Method for synthesizing UDP-glucose through cooperation of UDP-glucose pyrophosphorylase and cellobiose phosphorylase and one-pot method

Abstract

The invention discloses a method for synthesizing UDP-glucose through cooperation of UDP-glucose pyrophosphorylase and cellobiose phosphorylase by a one-pot method, and belongs to the technical field of enzyme engineering. The method comprises the following steps: selecting UDP-glucose pyrophosphorylase (UGPase) from mycelia thermophilus and cellobiose phosphorylase (CBP) from clostridium thermocellum, and carrying out catalytic reaction by taking cellobiose and UTP as raw materials, so as to obtain UDP-glucose. Experimental results show that heat-resistant UGPase is coupled with CBP, substrates cellobiose and UTP can be efficiently and highly selectively converted into UDP-glucose under mild conditions, and accumulation of an intermediate product glucose-1-phosphoric acid is extremely low. According to the invention, a synergistic reaction system with matched conditions is constructed, and a novel and efficient method for synthesizing UDP-glucose by a one-pot method is provided.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and in particular to a one-pot method for the synergistic synthesis of UDP-glucose by UDP-glucose pyrophosphorylase and cellobiose phosphorylase. Background Technology

[0002] UDP-glucose is an important glyconucleotide in living organisms. As a glucose donor, it plays a central role in glycogen synthesis, glycoside synthesis, and various glycosylation reactions. Industrially, UDP-glucose is a key intermediate in the synthesis of various glycosyl drugs, functional oligosaccharides (such as steviol glycosides and ginsenosides), and glycosylated natural products.

[0003] Currently, the main methods for preparing UDP-glucose include chemical synthesis, microbial fermentation extraction, and enzyme catalysis. Chemical synthesis is cumbersome, has low yields, and causes significant environmental pollution; microbial fermentation extraction and purification are difficult and costly. Enzyme catalysis, due to its high efficiency, specificity, and environmental friendliness, has become a research hotspot.

[0004] UDP-glucose pyrophosphorylase (UGPase) (EC 3.2.1.4) is a key enzyme catalyzing the reaction “glucose-1-phosphate + UTP ↔ UDP-glucose + PPi”. However, most UGPase sources exhibit poor thermostability and are easily inactivated at higher temperatures. For example, the optimum temperature for UGPases derived from *Escherichia coli*, *Saccharomyces cerevisiae*, and *Arabidopsis thaliana* is 37°C, and their activity rapidly decreases at 50°C, severely limiting their application in industrial production.

[0005] On the other hand, glucose-1-phosphate, a direct substrate of UGPase, is expensive, leading to high production costs for UDP-glucose. Cellobiosesphosphorylase (CBP) (EC 2.4.1.20) can catalyze the reaction of cellobiose with inorganic phosphate to produce glucose-1-phosphate and glucose, thus providing an economical substrate source for the UGPase reaction. It originates from *Clostridium thermocellum* (…). Clostridium thermocellum CBP has good thermal stability, with an optimal reaction temperature of about 50℃, making it a promising candidate enzyme for industrial applications.

[0006] In the current technology, there are no reports of a specific combination of heat-resistant UGPase and CBP to design and construct a synergistic enzyme catalytic system that is temperature and pH matched for the efficient one-pot synthesis of UDP-glucose directly from cellobiose and inorganic phosphate. This specific enzyme combination and process innovation is of great significance for reducing production costs and promoting the development of related sugar biomanufacturing industries. Summary of the Invention

[0007] The purpose of this invention is to provide a one-pot method for the synthesis of UDP-glucose by synergistic interaction of UDP-glucose pyrophosphorylase and cellobiose phosphorylase, in order to solve the problems existing in the prior art. This invention constructs a condition-matched synergistic reaction system that can be directly derived from inexpensive substrates, providing a novel and efficient one-pot method for the synthesis of UDP-glucose.

