A xylanase Pexyn116 derived from Pleurotus eryngii, its truncated form for removing CBM1, and its applications.

Abstract

This invention discloses a xylanase Pexyn116 derived from *Pleurotus eryngii*, its truncated form for removing CBM1, and its applications, belonging to the field of microbiology and biotechnology. This invention provides xylanase Pexyn116 and its truncated form Pexyn116ΔCBM1. The xylanase Pexyn116 is acid-resistant, with an optimal pH of 4.5, exhibiting good stability within the pH range of 4.5 to 7.5; its optimal temperature is 50℃, and it retains more than 50% of its enzyme activity after treatment at 50℃ for 1 hour. The truncated form Pexyn116ΔCBM1 is also acid-resistant, with an optimal pH of 4.5, exhibiting good stability within the pH range of 4.5 to 7.5; its optimal temperature is 50℃, and it retains more than 70% of its enzyme activity after treatment at 50℃ for 1 hour. The xylanase Pexyn116 and its truncated form Pexyn116ΔCBM1 provided by this invention have excellent enzymatic hydrolysis effects on beech xylan and corn cob xylan, and have high application prospects and promotion value.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and biotechnology, specifically relating to a xylanase Pexyn116 derived from Pleurotus eryngii, its truncated form for removing CBM1, and its applications. Background Technology

[0002] Xylan is a natural high-molecular-weight polymer found in plant cell walls, widely present in biological resources such as corn cobs, sugarcane bagasse, wheat bran, straw, and mulberry branches. Its structure contains abundant xylose units. During the decomposition of lignocellulose, xylose units are typically released through acid hydrolysis, alkaline hydrolysis, or biodegradation. Further processing and purification yield xylooligosaccharides. In addition, some agricultural byproducts such as corn cobs, sugarcane bagasse, and straw, as well as some industrial wastes, such as black liquor and wood pulp from the paper industry, contain abundant lignin and xylan. These byproducts, after appropriate processing and refining, can become important sources of xylooligosaccharides.

[0003] Xylanase (EC 3.2.1.8) is a key enzyme in the hydrolysis of xylan to produce xylooligosaccharides, playing a crucial role in the high-value utilization of lignocellulose. Xylanases are widely found, with genes related to xylanase production present in animals, plants, and microorganisms. Among fungi, xylanases from the genera *Aspergillus* and *Trichoderma* are the most common, originating from decaying tree branches and fallen leaves, and exhibiting higher specificity for natural lignocellulose substrates. Therefore, in industry, to obtain xylanases with different degradation characteristics, high-throughput screening methods are often used to select enzyme-producing strains with strong enzyme production capacity, rapid growth rate, and ease of cultivation in specific habitats.

[0004] Xylanase plays a crucial "pathfinder" role in the growth and development of edible fungi (macrofungi). Edible fungi are mostly wood-rotting fungi (such as shiitake, oyster, and king oyster mushrooms) or straw-rotting fungi (such as button mushrooms and giant oyster mushrooms). They must secrete xylanase to degrade hemicellulose in the substrate to obtain the carbon source needed for growth. Strains with high xylanase activity consume substrate quickly, establish mycelium early, and effectively inhibit contamination by other fungi. Adding exogenous xylanase to the cultivation substrate, or releasing xylooligosaccharides through fermentation pretreatment, can shorten the mycelial growth cycle and improve the weight and quality of fruiting bodies (due to more complete nutrient release). Utilizing the mycelial residue of edible fungi (such as king oyster and oyster mushrooms), through its residual xylanase activity, waste straw can be converted into animal feed rich in xylooligosaccharides, achieving secondary value-added processing of agricultural waste.

