WO1994000564A1 - Adsorbant specifique - Google Patents

Adsorbant specifique Download PDF

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Publication number
WO1994000564A1
WO1994000564A1 PCT/JP1993/000833 JP9300833W WO9400564A1 WO 1994000564 A1 WO1994000564 A1 WO 1994000564A1 JP 9300833 W JP9300833 W JP 9300833W WO 9400564 A1 WO9400564 A1 WO 9400564A1
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WO
WIPO (PCT)
Prior art keywords
maltotriosylglucamine
enzyme
present
solution
specific adsorbent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP1993/000833
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English (en)
Japanese (ja)
Inventor
Shigeaki Maruo
Yohji Ezure
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nippon Shinyaku Co Ltd
Original Assignee
Nippon Shinyaku Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nippon Shinyaku Co Ltd filed Critical Nippon Shinyaku Co Ltd
Priority to AU43568/93A priority Critical patent/AU4356893A/en
Publication of WO1994000564A1 publication Critical patent/WO1994000564A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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

Definitions

  • the present invention relates to an efficient improvement of affinity mouth chromatography which is effective for purifying a carbohydrate-related enzyme.
  • Affinity mouth chromatography is used in various fields involving biological materials for separation and purification, quantification, and elucidation of specific interaction mechanisms.
  • Separation and purification by affinity chromatography utilizes the interaction between two substances (A and B) having affinity (A) and binds (A) as a ligand to an insoluble support and immobilizes it. It is intended to separate and purify B by selectively adsorbing and bonding only one (B) from a mixed solution containing miscellaneous substances, and then extracting B.
  • ⁇ -amylase For ⁇ -amylase, ⁇ -amylase, dalcoamylase, etc., starch, dextran, and various derivatives thereof (such as Sephadex, hereinafter referred to as “starch-derived substances”) are used as adsorbents to obtain affinity. Separation and purification by two-way chromatography is performed.
  • an object of the present invention is to provide a method for purifying a large amount of a target substance with high purity when separating and purifying a carbohydrate-related enzyme by affinity mouth chromatography.
  • the present inventors have assiduously studied and found that the use of maltooligosylglucamine or an N-substituted product thereof as a ligand of affinity chromatography solves the above problems at once. This fact was confirmed by an experimental example of the present invention, and the present invention was completed.
  • the gist of the present invention is that maltooligosylglucamine or its N-substitute is applied as a ligand.
  • the present invention is a specific adsorbent itself having such a return.
  • the specific adsorbent of the present invention is composed of a ligand and an insoluble supporter.
  • the specific adsorbent of the present invention can have a spacer as a constituent element.
  • the ligand of the present invention must be maltooligosylglucamine or its N-form. As a result, the present invention can exhibit the effects unique to the present invention.
  • the maltooligosylglucamine according to the present invention can be represented by the following general formula [I].
  • R represents any of 1. to 5. of 1. hydrogen, 2. hydroxyl group, 3. alkyl, 4. aryl, 5. acyl, and n represents an integer of 1 to 3. .
  • the alkyl may be linear or branched and may have an unsaturated bond, and may be amino, imino, hydroxyl, alkoxy, It may have a functional group such as carbonyl or carboxylic acid.
  • the aryl may be substituted with a functional group such as amino, imino, hydroxyl, alkoxy, carbonyl, or carboxylic acid.
  • the derivative of maltooligosylglucamine according to the present invention may be as follows, for example, using an N-substituted compound of maltotriosylglucamine, which is one of the derivatives.
  • spacer is not limited to a specific substance, but is formed by binding an insoluble support to a ligand. (Or consequently sandwiched between the two).
  • the spacer has bifunctionality, the insoluble support is bound by one functional group and the ligand is bound by another functional group, but the two functional groups may be the same or different. It is possible. Further, there are cases where two or more compounds to be spacers are combined to form a part of the spacer.
  • a feature of the present invention resides in that maltooligosylglucamine or an N-substituted product thereof is used as a ligand, which is the most important constituent of affinity mouth chromatography.
  • the insoluble support and the spacer are not limited to the above, and appropriate substances widely known so far can be applied.
  • examples of such an insoluble support include cellulose, agarose, cross-linked dextran, cross-linked polyacrylamide, porous silica beads and the like.
  • the specific adsorbent of the present invention can be produced by a commonly used method.
  • the compound to be a spacer is a compound having a bifunctional group
  • 1,4-butanediol diglycidyl ether is directly reacted with an agarose-based insoluble support such as sepharose.
  • an agarose-based insoluble support such as sepharose.
  • maltooligosylglucamine of the present invention or its N-substituted product for example, sepharose and oxysilane are added to a 0.5 N sodium hydroxide solution in a 0.5 N sodium hydroxide solution, and the mixture is reacted at room temperature for about 10 hours. After washing with water, dalcosyl molanolins are similarly reacted. Thereby, the reaction can be completed.
  • the method for purifying the carbohydrate-related enzyme of the present invention can be carried out without using a special method, except for using the specific adsorbent for affinity chromatography of the present invention.
  • the specific adsorbent of the present invention is packed in a column, an appropriate solution (usually a buffer) is flowed, and then an appropriate solution (usually a buffer) is applied to elute non-adsorbed substances.
  • a target enzyme can be obtained by flowing a solution containing a substance having a releasing action (usually, a substrate or a substrate-related substance).
  • G 2 NH 2
  • G 3 NH 2
  • G 4 NH 2
  • G 5 -N H2 stands for darcosylglucamine, maltosylglucamine, maltotriosylglucamine and maltote traosylglucamine, respectively. means.
  • Each preparation method is exactly the same as the preparation method of maltotriosylglucamine shown in Reference Example 5.
  • Gl, G2, G3, G4, and G5 mean dalcoamylase, monoamylase, maltotriose-forming enzyme, maltotetraose-forming enzyme, and maltopentaose-forming enzyme, respectively.
  • the numbers in the table indicate ICs ) ig nil) value.
  • the inhibitory activity of maltooligosaccharides on the Gl, G2, G3, G4, and G5 enzymes was measured as follows.
  • Table 1 shows that the maltotriosylglucamine according to the present invention clearly inhibits only the G4 enzyme among maltooligosaccharide-forming enzymes.
  • N-substituted derivatives of maltotriosylglucamine for the G4 enzyme.
  • N-substituted derivatives also exhibit inhibitory activity, from which an appropriate ligand can be selected.
  • maltooligosylglucamine and the N-substituted derivative thereof according to the present invention also inhibit enzymes other than maltooligosaccharide-forming enzyme.
  • Table 3 shows that maltosylglucamine, maltotriosylglucamine and maltotetraosylglucamine exhibit inhibitory activity against pullulanase and isoamylase, and are useful as ligands.
  • G4 enzyme The activity of maltotetraose synthase (hereinafter referred to as “G4 enzyme”) was measured using the DNS (dinitrosalicylic acid) method.
  • DNS dinitrosalicylic acid
  • the protein was measured by the absorbance at 280 nm or the absorbance at 595 nm using a protein measurement kit “Protein Assay” manufactured by Biorad. Reference Example 2 (G4 culture of enzyme-producing bacteria)
  • Pseudomonas stutzeri IFO-3773 (Pseudomonas stutzeri IFO-3773) was added to the above medium.
  • the cells cultured for 24 hours at C were inoculated in the same medium as a preculture, and cultured at 30 ° C for 14 to 16 hours to obtain a mother liquor of G4 enzyme.
  • Suspension 10 g of dry Sepharose 6B (Pharmacia) is suspended in 10 ml of 0.6 N aqueous sodium hydroxide solution, and 10 ml of 1,4-butanediol ether is added thereto. Shake gently for 8 hours. The glass filter was sufficiently washed with water to obtain an epoxy-activated Sepha mouthpiece 6B.
  • maltotetraosylglucamine 880 mg was prepared in exactly the same manner as in Reference Example 5.
  • the activity of pullulanase was measured using a 1% pullulan solution as a substrate in 50 mM acetate buffer (pH 6.0) at 40 ° C for 30 minutes, and the reducing power was increased by the dinitrosalicylic acid method. was measured.
  • the column was filled with 10 g of the G4 enzyme-specific adsorbent obtained in Example 1, buffered with 0.01 M Tris-HCl buffer (pH 8.0), and then treated with crude G4 enzyme solution 2. After charging 0 ml (640 U, protein 6 mg, specific activity 107 UZmg protein), it was washed with 0.01 M Tris-HCl buffer (pH 8.0). When protein elution is no longer observed, Fujioligo 470 (manufactured by Nippon Shokuhin Kako Co., Ltd.), which is a mixture of oligosaccharides, is developed in a 0.1% M Tris buffer (pH 8.0) using a 5% solution. did. The results are shown in Figure 1.
  • a fraction having the same G4 enzyme activity and protein was obtained.
  • the specific activity of the G4 enzyme in the eluate was 3337 UZmg, which was about three times that of the protein.
  • the recovery of G4 enzyme was 100% between 200 and 300 ml of eluate.
  • Example 6 [Affinity chromatography with isoamylase-specific adsorbent using maltotetraosylglucamine]
  • FIG. 1 shows the results of Example 2.
  • FIG. 2 shows the results of Example 5.
  • FIG. 3 shows the results of Example 6.
  • FIG. 4 shows the results of Example 8.
  • the vertical bar indicates the enzymatic activity in each Example by the OD value of 53511111, and the amount of protein by the OD value at 595 nm.
  • ⁇ indicates enzyme activity, and mouth indicates protein content.
  • the horizontal axis shows the volume of the eluate (ml).

