Preparation method and application of peptide hydrogel reactor for efficiently packaging NADH (Nicotinamide Adenine Dinucleotide) dependent enzyme

Abstract

The invention discloses a preparation method of a peptide hydrogel reactor for efficiently packaging NADH (Nicotinamide Adenine Dinucleotide Hormone)-dependent enzyme, which comprises the following steps: S1, dissolving a screened peptide sequence under the condition of moderate pH, uniformly stirring with a pre-reaction solution containing a bismuth source reagent, centrifuging and washing to obtain a clean mixed solution containing a bismuth precursor; s2, assembling the mixed solution of the bismuth-containing precursor in pure water, fixing bismuth on the surface of an assembly in situ by using a reducing agent, uniformly stirring, centrifuging, washing to obtain a bismuth-containing polypeptide precipitate, and freezing the bismuth-containing polypeptide precipitate to obtain bismuth-containing polypeptide hydrogel; and S3, adding an enzyme reagent into the finally obtained bismuth-containing polypeptide hydrogel, stirring, and incubating for 10 minutes to finally obtain the enzyme-encapsulated peptide hydrogel reactor. The invention further discloses application of the peptide hydrogel reactor for efficiently packaging the NADH-dependent enzyme. The problems that NADH regeneration is low in catalytic efficiency and biological enzyme is poor in long-term stability and activity are solved.

Description

Technical Field

[0001] The invention relates to a preparation method and application of a peptide hydrogel reactor for efficiently encapsulating NADH-dependent enzymes. Background Art

[0002] In the face of global challenges in energy, resources and the environment, sustainable chemical processing has become necessary to minimize the use of natural resources, avoid toxic substances, low energy consumption and minimize waste and pollutants. Biocatalysis of essential biomolecules such as fatty acids, amino acids and nucleotides requires the formation of NADH (reduced coenzyme I) as an electron carrier to provide the necessary reducing equivalents. At present, several effective methods have been developed in industry to achieve NADH regeneration, including enzymes, chemistry, electrochemistry, etc., all of which have problems such as difficulty in separating coenzymes from substrates and by-products. Therefore, it is urgent to develop a sustainable method to replace the NADH regeneration system formed by photosynthesis in organisms. Using artificial methods to improve the efficiency of biocatalysis must be considered from the aspects of price, catalytic efficiency and stability.

[0003] At present, although electrocatalysis is one of the catalytic technologies of composite green concept, the low selectivity of electrocatalysts makes it difficult to convert the reaction 100%. Bioelectrocatalysis combines high selectivity and high yield biocatalysis with sustainable electrocatalysis to achieve the conversion of high value-added organic chemicals in vitro in an artificial sense. However, the higher potential and complex environment in the bioelectrocatalytic system will weaken the biological activity of the applied enzyme and reduce the catalytic efficiency. NADH coenzyme regeneration, as an important regulatory medium for downstream enzyme catalysis, needs to be realized in the bioelectrocatalytic process. It is also a huge challenge to reduce the production of by-products using efficient catalysts. Common conductive molecules are used as catalysts to transfer electrons and protons to achieve NADH regeneration, but their own toxicity and morphology will also hinder the bioelectrocatalytic NADH reaction. Nowadays, in order to protect bioenzyme materials from the poisoning of complex electrolytes, traditional conductive polymers form dense hydrogel films as a certain buffer area to encapsulate them. However, dense catalysts are often not conducive to material exchange and transmission, thereby affecting the downstream enzyme catalytic efficiency. Peptide materials have similar structural domains to proteases, such as NADH, which can provide a stable biological environment for bioactive molecules. The high water retention and high affinity of peptide hydrogels can more effectively encapsulate bioactive substances, but the low conductivity makes peptide hydrogels less studied in electrocatalysis. Therefore, the development of conductive peptide hydrogels as "buffer areas" to maintain high catalytic activity and long-term stability of enzymes provides a new idea for achieving NADH coenzyme regeneration and sustainable production of high value-added products required by organisms. Summary of the invention

[0004] The purpose of the present invention is to overcome the existing defects and provide a preparation method and application of a peptide hydrogel reactor for efficiently encapsulating NADH-dependent enzymes, which solves the problems of low catalytic efficiency of NADH regeneration and poor long-term stability and activity of biological enzymes.

