JPH01121342A - Immobilized lignin composite and use thereof - Google Patents

Abstract

PURPOSE:To provide the title composite having bonding affinity for proteins, thus suitable as an enzyme-immobilizing material and protein-eliminating one, containing a carrier consisting of polymeric solid granules and lignin matter layer immobilized on said carrier. CONSTITUTION:The objective composite containing (A) a carrier consisting of polymeric solid granules [pref. of cellulose (derivative), amino acid (derivative), polyacrylate, etc.] and (B) lignin matter immobilized on this carrier (pref. kraft lignin). Said polymeric solid granules are pref. 10-100mum for the size, being porous. In addition, it is preferable that said lignin matter be chemically or physically immobilized on the carrier and the content of said matter be 2-20wt.% based on the carrier.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to an immobilized lignin composition capable of immobilizing proteins, and more specifically, it can be used as an enzyme immobilization material, a protein removal material, etc. The present invention relates to useful immobilized lignin complexes.

[Problems to be solved by conventional technology and invention]

Enzymes are made of proteins and have a catalytic effect that promotes specific reactions with extremely high efficiency under mild conditions. Therefore of substances due to enzymes.

Production or decomposition is considered to be extremely beneficial from an industrial perspective, such as saving energy and utilizing biomass. By the way, if it were possible to immobilize enzymes on insoluble carriers, it would be possible to increase the physical and chemical stability of enzymes, which are expensive and relatively unstable proteins, and to give them reusability. It is possible to create many advantages such as:

For this reason, attempts have been made to immobilize enzymes on numerous carriers such as cellulose, plastic, metal, and silica. A wide variety of immobilization methods are proposed, including physical adsorption, entrapment, and chemical bonding methods. Among these,
Although the chemical bonding method is strong, this method has the problem that the enzyme may be denatured, deactivated, or reduced in activity during immobilization. The inclusion method has problems such as the relatively slow permeation of substances into the matrix and the fact that the reactants are limited to those with low molecular weights. Although physical adsorption methods have the great advantage of being simple, they generally have problems such as unavoidable desorption of enzymes due to changes in the environment and adsorption of reaction substrates and products.

To solve this problem, it is thought to be effective to use a polymer having a group that specifically binds proteins as a carrier. In this way, the groups that specifically bind to proteins include:
Possible examples include formyl group and isothiocyanate group. However, all of these have problems such as relatively low stability and, in the case of isothiocyanate groups, relatively complicated procedures for introduction. Furthermore, when a polymer having a specific bonding group is used as an immobilization carrier, there is a restriction that the active group must not be destroyed or denatured when the enzyme is bonded to the polymer.

For the above reasons, a material that is satisfactory as an enzyme immobilization material has not yet been found.

There is also a need for immobilization materials useful for selectively removing various proteins.

For example, analysis of components in blood, urine, and cerebrospinal fluid is one of the most important so-called clinical analyzes when diagnosing various diseases. In particular, blood, or blood plasma or serum that has been treated with blood removal methods, is most frequently analyzed, and the test targets include low-molecular components such as glucose, urea, and
Popular substances include uric acid.

During clinical testing of such low-molecular-weight components, detection is often hindered by high-molecular components contained in serum, especially fluorescent components such as albumin, globulin, and various proteases. For example, even when using the immobilized enzyme method, protein components may clog the enzyme column or cause decomposition or deactivation of the immobilized enzyme, so it is recommended to remove the protein in the sample in advance. This extends the life of the column and improves inspection accuracy.

Typical conventional protein removal methods include precipitation with water-soluble organic solvents such as acetone and acetonitrile, extraction of the test substance with water-insoluble organic solvents such as ethyl acetate, methylene chloride, and ether, and There are salting out methods using ammonium sulfate, sodium sulfate, sodium sulfite, etc., and precipitation methods using acids such as tungstic acid, phosphotungstic acid, and trichloroacetic acid. These methods are still often used today, for example, Po1in
- In the Wu method, 7 volumes of water are added to a 1:1:1 mixture of blood, 10% sodium tungstate, and 2/3N sulfuric acid, and after leaving it for 10 to 20 minutes, the mixture is heated at 2500 to 3000 rpm.
Protein can be removed by centrifugation at m for 5 to 10 minutes.

