WO2023087270A1 - 酶固定化载体及其制备方法、固定化酶及其制备方法 - Google Patents
酶固定化载体及其制备方法、固定化酶及其制备方法 Download PDFInfo
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- WO2023087270A1 WO2023087270A1 PCT/CN2021/131878 CN2021131878W WO2023087270A1 WO 2023087270 A1 WO2023087270 A1 WO 2023087270A1 CN 2021131878 W CN2021131878 W CN 2021131878W WO 2023087270 A1 WO2023087270 A1 WO 2023087270A1
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- Prior art keywords
- enzyme
- immobilized
- resin
- methacrylic resin
- reaction
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Links
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F20/00—Homopolymers and copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride, ester, amide, imide or nitrile thereof
- C08F20/02—Monocarboxylic acids having less than ten carbon atoms, Derivatives thereof
- C08F20/10—Esters
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F8/00—Chemical modification by after-treatment
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
- C12N11/087—Acrylic polymers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- 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.)
Definitions
- the invention relates to the field of biocatalysis, in particular to an enzyme-immobilized carrier and a preparation method thereof, an immobilized enzyme and a preparation method thereof.
- Biocatalysis has become an important part of green synthetic drugs, and it is one of the most promising and applied technologies for catalytic synthesis of drug structural units and intermediates, especially providing a unique and difficult-to-substitute method for chiral synthesis.
- More and more enzymes are used as catalysts in industrial processes, but due to the mildness of enzyme use conditions and its own variability, enzymes are very demanding on the environment and difficult to recycle, which greatly limits the use of enzymes in industry. Applications.
- the development and application of immobilized enzyme technology can effectively solve these problems, so immobilized enzymes, often called "biocatalysts", are widely used in industrial organic synthesis and biotransformation.
- Enzyme immobilization methods can be divided into two categories: physical methods and chemical methods. Physical methods mainly include adsorption and embedding methods, and chemical methods mainly include binding and cross-linking methods.
- Adsorption method also known as non-covalent method, is mainly a method of immobilizing enzymes and adsorption media through some non-covalent bond interactions such as hydrogen bonds, van der Waals forces, hydrophobic forces, and ionic bonds.
- Commonly used inorganic adsorption materials include silica gel, alumina, porous glass, diatomaceous earth, etc.
- organic adsorption materials mainly include natural alginate, chitin, chitosan, cellulose, starch, and synthetic organic materials include Polyurethane, macroporous resin, etc.
- the embedding method is to embed the free enzyme in the pores of a specific gel and other media for immobilization, such as polyacrylamide, calcium alginate and other gel media for embedding.
- the embedding method is simpler, and the structure of the embedded enzyme protein can basically remain unchanged, and the loss of enzyme activity is relatively small, but there are certain disadvantages, such as leakage of enzyme components and enzyme inactivation.
- the binding method is covalent immobilization, which is based on the reaction between the effective functional groups of the carrier material and the enzyme-related functional groups (such as amino, carboxyl, sulfhydryl, hydroxyl and other enzyme protein side chain groups) to combine with each other.
- Covalent immobilization makes the combination between the enzyme and the carrier stronger, and the strong chemical bond between the enzyme and the carrier can significantly reduce the loss of the enzyme and improve the reuse rate of the enzyme.
- the method has harsh conditions, severe reaction, and large loss of enzyme activity (generally, the residual enzyme activity is about 30%).
- the cross-linking method refers to a class of methods in which a covalent bond is formed for enzyme immobilization through a bifunctional or multifunctional reagent (cross-linking reagent) to carry out a cross-linking reaction between enzyme molecules, between an enzyme molecule and a carrier.
- cross-linking reagents are glutaraldehyde, hexamethylenediamine, maleic anhydride, bis-azobenzene, etc.
- CLMA cross-linked enzyme aggregates formed by cross-linking enzymes with cross-linking agents, such as CLEA immobilization of phenylalanine ammonia lyase and sucrose phosphorylase CLEA fixed.
- the above-mentioned traditional enzyme immobilization methods have many defects, such as the easy loss of adsorbed and immobilized enzymes, the difficulty of diffusing the substrates and products of enzymes after entrapment and immobilization, the covalent binding method easily leads to enzyme inactivation, and the mechanical properties of cross-linked enzymes Relatively poor and so on.
- affinity resins are mostly protein purification resins, and their matrices are generally cellulose, cross-linked dextran, agarose, polyacrylamide, porous glass beads, etc.
- agarose gel is the most widely used.
- the particle size of metal chelating affinity resin is small, it is difficult to recycle in large-scale application, and the particles are easily broken under high shear force, so it is not easy to apply in the traditional stirred tank reaction mode.