[0005] The technical solution to achieve the above purpose is: The present invention provides a method for preparing a peptide hydrogel reactor for efficiently encapsulating NADH-dependent enzymes, comprising: Step S1, dissolving the screened peptide sequence under a moderate pH condition, stirring evenly with the pre-reaction solution containing the bismuth source reagent, centrifuging, and washing to obtain a clean mixed solution containing the bismuth precursor; Step S2, assembling the mixed solution of bismuth-containing precursors in pure water, fixing bismuth in situ on the surface of the assembly with a reducing agent, stirring evenly, centrifuging, washing to obtain a bismuth-containing polypeptide precipitate, and freezing the bismuth-containing polypeptide hydrogel; Step S3, adding an enzyme reagent to the finally obtained bismuth-containing polypeptide hydrogel, stirring and incubating for 10 minutes to finally obtain an enzyme-encapsulated peptide hydrogel reactor.

[0006] Preferably, in step S1, the bismuth source reagent is one of bismuth trichloride hexahydrate, bismuth nitrate pentahydrate or bismuth sulfate.

[0007] Preferably, in step S1, the concentration of bismuth in the pre-reaction solution containing the bismuth source reagent is 0.1 μg / mL to 1.0 μg / mL.

[0008] Preferably, in step S1, the stirring speed ranges from 200 rap / min to 500 rap / min; In the step S2, the assembly time is 2 hours to 24 hours, and the stirring speed ranges from 200 rap / min to 500 rap / min.

[0009] Preferably, in step S2, the reducing agent is one of sodium borohydride or citric acid, and the peptide sequence in the mixed solution containing the bismuth precursor is one of Fmoc-FF, Fmoc-FFKG, Fmoc-FFKGC or Fmoc-FFGD; wherein the concentration of the reducing agent reaction solution is 10mM to 200mM, and the optimal concentration is 50mM to 120mM.

[0010] Preferably, in step S2, the molar ratio of the bismuth-containing polypeptide precipitate is 1-3:1-4:2-5, and the optimal molar ratio is 1-2:2-4:3-5.

[0011] Preferably, in step S3, the enzyme reagent is one of formate dehydrogenase, alcohol dehydrogenase or glucose dehydrogenase, wherein the activity of the enzyme in the bismuth-containing polypeptide hydrogel is 2U / mg to 50U / mg.

[0012] The second aspect of the present invention provides a method for preparing a peptide hydrogel reactor for efficiently encapsulating NADH-dependent enzymes. The peptide hydrogel reactor prepared is used in bioelectrocatalytic NADH conversion.

[0013] Preferably, the electrocatalytic NADH regeneration performance test is carried out on an electrochemical workstation, and the test is carried out using a three-electrode system, with electrocatalyst carbon paper coated with enzyme peptide as the working electrode, platinum sheet as the counter electrode, and Ag / AgCl electrode as the reference electrode; 20mM PBS (phosphate buffered saline) containing 1.5mM NAD+ and 3mM propionaldehyde is used as the electrolyte; an H-type glass electrolytic cell is used as the electrolytic reaction device, and cyclic voltammetry, time-current curve, and impedance spectrum are used to test the stability of the catalytic activity of bioelectrocatalytic NADH conversion, the conversion efficiency of NADH, the final yield of propanol, and the Faraday efficiency.