[0005] Beech wood chips are widely recognized as one of the highest-quality hardwood substrates for edible mushroom cultivation. Beech wood is hard and contains extremely high and well-balanced levels of cellulose, hemicellulose, and lignin. It provides a long-term, stable carbon source for mycelium, making it particularly suitable for varieties with long cultivation cycles and the need for multiple flushes of mushrooms. Unlike pine and fir wood, which contain large amounts of antibacterial resins, aromatic oils, and terpenes, beech wood contains very low levels of these natural antibacterial substances. Therefore, it does not require the long composting and aging process of pine chips (up to six months) to "degrease and detoxify," and usually only requires short-term aging or direct fermentation / sterilization before use. Beech wood chips do not easily become soft and gelatinous after fermentation or high-temperature sterilization, maintaining excellent porosity and ensuring a sufficient oxygen supply for mycelial growth (excellent aeration). Beech wood chips are a top-grade main material for the industrial cultivation of shiitake mushrooms (including *Pleurotus eryngii*, *Pleurotus ostreatus*, and *Pleurotus eryngii*). Beech wood chips can significantly increase the weight of individual shiitake mushrooms and the firmness of their flesh, producing shiitake mushrooms of far superior quality compared to those cultivated with cork (poplar) wood chips.

[0006] As a major agricultural country, my country possesses abundant straw resources. Corn cobs are a common agricultural byproduct, and they contain approximately 30% xylan, an important raw material for the industrial production of xylitol. Currently, there are no reports on xylanases derived from *Pleurotus eryngii* capable of degrading beechwood xylan and corn cob xylan. Summary of the Invention

[0007] Based on the above needs, the purpose of this invention is to provide a xylanase Pexyn116 derived from Pleurotus eryngii, its truncated form for removing CBM1, and its applications. This invention provides the amino acid sequences of xylanase Pexyn116 and its truncated form, as well as the nucleotide sequences of the encoding gene. Both Pexyn116 and its truncated form exhibit significant xylanase activity, capable of simultaneously degrading various plant polysaccharides, particularly showing good degradation effects on beech xylan and corn cob xylan.

[0008] To achieve the above-mentioned objectives of the present invention, the present invention provides the following technical solution:

[0009] This invention provides a xylanase Pexyn116 derived from Pleurotus eryngii, the amino acid sequence of which is shown in SEQ ID No. 1.

[0010] The present invention also provides a gene encoding xylanase Pexyn116, the nucleotide sequence of which is shown in SEQ ID No. 2.

[0011] The present invention also provides a truncated form of xylanase Pexyn116 with CBM1 removed, wherein the truncated form is xylanase Pexyn116 with CBM1 and signal peptide sequences removed, specifically Pexyn116ΔCBM1, and the amino acid sequence of Pexyn116ΔCBM1 is shown in SEQ ID No. 4.

[0012] The present invention also provides a coding gene for the truncated form, the nucleotide sequence of which is shown in SEQ ID No. 3.

[0013] The present invention also provides a recombinant strain containing the aforementioned coding gene, wherein the recombinant strain is Pichia pastoris GS115 of the Escherichia coli series.

[0014] Furthermore, the xylanase Pexyn116 obtained by heterologous expression of the encoding gene in Pichia pastoris GS115 competent strain has a molecular weight of 30-40 kDa, and its truncated form has a molecular weight of 20-30 kDa.

[0015] The present invention also provides the application of the xylanase Pexyn116 and / or the truncated form Pexyn116ΔCBM1 in the degradation of plant polysaccharides.

[0016] Furthermore, both the xylanase Pexyn116 and its truncated form possess xylanase activity and are capable of degrading plant polysaccharides.

[0017] Furthermore, the plant polysaccharide includes at least one of beech xylan and corn cob xylan.

[0018] Furthermore, the pH in the xylanase Pexyn116-catalyzed reaction is 4.5–7.5.

[0019] Furthermore, the pH is preferably 4.5 in the xylanase Pexyn116 catalytic reaction.

[0020] Furthermore, the optimal temperature for the xylanase Pexyn116-catalyzed reaction is 50 °C.

[0021] Furthermore, the optimal pH for the reaction catalyzed by the xylanase Pexyn116ΔCBM1 is 4.5.

[0022] Furthermore, the optimal temperature for the xylanase Pexyn116ΔCBM1-catalyzed reaction is 50 °C.

