Use of a xylanase AlXyn10A in the degradation of complex side chain containing xylans

Abstract

This invention belongs to the field of bioengineering technology, specifically disclosing the application of xylanase AlXyn10A in the degradation of xylans containing complex side chains. The amino acid sequence of the xylanase AlXyn10A provided by this invention is shown in SEQ ID NO:1, and this enzyme is derived from *Aspergillus foetida*. Aspergillus leporis The specific activity of the pure enzyme reached 306.4 U / mg, and Co 2+ It exhibits significant activation and excellent hydrolytic performance. Structural analysis shows that this xylanase can simultaneously recognize both FAX3 and MeGA substrates, effectively degrading xylans with complex side chains. It has broad application prospects in various fields such as food, agricultural waste resource utilization, textiles, papermaking, and environmental remediation. Applying this xylanase to the pear juice preparation process can significantly increase the juice yield and reducing sugar content while effectively preserving ascorbic acid.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology and relates to a xylanase, specifically the application of a xylanase AlXyn10A in the degradation of xylans containing complex side chains. Background Technology

[0002] Ferulaylarabinoxylan side chains (FAX3) and methylglucuronide xylan side chains (MeGA) are widely present in the cell walls of various plants. However, due to the relatively limited substrate specificity of existing xylanases, and the fact that most of them cannot effectively identify and efficiently hydrolyze xylans with complex side chains such as FAX3 and MeGA in the cell walls, multiple negative impacts are caused on product quality, processing efficiency, raw material utilization, and the release rate of functional components. In the food industry, for example, in the processing of fruit and vegetable juices such as pear juice and apple juice, FAX3 and MeGA contained in the cell walls will significantly reduce the clarification effect; the bran and endosperm of cereals such as wheat, corn, and oats are rich in FAX3. During flour processing and baking, undegraded FAX3 will not only limit the formation of gluten network, but also lead to smaller volume, uneven internal pores, rough texture, and rapid aging of flour products; in the extraction of plant proteins or starches, FAX3 and MeGA will lead to low yield, reduced whiteness and purity of the target product. In the field of biomass energy and biofuel production, using raw materials such as corn stalks, wheat stalks, and sugarcane bagasse as raw materials, the complex xylans can be degraded to release fermentable sugars, which can then be fermented to produce renewable energy sources such as bioethanol, biobutanol, and biomethane. However, undegraded FAX3 or MeGA can hinder enzyme-cellulose contact, and their strong hydrophilicity and steric hindrance can reduce saccharification efficiency. Furthermore, in the paper and pulp industry, bio-based material preparation, and agricultural waste resource utilization and organic fertilizer production, the efficient degradation of complex side chains such as FAX3 and MeGA plays a crucial role in improving product quality, raw material processing performance, and raw material utilization. Therefore, developing xylanases capable of efficiently hydrolyzing xylans containing complex side chains is particularly important.

[0003] With the upgrading of juice processing technology and the increasing health demands of consumers, pear juice, especially whole-pulp pear juice, is more competitive in the market than clarified pear juice because it can retain dietary fiber, vitamins, and other natural active nutrients in the raw materials to the greatest extent. However, traditional whole-pulp pear juice processing technology still faces many technical bottlenecks, which seriously restrict the improvement of product quality and processing efficiency. On the one hand, the cell wall structure of pears is complex, with a dense network formed by polysaccharides such as cellulose, pectin, and xylan, which encapsulates a large number of nutrients. This results in low raw material utilization during processing and incomplete release of core nutrients such as reducing sugars and vitamin C (Vc), causing waste of raw materials and reducing the nutritional added value of the product. On the other hand, whole-pulp pear juice is prone to stability problems such as sedimentation and stratification during storage, which not only damages the sensory appearance of the product but also affects consumer acceptance and limits its shelf life and market circulation. To solve these problems, enzyme technology, with its advantages of high efficiency, mildness, and environmental friendliness, has become a core auxiliary means in the field of juice processing. However, there are few reports on xylanases that can hydrolyze the side chains of fruit cell walls. Summary of the Invention

[0004] In view of the current situation where most xylanases in the prior art cannot efficiently degrade xylans with complex side chains in plant cell walls, one object of the present invention is to provide an application of xylanase AlXyn10A in the degradation of xylans with complex side chains. The present invention discovers that AlXyn10A can simultaneously recognize both FAX3 and MeGA side chains and specifically act on complex side chains, effectively degrading xylans with complex side chains.

[0005] Another objective of this invention is to provide a pear juice and its preparation method, which uses xylanase AlXyn10A or in combination with pectinase and cellulase to prepare pear juice, thereby achieving efficient release of reducing sugars, vitamin C and other nutrients in pear juice, while improving the stability and sensory quality of whole-fleshed pear juice. This is of great significance for promoting the technological upgrading of the pear juice processing industry and increasing the added value of products.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides the application of xylanase AlXyn10A in the degradation of xylan containing complex side chains, wherein the amino acid sequence of xylanase AlXyn10A is shown in SEQ ID NO.1; the xylan containing complex side chains includes at least one of xylan containing a ferulic acylated arabinoxylan side chain (FAX3) or xylan containing a methylglucuronic acid xylan side chain (MeGA).

