Cracking polysaccharide monooxygenase mutant and cellulose synergistic hydrolysis method

Abstract

The invention relates to a lytic polysaccharide monooxygenase mutant and a cellulose synergistic hydrolysis method. The invention belongs to the technical field of biology, aims to solve the problems of low efficiency and high cost of the traditional cellulose enzymolysis technology, and provides a mutant with higher catalytic activity and stability by performing site-specific mutagenesis on the second site of an amino acid sequence shown in SEQ ID No: 1, so that the cellulose hydrolysis efficiency is remarkably improved, and the cost is reduced. The method has wide application prospects in the fields of biological energy, papermaking, spinning and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology. More particularly, the present application relates to a lytic polysaccharide monooxygenase mutant, a DNA molecule, a recombinant expression vector, a host cell, a method for producing the lytic polysaccharide monooxygenase mutant, application of the lytic polysaccharide monooxygenase mutant in synergistic hydrolysis of cellulose, and a method for synergistic hydrolysis of cellulose. BACKGROUND

[0002] Lignocellulose such as straw is the most abundant renewable resource in nature, which can be degraded into six-carbon sugars and five-carbon sugars and other products by cellulase, and can further produce a variety of bio-based products including bioethanol. However, the cost of enzymatic degradation of lignocellulose accounts for about 1 / 4 of the cost of biofuels, so it is crucial to develop efficient lignocellulose-degrading enzymes and to engineer them to promote the benefits of lignocellulosic biomass. Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-dependent enzymes that can oxidize and cleave polysaccharides. LPMOs are key enzymes for synergistic cellulase degradation of lignocellulose, and the improvement of their performance is crucial to the overall hydrolysis effect of cellulase. How to accurately select mutation sites and predict the effect of mutations on enzyme function is the main content of the present application. SUMMARY

[0003] One object of the present application is to provide a lytic polysaccharide monooxygenase mutant. By site-directed mutagenesis at position 2 of the amino acid sequence shown in SEQ ID No: 1, the obtained LPMOs mutant has higher catalytic activity and hydrolysis effect, improves the catalytic reaction efficiency and product yield, and is more suitable for industrial application, which can significantly reduce the cost of enzymatic process.

[0004] Another object of the present application is to provide a DNA molecule. By codon optimization and sequence design, the problem of low gene expression efficiency in heterologous hosts is solved, and high-efficiency transcription and translation of the LPMOs mutant in the engineering bacteria are realized.

[0005] Another object of the present application is to provide a recombinant expression vector. By constructing an operable linkage structure comprising the DNA molecule and a high-efficiency expression regulatory element, stable transcription and induced expression of the LPMOs mutant gene in the host cell are ensured, and the expression amount and solubility of the recombinant protein are improved.

[0006] Another object of the present application is to provide a host cell. By screening suitable microbial hosts and introducing the above-mentioned recombinant expression vector, the problems of folding error and low secretion efficiency of natural LPMOs in traditional hosts are solved, and high-biomass expression and functional folding of the mutant enzyme are realized.

[0007] The present application also aims to provide a method for producing LPMOs, which significantly improves the yield and purity of LPMOs mutants, and meets the quality and cost requirements of enzyme preparations for industrial production.

[0008] The present application also aims to provide an application, which constructs an efficient enzymatic hydrolysis system by utilizing the synergistic mechanism of LPMOs mutants and cellulases, and improves the cellulose degradation efficiency.

[0009] The present application also aims to provide a method for synergistically hydrolyzing cellulose, which solves the problems of low efficiency and high cost of traditional cellulase hydrolysis technology by optimizing the synergistic conditions of mutant enzymes and other cellulases, and realizes the efficient conversion of lignocellulosic biomass.

[0010] In order to achieve these objects and other advantages according to the present application, a lytic polysaccharide monooxygenase mutant is provided, which is substituted with alanine at the glycine at position 2 of the amino acid sequence shown in SEQ ID No: 1, and the amino acid sequence of the lytic polysaccharide monooxygenase mutant is shown in SEQ ID No: 3.

[0011] A DNA molecule encoding the lytic polysaccharide monooxygenase mutant, the nucleotide sequence of which is shown in SEQ ID No: 4.

