Method for producing xylanase

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a method for producing xylanase. A production strain adopted by the method is specifically Trichoderma viride TP-1002, and the preservation number of the Trichoderma viride TP-1002 is CGMCC (China General Microbiological Culture Collection Center) No. 40415. The xylanase produced by the strain is resistant to heat, acid and alkali, high in enzyme activity and stable in performance, meanwhile, a corresponding fermentation mechanism is optimized, the fermentation mechanism is higher in fermentation activity, higher in extraction yield and lower in manufacturing cost, and the average fermentation enzyme activity is 29000 U / mL or above; the invention also develops a xylanase coating and granulating method, solves the problem that the existing xylanase is inconvenient to preserve and transport, improves the enzyme activity stability, is convenient to apply, greatly enhances the damp-heat capacity of the granules, and has wide application prospects.

Description

Technical field: The invention belongs to the technical field of biochemical engineering, relates to strain breeding of microbial fermentation, and specifically relates to a method for producing xylanase. Background technology: The earliest research on xylanase began in 1955, and it was initially named pentasaccharidase. In 1961, the International Society of Biochemistry and Molecular Biology officially named it xylanase, and its official name is endo-1,4-β-xylanase, but the commonly used names include xylanase, endo-xylanase, etc., which are essentially the same.

[0003] Xylanase is an important type of xylanase, and also an induced enzyme. It mainly includes a complex enzyme system composed of 1,4-β-xylanase, 1,4-β-D xylosidase, 1,4-β-D mannosidase, 1,4-β-D glucosidase, α-L-arabinosidase, α-D-glucuronidase, α-D-galactosidase, esterase and other enzymes. In this complex enzyme system, the synergistic action of multiple enzymes can degrade xylan into oligosaccharides and xylose. Xylanases are widely available and exist in marine and terrestrial bacteria, marine algae, fungi, rumen of ruminants, snails, crustaceans, terrestrial plant tissues and various invertebrates. Due to the low content of enzymes in animals and plants, the extraction and purification process is quite complicated and cannot meet the needs of research, so a large number of xylanases come from microorganisms. Enzyme-producing microorganisms include: Aspergillus, Trichoderma, Penicillium, yeast, Schizophyllum, Bacillus, etc.

[0004] The application of xylanase in the food industry is mainly reflected in the fact that the oligoxylose it produces has many physiological effects and can be used in the development of health foods; in winemaking, it can significantly improve the efficiency and yield of alcohol fermentation; when making baked goods, xylanase can also improve the stability and fluffiness of the dough, improve the elasticity of the gluten and the taste of the pasta; in the feed industry, after adding xylanase and other complex enzyme preparations, the feed can be fully converted into easily digestible and absorbable carbohydrates, thereby improving the utilization rate. In the papermaking industry, xylanase is added to pre-bleach the pulp, improve the bleaching efficiency, and effectively reduce the use of chemical reagents in the papermaking industry; in the pharmaceutical industry, xylanase can improve the balance of intestinal microorganisms, enhance the body's immunity, and prevent and treat diarrhea, constipation, tumors, etc. It has also been proven that xylanase has important physiological functions in plant tissues, which may be related to fruit softening, seed germination, and plant defense mechanisms. Especially in the process of studying microbial xylanase, some scholars were surprised to find that microbial xylanase can induce ethylene biosynthesis in plant cells and thus act as a trigger for the plant defense system. Therefore, current research mainly focuses on xylanase produced by microorganisms.

[0005] In recent decades, the application potential of xylanase has been the focus of many scholars. At present, the main problems of xylanase in industrial production are as follows: the activity of the produced xylanase itself is not high; the industrial production technology of xylanase is immature; transportation is inconvenient; the enzyme activity is not stable enough. The above problems lead to high production costs of xylanase. How to find strains with high xylanase production, improve the stability of xylanase, and obtain enzyme-producing strains that are not easy to inactivate and have a wide range of action have become the focus of research. Summary of the invention: To solve the above problems, the present invention aims to provide a green Trichoderma capable of producing xylanase, specifically Trichoderma ( Trichoderma viride )TP-1002, which was obtained by fast neutron irradiation and diethyl sulfate mutagenesis breeding, can greatly increase the production of xylanase, laying the foundation for low-cost, large-scale production of xylanase. The strain was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on November 8, 2022, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 40415.

