WO2023186186A1 - 一种高稳定性固定化酶的制备方法 - Google Patents

一种高稳定性固定化酶的制备方法 Download PDF

Info

Publication number
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
Authority
WO
WIPO (PCT)
Prior art keywords
linking agent
enzyme
lxte
cross
range cross
Prior art date
Application number
PCT/CN2023/095548
Other languages
English (en)
French (fr)
Inventor
饶经纬
王海勇
岳永力
沈艳阳
鲁飞
林立
于冰
Original Assignee
安庆朗坤药业有限公司
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 安庆朗坤药业有限公司 filed Critical 安庆朗坤药业有限公司
Publication of WO2023186186A1 publication Critical patent/WO2023186186A1/zh

Links

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
    • C12N11/08—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;

Landscapes

  • 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

一种高稳定性固定化酶的制备方法，在粗酶液中加入氨基树脂LXTE-700S过滤，然后加入硼酸缓冲液、磷酸吡哆醛，震荡反应；最后加入长程交联剂和短程交联剂，震荡反应后抽滤，即获得高稳定性固定化酶；该环氧基交联剂为长程交联剂聚乙二醇二缩水甘油醚及短程交联剂丙三醇三缩水甘油醚混合物，该固定化酶的制备方法操作简单，可显著延长其储存及使用寿命。

