Patent Application
20250236861
The present application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy is named_Sequence_Listing.txt and is 40,979 bytes in size, and the Sequence listing is identical to the international application No. PCT/CN2021/131878 filed on Nov. 19, 2021.
The present disclosure relates to the field of biological catalysis, and specifically, to an enzyme immobilization carrier and a preparation method therefor, and an immobilized enzyme and a preparation method therefor.
Biological catalysis has become an important part of green synthetic drugs, and is one of the most promising and applied technologies for catalytic synthesis of drug structure units and intermediates, especially for chiral synthesis, which provides a unique and irreplaceable method. Enzymes are increasingly used as catalysts in industrial processes. Since conditions of using the enzymes are mild, and the enzymes are easy to vary, requirements of the enzymes for environments are very strict, and the enzymes are difficult to recycle, such that the application of the enzymes in industry is greatly limited. The development and application of an immobilized enzyme technology may effectively solve these problems, such that an immobilized enzyme, which is often called “a biological catalyst”, is widely used in industrial organic synthesis and bioconversion.
An enzyme immobilization method may be classified into a physical method and a chemical method. The physical method mainly includes an adsorption method and an embedding method; and the chemical method mainly includes a binding method and a cross-linking method. The adsorption method is also called a non-covalent method, which is an immobilization method in which an enzyme is bound to an adsorbent medium mainly through interactions of several non-covalent bonds, such as a hydrogen bond, Van Der Waals force, hydrophobic interaction, and an ionic bond. Commonly used inorganic adsorption materials include silica gel, alumina, porous glass, diatomaceous earth, etc.; commonly used organic adsorption materials mainly include natural alginate, chitin, chitosan, cellulose, and starch; and synthetic organic materials include polyurethane, macroporous resins, etc. The embedding method is to embed free enzymes into the pores of a medium such as specific gel, to achieve immobilization, for example, performing embedding by using gel media such as polyacrylamide and calcium alginate. The embedding method is simpler, an embedded enzyme protein structure may remain basically unchanged, and the loss of enzyme activity is relatively small, but there are certain disadvantages, such as the leakage of enzyme components and enzyme inactivation. The binding method, also known as covalent immobilization, is based on a reaction between an effective functional group of a carrier material and an enzyme-related functional group (for example, enzyme-protein side-chain groups such as amino, carboxyl, sulfhydryl, and hydroxyl), to achieve mutual binding. The covalent immobilization makes the binding between the enzyme and the carrier stronger, and a strong chemical bond generated between the enzyme and the carrier may significantly reduce the loss of the enzyme, such that the reuse rate of the enzyme is improved. However, the method is strict in condition, intense in reaction, and large in enzyme activity loss (typical residual enzyme activity is around 30%). The cross-linking method refers to a method of performing a cross-linking reaction between enzyme molecules, and between the enzyme molecule and the carrier by means of a bifunctional or multifunctional reagent (a cross-linking reagent), to form a covalent bond for enzyme immobilization. Commonly used cross-linking reagents include glutaraldehyde, hexamethylene diamine, maleic anhydride, double azobenzene, etc. More research has been done on Cross-Linked Enzyme Aggregates (CLEA), which are formed by cross-linking the enzymes using a cross-linking agent, such as CLEA immobilization of a phenylalanine ammonia lyase, and CLEA immobilization of a sucrose phosphorylase. To sum up, the traditional immobilization methods have many disadvantages, for example, adsorption-immobilized enzymes are easily lost, substrates and products of the enzymes after embedding immobilization are uneasy to disperse, a covalent bonding method easily leads to enzyme inactivation, and the mechanical properties of cross-linked enzymes are relatively poor.
However, most of commercial affinity resins are protein purification resins, of which matrices are usually cellulose, cross-linked dextran, agarose, polyacrylamide, porous glass beads, etc., and agarose gels are the most widely used. For example, the small particle size of a metal-chelating affinity resin makes it difficult to recycle for large-scale application, and particles are easily broken under high shear, such that the resin is not easy to be applied in a traditional stirred tank reaction mode. Meanwhile, the matrix of the resin is very hydrophilic, and has a high requirement for a storage condition, such that the resin needs to be stored in an alcohol solution, and avoids drying as much as possible, causing the resin to be unsuitable for use as an immobilized enzyme. In addition, the industrial immobilized enzyme application of the resin is greatly affected due to high cost.