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Obtention avec un haut degré de pureté d'enzymes se rapportant aux sucres par chromatographie d'affinités mettant en ÷uvre un adsorbant spécifique dans lequel la malto-oligosylglucamine ou un dérivé N-substitué de cette substance sert de ligand.
PCT/JP1993/000833 1992-06-23 1993-06-22 Adsorbant specifique Ceased WO1994000564A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU43568/93A AU4356893A (en) 1992-06-23 1993-06-22 Specific adsorbent

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP18985892 1992-06-23
JP4/189858 1992-06-23
JP5/113755 1993-04-16
JP11375593 1993-04-16

Publications (1)

Publication Number Publication Date
WO1994000564A1 true WO1994000564A1 (fr) 1994-01-06

Family

ID=26452687

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP1993/000833 Ceased WO1994000564A1 (fr) 1992-06-23 1993-06-22 Adsorbant specifique

Country Status (2)

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AU (1) AU4356893A (fr)
WO (1) WO1994000564A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6030698A (ja) * 1983-07-28 1985-02-16 Wako Pure Chem Ind Ltd α−アミラ−ゼアイソザイムの新規な分別測定法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6030698A (ja) * 1983-07-28 1985-02-16 Wako Pure Chem Ind Ltd α−アミラ−ゼアイソザイムの新規な分別測定法

Also Published As

Publication number Publication date
AU4356893A (en) 1994-01-24

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