Mutant of xylanase with improved activity and use thereof

Abstract

This invention modifies the xylanase derived from *Trichoderma reesei* using molecular biology techniques to mutate amino acids, resulting in a class of mutants with high catalytic efficiency. These mutants include one or more mutations at amino acid positions 51, 54, 161, and 163. Specifically, asparagine at position 51 is mutated to aspartic acid or glutamic acid; histidine at position 54 is mutated to lysine, leucine, glutamine, arginine, or serine; isoleucine at position 161 is mutated to glutamic acid or glutamine; and threonine at position 163 is mutated to glutamine or valine. The mutants described in this invention possess excellent high catalytic efficiency, which helps reduce the cost of using xylanase and improves the conversion efficiency of producing xylooligosaccharides using this enzyme, thus showing broad industrial application prospects.

Description

Technical Field

[0001] This invention belongs to the field of functional gene modification technology, specifically relating to a class of mutants with enhanced xylanase activity and their applications. Background Technology

[0002] Agricultural waste is beneficial when used and harmful when discarded. As a key aspect of agricultural environmental governance, utilizing modern biotechnology can degrade the non-grain lignocellulose portion into soluble sugars, thereby producing bioenergy or bulk chemicals. This is of great strategic significance for protecting the ecological environment and achieving sustainable economic development. Lignocellulose is mainly composed of cellulose, hemicellulose, and lignin, with hemicellulose being the second most abundant component after cellulose. It is a heteropolymer composed of pentose and hexose sugars. Xylan is the main component of hemicellulose, and its main chain is formed by β-D-xylose units linked by β-1,4 glycosidic bonds. These glycosidic bonds can be hydrolyzed by xylanase to produce xylooligosaccharides. Xylooligosaccharides, with xylobiose, xylotriose, and xylotetraose as their main functional components, have a pure sweet taste and promote the growth of beneficial bacteria such as Bifidobacteria. They are currently considered the most stable, most functional, and least consumed beneficial bacteria among all functional sugars, and have broad application prospects in the food, pharmaceutical, and feed industries.

[0003] Xylanases from different sources exhibit varying substrate specificities, producing xylooligosaccharides with different degrees of polymerization. In large-scale industrial production, existing xylanases often suffer from limitations; low catalytic activity and poor stability are key challenges faced by natural enzymes in practical industrial applications. Therefore, modifying xylanases through enzyme engineering to improve their catalytic efficiency has become an important research direction. Summary of the Invention

[0004] This invention modifies the xylanase (original enzyme, WT) derived from *Trichoderma reesei*, whose main hydrolysis products are xylobiose and xylotriose. However, this enzyme exhibits low activity and poor thermal stability. To address these technical problems, this invention uses the original enzyme sequence for modeling, analyzes the hotspot amino acids around the gating points, and performs site-directed and combinatorial mutagenesis to identify activity-related hotspot amino acids. Through site-directed saturation mutagenesis and other techniques, the xylanase is rationally modified to obtain a high-specific-activity xylanase mutant, showing promising application prospects in biomass conversion and green chemical production.

[0005] The specific technical solution of this invention is as follows:

[0006] A xylanase mutant comprising one or more of the following amino acid mutations in positions 51, 54, 161, and 163 of the amino acid sequence shown in SEQ ID NO:1: wherein position 51 asparagine is mutated to aspartic acid or glutamic acid, position 54 histidine is mutated to lysine, leucine, glutamine, arginine, or serine, position 161 isoleucine is mutated to glutamic acid or glutamine, and position 163 threonine is mutated to glutamine or valine.

[0007] Preferably, the xylanase mutant of the present invention is a single point mutation, wherein histidine at position 54 is mutated to leucine (H54L), arginine (H54R) or serine (H54S) or isoleucine at position 161 is mutated to glutamine (I161Q).

[0008] Preferably, the xylanase mutant of the present invention is a two-site mutation, having two mutations among the mutations at amino acid positions 51, 54, 161, and 163.

