Mutants of lytic polysaccharide monooxygenase and methods of synergistic hydrolysis of cellulose

Abstract

The present application relates to a kind of lytic polysaccharide monooxygenase mutant and cellulose synergistic hydrolysis method.The present application belongs to the field of biotechnology, and traditional cellulose hydrolysis technology has the problems of low efficiency and high cost, the present application obtains mutant with higher catalytic activity and stability by site-directed mutagenesis to the 2nd amino acid sequence shown in SEQ ID No:1, significantly improves cellulose hydrolysis efficiency, and has wide application prospect in the fields of bioenergy, papermaking, textile and the like.

Description

Technical Field

[0001] This invention relates to the field of biotechnology. More specifically, this invention relates to a cleavable polysaccharide monooxygenase mutant, a DNA molecule, a recombinant expression vector, a host cell, a method for producing the cleavable polysaccharide monooxygenase mutant, the application of the cleavable polysaccharide monooxygenase mutant in the co-hydrolysis of cellulose, and a method for the co-hydrolysis of cellulose. Background Technology

[0002] Lignocellulose, such as straw, is one of the most abundant renewable resources in nature. It can be degraded into hexoses and pentoses by cellulases, which can be further used to produce various bio-based products, including bioethanol. However, the cost of enzymatic degradation of lignocellulose accounts for about one-quarter of the cost of biofuels. Therefore, developing efficient lignocellulose-degrading enzymes and engineering their modification is crucial to promoting the benefits of lignocellulose biomass. Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-dependent enzymes capable of oxidatively cleaving polysaccharides. LPMOs are key enzymes that work synergistically with cellulases to degrade lignocellulose, and improving their performance is crucial to the overall hydrolytic efficiency of cellulases. The main research content of this application is to accurately select mutation sites and predict the impact of mutations on enzyme function. Summary of the Invention

[0003] One objective of this invention is to provide a cleavable polysaccharide monooxygenase mutant. By performing a site-directed mutation on the second position of the amino acid sequence shown in SEQ ID No:1, the obtained LPMO mutant exhibits higher catalytic activity and hydrolysis efficiency, improves catalytic reaction efficiency and product yield, is more suitable for industrial applications, and can significantly reduce the cost of the enzymatic hydrolysis process.

[0004] Another objective of this invention is to provide a DNA molecule that, through codon optimization and sequence design, solves the problem of low gene expression efficiency in heterologous hosts and enables efficient transcription and translation of LPMO mutants in engineered bacteria.

[0005] Another objective of this invention is to provide a recombinant expression vector that, by constructing an operable linker structure containing the DNA molecule and a highly efficient expression regulatory element, ensures stable transcription and induced expression of LPMO mutant genes in host cells, thereby increasing the expression level and solubility of recombinant proteins.

[0006] Another objective of this invention is to provide a host cell that, by screening suitable microbial hosts and introducing the aforementioned recombinant expression vector, solves the problems of folding errors and low secretion efficiency of natural LPMOs in traditional hosts, thereby achieving high biomass expression and functional folding of mutant enzymes.

[0007] Another objective of this invention is to provide a method for producing LPMOs that significantly improves the yield and purity of LPMO mutants, meeting the quality and cost requirements of enzyme preparations for industrial production.

[0008] Another objective of this invention is to provide an application that utilizes the synergistic mechanism between LPMO mutants and cellulase to construct an efficient enzymatic hydrolysis system and improve cellulose degradation efficiency.

[0009] Another objective of this invention is to provide a synergistic hydrolysis method for cellulose, which solves the problems of low efficiency and high cost of traditional cellulose enzymatic hydrolysis technology by optimizing the synergistic interaction conditions of mutant enzymes and other cellulases, thereby achieving efficient conversion of lignocellulosic biomass.

