WO2023133957A1 - 固定化酶及其在连续化生产中的应用 - Google Patents

固定化酶及其在连续化生产中的应用 Download PDF

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WO2023133957A1
WO2023133957A1 PCT/CN2022/076232 CN2022076232W WO2023133957A1 WO 2023133957 A1 WO2023133957 A1 WO 2023133957A1 CN 2022076232 W CN2022076232 W CN 2022076232W WO 2023133957 A1 WO2023133957 A1 WO 2023133957A1
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carrier
enzyme
immobilized
dehydrogenase
immobilized enzyme
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PCT/CN2022/076232
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French (fr)
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洪浩
詹姆斯•盖吉
肖毅
张娜
潘龙
马利腾
崔瑜霞
高妍妍
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凯莱英生命科学技术(天津)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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
    • C12Y104/99—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with other acceptors (1.4.99)
    • C12Y104/99003—Amine dehydrogenase (1.4.99.3)

Definitions

  • the invention relates to the field of enzyme immobilization, in particular to an immobilized enzyme and its application in continuous production.
  • Chiral amines are important intermediates in the synthesis of chiral drugs, pesticides and fine chemicals.
  • Amine dehydrogenase refers to a class of enzymes that can catalyze the reduction of ketones to chiral amines.
  • amine dehydrogenases use cheap ammonium salts as ammonia sources and do not rely on expensive ammonia donors such as isopropylamine, alanine, etc.
  • Amine dehydrogenase is often associated with formate dehydrogenase (FDH) because the reduction process usually requires hydrogen delivery from reduced nicotinamide adenine dinucleotide phosphate (NADPH) or reduced nicotinamide adenine dinucleotide (NADH) combined to reduce production costs.
  • FDH formate dehydrogenase
  • NADPH reduced nicotinamide adenine dinucleotide phosphate
  • NADH nicotinamide adenine dinucleotide
  • water-soluble free enzymes face many problems in the production and application process, such as poor stability, inability to reuse, and problems of product separation and purification.
  • the main task of enzyme immobilization is to choose an appropriate immobilization method, so as to design a biological enzyme that meets both the catalytic requirements (selectivity, stability) and non-catalytic requirements (cost, separation process) of an application. catalyst.
  • the immobilization of enzymes by covalent bonding is widely used in production and application, and the commercial carriers used include amino-type carriers and epoxy-type carriers. Taking amino-type resin as an example, it usually needs to be activated by a bifunctional cross-linking agent and then bind to the enzyme through the terminal functional group of the cross-linking agent.
  • the most commonly used activation method is to use glutaraldehyde to activate the amino group, and then use the active aldehyde group to link the enzyme molecule to achieve immobilization. It has also been reported that after immobilization of enzymes with glutaraldehyde, post-modification with small molecule compounds such as amino acids or NaBH 4 can increase the stability of the immobilized enzymes.
  • the inventors found that the traditional glutaraldehyde-activated amino carrier or epoxy-type carrier cannot complete the immobilization of amine dehydrogenase well.
  • the obtained immobilized enzyme has the disadvantages of low catalytic activity and poor reusability. Adding protective agents such as mannitol or post-modifying the immobilized enzyme with small molecules during the immobilization process cannot overcome the above defects.
  • the main purpose of the present invention is to provide an immobilized enzyme and its application in continuous production, so as to improve the catalytic activity and reusability of the immobilized enzyme.
  • an immobilized enzyme is provided, the immobilized enzyme is an immobilized enzyme modified with polyethyleneimine, including: enzymes, including amine dehydrogenase and/or formic acid Dehydrogenase; and a carrier for immobilizing the enzyme, the carrier is an amino type carrier activated by cyanuric chloride.
  • amine dehydrogenase includes amine dehydrogenase shown in SEQ ID NO: 1 sequence;
  • formate dehydrogenase includes formate dehydrogenase shown in SEQ ID NO: 2 sequence;
  • amino Type carrier includes ECR8409, LX-1000HA or LX-1000EPHA; polyethyleneimine
  • polyethyleneimine Preferably, the molecular weight of polyethyleneimine is 1.8-10KDa.
  • a method for preparing an immobilized enzyme comprising: using cyanuric chloride to activate the amino-type carrier to obtain an activated carrier; mixing the enzyme with the activated carrier Incubate for immobilization, and then modify the immobilized product with polyethyleneimine to obtain immobilized enzymes; the above enzymes include amine dehydrogenase and/or formate dehydrogenase.
  • using cyanuric chloride to activate the amino-type carrier to obtain the activated carrier includes: dispersing the amino-type carrier in an organic solvent to obtain a carrier suspension; reacting the cyanuric chloride with the carrier suspension to activate the amino-type carrier , to obtain an activated carrier; preferably, the organic solvent is a polar organic solvent; preferably, the polar organic solvent includes any one or more of the following: tetrahydrofuran, acetone, dimethylformamide or dimethylsulfoxide; preferably , the activation temperature is 0-30°C, more preferably 0-5°C; preferably, the activation time is 2-5h; preferably, after activation, the activation carrier is washed with an organic solvent to remove unreacted cyanuric chloride; Preferably, after washing, blowing dry the organic solvent on the activated carrier with nitrogen gas; preferably, the amino-type carrier is a dry carrier; preferably, the amino-type carrier includes ECR8409, LX-1000HA or LX-1000
  • the preparation method also includes washing the activated carrier with a buffer to replace the organic solvent; preferably, the enzyme is prepared with a buffer; preferably, the buffer includes phosphate buffer, saline, Glycine buffer or borax buffer; preferably, the concentration of phosphate buffer is 20-50mM; preferably, the pH of phosphate buffer is 6.5-9.0; preferably, the immobilization temperature is 0-30°C, preferably Preferably it is 4-30°C; preferably, the immobilization time is 2-5 hours.
  • the buffer includes phosphate buffer, saline, Glycine buffer or borax buffer; preferably, the concentration of phosphate buffer is 20-50mM; preferably, the pH of phosphate buffer is 6.5-9.0; preferably, the immobilization temperature is 0-30°C, preferably Preferably it is 4-30°C; preferably, the immobilization time is 2-5 hours.
  • mixing and incubating the enzyme with an activated carrier for immobilization, and then modifying the immobilized product with polyethyleneimine to obtain an immobilized enzyme includes: mixing and incubating the enzyme with an activated carrier for immobilization to obtain an initial immobilized enzyme ; Use polyethyleneimine to post-modify the initial immobilized enzyme to obtain an immobilized enzyme; preferably, the enzyme includes amine dehydrogenase and/or formate dehydrogenase; preferably, the amine dehydrogenase includes such as SEQ ID NO: 1
  • the continuous reaction includes carrying out the continuous reaction in a packed bed reactor; preferably, the packed bed reactor is made by filling the immobilized enzyme in an empty chromatographic column or a stainless steel chromatographic column; preferably, the packed bed reaction
  • the volume of the container is 5-50mL; preferably, the filling method of the packed bed reactor is wet filling.
  • the continuous reaction includes: pumping the reaction system containing the substrate into the packed bed reactor from bottom to top, and remixing the reaction product into the reaction system; the pump is a constant flow pump, preferably, the constant flow pump Pressure ⁇ 20MPa; preferably, the flow rate of the continuous reaction is 0.01-10mL/min.
  • the enzyme is a main enzyme, or a mixed enzyme of a main enzyme and a coenzyme; preferably, the main enzyme is selected from amine dehydrogenase, and correspondingly, the coenzyme is selected from formate dehydrogenase; preferably, the immobilized enzyme includes immobilized A co-immobilization system of amine dehydrogenase, or amine dehydrogenase and formate dehydrogenase; preferably, amine dehydrogenase comprises the amine dehydrogenase shown in SEQ ID NO: 1 sequence; Preferably, formate dehydrogenase Including formate dehydrogenase as shown in SEQ ID NO: 2 sequence; Preferably, the reaction system includes a substrate or a mixture of a substrate and a coenzyme; Preferably, the immobilized enzyme is an immobilized amine dehydrogenase, and the reaction system includes Mixture of substrate and formate dehydrogenase.
  • Fig. 1 shows a schematic diagram of a continuous reaction device according to Example 5 of the present invention.
  • the traditional glutaraldehyde-activated amino carrier or epoxy carrier cannot complete the immobilization of amine dehydrogenase well.
  • the obtained immobilized enzyme has the disadvantages of low catalytic activity and poor reusability.
  • adding protective agents such as mannitol or post-modifying the immobilized enzyme with small molecules during the immobilization process cannot overcome the above defects.