- its resin matrix is very hydrophilic and requires high storage conditions. It needs to be stored in alcohol solution and should be kept dry as far as possible, so it is not suitable for immobilized enzymes. In addition, its high cost has greatly affected its application in industrial immobilized enzymes.
- the main purpose of the present invention is to provide an enzyme immobilization carrier and its preparation method, immobilized enzyme and its preparation method, to solve the poor compatibility of the enzyme immobilization carrier to the enzyme in the prior art, the immobilization effect is poor, and The problem of poor activity of immobilized enzymes and poor stability during repeated use.
- an enzyme-immobilized carrier comprises a resin sphere matrix, a -N(CH 2 COOH) 2 group linked to the resin sphere matrix by a chemical bond, And chelating metal ions adsorbed on the -N(CH 2 COOH) 2 group through coordination; wherein, the resin ball matrix is amino-type methacrylic resin or epoxy-type methacrylic resin, and the resin ball matrix is The particle size is 200-700 ⁇ m.
- the amino-type methacrylic resin has an amino functional group with a C2 length or a C6-length carbon chain arm, and the amino group content is 30-80 ⁇ mol/g; preferably, the amino-type methacrylic resin is One or more of Lifetech TM ECR8309, Lifetech TM ECR8409, ESR-1, ESR-3 or ESQ-1; more preferably amino-type methacrylic resin is or one or more of Lifetech TM ECR8309; preferably, the epoxy equivalent of the epoxy-based methacrylic resin is 2-5 ⁇ mol/g; more preferably, the epoxy-based methacrylic resin is EP200, One or more of Lifetech TM ECR8285, Lifetech TM ECR8204, Lifetech TM ECR8209, ES-1, ES103, ES-101, ReliZyme TM HFA403 or ReliZyme TM EC-HFA; more preferably epoxy-based methacrylic
- an immobilized enzyme comprises the above-mentioned enzyme immobilized carrier and the enzyme immobilized thereon.
- transaminase is selected from any one of transaminase, ketoreductase, alcohol dehydrogenase, formate dehydrogenase, glucose dehydrogenase, monooxygenase, ene reductase, imine reductase and amino acid dehydrogenase Or multiple;
- transaminase is derived from the transaminase of Chromobacterium violaceum DSM30191, or derived from the transaminase of Arthrobacter citreus, or derived from the transaminase of Actinobacteria, or derived from the transaminase of Sciscionella sp.SE31;
- Ketoreductase is derived from Acetobacter sp.
- the enzyme is glucose dehydrogenase from Lysinibacillus sphaericus G10; the monooxygenase is cyclohexanone monooxygenase from Rhodococcus sp.Phi1, or cyclohexanone monooxygenase from Rhodococcus ruber-SD1, or Cyclohexanone monooxygenase from Brachymonas petroleovorans; ene reductase from Saccharomyces cerevisiae, or ene reductase from Chryseobacterium sp.CA49; imine reductase from Streptomyces sp
- amino acid dehydrogenase is leucine dehydrogenase from Bacillus cereus, or phenylalanine dehydrogenase from Bacillus sphaericus, or from Thermoactinomyces Amino acid dehydrogenase from intermedius ATCC33205, or amino acid dehydrogenase from Thermosyntropha lipolytica.
- a method for preparing the above-mentioned enzyme immobilized carrier comprising the following steps: providing a resin sphere base; linking -N(CH 2 COOH) 2 groups on the resin sphere base through chemical bonds; Adsorb metal ions on the -N(CH 2 COOH) 2 group through coordination to obtain an enzyme-immobilized carrier; wherein, the resin ball matrix is amino-type methacrylic resin or epoxy-based methacrylic resin, and the resin ball The particle size of the matrix is 200-700 ⁇ m.
- the resin ball base is an amino-type methacrylic resin
- the amino-type methacrylic resin and sodium chloroacetate are mixed for a nucleophilic substitution reaction to link -N(CH 2 COOH ) 2 groups.
- the alkaline aqueous solution is sodium carbonate aqueous solution, sodium hydroxide solution or lithium hydroxide solution.
- the resin ball matrix is an epoxy-based methacrylic resin
- the epoxy-based methacrylic resin and disodium diacetate imide are mixed for an addition reaction, so that the epoxy-based methacrylic resin Link -N(CH 2 COOH) 2 groups.
- a metal salt solution is added to the reaction system to carry out a complexation reaction, so that the -N(CH 2 COOH) 2 groups
- the group adsorbs metal ions through coordination to obtain an enzyme-immobilized carrier; preferably, during the complexation reaction, the reaction temperature is 20-30°C, and the reaction time is 1-4h; preferably, the metal salt solution is nickel chloride Aqueous solution, copper sulfate aqueous solution, ferrous chloride aqueous solution or cobalt chloride aqueous solution; preferably, the mass ratio of the metal ion and the resin ball matrix in the metal salt solution is (0.05-0.1):1.