[0009] Preferably, the mutant is N51D / H54K, N51D / H54L, N51D / H54S, N51D / I161E, N51D / I161Q, or N51D / T163Q, N51E / H54L, N51E / H54Q, N51E / H54R, N51E / I161Q, N51E / T163V, I161Q / T163Q, I161Q / T163V, H54K / I161E, H54K / I161Q, H54L / I161E, H54L / I161Q, H54L / T163Q, H54Q / I161E, H54R / I161E, H54R / I161Q, H54R / T163Q, H54R / T163V, H54S / I161Q, H54S / T163Q.

[0010] More preferably, the mutant is a mutation of histidine at position 54 to leucine and isoleucine at position 161 to glutamine (H54L / I161Q).

[0011] Another object of the present invention is to provide a DNA molecule that encodes the xylanase mutant described in the present invention.

[0012] Another object of the present invention is to provide an expression vector for a xylanase mutant, expressing the xylanase mutant as described in the present invention. The expression vector contains a DNA molecule encoding the xylanase mutant described in the present invention.

[0013] The expression vector is a plasmid, bacteriophage, virus, or host cell.

[0014] The host cell can be a prokaryotic or eukaryotic cell, and can be Escherichia coli, yeast, Bacillus, lactobacillus, Aspergillus or Trichoderma, with Escherichia coli being preferred.

[0015] Another objective of this invention is to provide the application of the xylanase mutant, DNA molecule, or expression vector of the xylanase mutant described herein in the biodegradation of xylan. It can be used to prepare xylobiose, xylotriose, xylotetraose, etc.

[0016] Specifically, the xylan includes beech xylan, soluble wheat arabinoxylan, birch xylan, and insoluble wheat arabinoxylan. Advantages of this invention:

[0017] This invention involves the rational design of xylanase to improve its enzymatic properties. The obtained mutants showed significantly higher substrate activity for beech xylan than the bound weight (WT) mutants, with H54L / I161Q exhibiting the most significant improvement, reaching 4.5 times that of WT. Attached Figure Description

[0018] The accompanying drawings, which form part of this application, 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.

[0019] Figure 1 This is a three-dimensional structural diagram of the hot spot amino acids surrounding the WT-gated xylanase.

[0020] Figure 2 This is the result of saturation mutation of hotspot amino acids around the xylanase WT gate.

[0021] Figure 3 This is an SDS-PAGE image of xylanase WT and mutant H54L / I161Q.

[0022] Figure 4 The results show the relative enzyme activity of the xylanase double mutant.

[0023] Figure 5 The diagram shows the enzymatic properties of xylanase WT and the mutant H54L / I161Q at the optimal temperature.

[0024] Figure 6 The diagram shows the enzymatic properties of xylanase WT and the mutant H54L / I161Q—optimal pH.

[0025] Figure 7 The enzymatic properties of xylanase WT and mutant H54L / I161Q are shown in the diagram—temperature stability.

[0026] Figure 8The diagram shows the enzymatic properties of xylanase WT and the mutant H54L / I161Q—pH stability. Detailed Implementation

[0027] To enable those skilled in the art to better understand the present invention, the invention will be further described below with reference to the accompanying drawings and embodiments. It should be noted that these embodiments are only for explaining the invention and not for limiting its scope. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0028] Example 1: Construction of a xylanase mutant library

[0029] This invention selects typical endo-type xylanases derived from Trichoderma reesei, whose main hydrolysis products are X2 and X3, respectively. First, a structural model of the wild-type enzyme (WT) is constructed using AlphaFold 3. It has a unique three-dimensional spatial structure of GH11 family xylanases, resembling a half-clenched right hand.

[0030] In this structure, a significant contraction zone exists at the catalytic crack inlet, consistent with the conserved gating characteristics of the GH11 family. The narrowest channel is formed by Trp50 and Pro158, creating a WP-gated structure that allows for tunable catalytic crack entry. Based on spatial proximity, neighboring residues of W50 (N51, D52, G53, H54) and neighboring residues of P158 (S159, I160, I161, G162, T163) were selected for mutation studies. Furthermore, W50 and P158 themselves were also mutated to assess the feasibility of directly modifying the gating structure. Figure 1 ).