  • the inventors conducted in-depth research on immobilized enzymes, and tried many alternatives to glutaraldehyde-activated amino carriers, and found that only the alternatives activated by cyanuric chloride had excellent effects. That is, a kind of amino-type carrier activated by cyanuric chloride carrier activator was discovered, and an immobilized amine dehydrogenase with high catalytic activity and good operational stability was prepared by using this carrier, which is different from the immobilized amine dehydrogenase prepared by traditional immobilization method. Compared with immobilized amine dehydrogenase, it has higher catalytic activity and reusability.
  • an immobilized enzyme is provided, the immobilized enzyme is an immobilized enzyme modified with polyethyleneimine, including: an enzyme, the enzyme includes amine dehydrogenase and/or formate dehydrogenase ; and a carrier for immobilizing the enzyme, the carrier is an amino-type carrier activated by cyanuric chloride.
  • cyanuric chloride can activate the amino group on the amino carrier, providing a different resin structure from the traditional glutaraldehyde-activated amino resin, for those who cannot be effectively immobilized on the glutaraldehyde-activated amino resin due to structural and other factors
  • the enzyme provides a new carrier. After the enzyme is combined with the activated carrier to form an immobilized enzyme, in order to further improve the operational stability of the immobilized enzyme, the immobilized enzyme is modified by polyethyleneimine, and the polymer is used to "bind" the enzyme or protect the polymer Achieve more stable immobilization. Thereby obtaining the immobilized enzyme with improved catalytic activity and reusability.
  • the enzymes in the above-mentioned immobilized enzymes are mainly aimed at a class of enzymes that are difficult to be immobilized by glutaraldehyde-activated amino carriers. Specifically, it includes amine dehydrogenase, formate dehydrogenase or other enzymes and combinations of the above-mentioned enzymes.
  • the activation principle of the above-mentioned cyanuric chloride-activated amino-type carrier is: use cyanuric chloride to have three halogen active sites, first use one of the active sites to bind to the amino group during activation, and then the remaining active sites can be combined with the enzyme Amino, hydroxyl and other functional groups on the molecule react to complete the immobilization.
  • amine dehydrogenases include but are not limited to amine dehydrogenases AmDHs, and AmDHs in this application refers specifically to amine dehydrogenases whose amino acid sequence is SEQ ID NO: 1, rather than a variety of amine dehydrogenases; preferably , formate dehydrogenase includes but not limited to formate dehydrogenase AIY34662.1.
  • specific activated amino-type carriers include, but are not limited to, LX-1000EPHA, ECR8409 or LX-1000HA.
  • the molecular weight of polyethyleneimine is 1.8-10KDa.
  • amino acid sequence SEQ ID NO: 1 of the above-mentioned amine dehydrogenase AmDHs is as follows:
  • the above-mentioned formate dehydrogenase is AIY34662.1 wild type.
  • the sequence of the amino acid sequence SEQ ID NO: 2 is as follows:
  • Amine dehydrogenase refers to a class of enzymes that can catalyze the reduction of ketones to chiral amines. It uses cheap ammonium salts as ammonia sources and does not depend on expensive ammonia donors. Because the reduction process usually requires hydrogen delivery from reduced nicotinamide adenine dinucleotide phosphate (NADPH) or reduced nicotinamide adenine dinucleotide (NADH), amine dehydrogenase is often used in combination with formate dehydrogenase to reduce manufacturing cost. Compared with chemical catalytic synthesis, amine dehydrogenase not only has mild reaction conditions, but also has higher catalytic activity, selectivity and substrate specificity.
  • free enzymes especially water-soluble free enzymes
  • the immobilized enzyme can be one kind, or multiple enzymes can be immobilized at the same time, so as to achieve the purpose of joint use.
  • the traditional preparation method of immobilized enzyme carrier usually needs to be activated by a bifunctional cross-linking agent and then combined with the enzyme through the terminal functional group of the cross-linking agent.
  • the most commonly used activation method is to use glutaraldehyde to activate the amino group, and then use the active aldehyde group to link the enzyme molecule to achieve immobilization. It has also been reported that after immobilization of enzymes with glutaraldehyde, post-modification with small molecule compounds such as amino acids or NaBH 4 can increase the stability of the immobilized enzymes.
  • Cyanuric chloride has an activation effect on amino resins, and is suitable for amino resin carriers, providing a resin structure different from that of traditional glutaraldehyde activation. After cyanuric chloride is used to activate the amino resin, the remaining active halogen (chlorine) sites on the cyanuric chloride group can react with enzyme molecules, and different resin structures have relatively different binding abilities to different enzymes.
  • the use of support activators can help immobilize enzymes that are difficult to bind to traditional immobilized enzyme supports.
  • the amino carrier may include various commercially available amino resins such as ECR8409 resin produced by Purolite Company and LX-1000HA resin produced by Xi'an Lanxiao Technology Company.
  • Polyethyleneimine can be configured as an aqueous solution of appropriate concentration for use, and the final concentration during use can be adjusted according to the nature and content of the immobilized enzyme, and can be 0.5-3% W/V. According to the properties of different immobilized enzymes, polyethyleneimine with different molecular weights can be used for modification and protection, and the molecular weight of PEI can be 1.8-10KDa.
  • a method for preparing an immobilized enzyme includes: using cyanuric chloride to activate an amino-type carrier to obtain an activated carrier; mixing and incubating the enzyme with the activated carrier for immobilization and then modify the immobilized product with polyethyleneimine to obtain immobilized enzyme; the enzyme includes amine dehydrogenase and/or formate dehydrogenase.
  • the activated amino-type carrier prepared by using cyanuric chloride has a different structure and binding site from the traditional activated carrier, which provides a new alternative carrier for the preparation of immobilized enzymes, and helps the enzymes that are difficult to combine with traditional immobilized enzyme carriers to carry out Immobilized.
  • the immobilized enzyme can be obtained by immobilizing the enzyme and the activated carrier at a suitable temperature and rotation speed.
  • the above preparation method uses cyanuric chloride to activate the amino-type carrier, and obtaining the activated carrier includes: dissolving the amino-type carrier in an organic solvent to obtain a carrier suspension; Activation to obtain an activated carrier; preferably, the organic solvent can be a polar organic solvent; preferably, the polar organic solvent includes any one or more of the following: tetrahydrofuran, acetone, dimethylformamide or dimethyl sulfoxide;
  • the activation temperature is 0-30°C, more preferably 0-5°C; preferably, the activation time is 2-5h; preferably, after activation, the activated carrier is washed with an organic solvent to remove unreacted trimeric Cyanogen chloride; preferably, after washing, it further includes drying the activated carrier with nitrogen gas, that is, drying the organic solvent on the carrier; preferably, the amino-type carrier is a dry carrier; preferably, the amino-type carrier includes but is not limited to ECR8409, LX-1000HA or LX-1000
  • the activation temperature It can be 0-30°C, more preferably 0-5°C, and the activation time is 2-5 hours. Low-temperature activation can protect the activity of the carrier, and can also slow down the activation rate to prevent the activation rate from being too fast. A large amount of heat will affect the carrier. activation effect.
  • the activation time can be properly adjusted according to conditions such as enzyme, carrier or activation temperature to produce a better activation effect.
  • the amino-type carrier before activation can be a dry carrier to reduce the composition of the reaction organic system by reducing the residual water and other liquids in the carrier. The remaining liquid will contact or even wrap the amino groups on the carrier, which will also affect the activation efficiency.
  • use the same organic solvent to wash the activated carrier to remove unreacted cyanuric chloride. Further, nitrogen can also be used to dry the organic solvent on the activated carrier to avoid residual cyanuric chloride and organic solvents.
  • the enzyme activity will be affected, and the production yield and performance of the immobilized enzyme will be adversely affected.
  • the preparation method before mixing and incubating the enzyme with the activated carrier, that is, before immobilization, also includes washing the activated carrier with a buffer to replace the organic solvent; preferably, the enzyme is prepared with a buffer; preferably , the buffer includes but not limited to phosphate buffer, glycine buffer or borax buffer; preferably, the concentration of phosphate buffer is 20-50mM; preferably, the pH of phosphate buffer is 6.5-9.0; preferably , the immobilization temperature is 4-30° C.; preferably, the immobilization time is 2-5 hours.
  • the buffer includes but not limited to phosphate buffer, glycine buffer or borax buffer; preferably, the concentration of phosphate buffer is 20-50mM; preferably, the pH of phosphate buffer is 6.5-9.0; preferably , the immobilization temperature is 4-30° C.; preferably, the immobilization time is 2-5 hours.