However, all of the above methods are mainly used to process large amounts of serum, etc., and they also require steps such as centrifugation, separation, and filtration, which are disadvantageous to current clinical science. It is somewhat unsuitable for rapid protein removal treatment and subsequent analysis of trace amounts of samples. On the other hand, many of the currently used clinical diagnostic devices with immobilized enzyme columns use a dialysis machine to remove protein, but most of the target substance in the sample does not pass through the dialysis membrane and is wasted. As a result, large amounts of samples are required,
Many problems have been pointed out, such as sample dilution and large tailing.

Therefore, as an alternative to these conventional protein removal methods, a simple protein removal method using a pretreatment precolumn packed with a polymer resin that exhibits specific interaction with proteins is beginning to attract attention.

However, materials capable of fixing and removing proteins with simple operations and without difficulty have not yet been sufficiently developed.

On the other hand, although lignin is a substance that exists in large quantities in nature, there is currently no advanced way to use it, so it is currently incinerated daily as a fuel.

Although lignin substances have been known to have binding properties to proteins, their use as a protein immobilization material has not been attempted. This is because many of the lignin substances are water-soluble, which makes it difficult to completely remove the lignin substances from the protein-removed liquid.

Lignin is known to be harmless to the human body, animals and plants, and to have antibacterial effects.

The present inventors have conducted various studies on the effective use of lignin, which has the above-mentioned properties, and have attempted to fix it on solid polymer particles and use it as a material for protein fixation, thereby solving the above-mentioned problems. This problem has been solved and the present invention has been completed.

[Means for solving problems]

The immobilized lignin composite of the present invention is characterized by comprising a carrier made of solid polymer particles and a lignin material layer fixed to the carrier.

In the composite of the present invention, the solid polymer particles used as a carrier include, for example, cellulose, water-insoluble cellulose derivatives, such as acetylcellulose with a low degree of acetylation, nitrocellulose with a low degree of nitrification, chitosan, pulp, etc. Natural polymeric materials such as plant fibers, water-insoluble starches and their derivatives, and protein substances, as well as synthetic thermoplastic polymeric materials such as polyamides, polystyrene, polyacrylic esters, polymethacrylates, and synthetic thermosetting polymeric materials such as phenol-formaldehyde resin, melasin-formaldehyde resin, and urea-formaldehyde resin. Further, the water-insoluble polymer material may be an inorganic polymer material such as glass.

The lignin material used in the present invention is not particularly limited as long as it has binding properties to proteins, and includes, for example, alkaline lignins such as kraft lignin and soda lignin, as well as solvolysis lignin and monkey lignin. Phytolignin, blasted lignin, and the like can be used.

In the composite of the present invention, the lignin material layer may be fixed to the carrier by chemical bonding, or
It may be fixed by physical bonding.

In the fixation method by chemical bonding, a chemically active functional group may be introduced into at least one of the lignin material and the carrier material. As such a chemically active functional group, for example, a bond by ring opening of an epoxy group, or a bond by a condensation reaction with an amino group, a hydroxyl group, a carboxyl group, a carkynyl group, etc. can be used.

To introduce epoxy groups into a carrier material or a lignin material, an epoxidation reagent such as epichlorohydrin or glycidyl methacrylate is used under normal epoxidation reaction conditions.
For example, the reaction may be carried out by heating under alkaline conditions.

To utilize the condensation reaction group, functional groups in the lignin material,
For example, using a carboxyl group, a functional group that undergoes a condensation reaction with this functional group, such as an amino group, may be introduced into the carrier. Alternatively, amino groups, hydroxyl groups, carboxyl groups, carbonyl groups, etc. may be introduced into lignin according to the functional groups possessed by the carrier material according to a conventional method.