[0014] The beneficial effects of the present invention are: 1) The peptide hydrogel reactor prepared by the present invention can evenly distribute metal bismuth nanoparticles on the fibers of the peptide hydrogel through the process of polypeptide self-assembly, and the metal bismuth improves the conductivity of the peptide hydrogel reactor as an overall bioelectrocatalytic system and improves the H transfer catalytic activity to achieve NADH regeneration; 2) The residues of the peptide sequence and the protein-similar domains in the peptide hydrogel reactor prepared by the present invention provide attachment points for organic active molecules such as NADH, and the high biocompatibility and water retention encapsulate the alcohol dehydrogenase in the hydrogel, and quickly transfer the generated NADH to the catalytic active site of the alcohol dehydrogenase; 3) The peptide hydrogel reactor prepared by the present invention utilizes a similar biological microenvironment to form a stable enzyme activity environment, which is beneficial to the continuous generation of products. After 5 cycles for 50 hours, the regional biological microenvironment is constructed, and the NADH regeneration performance is only reduced by 20%, so that the long-term stability and catalytic activity of alcohol dehydrogenase are guaranteed. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a flow chart of a method for preparing a peptide hydrogel reactor for efficiently encapsulating NADH-dependent enzymes according to the present invention; Figure 2 : is the circular dichroism spectrum of Fmoc-FFKG before and after doping with Bi in Example 1 of the present invention; Figure 3 is a scanning electron microscope image of the Fmoc-FFKG self-assembled fiber structure in Example 1 of the present invention; Figure 4 is a transmission electron microscope image of the self-assembled fiber structure of Fmoc-FFKG before and after being doped with Bi in Example 2 of the present invention; Figure 5 This is a comparison diagram of enzyme activity test before and after Fmoc-FFKG encapsulation of alcohol dehydrogenase in Example 6 of the present invention; Figure 6 This is a laser confocal image of Fmoc-FFKG encapsulated alcohol dehydrogenase in Example 6 of the present invention. DETAILED DESCRIPTION

[0016] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. In the description of the present invention, it should be noted that the terms "center", "up", "down", "left", "right", "vertical", "horizontal", "inside", "outside" and the like indicate directions or positional relationships based on the directions or positional relationships shown in the accompanying drawings, which are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific direction, be constructed and operated in a specific direction, and therefore cannot be understood as a limitation on the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance.

[0017] The present invention will be further described below in conjunction with the accompanying drawings.

[0018] like Figure 1 As shown, a method for preparing a peptide hydrogel reactor for efficiently encapsulating NADH-dependent enzymes comprises: Step S1, dissolving the screened peptide sequence under a moderate pH condition, stirring evenly with the pre-reaction solution containing the bismuth source reagent, centrifuging, and washing to obtain a clean mixed solution containing the bismuth precursor.

[0019] In the embodiment, the bismuth source reagent is one of bismuth trichloride hexahydrate, bismuth nitrate pentahydrate or bismuth sulfate. In this embodiment, bismuth nitrate pentahydrate is used as the bismuth source reagent.

[0020] In the embodiment, the concentration of bismuth in the pre-reaction solution containing the bismuth source reagent is 0.1 μg / mL to 1.0 μg / mL.

[0021] In the embodiment, the stirring speed ranges from 200 rap / min to 500 rap / min.

[0022] Step S2, assembling the mixed solution of bismuth-containing precursors in pure water, fixing bismuth in situ on the surface of the assembly with a reducing agent, stirring evenly, centrifuging, washing to obtain a bismuth-containing polypeptide precipitate, and freezing it to obtain a bismuth-containing polypeptide hydrogel.

[0023] In the embodiment, the assembly time is 2 hours to 24 hours, and the stirring speed ranges from 200 rap / min to 500 rap / min.

[0024] In the embodiment, the reducing agent is one of sodium borohydride or citric acid, and the peptide sequence in the mixed solution containing bismuth precursor is one of Fmoc-FF, Fmoc-FFKG, Fmoc-FFKGC or Fmoc-FFGD; wherein the concentration of the reducing agent reaction solution is 10mM~200mM, and the optimal concentration is 50mM~120mM; in this embodiment, sodium borohydride is used as the reducing agent, and Fmoc-FFKG is used as the peptide sequence in the mixed solution containing bismuth precursor.

[0025] In the embodiment, the molar ratio of the bismuth-containing polypeptide precipitate is 1-3:1-4:2-5, and the optimal molar ratio is 1-2:2-4:3-5.

[0026] Step S3, adding an enzyme reagent to the finally obtained bismuth-containing polypeptide hydrogel, stirring and incubating for 10 minutes to finally obtain an enzyme-encapsulated peptide hydrogel reactor, that is, an enzyme-fixed Bi / Fmoc-FFKG peptide hydrogel reactor.

[0027] In the embodiment, the enzyme reagent is one of formate dehydrogenase, alcohol dehydrogenase or glucose dehydrogenase, wherein the activity of the enzyme in the bismuth-containing polypeptide hydrogel is 2U / mg to 50U / mg; in this embodiment, formate dehydrogenase is used as the enzyme reagent.

