A method for degrading chitin that is resistant to hydrogen peroxide

Abstract

The application discloses a method for degrading chitin resistant to hydrogen peroxide, and belongs to the technical field of chitin degradation. The method is as follows: under the condition of high-concentration hydrogen peroxide, chitin is degraded by using a lytic polysaccharide monooxygenase CdLPMO10A and a chitinase ChiB; the condition of high-concentration hydrogen peroxide refers to that the concentration of hydrogen peroxide is above 1.0 mM; the amino acid sequence of the lytic polysaccharide monooxygenase CdLPMO10A is shown in SEQ ID NO. 1; and the amino acid sequence of the chitinase ChiB is shown in SEQ ID NO. 3. The method for degrading chitin can degrade chitin for a long time under the condition of high-concentration hydrogen peroxide, and does not need to frequently reduce the concentration of hydrogen peroxide or add the lytic polysaccharide monooxygenase, which has important significance for the industrial degradation of chitin.

Description

Technical Field

[0001] This invention relates to a method for degrading chitin that is resistant to hydrogen peroxide, belonging to the field of chitin degradation technology. Background Technology

[0002] Chitin is one of the most abundant biomass resources on Earth, mainly found in the shells of crustaceans and the cell walls of fungi. Chitin oligosaccharides, a degradation product of chitin, possess various biological activities, and degrading chitin into smaller molecule products is an important way to achieve high-value utilization of chitin.

[0003] Lytic polysaccharide monooxygenases (LPMOs) are a class of enzymes that utilize molecular oxygen (usually oxygen gas or hydrogen peroxide) and copper ions as cofactors to introduce oxidative cleavage at the carbon-carbon (CC) or carbon-oxygen (CO) bonds of sugar chains. Unlike traditional glycoside hydrolases, LPMOs cleave polysaccharide chains through oxidation in the presence of the co-substrate hydrogen peroxide, producing chain ends or oxidized ends, thereby greatly enhancing the efficiency of subsequent hydrolytic enzymes.

[0004] Despite the significant potential of LPMO in chitin degradation, several challenges remain. For instance, the accumulation of hydrogen peroxide during the reaction disrupts LPMO stability. Hydrogen peroxide, as a co-substrate of LPMO, directly impacts its activity and substrate degradation efficiency during catalysis. However, excessive accumulation of hydrogen peroxide leads to oxidative damage to the LPMO structure, reducing its stability and catalytic lifetime. Studies have shown that reduced LPMO unbound to the substrate catalyzes the reduction of hydrogen peroxide, resulting in enzyme inactivation. Therefore, developing LPMOs capable of withstanding high concentrations of hydrogen peroxide, and methods for the sustained and stable degradation of chitin under high-concentration hydrogen peroxide conditions, is crucial for improving the stability and application prospects of chitin in industrial biotransformation. Summary of the Invention

[0005] In view of the above-mentioned prior art, the present invention provides a method for degrading chitin that is resistant to hydrogen peroxide, belonging to the field of chitin degradation technology.

[0006] This invention is achieved through the following technical solution: A method for degrading chitin resistant to hydrogen peroxide is as follows: under high concentration of hydrogen peroxide conditions, chitin is degraded using the cleaving polysaccharide monooxygenase CdLPMO10A and the chitinase ChiB. The high concentration hydrogen peroxide condition refers to a hydrogen peroxide concentration of 1.0 mM or higher; The amino acid sequence of the cleaving polysaccharide monooxygenase CdLPMO10A is shown in SEQ ID NO.1; The amino acid sequence of the chitinase ChiB is shown in SEQ ID NO.3.

[0007] Preferably, the high concentration of hydrogen peroxide condition refers to a hydrogen peroxide concentration of 2.0 mM to 15 mM, more preferably 5.0 mM to 15 mM, more preferably 10 mM to 15 mM, and even more preferably 2.0 mM, 5.0 mM, 10 mM or 15 mM.

