阅读说明：本技术 用于酶电催化还原的氧化还原酶电极及其制备方法和其酶电反应器 (Oxidoreductase electrode for enzyme electrocatalytic reduction, preparation method thereof and enzyme electric reactor thereof ) 是由 王世珍 刘凯泷 熊雨 于 2020-12-01 设计创作，主要内容包括：本发明涉及用于酶电催化还原的氧化还原酶电极及其制备方法和其酶电反应器。与酶电极检测检测限低、灵敏度高的要求相比,酶电催化要求能耐受高浓度底物、酶稳定性好,催化效率高。本发明的辅酶与酶共固定化在电极上,可实现辅酶原位再生,提高反应效率,可实现手性化合物的酶电催化高效制备,具有良好的应用前景。(The invention relates to an oxidoreductase electrode for enzyme electrocatalytic reduction, a preparation method thereof and an enzyme electric reactor thereof. Compared with the requirements of low detection limit and high sensitivity of enzyme electrode detection, the enzyme electrocatalysis is required to be capable of tolerating high-concentration substrates, and the enzyme has good stability and high catalysis efficiency. The coenzyme and the enzyme are co-immobilized on the electrode, so that the coenzyme can be regenerated in situ, the reaction efficiency is improved, the enzyme electrocatalytic high-efficiency preparation of the chiral compound can be realized, and the method has a good application prospect.)

1. An oxidoreductase electrode for enzymatic electrocatalytic reduction, comprising: comprises a substrate electrode, an electron conductor, an electron mediator, an enzyme and a coenzyme; the electronic conductor comprises graphene oxide, reduced graphene oxide, carbon nano-tubes, polydopamine, nanogold, MOF, polyethyleneimine or biomimetic mineralized TiO₂A combination of at least two of the materials; the coenzyme comprises at least one of NADH-IL or PEI-Fc-NADH; the coenzyme and the enzyme are jointly fixed on the enzyme electrode.

2. The oxidoreductase electrode of claim 1, wherein: the enzyme is an NADH-dependent or NADPH-dependent dehydrogenase.

3. The oxidoreductase electrode of claim 1, wherein: the substrate electrode comprises a carbon paper electrode, a glassy carbon electrode or a graphite electrode.

4. The oxidoreductase electrode of claim 1, wherein: the electron mediator includes at least one of 5-methylphenazinium methyl sulfate, toluidine blue, meldola blue, or methylene blue.

5. A method of preparing an oxidoreductase electrode according to any one of claims 1 to 4, wherein: and compounding the immobilized mixture of the enzyme and the electron conductor, the electron mediator and the coenzyme on the surface of the substrate electrode to obtain the oxidoreductase electrode.

6. The method of claim 5, wherein: the preparation method of the immobilized mixture of the enzyme and the electron conductor comprises the following steps:

the electronic conductor comprises graphene oxide and MOF, metal ions of the MOF are added into a graphene oxide solution, 0.1-100 mg/mL of the enzyme is further added, a ligand of the MOF is added under stirring at 0-50 ℃, reaction is carried out to obtain the MOF, and the MOF is immobilized in situ to obtain an immobilized mixture of the enzyme and the electronic conductor; or the like, or, alternatively,

the electronic conductor comprises a carbon nano tube, polydopamine and nanogold, and the polydopamine is added into a carbon nano tube solution to obtain a carbon nano tube-polydopamine solution; reacting the solution of the carbon nano tube-polydopamine with a nano-gold precursor to prepare a solution of the carbon nano tube-polydopamine containing nano-gold, and mixing the enzyme with the solution of the carbon nano tube-polydopamine containing nano-gold to obtain an immobilized mixture of the enzyme and an electronic conductor; or the like, or, alternatively,

the electronic conductor comprises reduced graphene oxide, polydopamine and nanogold, dopamine and the enzyme are added into a reduced graphene oxide solution, and the reduced graphene oxide solution reacts with a nanogold precursor and is immobilized to obtain an immobilized mixture of the enzyme and the electronic conductor; or the like, or, alternatively,

the electronic conductor comprises reduced graphene oxide, polyethyleneimine and biomimetic mineralized TiO₂A material comprising a reduced graphene oxide-polyethyleneimine to which the enzyme is coordinatively immobilized, followed by addition of Ti-BALDH to induce TiO via polyethyleneimine₂Biomimetic mineralization of the immobilized enzyme to obtain an immobilized mixture of the enzyme and the electronic conductor.

7. The method of claim 6, wherein: the ligand of the MOF comprises one of trimesic acid, 2,3,6,7,10, 11-hexaamino triphenyl hexahydrochloride, 2,3,6,7,10, 11-hexahydroxyl triphenyl, hexaamino benzene (3 hydrochloride), Nu-1006 or Nu-1007; the metal ions of the MOF comprise Co²⁺、Cu²⁺、Zn²⁺、Ni²⁺Or Zr⁴⁺At least one of (1).

8. The method of claim 5, wherein: the NADH-IL or PEI-Fc-NADH and the electronic mediator form a gel, and the immobilized mixture of the enzyme and the electronic conductor and the gel are compounded on the surface of the substrate electrode.

9. The method of claim 8, wherein: the electronic mediator, NADH-IL or PEI-Fc-NADH and ionic liquid form the gel; the ionic liquid comprises at least one of 1-ethyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium chloride, or N, N' - (methylene) bis (1- (3-vinylimidazole)) bromine.

10. The method of claim 5, wherein: the preparation method of the PEI-Fc-NADH comprises the following steps: respectively preparing 0.1-0.7 mmol/mL and 0.05-0.2 mmol/mL ethanol solutions of polyethyleneimine PEI and ferrocene formaldehyde Fc, dropwise adding the ethanol solution of Fc into the ethanol solution of PEI within 1-3 h, and continuously stirring for reaction for 1-3 h; adding sodium borohydride with the final concentration of 0.1-1.0 mmol/mL, and continuing to react for 1-4 h; adding 1-10 mL of water into the residue, and dialyzing for 10-14 h with water to obtain PEI-Fc; dissolving 10-100 mmol succinic anhydride in 6-30 mL dimethyl sulfoxide, and mixing 0.0745-0.5 g NAD⁺Adding the mixture into a succinic anhydride solution, and standing at room temperature for reaction for 10-14 h; adding 10-30 mL of acetone into the mixed solution, centrifuging to obtain a precipitate, adding 2-5 mL of phosphoric acid buffer solution containing 1-3 mmol of EDC and having pH of 4-6, and activating hydroxyl groups by EDC for 1-4 h; after activation is finished, adding 5-10 mL of PEI-Fc with pH of 4-6 into the solution, adjusting the pH to 4.6-4.8, and reacting at 0-4 ℃ for 12-24 hours; and after the reaction is finished, dialyzing for 10-14 h by using water to obtain PEI-Fc-NADH.

11. An enzymatic electric reactor for enzymatic electrocatalytic reduction, characterized by: the enzyme electric reactor adopts a three-electrode system, and takes the oxidoreductase electrode as claimed in any one of claims 1 to 4 as a working electrode; the reference electrode is selected from a saturated calomel electrode, a hydrogen electrode, a silver | silver chloride electrode or a mercury | mercury oxide electrode; the counter electrode is a platinum wire electrode or a carbon electrode.

12. A method for performing a catalytic reduction reaction using the enzyme electric reactor of claim 11, characterized in that: carrying out the reaction in a cyclic voltammetry and/or chronoamperometry manner; the potential scanning rate of the cyclic voltammetry is 1-500 mVS; adding 1-20 mg coenzyme NADH buffer solution, and introducing N before reaction₂The reaction substrates are ketoacid which is a substrate of amino acid dehydrogenase, ketone which is a substrate of amine dehydrogenase and ketone which is a substrate of alcohol dehydrogenase; the concentration range of the substrate is 5 mM-500 mM; the buffer solution is at least one of phosphate buffer solution, borate buffer solution, citrate buffer solution, carbonate buffer solution, Tris-HCl buffer solution and the like.

13. The method of claim 12, wherein: 1.5-100 mL of a three-electrode system is adopted, carbon paper fixed with enzyme and gel is used as a working electrode, a counter electrode is a platinum wire electrode, and Ag/AgCl is used as a reference electrode; reacting in 0.5-100 mL of phosphate buffer solution added with NADH-IL, and introducing N before the reaction₂In the test process, cyclic voltammetry is adopted, wherein a blank control is not added with a substrate dropwise; the scanning rate of the cyclic voltammetry test is 40-60 mV/s, the scanning range is-1V, the cyclic voltammetry test is carried out in a buffer solution with the pH value of 6.8-7.2, the timing amperometric test time is 30-1800 s, and the substrate sample adding is carried out every 25-35 s; the current density is regulated and controlled to be 0.1-1.5 mA/cm²Calculating the total conversion number of coenzyme regeneration and optimizing electrochemical regeneration conditions; the voltage is 1-200 mV/s, the scanning range is-1V, the detection is carried out in a buffer solution with the pH value of 6.0-12.0, the time of the chronoamperometric current test is 30-1800 s and 1140-1260 s, and the once substrate sample addition is 1 mM-500 mM. The current density is regulated and controlled to be 0.1-1.5 mA/cm²。

Technical Field

The invention relates to the technical field of biocatalysis, in particular to a high-efficiency enzyme bioelectrocatalysis reactor, a method for preparing a chiral compound by reduction of the reactor and application of the reactor.

Background

The bioelectrocatalysis synthesis has no by-product and high selectivity, and is concerned about the advantages of high efficiency, environmental protection and the like. The enzyme can realize various electrocatalysis processes by carrying out bidirectional electron transfer and energy metabolism with the external environment. At present, bioelectrocatalysis is mainly focused on a microbial electrosynthesis system, enzyme electrocatalysis reduction is less, and the bioelectrocatalysis is focused on a multi-enzyme coupling electrocatalysis system to catalyze and immobilize CO₂And nitrogen-fixing enzyme. And the research on asymmetric electroreduction of chiral compounds with high added values, such as unnatural chiral amino acid, chiral alcohol and the like, is less. Compared with the requirement of low detection limit and high sensitivity of an enzyme electrode, the enzyme electrocatalysis is required to be capable of tolerating high-concentration substrates, the enzyme stability is good, the catalysis efficiency is high, the catalytic preparation is emphasized, and the coenzyme is required to be regenerated efficiently. However, bottleneck problems existing in bioelectrocatalytic asymmetric reduction of the current NAD (P) H-dependent dehydrogenase include difficult coenzyme regeneration, poor enzyme stability on the surface of an electrode, limited enzyme load, difficulty in scale-up amplification and the like.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides an oxidoreductase electric reactor as well as a preparation method and application thereof. The novel oxidoreductase electric reactor is constructed, provides a good microenvironment for the electrochemical reaction of biomolecules, has high enzyme electrocatalytic reduction efficiency and good stability, and can be used for preparing chiral drug intermediates by bioelectrocatalysis.