Therefore, it is necessary to provide a new enzyme immobilization carrier with better compatibility to enzymes and better immobilization effects. In addition, in the chemical reaction application of an immobilized enzyme, the activity of the enzyme is reserved to a great extent, and the enzyme has higher stability during reuse.
The present disclosure is mainly intended to provide an enzyme immobilization carrier and a preparation method therefor, and an immobilized enzyme and a preparation method therefor, so as to solve the problems in the related art that an enzyme immobilization carrier is relatively poor in enzyme compatibility and poor in immobilization effect, and an immobilized enzyme is relatively poor in activity, and relatively poor in stability during repeated use.
In order to implement the above objectives, an aspect of the present disclosure provides an enzyme immobilization carrier. The enzyme immobilization carrier includes a resin ball matrix, an —N(CH2COOH)2 group linked on the resin ball matrix by means of a chemical bond, and a metal ion adsorbed on the —N(CH2COOH)2 group in a chelating manner through coordination. The resin ball matrix is an amino-type methacrylic resin or an epoxy-type methacrylic resin, and the resin ball matrix has a particle size of 200-700 km.
Further, in the amino-type methacrylic resin, the amino-type methacrylic resin has an amino functional group with a C2 or C6 length carbon chain arm, and the content of the amino is 30-80 μmol/g. Preferably, the amino-type methacrylic resin is one or more of Seplite®LX-1000HA, Seplite®LX-1000EPN, Seplite®LX-EPHA, Seplite®LX-1000EA, Lifetech™ECR8309, Lifetech™ECR8409, ESR-1, ESR-3, or ESQ-1. More preferably, the amino-type methacrylic resin is one or more of Seplite®LX-1000HA, Seplite®LX-1000EPN, Seplite®LX-EPHA, or Lifetech™ECR8309. Preferably, the epoxy-type methacrylic resin has an epoxide equivalent of 2-5 μmol/g. More preferably, the epoxy-type methacrylic resin is one or more of Seplite®LX-1000EP, Seplite®LX-103B, EP200, Seplite®LX-107B, Seplite®LX-1000SW, Seplite®LX-1000SD, Seplite®LX-109s, Seplite®LX-1000HFA, Lifetech™ECR8285, Lifetech™ECR8204, Lifetech™ECR8209, ES-1, ES103, ES-101, ReliZyme™HFA403, or ReliZyme™EC-HFA. More preferably, the epoxy-type methacrylic resin is one or more of Seplite®LX-1000EP, Seplite®LX-109s, Seplite®LX-1000HFA, or ReliZyme™EC-HFA. Preferably, the metal ion is a nickel ion, an iron ion, a copper ion, or a cobalt ion.
In order to implement the above objectives, an aspect of the present disclosure provides an immobilized enzyme. The immobilized enzyme includes the enzyme immobilization carrier, and an enzyme immobilized thereon.
