JPH03284693A - Carrier for gel-filtration - Google Patents

Abstract

PURPOSE:To provide the subject carrier containing a pectic acid cellulose gel, useful for the separation of a water-soluble polymeric substance, especially protein, etc., and enabling the separation and recovery of the substance in high yield. CONSTITUTION:The objective carrier is produced e.g. by extracting dried powder of a pectin-containing plant tissue such as peel of citrus fruit with a ketone, saponifying the insoluble material under alkaline condition, suspending in an alcohol, etc., crosslinking with a crosslinking reagent (preferably 2-5mol/sugar residue) to obtain a pectic acid cellulose gel having particle diameter of preferably 200-300mum and finally blocking the gel with carboxyl group originated from uronic acid by the covalent bond of ester, amide, etc., or equilibrating the gel in a solution containing a counter ion such as sodium chloride.

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention relates to a carrier for gel filtration made of cellulose pectate gel, particularly a carrier for filtration, and a method for purifying water-soluble polymeric substances, especially proteins, using the carrier. It is related to.

[Prior art and problems with the invention]

Background of the Invention Separation and purification of water-soluble polymeric substances, especially proteins, is widely carried out in various biochemical research fields.

Obtaining isolated and purified proteins is important for studying the properties of the proteins, and it is also important to apply the isolated and purified proteins to various biochemical reactions. . In addition, in recent years, industrial demand for proteins and enzymes as functional polymers has been increasing more and more in various fields such as food, pharmaceuticals, and chemistry.

Conventionally, chromatography has played a very important role as a protein separation and purification method, and gel filtration (gel chromatography) is one of the most frequently used methods. This method is a separation mode based on the molecular sieve effect based only on the molecular weight difference of globular proteins, and is carried out under conditions that exclude the effects of ionic and hydrophobic properties, so it can be applied to most proteins and enzymes simultaneously. This is a very versatile and sensitive separation method.

The carriers for gel chromatography used in this method can be roughly divided into two types: synthetic polymer-based and natural polysaccharide-based, but each carrier has its advantages and disadvantages, and these are important for protein purification by gel filtration. Therefore, it is often avoided due to its high degree of technical difficulty, which is thought to be the reason why its industrial use is limited to only a few high value-added products. That is, for example, synthetic polymer carriers are typically made into insolubilized gels by crosslinking water-soluble polymers such as polyvinyl alcohol and polyacrylamide resins.
Both resins have strong properties compared to carriers derived from natural polysaccharides, but since they are non-natural products, they have low affinity with proteins, hydrophobic adsorption, etc. Since yield reductions such as denaturation and deactivation due to non-specific adsorption of proteins tend to occur, there has been a problem in stable recovery of proteins from chromatography columns.

Furthermore, as natural polysaccharide carriers, microbial polysaccharides such as dextran gel, seaweeds such as agarose gel, celluloses, or crosslinked products thereof are most commonly used. A common characteristic of these carriers is that, compared to synthetic polymer systems, extraction from natural products of the carrier itself, purification, and gel preparation are costly, making them expensive overall. In addition, dextran and agarose carriers prepared for protein purification have a high swelling rate and are weak, making it impossible to flow the eluent at high speed through the gel filtration column. Cellulose-based carriers generally have the advantage of being relatively cheap and strong compared to other natural polysaccharides, but due to their highly crystalline structure, they have a higher molecular weight than gel filtration carriers that have a large network structure. It does not pass through the fractionation of substances very well. For these reasons, although gel filtration is an excellent separation and purification method, its industrial use is currently limited.

[Means to solve problems]

In view of the current situation, the inventors searched for a new material for gel filtration that is cheaper, has excellent physical and chemical strength, and has a higher affinity for unstable compounds such as proteins. As a result of diligent research, cellulose pectate gel was
It was discovered that it has very excellent properties in terms of molecular sieve properties, affinity with proteins, and physical and chemical strength, leading to the completion of the present invention.

That is, the first feature of the present invention is the use of pectic acid cellulose gel as a carrier for gel filtration, and the second feature is:
Use this gel to create water-soluble polymers! A'Jt, especially a method for separating and purifying proteins.