  • Washing the activated carrier with a buffer is also to replace the residual organic solvent in the activated carrier and prevent the residual organic solvent from affecting the enzyme activity.
  • the enzyme is prepared with a buffer, which can be commonly used phosphate buffer, glycine buffer or other common buffers, which can keep the pH of the solution relatively stable when adding a small amount of acid or alkali, and prevent the impact of products such as carbon dioxide produced by the reaction Solution pH, thereby maintaining enzyme activity.
  • the immobilization time can be properly adjusted according to conditions such as enzyme, carrier or immobilization temperature to produce a better immobilization effect.
  • mixing and incubating the enzyme with an activated carrier for immobilization, and then modifying the immobilized product with polyethyleneimine to obtain the immobilized enzyme includes: mixing and incubating the enzyme with an activated carrier for immobilization, obtaining Initially immobilized enzyme; using polyethyleneimine to post-modify the initially immobilized enzyme to obtain immobilized enzyme; preferably, the enzyme includes one or more of amine dehydrogenase, formate dehydrogenase or other enzymes; preferably, Amine dehydrogenases include amine dehydrogenase AmDHs shown in SEQ ID NO: 1 sequence; preferably, formate dehydrogenases include formate dehydrogenase AIY34662.1 shown in SEQ ID NO: 2 sequence; preferably, A solution of polyethyleneimine is added to the initial immobilized enzyme for post-modification; preferably, the molecular weight of polyethyleneimine is 1.8-10KDa; preferably, the pH of polyethyleneimine is 5-8; preferably
  • the enzyme and the activated carrier are mixed and incubated for immobilization, and the initially immobilized enzyme can be obtained.
  • the enzymes used can be selected from one or more kinds, and different kinds of enzymes can be mixed in any ratio to prepare a co-immobilized primary system of multiple enzymes.
  • polyethyleneimine can be used to post-modify the initially immobilized enzyme, and the operational stability of the immobilized enzyme can be further improved through the "tethering" or protection of the enzyme by the polymer.
  • Adding a certain volume of pre-dissolved and configured polyethyleneimine solution to the primary immobilized enzyme can efficiently mix and effectively control the final concentration of polyethyleneimine, and is also conducive to the uniform contact between the primary immobilized enzyme and polyethyleneimine, which can Improve the performance of the prepared immobilized enzyme.
  • Polyethyleneimine the molecular weight can be adjusted according to different carriers and enzymes to obtain better protection performance.
  • Polyethyleneimine can be formulated as a 10% w/v aqueous solution and adjusted with 1M hydrochloric acid solution. An appropriate amount of polyethyleneimine aqueous solution is added to the initial immobilized enzyme to make the final concentration 0.5-3% W/V.
  • an application of the above-mentioned immobilized enzyme or the immobilized enzyme prepared by the above-mentioned preparation method in continuous reaction is provided.
  • the above-mentioned immobilized enzymes have greatly improved catalytic activity and reusability compared with free enzymes or immobilized enzymes prepared by traditional processes, and can be used in continuous equipment for continuous reactions. Compared with batch reactions , easy to operate and high production efficiency.
  • the continuous reaction includes carrying out the continuous reaction in the packed bed reactor; preferably, the packed bed reactor is filled with the immobilized enzyme in the chromatographic empty column or stainless steel Made; Preferably, the volume of the packed bed reactor is 5-50mL; Preferably, the packing method of the packed bed reactor is wet packing.
  • Immobilized enzymes can be continuously reacted in pressure-resistant and corrosion-resistant packed bed reactors such as chromatographic empty columns and stainless steel chromatographic columns.
  • the pressure of the immobilized enzyme at the bottom of the reactor is too high, which affects the mechanical structure and enzyme activity of the immobilized enzyme.
  • the volume of the packed bed reactor can be 5-50mL.
  • the wet filling method can be selected to fill the immobilized enzyme into the filled reactor.
  • the continuous reaction includes: pumping the reaction system containing the substrate into the packed bed reactor from bottom to top, and re-mixing the reaction product into the reaction system; the pump is a constant flow pump; preferably, the constant flow
  • the pressure of the pump is ⁇ 20MPa; preferably, the flow rate of the continuous reaction is 0.01-10mL/min.
  • the reaction system containing the substrate is pumped into the packed bed reactor from bottom to top to prevent the reaction system from flowing too fast in the reactor due to gravity, resulting in low conversion yield.
  • the reaction product is remixed into the reaction system, and the reaction with a single catalytic yield is not high, which can be catalyzed in a cycle, and the uniformity of the reaction system is always maintained, which is conducive to the relative stability of the environment of the immobilized enzyme in the packed bed reactor; It is also beneficial to the detection of the reaction system, it is convenient to supplement raw materials such as ammonium formate, and it is also convenient to collect the final product, omitting the process of separating the final product from the immobilized enzyme required for batch reactions.
  • the pump used in the continuous reaction can be reasonably selected as a medium-pressure constant-flow pump or a low-pressure constant-flow pump according to the requirements of the packed bed reactor and flow rate, and the pressure tolerance of the immobilized enzyme. 20MPa.
  • the enzyme can be the main enzyme, or a mixed enzyme of the main enzyme and coenzyme; preferably, the main enzyme can be selected from amine dehydrogenase, and correspondingly, the coenzyme can be selected from formate dehydrogenase;
  • the immobilized enzyme includes Immobilized amine dehydrogenase, or the co-immobilization system of amine dehydrogenase and formate dehydrogenase;
  • amine dehydrogenase comprises the amine dehydrogenase AmDHs shown in SEQ ID NO:1 sequence;
  • formic acid Dehydrogenases include formate dehydrogenase AIY34662.1 as shown in the sequence of SEQ ID NO: 2;
  • the reaction system includes a substrate or a mixture of a substrate and a coenzyme;
  • the immobilized enzyme is an immobilized amine dehydrogenase Hydrogenase, the reaction system includes a mixture of substrate and formate de
  • the enzyme can be the main enzyme alone, or it can be a mixed enzyme of the main enzyme and the coenzyme, so that the product generated by the coenzyme can be used for the catalytic reaction of the main enzyme.
  • the amine dehydrogenase reduction process usually requires NADPH or NADH to transfer hydrogen, the use of amine dehydrogenase is often combined with formate dehydrogenase that can produce NADH to reduce production costs.
  • the immobilized enzyme can be a separate immobilized amine dehydrogenase, in which case additional formate dehydrogenase needs to be added to the reaction system;
  • the system can be a simple substrate solution.
  • Embodiment 1 ECR8409 immobilized amine dehydrogenase
  • Substrate preparation use 2-(1-adamantyl)-2-oxyacetic acid methyl ester shown in Reaction Formula 1 as the substrate, weigh 10g of the substrate in a 100mL beaker, add 50mL of purified water and 1.6g of K 2 HPO 4 ⁇ H 2 O, after stirring evenly, adjust the pH to 8.0 with 10M NaOH solution; weigh 1g of activated carbon and add it to the prepared solution, stir for 1h to absorb impurities and pigments; put the Buchner funnel on filter paper and add 2.5g of silicon alginate, add purified water and drain to obtain a filter cake.
  • the prepared substrate solution was suction filtered through the filter cake, and 20 mL of purified water was added to wash the filter cake to obtain a clear filtrate; 5.6 g of ammonium formate and 15 mg of dithiothreitol (DTT) were weighed and added to the filtrate, and used 10M NaOH solution to adjust the pH to 8.0. Add purified water to 82mL.
  • Carrier activation Take 10 grams of dry ECR8409 resin in a four-necked flask, add 250 mL of acetone under ice-bath conditions and stir for 0.5 h. Subsequently, 5 g of cyanuric chloride was added, and the stirring reaction was continued for 3 h under an ice bath. After the reaction was completed, the modified carrier was filtered and washed three times with acetone before use.
  • Enzyme immobilization After washing 0.5 g of the activated carrier three times with 0.1M pH 8.0 phosphate buffer, immediately add 4 mL of the prepared enzyme solution, and immobilize it in a shaker at 20°C and 100 rpm for 2 hours. Subsequently, 1 mL of 10% PEI (10 KDa) solution prepared in advance with a pH of 8.0 was added to continue the reaction overnight. Finally, the immobilized enzyme was filtered and washed three times with buffer to obtain the immobilized amine dehydrogenase.