In addition, in a method of fixing a lignin substance to a carrier by physical bonding, for example, solid particles of a polymer material are suspended in an aqueous solution of a lignin substance, and then irradiated with ultrasonic waves of 20 to 30 kHz for a required period of time, for example, 1 to 24 hours. And by this,
The lignin material can be physically bonded and fixed to the solid particle carrier.

In the composite of the present invention, the content of lignin material includes:
There is no particular limitation, but - 2 to 20
Preferably, it is within the range of % by weight.

Furthermore, the polymer material solid particles used in the present invention are lO
Preferably, it has a particle size of ~1000-1,000 -, and more preferably it is porous, in which case the pore size of its pores is preferably 0,02-0,2-.

The immobilized lignin complex of the present invention is useful as an active ingredient of enzyme immobilization materials.

This enzyme immobilization material includes urease, uricase, glucose oxidase, peroxidase, cholesterol esterase, alkaline phosphatase, β-galactosidase, chymotrypsin, carboxypeptidase,
and is useful for immobilizing enzymes such as thermolysin. Enzyme immobilization can be performed by conventional methods. For example, the immobilized lignin composition of the present invention is suspended in a solution of a predetermined enzyme, and the immobilized lignin composition of the present invention is suspended at a predetermined temperature, for example, 5 to 37°C.
The enzyme may be left to stand for a predetermined period of time, for example, 1 to 3 hours, and then the enzyme immobilized product may be separated and collected.

Furthermore, the immobilized lignin complex of the present invention is useful as an active ingredient of protein removal materials.

As mentioned above, the precipitation method and ultrafiltration method, which are typical conventional protein removal methods, require a large amount of sample. In order to solve this problem, a pre-column method was investigated, and as a result, excellent results were obtained using the composition of the present invention.

The switch method among the conventional pre-column methods requires the provision of a sample valve midway, and the venting method requires the provision of valves at three locations. By using the material of the present invention, a completely convenient method has become possible in which the detected substance itself passes through the column and only the protein is selectively adsorbed and removed by the column, rather than sending out the adsorbed substance. . In addition, in the conventional pre-column method, since a hydrophobic carrier is used, non-specific adsorption of proteins has to be used, but the composition of the present invention solves this problem. be able to.

There are batch methods and column methods for evaluating the protein removal effect, and the performance of the removal material can be determined based on the removal rate of the amount of introduced protein.

〔Example〕

The present invention will be further explained by examples.

In Example 1, cellulose microcrystals (^r
t, 2331) 5 g was suspended in 20-℃ water and heated to 5℃.
It was cooled to To this was added 75M1 of 6N sodium hydroxide, which had been prepared in advance and cooled to 5°C, and stirred to make it homogeneous. Condensate 1 in which this suspension was allowed to stand at 5°C for 1 hour
5(ld) was added and stirred at 60° C. for 1 hour. Epichlorohydrin 50° was added to the eel and stirred at 60° C. for 3 hours, and the obtained epoxy-activated cellulose was collected by filtration and washed with 500° C. water. The obtained epoxy activated cellulose was
The suspension was suspended in 150 ml of a 1.2% aqueous solution of hexamethylenediamine dihydrochloride adjusted to pH 12 with sodium hydroxide, and stirred at 60°C for 3 hours. The obtained aminohexylcellulose was collected by filtration, washed with 500 ml of water, and used in the next reaction.

The aminohexylcellulose was suspended in 200ml of 0.5N sodium hydroxide, stirred at 60°C for 1 hour, and then 401n1 of epichlorohydrin was added and stirred for an additional 2 hours. The obtained epoxy-activated aminohexyl cellulose was collected by filtration, washed with 500 d of water, and suspended in a 100% aqueous solution of 6% alkaline lignin (Tokyo Kasei L082) adjusted to pttll with sodium hydroxide and heated to 45°C. The mixture was stirred for 4 hours. The lignin-immobilized cellulose was collected by filtration and washed with water, 1/10N sodium hydroxide, and methanol to remove unreacted lignin, and the obtained lignin-immobilized cellulose was vacuum-dried.

