Natural enzyme-nano enzyme hybrid system and application thereof in lignin degradation
By constructing a natural enzyme-nanozyme hybrid system of λ-MnO2 nanoenzyme and CotA laccase, the problem of lignin being difficult to efficiently degrade under mild conditions in the prior art is solved, and efficient and directed lignin degradation and high proportion of aromatic compounds are achieved.
Patent Information
- Application Number
- CN202510270113.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-07
- Publication Date
- 2025-06-06
AI Technical Summary
The prior art is difficult to efficiently and directionally degrade lignin in complex structures under mild conditions, and traditional methods require harsh reaction conditions, high energy consumption and high cost.
Develop a natural enzyme-nanozyme hybrid system, including a hybrid system composed of λ-MnO2 nanoenzyme and CotA laccase, to achieve efficient degradation of lignin by adjusting pH and temperature.
Under mild conditions (30°C, pH 6.0), the hybrid system achieved efficient degradation of lignin, with a degradation rate of up to 32.4%, and the proportion of aromatic compounds in the product reached 51.7%, which significantly improved the degradation efficiency and product quality of lignin.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and in particular to a natural enzyme-nanozyme hybrid system and its application in lignin degradation. Background Art
[0002] With the growing global demand for sustainable resources, lignin, as a renewable aromatic polymer with considerable reserves in nature, is a potential green raw material for the preparation of aromatic compounds with wide applications and high added value. Lignin has a complex chemical structure, consisting of various phenylpropane units such as p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) connected by various CO and CC bonds such as α-O-4, β-O-4, 4-O-5, β-1, β-5, etc. The diversity of its constituent units and connection methods gives it natural structural complexity, heterogeneity and difficulty in degradation, making it a daunting challenge to degrade it into aromatic small molecule compounds.
[0003] Traditional lignin degradation technologies, including hydrogenolysis, hydrolysis, oxidation, liquefaction, supercritical alcoholysis and pyrolysis, have made some progress, but they often require harsh conditions such as external hydrogen sources, high temperature and high pressure, which not only consumes a lot of energy but also easily produce side reactions. 2 WO 8 Taking the catalyst as an example, its public document is "Chem. Eng. J. 2024, 485, 150020". The article shows that the efficient dispersion of Pt particles can be achieved by inducing oxygen vacancy formation through ammonium carbonate template and W doping to construct an ordered porous structure, which can simultaneously break β-O-4, β-1, α-1 and 5-5 bonds, and the monophenol yield reaches 49.08%. However, this technology still has significant limitations: the reaction requires 1.2MPa hydrogen pressure, relies on an external hydrogen source and has high energy consumption; the catalyst preparation requires high-temperature calcination, the process is complicated and the cost of metal Pt catalyst is high. In contrast, enzymatic lignin degradation has attracted much attention due to its mild reaction conditions and environmental friendliness. However, the protein properties of lignin-degrading enzymes, such as charge, hydrophilicity and size, limit their accessibility to specific areas in the complex structure of lignin, thereby failing to fully exert the catalytic efficiency of the enzyme.
[0004] Nanozymes, as a class of nanomaterials with enzyme-like activity, can catalyze reactions under mild conditions and have good synergistic potential with natural enzymes. Combining nanozymes with natural enzymes to construct a hybrid system is expected to integrate the advantages of both and achieve efficient and directional degradation of lignin. Therefore, it is of great practical significance to develop an efficient, green and precisely regulated lignin degradation method. Summary of the invention
[0005] In order to overcome the defects of the above-mentioned prior art, the purpose of the present invention is to provide a natural enzyme-nanozyme hybrid system and its application in lignin degradation, so as to achieve the degradation of lignin under mild conditions and increase the proportion of aromatic compounds in the degradation products, thereby providing a greener alternative for the high-value utilization of lignin.
[0006] In order to achieve the above object, the technical solution adopted by the present invention is:
[0007] The natural enzyme-nanozyme hybrid system comprises a mixed system obtained by mixing nanozymes, natural enzymes and phosphate buffer, and adjusting the pH of the mixed system to 3.0-8.5 with hydrochloric acid or NaOH solution to obtain the natural enzyme-nanozyme hybrid system.
[0008] The nanozyme is MnMOF, λ-MnO 2 or ε-MnO 2 .
[0009] The natural enzyme is laccase CotA from Bacillus subtilis.
