WO1991007427A1 - Agent vehiculeur pour chromatographie sur colonne echangeuse, procede de separation et de purification d'une substance polymere hydrosoluble au moyen d'un tel agent, nouveau gel a base d'acide pectique et de cellulose et procede de preparation d'un tel gel, et adsorbant pour chromatographie par affinite - Google Patents

Agent vehiculeur pour chromatographie sur colonne echangeuse, procede de separation et de purification d'une substance polymere hydrosoluble au moyen d'un tel agent, nouveau gel a base d'acide pectique et de cellulose et procede de preparation d'un tel gel, et adsorbant pour chromatographie par affinite Download PDF

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Publication number
WO1991007427A1
WO1991007427A1 PCT/JP1990/001493 JP9001493W WO9107427A1 WO 1991007427 A1 WO1991007427 A1 WO 1991007427A1 JP 9001493 W JP9001493 W JP 9001493W WO 9107427 A1 WO9107427 A1 WO 9107427A1
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Prior art keywords
gel
carrier
cellulose
protein
chromatography
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PCT/JP1990/001493
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English (en)
Japanese (ja)
Inventor
Kenjiro Makino
Tomoaki Yada
Masayuki Sakou
Chitoshi Hatanaka
Sawao Murao
Original Assignee
Nissei Chemical Industry Co., Ltd.
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Filing date
Publication date
Priority claimed from JP29620089A external-priority patent/JPH03157396A/ja
Priority claimed from JP2080256A external-priority patent/JPH03277967A/ja
Priority claimed from JP2082924A external-priority patent/JPH03284693A/ja
Priority claimed from JP2110609A external-priority patent/JPH048701A/ja
Application filed by Nissei Chemical Industry Co., Ltd. filed Critical Nissei Chemical Industry Co., Ltd.
Publication of WO1991007427A1 publication Critical patent/WO1991007427A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material