[0023] Compared with the prior art, the advantages and beneficial technical effects of the present invention are:

[0024] 1. This invention uses xylan and cellulose to induce the expression of *Pleurotus eryngii*, verifying a significant increase in the activities of cellulase and xylanase, indicating that xylan and cellulose can simultaneously induce the expression of both cellulase and xylanase. Through proteomics and transcriptomics analysis, this invention screened a xylanase, Pexyn116, from *Pleurotus eryngii*. The gene encoding Pexyn116 was heterologously expressed in *Pichia pastoris* GS115. After ammonium sulfate precipitation and nickel column affinity purification, SDS-PAGE results confirmed the secretory expression of a single protein band, with a molecular weight (MW) consistent with the theoretical value.

[0025] 2. This invention explores the enzymatic mechanism of xylanase catalyzing substrates from the perspective of CBM. After truncating and removing CBM1 from the xylan gene, it was heterologously expressed in Pichia pastoris GS115. After ammonium sulfate precipitation and nickel column affinity purification, SDS-PAGE results confirmed the secretory expression of a single protein band.

[0026] 3. This invention verifies that both the xylanase Pexyn116 and its truncated form possess xylanase activity, capable of simultaneously catalyzing beech xylan and corn cob xylan. Pexyn116 not only possesses the ability to hydrolyze short-chain xylans but also exhibits strong degradation capabilities for long-chain xylooligosaccharides. Compared to the full-length protein, the truncated form demonstrates stronger binding affinity and stability. This provides a basis for the development and application of enzyme preparations. Attached Figure Description

[0027] Figure 1 This study investigated the induction of xylanase in Pleurotus eryngii mycelium by cellulose.

[0028] Figure 2 This study investigated the induction of cellulase in Pleurotus eryngii mycelium by cellulose.

[0029] Figure 3 This is an agarose gel electrophoresis pattern of the PCR amplification product of the Pexyn116 gene.

[0030] Figure 4 The image shows the SDS-PAGE electrophoresis result of the purified xylanase.

[0031] Figure 5 This is an agarose gel electrophoresis pattern of the PCR amplification product of the Pexyn116ΔCBM1 gene.

[0032] Figure 6 The image shows the SDS-PAGE electrophoresis result of the purified xylanase truncated form Pexyn116ΔCBM1.

[0033] Figure 7 pH stability of xylanase Pexyn116.

[0034] Figure 8 Temperature stability of xylanase Pexyn116.

[0035] Figure 9 pH stability of the xylanase truncated form Pexyn116ΔCBM1.

[0036] Figure 10 Temperature stability of xylanase Pexyn116ΔCBM1.

[0037] Figure 11 The effects of xylanase truncated form on the release of reducing sugars from xylan are shown in Figure 1. a represents the effect of hydrolysis time on reducing sugar concentration; b represents the effect of enzyme dosage (activity) on reducing sugar concentration; and c represents the effect of substrate concentration on reducing sugar concentration. Data are expressed as mean ± standard deviation (mean ± SD, n=3). Detailed Implementation

[0038] The following detailed description of the invention's concept and technical solutions, in conjunction with specific embodiments, will provide a thorough understanding of the invention's purpose, results, and significance. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer; materials not explicitly named are commercially available products.

[0039] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and all materials and reagents used can be purchased from biological or chemical reagent companies. All molecular biology operations in the following examples were performed in accordance with "Molecular Cloning: A Laboratory Manual" (Science Press, 2002 edition).

[0040] The culture medium involved in this invention specifically includes the following:

[0041] YNB medium: 20 g / L glucose, 20 g / L agar, 13.4 g / L yeast nitrogen base.

[0042] MD medium: YNB 13.4 g / L, glucose 10 g / L, diluted to 1 L with distilled water, autoclaved at 121℃ for 20 min, then add 2 mL of 500×B-biotin.

[0043] YPD medium: 20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract, 400 μg / L biotin and 20 g / L agar.

[0044] BMGY liquid medium: YNB 13.4 g / L, yeast extract 10 g / L, peptone 20 g / L, glycerol 10 g / L, bring to volume with 0.1 M pH 6.0 phosphate buffer, autoclave at 121℃ for 20 min, and then add 2 mL of 500×B-biotin.

