High-efficiency degradation of straw powder by non-natural xylanase and application method thereof

Abstract

The application provides a kind of high-efficiency degradation straw powder non-natural xylanase and its application method, constructs non-natural xylanase StrXynB-GZ, the temperature stability of the non-natural xylanase at 65 DEG C has been significantly improved, can still maintain higher activity under high temperature condition of temperature > 65 DEG C, and then can be enzymolysis straw at 65 DEG C, the xylose output of the non-natural xylanase is significantly higher than that of unmodified natural enzyme and traditional sulfuric acid chemical method, and the enzymolysis process is environmentally friendly, no harmful waste liquid, no loss of cellulose, and the enzyme can be expressed and purified by prokaryotic system, suitable for industrial application, provides an efficient enzymolysis scheme for straw resource utilization.

Description

Technical Field

[0001] This invention relates to the field of straw degradation, and in particular to a non-natural xylanase that efficiently degrades straw powder and its application method. Background Technology

[0002] The main components of straw are cellulose, hemicellulose, and lignin, with cellulose being an important raw material in textiles, biomaterials, and other fields. Traditional methods of cellulose extraction use chemical methods, which generate large amounts of harmful waste liquid, causing serious environmental problems. This process also leads to structural damage and loss of effective components in cellulose, limiting extraction efficiency and quality. In contrast, utilizing specific enzymes to directionally degrade hemicellulose to achieve efficient separation of hemicellulose and cellulose is a more economical and environmentally friendly cellulose extraction technology. The core of this technology lies in the development of high-performance xylanase. The main component of hemicellulose is xylan, which is formed by the polymerization of xylose through β-1,4 glycosidic bonds to form polypentoses. Xylanase can specifically catalyze the breaking of β-1,4 glycosidic bonds, thereby degrading xylan into xylooligosaccharides and separating them from the straw, thus achieving efficient removal of hemicellulose.

[0003] From a protein structure classification perspective, the vast majority of xylanases belong to the GH10 and GH11 families. These two families of enzymes each have their own characteristics: GH10 family xylanases have larger molecular weights and lower isoelectric points; GH11 family xylanases have relatively smaller molecular weights and higher isoelectric points, and they are more specific to xylan substrates and have better catalytic targeting. At the same time, due to their simple structure, they have become the core research object for the artificial molecular modification of xylanases. However, GH11 family xylanases are generally mesophilic enzymes with poor thermal stability and cannot tolerate the high-temperature reaction environment in the straw degradation process, which has become the core bottleneck restricting their industrial application. Therefore, it is necessary to modify GH11 family xylanases to adapt to the needs of straw degradation scenarios.

[0004] Currently, the main methods for modifying xylanases include directed evolution, semi-rational design, and rational design. Directed evolution and semi-rational design require the construction of large-scale or small-scale mutant libraries, followed by high-throughput screening to obtain the target mutant enzyme. This approach suffers from problems such as large experimental workload, low screening efficiency, and strong uncertainty in modification effects. In contrast, rational design, which utilizes artificial intelligence technology, can precisely modify xylanase functional regions through simulation analysis, site-directed mutagenesis, and domain replacement / addition. This approach can directionally improve the enzyme's physicochemical properties and catalytic performance, making it an efficient and precise method for xylanase molecular modification and a research hotspot in this field.

[0005] Specifically, the core functional components of xylanase mainly include the GH catalytic domain and the CBM carbohydrate-binding domain. The main function of CBM is to promote the efficient binding of the enzyme to the xylan substrate, thereby improving catalytic efficiency, but its contribution to the thermal stability of the enzyme molecule itself is limited. For example, introducing the CBM6 family module into the GH11 family xylanase can increase its catalytic efficiency by 17%, and linking the C-terminus or N-terminus of the endoglucanase CelA to the C-terminus can increase the catalytic efficiency by 3-5 times compared to the unmodified original enzyme. Disulfide bonds are a classic molecular means of stabilizing the three-dimensional structure of proteins and improving thermal stability. They can maintain the native conformation of the enzyme molecule by reducing the entropy value of the unfolded state of the protein. Introducing a disulfide bond at the N-terminus of xylanase can significantly improve its thermal stability. For example, studies have reported that after constructing a disulfide bond at the N-terminus of Aspergillus oryzae-derived xylanase AoXyn11A, its optimal reaction temperature increased from 50℃ to 75℃. However, the introduction of disulfide bonds can only improve the enzyme's thermal stability, but cannot enhance its ability to recognize and bind substrates. In other words, how to rationally combine and optimize the functional elements of xylanase based on its structural characteristics to simultaneously improve its high-temperature tolerance and straw hydrolysis efficiency remains a critical technological gap to be addressed in this field. Summary of the Invention

[0006] The purpose of this invention is to provide a non-natural xylanase that is highly efficient at degrading straw powder and its construction method. The non-natural xylanase is obtained by modifying xylanases of eukaryotic or prokaryotic origin, so that it has both excellent high temperature tolerance and straw enzymatic hydrolysis efficiency.