Definitions

  • the present invention relates to a carrier for column chromatography, a method for separating and purifying a water-soluble polymer substance using the carrier, a novel cellulose monopectate gel and a method for producing the same, and an adsorption method for affinity mouth chromatography. About the body.
  • column chromatography which is the most suitable for separation from multi-component systems, has been used for protein separation and purification.
  • ion exchange chromatography gel filtration chromatography, adsorption chromatography, affinity chromatography, etc. are known. Have been.
  • ion exchange chromatography is the most orthodox.
  • a cation exchange resin or an anion exchange resin is selected as a carrier depending on the charge state of the protein to be separated and purified, and the protein is separated and purified by ion exchange.
  • many carriers currently supplied have a small ion exchange capacity, and the amount of protein processed is limited.
  • the recovery rate of the protein after chromatography was not good because there are many problems with the stability of the protein relative to the carrier. As a result, there were many problems, such as cost increase on the industrial scale.
  • the gel filtration method is one of the most widely used methods. This method is a separation mode based on the molecular sieve effect based solely on the difference in molecular weight of globular proteins, and is performed under conditions excluding the effects of ionic and hydrophobic properties. It is a very versatile and sensitive separation method that can be applied.
  • Carriers for gel filtration chromatography used in the method can be roughly classified into two types: synthetic polymer-based and natural polysaccharide-based.Each carrier has advantages and disadvantages. Purification This is a technical difficulty that tends to be avoided, which is the reason why industrial use is limited to only some high value-added products. That is, for example, as a synthetic polymer-based carrier, a water-soluble polymer such as polyvinyl alcohol or polyacrylamide resin, which is formed into an insolubilized gel by a bridge, is typical, and all resins are natural in terms of strength.
  • a synthetic polymer-based carrier a water-soluble polymer such as polyvinyl alcohol or polyacrylamide resin, which is formed into an insolubilized gel by a bridge, is typical, and all resins are natural in terms of strength.
  • the natural polysaccharide-based carrier most commonly used are microbial polysaccharides such as dextran gel, marine algae such as agarose gel and cellulose, or those obtained by crosslinking them.
  • microbial polysaccharides such as dextran gel, marine algae such as agarose gel and cellulose, or those obtained by crosslinking them.
  • a characteristic feature of these carriers is that they are more expensive to extract, purify, and prepare a gel of the carrier itself from a natural product than a synthetic polymer system, and are generally expensive.
  • dextran-based and agarose-based carriers prepared for protein purification have a high swelling rate and are weak, so that the eluent cannot be flowed at high speed in the gel permeation column.
  • Cellulose-based carriers generally have the advantage of being relatively inexpensive and having higher strength than other natural polysaccharides. Due to its high crystal structure, it is less suitable for fractionating high molecular weight substances than gel filtration carriers that have a huge network structure. For these reasons,
  • Another purification method is affinity chromatography.
  • one of two substances or groups of substances having biochemical specific affinity (affinity) is immobilized as a ligand on a water-insoluble carrier to form a stationary phase.
  • Chromatography that separates and purifies the target substance from impurities mixed with it using the difference in affinity for the stationary phase, and is widely used for separation and purification in the field of biochemistry, especially enzyme chemistry.
  • the carrier hydrophilic gel particles composed of polysaccharides such as dextran agarose and polyacrylamide have been generally used.
  • a first object of the present invention is to provide a carrier for column chromatography containing a cellulose cellulose pectate, wherein the carrier is ion-exchange chromatography. It can be used for both gel filtration and gel filtration. Further, a second object of the present invention is to provide a method for separating and purifying a water-soluble polymer substance, which comprises performing ion exchange chromatography or gel filtration chromatography using the carrier. It is in.
  • the cellulose pectate gel used in the present invention is prepared from a vector-containing plant.
  • a method for producing the gel is described in Japanese Patent Application No. 1-296199 filed by the present inventors. Is preferred.
  • a semi-continuous extraction of dry powder of a pectin-containing plant tissue such as citrus peel with ketones is used to produce an insoluble extract under alkaline conditions in the presence of ketones. After suspending in alcohols or ketones, it is crosslinked with a crosslinking reagent.
  • cellulose pectate gels obtained by other production methods can also be used, and the cellulose pectate gel used in the present invention is not particularly limited by the production method.
  • the gel obtained by the above method contains a large amount of pectic acid, and retains a high heavy metal capturing ability due to the effect of the carboxyl group derived from the ⁇ -D-galacturonic acid residue in pectic acid. It is known that it is useful as a resin for water treatment (JP-A-64-43501).
  • Ion exchange chromatography using the carrier of the present invention is a target, and a typical example thereof is a protein, for example, polygalacturonase.