[0008] To achieve the above objectives, the present invention provides the following solution: This invention provides the application of an enzyme composition in the synthesis of UDP-glucose, said enzyme composition comprising *Thermophilus thermophilus* (…). Myceliophthora thermophila UDP-glucose pyrophosphorylase (UGPase) from ) and Clostridium thermocellum ( Clostridium thermocellum Cellobiose phosphorylase (CBP) from ) source.

[0009] Furthermore, the amino acid sequence of the UDP-glucose pyrophosphorylase is shown in SEQ ID NO.1; The amino acid sequence of the cellobiose phosphorylase is shown in SEQ ID NO.2.

[0010] This invention provides a one-pot catalytic synthesis method for UDP-glucose, comprising the following steps: After mixing cellobiose, phosphate, auxiliary agent, and the above-mentioned enzyme composition to prepare a catalytic reaction system, a catalytic reaction was carried out to obtain UDP-glucose.

[0011] Furthermore, the phosphate is uridine triphosphate (UTP).

[0012] Furthermore, the adjuvants include sodium 3-morpholine propanesulfonate (MOPS-NaOH), magnesium chloride (MgCl2), bovine serum albumin (BSA), and phosphate buffered saline (PBS).

[0013] Furthermore, the catalytic reaction system includes: 50 mM sodium 3-morpholinopropanesulfonate, 10 mM magnesium chloride, 2.0 mM uridine triphosphate, 2.0 mM cellobiose, 0.2 mg / mL bovine serum albumin, 6.8 mM phosphate buffer solution, 5 μg UDP-glucose pyrophosphorylase and 5 μg cellobiose phosphorylase.

[0014] Furthermore, the temperature of the catalytic reaction is 40℃-60℃, and the pH is 6.0-8.0.

[0015] Furthermore, the catalytic reaction takes at least 10 minutes.

[0016] Furthermore, after the catalytic reaction is completed, the process also includes centrifugation, collection of supernatant, boiling of supernatant, cooling, secondary centrifugation, collection of supernatant, and filtration through a filter membrane.

[0017] The present invention discloses the following technical effects: Process Innovation and Simplification: This invention is the first to combine thermostable UGPase from thermophilic filamentous fungi with thermostable CBP (such as Clostridium thermophilum CBP) to successfully construct a one-pot reaction system. This system eliminates the need to separate the intermediate product glucose-1-phosphate, simplifying the multi-step reaction into a single step, greatly simplifying the process flow.

[0018] Condition matching and synergistic efficiency: The two enzymes selected in this invention have good thermal stability and similar optimal reaction temperature (about 50°C) and pH range, which ensures that they can maintain high activity in a single reaction system at the same time, realize seamless and efficient connection between reaction steps, and improve the overall conversion rate.

[0019] Significantly reduced production costs: The method provided by this invention uses cellobiose, a readily available lignocellulose hydrolysate, and inorganic phosphates instead of expensive glucose-1-phosphate as starting materials, which greatly reduces the synthesis cost of UDP-glucose.

[0020] Great potential for industrial application: The enzyme selected in this invention has good thermal stability, simple reaction conditions, and easy one-pot operation that is easy to control and scale up. It provides a highly competitive new technology solution for the large-scale green biomanufacturing of UDP-glucose and its downstream glycosylation products (such as glycopharmaceuticals and functional oligosaccharides).

[0021] Through the above technical solution, the present invention solves the problem that heat-resistant UGPase and CBP are not effectively combined for one-pot process in the prior art, and provides a new route for UDP-glucose synthesis that is efficient, economical and easy to scale up. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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.

[0023] Figure 1 Electrophoresis results of glucose pyrophosphorylase (MtUGP) from Thermophilus thermophilus before and after purification; where M is the protein molecular weight standard. Figure 2Electrophoresis results of cellobiose phosphorylase (CtCBP) from Clostridium thermophilum before and after purification; where M is the protein molecular weight standard. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] This invention constructs an efficient and low-cost one-pot synthesis process. The key lies in determining a heat-resistant UGPase that matches the thermal stability of CBP (optimal temperature 50°C), enabling the two to work efficiently and synergistically under the same reaction conditions, avoiding intermediate product separation and simplifying the process.