Description

一种高稳定性固定化酶的制备方法 技术领域
本申请属于酶工程技术领域，具体涉及一种高稳定性固定化酶的制备方法。
背景技术
酶作为重要的催化剂已经在食品、医药化工等领域广泛应用。相较于游离酶，固定化酶具有更好的工艺适应性，即更好的耐热性、试剂耐受性、可方便固液相分离及可回收重复利用等特性。因此，固定化酶已然成为现代工业催化领域中不可或缺的绿色催化剂。
固定化酶制备方法一般包括吸附法、共价结合法、交联法及包埋法。其中，吸附法利用载体与酶分子之间范德华力作用结合，使用过程中容易导致酶的脱落；共价结合法在载体与酶分子之间有共价键的形成，多次使用后酶也不易脱落；交联法则利用蛋白质交联剂，如戊二醛等在酶分子间形成共价键，进而将游离“单体酶”转变成具有三维结构的“多聚体酶”，进而增加酶的稳定性。而包埋法则利用物理包围方式，将酶分子包裹，使其活性中心不被破坏，同时具有相对坚固的机械物理防护。如专利CN113308457 A公开了一种在聚乙烯聚吡咯烷酮保护下，氨基配体与醛基配体反应形成有机骨架将酶分子包埋其中，该操作简单，但制备过程有强致癌化学物质二苯胺的参与，不利于生产应用。
以上现有固定化酶方法因利用原理、工艺各异及制备成本控制要求不同，在不同催化反应中均有规模化应用。但无论采用哪种制备方法，固定化酶的酶分子仍面临稳定性问题，常出现贮存失活，尤为严重的是在一些涉及高浓度有机试剂的重要的医药中间体合成过程随着使用批次增加底物转化率降低显著。
为解决酶失活问题，通常采用通过大量的基础实验，通过筛选不同的固定化方法以确定最优固定方法，如共价结合法中对不同的酶与载体进行固定化适配性优化以改善固定化酶的稳定性。如对选择不同的树脂种类、酶与载体配比等固定化过程进行参数优化。此外，对固定化酶做进一步的化学修饰也可以提高其稳定性。Hadjer Zaak等报道，利用聚乙烯亚胺和戊二醛进一步处理固定化脂酶，发现聚乙烯亚胺及戊二醛均可导致脂酶分子间形成共价键，进而在***将脂酶分子包裹住，防止酶分子脱落，显著增加了吸附法制备的固定化脂酶的稳定性(Zaak H,Fernandez-Lopez L,Otero C,et al.Improved stability of immobilized  lipases via modification with polyethylenimine and glutaraldehyde[J].Enzyme and Microbial Technology,2017,106:67-74.)。
生化研究表明，酶分子的空间结构对于酶的活性至关重要。通过定向进化技术对酶分子氨基酸种类进行改造是获得高稳定性酶的有效方法。比如，通过氨基酸的定向突变，改变酶的耐热性或其他抗性。其中，最为经典的是借助带有巯基的半胱氨酸成对的参入增加酶分子内部二硫键稳固酶的结构，进而提高其稳定性。但这种操作目前仍依赖于对蛋白质结构预测及耗时的突变体筛选过程，而且在引入多对二硫键以强化的过程中容易发生二硫键的错配，导致酶活性的大幅降低甚至丧失。中国专利CN 111117996A公开了一种固定化酶、其制备方法及应用，该方法采用聚乙二醇修饰戊二醛或醛基化右旋糖苷，最后形成分散有醛基、羟基的网状结构，通过共价键、离子键等作用力包裹酶，进而提升了酶的机械稳定性。但此过程需要优化醛与聚乙二醇的比例，在聚乙二醇分子上接入活性基团，再与酶蛋白上基团形成共价键，其机械稳定性提升依赖于酶分子间聚合及聚乙二醇大分子与酶分子互作，酶分子自身骨架稳定性仍有较大的提升空间。
环氧基与蛋白质中氨基较容易发生加成反应，当环氧基数量大于1时，蛋白分子间形成共价键，进而进行聚合。因此，环氧基交联剂在羊毛或蚕丝加工中应用，如溶解后单体聚合的纺丝制膜过程(张玉荣,刘建勇,王洁.乙二醇二缩水甘油醚交联羊毛角蛋白[J].材料导报,2013,27:230-232.)；也用来制备生物医学材料，如对血红蛋白进行分子交联，获得血红蛋白的功能性寡聚体，以期替代红细胞，同时，研究表明，环氧基化合物添加的浓度及反应体系的pH会影响蛋白质分子内还是分子间交联及其交联程度(徐宇红,路秀玲,郑春杨,等.乙二醇二缩水甘油醚交联血红蛋白的反应优化[J].高校化學工程學報,2006,20(3):401-406.)。目前，利用环氧基与酶蛋白共价结合固定酶的方法尚未见报道。
发明内容
针对传统固定化酶稳定性问题，本申请公开一种高稳定性固定化酶的制备方法，通过对交联剂的复配使用，提高酶的稳定性。
具体来说，以上目的是通过如下技术方案实现的：
一种高稳定性固定化酶的制备方法，其具体步骤如下：
(1)酶液制备
利用含有目标酶基因的重组菌(如重组大肠杆菌)100g～200g湿重/L溶于缓冲液(甘氨酸-NaOH缓冲液)经破碎、离心取上清，获得浓度为4.5～9U/ml的粗酶液；
本步骤粗酶液制备方法为本领域常规的酶液制备方法，如“魏东芝《酶工程》高等教育出版社，2020”书中公开的方法。
(2)按照质量体积比(g/ml)1：4向氨基树脂LXTE-700S中加入粗酶液，25℃，200rpm震荡混匀12h后，过滤获得LXTE-700S@酶，备用；
(3)按照质量体积百分比1:5-10(g/ml)向LXTE-700S@酶加入pH为8.0的硼酸缓冲液,再加入终浓度2mmol/L磷酸吡哆醛，震荡反应1h；最后加入长程交联剂和短程交联剂，37～55℃条件下震荡反应0.5～2.5h后抽滤，滤饼以纯水洗涤，即获得所述高稳定性固定化酶；
其中，长程交联剂的加入量为硼酸缓冲液体积的2-6％；短程交联剂的加入量为LXTE-700S@酶液酶液体积的的2-6％，且所加入的长程交联剂与短程交联剂的体积不能均为LXTE-700S@酶液体积的6％。
所述长程交联剂为环氧基交联剂为聚乙二醇二缩水甘油醚(215)；所述短程交联剂为乙二醇二缩水甘油醚(669)、丙三醇三缩水甘油醚(633)、1,6-己二醇二缩水甘油醚(632)中的至少一种。
优选的，所加入的长程交联剂与短程交联剂体积比为1:3～3:1，如1:1、1:2、1:3、2:1或3:1。
本申请在常规固定化酶制备的缓冲液体系中，利用聚乙二醇二缩水甘油醚的两个环氧基与酶蛋白共价结合，通过长程及短程环氧基交联剂协同作用下封装并加固酶分子骨架，同时短程交联剂形成分子内共价键，进一步提升酶分子骨架的稳定性，显著提升固定化酶储存及使用寿命，且制备方法简单易行，易于推广应用。