[0007] The present invention also provides the strain Trichoderma viride ( Trichoderma viride ) Application of TP-1002, especially in the production of xylanase; Furthermore, the present invention provides a method of using Trichoderma viride ( Trichoderma viride ) The liquid microbial fermentation production method of xylanase with high fermentation enzyme activity, high extraction yield and low manufacturing cost completed by TP-1002 is as follows: Fermentation tank culture: the seed liquid is transferred into the fermentation tank at a ratio of 8-12% (v / v) of the transfer amount. The culture conditions are initial pH 4.5-5.5, temperature 30-34°C, air volume: 0.1-0.25 vvm for the 0th to 24th hour, 0.3-0.6 vvm for the 25th to 48th hour, 0.6-1.0 vvm for the 49th hour to tank release, rotation speed 200-800 rpm. When the pH is lower than 4.5 during the fermentation period, ammonia is added to control the pH at 4.5-4.7. Furthermore, after 10 h of fermentation, the fed medium was fed to control the dissolved oxygen at 15-30%; Furthermore, the fermentation cycle is 150-160h, during which the enzyme activity in the fermentation tank increases slowly and the bacterial autolysis is serious. At the end of the fermentation, the enzyme activity of the xylanase in the fermentation liquid can reach more than 29000U / ml; Further, the fermentation enzyme production medium (w / v): corn cob powder 2%-5%, glucose 2%-3%, bran 0.5%-3%, MgSO4 0.02%-0.04%, (NH4)2SO4 0.4%-0.7%, K2HPO4 0.4%-0.6%, pH 5.0, and the rest is water.

[0008] Fermentation tank sterilization process: 121-123℃, 0.11-0.12MPa, sterilization for 30-35min.

[0009] Further, feed medium (w / v): glucose 40%-70%, ammonium sulfate 0.4%-0.6%, KH2PO4 0.3%-0.5%, corn steep liquor 2%-5%, and the rest water, pH 5.0.

[0010] Sterilization process of feed tank: Glucose is sterilized separately at 121-123℃, 0.11-0.12MPa for 30-35min; the remaining raw materials are sterilized together at 121-123℃, 0.11-0.12MPa for 30-35min, and then cooled to 40℃ and mixed.

[0011] Further, the extraction and purification method is as follows: The pH value of the fermentation liquid is adjusted to 7.0 with alkaline solution, and perlite filter aid is added to perform plate and frame filtration; diatomaceous earth is then added for fine filtration; the fine filtrate is ultrafiltered and concentrated with an ultrafiltration membrane; the concentrate is filtered and sterilized with a sterile membrane (stabilizers and preservatives may be added) to obtain a liquid finished enzyme preparation.

[0012] Further, the coating and granulation method of xylanase is as follows: Accurately measure the required liquid xylanase preparation product, add 30%-50% corn starch, 10%-20% talcum powder, and 2%-7% maltodextrin by weight of the liquid xylanase, and fully mix the above materials in a mixer; put them into a spinning extruder to extrude long strips of material with a mesh diameter of 0.6 mm; pour the extruded long strips of material into a spheronizer, adjust the spheronizer to a suitable speed to fully cut it, so that the enzyme particles are spherical; perform air drying on the spherical enzyme preparation particles, the air temperature is 45-55°C, the material temperature is 30-38°C, and the air drying is performed for 20-35 minutes to obtain the xylanase granule product.

[0013] The xylanase obtained by the present invention has the following enzymatic properties: (1) The optimum reaction temperature is 65°C. When kept at 80°C for 2.5 h, the enzyme activity can still be maintained at more than 85%, indicating good thermal stability. (2) The optimum reaction pH is 6.0. When treated at pH 3.0-10.0 for 2 h, the relative enzyme activity still remains above 80%.

[0014] Beneficial effects: 1. The present invention provides a green Trichoderma strain suitable for industrial production of xylanase, and optimizes the corresponding fermentation mechanism. The fermentation mechanism has higher fermentation activity and lower manufacturing cost, and the average fermentation enzyme activity is above 29000U / mL.

[0015] 2. The present invention provides a xylanase product which is heat-resistant, acid-resistant, alkali-resistant, has high enzyme activity, stable performance and low production cost. The product is easy to preserve, convenient to transport, has good enzyme activity stability and is easy to use. It greatly enhances the moisture and heat capacity of the particles and avoids many problems of liquid enzyme preparations. It can be widely used in food, feed, papermaking, medical and other fields and has broad application prospects. Description of the drawings: Figure 1 Optimum action temperature curve; Figure 2 Optimal pH curve; Figure 3 Temperature stability curve; Figure 4 pH stability curve. Specific implementation method: In order to make the purpose, technical solution and advantages of this patent more clear, this patent is further described in detail in combination with specific embodiments. It should be understood that the specific embodiments described here are only used to explain this patent and are not used to limit the present invention.

[0018] Example 1 Mutation breeding of strains 1. Culture medium Slant activation medium: PDA medium.