[0031] Saturation mutations were performed on the above candidate sites, and primers were designed using the degenerate codon NNK. The primer sequences are shown in the table below:

[0032]

[0033] Using the pET-28a(+) plasmid containing the xylanase WT gene sequence (SEQ ID NO: 2) as a template, and referring to the Vazyme biological products and operation manual, the site-directed mutant sequence was amplified from the whole plasmid using mutant primer pairs. The PCR product was digested with Dpn I. After template digestion, it was transformed into E. coli BL21(DE3) competent cells using the heat shock method and plated on LB agar plates containing 100 μg / ml kanamycin sulfate, and incubated overnight at 37°C. The mutation results were validated by sequencing by Anhui General Biotechnology Co., Ltd. The resulting mutant libraries of W50, N51, D52, G53, H54, P158, S159, I160, I161, G162, and T163 were successfully constructed through sequencing verification.

[0034] Example 2 Fermentation of recombinant xylanase mutant in Escherichia coli

[0035] Each of the mutants constructed above, as well as the original enzyme WT, was inoculated into 50 mL of LB liquid medium containing 100 μg / mL kanamycin sulfate and cultured overnight at 37 ℃ and 180 rpm. Seed culture was then inoculated into 50 mL of fresh LB liquid medium at a 2% inoculation rate and cultured at 37 ℃ and 180 rpm until OD (dose retardation). 600 When the concentration is 0.6–1.0, the sample is removed and cooled in an ice-water bath for 5 min. Then, IPTG (isopropyl-β-D-thiogalactoside) (final concentration 0.1 mmol / L) is added, and expression is induced at 20 ℃ and 150 rpm for 20 h.

[0036] The induced fermentation broth was centrifuged at 12,000 rpm for 20 min, the supernatant was discarded, and the cells were resuspended in 50 mM Na2HPO4-KH2PO4 (pH 7.0) buffer to wash the cells. The cells were then centrifuged again at 12,000 rpm for 20 min, the supernatant was discarded, and the cells were resuspended in the buffer again. The cells were then sonicated. The lysate was centrifuged at 12,000 rpm for 20 min, and the supernatant was used for SDS-PAGE electrophoresis. The stacking gel concentration was 4%, and the separating gel concentration was 12.5%. The sample and loading buffer were mixed at a 3:1 ratio, and the mixture was reacted in a boiling water bath for 5 min before loading for electrophoresis. The electrophoresis apparatus was set to an initial voltage of 120 V. When the sample moved to the separating gel, the voltage was increased to 230 V until the sample reached the bottom of the electrophoresis tank, at which point the electrophoresis was stopped.

[0037] Electrophoresis results showed that each lane had a distinct band at 24.5 kDa, indicating that the target protein was successfully expressed in each mutant.

[0038] Example 3: Screening Procedure for Enzyme Mutants

[0039] The enzyme activity changes of each mutant obtained in Example 2 were measured using beech xylan as a substrate. The measurement method is as follows:

[0040] Enzyme activity unit definition: One enzyme activity unit is defined as the amount of enzyme required to produce 1 mmol of reducing sugar from the substrate per minute under conditions of 60 ℃ and pH 7.0.

[0041] Accurately weigh 1 g of beech wood xylan and dissolve it in 100 mL of Na2HPO4-KH2PO4 buffer (50 mM, pH 7.0). Stir and mix well. Accurately pipette 1.0 mL into a test tube as the substrate for the enzyme reaction. After preheating at 60 ℃ for 5 min, add 0.5 mL of appropriately diluted protease solution. Place the tube in a 60 ℃ water bath and shake for 15 min. Add 3 mL of DNS (3,5-dinitrosalicylic acid) reagent. After reacting in a boiling water bath for 5 min, quickly cool to room temperature. Use the inactivated enzyme reaction solution as a control and measure the absorbance value at a wavelength of 540 nm.