[0010] To achieve these objectives and other advantages according to the present invention, a cleaving polysaccharide monooxygenase mutant is provided, wherein glycine at position 2 of the amino acid sequence shown in SEQ ID No:1 is replaced with alanine, and the amino acid sequence of the cleaving polysaccharide monooxygenase mutant is shown in SEQ ID No:3.

[0011] A DNA molecule encoding the cleavable polysaccharide monooxygenase mutant, the DNA molecule having the nucleotide sequence shown in SEQ ID No:4.

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

[0013] Preferably, the recombinant expression vector is pPICZαA.

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

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

[0016] The method for producing the cleavable polysaccharide monooxygenase mutant includes the following steps:

[0017] The host cells were cultured in a culture medium, and the cleavage polysaccharide monooxygenase mutant was induced to be expressed by the recombinant expression vector.

[0018] The application of the cleavable polysaccharide monooxygenase mutant, the DNA molecule, the recombinant expression vector, and the host cell in the synergistic hydrolysis of cellulose by cellulase.

[0019] A cellulose co-hydrolysis method involves adding the cleavage polysaccharide monooxygenase mutant and cellulase to cellulose for enzymatic hydrolysis.

[0020] The present invention has at least the following beneficial effects:

[0021] The enzyme provided by this invention has excellent cellulose degradation function, and its synergistic hydrolysis effect on cellulose can be improved by up to 2.8 times. The improved hydrolysis efficiency can reduce the amount of enzyme used to hydrolyze a certain amount of reducing sugar per unit time, and enhance the role of enzyme system in balancing enzyme system to improve the overall hydrolysis rate in enzyme system compounding. It has broad application prospects.

[0022] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0023] Figure 1 This is a gel electrophoresis image of the cleaving polysaccharide monooxygenase and its mutants after expression according to the present invention;

[0024] Figure 2 This is a bar chart of microcrystalline cellulose hydrolyzed by the mutant generated at position 2 of the cleaving polysaccharide monooxygenase of the present invention. Detailed Implementation

[0025] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0026] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0027] It should be noted that, unless otherwise specified, the experimental methods described in the following embodiments are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified, and therefore should not be construed as limiting the present invention.

[0028] 1. Experimental materials

[0029] Yeast Extract Peptone Dextrose Sorbitol (YPDS) Medium is an amino acid auxotrophic medium used for yeast hybridization and screening of genetic mutants. It was purchased from zeocin (Thermo Fisher Scientific, Runcorn, Cheshire, UK) and its formulation is: 1% yeast extract, 2% peptone, 2% glucose, 1M sorbitol, and 2% agar.

[0030] Buffered Glycerol Complex Medium is a buffered complex liquid culture medium containing glycerol, with the medium controlled at pH 6.0. It is used to optimize the expression of target proteins. Its formulation is: 1% yeast extract, 2% peptone, 100mM potassium phosphate (pH 6.0), 1.34% amino acid-free nitrogen source and 1% glycerol.

[0031] Buffered Minimal Methanol (BMMH) medium is a buffered basal medium containing methanol, used for methanol-induced protein expression in recombinant Pichia pastoris strains. Its formulation is: 100 mM potassium phosphate, pH 6.0, 1.34% amino acid-free nitrogen source, and 0.5% methanol.

[0032] <Example 1>

[0033] Using the amino acid sequence of the wild-type cleaving polysaccharide monooxygenase shown in SEQ ID No:1 as a template, sequence alignment and structural analysis identified the second amino acid as a potential mutation site that may affect substrate binding and catalytic efficiency.

[0034] Site-directed mutagenesis primers were designed based on the target mutation site. The primer sequences are as follows:

[0035] Second mutation primer:

[0036] G2A-F:TTAGTAGCAGGTCATGCTTTCGTTCAAAATATAGTCATTGATG

[0037] G2A-R: TATATTTTGAACGAAAGCATGACCTGCTACTAAAGATGCGGAT

[0038] 1. Wild-type gene cloning:

[0039] Wild-type lysin monooxygenase derived from thermophilic ascomycetes ( Thermoascus aurantiacus The secreted cleaving polysaccharide monooxygenase has the nucleotide sequence shown in SEQ ID No:2 and the encoded amino acid sequence shown in SEQ ID No:1. Using Ascomycetes thermophilic cDNA as a template, the gene fragment of SEQ ID No:2 was amplified by PCR and cloned into the EcoRI / XbaI site of the pPICZαA vector to construct pPICZ-LPMO-WT.