One of the technical schemes adopted by the invention for solving the technical problems is as follows:

an oxidoreductase electrode for enzymatic electrocatalytic reduction comprising a base electrode, an electron conductor, an electron mediator, an enzyme and a coenzyme; the electronic conductor comprises Graphene Oxide (GO), reduced graphene oxide (rGO), carbon nano-tubes (MWNT), Polydopamine (PDA), nanogold, MOF, Polyethyleneimine (PEI) or biomimetic mineralized TiO₂A combination of at least two of the materials; the coenzyme comprises at least one of the ionic coenzymes NADH-IL or PEI-Fc-NADH; the coenzyme and the enzyme are jointly fixed on the enzyme electrode.

Wherein the enzyme is an NADH-dependent or NADPH-dependent dehydrogenase, preferably comprising at least one of an amino acid dehydrogenase, an amine dehydrogenase, an alcohol dehydrogenase or a ketoreductase.

The substrate electrode comprises a carbon paper electrode (CP), a Glassy Carbon Electrode (GCE), a graphite electrode and the like, and is preferably a substrate electrode with a large specific surface area, such as a carbon paper electrode and a pyrolytic graphite electrode.

Wherein the electronic mediator comprises at least one of 5-methylphenazinium methyl sulfate, toluidine blue, meldola blue, or methylene blue.

Wherein the electronic conductor may be a combination of: the electronic conductor comprises GO and MOF; or the electronic conductor comprises carbon nanotubes, PDA and nanogold; or the electronic conductor comprises rGO, PDA and nanogold; or the electron conductor comprises rGO, PEI and biomimetic mineralized TiO₂A material.

In the oxidoreductase electrode, the coenzyme and the enzyme are co-immobilized on the electrode, so that the in-situ regeneration of the coenzyme can be realized, and the catalytic efficiency is improved. The ionic liquid can be used as a modifier and a binder of a traditional glassy carbon electrode, the problem that the graphene added with ILs is difficult to directly modify on the surface of the electrode can be solved, and the electron transfer between coenzyme NAD (P) H and the electrode is accelerated by the high conductivity of the IL. A substrate electrode having a large specific surface area such as carbon paper increases the enzyme loading on the electrode surface. The redox medium organic micromolecules accelerate electron shuttling and improve the biocompatibility of the electrode material under the action of a molecular bridge between the electrochemical active protein and the inorganic material electrode.

The second technical scheme adopted by the invention for solving the technical problems is as follows:

the preparation method of the oxidoreductase electrode comprises the step of compounding an immobilized mixture of enzyme and an electron conductor, an electron mediator and coenzyme on the surface of the substrate electrode to obtain the oxidoreductase electrode.

Specifically, the preparation method of the immobilized mixture of the enzyme and the electronic conductor comprises the following steps:

when the electronic conductor comprises GO and MOF, adding metal ions of MOF into a graphene oxide solution, further adding 0.1-100 mg/mL of the enzyme, adding a ligand of MOF under stirring at 0-50 ℃, reacting to obtain a metal-organic framework (MOF), and carrying out in-situ immobilization on the metal-organic framework (MOF) to obtain an immobilized mixture of the enzyme and the electronic conductor; further, the reaction of the in-situ immobilized enzyme may be performed on a substrate electrode; or the like, or, alternatively,

when the electronic conductor comprises a carbon nano tube, PDA and nanogold, polydopamine is added into a carbon nano tube solution to obtain a carbon nano tube-polydopamine solution; reacting the solution of the carbon nano tube-polydopamine with a nano-gold precursor to prepare a solution of the carbon nano tube-polydopamine containing nano-gold, and mixing the enzyme with the solution of the carbon nano tube-polydopamine containing nano-gold to obtain an immobilized mixture of the enzyme and an electronic conductor; or the like, or, alternatively,

when the electronic conductor comprises rGO, PDA and nanogold, DA and the enzyme are added into the rGO solution, and the solution reacts with the nanogold precursor and is immobilized to obtain an immobilized mixture of the enzyme and the electronic conductor; or the like, or, alternatively,

the electronic conductor comprises rGO, PEI and biomimetic mineralized TiO₂When the material is prepared, the enzyme is coordinated and fixed on reduced graphene oxide-polyethyleneimine (rGO-PEI), then Ti-BALDH is added, and TiO is induced by PEI₂Biomimetic mineralization of the immobilized enzyme to obtain an immobilized mixture of the enzyme and the electronic conductor. Further, the enzyme is coordinated and fixed on rGO-PEI, then Ti-BALDH, an electron mediator and coenzyme are added, and TiO is induced by PEI₂Biomimetic mineralizationTo simultaneously immobilize the enzyme and the coenzyme. The biomimetic mineralization process of immobilized enzyme or immobilized enzyme and coenzyme at the same time can be carried out on a substrate electrode.

Further, the ligand of the metal-organic framework (MOF) comprises trimesic acid (H)₃BTC; one of bezene-1, 3,5-tricarboxylic acid), 2,3,6,7,10, 11-hexaamino triphenyl Hexahydrochloride (HITP), 2,3,6,7,10, 11-hexahydroxy triphenyl (HHTP), hexaamino benzene (3 Hydrochloride) (HAB), Nu-1006 or Nu-1007; the metal ions comprise Co²⁺、Cu²⁺、Zn²⁺、Ni²⁺Or Zr⁴⁺(CoCl₂、CuCl₂、ZnCl₂、NiCl₂、ZrCl₄Etc.). The MOF formed comprises Ni₃(HITP)₂，Co-HAB，Cu₃(HHTP)₂Ni-CAT, etc.

Further, the ionic coenzyme NADH-IL or the high molecular modified coenzyme (PEI-Fc-NADH) coenzyme and the electronic mediator form gel, the immobilized mixture of the enzyme and the electronic mediator and the gel are compounded on the surface of the substrate electrode, so that the dehydrogenase, the coenzyme and the electronic mediator are co-immobilized on the surface of the electrode, the coenzyme is regenerated in situ, and the catalytic efficiency is improved.

Further, the electronic mediator, coenzyme (NADH-IL or PEI-Fc-NADH) and ionic liquid (for solubilizing NADH-IL or PEI-Fc-NADH) form the gel; the ionic liquid comprises at least one of 1-ethyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium chloride, or N, N' - (methylene) bis (1- (3-vinylimidazole)) bromine.

Further, the preparation method of the PEI-Fc-NADH (coenzyme covalently linked with macromolecule) comprises the following steps: respectively preparing 0.1-0.7 mmol/mL and 0.05-0.2 mmol/mL ethanol solutions of Polyethyleneimine (PEI) and ferrocene formaldehyde (Fc), dropwise adding the ethanol solution of Fc into the ethanol solution of PEI within 1-3 h, and continuously stirring for reacting for 1-3 h. Adding sodium borohydride with the final concentration of 0.1-1.0 mmol/mL, and continuing to react for 1-4 h. And adding 1-10 mL of distilled water into the residue, and dialyzing for 12h by using the distilled water to obtain the PEI-Fc. The thickness of the mixture is 10-100 mmoDissolving succinic anhydride in 6-30 mL of dimethyl sulfoxide, and mixing 0.0745-0.5 g of NAD⁺Adding into DMSO solution of succinic anhydride, and standing at room temperature for reaction for 12 h. Adding 10-30 mL of acetone into the mixed solution, centrifuging to obtain a precipitate, adding 2-5 mL of phosphoric acid buffer solution with pH value of 4-6 containing 1-3 mmol of EDC, and activating hydroxyl groups by EDC for 1-4 h. And after the activation is finished, adding 5-10 mL of PEI-Fc (pH 4-6) into the solution, adjusting the pH to 4.7, and reacting at 0-4 ℃ for 12-24 h. And after the reaction is finished, dialyzing for 12 hours by using distilled water to obtain PEI-Fc-NADH.

The third technical scheme adopted by the invention for solving the technical problems is as follows:

an enzyme electric reactor for enzyme electrocatalytic reduction adopts a three-electrode system, and takes the oxidoreductase electrode as a working electrode; the reference electrode is selected from a saturated calomel electrode, a hydrogen electrode, a silver | silver chloride electrode or a mercury | mercury oxide electrode; the counter electrode is a platinum wire electrode or a carbon electrode.

The fourth technical scheme adopted by the invention for solving the technical problems is as follows:

a method for carrying out catalytic reduction reaction by using an enzyme electric reactor, which carries out reaction in a cyclic voltammetry and/or chronoamperometry mode; the potential scanning rate of the cyclic voltammetry is 1-500 mV/s; adding 1-20 mg coenzyme NADH buffer solution, and introducing N before reaction₂The reaction substrates are ketoacid which is a substrate of amino acid dehydrogenase, ketone which is a substrate of amine dehydrogenase and ketone which is a substrate of alcohol dehydrogenase; the concentration range of the substrate is 5 mM-500 mM; the buffer solution is at least one of phosphate buffer solution, borate buffer solution, citrate buffer solution, carbonate buffer solution, Tris-HCl buffer solution and the like.

Specifically, designing a bioelectrocatalysis microreactor based on 1.5-100 mL of disposable plastic test tubes; a reactor three-electrode system is designed, carbon paper (5 multiplied by 5 mm-5 multiplied by 5cm) fixed with enzyme and gel is used as a working electrode, a counter electrode is a platinum wire electrode, and Ag/AgCl is used as a reference electrode. Adding NADH-IL (NADH-ionic liquid) into 0.5-100 mL of phosphate buffer (1-500 mM, pH 5-13) for reaction, and introducing N before the reaction₂With circulation during the testVoltammetry, where the blank is not substrate drop wise. The scanning rate of the cyclic voltammetry test is 50mV/s, the scanning range is-1V, the cyclic voltammetry test is carried out in a buffer solution with the pH value of 7.0, the timing amperometric test time is 30-1800 s, and the substrate sample adding is carried out at intervals of 30 s. The current density is regulated and controlled to be 0.1-1.5 mA/cm²Calculating the total conversion number (TTN) of coenzyme regeneration and optimizing electrochemical regeneration conditions. The voltage is 1-200 mV/s, the scanning range is-1V, the detection is carried out in a buffer solution with the pH value of 6.0-12.0, the time of the chronoamperometric current test is 30-1800 s and 1200s, and the once substrate sample addition is 1 mM-500 mM. The current density is regulated and controlled to be 0.1-1.5 mA/cm²。

The equipment, reagents, processes, parameters and the like related to the invention are conventional equipment, reagents, processes, parameters and the like except for special description, and no embodiment is needed.

All ranges recited herein include all point values within the range.

As used herein, "about" or "about" and the like refer to a range or value within plus or minus 20 percent of the stated range or value.