- the mass ratio of sodium chloroacetate to amino-type methacrylic resin is (5-10):1.
- the mass ratio of disodium diacetate imide to epoxy-based methacrylic resin is (5-10):1.
- a method for preparing the above-mentioned immobilized enzyme comprising the following steps: performing an immobilization reaction on the above-mentioned enzyme-immobilized carrier and the enzyme to obtain the immobilized enzyme.
- the preparation method of immobilized enzyme comprises the following steps: dispersing the enzyme-immobilized carrier in a first solvent to form a dispersion; wherein, the first solvent is a mixed solution of phosphate buffer, sodium chloride aqueous solution and imidazole buffer; The dispersion liquid is reacted with the enzyme solution containing the enzyme, so that the enzyme and the enzyme-immobilized carrier undergo an immobilization reaction to obtain the immobilized enzyme.
- the first solvent is a mixed solution of phosphate buffer, sodium chloride aqueous solution and imidazole buffer
- the concentration of phosphate buffer is 0.1-0.2 mol/L; the concentration of sodium chloride aqueous solution is 0.5-1 mol/L; the concentration of imidazole buffer is 0.05-0.1 mol/L.
- the reaction temperature is 20-25°C
- the reaction time is 16-24 hours; preferably, each gram of the enzyme-immobilized carrier reacts with 4-8 mL of the enzyme liquid, and the enzyme
- the protein content in the liquid is 20-25mg/mL.
- the above-mentioned carrier of the present invention uses amino-type methacrylic resin or epoxy-type methacrylic resin as the resin ball matrix, and is connected with -N(CH 2 COOH) 2 groups on the resin ball matrix through chemical bonds. Adsorption There are metal ions attached to the -N(CH 2 COOH) 2 group.
- the carrier firstly, the particle size (200-700 ⁇ m) of the resin ball matrix is larger than that of the traditional affinity resin (20-80 ⁇ m), and the recyclability is better.
- its mechanical strength is higher, which avoids the problem of broken resin ball matrix under high shear force, and then in the traditional stirred tank reaction mode, the service life of the immobilized enzyme is longer and repeatable.
- the metal chelate affinity structure composed of nitrogen-containing diacetic acid groups and metal ions is compared with the general covalent binding carrier, and the carrier only binds to the His tag at the end of the protein, so it is more compatible with enzymes.
- the enzyme activity can be retained to the greatest extent while being immobilized, and the enzyme has higher stability for repeated use.
- resins of the above types are less costly.
- the above-mentioned carrier of the present invention has better compatibility with enzymes, better immobilization effect, and can retain enzyme activity to the greatest extent while being immobilized.
- the immobilized enzyme obtained based on its immobilization has better activity and higher stability during repeated use, and is more suitable for application in industrial immobilized enzymes. Based on this, the invention effectively solves the problems in the prior art that the enzyme immobilization carrier has poor compatibility with the enzyme, poor immobilization effect, poor activity of the immobilized enzyme, and poor stability during repeated use.
- the enzyme immobilization carrier has poor compatibility with the enzyme, poor immobilization effect, poor activity of the immobilized enzyme, and poor stability during repeated use.
- the present invention provides an enzyme-immobilized carrier, the enzyme-immobilized carrier comprises a resin sphere matrix, -N(CH 2 COOH) 2 groups linked on the resin sphere matrix by chemical bonds, and Chelate metal ions adsorbed on -N(CH 2 COOH) 2 groups; wherein, the resin ball matrix is amino-type methacrylic resin or epoxy-type methacrylic resin, and the particle size of the resin ball matrix is 200 ⁇ 700 ⁇ m.
- the above-mentioned carrier of the present invention uses amino-type methacrylic resin or epoxy-type methacrylic resin as the resin ball matrix, and is connected with -N(CH 2 COOH) 2 groups on the resin ball matrix through chemical bonds. Adsorption There are metal ions attached to the -N(CH 2 COOH) 2 group.
- the His tag at the end of the enzyme can be adsorbed on the metal ion through coordination.
- the -N(CH 2 COOH) 2 group forms a coordination bond with the metal ion, but the -N(CH 2 COOH) 2 group does not occupy all the coordination sites of the metal ion.
- the enzyme continues to occupy the remaining unoccupied coordination sites on the metal ion, and forms an immobilized enzyme through coordination with the carrier.