[0010] In the natural enzyme-nanozyme hybrid system, the ratio of natural enzyme to nanozyme is 4:1 to 1:4 according to the enzyme activity U / L.
[0011] In the natural enzyme-nanozyme hybrid system, the ratio of natural enzyme to nanozyme in terms of enzyme activity U / L is 1:4.
[0012] The phosphate buffer is a potassium dihydrogen phosphate-disodium hydrogen phosphate buffer with a concentration of 50 mM. The amount of phosphate buffer added ensures that the total enzyme concentration of the natural enzyme and the nanozyme is 20 U / L.
[0013] The application of the natural enzyme-nanozyme hybrid system in lignin degradation includes the following steps:
[0014] (1) adding lignin to the natural enzyme-nanozyme hybrid system to make the final concentration of lignin in the system reach 1 g / L, adjusting the temperature to 20-40° C., and placing it on a shaker at 150-250 rpm for at least 24 hours;
[0015] (2) After the reaction is completed, the final system of step (1) is centrifuged at 10,000 to 12,000 rpm for 2 to 5 minutes, and the supernatant is taken for lignin degradation rate determination and GC-MS product analysis.
[0016] Compared with the prior art, the beneficial effects of the present invention are embodied in:
[0017] 1. In terms of degradation efficiency, laccase CotA has a specific active center structure, which can attack active sites such as phenolic hydroxyl groups in lignin molecules through redox reactions, causing initial breakage; λ-MnO 2 Nanozymes have abundant surface oxygen vacancies and stable metal-support interactions. Their large specific surface area and abundant surface active sites can adsorb lignin molecules and further promote degradation. The synergistic effect of the two avoids the accumulation of intermediates and the occurrence of side reactions, making CotA+λ-MnO 2 The lignin degradation rate of the hybrid system was as high as 32.40%, which was much higher than that of laccase CotA (15.32%) or λ-MnO alone. 2 The system of nanozymes (14.90%) significantly improved the degradation efficiency of lignin.
[0018] 2. In terms of product conversion, CotA and λ-MnO 2 The complementary effect of the two breaks through the diffusion limitation of the single enzyme system (the large size and strong hydrophilicity of the protein make it difficult to contact the hydrophobic area of lignin). 2 The nanostructure provides a stable carrier interface for laccase and enhances the electron transfer efficiency, making the proportion of aromatic compounds in the hybrid system product reach 51.7%, which is significantly higher than that of CotA and λ-MnO 2 The single catalytic system increased the yields by 52.06% and 135%, respectively, which was beneficial to the conversion of lignin into high-value aromatic compounds.
[0019] 3. In terms of reaction conditions, laccase CotA and λ-MnO nanozyme have good biocompatibility and catalytic activity stability. CotA can maintain high activity under mild temperature and pH conditions, while λ-MnO 2 The surface properties and crystal structure of nanozymes enable them to play a good catalytic role in a relatively mild environment. The hybrid system composed of the two can achieve efficient degradation under mild conditions of 30°C and pH 6.0, avoiding the harsh reaction conditions in traditional methods, reducing energy consumption and costs, and has good application prospects.
[0020] In summary, the present invention achieves the degradation of lignin under mild conditions and increases the proportion of aromatic compounds in the degradation products, providing a greener alternative for the high-value utilization of lignin. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 Figure 2 is the lignin degradation rate and product distribution diagram of different nanozyme-natural enzyme hybrid systems, among which, Figure 1 (a) is a schematic diagram of lignin degradation rates of different nanozyme-natural enzyme hybrid systems. Figure 1(b) is a schematic diagram of the distribution of lignin degradation products in different nanozyme-natural enzyme hybrid systems.
[0022] Figure 2 CotA+λ-MnO 2 Lignin degradation rate and product distribution diagram of hybrid system and single enzyme system; Figure 2 (a) is CotA+λ-MnO 2 Schematic diagram of lignin degradation rate of hybrid system and single enzyme system, Figure 2 (b) is CotA+λ-MnO 2 Schematic diagram of the distribution of lignin degradation products in the hybrid system and the single enzyme system.
[0023] Figure 3 CotA+λ-MnO at different pH 2 Lignin degradation rate and product distribution diagram of the hybrid system; among them, Figure 3 (a) shows CotA+λ-MnO at different pH values. 2 Schematic diagram of lignin degradation rate in the hybrid system, Figure 3 (b) shows CotA+λ-MnO at different pH values. 2 Schematic diagram of the distribution of lignin degradation products in the hybrid system.