Further, the enzyme is selected from any one or more of a transaminase, a ketoreductase, an alcohol dehydrogenase, a formate dehydrogenase (FDH), a glucose dehydrogenase, a monooxygenase, an ene-reductase, an imine reductase, and an amino acid dehydrogenase. The transaminase is a transaminase derived from Chromobacterium violaceum DSM30191, or a transaminase derived from Arthrobacter citreus, or a transaminase derived from Actinobacteria, or a transaminase derived from Sciscionella sp. SE31. The ketoreductase is a carbonyl reductase derived from Acetobacter sp. CCTCCM209061, or a ketoreductase derived from Sporobolomyces salmonicolor. The alcohol dehydrogenase is derived from Thermoanaerobium brockii. The FDH is an FDH derived from Candida boidinii. The glucose dehydrogenase is a glucose dehydrogenase derived from Lysinibacillus sphaericus G10. The monooxygenase is a cyclohexanone monooxygenase derived from Rhodococcus sp. Phi1, or a cyclohexanone monooxygenase derived from Rhodococcus ruber-SD1, or a cyclohexanone monooxygenase derived from Brachymonas petroleovorans. The ene-reductase is an ene-reductase derived from Saccharomyces cerevisiae, or an ene-reductase derived from Chryseobacterium sp. CA49. The imine reductase is an imine reductase derived from Streptomyces sp., or an imine reductase derived from Bacillus cereus. The amino acid dehydrogenase is a leucine dehydrogenase derived from Bacillus cereus, or a phenylalanine dehydrogenase derived from Bacillus sphaericus; or an amino acid dehydrogenase derived from Thermoactinomyces intermedius ATCC33205, or an amino acid dehydrogenase derived from Thermosyntropha lipolytica.
Another aspect of the present disclosure provides a method for preparing the enzyme immobilization carrier. The method includes the following steps: providing a resin ball matrix; linking an —N(CH2COOH)2 group onto the resin ball matrix by means of a chemical bond; absorbing a metal ion onto the —N(CH2COOH)2 group in a chelating manner through coordination, so as to obtain the enzyme immobilization carrier. The resin ball matrix is an amino-type methacrylic resin or an epoxy-type methacrylic resin, and the resin ball matrix has a particle size of 200-700 m.
Further, when the resin ball matrix is the amino-type methacrylic resin, the amino-type methacrylic resin is mixed with sodium chloroacetate for nucleophilic substitution, so as to link the —N(CH2COOH)2 group onto the amino-type methacrylic resin.
Further, after an aqueous solution of the amino-type methacrylic resin and the sodium chloroacetate is stirred for 30-60 min at 20-25° C., the reaction system is regulated to a pH of 9-10 with 1-2 mol/L of an aqueous alkaline solution, and then heated up to 70-80° C. for reaction for 20-30 h at an N2 atmosphere, so as to link the —N(CH2COOH)2 group onto the amino-type methacrylic resin. Preferably, the aqueous alkali solution is a sodium carbonate solution, a sodium hydroxide solution, or a lithium hydroxide solution.
Further, when the resin ball matrix is the epoxy-type methacrylic resin, the epoxy-type methacrylic resin is mixed with Iminodiacetic acid disodium for an addition reaction, so as to link the —N(CH2COOH)2 group onto the epoxy-type methacrylic resin.
Further, after an aqueous solution of the epoxy-type methacrylic resin and the Iminodiacetic acid disodium is stirred for 30-60 min at 20-25° C., the reaction system is heated up to 60-70° C. for reaction for 18-24 h at an N2 atmosphere, so as to link the —N(CH2COOH)2 group onto the epoxy-type methacrylic resin.
Further, after the step of linking the —N(CH2COOH)2 group onto the resin ball matrix by means of the chemical bond, a metal salt solution is added to the reaction system for a complex reaction, so as to absorb the metal ion on the —N(CH2COOH)2 group in the chelating manner through coordination, thereby obtaining the enzyme immobilization carrier. Preferably, during the complex reaction, a reaction temperature is 20-30° C. and reaction time is 1-4 h. Preferably, the metal salt solution is an aqueous solution of nickel chloride, an aqueous solution of copper sulfate, an aqueous solution of ferrous chloride, or an aqueous solution of cobalt chloride. Preferably, in the metal salt solution, a mass ratio of the metal ion to the resin ball matrix is (0.05-0.1):1.
Further, a mass ratio of the sodium chloroacetate to the amino-type methacrylic resin ball is (5-10):1.
Further, a mass ratio of the Iminodiacetic acid disodium to the epoxy-type methacrylic resin ball is (5-10):1.
Another aspect of the present disclosure provides a method for preparing the immobilized enzyme. The method includes the following step: performing an immobilization reaction on the enzyme immobilization carrier, and an enzyme, so as to obtain the immobilized enzyme.