In the present invention,% is mass% and ratio is mass ratio unless otherwise specified or indicated in the field.

In the invention, the room temperature, namely the normal environment temperature, can be 10-30 ℃.

The invention has the following beneficial effects:

1. according to the invention, carbon paper, Graphene Oxide (GO), reduced graphene oxide (rGO), Carbon Nano Tubes (CNT) and other carbon nano materials, conductive polymers, ionic liquid and the like are selected as electron transfer materials to prepare the enzyme electrode, so that stable immobilization and efficient electron transfer of the enzyme are realized by superior electronic performance, chemical modification performance, good biocompatibility and the like. The functional electrode material with good biocompatibility is used for directional immobilization of enzyme, in-situ regeneration of coenzyme is realized, the loading capacity of an enzyme electrode is increased, and the stability of the enzyme is improved.

2. Amino acid dehydrogenase (AaDH) and alcohol dehydrogenaseAnd amine dehydrogenases and the like are important enzymes in the biological metabolic pathway. AaDH using NH₃The chiral amino acid can be used as an amino donor to catalyze asymmetric reductive amination reaction of keto acid, can synthesize a series of chiral natural amino acids, unnatural amino acids, chiral amines and the like, and is used as an important chiral building block of fine chemicals such as medicines. The enzyme electric reactor can successfully catalyze the asymmetric electric reduction of various dehydrogenases, efficiently obtain various chiral compounds, and has good application prospect in the fields of preparation of chiral drug intermediates and the like.

Drawings

The invention is further illustrated by the following figures and examples.

FIG. 1 is a schematic diagram (left) of the reactor structure and a photograph (right) of an actual reactor in example 1 of the present invention.

FIG. 2 is a photograph showing a carbon paper-immobilized enzyme in example 1 of the present invention.

FIG. 3 shows immobilized enzymes CP/Cu obtained in example 1 of the present invention₃(BTC)₂Electron micrograph of/GO-AaDH.

FIG. 4 is a coenzyme regeneration curve in example 1 of the present invention.

FIG. 5 is a photograph of an enzyme electrocatalytic reactor in example 3 of the present invention.

FIG. 6 is an SEM representation of MWNTs-PDA in example 3 of the present invention.

Detailed Description

The present invention will be described in detail by examples.

Example 1

(1) Preparation of crude enzyme of amino acid dehydrogenase: based on the original sequence of amino acid dehydrogenase (AaDH) shown as SEQ ID No.1, the gene sequence of amino acid dehydrogenase was synthesized by a commercial company (Shanghai bioengineering Co., Ltd.) by PCR method for ligation to the vector pET28a to construct a plasmid.

Ligation of the PCR product (gene sequence of amino acid dehydrogenase) with the vector pET28 a: the PCR product and the vector pET28a were digested simultaneously with NdeI and XhoI, and the resulting product was purified and reacted with T4 DNA Ligase overnight at 4 ℃ to obtain a plasmid for transformation. Preparation of Competent cells was accomplished using the component Cel preference Kit. 10ng of plasmid for transformation was added to the competent cells, and after gently mixing, the mixture was left on ice for 30 minutes, and then left in a water bath at 42 ℃ for 45 seconds, and immediately left on ice for 1 to 2 minutes. Adding LB culture medium pre-warmed at 37 ℃, shaking and culturing at 37 ℃ for 1 hour, taking a proper amount of bacterial liquid, coating an LB solid plate with kanamycin resistance, culturing at 37 ℃ overnight, and picking out a single colony to obtain the recombinant escherichia coli (E.coli BL21(DE3)/pET28 a).

Cultivation of recombinant E.coli BL21(DE3)/pET28 a: the strain was inoculated into 200mL of LB medium at an inoculum size of 1%. The composition of LB culture medium is 10.0g/L tryptone, 5.0g/L yeast powder, 10g/L NaCl. The culture conditions were: the initial pH value is 7.0, the liquid loading volume fraction is 10%, the culture temperature is 37 ℃, the rotating speed of a shaking table is 200rpm, and the culture time is 6 hours. The inducer IPTG was added to a final concentration of 10mg/mL, and the culture was continued at 30 ℃ and 200rpm for 12 hours.

Preparation of crude enzyme solution: and (3) centrifuging the fermentation liquor obtained after the culture is finished in a refrigerated centrifuge (4 ℃, 8000rpm, 15min) to obtain cells, discarding supernate, resuspending the precipitate with Tris-HCl buffer solution (pH 7.5), fully washing, centrifuging, and repeating the operation for 3 times to obtain the cells. The cell fluid is treated by an ultrasonic disruptor, the probe of the cell disruptor is arranged 1cm below the liquid level, the disruption conditions are ultrasonic for 2 seconds at intervals of 4 seconds, ultrasonic is carried out for 40 times, and the power is 200W. Then, the mixture is centrifuged at 12,000rpm for 15min at 4 ℃ to remove insoluble cell debris, and the supernatant is the crude enzyme solution of the amino acid dehydrogenase.

(2) Preparation of pure enzyme of amino acid dehydrogenase: the crude enzyme solution of the amino acid dehydrogenase was separated and purified by using His Trap nickel column from GE, and then desalted by ultrafiltration using 10K ultrafiltration centrifuge tube from PALL. The purification column adopted in the purification process is a HisTrap HP column capable of specifically purifying the protein with the His-tagged label, and the purification process comprises the steps of balancing, loading, balancing, eluting and column regeneration; collecting the eluted part and desalting by using an ultrafiltration centrifugal tube; the liquid obtained after desalting is the pure enzyme (AaDH with His-tag) liquid of the amino acid dehydrogenase. Adding Tris-HCl (0.05M, pH 7.0) buffer solution into the mixture to prepare aqueous solution containing the amino acid dehydrogenase, and adjusting the concentration of the amino acid dehydrogenase to be 0.05-50 mg/mL according to needs for later use.

(3) Synthesis of ionic coenzyme NADH-IL: the ionic coenzyme in the form of ionic liquid with nicotinamide coenzyme NADH as anion and different imidazoles as cation is designed and synthesized. N-methylimidazole (33mL, 0.31M) and N-butyl chloride (20mL, 0.25M) were added in a molar ratio of about 1.2: 1, refluxing for 24 hours at 90 ℃ under the protection of nitrogen and magnetic stirring conditions to prepare 1-butyl-3-methylimidazole chloride, and purifying. Dissolving 1-butyl-3-methylimidazole chloride ionic liquid in water, and slowly passing through a column filled with 717 type anion exchange resin (the 717 type anion exchange resin uses Cl as the anion before^-) Adding equal-volume equimolar NADH aqueous solution, stirring at room temperature for 48h, purifying and vacuum drying to obtain the ionic coenzyme NADH-IL. Nuclear magnetic characterization of NADH-IL:¹H NMR(400Hz,D₂O)δ＝0.86(t,3H),1.27(m,2H),1.75(m,2H),3.84(s,3H),4.12(t,2H),4.21_4.71(m,15H),5.92(d,1H),5.97(d,1H),7.26(s,1H),7.31(s,1H),8.05(s,1H),8.12(m,1H),8.34(s,1H),8.55(s,1H),8.73(s,1H),8.76(s,1H),9.09(d,1H),9.24(s,1H)。

(4) carbon cloth immobilized enzyme: carbon paper TGP-H-060 special for fuel cells of Dongli corporation of Japan was used as a carrier. Graphene oxide is prepared by a Hummer method, and a uniformly dispersed GO solution is obtained after ultrasonic dispersion for 3 hours. 50mM and 15mM of Cu (NO) were prepared separately₃)₂·3H₂O aqueous solution and 25mM,8mM H₃BTC ethanol solution. Mixing GO solution with Cu (NO)₃)₂Aqueous solution and H₃Adding a His-tag solution obtained in the step (2) into a BTC ethanol solution (specifically, for example, adding Cu (NO) into a GO solution₃)₂An aqueous solution, and adding the solution of AaDH having His-tag obtained in the step (2) thereto, and finally adding ligand H₃BTC ethanol solution), soaking carbon paper TGP-H-060 into the mixed solution, and performing in-situ assembly by utilizing electrostatic action and metal coordination drive to obtain CP/Cu₃(BTC)₂(ii) GO-AaDH, as in FIG. 3; taking a mixture comprising methylene green, NADH-IL and [ EMIM]BF₄The gel of (A) is dropped on the above CP/Cu₃(BTC)₂And drying the surface of the/GO-AaDH at the temperature of 4 ℃ to obtain the working modified electrode, as shown in figure 2. Cu₃(BTC)₂The particles adsorbed on the GO sheet haveExcellent adhesion, and can be fixed on carbon paper to prepare three-dimensional electrode materials. The loading amount is 20mg of enzyme per 1g of composite material.

(5) Design and performance characterization of the bioelectrocatalysis reactor: 50mL of bioelectrocatalytic microreactor is designed based on a disposable plastic test tube. Designing a three-electrode system of the reactor, using the carbon paper (5X 5cm) with the enzyme and the gel immobilized prepared in the step (4) as a Working Electrode (WE), a Counter Electrode (CE) as a platinum wire electrode and Ag/AgCl as a Reference Electrode (RE), and showing in figure 1. The reaction was carried out in 10mL of phosphate buffer (0.02mM, pH 7.4) to which NADH-IL was added, and N was added before the reaction₂And cyclic voltammetry is adopted in the test process, wherein the blank is not added with the substrate dropwise. The scanning rate of the cyclic voltammetry test is 50mV/s, the scanning range is-1V, the cyclic voltammetry test is carried out in a buffer solution with the pH value of 8.0, and the substrate phenylpyruvic acid is added with the concentration of 100 mM. The current density is regulated and controlled to be 0.8mA/cm²And reacting for 8h to obtain the L-phenylalanine concentration of 85 mM. The coenzyme regeneration performance test of the obtained enzyme bioelectricity reactor is shown in FIG. 4.

Example 2

(1) Examples 1 to 2 are the same as examples 2.

(3) Preparation of high molecular covalently linked coenzyme (PEI-Fc-NADH): respectively preparing 0.7mmol/mL and 0.2mmol/mL ethanol solutions of Polyethyleneimine (PEI) and ferrocene formaldehyde (Fc), dropwise adding the ethanol solution of Fc into the ethanol solution of PEI within 3h, and continuously stirring for reacting for 3 h. Sodium borohydride was added to a final concentration of 1.0mmol/mL and the reaction was continued for 4 h. And (4) performing solid-liquid separation, adding 10mL of distilled water into the residue, and dialyzing for 12h by using the distilled water to obtain the PEI-Fc. 100mmol succinic anhydride was dissolved in 30mL dimethyl sulfoxide, 0.5g NAD was added⁺Adding into DMSO solution of succinic anhydride, and standing at room temperature for reaction for 12 h. To the mixed solution, 30mL of acetone was added, and after centrifugation, a precipitate was obtained, and 5mL of a phosphate buffer solution having pH 6 containing 3mmol of EDC was added, to activate the hydroxyl group with EDC for 4 h. After completion of activation, 10mL of PEI-Fc (pH 6) was added to the above solution, and the pH was adjusted to 4.7, followed by reaction at 4 ℃ for 24 hours. And after the reaction is finished, dialyzing for 12 hours by using distilled water to obtain PEI-Fc-NADH.