[0030] Example 1 1. Obtaining UGPase (MtUGP) from *Thermophilus pyriformis* 1.1 Synthesis and Cloning of the MtUGP Gene The coding gene sequence of the MtUGP protein (UniProt accession number G2Q357, amino acid sequence as shown in SEQ ID NO.1) was synthesized by Nanjing Genscript Biotech Co., Ltd., and then cloned into the pET-28b(+) vector to obtain the pET-28b-MtUGP plasmid.

[0031] SEQ ID NO.1: .

[0032] 1.2 Transformation of Escherichia coli BL21(DE3) Competent *E. coli* BL21(DE3) were removed from a -80°C freezer and immediately placed on ice for 3 min. During the freeze-thaw process, 3 μL of pET-28b-MtUGP plasmid was added, and the cells were placed on ice for 30 min. The centrifuge tubes were then rapidly placed in a 42°C water bath for 45 s heat shock, followed immediately by an ice bath for 2 min. 700 μL of antibiotic-free LB medium was added to the centrifuge tubes, and the cells were incubated at 37°C with shaking at 200 rpm for 1.5 h to allow cell recovery. After centrifugation at 8000 rpm for 1 min, most of the supernatant was discarded, and 200 μL of the supernatant was used to resuspend the cells. 50 μL of the bacterial suspension was then spread onto LB agar plates containing kanamycin (100 μg / mL) and incubated overnight at 37°C with the plates inverted.

[0033] 1.3 Culture of recombinant strains expressing MtUGP Take colonies from LB agar plates and inoculate them into 5 mL of antibiotic-free LB medium. Incubate at 37°C, 200 rpm, and shake for 1.5 h. Take the cultured bacterial solution and inoculate it into 50 mL of kanamycin-containing LB medium at a 1% inoculation rate. Incubate at 37°C with shaking until the absorbance OD reaches a certain level. 600 0.6-0.8. Add 200 µL of 100 mM IPTG stock solution and incubate at 16°C and 180 rpm for 16-20 h.

[0034] 1.4 Purification of recombinant MtUGP protein Pre-cool the centrifuge to 4°C. Centrifuge at 8000 rpm for 10 min at 4°C, discarding the supernatant. Resuspend the cells in 5 mL of lysis buffer (50 mM Tris, 500 mM NaCl, 100 μg / mL DNase, 100 μg / mL RNase, 200 μg / mL protease inhibitor, pH 7.0). Disrupt the cells using an ultrasonic cell disruptor. Keep the centrifuge tubes containing the cell suspension on ice during the ultrasonic disruption step.

[0035] The parameters for the ultrasonic cell disruptor were set as follows: ultrasonic power: 150 W, operating time: 15 min, ultrasonic on time: 1 s, ultrasonic off time: 2 s, alarm temperature: 25℃. After cell disruption, the cells were centrifuged at 8000 rpm for 10 min at 4℃, and the supernatant was filtered through a 0.22 μm filter membrane. All solutions and samples used in the purification process were filtered through the filter membrane. The stopper of the nickel column was removed, and the HisTrap HP chromatography column was rinsed with 5 mL of distilled water at a flow rate of 1 mL / min, followed by equilibration with 5 mL of equilibration solution (50 mM Tris, 500 mM NaCl, pH 7.0). The amount of protein to be purified was loaded, depending on the protein concentration. The column was rinsed with 5 mL of equilibration solution to remove contaminating proteins. Finally, linear elution was performed with elution buffer containing 500 mM imidazole (50 mM Tris, 500 mM NaCl, 500 mM imidazole, pH 7.0), and the protein was collected. The collected protein was placed in a dialysis bag (the dialysis bag was boiled in boiling water for 10 min and then rinsed with distilled water), and dialyzed with dialysis solution (50 mM Tris, 150 mM NaCl, pH 7.0) for 1 to 2 days, changing the dialysis solution in between. The purification effect of the protein was then assessed by SDS-PAGE.