[0007] The xylanase AlXyn10A provided by this invention is derived from rabbit cod aspergillus. Aspergillus leporisBelonging to the GH10 family, xylanase AlXyn10A consists of 327 amino acids, with a theoretical molecular weight of 33.34 kDa and an isoelectric point (pI) of 7.73. The purified xylanase AlXyn10A exhibited optimal activity at pH 6.5 and 50℃, showing significant hydrolytic activity against both beech xylan and steam-exploded corn cob xylan, with a specific activity of 306.4 U / mg after purification. Structural analysis revealed that xylanase AlXyn10A can simultaneously recognize both FAX3 and MeGA substrates. Specifically, Glu48, Glu72, and Asp299 form hydrogen bonds with the arabinose and ferulic acid side chains of FAX3, respectively, while Glu161 and Tyr200 specifically interact with the glucuronic acid side chain of MeGA. Through these hydrogen bonds or specific interactions, the substrate can be precisely anchored at the enzyme's active site, fixing the substrate conformation and creating the necessary space for subsequent hydrolysis of the two complex side chains.

[0008] The xylanase AlXyn10A provided by this invention can be applied to many fields such as food, agricultural waste resource utilization, textiles, papermaking and environmental protection, and has broad application prospects.

[0009] Preferably, the plant containing xylan with complex side chains includes at least one of pear, corn, wheat, rye, oats, pine, birch or cedar.

[0010] Preferably, when using xylanase AlXyn10A to degrade xylan containing complex side chains, a cobalt salt is added to the reaction system.

[0011] For example, Co in the reaction system 2+ The final concentration is 0.8mM-1.2mM.

[0012] Compared to other metal ions or reagents, only Co... 2+ It exhibits an activating effect on xylanase AlXyn10A, with a relative enzyme activity of 109%.

[0013] More preferably, the application includes its use in the preparation of pear juice.

[0014] In the production of whole-pear juice, compared with other xylanases, the xylanase AlXyn10A provided by this invention can significantly increase the juice yield due to its ability to hydrolyze the complex side chains in the pear cell wall, while promoting the release of nutrients from the pulp cells and effectively preserving the natural quality of the juice. Furthermore, this enzyme can also promote the hydrolysis of side chains to generate reducing sugars, further enhancing the storage stability of the pear juice and possessing significant application advantages.

[0015] Preferably, the preparation method of the xylanase AlXyn10A includes the following steps: recombinant Pichia pastoris expressing the xylanase AlXyn10A is cultured in high-density fermentation and methanol-induced fermentation to obtain xylanase AlXyn10A.

[0016] Furthermore, the nucleotide sequence of the gene encoding the xylanase AlXyn10A expressed in the recombinant Pichia pastoris is shown in SEQ ID NO:2.

[0017] For example, the codon-optimized nucleotide sequence was amplified by PCR, double-digested with enzymes, and ligated into the pPIC9K vector to obtain the recombinant expression plasmid pPIC9K-AlXyn10A. The plasmid construct was digested with restriction endonucleases to produce linearized DNA, which was then introduced into Pichia pastoris GS115 via electroporation to obtain recombinant Pichia pastoris.

[0018] The nucleotide sequence shown in SEQ ID NO:2 provided by this invention is a codon-optimized sequence.

[0019] Furthermore, the culture medium for the high-density fermentation culture includes BSM medium.

[0020] Furthermore, the high-density fermentation culture is carried out at a temperature of 27℃-33℃, a rotation speed of 580rpm-620rpm, and a system pH of 3.8-5.2.

[0021] Furthermore, during the high-density fermentation culture, when the wet cell biomass concentration is 180g / L-220g / L, the stirring speed is adjusted to 500rpm-1000rpm, the pH of the system is adjusted to 4-6, methanol is added, and the culture is induced for 72h-144h. The supernatant is then separated from the solid to obtain the crude xylanase AlXyn10A enzyme solution. Furthermore, the crude xylanase AlXyn10A solution was purified by SPFF chromatography to obtain xylanase AlXyn10A.

[0022] Secondly, the present invention provides a method for preparing pear juice, the method comprising the following steps: mixing pear pulp with water, adding xylanase AlXyn10A for enzymatic hydrolysis to obtain pear juice; wherein the amino acid sequence of the xylanase AlXyn10A is shown in SEQ ID NO:1.

[0023] The pear juice preparation method provided by the present invention can significantly improve the pear juice yield, and the content of reducing sugar, ascorbic acid and titratable acidity in the obtained pear juice is significantly increased.

[0024] Furthermore, during the enzymatic hydrolysis treatment, at least one of pectinase or cellulase is added.

[0025] Using AlXyn10A alone can increase the juice yield of pear juice by 4.38%. When xylanase AlXyn10A is used in combination with pectinase and cellulase, the juice yield can reach up to 95.53%, significantly improving the utilization rate of raw materials.

[0026] Furthermore, after the enzymatic hydrolysis, the pear juice is obtained by ultra-high pressure sterilization.

[0027] Pear juice produced using a combined enzymatic hydrolysis and ultra-high pressure process has a lower pH, higher titratable acidity, and higher ascorbic acid content compared to traditional pasteurized products, achieving an effective balance between production efficiency and natural quality.

[0028] Furthermore, the pear juice is made from whole pear pulp.

[0029] Whole-fruit pulp beverages are drinks made primarily from fruit, retaining all or most of the fruit pulp through processing. Unlike regular fruit juice (which filters out the pulp), they are characterized by their thick, full-bodied texture and rich content of dietary fiber and natural fruit nutrients. Pears, as an important agricultural product in my country, suffer from insufficient processing and severe product homogenization. Based on the advantages of whole-fruit pulp pear juice beverages, such as the limited variety of such products and the reduction of residue waste, this invention provides a whole-fruit pulp pear juice beverage that, while ensuring good taste and reducing fruit residue waste, also efficiently retains its nutrients and increases reducing sugar content, providing a theoretical basis for the deep processing and high-value utilization of pears in my country.