附图说明
图1为实施例几种固定化酶贮存稳定性比较结果图。
图2为环氧基交联剂处理制备的转氨酶固定化酶20次催化效率比较实验结果图。
具体实施方式
除非特别说明，以下实施例使用的原料和试剂均为市售，其中：交联剂聚乙二醇二缩水甘油醚(215)购自上海麦克林生化科技有限公司；乙二醇二缩水甘油(669)、丙三醇三缩水甘油醚(633)、1,6-己二醇二缩水甘油醚(632)均购自广州远达新材料有限公司；
ETDuet-1大肠杆菌购自九天基因。
实施例中涉及的培养基配方：
发酵培养基(g/L)：葡萄糖10g、酵母粉15g、蛋白胨20g、NaCl 10g、(NH4)2SO43g、K2HPO4·3H2O 2.28g、KH2PO41.36g、MgSO42.0g，加水补足至1L。
补料培养基(g/L)：甘油350g、酵母提取物50g、蛋白胨50g，加水补足1L。
实施例1转氨酶工程菌构建及发酵产酶
(1)全合成脱氧核糖核酸序列如SEQ ID NO.1所示的模板；
(2)将SEQ ID NO.1序列克隆到商业化的pETDuet-1载体的NcoI/XhoI之间，得到载体pETDuet-1-T；
(3)将pETDuet-1-T转化商业化的大肠杆菌BL21(λDE3)，得到转氨酶工程菌BL21-T；
(4)挑取BL21-T单菌落，接种于5ml含有100μg/ml氨苄青霉素的LB培养基中，于37℃，250rpm震荡培养8～12h；
(5)取步骤(4)所得培养物2ml接种于200ml含有100μg/ml氨苄青霉素的LB培养基中，于37℃，250rpm震荡培养8～10h；
(6)步骤(5)所得培养物作为种子，接种于5L含有100μg/ml氨苄青霉素的发酵培养基中，DO值恒定25％，搅拌与DO联动，培养至5h，pH值上升后，指数补料，至26～28h发酵结束，菌体湿重200～250g/L。
本实施例中所涉及的工程菌构建为本领域常规方法，如“萨姆布鲁克《分子克隆实验指南》，第四版，上册.科学出版社,2017”工具书中所公开的方法。
本实施例根据文献“Savile C K,Janey J M,Mundorff E C,et al.Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture[J].Science,2010,329(5989):305-309.”公开的氨基酸序列，经密码子优化得到SEQ ID NO.1所示的核苷酸序列。
实施例2制备转氨酶粗酶液
(1)4℃、8000g，5min离心收集实施例1所得菌体，称湿菌体质量；
(2)步骤(1)所得湿菌体，按质量体积比1:10加入0℃预冷的pH9.0甘氨酸-NaOH缓冲液，重悬菌体制备菌悬液；
(3)步骤(2)菌悬液置于超声波破碎仪中，破碎菌体，得菌体匀浆；
(4)步骤(3)菌体匀浆在4℃、9000g，10min离心，收集上清，得转氨酶粗酶液，转氨酶浓度为4.5U/ml(在具体实施例中，该酶液依据常规方法制备，浓度在4.5-9U/ml范围内，均可实现申请之目的。)
以上转氨酶粗酶液制备方法为本领域常规方法。
实施例3转氨酶固定化酶制备及西他列汀的转化
(1)树脂活化
称取环氧树脂LXTE-707(西安蓝晓科技新材料股份有限公司)、LXTE-600(西安蓝晓科技新材料股份有限公司)及ES-102(天津南开和成科技有限公司)和氨基树脂ESQ-1(天津南开和成科技有限公司)、LXTE-700S(西安蓝晓科技新材料股份有限公司)及LXTE-703(西安蓝晓科技新材料股份有限公司)各10g，其中环氧树脂用100ml蒸馏水室温下震荡浸泡12h后滤出，4℃保存备用；氨基树脂用100ml 0.1M pH8.5磷酸盐缓冲液室温下震荡浸泡2h后滤出，重新加入100ml 0.1M pH8.5磷酸盐缓及5ml 50％戊二醛室温震荡1h，滤出，4℃保存备用；
(2)在各活化树脂中，按照质量体积比(g/ml)1：4加入实施例2所制备的粗酶液，25℃，200rpm震荡混匀12h；
(3)步骤(2)的沉淀布氏漏斗抽滤，得4种不同树脂为载体的固定化转氨酶进行催化实验；
(4)催化方法：底物双酮(CAS:769195-26-8)浓度50g/L，体积百分比50％DMSO，2.5mmol/L磷酸吡哆醛，5mol/L异丙胺盐酸盐，与底物双酮质量比1:1.5加入固定化酶，50℃，搅拌15h，取反应液，HPLC检测(液相色谱条件：Agilent 1260 infinity II，氰基柱(C18 4.6*150mm 5μm)，流动相：乙腈：缓冲液＝15:85缓冲液：1.36g磷酸二氢钾全溶解于1000ml纯化水中，用磷酸调pH＝2.0。流动相等度洗脱，检测波长：205nm，流速： 1.0ml/min，柱温：30℃，下同)测定后计算底物转化率(产物峰面积/(产物峰面积+底物峰面积)*100％，下同)，结果见表1。
表1不同树脂固定化酶底物转化率
结果表明，氨基树脂LXTE-700S载体制备的固定化酶相对酶活最高，所得固定化酶命名为：LXTE-700S@转氨酶，用于后续实验。
实施例4不同交联剂处理LXTE-700S@转氨酶及西他列汀的转化
(1)按照实施例3方法制备LXTE-700S@转氨酶24份，每份10g，分别加入100ml 50mmol/L pH＝8.0的硼酸缓冲液；
(2)分别加入终浓度2mmol/L磷酸吡哆醛，200～300rpm震荡1h；
(3)步骤(2)所得24份体系中分别加入2ml、4ml、6ml、8ml、10ml及12ml的交联剂聚乙二醇二缩水甘油醚(215)、乙二醇二缩水甘油(669)、丙三醇三缩水甘油醚(633)及1,6-己二醇二缩水甘油醚(632)；
(4)步骤(3)所得体系置于37℃震荡温育2.5h后，抽滤并用至少50ml纯水洗后得固定化酶；
(5)催化方法：底物双酮(CAS:769195-26-8)浓度50g/L，体积百分比50％DMSO，5mol/L异丙胺盐酸盐，与底物双酮质量比1:1.5加入固定化酶，50℃，搅拌反应15h，取反应液过滤，HPLC测定并计算催化转化率。结果见表2。
表2不同交联剂处理LXTE-700S@转氨酶及西他列汀的转化率