[0007] To achieve the above objectives, this scheme modifies and verifies a non-natural xylanase that efficiently degrades straw powder, providing some theoretical support for subsequent experiments. Its amino acid sequence is shown in SEQ ID NO.1.

[0008] The modified and validated non-natural xylanase for efficiently degrading straw powder in this scheme is recombinant SviXyn10A-CBM22, in which the parent enzyme of recombinant SviXyn10A-CBM22 is SviXyn10A, and CBM22 is replaced in SviXyn10A.

[0009] This highly efficient non-natural xylanase for degrading straw powder was developed using SviXyn10A (Saccharomonospora viridis), a xylanase derived from *Saccharomonospora viridis*, as the template. The amino acid sequence of SviXyn10A is shown in SEQ ID NO.2. SviXyn10A is a prokaryotic xylanase composed of a GH10 domain and a C-terminal CBM domain, representing a relatively classic xylanase structure.

[0010] This method replaces the original CBM domain at the C-terminus of SviXyn10A xylanase with the CBM22 domain derived from the thermophilic bacterium *Acetivibrio thermocellus*. This is because CBM22 significantly improves the temperature stability of xylanase. It should be noted that while the CBM domain family is extensive, not all CBM domains enhance the thermostability of GH10 family xylanases. For example, xylanases with the CBM6 domain linked to the N-terminus or C-terminus of GH10 family xylanases actually lose 17% and 11% of their activity at 60℃, respectively.

[0011] Specifically, the amino acid sequence at positions 305-410 of the amino acid sequence shown in SEQ ID NO.2 is replaced with the amino acid sequence shown in SEQ ID NO.3.

[0012] Furthermore, the linker between the CBM and GH domains has a significant impact on the fusion between the domains and the overall properties of xylanase. In this scheme, a flexible linker (GGGGS)2 is used to link the CBM22 and GH10 domains to improve their thermostability. Specifically, the parent enzyme of the recombinant SviXyn10A-CBM22 in this scheme is SviXyn10A, and CBM22 is added to the GH10 domain of SviXyn10A. The CBM22 and GH10 domains are linked by a flexible linker (GGGGS)2.

[0013] Based on the above results, it was determined that the replacement of the CBM domain of thermophilic bacteria can improve the thermal stability of xylanase to a certain extent. Based on the above conclusion, firstly, this technical solution provides a non-natural xylanase that efficiently degrades straw powder, the amino acid sequence of which is shown in SEQ ID NO.4.

[0014] The non-natural xylanase for efficiently degrading straw powder provided by this scheme is recombinant StrXynB-GZ, in which the parent enzyme of recombinant StrXynB-GZ is StrXynB27, and the disulfide bond in the non-parent enzyme is replaced by a homologous fragment at the N-terminus of the GH11 catalytic domain of StrXynB27, and the N-terminus of the GH11 catalytic domain is connected to the CBM9 domain.

[0015] The amino acid sequences of StrXynB27 and StrXynB-GZ are as follows: Figure 5 As shown, the three-dimensional structures and disulfide bond positions of StrXynB27 and StrXynB-GZ are illustrated in the diagram. Figure 7 As shown. Figure 5The image shows a comparison of the amino acid sequences of StrXynB27 and the modified StrXynB-GZ. A represents StrXynB, and B represents StrXynB-GZ. Figure 7 The image shows a comparison of the amino acid sequences of StrXynB27 and the modified StrXynB-GZ. A represents StrXynB and B represents StrXynB-GZ. The positions of the disulfide bonds are marked in B.

[0016] As mentioned in the background, there is a natural balance between the high-temperature tolerance of xylanase and its enzymatic hydrolysis ability in straw. The catalytic activity of xylanase depends on its specific flexible three-dimensional active conformation. However, improving its high-temperature tolerance requires modifying the enzyme molecule to be more rigid and stable. This leads to an overemphasis on structural rigidity, which reduces the flexibility of the active center. Consequently, the binding of xylanase to xylan substrates and the conformational changes in the catalytic reaction are hindered, directly weakening the hydrolysis efficiency. Furthermore, if thermal stability is improved only by increasing hydrophobic interactions and constructing disulfide bonds without considering the structure of the substrate-binding region, the enzyme's substrate recognition ability will decrease. Even if the structure remains undegraded at high temperatures, it will be unable to efficiently bind xylan in straw, resulting in a significant reduction in hydrolysis ability. Conversely, if the pursuit of hydrolysis ability is pursued at all costs (such as retaining a highly flexible active conformation and strengthening the substrate-binding ability of the CBM domain), the enzyme molecule will rapidly undergo conformational unfolding at high temperatures, destroying the active center and drastically losing its hydrolysis ability at high temperatures. In other words, constructing disulfide bonds alone can only improve thermal stability but not enzymatic hydrolysis ability, while linking CBM domains alone can only improve enzymatic hydrolysis ability but not thermal stability. This scheme breaks this contradiction by introducing disulfide bonds at the N-terminus of xylanase from eukaryotes to improve its high-temperature tolerance, and simultaneously linking CBM domains from thermophilic bacteria to improve enzymatic hydrolysis ability. This combination of modification allows xylanase to maintain structural stability at temperatures of 65°C and above, and to enhance substrate binding through the CBM domain, thus achieving highly efficient enzymatic hydrolysis at high temperatures.

