CN1075663A - Adsorbent and process for purification - Google Patents
Adsorbent and process for purification Download PDFInfo
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- CN1075663A CN1075663A CN 93101782 CN93101782A CN1075663A CN 1075663 A CN1075663 A CN 1075663A CN 93101782 CN93101782 CN 93101782 CN 93101782 A CN93101782 A CN 93101782A CN 1075663 A CN1075663 A CN 1075663A
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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
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
- C12N9/2414—Alpha-amylase (3.2.1.1.)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2445—Beta-glucosidase (3.2.1.21)
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- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01001—Alpha-amylase (3.2.1.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01021—Beta-glucosidase (3.2.1.21)
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Abstract
The present invention relates to a kind of special adsorbent that affinity chromatography separates that is used for, the ligand in this special adsorbent is a glucityl glucosides class, oligomeric glucityl sugar glucosides class or its mixture.The invention still further relates to the described special adsorbent of a kind of usefulness and carry out the method that affinity chromatography separates and makes with extra care.
Description
The present invention relates to a kind of adsorbent and process for purification, particularly a kind of special adsorbent that is used for the affinity chromatography separation, and use this special adsorbent to carry out the method for affinity chromatography separation and purification.
The invention relates to utilize affinity chromatography effectively with high-purity make with extra care beta amylase, malto-oligosaccharide and generate the enzymes relevant such as enzyme with carbohydrate.
In containing the various fields of organism material, affinity chromatography is used to organism is separated, makes with extra care and quantitative analysis, particularly with the special interaction function between its explanation organism.
The separation and purification of affinity chromatography is the interaction that utilizes between the two kinds of materials (A and B) with affinity, make a kind of material (A) combine with insoluble carrier, fix with the ligand form, and from the mixed liquor that contains multiple material, only optionally absorb in conjunction with another kind of material (B), then, by taking out substance B, and reach the purpose of separation and purification B.
For AMS, beta amylase, glucoamylase etc., can utilize starch, various derivatives (the cross-link dextran class etc. of contract glucose and these materials more, to call " from starch material " in the following text) as adsorbent, utilize affinity chromatography to carry out separation and purification.
Affinity chromatography is applied to industry refining the time, requires to adsorb a large amount of enzymes as far as possible for adsorbent from the Unit Weight of starch material; If be applied to breadboardly when refining, also be necessary on the purity that improves the purified starch enzyme, to put in a lot of time and effort.
, on commercial scale, need the high enzyme of purity at present, and also need a large amount of purpose product of high-purity ground separation and purification.
Use and above-mentionedly can not satisfy these requirements as the affinity chromatogram of adsorbent from starch material.
In view of above present situation, the objective of the invention is when utilizing affinity chromatography that relevant carbohydrate enzyme is carried out separation and purification, a kind of special adsorbent and high-purity are provided made with extra care the method for a large amount of purpose products.
Present inventors are through with keen determination research, found that use glucityl glucosides (Glucosyl Moranolines) class, oligomeric glucityl glucosides (Oligo-glucosyl Moranolines) class or its mixture (below be generically and collectively referred to as " glucityl glucosides class etc. ") have solved above-mentioned problem at one stroke as the ligand of affinity chromatography, have finished the present invention.
Main idea of the present invention just is with glucityl glucosides class as ligand.The invention provides a kind of affinity chromatography method for refining that has the special adsorbent of ligand and use this special adsorbent.
In the present invention with glucityl glucosides class etc. as the special adsorbent of the ligand of affinity chromatography before this without any report, so the inventive method is a kind of new method.In addition, generate enzyme about maltotriose and separate before this also not success example with affinity chromatography, by the present invention just realization.
Below describe the present invention.
Special adsorbent of the present invention is by ligand and insoluble carrier and constitute.Special adsorbent of the present invention can contain the interval base (Spacer) as inscape.
Ligand of the present invention is a glucityl glucosides class.Can reach peculiar effect of the present invention by them.
In addition, so-called glucityl glucosides class is meant the glucityl material or the oligomeric glucityl material of the glycosides derivatives of glucosides (Moranoline), N-replacement glucosides etc.