- the enzyme main body is destroyed due to the chemical reaction between the enzyme main body and the carrier during the binding process, and the rigidity of the immobilized enzyme becomes stronger, resulting in lower enzyme activity.
- the present invention uses the His tag at the end of the enzyme and the metal ion in the carrier to coordinate and combine, so there is no reactive contact between the carrier and the main body of the enzyme, and the loss of enzyme activity is smaller.
- the aforementioned resin ball base of the present invention is amino-type methacrylic resin and/or epoxy-type methacrylic resin. Based on this, first, the particles of the resin ball matrix are larger than traditional affinity resins, and the recyclability is better. At the same time, its mechanical strength is higher, which avoids the problem of broken resin ball matrix under high shear force, and then in the traditional stirred tank reaction mode, the service life of the immobilized enzyme is longer and the recyclability is better. Second, the carrier composed of it has better compatibility with enzymes, can retain enzyme activity to the greatest extent while immobilizing, and has higher stability for repeated use of enzymes. In addition, the aforementioned types of resins are less costly.
- the above-mentioned carrier of the present invention has better compatibility with enzymes, better immobilization effect, and can retain enzyme activity to the greatest extent while being immobilized. Furthermore, the immobilized enzyme obtained based on its immobilization has better activity and higher stability during repeated use, and is more suitable for application in industrial immobilized enzymes.
- the present invention effectively solves the problems in the prior art that the enzyme immobilization carrier has poor compatibility with the enzyme, poor immobilization effect, poor activity of the immobilized enzyme, and poor stability during repeated use.
- the amino-type methacrylic resin has an amino functional group with a carbon chain arm of C2 length or C6 length, and the amino group content is 30-80 ⁇ mol/g; preferably, the amino-type methacrylic resin is One or more of Lifetech TM ECR8309, Lifetech TM ECR8409, ESR-1, ESR-3 or ESQ-1; more preferably amino-type methacrylic resin is or one or more of Lifetech TM ECR8309;
- the epoxy equivalent of the epoxy-based methacrylic resin is 2-5 ⁇ mol/g; more preferably, the epoxy-based methacrylic resin is EP200, One or more of Lifetech TM ECR8285, Lifetech TM ECR8204, Lifetech TM ECR8209, ES-1, ES103, ES-101, ReliZyme TM HFA403 or ReliZyme TM EC-HFA; more preferably epoxy-based methacrylic resin is or one or more of ReliZyme TM EC-HFA.
- the resin ball matrix is selected from the above types. On the one hand, it has higher mechanical strength, larger particles than traditional affinity resins, and lower cost, so it is easier to apply on a large scale and has better prospects for industrial production. On the other hand, the carrier composed of it has better compatibility with enzymes, can retain enzyme activity to the greatest extent while immobilizing, and has higher stability for repeated use of enzymes.
- the above resin sources are as follows:
- the metal ion is nickel ion, iron ion, copper ion or cobalt ion, more preferably the metal ion is nickel ion, copper ion or cobalt ion.
- the present invention also provides an immobilized enzyme, which comprises the above-mentioned enzyme immobilized carrier and the enzyme immobilized thereon.
- the carrier of the present invention has better compatibility with enzymes, better immobilization effect, and can retain enzyme activity to the greatest extent while immobilized. Furthermore, the immobilized enzyme obtained based on its immobilization has better activity and higher stability during repeated use, and is more suitable for application in industrial immobilized enzymes.
- the immobilized enzyme of the present invention has wide applicability to enzymes, for example, enzymes include but not limited to transaminase, ketoreductase, alcohol dehydrogenase, formate dehydrogenase, glucose dehydrogenase, monooxygenase, ene reduction Any one or more of enzymes, imine reductases and amino acid dehydrogenases; wherein the following enzymes are more suitable, the transaminases are transaminases derived from Chromobacterium violaceum DSM30191, or derived from Arthrobacter citreus, or derived from The transaminase of Actinobacteria, or the transaminase derived from Sciscionella sp.SE31; the ketoreductase is the carbonyl reductase derived from Acetobacter sp.CCTCC M209061, or the ketoreductase derived from Sporobolomyces salmonicolor; the alcohol dehydrogena
- the matrix resin is one or more of LX-1000HA, LX-1000EPN, LX-EPHA or LX-109s;
- the matrix resin is one or more of LX-1000EPN, LX-109s, LX-1000HFA, ECR8285 or EC-HFA;
- the matrix resin is LX- One or more of 109s, LX-EPHA, ECR8285, LX-1000HA or ECR8409;
- the matrix resin is a monooxygenase, the matrix resin is one or more of LX-109s, LX-EPHA or LX-1000HA species;
- the enzyme is imine reductase, the matrix resin is LX-109s and/or LX-EPHA; when the enzyme is amino acid dehydrogenase, alcohol dehydrogen
- the present invention also provides a method for preparing the above-mentioned enzyme immobilized carrier, comprising the following steps: providing a resin sphere matrix; connecting -N(CH 2 COOH) 2 groups through chemical bonds on the resin sphere matrix; 2 COOH) 2 groups adsorb metal ions through coordination to obtain enzyme immobilized carriers.