[0008] Furthermore, the chitin is α-chitin.

[0009] Furthermore, the time for degrading chitin is more than 12 hours.

[0010] Preferably, the specific steps are as follows: α-chitosan, chitinase ChiB, cleaving polysaccharide monooxygenase CdLPMO10A, vitamin C, and hydrogen peroxide are added to 10 mM pH 6.0 phosphate buffer. The concentration of α-chitosan is 5.0 mg / mL, the concentration of chitinase ChiB is 2.0 μM, the concentration of cleaving polysaccharide monooxygenase CdLPMO10A is 3.0 μM, the concentration of vitamin C is 1.0 mM, and the concentration of hydrogen peroxide is 1.0–15.0 mM; the mixture is then incubated at 37°C and 220 rpm in a shaker for 12 hours.

[0011] This invention uncovered the cleaving polysaccharide monooxygenase CdLPMO10A. Experimental studies unexpectedly revealed that it can tolerate high concentrations of hydrogen peroxide; after incubation at 1.0 mM hydrogen peroxide for 24 hours, the residual enzyme activity remained at 90%, demonstrating its superior suitability for prolonged chitin degradation. The chitin degradation method of this invention allows for long-term chitin degradation under high-concentration hydrogen peroxide conditions without frequent reductions in hydrogen peroxide concentration or the addition of cleaving polysaccharide monooxygenase, which is of significant importance for the industrial degradation of chitin.

[0012] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0013] Figure 1 The results of SDS-PAGE protein electrophoresis of the purified enzyme solution are shown. The left band is the protein marker, and the right band is the cleaving polysaccharide monooxygenase CdLPMO10A.

[0014] Figure 2 : The results of the optimal temperature determination.

[0015] Figure 3Results of temperature stability measurements.

[0016] Figure 4 : Results of the determination of the optimal pH.

[0017] Figure 5 Results of pH stability measurements.

[0018] Figure 6 Mass spectrum of enzymatic hydrolysis products when the substrate is α-chitin.

[0019] Figure 7 Mass spectrum of enzymatic hydrolysis products when the substrate is β-chitin.

[0020] Figure 8 Mass spectrum of enzymatic hydrolysis products when the substrate is chitosan.

[0021] Figure 9 Mass spectrum of enzymatic hydrolysis products when the substrate is cellulose.

[0022] Figure 10 Results of the determination of the optimal H2O2 concentration.

[0023] Figure 11 Results of H2O2 tolerance test.

[0024] Figure 12 Results of relative enzyme activity determination under different concentrations of hydrogen peroxide. Detailed Implementation

[0025] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0026] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0027] The α-chitosan used in this invention was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., the chitosan from Shanghai Yuanye Biotechnology Co., Ltd., and the cellulose from Shanghai Maclean Biochemical Technology Co., Ltd.

[0028] Example 1: Cleavage Polysaccharide Monooxygenase Cd Discovery of the LPMO10A gene A cleaving polysaccharide monooxygenase fragment (GenBank: WP_034572838.1) was mined from the NCBI database, which originated from *Clostridium difficile*. Carnobacterium divergens Based on phylogenetic analysis and multiple sequence alignment, the cleaving polysaccharide monooxygenase expressed by this gene fragment was found to belong to the chitin-active branch of the Coenzyme 10 family. Codon optimization was performed on the sequence of this gene fragment according to the codon bias of the host *E. coli* for efficient expression in *E. coli*. The optimized gene sequence is shown in SEQ ID NO.2, and the expressed cleaving polysaccharide monooxygenase is named cleaving polysaccharide monooxygenase CdLPMO10A, with its amino acid sequence shown in SEQ ID NO.1.

[0029] Example 2 Heterologous expression of the polysaccharide monooxygenase CdLPMO10A The steps are as follows: (1) The gene fragment shown in SEQ ID NO.2 was artificially synthesized. The artificially synthesized gene fragment was used as a template for conventional PCR amplification. The PCR product fragment of the correct size was recovered after agarose gel electrophoresis.