Glucityl glucosides class of the present invention does not have special restriction, but its typical example is as follows, promptly uses the compound of general formula [I] expression.
In the formula, n represents 0～30 integer.R represents hydrogen, alkyl, phenylalkyl, phenyl thiazolinyl, phenyl alkynyl, benzene oxyalkyl, benzene oxy alkylene, benzene oxycetylene base etc.Above-mentioned R, when containing alkyl as the part of its structure, its alkyl is a straight or branched, and unsaturated bond can be arranged, and also functional groups such as amino, imino group, hydroxyl, alkoxyl, carbonyl or carboxyl can be arranged.Perhaps above-mentioned R, when containing phenyl as the part of its structure, this phenyl also can replace with functional groups such as amino, imino group, hydroxyl, alkoxyl, carbonyl or carboxyls.
In the present invention, there is no particular limitation to so-called interval base, and it is (or be clipped in both the centre) inscape that makes that insoluble carrier and ligand combine.When base has two functional groups at interval, combine with insoluble carrier by a functional group, combine reason by another functional group with ligand, these two functional groups can be identical, also can be different.After also can being the interval based compound combination more than two in addition, constitute the part of base at interval.
Among the present invention, the method with glucityl glucosides class etc. is made the separatory special adsorbent of affinity chromatography as ligand can adopt known general method for making.These conventional methods have been documented in (for example, thousand cigarettes, one youth: affinity chromatography partition method, lecture report in a lot of document or various report.Stand erect really etc. in the mountain: affinity chromatography partition method, lecture report).
What is called among the present invention enzyme relevant with carbohydrate can be enumerated, the decomposition of polysaccharide and the enzyme of generation, for example, the amylase of AMS, beta amylase, glucoamylase, isoamylase, alpha-Glucosidase, β-Pu Tangganmei, amylopectase, malto-oligosaccharide generation (amylase that the amylase that the amylase that maltotriose generates, maltotetraose generate, maltopentaose generate etc.), cyclodextrin glucosyltransferase etc.
Feature of the present invention is to be to be to use glucityl glucosides class etc. as most important inscape-ligand in the affinity chromatography.
In the special adsorbent of the present invention, have no particular limits, can use widely used suitable material for insoluble carrier and interval base.
As these insoluble carriers, cellulose, agarose, crosslinked contract glucose, cross-linked polyacrylamide, porous silica beads etc. can have been enumerated more.
Special adsorbent of the present invention can be by the usual way manufacturing.The example with two functional group's compounds that can become the interval based compound is as follows, for example can be with 1,4-butanediol diglycidyl ethers etc. are that insoluble carrier reacts with the agarose of Ago-Gel etc. directly, after removing superfluous oxirane by washing, react with glucityl glucosides class of the present invention and obtain.At this moment, can in the sodium hydroxide solution of 0.5N, add Ago-Gel, oxirane, insoluble carrier was reacted about 10 hours, after the washing, itself and glucityl glucosides class be reacted prepare.
For the process for purification of the enzyme relevant of the present invention, except using the special adsorbent that affinity chromatography of the present invention uses, need not adopt other adhoc approach also can implement with carbohydrate.
For example, special adsorbent of the present invention is filled in the post, after flowing through suitable solution (being generally buffer solution), flow through suitable solution (being generally buffer solution) again, after wash-out goes out non-adsorbent, flow through the solution that contains the material (being generally matrix or matrix similar substance) that can adsorb the enzyme that from adsorbent, dissociates again, just can obtain desired enzyme.
Embodiment
Below further describe the present invention by reference example of the present invention and embodiment.
Reference example 1 maltotriose generates the determination of activity of enzyme
Maltotriose generates the determination of activity of enzyme (hereinafter referred to as " G3 enzyme ") and has used the DNS(dinitrosalicylic acid) method.In 400 μ, 12% soluble starch, add 100 μ 1G3 enzymes, after 10 minutes, add 1ml DNS, seethed with excitement 5 minutes at 40 ℃ of constant temperature.After the water-cooled, add 4.5ml water, measure its absorbance at 535nm.To in 1 minute, generate the enzyme amount of 1 micromolar maltotriose in addition as 1U.Protein is by the absorbance that is determined at 280nm or uses the Instrument measuring of measuring bio-absorbable amount (biorad) to finish in the absorbance of 595nm.