In addition, in Example 2, Toyo Roshi type cellulose powder (filter paper powder A, 100 to 200 mesochetes) was used in Example 1.
Lignin was fixed in the same manner. The obtained lignin-immobilized cellulose was classified using a standard sieve and
350 meshes were collected.

The method for measuring the lignin content of lignin-immobilized cellulose is shown below. The lignin content of the obtained lignin-immobilized cellulose was measured using the acetyl bromide method as described below.

A 25% acetic acid solution of acetyl bromide was added to 2 parts of the obtained lignin-immobilized cellulose and heated to 70°C for 30 minutes to dissolve the lignin-immobilized cellulose. After cooling this in a water bath, add 0.9 m of ZN-sodium hydroxide.
and 5 m of glacial acetic acid were added and mixed. To this was added 0.1 mg of 7.5M hydroxylamine hydrochloride solution, and the mixture was shaken while cooling. After adding glacial acetic acid to make the total volume 20-280n
The lignin content was determined by measuring the absorbance of s.

In addition, unmodified cellulose was used as a sample for comparison of the catalytic activity of the obtained lignin-fixed sample.

The lignin contents of the obtained lignin-immobilized samples of Examples 1 and 2 were 11% and 5.7%, respectively, based on the weight of the cellulose carrier.

Alkaline phosphatase derived from calf small intestine (Sigma P-
3877, 1,40/*) 15mg, 3d(7)
To this solution dissolved in pl (8,0° 0.5M, 2-(cyclohexylamino)ethanesulfonic acid (CHES) buffer) was added about 0.3 g of the solution, which had been previously degassed in the same buffer. The lignin fixed sample was suspended and fixed by standing at 25° C. for 1 hour.

After performing an operation to immobilize the enzyme, the enzyme-immobilized sample was thoroughly washed with the same buffer solution used for immobilization and packed into a thermostatically jacketed glass column with an inner diameter of 10 mm. In addition, the pH
9,o CHESil solution (MgC1z 80r@m
ol/l) dissolved in 1 1m moff /7! Phosphoric acid p-nitrophenyl ester disodium salt at 25°C
Perfusion was performed at a rate of 0.05-/ff1 in. The catalytic activity of the column was examined by measuring the absorbance change at 430 nm resulting from the product p-nitrophenol anion in the effluent solution.

When the lignin-fixed sample and enzyme were incubated for 1 hour, the amount of enzyme immobilized was 5.4 μ/g-gel for both lignin-immobilized cellulose (Examples 1 and 2) and unmodified cellulose alone. However, their catalytic activity was 5 to 7 times higher in the case of lignin-immobilized cellulose (Example 1.2) than in the case of unmodified cellulose alone. This is because the enzyme is immobilized on the carrier via lignin, which is more advantageous for the enzyme's catalytic reaction than when it is directly adsorbed on the cellulose surface. Conceivable.

Furthermore, when alkaline phosphatase was immobilized on lignin-immobilized cellulose (Example 1.2), the half-life of the enzyme activity was as good as about 7 days, and it was clear that the enzyme was difficult to detach from lignin-immobilized cellulose. .

The lignin-fixed samples described in Examples 1 and 2 were used as protein removal materials as described below.

1) Batch method A lignin-fixed sample was suspended in a solution of bovine serum albumin or bovine serum γ-globulin in 1/30M phosphate buffer. After stirring at 25°C, membrane filter (Toyo Roshi DISMIC25cs, pore size 0
．． 45m), and the protein concentration in the filtrate was measured by the Lowry method to determine the amount of protein bound to the resin.