[0030] Thirdly, the present invention provides a pear juice prepared by the above-described method for preparing pear juice.

[0031] This invention applies xylanase AlXyn10A to the preparation of pear juice. Leveraging the enzyme's unique substrate recognition and catalytic properties, it achieves a dual improvement in the efficiency and quality of pear juice preparation. Compared to commonly used xylanase preparations in the prior art, it has the following significant advantages: 1. Nutrient release rate is significantly improved. Xylanase AlXyn10A can specifically recognize and hydrolyze key residues (Glu48, Glu72, Asp299, Glu161, Tyr200) of the complex side chains FAX3 and MeGA in pear cell walls. It can efficiently destroy the dense structure of pear cell walls, promote the full dissolution of nutrients such as reducing sugars and organic acids stored in the cells, and ensure the efficient release of nutrients in pear juice from the mechanism of action.

[0032] Compared with other xylanases, the preparation of pear juice using the xylanase AlXyn10A described in this invention can not only significantly increase the juice yield and reduce raw material loss, but also significantly improve the reducing sugar content, vitamin C content and titratable acidity of the obtained pear juice, effectively improving the flavor and nutritional value of the pear juice.

[0033] 2. More stable product quality The catalytic reaction conditions of xylanase AlXyn10A are mild and controllable. While promoting the release of nutrients, it will not damage the heat-sensitive nutrients in pear juice, nor will it easily cause adverse phenomena such as turbidity or layering in pear juice. It can effectively improve the batch consistency of pear juice products and ensure the stability of product quality.

[0034] The xylanase AlXyn10A provided by this invention can simultaneously recognize both FAX3 and MeGA side chains and specifically act on complex side chains, effectively degrading xylan containing complex side chains. It has good prospects for industrial application in many fields such as food, agricultural waste resource utilization, textiles, papermaking and environmental governance. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 The results of measuring wet cell biomass, xylanase activity, and extracellular protein concentration in the supernatant during the methanol induction stage of Example 1 of this invention are as follows: Figure 2 The above are the SDS-PAGE results of the fermentation supernatant at different induction times during the methanol induction stage in Example 1 of this invention. Figure 3 The SDS-PAGE result of purified xylanase AlXyn10A in Example 1 of this invention; Figure 4 The results of the enzymatic characterization of xylanase AlXyn10A in Example 2 of this invention are shown; wherein Figure 4 A represents the optimal pH measurement curve. Figure 4 B is the pH stability measurement curve. Figure 4 C represents the optimal temperature measurement curve. Figure 4 D represents the thermal stability curve; Figure 5The above describes the thin-layer chromatography results of xylanase AlXyn10A on the hydrolysate of steam-exploded corn cob and beech xylan in Example 2 of this invention. Figure 5 A represents corn cob bursting fluid. Figure 5 B represents beech wood xylan; Figure 6 This is a schematic diagram illustrating the three-dimensional structural simulation of xylanase AlXyn10A and its interaction with the substrate in Example 2 of the present invention; wherein, Figure 6 A represents the overall folding pattern of the protein. Figure 6 B represents the structural comparison of AlXyn10A (blue) and SoXyn10A (gold) with FAX3 and Araf, respectively, highlighting the interaction of key residues; Figure 6 C represents a comparison of the structures and related interactions of the complexes formed by AlXyn10A (blue) and SoXyn10A (gold) with MeGA. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0038] To better illustrate the embodiments provided by the present invention, further examples are given below.

[0039] In the following embodiments, the experimental methods used are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0040] I. Reagents or raw materials used in this invention Pichia pastoris GS115 (His4), Escherichia coli DH5α, and pPIC9K vector were purchased from Beijing Qingke Biotechnology Co., Ltd.; DNA polymerase was purchased from TransGen Biotech (Beijing, China); restriction endonucleases EcoRI and NotRI, and T4 DNA ligase were provided by Sangon Biotech (Shanghai) Co., Ltd.; food-grade pectinase (300,000 U / g) and food-grade cellulase (500,000 U / g) were purchased from Shandong Longket Enzyme Preparation Co., Ltd.; beech xylan was purchased from Megazyme (Bree, Ireland). Commercially available food-grade xylanase (290,000 U / g) was purchased from Shandong Longket Enzyme Preparation Co., Ltd.

[0041] In this invention, the preparation of pear juice is illustrated using Jinzhou Ya pear as an example. The Jinzhou Ya pears used in this invention are all provided by Chenwang Fruit Products Co., Ltd. of Jinzhou City.

[0042] II. Relevant Experimental Methods 1. Enzyme activity assay The reaction system containing a suitable substrate solution and diluted enzyme solution was incubated under specified conditions. After incubation, 1 mL of DNS reagent was added to terminate the reaction. The reaction solution was then boiled for 15 min, and 1 mL of 40% (w / v) potassium sodium tartrate solution was added and mixed thoroughly. After cooling to room temperature, the absorbance was measured at a wavelength of 540 nm. In this assay, xylanase activity units (U) are defined as the amount of enzyme required to catalyze the production of 1 μmol of reducing sugar per minute under specified conditions. All experiments were performed in triplicate.

[0043] 2. Protein concentration determination Protein concentration was determined using the Lowry method, following the standard procedure for this assay.