Reference example 2 G3 enzymes produce the cultivation of bacterium
The composition of the culture medium glucose 3% that contracts more
Soy meal 2%
Yeast decoction 0.1%
Many peptones 0.1%
Potassium phosphate 0.3%
Calcium chloride 0.5%
Ammonium sulfate 0.1%
Connect mould Pseudomonas griseus NA-468(Streptomyces griseus NA-468 at above-mentioned inoculation of medium) little worker grinds bacterium and deposits No. 2227), be 7,27 ℃ of shaken cultivation 4 days at pH.
The preparation of reference example 3 thick G3 enzymes
Above-mentioned medium centrifugal is separated (3000rpm, 10 minutes), after adding ammonium sulfate makes it to reach 40% degree of saturation in the centrifugation supernatant that obtains, carry out centrifugation (10000rpm, 10 minutes) again, in supernatant, add ammonium sulfate and make it to reach 60% degree of saturation.After carrying out centrifugation (10000rpm, 10 minutes), being dissolved in precipitation in the 0.5M acetate buffer solution (pH5.8), put in the dialysis membrane, use 0.01M acetic acid (pH5.8) to dialyse as outer liquid.With its centrifugation supernatant as thick G3 enzyme solutions.
The preparation of reference example 4 epoxy activate Ago-Gel 6B
Ago-Gel 6B(PHARMASIA society system with suction dried) 10g is suspended in the 0.6N sodium hydrate aqueous solution of 10ml, adds 10ml 1, and the 4-butanediol ethere under 25 ℃, slightly vibrated 8 hours.On glass filter, fully wash, so just obtain epoxy activate Ago-Gel 6B.
The preparation of reference example 5 4-O-α-D-glycopyranosyl glucosides
According to Jiang Lian etc. at (Y, Ezure Agnic, Biol, chem, 491 2159(1985)) in the method modulation described.Following 4-O-α-D-glycopyranosyl glucosides is abbreviated as the glucityl glucosides.
The beta amylase of reference example 6 oligomeric glucityl glucosides cuts off the preparation of residue
Will be according to the method (Y of Jiang Lian etc., Ezore Agnic, Biol, Chem 49,2159(1985) Zhi Bei oligomeric glucityl glucosides is made into the solution of 50mg/ml, after regulating pH and be 5.0 with 1N hydrochloric acid, to the beta amylase aqueous solution (biochemical industrial society system) that wherein adds from sweet potato, in an amount equivalent to 0.5% of load responsive fluid, reaction is spent the night under 40 ℃.Reactant liquor is adsorbed on the strong-acid ion exchange resin (Dao Kesi 50W * 2(H of appropriate amount
+)) on, fully after the washing, with the ammoniacal liquor wash-out of 1N.The beta amylase that the drying under reduced pressure eluent obtains oligomeric glucityl glucosides cuts off residue.With the result of high-speed liquid phase chromatogram analysis, this material is by glucosides 42%, glucityl glucosides 31.9%, malt-base glucosides 23.5%, maltotriose glycosyl glucosides 2.5% and the mixture of forming.
Reference example 7 maltopentaoses generate the determination of activity of enzyme
Maltopentaose generates the determination of activity of enzyme (to call " G5 enzyme " in the following text) can use the DNS method.Promptly in 250 μ l, 2% soluble starch (0.1M acetate buffer solution, pH5.8), add 250 μ lG5 enzymes, under 40 ℃, constant temperature added DNS solution 1ml after 20 minutes, and heating is 5 minutes in boiling water.After the water-cooled, add 4.5ml water, measure absorbance at 535nm.Measure the zymoprotein amount according to reference example 1.
Reference example 8 G5 enzymes produce the cultivation of bacterium
Culture medium is formed meat extract 0.8%
Ammonium sulfate 1.0%
Maltose 0.8%
Agar 1.9%
In the slant medium of above composition with false single-cell bacteria SP.KO-8940(FERM P-7456) cultivated 2 hours at 40 ℃.1 platinum coil is inserted into (マ イ ヤ-To) among the 500ml wheat Ye Shi, add forms identical 100ml fluid nutrient medium, 40 ℃ of shaken cultivation 3 days.