[0036] The electrophoresis results of the purified MtUGP protein are as follows: Figure 1 As shown.

[0037] 1.5 Determination of Recombinant MtUGP Protein Concentration The concentration of purified protein was determined using the Bradford Protein Assay Kit. A standard curve for protein concentration was prepared according to the kit instructions. The purified recombinant UGPase sample was diluted to an appropriate concentration. 20 μL of sample and 200 μL of Bradford solution were added to a microplate tube, and the mixture was gently pipetted to mix. After reacting for 5 min, the OD value was measured at 595 nm. The blank control consisted of 20 μL of UGPase-free buffer and 200 μL of Bradford solution. The protein concentration in the fermentation broth was calculated based on the standard curve.

[0038] 2. Obtaining CBP protein (CtCBP) from Clostridium thermocellum 2.1 Synthesis and Cloning of the CtCBP Gene The coding gene sequence of CBP protein (UniProt accession number O66383, amino acid sequence shown in SEQ ID NO.2) from Clostridium thermophilum was synthesized by Nanjing Genscript Biotech Co., Ltd., and then cloned into the pET-28b(+) vector to obtain the pET-28b-CtCBP plasmid.

[0039] SEQ ID NO.2: MKFGFFDDANKEYVITVPRTPYPWINYLGTENFFSLISNTAGGYCFYRDARLRRITRYRYNNVPIDMGGRYFYIYDNGDFWSPGWSPVKRELESYECRHGLGYTKIAGKRNGIKAEVTFFVPLNYNGEVQKLILKNEGQDKKKITLFSFIEFCLWNAYDDMTNFQRNFSTGEVEIEGSVIYHKTEYRERRNHYAFYSVNAKISGFDSDRDSFIGLYNGFDAPQAVVNGKSNNSVADGWAPIASHSIEIELNPGEQKEYVFIIGYVENKDEEKWESKGVINKKKAYEMIEQFNTVEKVDKAFEELKSYWNALLSKYFLESHDEKLNRMVNIWNQYQCMVTFNMSRSASYFESGIGRGMGFRDSNQDLLGFVHQIPARARERLLDLAATQLEDGGAYHQYQPLTKKGNNEIGSNFNDDPLWLILATAAYIKETGDYSILKEQVPFNNDPSKADTMFEHLTRSFYHVVNNLGPHGLPLIGRADWNDCLNLNCFSTVPDESFQTTTSKDGKVAESVMIAGMFVFIGKDYVKLCEYMGLEEEARKAQQHIDAMKEAILKYGYDGEWFLRAYDDFGRKVGSKENEEGKIFIESQGFCVMAEIGLEDGKALKALDSVKKYLDTPYGLVLQNPAFTRYYIEYGEISTYPPGYKENAGIFCHNNAWIICAETVVGRGDMAFDYYRKIAPAYIEDVSDIHKLEPYVYAQMVAGKDAKRHGEAKNSWLTGTAAWNFVAISQWILGVKPDYDGLKIDPCIPKAWDGYKVTRYFRGSTYEITVKNPNHVSKGVAKITVDGNEISGNILPVFNDGKTHKLK。

[0040] 2.2 Transformation of Escherichia coli BL21(DE3) Competent *E. coli* BL21(DE3) were removed from a -80°C freezer and immediately placed on ice for 3 min. During the freeze-thaw process, 3 μL of pET-28b-CtCBP plasmid was added, and the cells were placed on ice for 30 min. The centrifuge tubes were then rapidly placed in a 42°C water bath for 45 s heat shock, followed immediately by an ice bath for 2 min. 700 μL of antibiotic-free LB medium was added to the centrifuge tubes, and the cells were incubated at 37°C with shaking at 200 rpm for 1.5 h to allow cell recovery. After centrifugation at 8000 rpm for 1 min, most of the supernatant was discarded, and 200 μL of the supernatant was used to resuspend the cells. 50 μL of the bacterial suspension was then spread onto LB agar plates containing kanamycin (100 μg / mL) and incubated overnight at 37°C with the plates inverted.

