EP0000486B1 - Procédé de préparation de protéines stabilisées fixées sur des supports - Google Patents

Procédé de préparation de protéines stabilisées fixées sur des supports Download PDF

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Publication number
EP0000486B1
EP0000486B1 EP78100312A EP78100312A EP0000486B1 EP 0000486 B1 EP0000486 B1 EP 0000486B1 EP 78100312 A EP78100312 A EP 78100312A EP 78100312 A EP78100312 A EP 78100312A EP 0000486 B1 EP0000486 B1 EP 0000486B1
Authority
EP
European Patent Office
Prior art keywords
groups
bound
carrier
mercapto
protein
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.)
Expired
Application number
EP78100312A
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German (de)
English (en)
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EP0000486A1 (fr
Inventor
Dieter Dr. Krämer
Klaus Dr. Sauber
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.)
Roehm GmbH Darmstadt
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Roehm GmbH Darmstadt
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Publication of EP0000486A1 publication Critical patent/EP0000486A1/fr
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Publication of EP0000486B1 publication Critical patent/EP0000486B1/fr
Expired legal-status Critical Current

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Classifications

    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • C12N11/087—Acrylic polymers
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
    • Y10S530/812—Peptides or proteins is immobilized on, or in, an organic carrier
    • Y10S530/813—Carrier is a saccharide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
    • Y10S530/812—Peptides or proteins is immobilized on, or in, an organic carrier
    • Y10S530/815—Carrier is a synthetic polymer
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
    • Y10S530/812—Peptides or proteins is immobilized on, or in, an organic carrier
    • Y10S530/815—Carrier is a synthetic polymer
    • Y10S530/816—Attached to the carrier via a bridging agent