[0024] Figure 4 CotA+λ-MnO at different temperatures 2 Lignin degradation rate and product distribution diagram of the hybrid system; among them, Figure 4 (a) shows CotA+λ-MnO at different temperatures 2 Schematic diagram of lignin degradation rate in the hybrid system, Figure 4 (b) shows CotA+λ-MnO at different temperatures 2 Schematic diagram of the distribution of lignin degradation products in the hybrid system.
[0025] Figure 5 For different CotA and λ-MnO 2 Ratio of CotA+λ-MnO 2 Lignin degradation rate and product distribution diagram of the hybrid system; among them, Figure 5 (a) shows different ratios of CotA+λ-MnO 2 Schematic diagram of lignin degradation rate in the hybrid system, Figure 5 (b) shows different ratios of CotA+λ-MnO 2 Schematic diagram of the distribution of lignin degradation products in the hybrid system. DETAILED DESCRIPTION
[0026] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.
[0027] The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged.
[0028] Embodiment 1
[0029] Degradation of lignin by different nanozyme-natural enzyme hybrid systems
[0030] 1. Construction of different nanozyme-natural enzyme hybrid systems
[0031] Weigh three nanozymes, MnMOF, λ-MnO 2 and ε-MnO 2 , add the natural enzyme - laccase CotA, the natural enzyme and nanozyme are dispersed in 50mM PBS buffer solution at a ratio of 1:4 according to the enzyme activity, and three natural enzyme-nanozyme hybrid systems with a concentration of 1mg / mL are prepared, and ultrasonicated for 30min for use.
[0032] 2. Lignin degradation reaction
[0033] (1) Take lignin respectively, dissolve it in the above three natural enzyme-nanozyme hybrid systems, adjust the final lignin concentration to 1 mg / mL, adjust the temperature to 20-40°C, place it on a shaker at 200 rpm for 24 h, and centrifuge it at 12000 rpm for 4 min to use as the reaction substrate solution.
[0034] (2) The enzyme activity of nanozymes and natural enzymes was measured to determine the amount to be added. Using ABTS as the substrate, the absorbance change was measured at a specific wavelength to calculate the enzyme activity. The total system was 1 mL. 980 μL of phosphate buffer (pH = 5.6), 10 μL of ABTS mother solution (final concentration of 1 mM) and 10 μL of nanozyme solution were added to a 1.5 mL EP tube in sequence. The tube was incubated at 30°C and shaken at 200 rpm for 5 minutes. Three parallel groups were set up for each group. After the reaction was completed, the tube was precooled on ice until it was measured. The supernatant without enzyme (PBS replacement) was used as the background control. The measurement wavelength was 420 nm.
[0035] The enzyme activity was calculated by the following formula:
[0036]
[0037] Note: V R represents the total volume of the reaction system (mL); Ve represents the volume of the enzyme solution in the system (μL); ε represents the molar extinction coefficient (L·mol -1 cm -1 ), here is 36000; c Pro protein concentration; d represents the optical path width (cm).
[0038] (5) In a 1.5 mL centrifuge tube, add lignin (final concentration 1 mg / mL), nanozyme (10 U / L), and CotA (10 U / L) enzyme solution in sequence and mix gently. Place the reaction tube in a constant temperature oscillator at 30 °C and 200 rpm for 24 h.
[0039] 3. Determination of lignin degradation rate
[0040] (1) Prepare a series of lignin standard solutions with different concentrations ranging from 0 to 5 mg / mL.
[0041] (2) Take 200 μL of each concentration standard solution, add 800 μL of 100 mM PBS (pH 5.6) buffer, mix well and measure its absorbance at 280 nm. Draw a standard curve with lignin concentration as the horizontal axis and absorbance as the vertical axis to obtain the standard curve equation.
[0042] (3) Take 200 μL of the lignin degradation solution after the reaction, add 800 μL of 100 mM PBS (pH 5.6) buffer, measure its absorbance at 280 nm, and calculate the concentration of lignin after the reaction according to the standard curve equation.
[0043] (3) The calculation formula of lignin degradation rate is: degradation rate = (initial lignin concentration - lignin concentration after reaction) / initial lignin concentration × 100%.
[0044] 4. Analysis of degradation product composition by gas chromatography-mass spectrometry (GC-MS)
[0045] (1) After the reaction is completed, the reaction solution is transferred to a centrifuge tube, centrifuged at 4°C and 12,000 rpm for 10 min, and the supernatant is collected.
