A method for selective catalytic oxidation of non-active c-h bonds of polysaccharides by a photoenzyme

Abstract

This invention discloses a method for the photoenzymatic selective catalytic oxidation of inactive C-H bonds in polysaccharides, belonging to the field of photoenzyme-coupled catalysis technology. This invention employs a photocatalyst that enhances visible light absorption and efficiently generates electrons and H2O2, continuously and gently providing the necessary electrons and co-substrate for the LPMO reaction under illumination. Furthermore, by introducing a dopamine functional layer onto the catalyst surface, LPMO is enriched on the catalyst surface, forming a proximity effect that effectively improves the transfer efficiency of electrons and H2O2 from the photocatalyst to the LPMO, significantly enhancing the catalytic activity of the LPMO and achieving efficient oxidation of inactive C-H bonds in polysaccharides. Compared with traditional systems that require continuous replenishment of exogenous electron donors and H2O2, this invention is simpler to operate, lower in cost, and can be further extended to other peroxidase systems that rely on electron donors and H2O2. Overall, it has green, low-carbon, high-efficiency characteristics and promising prospects for industrial application.

Description

Technical Field

[0001] This invention relates to a method for selectively catalyzing the oxidation of inactive CH bonds in polysaccharides using photoenzymes, belonging to the field of photoenzyme-coupled catalysis technology. Background Technology

[0002] Selective catalytic oxidation of inactive C–H bonds is considered the "holy grail reaction" in modern chemistry. For polysaccharide natural polymers, their C–H bonds are located within a highly crystalline structure with tightly packed hydrogen bonds, exhibiting extremely high chemical inertness. Therefore, achieving efficient and precise oxidation of these inactive C–H bonds in the conversion, functionalization, and high-value utilization of polysaccharides such as cellulose and chitin remains a significant challenge in industrial biomass processing and material modification. Although existing chemical methods can achieve polysaccharide oxidation under specific conditions, they generally suffer from poor selectivity, demanding reaction conditions, reliance on high temperatures, strong oxidants or noble metal catalysts, numerous side reactions, and a tendency to damage the molecular framework. These methods struggle to achieve mild, controllable, site-selective oxidation, falling significantly short of the development direction of green chemistry. Lysaturase monooxygenases (LPMOs) are a class of copper-dependent oxidases widely distributed in the AA9-AA11 and AA13-AA16 families in the CAZy database. These enzymes can oxidize the surfaces of natural polysaccharides such as cellulose, chitin, and pectin under mild conditions by selectively hydroxylating the inactive C–H bonds at the C1 and / or C4 or C6 positions of the glycounit. However, the catalytic process of LPMOs is highly complex, relying on an exogenous electron donor and the co-substrate H2O2. On the one hand, the exogenous electron donor is continuously consumed with the reaction, increasing system costs; on the other hand, H2O2 has a dual effect in LPMO catalysis, with its concentration only positively correlated with enzyme activity within a narrow range. Excessive H2O2 can induce non-productive conversion of LPMOs, leading to rapid enzyme inactivation. Ideally, H2O2 should be supplied continuously in a slow and stable manner to maintain efficient enzyme catalysis, but precise, gradual supply is difficult to achieve under actual experimental or industrial conditions. The current common method is to add small amounts of H2O2 multiple times during the reaction process. Although this can improve the reaction efficiency to some extent, the operation is cumbersome and it is difficult to precisely control the H2O2 concentration, which often leads to a "step-like" fluctuation in LPMO activity.

[0003] Photoenzyme-coupled catalysis offers a solution for the selective oxidation of inactive C–H bonds in polysaccharides. In this approach, the photocatalyst continuously provides electrons and H₂O₂ to the LPMO in situ under visible light irradiation, achieving mild and sustainable oxidation. However, current photoenzyme-coupled systems for oxidizing inactive C–H bonds in polysaccharides suffer from several problems, including poor enzyme-photocatalyst compatibility, narrow light absorption range, and a mismatch between electron supply capacity and H₂O₂ generation capacity and the catalytic requirements of LPMO. This results in insufficient enzyme activity and hinders the efficient oxidation of polysaccharide C–H bonds. Therefore, constructing a highly compatible photoenzyme-coupled system with good LPMO compatibility, strong visible light response, and the ability to stably and continuously provide electrons and H₂O₂ remains a key technological bottleneck for achieving efficient oxidation of inactive C–H bonds in polysaccharides. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a photoenzyme reaction system with high enzyme adaptability for the catalytic oxidation of inactive CH bonds in polysaccharides. This system employs a photocatalyst that enhances visible light absorption and efficiently generates electrons and H₂O₂, continuously and gently providing LPMO with the necessary electrons and co-substrate under illumination. Simultaneously, by introducing a dopamine functional layer onto the catalyst surface, LPMO is enriched on the catalyst surface, forming a proximity effect. This effectively improves the transfer efficiency of electrons and H₂O₂ from the photocatalyst to LPMO, significantly enhancing the catalytic activity of LPMO and achieving highly efficient oxidation of inactive CH bonds in polysaccharides.