[0045] BSNM liquid medium: peptone 2.5 g / L, yeast extract 2.5 g / L, K2HPO4 1.5 g / L, KH2PO4 3.5 g / L, NaCl 1.0 g / L, CaCl2 0.1 g / L, MgSO4 0.2 g / L, FeSO4·7H2O 0.005 g / L, CoCl2 0.002 g / L, MnSO4 0.002 g / L, ZnSO4·7H2O 0.002 g / L, CuSO4·5H2O 0.005 g / L, Na2MoO4 0.001 g / L, H3BO3 0.001 g / L, KAl(SO4)2 0.001 g / L.

[0046] The king oyster mushroom (P. eryngii) described in this invention is obtained through commercial channels.

[0047] Example 1: Cellulose-induced expression of xylanase in Pleurotus eryngii

[0048] Mycelia of *Pleurotus eryngii* were scraped from agar plates and inoculated into BSNM liquid medium. The culture was incubated at 25 °C and 125 rpm for 8–10 days to obtain a seed culture. After homogenization, the culture was transferred to fresh BSNM medium at an initial inoculum of 3.5%. After 5 days of incubation, cellulose powder was added to a final concentration of 5 g / L to initiate induction.

[0049] Samples were collected at 0, 6, 12, 24, 36, and 48 hours after induction. The culture supernatant and mycelial precipitate were separated using sterile gauze. The supernatant was used for enzyme activity assay, while the mycelium was stored at -80 °C for subsequent analysis. Results showed that xylanase activity significantly increased 24 hours after cellulose induction; the increase was even more pronounced after 36 hours of induction, reaching 7.52 ± 0.23 U / mL. Figure 1 Simultaneously, cellulase activity also increased to 0.09 ± 0.00 U / mL. Figure 2 Xylan has no inducing effect on xylanase in Pleurotus eryngii, but cellulose has a strong inducing effect on both xylanase and cellulase in Pleurotus eryngii.

[0050] Enzyme activity assay: Xylanase activity was determined using the 3,5-dinitrosalicylic acid (DNS) method (1, 2), with beechwood xylan as the substrate. The reaction system contained 100 μL of appropriately diluted purified enzyme solution and 0.4% (w / v) xylan substrate dissolved in 50 mM acetate buffer (pH 4.8). The reaction system was incubated at 50 °C for 30 min, followed by the addition of an equal volume of DNS reagent to terminate the reaction, and then heated at 100 °C for 10 min to develop color. After color development, the reaction solution was rapidly cooled to room temperature in an ice-water bath, and its absorbance was measured at 540 nm. A reaction system without enzyme solution was used as a blank control. All assays were performed in triplicate. One unit of xylanase activity (U) was defined as the amount of enzyme required to catalyze the release of 1 μmol xylose per minute from the substrate under the above assay conditions.

[0051] Cellulase activity was determined using the DNS method with sodium carboxymethyl cellulose (CMC-Na) as the substrate. The reaction system composition was the same as that for xylanase activity determination, except that the substrate was replaced with 0.4% (w / v) CMC-Na. The reaction system was incubated at 50 °C for 120 min, and the reaction was terminated by adding DNS reagent. Subsequent processing steps were the same as above. The absorbance was measured at 540 nm, with the reaction system without enzyme solution serving as a blank control. All measurements were performed in triplicate. One unit of cellulase activity (U) was defined as the amount of enzyme required to catalyze the release of 1 μmol of glucose per minute from the substrate under the above measurement conditions.