[0028] A peptide hydrogel reactor for efficiently encapsulating NADH-dependent enzymes is prepared and the prepared peptide hydrogel reactor is used in bioelectrocatalytic NADH conversion.

[0029] In the embodiment, the electrocatalytic NADH regeneration performance test is carried out on an electrochemical workstation, and the test is carried out using a three-electrode system, with electrocatalyst carbon paper coated with enzyme peptide as the working electrode, platinum sheet as the counter electrode, and Ag / AgCl electrode as the reference electrode; 20mM PBS containing 1.5mM NAD+ and 3mM propionaldehyde is used as the electrolyte; an H-type glass electrolytic cell is used as the electrolytic reaction device, and cyclic voltammetry, time-current curve, and impedance spectrum are used to test the stability of the catalytic activity of bioelectrocatalytic NADH conversion, the conversion efficiency of NADH, the final yield of propanol, and the Faraday efficiency.

[0030] Zeta potential and dynamic light scattering analysis, nuclear magnetic resonance hydrogen spectrum, infrared spectrum, circular dichroism spectrum, ultraviolet-visible absorption spectrum, inductively coupled plasma atomic emission spectrum, transmission electron microscope, nuclear magnetic resonance imaging analyzer and other means were used to characterize the physical and chemical properties of the peptide hydrogel reactor. The bioelectrocatalytic NADH conversion efficiency was evaluated by electrochemical workstation analysis and applied to various enzyme-catalyzed syntheses.

[0031] The special structure of the Bi / Fmoc-FFKG peptide hydrogel reactor with immobilized enzyme prepared by the present invention can provide more adsorption sites for electrochemical NADH regeneration, and the metal Bi nanoparticles uniformly fixed on the surface of the fiber material can better play the electron-proton coupling mechanism in the electrocatalytic NADH regeneration process; the Bi / Fmoc-FFKG peptide hydrogel reactor with immobilized enzyme improves the enzyme catalysis and provides good biological activity, providing a new direction for bioelectrocatalysis to achieve NADH regeneration. Therefore, the peptide hydrogel reactor of the present invention is widely applicable to the field of bioelectrocatalysis and has good application prospects.

[0032] The present invention will be further described below with reference to the embodiments.

[0033] Example 1 Step 1: 10 mg of Fmoc-FFKG was ultrasonically dissolved in 120 μL of DMSO, 2 mg of bismuth nitrate pentahydrate was added to the above solution, and after ultrasonic treatment for 10 min, it was added to PBS with a pH of 10. After stirring for 2 h, the bismuth precursor nanosuspension was washed and collected by centrifugation.

[0034] Step 2: Stir the collected bismuth-containing precursor suspension in pure water for 10 minutes to evenly disperse the bismuth ions in the peptide solution. Take 200 mL of 100 mM solution of sodium borohydride and add it dropwise to the stirred bismuth-containing peptide solution. After stirring for 2 hours, bismuth is fixed in situ on the peptide fibers. After multiple washings with distilled water, the precipitate is collected by centrifugation, frozen in the refrigerator overnight, and then freeze-dried for 72 hours to finally obtain the bismuth-containing peptide hydrogel.

[0035] Step 3: Add the final bismuth-containing peptide hydrogel to 1 mg / ml alcohol dehydrogenase in PBS and stir for 12 hours to obtain the enzyme-encapsulated Bi / Fmoc-FFKG peptide hydrogel.

[0036] Step 4: The obtained Bi / Fmoc-FFKG peptide hydrogel was subjected to a bioelectrocatalytic NADH conversion performance test. The circular dichroism spectra of Fmoc-FFKG before and after Bi doping are shown in Figure 4. Figure 2 The scanning electron microscopy image of the Fmoc-FFKG self-assembled fiber structure is shown in Figure 3 shown.

[0037] Weigh 2 mg of Bi / Fmoc-FFKG peptide hydrogel powder and mix it with 950 μL of a mixed solvent of ethanol and water (1:1), add 50 μL of Nafion solution, and ultrasonicate for 1 hour to obtain a uniform dispersion; take 20 μL of the above dispersion and drop it on the surface of a clean and dry carbon paper, where the surface area of ​​the carbon paper is controlled to be 1 cm×1 cm, and use it as a working electrode after natural drying; on an electrochemical workstation, a three-electrode standard system is used, the electrolyte is 3 mM propionaldehyde and 1.5 mM NAD+ solution, and an H-type glass electrolytic cell is used as the reaction device; cyclic voltammetry, time-current curves, impedance spectroscopy, etc. are used to test the stability of the catalytic activity of bioelectrocatalytic NADH conversion, the conversion efficiency of NADH, the final yield of propanol, and the Faraday efficiency.