  • Reaction conditions Weigh 0.15 g of immobilized amine dehydrogenase, add 0.82 mL of prepared substrate solution, 25 ⁇ L of 1 mg/mL NAD + solution and 50 ⁇ L of FDH. React at 40°C for 18 hours in a shaker at 100 rpm. The supernatant of the reaction system was taken, and the conversion rate of the reaction was determined by HPLC.
  • the carrier activation steps and preparation of the enzyme solution were the same as in Example 1, except that 50 ⁇ L of formate dehydrogenase (FDH) AIY34662.1 crude enzyme solution was added during the enzyme immobilization process. Then use the same immobilization method and post-modification process to complete the immobilization.
  • FDH formate dehydrogenase
  • Carrier activation Take 10 grams of dry LX-1000HA resin in a four-necked flask, add 200 mL of tetrahydrofuran under ice-cooling conditions and stir for 0.5 h. Subsequently, 2.5 g of cyanuric chloride was added, and the stirring reaction was continued for 3 h under an ice bath. After the reaction was completed, the modified carrier was filtered and washed three times with tetrahydrofuran before use.
  • Embodiment 4 LX-1000EPHA immobilized amine dehydrogenase
  • Carrier activation take 10 grams of dry LX-1000EPHA resin in a four-necked flask, add 200 mL of tetrahydrofuran under ice-cooling conditions and stir for 0.5 h. Subsequently, 2.5 g of cyanuric chloride was added, and the stirring reaction was continued for 3 h under an ice bath. After the reaction was completed, the modified carrier was filtered and washed three times with tetrahydrofuran before use.
  • Embodiment 5 Application of immobilized amine dehydrogenase in continuous production
  • system is connected in the continuous equipment. After 24 hours of reaction, the conversion rate of the substrate reached 94%, and the conversion rate still reached 84% after repeated use ten times.
  • Comparative example 1 LX-1000HA glutaraldehyde activates immobilized amine dehydrogenase
  • LX-1000HA glutaraldehyde activation take 1g of wet weight LX-1000HA and add 4mL 2% (w/v) glutaraldehyde solution, the glutaraldehyde solution is prepared with 20mM phosphate buffer with pH 7.0. Activated in a shaker at 30°C for 1h.
  • Comparative example 2 LX-1000HA hexamethylene diisocyanate activates immobilized amine dehydrogenase
  • LX-1000HA resin hexamethylene diisocyanate take 10 grams of dry LX-1000HA resin in a four-necked flask, add 200 mL of dimethylformamide under ice-bath conditions and stir for 0.5 h. Subsequently, 2.3 g of hexamethylene diisocyanate was added, and the stirring reaction was continued for 3 h under ice-cooling. After the reaction was completed, the modified carrier was filtered and washed three times with DMF before use.
  • LX-1000HA resin p-benzoquinone activation take 1g of wet weight LX-1000HA and add 4mL 0.02g/mL p-benzoquinone ethanol solution, p-benzoquinone ethanol solution is 20% (V/V) ethanol water solution. Activated in a shaker at 30°C for 1h. Subsequently, the activated carrier was filtered and washed successively with 20% (V/V) ethanol aqueous solution and 0.1M pH 8.0 phosphate buffer to obtain p-benzoquinone-activated LX-1000HA.
  • the above-mentioned activated carrier was added to 4 mL of prepared enzyme solution (the enzyme solution prepared in the same way as in Example 1), and immobilized in a shaker at 20° C. and 100 rpm for 20 h. Finally, the immobilized enzyme was filtered and washed three times with buffer to obtain the immobilized amine dehydrogenase.
  • the steps of carrier activation and preparation of enzyme solution are the same as those in Example 3, except that the post-modification process is not performed during the process of enzyme immobilization. After immobilization, it is directly filtered and washed with buffer three times to obtain the primary immobilized amine dehydrogenase.
