• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    Die Erfindung betrifft oberflächenreiche Systeme mit reaktiven Einheiten zur Fixierung von nucleophile Gruppen enthaltenden Substraten, wobei die reaktiven Einheiten Bestandteile eines Polymerlatex sind, der selbst zu einem oberflächenreichen System aggregiert und/oder an einem oberflächenreichen Trägermaterial fixiert ist, und deren Verwendung.
    公开号:SU1655301A3
    申请号:SU823470942
    申请日:1982-08-04
    公开日:1991-06-07
    发明作者:Зиоль Вернер；Кремер Дитер；Зюттерлин Норберт；Феиль Корнелия；Маркерт Герхард；Шустер Эрвин
    申请人:Рем Гмбх Хемише Фабрик (Фирма)；
    IPC主号:
    专利说明:

    This invention relates to the field of creating immobilized enzymes.
    The purpose of the invention is to increase the specific activity of the immobilized enzyme.
    The following examples illustrate the invention, while examples 1-11 illustrate the preparation of the polymer used in the proposed method, and examples 12-29 illustrate the implementation of the proposed method.
    Example 1. In a polymerizer equipped with a reflux condenser, a stirrer and a thermostat, a solution of the following composition is heated to 80 ° C, g: phosphate buffer solution (pH 7.0) 10; natri.np salt of 4,4 -azobis- (4 dipovanaleric acid) 0.4; sodium lauryl sulfate 0.3; distilled water 555.
    An emulsion containing, g: methyl methacrylate 360; butyl acrylate 210; glycium-.
    dilmetacrylate 30; sodium salt 4,4 azobis-, (4-cyanovaleric acid) 2;
    sodium lauryl sulfate 3; distilled hours
    bath water 840.
    Then it is stirred for another 12 hours at 80 ° C, cooled to room temperature and filtered. A coagulant-free dispersion is obtained. The solids content is
    OE
    cl cn
    with

    cm
    80 ° C
    about 30%, pH value 7.3, viscosity 2. mPa “s.
    Example 2. In a polymerizer equipped with a reflux condenser and thermostat, serves a solution of the following composition, g: phosphate buffer solution (pH 7.0) 50; sodium lauryl sulfate 0.3; sodium salt of 4,4 -azobis- (4-cyanovaleric acid) 0.3; distilled water 515, and heated to 80 ° C.
    To this solution is added at
    within 4 hours, an emulsion containing, g: methyl methacrylate 300; butyl acrylate 210; glycidyl methacrylate 90; sodium salt of 4,4 -azobis- (4-cyanovaleric acid) 2; sodium lauryl sulfate 3; distilled water 840.
    Then stirred for 90 min at 80 ° C, cooled to room temperature and filtered. A well-filtered, non-coagulated dispersion is obtained. The solids content is about 30%, pH value, 1, viscosity 1.
    Dispersion contains the polymer specified in example 1 composition.
    Example A solution of the following composition is fed to the polymerizer equipped with a reflux condenser, a stirrer and a thermostat, g: sodium lauryl sulphate 6.5; sodium salt of 4,4 -azobis- (4-cyanovaleric acid) 0.6; phosphate buffer solution (pH 7.0) 10.0; distilled water 600.0, and heated to
    80 ° C.
    To this solution is added dropwise at 80 ° C for 3 hours an emulsion containing, g: methacrylamide 18; ethylene glycol dimethacrylate 11; methyl methacrylate 150; glycidyl methacrylate 180; sodium lauryl sulfate 1.5; sodium salt from 4,4 -azobis- (4-cyanovaleric acid) 2.0; distilled water 900.
    Then it is stirred for 30 minutes at 80 ° C, cooled to room temperature and filtered. A well-filtered, non-coagulated dispersion is obtained. The solids content is 19.4%, the pH value is 7.7, the viscosity is L MPa-s.
    EXAMPLE 4 Example 3 is repeated with the warrior that an emulsion of the following composition is added, g; methylcrylamide 13; ethylene glycol dimetmicry tat 5o;

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    methyl methacrylate 125; glycidyl methacrylate 180.