(4) Carbon cloth immobilized enzyme: carbon paper TGP-H-060 special for fuel cells of Dongli corporation in Japan is adopted as a carrier. Graphene oxide is prepared by a Hummer method, and a uniformly dispersed GO solution is obtained after ultrasonic dispersion for 3 hours. 50mM and 15mM of Cu (NO) were prepared separately₃)₂·3H₂O aqueous solution and 25mM,8mM H₃BTC ethanol solution. Adding Cu (NO) to GO solution₃)₂Adding the solution of His-tag AaDH obtained in step (2) to the aqueous solution, and adding ligand H₃BTC ethanol solution; immersing carbon paper TGP-H-060 in the mixed solution, and obtaining CP/Cu by utilizing electrostatic action and metal coordination drive in-situ assembly₃(BTC)₂(ii) GO-AaDH; will comprise methylene green, PEI-Fc-NADH and [ EMIM]BF₄The gel is dropped on CP/Cu₃(BTC)₂And drying the surface of the/GO-AaDH at the temperature of 4 ℃ to obtain the working modified electrode.

(5) Design and performance characterization of the bioelectrocatalysis reactor: 50mL of bioelectrocatalytic microreactor is designed based on a disposable plastic test tube. And (3) designing a three-electrode system of the reactor, taking the carbon paper (5 multiplied by 5cm) which is prepared in the step (4) and is fixed with the enzyme and the PEI-Fc-NADH as a working electrode, taking a counter electrode as a platinum wire electrode, and taking Ag/AgCl as a reference electrode. The reaction was carried out in 10mL of phosphate buffer (0.02mM, pH 7.4) to which NADH-IL was added, and N was added before the reaction₂And cyclic voltammetry is adopted in the test process, wherein the blank is not added with the substrate dropwise. The scanning rate of the cyclic voltammetry test is 50mV/s, the scanning range is-1V, the cyclic voltammetry test is carried out in a buffer solution with the pH value of 8.0, and the substrate phenylpyruvic acid is added with the concentration of 100 mM. The current density is regulated and controlled to be 0.8mA/cm²And reacting for 8h to obtain the L-phenylalanine concentration of 85 mM.

Example 3

(1) Preparation of crude amine dehydrogenase: the gene sequence of the amine dehydrogenase shown as SEQ ID NO.2 was synthesized by a commercial company (Shanghai bioengineering Co., Ltd.) by PCR based on the original sequence of the amine dehydrogenase, and used for ligation with the vector pET28a to construct a plasmid.

Ligation of the PCR product (gene sequence of amine dehydrogenase) with the vector pET28 a: the PCR product and the vector pET28a were digested simultaneously with NdeI and XhoI, and the resulting product was purified and reacted with T4 DNA ligase overnight at 4 ℃ to obtain a plasmid for transformation. Preparation of Competent cells was accomplished using the component Cell preference Kit. 10ng of plasmid for transformation was added to the competent cells, and after gently mixing, the mixture was left on ice for 30 minutes, and then left in a water bath at 42 ℃ for 45 seconds, and immediately left on ice for 1 to 2 minutes. Adding LB culture medium pre-warmed at 37 ℃, shaking and culturing at 37 ℃ for 1 hour, taking a proper amount of bacterial liquid, coating an LB solid plate with kanamycin resistance, culturing at 37 ℃ overnight, and picking out a single colony to obtain the recombinant escherichia coli (E.coli BL21(DE3)/pET28 a).

Cultivation of recombinant E.coli BL21(DE3)/pET28 a: the strain was inoculated into 200mL of LB medium at an inoculum size of 1%. The composition of LB culture medium is 10.0g/L tryptone, 5.0g/L yeast powder, 10g/L NaCl. The culture conditions were: the initial pH value is 7.0, the liquid loading volume fraction is 10%, the culture temperature is 37 ℃, the rotating speed of a shaking table is 200rpm, and the culture time is 6 hours. The inducer IPTG was added to a final concentration of 10mg/mL, and the culture was continued at 30 ℃ and 200rpm for 2 hours.

Preparation of crude enzyme solution: and (3) centrifuging the fermentation liquor obtained after the culture is finished in a refrigerated centrifuge (4 ℃, 8000rpm, 15min) to obtain cells, discarding supernate, resuspending the precipitate with Tris-HCl buffer solution (pH 7.5), fully washing, centrifuging, and repeating the operation for 3 times to obtain the cells. The cell fluid is treated by an ultrasonic disruptor, the probe of the cell disruptor is arranged 1cm below the liquid level, the disruption conditions are ultrasonic for 2 seconds at intervals of 4 seconds, ultrasonic is carried out for 40 times, and the power is 200W. Then, the insoluble cell debris is removed by centrifugation at 12,000rpm for 15min at 4 ℃, and the supernatant is the crude enzyme solution of the amine dehydrogenase.

(2) Preparation of pure amine dehydrogenase: the crude enzyme solution of amine dehydrogenase was separated and purified by using His Trap nickel column from GE company, and then desalted by ultrafiltration using 10K ultrafiltration centrifuge tube from PALL company. The purification column adopted in the purification process is a HisTrap HP column capable of specifically purifying the protein with the His-tagged label, and the purification process comprises the steps of balancing, loading, balancing, eluting and column regeneration; collecting the eluted part and desalting by using an ultrafiltration centrifugal tube; the liquid obtained after desalting is the pure enzyme liquid of the amine dehydrogenase. Adding Tris-HCl (0.05M, pH 7.0) buffer solution to prepare amine dehydrogenase solution containing amine dehydrogenase, and adjusting the concentration of the amine dehydrogenase to be 0.05-50 mg/mL as required for later use.

(3) Preparation of immobilized enzyme material: adding 20mg of multi-walled carbon nanotubes (MWNTs) (Nanjing Ginko nanotechnology Co., Ltd., multi-walled carbon nanotubes JCMT-95-11-10) into 20mL of water, performing ultrasonic dispersion for 3h to obtain a uniformly dispersed 1mg/mL MWNTs solution, adding 40mg of PDA while stirring, stirring for 12h at room temperature, centrifuging, and re-suspending to prepare a 1mg/mL MWNTs-PDA solution, wherein the SEM image of MWNTs-PDA is shown in FIG. 6. MWNTs-PDA solution and NaAuCl₄The solution is mixed and reacted to obtain MWNTs-PDA solution containing AuNPs.

(4) Preparation of MWNTs-PDA-AuNPs-AmDH: mixing the amine dehydrogenase solution with the enzyme concentration of 5mg/mL obtained in the step (2) with MWNTs-PDA solution containing AuNPs at the temperature of 4 ℃ in the ratio of 2:1:1, centrifuging at 8000rpm, discarding supernatant, and precipitating to obtain an amine dehydrogenase immobilized mixture MWNTs-PDA-AuNPs-AmDH; the mixture was then centrifuged at 8000rpm for 15 minutes, washed, and resuspended in 0.05M ammonium chloride-ammonia buffer pH 9.0 to prepare a MWNTs-PDA-AuNPs-AmDH dispersion containing 1mg/mL of amino acid dehydrogenase.

(5) And (3) dripping 10 mu L of MWNTs-PDA-AuNPs-AmDH dispersion liquid obtained in the step (4) and gel containing methylene green, NADH-IL and [ BMIM ] Cl on the surface of carbon paper, and airing at 4 ℃ to obtain a working electrode which is recorded as MWNTs-PDA-AuNPs-AmDH-CP. The loading of the enzyme is 10mg of enzyme per 1g of composite material.

(6) Design and performance characterization of the bioelectrocatalysis reactor: and (5) taking the amine dehydrogenase electrode MWNTs-PDA-AuNPs-AmDH-CP obtained in the step (5) as a working electrode, a platinum electrode as a counter electrode and a calomel electrode as a reference electrode, and performing an electrochemical experiment at room temperature. The MWNTs-PDA-AuNPs-AmDH-CP tests are all carried out in 10mL of phosphate buffer (0.02M, pH 7.0.0) added with the electronic mediator toluidine blue and 20mg of coenzyme NADH, and N is introduced before the tests₂And the cyclic voltammetry is adopted in the testing process, wherein the substrate amino acid is not added dropwise to the blank control. The scanning rate of the cyclic voltammetry test is 100mV/s, the scanning range is-1.0V, the cyclic voltammetry test is carried out in a buffer solution with the pH value of 7.0, the substrate alpha-ketoglutaric acid is added with the concentration of 200mM, and the current density is regulated and controlled to be 1.2mA/cm²After 20 hours of reaction, the concentration of L-glutamic acid was 185 mM.

Example 4

(1) Preparation of aromatic alcohol dehydrogenase: according to the original sequence of aromatic alcohol dehydrogenase shown as SEQ ID NO.3, the gene sequence of aromatic alcohol dehydrogenase was synthesized by commercial company (Shanghai bioengineering Co., Ltd.) by PCR method and used for ligation with the vector pET28a to construct a plasmid. Ligation of the PCR product with the vector pET28 a: the PCR product and the vector pET28a were digested simultaneously with NdeI and XhoI, and the resulting product was purified and reacted with T4 DNA Ligase overnight at 4 ℃ to obtain a plasmid for transformation. Preparation of Competent cells was accomplished using the component Cel preference Kit. 10ng of plasmid for transformation was added to the competent cells, and after gently mixing, the mixture was left on ice for 30 minutes, and then left in a water bath at 42 ℃ for 45 seconds, and immediately left on ice for 1 to 2 minutes. Adding LB culture medium pre-warmed at 37 ℃, shaking and culturing at 37 ℃ for 1 hour, taking a proper amount of bacterial liquid, coating an LB solid plate with kanamycin resistance, culturing at 37 ℃ overnight, and picking out a single colony to obtain the recombinant escherichia coli (E.coli BL21(DE3)/pET28 a).

Cultivation of recombinant E.coli BL21(DE3)/pET28 a: the strain was inoculated into 200mL of LB medium at an inoculum size of 1%. The composition of LB culture medium is 10.0g/L tryptone, 5.0g/L yeast powder, 10g/L NaCl. The culture conditions were: the initial pH value is 7.0, the liquid loading volume fraction is 10%, the culture temperature is 37 ℃, the rotating speed of a shaking table is 200rpm, and the culture time is 6 hours. The inducer IPTG was added to a final concentration of 10mg/mL, and the culture was continued at 30 ℃ and 200rpm for 12 hours.