[0044] Example 1 This invention uses Aspergillus feces from rabbit feces Aspergillus leporis A xylanase derived from this source was named xylanase AlXyn10A, with its amino acid sequence shown in SEQ ID NO:1. Its theoretical molecular weight is 33.34 kDa, and its isoelectric point is 7.73. BLAST analysis of the AlXyn10A amino acid sequence against the NCBI database showed that it is homologous to several fungal GH10 family xylanases, and also homologous to xylanases derived from [a specific fungus / organism / source]. Penicillium simplicissimum The highest homology was 89.74% with xylanase (GenBank accession number P56588.1). Furthermore, the homology of this xylanase with other GH10 family xylanases is as follows: 87.16% homology with *Aspergillus niger* (A2QFV7.1), 86.24% homology with *Aspergillus ryukyuensis* IFO 4308 (P33559.2), 81.25% homology with *Penicillium chrysogenum* (Q6PRW6.1), 73.38% homology with *Thermophilus auricula-judae* (P23360.4), 70.03% homology with *Aspergillus fumigatus* Af293 (Q0H904.2), 69.11% homology with *Aspergillus oryzae* RIB40 (Q96VB6.1), and 63.91% homology with *Bacillus oryzae* 70-15 (G4MPQ7.1). Multiple sequence alignment based on CAZy and NCBI databases confirmed that AlXyn10A belongs to the GH10 xylanase family.

[0045] This invention provides a method for preparing and purifying xylanase AlXyn10A. The specific details are as follows: 1.1 Construction of recombinant strains The coding gene for xylanase AlXyn10A (nucleotide sequence shown in SEQ ID NO:2), optimized with codons, was amplified by PCR and cloned into the pPIC9K vector to construct the pPIC9K-AlXyn10A vector. The linearized recombinant plasmid was digested with SacI and then transformed into Pichia pastoris GS115 via electroporation. After culturing on MD agar plates at 30°C for 72 hours, transformants were initially screened using YPD agar medium containing a final concentration of G418 of 2.0 mg / mL, yielding 35 recombinant Pichia pastoris strains. Positive clones were then amplified in BMGY medium to accumulate biomass, followed by adaptation with 0.5% methanol added at 24-hour intervals in BMGY medium. After three days of methanol-induced expression, recombinant Pichia pastoris 002 was identified as having the strongest xylanase production capacity, and this strain was used for subsequent studies.

[0046] During PCR amplification, the nucleotide sequences of the amplification primer pair AlXyn10A-F / R are shown in SEQ ID NO:3~4.

[0047] The nucleotide sequence of AlXyn10A-F is shown in SEQ ID NO:3, specifically: 5′-ATCGAG GAATTC GGCTCTGTTGAAAGCCG-3′; The nucleotide sequence of AlXyn10A-R is shown in SEQ ID NO:4, specifically as follows: 5′-TATATATTAT GCGGCC G CCAGAGCGTTTGCGATAGC-3′.

[0048] In the primer sequences shown in SEQ ID NO:3~4, the underlined bases represent EcoRI and NotI restriction enzyme sites.

[0049] 1.2 Preparation and purification of xylanase AlXyn10A (1) Fermentation and induction culture The recombinant Pichia pastoris 002 was cultured in YPD medium with shaking at 30°C and 200 rpm until the optical density at 600 nm reached 10.0, yielding a seed culture. Subsequently, the seed culture was inoculated at a 10% inoculum into a 5L bioreactor containing BSM medium for high-density fermentation. During high-density fermentation, the pH was adjusted to 4.0 by controlled addition of ammonia, while the temperature and stirring speed were maintained at 30°C and 600 rpm, respectively. After complete glycerol consumption, the pH was adjusted to 5.0, and a 50% (v / v) glycerol-water solution was added in batches, maintaining the dissolved oxygen (DO) at 20%-30%, until the biomass concentration reached the range of 180 g / L-220 g / L. In this invention, this stage of cultivation ended when the biomass concentration reached 200 g / L. The original culture parameters were then maintained for 1 hour for metabolic adaptation culture. After that, the operating parameters were modified: the stirring speed was increased to 800 rpm, the pH was readjusted to 6.0, and methanol was added to a final concentration of 0.5% for 144 hours to induce culture. Methanol was added at 24-hour intervals to maintain a final concentration of 0.5% in order to promote enzyme synthesis.

[0050] Throughout the fermentation process, samples were taken every 12 hours to quantitatively determine wet cell biomass, xylanase activity, and extracellular protein concentration in the supernatant.

[0051] During the methanol induction phase, the results of wet cell biomass, xylanase activity, and extracellular protein concentration in the supernatant at different induction times are as follows: Figure 1 As shown. During the methanol induction stage, the SDS-PAGE results of the fermentation supernatant at different induction times are shown below. Figure 2 As shown, lanes 1-8 are samples collected at 0, 24, 48, 72, 96, 120, 132 and 144 hours, respectively.

[0052] The supernatant (crude enzyme solution) obtained after centrifuging the fermentation broth following 144 hours of methanol induction showed an enzyme activity of 98.7 U / mL, a protein concentration of 4.1 mg / mL, and a wet cell weight of 340 g / L. SDS-PAGE analysis showed that the target protein band gradually increased with increasing induction time, indicating a positive correlation between xylanase content and fermentation time. Analysis revealed that the expression level of AlXyn10A was higher than that of xylanases reported from *Trichoderma* (15.6 U / mL) and *Aspergillus nidulans* (80.5 U / mL), demonstrating potential for industrial production.