[0017] Our team screened and compared multiple GH11 family xylanases in the literature and found that the optimal temperature for these xylanases was generally 40-60℃, and their temperature stability was <60℃, indicating that they were not tolerant of high-temperature environments. Ultimately, we selected the xylanase StrXynB27 from *Streptomyces sp. S27* as the parent enzyme for modification. The amino acid sequence of *Streptomyces sp. S27* xylanase StrXynB27 is shown in SEQ ID NO. 5. This is because this xylanase is a eukaryotic GH11 family xylanase, containing only one GH11 catalytic domain, with a simple structure. According to the literature, the optimal temperature for StrXynB27 is 65℃, and its stability is poor above 60℃, indicating significant potential for modification.

[0018] It should be noted that, from a protein structure perspective, the vast majority of xylanases belong to the GH10 and GH11 families. GH10 family xylanases have larger molecular weights and lower isoelectric points, while GH11 family xylanases have relatively smaller molecular weights, higher isoelectric points, and better substrate specificity. However, GH11 family xylanases are mostly mesophilic enzymes, unable to tolerate the high-temperature environment of xylan decomposition, and their small molecular weight and simple structure make them the preferred targets for artificial modification of xylanases. In terms of origin, xylanases belong to a broad family of glycoside hydrolases, mostly derived from prokaryotic and eukaryotic microorganisms. Eukaryotic xylanases typically have an optimal pH that is slightly acidic and a more complex structure, which may lead to problems such as abnormal glycosylation and low secretion efficiency when expressed in heterologous systems. Prokaryotic xylanases, on the other hand, often have an optimal pH that is neutral or weakly alkaline, a simple structure, and are suitable for heterologous expression systems. Xylanase StrXynB27 combines the advantages of both the GH11 family and eukaryotic sources.

[0019] Furthermore, the non-parental enzyme is the engineered thermostable enzyme xyn11Ts, that is, in this scheme, the disulfide bond of the engineered thermostable enzyme xyn11Ts is introduced into StrXynB27.

[0020] Furthermore, by replacing the first 35 amino acids at the N-terminus of the engineered thermostable enzyme xyn11Ts with the first 35 amino acids at the N-terminus of StrXynB27, a natural disulfide bond is introduced into the engineered thermostable enzyme. Specifically, the amino acid sequence from position 1 to 73 of the amino acid sequence shown in SEQ ID NO.5 is replaced with the amino acid sequence shown in SEQ ID NO.6. The amino acid sequence alignment results of StrXynB27 and the engineered thermostable enzyme xyn11Ts are shown in the figure below. Figure 6 As shown in the figure, the first 35 amino acid sequences of xyn11TsN have low similarity to StrXynB27, and this region contains disulfide bonds, which are a region that contributes significantly to the thermostability of xylanase. Therefore, the first 35 amino acids of xyn11TsN were selected for fragment replacement.

[0021] Disulfide bonds are covalent bonds formed between sulfur atoms when the -SH residues between two cysteine ​​residues in a protein molecule are oxidized. They play a crucial role in the stability of protein structure. Our team discovered through sequence alignment that some GH11 family xylanases contain disulfide bonds, which explains their high-temperature tolerance. The N-terminal region is the initiation region for the unfolding of GH11 family xylanase molecules and is closely related to their thermostability. Furthermore, our team found through sequence alignment that the engineered thermostable enzyme xyn11Ts has two natural disulfide bonds at its N-terminus. Moreover, the N-terminus of the engineered thermostable enzyme xyn11Ts differs from that of xylanase StrXynB27. These differences are the basis for the high-temperature resistance of the engineered thermostable enzyme xyn11Ts. Therefore, our approach involves replacing the corresponding amino acids at the N-terminus of xylanase StrXynB27 with the first 35 amino acids of the engineered thermostable enzyme xyn11Ts to artificially introduce disulfide bonds.