- the carrier of the present invention has better compatibility with enzymes, better immobilization effect, and can retain enzyme activity to the greatest extent while immobilized. Furthermore, the immobilized enzyme obtained based on its immobilization has better activity and higher stability during repeated use, and is more suitable for application in industrial immobilized enzymes.
- the resin ball matrix is an amino-type methacrylic resin
- the amino-type methacrylic resin and sodium chloroacetate are mixed to carry out a nucleophilic substitution reaction to link the amino-type methacrylic resin -N(CH 2 COOH) 2 group.
- the alkali aqueous solution is sodium carbonate aqueous solution, dilute sodium hydroxide solution or lithium hydroxide solution. Based on this, the conversion rate of the amino-type methacrylic resin ball matrix of the present invention is higher, and the reaction process is more stable.
- the resin ball matrix is an epoxy-based methacrylic resin
- the epoxy-based methacrylic resin and disodium diacetate imide are mixed for an addition reaction to -N(CH 2 COOH) 2 groups are linked on the base type methacrylic resin. More preferably, stir the aqueous solution of epoxy-based methacrylic resin and disodium diacetate imide at 20-25°C for 30-60 minutes, then raise the temperature to 60-70°C, and react under N2 atmosphere for 18- 24h to link -N(CH 2 COOH) 2 groups on the epoxy-based methacrylic resin. Based on this, the conversion rate of the epoxy-based methacrylic resin ball matrix of the present invention is higher, and the reaction process is more stable.
- a metal salt solution is added to the reaction system to carry out a complexation reaction, so that -N(CH 2 COOH) 2 groups adsorb metal ions through coordination to obtain enzyme immobilized carriers.
- the reaction temperature is 2-30° C., and the reaction time is 1-4 hours; the metal salt solution is nickel chloride, copper sulfate, ferrous chloride, cobalt chloride and the like. Based on this, the reaction conditions are more suitable, which promotes higher yield and higher purity of the enzyme-immobilized carrier obtained from the reaction. More preferably, the mass ratio of the metal ion to the resin ball matrix in the metal salt solution is (0.05-0.1):1.
- the mass ratio of sodium chloroacetate to amino-type methacrylic resin is preferably (5-10):1.
- the mass ratio of disodium diacetate imide to epoxy-based methacrylic resin is preferably (5-10):1.
- the present invention also provides a method for preparing the above-mentioned immobilized enzyme, comprising the following steps: performing an immobilization reaction on the above-mentioned enzyme-immobilized carrier and the enzyme to obtain the immobilized enzyme.
- the carrier of the present invention has better compatibility with enzymes, better immobilization effect, and can retain enzyme activity to the greatest extent while immobilized. Furthermore, the immobilized enzyme obtained based on its immobilization has better activity and higher stability during repeated use, and is more suitable for application in industrial immobilized enzymes.
- the preparation method of immobilized enzyme comprises the following steps: dispersing the enzyme-immobilized carrier in a first solvent to form a dispersion; wherein, the first solvent is a mixed solution of phosphate buffer, sodium chloride aqueous solution and imidazole buffer.
- the dispersion liquid is reacted with the enzyme solution containing the enzyme, so that the enzyme and the enzyme-immobilized carrier undergo an immobilization reaction to obtain the immobilized enzyme.
- the first solvent creates a milder and more suitable environment for immobilization, which can protect the activity of the enzyme, enhance the immobilization effect of the enzyme, and prevent non-target proteins in the enzyme solution from being immobilized. Based on this, the compatibility of the above-mentioned carrier to the enzyme is further improved, and the enzyme immobilization effect is better and the activity is better.
- the concentration of phosphate buffer is 0.1 ⁇ 0.2mol/L; preferably, the concentration of sodium chloride aqueous solution is 0.5 ⁇ 1mol/L; preferably Preferably, the concentration of the imidazole buffer solution is 0.05-0.1 mol/L.
- the reaction temperature is 20-25° C.
- the reaction time is 16-24 hours.
- each gram of the enzyme-immobilized carrier corresponds to 4-8 mL of enzyme solution, and the protein content in the enzyme solution is 20-25 mg/mL. This is beneficial to promote the immobilization of the enzyme more fully, provide a loading capacity, and enable the immobilized enzyme to have higher catalytic activity.