结果表明，LXTE-700S@转氨酶经2％～6％的交联剂215处理或经2％～6％的交联剂633处理或经2％的交联剂632处理，其催化转化率均大于90％。其中，经6％交联剂215处理、经6％的交联剂633处理的LXTE-700S@转氨酶分别命名为：LXTE-700S@转氨酶@215和LXTE-700S@转氨酶@633。
实施例5交联剂215及633复合处理LXTE-700S@转氨酶
(1)按照实施例3方法制备LXTE-700S@转氨酶9份，每份5g，分别加入50ml 50mmol/L pH8.0硼酸缓冲液；
(2)分别加入终浓度2mmol/L磷酸吡哆醛，充分震荡1h；
(3)向步骤(2)所得9份体系中分别加入1ml、2ml及3ml的交联剂215和交联剂633的组合；
(4)催化方法：底物双酮(CAS:769195-26-8)浓度50g/L，体积百分比50％DMSO，5mol/L异丙胺盐酸盐，与底物双酮质量比1:1.5加入固定化酶，50℃，搅拌反应15h，取反应液过滤，HPLC测定并计算催化转化率。结果见表3(表2中横向为交联剂215的加入体积比)。
表3交联剂215及633复合处理LXTE-700S@转氨酶底物转化率
结果表明，除6％交联剂633与6％交联剂215的组合处理外，所得固定化酶的催化转化率均大于90％。其中，4％交联剂633与4％交联剂215处理后固定化酶命 名为：LXTE-700S@转氨酶@215-633。
实施例6固定化酶贮存稳定性比较实验
(1)LXTE-700S@转氨酶、LXTE-700S@转氨酶@215、LXTE-700S@转氨酶@633及LXTE-700S@转氨酶@215-633分别置于室温、4℃及-20℃条件下30天；
(2)催化方法：底物双酮(CAS:769195-26-8)浓度50g/L，体积百分比50％DMSO，5mol/L异丙胺盐酸盐，与底物双酮质量比1:1.5加入固定化酶，50℃，搅拌反应15h，取反应液经0.22um的有机系滤膜过滤，HPLC测定催化转化率。
不同温度处理后的催化结果见图1。结果表明，与常规固定化酶相比，经环氧基交联剂处理后的转氨酶固定化酶在室温、冷藏(4℃)及冷冻(-20℃)等三种储存环境下，其稳定性都有显著的提升。
实施例7固定化酶20批次催化效率比较实验
(1)称取LXTE-700S@转氨酶、LXTE-700S@转氨酶@215、LXTE-700S@转氨酶@633及LXTE-700S@转氨酶@215-633三种固定化酶各75g；
(2)催化方法：底物双酮(CAS:769195-26-8)浓度50g/L，体积百分比50％DMSO，5mol/L异丙胺盐酸盐，与底物双酮质量比1:1.5加入固定化酶，50℃，搅拌反应15h，取反应液过滤，HPLC测定并计算催化转化率；
(3)分别滤出三种固定化酶，至少750ml纯水洗后滤出备用；
(4)重复步骤(2)和(3)18次。
催化效率实验结果如图2所示。结果表明，LXTE-700S@转氨酶@215-633具有显著的优势，经过20批次的使用，在给定条件下，其催化转化率仍高于92％，在西他列汀酶法制备中，具有良好的应用前景。
以上所述仅为本申请技术方案的代表性实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (4)