[0022] Furthermore, the N-terminus of the GH11 catalytic domain is linked to the CBM9 domain derived from thermophilic anaerobic bacteria via a flexible linker peptide.

[0023] Furthermore, the N-terminus of the GH11 catalytic domain is linked to the CBM9 domain derived from *Thermotoga maritima* via a flexible linker peptide. *Thermotoga maritima* is an extreme thermophilic microorganism with an optimal growth temperature of approximately 80–85°C and can survive stably in high-temperature environments of 70–90°C. Its proteins (including enzymes and domains) inherently possess structural features adapted to high temperatures (such as a more compact three-dimensional conformation and more hydrophobic interactions), which is the core reason for its use in modifying the thermostability of xylanases. Linking the thermostable CBM domain of *Thermotoga maritima* to the N-terminus of the GH11 family xylanase StrXynB27 directly endows the modified enzyme with stronger high-temperature tolerance without significantly affecting its catalytic activity.

[0024] Furthermore, since the thermal degradation of xylanase begins from the N-terminus, our team linked the CBM9 domain and the GH11 domain with a (GGGGS)3 flexible linker before linking the CBM9 domain to the N-terminus of StrXynB27. That is, the amino acid sequence shown in SEQ ID NO. 7 is linked with a (GGGGS)3 flexible linker before the first amino acid sequence shown in SEQ ID NO. 5.

[0025] Secondly, this scheme provides a recombinant plasmid comprising a nucleic acid sequence encoding an amino acid sequence of a non-natural xylanase as shown in SEQ ID NO.4.

[0026] In some embodiments, the recombinant plasmid is pET-28a (+).

[0027] Thirdly, this solution provides a method for preparing a non-natural xylanase that efficiently degrades straw powder, comprising introducing a recombinant plasmid containing a nucleic acid sequence encoding an amino acid sequence of the non-natural xylanase shown in SEQ ID NO.4 into a prokaryotic host cell, and after inducing expression, separating and purifying the non-natural xylanase to obtain it.

[0028] Further, the nucleic acid sequence encoding the non-natural xylanase of the first aspect is cloned into an expression vector to obtain a recombinant plasmid; the recombinant plasmid is introduced into a host cell to obtain a positive transformant; the positive transformant is cultured and the expression of the non-natural xylanase is induced; the non-natural xylanase is isolated and purified.

[0029] Fourthly, this solution provides the application of xylanase with the amino acid sequence shown in SEQ ID NO.4 in the high-temperature enzymatic hydrolysis of xylan.

[0030] In some embodiments, xylanase with an amino acid sequence as shown in SEQ ID NO.4 is used to degrade hemicellulose in straw.

[0031] Fifthly, this scheme provides a method for degrading hemicellulose in straw, comprising: using an amino acid sequence of a non-natural xylanase, as shown in SEQ ID NO.4, at a temperature of 65°C.

[0032] The sequence lists involved in this scheme are summarized in Table 1 below: Table 1 Sequence List

[0033] Compared with existing technologies, this technical solution has the following characteristics and beneficial effects: This scheme precisely modifies GH10 family xylanases through rational design, constructing the non-natural xylanase StrXynB-GZ. This scheme overcomes the limitations of single-modification approaches; by replacing the N-terminal fragment of the GH11 family enzyme with a disulfide bond and incorporating the thermophilic CBM9 domain, it simultaneously improves thermal stability and catalytic efficiency. The non-natural xylanase obtained by this scheme exhibits significantly improved temperature stability at 65℃, maintaining high activity even at temperatures above 65℃. This allows for the enzymatic hydrolysis of straw at 65℃. The xylose yield of the two non-natural xylanases is significantly higher than that of the unmodified natural enzyme and the traditional sulfuric acid chemical method. Furthermore, the enzymatic hydrolysis process is environmentally friendly, producing no harmful waste liquid and without cellulose loss. Simultaneously, the enzymes can be efficiently expressed and purified using a prokaryotic system, making them suitable for industrial applications and providing an efficient enzymatic hydrolysis solution for the resource utilization of straw. Attached Figure Description

[0034] Figure 1 These are colony PCR results for SviXyn10A, SviXyn10A-CBM22, and StrXynB-GZ.

[0035] Figure 2 These are SDS-PAGE results for SviXyn10A, SviXyn10A-CBM22, and StrXynB-GZ.

[0036] Figure 3 The graph shows the optimal temperature results for SviXyn10A, SviXyn10A-CBM22, and StrXynB-GZ.

[0037] Figure 4 The graph shows the temperature stability results of SviXyn10A, SviXyn10A-CBM22 and StrXynB-GZ.

[0038] Figure 5 This is the amino acid sequence diagram of StrXynB27 and StrXynB-GZ.

[0039] Figure 6 This is a diagram showing the amino acid sequence alignment results of StrXynB27 and xyn11Ts.