[0042] Enzyme activity X = (reducing sugar content / 150 / 15) / n

[0043] in:

[0044] X – Enzyme activity, U / mg

[0045] 150 — Reducing sugar converted from milligrams to micromoles

[0046] 15 — Reaction Time

[0047] n – Reactive protein content, mg

[0048] For the constructed mutants W50, N51, D52, G53, H54, P158, S159, I160, I161, G162, and T163, the changes in enzyme activity were measured using beech xylan as a substrate. The relative enzyme activities of each mutant are as follows: Figure 2 As shown in the figure. The results showed that replacing asparagine at position 51 with aspartic acid and glutamic acid increased enzyme activity by 1.2 and 1.3 times, respectively; replacing histidine at position 54 with lysine, leucine, arginine, glutamine, and serine increased enzyme activity by 1.5 to 2 times, respectively; replacing isoleucine at position 161 with glutamic acid and glutamine increased enzyme activity by 1.6 and 2.4 times, respectively; and replacing threonine at position 163 with glutamine and valine increased enzyme activity by approximately 1.4 times.

[0049] Based on the above results, the mutants N51D, N51E, H54K, H54L, H54Q, H54R, H54S, I161E, I161Q, T163Q, and T163V were combined in pairs to obtain 42 double mutants. The corresponding mutants were prepared according to the method in Example 2. The templates and primers used for preparing each combination mutant are shown in the table below. SDS-PAGE electrophoresis results of the crude enzyme solutions of each mutant showed a clear band at 24.5 kDa in each lane, indicating successful expression of the target protein in each mutant. The SDS-PAGE electrophoresis results of the double mutant S24D / K137S are shown below. Figure 3 As shown.

[0050]

[0051]

[0052] For each constructed double mutant, the enzyme activity changes were measured using beech xylan as a substrate. The relative enzyme activity results for each mutant are as follows: Figure 4 As shown, the results indicate that compared with the control WT, except for the lower enzyme activity of the combined mutant H54Q / T163Q, the combined mutants N51D / H54K, N51D / H54L, N51D / H54S, N51D / I161E, N51D / I161Q, and N51D / T163Q, N51E / H54L, N51E / H54Q, N51E / H54R, N51E / I161Q, N51E / T163V, I161Q / T163Q, I161Q / T163V, H54K / I161E, H54K / I161Q, H54L / I161E, H54L / I161Q, H54L / T163Q, H54Q / I161E, H54R / I161E, H54R / I161Q, H54R / T163Q, H54R / T163V, H54S / Both I161Q and H54S / T163Q showed varying degrees of improvement, with H54L / I161Q exhibiting the highest enzyme activity, which was 4.5 times that of the original enzyme and superior to other combinations.

[0053] Example 4: Enzymatic Properties Analysis of Enzyme Mutants

[0054] 1. Optimal temperature and temperature stability

[0055] To determine the optimal temperatures for the mutant and the original enzyme, the enzyme solutions of the mutant H54L / I161Q and the original enzyme WT obtained in Example 3 were diluted to a certain concentration, and 0.5 ml of each solution was added to a test tube containing 1.0 mL of beech xylan substrate. The reactions were carried out at 40 °C, 50 °C, 60 °C, 70 °C, and 80 °C for 15 min, respectively. After the reaction, 3 mL of DNS solution was added and the mixture was boiled for 5 min. The enzyme activity was then measured. Using the highest enzyme activity as 100%, the relative enzyme activity was calculated sequentially, and a curve showing the change in enzyme activity with temperature was plotted. The results are shown below. Figure 5 As shown, the optimal temperature for both the original enzyme WT and the mutant H54L / I161Q is 50℃.

[0056] To determine the temperature stability of the mutant and the original enzyme, enzyme solutions of the mutant H54L / I161Q and the original enzyme WT were incubated in water baths at 40 °C, 50 °C, 60 °C, 70 °C, and 80 °C for 2 h, respectively. Enzyme activity changes were then measured according to the method described in Example 3. Using the highest enzyme activity as 100%, relative enzyme activities were calculated sequentially, and enzyme activity change curves under different incubation conditions were plotted. The results are shown below. Figure 6 As shown, there is no significant difference in temperature stability between the original enzyme WT and the mutant H54L / I161Q.