[0038] Example 2 Step 1: 15 mg of Fmoc-FFKG was ultrasonically dissolved in 120 μL of DMSO, 2 mg of bismuth nitrate pentahydrate was added to the above solution, and after ultrasonic treatment for 10 min, it was added to PBS with a pH of 10. After stirring for 2 h, the bismuth precursor nanosuspension was washed and collected by centrifugation.

[0039] Step 2: Stir the collected bismuth-containing precursor suspension in pure water for 10 minutes to evenly disperse the bismuth ions in the peptide solution. Take 200 mL of 100 mM solution of sodium borohydride and add it dropwise to the stirred bismuth-containing peptide solution. After stirring for 2 hours, bismuth is fixed in situ on the peptide fibers. After multiple washings with distilled water, the precipitate is collected by centrifugation, frozen in the refrigerator overnight, and then freeze-dried for 72 hours to finally obtain the bismuth-containing peptide hydrogel.

[0040] Step 3: Add the final bismuth-containing peptide hydrogel to 1 mg / ml alcohol dehydrogenase in PBS and stir for 12 hours to obtain the enzyme-encapsulated Bi / Fmoc-FFKG peptide hydrogel.

[0041] Step 4: The obtained Bi / Fmoc-FFKG peptide hydrogel was subjected to a bioelectrocatalytic NADH conversion performance test. The transmission electron microscopy images of the self-assembled fiber structure of Fmoc-FFKG before and after Bi doping are shown in Figure 4. Figure 4 shown.

[0042] Weigh 2 mg of Bi / Fmoc-FFKG peptide hydrogel powder and mix it with 950 μL of a mixed solvent of ethanol and water (1:1), add 50 μL of Nafion solution, and ultrasonicate for 1 hour to obtain a uniform dispersion; take 20 μL of the above dispersion and drop it on the surface of a clean and dry carbon paper, where the surface area of ​​the carbon paper is controlled to be 1 cm×1 cm, and use it as a working electrode after natural drying; on an electrochemical workstation, a three-electrode standard system is used, the electrolyte is 3 mM propionaldehyde and 1.5 mM NAD+ solution, and an H-type glass electrolytic cell is used as the reaction device; cyclic voltammetry, time-current curves, impedance spectroscopy, etc. are used to test the stability of the catalytic activity of bioelectrocatalytic NADH conversion, the conversion efficiency of NADH, the final yield of propanol, and the Faraday efficiency.

[0043] Example 3 Step 1: 10 mg of Fmoc-FFKG was ultrasonically dissolved in 120 μL of DMSO, 3 mg of bismuth nitrate pentahydrate was added to the above solution, and after ultrasonic treatment for 30 min, it was added to PBS with a pH of 10. After stirring for 2 h, the bismuth precursor nanosuspension was washed and collected by centrifugation.

[0044] Step 2: Stir the collected bismuth-containing precursor suspension in pure water for 10 minutes to evenly disperse the bismuth ions in the peptide solution. Take 200 mL of 100 mM solution of sodium borohydride and add it dropwise to the stirred bismuth-containing peptide solution. After stirring for 2 hours, bismuth is fixed in situ on the peptide fibers. After multiple washings with distilled water, the precipitate is collected by centrifugation, frozen in the refrigerator overnight, and then freeze-dried for 72 hours to finally obtain the bismuth-containing peptide hydrogel.

[0045] Step 3: Add the final bismuth-containing peptide hydrogel to 1 mg / ml alcohol dehydrogenase in PBS and stir for 12 hours to obtain the enzyme-encapsulated Bi / Fmoc-FFKG peptide hydrogel.

[0046] Step 4: Test the bioelectrocatalytic NADH conversion performance of the obtained Bi / Fmoc-FFKG peptide hydrogel.