[0052] Example 2: Cloning of the xylan gene and its expression and preparation in Pichia pastoris

[0053] The Pexyn116 gene was amplified using cDNA from *Pleurotus eryngii* as a template. An N-terminal His tag was introduced during PCR amplification to facilitate subsequent protein purification and downstream applications. The total volume of the PCR reaction system was 30 μL, containing: 15 μL of 2× Phanta Flash Master Mix, 1 μL each of forward and reverse primers (10 μM), 1 μL of template (cDNA), and 12 μL of nuclease-free water. The PCR amplification program was: 98 ℃ pre-denaturation for 30 s; followed by 40 cycles (98 ℃ denaturation for 15 s, 56 ℃ annealing for 15 s, 72 ℃ extension for 30 s); and finally, a complete extension at 72 ℃ for 3 min. The pPIC9K expression vector was linearized using EcoRI and NotI restriction endonucleases. Subsequently, using the C115 homologous recombination kit (Vazyme, Nanjing, China), the purified Pexyn116 PCR product was seamlessly cloned and assembled with a linearized vector. The resulting recombinant plasmid was transformed into E. coli DH5α competent cells for plasmid amplification and positive clone screening.

[0054] The constructed pPIC9K-Pexyn116 plasmid was linearized using SacI restriction endonuclease, and then introduced into Pichia pastoris GS115 competent cells via electroporation using a MicroPulser electroporator (Bio-Rad) at 1.5 kV and 5 ms. After incubation at 30°C, transformants were initially screened on MD agar plates containing YNB and 400 μg / L biotin. A second round of screening was then performed on YPD agar plates supplemented with 0, 2, or 4 mg / mLG418. Positive transformants were validated by colony PCR and sequencing analysis (primers are shown in Table 1).

[0055] Table 1 Primers required for cloning

[0056]

[0057] The positive recombinant strain was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of BMGY medium and cultured at 30 °C and 200 rpm for 24 h. The bacterial cells were collected by centrifugation at 5,000 × g for 5 min, washed twice with 0.1 M potassium phosphate buffer (pH 6.0), and the bacterial pellet was resuspended in 50 mL of BMMY medium to allow the initial absorbance (OD) value to be measured. 600The expression level reached 1.0. The mixture was cultured under the same conditions with shaking, and methanol was added every 24 hours to maintain the induction of recombinant protein expression.

[0058] Xylanase Pexyn116 was prepared after ammonium sulfate precipitation and nickel column affinity purification. SDS-PAGE results confirmed the secretory expression of a single protein band, with a molecular weight (MW) of 30 kDa, consistent with the theoretical value. The amino acid sequence of xylanase Pexyn116 is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.2. Figure 3 This is an agarose gel electrophoresis pattern of the PCR amplification products of the Pexyn116 gene. Figure 4 The image shows the SDS-PAGE electrophoresis result of the purified xylanase.

[0059] Example 3: Cloning of a truncated xylan gene and its expression and preparation in Pichia pastoris

[0060] The Pexyn116ΔCBM1 gene was amplified using cDNA from *Pleurotus eryngii* as a template. An N-terminal His tag was introduced during PCR amplification to facilitate subsequent protein purification and downstream applications. The total PCR reaction volume was 30 μL, containing: 15 μL 2× Phanta Flash Master Mix, 1 μL each of forward and reverse primers (10 μM), 1 μL template (cDNA), and 12 μL nuclease-free water. The PCR amplification program was: 98 ℃ pre-denaturation for 30 s; followed by 40 cycles (98 ℃ denaturation for 15 s, 56 ℃ annealing for 15 s, 72 ℃ extension for 30 s); and finally, a complete extension at 72 ℃ for 3 min. The pPIC9K expression vector was linearized using EcoRI and NotI restriction endonucleases. Subsequently, using the C115 homologous recombination kit (Vazyme, Nanjing, China), the purified Pexyn116ΔCBM1 PCR product was seamlessly cloned and assembled with a linearized vector. The resulting recombinant plasmid was transformed into E. coli DH5α competent cells for plasmid amplification and positive clone screening.

[0061] The constructed pPIC9K-Pexyn116ΔCBM1 plasmid was linearized using SacI restriction endonuclease, and then introduced into Pichia pastoris GS115 competent cells via electroporation at 1.5 kV and 5 ms using a MicroPulser electroporator (Bio-Rad). After incubation at 30°C, transformants were initially screened on YNB and MD agar plates containing 400 μg / L biotin. A second round of screening was then performed on YPD agar plates containing 0, 2, or 4 mg / mL genimycin. Positive transformants were validated by colony PCR and sequencing analysis (primers are shown in Table 2).