[0012] A recombinant expression vector containing the DNA molecule and a regulatory sequence for expression operably linked to the DNA molecule.

[0013] Preferably, the recombinant expression vector is pPICZ alpha A.

[0014] A host cell containing the DNA molecule or the recombinant expression vector, which is a non-animal and non-plant cell.

[0015] Preferably, the host cell is a Pichia pastoris cell.

[0016] A method for producing the lytic polysaccharide monooxygenase mutant, comprising the following steps: Culturing the host cell in a culture medium to induce the expression of the lytic polysaccharide monooxygenase mutant by the recombinant expression vector.

[0017] The lytic polysaccharide monooxygenase mutant, the DNA molecule, the recombinant expression vector, and the host cell in the application of synergistically hydrolyzing cellulose with cellulases.

[0018] The cellulose synergistic hydrolysis method comprises adding the lytic polysaccharide monooxygenase mutant and cellulase into cellulose for enzymolysis.

[0019] The present application at least includes the following beneficial effects: The enzyme provided by the present application has good cellulose degradation function, and the cellulose synergistic hydrolysis effect can be improved by 2.8 times. The improvement of the hydrolysis efficiency can reduce the enzyme usage amount for hydrolyzing a certain amount of reducing sugar in a unit of time, improve the role of balancing the enzyme system in enzyme system compounding, and improve the overall hydrolysis rate, and has wide application prospect.

[0020] Other advantages, objects and features of the present application will be apparent from the following description, and will be understood by those skilled in the art through the study and practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The figure is a gel electrophoresis chart of the lytic polysaccharide monooxygenase and its mutant expressed by the present application; Figure 2 The figure is a column chart of the hydrolysis of microcrystalline cellulose by the mutant produced by the second mutation of the lytic polysaccharide monooxygenase of the present application. DETAILED DESCRIPTION

[0022] The present application will be further described in detail below with reference to the accompanying drawings, so that those skilled in the art can implement the present application according to the description.

[0023] It should be understood that the terms such as "have", "contain" and "include" used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0024] It should be noted that the experimental methods in the following embodiments are all conventional methods, and the reagents and materials can be obtained from commercial channels unless otherwise specified, so they cannot be understood as limiting the present application.

[0025] 1. Experimental materials Yeast Extract Peptone Dextrose Sorbitol (YPDS) Medium is an amino acid deficient medium, which is used for yeast hybridization and screening of genetic mutant strains, and is purchased from zeocin (ThermoFisher Scientific, Runcorn, Cheshire, UK), and the formula is: 1% yeast extract, 2% peptone, 2% glucose, 1M sorbitol, and 2% agar.

[0026] Buffered Glycerol Complex Medium is a buffered complex liquid culture medium containing glycerol. The culture medium is controlled at pH 6.0 and is used to optimize the expression of the target protein. Its formula is: 1% yeast extract, 2% peptone, 100mM potassium phosphate (pH 6.0), 1.34% amino acid-free nitrogen source, and 1% glycerol.

[0027] Buffered Minimal Methanol (BMMH) medium is a methanol-containing buffered basal medium used for methanol-induced protein expression in recombinant Pichia pastoris strains. Its formula is: 100 mM potassium phosphate pH 6.0, 1.34% amino acid-free nitrogen source, and 0.5% methanol.

[0028] <Example 1> Using the wild-type lytic polysaccharide monooxygenase amino acid sequence shown in SEQ ID No: 1 as a template, through sequence alignment and structural analysis, the second amino acid was identified as a potential mutation site, which may affect substrate binding and catalytic efficiency.

[0029] Design site-directed mutagenesis primers based on the target mutation site. The primer sequences are as follows: Second mutation primer: G2A-F:TTAGTAGCAGGTCATGCTTTCGTTCAAAATATAGTCATTGATG G2A-R: TATATTTTGAACGAAAGCATGACCTGCTACTAAAGATGCGGAT 1. Wild-type gene cloning: Wild-type lytic polysaccharide monooxygenase from Thermoascus ascomycetes ( Thermoascus aurantiacus ), a lytic polysaccharide monooxygenase secreted by a bacterium (B. aureus), whose nucleotide sequence is shown in SEQ ID No: 2 and its encoded amino acid sequence is shown in SEQ ID No: 1. Using a Thermoascus aurantiacus cDNA as a template, the gene fragment of SEQ ID No: 2 was amplified by PCR and cloned into the EcoR I / Xba I sites of the pPICZαA vector to construct pPICZ-LPMO-WT.