[0040] 2. Site-directed mutation:

[0041] Using the overlap extension PCR method, primers containing mutation sites (such as G2A: G2A-F, G2A-R) were designed to construct mutant plasmids such as pPICZ-LPMO-G2A.

[0042] 3. Pichia pastoris transformation and screening:

[0043] The obtained wild-type polysaccharide monooxygenase strain and its mutant strains were plated into an amino acid auxotrophic medium containing 100 μg / mL and cultured at 30°C for 3 days. Single clones were picked and inoculated into a buffered complex liquid medium containing glycerol and cultured overnight at 30°C with shaking at 240 rpm.

[0044] 4. Induced expression:

[0045] When the bacterial concentration reaches OD 600 At 2-6, using a UNICOUV2102 UV-Vis spectrophotometer, with a buffered complex culture medium containing glycerol as a blank control, the bacterial culture was centrifuged at 4000g, the supernatant was poured off, and the bacterial cells were resuspended in a buffered basal culture medium containing methanol. Then, 0.5% methanol was added every 12 hours, and the culture was shaken at 240rpm and 30℃ for 3 days.

[0046] 5. Protein purification:

[0047] The crude enzyme solution was purified by Ni-NTA column chromatography. Elution was performed with 300 mM imidazole elution buffer for 10 column volumes. The target protein peak was collected, and the obtained protein was then analyzed by SDS-PAGE to determine the protein purity.

[0048] The above method was used to obtain the cleavable polysaccharide monooxygenase mutant G2A (amino acid sequence as shown in SEQ ID No:3, nucleotide sequence encoding G2A as shown in SEQ ID No:4).

[0049] like Figure 1The cleavable polysaccharide monooxygenase WT and its mutant G2A are shown, demonstrating the efficient expression and purification of WT and its mutant.

[0050] Molecular weight analysis:

[0051] The main band of the target protein was located at 43-55 kDa, which is within the expected molecular weight range of LPMO, indicating that the size of the gene expression product is normal.

[0052] Purity and expression level:

[0053] As a control, the main band of G2A was clear and single with few extraneous bands, indicating that the expression level of G2A protein was high and the purity was good. The brightness of the G2A band was slightly higher than that of WT, suggesting that the G2A mutant was stably expressed in Pichia pastoris and that the protein expression level was not significantly affected by single-point mutation.

[0054] <Example 2>

[0055] The determination of reducing sugar yield of substrates by a cleavable polysaccharide monooxygenase mutant in synergistic cellulase degradation of the substrate using microcrystalline cellulose as a substrate is as follows:

[0056] Preheat 1% (w / v) microcrystalline cellulose in a 60°C constant-temperature metal bath for 5-10 min, then add 1.33 μM enzyme dilution and cellulase (the mass ratio of cleaving polysaccharide monooxygenase to cellulase is 1:3). Place the well-mixed enzyme containing the mutant and the control group simultaneously in a 60°C constant-temperature metal bath and react for 60 h. At the end of the reaction, boil the reactants in a boiling water bath for 5 min to inactivate the enzyme.