[0053] (2) Isolation and purification of xylanase AlXyn10A The fermentation broth, after 144 h of methanol-induced culture, was centrifuged at 12000 rpm for 5 min, and the supernatant was collected to obtain crude xylanase AlXyn10A enzyme solution. 10 mL of the crude enzyme solution was centrifuged at 10000 rpm for 5 min to remove insoluble components, and then dialyzed against 20 mM MES buffer (pH 6.0) to obtain a clear supernatant. This supernatant was then loaded onto an SPFF chromatography column at a constant flow rate of 0.5 mL / min. Enzyme purification was performed on an ÄKTA purification system using a linear NaCl gradient (0-500 mM) in 20 mM MES buffer (pH 6.0) at a flow rate of 1 mL / min, with an elution volume of 9 column volumes. The fraction containing the target enzyme was collected and eluted at 4 °C with the same linear gradient of buffer containing 0-500 mM NaCl. The active peak was collected, and the solution was dialyzed to MES buffer (pH 6.5) to obtain purified xylanase AlXyn10A. The purity of the purified xylanase was assessed by SDS-PAGE.

[0054] The SDS-PAGE results of the purified xylanase AlXyn10A are as follows: Figure 3 As shown in Table 1, the statistical table of parameters of recombinant xylanase AlXyn10A before and after purification is presented.

[0055] Table 1

[0056] Depend on Figure 3 The SDS-PAGE results showed a single, clear band with high expression intensity and a theoretical molecular weight of 33.34 kDa. The specific activity of the purified enzyme increased from 26.1 U / mg to 306.4 U / mg, with a recovery rate of 88%. The specific activity of the purified AlXyn10A was 11.7 times higher than that of the crude enzyme, indicating that this purification method is suitable for AlXyn glycosylase. After purification, AlXyn10A exhibited high specific activity and substrate specificity for beech xylan, which may be attributed to its capacity to accommodate the catalytic cleavage of side chains and its strong polysaccharide binding ability.

[0057] Example 2 This invention explores the enzymatic properties of xylanase AlXyn10A and analyzes its structure through three-dimensional structural modeling.

[0058] I. Study on Enzymatic Properties 1. pH optimum and stability analysis The optimal pH for AlXyn10A was determined by measuring enzyme activity at suitable temperatures using different buffers (pH range 4.0–11.0). The buffers used included citrate-sodium citrate (pH 4.0–6.0), MES buffer (pH 5.5–6.5), MOPS buffer (pH 6.5–7.5), Tris-HCl buffer (pH 7.0–9.0), CHES buffer (pH 8.0–10.0), and Na₂HPO₄-NaOH buffer (pH 10.5–11.0). The baseline enzyme activity of the natural enzyme preparations in all comparative analyses was designated as 100%.

[0059] 2. Optimal temperature and thermal stability assessment The optimal temperature for xylanase AlXyn10A was assessed by measuring its enzyme activity within a temperature range of 25°C to 80°C using purified enzyme dissolved in 50 mM MES buffer (pH 6.5). To assess thermostability, the purified enzyme was incubated in 50 mM MES buffer (pH 6.5) at 25–80°C for 30 min, cooled in an ice bath for 30 min, and then residual activity was measured. A natural enzyme preparation was used as a control group.

[0060] The results of the enzymatic characterization of xylanase AlXyn10A are as follows: Figure 4 As shown, where Figure 4 A represents the optimal pH measurement curve. Figure 4 B is the pH stability measurement curve. Figure 4 C represents the optimal temperature measurement curve. Figure 4 D represents the thermal stability curve. (From...) Figure 4 It was found that xylanase AlXyn10A exhibited maximum enzyme activity at pH 6.5, and after incubation for 30 min at different pH levels (pH 5.5-7.0), the residual enzyme activity remained above 50%. Its optimal temperature was 50℃, and after incubation at 40℃ for 30 min, more than 50% of the residual enzyme activity was retained.

[0061] 3. Effects of metal ions and compounds on xylanase AlXyn10A The purified xylanase AlXyn10A formulation was incubated with various metal ions and chemical compounds (final concentration 1 mM). The reaction system was maintained at 35°C for 30 min, and then immediately transferred to an ice bath for thermal stabilization for the same time. The control group consisted of enzyme incubated with the same concentration of MES buffer. Residual enzyme activity was determined under optimal conditions following the steps in Section 2.5.1. Relative and specific enzyme activities were calculated, with the enzyme activity in the absence of any metal ions or chemical reagents defined as 100% (control). The effects of the following substances were investigated: Ni 2+ Na +Mn 2+ Fe 2+ Mg 2+ Zn 2+ K + Co 2+ Ca 2+ Ba² + Cu 2 + Fe 3+ EDTA, SDS and β-mercaptoethanol.

[0062] The effects of different metal ions and compounds on recombinant xylanase AlXyn10A are shown in Table 2.

[0063] Table 2

[0064] Note: Different lowercase letters in the same group (such as a, b, c...) indicate significant differences within the group (P < 0.05).

[0065] As shown in Table 2, among the tested metal ions and reagents, only Co... 2+ It exhibited activating activity, with a relative enzyme activity of 109%. In contrast, Fe... 3+ The activity of AlXyn10A was completely suppressed (0% residual activity), while Cu 2+ Significant inhibition was observed (41% residual activity). Both SDS and β-mercaptoethanol strongly inhibited the enzyme, with residual activities of 3% and 6%, respectively. SDS, as a strong anionic surfactant, may have disrupted the enzyme's hydrophobic structure, while β-mercaptoethanol may have disrupted disulfide bonds, leading to conformational disorder. EDTA, a metal ion chelating agent, reduced the residual activity to 52%, indicating that the activity of AlXyn10A may be dependent on metal ions to some extent. + and Ba 2+ Slight inhibition of the enzyme (residual activity of 63%), while other metal ions, including Ni... 2+ Mn 2+ Fe 2+ Mg 2+ Zn 2+ and K + These showed varying degrees of inhibition, but their effects were weaker than the aforementioned strong inhibitors. Fe 3+ The significant inhibitory effect may be attributed to the interference with the microenvironment of tryptophan residues.

