CN113564154A - Method for coordinating and immobilizing oxidoreductase by using graphene oxide-wool keratin and metal ions - Google Patents

Method for coordinating and immobilizing oxidoreductase by using graphene oxide-wool keratin and metal ions Download PDF

Info

Publication number
CN113564154A
CN113564154A CN202110937217.5A CN202110937217A CN113564154A CN 113564154 A CN113564154 A CN 113564154A CN 202110937217 A CN202110937217 A CN 202110937217A CN 113564154 A CN113564154 A CN 113564154A
Authority
CN
China
Prior art keywords
amino acid
acid dehydrogenase
enzyme
graphene oxide
oxidoreductase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202110937217.5A
Other languages
Chinese (zh)
Inventor
王世珍
王琪琪
江亮
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Xiamen University
Original Assignee
Xiamen University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Xiamen University filed Critical Xiamen University
Priority to CN202110937217.5A priority Critical patent/CN113564154A/en
Publication of CN113564154A publication Critical patent/CN113564154A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)

Abstract

The invention discloses a method for coordinating and immobilizing oxidoreductase by using graphene oxide-wool keratin and metal ions, and belongs to the technical field of immobilized enzymes. The graphene oxide has stable structure and good biocompatibility, and is an excellent enzyme immobilization carrier. The graphene oxide with the lamellar structure has high specific surface area, high flexibility and excellent processability, and is an excellent enzyme immobilization carrier. The wool keratin has an extremely dense structure formed by random coil, alpha-helix and beta-folding parallel polypeptide conformation, has effective functional groups, and has strong binding capacity with graphene oxide. The pH stability and the repeated use stability of the immobilized oxidoreductase obtained by the invention are obviously enhanced, and the immobilized oxidoreductase has higher enzyme activity recovery rate. The invention has the advantages of simple process, mild preparation conditions and high immobilization rate.