  • an amino-type carrier activated by cyanuric chloride is used to prepare an immobilized amine with high catalytic activity and good operational stability.
  • the dehydrogenase and the immobilized formate dehydrogenase have higher catalytic activity and reusability than the immobilized enzyme prepared by the traditional immobilization method.

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Abstract

提供了一种固定化酶及其在连续化生产中的应用。其中，固定化酶为聚乙烯亚胺修饰的固定化酶，包括：酶，酶包括胺脱氢酶和/或甲酸脱氢酶；以及载体，该载体为三聚氯氰活化的氨基型载体。解决了现有技术中固定化酶性能较差的问题，提高了固定化酶的催化活性和重复使用性，适用于酶固定化领域。

Description

固定化酶及其在连续化生产中的应用 技术领域
本发明涉及酶固定化领域，具体而言，涉及一种固定化酶及其在连续化生产中的应用。
背景技术
手性胺是合成手性药物、农药品和精细化学品等的重要中间体。近年来，为避开制备关键化合物的许多化学合成过程，满足绿色化学的要求，胺脱氢酶(AmDH)的使用在不断增加。胺脱氢酶是指能催化酮类化合物还原为手性胺的一类酶。与转氨酶不同的是，胺脱氢酶以廉价的铵盐作为氨源，不依赖于昂贵的氨供体如异丙胺，丙氨酸等。因还原过程通常需要还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)或还原型烟酰胺腺嘌呤二核苷酸(NADH)递氢，故胺脱氢酶经常与甲酸脱氢酶(FDH)联用以降低生产成本。相较于化学催化合成，胺脱氢酶不仅反应条件温和，且拥有更高的催化活性、选择性以及底物专一性。然而水溶性的游离酶在生产应用过程中面临着许多问题，如稳定性差、无法重复使用以及产物分离纯化的问题。
酶固定化的主要任务是选择一种适当的固定化方法，从而设计出一种既满足某一应用的催化需求(选择性、稳定性)又能满足非催化需求(成本、分离工艺)的生物催化剂。目前，生产应用中使用较多的为共价结合法固定化酶，所用到的商业化载体包括氨基型载体和环氧型载体。以氨基型树脂为例，其通常需要经过双功能的交联剂活化之后再通过交联剂的末端官能团与酶结合。最常用的活化方法是使用戊二醛对氨基进行活化，随后利用活性醛基与酶分子进行连接以实现固定化。也有报道指出在使用戊二醛对酶进行固定化后，用小分子化合物如氨基酸或NaBH 4进行后修饰以增加固定化酶的稳定性。
在对胺脱氢酶固定化研究过程中，发明人发现传统的戊二醛活化的氨基载体或环氧型载体不能很好的完成胺脱氢酶的固定化。所得到的固定化酶存在催化活性低，重复使用性差的缺点。在固定化过程中添加甘露醇等保护剂或对得到的固定化酶进行小分子后修饰也不能克服上述缺陷。
发明内容
本发明的主要目的在于提供一种固定化酶及其在连续化生产中的应用，以提高固定化酶的催化活性和重复使用性。
为了实现上述目的，根据本发明的第一个方面，提供了一种固定化酶，该固定化酶为聚乙烯亚胺修饰的固定化酶，包括：酶，包括胺脱氢酶和/或甲酸脱氢酶；以及固定酶的载体，载体为三聚氯氰活化的氨基型载体。
进一步地，胺脱氢酶包括如SEQ ID NO：1序列所示的胺脱氢酶；优选地，甲酸脱氢酶包括如SEQ ID NO：2序列所示的甲酸脱氢酶；优选地，氨基型载体包括ECR8409、LX-1000HA或LX-1000EPHA；聚乙烯亚胺优选地，聚乙烯亚胺的分子量为1.8～10KDa。
为了实现上述目的，根据本发明的第二个方面，提供了一种固定化酶的制备方法，该制备方法包括：利用三聚氯氰活化氨基型载体，获得活化载体；将酶与活化载体混合孵育以进行固定化，然后采用聚乙烯亚胺对固定化产物进行修饰，得到固定化酶；上述酶包括胺脱氢酶和/或甲酸脱氢酶。
进一步地，利用三聚氯氰活化氨基型载体，获得活化载体包括：将氨基型载体分散于有机溶剂中，得到载体悬浮液；将三聚氯氰与载体悬浮液反应以对氨基型载体进行活化，得到活化载体；优选地，有机溶剂为极性有机溶剂；优选地，极性有机溶剂包括如下任意一种或多种：四氢呋喃、丙酮、二甲基甲酰胺或二甲基亚砜；优选地，活化的温度为0～30℃，更优选为0～5℃；优选地，活化的时间为2～5h；优选地，活化后采用有机溶剂洗涤活化载体以去除未反应的三聚氯氰；优选地，在洗涤之后，进一步包括采用氮气吹干活化载体上的有机溶剂；优选地，氨基型载体为干燥载体；优选地，氨基型载体包括ECR8409、LX-1000HA或LX-1000EPHA。
进一步地，在将酶与活化载体混合孵育之前，制备方法还包括采用缓冲液洗涤活化载体以置换有机溶剂；优选地，酶采用缓冲液配制而成；优选地，缓冲液包括磷酸盐缓冲液、甘氨酸缓冲液或硼砂缓冲液；优选地，磷酸盐缓冲液的浓度为20～50mM；优选地，磷酸盐缓冲液的pH为6.5～9.0；优选地，固定化的温度为0～30℃，更优选为4～30℃；优选地，固定化的时间为2～5h。
进一步地，将酶与活化载体混合孵育以进行固定化，然后采用聚乙烯亚胺对固定化产物进行修饰，得到固定化酶包括：将酶与活化载体混合孵育以进行固定化，得到初固定酶；采用聚乙烯亚胺对初固定酶进行后修饰，得到固定化酶；优选地，酶包括胺脱氢酶和/或甲酸脱氢酶；优选地，胺脱氢酶包括如SEQ ID NO：1序列所示的胺脱氢酶；优选地，甲酸脱氢酶包括如SEQ ID NO：2序列所示的甲酸脱氢酶；优选地，向初固定化酶中加入聚乙烯亚胺的溶液进行后修饰；优选地，聚乙烯亚胺包括聚乙烯亚胺；优选地，聚乙烯亚胺的分子量为1.8～10KDa；优选地，聚乙烯亚胺的溶液的pH为5～8；优选地，加入的聚乙烯亚胺的终浓度为0.5～3％W/V；优选地，后修饰的温度为0～30℃，更优选为10～30℃；优选地，后修饰的时间为8～24h。
为了实现上述目的，根据本发明的第三个方面，提供了一种上述固定化酶、或根据上述制备方法制备的固定化酶在连续化反应中的应用。
进一步地，连续化反应包括在填充床反应器中进行连续化反应；优选地，填充床反应器通过将固定化酶填充于层析空柱或不锈钢色谱柱中制成；优选地，填充床反应器的容积为5～50mL；优选地，填充床反应器的填充方法为湿法填装。
进一步地，连续化反应包括：将含底物的反应体系自下而上泵入填充床反应器中，并将反应产物重新混入反应体系中；泵为恒流泵，优选地，恒流泵的压力≤20MPa；优选地，连续化反应的流速为0.01～10mL/min。
进一步地，酶为主酶，或者为主酶和辅酶的混合酶；优选地，主酶选自胺脱氢酶，相应地，辅酶选自甲酸脱氢酶；优选地，固定化酶包括固定化胺脱氢酶、或胺脱氢酶与甲酸脱氢酶的共固定体系；优选地，胺脱氢酶包括如SEQ ID NO：1序列所示的胺脱氢酶；优选地，甲酸脱氢酶包括如SEQ ID NO：2序列所示的甲酸脱氢酶；优选地，反应体系包括底物或者包括底物与辅酶的混合物；优选地，固定化酶为固定化胺脱氢酶，反应体系包括底物和甲酸脱氢酶的混合物。
应用本发明的技术方案，利用三聚氯氰活化的氨基型载体，制备了一种催化活性高、操作稳定性好的固定化胺脱氢酶和甲酸脱氢酶，与利用传统固定化方法制备的固定化酶相比，具有更高的催化活性及重复使用性。
附图说明
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1示出了根据本发明实施例5的连续化反应装置示意图。
具体实施方式
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将结合实施例来详细说明本发明。
如背景技术所提到的，传统的戊二醛活化的氨基载体或环氧型载体不能很好的完成胺脱氢酶的固定化。所得到的固定化酶存在催化活性低，重复使用性差的缺点。同样的，在固定化过程中添加甘露醇等保护剂或对得到的固定化酶进行小分子后修饰也不能克服上述缺陷。