Further, the method for preparing the immobilized enzyme includes the following steps: dispersing the enzyme immobilization carrier into a first solvent to form dispersion liquid, where the first solvent is a mixed solution of a phosphate buffer solution, a sodium chloride aqueous solution, and an imidazole buffer solution; and reacting the dispersion liquid with an enzyme solution containing the enzyme, to cause the enzyme and the enzyme immobilization carrier to be subjected to an immobilization reaction, so as to obtain the immobilized enzyme.
Further, in the first solvent, the phosphate buffer solution has the concentration of 0.1-0.2 mol/L; the sodium chloride aqueous solution has the concentration of 0.5-1 mol/L; and the imidazole buffer solution has the concentration of 0.05-0.1 mol/L.
Further, during the reaction process of the dispersion liquid and the enzyme solution, a reaction temperature is 20-25° C., and a reaction time is 16-24 h. Preferably, per gram of the enzyme immobilization carrier is reacted with 4-8 mL of the enzyme solution, and the enzyme solution has a protein content of 20-25 mg/mL.
In the carrier of the present disclosure, the amino-type methacrylic resin or the epoxy-type methacrylic resin is used as the resin ball matrix; the —N(CH2COOH)2 group is linked onto the resin ball matrix by means of the chemical bond; and the metal ion adsorbed on the —N(CH2COOH)2 group through coordination is also linked to the resin ball matrix. By using the carrier, first, the particle size (200-700 μm) of the resin ball matrix is larger than that (20-80 μm) of traditional affinity resin, such that better repeatable recyclability is achieved. In addition, the resin ball matrix has higher mechanical strength than general affinity resins, so as to prevent the resin ball matrix from being broken under high shear, such that under a traditional stirred tank reaction mode, the immobilized enzyme is longer in service life, and better in repeatable recyclability. Second, compared with a general covalent binding carrier, for a formed metal-chelating affinity structure containing a nitrogen diacetate group and the metal ion, the carrier only binds to a His tag at the end of the protein, such that the carrier is better in enzyme compatibility, the activity of the enzyme can be reserved to a great extent during immobilization, and the stability of the enzyme during reuse is higher. In addition, the resin of the above type is lower in cost. Based on this, the carrier of the present disclosure is better in enzyme compatibility and better in immobilization effect, and the activity of the enzyme can be reserved to a great extent during immobilization. Therefore, the immobilized enzyme obtained through immobilization is better in activity and higher in stability during reuse, and is more suitable for application in industrialized immobilized enzymes. Based on this, the present disclosure effectively solves the problems in the related art that an enzyme immobilization carrier is relatively poor in enzyme compatibility and poor in immobilization effect, and an immobilized enzyme is relatively poor in activity, and relatively poor in stability during repeated use.
It is to be noted that the embodiments in the present application and the features in the embodiments may be combined with one another without conflict. The present disclosure will be described below in detail with reference to the embodiments.
As described in the Background, the problems that an enzyme immobilization carrier is relatively poor in enzyme compatibility and poor in immobilization effect, and an immobilized enzyme is relatively poor in activity, and relatively poor in stability during repeated use exist in the related art.
In order to solve the above problems, the present disclosure provides an enzyme immobilization carrier. The enzyme immobilization carrier includes a resin ball matrix, an —N(CH2COOH)2 group linked on the resin ball matrix by means of a chemical bond, and a metal ion adsorbed on the —N(CH2COOH)2 group in a chelating manner through coordination. The resin ball matrix is an amino-type methacrylic resin or an epoxy-type methacrylic resin, and the resin ball matrix has a particle size of 200-700 μm.
