WO2023186186A1 - Procédé de préparation d'une enzyme immobilisée ayant une forte stabilité - Google Patents

Procédé de préparation d'une enzyme immobilisée ayant une forte stabilité Download PDF

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WO2023186186A1
WO2023186186A1 PCT/CN2023/095548 CN2023095548W WO2023186186A1 WO 2023186186 A1 WO2023186186 A1 WO 2023186186A1 CN 2023095548 W CN2023095548 W CN 2023095548W WO 2023186186 A1 WO2023186186 A1 WO 2023186186A1
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linking agent
enzyme
lxte
cross
range cross
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PCT/CN2023/095548
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English (en)
Chinese (zh)
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饶经纬
王海勇
岳永力
沈艳阳
鲁飞
林立
于冰
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安庆朗坤药业有限公司
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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
    • 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
    • C12N11/091—Phenol resins; Amino resins
    • 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
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y206/00—Transferases transferring nitrogenous groups (2.6)
    • C12Y206/01—Transaminases (2.6.1)
    • 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
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals

Definitions

  • This application belongs to the technical field of enzyme engineering, and specifically relates to a method for preparing a highly stable immobilized enzyme.
  • immobilized enzymes As important catalysts, enzymes have been widely used in food, pharmaceutical, chemical and other fields. Compared with free enzymes, immobilized enzymes have better process adaptability, that is, better heat resistance, reagent tolerance, convenient solid-liquid phase separation, and recyclability and reuse. Therefore, immobilized enzymes have become indispensable green catalysts in the field of modern industrial catalysis.
  • Immobilized enzyme preparation methods generally include adsorption methods, covalent binding methods, cross-linking methods and embedding methods.
  • the adsorption method uses the van der Waals force between the carrier and the enzyme molecule to combine, which can easily cause the enzyme to fall off during use
  • the covalent binding method has the formation of a covalent bond between the carrier and the enzyme molecule, and the enzyme is not easy to use after multiple uses.
  • Shedding uses protein cross-linking agents, such as glutaraldehyde, to form covalent bonds between enzyme molecules, thereby converting the free "monomeric enzyme” into a "polymeric enzyme” with a three-dimensional structure, thereby increasing the enzyme's stability.
  • patent CN113308457 A discloses a method in which amino ligands react with aldehyde ligands under the protection of polyvinyl polypyrrolidone to form an organic skeleton and embed enzyme molecules in it. The operation is simple, but the preparation process involves the strong carcinogenic chemical diphenylamine. Participation is not conducive to production applications.
  • the immobilization adaptability of different enzymes and carriers is optimized to improve Stability of immobilized enzymes.
  • parameter optimization of the immobilization process such as selecting different resin types, enzyme and carrier ratios, etc.
  • further chemical modification of the immobilized enzyme can also improve its stability. Hadjer Zaak et al.
  • Chinese patent CN 111117996A discloses an immobilized enzyme, its preparation method and application.
  • This method uses polyethylene glycol to modify glutaraldehyde or aldehyde-based dextran, and finally forms a network structure dispersed with aldehyde groups and hydroxyl groups.
  • the enzyme is wrapped by covalent bonds, ionic bonds and other forces, thereby improving the mechanical stability of the enzyme.
  • this process requires optimizing the ratio of aldehydes to polyethylene glycol, adding active groups to the polyethylene glycol molecules, and then forming covalent bonds with the groups on the enzyme protein.
  • the improvement of mechanical stability depends on the polymerization of enzyme molecules. As well as the interaction between polyethylene glycol macromolecules and enzyme molecules, there is still much room for improvement in the stability of the enzyme molecule's own skeleton.
  • Epoxy groups are more likely to undergo addition reactions with amino groups in proteins. When the number of epoxy groups is greater than 1, covalent bonds are formed between protein molecules and polymerization proceeds. Therefore, epoxy cross-linking agents are used in wool or silk processing, such as the spinning and film-making process of monomer polymerization after dissolution (Zhang Yurong, Liu Jianyong, Wang Jie. Ethylene glycol diglycidyl ether cross-linked wool keratin [J ]. Materials Herald, 2013, 27: 230-232.); is also used to prepare biomedical materials, such as molecular cross-linking of hemoglobin to obtain functional oligomers of hemoglobin in order to replace red blood cells.
  • this application discloses a preparation method of high-stability immobilized enzymes, which improves the stability of the enzyme through the compound use of cross-linking agents.
  • recombinant bacteria such as recombinant Escherichia coli
  • buffer glycine-NaOH buffer
  • centrifuge to take the supernatant to obtain a crude solution with a concentration of 4.5 ⁇ 9U/ml. enzyme solution
  • the preparation method of the crude enzyme solution in this step is a conventional enzyme solution preparation method in this field, such as the method disclosed in the book "Wei Dongzhi "Enzyme Engineering” Higher Education Press, 2020”.