[0030] (2) Construction of recombinant expression vector: The gene fragment amplified above was ligated with the pET-22b linearized plasmid using seamless cloning technology. The ligation product was transformed into E. coli DH5α competent cells and plated on LB agar plates (containing 100 μg / mL ampicillin). The plates were incubated at 37°C for 16 hours, and single colonies with correct morphology and size were selected for positive clone verification. The verified colonies were inoculated into LB liquid medium (containing 100 μg / mL ampicillin) and incubated at 37°C and 220 rpm for 12 hours. A small amount of bacterial culture was sequenced, and then the recombinant plasmid was extracted from the correctly sequenced bacterial cultures. The extracted plasmid was stored at -20°C for later use.

[0031] (3) Construction of recombinant engineered bacteria: The plasmid extracted above was transformed into E. coli BL21 competent cells, spread on LB medium solid plates (containing 100 μg / mL ampicillin), and cultured in an incubator at 37℃ for 16 hours to obtain recombinant engineered bacteria.

[0032] (4) Expression of the lysin monooxygenase CdLPMO10A: The recombinant engineered strain was activated by inoculating it into LB liquid medium (containing 100 μg / mL ampicillin). Then, the activated bacterial solution was inoculated into LB liquid medium (containing 100 μg / mL ampicillin) at a volume ratio of 1% and cultured at 37℃ and 220 rpm until OD. 600The concentration was 0.6–0.8. Isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.1 mM as an inducer, and the mixture was cultured at 20°C and 200 rpm for 20 hours to express the cleaving polysaccharide monooxygenase CdLPMO10A.

[0033] (5) Extraction and purification of lysin monooxygenase CdLPMO10A: After the culture was completed, the fermentation broth was centrifuged at 8000 g for 10 minutes, the cells were collected, resuspended in 50 mM pH 8.0 Tirs-HCl buffer, and then sonicated in an ice water bath for 20 minutes (350 W), centrifuged at 8000 g for 15 minutes, and the supernatant was collected, which is the crude enzyme solution.

[0034] During the construction of the recombinant plasmid, a His tag was added to the C-terminus of the protein for crude enzyme purification. First, the column was equilibrated with 10 mM imidazole solution (10 mM imidazole, 500 mM NaCl, 50 mM pH 8.0 Tris-HCl). Then, weakly binding proteins were eluted with 50 mM imidazole solution (50 mM imidazole, 500 mM NaCl, 50 mM pH 8.0 Tris-HCl), followed by elution of the target protein with 150 mM imidazole solution (150 mM imidazole, 500 mM NaCl, 50 mM pH 8.0 Tris-HCl). The purified enzyme solution was collected. SDS-PAGE protein electrophoresis was performed to determine protein purity and molecular weight. The SDS-PAGE results of the purified enzyme solution are shown below. Figure 1 As shown, the molecular weight of the obtained protein was approximately 53 kDa, consistent with the predicted result. The protein concentration was determined using the Bradford method, and the protein concentration in the purified enzyme solution was 625.89 mg / L. The imidazole in the enzyme solution was replaced with ultrapure water via ultrafiltration, followed by the addition of CuSO4 at three times the protein molar concentration, and incubation at 4°C for 3 hours to activate the polysaccharide-cleaving monooxygenase CdLPMO10A. Excess Cu was then replaced again via ultrafiltration. 2+ , thus obtaining pure enzyme solution.

[0035] Example 3: Determination of the specific activity of the polysaccharide-lysing monooxygenase CdLPMO10A The standard method for determining the activity of the polysaccharide-lysing monooxygenase CdLPMO10A is as follows: H2O2, 2,6-DMP, and pure enzyme solution (prepared in Example 2) are added to 10 mM pH 6.0 phosphate buffer. After addition, the concentration of H2O2 is 0.1 mM, the concentration of 2,6-DMP is 1.0 mM, and the concentration of polysaccharide-lysing monooxygenase CdLPMO10A is 2.0 μM. The change in absorbance at 469 nm is measured within 300 s at 30°C, and the amount of xylenoid generated is used as the detection standard.

