Beta-xylosidase mutants, genes, recombinant plasmids, modified cells and uses

Abstract

The application discloses a beta-xylosidase mutant from Aspergillus brasiliensis, genes, a recombinant plasmid, modified cells and applications, and relates to the technical field of biological genetic engineering. The beta-xylosidase mutant is subjected to amino acid mutation on the basis of the sequence shown in SEQ ID NO:1, and the amino acid mutation is single-point mutation or combined mutation of S175F, V293I, Q390E, A449S, N526G, Y565K and N576A. Through combined mutation of specific sites of the beta-xylosidase, the application realizes the synergistic improvement of enzyme activity and thermal stability, and significantly improves the yield of xylose when the beta-xylosidase is used in combination with xylanase for hydrolysis of lignocellulose.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to β-xylosidase mutants, genes, recombinant plasmids, modified cells, and their applications. Background Technology

[0002] Xylan is a major component of hemicellulose and the second largest renewable polysaccharide resource in nature after cellulose. It is widely found in agricultural and forestry byproducts and has significant applications in biomass energy development, food processing, papermaking, and animal feed. Due to its complex structure, efficient xylan degradation typically relies on the synergistic action of multiple enzymes. Endoxylanases are responsible for breaking down the xylan backbone to generate xylo-oligosaccharides, while β-xylosidases further hydrolyze the xylo-oligosaccharides into xylose, making them one of the key enzymes for achieving complete xylan degradation.

[0003] In existing technologies, performance modification of β-xylosidase mainly focuses on improving its enzyme activity or thermal stability through single-point mutations. However, this usually only achieves an improvement in one specific performance, and there is often a trade-off between enzyme activity and thermal stability. Furthermore, in practical applications, β-xylosidase needs to work synergistically with endoxylanase to achieve efficient hydrolysis of lignocellulose. The synergistic efficiency of existing β-xylosidases in complex systems still needs improvement, making it difficult to fully realize their application potential in complex substrate systems.

[0004] Therefore, there is an urgent need to provide a molecularly modified β-xylosidase that can improve enzyme activity while maintaining thermal stability, and further enhance the hydrolysis efficiency of lignocellulose when working synergistically with xylanase. Summary of the Invention

[0005] One objective of the first aspect of this invention is to provide a β-xylosidase mutant derived from Aspergillus brasiliensis, thereby solving the technical problem in the prior art that the β-xylosidase has low enzyme activity and insufficient thermal stability, resulting in low synergistic efficiency during the hydrolysis of lignocellulose.

[0006] According to a first aspect of the present invention, the present invention provides a β-xylosidase mutant derived from Aspergillus brasiliensis, wherein the β-xylosidase mutant undergoes an amino acid mutation based on the sequence shown in SEQ ID NO: 1, and the amino acid mutation is a single-point mutation or a combination of mutations of S175F, V293I, Q390E, A449S, N526G, Y565K and N576A.

[0007] Optionally, the amino acid mutation is a combination mutation formed by combining N576A with any single-point mutation among V293I, Q390E, N526G or Y565K.

[0008] Optionally, the β-xylosidase mutant is obtained through directed evolution screening, the directed evolution method comprising:

[0009] Using a recombinant plasmid containing the β-xylosidase encoding gene as a template, random mutagenesis PCR was used for amplification to construct a mutant library. The mutant library was then transformed into host cells for expression, and β-xylosidase mutants with enhanced enzyme activity were obtained through enzyme activity screening.

[0010] In a second aspect, the present invention also provides a gene encoding a β-xylosidase mutant as described in any of the preceding claims.

[0011] Thirdly, the present invention also provides a recombinant plasmid containing the gene encoding the above-mentioned β-xylosidase mutant.

[0012] Fourthly, the present invention also provides a genetically engineered bacterium, including the genetically engineered bacterium encoding the above-mentioned β-xylosidase mutant gene.

[0013] Fifthly, the present invention also provides a modified cell comprising the above-mentioned recombinant plasmid.

[0014] In a sixth aspect, the present invention also provides the application of the above-mentioned β-xylosidase mutant, genetically engineered bacteria or modified cells in the production of xylose from lignocellulose hydrolysis.