[0005] This invention is achieved through the following technical solution: The first objective of this invention is to provide a photocatalyst prepared by the following method: S1. Urea, melamine and cobalt salt are ball-milled and mixed. After the reaction, the mixture is calcined. After calcination, the metal oxide is removed to obtain cobalt-doped porous micro-nano tubular g-C3N4 / Co. S2. The porous micro-nanotube g-C3N4 / Co is dispersed in a buffer solution, dopamine is added and stirred to react, and after the reaction, it is washed and dried to obtain the porous micro-nanotube g-C3N4 / Co / DA, which is the photocatalyst.

[0006] In one embodiment of the present invention, the mass ratio of urea to melamine is 8-12:1.

[0007] In one embodiment of the present invention, the cobalt salt is cobalt nitrate, and its amount is 1wt%-5wt% of the mass of the mixture. Preferably, its amount is 2wt% of the mass of the mixture.

[0008] In one embodiment of the present invention, the ball milling mixing time is 10-15 hours. In this invention, the ball milling mixing method allows for more thorough mixing, which is beneficial for preparing tubular porous micro / nano structures.

[0009] In one embodiment of the present invention, calcination is carried out by heating to 500-600°C at a heating rate of 4-6°C / min, holding at that temperature for 3-5 hours, and then naturally cooling to 20-30°C.

[0010] In one embodiment of the present invention, the removal of metal oxides involves placing the calcined product in a 0.05-0.5M hydrochloric acid solution and stirring for 10-20 hours, followed by centrifugation and washing with water to obtain porous micro / nanotube-shaped g-C3N4 / Co.

[0011] In one embodiment of the present invention, the mass-to-volume ratio of porous micro / nanotube g-C3N4 / Co to the buffer solution is 1:200-800 g / mL.

[0012] In one embodiment of the present invention, the buffer solution is a Tris-HCl buffer solution with a concentration of 10-30 mM and a pH of 8-9.

[0013] In one embodiment of the present invention, the mass ratio of dopamine to porous micro / nanotubular g-C3N4 / Co is 1:100-300. Preferably, the mass ratio of dopamine to porous micro / nanotubular g-C3N4 / Co is 1:200.

[0014] In one embodiment of the present invention, in step S2, the stirring reaction is carried out at 20-30°C for 20-30 hours.

[0015] In one embodiment of the present invention, drying is performed under vacuum at 50-70°C for 10-15 hours.

[0016] The second objective of this invention is to provide a method for photocatalytic selective oxidation of inactive CH bonds in polysaccharides. The method uses a polysaccharide-cleaving monooxygenase as a biocatalyst and natural polysaccharides as substrates. In the presence of the photocatalyst, a light source is used to catalyze the reaction, thereby selectively catalytically oxidizing the inactive CH bonds in polysaccharides.

[0017] In one embodiment of the present invention, the amount of the photocatalyst used is 1-10 mg / mL.

[0018] In one embodiment of the present invention, the polysaccharide cleaving monooxygenase is one or more of the polysaccharide cleaving monooxygenases of the AA9, AA10, AA11, AA13, AA14, AA15, and AA16 families.

[0019] In one embodiment of the present invention, the amount of the polysaccharide cleaving monooxygenase is 1-10 U.

[0020] In one embodiment of the present invention, the natural polysaccharide is one or more of cellulose, hemicellulose, chitin, starch and pectin.

[0021] In one embodiment of the present invention, the amount of natural polysaccharide added is 1%-10% of the total volume of the reaction system.