  1. 一种高稳定性固定化酶的制备方法，其特征在于，具体步骤如下：
    1)酶液制备
    利用含有目标酶基因的重组菌溶于缓冲液中，经破碎、离心取上清，获得浓度为4.5～9U/ml的粗酶液，备用；
    2)向氨基树脂LXTE-700S中加入步骤1)获得的粗酶液，25℃，200rpm震荡混匀12h后，过滤，获得LXTE-700S@酶，备用；
    3)向LXTE-700S@酶中加入硼酸缓冲液,再加入终浓度为2mmol/L的磷酸吡哆醛，震荡反应1h；最后加入长程交联剂和短程交联剂，37～55℃条件下震荡反应0.5～2.5h后抽滤，滤饼即为所述高稳定性固定化酶；
    其中，长程交联剂的加入量为硼酸缓冲液体积的2-6％；短程交联剂的加入量为LXTE-700S@酶液酶液体积的的2-6％，且所加入的长程交联剂与短程交联剂的体积不能均为LXTE-700S@酶液体积的6％；
    所述长程交联剂为环氧基交联剂为聚乙二醇二缩水甘油醚；所述短程交联剂包括乙二醇二缩水甘油醚、丙三醇三缩水甘油醚、1,6-己二醇二缩水甘油醚中的至少一种。
  2. 根据权利要求1所述高稳定性固定化酶的制备方法，其特征在于，步骤2)中氨基树脂LXTE-700S与粗酶液的质量体积比为1：4，质量体积比单位为g/ml。
  3. 根据权利要求1所述高稳定性固定化酶的制备方法，其特征在于，步骤3)中LXTE-700S@酶与硼酸缓冲液质量体积比为1:5-10,质量体积比单位为g/ml。
  4. 根据权利要求1-3任一所述高稳定性固定化酶的制备方法，其特征在于，所加入的长程交联剂与短程交联剂体积比为1:3～3:1。
PCT/CN2023/095548 2022-03-28 2023-05-22 一种高稳定性固定化酶的制备方法 WO2023186186A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210330110.9 2022-03-28
CN202210330110.9A CN114657170B (zh) 2022-03-28 2022-03-28 一种高稳定性固定化酶的制备方法