Description

Method for coordinating and immobilizing oxidoreductase by using graphene oxide-wool keratin and metal ions
Technical Field
The invention relates to a method for immobilizing oxidoreductase, belonging to the field of enzyme immobilization.
Background
Oxidoreductases are a generic term for a class of enzymes that catalyze the redox reaction between two molecules, including a variety of important enzymes. Wherein, the amino acid dehydrogenase (AaDH) can catalyze reversible amino acid oxidative deamination and ketonic acid reductive amination reaction. For reductive amination of non-natural substrates, the substrate ethyl 2-oxo-4-phenylbutyrate (EOPB) was selected in an ammonium salt containing system (providing NH)4 +Acceptor) to produce L-homophenylalanine (LHPA) with high selectivity. The AaDH can be used for synthesizing unnatural amino acid L-homophenylalanine (LHPA), so that the AaDH can be widely applied to the fields of medicines, novel biological materials, cosmetics and the like.
Since free amino acid dehydrogenases have poor stability, the use of immobilization methods can be improved. The performance of an immobilized enzyme depends on the nature of the support material used for the immobilized enzyme and the immobilization method. The enzyme immobilization material includes inorganic, organic polymers, gels, biomaterials, and the like. The prior art for immobilizing oxidoreductases such as amino acid dehydrogenase has the problems of poor stability of immobilized enzyme, low recycling activity and the like. Therefore, further studies on the immobilization carrier and the method are also necessary.
Disclosure of Invention
The invention provides a method for coordinating and immobilizing oxidoreductase by using graphene oxide-wool keratin and metal ions. The oxidoreductase can be used in this method, such as amino acid dehydrogenase.
One of the technical schemes adopted by the invention for solving the technical problems is as follows:
a method for coordinating and immobilizing oxidoreductase by using graphene oxide-wool keratin and metal ions comprises the steps of adding oxidoreductase into a wool keratin solution with the concentration of 0.1-50 mg/mL, and enabling the oxidoreductase to beThe final concentration of (A) is 0.1-100 Mg/mL, and after the mixture is uniformly mixed at 0-50 ℃, metal ions (Mg) are added2+、Zn2+、Ni2+、Mn2+、Cu2+And the like) and the final concentration of the graphene oxide solution is 0.8-40 mM for coordination, then the graphene oxide solution is added and the final concentration of the graphene oxide solution is 0.01-100 mg/mL, and the mixture is uniformly mixed at 0-50 ℃ to obtain the graphene oxide-wool keratin and metal ion coordination composite material immobilized oxidoreductase.
Further, the oxidoreductase includes an amino acid dehydrogenase.
Preferably, the amino acid sequence of the amino acid dehydrogenase is shown as SEQ ID No.1, and the amino acid dehydrogenase is provided with a His-tag label.
Further, the wool keratin is prepared by dissolving wool by a urea/sodium sulfide/Sodium Dodecyl Sulfate (SDS) process; the mass ratio of the wool, the urea, the sodium sulfide nonahydrate and the sodium dodecyl sulfate is (4-20): 22-50: 5-20: 1.4-2, the dissolving temperature is 25-65 ℃, and the reaction time is 2-20 h.
Preferably, the concentration of the wool keratin solution is preferably 0.1-0.16 mg/mL, and the enzyme activity of the immobilized enzyme is highest when the concentration of the wool keratin solution is 0.14-0.16 mg/mL.
Graphene Oxide (GO) is an important derivative of Graphene, has good biocompatibility, and has wide application prospects in the fields of biological medicines, drug sustained release, packaging materials and the like. GO has a two-dimensional lamellar structure similar to graphene, and the oxidation degree can be regulated and controlled by controlling the chemical oxidation time. GO with different oxidation degrees has different numbers of oxygen-containing groups such as-OH, -COOH and-O-on the two-dimensional plane and the edge of the carbon skeleton, and has different hydrophobic properties, so that the GO has good biocompatibility.
The macromolecular chain of Wool Keratin (WK) contains a large number of peptide bond structures, which are the basic skeleton structures in the macromolecular chain structure of protein substances. Besides, the surface of the macromolecular chain also has a large number of functional groups such as amino, carboxyl and the like. Wool keratin is a very dense structure composed of random coil, alpha-helix and beta-sheet parallel polypeptide conformations together.
According to researches, the interaction of the wool keratin, the graphene oxide and the amino acid dehydrogenase can form a composite material, so that the composite material has good biocompatibility and is beneficial to enzyme immobilization.
In a particular embodiment, the method comprises the steps of:
1) preparing a crude enzyme solution: inoculating a recombinant expression strain capable of expressing the amino acid dehydrogenase with the His-tag label into an LB culture medium containing kanamycin for culture, and adding a lactose inducer IPTG after culturing for a period of time; culturing the obtained bacterial liquid, centrifuging to obtain cells, and preparing into cell suspension; carrying out ultrasonic crushing and centrifugation, and collecting supernatant, namely crude enzyme liquid containing amino acid dehydrogenase with a His-tag label;
wherein, the method for constructing the recombinant expression strain capable of expressing the amino acid dehydrogenase with the His-tag label comprises the following steps: the gene sequence of the amino acid dehydrogenase is shown as SEQ ID NO. 1; carrying out double enzyme digestion on the amino acid dehydrogenase gene and the pET28a plasmid respectively by NdeI and Xhol, and carrying out ligation transformation to obtain a pET28a-AaDH plasmid; the plasmid is transformed into E.coli BL21(DE3), and a recombinant expression strain E.coli BL21(DE3)/pET28a capable of expressing His-tag-labeled amino acid dehydrogenase is obtained.