  • the carrier of the present invention can be used for separating and purifying cells such as lymphocyte T cells in addition to proteins.
  • the amount of pectic acid-cellulose gel in the column is determined in consideration of the type and amount of the protein to be separated and purified, the ion exchange capacity of the gel, and the like.
  • Buffers used for equilibration include phosphate buffer, acetate buffer, Tris-HCl buffer, etc., and the type and pH etc. of the protein for separation and purification are stable and stable.
  • the sample containing the protein to be separated and purified must be equilibrium-dialyzed against a buffer or the like in advance.
  • the type and amount of salt added to the buffer are also determined by the column.
  • the above pre-treatment of the sample was carried out under salt coexist to aggregate, because it must be applied to remove after column 0
  • the target protein binds to the pectic acid-cellulose gel.
  • the recovery rate and specific activity are calculated from the enzyme activity and protein in the obtained fraction.
  • the above-mentioned effect of the cellulose pectate gel results from the fact that the gel retains the three-dimensional structure of the cell wall component of the raw material plant in its natural state. In other words, it has a place for active and passive substance transport in living organisms This suggests that delicate biological substances such as proteins can move and move in a stable state.
  • a gel using a crosslinking agent of 1 to 10 (molar sugar residues), preferably 2 to 5 (molar sugar residues) is suitable for separating proteins.
  • the particle size is desirably 100 to 500 zm (dry state), preferably 200 to 300 zm.
  • this gel retains a large pore size, it is quite hard and has a low swelling property. For swelling, immersion in an eluent for 1 hour at room temperature is sufficient. It is also possible with an autocrap (121, 15 minutes).
  • the method of packing, washing, and adding the sample to the chromatographic column of this gel can be the same as that of a conventional hard gel filtration agent on the market.
  • a buffer in a region where the present gel having a pH of 3 to 10 is stable can be used, and it is preferable to perform the chromatography in a stable PH region of the target substance. If the gel does not have carboxyl groups blocked by chemical treatment such as esterification or amidation as described above, add 0.1M to 0M NaCl to the eluate. It is preferable to do so.
  • this gel is a hard gel and its volume change is small, for example, when a 2.5 ⁇ 30 cm column is used, a high flow rate of maximum SV 5 / hr l SOml 'cnr ⁇ hr) It is possible, but it may also be pumped by the pump as necessary (maximum operating pressure 300 cm H 2 0 or more).
  • the eluate from the column is sequentially collected in a fixed amount using a fraction collector or the like.
  • the target fraction collected by the above procedure is desalted and concentrated by ordinary processing, and after various chromatographic operations are repeated as necessary, it is purified by electrophoretic homogenization or crystallization. Complete.
  • the cellulose pectate gel used in the present invention is suitable as a carrier for gel filtration of a water-soluble polymer substance, particularly, a protein.
  • This gel is inactive against proteins and enzymes, and it is capable of performing mass transfer in a stable state in this gel, so that it can be recovered with a yield of almost 100% from the column.
  • this gel Has a robust matrix structure and is extremely strong as a natural polysaccharide gel, so it can be run at high flow rates and can be used for high-performance liquid chromatography.
  • macromolecules such as viruses can be fractionated by adjusting the pore size by the degree of crosslinking. As shown by the inventors in Japanese Patent Application No.
  • the present gel is made from pectin-containing plant tissues such as citrus, and has a great advantage in terms of cost. From these results, it was clarified that cellulose pectate gel is a useful carrier not only for laboratory use but also for industrial-scale water-soluble polymer substances, especially for protein purification.
  • cellulose monopectate gel it is preferable to use a cellulose acetate-cellulose gel having a very small amount of residual protein in order to prevent nonspecific adsorption due to proteins present as impurities in the gel.
  • a third object of the present invention is to provide a novel pectic acid-cellulose gel from which a structural protein and a cell wall binding protein are removed, and a fourth object of the present invention is to provide the novel pectic acid monosaccharide.
  • An object of the present invention is to provide a method for producing a cellulose gel. The novel vecitrate monocellulose gel can be obtained while maintaining the structure of the plant cell wall.
  • Plant cell walls are composed of polysaccharides such as cellulose microfibril, xyloglycan, galactan, and arabinogalactan, which are structural proteins (extensin'arabinogaratan protein), It is composed of a matrix that contains cell wall binding enzymes (lingoate dehydrogenase, peroxidase, phosphatase, and polysaccharide hydrolases) in a layered manner. It is thought that non-specific adsorption occurs due to this.
  • the pectic acid-cellulose gel of the present invention is produced, for example, according to the following method. That is, powder derived from a pectin-containing plant, preferably an alcohol-insoluble substance, is used.
  • the amount of the buffer used is usually 10 to 30 ml, preferably 15 to 20 ml, per lg of the alcohol-insoluble matter. If less than 10ml In this case, the slurry concentration is high and the stirring becomes insufficient, and if it exceeds 30 ml, the processing efficiency decreases.
  • the buffer may be any one as long as it can stabilize the protease.