Publications (1)

Publication Number Publication Date
WO2023186186A1 true WO2023186186A1 (zh) 2023-10-05

Family

ID=82033886

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/095548 WO2023186186A1 (zh) 2022-03-28 2023-05-22 一种高稳定性固定化酶的制备方法

Country Status (2)

Country Link
CN (1) CN114657170B (zh)
WO (1) WO2023186186A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114657170B (zh) * 2022-03-28 2023-04-07 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

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110964710A (zh) * 2019-12-25 2020-04-07 吉林凯莱英医药化学有限公司 固定化酶、其制备方法及其应用
WO2021128086A1 (zh) * 2019-12-25 2021-07-01 吉林凯莱英医药化学有限公司 固定化酶、其制备方法及其应用
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

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110241107A (zh) * 2019-06-11 2019-09-17 中国科学院南海海洋研究所 一种使用氨基树脂固定化脂肪酶的方法及由该方法制得的固定化脂肪酶

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110964710A (zh) * 2019-12-25 2020-04-07 吉林凯莱英医药化学有限公司 固定化酶、其制备方法及其应用
WO2021128086A1 (zh) * 2019-12-25 2021-07-01 吉林凯莱英医药化学有限公司 固定化酶、其制备方法及其应用
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

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MINGHUA QIAN, ZHANG JIFU, ZHANG YUN, SUN AIJUN, YU RONGMIN: "Immobilization of lipase on macroporous resin by adsorption-crosslinking method ", JOURNAL OF SOUTH CHINA AGRICULTURAL UNIVERSITY, CN, vol. 40, no. 2, 30 January 2019 (2019-01-30), CN , pages 103 - 110, XP093095373, ISSN: 1001-411X, DOI: 10.7671/j.issn.1001-411X.201804031 *

Also Published As

Publication number Publication date
CN114657170A (zh) 2022-06-24
CN114657170B (zh) 2023-04-07

Similar Documents

Publication Publication Date Title
CN108384767B (zh) 转氨酶突变体及其应用
US11535839B2 (en) Encoding genes of nitrilase mutants and application thereof
WO2023186186A1 (zh) 一种高稳定性固定化酶的制备方法
JP7105307B2 (ja) トランスアミナーゼ突然変異体及びその応用
CN106978368B (zh) 解鸟氨酸拉乌尔菌及其应用
US11987826B2 (en) Nitrilase mutant and application thereof in the synthesis of an anti-epileptic drug intermediate
IT8322155A1 (it) Procedimento per immobilizzare materie biologiche e composto di materie biologiche adsorbite in vermiculite
CN105624128B (zh) 一种固定化单胺氧化酶及其在合成手性氮杂双环化合物中的应用
EP3533862B1 (en) Methylopila sp. and use thereof in selective resolution and preparation of (s)-alpha-ethyl-2-oxo-1 -pyrrolidine acetate
Cabirol et al. Linum usitatissimum hydroxynitrile lyase cross‐linked enzyme aggregates: a recyclable enantioselective catalyst
WO2019210606A1 (zh) 一种酸性磷酸酶突变体、其应用及其制备烟酰胺核糖的方法
WO2023004969A1 (zh) 酯酶突变体及其应用
CN113817699B (zh) 转氨酶突变体及其应用
CN109593739B (zh) 重组酮酸还原酶突变体、基因、工程菌及其应用
CN117778371B (zh) 苯丙酮酸脱羧酶和醇脱氢酶的共固定化酶、制备及应用
CN112143718B (zh) 一种双功能酶生物催化剂及其制备方法和应用
US20220204953A1 (en) Mutant of Acid Phosphatase and Application Thereof
CN107118977B (zh) 粘质沙雷氏菌及其应用
CN116574704A (zh) 醛酮还原酶-葡萄糖脱氢酶共固定化酶及在他汀类药物手性中间体连续流合成中的应用
CN116814574A (zh) 一种亚胺还原酶突变体、重组基因工程菌及其在(r)-2-甲基吡咯烷合成中的应用
JP2008061501A (ja) 遊離カルボン酸・アミンの直接縮合反応を触媒する酵素の設計方法及びナイロンオリゴマーの酵素的製造方法
CN117025582A (zh) 一种固定醛缩酶的方法
CN117867050A (zh) 一种体外多酶级联催化合成丁内酰胺的方法
CN104694543A (zh) 特异性识别β-葡萄糖醛酸苷酶的核酸适配体序列及其应用
CN116987650A (zh) 一种产四氢嘧啶的嗜甲烷工程菌及其构建方法和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23778548

Country of ref document: EP

Kind code of ref document: A1