The preparation of reference example 9 thick G5 enzymes
The nutrient solution that centrifugation (9000rpm 20 minutes) reference example 8 obtains, in the supernatant that obtains, add ammonium sulfate up to 50% saturated till, centrifugation (10000rpm15 minute), collecting precipitation, precipitation is dissolved in the 0.5M acetate buffer solution (pH5.8), uses 0.01M acetate buffer solution (pH5.8) to dialyse as outer liquid.Its centrifuged supernatant is thick G5 enzyme liquid.
The G5 enzyme of reference example 10 oligomeric glucityl glucosides cuts off the preparation of residue
Modulate according to identical with reference example 6 basically method, be about to (pH does not adjust) in the water that oligomeric glucityl glucosides 10g is dissolved in 100ml, be added in the G5 enzyme liquid 50ml of reference example 9 modulation, 30 ℃ of reactions 20 hours.This reactant liquor is adsorbed on (Dao Kesi 50W * 2) on an amount of storng-acid cation exchange resin, and water fully washs.With behind the 1N ammoniacal liquor wash-out, decompression concentrates, and obtains the G5 cut-out residue of the oligomeric glucityl glucosides of about 2.6g.
Use 212g Ago-Gel 6B to obtain being in the epoxy activate Ago-Gel 6B of the state of blotting according to the method for reference example 4 records, again it is suspended in the sodium hydrate aqueous solution of 300ml0.1N, add 15g glucityl glucosides, under 40 ℃, vibrated gently 27 hours.On glass filter, wash, wash, wash, use 0.1M borate buffer (pH8.0) 1000ml washing, fully washing then with 0.1M acetate buffer solution (pH4.0) 1000ml.This gel is suspended in the 1M monoethanolamine of 400ml, at room temperature, slow shaken overnight.On glass filter, wash,, obtained using the G3 enzyme special adsorbent of glucityl glucosides with 0.1M acetate buffer solution (pH4.0) the 1000ml washing, the washing that contain 0.5M sodium chloride.
Use the beta amylase of oligomeric glucityl glucosides to cut off residue replacement glucityl glucosides, obtain using the diastatic G3 special adsorbent of oligomeric glucityl glucosides β according to the method for embodiment 1.
The affinity chromatography of the special adsorbent of the G3 enzyme of embodiment 3 use glucityl glucosides separates
The 250mlG3 enzyme special adsorbent that embodiment 1 is obtained is filled in the post, after 0.02M acetate buffer solution (pH5.8) buffering, add 165ml thick G3 enzyme liquid (25493U, protein 523mg, specific activity 48.7U/mg protein), after 0.62M acetate buffer solution (pH5.8) the 2150ml expansion that contains 0.2M sodium chloride, launch with the 1% maltotriose aqueous solution.Its result is illustrated among Fig. 1.Obtain the G3 enzymatic activity flow point consistent with protein.The specific activity of the G3 enzyme of flow point 9 is a 286.4U/mg protein, rises to about 5.9 times.The rate of recovery of G3 enzyme, only flow point 9 is 82.6%.
In Fig. 1, represented the result of embodiment 3.The longitudinal axis is represented G3 enzyme effectiveness (U/ml) and albumen quality (O.D.595).Transverse axis represents to flow branch.Represent among the figure that absorbance, oral thermometer show enzyme effectiveness.Flow point 1～6th, 300ml, flow point 7 are that 250ml, flow point 9～10 are 300ml for 350ml, flow point.
The affinity chromatography that the G3 enzyme special adsorbent that embodiment 4 uses oligomeric glucityl glucosides beta amylase to cut off residue carries out separates
The G3 enzyme special adsorbent 5ml that embodiment 2 is obtained is filled in the post, after 0.2M acetate buffer solution (pH5.8) buffering, add thick G3 enzyme solutions 5ml(773U, protein 18.5mg), fully launch with the 0.02M acetate buffer solution (pH5.8) that contains 1.0M sodium chloride.Then launch with 3% maltose solution.The result is illustrated among Fig. 2.