因而，在本申请中发明人对固定化酶进行深入研究，并尝试了很多种戊二醛活化氨基载体的替代方案，并发现仅三聚氯氰活化后的替代方案效果优异。即发现了一种利用三聚氯氰载体活化剂活化的氨基型载体，利用此载体制备了一种催化活性高、操作稳定性好的固定化胺脱氢酶，与利用传统固定化方法制备的固定化胺脱氢酶相比，具有更高的催化活性及重复使用性。此前虽有利用三聚氯氰活化树脂的报道，但在酶的固定化领域，仅在上世纪70年代有少数文献报道用三聚氯氰活化携带羟基载体如多糖基载体进行固定化酶，暂无相关利用三聚氯氰活化氨基树脂进行酶固定化的报道。因而提出了本申请的一系列保护方案。
在本发明第一种实施方式中，提供了一种固定化酶，该固定化酶为聚乙烯亚胺修饰的固定化酶，包括：酶，酶包括胺脱氢酶和/或甲酸脱氢酶；以及固定酶的载体，载体为三聚氯氰活化的氨基型载体。
利用三聚氯氰，能够活化氨基载体上的氨基基团，提供了与传统的戊二醛活化氨基树脂不同的树脂结构，为那些由于结构等因素无法在戊二醛活化氨基树脂上有效固定化的酶提供了新的载体。酶与活化后的载体结合，形成固定化酶后，为进一步提升固定化酶的操作稳定性，通过聚乙烯亚胺对固定化酶进行修饰，利用高分子对酶的“束缚”或保护高分子实现更稳定的固定化。从而获得催化活性和重复使用性得到提升的固定化酶。
上述固定化酶中的酶主要针对一类难以通过戊二醛活化的氨基载体实现固定化的酶。具体地，包括胺脱氢酶、甲酸脱氢酶或其他酶和相应上述多种酶的组合。上述三聚氯氰活化氨基型载体的活化原理为：利用三聚氯氰具有三个卤素活性位点，在活化时首先利用其中一个活性位点与氨基结合，随后剩余的活性位点可以与酶分子上氨基、羟基等官能团反应完成固定化。优选地，胺脱氢酶包括但不限于胺脱氢酶AmDHs，本申请中的AmDHs，专指氨基酸序列为SEQ ID NO：1的胺脱氢酶，而非多种胺脱氢酶；优选地，甲酸脱氢酶包括但不限于甲酸脱氢酶AIY34662.1。优选地，具体活化的氨基型载体包括但不限于LX-1000EPHA、ECR8409或LX-1000HA。优选地，聚乙烯亚胺的分子量为1.8～10KDa。
上述胺脱氢酶AmDHs的氨基酸序列SEQ ID NO：1的序列如下：
Figure PCTCN2022076232-appb-000001
上述甲酸脱氢酶为AIY34662.1野生型。氨基酸序列SEQ ID NO：2的序列如下：
Figure PCTCN2022076232-appb-000002
胺脱氢酶是指能催化酮类化合物还原为手性胺的一类酶，以廉价的铵盐作为氨源，不依赖于昂贵的氨供体。因还原过程通常需要还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)或还原型烟酰胺腺嘌呤二核苷酸(NADH)递氢，故胺脱氢酶经常与甲酸脱氢酶联用以降低生产成本。较于化学催化合成，胺脱氢酶不仅反应条件温和，且拥有更高的催化活性、选择性以及底物专一性。利用上述活化载体，可以将游离酶，尤其是水溶性的游离酶固定，形成固定化酶，从而克服游离酶稳定性差、无法重复使用、产物分离纯化困难等问题。进行固定化的酶可以为一种，也可以多种酶同时进行固定化，以达到联合使用的目的。
传统的固定化酶载体的制备方法，以氨基型树脂为例，通常需要经过双功能的交联剂活化之后再通过交联剂的末端官能团与酶结合。最常用的活化方法是使用戊二醛对氨基进行活 化，随后利用活性醛基与酶分子进行连接以实现固定化。也有报道指出在使用戊二醛对酶进行固定化后，用小分子化合物如氨基酸或NaBH 4进行后修饰以增加固定化酶的稳定性。三聚氯氰对于氨基树脂有活化作用，适合用于氨基树脂载体，提供与传统戊二醛活化不同的树脂结构。利用三聚氯氰活化氨基树脂后，三聚氯氰基团上剩余的活性卤素(氯)位点可以与酶分子反应，不同的树脂结构对不同的酶的结合能力差异较大。利用载体活化剂可以帮助难以与传统固定化酶载体结合酶进行固定化。氨基载体可以包括Purolite公司生产的ECR8409树脂、西安蓝晓科技公司生产的LX-1000HA树脂等多种市售氨基树脂。
聚乙烯亚胺(PEI)，可以配置为适宜浓度的水溶液进行使用，使用时终浓度可以根据固定化酶的性质、含量等因素进行调节，可以为0.5～3％W/V。针对不同固定化酶的性质，可选用不同分子量的聚乙烯亚胺进行修饰保护，PEI的分子量可以为1.8～10KDa。
在本发明第二种实施方式中，提供了一种固定化酶的制备方法，该制备方法包括：利用三聚氯氰活化氨基型载体，获得活化载体；将酶与活化载体混合孵育以进行固定化，然后采用聚乙烯亚胺对固定化产物进行修饰，得到固定化酶；酶包括胺脱氢酶和/或甲酸脱氢酶。
利用三聚氯氰制备的活化氨基型载体，具有与传统活化载体不同的结构和结合位点，为固定化酶的制备提供了新的可选择载体，帮助难以与传统固定化酶载体结合酶进行固定化。将酶与活化载体在适宜温度、转速等进行固定化，即可得到固定化酶。
上述制备方法，利用三聚氯氰活化氨基型载体，获得活化载体包括：将氨基型载体溶于有机溶剂中，得到载体悬浮液；将三聚氯氰与载体悬浮液混合以对氨基型载体进行活化，得到活化载体；优选地，有机溶剂可以为极性有机溶剂；优选地，极性有机溶剂包括如下任意一种或多种：四氢呋喃、丙酮、二甲基甲酰胺或二甲基亚砜；优选地，活化的温度为0-30℃，更优选的为0～5℃；优选地，活化的时间为2～5h；优选地，活化后采用有机溶剂洗涤活化载体以去除未反应的三聚氯氰；优选地，在洗涤之后，进一步包括采用氮气吹干活化载体，即吹干载体上的有机溶剂；优选地，氨基型载体为干燥载体；优选地，氨基型载体包括但不限于ECR8409、LX-1000HA或LX-1000EPHA。
利用四氢呋喃、丙酮、二甲基甲酰胺或二甲基亚砜等极性有机溶剂分散氨基型载体，利用三聚氯氰对氨基型载体进行活化，根据不同的酶、载体等因素，活化的温度可以为0～30℃，更优选可以为0～5℃，活化时间为2～5h，低温活化可以保护载体的活性，也可以减慢活化速率，防止活化速率过快，大量产热影响载体活化效果。活化时间可以根据酶、载体或活化温度等条件进行适当的调整，以产生较好的活化效果。活化前的氨基型载体可以为干燥载体，减少载体中残留的水等液体对于反应有机体系的组成，残留的液体与载体上的氨基基团接触甚至包裹，也会对活化效率产生影响。活化后采用同样的有机溶剂洗涤活化载体，去除未反应的三聚氯氰，进一步也可以采用氮气吹干活化载体上的有机溶剂，避免三聚氯氰和有机溶剂残留，在后续的固定化步骤中，影响酶活，对固定化酶的制备产率和性能产生不良影响。
在上述制备方法中，在将酶与活化载体混合孵育之前，即固定化之前，该制备方法还包括采用缓冲液洗涤活化载体以置换有机溶剂；优选地，酶采用缓冲液配制而成；优选地，缓 冲液包括但不限于磷酸盐缓冲液、甘氨酸缓冲液或硼砂缓冲液；优选地，磷酸盐缓冲液的浓度为20～50mM；优选地，磷酸盐缓冲液的pH为6.5～9.0；优选地，固定化的温度为4～30℃；优选地，固定化的时间为2～5h。
采用缓冲液洗涤活化载体，也是为了将活化载体中残留的有机溶剂置换出来，防止残留的有机溶剂对酶活产生影响。酶采用缓冲液配制而成，缓冲液可以为常用的磷酸盐缓冲液、甘氨酸缓冲液或其他常用缓冲液，能够在加入少量酸或碱时保持溶液pH相对稳定，防止反应产生的二氧化碳等产物影响溶液pH值，从而保持酶活性。固定化的时间可以根据酶、载体或固定化温度等条件进行适当的调整，以产生较好的固定效果。
在上述制备方法中，将酶与活化载体混合孵育以进行固定化，然后采用聚乙烯亚胺对固定化产物进行修饰，得到固定化酶包括：将酶与活化载体混合孵育以进行固定化，得到初固定酶；采用聚乙烯亚胺对初固定酶进行后修饰，得到固定化酶；优选地，酶包括胺脱氢酶、甲酸脱氢酶或其他酶类的一种或多种；优选地，胺脱氢酶包括如SEQ ID NO：1序列所示的胺脱氢酶AmDHs；优选地，甲酸脱氢酶包括如SEQ ID NO：2序列所示的甲酸脱氢酶AIY34662.1；优选地，向初固定化酶中加入聚乙烯亚胺的溶液进行后修饰；优选地，聚乙烯亚胺的分子量为1.8～10KDa；优选地，聚乙烯亚胺的pH为5～8；优选地，聚乙烯亚胺的终浓度为0.5～3％W/V；优选地，后修饰的温度可以为10～30℃；优选地，后修饰的时间可以为8～24h。
将酶与活化载体混合孵育进行固定化，即可得到初固定酶。所用酶可以选自一种或多种，不同种酶可以以任意比例混合，制备多种酶的共固定初体系。为了提高固定化酶的稳定性可以进一步利用聚乙烯亚胺对初固定酶进行后修饰，通过高分子对酶的“束缚”或保护进一步提升固定化酶的操作稳定性。向初固定酶中加入一定体积的预先溶解、配置好的聚乙烯亚胺溶液，可以高效混合并有效控制聚乙烯亚胺的终浓度，也利于初固定化酶与聚乙烯亚胺均匀接触，可以提高制备得到的固定化酶的性能。聚乙烯亚胺，分子量可以根据不同的载体和酶进行相应调整，以获得较好的保护性能。聚乙烯亚胺可以配置为10％W/V的水溶液，并用1M盐酸溶液调节。向初固定化酶中加入适量聚乙烯亚胺水溶液，使其终浓度为0.5～3％W/V。