The recombinant expression strain E.coli BL21(DE3)/pET28a capable of expressing the His-tag-labeled amino acid dehydrogenase is inoculated into LB culture medium containing kanamycin for culture, and after a period of culture, a lactose inducer IPTG is added. The inoculation amount is 1-3%, and the LB culture medium comprises the following components: 5.0-15.0 g/L tryptone, 1.0-10.0 g/L yeast extract and 0.0-15.0 g/L NaCl, adjusting the pH value to 7.0-7.5, and adding kanamycin before inoculation to make the final concentration to be 50-150 mu g/mL; the culture conditions are 36-38 ℃, 150-250 rpm culture is carried out for 1.5-6 h, then inducer IPTG is added to enable the final concentration to be 5-15 mg/mL, and the culture is continued for 2-12 h at 25-30 ℃ and 150-250 rpm. Culturing the obtained bacterial liquid, centrifuging in a refrigerated centrifuge (preferably at 4 ℃, 8000rpm, 15min) to obtain cells, discarding supernatant, resuspending the precipitate with phosphate buffer (pH 7-7.5), fully washing, centrifuging, and repeating the operation for 3 times. Preparing cell suspension with the concentration of 50-150 g/L by using phosphate buffer solution (pH 6.5-8.0).
And placing the prepared cell suspension in an ice bath, placing a probe of an ultrasonic cell disruptor below the liquid level by 1cm, performing ultrasonic treatment at a power of 200W for 3 seconds at intervals of 6 seconds, and performing ultrasonic treatment for 20-60 times. Then, the mixture is centrifuged at 12000rpm for 15min at 4 ℃ to remove insoluble cell debris, and the supernatant is the crude enzyme solution containing the amino acid dehydrogenase with the His-tag label.
2) Preparation of pure enzyme: purifying the crude enzyme solution obtained in the step 1) by using a nickel column and desalting; the adopted purification column is a HisTrap HP column capable of specifically purifying the protein with the His-tagged label, and the steps comprise balancing, loading, balancing, eluting and column regeneration; and collecting the eluted part, desalting by using an ultrafiltration centrifugal tube, and obtaining a liquid after desalting, namely a pure enzyme solution of the amino acid dehydrogenase with the His-tag label.
3) Preparation of wool keratin solution: firstly, weighing 6 g of sodium sulfide nonahydrate, 24 g of urea and 1.44 g of sodium dodecyl sulfate, mixing, adding into a reaction bottle, adding ultrapure water to make the volume of the solution reach 50mL, and putting the reaction bottle into an ultrasonic cleaning machine for completely dissolving solid substances by ultrasonic. Weighing 5 g of wool, adding the wool into a blue-mouth bottle, completely immersing the wool into the solution, and then putting the blue-mouth bottle into a constant-temperature drying oven at 60 ℃ for heat preservation for 8 hours to completely dissolve the wool. The keratin solution at this time contains not only keratin but also chemical agents previously added, which can be removed by deionized water using a dialysis method. The wool keratin solution obtained by preliminary dissolution is balanced in pairs and put into a centrifuge, the centrifugation is carried out at the rotating speed of 9000 r/min, the yellowish keratin clear solution is obtained after the centrifugation and the filtration, then a dialysis bag with the molecular weight cutoff of 3500Da is selected for dialyzing the keratin, the deionized water is replaced every 2 hours, the dialysis time is 3 days, thus the chemical reagent in the keratin solution can be removed, and finally the purified wool keratin solution is obtained;
4) preparing graphene oxide-wool keratin and metal ion coordination immobilized amino acid dehydrogenase: adding the diluted solution of the wool keratin solution obtained in the step 3) into the His-t-carrying solution obtained in the step 2)ag-labeled amino acid dehydrogenase, and the final concentration of His-tag-labeled amino acid dehydrogenase is 0.1-100 Mg/mL, the mixture is oscillated in a 0-50 ℃ constant temperature oscillator, and after the wool keratin and the amino acid dehydrogenase are combined, 0.8-40 mM metal ions (Mg) are added2+、Zn2+、Ni2+、Mn2 +、Cu2+And the like), adding the prepared graphene oxide solution (0.01-100 mg/mL) after chelating metal ions, uniformly mixing, and oscillating in a constant temperature oscillator at 4 ℃ to obtain the graphene oxide-wool keratin and metal ion coordination composite material immobilized amino acid dehydrogenase.
5) The detection method of the enzyme activity comprises the following steps: the activity determination reaction system of the amino acid dehydrogenase comprises 10 mu L of ammonium chloride-ammonia water with the concentration range of 50-200 mM, 10 mu L of substrate solution with the concentration range of 8-12 and the concentration range of 160 mu L of 4mM NADH as an amino donor, 10 mu L of 40mM and 20 mu L of enzyme solution, wherein the substrate comprises 2-oxo-ethyl butyrate, phenylpyruvic acid or alpha-ketoglutaric acid, and the activity of the enzyme is determined at the wavelength of 340 nm. The enzyme activity is defined as the amount of enzyme required to consume or generate 1. mu. mol NADH by oxidation per minute under the above conditions as one unit of enzyme activity. The second technical scheme adopted by the invention for solving the technical problems is as follows:
the invention also provides the graphene oxide-wool keratin and metal ion coordination composite material immobilized oxidoreductase prepared by the method.