Represent G3 enzyme effectiveness (O.D.535) and albumen quality (O.D.280) from axle.Transverse axis represents to flow branch.The absorbance, the oral thermometer that are illustrated in 280nm among the figure are shown in 535 absorbance.The about 5ml of each flow point.
Use the G5 enzyme of oligomeric glucityl glucosides to cut off residue and replace the method for glucityl glucosides, the G5 of oligomeric glucityl glucosides is cut off residue as ligand, make G5 enzyme special adsorbent according to embodiment 1.
Embodiment 6 uses the G5 enzyme of oligomeric glucityl glucosides to cut off the G5 enzyme special adsorbent of residue and the affinity chromatography that carries out separates
To be filled in the post at the G5 enzyme special adsorbent 5ml that embodiment 5 obtains, after 0.2M acetate buffer solution (pH5.8) buffering, be added in the thick G5 enzyme liquid 5ml that reference example 9 obtains, fully launch with same buffer solution.Then, join loose industrial society system with the Sun oligomer 5.6(of oligosaccharide mixture) 3% solution when carrying out wash-out, confirm that wash-out has gone out the G5 enzyme.The result is illustrated among Fig. 3.
The longitudinal axis is represented G5 enzyme effectiveness (O.D.535) and albumen quality (O.D.595).Transverse axis is represented wash-out liquid measure (ml).Be illustrated in the absorbance of 595nm, the absorbance that oral thermometer is shown in 535nm among the figure.
Embodiment 7[N-(5-carboxy pentyl) coupling of glucityl glucosides and EAH-Ago-Gel
With the N-(5-carboxy pentyl) glucityl glucosides 100mg and EDC385mg be dissolved in the 5ml water, regulating pH is 4.5, adds 5mlEAH-Ago-Gel 4B(PHARMSIA society system), at room temperature, vibrated 24 hours, and used the 400ml water washing, obtain the AMS special adsorbent.
The AMS special adsorbent 5ml that embodiment 7 is obtained is filled in the post, after 0.01M acetate buffer solution (pH6.0) buffering, to wherein adding AMS (the liquefaction type is from Bacillus subtilis) solution, fully launch with above-mentioned buffer solution.Then, launch with 2% soluble starch solution (0.01M acetate buffer solution, pH6.0).In the first stream of starch solution, affirmation AMS wash-out comes out.
Claims (2)
1, the separatory special adsorbent of a kind of affinity chromatography is characterized in that the ligand in the described special adsorbent is glucityl glucosides class, oligomeric glucityl glucosides class or its mixture.
2, a kind of process for purification of the enzyme relevant with carbohydrate is characterized in that using the special adsorbent of claim 1 record to carry out affinity chromatography and separates.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP72676/92 | 1992-02-21 | ||
| JP7267692 | 1992-02-21 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1075663A true CN1075663A (en) | 1993-09-01 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 93101782 Pending CN1075663A (en) | 1992-02-21 | 1993-02-22 | Adsorbent and process for purification |
Country Status (3)
| Country | Link |
|---|---|
| CN (1) | CN1075663A (en) |
| AU (1) | AU4672893A (en) |
| WO (1) | WO1993017100A1 (en) |
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|---|---|---|---|---|
| CN109371078B (en) * | 2018-10-18 | 2022-03-11 | 山东福田药业有限公司 | Preparation process of high-purity maltose |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6024798B2 (en) * | 1980-06-06 | 1985-06-14 | 日本新薬株式会社 | Moranoline derivatives and their production method |
| JPS5681595A (en) * | 1979-12-08 | 1981-07-03 | Nippon Shinyaku Co Ltd | Moranoline derivative and its preparation |
-
1993
- 1993-02-19 WO PCT/JP1993/000202 patent/WO1993017100A1/en active Application Filing
- 1993-02-19 AU AU46728/93A patent/AU4672893A/en not_active Abandoned
- 1993-02-22 CN CN 93101782 patent/CN1075663A/en active Pending
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| Publication number | Publication date |
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| AU4672893A (en) | 1993-09-13 |
| WO1993017100A1 (en) | 1993-09-02 |
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