Definitions

  • the invention relates to a method for producing stabilized carrier-bound proteins.
  • the proteins are advantageously used in the form of a material bound to a solid support.
  • the protein can thereby be separated from the treated substrate solution by simple filtration.
  • Carrier-bound proteins are important in affinity chromatography and especially in carrying out enzymatic processes, the protein bound by the carrier being an enzyme.
  • carrier-bound proteins are generally characterized by increased stability against inactivation due to pH shift, high temperatures or autoxidation. Nevertheless, the bio-specific effectiveness drops, e.g. in the case of enzymes, the enzymatic activity, with repeated reuse of the carrier-bound protein at each stage of use somewhat.
  • loss of activity of proteins can be stopped or reversed by treating them with mercaptans (see dissertation Jan Carlsson, Uppsala 1974, p. 8, abstract).
  • the effect of the mercaptans is attributed to the fact that they break down disulfide bridges within the protein molecule which have arisen from thiol groups by autoxidation. This auto-oxidation is considered to be the cause of the reversible loss of activity.
  • the object of the present invention was to produce carrier-bound proteins with increased stability against gradual loss of activity in aqueous solution by reacting the protein with a water-insoluble carrier material.
  • the object is achieved according to the invention in that a carrier material free of mercapto groups is used which contains oxirane groups as binding-active groups, and in that hydrogen sulfide or a compound having a molecular weight below 5000 with at least one mercapto group and at least one further mercapto group is added to the protein bound to the carrier. , Hydroxyl or primary or secondary amino group.
  • the sterically readily accessible oxirane groups which are active against binding to the protein are completely available for binding the protein in the process according to the invention, while the sterically hindered oxirane groups which are inaccessible to the large protein molecules are still present after the enzyme has been bound and for reaction with the relatively small molecules of the Sulfur compound are capable.
  • Penicillin acylase compared the loss of activity with multiple reuse with and without mercapto groups on the wearer.
  • the mercapto groups were introduced by binding dithioerythritol to an enzyme carrier containing epoxide groups.
  • the carrier material was a crosslinked acrylamide polymer with epoxy groups, which had first been reacted with penicillin acylase and then optionally with 50 or 500 mg of dithioerythritol per gram of the moist carrier material.
  • the activity of the preparation against 6-amino-penicillinic acid was determined in a repeated reaction of 1 to 2 g of the preparation, each with 5% substrate in 200 ml of substrate solution (0.01 M phosphate buffer, pH 7.5) at 37 ° C. by automatic titration Determined 1.0 N NaOH. The initial activity when used for the first time was designated as 100%.
  • the stabilization achieved in accordance with the invention is based on the binding of hydrogen sulfide or a compound containing at least one mercapto group and at least one further mercapto, hydroxyl or primary or secondary amino group to the binding-active oxirane groups of the carrier material.
  • the mercapto, hydroxyl or amino groups of the compounds mentioned react with the oxirane groups of the support material. Since the compounds have a molecular weight below 5000, they are still able to react with the oxirane groups of the carrier material due to their small molecular size, which are not accessible to protein molecules for reasons of steric hindrance.
  • the oxirane groups react more easily with hydrogen sulfide or with mercapto, hydroxyl and amino groups than with water, so that it is possible to react both the oxirane groups with the protein and the subsequent reaction with the mercapto and optionally hydroxyl or amino groups Make connection in the aqueous phase. Since the oxirane groups are nevertheless gradually decomposed by water, there should not be an unnecessarily long period of time between the first and the second reaction stages of the process according to the invention.
  • the desired stabilizing effect occurs when at least one mercapto group per bound molecule is retained in free form and the further mercapto, hydroxyl or amino group reacts with the oxirane group of the carrier. Since the carrier consists of an insoluble solid matrix and has a correspondingly low molecular mobility, the compound containing mercapto groups is largely bound to the carrier only with one of its functional groups. If this group is a hydroxyl or amino group, a mercapto group is definitely preserved. When using a compound with only one mercapto group and one or more amino or hydroxy groups, the mercapto group can react with the oxirane group of the support, so that only amino or hydroxyl groups remain. In this case the bound molecule does not contribute to the stabilization. However, since some of the molecules do not react with the mercapto but with an amino or hydroxyl group, the statistical average remains sufficiently stabilizing mercapto groups.
  • the amount of the mercapto group-containing compound is generally such that at least 0.3% by weight, based on the wet weight of the carrier enzyme product, is bound to free SH groups.
  • Hydrogen sulfide and compounds with 2 or more mercapto groups contain two (or more) hydrogen atoms bound to sulfur, so that after their binding to the Carrier definitely has at least one free Merkapto group left.
  • Compounds with two or more SH groups are particularly preferred.
  • dithioethylene glycol dithiopropylene glycol or dithiobutylene glycol
  • 1,12-dimerkaptododecane di-mercapto-acetic ester of ethylene glycol
  • dodecanediol dodecanediol
  • neopentyl glycol or 2,2'-bis (4-hydroxy-cyclohexyl) propane pentethylolaptopropionate
  • trimethylolaprophrione tri mercaptoacetate
  • -tetra-mercaptopropionate -tetra-mercaptocaproic acid ester or -tetra-mercaptoundecanoic acid ester
  • compounds with further functional groups such as dithioerythritol.
  • Merkaptoethylamine and cysteine may be mentioned as compounds with one amino and one mercapto group.
  • Compounds with only one mercapto and one or more hydroxyl groups are the least preferred because the mercapto groups generally react more easily than the hydroxyl groups with the oxirane groups of the support. Examples of this type of compound are monothioethylene glycol and monothiopropylene glycol.
  • a large number of water-insoluble carrier materials with oxirane groups which are active against proteins are known. An overview of such carriers can be found in 0. Zaborsky, "Immobilized Enzymes", Cleveland 1973.
  • the most important carrier materials are synthetic crosslinked polymers based on acrylamide, maleic anhydride, methacrylic acid, styrene or polypeptides. Carbohydrates such as agarose, cellulose, cross-linked dextran or starch are mentioned as natural carriers. Glass and other inorganic substances can also be used as carriers.
  • the number of oxirane groups should be large enough to bind at least 10 mg, preferably 100 mg or more protein per gram of carrier material.
  • Oxirane groups can already be incorporated in the preparation of a crosslinked acrylamide polymer, preferably a bead polymer produced by reverse suspension polymerization, by using comonomers containing glycidyl groups, such as glycidyl acrylate or methacrylate.
  • glycidyl groups can e.g. by reaction with epichlorohydrin or 1,4-bis (2,3-epoxypropyl) butane.
  • Pearl-shaped carriers with pearl diameters of 5 to 5000 ⁇ m are particularly advantageous because of their easy handling and their flow permeability.
  • a protein is bound to the support, which can interact in a biospecific manner with a component of a substrate mixture to be separated. This component is temporarily held on the carrier-bound protein.
  • Enzymes are preferably attached to the support, e.g. Penicillin acylase, ribonuclease, amylase or catalase. Penicillin acylase bound and stabilized in the sense of the invention is of particular technical importance because this is the only way to economically reuse this valuable enzyme on an industrial scale.
  • Some enzymes are sensitive to hydrogen sulfide or mercaptans; they are then generally not suitable for the process of the invention. Enzymes which are already stabilized or reactivated by hydrogen sulfide or free mercaptans can be converted into enzyme products of increased stability by the process of the invention.
  • penicillin acylase E. coli, specific activity 12.1 U / mg
  • 40 ml 0.1 M phosphate buffer pH 8, 0.02% NaN,
  • 10 g of the product (1a) given The mixture is left at 23 ° C. for 72 hours stand and then wash three times with 1000 ml of 1 M aqueous NaCl solution and twice with 0.05 M phosphate buffer (pH 7.5; 0.02% NAN 3 ). Yield: 37 g of moist product (after suction on glass frit).
  • Enzymatic activity 237 U / g wet product.
  • penicillin-G (potassium salt) are dissolved in 90 ml phosphate buffer (pH 7.5; 0.05 M) and the volume is brought to 100 ml with this buffer.
  • the activity is determined graphically from the linear starting area of the sales curve; 1 enzyme unit (U) corresponds to 1 ⁇ mol of cleaved substrate per minute.
  • Example 1 The product is tested as in Example 1 (a). Loss of activity after 20 repeated uses: 42%.
  • 1,4-dithioerythritol 50 mg are dissolved in 1 ml of H 2 O and the pH is adjusted to 7.5 by adding NaOH.
  • the solution is added to 2 g of carrier-bound penicillin acylase according to Example 1 (b) and the mixture is left to stand at 23 ° C. for 48 hours. Then it is washed with water (five times) and buffer (twice).