  • the adding amount of long-range cross-linking agent is 2-6% of the volume of boric acid buffer; the adding amount of short-range cross-linking agent is 2-6% of the volume of LXTE-700S@enzyme solution, and the added long-range cross-linking agent
  • the volumes of linking agent and short-range cross-linking agent cannot both be 6% of the volume of LXTE-700S@enzyme liquid.
  • the long-range cross-linking agent is an epoxy-based cross-linking agent, which is polyethylene glycol diglycidyl ether (215); the short-range cross-linking agent is ethylene glycol diglycidyl ether (669), glycerol triglycidyl ether At least one of ether (633) and 1,6-hexanediol diglycidyl ether (632).
  • the volume ratio of the added long-range cross-linking agent to the short-range cross-linking agent is 1:3 to 3:1, such as 1:1, 1:2, 1:3, 2:1 or 3:1.
  • This application uses the two epoxy groups of polyethylene glycol diglycidyl ether to covalently bind to the enzyme protein in a buffer system prepared by conventional immobilized enzymes, and encapsulates them through the synergistic action of long-range and short-range epoxy cross-linking agents. It also strengthens the enzyme molecular skeleton, and at the same time, the short-range cross-linking agent forms intramolecular covalent bonds, further improving the stability of the enzyme molecular skeleton, significantly increasing the storage and service life of the immobilized enzyme, and the preparation method is simple and easy to promote and apply.
  • Figure 1 is a diagram showing the storage stability comparison results of several immobilized enzymes in Examples.
  • Figure 2 is a graph showing the results of 20 experiments comparing the catalytic efficiency of transaminase immobilized enzymes prepared by treatment with epoxy cross-linking agents.
  • the raw materials and reagents used in the following examples are all commercially available, among which: the cross-linking agent polyethylene glycol diglycidyl ether (215) was purchased from Shanghai McLean Biochemical Technology Co., Ltd.; ethylene glycol diglycidyl ether (215) was purchased from Shanghai McLean Biochemical Technology Co., Ltd. 669), glycerol triglycidyl ether (633), and 1,6-hexanediol diglycidyl ether (632) were purchased from Guangzhou Yuanda New Materials Co., Ltd.;
  • ETDuet-1 E. coli was purchased from Jiutian Gene.
  • Fermentation medium 10g glucose, 15g yeast powder, 20g peptone, 10g NaCl, (NH 4 ) 2 SO 4 3g, K 2 HPO 4 ⁇ 3H 2 O 2.28g, KH 2 PO 4 1.36g, MgSO 4 2.0g, add water to make up to 1L.
  • Feed medium 350g glycerol, 50g yeast extract, 50g peptone, add water to make up 1L.
  • step (4) Take 2 ml of the culture obtained in step (4) and inoculate it into 200 ml of LB medium containing 100 ⁇ g/ml ampicillin, and culture it at 37°C and shaking at 250 rpm for 8 to 10 hours;
  • step (6) The culture obtained in step (5) is used as a seed and inoculated into 5L fermentation medium containing 100 ⁇ g/ml ampicillin.
  • the DO value is constant at 25%. Stir and DO are linked. Cultivate for 5 hours. After the pH value rises, the index is supplemented. When the fermentation is completed in 26 to 28 hours, the wet weight of the cells will be 200 to 250g/L.
  • the engineering bacteria involved in this example were constructed using conventional methods in the field, such as the methods disclosed in the reference book “Sam Brooke's “Molecular Cloning Experiment Guide,” Fourth Edition, Volume 1. Science Press, 2017.
  • step (2) Add the wet bacterial cells obtained in step (1) to 0°C pre-cooled pH 9.0 glycine-NaOH buffer at a mass-to-volume ratio of 1:10, and resuspend the bacterial cells to prepare a bacterial suspension;
  • step (2) the bacterial suspension is placed in an ultrasonic crusher to crush the bacterial cells to obtain a homogenate of the bacterial cells;
  • step (3) The bacterial homogenate in step (3) was centrifuged at 4°C and 9000g for 10 minutes, and the supernatant was collected to obtain a crude transaminase enzyme solution with a transaminase concentration of 4.5 U/ml (in specific embodiments, the enzyme solution was prepared according to a conventional method Preparation, the concentration is within the range of 4.5-9U/ml, and the purpose of application can be achieved.)
  • the above method for preparing crude aminotransferase enzyme solution is a conventional method in this field.
  • step (3) The precipitated Buchner funnel of step (2) is suction-filtered to obtain four types of immobilized aminotransferases using different resins as carriers for catalytic experiments;
  • the mobile phase elutes isocratic, detection wavelength: 205nm, flow rate: 1.0ml/min, column temperature: 30°C, the same below) and calculate the substrate conversion rate (product peak area/(product peak area + substrate peak area)*100%, the same below) after measurement.
  • the results are shown in Table 1.
  • the resulting immobilized enzyme was named: LXTE-700S@transaminase and was used for subsequent experiments.
  • step (3) Add 2ml, 4ml, 6ml, 8ml, 10ml and 12ml of cross-linking agents polyethylene glycol diglycidyl ether (215) and ethylene glycol diglycidyl (669) to the 24 systems obtained in step (2) respectively. , glycerol triglycidyl ether (633) and 1,6-hexanediol diglycidyl ether (632);
  • step (3) The system obtained in step (3) is incubated with shaking at 37°C for 2.5 hours, then filtered and washed with at least 50 ml of pure water to obtain the immobilized enzyme;
  • LXTE-700S@transaminase is treated with 2% to 6% cross-linking agent 215 or 2% to 6% cross-linking agent 633, its catalytic conversion rate is greater than 90%.
  • LXTE-700S@transaminase treated with 6% cross-linking agent 215 and 6% cross-linking agent 633 are named: LXTE-700S@transaminase@215 and LXTE-700S@transaminase@633 respectively.
  • LXTE-700S@transaminase, LXTE-700S@transaminase@215, LXTE-700S@transaminase@633 and LXTE-700S@transaminase@215-633 were placed at room temperature, 4°C and -20°C for 30 days respectively;