[0019] Wash and peel the potatoes, weigh 200g of potatoes, cut into small pieces, add water and boil for 20-30 minutes, filter with eight layers of gauze, add 20g of agar, continue heating and stirring to mix, after the agar is dissolved, add 20g of glucose, stir evenly, cool slightly, then add water to 1000ml, divide into packages, add stoppers and bandages, and sterilize at 121℃ for 20 minutes.

[0020] Seed activation medium: bran 2%, glucose 1%, (NH4)2SO4 0.4%, MgSO4 0.2%, K2HPO4 0.1%, KCl 0.05%, FeSO4 0.01%, and the rest is water, pH 5.0-5.2.

[0021] Primary screening culture medium: sodium carboxymethyl cellulose 1%, KH2PO4 0.5%, FeSO4 0.01%, MgSO4 0.2%, peptone 1%, sodium deoxycholate 0.2%, Congo red 0.03%, agar powder 2.0%, and the rest is water, pH 5.0-5.2.

[0022] Shake flask fermentation medium: corn cob 3%, bran 2.0%, glucose 3%, cottonseed protein 1%, KH2PO4 0.3%, MgSO4 0.2%, CaCl2 0.05%, (NH4)2SO4 0.4%, pH 5.0-5.2.

[0023] 2. Preparation of bacterial suspension: The green Trichoderma spores in the laboratory-preserved slant medium were washed off with physiological saline, placed in a triangular flask with glass beads, and shaken on a shaker at 180 r / min for 1.5 h to make the dispersion rate of the bacteria reach more than 90%. The spore suspension was filtered with sterile gauze, the number of spores was counted with a hemocytometer, and diluted with physiological saline to a certain multiple to keep the spore concentration at 10 7 -10 8 cfu / ml.

[0024] 3. Mutagenesis lethality and positive mutation rate Lethality rate (%) = [(number of colonies on the control plate - number of colonies on the plate after mutagenesis) / number of colonies on the control plate] × 100% Positive mutation rate (%) = number of mutants with a hydrolysis zone diameter greater than that of the control group / total number of colonies × 100% 4. Fast neutron irradiation mutagenesis Strain mutagenesis treatment: Place the bacterial suspension in a sterile culture dish and treat the bacterial suspension at a dose rate of 0.003 Gy / s, a vertical distance of 10 cm, and a dose of 0.2 Gy, 0.3 Gy, 0.5 Gy, 0.7 Gy, 1 Gy, 1.5 Gy, and 2.25 Gy. Dilute the irradiated bacterial suspension to 10 -3 Up to 10 -6 , 0.2 ml of the bacterial suspension at different dilution multiples was taken and spread on the primary screening culture medium, and cultured in a 34°C incubator. The bacterial suspension that had not been irradiated and mutagenized was diluted and spread on the plate as a control.

[0025] Table 1 Lethality and positive mutation rate of strains induced by fast neutron irradiation

[0026] As shown in the table, the lethality rate gradually increases with the increase of irradiation dose, and the positive mutation rate increases first and then decreases. At a dose of 1Gy, the positive mutation rate is the highest at 45.2% and the lethality rate is 39.6%. Determine the irradiation dose of 1Gy.

[0027] 5. Diethyl sulfate (DES) mutagenesis Take 1 ml of diethyl sulfate and add it to 9 ml of 95% ethanol to make diethyl sulfate dilution. Mix the diluted diethyl sulfate solution with the bacterial suspension in a certain ratio (diethyl sulfate dilution: bacterial suspension), and mix them into the following ratios: 1:400, 1:250, 1:150, 1:80, respectively. Shake at 30℃ for 15min, 20min, 30min, 40min, add 25% sodium thiosulfate solution to terminate the reaction, and dilute to 10 -4 , 10 -5 , 10 -6 , take 0.2 mL of each of them and spread it on the primary screening culture medium, place it in a 34°C incubator for culture, and use the bacterial solution that has not been mutagenized with diethyl sulfate to dilute and spread it on the plate as a control.

[0028] Table 2 Lethality and positive mutation rate of strains induced by diethyl sulfate

[0029] As shown in the table, the ratio of DES solution to bacterial suspension was 1:250, the mutagenesis time was 20 min, and the highest positive mutation rate was 43.3%. The optimal conditions for diethyl sulfate mutagenesis were determined as follows: the ratio of DES solution to bacterial suspension was 1:250, and the mutagenesis time was 20 min.

[0030] 6. Compound mutagenesis After the above two mutagenesis, the mutagenesis methods of the two mutagenesis were determined, and a composite mutagenesis was carried out by combining fast neutron mutagenesis and diethyl sulfate mutagenesis. Seven mutant strains with large hydrolysis circles were selected and cultured in shake flasks for 48 hours. The xylanase activity was measured and the original starting strain was used as a control. The results are shown in the table.