Taking the amino acid dehydrogenase shown as SEQ ID No.1 as an example, the temperature stability and the repeated use times of the amino acid dehydrogenase can be improved by immobilizing the amino acid dehydrogenase by using graphene oxide and wool keratin as immobilization carriers. The immobilized amino acid dehydrogenase prepared by the method has the optimum pH of 9 and the optimum temperature of 60-70 ℃; the immobilized amino acid dehydrogenase has good tolerance to acetic acid and cyclohexane.
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.
The invention has the following beneficial effects:
the graphene oxide with the lamellar structure is an excellent immobilized carrier, and the surface of the graphene oxide is lack of functional groups such as epoxy groups, carbonyl groups, carboxyl groups and the like, so that the load area is large. In the immobilization process, the graphene oxide immobilized amino acid dehydrogenase is mainly hydrophobic. The wool keratin has a very compact structure consisting of random coil, alpha-helix and beta-fold parallel polypeptide conformation, and has strong binding capacity with graphene oxide due to effective chelating and functional groups (amino, carboxyl, thiol and the like). By utilizing the interaction of Wool Keratin (WK), graphene oxide and amino acid dehydrogenase and regulating the temperature and pH of an immobilized enzyme system, the hydrophobic interaction and hydrogen bond acting force between the enzyme and GO and WK can be controlled, the controllable immobilization of the enzyme is realized, and the immobilized enzyme effect is enhanced. Meanwhile, metal ions are introduced for coordination, so that the stability is enhanced, the method has less structural interference on enzyme molecules, and the enzyme activity can be well maintained. The invention has the advantages of simple process, mild preparation conditions and high immobilization rate. The pH stability and the repeated use stability of the obtained immobilized enzyme are obviously enhanced, and the recovery rate of the enzyme activity is higher.
Drawings
FIG. 1 shows the immobilized enzyme (metal ion Cu) of keratin pair at different concentrations in example 12+) The loading capacity and the enzyme activity.
FIG. 2 shows the immobilized enzyme (metal ion Cu) in example 12+) The activity at different pH values was compared with that of the free amino acid dehydrogenase.
FIG. 3 shows the immobilized enzyme (metal ion Cu) in example 22+) Electron micrographs of (A).
FIG. 4 shows the immobilized enzyme (metal ion Cu) in example 22+) The recovery rate of enzyme activity is improved.
FIG. 5 shows the immobilized enzyme (metal ion Mg) in example 52+) Picture of
Detailed Description
The invention is further illustrated by the following figures and examples.
Example 1
(1) Preparation of crude enzyme solution:
constructing a recombinant expression strain capable of expressing the amino acid dehydrogenase with the His-tag label: the gene sequence of the amino acid dehydrogenase (NTAaDH) is shown in SEQ ID NO. 1. And (3) carrying out 1% agarose gel electrophoresis identification on the PCR amplification product, carrying out gel recovery on the NTAaDH gene fragment, carrying out double digestion by using NdeI and XhoI enzyme digestion enzymes, recovering the enzyme digestion product, connecting the enzyme digestion product with pET-28a plasmid (with His-tag label) subjected to double digestion, and transforming the connected plasmid into escherichia coli BL21(DE3) to obtain pET28a-NTAaDH plasmid. The plasmid is transformed into E.coli BL21(DE3), and a recombinant expression strain E.coli BL21(DE3)/pET28a capable of expressing His-tag-labeled amino acid dehydrogenase is obtained.
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 was centrifuged at 12,000rpm for 15min at 4 ℃ to remove insoluble cell debris, and the supernatant was a crude enzyme solution containing the His-tag-tagged amino acid dehydrogenase.
(2) Preparation of pure enzyme of amino acid dehydrogenase: his Trap nickel column (Histrap) from GE corporation is usedTMHP, 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. What is needed isThe 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 purified His-tag-labeled amino acid dehydrogenase.
(3) Preparation of wool keratin solution: firstly, weighing 6 g of sodium sulfide nonahydrate, 24 g of urea and 1.44 g of sodium dodecyl sulfate, mixing, adding into a reaction bottle, adding ultrapure water to make the volume of the solution reach 50mL, and putting the reaction bottle into an ultrasonic cleaning machine for completely dissolving solid substances by ultrasonic. Weighing 5 g of wool, adding the wool into a blue-mouth bottle, completely immersing the wool into the solution, and then putting the blue-mouth bottle into a constant-temperature drying oven at 25 ℃ for heat preservation for 20 hours to completely dissolve the wool. The keratin solution at this time contains not only keratin but also chemical agents previously added, which can be removed by deionized water using a dialysis method. And (2) balancing the wool keratin solution obtained by preliminary dissolution in pairs, putting the wool keratin solution into a centrifugal machine, centrifuging at the rotating speed of 9000 r/min, filtering after centrifugation to obtain a light yellow clarified keratin solution, dialyzing the keratin by using a dialysis bag with the molecular weight cutoff of 3500Da, replacing deionized water every 2 hours, and dialyzing for 3 days, so that chemical reagents in the keratin solution can be removed, and finally obtaining the purified wool keratin solution.