[0062] Table 2 Primers required for cloning

[0063]

[0064] The positive recombinant strain was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of BMGY medium and cultured at 30 °C and 200 rpm for 24 h. The bacterial cells were collected by centrifugation at 5,000 × g for 5 min, washed twice with 0.1 M potassium phosphate buffer (pH 6.0), and the bacterial pellet was resuspended in 50 mL of BMMY medium to allow the initial absorbance (OD) value to be measured. 600 The expression level reached 1.0. The mixture was cultured under the same conditions with shaking, and methanol was added every 24 hours to maintain the induction of recombinant protein expression.

[0065] After ammonium sulfate precipitation and nickel column affinity purification, a truncated form of xylanase Pexyn116 (Pexyn116ΔCBM1) was prepared. SDS-PAGE results confirmed the secretory expression of a single protein band, with a molecular weight (MW) of 25 kDa, consistent with the theoretical value. Figure 5 This is an agarose gel electrophoresis pattern of the PCR amplification products of the Pexyn116ΔCBM1 gene. Figure 6 SDS-PAGE electrophoresis image of the purified xylanase truncated form Pexyn116ΔCBM1.

[0066] The amino acid sequence of the truncated form of CBM1 removed by the xylanase Pexyn116 is shown in SEQ ID NO.3, and its nucleotide sequence is shown in SEQ ID NO.4.

[0067] Example 4: Effects of pH and temperature on the activity of xylanase Pexyn116

[0068] pH and temperature are important factors affecting enzyme catalytic activity. The catalytic activity of the xylanase Pexyn116 in Example 2 of this invention was detected using beech xylan as a substrate. The enzyme activity detection method is described in Example 1.

[0069] 1. pH stability

[0070] like Figure 7 As shown, the optimal pH for xylanase Pexyn116 is 4.5, which is acidic, and it exhibits significant stability over a wide pH range (pH 4.5–7.5), retaining >60% of its relative enzyme activity after 24 hours of treatment. This indicates that xylanase Pexyn116 has good pH stability under acidic conditions.

[0071] 2. Temperature stability

[0072] like Figure 8 As shown, xylanase Pexyn116 is a mesophilic enzyme with an optimum temperature of 50 °C. After incubation at 50 °C for 40 minutes, it retains >50% of its activity.

[0073] Example 5: Effects of pH and temperature on the activity of the xylanase truncated form Pexyn116ΔCBM1

[0074] pH and temperature are important factors affecting enzyme catalytic activity. Using beech xylan as a substrate, the catalytic activity of the truncated xylanase Pexyn116ΔCBM1 in Example 3 of this invention was detected. The enzyme activity detection method is described in Example 1.

[0075] 1. pH stability

[0076] The optimal pH for Pexyn116ΔCBM1 is 4.5, which is acidic. Figure 9 Furthermore, it exhibits significant stability over a wide pH range (pH 4.5–7.5), retaining >60% of its relative enzyme activity after 24 hours of treatment. This indicates that xylanase Pexyn116ΔCBM1 possesses good pH stability under acidic conditions.

[0077] 2. Temperature stability

[0078] like Figure 10 As shown, Pexyn116ΔCBM1 is a mesothermal enzyme with an optimum temperature of 50 °C. After incubation at 50 °C for 40 minutes, it retains >50% of its activity. Compared to the full-length Pexyn116 protein, the truncated xylanase Pexyn116ΔCBM1 exhibits better temperature tolerance.