The cellulose pectate gel used in the present invention is prepared from pectin-containing plants, and the method for its production is, for example, disclosed in the patent application filed by the present inventors previously (Japanese Patent Application No.
No. 96199) method is preferred.

In this method, for example, dry powder of pectin-containing plant tissue such as citrus peel is semi-continuously extracted with ketones, and the extracted insoluble matter is saponified under alkaline conditions in the presence of ketones.
Next, it is suspended in lower alcohols or ketones, and then crosslinked with a crosslinking reagent.

However, cellulose pectate gel obtained by other manufacturing methods can also be used, and the cellulose pectate gel used in the present invention is not particularly limited by its manufacturing method.

The gel obtained by the production method described in L contains a large amount of pectic acid, and has a high heavy metal scavenging ability due to the effect of carboxyl groups derived from α-D-galacturonic acid residues in pectic acid. It is known that cellulose pectate gel is useful as a water treatment resin (Japanese Unexamined Patent Publication No. 64-43501). Cellulose pectate gel can also be used as a carrier for ion exchange chromatography and can be separated and recovered in very high yields. The inventors have already filed an application for a protein separation and purification method using this method (Japanese Patent Application No. 1-296200).

The effects of cellulose pectate gel as described above are thought to be due to the fact that the gel retains the three-dimensional structure of the cell wall components of the raw material plant in its natural state. Hμ, it is thought that delicate biological substances such as proteins can be transported in a stable state by having a field for active and passive substance transport in living organisms.

It was found that this property works effectively when used directly as a carrier for gel filtration. In other words, chromatography is performed in an eluent to which counterions such as sodium chloride are added to suppress ion exchange effects, or carboxyl groups derived from uronic acid are blocked by covalent bonds such as esters and amides. It is possible to obtain a molecular sieve with almost no ionic interactions.

Next, a case in which gel filtration chromatography of proteins is actually performed using a column will be explained.

The grade of the cellulose pectate gel used is 1 to 10 (mol/!R1 residue), preferably 2 to 5
(mol/sugar residue) gel using a crosslinking agent is suitable for protein separation. The particle size is preferably 100 to 500 μm (dry state), preferably 200 to 300 μm.Although the zero gel maintains a large pore size,
Since it is quite hard and has low swelling properties, it is sufficient to immerse it in an eluent at room temperature for one hour or less for swelling. Also,
It is also possible by autoclaving (121°C for 115 minutes). How to fill this gel into a chromatography column, how to wash it,
The method for adding the sample may be the same as for conventional commercially available hard gel filtration agents.

As for the eluent, a buffer with a pH of 3 to 10 in which the present gel is stable can be used, and chromatography is preferably performed in a stable pH range of the target substance. When using the gel whose carboxyl groups have not been blocked by chemical treatments such as esterification and amidation as described above, the eluent contains 0.1M to 1.0M. Preferably, leaf NaCl is added.

Next, regarding the flow rate, since this gel is a hard gel and has little volume change, for example, when using a 2.5 x 30C111 column, the maximum SV5/hr (150ae・
high flow rates (cm-'-hr) are possible, and may be pumped if necessary (maximum operating pressure 300 cm).
H, 0 or more).

The eluate from the column is sequentially collected in fixed amounts using a fraction collector or the like.

The target fraction collected through the above operations is desalted and concentrated through normal processing, and after repeating various chromatographic operations as necessary, is electrophoretically homogenized or crystallized to complete purification. do.

[Effects of the Invention] As explained above, the present invention shows that cellulose pectate gel is suitable as a carrier for gel filtration of water-soluble polymer substances, especially proteins. This gel is inert to proteins and enzymes, and moreover, substances can be transferred in a stable state within this gel, so it is possible to recover at a yield close to 100% compared to a column. In addition, this gel has a robust matrix structure and is extremely strong for a natural polysaccharide gel, so it can be flowed at high flow rates.
It can also be used for high performance liquid chromatography. Furthermore, by adjusting the pore size, it is also possible to fractionate macromolecules such as viruses.