[0015] The β-xylosidase mutants obtained through directed evolution in this invention exhibit improved enzymatic and application performance. Some mutants enhance enzyme activity while simultaneously improving thermostability; in particular, certain combinations of mutants achieve both high enzyme activity and a long half-life. Furthermore, when these mutants are combined with xylanase for the hydrolysis of lignocellulose, they effectively increase xylose yield, demonstrating a good synergistic effect. Moreover, different combinations of mutation sites have varying effects on enzyme performance, providing diverse options for subsequent enzyme molecule modification and industrial applications.

[0016] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0017] The following sections will describe some specific embodiments of the invention in detail by way of example and not limitation, with reference to the accompanying drawings. The same reference numerals in the drawings denote the same or similar parts or portions. Those skilled in the art should understand that these drawings are not necessarily drawn to scale. In the drawings:

[0018] Figure 1 This is a bar chart showing the specific enzyme activity of β-xylosidase with different combinations of mutations according to Example 3 of the present invention. Detailed Implementation

[0019] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0020] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, the specific embodiments of this application will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of this application. Furthermore, it should be noted that, for ease of description, only the parts relevant to this application are shown in the accompanying drawings, not the entire structure. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this application.

[0021] The terms “comprising” and “having”, and any variations thereof, used in this application are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or apparatus that includes a series of steps or units is not limited to the steps or units listed, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to such process, method, product, or apparatus.

[0022] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0023] Some of the experimental materials and reagents used in this invention:

[0024] BMMY medium was prepared by autoclaving 1% (w / v) yeast extract, 2% (w / v) tryptone, 1.34% (w / v) amino-free yeast nitrogen source, and 10% (v / v) 1M potassium phosphate buffer (pH 6.0) at 115°C for 20 min, followed by the addition of 1% (v / v) methanol and thorough mixing.

[0025] This invention provides a β-xylosidase mutant derived from Aspergillus brasiliensis, namely β-xylosidase Abxyl, which is a protein containing the amino acid sequence shown in SEQ ID NO:2. The β-xylosidase protein sequence number published by the National Center for Biotechnology Information in the United States is GKZ37584.1.

[0026] Example 1

[0027] Construction of the recombinant plasmid pPIC9K-Abxyl of the wild-type β-xylosidase Abxyl gene and the genetically engineered strain GS115 / pPIC9K-Abxyl.

[0028] First, the amino acid sequence of Abxyl was analyzed using the signal peptide prediction tool SignalP-6.0 (https: / / services.healthtech.dtu.dk / services / SignalP-6.0 / ). It was found that the first 26 amino acids expressed an endogenous signal peptide. Since there is an α-factor secretion signal peptide on the expression plasmid pPIC9K, in order to prevent the two from conflicting and affecting the secretory expression of the protein in the Pichia pastoris system, the amino acid sequence referenced when synthesizing the whole Abxyl genome was a truncated sequence with the first 26 amino acids removed, namely the original amino acid sequence of the β-xylosidase mutant, SEQ ID NO:1.

[0029] The synthetic gene encoding the β-xylosidase mutant was optimized according to the codon preference of Pichia pastoris, resulting in the gene sequence SEQ ID NO:3. The synthesized gene sequence SEQ ID NO:3 was then inserted between the EcoRI and NotI restriction sites of the pPIC9K plasmid, thereby obtaining the recombinant plasmid pPIC9K-Abxyl expressing the wild-type β-xylosidase Abxyl. The plasmid pPIC9K-Abxyl was linearized with the restriction endonuclease SalI, and the linearized vector was purified and recovered. It was then electroporated into Pichia pastoris GS115 competent cells (Ingenie Life Sciences, Inc., C18100, USA) and cultured inverted at 30°C. Positive transformants were identified, and the recombinant strain GS115 / pPIC9K-Abxyl expressing the wild-type β-xylosidase Abxyl was obtained.