[0041] 2.3 Culture of recombinant strains expressing CtCBP Colonies were picked from LB agar plates and inoculated into 5 mL of antibiotic-free LB medium. The plates were then incubated at 37°C and 200 rpm with shaking for 1.5 h. The incubated bacterial solution was then inoculated into 50 mL of kanamycin-containing LB medium at a 1% inoculation rate and cultured at 37°C with shaking until the absorbance OD reached a certain level. 600 0.6-0.8. Add 200 µL of 100 mM IPTG stock solution and incubate at 16°C and 180 rpm for 16-20 h.

[0042] 2.4 Purification of recombinant CtCBP protein Pre-cool the centrifuge to 4°C. Centrifuge at 8000 rpm for 10 min at 4°C, discarding the supernatant. Resuspend the cells in 5 mL of lysis buffer (50 mM Tris, 500 mM NaCl, 100 μg / mL DNase, 100 μg / mL RNase, 200 μg / mL protease inhibitor, pH 7.0). Disrupt the cells using an ultrasonic cell disruptor. Keep the centrifuge tubes containing the cell suspension on ice during the ultrasonic disruption step.

[0043] The parameters for the ultrasonic cell disruptor were set as follows: ultrasonic power: 150 W, operating time: 15 min, ultrasonic on time: 1 s, ultrasonic off time: 2 s, alarm temperature: 25℃. After cell disruption, the cells were centrifuged at 8000 rpm for 10 min at 4℃, and the supernatant was filtered through a 0.22 μm filter membrane. All solutions and samples used in the purification process were filtered through the filter membrane. The stopper on the nickel column was removed, and the HisTrap HP chromatography column was rinsed with 5 mL of distilled water at a flow rate of 1 mL / min, followed by equilibration with 5 mL of equilibration solution (50 mM Tris, 500 mM NaCl, pH 7.0). The amount of protein to be purified was loaded, depending on the protein concentration. The column was rinsed with 5 mL of equilibration solution to remove contaminating proteins. Finally, linear elution was performed with elution buffer containing 500 mM imidazole (50 mM Tris, 500 mM NaCl, 500 mM imidazole, pH 7.0), and the protein was collected. The collected protein was placed in a dialysis bag (the dialysis bag was boiled in boiling water for 10 min and then rinsed with distilled water), and dialyzed with dialysis solution (50 mM Tris, 150 mM NaCl, pH 7.0) for 1 to 2 days, changing the dialysis solution in between. The purification effect of the protein was then assessed by SDS-PAGE.

[0044] The electrophoresis results of the purified CtCBP protein are as follows: Figure 2 As shown.

[0045] 2.5 Determination of recombinant CtCBP protein concentration The concentration of purified protein was determined using the Bradford Protein Assay Kit. A standard curve for protein concentration was prepared according to the kit instructions. The purified recombinant CBP protein sample was diluted to an appropriate concentration. 20 μL of sample and 200 μL of Bradford solution were added to a microplate tube, and the mixture was gently pipetted to mix. After reacting for 5 min, the OD value was measured at 595 nm. The blank control consisted of 20 μL of buffer containing no CBP protein and 200 μL of Bradford solution. The protein concentration in the fermentation broth was calculated based on the standard curve.

[0046] 3. One-pot method for preparing UDP-glucose using MtUGP and CtCBP 5 μg each of purified MtUGP and CtCBP were added to a solution of 50 mM MOPS-NaOH (pH 7.0), 10 mM MgCl2, 2.0 mM UTP, 2.0 mM cellobiose, 0.2 mg / mL BSA, and 6.8 mM PBS. The solution was then incubated in a 50°C water bath for 10 min, followed by centrifugation at 12000 rpm for 1 min. The solution was then boiled for 15 min, cooled, and centrifuged at 1200 rpm for 5 min. The supernatant was filtered through a 0.22 μm filter to prepare a liquid chromatography vial sample.

[0047] 4. Liquid Chromatography Detection Conditions All the following ratios are volume ratios.