[0040] Figure 7 This is a diagram showing the three-dimensional structure and disulfide bond positions of StrXynB27 (A) and StrXynB-GZ (B). Detailed Implementation

[0041] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention are within the scope of protection of the present invention.

[0042] Example 1: Performance testing of SviXyn10A and SviXyn10A-CBM22: 1.1 Identification of positive transformants of xylanase SviXyn10A and SviXyn10A-CBM22 1) The xylanase gene SviXyn10A and its fusion gene SviXyn10A-CBM22 were synthesized in their entirety by Sangon Biotech (Shanghai) Co., Ltd. and cloned into the pET-28a(+) expression vector. The amino acid sequence of SviXyn10A-CBM22 is shown in SEQ ID NO.5. 2) Place 100 μL of thawed competent cells on ice, add 2-3 μL of plasmid DNA (concentration of approximately 10 ng / μL), gently mix, and incubate on ice for 10 minutes to obtain a mixture. The plasmid DNA is a recombinant pET-28a (+) plasmid that has been artificially modified and carries xylanase SviXyn10A or SviXyn10A-CBM22. 3) Heat shock the mixture in a 42°C water bath for 60 seconds, then transfer it to ice to cool for 3 minutes. Then add 600 μL of antibiotic-free LB liquid medium and place it in a 37°C shaker at 200 rpm for 45 minutes to obtain the revived bacterial culture. 4) Spread 80 μL of the resuscitated bacterial solution evenly onto an LB agar plate containing kanamycin (final concentration 50 ng / μL), and invert the plate in a 37°C incubator for about 12 hours. 5) Pick a single colony from the plate and inoculate it into a centrifuge tube containing 50 ng / μL kanamycin LB liquid medium. Pre-incubate at 37°C and 200 rpm for 2 hours. Use this bacterial culture as a template for colony PCR amplification. The colony PCR results for SviXyn10A and SviXyn10A-CBM22 are as follows: Figure 1 As shown, Figure 1 In the diagram, A corresponds to SviXyn10A, and B corresponds to SviXyn10A-CBM22. The amplification products were analyzed by 1% agarose gel electrophoresis to identify positive transformants containing the target plasmid. Figure 1 As can be seen, the colony PCR results show that SviXyn10A and SviXyn10A-CBM22 were successfully ligated.

[0043] 1.2 Xylanase SviXyn10A and SviXyn10A-CBM22 induced expression and SDS-PAGE verification: 1) Inoculate the positive bacterial culture into 10 ml of liquid medium with anti-LB, and culture overnight at 37°C with shaking at 200 rpm. After culture, take a certain amount of bacterial culture and add glycerol to a final concentration of 15% to preserve the culture. 2) Add 100 ml of liquid culture medium with LB resistance to the conical flask, inoculate the overnight culture solution at a ratio of 1:100, and then incubate at 37°C and 200 rpm until the OD600 = 0.6. 3) After removing and cooling the conical flask, add IPTG to the bacterial culture to a final concentration of 1 mM, and induce for 4 h at 25°C and 150 rpm in a constant temperature shaker. 4) Collect the bacterial culture by centrifugation at 4℃, 4000 rpm for 10 min, discard the supernatant, wash with lysis buffer (50 mM Tris, 500 mM NaCl, pH=7), and then concentrate and resuspend at a ratio of 10:1. 5) Place the bacterial culture in an ice-water bath and sonicate to disrupt the bacterial culture, 60% power, 2 s / 3 s, 30 min; 6) Centrifuge at 4℃, 12000 rpm, for 15 min and collect the supernatant; 7) The supernatant was purified by nickel ion affinity chromatography, using a gradient elution buffer containing imidazole. The results were then analyzed by SDS-PAGE, and the protein content was determined using the Bradford Protein Assay Kit. SDS-PAGE results for SviXyn10A and SviXyn10A-CBM22 are as follows: Figure 2 As shown, Figure 2 In the diagram, A corresponds to SviXyn10A, and B corresponds to SviXyn10A-CBM22. SDS-PAGE results show that the purified product contains the target band, indicating successful induction of expression. 8) If inclusion body expression is performed, after step 6), the precipitate is resuspended in lysis buffer containing 8 M urea, allowed to stand at room temperature for 1 h, centrifuged at 12000 rpm for 30 min, the supernatant is collected and purified and refolded.