[0057] Optimal pH and pH stability

[0058] 2. Optimal pH and pH stability

[0059] Buffers and substrates with different pH values ​​were prepared: citric acid-sodium citrate (pH 4.0-6.0), Na2HPO4-KH2PO4 (pH 6.0-8.0), and glycine-sodium hydroxide (pH 8.0-9.0), respectively, to prepare beech xylan substrates under different pH conditions.

[0060] The mutant and the original enzyme solution obtained in Example 3 were diluted to specific concentrations and added to beech xylan substrate reaction systems prepared with different pH buffers. The reaction was carried out at 50 °C for 15 min. After the reaction, 3 mL of DNS solution was added and the mixture was boiled for 5 min. The enzyme activity was then measured. Using the highest enzyme activity as 100%, the relative enzyme activity was calculated sequentially, and a curve showing the change in enzyme activity with substrate pH was plotted. The results are as follows: Figure 7 As shown by the optimal pH, both the original enzyme and the mutant exhibit the highest activity at pH 5.0.

[0061] The mutant and original enzyme solutions were diluted with buffers of different pH values ​​and incubated on ice at 4 °C for 2 h, after which enzyme activity was measured. Using the highest enzyme activity as 100%, relative enzyme activities were calculated sequentially, and enzyme activity change curves under different incubation conditions were plotted. Figure 8The pH stability plot shows that the pH stability of the mutant and the original enzyme is not significantly different; both enzyme activities decrease with increasing or decreasing pH. The mutant and the original enzyme are relatively stable at pH 5-7, retaining more than 50% of their activity.

[0062] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A xylanase mutant, characterized in that... The mutant contains one or more of the following amino acid mutations in positions 51, 54, 161, and 163 of the amino acid sequence shown in SEQ ID NO:1: wherein position 51 asparagine is mutated to aspartic acid or glutamic acid, position 54 histidine is mutated to lysine, leucine, glutamine, arginine, or serine, position 161 isoleucine is mutated to glutamic acid or glutamine, and position 163 threonine is mutated to glutamine or valine.

2. The xylanase mutant according to claim 1, characterized in that... The mutant has two mutations among the mutations at amino acid positions 51, 54, 161, and 163.

3. The xylanase mutant according to claim 2, characterized in that... The mutants are N51D / H54K, N51D / H54L, N51D / H54S, N51D / I161E, N51D / I161Q, and N51D / T163Q, N51E / H54L, N51E / H54Q, N51E / H54R, N51E / I161Q, N51E / T163V, I161Q / T163Q, I161Q / T163V, H54K / I161E, H54K / I161Q, H54L / I161E, H54L / I161Q, H54L / T163Q, H54Q / I161E, H54R / I161E, H54R / I161Q, H54R / T163Q, H54R / T163V, H54S / I161Q, H54S / T163Q.

4. A DNA molecule, characterized in that, The DNA molecule encodes the xylanase mutant according to any one of claims 1-3.

5. An expression vector for a xylanase mutant, characterized in that... Express the xylanase mutant according to claim 1 or 2.

6. The expression vector as described in claim 5, characterized in that... It contains the DNA molecule as described in claim 3.

7. A host cell expressing the xylanase mutant of claim 1 or 2, wherein the host cell is a prokaryotic cell or a eukaryotic cell.

8. The host cell according to claim 7, characterized in that... The host cells are selected from Escherichia coli, yeast, Bacillus, Lactobacillus, Aspergillus, or Trichoderma.

9. The use of the xylanase mutant according to any one of claims 1-3, the DNA molecule according to claim 4, the expression vector of the xylanase mutant according to claim 5 or 6, or the host cell of the xylanase mutant according to claim 7 or 8 in xylan biodegradation.

10. The application according to claim 9, wherein the xylan comprises beech xylan, soluble wheat arabinoxylan, birch xylan, and insoluble wheat arabinoxylan.

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