[0066] 4. Hydrolytic characteristics of xylanase AlXyn10A This invention establishes a reaction system consisting of 10 mg / mL beech xylan or steam-exploded corn cob hydrolysate (dissolved in 50 mM MES buffer, pH 6.5). The mixture was incubated with purified enzyme (5 U / mL) at a controlled temperature of 35°C. Samples were taken at specified time points and subjected to heat denaturation by boiling. After cooling and centrifugation, the resulting supernatant was analyzed by thin-layer chromatography (TLC), using commercial xylan and xylooligosaccharides as reference standards.

[0067] The thin-layer chromatography results of xylanase AlXyn10A on the hydrolysate of steam-exploded corn cob and beech xylan are shown in Figure 5. Figure 5 A represents corn cob bursting fluid. Figure 5 B represents beechwood xylan. Lane M: Xylooligosaccharide standard; X1 is xylose, X2 is xylobiose, and X3 is xylotriose. Figure 5 It was found that the xylanase AlXyn10A hydrolyzes beech xylan to produce xylobiose and xylotriose. During the hydrolysis of steam-exploded corn cob hydrolysate, changes in the concentrations of highly polymerized xylo-oligosaccharides were observed with increasing reaction time (0, 0.5, 1, 2, and 4 h), along with corresponding changes in the concentrations of xylobiose and xylotriose. The bands corresponding to xylobiose and xylotriose showed dynamic changes over time, indicating the enzymatic performance of AlXyn10A on corn cob substrates. As the reaction proceeded, low-polymerization-degree xylo-oligosaccharides accumulated, while high-polymerization-degree xylo-oligosaccharides were gradually consumed, demonstrating the superior performance of AlXyn10A in the enzymatic hydrolysis of corn cob substrates.

[0068] II. Three-dimensional structural simulation and substrate recognition mechanism analysis This invention uses the AlphaFold2 server to predict the three-dimensional structure of the xylanase AlXyn10A and validates it through template homology comparison in the SWISS-MODEL workspace. The tertiary structure of AlXyn10A is generated using an automated homology modeling platform. The substrate binding pocket is then analyzed using PyMOL software. This computational characterization identifies key residues involved in ligand interactions.

[0069] A three-dimensional structural simulation of xylanase AlXyn10A and a schematic diagram of its interaction with the substrate are shown below. Figure 6 As shown. Figure 6 A represents the overall folding pattern of the protein. Figure 6 B represents the structural comparison of AlXyn10A (blue) and SoXyn10A (gold) with FAX3 and Araf, respectively, highlighting the interaction of key residues; Figure 6 C represents a comparison of the structures and related interactions of the complexes formed by AlXyn10A (blue) and SoXyn10A (gold) with MeGA.

[0070] The catalytic domain of GH10 xylanase adopts a typical (β / α)8TIM barrel fold, consisting of eight β-sheets and eight α-helices. The eight β-sheets form a 30 Å cylindrical structure within a hydrophobic core, surrounded by a 45 Å barrel framework of eight α-helices. The overall structure resembles a "salad bowl." The active site is located in a shallow groove on the molecular surface near the C-terminus of the β-sheets. Two conserved catalytic glutamate residues, crucial for the retention mechanism, are found near the C-termini of β-sheets 4 and 7. In the three-dimensional structure of the recombinant xylanase AlXyn10A, two key catalytic glutamates were identified as Glu157 and Glu263. The active site groove contains multiple subsites (typically -4 to +2). Among these, the -2 and -1 subsites are highly conserved high-affinity sites that tightly bind xylose residues via multiple hydrogen bonds and are crucial for enzyme activity. Compared to the conservative main chain framework, key side chain residues that determine the specificity of complex xylan side chains (such as ferulic acid and glucuronic acid) have been less explored.

[0071] This invention focuses on the interaction mechanism between AlXyn10A and complex substrate side chains. Typical GH10 xylanases usually possess four to seven substrate binding sites, with five or six being most common. The catalytic groove of xylanase AlXyn10A contains six substrate binding sites. The structural superposition of AlXyn10A with the SoXyn10A-FAX3 complex suggests its potential ability to recognize FAX3. Specifically, Glu48, Glu72, and Asp299 in AlXyn10A form stable hydrogen bonds with the arabinose and ferulic acid side chains of FAX3. Further structural comparison with the GH10 xylanase mutant Xyn10B (E262S) reveals a possible MeGA recognition mechanism. In this case, Glu161 and Tyr200 form hydrogen bonds with the glucuronic acid side chain. Furthermore, the uronic acid interacts with the enzyme at the -2 subsite via hydrogen bonding and hydrophobic interactions, indicating that the sugar modifications in glucuronic acid xylan are primarily targeted at this proximal glycosyl binding site. These findings collectively reveal the crucial role of the side chain residues at the AlXyn10A active site in recognizing complex xylan side chains, providing a structural basis for its broad substrate adaptability.