[0030] Example 2

[0031] Based on the synthesized Abxyl gene sequence SEQ ID NO:3, primers Abxyl-1 (nucleotide sequence SEQ ID NO:4) and Abxyl-2 (nucleotide sequence SEQ ID NO:5) were designed. Using the recombinant plasmid pPIC9K-Abxyl as a template, PCR amplification was performed using the GeneMorph II random mutagenesis PCR kit (Agilent Technologies (China) Co., Ltd., 200550). After gel purification, the PCR product was obtained. The PCR product and plasmid pPIC9K were digested with restriction endonucleases EcoRI and NotI, respectively. The digested PCR product fragment was ligated to the pPIC9K plasmid fragment, and the ligation product was transformed into Top10 competent cells (Beijing Zhuangmeng International Biotechnology Co., Ltd., ZC104). The cells were plated on LBK kanamycin-resistant plates and incubated upside down at 37°C. After the transformants appeared, all colonies were washed off with sterile water and the bacterial cells were collected. The plasmid in the bacterial cells was extracted and named pPIC9K-Abxyl-mutant. This plasmid is a mixed plasmid containing multiple Abxyl gene mutations.

[0032] The plasmid pPIC9K-Abxyl- mutant was linearized using the restriction endonuclease SalI. The linearized vector was purified, recovered, and electroporated into Pichia pastoris GS115 competent cells (Ingenie Life Sciences, Inc., C18100). Positive transformants were screened on histidine auxotrophic plates. After the transformants appeared, they were picked one by one with a toothpick and transferred to 96-well plates. 0.2 mL of YPD medium was added to each well. At the same time, single colonies of the recombinant strain GS115 / pPIC9K-Abxyl were streaked onto histidine auxotrophic plates and inoculated into 96-well plates under the same conditions.

[0033] Next, after culturing the 96-well plate at 30°C and 200 rpm for 24 h, 10 µL of culture from each well was added to a new 96-well deep plate. 1 mL of BMMY medium containing 1% (v / v) methanol was added to each well of the deep plate. After culturing at 30°C and 200 rpm for 60 h, the supernatant from each well was collected by centrifugation.

[0034] The enzyme activity of the supernatant was measured, revealing that the enzyme activity of the vast majority of mutants remained unchanged or decreased compared to the wild-type Abxyl. Through 10 rounds of random mutation and screening, a total of 950 mutant transformants were screened. Among them, 878 mutants showed decreased activity, accounting for 92.4%; 65 mutants showed essentially unchanged activity, accounting for 6.84%; and 7 mutants showed increased activity, accounting for 0.74%. These screening results indicate that the enhancement of β-xylosidase activity cannot be achieved simply through conventional mutations, but rather requires screening from a large number of ineffective or even negatively effective mutations.

[0035] The seven β-xylosidase mutants with enhanced activity were Abxyl-S175F, Abxyl-V293I, Abxyl-Q390E, Abxyl-A449S, Abxyl-N526G, Abxyl-Y565K, and Abxyl-N576A. Specific enzyme activity data are shown in Table 1.

[0036] Table 1. Enzyme activity test results of β-xylosidase Abxyl mutants corresponding to different mutation sites

[0037]

[0038] The method for determining β-xylosidase activity is as follows: Using 4-nitrophenyl-β-D-xylopyranoside (pNPX) as a substrate, 50 µL of enzyme solution and 150 µL of pNPX substrate buffer are mixed and reacted at 60 °C for 15 min. 1 mL of 1 mol / L Na₂CO₃ solution is added to terminate the reaction. 200 µL of the reaction solution is then measured at 405 nm. The enzyme activity unit (U) is defined as the amount of enzyme required to generate 1 μmol of pNP per minute under optimal conditions.

[0039] Meanwhile, the corresponding β-xylosidase Abxyl single-point mutant protein expression strains were obtained as follows: GS115 / pPIC9K-Abxyl-S175F, GS115 / pPIC9K-Abxyl-V293I, GS115 / pPIC9K-Abxyl-Q390E, GS115 / pPIC9K-Abxyl-A449S, GS115 / pPIC9K-Abxyl-N526G, GS115 / pPIC9K-Abxyl-Y565K, and GS115 / pPIC9K-Abxyl-N576A.

[0040] Example 3

[0041] The construction of recombinant plasmids carrying the β-xylosidase Abxyl double-point mutant gene and genetically engineered strains involves superimposing other mutation sites that can enhance Abxyl activity onto the β-xylosidase mutant Abxyl-N576A, which has the greatest increase in enzyme activity, to construct the β-xylosidase Abxyl double-point mutant.