2) Column method Approximately 0.2 g of a fixed lignin sample packed in a glass column with an internal diameter of 4.7 mm and a length of 5511 m was incorporated into a conventional low-pressure chromatography device. Mobile phase pH 5,5, 1/
A 30M phosphate buffer was used and the flow rate was 1-/sin. A bovine serum albumin solution with a concentration of 2.5 μ/- prepared using the same buffer as the mobile phase was repeatedly injected into the analyzer using an autoinjector at 1OI every 10 minutes.

The amount of protein binding was determined by tracking the protein in the solution flowing out from the column using a UV monitor and determining the peak area using an integrator.

Ino-Raiji describes the results of protein binding to lignin-immobilized cellulose.

Lignin fixed to cellulose microcrystals (Example 3
．． When the lignin content (11%) was evaluated by a batch method, the amount of bound bovine serum albumin increased compared to the cellulose carrier alone. Especially pu
This tendency was remarkable under weakly acidic conditions of s, o ~ 5.5, with the amount of binding increasing 6 to 30 times (Table 1). In this way, the isoelectric point of protein (the isoelectric point of bovine serum albumin 4
．． 7-=4.9), the tendency for the amount of protein binding to the lignin-fixed sample to increase is also seen in other protein adsorbents. pos, o near the protein isoelectric point
Under conditions of ~5.5, the hydrophobicity of the protein increases, and it is thought that the hydrophobic interaction between the protein and the lignin-fixed sample increases.

For this reason, proteins are likely to be attracted to the surface of the lignin-fixed sample particles (conditions are favorable for protein binding to the resin, and it is thought that the amount of protein binding increases in such regions. In the case of bovine serum T-globulin) By immobilizing lignin, the amount of protein binding increased significantly (Table 2).The isoelectric point of bovine serum γ-globulin is 5.7 to 6.8, but this time the amount of binding increased at around pH 5.5. Since T-globulin is a protein that easily associates, it is thought that it becomes large associated particles in the pH range of 6 to 7.For this reason, protein particles enter the micropores that are thought to exist on the surface of cellulose crystals. The diffusion of T-
It is thought that the amount of globulin bound decreased.

Moreover, the amount of bovine serum albumin bound increased significantly (Table 3)
. When we performed an evaluation using a column method using this cellulose powder with lignin fixed, we found that in the case of only cellulose powder, protein elution was observed from the first protein solution injection, but until the 9th injection. No elution of protein was observed, and the number of times protein solution was injected until the efflux curve reached saturation increased, resulting in a large increase in the amount of protein bound (Figure 1). Additionally, in this column method, the column is constantly washed with a mobile phase solvent during repeated injections of protein solutions, so only irreversible protein binding, not reversible binding, is measured. Therefore, the large increase in the number of protein solution injections until protein elution begins and the number of injections until the efflux curve reaches saturation indicates that the filler in which lignin is fixed to cellulose strongly binds to protein.

Margin table below 1 Amount of bovine serum albumin bound to lignin-immobilized cellulose (Example 3) BSA binding!
(mg/g-ge4)5.0 180
5.45, 5 147 1
1.86, O35,811,36,518,85,37,013,90 Experimental conditions Initial protein concentration: 0.48/-11/30M phosphoric acid.

Buffer solution/liquid ratio: 5+++ffi/15■ Adsorbent, 25°C, stirring for 12 hours Table 2 Amount of bovine serum T-globulin bound to lignin-immobilized cellulose (Example 3) B-r-G
Binding amount (■/g-gel) 5,0 389
91.55, 5 406
1526, 5 276 29.
4? , 0 139 22.1 Experimental conditions Initial protein concentration: 0.48 mg/ml, 1/30M phosphate buffer solution ratio: 5d/15■ Adsorbent, 25°C, 12 hours stirring table
3 Cellulose powder to lignin-immobilized cellulose powder 16.3 Experimental conditions Initial protein concentration; 0.48■/-1pl+5.5.1
/30M phosphate buffer solution ratio: 5mf/15■ Adsorbent, stirred at 25°C for 112 hours Chitopal CHM3001 manufactured by Fujibo Co., Ltd. (trademark of chitosan, particle size 74-1771M1 pore size 500-2500)
10 g (wet weight) was suspended in a 10% aqueous lignin solution whose pH was adjusted to 12 with sodium hydroxide, and irradiated with ultrasonic waves for 18 hours. The obtained immobilized lignin was collected by filtration and washed with water, 1/10N hydrochloric acid, 1/10N sodium hydroxide, and methanol to remove unreacted lignin.