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Claims (8)

1. Procédé pour la préparation de protéines stabilisées, fixées sur un support, par réaction d'une protéine en solution aqueuse et d'un stabilisant contenant des groupes SH avec une matière de support insoluble dans l'eau qui contient des groupes ayant, à l'égard des protéines, une action de fixation, caractérisé en ce qu'on met en oeuvre une matière de support exempte de groupes mercapto qui contient des groupes oxirane en tant que groupes ayant un pouvoir de fixation, et en ce qu'on fait agir, sur la protéine fixée sur le support, de l'acide sulfhydrique ou un composé ayant un poids moléculaire inférieur à 5000 et comportant au moins un groupe mercapto et au moins un autre groupe mercapto, un groupe hydroxy ou un groupe amino primaire ou secondaire.
2. Procédé selon la revendication 1, caractérisé en ce qu'on utilise une matière de support à base d'un polyacrylamide ou de dérivés d'hydrates de carbone.
3. Procédé selon la revendication 1 ou 2, caractérisé en ce qu'on met en oeuvre une matière de support en perles, ayant un diamètre des perles compris entre 5 et 1000 microns, à base d'un polyacrylamide ramifié comportant des groupes oxirane.
4. Procédé selon l'une quelconque des revendications 1 à 3, caractérisé en ce qu'on met en oeuvre, en tant que composé comportant au moins un groupe mercapto, un composé aliphatique à 2 à 8 atomes de carbone.
5. Procédé selon l'une quelconque des revendications 1 à 4, caractérisé en ce que la protéine fixée est un enzyme.
6. Procédé selon la revendication 5, caractérisé en ce que l'enzyme fixé est une pénicilline- acylase, une ribonucléase, un amylase ou une catalase.
7. Procédé selon la revendication 6, caractérisé en ce que l'enzyme fixé est une pénicilline- acylase.
8. Procédé selon l'une quelconque des revendications 1 à 7, caractérisé en ce qu'au moins 0,396 en poids (sur la base du poids humide du produit final) de groupes mercapto libres sont fixés par addition à la matière de support.
EP78100312A 1977-07-16 1978-07-06 Procédé de préparation de protéines stabilisées fixées sur des supports Expired EP0000486B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE2732301A DE2732301C3 (de) 1977-07-16 1977-07-16 Verfahren zur Herstellung von stabilisierten trägergebundenen Proteinen
DE2732301 1977-07-16