[0044] 1.3 Detection of xylanase SviXyn10A and SviXyn10A-CBM22 enzyme activities: 1) Weigh 1 g of xylan and dissolve it in an appropriate amount of citric acid-Na2HPO4 (0.1 M citric acid, 0.2 M Na2HPO4, pH=6.5) buffer. After boiling in a water bath for 20 min, bring the volume up to 100 ml. Then centrifuge at 10,000 rpm for 10 min and take the supernatant as the xylan base for enzyme activity detection. 2) Take 900 μl of the xylan solution prepared in step 1) into a centrifuge tube, add 100 μl of diluted enzyme solution containing xylanase SviXyn10A or SviXyn10A-CBM22, react accurately in a 60℃ water bath for 10 min, add 2 ml of DNS reagent to terminate the reaction, boil in a water bath for 5 min, add 9 ml of distilled water, mix well, and measure the A540 absorbance. Then calculate the reducing sugar concentration and enzyme activity according to the standard curve.

[0045] 1.4 Optimal Temperature Experiment of Xylanase SviXyn10A and SviXyn10A-CBM22: The diluted enzyme solution (containing the same mass ratio of xylanase SviXyn10A or SviXyn10A-CBM22) was mixed with an equal volume of xylan substrate solution preheated to the corresponding temperature. The mixture was immediately placed in a water bath with a series of temperature gradients (30–80°C) and reacted precisely for 10 minutes. After the reaction, samples were immediately taken, and the reducing sugar content generated at each temperature was determined using the DNS method. The enzyme activity was calculated, with the highest enzyme activity measured at each temperature taken as 100%. The relative enzyme activities at different temperatures were calculated and compared to determine the optimal reaction temperature for the two enzymes. The experimental results are shown below. Figure 3 As shown, Figure 3 In the diagram, A represents xylanase SviXyn10A and B represents SviXyn10A-CBM22. The experimental results show that the optimal temperature of the modified xylanase SviXyn10A-CBM22 increased from 60℃ to 70℃ compared with the unmodified SviXyn10A, indicating that its tolerance to high temperature has been improved.

[0046] 1.5 Temperature stability experiment of xylanase SviXyn10A and SviXyn10A-CBM22 The diluted enzyme solution was heat-treated in constant temperature water baths at 60℃, 65℃, and 70℃. At time points 0 (starting time), 1, 2, 3, and 4 hours, equal amounts of samples were taken and rapidly cooled on ice to terminate the heat treatment. Subsequently, the samples at each time point were subjected to standard enzyme activity assays with xylan substrate at the optimum temperature. The enzyme activity measured at the optimum temperature of the untreated original enzyme solution was defined as 100%, and the residual relative enzyme activity of the samples at each time point was calculated. The difference in thermal stability between the two enzymes was evaluated by comparing the curves of the decrease in residual enzyme activity over time at different temperatures.

[0047] Experimental results are as follows Figure 4 As shown, Figure 4 In the diagram, A represents xylanase SviXyn10A, and B represents SviXyn10A-CBM22. Experimental results show that both the unmodified xylanase SviXyn10A and the modified SviXyn10A-CBM22 exhibit stability at 60℃ but instability at 70℃. However, SviXyn10A-CBM22 demonstrates superior temperature stability at 65℃ compared to SviXyn10A. SviXyn10A lost over 50% of its relative enzyme activity after 2 hours of incubation at 65℃, while SviXyn10A-CBM22 retained nearly 50% of its relative enzyme activity after 4 hours of incubation under the same conditions.

[0048] Example 2: Performance testing of StrXynB-GZ: 2.1 Identification of positive transformants of xylanase StrXynB-GZ 1) The xylanase gene StrXynB-GZ was synthesized in its entirety by Sangon Biotech (Shanghai) Co., Ltd. and cloned into the pET-28a(+) expression vector. The amino acid sequence of StrXynB-GZ is shown in SEQ ID NO.1. 2) Take 100 μL of thawed competent cells and place them on ice. Add 2-3 μL of plasmid DNA (concentration of about 10 ng / μL), mix gently, and incubate on ice for 10 minutes. The plasmid DNA is a recombinant pET-28a (+) plasmid that has been artificially modified and carries xylanase StrXynB-GZ. 3) Heat shock the mixture in a 42°C water bath for 60 seconds, then transfer it to ice to cool for 3 minutes. Add 600 μL of antibiotic-free LB liquid medium and incubate at 37°C with a shaker at 200 rpm for 45 minutes to obtain the revived bacterial culture. 4) Take 80 μL of resuscitated bacterial solution and spread it evenly on an LB agar plate containing kanamycin (final concentration 50 ng / μL). Invert the plate and incubate it in a 37°C incubator for about 12 hours. 5) Pick a single colony from the plate and inoculate it into a centrifuge tube containing 50 ng / μL kanamycin LB liquid medium. Pre-incubate at 37°C and 200 rpm for 2 hours. Use this bacterial culture as a template for colony PCR amplification. The colony PCR results for StrXynB-GZ are as follows: Figure 1 As shown, Figure 1 The C in the formula corresponds to StrXynB-GZ. The amplification products were analyzed by 1% agarose gel electrophoresis to identify positive transformants containing the target plasmid. Figure 1As can be seen, the colony PCR results show that StrXynB-GZ ligation was successful.