Preparation of crude enzyme solution: and (3) centrifuging the fermentation liquor obtained after the culture is finished in a refrigerated centrifuge (4 ℃, 8000rpm, 15min) to obtain cells, discarding supernate, resuspending the precipitate with Tris-HCl buffer solution (pH 7.5), fully washing, centrifuging, and repeating the operation for 3 times to obtain the cells. The cell fluid is treated by an ultrasonic disruptor, the probe of the cell disruptor is arranged 1cm below the liquid level, the disruption conditions are ultrasonic for 2 seconds at intervals of 4 seconds, ultrasonic is carried out for 40 times, and the power is 200W. Then, the mixture is centrifuged at 12,000rpm for 15min at 4 ℃ to remove insoluble cell debris, and the supernatant is crude enzyme solution of the aromatic alcohol dehydrogenase.

(2) Preparing pure enzyme of aromatic alcohol dehydrogenase: the aromatic alcohol dehydrogenase was isolated and purified by using His Trap nickel column from GE, and then desalted by ultrafiltration using 10K ultrafiltration centrifuge tube from PALL. The purification column adopted in the purification process is a HisTrap HP column capable of specifically purifying the protein with the His-tagged label, and the purification process comprises the steps of balancing, loading, balancing, eluting and column regeneration; collecting the eluted part and desalting by using an ultrafiltration centrifugal tube; the liquid obtained after desalting is the pure (ArylDH with His-tag) enzyme liquid of the aromatic alcohol dehydrogenase. Adding Tris-HCl (0.05M, pH 7.0) buffer solution into the mixture to prepare aqueous solution containing the aromatic alcohol dehydrogenase, and adjusting the concentration of the aromatic alcohol dehydrogenase to be 0.05-50 mg/mL according to needs for later use.

(3) Synthesis of ionic coenzyme NADH-IL: as in step (3) of example 1.

(4) Carbon cloth immobilized enzyme: carbon paper carbon cloth TGP-H-060 special for a fuel cell of Dongli corporation in Japan is adopted as a carrier. Graphene oxide is prepared by a Hummer method, and a uniformly dispersed GO solution is obtained after ultrasonic dispersion for 3 hours. 50mM and 15mM Zn (NO) were prepared separately₃)₂·3H₂O aqueous solution and 25mM,8mM H₃BTC ethanol solution. Adding Zn (NO) to GO solution₃)₂Adding the solution of aromatic alcohol dehydrogenase (ArylDH) with His-tag obtained in step (2) to the aqueous solution, and adding ligand H₃BTC ethanol solution; immersing carbon paper TGP-H-060 in the mixed solution, and obtaining CP/Zn by using electrostatic action and metal coordination driving in-situ assembly₃(BTC)₂Per GO-ArylDH, 10. mu.L of a mixture containing methylene green, NADH-IL and [ EMIM ]]BF₄The gel is dropped on CP/Zn₃(BTC)₂And drying the surface of the/GO-ArylDH at the temperature of 4 ℃ to obtain the working modified electrode. Zn₃(BTC)₂The particles are adsorbed on the GO sheet and have excellent adhesion, and then the particles are fixed on carbon paper to prepare the three-dimensional electrode material. The loading amount is 30mg of enzyme per 1g of composite material.

(5) Bioelectrocatalysis reaction: a bioelectrocatalytic microreactor with a capacity of 100mL was designed based on a disposable plastic cuvette. Designing a three-electrode system of the reactor, taking the carbon paper (3 multiplied by 3cm) fixed with the enzyme and the gel prepared in the step (4) as a working electrode, taking a graphite electrode as a counter electrode and taking Ag/AgCl as a reference electrode. The reaction was carried out in 10mL of phosphate buffer (0.02mM, pH 7.4) to which NADH-IL was addedFront introduction of N₂And cyclic voltammetry is adopted in the test process, wherein the blank is not added with the substrate dropwise. The scanning rate of the cyclic voltammetry test is 50mV/s, the scanning range is-0.8V, the cyclic voltammetry test is carried out in a buffer solution with the pH value of 7.5, the time of the chronoamperometric test is 1200s, and the adding concentration of the acetophenone is 500 mM. The current density is regulated and controlled to be 2.5mA/cm²The reaction is carried out for 20h at 40 ℃ to obtain the product of 450mM of phenethyl alcohol.

Example 5

(1) Preparation of crude malate dehydrogenase: the gene sequence of malate dehydrogenase was synthesized by a commercial company (Shanghai bioengineering Co., Ltd.) by PCR based on the malate dehydrogenase original sequence shown in SEQ ID NO.4, and used for ligation with the vector pET28a to construct a plasmid.

Ligation of the PCR product (gene sequence of malate dehydrogenase) with the vector pET28 a: the PCR product and the vector pET28a were digested simultaneously with NdeI and XhoI, and the resulting product was purified and reacted with T4 DNA ligase overnight at 4 ℃ to obtain a plasmid for transformation. Preparation of Competent cells was accomplished using the component Cell preference Kit. 10ng of plasmid for transformation was added to the competent cells, and after gently mixing, the mixture was left on ice for 30 minutes, and then left in a water bath at 42 ℃ for 45 seconds, and immediately left on ice for 1 to 2 minutes. Adding LB culture medium pre-warmed at 37 ℃, shaking and culturing at 37 ℃ for 1 hour, taking a proper amount of bacterial liquid, coating an LB solid plate with kanamycin resistance, culturing at 37 ℃ overnight, and picking out a single colony to obtain the recombinant escherichia coli (E.coli BL21(DE3)/pET28 a).

Cultivation of recombinant E.coli BL21(DE3)/pET28 a: the strain was inoculated into 200mL of LB medium at an inoculum size of 1%. The composition of LB culture medium is 10.0g/L tryptone, 5.0g/L yeast powder, 10g/L NaCl. The culture conditions were: the initial pH value is 7.0, the liquid loading volume fraction is 10%, the culture temperature is 37 ℃, the rotating speed of a shaking table is 200rpm, and the culture time is 6 hours. The inducer IPTG was added to a final concentration of 10mg/mL, and the culture was continued at 30 ℃ and 200rpm for 2 hours.

Preparation of crude enzyme solution: and (3) centrifuging the fermentation liquor obtained after the culture is finished in a refrigerated centrifuge (4 ℃, 8000rpm, 15min) to obtain cells, discarding supernate, resuspending the precipitate with Tris-HCl buffer solution (pH 7.5), fully washing, centrifuging, and repeating the operation for 3 times to obtain the cells. The cell fluid is treated by an ultrasonic disruptor, the probe of the cell disruptor is arranged 1cm below the liquid level, the disruption conditions are ultrasonic for 2 seconds at intervals of 4 seconds, ultrasonic is carried out for 40 times, and the power is 200W. Then, the mixture is centrifuged at 12,000rpm for 15min at 4 ℃ to remove insoluble cell debris, and the supernatant is the crude malate dehydrogenase enzyme solution.

(2) Preparation of malate dehydrogenase pure enzyme: the crude malate dehydrogenase enzyme solution was separated and purified using His Trap nickel column from GE, and then desalted by ultrafiltration using 10K ultrafiltration tube from PALL. The purification column adopted in the purification process is a HisTrap HP column capable of specifically purifying the protein with the His-tagged label, and the purification process comprises the steps of balancing, loading, balancing, eluting and column regeneration; collecting the eluted part and desalting by using an ultrafiltration centrifugal tube; the liquid obtained after the desalting is the malic dehydrogenase pure enzyme liquid. Adding Tris-HCl (0.05M, pH 7.0) buffer solution into the solution to prepare a malate dehydrogenase solution containing malate dehydrogenase, and adjusting the concentration of the malate dehydrogenase to be 0.05-50 mg/mL according to needs for later use.

(3) Preparing an enzyme electrode modified material carbon nano tube MWNTs-PDA: 30mg of multi-walled carbon nanotubes (MWNTs) (Nanjing Ginko nanotechnology Co., Ltd., multi-walled carbon nanotube JCMT-95-11-10) is added with 30mL of water and ultrasonically dispersed for 3h to obtain a uniformly dispersed 1mg/mL MWNTs solution, 40mg of PDA is added under stirring, the mixture is stirred at room temperature for 12h and centrifuged and resuspended to prepare a 1mg/mL MWNTs-PDA solution, and the SEM image of the MWNTs-PDA is shown in figure 6. MWNTs-PDA solution and NaAuCl₄The solution is mixed and reacted to obtain MWNTs-PDA solution containing AuNPs.

(4) MWNTs-PDA-AuNPs-MDH preparation: mixing the malate dehydrogenase solution with the enzyme concentration of 5mg/mL obtained in the step (2) with MWNTs-PDA solution containing AuNPs at the temperature of 4 ℃ in the ratio of 2:1:1, centrifuging at 8000rpm, discarding supernatant, and precipitating to form malate dehydrogenase immobilized mixture MWNTs-PDA-AuNPs-MDH; the mixture was then centrifuged at 8000rpm for 15 minutes, washed, and resuspended in 0.08M aqueous ammonia-ammonium chloride buffer pH 9.0 to prepare a MWNTs-PDA-AuNPs-MDH dispersion containing 1mg/mL malate dehydrogenase.

(5) And (3) dripping 10 mu L of MWNTs-PDA-AuNPs-MDH dispersion liquid obtained in the step (4) and gel containing methylene green, NADH-IL and [ BMIM ] Cl on the surface of a Glassy Carbon Electrode (GCE), and airing at 4 ℃ to obtain a modified electrode which is recorded as MWNTs-PDA-AuNPs-MDH-GCE. As a control, MWNTs modified electrodes were prepared by drop-coating and are designated as MWNTs-GCE.

(6) Bioelectrocatalysis reaction: designing a three-electrode system of the reactor based on the glass bioelectrocatalysis microreactor, taking the GCE immobilized with the enzyme obtained in the step (5) as a working electrode, taking a graphite electrode as a counter electrode and taking Ag/AgCl as a reference electrode. The reaction was carried out in 10mL of phosphate buffer (0.02mM, pH 7.4) to which NADH-IL was added, and N was added before the reaction₂And cyclic voltammetry is adopted in the test process, wherein the blank is not added with the substrate dropwise. The sweep rate of the cyclic voltammetry test was 50mV/s, the sweep range was-0.8V, the test was run in a buffer solution at pH 9.0, the chronoamperometric test was 1200s, and the oxaloacetate addition concentration was 250 mM. The current density is regulated and controlled to be 1.5mA/cm²The reaction was carried out at 10 ℃ for 5 hours, and the concentration of L-malic acid was 220 mM.

Example 6

(1) Examples 5 and 2 are as described in example 5.