As the inventors have shown in Japanese Patent Application No. 1-296199, this gel is made from Mi fibers of pectin-containing plants such as citrus fruits, and has a large cost advantage. These results revealed that cellulose pectate gel is a useful carrier not only for laboratory use but also for industrial-scale water-soluble polymeric substances, especially protein purification.

The present invention will be explained in more detail below with reference to Examples. Note that the present invention is not limited to these examples.

(Reference Example) A method for preparing a cellulose pectate gel for gel filtration containing 2.6 mol of crosslinking agent/sugar residue will be described.

41 g of dry powder of Satsuma mandarin peel was subjected to Soxhlet extraction for about 6 hours using a 45% acetone aqueous solution heated to 60°C with 240 paper, and 28 g of acetone insoluble matter remained.
(mixed crystal) was collected by filtration. This is mixed with a 5:l (volume ratio) of 50% by weight acetone aqueous solution and epichlorohydrin.
50 d and maintained at 40°C under stirring. Next, 5M
50 d of NaOH was added thereto, a crosslinking reaction was carried out at 40''C for 2 hours, and the water-insoluble gel was collected by filtration.

This was added to 500 d of water, boiled for 30 minutes, and then filtered to remove hot water soluble components. After drying the insoluble matter with a blow dryer,
23.0 g of dried cellulose pectate gel was obtained.

(Yield: 11.5%) (Example 1) Endopolygalacturonase (hereinafter abbreviated as PG) was obtained from Chryphromyces fragilis IFO1963 culture solution.

) is shown below.

After culturing Cryplomyces fragilis strain IF01963, the bacterial cells were removed by centrifugation to obtain a supernatant.

Partially purified PG was obtained from the wood supernatant by ultraconcentration and ion exchange chromatography. The total activity of this thing is 134
．． 9 units, specific activity 131.0 units/mg
It was protein.

A cellulose pectate gel containing 2.6 mol of cross-linking agent/sugar residue was prepared by a conventional method, and 0.1 mol containing 0.5 M NaCl was added.
h After equilibration with acetate buffer (pH 4, 5), 2.5
A x40C1O column was created. After adding the partially purified PG sample to the column, it was eluted with the buffer. The eluate was fractionated using a fraction collector, and the enzyme activity and protein content (A210) of each fraction were measured. After the active fractions were combined, NaCl was removed by dialysis to obtain a purified PG solution.

This solution has a total activity of 133.8υn1ts and a specific activity of 180.2
units/■protein, recovery rate 99.2
%, the increase in specific activity was 1.38 times.

(Control Example 1) The same partially purified PG sample used in Example 1 was prepared using Sephadex G that had been equilibrated in advance with 0.1M acetate buffer (pH 4, 5) containing 0.5M NaCl.
After passing through a -75 column (2,5×40C1), analysis was carried out in the same manner as in Example 1. The total activity of the sample obtained after combining the active fractions and desalting was 127.6 units, the specific activity was 177.5 units/pg proteln,
The recovery rate was 94.6%, and the specific activity increased by 1.35 times.

(Example 2) Molecular weight measurement kits (manufactured by Pharmacia LKB, aldolase: MW 158,000, catalase: Sin 23) were added to the cellulose pectate column used in Example 1.
After carefully adding 1 Ini from the top of the column, 0.1 hNaCl was added. Containing 0.05M potassium phosphate buffer (pH 6,8) at a flow rate of 1I11/win?
It was buried. A fraction of 2 d was collected using a fraction collector, and the protein content (A28°) was measured. The results are shown in the figure.

As can be seen from the figure, it was found that this gel column can be applied to the measurement of the molecular weight of water-soluble polymers such as proteins if it is calibrated in advance.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the separation of proteins using pectate cellulose gel.

Claims (1)

[Claims] 1. A carrier for gel filtration containing pectic acid cellulose gel. 2. A method for purifying a water-soluble polymer substance, which comprises performing gel filtration using the carrier according to claim 1.