In the carrier of the present disclosure, the amino-type methacrylic resin or the epoxy-type methacrylic resin is used as the resin ball matrix; the —N(CH2COOH)2 group is linked onto the resin ball matrix by means of the chemical bond; and the metal ion adsorbed on the —N(CH2COOH)2 group through coordination is also linked to the resin ball matrix. By using the carrier, in the subsequent reaction process of the immobilized enzyme, a His tag at the end of the enzyme may be adsorbed on the metal ion through coordination. Specifically, the N(CH2COOH)2 group and the metal ion form a coordinate bond. However, the —N(CH2COOH)2 group does not completely occupy the coordination site of the metal ion, the enzyme with the His tag continues to occupy the remaining coordination sites, which are not occupied, on the metal ion, and the immobilized enzyme is formed with the carrier through coordination. Compared with covalent bonding, a chemical reaction between an enzyme body and the carrier leads to destruction of the enzyme body, and the rigidity of the immobilized enzyme becomes stronger, resulting in lower enzyme activity. However, unlike the above, in the present disclosure, by using the form of coordination binding of the His tag at the end of the enzyme and the metal ion in the carrier, there is no reaction contact between the carrier and the enzyme body, such that the loss of enzyme activity is small.
In particular, the resin ball matrix of the present disclosure is an amino-type methacrylic resin and/or an epoxy-type methacrylic resin. Based on this, first, the particle size of the resin ball matrix is larger than that of traditional affinity resin, such that better repeatable recyclability is achieved. In addition, the resin ball matrix has higher mechanical strength, so as to prevent the resin ball matrix from being broken under high shear, such that under a traditional stirred tank reaction mode, the immobilized enzyme is longer in service life, and better in repeatable recyclability. Second, the formed carrier is better in enzyme compatibility, the activity of the enzyme can be reserved to a great extent during immobilization, and the stability of the enzyme during reuse is higher. In addition, the resin of the above type is lower in cost. Based on this, the carrier of the present disclosure is better in enzyme compatibility and better in immobilization effect, and the activity of the enzyme can be reserved to a great extent during immobilization. Therefore, the immobilized enzyme obtained through immobilization is better in activity and higher in stability during reuse, and is more suitable for application in industrialized immobilized enzymes.
In conclusion, the present disclosure effectively solves the problems in the related art that an enzyme immobilization carrier is relatively poor in enzyme compatibility and poor in immobilization effect, and an immobilized enzyme is relatively poor in activity, and relatively poor in stability during repeated use.
Preferably, the amino-type methacrylic resin has an amino functional group with a C2 or C6 length carbon chain arm, and a content of the amino is 30-80 μmol/g. Preferably, the amino-type methacrylic resin is one or more of Seplite®LX-1000HA, Seplite®LX-1000EPN, Seplite®LX-EPHA, Seplite®LX-1000EA, Lifetech™ECR8309, Lifetech™ECR8409, ESR-1, ESR-3, or ESQ-1. More preferably, the amino-type methacrylic resin is one or more of Seplite®LX-1000HA, Seplite®LX-1000EPN, Seplite®LX-EPHA, or Lifetech™ECR8309.
Preferably, the epoxy-type methacrylic resin has an epoxide equivalent of 2-5 μmol/g. More preferably, the epoxy-type methacrylic resin is one or more of Seplite®LX-1000EP, Seplite®LX-103B, EP200, Seplite®LX-107B, Seplite®LX-1000SW, Seplite®LX-1000SD, Seplite®LX-109s, Seplite®LX-1000HFA, Lifetech™ECR8285, Lifetech™ECR8204, LifetechTMECR8209, ES-1, ES103, ES-101, ReliZyme™HFA403, or ReliZyme™EC-HFA. More preferably, the epoxy-type methacrylic resin is one or more of Seplite®LX-1000EP, Seplite®LX-109s, Seplite® LX-1000HFA, or ReliZyme™EC-HFA.
The resin ball matrix is selected from the above types. On the one hand, the resin ball matrix is higher in mechanical strength, larger in particle than that of the traditional affinity resin, and lower in cost, such that the resin ball matrix is more liable to apply on a large scale, and has better prospects in industrial production. On the other hand, the formed carrier is better in enzyme compatibility, the activity of the enzyme can be reserved to a great extent during immobilization, and the stability of the enzyme during reuse is higher. Sources of the resins are shown below:
In order to further improve the bonding stability of the enzyme and the carrier, preferably, the metal ion is a nickel ion, an iron ion, a copper ion, or a cobalt ion; and more preferably, the metal ion is the nickel ion, the copper ion, or the cobalt ion.