  • the buffer is not particularly limited. After sufficiently immersing in a buffer, a protease such as papain or pronase is added. Since the specific activity of the reagent enzyme and the like vary, the amount of addition cannot be specified unconditionally.
  • the concentration in the system is preferably 0.1 to 1%, and more preferably 0.4 to 0.6%.
  • the treatment temperature should be the optimal temperature for protease (generally around 30 ° C).
  • the processing time is preferably 24 to 48 hours, but is not particularly limited. This treatment removes 95% of the total protein in pectic acid-cellulose.
  • the sample is not limited to alcohol-insoluble matter, but any sample can be used as long as it is a sample before crosslinking.
  • the same polar solvent eg, methanol, acetone
  • Separation is carried out using a solid-liquid separation device such as a filtration machine, washed with a polar solvent (eg, methanol, acetone) and dehydrated to obtain a protein-removed alcohol-insoluble matter.
  • a polar solvent eg, methanol, acetone
  • This may be used as it is in the cross-linking step, or may be used after the drying step in the cross-linking step.
  • JP-A-63-43501 According to the above, a pectic acid-monocellulose gel of the present invention is obtained by crosslinking by a crosslinking reaction.
  • a fifth object of the present invention is to provide an adsorbent for affinity chromatography, wherein a ligand is immobilized by covalent bonding on a carrier made of pectic acid-single cell gel.
  • the adsorbent and the ligand can be bonded by a covalent bond via a spacer group.
  • the spacer group may be bound to the adsorbent in advance, and then the ligand may be bound to the spacer group. Alternatively, the spacer group may be bound to the ligand in advance. This may be combined with the adsorbent. Further, if necessary, a spacer group may be previously bonded to both the adsorbent and the ligand, and these may be mutually bonded.
  • the compound that can be used as the spacer group is at least a bifunctional organic compound and does not exclude a polyfunctional polymer, but in particular, a carbon chain having 1 to 12 carbon atoms.
  • Bifunctional organic compounds having a group are preferred.
  • Specific examples of such a compound that functions as a spacer group include, for example, diamines such as hexamethylene diamine, dodecamethylene diamine, xylylene diamine, glycine, / S-amino propionic acid , Aminoaminobutyric acid, ⁇ -aminocaproic acid, aminoalkylcarboxylic acids such as ⁇ -aminocaprylic acid, lysine, glutamic acid, and amino acids such as S-alanine and arginine are preferably used.
  • the present invention is not limited to these.
  • Ligand is directly bonded to the above-mentioned cellulose acid cellulose particle by covalent bond, or a spacer group is bonded to the cellulose acid-cellulose gel particle, and the ligand is added to this spacer group.
  • the method for bonding by a covalent bond is not particularly limited, and any conventionally known method can be used.
  • one of preferred methods is a method using water-soluble carbodiimide as a binding reagent.
  • aminoalkyl carboxylic acid is bound to cellulose cellulose pectate gel particles, and then the aminoalkyl carboxylic acid bound to the cellulose cellulose pectate gel is subjected to water-soluble carposide by using a water-soluble carposide.
  • the ligand can be covalently bonded.
  • water-soluble carbohydrate used in this method examples include, for example, 1-ethyl-3- (3-dimethylamido).
  • a water-soluble carpoimide through a covalent bond directly or without a spacer group, conventionally, It can be according to the usual methods and conditions known.
  • the reactant is reacted with cyanogen bromide to activate the functional group, thereby bonding a spacer group, and then bonding the ligand through a covalent bond in the same manner as described above. be able to.
  • the ligand can be directly bound to the pectic acid-cellulose gel particles.
  • the adsorbent for affinity mouth chromatography is obtained by a conventional separation method such as filtration and centrifugation. It can be separated and purified.
  • the adsorbent for affinity chromatography according to the present invention can be used in the same manner as a swollen gel in conventional affinity chromatography. Therefore, the adsorbent can be used for both the column method and the batch method.
  • the affinity chromatography of the present invention The adsorbent for fibrous use uses pectic acid-cellulose gel particles as a carrier, on which a ligand is covalently bonded. Therefore, such an adsorbent is excellent in stability to a biological substance because it uses a pectic acid-cellulose gel which holds a cell wall matrix as a carrier of the biological substance as a carrier.
  • this carrier is the same or more than the conventional gel despite the crushed type Flow velocity can be obtained.
  • conventional gels are expensive in terms of cost and have been difficult to introduce on an industrial scale.
  • this carrier can be provided at very low cost, it is natural that affinity chromatography using this carrier can be performed.
  • the adsorbent for use is also inexpensive, and can provide great advantages when this adsorbent is introduced on an industrial scale.
  • Fig. 1 shows a chromatogram of PG using a cellulose acetate-cellulose gel
  • Fig. 2 shows a chromatogram of PG using Sephadex
  • Fig. 3 shows a chromatogram of exo-PGL using a cellulose acetate-cellulose gel
  • Fig. 4 shows a chromatogram of carrot exo-PG using a cellulose pectate gel
  • Fig. 5 shows a separation of proteins using a cellulose pectate gel.
  • Fig. 6 is a diagram of column chromatography of BSA, GPDH, and LDH using a NAD adsorbent derived from vectic acid-cellulose gel.
  • PG endogenous galacturonidase
  • a cell-free culture solution of C. fragilis (IF0 1963) was concentrated 1/10 using an Amicon ultrafiltration apparatus equipped with a YM-10 membrane. The concentrate was then diluted with 4 volumes of 0.1M acetate buffer at pH 4.0 and reconcentrated again by ultrafiltration to 1/4. This operation was repeated twice, and the finally obtained concentrate was used as partially purified PG.