[0022] In one embodiment of the present invention, the reaction system selectively catalytically oxidizes the C1 and / or C4 or C6 positions of cellulose; the C1 and / or C4 positions of chitin and starch; and the C1 position of hemicellulose and pectin.

[0023] In one embodiment of the present invention, the wavelength of the light source is 390-503nm.

[0024] The beneficial effects of this invention are: The photocatalytic material of this invention is based on carbon nitride, exhibiting excellent biocompatibility and minimal risk of direct damage to enzymes. A metal-doped porous nanotube-shaped g-C3N4 composite structure is prepared by a one-step method involving the direct calcination of a ball-milled carbon nitride precursor with a metal nitrate. The nitrogen-rich triazine units in the g-C3N4 framework form stable coordination with the metal, resulting in a simple and reproducible preparation process that significantly increases the specific surface area of ​​the material, effectively expanding the enzyme-catalyst interface and accelerating the reaction rate. Furthermore, a biocompatible dopamine functional layer is coated onto the material surface. The resulting proximity effect promotes the enrichment of LPMO on the catalyst surface, significantly shortening the electron and H2O2 transport pathways, thereby greatly improving the local transfer efficiency of electrons and H2O2 to LPMO and significantly enhancing the catalytic activity of LPMO. After metal regulation and dopamine surface functionalization, this porous nanotube material can mildly and continuously generate H2O2 under light conditions, which is highly matched to the enzyme reaction requirements. This avoids enzyme activity reduction or inactivation caused by insufficient or excessive H2O2, and the reaction system does not require the addition of additional sacrificial agents. Simultaneously, the modified material can more efficiently generate and transfer electrons to the enzyme active site, increasing the electron transfer rate by approximately 2.5 times. Its light absorption range extends to approximately 503 nm, allowing for more efficient use of visible light and achieving low-energy, high-efficiency catalytic drive. It exhibits excellent cycle stability, maintaining a high level of enzyme activity even after multiple reuses. Compared to traditional systems that require continuous replenishment of exogenous electron donors and H2O2, this invention is simpler to operate, lower in cost, and can be further extended to other peroxidase systems that rely on electron donors or H2O2. Overall, it has green, low-carbon, high-efficiency, and promising prospects for industrial application. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The activity of LPMO after 24 hours of reaction with different catalysts according to the present invention; Figure 2 EPR images of the porous nanotubular g-C3N4, porous nanotubular g-C3N4 / Co, and porous nanotubular g-C3N4 / Co / DA catalysts of the present invention are shown. Figure 3 This is a SEM image of the porous nanotubular g-C3N4 / Co catalyst described in this invention; Figure 4 This is a TEM image of the g-C3N4 / Co / DA catalyst described in this invention; Figure 5 This is the XPS image of the g-C3N4 / Co / DA catalyst described in this invention. Detailed Implementation

[0027] The present invention will be further illustrated below with specific examples. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0028] Source of raw materials Commercially available TiO2 (anatase type) and CdS nanopowder were purchased from: TiO2 (anatase type) from Yuan Ye; CdS nanopowder from Sinopharm Wokai.

[0029] Detection method: H2O2 concentration detection: The photocatalyst (5 mg / mL) was added to a reaction system containing 1% (w / v) phosphate-swelled cellulose and 50 mM phosphate buffer (pH 6.0), and reacted at 50°C under 395 nm LED illumination for 24 h. After the reaction, the supernatant was collected by centrifugation at 8000 rpm for 5 min as the test sample. 50 µL of the reaction supernatant was mixed with 50 µL of premix, which consisted of horseradish peroxidase (HRP, 10 U / mL) and Amplex Red (200 µM, 2% DMSO), prepared with 50 mM sodium phosphate buffer at pH 7.5. The resulting reaction mixture was incubated at 25°C in the dark for 20 min, and the absorbance was measured at 560 nm to calculate the H2O2 concentration in the system.

[0030] Electron transfer capacity test: 2 mg of catalyst was weighed and added to a solution containing 100 μL of 1 mM methyl viologen and 900 μL of 100 mM phosphate buffer (pH 7.4). The mixture was thoroughly mixed under anaerobic conditions and then reacted for 1 min under 395 nm LED light source irradiation. After the reaction, the absorbance of the system was measured at 603 nm to characterize the ability of photogenerated electrons to transfer to methyl viologen.