[0030] 2. Site-directed mutagenesis: Overlap extension PCR was used to design primers containing mutation sites (e.g., G2A: G2A-F, G2A-R) and construct mutant plasmids such as pPICZ-LPMO-G2A.

[0031] 3. Pichia transformation and screening: The obtained wild-type lytic polysaccharide monooxygenase strain and its mutant strain were inoculated on an amino acid-deficient medium containing 100 μg / mL, and cultured at 30°C for 3 days. Single colonies were inoculated in a buffered complex liquid medium containing glycerol and cultured at 30°C with shaking at 240 rpm overnight.

[0032] 4. Inducing expression: When the concentration of the bacterial solution reached OD 600 =2-6, the bacterial solution was centrifuged at 4000 g, and the supernatant was poured out. The bacterial body was resuspended with a buffered basic culture medium containing methanol. Then, 0.5% methanol was supplemented every 12 hours, and the culture was shaken at 240 rpm at 30°C for 3 days.

[0033] 5. Protein purification: The crude enzyme solution was purified by Ni-NTA column chromatography, eluted with an eluent containing 300 mM imidazole, and eluted for 10 column volumes. The target protein peak was collected, and then the obtained protein was detected by SDS-PAGE to detect the purity of the protein.

[0034] The lytic polysaccharide monooxygenase mutant G2A (the amino acid sequence is shown as SEQ ID No: 3, and the nucleotide sequence encoding G2A is shown as SEQ ID No: 4) was obtained by the above method.

[0035] As shown in the lytic polysaccharide monooxygenase WT and its mutant G2A, the efficient expression and purification of the WT and the mutant were exhibited. Figure 1

[0036] Molecular weight analysis: The main band of the target protein was located at 43-55 kDa, which was consistent with the expected molecular weight range of LPMO, indicating that the size of the gene expression product was normal.

[0037] Purity and expression amount: WT as a control, G2A main band clear, single, few bands, indicating that the expression amount of G2A protein is high and the purity is good, the brightness of G2A band is slightly higher than that of WT, which indicates that the G2A mutant is stably expressed in Pichia pastoris, and the single-point mutation does not significantly affect the protein expression level.

[0038] <Example 2> The determination of the lytic polysaccharide monooxygenase mutant in cooperation with cellulase to degrade the substrate was carried out with microcrystalline cellulose as the substrate, and the specific method and steps were as follows: ​Preheat 1% (w / v) microcrystalline cellulose in a 60°C constant temperature metal bath for 5-10 min, add 1.33 μM enzyme diluent and cellulase (mass ratio of lytic polysaccharide monooxygenase to cellulase is 1:3). The mixed enzyme containing the mutant and the control group are placed in a 60°C constant temperature metal bath at the same time, and the reaction is carried out for 60 h. At the end of the reaction, the reactants are boiled in a boiling water bath for 5 min to inactivate the enzyme.

[0039] The detection method adopts DNS method (3,5-dinitrosalicylic acid) to detect reducing sugar (converted into glucose equivalent) and calculate the yield (mg / mL). Prepare glucose standard solution with concentration gradient of 0.15, 0.30, 0.45, 0.60, 0.75, 0.90, 1.00 mg / mL, respectively take 115 μL of each glucose standard solution and the sample to be tested in a 2 mL centrifuge tube, add 85 μL of DNS reagent, mix thoroughly, then boil in a boiling water bath for 5 min, cool to room temperature, add 200 μL of distilled water, mix thoroughly, then take 200 μL to a 96-well plate, measure the absorbance value at 540 nm. Draw the glucose standard curve with glucose content as the abscissa and OD 540 as the ordinate, and substitute the absorbance value of the sample to be tested into the glucose standard curve to obtain the reducing sugar yield of the sample.