[0057] The detection method used was the DNS method (3,5-dinitrosalicylic acid) to determine reducing sugars (converted to glucose equivalents), and the yield (mg / mL) was calculated. Glucose standard solutions with concentration gradients of 0.15, 0.30, 0.45, 0.60, 0.75, 0.90, and 1.00 mg / mL were prepared. 115 µL of each glucose standard solution and the sample to be tested were respectively placed in a 2 mL centrifuge tube, 85 µL of DNS reagent was added, and the mixture was thoroughly mixed. The mixture was then boiled in a water bath for 5 min, cooled to room temperature, and 200 µL of distilled water was added. After mixing, 200 µL was pipetted into a 96-well plate, and the absorbance was measured at 540 nm. The results were plotted with glucose content as the x-axis and OD value as the y-axis. 540 Plot a glucose standard curve with the vertical axis as the ordinate. Substitute the absorbance value of the sample to be tested into the glucose standard curve to calculate the reducing sugar yield of the sample.

[0058] like Figure 2The bar chart shown illustrates the hydrolysis of microcrystalline cellulose by a mutant of the cleaving polysaccharide monooxygenase at position 2. In the figure, G2A refers to the complex enzyme system of cellulase with glycine mutated to alanine at position 2 of the amino acid sequence shown in SEQ ID No:1 (G2A). This system is used to compare the oxidative degradation activity of the complex enzyme system of the thermophilic ascomycete-derived cleaving polysaccharide monooxygenase on microcrystalline cellulose after mutation at the G2 site.

[0059] Control was used as an enzyme-free control, and WT was a complex enzyme system of cleaving polysaccharide monooxygenase and cellulase. G2A had the highest yield (>2.8 mg / mL), and the mutation significantly enhanced enzyme activity. In the complex enzyme system, the high activity of G2A can optimize substrate binding or catalytic efficiency. The G2A mutation introduces a methyl side chain (hydrophobic small group), which stabilizes the conformation of the active site of the enzyme, promotes substrate binding and oxidative cleavage, and more efficiently destroys the cellulose crystalline region. The cleaving polysaccharide monooxygenase mutant and cellulase work synergistically to reduce enzyme dosage, shorten reaction time, and improve process efficiency, which can significantly improve the degradation efficiency of the substrate microcrystalline cellulose.

[0060] As described above, according to the present invention, since enzymes and their compositions with enhanced enzyme activity are provided, the yield of reducing sugars generated by the degradation of lignocellulose per unit time can be increased, and the role of balancing enzyme systems in improving the overall hydrolysis rate can be enhanced in enzyme system compounding, which has broad application prospects.

[0061] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the invention will be readily apparent to those skilled in the art.

[0062] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

[0063] WT (Tonal-Cosmetic Polysaccharide Monooxygenase):

[0064] Amino acid sequence (SEQ ID No:1):

[0065] HGFVQNIVIDGKNYGGYLVNQYPYMSNPPEVIAWSTTATDLGFVDGTGYQTPDIICHRGAKPGALTAPVSPGGTVELQWTPWPDSHHGPVINYLAPCNGDCSTVDKTQLEFFKIAESGLINDDNPPGIWASDNLIAANNSWTVTIPTTIAPGNYVLRHEIIALHSAQNQDGAQNYPQCINLQVTGGGSDNPAGTLGTALYHDTDPGILINIYQKLSSYIIPGPPLYTG

[0066] Nucleotide sequence (SEQ ID No:2):

[0067] CATGGTTTCGTTCAAAATATAGTCATTGATGGTAAAAATTACGGAGGATACTTAGTTAATCAGTATCCTTATATGTCTAACCCACCCGAGGTTATAGCCTGGTCCACCACAGCCACGGATCTAGGGTTCGTCGATGGTACAGGATACCAGACTCCAGATATAATATGTCATAGAGGTGCAAAGCCCGGTGCCCTTACAGCACCAGTTTCCCCAGGTGGTACTGTCGAATTGCAGTGGACGCCTTGGCCAGACTCGCATCACGGACCTGTAATTAACTATTTGGCTCCCTGCAATGGAGACTGTTCGACAGTCGATAAAACCCAACTTGAGTTCTTTAAGATTGCCGAGAGTGGCTTGATTAATGACGACAATCCACCAGGGATATGGGCTTCCGATAATTTAATTGCTGCCAATAACAGCTGGACGGTCACAATTCCAACAACCATTGCACCTGGTAATTACGTTTTGCGACATGAAATCATCGCATTGCATAGTGCTCAAAATCAGGATGGAGCCCAAAACTATCCTCAATGCATAAACTTGCAGGTTACAGGAGGTGGCTCTGACAACCCTGCTGGAACTTTGGGCACTGCATTATACCATGATACGGACCCTGGAATACTGATCAACATCTATCAGAAGCTTAGTTCATATATTATTCCTGGTCCTCCTTTGTACACAGGA