The present disclosure further provides an immobilized enzyme. The immobilized enzyme includes the enzyme immobilization carrier, and an enzyme immobilized thereon.
Based on various reasons mentioned earlier, the carrier of the present disclosure is better in enzyme compatibility and better in immobilization effect, and the activity of the enzyme can be reserved to a great extent during immobilization. Therefore, the immobilized enzyme obtained through immobilization is better in activity and higher in stability during reuse, and is more suitable for application in industrialized immobilized enzymes.
The immobilized enzyme of the present disclosure has wide applicability to the enzymes. For example, the enzyme includes, but is not limited to, any one or more of a transaminase, a ketoreductase, an alcohol dehydrogenase, a FDH, a glucose dehydrogenase, a monooxygenase, an ene-reductase, an imine reductase, and an amino acid dehydrogenase. It is more suitable for the following enzymes, the transaminase is a transaminase derived from Chromobacterium violaceum DSM30191, or a transaminase derived from Arthrobacter citreus, or a transaminase derived from Actinobacteria, or a transaminase derived from Sciscionella sp. SE31; the ketoreductase is a carbonyl reductase derived from Acetobacter sp. CCTCCM209061, or a ketoreductase derived from Sporobolomyces salmonicolor; the alcohol dehydrogenase is derived from Thermoanaerobium brockii; the FDH is a FDH derived from Candida boidinii; the glucose dehydrogenase is a glucose dehydrogenase derived from Lysinibacillus sphaericus G10; the monooxygenase is a cyclohexanone monooxygenase derived from Rhodococcus sp. Phi1, or a cyclohexanone monooxygenase derived from Rhodococcus ruber-SD1, or a cyclohexanone monooxygenase derived from Brachymonas petroleovorans; the ene-reductase is an ene-reductase derived from Saccharomyces cerevisiae, or an ene-reductase derived from Chryseobacterium sp. CA49; the imine reductase is an imine reductase derived from Streptomyces sp., or an imine reductase derived from Bacillus cereus; and the amino acid dehydrogenase is a leucine dehydrogenase derived from Bacillus cereus, or a phenylalanine dehydrogenase derived from Bacillus sphaericus; or an amino acid dehydrogenase derived from Thermoactinomyces intermedius ATCC33205, or an amino acid dehydrogenase derived from Thermosyntropha lipolytica. The enzyme numbers, sources, and enzyme sequence information for the above enzymes of the present disclosure are shown below:
Chromobacterium
violaceum
citreus
Thermoanaerobium
brockii
Sporobolomyces
salmonicolor
Candida boidinii
Lysinibacillus
sphaericus G10
ruber-SD1
petroleovorans
Chryseobacterium
Saccharomyces
cerevisiae
Bacillus cereus
Bacillus cereus
Bacillus sphaericus
Thermoactinomyces
intermedius
Thermosyntropha
lipolytica
It is to be noted that, the above enzyme sequences are all disclosed enzymes in the related art, and the present disclosure only uses the above enzymes for the verification of the performance of the enzyme immobilization carrier of the present disclosure.
In order to further improve the activity and reuse stability of the immobilized enzyme, more preferably, when the enzyme is the transaminase, the matrix resin is one or more of LX-1000HA, LX-1000EPN, LX-EPHA, or LX-109s; when the enzyme is the FDH, the matrix resin is one or more of LX-1000EPN, LX-109s, LX-1000HFA, ECR8285, or EC-HFA; when the enzyme is the ketoreductase, the matrix resin is one or more of LX-109s, LX-EPHA, ECR8285, LX-1000HA, or ECR8409; when the enzyme is the monooxygenase, the matrix resin is one or more of LX-109s, LX-EPHA, or LX-1000HA; when the enzyme is the imine reductase, the matrix resin is LX-109s and/or LX-EPHA; and when the enzyme is the amino acid dehydrogenase, the alcohol dehydrogenase, or the ene-reductase, the matrix resin is one or more of LX-109s, LX-1000EPN, LX-EPHA, or LX-1000HA.