[0040] As Figure 2 shown in the bar chart of the mutant at position 2 of the lytic polysaccharide monooxygenase hydrolyzing microcrystalline cellulose, G2A in the figure refers to the complex enzyme system of the glycine at position 2 of the amino acid sequence shown in SEQ ID No: 1 being mutated to alanine (G2A) and cellulase, which is used to compare the oxidative degradation activity of the complex enzyme system to microcrystalline cellulose after the lytic polysaccharide monooxygenase from thermophilic ascomycetes is mutated at G2 site.

[0041] Control as the enzyme-free control, WT as the complex enzyme system of lytic polysaccharide monooxygenase and cellulase, G2A has the highest yield (>2.8 mg / mL), and the mutation significantly enhances the enzyme activity. In the complex enzyme system, the high activity of G2A can optimize the substrate binding or catalytic efficiency. The G2A mutation introduces a methyl side chain (a small hydrophobic group) to stabilize the active center conformation of the enzyme, promote substrate binding and oxidative cleavage, and more efficiently destroy the crystalline region of cellulose. The synergistic effect of the lytic polysaccharide monooxygenase mutant and cellulase can reduce enzyme dosage, shorten reaction time, improve process efficiency, and significantly improve the degradation efficiency of the substrate microcrystalline cellulose.

[0042] As described above, according to the present application, since the enzyme with improved enzyme activity and the composition thereof are provided, the yield of reducing sugar generated by the degradation of lignocellulose per unit time can be improved, the effect of balancing the enzyme system to improve the overall hydrolysis rate in the enzyme system recombination can be improved, and it has a wide application prospect.

[0043] The number of devices and processing stages described herein are used to simplify the description of the application. Applications, modifications and variations of the application will be apparent to those skilled in the art without departing from the general concept of the application.

[0044] While the embodiments of the application have been disclosed as above, it is not limited to the applications listed in the specification and the embodiments, but can be applied to various fields suitable for the application, and additional modifications can be easily made by those skilled in the art, and thus the application is not limited to specific details and the figures shown and described herein, without departing from the general concept defined by the claims and the equivalent scope.