(3) Preparation of immobilized enzyme CP-rGO-PDA-AuNPs-MDH: graphene oxide is prepared by a Hummer method, and a uniformly dispersed GO solution is obtained after ultrasonic dispersion for 3 hours. 0.5g ascorbic acid was added to 1mg/mL GO for reduction for 12h, centrifuged at 12000rpm, the supernatant removed, washed once with 10mL ultrapure water, centrifuged again, and then 10mL of 0.1M citric acid buffer (pH 7.0) was added to prepare a 1mg/mL rGO solution. To 10mL of a 1mg/mL rGO solution were added 20mg Dopamine (DA) and 5mL of 2mg/mL MDH, and 3.0mM NaAuCl was added with slow stirring at room temperature₄Carrying out chemical oxidative polymerization and immobilizing malate dehydrogenase, reacting for 5 hours, centrifuging the obtained dispersion liquid at 10000rpm, removing supernatant, and dispersing the precipitate (namely malate dehydrogenase immobilization mixture rGO-PDA-AuNPs-MDH) into 10mL PBS.

(4) And (4) dripping 10 mu L of rGO-PDA-AuNPs-MDH dispersion liquid obtained in the step (3) and gel containing methylene green, NADH-IL and [ BMIM ] Cl on the surface of carbon paper, and airing at 4 ℃ to obtain a modified electrode, which is recorded as CP-rGO-PDA-AuNPs-MDH.

(5) Catalyzing reaction by using an enzyme electric reactor: designing a three-electrode system of the reactor based on a glass bioelectrochemical microreactor, taking the carbon paper CP-rGO-PDA-AuNPs-MDH (2 multiplied by 2cm) fixed with the enzyme and the multifunctional gel obtained in the step (4) as a working electrode, taking a graphite electrode as a counter electrode and taking Ag/AgCl as a reference electrode. The reaction was carried out in 10mL of phosphate buffer (0.02mM, pH 7.4) to which NADH-IL was added, and N was added before the reaction₂And cyclic voltammetry is adopted in the test process, wherein the blank is not added with the substrate dropwise. The sweep rate of the cyclic voltammetry test was 40mV/s, the sweep range was-0.8V, the test was run in a buffer solution at pH 9.5, the chronoamperometric test was 1200s, and the oxaloacetate addition concentration was 250 mM. The current density is regulated to be 1.5mA/cm2, the reaction is carried out for 5 hours at the temperature of 10 ℃, and the concentration of the L-malic acid is 120 mM.

Example 7

(1) Preparation of geraniol dehydrogenase crude enzyme: the gene sequence of geraniol dehydrogenase was synthesized by a commercial company (Shanghai bioengineering Co., Ltd.) by PCR method based on the original sequence of geraniol dehydrogenase shown as SEQ ID NO.5, and used for ligation with the vector pET28a to construct a plasmid.

Ligation of the PCR product (geraniol dehydrogenase gene sequence) to the vector pET28 a: the PCR product and the vector pET28a were digested simultaneously with NdeI and XhoI, and the resulting product was purified and reacted with T4 DNA ligase overnight at 4 ℃ to obtain a plasmid for transformation. Preparation of Competent cells was accomplished using the component Cell preference Kit. 10ng of plasmid for transformation was added to the competent cells, and after gently mixing, the mixture was left on ice for 30 minutes, and then left in a water bath at 42 ℃ for 45 seconds, and immediately left on ice for 1 to 2 minutes. Adding LB culture medium pre-warmed at 37 ℃, shaking and culturing at 37 ℃ for 1 hour, taking a proper amount of bacterial liquid, coating an LB solid plate with kanamycin resistance, culturing at 37 ℃ overnight, and picking out a single colony to obtain the recombinant escherichia coli (E.coli BL21(DE3)/pET28 a).

Cultivation of recombinant E.coli BL21(DE3)/pET28 a: the strain was inoculated into 200mL of LB medium at an inoculum size of 1%. The composition of LB culture medium is 10.0g/L tryptone, 5.0g/L yeast powder, 10g/L NaCl. The culture conditions were: the initial pH value is 7.0, the liquid loading volume fraction is 10%, the culture temperature is 37 ℃, the rotating speed of a shaking table is 200rpm, and the culture time is 6 hours. The inducer IPTG was added to a final concentration of 10mg/mL, and the culture was continued at 30 ℃ and 200rpm for 2 hours.

Preparation of crude enzyme solution: and (3) centrifuging the fermentation liquor obtained after the culture is finished in a refrigerated centrifuge (4 ℃, 8000rpm, 15min) to obtain cells, discarding supernate, resuspending the precipitate with Tris-HCl buffer solution (pH 7.5), fully washing, centrifuging, and repeating the operation for 3 times to obtain the cells. The cell fluid is treated by an ultrasonic disruptor, the probe of the cell disruptor is arranged 1cm below the liquid level, the disruption conditions are ultrasonic for 2 seconds at intervals of 4 seconds, ultrasonic is carried out for 40 times, and the power is 200W. Then, the mixture is centrifuged at 12,000rpm for 15min at 4 ℃ to remove insoluble cell debris, and the supernatant is the crude enzyme solution of geraniol dehydrogenase.

(2) Preparation of pure enzyme of geraniol dehydrogenase: the geraniol dehydrogenase crude enzyme solution was separated and purified by using His Trap nickel column from GE company, and then subjected to ultrafiltration desalting by using 10K ultrafiltration centrifugal tube from PALL company. The purification column adopted in the purification process is a HisTrap HP column capable of specifically purifying the protein with the His-tagged label, and the purification process comprises the steps of balancing, loading, balancing, eluting and column regeneration; collecting the eluted part and desalting by using an ultrafiltration centrifugal tube; the liquid obtained after desalting is the pure geraniol dehydrogenase liquid. Adding Tris-HCl (0.1M, pH 6.5) buffer solution into the mixture to prepare geraniol dehydrogenase solution containing geraniol dehydrogenase, and adjusting the concentration of the geraniol dehydrogenase to be 0.05-50 mg/mL for later use according to needs.

(3) Carbon cloth immobilized enzyme: carbon paper TGP-H-060 special for fuel cells of Dongli corporation of Japan was used as a carrier. Graphene oxide is prepared by a Hummer method, and a uniformly dispersed GO solution is obtained after ultrasonic dispersion for 3 hours. 50mM and 15mM NiCl are respectively prepared₂Aqueous solution and 25mM,8mM HITP solution. Adding NiCl into GO solution₂Adding geraniol dehydrogenase (GerDH) solution with His-tag obtained in the step (2) into the aqueous solution, immersing carbon paper TGP-H-060 into the mixed solution, and finally adding ligand HITP solution; CP/Ni production by electrostatic interaction and metal coordination driven assembly₃(HITP)₂(GO-GerDH) containingMethyl Green, NADH-IL and [ EMIM]BF₄The gel is dropped on CP/Ni₃(HITP)₂And drying the surface of the/GO-GerDH at 4 ℃ to obtain the working modified electrode. Ni₃(HITP)₂The particles are adsorbed on the GO sheet and have excellent adhesion, and then the particles are fixed on carbon paper to prepare the three-dimensional electrode material. The loading of the enzyme is 40mg of enzyme per 1g of composite material.

(4) Electro-biocatalytic reaction:

a bioelectrocatalytic microreactor with a capacity of 100mL was designed based on a disposable plastic cuvette. And (3) designing a three-electrode system of the reactor, taking the carbon paper (3 multiplied by 3cm) fixed with the enzyme and the gel obtained in the step (3) as a working electrode, taking a graphite electrode as a counter electrode, and taking Ag/AgCl as a reference electrode. The reaction was carried out in 10mL of phosphate buffer (0.02mM, pH 7.4) to which NADH-IL was added, and N was added before the reaction₂And cyclic voltammetry is adopted in the test process, wherein the blank is not added with the substrate dropwise. The scanning rate of the cyclic voltammetry test is 50mV/s, the scanning range is-0.8V, the cyclic voltammetry test is carried out in a buffer solution with the pH value of 7.0, the time of the chronoamperometric test is 1200s, and the adding concentration of the L-phenylpyruvic acid is 300 mM. The current density is regulated and controlled to be 1.5mA/cm²The reaction was carried out at 50 ℃ for 12 hours to obtain a geraniol concentration of 263 mM.

Example 8

(1) Preparing a crude enzyme solution of the homophenylalanine dehydrogenase: according to the homophenylalanine dehydrogenase sequence shown as SEQ ID No.6, a gene sequence was synthesized from a commercial company (Shanghai bioengineering Co., Ltd.) by a PCR method in a whole gene; amplifying the gene sequence and ligating it to pET28a vector, after which the plasmid was introduced into e.coli bl21 DE 3; cultivation of recombinant E.coli BL21(DE3)/pET28 a: the strain was inoculated into 200mL of LB medium at an inoculum size of 1%. The composition of LB culture medium is 10.0g/L tryptone, 5.0g/L yeast extract, 10g/L NaCl. The culture conditions were: the initial pH value is 7.0, the liquid loading volume fraction is 10%, the culture temperature is 37 ℃, the rotating speed of a shaking table is 200rpm, and the culture time is 6 hours. The inducer IPTG was added to a final concentration of 10mg/mL, and the culture was continued at 25 ℃ and 200rpm for 12 hours. And (3) centrifuging the fermentation liquor obtained after the culture is finished in a refrigerated centrifuge (4 ℃, 8000rpm, 15min) to obtain cells, discarding supernatant, re-suspending the precipitate with phosphate buffer (pH 7-7.4), fully washing, centrifuging, repeating the operation for 3 times, and preparing cell suspension with the phosphate buffer (pH 7-7.4) to obtain cell suspension with the concentration of 50-150 g/L. And (3) placing the prepared cell suspension in an ice bath, treating cell sap by using an ultrasonic disruptor, placing a probe of the cell disruptor below the liquid level by 1cm, and carrying out ultrasonic treatment for 60 times at 6-second intervals under the disrupting conditions of 3 seconds of ultrasonic treatment and 200W of power. Then, the mixture is centrifuged at 12,000rpm for 15min at 4 ℃ to remove insoluble cell debris, and the supernatant is the crude enzyme solution containing the His-tag-labeled high phenylalanine dehydrogenase.

(2) Preparing pure enzyme of homophenylalanine dehydrogenase: his Trap nickel column (Histrap) from GE corporation is used^TMHP, 5mL) was used to separate and purify the crude enzyme solution obtained in step (1), and ultrafiltration was performed using a 10K ultrafiltration centrifuge tube from PALL corporation to remove salts. The purification column adopted in the purification process is a HisTrap HP column capable of specifically purifying the protein with the His-tagged label, and the purification process comprises the steps of balancing, loading, balancing, eluting and column regeneration; collecting the eluted part and desalting by using an ultrafiltration centrifugal tube; the liquid obtained after desalting is a purified enzyme solution of the His-tag-tagged homophenylalanine dehydrogenase shown in FIG. 2.