[0031] Light absorption range detection: The light absorption range of the solid powder photocatalyst was tested using a UV-2700 UV-Vis diffuse reflectance spectrometer manufactured by Shimadzu Corporation of Japan. BaSO4 was selected as the internal standard for the sample test. The wavelength of the light source was 200 ~ 800 nm, and the bandwidth was 5 nm. The UV-Vis diffuse reflectance spectrum was plotted based on the data.

[0032] Enzyme stability test: After the enzymatic hydrolysis reaction was completed, the catalyst was recovered by centrifugation at 8000 rpm for 10 min. The obtained catalyst was dried in a 60℃ oven and then added back to the same enzymatic hydrolysis system for the next round of catalytic reaction (reaction conditions were the same as in Example 1). The above cycle was repeated 5 times, with each reaction time being 3–24 h. The cycle stability of the catalyst was evaluated by detecting the change in the amount of reducing sugar generated during each cycle.

[0033] The technical solution of the present invention will be described in detail below with reference to specific embodiments. In the following embodiments, unless otherwise specified, the reagents, materials and equipment used can be purchased commercially, prepared by conventional methods, or commonly used in the industry.

[0034] Example 1: The porous micro / nanotubular g-C3N4 / Co / DA catalyst and its preparation method in this embodiment specifically include the following steps: Urea and melamine were weighed at a mass ratio of 10:1, with 150 mg of urea used, and mixed with 2 wt% Co(NO3)2. The mixture was then ball-milled for 12 h. Subsequently, the resulting mixture was placed in a muffle furnace and heated to 550 °C at a heating rate of 5 °C / min, held at that temperature for 4 h, and then naturally cooled to room temperature to obtain CoO. x / Co-doped porous micro / nanotube g-C3N4; the product was then placed in 0.1M hydrochloric acid solution and stirred overnight, followed by centrifugation at 8000 rpm for 10 min, and washing five times with deionized water to remove metal oxides, finally obtaining Co-doped porous micro / nanotube g-C3N4 / Co. Its SEM image is shown below. Figure 3 As shown.

[0035] Then, 1g of the porous micro / nanotube g-C3N4 / Co powder prepared above was dispersed in 500mL of a 20mM Tris-HCl buffer solution with a pH of 8.5; 5mg of dopamine was added, and the mixture was stirred at room temperature for 24h; after the reaction was completed, the resulting solid was washed three times with deionized water, filtered, and dried in a vacuum drying oven at 60℃ for 12h to obtain porous micro / nanotube g-C3N4 / Co / DA. Its TEM image is shown below. Figure 4 As shown in the figure, the XPS graph is as follows Figure 5 As shown, its 24h H2O2 production concentration was 9.6±0.3uM, and its protein adsorption capacity was 1.6-2.0 times that of g-C3N4 / Co material.

[0036] Porous micro-nanotube g-C3N4, porous micro-nanotube g-C3N4 / Co, and porous micro-nanotube g-C3N4 / Co / DA were added as catalysts (5 mg / mL) to an enzymatic hydrolysis system containing 0.1 U LPMO, 1% (w / v) phosphate-swelled cellulose, and 50 mmol / L phosphate buffer (pH 6.0). The reaction was carried out under 395 nm LED illumination at 50°C for 24 h. After the reaction, the supernatant was obtained by centrifugation at 8000 rpm for 5 min. The oxidation products were detected by high-performance anion exchange chromatography (HPAEC). The results showed that the concentration of the oxidation product obtained from porous micro-nanotubular g-C3N4 was 0.358±0.01 g / L; the concentration of the oxidation product obtained from porous micro-nanotubular g-C3N4 / Co was 0.489±0.02 g / L; and the concentration of the oxidation product obtained from porous micro-nanotubular g-C3N4 / Co / DA was 0.627±0.01 g / L.

[0037] Electron transfer capacity tests were conducted on each catalyst. The results showed that the absorbance of the g-C3N4 catalyst was 0.308, while that of the g-C3N4 / Co / DA catalyst was 0.761, indicating that the electron transfer capacity of the material was significantly enhanced after Co regulation and dopamine modification. The light absorption range of each catalyst was tested, and the light absorption range of the g-C3N4 / Co / DA catalyst was extended to approximately 503 nm, allowing for more efficient use of visible light and achieving low-energy, high-efficiency catalytic drive. Enzyme stability tests revealed that the enzyme exhibited excellent cycle stability when used in conjunction with the porous micro / nanotube g-C3N4 / Co / DA, maintaining a high enzyme activity level even after repeated use.