[0048] 4.1 Preparation of mobile phase Mobile phase A: 8 mM tetrabutylammonium bisulfate (pH 6.5), mobile phase B: 8 mM tetrabutylammonium bisulfate (pH 6.5): methanol = 7:3; both mobile phases A and B need to be filtered through a 0.22 μm organic filter membrane, and then sonicated for 10 min to remove air bubbles.

[0049] 4.2 Testing Conditions High performance liquid chromatograph (Shimadzu LC-20A), ultraviolet detector (SPD-20A), Sepax GP-C18 column (250×4.6 mm); isocratic elution: A:B=23:77, flow rate: 0.6 mL / min, column temperature 40℃; detection wavelength: 260 nm.

[0050] 5. Synthesis and Catalytic Efficiency Evaluation of UDP-Glucose Using the method described in "3. One-pot preparation of UDP-glucose using MtUGP and CtCBP", 5 μg each of purified MtUGP and CtCBP were added to a reaction system consisting of 50 mM MOPS-NaOH (pH 7.0), 10 mM MgCl2, 2.0 mM UTP, 2.0 mM cellobiose, 0.2 mg / mL BSA, and 6.8 mM PBS. The reaction was carried out at 50 °C for 10 min. After the reaction, the sample was treated as described in "3. One-pot preparation of UDP-glucose using MtUGP and CtCBP", and detected using the conditions described in "4. Liquid Chromatography Detection Conditions".

[0051] After 10 min of reaction, the UTP conversion rate reached 71.4%, the UDP-glucose concentration in the reaction solution was 1.36 mM, the molar yield of consumed UTP was 95.0%, and the formation rate was 0.136 mM / min. Experimental results show that the heat-resistant UGPase-CBP coupling system provided by this invention can efficiently and selectively convert inexpensive substrates cellobiose and UTP into UDP-glucose under mild conditions, with minimal accumulation of the intermediate product glucose-1-phosphate, demonstrating the excellent thermal compatibility and catalytic synergy between the two.

[0052] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of an enzyme composition in the synthesis of UDP-glucose, characterized in that, The enzyme composition includes UDP-glucose pyrophosphorylase from *Thermophilus hygrophytes* and cellobiose phosphorylase from *Clostridium thermophilum*.

2. The application as described in claim 1, characterized in that, The amino acid sequence of the UDP-glucose pyrophosphorylase is shown in SEQ ID NO.1; The amino acid sequence of the cellobiose phosphorylase is shown in SEQ ID NO.

2.

3. A one-pot catalytic synthesis method for UDP-glucose, characterized in that, Includes the following steps: After mixing cellobiose, phosphate, auxiliaries and the enzyme composition described in claim 1 or 2 to prepare a catalytic reaction system, a catalytic reaction is carried out to obtain UDP-glucose.

4. The method as described in claim 3, characterized in that, The phosphate is uridine triphosphate.

5. The method as described in claim 3, characterized in that, The adjuvants include sodium 3-morpholine propanesulfonate, magnesium chloride, bovine serum albumin, and phosphate buffer solution.

6. The method as described in claim 3, characterized in that, The catalytic reaction system includes: 50 mM sodium 3-morpholinopropanesulfonate, 10 mM magnesium chloride, 2.0 mM uridine triphosphate, 2.0 mM cellobiose, 0.2 mg / mL bovine serum albumin, 6.8 mM phosphate buffered saline, 5 μg UDP-glucose pyrophosphorylase and 5 μg cellobiose phosphorylase.

7. The method as described in claim 3, characterized in that, The catalytic reaction is carried out at a temperature of 40℃-60℃ and a pH of 6.0-8.

0.

8. The method as described in claim 3, characterized in that, The catalytic reaction time shall be no less than 10 min.

9. The method as described in claim 3, characterized in that, After the catalytic reaction is completed, the process also includes centrifugation, collection of supernatant, boiling of supernatant, cooling, secondary centrifugation, collection of supernatant, and filtration through a filter membrane.

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