(4) Preparation of graphene oxide and wool keratin immobilized amino acid dehydrogenase: diluting the wool keratin solution obtained in the step 3) to final concentrations of 0.30mg/mL, 0.25mg/mL, 0.20mg/mL, 0.15mg/mL and 0.10mg/mL respectively, adding the diluted solution into the amino acid dehydrogenase with the His-tag label obtained in the step 2), enabling the final concentration of the amino acid dehydrogenase with the His-tag label to be 0.1-10 mg/mL, oscillating in a 4 ℃ constant temperature oscillator for 10min, adding 10mM metal ion Cu after the wool keratin and the amino acid dehydrogenase are combined2+Coordinating for 30min, chelating metal ions, adding the prepared graphene oxide solution (0.1mg/mL), mixing, and oscillating in a constant temperature oscillator at 4 ℃ for 4h to obtain graphene oxide-Immobilized amino acid dehydrogenase GO-WK-Cu of wool keratin and metal ion coordination composite material2+-NTAaDH. When the concentration of the wool keratin solution is less than 0.20mg/mL, the loading capacity of the graphene oxide and wool keratin immobilized enzyme is 100%, and the obtained immobilized enzyme has the highest enzyme activity when the concentration of the wool keratin solution is 0.15mg/mL, as shown in the attached figure 1 of the specification.
(5) The detection method of the enzyme activity comprises the following steps: activity measurement of amino acid dehydrogenase the reaction system contained 10. mu.L of 4mM NADH, 160. mu.L of 0.2mol/L ammonium chloride-aqueous ammonia buffer solution (pH 9.5), 10. mu.L of 40mM ethyl-2-oxo-butyrate solution and 20. mu.L of enzyme solution, and the enzyme activity of L-homophenylalanine was measured at a wavelength of 340nm at 20 ℃. The enzyme activity is defined as the amount of enzyme required to oxidatively consume (or generate) 1. mu. mol NADH per minute under the above conditions as one unit of enzyme activity.
The enzyme activity measuring system of the amino acid dehydrogenase is buffer solutions with different pH values (the pH value of the buffer solution comprises 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5 and 11.0), and the action of the enzyme activity measuring system on free amino acid dehydrogenase and immobilized amino acid dehydrogenase under different pH systems is examined. The results are shown in figure 2 of the specification.
Example 2
(1) The experimental procedures (3) were as in the procedures (1) to (3) of example 1.
(4) Preparation of graphene oxide and wool keratin immobilized amino acid dehydrogenase: respectively diluting the wool keratin solution obtained in the step 3) to a final concentration of 0.15mg/mL, adding the diluted solution into the amino acid dehydrogenase with the His-tag label obtained in the step 2), enabling the final concentration of the amino acid dehydrogenase with the His-tag label to be 0.1-10 mg/mL, oscillating for 10min in a constant temperature oscillator at 4 ℃, and adding 10mM metal ion Cu after the wool keratin and the amino acid dehydrogenase are combined2+Coordinating for 30min, chelating metal ions, adding the prepared graphene oxide solution (0.1mg/mL), mixing, and oscillating in a constant temperature oscillator at 4 deg.C for 4h to obtain graphene oxide-wool keratin and metal ion coordination composite immobilized amino acid dehydrogenase GO-WK-Cu2+-NTAaDH. The electron micrograph is shown in FIG. 3.
(5) The detection method of the enzyme activity comprises the following steps: activity measurement of amino acid dehydrogenase the reaction system contained 10. mu.L of 4mM NADH, 160. mu.L of 0.2mol/L ammonium chloride-aqueous ammonia buffer solution (pH 9.5), 10. mu.L of 40mM ethyl-2-oxo-butyrate solution and 20. mu.L of enzyme solution, and the enzyme activity catalyzing the preparation of L-homophenylalanine was measured at 340nm wavelength at 20 ℃. The enzyme activity is defined as the amount of enzyme required to oxidatively consume (or generate) 1. mu. mol NADH per minute under the above conditions as one unit of enzyme activity. The activity value of the immobilized enzyme is 1.26 times of that of the free enzyme, as shown in the attached figure 4 of the specification.
Example 3
(1) The experimental procedures (3) were as in the procedures (1) to (3) of example 1.
(4) Preparation of graphene oxide and wool keratin immobilized amino acid dehydrogenase: respectively diluting the wool keratin solution obtained in the step 3) to a final concentration of 0.15mg/mL, adding the diluted solution into the His-tag-labeled amino acid dehydrogenase obtained in the step 2), enabling the final concentration of the His-tag-labeled amino acid dehydrogenase to be 0.1-10 mg/mL, oscillating for 10min in a 4 ℃ constant temperature oscillator, and adding 10mM metal ions Mn after the wool keratin and the amino acid dehydrogenase are combined2+Coordinating for 30min, chelating metal ions, adding the prepared graphene oxide solution (0.1mg/mL), mixing, and oscillating in a constant temperature oscillator at 4 deg.C for 4h to obtain graphene oxide-wool keratin and metal ion coordination composite immobilized amino acid dehydrogenase GO-WK-Mn2+-NTAaDH。
(5) The detection method of the enzyme activity comprises the following steps: activity measurement of amino acid dehydrogenase the reaction system contained 10. mu.L of 4mM NADH, 160. mu.L of 0.2mol/L ammonium chloride-ammonia buffer solution (pH 9.5), 10. mu.L of 40mM alpha-ketoglutarate solution and 20. mu.L of enzyme solution, and the enzyme activity for catalytically preparing L-glutamic acid was measured at 340nm wavelength at 20 ℃. The enzyme activity is defined as the amount of enzyme required to oxidatively consume (or generate) 1. mu. mol NADH per minute under the above conditions as one unit of enzyme activity.
The amino acid dehydrogenase enzyme activity determination system is buffer solutions at different temperatures (the buffer solution temperature comprises 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃), the action of the free amino acid dehydrogenase and the immobilized amino acid dehydrogenase under different temperature systems is examined, the immobilized enzyme and the free enzyme reach the maximum enzyme activity at 70 ℃, and the enzyme activity of the immobilized enzyme is about 1.25 times of that of the free enzyme.