    The auxiliary substances, the polymerization time and the polymerization temperature are the same as in Example 3. A well-filtered, non-coagulated dispersion is obtained. Solid content 19.7%, pH 7.6, viscosity 1 mPa s. v Dispersion contains the polymer described in example 3.
    PRI me R 5.
    A. In a reactor equipped with a reflux condenser, a stirrer and a thermometer, 16 DO g of water are fed and heated to 80 ° C. After adding 3 g of isobutyl methacrylate, 3 g of methyl methacrylate, 0.3 g of ethylene glycol dimethacrylate and 0.8 g of sodium lauryl sulfate, 4 g of ammonium persulfate dissolved in 36 g of water. A mixture of 200 g of isobutyl methacrylate, 200 g of methyl methacrylate and 20 g of ethylene glycol dimethacrylate is then added dropwise over 80 hours at 80 ° C.
    After the addition of the monomer, the mixture is stirred for 1 hour at 80 ° C. A coagulum-free, well-filtered, low-viscosity 20% dispersion is obtained.
    b. 330 g of water are supplied to a reactor equipped with a reflux condenser, a stirrer and a thermometer. Then add
    10 ml of phosphate buffer solution with a pH value of 7 (Titrisol from Merck) and 160 g of the initial dispersion a. After heating to 80 ° C, 0.4 g of 4,4-aazobis- (4-cyanovaleric acid) sodium salt is added to 4 ml of water. Chatham is added over a period of 3 hours at 80 s an emulsion consisting of 1000 g of water, 1 g of sodium lauryl sulfate, 2 g of sodium salt of 4,4-aa-bis- (4-cyanovaleric acid), 160 g of ethyl acetate and 145 g of isobutyl methacrylate .
    A solution of 10 g of methacrylic acid amide and 0.6 g of 4,4 -azo-bis (4-cyanovaleric acid) sodium salt in 300 g of water is then added over 60 minutes, as well as a monomer mixture consisting of 50 g ethyl acrylate, 45 g of glycidyl methacrylate. Then it is stirred for an additional 60 minutes at 80 C. A coagulum-free low dispersion dispersion is obtained with a solids content. 0%.
    516
    II p and me 6. Example 5 is repeated with the difference that an emulsion consisting of 1000 g of oxen, 1 g of sodium lauryl sulfate, 2 g of sodium salt of 4,4 -azobis- (4-cyanovaleric acid) is fed for 3 hours at 80 ° C. , 160 g of ethyl acrylate, 145 g of isobutyl methacrylate and 1.5 g of ethylene glycol dimethacrylate.
    A solution of Yu g of methacrylic acid amide and 0.6 g of 4,4 -azo-bis (4-cyanovaleric acid) sodium salt in 300 g of water and a monomer mixture consisting of 50 g of ethyl acrylate are then added over 60 minutes. , 45 g of glycidyl methacrylate and 0.5 g of ethylene glycol dimethacrylate. Then mix up
    for another 60 minutes at 80 C.
    A low viscosity dispersion free of coagulum with a solids content of 20% is obtained.
    Lisperse contains a polymer of composition given in example 5.
    Example 7, Example 5 is repeated with the difference that the solution used in the last stage of polymerization contains 25 g of hydroxyethyl methacrylate (instead of 10 g of methacrylamide), 70 g of ethyl acrylate (instead of 50 g) and 5 g of methacrolein (in experiment A) or 5 g of N-oxysuccinimide methacrylate (in experiment B) instead of 45 g of glycidyl methacrylate. In both cases, a coagulum-free, low-viscosity dispersion is obtained.