[0047] Weigh 2 mg of Bi / Fmoc-FFKG peptide hydrogel powder and mix it with 950 μL of a mixed solvent of ethanol and water (1:1), add 50 μL of Nafion solution, and ultrasonicate for 1 hour to obtain a uniform dispersion; take 20 μL of the above dispersion and drop it on the surface of a clean and dry carbon paper, where the surface area of ​​the carbon paper is controlled to be 1 cm×1 cm, and use it as a working electrode after natural drying; on an electrochemical workstation, a three-electrode standard system is used, the electrolyte is 3 mM propionaldehyde and 1.5 mM NAD+ solution, and an H-type glass electrolytic cell is used as the reaction device; cyclic voltammetry, time-current curves, impedance spectroscopy, etc. are used to test the stability of the catalytic activity of bioelectrocatalytic NADH conversion, the conversion efficiency of NADH, the final yield of propanol, and the Faraday efficiency.

[0048] Example 4 Step 1: 10 mg of Fmoc-FFKG was ultrasonically dissolved in 120 μL of DMSO, 2 mg of bismuth nitrate pentahydrate was added to the above solution, and after ultrasonic treatment for 10 min, it was added to PBS with a pH of 10. After stirring for 2 h, the bismuth precursor nanosuspension was washed and collected by centrifugation.

[0049] Step 2: Stir the collected bismuth-containing precursor suspension in pure water for 10 minutes to evenly disperse the bismuth ions in the peptide solution. Take 200 mL of 150 mM solution of sodium borohydride and add it dropwise to the stirred bismuth-containing peptide solution. After stirring for 2 hours, bismuth is fixed in situ on the peptide fibers. After multiple washings with distilled water and centrifugation, the precipitate is collected and frozen in the refrigerator overnight and freeze-dried for 72 hours to finally obtain the bismuth-containing peptide hydrogel.

[0050] Step 3: Add the final bismuth-containing peptide hydrogel to 1 mg / ml alcohol dehydrogenase in PBS and stir for 12 hours to obtain the enzyme-encapsulated Bi / Fmoc-FFKG peptide hydrogel.

[0051] Step 4: Test the bioelectrocatalytic NADH conversion performance of the obtained Bi / Fmoc-FFKG peptide hydrogel.

[0052] Weigh 2 mg of Bi / Fmoc-FFKG peptide hydrogel powder and mix it with 950 μL of a mixed solvent of ethanol and water (1:1), add 50 μL of Nafion solution, and ultrasonicate for 1 hour to obtain a uniform dispersion; take 20 μL of the above dispersion and drop it on the surface of a clean and dry carbon paper, where the surface area of ​​the carbon paper is controlled to be 1 cm×1 cm, and use it as a working electrode after natural drying; on an electrochemical workstation, a three-electrode standard system is used, the electrolyte is 3 mM propionaldehyde and 1.5 mM NAD+ solution, and an H-type glass electrolytic cell is used as the reaction device; cyclic voltammetry, time-current curves, impedance spectroscopy, etc. are used to test the stability of the catalytic activity of bioelectrocatalytic NADH conversion, the conversion efficiency of NADH, the final yield of propanol, and the Faraday efficiency.

[0053] Example 5 Step 1: 10 mg of Fmoc-FFKG was ultrasonically dissolved in 120 μL of DMSO, 2 mg of bismuth nitrate pentahydrate was added to the above solution, and after ultrasonic treatment for 10 min, it was added to PBS with a pH of 10. After stirring for 2 h, the bismuth precursor nanosuspension was washed and collected by centrifugation.

[0054] Step 2: Stir the collected bismuth-containing precursor suspension in pure water for 10 minutes to evenly disperse the bismuth ions in the peptide solution. Take 200 mL of 100 mM solution of sodium borohydride and add it dropwise to the stirred bismuth-containing peptide solution. After stirring for 6 hours, bismuth is fixed in situ on the peptide fibers. After multiple washings with distilled water, the precipitate is collected by centrifugation, frozen in the refrigerator overnight, and then freeze-dried for 72 hours to finally obtain the bismuth-containing peptide hydrogel.

[0055] Step 3: Add the final bismuth-containing peptide hydrogel to 1 mg / ml alcohol dehydrogenase in PBS and stir for 12 hours to obtain the enzyme-encapsulated Bi / Fmoc-FFKG peptide hydrogel.