[0038] Comparative Example 1: In Example 1, Co(NO3)2 was replaced with AgNO3, Pd(NO3)2, and Mn(NO3)2 to prepare the corresponding g-C3N4 / Ag, g-C3N4 / Pd, and g-C3N4 / Mn catalysts. The preparation methods and subsequent dopamine modification steps were the same as in Example 1. Subsequently, the obtained catalysts were used for the enzymatic hydrolysis of LPMO. The results showed that when using g-C3N4 / Ag / DA, g-C3N4 / Pd / DA, and g-C3N4 / Mn / DA as catalysts, the concentrations of the oxidation products were 0.472±0.05 g / L, 0.401±0.05 g / L, and 0.386±0.05 g / L, respectively. This phenomenon may be attributed to the relatively limited H2O2 generation capacity of these metal-doped catalysts, resulting in insufficient supply of co-substrate to LPMO, thereby limiting the improvement of catalytic efficiency.

[0039] Comparative Example 2: Take 100 mg of porous micro-nanotube g-C3N4 and porous micro-nanotube g-C3N4 / Co from Example 1, and mix them with 10 mg of 2-anthraquinone-2-carboxylic acid, 7.7 mg of diisopropylethylamine, 8.1 mg of 1-hydroxybenzotriazole hydrate, and 11.5 mg of N-(3-dimethylaminopropyl)-N 1-Ethylcarbodiimide hydrochloride was mixed in 50 mL of dichloromethane, sonicated for 5 min, and stirred for 48 h. After centrifugation, it was washed successively with dichloromethane and water, and dried at 80°C to obtain porous micro / nanotube g-C3N4 / AQ and porous micro / nanotube g-C3N4 / Co / AQ. The H2O2 concentrations produced after 24 h were 12.5 ± 0.1 μM and 12.7 ± 0.6 μM, respectively.

[0040] g-C3N4 / AQ and g-C3N4 / Co / AQ were added as catalysts (5 mg / mL) to an enzymatic hydrolysis system containing 0.1 U LPMO, 1% (w / v) phosphate-swelled cellulose, and 50 mmol / L phosphate buffer (pH 6.0), respectively, and reacted at 50°C under 395 nm LED illumination for 24 h. After the reaction, the supernatant was obtained by centrifugation at 8000 rpm for 5 min. The oxidation products were detected by high-performance anion exchange chromatography (HPAEC). The results showed that the concentration of oxidation products obtained by g-C3N4 / AQ was 0.388 ± 0.02 g / L; the concentration of oxidation products obtained by g-C3N4 / Co / AQ was 0.412 ± 0.03 g / L. This was mainly due to the high concentration of H2O2 generated in situ by g-C3N4 / AQ and g-C3N4 / Co / AQ, which led to enzyme inactivation.

[0041] Comparative Example 3: Using commercially available TiO2 (anatase type) and CdS nanoparticles, TiO2, TiO2 / Co, TiO2 / DA, TiO2 / DA / Co and CdS, CdS / Co, CdS / DA, and CdS / DA / Co catalytic materials were obtained according to the method described in Example 1. For example, 165 mg of TiO2 powder was mixed with 2 wt% Co(NO3)2 and ball-milled for 12 h. The resulting mixture was then placed in a muffle furnace and heated to 550 °C at a heating rate of 5 °C / min, held at that temperature for 4 h, and then naturally cooled to room temperature to obtain CoO2 / Co ... x / Co-doped TiO2; then the product was placed in 0.1M hydrochloric acid solution and stirred overnight, then centrifuged at 8000 rpm for 10 min, and washed 5 times with deionized water to remove metal oxides, finally obtaining Co-doped TiO2.

[0042] The H2O2 concentrations produced by the above-mentioned catalysts over 24 hours were 3.3±0.1 uM, 3.3±0.1 uM, 3.1±0.0 uM, 3.2±0.0 uM and 10.6±0.3 uM, 11.8±0.6 uM, 11.1±0.2 uM, 12.0±0.4 uM, respectively.