- Immobilization of transaminase on epoxy-based methacrylic resin Weigh 1 g of epoxy-based resin and wash with 0.2M phosphate buffer (pH7.0) and 0.8M NaCl buffer for several times, remove the buffer, and keep the resin for use . Then add 4mL enzyme solution (with cofactor pyridoxal phosphate PLP) prepared by 0.2M phosphate buffer (pH7.0) and 0.8MNaCl buffer and 0.1g enzyme powder, and incubate at 20°C for 36-48h, Remove buffer. Wash 3 times with 0.1M phosphate buffer (pH 7.0) and 0.5M NaCl buffer, remove the buffer, and keep the immobilized enzyme for use.
- enzyme solution with cofactor pyridoxal phosphate PLP
- Immobilization of transaminase on amino-type methacrylic resin Weigh 1 g of amino-type resin and wash it with 0.1M phosphate buffer 7.0 for several times, remove the buffer, add 0.1M phosphate buffer (pH7.0) and the final concentration is 2 % glutaraldehyde solution. Then add 4mL enzyme solution (with cofactor PLP) prepared by 0.1M phosphate buffer 7.0 and 0.1g enzyme powder, and incubate at 20°C for 16-24h, and remove the buffer. Wash 3 times with 20 mM phosphate buffer 7.0 containing 0.5 M NaCl, remove the buffer, and keep the immobilized enzyme for use.
- Example 1 The difference from Example 1 is that the equivalent mass of transaminase is replaced by formate dehydrogenase, and the cofactor is replaced by equal mass of NAD + .
- ketoreductase Immobilization and application of ketoreductase on enzyme-immobilized carrier.
- Example 1 The difference from Example 1 is that the same amount of transaminase is replaced by ketoreductase, and the cofactor is replaced by an equal amount of NAD + .
- Example 1 The difference from Example 1 is that the same amount of transaminase is replaced by monooxygenase, and the cofactor is replaced by equal mass of NADP + .
- Example 1 The difference from Example 1 is that the same amount of transaminase is replaced by imine reductase, and the cofactor is replaced by NAD + of equal mass.
- Example 1 The difference from Example 1 is that the same amount of transaminase is replaced by glutamate dehydrogenase, and the cofactor is replaced by equal mass of NADP + .
- Example 1 The difference from Example 1 is that the same amount of transaminase is replaced by alcohol dehydrogenase, and the cofactor is replaced by an equal amount of NADP + .