[0056] Step 4: The obtained Bi / Fmoc-FFKG peptide hydrogel was subjected to a bioelectrocatalytic NADH conversion performance test. The circular dichroism spectra of Fmoc-FFKG before and after Bi doping are shown in Figure 4. Figure 2 The scanning electron microscopy image of the Fmoc-FFKG self-assembled fiber structure is shown in Figure 3 shown.

[0057] Weigh 2 mg of Bi / Fmoc-FFKG peptide hydrogel powder and mix it with 950 μL of a mixed solvent of ethanol and water (1:1), add 50 μL of Nafion solution, and ultrasonicate for 1 hour to obtain a uniform dispersion; take 20 μL of the above dispersion and drop it on the surface of a clean and dry carbon paper, where the surface area of ​​the carbon paper is controlled to be 1 cm×1 cm, and use it as a working electrode after natural drying; on an electrochemical workstation, a three-electrode standard system is used, the electrolyte is 3 mM propionaldehyde and 1.5 mM NAD+ solution, and an H-type glass electrolytic cell is used as the reaction device; cyclic voltammetry, time-current curves, impedance spectroscopy, etc. are used to test the stability of the catalytic activity of bioelectrocatalytic NADH conversion, the conversion efficiency of NADH, the final yield of propanol, and the Faraday efficiency.

[0058] Embodiment 6 Step 1: 10 mg of Fmoc-FFKG was ultrasonically dissolved in 120 μL of DMSO, 2 mg of bismuth nitrate pentahydrate was added to the above solution, and after ultrasonic treatment for 10 min, it was added to PBS with a pH of 10. After stirring for 2 h, the bismuth precursor nanosuspension was washed and collected by centrifugation.

[0059] Step 2: Stir the collected bismuth-containing precursor suspension in pure water for 10 minutes to evenly disperse the bismuth ions in the peptide solution. Take 200 mL of 100 mM solution of sodium borohydride and add it dropwise to the stirred bismuth-containing peptide solution. After stirring for 2 hours, bismuth is fixed in situ on the peptide fibers. After multiple washings with distilled water, the precipitate is collected by centrifugation, frozen in the refrigerator overnight, and then freeze-dried for 72 hours to finally obtain the bismuth-containing peptide hydrogel.

[0060] Step 3: Add the final bismuth-containing peptide hydrogel to 0.5 mg / ml alcohol dehydrogenase in PBS and stir for 12 hours to obtain the enzyme-encapsulated Bi / Fmoc-FFKG peptide hydrogel.

[0061] Step 4: The obtained Bi / Fmoc-FFKG peptide hydrogel was subjected to a bioelectrocatalytic NADH conversion performance test. The comparison of the enzyme activity test before and after Fmoc-FFKG encapsulation of alcohol dehydrogenase is shown in the figure below. Figure 5 As shown, the laser confocal microscopy image of Fmoc-FFKG encapsulated alcohol dehydrogenase is as follows: Figure 6 shown.

[0062] Weigh 2 mg of Bi / Fmoc-FFKG peptide hydrogel powder and mix it with 950 μL of a mixed solvent of ethanol and water (1:1), add 50 μL of Nafion solution, and ultrasonicate for 1 hour to obtain a uniform dispersion; take 20 μL of the above dispersion and drop it on the surface of a clean and dry carbon paper, where the surface area of ​​the carbon paper is controlled to be 1 cm×1 cm, and use it as a working electrode after natural drying; on an electrochemical workstation, a three-electrode standard system is used, the electrolyte is 3 mM propionaldehyde and 1.5 mM NAD+ solution, and an H-type glass electrolytic cell is used as the reaction device; cyclic voltammetry, time-current curves, impedance spectroscopy, etc. are used to test the stability of the catalytic activity of bioelectrocatalytic NADH conversion, the conversion efficiency of NADH, the final yield of propanol, and the Faraday efficiency.

[0063] In summary, the present invention designs a synthetic peptide sequence to self-assemble to form fibers that uniformly load Bi nanoparticles to finally form a Bi / Fmoc-FFKG hydrogel reactor with good safety and biocompatibility, and conducts a bioelectrocatalytic NADH conversion performance test to improve the catalytic efficiency of biological enzymes in vitro, and also provides a new direction for artificially synthesized bioelectrocatalytic systems. Therefore, the peptide hydrogel reactor for efficiently encapsulating NADH-dependent enzymes provided by the present invention has broad application prospects in the field of bioelectrocatalysis.