[0049] 2.2 Xylanase StrXynB-GZ induced expression and SDS-PAGE verification: 1) Inoculate the positive bacterial culture into 10 ml of liquid medium with anti-LB, and culture overnight at 37°C with shaking at 200 rpm. After culture, take a certain amount of bacterial culture and add glycerol to a final concentration of 15% to preserve the culture. 2) Add 100 ml of liquid culture medium with LB resistance to the conical flask, inoculate the overnight culture solution at a ratio of 1:100, and then incubate at 37°C and 200 rpm until the OD600 = 0.6. 3) After removing and cooling the conical flask, add IPTG to the bacterial culture to a final concentration of 1 mM, and induce for 4 h at 25°C and 150 rpm in a constant temperature shaker. 4) Collect the bacterial culture by centrifugation at 4℃, 4000 rpm for 10 min, discard the supernatant, wash with lysis buffer (50 mM Tris, 500 mM NaCl, pH=7), and then concentrate and resuspend at a ratio of 10:1. 5) Place the bacterial culture in an ice-water bath and sonicate to disrupt the bacterial culture, 60% power, 2 s / 3 s, 30 min; 6) Centrifuge at 4℃, 12000 rpm, for 15 min and collect the supernatant; 7) The supernatant was purified by nickel ion affinity chromatography, using a gradient elution buffer containing imidazole. The results were then analyzed by SDS-PAGE, and the protein content was determined using the Bradford Protein Assay Kit. The SDS-PAGE results for StrXynB-GZ are as follows: Figure 2 As shown, Figure 2 C in the formula corresponds to StrXynB-GZ. SDS-PAGE results showed that the purified product contained the target band, indicating successful induction of expression. 8) If inclusion body expression is performed, after step 6), the precipitate is resuspended in lysis buffer containing 8 M urea, allowed to stand at room temperature for 1 h, centrifuged at 12000 rpm for 30 min, the supernatant is collected and purified and refolded.

[0050] 2.3 Xylanase StrXynB-GZ enzyme activity assay 1) Weigh 1 g of xylan and dissolve it in an appropriate amount of citric acid-Na2HPO4 (0.1 M citric acid, 0.2 M Na2HPO4, pH=6.5) buffer. After boiling in a water bath for 20 min, bring the volume up to 100 ml. Then centrifuge at 10,000 rpm for 10 min and take the supernatant as the xylan base for enzyme activity detection. 2) Take 900 μl of the xylan solution prepared in step 1) into a centrifuge tube, add 100 μl of diluted enzyme solution containing StrXynB-GZ, react accurately in a 60℃ water bath for 10 min, add 2 ml of DNS reagent to terminate the reaction, boil in a water bath for 5 min, add 9 ml of distilled water, mix well, measure the absorbance at A540, and then calculate the reducing sugar concentration and enzyme activity according to the standard curve.

[0051] 2.4 Experiment on the optimal temperature of xylanase StrXynB-GZ The diluted enzyme solution (containing xylanase StrXynB-GZ in the same mass ratio as xylanase SviXyn10A-CBM22) was mixed with an equal volume of xylan substrate solution preheated to the appropriate temperature. The mixture was immediately placed in a water bath with a series of temperature gradients (30-90℃) and reacted precisely for 10 minutes. After the reaction, samples were immediately taken, and the reducing sugar content generated at each temperature was determined using the DNS method, and the enzyme activity was calculated. The highest enzyme activity measured at each temperature was taken as 100%, and the relative enzyme activities at different temperatures were calculated and compared to determine the optimal reaction temperature. Simultaneously, the results were compared with data from unmodified pristine xylanase reported in published literature to clarify the modification effect.

[0052] Experimental results are as follows Figure 3 As shown, Figure 3 In the figure, C represents xylanase StrXynB-GZ. As shown in the figure, the experimental results indicate that the optimal temperature for both the modified xylanase StrXynB-GZ and the unmodified original enzyme in the literature is 65℃. Above 65℃, the activity of the unmodified original enzyme drops significantly, with less than 20% relative enzyme activity remaining at 80℃, and complete inactivation at 90℃. In contrast, the modified xylanase StrXynB-GZ retains 97.8% and 71.9% relative enzyme activity at 80℃ and 90℃, respectively, demonstrating improved tolerance to high temperatures.