在本发明第三种实施方式中，提供了一种上述固定化酶，或上述制备方法制备的固定化酶在连续化反应中的应用。上述的固定化酶，其催化活性和重复使用性相比于游离酶或传统工艺制备的固定化酶得到极大的提升，可以用于连续化设备中进行连续化反应，与批次反应相比，操作简便，生产效率高。
上述固定化酶在连续化反应中的应用，连续化反应包括在填充床反应器中进行连续化反应；优选地，填充床反应器通过将固定化酶填充于层析空柱或不锈钢色谱柱中制成；优选地，填充床反应器的容积为5-50mL；优选地，填充床反应器的填充方法为湿法填装。
固定化酶可以在层析空柱、不锈钢色谱柱等耐压耐腐蚀的填充床反应器中进行连续化反应，为了防止填充量过多造成用于催化的固定化酶的浪费，以及对填充床反应器底部的固定化酶压力过大，影响固定化酶的机械结构和酶活，填充床反应器的容积可以为5-50mL。为了保证固定化酶的活性和填充均匀性，可以选择湿法填装的方式向填充反应器中填装固定化酶。
在上述应用中，连续化反应包括：将含底物的反应体系自下而上泵入填充床反应器中，并将反应产物重新混入反应体系中；泵为恒流泵；优选地，恒流泵的压力≤20MPa；优选地，连续化反应的流速为0.01～10mL/min。
含有底物的反应体系自下而上泵入填充床反应器，防止反应体系由于重力的作用在反应器中流速过快，导致转化产率低。将反应产物重新混入反应体系中，对于单次催化产率不高的反应，可以进行循环催化，始终保持反应体系的均一性，利于填充床反应器中的固定化酶所处环境的相对稳定；也有利于对于反应体系的检测，便于补充如甲酸铵等原料，也便于终产物的收集，省略了批次反应所需的对终产物与固定化酶进行分离的工艺流程。连续化反应所用的泵，可以根据填充床反应器和流速的需求，以及固定化酶对于压力的耐受能力，合理选择中压恒流泵或低压恒流泵，提供的压力范围可以为0～20MPa。
在上述应用中，酶可以为主酶，或者为主酶和辅酶的混合酶；优选地，主酶可选自胺脱氢酶，相应地，辅酶可选自甲酸脱氢酶；固定化酶包括固定化胺脱氢酶、或胺脱氢酶与甲酸脱氢酶的共固定体系；优选地，胺脱氢酶包括如SEQ ID NO：1序列所示的胺脱氢酶AmDHs；优选地，甲酸脱氢酶包括如SEQ ID NO：2序列所示的甲酸脱氢酶AIY34662.1；优选地，反应体系包括底物或者包括底物与辅酶的混合物；优选地，固定化酶为固定化胺脱氢酶，反应体系包括底物和甲酸脱氢酶的混合物。
在固定化酶连续化反应的应用中，酶可以单独为主酶，也可以是主酶和辅酶的混合酶，从而利用辅酶生成的产物供给主酶的催化反应。因为胺脱氢酶还原过程通常需要NADPH或NADH递氢，所以使用胺脱氢酶时经常与能够生产NADH的甲酸脱氢酶联用，以降低生产成本。固定化酶可以为单独的固定化胺脱氢酶，此时反应体系中需要额外添加甲酸脱氢酶；固定化酶也可以为胺脱氢酶与甲酸脱氢酶的共固定体系，此时反应体系则可以是单纯的底物溶液。
下面将结合具体的实施例来进一步详细解释本申请的有益效果。
实施例1：ECR8409固定化胺脱氢酶
Figure PCTCN2022076232-appb-000003
底物配制：利用反应式1所示的2-(1-金刚烷基)-2-氧化乙酸甲酯作为底物，称取10g底物于100mL烧杯中，加入50mL纯化水及1.6g的K 2HPO 4·H 2O，搅拌均匀后用10M的NaOH溶液调pH至8.0；称取1g活性炭加至配制溶液中，搅拌1h以吸附杂质及色素；将布氏漏斗垫上滤纸后加入2.5g硅藻土，加纯化水抽干后得到滤饼。将所配制的底物溶液经过滤饼抽滤，并补加20mL纯化水洗涤滤饼，最后得到澄清滤液；称取5.6g甲酸铵及15mg的二硫苏糖醇(DTT)加入滤液中，并用10M的NaOH溶液调pH至8.0。补加纯化水至82mL。
载体活化：取10克干燥的ECR8409树脂于四口瓶中，冰浴条件下加入250mL丙酮并搅拌0.5h。随后加入5g三聚氯氰，冰浴下继续搅拌反应3h。反应完成后，将修饰的载体过滤，并用丙酮洗涤三次后备用。
酶液配制：将冷冻保存的胺脱氢酶AmDHs酶液室温化冻后，用0.1M pH 8.0的磷酸盐缓冲液稀释至8.5mg/mL，备用。
酶固定化：将活化后的载体0.5g用0.1M pH 8.0的磷酸盐缓冲液洗涤三次后，立即加入4mL配置好的酶液，于摇床中20℃，100rpm下固定2h。随后加入1mL预先配置好pH为8.0的10％PEI(10KDa)溶液继续反应过夜。最后将固定化酶过滤并用缓冲液洗涤三次，得到固定化胺脱氢酶。
反应条件：称取0.15g固定化胺脱氢酶，加入0.82mL配制好的底物溶液、25μL 1mg/mL的NAD +溶液以及50μL FDH。于40℃，100rpm摇床中反应18h。取反应体系的上清液，用HPLC测定反应转化率。
固定化胺脱氢酶的活性验证结果如表1所示，每批次反应完成后，均对固定化胺脱氢酶进行过滤分离，并用0.1M pH 8.0的磷酸盐缓冲液洗涤三次。
表1 ECR8409固定化胺脱氢酶对底物的转化率
循环次数 1st 2nd 3rd 4th 5th
转化率(％) 99 99 95 93 91
。
实施例2：胺脱氢酶与甲酸脱氢酶共固定化于ECR8409
载体活化步骤及酶液配制同实施例1，所不同的是在酶固定化的过程中额外加入50μL的甲酸脱氢酶(FDH)AIY34662.1粗酶液。随后用同样的固定化方法及后修饰过程完成固定化。
胺脱氢酶与FDH共固定于ECR8409，活性验证结果如表2所示，详细操作条件见实施例1，不同的是，反应体系中不再添加FDH粗酶液。
表2 ECR8409共固定化胺脱氢酶及FDH对底物的转化率
循环次数 1st 2nd 3rd 4th 5th
转化率(％) 99 99 98 96 96
。
实施例3：LX-1000HA固定化胺脱氢酶
载体活化：取10克干燥的LX-1000HA树脂于四口瓶中，冰浴条件下加入200mL四氢呋喃并搅拌0.5h。随后加入2.5g三聚氯氰，冰浴下继续搅拌反应3h。反应完成后，将修饰的载体过滤，并用四氢呋喃洗涤三次后备用。
酶液配制、固定化过程和活性验证同实施例1。
固定化胺脱氢酶的活性验证结果如表3所示。
表3 LX-1000HA固定化胺脱氢酶对底物的转化率
循环次数 1st 2nd 3rd 4th 5th
转化率(％) 99 96 93 87 84
。
实施例4：LX-1000EPHA固定化胺脱氢酶
载体活化：取10克干燥的LX-1000EPHA树脂于四口瓶中，冰浴条件下加入200mL四氢呋喃并搅拌0.5h。随后加入2.5g三聚氯氰，冰浴下继续搅拌反应3h。反应完成后，将修饰的载体过滤，并用四氢呋喃洗涤三次后备用。
酶液配制、固定化过程和活性验证同实施例1。
固定化胺脱氢酶的活性验证结果如表4所示。
表4 LX-1000EPHA固定化胺脱氢酶对底物的转化率
循环次数 1st 2nd 3rd 4th 5th
转化率(％) 98 92 89 88 82
。
实施例5：固定化胺脱氢酶在连续化生产中的应用
反应体系配制：量取80mL底物溶液，底物溶液的详细配制步骤见实施例1。随后向底物溶液中加入2.5mg NAD +及5mL FDH粗酶液。将体系放置在磁力搅拌器上混匀，并在反应过程中保持缓慢搅拌。
称取3.5克实施例1制备得到的固定化胺脱氢酶，按照层析柱装柱操作流程将其填充至5mL层析空柱，并将层析柱置于40℃水浴中。
将CP-M205精密恒流泵连入反应系统，将反应体系以0.4mL/min流速自下而上泵入装有固定化酶的层析柱，并使得流出液重新流回反应体系，形成闭环回路，连续化反应装置如图1所示。反应24h后，取反应体系的上清液送样检测，底物转化率达到95％。随后更换为新配制的反应体系，将连续化投入下一循环反应。经测定，经过十次循环后，反应的转化率仍能达到89％。
通过上述连续化系统的使用，不仅使得生产操作更为简单，处理量更大，而且也增加了固定化胺脱氢酶的操作稳定性。
实施例6：共固定化胺脱氢酶及FDH在连续化生产中的应用
反应体系配制：量取80mL底物溶液，底物溶液的详细配制步骤见实施例1。随后向底物溶液中加入2.5mg NAD +。将体系放置在磁力搅拌器上混匀，并在反应过程中保持缓慢搅拌。
称取3.8克实施例2制备得到的固定化胺脱氢酶及FDH，按照层析柱装柱操作流程将其填充至5mL层析空柱，并将层析柱置于40℃水浴中。
同实施例5将体系连入连续化设备中。反应24h后，底物转化率达到94％，并且在重复使用十次后转换率仍能达到84％。
通过上述连续化系统的使用，不仅使得生产操作更为简单，处理量更大，而且也增加了固定化胺脱氢酶和FDH的操作稳定性。
对比例1：LX-1000HA戊二醛活化固定化胺脱氢酶
LX-1000HA戊二醛活化：取1g湿重LX-1000HA加入4mL 2％(w/v)的戊二醛溶液，戊二醛溶液为用20mM pH为7.0的磷酸盐缓冲液配制。于30℃摇床中活化1h。
