

As a result of intensive studies on the immobilization of lipase, the present inventors found that when lipase is adsorbed to water-insoluble polyacrylic acid or its derivatives, the adsorbed lipase is immobilized while retaining its activity. I found out. The present invention is based on this knowledge.

The present invention will be explained in detail below.

In the present invention, polyacrylic acid used as a carrier for immobilizing lipase has extremely low solubility in water, that is, it is substantially insoluble in water. In general, polyacrylic acid has different properties depending on the degree of polymerization or the presence or absence of crosslinking, but polyacrylic acid that is substantially soluble in water cannot be used because it is not suitable for the purpose of the present invention. Can not.

Polyacrylic acid is usually used in the form of a free carboxyl group, but it may also be partially used in the form of a salt, or in the form of derivatives such as amides and esters.

Furthermore, this polyacrylic acid may be a copolymer of acrylic acid and other heptamers, and the carboxyl groups may partially be derivatives such as amides.

The lipase-immobilized polyacrylic acid material in the present invention is
Lipase can be obtained by immobilizing lipase on these polyacrylic acid-based materials. In this case, immobilization methods include physical bonding, such as adsorption, ionic bonding, etc. Other methods include chemical bonding, such as immobilization by amide bonding.

Lipase immobilization in the present invention may be performed by any of the methods described above, and is not limited to a specific method.

Fixation by chemical bonding is a desirable method when used in pharmaceutical products, etc., where material purity is particularly required. An example of such a method is to fix polyacrylic acid by converting it into an active ester and reacting it with lipase. As the esterifying agent in this case, water-soluble carbodiimide is particularly preferably used.

Polyacrylic acid as an immobilization carrier sufficiently adsorbs lipase in an aqueous solution even when its concentration is extremely low, and the adsorbed lipase does not lose its original activity even after being adsorbed. . Therefore, the activity of lipase is
It is mostly fixed as is. This was discovered for the first time by the present inventors. There are many carriers that simply adsorb lipase from an aqueous solution, such as activated carbon, zeolite, talc powder, DEAE cellulose,
Cellulose phosphate and the like can be used as carriers that simply adsorb lipase, but in these cases, the adsorbed lipase does not exhibit any lipase activity. What is even more surprising is that polyacrylic acid also has the property of retaining the activity of adsorbed lipase for a long period of time.

Since the lipase-immobilized polyacrylic acid material according to the present invention can decompose fats and oils under extremely mild conditions,
It is extremely useful for this purpose. Conventionally, fats and oils were decomposed using high-temperature, high-pressure water vapor gas under conditions such as tens of atmospheres of 200°C, but it is now possible to decompose fats and oils under extremely mild conditions without relying on such harsh conditions. Decomposition can be carried out, which is particularly useful for the decomposition of fats and oils consisting of highly unsaturated fatty acids. In the field of cleaning chemistry, it is particularly useful for glycerides, especially triglycerides, which are difficult to remove and clean.

It was confirmed that the polyacrylic acid-immobilized lipase of the present invention exhibits lipase activity not only in the above-mentioned aqueous system but also in a non-aqueous system. That is, it exhibited glyceride decomposition activity for oils and fats dissolved in either petroleum-based solvents or chlorinated solvents (Example 7.8). By utilizing this property, it becomes possible to purify cleaning solvents in the dry cleaning field. Solvents containing dirt components eluted from dirty clothes are usually purified by adsorption using activated carbon or activated clay and then recycled for reuse. However, glycerides, one of the dirt components, are difficult to adsorb and therefore are not used in cleaning solvents. This has been an obstacle in refining and reuse. This objective can be achieved by using the polyacrylic acid-immobilized lipase of the present invention to decompose glyceride into fatty acid and glycerin, and then adsorbing the mixture after increasing its polarity.

Furthermore, the lipase-immobilized polyacrylic acid material according to the present invention can be added to polyfilaments commonly used as detergents to increase the cleaning effect.

It goes without saying that the amount of the lipase-immobilized polyacrylic acid material according to the present invention to be contained in a detergent will vary depending on the strength of the activity of the lipase used. , the desirable lipase content is o, oi to 1, h/lOh detergent.

Next, specific examples will be given and explained.

[Example 1] Amberlite CG-50 (H”) as polyacrylic acid
6.84 g of this was injected into lipase (Toyo Jozo Co., Ltd. chrom).
Aqueous solution 15 containing 30 mg of 120.0 units/mg isolated from obacter ium viscosum
01IQ, stirred at room temperature for about 60 minutes, and then centrifuged (3000 rpm x 6ml).

Subsequently, the activity of the hydrous polyacrylic acid adsorbed lipase (2,119) obtained by repeating centrifugation twice and washing with water was measured by the method described in the reference side below, and it was found that 30
It was 0 units/g.