(6) Determination of temperature stability of immobilized enzyme: free enzyme and immobilized enzyme GO-WK-Mn2+The temperature of NTAaDH in water bath is kept constant for 2h at 60 ℃, and the enzyme activity is measured every 30min in the process. Compared with free enzyme, the temperature stability of the immobilized enzyme is obviously improved, the initial enzyme activity of the immobilized enzyme is 100 percent, and the immobilized enzyme GO-WK-Mn is obtained after 2 hours of constant temperature water bath at 60 DEG C2+-NTAaDH with a relative enzyme activity of 70.26%.
Example 4
(1) The experimental procedures (3) were as in the procedures (1) to (3) of example 1.
(4) Preparation of graphene oxide and wool keratin immobilized amino acid dehydrogenase: respectively diluting the wool keratin solution obtained in the step 3) to a final concentration of 0.15mg/mL, adding the diluted solution into the amino acid dehydrogenase with the His-tag label obtained in the step 2), enabling the final concentration of the amino acid dehydrogenase with the His-tag label to be 0.1-10 mg/mL, oscillating for 10min in a constant temperature oscillator at 4 ℃, and adding 10mM metal ions Ni after the wool keratin and the amino acid dehydrogenase are combined2+Coordinating for 30min, chelating metal ions, adding the prepared graphene oxide solution (0.1mg/mL), mixing, and oscillating in a constant temperature oscillator at 4 deg.C for 4h to obtain graphene oxide-wool keratin and metal ion coordination composite immobilized amino acid dehydrogenase GO-WK-Ni2+-NTAaDH。
(5) The detection method of the enzyme activity comprises the following steps: the reaction system for measuring the activity of the amino acid dehydrogenase comprises 10 mu L of 4mM NADH, 112 mu L of 0.2mol/L ammonium chloride-ammonia water buffer solution (pH 9.5), 48 mu L of organic solvent (methanol, ethanol, acetic acid, acetonitrile acetitrile, acetone, isopropanol, cyclohexane cycloxane, dimethyl sulfoxide DMSO, tert-amyl alcohol 2-methyl-2-butanol), 10 mu L of 40mM phenylketonic acid solution and 20 mu L of enzyme solution, and the enzyme activity for catalyzing the preparation of L-phenylalanine is measured at the temperature of 20 ℃ and the wavelength of 340 nm. The enzyme activity is defined as the amount of enzyme required to oxidatively consume (or generate) 1. mu. mol NADH per minute under the above conditions as one unit of enzyme activity. The immobilized enzyme has the highest enzyme activity under 10% of organic solvent cyclohexane, and the enzyme activity is about 2 times of that of free enzyme.
Example 5
(1) The experimental procedures (3) were as in the procedures (1) to (3) of example 1.
(4) Preparation of graphene oxide and wool keratin immobilized amino acid dehydrogenase: respectively diluting the wool keratin solution obtained in the step 3) to a final concentration of 0.15Mg/mL, adding the diluted solution into the amino acid dehydrogenase with the His-tag label obtained in the step 2), enabling the final concentration of the amino acid dehydrogenase with the His-tag label to be 0.1-10 Mg/mL, oscillating for 10min in a constant temperature oscillator at 4 ℃, and adding 10mM metal ions Mg after the wool keratin and the amino acid dehydrogenase are combined2+Coordinating for 30min, chelating metal ions, adding the prepared graphene oxide solution (0.1Mg/mL), mixing, and oscillating in a constant temperature oscillator at 4 deg.C for 4h to obtain graphene oxide-wool keratin and metal ion coordination composite immobilized amino acid dehydrogenase GO-WK-Mg2+-NTAaDH. Zeta potential (mV) of free enzyme NTAaDH measured by Malvern particle size analyzer-3.67, GO-WK-Mg2+Zeta potential (mV) of NT-8.04, and the state of the immobilized enzyme as determined is shown in FIG. 5. After 5 times of repeated utilization, the enzyme activity of the immobilized enzyme can still keep 73.46 percent of activity.
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> method for coordinating and immobilizing oxidoreductase by using graphene oxide-wool keratin and metal ions
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 358
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Met Ser Val Lys Ile Phe Glu Glu Met Glu Lys His Gly His Glu Gln
1 5 10 15
Val Ile Phe Asn Tyr Asp Lys Thr Thr Gly Leu Lys Ala Ile Ile Ala
20 25 30
Val His Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Cys Arg Met Leu
35 40 45
Pro Tyr Glu Thr Glu Glu Glu Ala Leu Asp Asp Val Leu Arg Leu Ser
50 55 60
Lys Gly Met Thr Tyr Lys Cys Ser Ile Ser Glu Val Asp Tyr Gly Gly
65 70 75 80
Gly Lys Thr Val Ile Ile Gly Asp Pro Glu Lys Asp Lys Ser Glu Gly
85 90 95
Met Phe Arg Ala Leu Gly Arg Phe Val Gly Thr Met Lys Gly Arg Tyr
100 105 110
Tyr Thr Gly Thr Asp Val Gly Thr Val Pro Asp Asp Phe Val His Ser
115 120 125
Tyr Arg Glu Ser Asp Tyr Phe Val Gly Leu Pro Glu Glu Phe Gly Gly
130 135 140
Ser Gly Asn Ser Ala Ile Asn Thr Ala Phe Gly Thr Leu Met Gly Ile
145 150 155 160
Lys Ala Cys Val Lys Glu His Phe Gly Gln Val Asn Leu Glu Gly Lys
165 170 175
Lys Ile Ala Ile Gln Gly Leu Gly Lys Val Gly Gln Asn Leu Val Asp
180 185 190
Phe Leu Met Glu Glu Gly Ala His Ile Ile Ala Thr Asp Ile Ser Lys
195 200 205
Asp Asn Ile Asn Gln Val Lys Glu Arg His Ser Ser Val Glu Met Val
210 215 220
Glu Pro Asp Ala Ile Tyr Glu Val Asp Cys Asp Ile Phe Ser Pro Asn
225 230 235 240
Ala Leu Gly Ala Val Ile Asn Asp Phe Thr Val Glu Lys Leu Lys Cys
245 250 255
Ser Ile Ile Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Glu Lys His
260 265 270
Gly Arg Met Leu Phe Asp Lys Gly Ile Leu Tyr Ala Pro Asp Tyr Ile
275 280 285
Val Asn Ala Gly Gly Leu Ile Gln Val Ser Asp Glu Ile Gly Gly His
290 295 300
Asn Lys Asp Arg Ile Arg Lys Lys Thr Glu Arg Ile Tyr Asp Ile Leu
305 310 315 320
Leu Gln Val Phe Lys Ile Ser Arg Glu Glu Asn Ile Thr Pro Gln Glu
325 330 335
Ala Ser Asp Lys Leu Val Glu Glu Arg Ile Ala Thr Val Lys Gly Leu
340 345 350
Gln Ser Asn Tyr Met Gly
355