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

Procédé de préparation d'une enzyme immobilisée stable. Le procédé consiste à ajouter la résine aminée LXTE-700S dans une solution d'enzyme brute pour la filtrer, puis à ajouter une solution tampon d'acide borique et du phosphate de pyridoxal, et à effectuer une réaction d'oscillation; et enfin, à ajouter un agent de réticulation à longue portée et un agent de réticulation à courte portée, à effectuer une réaction d'oscillation, puis à effectuer une filtration sous vide pour obtenir l'enzyme immobilisée présentant une forte stabilité. L'agent de réticulation à base d'époxy est un mélange d'un agent de réticulation à longue portée de l'éther diglycidylique du polyéthylène glycol et d'un agent de réticulation à courte portée de l'éther triglycidylique du glycérol. Le procédé de préparation de l'enzyme immobilisée comporte des étapes simples et permet de prolonger sensiblement la durée de stockage et d'utilisation de l'enzyme immobilisée.
PCT/CN2023/095548 2022-03-28 2023-05-22 Procédé de préparation d'une enzyme immobilisée ayant une forte stabilité WO2023186186A1 (fr)

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WO2021128086A1 (fr) * 2019-12-25 2021-07-01 吉林凯莱英医药化学有限公司 Enzyme immobilisée, procédé de préparation et application associés
CN113817699A (zh) * 2021-09-28 2021-12-21 凯莱英医药集团（天津）股份有限公司 转氨酶突变体及其应用
CN114657170A (zh) * 2022-03-28 2022-06-24 安庆朗坤药业有限公司 一种高稳定性固定化酶的制备方法

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WO2021128086A1 (fr) * 2019-12-25 2021-07-01 吉林凯莱英医药化学有限公司 Enzyme immobilisée, procédé de préparation et application associés
CN113817699A (zh) * 2021-09-28 2021-12-21 凯莱英医药集团（天津）股份有限公司 转氨酶突变体及其应用
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