[0068] G2A, a mutant of polysaccharide monooxygenase:

[0069] Amino acid sequence (SEQ ID No:3):

[0070] HAFVQNIVIDGKNYGGYLVNQYPYMSNPPEVIAWSTTATDLGFVDGTGYQTPDIICHRGAKPGALTAPVSPGGTVELQWTPWPDSHHGPVINYLAPCNGDCSTVDKTQLEFFKI AESGLINDDNPPGIWASDNLIAANNSWTVTIPTTIAPGNYVLRHEIIALHSAQNQDGAQNYPQCINLQVTGGGSDNPAGTLGTALYHDTDPGILINIYQKLSSYIIPGPPLYTG

[0071] Nucleotide sequence (SEQ ID No:4):

[0072] CATGCTTTCGTTCAAAATATAGTCATTGATGGTAAAAATTACGGAGGATACTTAGTTAATCAGTATCCTTATATGTCTAACCCACCCGAGGTTATAGCCTGGTCCACCACAGCCACGGATCTAGGGTTCGTCGATGGTACAGGATACCAGACTCCAGATATAATATGTCATAGAGGTGCAAAGCCCGGTGCCCTTACAGCACCAGTTTCCCCAGGTGGTACTGTCGAATTGCAGTGGACGCCTTGGCCAGACTCGCATCACGGACCTGTAATTAACTATTTGGCTCCCTGCAATGGAGACTGTTCGACAGTCGATAAAACCCAACTTGAGTTCTTTAAGATTGCCGAGAGTGGCTTGATTAATGACGACAATCCACCAGGGATATGGGCTTCCGATAATTTAATTGCTGCCAATAACAGCTGGACGGTCACAATTCCAACAACCATTGCACCTGGTAATTACGTTTTGCGACATGAAATCATCGCATTGCATAGTGCTCAAAATCAGGATGGAGCCCAAAACTATCCTCAATGCATAAACTTGCAGGTTACAGGAGGTGGCTCTGACAACCCTGCTGGAACTTTGGGCACTGCATTATACCATGATACGGACCCTGGAATACTGATCAACATCTATCAGAAGCTTAGTTCATATATTATTCCTGGTCCTCCTTTGTACACAGGA。

Claims

1. Mutants of lytic polysaccharide monooxygenase characterized in that, The lysing polysaccharide monooxygenase mutant 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 lysing polysaccharide monooxygenase mutant is shown in SEQ ID No:

3.

2. A DNA molecule characterised in that, The DNA molecule encodes the lysing polysaccharide monooxygenase mutant of 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 of claim 2 and a regulatory sequence for expression operably linked to the DNA molecule.

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

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

6. The host cell of 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 method comprises the following steps: The host cell of claim 5 or 6 is cultured in a culture medium, and the lysing polysaccharide monooxygenase mutant of claim 1 is induced to express by the recombinant expression vector of claim 3 or 4.

8. Use of the lysing polysaccharide monooxygenase mutant of claim 1, the DNA molecule of claim 2, the recombinant expression vector of claim 3 or 4, or the host cell of claim 5 or 6 in synergistic cellulase hydrolysis of cellulose.

9. A method for the synergistic hydrolysis of cellulose, characterized in that, The lysing polysaccharide monooxygenase mutant of claim 1 is added to cellulase for enzymatic hydrolysis of cellulose.

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