[0072] Example 3 This invention provides a method for preparing whole pear juice, the specific preparation method is as follows: Take 40g of Jinzhou pear pulp, mix it with distilled water at a ratio of 1:1.5 (w / v), add 5% (v / v) lemon juice, add 2mL of crude xylanase AlXyn10A prepared in Example 1, mix and incubate at 45℃ for 30min for enzymatic hydrolysis, homogenize for 1min after enzymatic hydrolysis, package, and sterilize under ultra-high pressure (UHP) at 500MPa for 10min to obtain whole pear pulp juice beverage.

[0073] Example 4 This invention provides a method for preparing whole pear juice, the specific preparation method is as follows: Take 40g of Jinzhou pear pulp and mix it with distilled water at a ratio of 1:1.5 (w / v). Add 5% (v / v) lemon juice. Add 3mL of the crude xylanase AlXyn10A enzyme solution prepared in Example 1, and add pectinase and cellulase to make the final concentration of pectinase 100U / mL pear juice and the final concentration of cellulase 50U / mL pear juice. After mixing, incubate at 45℃ for 30min for enzymatic hydrolysis. After enzymatic hydrolysis, homogenize for 1 minute, package, and sterilize under ultra-high pressure at 500MPa for 10min to obtain whole pear pulp pear juice beverage.

[0074] Example 5 This invention provides a method for preparing whole pear juice, the specific preparation method is as follows: Take 40g of Jinzhou pear pulp and mix it with distilled water at a ratio of 1:1.4 (w / v). Add 4.5% (v / v) of lemon juice. Add 3mL of the crude xylanase AlXyn10A enzyme solution prepared in Example 1, and add pectinase and cellulase to make the final concentration of pectinase 95U / mL pear juice and the final concentration of cellulase 45U / mL pear juice. After mixing, incubate at 45℃ for 30min for enzymatic hydrolysis. After enzymatic hydrolysis, homogenize for 1 minute, package, and sterilize under ultra-high pressure at 600MPa for 8min to obtain whole pear pulp pear juice beverage.

[0075] Example 6 This invention provides a method for preparing whole pear juice, which is basically the same as that in Example 4, except that the pressure during ultra-high pressure sterilization is 300 MPa, and the other steps and parameters are the same as in Example 4.

[0076] Example 7 This invention provides a method for preparing whole pear juice, which is basically the same as that in Example 4, except that the ultra-high pressure sterilization time is 15 minutes, and the other steps and parameters are the same as in Example 4.

[0077] Comparative Example 1 Comparative Example 1 of this invention provides a method for preparing whole pear juice, which is basically the same as that of Example 3, except that xylanase AlXyn10A is not added. The remaining steps and parameters are the same as those of Example 3.

[0078] Comparative Example 2 Comparative Example 2 of this invention provides a method for preparing whole pear juice, which is basically the same as that of Example 3, except that xylanase AlXyn10A is replaced with xylanase PcXyn11A, so that the enzyme activity of xylanase PcXyn11A in pear juice is equal to that of xylanase AlXyn10A. The remaining steps and parameters are the same as those in Example 3. Xylanase PcXyn11A was first disclosed in Chinese Patent CN202411878848.4.

[0079] Comparative Example 3 Comparative Example 3 of the present invention provides a method for preparing whole pear juice, which is basically the same as that of Example 3, except that xylanase AlXyn10A is replaced with commercially available xylanase, so that the enzyme activity of commercially available xylanase and xylanase AlXyn10A in pear juice is equal. The remaining steps and parameters are the same as those of Example 3.

[0080] Comparative Example 4 Comparative Example 4 of this invention provides a method for preparing whole pear juice, which is basically the same as that of Example 4, except that xylanase AlXyn10A is not added. The remaining steps and parameters are the same as those of Example 4.

[0081] Comparative Example 5 Comparative Example 5 of the present invention provides a method for preparing whole pear juice, which is basically the same as that of Example 4, except that xylanase AlXyn10A is replaced with xylanase PcXyn11A, so that the enzyme activity of xylanase PcXyn11A and xylanase AlXyn10A in pear juice is equal. The remaining steps and parameters are the same as those of Example 4.

[0082] Comparative Example 6 Comparative Example 6 of the present invention provides a method for preparing whole pear juice, which is basically the same as that of Example 4, except that xylanase AlXyn10A is replaced with commercially available xylanase, so that the enzyme activity of commercially available xylanase and xylanase AlXyn10A in pear juice is equal. The remaining steps and parameters are the same as those of Example 4.

[0083] Example of effect To evaluate the advantages and disadvantages of different methods for preparing whole-pear juice, this invention measured the juice yield, reducing sugar content, pH, total titratable acidity (TTA), and ascorbic acid content of the pear juice obtained by different methods. Specifically, pH was measured using a calibrated pH meter; TTA was analyzed using acid-base titration according to GB 12456-2021, with phenolphthalein as an indicator, to quantitatively determine the titratable acidity in the whole-pear juice; ascorbic acid content was determined using method 3 in GB 5009.86-2016, using the 2,6-dichlorophenolindophenol titration method. Reducing sugar content was determined using the DNS method, measuring absorbance at 540 nm and referring to a glucose standard curve.

[0084] In this invention, one-tenth of the systems prepared in Examples 3-4 and Comparative Examples 1-4 were used for determination. The results of the determination of juice yield, reducing sugar content, pH, total titratable acidity and ascorbic acid content in different systems are shown in Table 3.

[0085] Table 3

[0086] Note: Different lowercase letters in the same group (such as a, b, c...) indicate significant differences within the group (P < 0.05).