Publications (2)

Publication Number Publication Date
EP0000486A1 EP0000486A1 (fr) 1979-02-07
EP0000486B1 true EP0000486B1 (fr) 1981-07-29

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EP78100312A Expired EP0000486B1 (fr) 1977-07-16 1978-07-06 Procédé de préparation de protéines stabilisées fixées sur des supports

Country Status (6)

Country Link
US (1) US4247643A (fr)
EP (1) EP0000486B1 (fr)
JP (1) JPS5419901A (fr)
AT (1) AT358516B (fr)
DE (2) DE2732301C3 (fr)
IT (1) IT1108114B (fr)

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE7396T1 (de) * 1979-09-21 1984-05-15 Imperial Chemical Industries Plc Schnell kristallisierbare zusammensetzung auf der basis eines blockcopolyesters.
US4442212A (en) * 1980-11-10 1984-04-10 E. I. Du Pont De Nemours & Company Monothioglycerol as thiol-protector in lyophilized materials
DE3136940A1 (de) * 1981-09-17 1983-03-31 Hoechst Ag, 6230 Frankfurt "verfahren zur herstellung von 5'-ribonucleotiden"
DE3334848A1 (de) * 1983-09-27 1985-04-04 Hoechst Ag, 6230 Frankfurt Verfahren zur herstellung der optischen antipoden von tert.-leucin
DE3435156A1 (de) * 1984-09-25 1986-04-03 Boehringer Mannheim Gmbh, 6800 Mannheim Verfahren zur herstellung der stereoisomeren von 1-amino-alkylphosphonsaeuren oder 1-aminoalkylphosphinsaeuren
DE3726454A1 (de) * 1987-08-08 1989-02-16 Behringwerke Ag Thiolgruppenhaltige polymere, verfahren zu ihrer herstellung und ihre verwendung
KR0152667B1 (ko) * 1990-04-18 1998-10-01 한스 월터 라벤 페니실린 g 아실라아제, 이 효소를 코드화한 유전자 및 이 효소의 제조방법
DE4123014A1 (de) * 1991-07-11 1993-01-14 Kaltenbach & Voigt Bearbeitungsgeraet zum bearbeiten von feinwerktechnischen, insbesondere dentaltechnischen werkstuecken in einem von einem gehaeuse umgebenen bearbeitungsraum
DE19620036C2 (de) * 1996-05-17 1998-03-12 Roehm Gmbh Gewinnung von wasserfreiem 2-Trimethylammoniumethylmethacrylatchlorid aus wäßriger Lösung
DE19804518C2 (de) * 1998-02-05 2000-10-05 Roehm Gmbh Verfahren zur Herstellung von perlförmigen Mischpolymerisaten auf Acrylatbasis, danach hergestellte Trägerpolymermaterialien und deren Verwendung
JP7159617B2 (ja) 2018-05-25 2022-10-25 富士電機株式会社 冷却装置、半導体モジュール、車両および製造方法

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SE337223B (fr) * 1967-05-23 1971-08-02 Pharmacia Ab
US3853708A (en) * 1970-01-23 1974-12-10 Exploaterings Ab Tbf Coupling biologically active substances to oxirane-containing polymers
DE2215509C3 (de) * 1972-03-30 1980-06-26 Bayer Ag, 5090 Leverkusen Vernetzte Copolymerisate
SE420733B (sv) * 1974-05-30 1981-10-26 Exploaterings Ab Tbf Gelprodukt, innehallande tiosulfatgrupper samt sett for framstellning derav
US4008126A (en) * 1976-03-15 1977-02-15 Owens-Illinois, Inc. Immobilization of proteins by in-situ polymerization

Also Published As

Publication number Publication date
DE2732301A1 (de) 1979-01-25
DE2732301C3 (de) 1980-12-11
JPS5419901A (en) 1979-02-15
DE2732301B2 (de) 1980-03-27
IT1108114B (it) 1985-12-02
DE2860880D1 (en) 1981-10-29
IT7868683A0 (it) 1978-07-14
ATA424978A (de) 1980-02-15
JPS6239997B2 (fr) 1987-08-26
EP0000486A1 (fr) 1979-02-07
US4247643A (en) 1981-01-27
AT358516B (de) 1980-09-10

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