[0053] 2.5 Temperature stability experiment of xylanase StrXynB-GZ The diluted enzyme solution was heat-treated in a constant temperature water bath at 65℃, 70℃, 80℃, and 90℃. At time points 0 (start time), 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 h, equal amounts of samples were taken and rapidly cooled on ice to terminate the heat treatment. Subsequently, the samples at each time point were subjected to standard enzyme activity assays with the xylan substrate at the optimum temperature. The enzyme activity measured at the optimum temperature using the untreated original enzyme solution was defined as 100%, and the residual relative enzyme activity of the samples at each time point was calculated. The results were compared with data from previously published literature on the unmodified original xylanase to clarify the modification effect.

[0054] Experimental results are as follows Figure 4 As shown, Figure 4 The "C" in the figure represents xylanase StrXynB-GZ. Experimental results show that the unmodified original enzyme in the literature had a relative enzyme activity of less than 20% after incubation at 65℃ for 1 h, and was completely inactivated after incubation at 70℃ for 20 min. The modified xylanase StrXynB-GZ had an enzyme activity loss of less than 10% after incubation at 65℃ for 1 h, which is about 5 times higher than the unmodified original enzyme. After 4 h, only 29.14% of the enzyme activity was lost. Enzyme activity could be detected within 2.5 h of incubation at 70℃. At extreme temperatures of 80 and 90℃, the enzyme activity was still retained for at least 30 min, indicating that the modified xylanase StrXynB-GZ has a significantly improved temperature stability compared to the unmodified enzyme.

[0055] Example 3: Tests on the degradation of hemicellulose in straw by SviXyn10A, SviXyn10A-CBM22, and StrXynB-GZ: 1. Determination of key parameters for enzymatic removal of hemicellulose from straw Based on literature review and previous experimental results, the main process parameters for enzymatic hydrolysis of straw were determined as follows: solid-liquid ratio: 1:5.5-1:6; enzyme dosage: 4000 U / kg dry straw weight; temperature: 65℃; reaction time: 4 h.

[0056] 2. Comparison of the straw hydrolysis capabilities of xylanases SviXyn10A, SviXyn10A-CBM22, and StrXynB-GZ: An appropriate amount of enzyme solution (containing the same mass ratio of SviXyn10A, SviXyn10A-CBM22, or StrXynB-GZ) and 1.4% sulfuric acid were mixed evenly with 10 mg of straw powder and then placed in a 65℃ water bath for 30 min. After filtration, the supernatant was taken for testing and the xylose content was calculated and compared. The results are shown in Table 2 below: Table 2. Results of Xylose Content Test .

[0057] The results showed that the SviXyn10A-CBM22 system had the highest xylose content, followed by StrXynB-GZ and SviXyn10A, and finally the unmodified wild-type xylanase rRuXyn024 and the 1.4% sulfuric acid group reported in the literature. As a representative of traditional chemical treatment methods, the 1.4% sulfuric acid treatment of straw was less effective in decomposing hemicellulose than the modified enzymatic hydrolysis group, and it also caused cellulose loss. The two unmodified xylanases, rRuXyn024 and SviXyn10A, were also less effective in decomposing straw hemicellulose than the modified SviXyn10A-CBM22 and StrXynB-GZ.

[0058] Experimental results show that modifying the xylanase molecule improves its ability to degrade hemicellulose in straw.

[0059] Those skilled in the art should understand that 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 have been 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.

[0060] The above embodiments are merely illustrative of several implementation methods of this application, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this application should be determined by the appended claims.

Claims

1. A highly efficient non-natural xylanase for degrading straw powder, characterized in that, The amino acid sequence is shown in SEQ ID NO.

4.

2. A recombinant plasmid, characterized in that, Nucleic acid sequences including those encoding amino acid sequences of non-natural xylanases as shown in SEQ ID NO.

4.

3. A method for preparing a non-natural xylanase that efficiently degrades straw powder, characterized in that, The method involves introducing a recombinant plasmid containing a nucleic acid sequence encoding an amino acid sequence of a non-natural xylanase as shown in SEQ ID NO.4 into a prokaryotic host cell, followed by induction of expression, and then isolating and purifying the non-natural xylanase.

4. The method for preparing a non-natural xylanase that efficiently degrades straw powder according to claim 3, characterized in that, The nucleic acid sequence of the non-natural xylanase shown in SEQ ID NO.4 was cloned into an expression vector to obtain a recombinant plasmid. The recombinant plasmid was introduced into a host cell to obtain a positive transformant. The positive transformant was cultured and induced to express the non-natural xylanase. The non-natural xylanase was then isolated and purified.

5. The application of a xylanase with the amino acid sequence shown in SEQ ID NO.4 in the enzymatic hydrolysis of xylan, characterized in that, Xylanase hydrolyzes xylan at temperatures above 65°C.

6. A method for degrading hemicellulose in straw, characterized in that, include: At a temperature of 65°C, a non-natural xylanase with an amino acid sequence as shown in SEQ ID NO.4 was used.

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