    EXAMPLE 8 At a temperature of 80 ° C, 530 g of water, 0.5 g of sodium salt of 4,4-dzobis- (4-cyanovaleric acid) and T, 15 g of sodium lauryl sulfate are dissolved in a polymerizer. To this solution, an emulsion consisting of 156 g of ethyl acrylate, 14.5 g of isobutyl methacrylate, 1.5 g of ethylene glycol dimethacrylate, 0.9 g of sodium lauryl sulfate, 1.8 g of sodium salt 4 , 4 -azobis- (4-cyanovaleric acid) and 90;) g of water. I
    An emulsion consisting of 70 g of ethyl acrylate, 25 g of methyl methacrylate, 5 g of glycidyl methacrylate, 0.6 g of 4,4 -azobis- (4-cyanovaleric acid) sodium salt and 300 g of water.
    15 minutes after the feed, the dispersion is cooled to room temperature.
    The dry matter content is 20%, the pH value is 7.1.
    sixteen
    II p and m er. 15, according to Example 8, the dispersion is immersed in polystyrene beads (diameter O / mm) with forceps for 3 hours. After taking out, these ariki are placed on straw paper and dried for 3 hours at room temperature. Dry beads are stored in a refrigerator at -20 ° C, 32 µg of dry varnish is applied to the beads.
    Example 10. Example 1 is repeated with the difference that the monomer of the composition is used, g: methyl methacrylate 360; butyl acrylate 200; N-oxysuccinimide ester of 6-methacryloylamino-hexanoic acid 40.
    A dispersion is obtained with a solids content of about 30% by weight. Example 11
    Example 1 is repeated with the difference that the monomer of the composition is used, t: methyl methacrylate 380; butyl acrylate 200; adduct of acrylic acid 2-hydroxyethyl ester to 1,6-hexadiisothiacyanate 20.
    A dispersion is obtained with a solids content of about 30%.
    Example 12. Immobilization of the enzyme ribonuclease.
    100 mg of ribonuclease (E.C. 2..7.7,16) from pancrease (specific activity of 14.6 U / mg) with ribonucleic acid as substrate is dissolved in 1 ml of 0.05 M phosphate buffer with pH 7.5. Under stirring, 5 ml of the dispersion according to Example 2 is added to this solution. Then it is settled for 3 days at 23 ° C.
    The processing was carried out by suspending three times in 50 ml of a solution of 1 M sodium chloride and then centrifuging. Then another 2 times washed with 50 ml of 0.05 M phosphate buffer. Yield 1.1 g wet product.
    According to the analysis of the supernatant and wash water, the yield of the immobilized product is 50%.
    Enzyme activity is determined by alkalimetric titration at 37 ° C and pH 7.5 with ribonucleic acid as a substrate.
    The results are summarized in table 1.
    i
    Example 13. Immobilization of this trypsin.
    Example 12 is repeated with the difference that the enzyme used is
    716
    1i) 0 mg of trypsin (EG 3.4.4 .4) from te-1 Lenka (specific activity 144 / mg using casein as a substrate and 51.4 U / mg with CAEE — ethyl ester hydrochloride — N - benzoyl-arginine - as substrate).
    According to the analysis of the supernatant and wash water, the yield of the immobilized product is 52%.
    Determination of enzyme activity is carried out by calimetric titration at 3 / bS and pH 7.5 (BAEE) or pH 8.0 (casein).
    The results are summarized in table. 2
    Examples 14-19. Immobilization of the enzyme trypsin.
    Example 13 is repeated with the difference that the enzyme and latex are taken in the ratio indicated in Table. 3
    According to the analysis of the supernatant and wash water, the yield of the immobilized product in Examples 14-19 is 80, 74, 88, 62, 50 and 58%, respectively.
    Example 20. Immobilization of the penicillin amidase enzyme.
    Example 12 is repeated, with the difference that 100 mg of penicillin amidase (E.G. 3.5.1.11) Escherichia coli (specific activity 10 U / mg) is used as the enzyme.
    According to the analysis of the supernatant and wash water, the immobilized yield:. product is 55%.
    Enzyme activity is determined by measuring at 37 ° C and pH 7.8 (substrate potassium salt of penicillin).
    The results are summarized in table 4.