[Example 2] The polyacrylic acid-adsorbed lipase obtained in Example 1 was kept at 25°C for 75 days, and the lipase activity was measured in the same manner as in Example 1. The rate of decrease in the activity was approximately 5%. It stopped at

[Example 3] 511 g of l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride was dissolved in water 411112, and Amberlite CG-50 (lo
o ~ 200mesh) 1.09 was added, and after 30 seconds, a lipase aqueous solution (30 mg/l m12) was added, and then 3
Stir for 0 minutes. The obtained polyacrylic acid-bonded immobilized lipase was prepared according to the reference example described later, and its activity was measured, and it showed an activity yield of 32.8%.

[Example 4] When the polyacrylic acid-bound immobilized lipase obtained in Example 3 was left at 35°C for 175 days, the activity was measured according to Example 1, and the rate of decrease in activity was 13%.

[Example 5] A contaminated cotton cloth (standard cotton cloth of the Laundry Association) was wrapped around the wrist of an adult male at about 200 o'clock in the winter, and the contaminated cotton cloth was shredded and used as a sample. 2001 g of the sample was dispersed in 10 g of water, polyacrylic acid adsorbed lipase 50119 obtained in Example 1 was added, and the decomposition of triglyceride contained in the contaminated cotton cloth was measured according to the reference example described later.

The amount of triglyceride decomposed per minute reached 16 μmo (le) based on the amount of alkali consumed for decomposing triglyceride.

[Example 6] 1.0 g of olive oil was dissolved in n-hexane 9,0 g,
Polyacrylic acid adsorbed lipase 50 obtained in Example 1
mg was added and the decomposition of n-hexane-dissolved olive oil was measured according to the Reference Example described later. The amount of triglycerides in olive oil decomposed per minute is 3.3μt compared to the amount of alkali consumed.
It is aoQe.

[Example 7] Olive oil 1. (h is 1.1.1-) Dissolved in 9 mQ of dichlorethane, added 5011 g of polyacrylic acid adsorbed lipase obtained in Example 1,
1.1- Decomposition of trichloroethane dissolved olive oil was measured. The amount of triglycerides in olive oil decomposed per minute is 3.4 μmol 12e based on the amount of alkali consumed.

[Example 8] 200 m of the same shredded contaminated cotton cloth used in Example 5
g to 10ml of 0.1% sodium stearate aqueous solution
The polyacrylic acid-adsorbing lipase 10119 obtained in Example 1 was added thereto, and the mixture was stirred for 22 minutes while being maintained at 30° C. and pH 9.0. The reaction solution was filtered and the resulting solution was analyzed for decomposed free fatty acids using reverse phase liquid chromatography.

On the other hand, free fatty acids in the reaction solution obtained by conducting the same experiment as above without adding polyacrylic acid-adsorbing lipase were analyzed. When comparing the two, when polyacrylic acid-adsorbing lipase was added, compared to when it was not added,
3.1 times the amount of free fatty acids was confirmed.

As a result, the obtained lipase adsorption carrier was prepared by adding 2.28 g of diethylaminoethyl cellulose (DEAE cellulose) to 2.2 g of water (JaQ) and adjusting the resulting suspension to below 10°C and adjusting the pH to 8 while stirring. keep.

Add lipase (chromo produced by Toyo Jozo Co., Ltd.) to this suspension.
-120.0 units/1119) IO+mg isolated from bacterium viscosum was added, stirred for about 20 minutes, and then centrifuged (3000 rpm 86 mL)
L. Obtain supernatant and precipitate.

The precipitated portion was washed with water by repeated centrifugation twice, and the water-containing D
EAE cellulose adsorbed lipase 3.969 is obtained. When lipase activity was measured at 30°C at pH 9.0 using olive oil as a substrate and gum arabic as an emulsifier using a pHstat, no activity was observed in either the supernatant liquid or DEAE cellulose-adsorbed lipase.

A similar experiment was carried out using activated carbon (NoritA) as an adsorption carrier.
C-talc powder (manufactured by Osaka Ceramics Fireproof Co., Ltd.), zeolite (
No activity was observed in selected products such as Shichicard Pi1 (manufactured by Kasei Co., Ltd.), cellulose phos-7ate (P-cellulose), and carboxymethyl cellulose (CMC).

Patent applicant: Esthe Chemical Co., Ltd.

Claims (1)

[Scope of Claims] 1) A lipase-immobilized polyacrylic acid material, characterized in that a water-insoluble polyacrylic acid or a derivative thereof is used as a carrier, and lipase is immobilized thereon. 2) An immobilized lipase-containing cleaning agent comprising the lipase-immobilized polyacrylic acid material according to claim 1.