The present disclosure further provides a method for preparing the enzyme immobilization carrier. The method includes the following steps: providing a resin ball matrix; linking an —N(CH2COOH)2 group onto the resin ball matrix by means of a chemical bond; absorbing a metal ion onto the —N(CH2COOH)2 group in a chelating manner through coordination, so as to obtain the enzyme immobilization carrier.
Based on various reasons mentioned earlier, the carrier of the present disclosure is better in enzyme compatibility and better in immobilization effect, and the activity of the enzyme can be reserved to a great extent during immobilization. Therefore, the immobilized enzyme obtained through immobilization is better in activity and higher in stability during reuse, and is more suitable for application in industrialized immobilized enzymes.
In a preferred implementation solution, when the resin ball matrix is the amino-type methacrylic resin, the amino-type methacrylic resin is mixed with sodium chloroacetate for nucleophilic substitution, so as to link the —N(CH2COOH)2 group onto the amino-type methacrylic resin. More preferably, after an aqueous solution of the amino-type methacrylic resin and the sodium chloroacetate is stirred for 30-60 min, the reaction system is regulated to a pH of 9-10 with 1-2 mol/L of an aqueous alkaline solution, and then heated up to 70-80° C. for reaction for 20-30 h at an N2 atmosphere, so as to link the —N(CH2COOH)2 group onto the amino-type methacrylic resin. Preferably, the aqueous alkali solution is a sodium carbonate solution, a dilute sodium hydroxide solution, or a lithium hydroxide solution. Based on this, the amino-type methacrylic resin ball matrix of the present disclosure is higher in conversion rate, and more stable in reaction process.
In a preferred implementation solution, when the resin ball matrix is the epoxy-type methacrylic resin, the epoxy-type methacrylic resin is mixed with Iminodiacetic acid disodium for an addition reaction, so as to link the —N(CH2COOH)2 group onto the epoxy-type methacrylic resin. More preferably, after an aqueous solution of the epoxy-type methacrylic resin and the Iminodiacetic acid disodium is stirred for 30-60 min at 20-25° C., the reaction system is heated up to 60-70° C. for reaction for 18-24 h at an N2 atmosphere, so as to link the —N(CH2COOH)2 group onto the epoxy-type methacrylic resin. Based on this, the epoxy-type methacrylic resin ball matrix of the present disclosure is higher in conversion rate, and more stable in reaction process.
In a preferred implementation solution, after the step of linking the —N(CH2COOH)2 group onto the resin ball matrix by means of the chemical bond, a metal salt solution is added to the reaction system for a complex reaction, so as to absorb the metal ion on the —N(CH2COOH)2 group in the chelating manner through coordination, thereby obtaining the enzyme immobilization carrier. Preferably, during the complex reaction, a reaction temperature is 2-30° C. and reaction time is 1-4 h; and the metal salt solution is an aqueous solution of nickel chloride, an aqueous solution of copper sulfate, an aqueous solution of ferrous chloride, or an aqueous solution of cobalt chloride. Based on this, the reaction condition is more appropriate, such that the enzyme immobilization carrier obtained through reaction is higher in yield and higher in purity. More preferably, in the metal salt solution, a mass ratio of the metal ion to the resin ball matrix is (0.05-0.1):1.
In order to further improve the conversion rate of the amino-type matrix resin, preferably, a mass ratio of the sodium chloroacetate to the amino-type methacrylic resin ball is (5-10):1.
In order to further improve the conversion rate of the epoxy-type matrix resin, preferably, a mass ratio of the Iminodiacetic acid disodium to the epoxy-type methacrylic resin ball is (5-10):1.
The present disclosure further provides a method for preparing the immobilized enzyme. The method includes the following step: performing an immobilization reaction on the enzyme immobilization carrier, and an enzyme, so as to obtain the immobilized enzyme.