    Example 21. Immobilization of the enzyme ribonuclease.
    Example 12 is repeated, with the difference that latex is used according to example 3.
    According to the analysis of the supernatant and wash water, the yield of the immobilized product is 56%.
    The results are summarized in table. five.
    Example 22. The enzyme fixation ribonuclease on paper activated by impregnation of the dispersion of example 1.
    but. 100 ml of the dispersion according to Example 1 is diluted with 500 ml of distilled water. Paper of 100 cm2 is impregnated with this 5% dispersion.
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    L
    It is then drained, left to stand for 1 hour at room temperature and then dried at 80 ° C for 30 minutes. The paper thus treated contains 20 g of polymer per mg. This reactive paper can be stored at temperatures below -15 ° C for at least 12 months.
    b. 100 mg of ribonuclease (from pacrase) is dissolved in 0.05 M phosphate buffer (pH 7.5). The paper treated in step a is impregnated with this solution and then left to stand for 72 hours at 23 ° C. Then squeeze and washed 3 times with 1N. a solution of sodium chloride and 2 times a 0.05 M solution of phosphate buffer (pH 7.5). The activity (the third application) is 2 U / g wet paper.
    Conditions for determining the activity: temperature 37 ° C, pH 7 (5. Activity yield of immobilized product 0.4%.
    . Example 23: Example 12 is repeated. With the difference that the enzyme trypsin is used. The activity (the third application, the BAEE substrate, pH 7.5, temperature 37 ° C) is 2.5 U / g wet paper.
    The yield of the immobilized product is 58%, and its activity is 0.1%.
    Example 24. Example 22 is repeated with the difference that a lower concentration dispersion is used for paper impregnation (100 ml of dispersion according to example 1, diluted with 200 ml of distilled water).
    Drying conditions: 12 hours at 25 ° C in a tumble dryer with circulation. of the spirit.
    Polymer solids / m2 boom - g
    The enzyme trypsin is then fixed as in Example 22.
    The activity (the third application) is 4.1 U / g wet paper using the BAEE substrate with a pH value of 7.5 at 37 ° C, and the activity yield of the immobilized product is 0.15%.
    Example 25. Example 24 is repeated, with the difference that latex is used for impregnation according to Example 2 (diluted as Example 24: 200 ml of dispersion in 200 ml of distilled water).
    Polymer solids / m2 bu-g. The activity (the third application) is 7 U / r wet paper (BAEE substrate, pH 7.5, temperature 37 ° C), and the activity yield of the immobilized product is Oj25%.
    EXAMPLE 26 Example 22 is repeated with the difference that the impregnation of JQ paper is carried out with the dispersion of Example 10, brought to a solid content of 10% by adding distilled water, and trypsin is used as the enzyme. 15
    The activity (the third application) is 5.2 and 2.5 U / r wet paper, respectively.
    Approx. 27. Immobilization of Escherichia coli.20
    To 10 ml of a 20% suspension of Escherichia coli in physiological saline solution, add 10 ml of dispersion according to example 4. Then it is left to stand for 25 hours at 25 room temperature. Net material is obtained, which is purified by centrifugation.
    The yield of the wet product is 4.5 g, and the yield of the immobilized product .. 30 100% (no longer detected in the supernatant and in the wash water).
    Data on the activity of the wet product and the yield of activity of the immobilized product are summarized in table. tab. 6
    Example 28. Prior 13 was repeated with the difference that the version of examples 5-7 was used. In this case, the reactivity (the third application, casein as a substrate) is 30.5; 29.8 and 31.2 and / g, respectively.
    Example 29. To 100 beads according to example 9, 20 ml of andd titers obtained from antiserum (from goats) against human globuin are poured by affinity chromatography and subsequent dilution with 0.5 M of phosphate buffer (pH 7.5) to a concentration of 5Q 30 µg / ml.
    The mixture was allowed to stand overnight at 23 ° C without shaking and without stirring. Then it is decanted and washed 3 times with 0.5 M phosphate bu35.