[0042] Based on the gene sequence of the synthesized wild-type xylanase, corresponding site-directed mutagenesis primers were designed. Each set of site-directed mutagenesis primers includes a first primer and a second primer. When the same site is mutated to different amino acids, the same first primer and different second primers are used. The primer sequences are shown in Table 2.

[0043] Table 2. Primer sequences corresponding to different mutation sites

[0044]

[0045] Using plasmid pPIC9K-Abxyl as a template, PCR amplification was performed using KOD Fx Neo high-fidelity DNA polymerase (Toyobo Shanghai Biotechnology Co., Ltd., KFX-201) with primers N576A-1 and N576A-2, respectively. The PCR products were digested using DpnI enzyme, and 10 μL of the digested product was transformed into *E. coli* Top10 competent cells (Beijing Zhuangmeng International Biotechnology Co., Ltd., ZC104). The cells were then plated on LBK kanamycin-resistant plates for positive transformant selection. Transformants that had been correctly identified by colony PCR were then inoculated into LB medium, and plasmids were extracted. Sequencing verification by Shanghai Sangon Biotech Co., Ltd. yielded the correct recombinant plasmid pPIC9K-Abxyl-N576A carrying the Abxyl-N576A mutant gene.

[0046] Using plasmid pPIC9K-Abxyl-N576A as a template, PCR amplification was performed using KOD Fx Neo high-fidelity DNA polymerase (Toyobo Shanghai Biotechnology Co., Ltd., KFX-201) with multiple primer pairs listed in Table 2: S175F-1 and S175F-2, V293I-1 and V293I-2, Q390E-1 and Q390E-2, A449S-1 and A449S-2, N526G-1 and N526G-2, and Y565K-1 and Y565K-2.

[0047] The PCR products were digested using DpnI enzyme, and 10 μL of the digested product was transformed into *E. coli* Top10 competent cells (Beijing Zhuangmeng International Biotechnology Co., Ltd., ZC104). The transformed cells were then plated on LBK kanamycin-resistant plates for positive transformant selection. Transformants identified by colony PCR were then inoculated into LB medium, and plasmids were extracted. Sequencing by Shanghai Sangon Biotech Co., Ltd. yielded the correct recombinant plasmids carrying the double-point mutant gene: pPIC9K-Abxyl-N576A-S175F, pPIC9K-Abxyl-N576A-V293I, pPIC9K-Abxyl-N576A-Q390E, pPIC9K-Abxyl-N576A-A449S, pPIC9K-Abxyl-N576A-N526G, and pPIC9K-Abxyl-N576A-Y565K.

[0048] The six correct recombinant plasmids carrying the Abxyl double-point mutant gene were linearized using the restriction endonuclease SalI. The six linearized vectors were purified and recovered, and then electroporated into Pichia pastoris. GS115 competent cells (Ingenie Life Sciences, Inc., C18100, USA) were screened for positive transformants on histidine auxotrophic plates to obtain recombinant strains expressing the corresponding Abxyl double-point mutant proteins: GS115 / pPIC9K-Abxyl-N576A-S175F, GS115 / pPIC9K-Abxyl-N576A-V293I, GS115 / pPIC9K-Abxyl-N576A-Q390E, GS115 / pPIC9K-Abxyl-N576A-A449S, GS115 / pPIC9K-Abxyl-N576A-N526G, and GS115 / pPIC9K-Abxyl-N576A-Y565K.

[0049] The recombinant strain GS115 / pPIC9K-Abxyl-N576A, as well as the aforementioned recombinant strains GS115 / pPIC9K-Abxyl-N576A-S175F, GS115 / pPIC9K-Abxyl-N576A-V293I, GS115 / pPIC9K-Abxyl-N576A-Q390E, GS115 / pPIC9K-Abxyl-N576A-A449S, GS115 / pPIC9K-Abxyl-N576A-N526G, and GS115 / pPIC9K-Abxyl-N576A-Y565K, were inoculated into BMGY medium and cultured at 30℃ and 250rpm for 20h. Cells were collected by centrifugation at 6000 rpm and 4°C for 5 min. The collected cells were then resuspended in BMMY medium until the initial OD600 approached 0.5. The cells were then cultured at 30°C with shaking at 250 rpm, with 1% methanol added daily to achieve a final concentration. After five days of fermentation, the fermentation supernatant was recovered by centrifugation at 6000 rpm and 4°C for 5 min, and β-xylosidase activity was measured. The results are shown in Table 3. Figure 1 The enzyme activity test results are shown. Here, three sets of parallel experiments were performed for each combination of β-xylosidase mutants.