[0045] Lytic polysaccharide monooxygenase WT: Amino acid sequence (SEQ ID No: 1): HGFVQNIVIDGKNYGGYLVNQYPYMSNPPEVIAWSTTATDLGFVDGTGYQTPDIICHRGAKPGALTAPVSPGGTVELQWTPWPDSHHGPVINYLAPCNGDCSTVDKTQLEFFKIAESGLINDDNPPGIWASDNLIAANNSWTVTIPTTIAPGNYVLRHEIIALHSAQNQDGAQNYPQCINLQVTGGGSDNPAGTLGTALYHDTDPGILINIYQKLSSYIIPGPPLYTG Nucleotide sequence (SEQ ID No: 2): CATGGTTTCGTTCAAAATATAGTCATTGATGGTAAAAATTACGGAGGATACTTAGTTAATCAGTATCCTTATATGTCTAACCCACCCGAGGTTATAGCCTGGTCCACCACAGCCACGGATCTAGGGTTCGTCGATGGTACAGGATACCAGACTCCAGATATAATATGTCATAGAGGTGCAAAGCCCGGTGCCCTTACAGCACCAGTTTCCCCAGGTGGTACTGTCGAATTGCAGTGGACGCCTTGGCCAGACTCGCATCACGGACCTGTAATTAACTATTTGGCTCCCTGCAATGGAGACTGTTCGACAGTCGATAAAACCCAACTTGAGTTCTTTAAGATTGCCGAGAGTGGCTTGATTAATGACGACAATCCACCAGGGATATGGGCTTCCGATAATTTAATTGCTGCCAATAACAGCTGGACGGTCACAATTCCAACAACCATTGCACCTGGTAATTACGTTTTGCGACATGAAATCATCGCATTGCATAGTGCTCAAAATCAGGATGGAGCCCAAAACTATCCTCAATGCATAAACTTGCAGGTTACAGGAGGTGGCTCTGACAACCCTGCTGGAACTTTGGGCACTGCATTATACCATGATACGGACCCTGGAATACTGATCAACATCTATCAGAAGCTTAGTTCATATATTATTCCTGGTCCTCCTTTGTACACAGGA Lytic polysaccharide monooxygenase mutant G2A: Amino acid sequence (SEQ ID No: 3): HAFVQNIVIDGKNYGGYLVNQYPYMSNPPEVIAWSTTATDLGFVDGTGYQTPDIICHRGAKPGALTAPVSPGGTVELQWTPWPDSHHGPVINYLAPCNGDCSTVDKTQLEFFKIAESGLINDDNPPGIWASDNLIAANNSWTVTIPTTIAPGNYVLRHEIIALHSAQNQDGAQNYPQCINLQVTGGGSDNPAGTLGTALYHDTDPGILINIYQKLSSYIIPGPPLYTG Nucleotide sequence (SEQ ID No: 4): CATGCTTTCGTTCAAAATATAGTCATTGATGGTAAAAATTACGGAGGATACTTAGTTAATCAGTATCCTTATATGTCTAACCCACCCGAGGTTATAGCCTGGTCCACCACAGCCACGGATCTAGGGTTCGTCGATGGTACAGGATACCAGACTCCAGATATAATATGTCATAGAGGTGCAAAGCCCGGTGCCCTTACAGCACCAGTTTCCCCAGGTGGTACTGTCGAATTGCAGTGGACGCCTTGGCCAGACTCGCATCACGGACCTGTAATTAACTATTTGGCTCCCTGCAATGGAGACTGTTCGACAGTCGATAAAACCCAACTTGAGTTCTTTAAGATTGCCGAGAGTGGCTTGATTAATGACGACAATCCACCAGGGATATGGGCTTCCGATAATTTAATTGCTGCCAATAACAGCTGGACGGTCACAATTCCAACAACCATTGCACCTGGTAATTACGTTTTGCGACATGAAATCATCGCATTGCATAGTGCTCAAAATCAGGATGGAGCCCAAAACTATCCTCAATGCATAAACTTGCAGGTTACAGGAGGTGGCTCTGACAACCCTGCTGGAACTTTGGGCACTGCATTATACCATGATACGGACCCTGGAATACTGATCAACATCTATCAGAAGCTTAGTTCATATATTATTCCTGGTCCTCCTTTGTACACAGGA.

Claims

1. A lytic polysaccharide monooxygenase mutant, characterized in that In the lytic polysaccharide monooxygenase mutant, the glycine at position 2 of the amino acid sequence shown in SEQ ID No: 1 is substituted with alanine. The amino acid sequence of the lytic polysaccharide monooxygenase mutant is shown in SEQ ID No:

3.

2. A DNA molecule, characterized in that The DNA molecule encodes the lytic polysaccharide monooxygenase mutant according to claim 1, and the nucleotide sequence of the DNA molecule is shown in SEQ ID No:

4.

3. A recombinant expression vector, characterized in that: The recombinant expression vector contains the DNA molecule according to claim 2 and a regulatory sequence for expression operably linked to the DNA molecule.

4. The recombinant expression vector according to claim 3, wherein The recombinant expression vector is pPICZαA.

5. A host cell, characterized in that The host cell contains the DNA molecule according to claim 2 or the recombinant expression vector according to claim 3 or 4, and the host cell is a non-animal or plant cell.

6. The host cell according to claim 5, wherein The host cell is a Pichia pastoris cell.

7. The method for producing a lytic polysaccharide monooxygenase mutant according to claim 1, wherein: The steps include: The host cell according to claim 5 or 6 is cultured in a culture medium, and the recombinant expression vector according to claim 3 or 4 is used to induce expression of the lytic polysaccharide monooxygenase mutant according to claim 1.

8. Use of the lytic polysaccharide monooxygenase mutant according to claim 1, the DNA molecule according to claim 2, the recombinant expression vector according to claim 3 or 4, and the host cell according to claim 5 or 6 in synergistically hydrolyzing cellulose with cellulase.

9. A method for the coordinated hydrolysis of cellulose, characterized in that: The lytic polysaccharide monooxygenase mutant according to claim 1 and cellulase are added to cellulose for enzymatic hydrolysis.

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