[0043] TiO2, TiO2 / Co, TiO2 / DA, TiO2 / DA / Co, CdS, CdS / Co, CdS / DA, and CdS / DA / Co were added as catalysts (5 mg / mL) to an enzymatic hydrolysis system containing 0.1 U LPMO, 1% (w / v) phosphate-swelled cellulose, and 50 mmol / L phosphate buffer (pH 6.0), respectively. The reaction was carried out under 380 nm LED illumination at 50°C for 24 h. After the reaction, the supernatant was obtained by centrifugation at 8000 rpm for 5 min. The oxidation products were detected by high-performance anion exchange chromatography (HPAEC). The results showed that the concentrations of oxidation products obtained from TiO2 were 0.163±0.01 g / L; those from TiO2 / Co were 0.290±0.04 g / L; those from TiO2 / DA were 0.185±0.01 g / L; and those from TiO2 / DA / Co were 0.330±0.04 g / L, significantly lower than those from the g-C3N4-based catalyst. This is mainly due to the low H2O2 production of the TiO2-based catalyst, leading to low LPMO activity. Similarly, the concentrations of oxidation products obtained from CdS were 0.237±0.00 g / L; those from CdS / Co were 0.241±0.01 g / L; those from CdS / DA were 0.235±0.02 g / L; and those from CdS / DA / Co were 0.252±0.04 g / L, also significantly lower than those from the g-C3N4-based catalyst. This is mainly because CdS readily generates various reactive oxygen species (ROS) under light, which can damage LPMO and thus inhibit overall catalytic efficiency.

[0044] Comparative Example 4: 10g of wheat straw biomass, passed through a 60-mesh sieve, was mixed evenly with 150g of glycerol. The mixture was then placed in a heating mantle and reacted at 240℃ and 180rpm for 30min. After the reaction, the mixture was separated into solid and liquid phases using a sintered glass funnel. The solid residue containing residual lignin was washed with 600mL of hot water. The filtrate obtained from the solid-liquid separation and the washing filtrate were combined, and 1500mL of tap water was added. The mixture was allowed to stand overnight at room temperature. The lignin-containing precipitate was collected by centrifugation at 8000rpm for 15min. The precipitate was then washed three times with 200mL of tap water to remove residual glycerol and sugar compounds. Finally, the treated precipitate was freeze-dried to obtain wheat straw lignin. The 24h H2O2 concentration was 152±12.1uM.

[0045] The wheat straw lignin prepared above was added as a catalyst (5 mg / mL) to an enzymatic hydrolysis system containing 0.1 U LPMO, 1% (w / v) phosphate-swelled cellulose, and 50 mmol / L phosphate buffer (pH 6.0). The reaction was carried out under 395 nm LED illumination at 50°C for 24 h. After the reaction, the supernatant was obtained by centrifugation at 8000 rpm for 5 min. The oxidation products were detected by high-performance anion exchange chromatography (HPAEC). The results showed that the concentration of the obtained oxidation products was 0.453 ± 0.09 g / L. In addition, lignin undergoes photolysis and auto-oxidation under light conditions, which are easily accompanied by ineffective consumption and side reactions, making it difficult to achieve sustained and efficient enhancement of LPMO catalytic activity under mild conditions.

[0046] Comparative Example 5: 11 g of urea and 1.1 g of melamine were mixed with 0.6 wt% and 5 wt% Co(NO3)2, respectively. Then, porous micro / nanotube g-C3N4 / Co catalysts with different Co doping amounts were prepared according to the method in Example 1. The obtained catalysts were then used for the enzymatic hydrolysis reaction with LPMO. The results showed that when the Co doping amount was 0.6 wt%, the concentration of the resulting oxidation product was 0.407 ± 0.04 g / L; when the Co doping amount was 5 wt%, the concentration of the resulting oxidation product was 0.432 ± 0.03 g / L.

[0047] Comparative Example 6: After preparing porous micro / nanotube g-C3N4 / Co catalysts according to the method in Example 1, the amount of dopamine added was varied in the dopamine modification step, with 1 mg, 2 mg, 3 mg, and 10 mg of dopamine added respectively to obtain g-C3N4 / Co / DA catalysts with different dopamine loadings. The obtained catalysts were then used for the enzymatic hydrolysis reaction with LPMO. The results showed that when the dopamine addition was 1 mg, 2 mg, 3 mg, and 10 mg, the concentrations of the resulting oxidation products were 0.525 ± 0.03 g / L, 0.558 ± 0.02 g / L, 0.562 ± 0.00 g / L, and 0.614 ± 0.01 g / L, respectively. This indicates that appropriate dopamine modification can significantly improve the catalytic activity of LPMO, while too low a loading makes it difficult to form an effective proximity effect, and too high a loading tends to saturate the catalytic enhancement effect.