- Example 1 The difference from Example 1 is that the same amount of transaminase is replaced by ene reductase, and the cofactor is replaced by an equal amount of NAD(P) + .
- Example 1 The difference from Example 1 is that the transaminase is replaced by amino acid dehydrogenase in equal amount, and the cofactor is replaced by NAD + in equal mass.
- the immobilization and application of transaminases on amino- and epoxy-type methacrylic resins modified with the affinity of different metal ions differ from Example 1 only in that the equimolar amounts of nickel ions are replaced by cobalt ions and copper ions, respectively.
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Abstract
Description
品牌 | 树脂名称 | 基质 | 官能团 |
蓝晓科技 | LX-1000HA | 甲基丙烯酸酯 | 长链氨基(C6) |
蓝晓科技 | LX-1000EPN | 甲基丙烯酸酯 | 短链氨基(C2) |
蓝晓科技 | LX-1000EA | 甲基丙烯酸酯 | 短链氨基(C2) |
蓝晓科技 | LX-EPHA | 甲基丙烯酸酯 | 短链氨基(C2) |
漂莱特 | ECR8309 | 甲基丙烯酸酯 | 短链氨基(C2) |
漂莱特 | ECR8409 | 甲基丙烯酸酯 | 长链氨基(C6) |
南开和成 | ESR-1 | 甲基丙烯酸酯 | 氨基(亲水) |
南开和成 | ESR-3 | 甲基丙烯酸酯 | 氨基(憎水) |
南开和成 | ESQ-1 | 甲基丙烯酸酯 | 氨基(憎水) |
蓝晓科技 | LX-1000EP | 甲基丙烯酸酯 | 环氧基 |
蓝晓科技 | LX-103B | 甲基丙烯酸酯 | 环氧基 |
蓝晓科技 | EP200 | 甲基丙烯酸酯 | 环氧基 |
蓝晓科技 | LX-107B | 甲基丙烯酸酯 | 环氧基 |
蓝晓科技 | LX-1000SW | 甲基丙烯酸酯 | 环氧基 |
蓝晓科技 | LX-1000SD | 甲基丙烯酸酯 | 环氧基 |
蓝晓科技 | LX-109s | 甲基丙烯酸酯 | 环氧基 |
蓝晓科技 | LX-1000HFA | 甲基丙烯酸酯 | 氨基-环氧基 |
漂莱特 | ECR8285 | 甲基丙烯酸酯 | 环氧基 |
漂莱特 | ECR8204 | 甲基丙烯酸酯 | 环氧基 |
漂莱特 | ECR8209 | 甲基丙烯酸酯 | 环氧基 |
南开和成 | ES-1 | 甲基丙烯酸酯 | 环氧基(亲水) |
南开和成 | ES103 | 甲基丙烯酸酯 | 环氧基(憎水) |
三菱化工 | ES-101 | 甲基丙烯酸酯 | 环氧基(憎水) |
三菱化工 | HFA403 | 甲基丙烯酸酯 | 环氧基 |
三菱化工 | EC-HFA | 甲基丙烯酸酯 | 环氧基 |
品牌 | 树脂名称 | 基质 | 官能团 |
漂莱特 | MIDA-Ni | 甲基丙烯酸酯 | Ni + |
伯乐 | IMAC-Ni | 甲基丙烯酸酯 | Ni + |
Claims (16)
- 一种酶固定化载体,其特征在于,所述酶固定化载体包含树脂球基体、通过化学键链接在所述树脂球基体上的-N(CH 2COOH) 2基团、以及通过配位作用螯合吸附在所述-N(CH 2COOH) 2基团上的金属离子;其中,所述树脂球基体为氨基型甲基丙烯酸树脂或环氧基型甲基丙烯酸树脂,所述树脂球基体的粒径为200~700μm。
- 根据权利要求1所述的酶固定化载体,其特征在于,所述氨基型甲基丙烯酸树脂具有C2长度或C6长度碳链手臂的氨基官能团,且氨基含量为30~80μmol/g;优选地,所述氨基型甲基丙烯酸树脂为 Lifetech TMECR8309、Lifetech TMECR8409、ESR-1、ESR-3或ESQ-1中的一种或多种;更优选所述氨基型甲基丙烯酸树脂为 或Lifetech TMECR8309中的一种或多种;优选地,所述环氧基型甲基丙烯酸树脂的环氧当量为2~5μmol/g;更优选地,所述环氧基型甲基丙烯酸树脂为 EP200、 Lifetech TMECR8285、Lifetech TMECR8204、Lifetech TMECR8209、ES-1、ES103、ES-101、ReliZyme TMHFA403或ReliZyme TMEC-HFA中的一种或多种;更优选所述环氧基型甲基丙烯酸树脂为 或ReliZyme TMEC-HFA中的一种或多种;优选地,所述金属离子为镍离子、铁离子、铜离子或钴离子。
- 一种固定化酶,其特征在于,所述固定化酶包括权利要求1或2所述的酶固定化载体和固定在其上的酶。