将活化后的载体用0.1M pH 8.0的磷酸盐缓冲液洗涤三次后，加入4mL配置好的酶液(同实施例1配制酶液)，于摇床中20℃，100rpm下固定化20h。最后将固定化酶过滤并用缓冲液洗涤三次，得到固定化胺脱氢酶。
固定化胺脱氢酶的活性验证结果如表5所示。
表5 LX-1000HA戊二醛固定化胺脱氢酶对底物的转化率
循环次数 1st 2nd 3rd 4th 5th
转化率(％) 55 36 18 11 6
。
对比例2：LX-1000HA六亚甲基二异氰酸酯活化固定化胺脱氢酶
LX-1000HA树脂六亚甲基二异氰酸酯活化：取10克干燥的LX-1000HA树脂于四口瓶中，冰浴条件下加入200mL二甲基甲酰胺并搅拌0.5h。随后加入2.3g六亚甲基二异氰酸酯，冰浴下继续搅拌反应3h。反应完成后，将修饰的载体过滤，并用DMF洗涤三次后备用。
酶液配制、固定化过程和活性验证同实施例1。
固定化胺脱氢酶的活性验证结果如表6所示。
表6 LX-1000HA六亚甲基二异氰酸酯活化固定化胺脱氢酶对底物的转化率
循环次数 1st 2nd 3rd 4th 5th
转化率(％) 91 84 81 65 52
。
对比例3：LX-1000HA对苯醌活化固定化胺脱氢酶
LX-1000HA树脂对苯醌活化：取1g湿重LX-1000HA加入4mL 0.02g/mL对苯醌乙醇溶液，对苯醌乙醇溶液为20％(V/V)乙醇水溶液。于30℃摇床中活化1h。随后将活化后的载体过滤并依次用20％(V/V)乙醇水溶液、0.1M pH 8.0的磷酸盐缓冲液依次清洗，得到对苯醌活化的LX-1000HA。
将上述活化后的载体加入4mL配置好的酶液(同实施例1配制酶液)，于摇床中20℃，100rpm下固定化20h。最后将固定化酶过滤并用缓冲液洗涤三次，得到固定化胺脱氢酶。
固定化胺脱氢酶的活性验证结果如表7所示。
表7对苯醌活化的LX-1000HA固定化胺脱氢酶对底物的转化率
循环次数 1st 2nd 3rd 4th 5th
转化率(％) 37 19 11 9 2
。
对比例4：LX-1000HA固定化胺脱氢酶(无后修饰)
载体活化步骤及酶液配制同实施例3，所不同的是在酶固定化的过程不进行后修饰过程，固定化后直接过滤并用缓冲液洗涤三次，得到初固定化胺脱氢酶。
固定化胺脱氢酶的活性验证结果如表8所示。
表8 LX-1000HA固定化胺脱氢酶(无后修饰)对底物的转化率
循环次数 1st 2nd 3rd 4th 5th
转化率(％) 98 63 27 20 13
。
对比例5：ECR8409共固定化胺脱氢酶及FDH(无后修饰)
载体活化步骤及酶液配制同实施例2，所不同的是在酶固定化的过程不进行后修饰过程，固定化后直接过滤并用缓冲液洗涤三次，得到胺脱氢酶与FDH共固定初体系。
固定化胺脱氢酶的活性验证结果如表9所示。
表9 ECR8409共固定化胺脱氢酶及FDH(无后修饰)对底物的转化率
循环次数 1st 2nd 3rd 4th 5th
转化率(％) 92 77 58 45 30
。
从以上的描述中，可以看出，本发明上述的实施例实现了如下技术效果：利用三聚氯氰活化的氨基型载体，利用此载体制备了催化活性高、操作稳定性好的固定化胺脱氢酶和固定化甲酸脱氢酶，与利用传统固定化方法制备的固定化酶相比，具有更高的催化活性及重复使用性。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。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 (10)

  1. 一种固定化酶，其特征在于，所述固定化酶为聚乙烯亚胺修饰的固定化酶，包括：
    酶，所述酶包括胺脱氢酶和/或甲酸脱氢酶，以及
    载体，所述载体为三聚氯氰活化的氨基型载体。
  2. 根据权利要求1所述的固定化酶，其特征在于，所述胺脱氢酶包括如SEQ ID NO：1序列所示的胺脱氢酶；
    优选地，所述甲酸脱氢酶包括如SEQ ID NO：2序列所示的甲酸脱氢酶；
    优选地，所述氨基型载体包括ECR8409、LX-1000HA或LX-1000EPHA；
    优选地，所述聚乙烯亚胺的分子量为1.8～10KDa。
  3. 一种固定化酶的制备方法，其特征在于，所述制备方法包括：
    利用三聚氯氰活化氨基型载体，获得活化载体；
    将酶与所述活化载体混合孵育以进行固定化，然后采用聚乙烯亚胺对固定化产物进行修饰，得到所述固定化酶；
    所述酶包括胺脱氢酶和/或甲酸脱氢酶。
  4. 根据权利要求3所述的制备方法，其特征在于，利用所述三聚氯氰活化氨基型载体，获得活化载体包括：
    将所述氨基型载体分散于有机溶剂中，得到载体悬浮液；
    将所述三聚氯氰与所述载体悬浮液反应以对所述氨基型载体进行活化，得到所述活化载体；
    优选地，所述有机溶剂为极性有机溶剂；
    优选地，所述极性有机溶剂包括如下任意一种或多种：四氢呋喃、丙酮、二甲基甲酰胺或二甲基亚砜；
    优选地，所述活化的温度为0～30℃，更优选为0～5℃；
    优选地，所述活化的时间为2～5h；
    优选地，所述活化后采用所述有机溶剂洗涤所述活化载体以去除未反应的所述三聚氯氰；
    优选地，在所述洗涤之后，进一步包括采用氮气吹干所述活化载体；
    优选地，所述氨基型载体为干燥载体；
    优选地，所述氨基型载体包括ECR8409、LX-1000HA或LX-1000EPHA。
  5. 根据权利要求4所述的制备方法，其特征在于，在所述固定化之前，所述制备方法还包括采用缓冲液洗涤所述活化载体以置换所述有机溶剂；
    优选地，所述酶采用所述缓冲液配制而成；
    优选地，所述缓冲液包括磷酸盐缓冲液、甘氨酸缓冲液或硼砂缓冲液；
    优选地，所述磷酸盐缓冲液的浓度为20～50mM；
    优选地，所述磷酸盐缓冲液的pH为6.5～9.0；
    优选地，所述固定化的温度为0～30℃，更优选为4～30℃；
    优选地，所述固定化的时间为2～5h。
  6. 根据权利要求3至5中任一项所述的制备方法，其特征在于，将所述酶与所述活化载体混合孵育以进行固定化，然后采用聚乙烯亚胺对固定化产物进行修饰，得到所述固定化酶包括：
    将所述酶与所述活化载体混合孵育以进行固定化，得到初固定酶；
    采用所述聚乙烯亚胺对所述初固定酶进行后修饰，得到所述固定化酶；
    优选地，所述胺脱氢酶包括如SEQ ID NO：1序列所示的胺脱氢酶；
    优选地，所述甲酸脱氢酶包括如SEQ ID NO：2序列所示的甲酸脱氢酶；
    优选地，向所述初固定化酶中加入所述聚乙烯亚胺的溶液进行所述后修饰；
    优选地，所述聚乙烯亚胺的分子量为1.8～10KDa；
    优选地，所述聚乙烯亚胺的溶液的pH为5～8；
    优选地，加入的所述聚乙烯亚胺的终浓度为0.5～3％W/V；
    优选地，所述后修饰的温度为0～30℃，更优选为10～30℃；
    优选地，所述后修饰的时间为8～24h。
  7. 权利要求1或2所述的固定化酶，或根据权利要求3至6中任一项所述制备方法制备的所述固定化酶在连续化反应中的应用。
  8. 根据权利要求7所述的应用，其特征在于，所述连续化反应包括在填充床反应器中进行所述连续化反应；
    优选地，所述填充床反应器通过将所述固定化酶填充于层析空柱或不锈钢色谱柱中制成；
    优选地，所述填充床反应器的容积为5～50mL；
    优选地，所述填充床反应器的填充方法为湿法填装。
  9. 根据权利要求8所述的应用，其特征在于，所述连续化反应包括：
    将含底物的反应体系自下而上泵入所述填充床反应器中，并将反应产物重新混入所述反应体系中；
    所述泵为恒流泵，优选地，所述恒流泵的压力≤20MPa；
    优选地，所述连续化反应的流速为0.01～10mL/min。
  10. 根据权利要求9所述的应用，其特征在于，所述固定化酶包括固定化胺脱氢酶、或胺脱氢酶与甲酸脱氢酶的共固定体系；
    优选地，所述胺脱氢酶包括如SEQ ID NO：1序列所示的胺脱氢酶；
    优选地，所述甲酸脱氢酶包括如SEQ ID NO：2序列所示的甲酸脱氢酶；
    优选地，所述反应体系包括底物或者包括底物与所述甲酸脱氢酶的混合物；
    优选地，所述固定化酶为所述固定化胺脱氢酶，所述反应体系包括底物和所述甲酸脱氢酶的混合物。
PCT/CN2022/076232 2022-01-12 2022-02-14 固定化酶及其在连续化生产中的应用 WO2023133957A1 (zh)

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