[0048] Comparative Example 7: Urea and melamine were weighed at a mass ratio of 10:1, with 150 mg of urea used. The mixture was placed in a ceramic crucible and heated to 550°C in a muffle furnace at a heating rate of 5°C / min, and held for annealing for 4 hours. After the reaction, the mixture was allowed to cool naturally to room temperature. The resulting light yellow lumpy product was then ground into powder to obtain g-C3N4. 1 g of the obtained g-C3N4 powder was dispersed in 500 mL of a 20 mM Tris-HCl buffer solution (pH 8.5), and 5 mg of dopamine was added. The mixture was magnetically stirred at room temperature for 24 hours. After the reaction, the resulting solid was washed three times with deionized water, filtered, and dried in a vacuum oven at 60°C for 12 hours to obtain dopamine-modified g-C3N4 / DA material. Subsequently, 160 mg of g-C3N4 / DA was dispersed in 50 mL of deionized water and sonicated for 30 minutes. 1.5 mL of a 2% dopamine buffer solution was added. A Co(NO3)2 solution of g / L was stirred at 60°C for 18 h. After the reaction, the mixture was centrifuged, dried at 80°C overnight, and then annealed at 400°C for 2 h. After cooling and grinding, g-C3N4 / DA / Co was obtained. The catalyst was then used for the enzymatic hydrolysis of LPMO according to the method in Example 1. The results showed that the concentration of oxidation products obtained by g-C3N4 / DA was 0.412±0.04 g / L, and the concentration of oxidation products obtained by g-C3N4 / DA / Co was 0.423±0.02 g / L. Both were lower than the catalytic effect of the catalyst obtained by first doping with Co and then modifying with dopamine. This indicates that the introduction of dopamine before loading with Co may lead to spatial separation between the metal active sites and the photogenerated carrier transport channels of g-C3N4, thereby weakening the system's synergistic ability to provide electrons to LPMO and the co-substrate H2O2.

[0049] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A photocatalyst, characterized in that, It is prepared by the following method: S1. Urea, melamine and cobalt salt are ball-milled and mixed. After the reaction, the mixture is calcined. After calcination, the metal oxide is removed to obtain cobalt-doped porous micro-nano tubular g-C3N4 / Co. S2. The porous micro-nanotube g-C3N4 / Co is dispersed in a buffer solution, dopamine is added and stirred to react, and after the reaction, it is washed and dried to obtain the porous micro-nanotube g-C3N4 / Co / DA, which is the photocatalyst.

2. The photocatalyst according to claim 1, characterized in that, The mass ratio of urea to melamine is 8-12:

1.

3. The photocatalyst according to claim 1, characterized in that, The cobalt salt is cobalt nitrate, and its amount is 1wt%-5wt% of the mass of the mixture.

4. The photocatalyst according to any one of claims 1-3, characterized in that, Calcination involves heating to 500-600℃ at a heating rate of 4-6℃ / min, holding at that temperature for 3-5 hours, and then naturally cooling to 20-30℃.

5. The photocatalyst according to claim 1, characterized in that, The mass ratio of dopamine to porous micro / nanotube g-C3N4 / Co is 1:100-300.

6. A method for photoenzymatic selective catalytic oxidation of inactive CH bonds in polysaccharides, characterized in that, The method uses polysaccharide monooxygenase as a biocatalyst and natural polysaccharides as substrates. In the presence of the photocatalyst described in any one of claims 1-5, a light source is used to catalyze the reaction, selectively catalyzing the oxidation of inactive CH bonds in polysaccharides.

7. The method according to claim 6, characterized in that, The amount of the photocatalyst used is 1-10 mg / mL.

8. The method according to claim 6, characterized in that, The amount of the polysaccharide-lysing monooxygenase used is 1-10 U.

9. The method according to claim 6, characterized in that, The natural polysaccharide is one or more of cellulose, hemicellulose, chitin, starch, and pectin; the amount of the natural polysaccharide added is 1%-10% of the total volume of the reaction system.

10. The method according to claim 6, characterized in that, The wavelength of the light source is 390-503nm.

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