Claims (10)

1. A method for immobilizing oxidoreductase by coordination of graphene oxide-wool keratin and metal ions is characterized by comprising the following steps: adding oxidoreductase into a wool keratin solution with the concentration of 0.1-50 Mg/mL, enabling the final concentration of the oxidoreductase to be 0.1-100 Mg/mL, uniformly mixing at 0-50 ℃, adding metal ions and enabling the final concentration to be 0.8-40 mM for coordination, wherein the metal ions comprise Mg2+、Zn2+、Ni2+、Mn2+Or Cu2+Then adding at least one of the graphene oxide solutions to a final concentration of 0.01-100 mg/mL, and uniformly mixing at 0-50 ℃ to obtain the graphene oxide-wool keratin and metal ion coordination composite material immobilized oxidoreductase.
2. The method of claim 1, wherein: the oxidoreductase enzyme comprises an amino acid dehydrogenase.
3. The method of claim 2, wherein: the amino acid sequence of the amino acid dehydrogenase is shown as SEQ ID No. 1.
4. The method of claim 1, wherein: the wool keratin is prepared by dissolving wool in a urea/sodium sulfide/sodium dodecyl sulfate process; the mass ratio of the wool, the urea, the sodium sulfide nonahydrate and the sodium dodecyl sulfate is (4-20): 22-50: 5-20: 1.4-2, the dissolving temperature is 25-65 ℃, and the reaction time is 2-20 h.
5. The method of claim 1, wherein: the concentration of the wool keratin solution is 0.1-0.16 mg/mL.
6. The method of claim 3, wherein: the amino acid dehydrogenase with the amino acid sequence shown as SEQ ID No.1 is prepared by a recombinant expression mode, a recombinant expression strain capable of expressing the amino acid dehydrogenase is inoculated into an LB culture medium containing kanamycin for culture, and a lactose inducer IPTG is added after the culture is carried out for a period of time; culturing the obtained bacterial liquid, centrifuging to obtain cells, and preparing into cell suspension; carrying out ultrasonic crushing and centrifugation, and collecting supernatant, namely crude enzyme liquid containing amino acid dehydrogenase with a His-tag label; and purifying and desalting the obtained crude enzyme solution by using a nickel column to obtain the amino acid dehydrogenase with the His-tag label.
7. The method of claim 6, wherein: the construction method of the recombinant expression strain capable of expressing the amino acid dehydrogenase comprises the following steps: carrying out double enzyme digestion on the amino acid dehydrogenase gene and the pET28a plasmid respectively by NdeI and Xhol, and carrying out ligation transformation to obtain a pET28a-NTAaDH plasmid; the plasmid is transformed into E.coli BL21(DE3), and a recombinant expression strain E.coli BL21(DE3)/pET28a capable of expressing His-tag-labeled amino acid dehydrogenase is obtained.
8. The method of claim 7, wherein: inoculating the E.coli BL21(DE3)/pET28a into the LB culture medium containing kanamycin in an inoculation amount of 1-3% for culture, wherein the LB culture medium comprises: 5.0-15.0 g/L tryptone, 1.0-10.0 g/L yeast extract and 0.0-15.0 g/L NaCl, adjusting the pH value to 7.0-7.5, and adding kanamycin before inoculation to make the final concentration to be 50-150 mu g/mL; culturing at the temperature of 36-38 ℃ at 150-250 rpm for 1.5-6 h, adding an inducer IPTG (isopropyl-beta-D-thiogalactoside) to enable the final concentration to be 5-15 mg/mL, and continuously culturing at the temperature of 25-30 ℃ at 150-250 rpm for 2-12 h; culturing the obtained bacterial liquid, centrifuging to obtain cells, removing supernatant, resuspending the precipitate with phosphate buffer solution with pH 7-7.5, fully washing, centrifuging, repeating the operation for a plurality of times, and preparing the cell suspension with the concentration of 50-150 g/L by using the phosphate buffer solution with pH 6.5-8.0.
9. The method of claim 1, wherein: the method also comprises a method for detecting the enzyme activity: the activity determination reaction system of the amino acid dehydrogenase comprises 10 mu L of ammonium chloride-ammonia water with the concentration range of 50-200 mM, 10 mu L of substrate solution with the concentration range of 8-12 and the concentration range of 160 mu L of 4mM NADH as an amino donor, 10 mu L of 40mM and 20 mu L of enzyme solution, wherein the substrate comprises 2-oxo-ethyl butyrate, phenylpyruvic acid or alpha-ketoglutaric acid, and the enzyme activity is determined at the wavelength of 340 nm; the enzyme activity is defined as the amount of enzyme required to consume or generate 1. mu. mol NADH by oxidation per minute under the above conditions as one unit of enzyme activity.
10. An oxidized graphene-wool keratin and metal ion coordination complex immobilized oxidoreductase prepared according to the method of any one of claims 1 to 9.
CN202110937217.5A 2021-08-16 2021-08-16 Method for coordinating and immobilizing oxidoreductase by using graphene oxide-wool keratin and metal ions Pending CN113564154A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110937217.5A CN113564154A (en) 2021-08-16 2021-08-16 Method for coordinating and immobilizing oxidoreductase by using graphene oxide-wool keratin and metal ions