    55
    fera. Then bovine serum albumin (1% in O, 1 M phosphate buffer, pH 7.5) is incubated for 3 hours at room temperature,
    Jq 15
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    $ Q
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    once again and then once again was washed with 0.1 M phosphate buffer (pH /, 5, containing 0.1 wt.% sodium azide). One of the balls treated in this manner is fed to a test tube, the diameter of which is 1 mm larger than the diameter of the wig. 0.2 ml of diluted 0.02 M phosphate buffer CpH /, 5, 0.15 M NaCl) to 1: 10,000 human serum is fed into it and incubated for 30 minutes at room temperature. The bead is then washed with 0.02 M NaCl-containing phosphate buffer, and 0.2 ml of the product from peroxidase and anti-human globulin (from goats), which is prediluted with NaCl-containing phosphate buffer, is added to it and incubated for 1 hour at room temperature. . It is then washed well with phosphate buffer containing NaCl, after which 0.2 ml of a substrate solution consisting of a mixture of 5% pyrogallol solution in water and hydrogen peroxide solution (5% in O, 1 M phosphate buffer, pH 6) is added to the bead. , 0). The bead is incubated in the substrate at room temperature. Already after 15 minutes a clear yellow color of the ball is observed, which turns into orange for a further 45 minutes. Yellow and orange colorations are caused by pyrogallol formed by enevoltagallin, which occurs at the place of its formation (i.e., on the surface of the ball) in an undissolved form.
    Comparative experiences. Trypsin, ribonuclease and penicillin amidase were fixed on the following carriers:
    I- product G-Gel-70 (a copolymer based on glycidyl methacrylate and ethylene glycol dimethacrylate), obtained from a group of Czechoslovak researchers led by Professor Szczawk;
    II - bead polymer based on N, N - methylene bis- (methacrylamide), methacrylamide, methacrylic acid, glycidyl methacrylate and simple allyl glycidyl ether in a weight ratio
    39.1: 18.6: 3.3: 19.5: 19.5 (obtained according to Example 2 of the German application N 2722751); III - polymer latex of example 4.
    The experiments were carried out as follows.
    Experience A. Fixes trypsin.
    240 mg of trypsin (from cattle, activity 3.5 U / mg)
    dissolved in 4 ml of O, 1M sodium phosphate buffer with a temperature of 8 ° C and a pH of 8.6 and added to 1000 mg of these polymers I, II and III (Polymer III was used as a latex). The mixture is stirred, settled at 23 ° C for 72 hours, washed successively with 2 times 1 M sodium chloride and 2 times with 0.05 And phosphate buffer with pH 7.5, and then the enzymatic activity of the U / r of the wet product (μmol hydrolyzed substrate / min) by alkaline titration at 37 ° C and pH 7.5 (BAEE as substrate) or pH 8.0 (casein as substrate).
    The following results were obtained: 22.0 and 2.4 using polymer I, 194 and 11 using polymer II, and 276 and 28.5 using polymer III (in the form of latex).
    Experience B. Fixation of ribonuclease.
    50 mg of ribonuclease (5 times crisp., High-purity, from serum) are dissolved in 2.0 ml of 0.2 m sodium hydrogenphosphate at room temperature and 250 mg of the polymerization U / g of the wet product is added (in micromols of hydrolyzed substrate / min ) using the potassium salt of penicillin G as a substrate.
    The following results were obtained: 3.1 using polymer I, 21 / using polymer II and 234 using polymer III (in the form of latex).
    Experiment G. Experiment A is repeated with the difference that trypsin is acylated with acrylic acid chloride and then copolymerized with acrylamide under the conditions of Example 1 auth. St. No. 545648. The activity of the resulting copolymer of acylated trypsin with acrylamide is 212 and 15.8 U / g, respectively.
    Experiment D. Example 29 is repeated, with the difference that a bead coated with the product G-Gel-70 and a beaded polymer are used.
    In both cases, only a slightly yellowish color of the ball is observed.