[0050] Table 3. Enzyme activity test results of combined mutant β-xylosidase

[0051]

[0052] As shown in Table 3 and Figure 1As shown, some combinations of mutation sites exhibit conflicting effects. For example, the activity of the combined mutant N576A-A449S is reduced by 26% compared to the single-site mutant N576A, from 58.1 U / mL to 42.8 U / mL. Some combinations of sites failed to further improve activity; for instance, the activity of the double mutant N576A-N526G is 57.4 U / mL, essentially the same as the single mutant activity. However, most combinations of mutation sites show a significant synergistic effect in improving enzyme activity. For example, N576A-S175F, N576A-V293I, N576A-Q390E, and N576A-Y565K show an 11%-75% increase in activity compared to the single-site mutant N576A, with the double-site mutant N576A-Y565K exhibiting the highest activity of 101.9 U / mL.

[0053] Furthermore, the thermostability of the combined mutant β-xylosidase protein was tested. The test method was as follows: 500 μL of the fermentation supernatant enzyme solution of different recombinant strains prepared in the above steps was taken into 1.5 mL centrifuge tubes and placed in a constant temperature mixer at 60℃ for different incubation times. After incubation, the precipitate was separated by centrifugation at 13300 rpm for 2 min. Then, an appropriate amount of supernatant was taken for β-xylosidase activity detection. The remaining enzyme activity was calculated using the activity of the enzyme solution that had not undergone heat incubation as a control. The thermostability test results of β-xylosidase are shown in Table 4.

[0054] Table 4. Results of thermostability tests for combined mutant β-xylosidase

[0055]

[0056] Table 4 shows that, compared to the wild-type β-xylosidase Abxyl protein, the mutant N576A exhibits slightly improved thermostability, with its half-life at 60℃ increasing from 2.44 h to 2.66 h. However, the effects of combining other sites on protein thermostability are more complex than those of the single-site mutant N576A. The half-lives of N576A-S175F and N576A-A449S decreased by 37.5% and 71.3%, respectively, to 1.66 h and 0.76 h. In contrast, the thermostability of N576A-V293I, N576A-Q390E, N576A-N526G, and N576A-Y565K is significantly improved, with the mutant N576A-Y565K showing the most significant effect, increasing the half-life to 13.62 h, a 411% improvement compared to the wild-type β-xylosidase Abxyl. Furthermore, as shown in Table 3, the mutant N576A-Y565K also exhibited the highest activity, indicating that in this embodiment, by performing combined mutations on β-xylosidase, the enzyme activity and thermostability of β-xylosidase can be simultaneously and significantly improved.

[0057] Example 4

[0058] To verify the ability of the β-xylosidase Abxyl mutant to hydrolyze lignocellulose, wild-type β-xylosidase Abxyl and the β-xylosidase Abxyl mutant were combined with endoxylanase, and the final xylose yield was measured.

[0059] 2.616 g of wheat straw was weighed and pretreated with delignification to obtain 1 g of dry matter. Water was added and mixed thoroughly. Then, 2.5 mL of 1 mol / L acetate-sodium acetate buffer and 500 U of endoxylanase (referring to the xylanase PuXyn-WT disclosed in patent publication number CN120173917A) were added, and water was added to bring the system to 50 mL, making the dry matter content 2%, thus obtaining hydrolysis system 1. Based on the above hydrolysis system 1, 500 U of wild-type β-xylosidase Abxyl-WT from Example 1, the single-point mutant N576A from Example 2, or any one of the double-point mutants from Example 3 were added sequentially to obtain hydrolysis systems 2 through 9.

[0060] The above hydrolysis systems 1-9 were placed in a 60℃ water bath shaker for 5 hours for enzymatic hydrolysis. The solutions were then removed and separated using a 300-mesh mesh bag to obtain a first hydrolysate. An equal volume of 8% sulfuric acid (m / v) was added to the first hydrolysate, and the solution was acid-hydrolyzed at 121℃ for 60 minutes in an autoclave. Sodium hydroxide was added to the acid hydrolysate to adjust the pH to the range of 5-7. The sugar content in the obtained complete hydrolysate was determined according to the method in GB / T 35545-2017, and the sugar yield was calculated. The formula for calculating the xylose yield is as follows:

[0061] Xylose yield = xylose content / dry matter content × 100%.