[0036] Enzyme activity is defined as the amount of enzyme that converts 1 μM xylenoid into quinone in 1 minute under standard conditions.

[0037] Results: The enzyme activity of the purified cleaving polysaccharide monooxygenase CdLPMO10A was determined to be 10.64 U / g.

[0038] Example 4: Substrate specificity of the cleaving polysaccharide monooxygenase CdLPMO10A The substrate binding specificity of the cleaving polysaccharide monooxygenase CdLPMO10A was determined using α-chitin, β-chitin, colloidal chitin, chitosan, and cellulose as substrates, respectively. The procedure was as follows: Substrate (10.0 mg / mL) and cleaving polysaccharide monooxygenase CdLPMO10A (3.0 μM) were added to 10 mM pH 6.0 phosphate buffer and incubated at 37 °C and 220 r / min for 3 h. Then, the mixture was centrifuged at 12000 g for 5 min to separate the precipitate and supernatant. The precipitate was washed three times with 10 mM pH 6.0 phosphate buffer. Subsequently, SDS-PAGE protein electrophoresis was used to detect the proteins in the precipitate and supernatant.

[0039] Results: In the precipitates of reaction systems using α-chitin, β-chitin, colloidal chitin, and chitosan as substrates, protein bands with a molecular weight of approximately 53 kDa were detected, while no protein bands were detected in the supernatant. This indicates that the polysaccharide-cleaving monooxygenase CdLPMO10A has binding activity for α-chitin, β-chitin, colloidal chitin, and chitosan. In the precipitates of reaction systems using cellulose as substrates, no protein bands were detected, but a protein band with a molecular weight of approximately 53 kDa was detected in the supernatant. This indicates that the polysaccharide-cleaving monooxygenase CdLPMO10A did not exhibit binding activity for cellulose.

[0040] The β-chitosan was extracted from the squid capsid, and the extraction method was as follows: The squid capsid was repeatedly washed with distilled water to remove surface impurities; after washing, it was dried in an oven at 60°C for 10 hours, pulverized using a pulverizer, and added to hydrochloric acid (1 M concentration), with 1 mg of powder added per 10 ml of hydrochloric acid. The mixture was stirred for 12 hours to remove minerals; the precipitate was filtered and washed with distilled water until neutral. Then, it was added to NaOH solution (2 M concentration) for deproteinization treatment, with 1 mg of precipitate added per 10 ml of NaOH solution. The mixture was stirred at 80°C for 2 hours; the precipitate was filtered and repeatedly washed with distilled water until neutral, then freeze-dried to obtain β-chitosan powder.

[0041] The gelatinous chitin is prepared by acid treatment of α-chitin. The preparation method is as follows: α-chitin powder is added to concentrated hydrochloric acid (concentration 38%), 1 mg of α-chitin powder is added for every 20 ml of concentrated hydrochloric acid, stirred evenly, refrigerated at 4°C for 24 hours, and then sufficient ethanol is added to precipitate the gelatinous chitin. The precipitate is repeatedly washed with distilled water until neutral, and then freeze-dried to obtain gelatinous chitin powder.

[0042] Example 5 Determination of optimal reaction conditions for the cleaving polysaccharide monooxygenase CdLPMO10A (1) Determination of the optimal temperature The polysaccharide-lysing monooxygenase CdLPMO10A (concentration 2.0 μM) was added to 10 mM pH 6.0 phosphate buffer and incubated for 15 min at 20℃, 30℃, 40℃, 50℃, 60℃, and 70℃, respectively. Then, 0.1 mM H2O2 and 1.0 mM 2,6-DMP were added, and the activity was detected using the assay method described in Example 3 (each experiment was repeated three times). The relative enzyme activity at each temperature condition was calculated with the highest enzyme activity as 100%.