[0046] (2) Add three times the volume of ethyl acetate to the supernatant, shake and mix, and extract the organic phase. Transfer the organic phase to a new centrifuge tube and wash with anhydrous Na 2 SO 4 Dry to remove any residual moisture.
[0047] (3) Take 100 μL of the dried organic phase, add 10 μL of pyridine and 50 μL of N,O-bis(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane (99% BSTFA + 1% TMCS), vortex mix, and heat in a 60°C water bath for 30 min with regular shaking to carry out the derivatization reaction.
[0048] (4) The derivatized sample was cooled to room temperature, and 1 μL was injected into a gas chromatograph-mass spectrometer (ThermoFisher Scientific Trace ISQ, America). An HP-5 capillary column was used, the carrier gas was helium, and the flow rate was 1 mL / min. The injection port temperature was 280°C, and the split ratio was 10:1. Column temperature program: initial temperature 50°C, maintained for 2 min, increased to 280°C at 10°C / min, and maintained for 10 min. Mass spectrometry conditions: electron energy 70 eV, ion source temperature 230°C, scanning range m / z 40-600. According to the GC-MS spectra and database search results, the types and relative contents of lignin degradation products were analyzed.
[0049] Depend on Figure 1 The degradation of lignin by different nanozyme-natural enzyme systems provided by CotA+λ-MnO 2 The degradation rate of the system reached 25.15%, and the proportion of aromatic products reached 48%, which was significantly higher than that of CotA+MnMOF and CotA+ε-MnO 2 Therefore, CotA+λ-MnO 2 Further research on the system.
[0050] Embodiment 2
[0051] CotA+λ-MnO 2 Degradation of lignin by hybrid system and single enzyme system
[0052] (1) Prepare different enzyme solutions: CotA laccase solution (20 U / L), λ-MnO 2 Nanozyme solution (20U / L) and CotA (10U / L) with λ-MnO 2 (10U / L) combined mixed enzyme solution.
[0053] (2) In a 1.5 mL centrifuge tube, add lignin (final concentration 1 mg / mL) and enzyme solution in sequence and mix gently. Place the reaction tube in a constant temperature oscillator at 30°C and react at 200 rpm for 24 h.
[0054] Depend on Figure 2 The degradation of lignin by different systems provided in (a) shows that CotA+λ-MnO 2The degradation rate of the hybrid system (25.15%) was much higher than that of laccase CotA (15.32%) or λ-MnO 2 The system of nanozymes (14.90%) strongly indicates that there is a significant synergistic effect between the two.
[0055] Depend on Figure 2 The GC-MS spectra and the proportion of aliphatic and aromatic products provided in (b) show that the product types of the hybrid system are more concentrated and the proportion of aromatic compounds is the highest, reaching 48%, which is significantly higher than CotA (34%) or λ-MnO 2 Catalytic system (22%). This is mainly due to the synergistic catalytic effect of natural enzymes and nanozymes.
[0056] Embodiment 3
[0057] Effect of different pH on CotA+λ-MnO 2 Effect of hybrid system on lignin degradation
[0058] This example focuses on the effect of pH factor on the lignin degradation process. Compared with Example 2, the core difference lies in the change of pH conditions. The specific experimental operation is as follows:
[0059] A series of phosphate buffer solutions with pH values of 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5 were precisely prepared to construct reaction environments of different pH values. CotA and λ-MnO were reacted at the same temperature while keeping other reaction conditions (such as reaction temperature, enzyme and substrate concentrations, reaction time, etc.) exactly the same. 2 The buffer solutions with different pH values were added to form a reaction system with lignin. The degradation of lignin and the changes in product distribution at different temperatures were analyzed according to the above lignin degradation rate determination method, GC-MS analysis and other methods.
[0060] Figure 3 The results show that when the pH of the reaction system is 6.0, the lignin degradation rate is the highest, reaching 23.86%, and the aromatic products account for 49.6%.