  • the active fraction is mixed and concentrated, and as described above, calcium chloride is removed by ultrafiltration to obtain a purified PG solution.
  • the results of the enzyme purification are summarized in Table 1.
  • the purified PG solution was purified 86-fold in the medium, and the recovery was 91.7%.
  • exopolygalacturate lyase Exp-PGL
  • Erwinia carotovora subsp. Carorovora IFO 13921.
  • the cells should be centrifuged at 10,000 xg for 15 min from the culture. And washed twice with a pH 7.0 0.02 M phosphate buffer. All operations were performed at 0 to 6 ° C. Approximately 100 g of cell paste was suspended in 200 ml of the same buffer, and crushed with an ultrasonic oscillator 100W, 20KHZ. As a crushing method, apply 50 ml of the suspension 5 times at 2-minute intervals for a total of 10 min. The cell debris was removed by centrifugation at 18,000 xg for 15 min, and the supernatant was dialyzed overnight with a PH7.0 0.02 M phosphate buffer, and the dialyzed product was used as a crude enzyme product.
  • the crude enzyme solution was treated with solid ammonium sulfate, and the precipitate having a saturation level of 0.3 to 0.8 was collected by centrifugation at 18,000 xg for 15 minutes.
  • the collected precipitate was dissolved in 150 ml of a pH 8.0 0.04 M phosphate buffer, and dialyzed overnight against the same buffer. Subsequently, the dialysate was adsorbed on a DEAE-cellulose column (3.8 x 24 cm) that had been equilibrated with a pH 8.0 0.04 M phosphate buffer. After washing with the same buffer, the active fraction was pooled to obtain a partially purified enzyme.
  • the activity of the partially purified enzyme (60 ml, 120 units) was completely adsorbed on a pectic acid-cellulose gel column in a 0.04 M phosphate buffer at pH 6.3. Most of the contaminating proteins passed through the column. Significant amounts of protein impurities still remain on the column, but they are all eluted with 0.04M phosphate buffer, pH 7.6, after which the enzyme activity is reduced to 0.04M at pH 8.5, 5 ° C. It was eluted with Tris-HCl buffer (Fig. 3). The results of the enzyme purification are summarized in Table 2. By this method, purified exo-PGL was obtained with a specific activity of 469 times and a recovery of 43%. Purified exo-PGU, polyacrylamide gel electrophoresis and SDS-polyacrylamide gel-slab electrophoresis confirmed that it was a single protein.
  • Crosslinking agent 2 This section describes the preparation method of pectic acid cellulose gel for gel filtration of 2.6 mol / sugar residue.
  • Soxhlet extraction was carried out for about 6 hours using 41 g of dried powder of Satsuma mandarin peel using 240 ml of a 45% by weight aqueous acetone solution heated to 60 ° C, and the remaining 28 g of insoluble acetone (wet product) was filtered. I took it. This was added to 350 ml of a 5: 1 (volume ratio) mixture of a 50% by weight aqueous solution of acetone and epihydric luhydrin, and kept at 40 ° C. with stirring. Next, 50 ml of 5 M NaOH was added thereto, and a crosslinking reaction was performed at 40 ° C. for 2 hours, and a water-insoluble gel was collected by filtration.
  • Crosslinking agent Cellulose pectate gel of 2.6 mol Z sugar residue is prepared by a conventional method, equilibrated with 0.1 M acetic acid buffer solution (PH4.5) containing 0.5 M NaCl, and then a 2.5 ⁇ 40 cm column is prepared. It was created. After the partially purified PG sample was added to the column, it was eluted with the buffer. The eluate was fractionated by a fraction collector, and the respective enzyme activities and protein contents (A28 () ) were measured. After the active fractions were combined, NaCl was removed by dialysis to obtain a purified PG solution. This solution had a total activity of 133.8 units and a specific activity of 180.2 units Zmg protein. The recovery rate was 99.2% and the specific activity increased 1.38 times.
  • PH4.5 acetic acid buffer solution
  • Example 6 The same as the partially purified PG sample used in Example 5 was pre-equilibrated with 0.1 M acetate buffer (PH4.5) containing 0.5 M NaCl, and Sephadex G-75 force ram (2.5 X 40 cm) and analyzed in the same manner as in Example 5. went. The active fractions were combined and the total activity of the sample obtained after desalting was 127.6 units, the specific activity was 177.5 units tsZmg protein, the recovery was 94.6%, and the specific activity increased 1.35 times. (Example 6)
  • the gel column was calibrated in advance, it could be applied to the measurement of the molecular weight of water-soluble polymers such as proteins.
  • the methods used were 0.1%, 0.5% and 1.0% papine, and 0.5% pronase.
  • the treatment time was 24 hours and 48 hours.
  • the evaluation was carried out using a protein-removed alcohol-insoluble material. The results are shown in Table 6.
  • the alcohol-insoluble matter was immersed in 60% methanol, and then a 5 M NaOH aqueous solution or a 17.5% HC1 aqueous solution was added thereto, followed by protein treatment.
  • NAD nicotinamide adenyline dinucleotide
  • Pectin acid - cellulose gel 50ml dry weight 8 g activated with pHll with CNBr in 40 g, washed, this .epsilon. solution of amino Nokapuron acid (.epsilon. amino Nokapuron acid 20g of 0.1 M NaHCO 3 200 ml aqueous solution The solution was washed sequentially with 0.01M HCK and 0.5M NaCl solution, and finally washed thoroughly with water.
  • the NAD adsorbent obtained above was applied to a 10 ⁇ 103 ml column, and the sample was: 0.1 M phosphate buffer (pH 7.0) containing GPDH (4.1 U), LDH (3711) and 0.9 mg of BSA (pH 7.0) 0.2 ml
  • the column was washed with a 0.1 M phosphate buffer, and then eluted sequentially with 0.15 mM NADH-IOmM NADH.
  • the flow rate was 1 mlZ 8 min.
  • Figure 6 shows the results of the column chromatography.