[0087] As shown in Table 3, in the overall process of pear juice preparation, the pear juice prepared by the enzyme-added group was significantly better than that prepared by the non-enzyme-added group in terms of juice yield, reducing sugar content, and ascorbic acid content. At the same time, the pear juice prepared by the enzyme-added group had a lower pH value and a higher total titratable acidity, indicating that the pear juice prepared by the enzyme-added group was more acidic.

[0088] In the groups with only a single xylanase added, the effects of different xylanases varied significantly: the juice yield of the AlXyn10A xylanase group was higher than that of the commercially available xylanase group and the PcXyn11A xylanase group. Furthermore, the ascorbic acid and reducing sugar content in the pear juice produced by this group was significantly higher than that of the commercially available xylanase group. At the same time, the reducing sugar content, ascorbic acid content, and total titratable acidity were also higher than those of the PcXyn11A xylanase group, indicating a better overall effect.

[0089] In the group of pear juice preparations using xylanase combined with pectinase and cellulase, AlXyn10A xylanase showed the best results when combined with pectinase and cellulase, achieving a juice yield of 95.53% and reducing sugar, total titratable acidity, and ascorbic acid content of 124.3 g / L, 4.208 g / L, and 210.0 mg / 100g, respectively. The reducing sugar and ascorbic acid contents were significantly higher than those of other xylanase-pectinase-cellulase combinations, while the juice yield and total titratable acidity were also higher, further demonstrating the advantages of AlXyn10A xylanase in pear juice preparation.

[0090] In summary, compared with other xylanases, AlXyn10A xylanase, whether used alone or in combination with pectinase and cellulase, can effectively increase the juice yield of pear juice. It can also increase the content of reducing sugar, total titratable acidity and ascorbic acid in pear juice, thus significantly improving the nutritional value of pear juice and the overall utilization rate of pear fruit.

[0091] Structural analysis of this invention shows that xylanase AlXyn10A can simultaneously recognize both FAX3 and MeGA substrates, effectively degrading xylans with complex side chains. In pear juice preparation, compared to other xylanase groups, the pear juice prepared with added xylanase AlXyn10A showed increased reducing sugar content, further confirming the effectiveness of AlXyn10A in degrading xylans with complex side chains. Therefore, xylanase AlXyn10A can be widely applied in various fields such as food, agricultural waste resource utilization, textiles, papermaking, and environmental remediation.

[0092] 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 or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a xylanase AlXyn10A in the degradation of xylans containing complex side chains, characterized in that: The amino acid sequence of the xylanase AlXyn10A is shown in SEQ ID NO:1; The xylan containing complex side chains includes at least one of xylan containing ferulic acylated arabinoxylan side chains or xylan containing methylglucuronic acid xylan side chains.

2. The application of the xylanase AlXyn10A as described in claim 1 in the degradation of xylans containing complex side chains, characterized in that: The plants containing xylan with complex side chains include at least one of pear, corn, wheat, rye, oats, pine, birch, or cedar; and / or When using xylanase AlXyn10A to degrade xylan containing complex side chains, cobalt salt is added to the reaction system.

3. The application of the xylanase AlXyn10A as described in claim 1 in the degradation of xylans containing complex side chains, characterized in that: The application includes its use in the preparation of pear juice.

4. The application of xylanase AlXyn10A as described in any one of claims 1-3 in the degradation of xylan containing complex side chains, characterized in that: The preparation method of the xylanase AlXyn10A includes the following steps: recombinant Pichia pastoris expressing the xylanase AlXyn10A is cultured in high-density fermentation and methanol-induced fermentation to obtain xylanase AlXyn10A.

5. The application of the xylanase AlXyn10A as described in claim 4 in the degradation of xylan containing complex side chains, characterized in that: The nucleotide sequence of the gene encoding the xylanase AlXyn10A expressed in the recombinant Pichia pastoris is shown in SEQ ID NO:

2.

6. The application of the xylanase AlXyn10A as described in claim 4 in the degradation of xylan containing complex side chains, characterized in that: The high-density fermentation culture medium includes BSM medium; and / or The high-density fermentation culture was carried out at a temperature of 27℃-33℃, a rotation speed of 500rpm-1000rpm, and a pH of 4-6.

7. The application of the xylanase AlXyn10A as described in claim 4 in the degradation of xylan containing complex side chains, characterized in that: During the high-density fermentation culture, when the wet cell biomass concentration is 180g / L-220g / L, the stirring speed is adjusted to 780rpm-820rpm, the pH of the system is adjusted to 5.8-6.2, methanol is added, and the culture is induced for 72h-144h. The supernatant is then separated from the solid to obtain the crude xylanase AlXyn10A enzyme solution. The crude xylanase AlXyn10A solution was purified to obtain xylanase AlXyn10A.

8. A method for preparing pear juice, characterized in that: The preparation method includes the following steps: mixing pear pulp with water, adding xylanase AlXyn10A for enzymatic hydrolysis to obtain pear juice; wherein, the amino acid sequence of the xylanase AlXyn10A is shown in SEQ ID NO:

1.

9. The method for preparing pear juice as described in claim 8, characterized in that: During the enzymatic hydrolysis treatment, at least one of pectinase or cellulase is also added; and / or After the enzymatic hydrolysis, the pear juice is obtained by ultra-high pressure sterilization; and / or The pear juice is made from whole pear pulp.

10. A pear juice, characterized in that: It is prepared by the method for preparing pear juice as described in claim 8 or 9.

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