[0062] It should be noted that, due to solvent peak interference observed in the HPLC analysis of the xylooligosaccharide peak position in the first hydrolysis solution, the above-mentioned acid hydrolysis method was used to hydrolyze xylooligosaccharide into xylose to characterize the degree of hemicellulose degradation. Therefore, the xylose yield after the first hydrolysis is the sum of the xylooligosaccharide yield and the xylose yield. The xylose yields of different hydrolysis systems are shown in Table 5.

[0063] Table 5. Xylose yield test results for hydrolysis systems 1-9

[0064]

[0065] As shown in Table 5, compared to the hydrolysis system with only xylanase, the combination of xylanase and xylosidase significantly improved the xylose yield. The combination of wild-type β-xylosidase Abxyl-WT and xylanase PuXyn-WT increased the sugar yield by 1.51 times, while the combination of the single-point mutant N576A and xylanase PuXyn-WT increased the sugar yield by 1.83 times. Compared to the single-point mutant, the double-point mutants (excluding N576A-A449S) further improved the sugar yield during lignocellulose hydrolysis. Among them, the double-point mutant N576A-Y565K, with the highest enzyme activity, also showed the best hydrolysis effect, with a sugar yield 1.70 times higher than that of wild-type β-xylosidase Abxyl-WT and 1.41 times higher than that of the single-point mutant N576A. This indicates that the β-xylosidase mutant obtained in this embodiment not only has higher enzyme activity, but also exhibits a significantly enhanced synergistic effect when it hydrolyzes lignocellulose in conjunction with xylanase, thereby achieving a significant increase in xylose yield.

[0066] In summary, the β-xylosidase mutants obtained through directed evolutionary screening in this invention exhibit significant optimization effects across multiple performance dimensions. On one hand, single-point mutants generally improve enzyme activity, with N576A showing the largest increase. Further analysis of combinatorial mutations revealed complex interactions between different sites, potentially producing both negative conflict effects and significant synergistic effects. The double-point mutant N576A-Y565K exhibited the best performance, with its enzyme activity significantly increased to 101.9 U / mL. On the other hand, regarding thermostability, the mutants also exhibited a non-linear variation, with some combinations leading to decreased stability. N576A-Y565K, however, achieved a simultaneous and substantial increase in both enzyme activity and thermostability, with its half-life more than four times longer than the wild type, demonstrating excellent overall performance. Furthermore, in practical applications, the aforementioned mutants, when combined with xylanase for the hydrolysis of lignocellulose, significantly improved xylose yield, with the increase consistent with changes in their enzymatic properties. In particular, N576A-Y565K exhibited the best synergistic hydrolysis effect. Therefore, the mutants obtained in this invention not only optimize single enzymatic indicators but also demonstrate significant application advantages in complex substrate systems, and the effects of different mutant site combinations are noticeably unpredictable.

[0067] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0068] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A β-xylosidase mutant of Aspergillus brasiliensis origin, characterized in that, The β-xylosidase mutant is based on the sequence shown in SEQ ID NO: 1, and the amino acid mutation is a single-point mutation of N576A.

2. A β-xylosidase mutant of Aspergillus brasiliensis origin, characterized in that, The β-xylosidase mutant is based on the sequence shown in SEQ ID NO: 1, and the amino acid mutation is a combination mutation of N576A combined with any one of V293I, Q390E, N526G or Y565K.

3. The β-xylosidase mutant encoding gene according to any one of claims 1-2.

4. A recombinant plasmid containing the β-xylosidase mutant encoding gene according to claim 3.

5. A genetically engineered bacterium, characterized by, 5. A genetically engineered bacterium comprising the β-xylosidase mutant encoding gene according to claim 3.

6. A modified cell, wherein, 6. A modified cell comprising the recombinant plasmid according to claim 4.

7. Use of the β-xylosidase mutant according to any one of claims 1-2, or the genetically engineered bacterium according to claim 5, or the modified cell according to claim 6 in the production of xylose from lignocellulose hydrolysis.

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