[0064] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may still be modified, or some or all of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a peptide hydrogel reactor for efficiently encapsulating NADH-dependent enzymes. It is characterized in that include: Step S1, dissolving the screened peptide sequence under a moderate pH condition, stirring evenly with the pre-reaction solution containing the bismuth source reagent, centrifuging, and washing to obtain a clean mixed solution containing the bismuth precursor; Step S2, assembling the mixed solution of bismuth-containing precursors in pure water, fixing bismuth in situ on the surface of the assembly with a reducing agent, stirring evenly, centrifuging, washing to obtain a bismuth-containing polypeptide precipitate, and freezing the bismuth-containing polypeptide hydrogel; Step S3, adding an enzyme reagent to the finally obtained bismuth-containing polypeptide hydrogel, stirring and incubating for 10 minutes to finally obtain an enzyme-encapsulated peptide hydrogel reactor.

2. A method for preparing a peptide hydrogel reactor for efficiently encapsulating NADH-dependent enzymes according to claim 1, It is characterized in that In step S1, the bismuth source reagent is one of bismuth trichloride hexahydrate, bismuth nitrate pentahydrate or bismuth sulfate.

3. The method for preparing a peptide hydrogel reactor for efficiently encapsulating NADH-dependent enzymes according to claim 1, It is characterized in that In the step S1, the concentration of bismuth in the pre-reaction solution containing the bismuth source reagent is 0.1 μg / mL to 1.0 μg / mL.

4. The method for preparing a peptide hydrogel reactor for efficiently encapsulating NADH-dependent enzymes according to claim 1, It is characterized in that In the step S1, the stirring speed ranges from 200 rap / min to 500 rap / min; In the step S2, the assembly time is 2 hours to 24 hours, and the stirring speed ranges from 200 rap / min to 500 rap / min.

5. The method for preparing a peptide hydrogel reactor for efficiently encapsulating NADH-dependent enzymes according to claim 1, It is characterized in that In step S2, the reducing agent is one of sodium borohydride or citric acid, and the peptide sequence in the mixed solution containing the bismuth precursor is one of Fmoc-FF, Fmoc-FFKG, Fmoc-FFKGC or Fmoc-FFGD; wherein the concentration of the reducing agent reaction solution is 10mM to 200mM, and the optimal concentration is 50mM to 120mM.

6. The method for preparing a peptide hydrogel reactor for efficiently encapsulating NADH-dependent enzymes according to claim 1, It is characterized in that In step S2, the molar ratio of the bismuth-containing polypeptide precipitate is 1-3:1-4:2-5, and the optimal molar ratio is 1-2:2-4:3-5.

7. The method for preparing a peptide hydrogel reactor for efficiently encapsulating NADH-dependent enzymes according to claim 1, It is characterized in that In step S3, the enzyme reagent is one of formate dehydrogenase, alcohol dehydrogenase or glucose dehydrogenase, wherein the activity of the enzyme in the bismuth-containing polypeptide hydrogel is 2 U / mg to 50 U / mg.

8. A peptide hydrogel reactor prepared according to the method for preparing a peptide hydrogel reactor for efficiently encapsulating NADH-dependent enzymes according to claims 1-7 is used in bioelectrocatalytic NADH conversion.

9. The use according to claim 8, It is characterized in that The electrocatalytic NADH regeneration performance test was carried out on an electrochemical workstation using a three-electrode system, with electrocatalyst carbon paper coated with enzyme peptide as the working electrode, platinum sheet as the counter electrode, and Ag / AgCl electrode as the reference electrode; 20 mM PBS containing 1.5 mM NAD+ and 3 mM propionaldehyde was used as the electrolyte; An H-type glass electrolytic cell was used as the electrolytic reaction device. Cyclic voltammetry, time-current curve, and impedance spectrum were used to test the stability of the catalytic activity of bioelectrocatalytic NADH conversion, the conversion efficiency of NADH, the final yield of propanol, and the Faraday efficiency.

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