(3) And (3) enzyme activity detection: the catalytic reaction system comprises 40mM NADH, 0.2mol/L glycine-sodium hydroxide buffer solution (pH 9.5), 20% isopropanol as a cosolvent, 40mM ethyl 2-oxo-4-phenylbutyrate and 20mg/mL enzyme, and the reaction is carried out at 37 ℃ and the enzyme activity is measured at a wavelength of 340 nm. The enzyme activity is defined as the consumption (or formation) of 1. mu. mol NAD per minute by oxidation under the above conditions⁺The required enzyme amount is one enzyme activity unit. The enzyme activity was measured to be 0.192U/mg.

(4) And (3) enzyme activity detection: the catalytic reaction system comprises 200mM NADH, 0.5mol/L glycine-sodium hydroxide buffer solution (pH 11), a cosolvent of 10% acetonitrile, 80mM L-2-carbonyl-5-phenylpentanoic acid and 60mg/mL enzyme, and the reaction is carried out at 40 ℃ and the enzyme activity is measured at a wavelength of 340 nm. The enzyme activity is defined as the consumption (or formation) of 1. mu. mol NAD per minute by oxidation under the above conditions⁺The required enzyme amount is one enzyme activity unit. The enzyme activity was measured to be 0.223U/mg.

(5) In-situ mineralization and immobilization enzyme of porous composite carbon material

Immobilizing enzyme and gel on porous carbon paper, and performing biomimetic mineralization to construct an electrode/electronic mediator/coenzyme/enzyme integrated composite system. Firstly, preparing a 100mg/mL 500kDa branched polyethyleneimine solution, preparing a graphene oxide-polyethyleneimine (GO-PEI) composite material, and reducing by a hydrothermal method to obtain rGO-PEI. Secondly, the homophenylalanine dehydrogenase adds Mn²⁺Metal salt solution and brought to a final concentration of 50mmol/L by MnCl₂Metal coordination is immobilized on an rGO-PEI carrier, 2mg/mL high phenylalanine dehydrogenase solution is added into the rGO-PEI, and PEI and Mn are utilized²⁺And (3) immobilizing the enzyme by using ion coordination force. Adding a solution of titanium di- (2-hydroxypropionic acid) diammonium dihydroxide (Ti-BALDH) as a precursor of titanium at a concentration of 0.5mol/L and a pH in the range of 10.0, and a solution containing methylene green, NADH-IL and [ EMIM ]]BF₄The multifunctional conductive gel of (1), PEI-induced TiO₂Biomimetic mineralization realizes in-situ immobilization of enzyme and coenzyme on porous carbon paper.

(6) Electrocatalytic reaction: and (3) enzyme activity detection: the catalytic reaction system comprises 100mM NADH, 0.6mol/L glycine-sodium hydroxide buffer solution (pH 11), 30% propanol as a cosolvent, 40mM L-2-carbonyl-3-phenylbutyric acid and 80mg/ml enzyme, and the reaction is carried out at 35 ℃ and the enzyme activity is measured at a wavelength of 340 nm. The enzyme activity is defined as the consumption (or formation) of 1. mu. mol NAD per minute by oxidation under the above conditions⁺The required enzyme amount is one enzyme activity unit. The enzyme activity was determined to be 0.22U/mg. A bioelectrocatalytic microreactor with a capacity of 50mL was designed based on a disposable plastic cuvette. A reactor three-electrode system is designed, carbon paper (5 multiplied by 5cm) fixed with enzyme and gel is used as a working electrode, a counter electrode is a graphite electrode, and Ag/AgCl is used as a reference electrode. The reaction was carried out in 10mL of phosphate buffer (0.02mM, pH 7.4) to which NADH-IL was added, and N was added before the reaction₂And cyclic voltammetry is adopted in the test process, wherein the blank is not added with the substrate dropwise. The sweep rate of the cyclic voltammetry test was 50mV/s, the sweep range was-0.8V, the test was performed in a buffer solution at pH 9.5, the amperometric assay was timed at 1200s, and L-2-carbonyl-3-phenylbutyric acid was added at a concentration of 40 mM. The current density is regulated and controlled to be 1.5mA/cm²20 degree reactionAfter 10h, 33.7mM of L-2-amino-3-phenylbutyric acid are obtained. Product analysis was performed by NMR to obtain NMR spectrum 1H NMR:δ1.26(3H, d, J ═ 6.7Hz),3.18(1H, dq, J ═ 7.1,6.7Hz),3.64(1H, d, J ═ 7.1Hz),7.21(2H, dddd, J ═ 7.8,1.3,1.2,0.5Hz),7.28-7.40(3H,7.35(dddd, J ═ 7.8,7.7,1.9,0.5Hz),7.31(tt, J ═ 7.7,1.3Hz)). conforming to the characteristics of the product L-2-amino-3-phenylbutyric acid.

The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims and their equivalents.

Sequence listing

<110> university of mansion

<120> oxidoreductase electrode for enzymatic electrocatalytic reduction, method for preparing same, and enzymatic bioreactor using same

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 354

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Lys Ile Leu Glu Thr Met Lys Ala Ser Asp Tyr Glu Gln Leu Val

1 5 10 15

Phe Cys Gln Asp Glu Lys Thr Gly Leu Lys Gly Ile Ile Ala Ile His

20 25 30

Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Thr Tyr

35 40 45

Asp Asn Glu Glu Glu Ala Ile Glu Asp Val Leu Arg Leu Ala Arg Gly

50 55 60

Met Thr Tyr Lys Ser Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys

65 70 75 80

Gly Val Ile Ile Gly Asp Pro Lys Lys Asp Lys Ser Glu Glu Met Trp

85 90 95

Arg Ala Phe Gly Arg Phe Val Gln Ser Leu Asn Gly Arg Tyr Ile Thr

100 105 110

Ala Glu Asp Val Gly Val Arg Glu Thr Asp Leu Glu Ile Val Asn Thr

115 120 125

Glu Thr Asp Phe Ala Val Gly Leu Pro Gly Lys Ser Gly Asn Pro Ser

130 135 140

Pro Ala Thr Ala Tyr Gly Val Tyr Ser Gly Ile Lys Ala Val Ala Asp

145 150 155 160

Glu Ile Trp Gly Ser Ala Asp Leu Asn Gly Lys Thr Ile Ala Ile Gln

165 170 175

Gly Ala Gly Ser Val Gly Tyr Tyr Leu Ser Glu Leu Leu His Lys Asp

180 185 190

Gly Ala Lys Leu Ile Val Thr Asp Ile Asp Lys Glu Ala Val Asp Lys

195 200 205

Leu Val Ser Asp Phe Gly Ala Thr Ala Val Glu Thr Asp Glu Ile Tyr

210 215 220

Glu Gln Glu Ala Asp Ile Phe Ala Pro Cys Ala Leu Gly Ala Ile Leu

225 230 235 240

Asn Asp Glu Thr Ile Pro Lys Leu Lys Val Lys Ala Val Ala Gly Ala

245 250 255

Ala Asn Asn Gln Leu Glu Asp Glu Lys Arg His Ala Glu Glu Leu Lys

260 265 270

Lys Arg Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala Gly Gly

275 280 285

Val Ile Asn Val Ser Phe Glu Leu Thr Gly Tyr Asp Glu Glu Arg Ala

290 295 300

Tyr Arg Lys Ile Ser Thr Ile Tyr Asp Asn Ile Lys Lys Ile Phe Asn

305 310 315 320

Ile Ala Asn Arg Asp Asp Ile Thr Ser His Glu Ala Ala Asn Arg Met

325 330 335

Ala Glu Glu Arg Ile Glu Ala Ile Lys His Val Lys Thr Ser Tyr Ile

340 345 350

Asn Lys

<210> 2

<211> 1152

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

catatgagct tagtagaaaa aacatccatc ataaaagatt tcactctttt tgaaaaaatg 60

tctgaacatg aacaagttgt tttttgcaac gatccggcga caggactaag ggccattatc 120

gctattcatg acaccacact cggacctgcg ctcggcggct gccgcatgca gccttataac 180

agtgtggaag aagcattgga agatgctctt cgcctttcca aaggaatgac ttactcttgc 240

gcggcgtccg atgtcgactt tggcggcgga aaagcagtca ttatcggtga tccgcagaaa 300

gataaatctc cagaactgtt ccgcgcgttt ggccaatttg ttgattcgct tggcggccgt 360

ttctatacag gtactgatat gggaacgaat atggaagatt tcattcacgc catgaaagaa 420

acaaactgca ttgttggggt gccggaagct tacggcggcg gcggagattc ctctattcca 480

actgccatgg gtgtcctgta cggcattaaa gcaaccaaca aaatgttgtt tggcaaggac 540

gatcttggcg gcgtcactta tgccattcaa ggacttggca aagtaggcta caaagtagcg 600

gaagggctgc tcgaagaagg tgctcattta tttgtaacgg atattaacga gcaaacgttg 660

gaggctatcc aggaaaaagc aaaaacaaca tccggttctg tcacggtagt agcgagcgat 720

gaaatttatt cccaggaagc cgatgtgttc gttccgtgtg catttggcgg cgttgttaat 780

gatgaaacga tgaagcagtt caaggtgaaa gcaatcgccg gttcagccct gaatcagctg 840

cttacggagg atcacggcag acaccttgca gacaaaggca ttctgtatgc tccggattat 900

attgttaact ctggcggtct gatccaagta gccgacgaat tgtatgaggt gaacaaagaa 960

cgcgtgcttg cgaagacgaa gcatatttac gacgcaattc ttgaagtgta ccagcaagcg 1020

gaattagatc aaatcaccac aatggaagca gccaacagaa tgtgtgagca aagaatggcg 1080

gcaagaggcc gacgcaacag cttctttact tcttctgtta agccaaaatg ggatattcgc 1140

aactaactcg ag 1152

<210> 3

<211> 366

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Glu Ile Lys Ala Ala Ile Val Arg Gln Lys Asn Gly Pro Phe Leu