    The results of comparative experiments indicate that the proposed method allows to increase the quality
    35
    40
    Levels 1, II and III (Polymer III is a fixation agent, and therefore contains a latex). The mixture is then stirred and set at 23 ° C for 72 hours. Next, the mixture is successively washed 5 times with 1 M sodium chloride and 2 times with 0.05 M phosphate buffer with pH /, 5, after which the enzymatic activity U / r of the wet product is determined in μmol hydrolyzed substrate / min) by alkaline titration at 37 ° C and pH 7.5 (ribonucleic acid as substrate).
    The following results were obtained: 9.4 using polymer I, 94 using polymer II and 105 using polymer-Ill (in the form of latex).
    Experience V. Fixation penitshshinovoy amidase.
    权利要求:
    Claims (1)
    [1]
    60 g of the enzyme (E. coli, 23.6 U / mg activity) is dissolved in 2.0 ml of O, 1M sodium phosphate buffer pH 8.0 and added to 500 mg of the indicated polymers I, II and III (polymer III is applied as a latex). The mixture is exchanged, settled at 23 ° C for 72 hours and washed 2 times with 0.05 M sodium phosphate buffer pH 7.5. The enzymatic active nucleophilic group is then determined to exhibit increased activity. Invention Formula
    The method of obtaining the immobilized enzyme by contacting the enzyme containing nucleophilic groups with a copolymer of acrylic esters and glycidyl methacrylate is different in that, in order to increase the specific activity of the immobilized enzyme, the acrylic copolymer based on monomers selected from the group consisting of 45 methyl methacrylate, butyl acrylate, methacrylamide, isobutyl methacrylate, ethyl methacrylate, hydroxyethyl methacrylate, Ta, ethyl acrylate, and monomers selected from the group with from ethylene glycol dimethacrylate, glycycylmoacrylate, methacrolein, N-hydroxysuccinimide metrylate, N-hydroxy succinimide ester of 6-methacryloyl aminohexanoic acid, acetic acid 2-acrylate acid, -hexadiisothiocyanate with a mass ratio of enzyme and copolymer of 1: 1-1: 330.
    50
    55
    thanks to what the
    The substance harboring nucleophilic groups exhibits increased activity. Invention Formula
    The method of obtaining the immobilized enzyme by contacting the enzyme containing nucleophilic groups with a copolymer of acrylic esters and glycidyl methacrylate is different in that, in order to increase the specific activity of the immobilized enzyme, the acrylic copolymer based on monomers selected from the group consisting of methyl methacrylate, butyl acrylate, methacrylamide, isobutyl methacrylate, ethyl methacrylate, hydroxyethyl methacrylate - Ta, ethyl acrylate, and monomers selected from the group that of ethylene glycol dimethacrylate, glycycylmoacrylate, methacrolein, N-oxysuccinimide methylate, N-hydroxy succinimide 6-methacryloyl-aminohexanoic acid methylate, a product of addition of 2-hydroxy-ethyl ester of acrylic acid -hexadiisothiocyanate with a mass ratio of enzyme and copolymer of 1: 1-1: 330.
    I j
     III corresponds to 1 µmol / min (at initial speed).
    Table
    The activity is determined each time in the third application.
    1655301
    Table 1
    1h
    1655101
    Table4
    Application Activity, Active output - U / r of the suspension of the immo-wet bilized product,%
    The first 42.7 4.06 The second 4., About 4.0 The third 41.6 3.96
    Table5
    Application Activity, Active yield - 11 / g of the weight of the immobidant product,%
    First 68,55,12 Second 61,84,62 Third 61,84,62 Fourth 61, 04,56
    Table
    Application Activity, Yield ak-U / r of the wetness of the product of the immobilized product,%
    First11891
    Second6685
    Third5588
    Fourth 5380
    At 3-7 ° C using the potassium salt of 2% penicillin.
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    同族专利:
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    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    DE3130924A|DE3130924C2|1981-08-05|1981-08-05|
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