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110937217.5A CN113564154A (en) 2021-08-16 2021-08-16 Method for coordinating and immobilizing oxidoreductase by using graphene oxide-wool keratin and metal ions

Publications (1)

Publication Number Publication Date
CN113564154A true CN113564154A (en) 2021-10-29

Family

ID=78171700

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110937217.5A Pending CN113564154A (en) 2021-08-16 2021-08-16 Method for coordinating and immobilizing oxidoreductase by using graphene oxide-wool keratin and metal ions

Country Status (1)

Country Link
CN (1) CN113564154A (en)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012032154A1 (en) * 2010-09-09 2012-03-15 Fidia Farmaceutici S.P.A. Process for the production of hyaluronic acid in escherichia coli or bacillus subtilis
CN103409472A (en) * 2013-08-13 2013-11-27 厦门大学 Method for improving 1,3-propylene glycol fermentation performance
CN106191025A (en) * 2016-07-11 2016-12-07 厦门大学 A kind of method utilizing graphene oxide metallic ion coordination immobilized enzyme
CN109456960A (en) * 2018-12-28 2019-03-12 厦门大学 A kind of method of redox graphene immobilization Phenylalanine dehydrogenase
CN111990409A (en) * 2020-07-08 2020-11-27 厦门大学 Broad-spectrum antiviral material and preparation method and application thereof
CN113150337A (en) * 2021-04-02 2021-07-23 东华大学 Regenerated cellulose membrane based on phosphoric acid solution and preparation method thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012032154A1 (en) * 2010-09-09 2012-03-15 Fidia Farmaceutici S.P.A. Process for the production of hyaluronic acid in escherichia coli or bacillus subtilis
CN103409472A (en) * 2013-08-13 2013-11-27 厦门大学 Method for improving 1,3-propylene glycol fermentation performance
CN106191025A (en) * 2016-07-11 2016-12-07 厦门大学 A kind of method utilizing graphene oxide metallic ion coordination immobilized enzyme
CN109456960A (en) * 2018-12-28 2019-03-12 厦门大学 A kind of method of redox graphene immobilization Phenylalanine dehydrogenase
CN111990409A (en) * 2020-07-08 2020-11-27 厦门大学 Broad-spectrum antiviral material and preparation method and application thereof
CN113150337A (en) * 2021-04-02 2021-07-23 东华大学 Regenerated cellulose membrane based on phosphoric acid solution and preparation method thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
孙小娟等: "羊毛角蛋白/氧化石墨烯复合材料的制备及其吸附性能", 毛纺科技, vol. 49, no. 1 *

Similar Documents

Publication Publication Date Title
Nahalka et al. Fusion to a pull‐down domain: a novel approach of producing Trigonopsis variabilisd‐amino acid oxidase as insoluble enzyme aggregates
CN109593750B (en) Nitrile hydratase mutant, genetic engineering bacterium containing same and application thereof
Singh et al. Extraction, purification, kinetic characterization and immobilization of urease from Bacillus sphaericus MTCC 5100
CN106191025B (en) A method of utilizing graphene oxide-metallic ion coordination immobilised enzymes
CN112662656B (en) Enzyme immobilization carrier and preparation method thereof, immobilized enzyme and preparation method thereof
CN114317642A (en) Method for preparing chitosan oligosaccharide, chitosanase, gene thereof, enzyme preparation and application
CN112921023B (en) Recombinant aspartate lyase and method for preparing R-3-aminobutyric acid with high repeated utilization rate
CN113061158A (en) Method for purifying and immobilizing protein by using divalent metal ions and histidine tag and application thereof
CN105063010A (en) Multi-enzyme system with immobilized polyethylenimine and metal coordination and method for preparing multi-enzyme system
CN113564154A (en) Method for coordinating and immobilizing oxidoreductase by using graphene oxide-wool keratin and metal ions
CN112708612A (en) Oxidoreductase electrode for enzyme electrocatalytic reduction, preparation method thereof and enzyme electric reactor thereof
CN112266905B (en) Polypeptide modified amino acid dehydrogenase and preparation and immobilization method thereof
US7846684B2 (en) Enzyme system comprising an enzyme bonded in a porous matrix
CN113801239B (en) Polypeptide tag, highly soluble recombinant nitrilase and application thereof in synthesis of medicinal chemicals
CN114657170A (en) Preparation method of high-stability immobilized enzyme
CN113817697A (en) Phenylalanine dehydrogenase mutant and application thereof in synthesis of L-homophenylalanine
CN111254181B (en) Method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid by chemical enzyme method
CN111254180B (en) Method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid by enzymatic resolution
CN114107274A (en) Preparation method of wool immobilized enzyme and wool immobilized enzyme column reactor
CN113621603A (en) Co-immobilization continuous catalysis method for cofactor and cofactor-dependent enzyme
JP3819969B2 (en) Recombinant fructosyl amino acid oxidase
CN110241063B (en) Method for enhancing salt tolerance of glutaminase
CN114807094B (en) Chitosan SvChiAJ54 and encoding gene and application thereof
Goimawala et al. Fermentation, immobilization, and application of de-acylase for biosynthesis of anti-fungal intermediates
KR101193943B1 (en) A method for pretreating cellobiose dehydrogenase before immobilizing the same on carrier

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination