Lytic polysaccharide monooxygenase slpmo10 and use thereof

Abstract

The application discloses a kind of lytic polysaccharide monooxygenase SLPMO10 and application thereof, belong to functional enzyme technical field.SLPMO10 amino acid sequence is as shown in SEQ ID NO.1, the nucleotide sequence of its encoding gene is as shown in SEQ ID NO.2.The enzyme belongs to AA10 family, optimum reaction temperature is 60 DEG C, optimum pH is 8.0, by C1 oxidation mode cleavage cellulose, and specifically produce polymeric degree 6 and 8 chitooligosaccharide acid.SLPMO10 and endoglucanase or complex cellulase synergistic effect, can significantly improve the degradation efficiency of cellulose;Especially with complex cellulase synergistic treatment seaweed powder, reducing sugar yield increases 23.5%.The application has important industrial application value in enzyme method treatment cellulose and seaweed biomass conversion.

Description

Technical Field

[0001] This invention relates to the cleavage polysaccharide monooxygenase SLPMO10 and its applications, belonging to the field of functional enzyme technology. Background Technology

[0002] Cellulose is the most abundant organic polymer on Earth, widely found in plants, algae, and other organisms. The breakdown of cellulose is one of the most important reactions in nature and is central to the conversion of biomass fuels and chemicals. Although cellulose is composed entirely of glucose residues, its crystalline microfibrillary structure and its binding with lignin and hemicellulose make it highly resistant to enzymatic degradation.

[0003] Recent studies have discovered a class of cleaving polysaccharide monooxygenases (LPMOs) that can disrupt the polymeric structure of insoluble polysaccharides through redox reactions. These LPMOs primarily work by cleaving glycosidic bonds in crystalline regions, loosening the substrate structure and making the crystalline regions amorphous, thereby promoting polysaccharide depolymerization. The significant advantage of LPMOs in depolymerizing resistant polysaccharides provides a new approach for the enzymatic breakdown of cellulose.

[0004] The synergistic effect of LPMO and glycoside hydrolases depends on the type of enzyme, substrate, and synergistic mechanism. Current research is largely limited to verifying the synergistic effect of LPMO and hydrolases, and mainly focuses on the conversion of cellulose biomass from terrestrial plants. For example, patent CN120536525A studied the synergistic enzymatic hydrolysis of corn straw by red pleurotus erythrorhizon polysaccharide-cleaving monooxygenases PdLPMO9A and PdLPMO9B and cellulase, increasing glucose content by approximately 31%; patent CN113832121A studied the 28% increase in the efficiency of sugarcane bagasse degradation by NeLPMO10A and commercial enzymes. Unlike terrestrial plants, cellulose in seaweed is often intertwined with marine characteristic polysaccharides such as alginate and mannitol, resulting in a more complex crystalline structure, which places different demands on the substrate compatibility and synergistic mechanism of LPMO. A search reveals that publicly available reports on the degradation of LPMO and its applications in marine biomass such as seaweed are currently limited.

[0005] Therefore, exploring LPMOs with high cellulose cleavage activity and suitable for seaweed degradation is particularly important for the conversion of marine-derived biomass. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention aims to provide a novel cleavable polysaccharide monooxygenase SLPMO10, its encoding gene, recombinant vector, recombinant engineered bacteria, and its application in the degradation of cellulose and seaweed, thereby solving the problems of limited application of LPMO in the degradation of marine biomass (such as seaweed) and insufficient thermal stability in existing technologies.

[0007] The technical solution adopted in this invention is as follows: In a first aspect, the present invention provides a cleavable polysaccharide monooxygenase SLPMO10, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] Furthermore, the cleaving polysaccharide monooxygenase SLPMO10 is derived from Streptomyces ( ). Streptomyces sp. It belongs to the 10th family of co-active compounds (AA10).

[0009] In a second aspect, the present invention provides a gene encoding the cleavable polysaccharide monooxygenase SLPMO10 described in the first aspect, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0010] Furthermore, the nucleotide sequence is an optimized sequence based on the codon preference of the host cell, and the host cell is Escherichia coli.

[0011] Thirdly, the present invention provides a recombinant expression vector carrying the gene encoding the cleaving polysaccharide monooxygenase SLPMO10 as described in the second aspect.

[0012] Furthermore, the recombinant expression vector is pET22b-SLPMO10.

[0013] Fourthly, the present invention provides a recombinant engineered bacterium whose genome contains the gene encoding the cleaving polysaccharide monooxygenase SLPMO10 as described in the second aspect.

[0014] Furthermore, the recombinant engineered bacteria is E. coli BL21(DE3) / SLPMO10.

[0015] Fifthly, the present invention provides the application of the cleaving polysaccharide monooxygenase SLPMO10 described in the first aspect in the degradation of seaweed, wherein the degradation is a synergistic degradation of seaweed powder with a complex cellulase.

[0016] Further, the degradation conditions are as follows: seaweed powder concentration of 5.0~20.0 mg / mL, SLPMO10 enzyme dosage of 0.05~1.0 µM, compound cellulase dosage of 0.1~1.0 mg / mL, reaction temperature of 45~55℃, pH of 5.5~6.5, and reaction time of 6~24 h.

[0017] Furthermore, the degradation conditions are as follows: seaweed powder concentration 10.0 mg / mL, SLPMO10 enzyme dosage 0.1 µM, compound cellulase enzyme dosage 0.4 mg / mL, temperature 50℃, pH 6, and reaction time 12 h.

[0018] In a sixth aspect, the present invention provides the application of the cleaving polysaccharide monooxygenase SLPMO10 described in the first aspect in the cleavage of cellulose.

[0019] Furthermore, cellulose is cleaved to obtain cellulose oligosaccharides with degrees of polymerization of 6 and 8, namely cellulose hexasaccharide and cellulose octasaccharide.

[0020] In a seventh aspect, the present invention provides the application of the cleaving polysaccharide monooxygenase SLPMO10 described in the first aspect in the synergistic degradation of cellulose, wherein the cleaving polysaccharide monooxygenase SLPMO10 works synergistically with endoglucanase or complex cellulase.

[0021] Preferably, the substrate for degradation is microcrystalline cellulose, and the degradation conditions are as follows: microcrystalline cellulose concentration 5.0~10.0 mg / mL, SLPMO10 enzyme dosage 0.5~5 µM, endoglucanase dosage 5 µM or complex cellulase dosage 0.4 mg / mL, temperature 50℃, pH 6, and reaction time 12 h.

[0022] In an eighth aspect, the present invention provides an enzyme preparation comprising the cleaving polysaccharide monooxygenase SLPMO10 described in the first aspect.

[0023] Furthermore, the enzyme preparation also contains pharmaceutically or industrially acceptable carriers, auxiliaries, or excipients.

[0024] The present invention also provides the application of the recombinant expression vector described in the third aspect or the recombinant engineered bacteria described in the fourth aspect in the conversion of fibrous biomass. The present invention also provides a method for degrading seaweed, comprising co-treating seaweed powder with the cleaving polysaccharide monooxygenase SLPMO10 described in the first aspect and a complex cellulase.

[0025] Further, the treatment conditions are as follows: seaweed powder concentration is 5.0~20.0 mg / mL, SLPMO10 enzyme dosage is 0.05~1.0 µM, compound cellulase dosage is 0.1~1.0 mg / mL, reaction temperature is 45~55℃, pH is 5.5~6.5, and reaction time is 6~24 h.

[0026] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects: (1) A novel AA10 family LPMO is provided: This invention expresses and studies for the first time the lysogenic polysaccharide monooxygenase SLPMO10 derived from Streptomyces, enriching the enzyme resource library of LPMO.

[0027] (2) Excellent enzymatic properties and good thermal stability: The optimal reaction temperature of SLPMO10 is 60℃ and the optimal pH is 8. It has good thermal stability and acid and alkali tolerance.

[0028] (3) Clear pyrolysis products: MALDI-TOF-MS results showed that SLPMO10 pyrolyzes cellulose via C1 oxidation, specifically producing cellulose oligosaccharides with polymerization degrees of 6 and 8.

[0029] (4) Significant synergistic degradation of cellulose: SLPMO10 and endoglucanase Cel5A synergistically degrade microcrystalline cellulose, increasing the yield of reducing sugar by 33.5%; and synergistically degrade microcrystalline cellulose with complex cellulase, increasing the yield of reducing sugar by 19.5%.

[0030] (5) Filling the gap in the application of LPMO for seaweed degradation: There are few public reports on LPMO for the degradation of marine biomass such as seaweed and its applications. This invention is the first to apply SLPMO10 to seaweed degradation, and it works in synergistically with compound cellulase to treat seaweed powder, increasing the yield of reducing sugar by 23.5%.

[0031] (6) High industrial application value: The SLPMO10 of the present invention has important industrial application value and economic value in the enzymatic treatment of cellulose and seaweed biomass conversion. Attached Figure Description

[0032] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0033] Figure 1 SDS-PAGE electrophoresis image of purified polysaccharide monooxygenase SLPMO10, where M is the standard protein marker, lane 1 is the crude enzyme solution, lane 2 is the pure enzyme SLPMO10, and lane 3 is the concentrated pure enzyme SLPMO10.

[0034] Figure 2 Schematic diagram of the effect of temperature change on relative enzyme activity.

[0035] Figure 3 Schematic diagram of the effect of pH change on relative enzyme activity.

[0036] Figure 4 : Matrix-assisted time-of-flight mass spectra of the enzymatic hydrolysis products, where DP6ox and DP8ox represent the C1 oxidation products cellulose hexasaccharide (degree of polymerization 6) and cellulose octasaccharide (degree of polymerization 8), respectively.

[0037] Figure 5: The effect of synergistic degradation of cellulose by polysaccharide monooxygenase SLPMO10 and endoglucanase Cel5A. The error bars represent the standard deviation of three replicates.

[0038] Figure 6 : Graph showing the effect of synergistic degradation of cellulose by cleaving polysaccharide monooxygenase SLPMO10 and complex cellulase.

[0039] Figure 7 : Effect of the synergistic degradation of seaweed by the polysaccharide monooxygenase SLPMO10. Detailed Implementation

[0040] 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 premise of the invention.

[0041] 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.

[0042] Example 1 Cloning of the gene encoding the polysaccharide monooxygenase SLPMO10 The inventors of this application discovered Streptomyces from the NCBI database. Streptomyces sp. The polysaccharide cleavage monooxygenase of NPDC046203 (GenBank accession number: WP_361238814.1).

[0043] Phylogenetic analysis with known LPMOs confirmed that the protein belongs to family 10 of accessory activity (AA10), and it is named SLPMO10, a cleaving polysaccharide monooxygenase. This invention is the first to express, purify and study this enzyme.

[0044] The inventors optimized the gene sequence based on the codon preference of the host *E. coli* for efficient expression in *E. coli*. The optimized gene encoding the cleavable polysaccharide monooxygenase SLPMO10 contains 1020 bases, as shown in SEQ ID NO.2, and encodes 340 amino acids, as shown in SEQ ID NO.1 (signal peptide removed).

[0045] The gene encoding the cleavage polysaccharide monooxygenase SLPMO10 shown in SEQ ID NO.2 was artificially synthesized.

[0046] Example 2 Construction of recombinant expression vector Using the artificially synthesized gene fragment shown in SEQ ID NO.2 from Example 1 as a template, and employing the upstream primer SLPMO10-F (sequence: 5'-GATGGC)... CCATGG TGTTGCGATGGCAC-3' (underlined is the NcoI restriction site, as shown in SEQ ID NO. 4) and downstream primer SLPMO10-R (sequence: 5'-GTGGTG CTCGAG PCR amplification was performed using ACTAGCTGTACAACTCACGCTAC-3' (the underlined part indicates the XhoI restriction site, as shown in SEQ ID NO. 5). The PCR reaction conditions were: 95°C pre-denaturation for 3 min; 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min, for a total of 30 cycles; and a final extension at 72°C for 10 min.

[0047] After separation by 1% agarose gel electrophoresis, the PCR products were purified using an OMEGA Gel Extraction Kit. Simultaneously, the pET-22b(+) empty vector was double-digested with NcoI and XhoI, and the digestion products were also purified using gel extraction. The purified PCR products were mixed with the linearized pET-22b(+) vector at a molar ratio of 3:1, and seamless cloning was performed using a homologous recombinase (ClonExpress II One Step Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd.) at 50°C for 15 min.

[0048] Take 5 µL of the ligation product and convert the ligation product to... E. coli In DH5α competent cells, the cells were incubated on ice for 30 min, heat-shocked at 42°C for 90 s, immediately incubated on ice for 2 min, and then thawed in 500 µL of LB liquid medium at 37°C and 200 rpm for 1 h. The thawed bacterial culture was spread onto LB agar plates containing 100 µg / mL ampicillin and incubated at 37°C for 16 hours. Single colonies were then picked and cultured in LB liquid medium containing 100 µg / mL ampicillin at 37°C for 12 hours using a shaker at 220 rpm. Plasmids were extracted using a plasmid miniprep kit and verified by double enzyme digestion (NdeI and XhoI). Electrophoresis of the digestion products showed that plasmids releasing a target band of approximately 1020 bp were positive recombinant plasmids. The positive recombinant plasmids were sent to a sequencing company for sequencing verification. The sequencing results showed that the inserted fragment sequence was completely identical to SEQ ID NO.2. The successfully validated recombinant plasmid was named pET22b-SLPMO10 and stored at -20℃ for later use.

[0049] Example 3 Construction of recombinant engineered bacteria The recombinant plasmid pET22b-SLPMO10, which was constructed and sequenced correctly in Example 2, was transformed into the expression host cell via heat shock. E. coli Transformation in BL21 competent cells. The transformation steps are as follows: Add 1 µL of plasmid (about 50 ng) to 50 µL of BL21(DE3) competent cells thawed in an ice bath, mix gently, and incubate on ice for 30 min; heat shock in a water bath at 42℃ for 90 s, then immediately incubate on ice for 2 min; add 500 µL of LB liquid medium, and revive and culture at 37℃ and 200 rpm for 1 h.

[0050] Take 100 µL of the revived bacterial culture and spread it onto an LB agar plate containing 100 µg / mL ampicillin. Incubate overnight at 37°C with the plate inverted. The next day, pick a single colony growing on the plate and inoculate it into LB liquid medium containing 100 µg / mL ampicillin. Extract the plasmid and perform double enzyme digestion verification to confirm successful transformation. The verified positive clone is the recombinant engineered bacteria, named [name missing]. E. coli BL21(DE3) / pET22b-SLPMO10. This engineered strain was added to a final concentration of 15% glycerol and stored at -80°C for later use.

[0051] Example 4 Preparation of the cleaving polysaccharide monooxygenase SLPMO10 Recombinant Escherichia coli was activated in 5 mL LB liquid medium (containing 100 µg / mL ampicillin) and then inoculated at a rate of 1% into LB medium containing 100 µg / mL ampicillin. The culture was carried out at 37°C and 200 rpm for 4 h. When the OD (600) value of the bacterial culture was 0.6, 1‰ IPTG (100 mM, final concentration 0.1 mM) was added and the culture was induced at 20°C for 16 h to express cleaving polysaccharide monooxygenase.

[0052] After fermentation, the fermentation broth was centrifuged at 8000 g for 10 min, and the cells were collected. The cells were resuspended in 50 mM pH 8.0 Tris-HCl buffer and then sonicated in an ice-water bath at 200 W for 3 s on and 3 s off for a total of 25 min. The cells were then centrifuged again at 8000 g for 10 min, and the supernatant was collected as the crude enzyme solution.

[0053] Based on His-tag fusion, the crude enzyme solution was purified by affinity chromatography using a Ni-NTA column. The column was equilibrated with a low concentration of 10 mM imidazole solution (10 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl), followed by elution of weakly binding contaminating proteins with 20 mM imidazole solution (20 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl). The target protein was then eluted with 120 mM imidazole solution (120 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl). This eluent was collected to obtain a purified solution of recombinant cleavable polysaccharide monooxygenase. Protein purity and molecular weight were determined by SDS-PAGE (results shown in Figure 1). Figure 1 (As shown) Protein concentration was determined using the Bradford method. Results showed that the recombinant protein, after affinity column purification, yielded electrophoretically purified protein with a molecular weight of approximately 38 kDa, comparable to the cleavable polysaccharide monooxygenase SLPMO10. The imidazole in the eluent was replaced with water, and the purified protein was mixed with 3 molar amounts of copper sulfate solution and incubated at 4°C for 30 min. Excess copper ions were then removed by ultrafiltration using a 10 kDa molecular weight cutoff tube, yielding copper-saturated SLPMO10 pure enzyme for later use.

[0054] Example 5: Determination of the specific enzyme activity of the cleaving polysaccharide monooxygenase SLPMO10 The standard method for determining the activity of the polysaccharide monooxygenase SLPMO10 is as follows: The total reaction volume is 200 µL, containing: 50 mM phosphate buffer (pH 6), 0.1 mM H2O2, and 1 mM 2,6-DMP. The reaction solution is incubated at 50 °C for 10 min, then the purified SLPMO10 enzyme prepared in Example 4 (final concentration 2.5 µM) is added, and the absorbance change at 469 nm within 300 s is measured using a microplate reader. The control reaction uses an equal volume of inactivated enzyme (boiled for 10 min) instead of the active enzyme.

[0055] Enzyme activity unit (U) is defined as the amount of enzyme (using 2,6-DMP as substrate) required to cause an increase of 0.001 in absorbance at 469 nm per minute under conditions of 50°C and pH 6.0.

[0056] Example 6 Determination of optimal reaction conditions The effects of temperature and pH on the activity of SLPMO10 enzyme were determined using the method described in Example 5.

[0057] Determination of optimal temperature: At temperatures of 20℃, 30℃, 40℃, 50℃, 60℃, and 70℃, the reaction components (buffer, H2O2, 2,6-DMP) except for the enzyme were pre-incubated for 5 min at each test temperature. Then, the purified SLPMO10 enzyme prepared in Example 4 (final concentration 2.5 µM) was added to initiate the reaction, and enzyme activity was measured according to the method in Example 5. The relative enzyme activity at each temperature was calculated with the highest enzyme activity defined as 100%. The results are as follows: Figure 2 As shown, the optimal reaction temperature for SLPMO10 is 60℃.

[0058] Determination of optimal pH: The effect of different pH values ​​on enzyme activity was determined at 60°C. Buffer solutions with pH values ​​ranging from 4.0 to 10.0 were selected as the different pH buffers for the reaction. Enzyme activity was determined according to the method in Example 5, and the relative enzyme activity at each pH was calculated with the highest enzyme activity as 100%. The results are as follows: Figure 3 As shown, the optimal reaction pH for SLPMO10 is 8.0 (phosphate buffer).

[0059] Selection of reaction temperature: Since SLPMO10 needs to be used in conjunction with cellulase (the optimal reaction temperature is about 50℃) in practical applications, and long-term reaction at 60℃ may lead to a decrease in enzyme activity, 50℃ was selected as the reaction temperature for subsequent co-degradation experiments.

[0060] Example 7: Determination of degradation products of the cleaving polysaccharide monooxygenase SLPMO10 Add microcrystalline cellulose and ascorbic acid to 10 mM phosphate buffer (pH 8.0), vortex thoroughly to suspend the microcrystalline cellulose evenly, and mix well to obtain the substrate solution. The final concentration of microcrystalline cellulose is 20 mg / mL, and the final concentration of ascorbic acid is 1 mM.

[0061] The purified SLPMO10 enzyme from Example 4 was added to the above substrate solution (final concentration of SLPMO10 was 2.5 µM, total reaction volume was 500 µL), and the reaction was carried out at 50 °C for 24 h.

[0062] After the reaction was completed, 1 μL of the reaction solution was mixed with 1 µL of 2,5-dihydroxybenzoic acid (2,5-DHB) matrix solution (10 mg / mL, dissolved in 50 v / v% acetonitrile / 0.1 v / v% trifluoroacetic acid), and the enzymatic hydrolysis products were detected using matrix-assisted laser desorption / ionization mass spectrometry (positive ion reflectance mode, mass scan range m / z 500-2000).

[0063] The results are as follows Figure 4As shown, the main ion peaks in the mass spectrum correspond to the C1 oxidation products cellulose hexasaccharide (DP6) and cellulose octasaccharide (DP8), respectively, indicating that the cleaving polysaccharide monooxygenase SLPMO10 cleaves cellulose via C1 oxidation, specifically producing cellulose oligosaccharide acids with a degree of polymerization of 6 and 8 (no products with other degrees of polymerization were detected).

[0064] Example 8: Synergistic degradation of cellulose by the polysaccharide monooxygenase SLPMO10 and endoglucanase. The purified SLPMO10 enzyme prepared in Example 4 and the endoglucanase Cel5A were simultaneously added to the substrate solution (10 mM phosphate buffer (pH 8.0) containing 20 mg / mL microcrystalline cellulose and 1 mM ascorbic acid, prepared in Example 7). In the reaction system, the final concentrations of SLPMO10 and Cel5A were 5 µM, and the total reaction volume was 500 μL. The reaction was carried out at 50 °C for 12 h. Simultaneously, under the same reaction time and conditions, Cel5A (5 µM) was added alone as a control for comparison.

[0065] After the reaction, the reducing sugar content was determined using the DNS (3,5-dinitrosalicylic acid) method: 100 µL of enzymatic hydrolysate was mixed with 100 µL of DNS reagent, boiled in a water bath for 5 min, and the absorbance was measured at 540 nm after cooling. A standard curve was prepared using glucose as a standard, and the reducing sugar yield (mmol / L) was calculated. Each experimental group was tested in triplicate, and the results are expressed as mean ± standard deviation.

[0066] Endoglucanase Cel5A comes from Cellulomonas bogoriensis 69B4 T It encodes 599 amino acids (the mature protein with the signal peptide removed), as shown in SEQ ID NO.3.

[0067] The enzyme preparation Cel5A was prepared according to the genetic engineering method described in the literature (AMB Express. 2020 Mar 10;10(1):44.).

[0068] The results are as follows Figure 5 As shown, the amount of reducing sugar produced by the synergistic group with the addition of 5 µM SLPMO10 increased by 33.5% compared with the addition of Cel5A alone, indicating that SLPMO10 can significantly improve the degradation ability of endoglucanase on cellulose.

[0069] Example 9: Synergistic degradation of cellulose by the cleaving polysaccharide monooxygenase SLPMO10 and a complex cellulase. The SLPMO10 purified enzyme prepared in Example 4 and the compound cellulase were simultaneously added to the substrate solution (10 mM phosphate buffer (pH 8.0) containing 20 mg / mL microcrystalline cellulose and 1 mM ascorbic acid prepared in Example 7). Different concentrations of SLPMO10 were set: 0.1 µM, 0.25 µM, 0.5 µM, 1 µM, 2 µM, and 5 µM. The concentration of the compound cellulase was 0.4 mg / mL, and the total reaction volume was 500 μL. The reaction was carried out at 50 °C for 12 h. Simultaneously, under the same reaction time and conditions, the addition of the compound cellulase alone (0.4 mg / mL) was used as a control for comparison. The reducing sugar content was determined according to the DNS method in Example 8. Each experimental group was repeated three times, and the results are expressed as mean ± standard deviation.

[0070] The compound cellulase preparation (product model: Cel L-15, sourced from Qingdao Weilan Biotechnology Co., Ltd.) has an enzyme activity of 4000 FPU / g. According to the product instructions, this compound cellulase mainly contains exoglucanase, endoglucanase, and β-glucosidase.

[0071] The results are as follows Figure 6 As shown, when the amount of SLPMO10 added was 0.5 µM, the amount of reducing sugar produced by the synergistic group in the degradation of cellulose was increased by 19.5% compared with the use of the composite cellulase alone, indicating that the polysaccharide monooxygenase SLPMO10 can significantly improve the degradation ability of the composite cellulase on microcrystalline cellulose.

[0072] Example 10: Synergistic degradation of seaweed by the cleaving polysaccharide monooxygenase SLPMO10 and a complex cellulase. Preparation of seaweed powder: Giant kelp Lessonia nigrescens (LN) The sample was obtained from Qingdao Mingyue Seaweed Group. The giant kelp was washed with distilled water to remove salt, dried at 60°C to constant weight, pulverized using a pulverizer, and passed through a 60-mesh sieve (pore size 250 µm). The powder that passed through the sieve was collected and sealed for later use.

[0073] Enzymatic hydrolysis: The substrate solution was a 50 mM phosphate buffer (pH 6.0) containing 20 mg / mL seaweed powder and 1 mM ascorbic acid. The seaweed powder was thoroughly vortexed to ensure uniform suspension. The SLPMO10 purified enzyme prepared in Example 4 and the composite cellulase were simultaneously added to the substrate solution. Different SLPMO10 concentrations were set: 0.1 µM, 0.25 µM, 0.5 µM, and 1 µM. The composite cellulase concentration was 0.4 mg / mL. The total reaction volume was 500 μL, and the reaction was carried out at 50 °C for 12 h. Simultaneously, under the same reaction time and conditions, the composite cellulase alone (0.4 mg / mL) was used as a control for comparison. The reducing sugar content was determined according to the DNS method in Example 8. Each experimental group was repeated three times, and the results are expressed as mean ± standard deviation.

[0074] The compound cellulase preparation used is the same as in Example 9.

[0075] Result: As Figure 7 As shown, when the amount of SLPMO10 added was 0.1 µM, the amount of reducing sugar produced by the synergistic degradation of seaweed powder increased by 23.5% compared with the use of compound cellulase alone, indicating that the cleaving polysaccharide monooxygenase SLPMO10 has great potential in synergistic degradation of seaweed substrates with other enzymes.

[0076] In summary, the cleavable polysaccharide monooxygenase SLPMO10 of the present invention can synergistically work with endonucleases or complex cellulases to improve the degradation efficiency of microcrystalline cellulose; at the same time, SLPMO10 of the present invention has greater application potential in the synergistic degradation of marine-derived biomass such as algae.

[0077] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. SLPMO10, a polysaccharide-lysing monooxygenase, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

1.

2. The gene encoding the cleaving polysaccharide monooxygenase SLPMO10 of claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that, Carrying the gene as described in claim 2.

4. A recombinant engineered bacterium, characterized in that, The genome contains the gene described in claim 2.

5. The application of the cleaving polysaccharide monooxygenase SLPMO10 according to claim 1 in the degradation of seaweed, characterized in that, The degradation process involves the synergistic degradation of seaweed powder with a complex cellulase.

6. The application according to claim 5, characterized in that, The degradation conditions were as follows: seaweed powder concentration of 5.0~20.0 mg / mL, SLPMO10 enzyme dosage of 0.05~1.0 µM, compound cellulase dosage of 0.1~1.0 mg / mL, reaction temperature of 45~55℃, pH of 5.5~6.5, and reaction time of 6~24 h.

7. The application of the cleaving polysaccharide monooxygenase SLPMO10 of claim 1 in cleaving cellulose.

8. The application according to claim 7, characterized in that, Cellulose was cleaved to obtain cellulose oligosaccharides with degrees of polymerization of 6 and 8.

9. The application of the cleaving polysaccharide monooxygenase SLPMO10 according to claim 1 in the synergistic degradation of cellulose, characterized in that, The cleaving polysaccharide monooxygenase SLPMO10 works synergistically with endoglucanase or complex cellulase.

10. An enzyme preparation, characterized in that, It contains the cleaving polysaccharide monooxygenase SLPMO10 as described in claim 1.

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