- 根据权利要求3所述的固定化酶,其特征在于,所述酶选自转氨酶、酮还原酶、醇脱氢酶、甲酸脱氢酶、葡萄糖脱氢酶、单加氧酶、烯还原酶、亚胺还原酶和氨基酸脱氢酶中的任意一种或多种;其中,所述转氨酶为来源于Chromobacterium violaceum DSM30191的转氨酶,或者来源于Arthrobacter citreus的转氨酶,或者来源于Actinobacteria的转氨酶,或者来源于Sciscionella sp.SE31的转氨酶;所述酮还原酶为来源于Acetobacter sp.CCTCC M209061的羰基还原酶,或者来源于Sporobolomyces salmonicolor的酮还原酶;所述醇脱氢酶为来源于Thermoanaerobium brockii的醇脱氢酶;所述甲酸脱氢酶为来源于Candida boidinii的甲酸脱氢酶;所述葡萄糖脱氢酶为来源于Lysinibacillus sphaericus G10的葡萄糖脱氢酶;所述单加氧酶为来源于Rhodococcus sp.Phi1的环己酮单加氧酶,或者来源于Rhodococcus ruber-SD1的环己酮单加氧酶,或者来源于Brachymonas petroleovorans的环己酮单加氧酶;所述烯还原酶为来源于Saccharomyces cerevisiae的烯还原酶,或者来源于Chryseobacterium sp.CA49的烯还原酶;所述亚胺还原酶为来源于Streptomyces sp.的亚胺还原酶,或者来源于Bacillus cereus的亚胺还原酶;所述氨基酸脱氢酶为来源于Bacillus cereus的亮氨酸脱氢酶,或者来源于Bacillus sphaericus的苯丙氨酸脱氢酶,或者来源于Thermoactinomyces intermedius ATCC33205的氨基酸脱氢酶,或者来源于Thermosyntropha lipolytica的氨基酸脱氢酶。
- 一种权利要求1或2所述的酶固定化载体的制备方法,其特征在于,包括以下步骤:提供树脂球基体;在所述树脂球基体上通过化学键链接-N(CH 2COOH) 2基团;在所述-N(CH 2COOH) 2基团上通过配位作用螯合吸附金属离子,得到所述酶固定化载体;其中,所述树脂球基体为氨基型甲基丙烯酸树脂或环氧基型甲基丙烯酸树脂,所述树脂球基体的粒径为200~700μm。
- 根据权利要求5所述的酶固定化载体的制备方法,其特征在于,所述树脂球基体为氨基型甲基丙烯酸树脂时,将所述氨基型甲基丙烯酸树脂和氯乙酸钠混合,进行亲核取代反应,以在所述氨基型甲基丙烯酸树脂上链接所述-N(CH 2COOH) 2基团。
- 根据权利要求6所述的酶固定化载体的制备方法,其特征在于,将所述氨基型甲基丙烯酸树脂和所述氯乙酸钠的水溶液在20~25℃下搅拌30~60min后,用1~2mol/L的碱水溶液调节反应体系pH为9~10,然后升温至70~80℃,并在N 2氛围下反应20~30h,以在所述氨基型甲基丙烯酸树脂上链接所述-N(CH 2COOH) 2基团;优选地,所述碱水溶液为碳酸钠水溶液、氢氧化钠溶液或氢氧化锂溶液。
- 根据权利要求5所述的酶固定化载体的制备方法,其特征在于,所述树脂球基体为环氧基型甲基丙烯酸树脂时,将所述环氧基型甲基丙烯酸树脂和二乙酸亚胺二钠混合,进行加成反应,以在所述环氧基型甲基丙烯酸树脂上链接所述-N(CH 2COOH) 2基团。
- 根据权利要求8所述的酶固定化载体的制备方法,其特征在于,将所述环氧基型甲基丙烯酸树脂和所述二乙酸亚胺二钠的水溶液在20~25℃下搅拌30~60min后,升温至60~70℃,并在N 2氛围下反应18~24h,以在所述环氧基型甲基丙烯酸树脂上链接所述 -N(CH 2COOH) 2基团。
- 根据权利要求5至9中任一项所述的酶固定化载体的制备方法,其特征在于,在所述树脂球基体上通过化学键链接所述-N(CH 2COOH) 2基团的步骤后,在反应体系中加入金属盐溶液,进行络合反应,以在所述-N(CH 2COOH) 2基团上通过配位作用螯合吸附所述金属离子,得到所述酶固定化载体;优选地,所述络合反应过程中,反应温度为20~30℃,反应时间为1~4h;优选地,所述金属盐溶液为氯化镍水溶液、硫酸铜水溶液、氯化亚铁水溶液或氯化钴水溶液;优选地,所述金属盐溶液中金属离子和所述树脂球基体的质量比为(0.05~0.1):1。
- 根据权利要求6所述的酶固定化载体的制备方法,其特征在于,所述氯乙酸钠与所述氨基型甲基丙烯酸树脂球的质量比为(5~10):1。
- 根据权利要求8所述的酶固定化载体的制备方法,其特征在于,所述二乙酸亚胺二钠与所述环氧基型甲基丙烯酸树脂球的质量比为(5~10):1。
- 一种权利要求3或4所述的固定化酶的制备方法,其特征在于,包括以下步骤:将权利要求1或2所述的酶固定化载体和酶进行固定化反应,得到所述固定化酶。
- 根据权利要求13所述的固定化酶的制备方法,其特征在于,所述固定化酶的制备方法包括以下步骤:将所述酶固定化载体分散于第一溶剂中形成分散液;其中,所述第一溶剂为磷酸缓冲液、氯化钠水溶液及咪唑缓冲液的混合溶液;将所述分散液与含所述酶的酶液进行反应,以使所述酶与所述酶固定化载体进行所述固定化反应,得到所述固定化酶。
- 根据权利要求14所述的固定化酶的制备方法,其特征在于,所述第一溶剂中,所述磷酸缓冲液的浓度为0.1~0.2mol/L;所述氯化钠水溶液的浓度为0.5~1mol/L;所述咪唑缓冲液的浓度为0.05~0.1mol/L。
- 根据权利要求14所述的固定化酶的制备方法,其特征在于,所述分散液与所述酶液进行反应的过程中,反应温度为20~25℃,反应时间为16~24h;优选地,每克所述酶固定化载体与4~8mL的所述酶液进行反应,且所述酶液中的蛋白含量为20~25mg/mL。
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