[0043] The results of the optimal temperature determination are as follows: Figure 2 As shown, the optimal temperature is 35℃.

[0044] (2) Determination of temperature stability The method is the same as (1) above, except that the incubation time is 24 hours.

[0045] The results of the temperature stability test are as follows: Figure 3 As shown, the temperature stability is highest at 20℃. After incubation at 30-40℃ for 24 hours, the enzyme activity residue is 60%-80%, and after incubation at 50-60℃ for 24 hours, the enzyme activity residue is about 20%.

[0046] (3) Determination of optimal pH The polysaccharide monooxygenase CdLPMO10A (2.0 μM) was added to buffer solutions of different pH values ​​and incubated for 15 min. The buffer solutions used were as follows: citrate buffer at pH 4.0, 5.0, and 6.0; phosphate buffer at pH 6.0, 7.0, and 8.0; Tris-HCl buffer at pH 8.0 and 9.0; and Gly-NaOH buffer at pH 9.0 and 10.0. Then, 0.1 mM H2O2 and 1.0 mM 2,6-DMP were added, and the activity was measured using the method described in Example 3 (each experiment was repeated three times). The relative enzyme activity under each pH condition was calculated with the highest enzyme activity as 100%.

[0047] The results of the optimal pH determination are as follows Figure 4 As shown, the highest enzyme activity is observed at pH 8.0.

[0048] (4) Determination of pH stability The method is the same as above (3), except that the incubation time is 24 hours.

[0049] The results of the pH stability test are as follows: Figure 5 As shown, after 24 hours of incubation, the residual enzyme activity was highest in Tris-HCl buffer at pH 8.0; while the enzyme activity did not change much in phosphate buffer, and the enzyme activity was 90.9% in phosphate buffer at pH 7.0.

[0050] Example 6: Analysis of enzymatic hydrolysis products of different substrates The enzymatic hydrolysis products of the polysaccharide monooxygenase CdLPMO10A were analyzed using α-chitin, β-chitin, chitosan, and cellulose as substrates, respectively.

[0051] Vitamin C, acting as an electron donor, reacts with polysaccharide-cleaving monooxygenase (CdLPMO10A) to generate hydrogen peroxide, providing a stable hydrogen peroxide system for the reaction. The substrate, vitamin C, and CdLPMO10A were added to 10 mM pH 6.0 phosphate buffer. After addition, the concentrations were: substrate 10.0 mg / mL, vitamin C 1.0 mM, and CdLPMO10A 2.0 μM. The reaction was carried out at 37°C and 220 r / min for 24 hours. The reaction was terminated by heating in a boiling water bath for 10 min, followed by centrifugation at 12000 g for 5 min, and the supernatant was retained.

[0052] The supernatant was filtered through a 0.22 μm filter membrane, and the product was detected using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOFMS). The signal in the mass range of 100–3500 Da was monitored in positive ion mode using 2,5-dihydroxybenzoic acid (2,5-DHB) as the matrix.

[0053] The mass spectrum of the enzymatic hydrolysis products when the substrate is α-chitin is shown below. Figure 6 As shown, the mass spectrum of the enzymatic hydrolysis products when the substrate is β-chitin is as follows. Figure 7 As shown, the mass spectrum of the enzymatic hydrolysis products when the substrate is chitosan is as follows. Figure 8 As shown, the mass spectrum of the enzymatic hydrolysis products when the substrate is cellulose is as follows. Figure 9As shown, when the substrate is α-chitin, uronic acids with a degree of polymerization of 2 to 6 are generated; when the substrate is β-chitin, uronic acids with a degree of polymerization of 2 to 6 are generated, similar to the composition of the enzymatic hydrolysis products of α-chitin; however, when the substrate is chitosan or cellulose, no obvious uronic acid generation was detected in the enzymatic hydrolysis products.