[0061] Embodiment 4
[0062] Effect of different temperatures on CotA+λ-MnO 2 Effect of hybrid system on lignin degradation
[0063] This example focuses on the effect of temperature factors on the lignin degradation process. Compared with Example 3, the core difference lies in the change of temperature conditions. The specific experimental operation is as follows:
[0064] Under the premise of ensuring that other reaction conditions (such as substrate concentration, enzyme concentration, reaction time, etc.) are strictly consistent, the reaction tubes containing lignin and related reaction reagents are placed in a precisely temperature-controlled constant temperature oscillator, and the set temperatures are 20°C, 25°C, 30°C, 35°C, and 40°C, respectively, and the pH value of the reaction system is 6.0. According to the above-mentioned lignin degradation rate determination method, GC-MS analysis and other methods, the degradation of lignin and the changes in product distribution at different temperatures are analyzed.
[0065] Figure 4 The results show that when the reaction system temperature is 30℃, the lignin degradation rate is the highest, reaching 28.67%, and the aromatic products account for 50.2%.
[0066] Embodiment 5
[0067] Different CotA and λ-MnO 2 The ratio of CotA+λ-MnO 2 Effect of hybrid system on lignin degradation
[0068] This example focuses on the study of CotA and λ-MnO 2 The effect of different ratios on the lignin degradation process is mainly different from Example 4 in that the ratio of CotA to λ-MnO is changed. 2 The proportional relationship in the hybrid system. The specific experimental operation is as follows:
[0069] Constructed 2 A hybrid system with a ratio of (4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4). While maintaining the same other reaction conditions, the lignin degradation reaction was carried out at 30°C and pH 6.0. According to the above-mentioned lignin degradation rate determination method, GC-MS analysis and other methods, the degradation of lignin and the changes in product distribution at different temperatures were analyzed.
[0070] Figure 5 CotA and λ-MnO 2 Effects of different ratios on lignin degradation rate and product distribution. The results showed that when CotA and λ-MnO 2 When the ratio was 1:4, the lignin degradation rate was the highest, reaching 32.4%, and the proportion of aromatic products reached 51.7%.
[0071] In summary, the present invention discloses a natural enzyme-nanozyme hybrid system for lignin degradation, which consists of λ-MnO 2Nanozyme is composed of CotA laccase, which shows significant advantages in lignin degradation. It can achieve lignin degradation under mild conditions (30°C, pH 6.0), with a degradation rate of up to 32.4%. The proportion of aromatic compounds in the product can reach 51.7%, which is much better than a single enzyme system. The present invention provides a new way for the high-value utilization of lignin, has broad application prospects in the field of biotechnology, and is expected to promote the development of related industries and realize the sustainable utilization of resources.
Claims
1. A natural enzyme-nanozyme hybrid system, characterized in that: The invention comprises a mixed system obtained by mixing nanozymes, natural enzymes and phosphate buffer, wherein the pH value of the mixed system is adjusted to 3.0-8.5 with hydrochloric acid or NaOH solution to obtain a natural enzyme-nanozyme hybrid system.
2. The natural enzyme-nanozyme hybrid system according to claim 1, characterized in that: The nanozyme is MnMOF, λ-MnO2 or ε-MnO2.
3. The natural enzyme-nanozyme hybrid system according to claim 1, characterized in that: The natural enzyme is laccase CotA from Bacillus subtilis.
4. The natural enzyme-nanozyme hybrid system according to claim 1, characterized in that: In the natural enzyme-nanozyme hybrid system, the ratio of natural enzyme to nanozyme is 4:1 to 1:4 according to the enzyme activity U / L.
5. The natural enzyme-nanozyme hybrid system according to claim 4, characterized in that: In the natural enzyme-nanozyme hybrid system, the ratio of natural enzyme to nanozyme in terms of enzyme activity U / L is 1:
4.
6. The natural enzyme-nanozyme hybrid system according to claim 1, characterized in that: The phosphate buffer is a potassium dihydrogen phosphate-disodium hydrogen phosphate buffer with a concentration of 50 mM. The amount of phosphate buffer added ensures that the total enzyme concentration of the natural enzyme and the nanozyme is 20 U / L.
7. The use of the natural enzyme-nanozyme hybrid system in lignin degradation according to any one of claims 1 to 6, characterized in that: The following steps are involved: (1) adding lignin to the natural enzyme-nanozyme hybrid system to make the final concentration of lignin in the system reach 1 g / L, adjusting the temperature to 20-40° C., and placing it on a shaker at 150-250 rpm for at least 24 hours; (2) After the reaction is completed, the final system of step (1) is centrifuged at 10,000 to 12,000 rpm for 2 to 5 minutes, and the supernatant is taken for lignin degradation rate determination and GC-MS product analysis.