[0079] Example 6: Enzymatic hydrolysis of beech xylan and corn cob xylan by xylan Pexyn116ΔCBM1

[0080] This embodiment provides the characteristics of xylan Pexyn116ΔCBM1 in the degradation of plant polysaccharides, specifically including the following:

[0081] 1. Specific degradation of plant polysaccharides

[0082] The experimental method included the following: 500 μL of sodium acetate buffer (50 μm, pH 4.8) was added to a 1.5 mL centrifuge tube as the zeroing group; 300 μL of sodium acetate buffer + 200 μL of xylan substrate was added to a 1.5 mL centrifuge tube as group C1; 400 μL of sodium acetate buffer + 100 μL of enzyme solution was added to a 1.5 mL centrifuge tube as group C2; and 200 μL of substrate + 200 μL of sodium acetate buffer + 100 μL of enzyme solution was added to a 1.5 mL centrifuge tube as the experimental group. All groups were reacted at 50 °C for 30 min, followed by the addition of 500 μL of DNS, and then reacted at 100 °C for 10 min before being cooled in ice water. The absorbance was measured at 540 nm using a UV spectrophotometer with a mixture of 2.4 mL water and 0.6 mL reaction solution.

[0083] Pexyn116ΔCBM1 exhibits strict substrate specificity for xylan, showing the highest affinity for beech xylan, followed by corn cob xylan (Table 3), while showing no activity towards CMC-Na or starch.

[0084] Table 3. Substrate specificity of xylanase Pexyn116ΔCBM1

[0085]

[0086] 2. Effect on reducing sugar release

[0087] This embodiment uses the 3,5-dinitrosalicylic acid (DNS) method to detect reducing sugars. The specific detection method is as follows:

[0088] Experimental group: Add 0.25 mL of the sample to be tested (if the sample is not particularly precious, it is best to measure two concentrations; the reducing sugar in the sample should not exceed 4 mg / mL) and 0.25 mL of DNS solution. Control (for zeroing): Add 0.25 mL of water + 0.25 mL of DNS solution. Negative control: Add 0.25 mL of the blank sample to be tested + 0.25 mL of DNS solution.

[0089] React in a metal bath at 100℃ for 10 min. After the reaction is complete, quickly remove the centrifuge tube, without removing the explosion-proof clip, place it on the float, and gently agitate it in an ice-water bath to cool to 0℃. Pipette 0.3 mL of the reaction solution into a cuvette, add 2.7 mL of distilled water, and mix thoroughly by inverting. Zero the instrument using the control group, measure and record the absorbance at 540 nm.

[0090] The concentration of reducing sugars was calculated using a standard curve.

[0091] The standard curve is: y = 2.5904x - 0.1882

[0092] x is the absorbance value, y is 0.25 mL, and mL is the glucose concentration in the sample, in mg / mL.

[0093] The release of reducing sugars reached a plateau (1.79 mg / mL) after 3 hours. Figure 11 As shown in Figure a). Optimization of enzyme dosage showed that saturation was achieved at 3.6 U (as shown in Figure a). Figure 11 As shown in Figure b). At a fixed enzyme dosage (2.4 U), the reducing sugar yield increased linearly with increasing substrate concentration until saturation was reached at 2.18 mg / mL. Figure 11 (As shown in c). This hydrolysis characteristic is similar to that of commercial benchmark products derived from *Pexyn116ΔCBM1*, demonstrating its potential for industrial applications.

[0094] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A xylanase Pexyn116 derived from Pleurotus eryngii, characterized in that, The amino acid sequence of the xylanase Pexyn116 is shown in SEQ ID No.

1.

2. The gene encoding the xylanase Pexyn116 according to claim 1, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID No.

2.

3. A xylanase Pexyn116 for removing truncated CBM1, characterized in that, The truncated form is obtained by removing the CBM1 fragment from the xylanase Pexyn116 according to claim 1, specifically Pexyn116ΔCBM1, and the amino acid sequence of Pexyn116ΔCBM1 is shown in SEQ ID No.

3.

4. The coding gene of the truncated form according to claim 3, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID No.

4.

5. A recombinant expression vector comprising the coding gene of claim 2 or 4.

6. A recombinant strain comprising the encoding gene of claim 2 or 4, characterized in that, The recombinant strain is an Escherichia coli series. Pichia pastoris GS115 competent strain.

7. The use of the xylanase Pexyn116 of claim 1 and / or the truncated form Pexyn116ΔCBM1 of claim 3 in the degradation of plant polysaccharides.

8. The application according to claim 7, characterized in that, The degraded plant polysaccharides include degraded fir and beech xylan and / or corn cob xylan.

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