[0054] Example 7: Synergistic hydrolysis of polysaccharide-lysing monooxygenase CdLPMO10A and chitinase (1) Stepwise method: α-Chitosan, polysaccharide-lysing monooxygenase CdLPMO10A, and vitamin C were added to 10 mM pH 6.0 phosphate buffer. After addition, the concentration of α-chitosan was 5.0 mg / mL, the concentration of polysaccharide-lysing monooxygenase CdLPMO10A was 3.0 μM, and the concentration of vitamin C was 1.0 mM. The mixture was incubated at 37℃ and 220 rpm for 12 hours, and the reaction of polysaccharide-lysing monooxygenase was terminated by boiling for 20 minutes. Then, chitinase ChiB (concentration 2.0 μM) was added, and the mixture was incubated at 37℃ and 220 rpm for another 12 hours. Polysaccharide-lysing monooxygenase OsLPMO10A was used as a control.

[0055] The cleaving polysaccharide monooxygenase OsLPMO10A and chitinase ChiB are both known enzymes in the prior art, and are described in invention patent CN 116790696 A (the chitinase SmChiB involved in this patent is the chitinase ChiB of this invention). The amino acid sequence of chitinase ChiB is shown in SEQ ID NO.3.

[0056] (2) One-pot method: α-Chitosan, chitinase ChiB, cleaving polysaccharide monooxygenase CdLPMO10A, and vitamin C were added to 10 mM pH 6.0 phosphate buffer. After addition, the concentrations of α-chitosan were 5.0 mg / mL, chitinase ChiB was 2.0 μM, cleaving polysaccharide monooxygenase CdLPMO10A was 3.0 μM, and vitamin C was 1.0 mM. The mixture was incubated at 37℃ and 220 rpm for 24 hours. Cleaving polysaccharide monooxygenase OsLPMO10A was used as a control.

[0057] (3) After the reaction, the reducing sugar content generated by the reaction was determined using the DNS method. The relative enzyme activity was calculated with the highest enzyme activity as 100%. Results: When the cleaving polysaccharide monooxygenase CdLPMO10A was co-hydrolyzed with chitinase ChiB, the relative enzyme activity of the stepwise method was 27.4%, and that of the one-pot method was 85.0%. When the cleaving polysaccharide monooxygenase OsLPMO10A was co-hydrolyzed with chitinase ChiB, the relative enzyme activity of the stepwise method was 51.6%, and that of the one-pot method was 100%. This indicates that the synergistic reaction of the one-pot method is more effective than that of the stepwise method, and that the synergistic effect of the cleaving polysaccharide monooxygenase OsLPMO10A is better than that of the cleaving polysaccharide monooxygenase CdLPMO10A without the addition of hydrogen peroxide.

[0058] Example 8 Determination of optimal H2O2 concentration and H2O2 tolerance (1) Determination of optimal H2O2 concentration: CdLPMO10A (2.0 μM) of lysinic polysaccharide monooxygenase and H2O2 at concentrations of 0 M, 0.05 mM, 0.1 mM, 0.2 mM, 0.5 mM, 2.0 mM, and 5.0 mM were added to 10 mM pH 6.0 phosphate buffer. Then, 2,6-DMP (1.0 mM) was added, and the reaction was carried out at 37°C for 300 s. Enzyme activity was determined according to the method in Example 3. OsLPMO10A of lysinic polysaccharide monooxygenase was used as a control. The relative enzyme activity of each enzyme was calculated with the highest enzyme activity as 100%.