Based on various reasons mentioned earlier, the carrier of the present disclosure is better in enzyme compatibility and better in immobilization effect, and the activity of the enzyme can be reserved to a great extent during immobilization. Therefore, the immobilized enzyme obtained through immobilization is better in activity and higher in stability during reuse, and is more suitable for application in industrialized immobilized enzymes.
Preferably, the method for preparing the immobilized enzyme includes the following step: dispersing the enzyme immobilization carrier into a first solvent to form dispersion liquid, where the first solvent is a mixed solution of a phosphate buffer solution, a sodium chloride aqueous solution, and an imidazole buffer solution; and reacting the dispersion liquid with an enzyme solution containing the enzyme, to cause the enzyme and the enzyme immobilization carrier to be subjected to an immobilization reaction, so as to obtain the immobilized enzyme. The first solvent creates a milder and more appropriate environment for immobilization, such that the activity of the enzyme may be protected, an enzyme immobilization effect is improved, and non-target proteins in an enzyme solution are prevented from being immobilized. Based on this, the enzyme compatibility of the carrier is further improved, and the enzyme immobilization effect is better, and the activity is better.
In order to further improve the activity and reuse stability of the immobilized enzyme, preferably, in the first solvent, the phosphate buffer solution has the concentration of 0.1-0.2 mol/L; preferably, the sodium chloride aqueous solution has the concentration of 0.5-1 mol/L; and preferably, the imidazole buffer solution has the concentration of 0.05-0.1 mol/L.
In order to improve the stability during reaction while improving reaction efficiency, preferably, during the reaction process of the dispersion liquid and the enzyme solution, a reaction temperature is 20-25° C., and reaction time is 16-24 h. More preferably, per gram of the enzyme immobilization carrier corresponds to 4-8 mL of the enzyme solution, and the enzyme solution has protein content of 20-25 mg/mL. Therefore, the enzyme is immobilized more fully, a carrying capacity is provided, and the immobilized enzyme has higher catalytic activity.
The present application is further described in detail below with reference to specific embodiments, and the embodiments cannot be construed as limiting the scope of protection claimed in the present application.
Preparation of the enzyme immobilization carrier by using an amino-type methacrylic resin as a resin ball matrix:
Preparation of the enzyme immobilization carrier by using an epoxy-type methacrylic resin as a resin ball matrix:
Immobilization of the transaminase on the enzyme immobilization carrier using the amino-type methacrylic resin as the resin ball matrix:
Immobilization of the transaminase on the enzyme immobilization carrier using the epoxy-type methacrylic resin as the resin ball matrix:
Immobilization of the transaminase on the amino-type methacrylic resin: 1 g of an amino-type resin was weighed, and washed for a plurality of times by using the 0.1M phosphate buffer solution 7.0; the buffer solution was removed; and the 0.1M phosphate buffer solution (pH7.0), and a glutaraldehyde solution with a final concentration being 2% were added. Then 4 mL of an enzyme solution (having the cofactor PLP), which was prepared by using the 0.1M phosphate buffer solution 7.0 and 0.1 g of the enzyme powder, was added; incubation was performed for 16-24 h at 20° C., and the buffer solution was removed. Washing was performed for 3 times by using the 20M phosphate buffer solution 7.0 containing 0.5M NaCl; the buffer solution was removed; and the immobilized enzyme was maintained to be used.
Immobilization of the transaminase on a commercial affinity chromatography resin in the related art:
Immobilized transaminase activity and reusability test:
Immobilization and Application of a FDH on an Enzyme Immobilization carrier.
The difference between this embodiment and Embodiment 1 lied in that, the transaminase was replaced with the FDH with equal mass, and the cofactor was replaced with NAD+ with equal mass.
Immobilized FDH activity and reusability test:
The difference between this embodiment and Embodiment 1 lied in that, the transaminase was equivalently replaced with the ketoreductase, and the cofactor was replaced with NAD+ with equal mass.
Immobilized ketoreductase activity and reusability test.
The present application is a U.S. National Stage of International Patent Application No: PCT/CN2021/131878 filed on Nov. 19, 2021, the entire content of which is incorporated in this application by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/131878 | 11/11/2019 | WO |