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Abstract

L'invention se rapporte à un agent véhiculeur pour chromatographie sur colonne échangeuse et à ses applications. L'agent véhiculeur se caractérise en ce qu'il contient un gel à base d'acide pectique et de cellulose. Lorsque cet agent est utilisé pour la chromatographie par échange d'ions et pour la chromatographie par filtrage par gel, une substance polymère hydrosoluble, telle que notamment une protéine, peut être séparée à l'état stable et purifiée, ce qui améliore la récupération. En outre, le retrait d'une protéine structurelle et d'une protéine se liant aux parois cellulaires dans le gel à base d'acide pectique et de cellulose permet de réduire l'adsorption non spécifique par les protéines restantes.
PCT/JP1990/001493 1989-11-16 1990-11-15 Agent vehiculeur pour chromatographie sur colonne echangeuse, procede de separation et de purification d'une substance polymere hydrosoluble au moyen d'un tel agent, nouveau gel a base d'acide pectique et de cellulose et procede de preparation d'un tel gel, et adsorbant pour chromatographie par affinite WO1991007427A1 (fr)

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
JP1/296200 1989-11-16
JP29620089A JPH03157396A (ja) 1989-11-16 1989-11-16 ペクチン酸―セルロースゲルによる蛋白質分離方法
JP2/80256 1990-03-27
JP2080256A JPH03277967A (ja) 1990-03-27 1990-03-27 アフィニティクロマトグラフィー用吸着体
JP2082924A JPH03284693A (ja) 1990-03-31 1990-03-31 ゲル濾過用担体
JP2/82924 1990-03-31
JP2/110609 1990-04-27
JP2110609A JPH048701A (ja) 1990-04-27 1990-04-27 新規ペクチン酸―セルロースゲル及びその製造法

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1043393A3 (fr) * 1999-04-09 2002-05-15 Universita' degli studi di Bologna Méthode de purification de lactoperoxidase
US8912117B2 (en) 2009-02-20 2014-12-16 Jnc Corporation Cellulose gel for purification of immunoglobulin

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS54146862A (en) * 1978-05-09 1979-11-16 Res Inst For Prod Dev Production of porous material
JPS6443501A (en) * 1987-08-07 1989-02-15 Nakahara Shokai Kk Preparation of pectic acid-cellulose gel

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS54146862A (en) * 1978-05-09 1979-11-16 Res Inst For Prod Dev Production of porous material
JPS6443501A (en) * 1987-08-07 1989-02-15 Nakahara Shokai Kk Preparation of pectic acid-cellulose gel

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
AGRICULTURAL AND BIOLOGICAL CHEMISTRY, Vol. 48, No. 3, (1984), SHIRO INOUE, YASUNORI NAGAMATSU, CHITOSHI HATANAKA, (Preparation of Cross-linked Pectate and Endopolygalacturonase of Kluyveromyces Fragilis), p. 633-640. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1043393A3 (fr) * 1999-04-09 2002-05-15 Universita' degli studi di Bologna Méthode de purification de lactoperoxidase
US8912117B2 (en) 2009-02-20 2014-12-16 Jnc Corporation Cellulose gel for purification of immunoglobulin

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