[0059] Results: The determination results of the optimal H2O2 concentration are as follows: Figure 10 As shown, the optimal H2O2 concentration for the cleaving polysaccharide monooxygenase CdLPMO10A is 0.5 mM. In higher concentrations (2.0 mM, 5.0 mM) of H2O2, the activity of the cleaving polysaccharide monooxygenase OsLPMO10A decreased significantly, but the activity of the cleaving polysaccharide monooxygenase CdLPMO10A did not change significantly. This indicates that the cleaving polysaccharide monooxygenase CdLPMO10A has good resistance to high concentrations of H2O2 in the system.

[0060] (2) Determination of H2O2 tolerance: The method is the same as (1) above, except that: after adding different concentrations of H2O2, incubate for 30 min, and then add 1.0 mM of 2,6-DMP. Calculate the relative enzyme activity of each enzyme after incubation for 30 min with the highest enzyme activity of each enzyme in (1) above as 100%.

[0061] Results: The results of the H2O2 tolerance test are as follows: Figure 11As shown, after incubation in a high-concentration (5.0 mM) H2O2 system for 30 min, the relative enzyme activity of the polysaccharide-lysing monooxygenase OsLPMO10A decreased to approximately 50%, while the enzyme activity of the polysaccharide-lysing monooxygenase CdLPMO10A remained relatively stable at 91.1%, with a small decrease. This indicates that the polysaccharide-lysing monooxygenase CdLPMO10A has good resistance to high concentrations of H2O2 in the system.

[0062] Example 9: Degradation of α-chitin by the polysaccharide-lysing monooxygenase CdLPMO10A in combination with chitinase ChiB. Add 3.0 μM of the polysaccharide-lysing monooxygenase CdLPMO10A and 1.0 mM of H2O2 to 10 mM pH 6.0 phosphate buffer and incubate for 24 hours. Then add α-chitin and chitinase ChiB at a concentration of 5.0 mg / mL and 2.0 μM respectively. React at 37 °C and 220 r / min for 12 hours. Terminate the reaction by heating in a boiling water bath for 20 min.

[0063] After the reaction was completed, the content of reducing sugar in the reaction system was measured. Using the enzyme activity of the cleaving polysaccharide monooxygenase CdLPMO10A without hydrogen peroxide incubation as 100%, the relative enzyme activity of the cleaving polysaccharide monooxygenase CdLPMO10A after incubation with 1.0 mM H2O2 for 24 hours was calculated.

[0064] The above procedure was performed using the polysaccharide cleaving monooxygenase OsLPMO10A as a control. The relative enzyme activity of OsLPMO10A after incubation with 1.0 mM H2O2 for 24 hours was calculated, with the enzyme activity of OsLPMO10A without hydrogen peroxide incubation being considered 100%.

[0065] Results: After 24 hours of incubation with 1.0 mM H2O2, the relative enzyme activity of the polysaccharide cleaving monooxygenase CdLPMO10A was 89.3%, with high residual enzyme activity, demonstrating excellent hydrogen peroxide tolerance and better adaptation to prolonged high-concentration H2O2 environments. In contrast, under the same treatment conditions, the relative enzyme activity of the polysaccharide cleaving monooxygenase OsLPMO10A was 52.3%, a significant decrease, indicating poor tolerance to hydrogen peroxide.

[0066] Example 10 Degradation of α-chitin under high concentration of hydrogen peroxide conditions Under laboratory conditions, hydrogen peroxide was added to create a high-concentration hydrogen peroxide environment to simulate the real reaction environment, and a certain amount of vitamin C was added (which will affect the generation and consumption of hydrogen peroxide in the reaction system). The relative enzyme activity of the polysaccharide monooxygenase CdLPMO10A combined with chitinase ChiB in the degradation of α-chitin was determined.

[0067] α-Chitosan, chitinase ChiB, cleaving polysaccharide monooxygenase CdLPMO10A, and vitamin C, along with different concentrations of H2O2, were added to 10 mM pH 6.0 phosphate buffer. After addition, the concentrations of α-chitosan were 5.0 mg / mL, chitinase ChiB was 2.0 μM, cleaving polysaccharide monooxygenase CdLPMO10A was 3.0 μM, vitamin C was 1.0 mM, and H2O2 concentrations were 0 mM, 1.0 mM, 2.0 mM, 5.0 mM, 10.0 mM, and 15.0 mM, respectively. The mixture was incubated at 37℃ and 220 rpm for 12 hours. The reaction was terminated by heating in a boiling water bath for 20 min.