1 Next, the binding of proteins to lignin-immobilized Chitopal will be described.

The time dependence of the amount of bovine serum albumin bound to lignin-immobilized Chitopal was measured by a batch method (Figure 2).
These results revealed that protein binding to lignin-immobilized Chitopal reached equilibrium in about 24 hours. Next, the dependence of adsorption amount on protein concentration was shown (Figure 3).

By immobilizing lignin, the amount of protein binding is increased compared to the case where only Chitopal is used as a carrier.1.
It was revealed that equilibrium was reached at a concentration of about 6/-. The equilibrium adsorption amount was approximately 500 .mu./g-gel, and it had sufficient protein binding ability as an adsorbent for protein removal pretreatment in clinical tests or as an adsorbent for treating protein-containing wastewater.

In addition, when approximately 0.1 g of lignin-immobilized Chitovar was packed into a glass column (55+nX 4.7 n+ID), bovine serum albumin was bound and washed with 1/10 N-acetic acid. Protein eluted, whereas lignin immobilized eluted 1
Only 1% of the lignin was eluted, indicating that lignin is firmly bound to proteins by immobilizing it on Chitopearl.

〔Effect of the invention〕

The immobilized lignin complex of the present invention has excellent binding properties to proteins and is useful as an enzyme immobilization material and a protein removal material. Activated carbon for isotope adsorption is pretreated because it is significantly interfered with by proteins).
It can also be effectively used in fields such as preventing turbidity in foods such as sake and beer.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the protein removal effect of the immobilized lignin complex according to the present invention prepared in Example 4, and FIG. 2 is a graph showing the protein removal effect of the immobilized lignin complex according to the present invention prepared in Example 5. FIG. 3 is a graph showing the temporal dependence of the protein binding amount of the complex, and FIG. 3 shows the relationship between the protein concentration and the protein binding amount of the immobilized lignin complex according to the present invention prepared in Example 5. It is a graph.

Claims (1)

[Scope of Claims] 1. An immobilized lignin composite comprising a carrier made of solid polymer particles and a lignin material layer fixed to the carrier. 2. The solid polymer particles are cellulose, cellulose derivatives, chitosan, chitosan derivatives, starch, starch derivatives, amino acids, amino acid derivatives, polyamides, polystyrene, polyacrylic polymers, phenol-formaldehyde polymers, melamine-formaldehyde polymers,
A composite according to claim 1, which is selected from particles of urea-formaldehyde polymers. 3. The lignin material is kraft lignin, soda lignin, solvolysis lignin, sulfite lignin,
A composite according to claim 1, selected from blasted lignin. 4. The composite according to claim 1, wherein the content of the lignin substance is within the range of 2 to 20% by weight based on the weight of the carrier. 5. The composite according to claim 1, wherein the polymer solid particles have a particle size of 10 to 1000 μm. 6. The composite according to claim 1, wherein the solid polymer particles are porous. 7. The composite according to claim 1, wherein the lignin material is chemically immobilized on the carrier. 8. The composite according to claim 1, wherein the lignin material is physically immobilized on the carrier. 9. An enzyme immobilization material whose active ingredient is an immobilized lignin complex comprising a carrier made of solid polymer particles and a lignin substance immobilized thereon. 10. A protein removal material whose active ingredient is an immobilized lignin complex comprising a carrier made of solid polymer particles and a lignin substance immobilized thereon.