1 5 10 15

Leu Glu His Val Ala Leu Asn Glu Pro Ala Glu Asp Gln Val Leu Val

20 25 30

Arg Leu Val Ala Thr Gly Leu Cys His Thr Asp Leu Val Cys Arg Asp

35 40 45

Gln His Tyr Pro Val Pro Leu Pro Met Val Phe Gly His Glu Gly Ala

50 55 60

Gly Val Val Glu Arg Val Gly Ser Ala Val Lys Lys Val Gln Pro Gly

65 70 75 80

Asp His Val Val Leu Thr Phe Tyr Thr Cys Gly Ser Cys Asp Ala Cys

85 90 95

Leu Ser Gly Asp Pro Thr Ser Cys Ala Asn Ser Phe Gly Pro Asn Phe

100 105 110

Met Gly Arg Ser Val Thr Gly Glu Cys Thr Ile His Asp His Gln Gly

115 120 125

Ala Glu Val Gly Ala Ser Phe Phe Gly Gln Ser Ser Phe Ala Thr Tyr

130 135 140

Ala Leu Ser Tyr Glu Arg Asn Thr Val Lys Val Thr Lys Asp Val Pro

145 150 155 160

Leu Glu Leu Leu Gly Pro Leu Gly Cys Gly Ile Gln Thr Gly Ala Gly

165 170 175

Ser Val Leu Asn Ala Leu Asn Pro Pro Ala Gly Ser Ala Ile Ala Ile

180 185 190

Phe Gly Ala Gly Ala Val Gly Leu Ser Ala Val Met Ala Ala Val Val

195 200 205

Ala Gly Cys Thr Thr Ile Ile Ala Val Asp Val Lys Glu Asn Arg Leu

210 215 220

Glu Leu Ala Ser Glu Leu Gly Ala Thr His Ile Ile Asn Pro Ala Ala

225 230 235 240

Asn Asp Pro Ile Glu Ala Ile Lys Glu Ile Phe Ala Asp Gly Val Pro

245 250 255

Tyr Val Leu Glu Thr Ser Gly Leu Pro Ala Val Leu Thr Gln Ala Ile

260 265 270

Leu Ser Ser Ala Ile Gly Gly Glu Ile Gly Ile Val Gly Ala Pro Pro

275 280 285

Met Gly Ala Thr Val Pro Val Asp Ile Asn Phe Leu Leu Phe Asn Arg

290 295 300

Lys Leu Arg Gly Ile Val Glu Gly Gln Ser Ile Ser Asp Ile Phe Ile

305 310 315 320

Pro Arg Leu Val Glu Leu Tyr Arg Gln Gly Lys Phe Pro Phe Asp Lys

325 330 335

Leu Ile Lys Phe Tyr Pro Phe Asp Glu Ile Asn Arg Ala Ala Glu Asp

340 345 350

Ser Glu Lys Gly Val Thr Leu Lys Pro Val Leu Arg Ile Gly

355 360 365

<210> 4

<211> 305

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Thr Lys Val Ser Val Val Gly Ala Ala Gly Thr Val Gly Ala Ala Ala

1 5 10 15

Gly Tyr Asn Ile Ala Leu Arg Asp Ile Ala Asp Glu Val Val Phe Val

20 25 30

Asp Ile Pro Asp Lys Glu Asp Asp Thr Val Gly Gln Ala Ala Asp Thr

35 40 45

Asn His Gly Ile Ala Tyr Asp Ser Asn Thr Arg Val Arg Gln Gly Gly

50 55 60

Tyr Glu Asp Thr Ala Gly Ser Asp Val Val Val Ile Thr Ala Gly Ile

65 70 75 80

Pro Arg Gln Pro Gly Gln Thr Arg Ile Asp Leu Ala Gly Asp Asn Ala

85 90 95

Pro Ile Met Glu Asp Ile Gln Ser Ser Leu Asp Glu His Asn Asp Asp

100 105 110

Tyr Ile Ser Leu Thr Thr Ser Asn Pro Val Asp Leu Leu Asn Arg His

115 120 125

Leu Tyr Glu Ala Gly Asp Arg Ser Arg Glu Gln Val Ile Gly Phe Gly

130 135 140

Gly Arg Leu Asp His Asn Arg Ala Lys Ala Gln Leu Ala Lys Lys Thr

145 150 155 160

Gly Thr Gly Val Asp Arg Ile Arg Arg Met Thr Val Ile Leu Gly Glu

165 170 175

His Gly Asp Ala Gln Val Pro Val Phe Ser Lys Val Ser Val Asp Gly

180 185 190

Thr Asp Pro Glu Phe Ser Gly Asp Glu Lys Glu Gln Leu Leu Gly Asp

195 200 205

Leu Gln Glu Ser Ala Met Asp Val Ile Glu Arg Lys Gly Ala Thr Glu

210 215 220

Trp Gly Pro Ala Arg Gly Val Ala His Met Val Glu Ala Ile Leu His

225 230 235 240

Asp Thr Gly Glu Val Leu Pro Ala Ser Val Lys Leu Glu Gly Glu Phe

245 250 255

Gly His Glu Asp Thr Ala Phe Gly Val Pro Val Ser Leu Gly Ser Asn

260 265 270

Gly Val Glu Glu Ile Val Glu Trp Asp Leu Asp Asp Tyr Glu Gln Asp

275 280 285

Leu Met Ala Asp Ala Ala Glu Lys Leu Ser Asp Gln Tyr Asp Lys Ile

290 295 300

Ser

305

<210> 5

<211> 373

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Met Asn Asp Thr Gln Asp Phe Ile Ser Ala Gln Ala Ala Val Leu Arg

1 5 10 15

Gln Val Gly Gly Pro Leu Ala Val Glu Pro Val Arg Ile Ser Met Pro

20 25 30

Lys Gly Asp Glu Val Leu Ile Arg Ile Ala Gly Val Gly Val Cys His

35 40 45

Thr Asp Leu Val Cys Arg Asp Gly Phe Pro Val Pro Leu Pro Ile Val

50 55 60

Leu Gly His Glu Gly Ser Gly Thr Val Glu Ala Val Gly Glu Gln Val

65 70 75 80

Arg Thr Leu Lys Pro Gly Asp Arg Val Val Leu Ser Phe Asn Ser Cys

85 90 95

Gly His Cys Gly Asn Cys His Asp Gly His Pro Ser Asn Cys Leu Gln

100 105 110

Met Leu Pro Leu Asn Phe Gly Gly Ala Gln Arg Val Asp Gly Gly Gln

115 120 125

Val Leu Asp Gly Ala Gly His Pro Val Gln Ser Met Phe Phe Gly Gln

130 135 140

Ser Ser Phe Gly Thr His Ala Val Ala Arg Glu Ile Asn Ala Val Lys

145 150 155 160

Val Gly Asp Asp Leu Pro Leu Glu Leu Leu Gly Pro Leu Gly Cys Gly

165 170 175

Ile Gln Thr Gly Ala Gly Ala Ala Ile Asn Ser Leu Gly Ile Gly Pro

180 185 190

Gly Gln Ser Leu Ala Ile Phe Gly Gly Gly Gly Val Gly Leu Ser Ala

195 200 205

Leu Leu Gly Ala Arg Ala Val Gly Ala Asp Arg Val Val Val Ile Glu

210 215 220

Pro Asn Ala Ala Arg Arg Ala Leu Ala Leu Glu Leu Gly Ala Ser His

225 230 235 240

Ala Leu Asp Pro His Ala Glu Gly Asp Leu Val Ala Ala Ile Lys Ala

245 250 255

Ala Thr Gly Gly Gly Ala Thr His Ser Leu Asp Thr Thr Gly Leu Pro

260 265 270

Pro Val Ile Gly Ser Ala Ile Ala Cys Thr Leu Pro Gly Gly Thr Val

275 280 285

Gly Met Val Gly Leu Pro Ala Pro Asp Ala Pro Val Pro Ala Thr Leu

290 295 300

Leu Asp Leu Leu Ser Lys Ser Val Thr Leu Arg Pro Ile Thr Glu Gly

305 310 315 320

Asp Ala Asp Pro Gln Arg Phe Ile Pro Arg Met Leu Asp Phe His Arg

325 330 335

Ala Gly Lys Phe Pro Phe Asp Arg Leu Ile Thr Arg Tyr Arg Phe Asp

340 345 350

Gln Ile Asn Glu Ala Leu His Ala Thr Glu Lys Gly Glu Ala Ile Lys

355 360 365

Pro Val Leu Val Phe

370

<210> 6

<211> 354

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Met Phe Glu Lys Ile Ser Gln His Glu Gln Val Val Phe Cys Asn Asp

1 5 10 15

Pro Ser Thr Gly Leu Lys Ala Ile Ile Ala Ile His Asn Thr Thr Leu

20 25 30

Gly Pro Ala Leu Gly Gly Cys Arg Met Arg Pro Tyr Gly Ser Val Asp

35 40 45

Glu Ala Leu Glu Asp Val Leu Arg Leu Ser Lys Gly Met Thr Tyr Lys

50 55 60

Cys Ala Gly Ala Asp Val Asp Phe Gly Gly Gly Lys Ser Val Ile Ile

65 70 75 80

Gly Asp Pro Met Thr Asp Arg Thr Pro Glu Leu Phe Arg Ala Phe Gly

85 90 95

Gln Phe Val Asp Ser Leu Asn Gly Arg Phe Tyr Thr Gly Thr Asp Met

100 105 110

Gly Thr Thr Pro Asp Asp Phe Met His Ala Leu Lys Glu Thr Asn Cys

115 120 125

Ile Val Gly Val Pro Glu Glu Tyr Gly Gly Ser Gly Asp Ser Ser Val

130 135 140

Pro Thr Ala Gln Gly Val Ile Tyr Gly Leu Gln Ala Thr Ile Gln Thr

145 150 155 160

Leu Glu Gly Thr Asp Glu Leu Ser Gly Lys Ser Tyr Ser Ile Gln Gly

165 170 175

Leu Gly Lys Val Gly Phe Lys Val Ala Glu Gln Leu Leu Ala Ala Gly

180 185 190

Ala Gln Ile Tyr Val Thr Asp Ile Asn Glu Lys Ala Leu Lys Met Ile

195 200 205

Gln Glu Arg Ala Glu Leu Leu Pro Gly Asn Val Glu Val Val Glu Gly

210 215 220

Ser Asp Ile Tyr Gly Val Asp Ala Asp Ile Phe Ile Pro Cys Ala Leu

225 230 235 240

Gly Gly Ile Ile His Asp Glu Thr Ile Glu Gln Leu Lys Val Lys Ala

245 250 255

Ile Val Gly Ser Ala Asn Asn Gln Leu Leu Glu Asp Lys His Gly Leu

260 265 270

Tyr Leu Gln Gln Lys Gly Ile Leu Tyr Gly Pro Asp Tyr Ile Val Asn

275 280 285

Ala Gly Gly Leu Ile Gln Val Ala Asp Glu Leu Tyr Gly Pro Asn Lys

290 295 300

Ala Arg Val Leu Thr Lys Thr Arg Ala Ile Tyr Asp Ser Leu Ile Gln

305 310 315 320

Ile Tyr Ser Glu Ser Thr Lys Asn Gln Ile Ser Thr Met Glu Ala Ala

325 330 335

Asn Leu Phe Cys Glu Glu Lys Leu Leu Ala Arg Ser Lys Arg Asn Ser

340 345 350

Phe Phe