[0068] After the reaction was completed, the content of reducing sugar in the reaction system was measured. Using the enzyme activity of the polysaccharide-cleaving monooxygenase CdLPMO10A at a hydrogen peroxide concentration of 0% as 100%, the relative enzyme activities of CdLPMO10A combined with chitinase ChiB for the degradation of α-chitin under different hydrogen peroxide concentrations were calculated.

[0069] The above procedures were performed using the polysaccharide-cleaving monooxygenase OsLPMO10A as a control. The enzyme activity of OsLPMO10A at a hydrogen peroxide concentration of 0% was taken as 100%, and the relative enzyme activity of OsLPMO10A combined with chitinase ChiB for the degradation of α-chitin under different hydrogen peroxide concentrations was calculated.

[0070] Results: The relative enzyme activity was measured under different concentrations of hydrogen peroxide as follows: Figure 12 As shown, after reacting for 12 hours under high-concentration hydrogen peroxide conditions, the relative activity of the polysaccharide-lysing monooxygenase CdLPMO10A was significantly increased. Under 10 mM hydrogen peroxide, the relative activity increased to 283.4%, and under 15 mM hydrogen peroxide, it increased to 174.4%, demonstrating excellent tolerance to high concentrations of hydrogen peroxide. In contrast, under the same treatment conditions, the relative activity of the polysaccharide-lysing monooxygenase OsLPMO10A was 50.6% under 2 mM hydrogen peroxide and 21.7% under 10 mM hydrogen peroxide, showing a significant decrease and poor tolerance to hydrogen peroxide.

[0071] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A method for degrading chitin resistant to hydrogen peroxide, characterized in that: α-Chitin, chitinase ChiB, cleaving polysaccharide monooxygenase CdLPMO10A, vitamin C, and hydrogen peroxide were added to 10 mM pH 6.0 phosphate buffer and cultured in a shaker at 37°C and 220 rpm for 12 hours. Under high concentration of hydrogen peroxide, chitin was degraded by cleaving polysaccharide monooxygenase CdLPMO10A and chitinase ChiB. The high concentration hydrogen peroxide condition refers to a hydrogen peroxide concentration of 1.0 mM to 15.0 mM; The concentration of α-chitosan was 5.0 mg / mL, the concentration of chitinase ChiB was 2.0 μM, the concentration of polysaccharide monooxygenase CdLPMO10A was 3.0 μM, and the concentration of vitamin C was 1.0 mM. The amino acid sequence of the cleaving polysaccharide monooxygenase CdLPMO10A is shown in SEQ ID NO.1; The amino acid sequence of the chitinase ChiB is shown in SEQ ID NO.

3.

2. The method for degrading chitin to withstand hydrogen peroxide according to claim 1, characterized in that: The high concentration of hydrogen peroxide conditions refers to a hydrogen peroxide concentration of 2.0 mM to 15 mM.

3. The method for degrading chitin to withstand hydrogen peroxide according to claim 2, characterized in that: The high concentration of hydrogen peroxide conditions refers to a hydrogen peroxide concentration of 5.0 mM to 15 mM.

4. The method for degrading chitin to withstand hydrogen peroxide according to claim 3, characterized in that: The high concentration of hydrogen peroxide condition refers to a hydrogen peroxide concentration of 10 mM to 15 mM.

5. The method for degrading chitin to withstand hydrogen peroxide according to claim 2, characterized in that: The high concentration hydrogen peroxide condition refers to a hydrogen peroxide concentration of 5.0 mM, 10 mM, or 15 mM.

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