[0031] Table 3 Xylanase activity of 7 mutant strains

[0032] After composite mutagenesis, mutagenic seed No. 3 was selected, and the enzyme activity reached 4258U / mL, which was 3.4 times higher than that of the original strain.

[0033] 7. Genetic stability The strain was used as the research object to determine its genetic stability, and the enzyme activity experiment was carried out by shaking flask fermentation. The culture period was 48 hours, and the culture was continuously subcultured 8 times. The enzyme activity remained stable. The results are shown in Table 4. The experimental results showed that the genetic traits were stable, and this strain could be used in large-scale industrial production. It was named Trichoderma viride ( Trichoderma viride )TP-1002, and was deposited on November 8, 2022 at the General Microbiology Center of China Microbiological Culture Collection Administration, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No.40415.

[0034] Table 4 Genetic stability results of strains

[0035] Example 2 Determination and definition of xylanase activity of the present invention 1. Definition of xylanase activity unit The amount of enzyme required to release 1 μmol of reducing sugar per minute at 50°C and pH 5.5 was defined as one unit of enzyme activity.

[0036] 2. Enzyme activity determination method Take 2 ml of diluted enzyme solution (equilibrated at 50°C for 10 min), add it to a graduated test tube, then add 5 mL of DNS reagent, and oscillate electromagnetically for 3s-5s. Then add 2 mL of 1% xylan substrate (prepared with pH 5.5 acetic acid-sodium acetate buffer), keep warm at 50°C for 30 min, and heat in a boiling water bath for 5 min. Cool to room temperature with tap water, add distilled water to make up to 25 ml, and oscillate electromagnetically for 3s-5s. Use the standard blank sample (4.0 mL of acetic acid-sodium acetate buffer solution, 5.0 mL of DNS reagent, heat in a boiling water bath for 5 min, cool, and make up to 25 mL) as a blank control, and measure the absorbance A at 540 nm. B .

[0037] Take 2 ml of diluted enzyme solution (equilibrated at 50°C for 10 min), add it to a graduated test tube, then add 2 ml of 1% xylan substrate (prepared with pH 5.5 acetic acid-sodium acetate buffer), electromagnetically oscillate for 3s-5s, and keep warm at 50°C for 30 min. Add 5 ml of DNS reagent and electromagnetically oscillate for 3s-5s to terminate the enzymatic reaction. Heat in a boiling water bath for 5 min. Cool to room temperature with tap water, add distilled water to make up to 25 ml, and electromagnetically oscillate for 3s-5s. Use the standard blank sample (4.0 ml of acetic acid-sodium acetate buffer solution, 5.0 ml of DNS reagent, heat in a boiling water bath for 5 min, cool, and make up to 25 ml) as a blank control, and measure the absorbance A at 540 nm. E .

[0038] Enzyme activity calculation formula:

[0039] Where: X D is the activity of xylanase in the enzyme solution, U / ml; A E is the absorbance of the enzyme reaction solution; A B is the absorbance of the enzyme blank sample; K is the slope of the xylose standard curve; C0 is the intercept of the standard curve; M is the molar mass of xylose, g / mol; t is the enzymatic reaction time, min; N is the enzyme solution dilution multiple; 1000 is the conversion factor, 1mmol=1000μmol.

[0040] Example 3 Production of xylanase by submerged fermentation of Trichoderma viride TP-1002 and product processing 1. Seed tank cultivation Seed tank culture medium: glucose 1.5%, bran 2.5%, (NH4)2SO4 1%, MgSO4 0.03%, CaCl2 0.1%, pH 5.0, and the rest is water.

[0041] Seed tank sterilization process: 121-123℃, 0.11-0.12MPa, sterilize for 30 minutes.

[0042] Seed tank culture: The seed liquid after shake flask fermentation was inoculated into the seed tank at a ratio of 6% of the inoculation amount for culture. The culture conditions were temperature 34°C, tank pressure 0.06Mpa, air volume 0.3vvm, and rotation speed 400rpm. When the pH dropped to 4.4 during the culture process, ammonia was added to control the pH at 4.5. The culture time was 24h, and more bacteria were observed under the microscope.

[0043] 2. Fermentation tank culture Fermentation tank enzyme production culture medium: corn cob powder 3%, glucose 2.5%, bran 1%, MgSO4 0.03%, (NH4)2SO4 0.5%, K2HPO4 0.5%, pH 5.0, and the rest is water.

[0044] Fermentation tank sterilization process: 121-123℃, 0.11-0.12MPa, sterilization for 35min.

[0045] Fermentation tank culture: the seed liquid in the seed tank was transferred into the fermentation tank at a ratio of 10% of the transplanting amount. The culture conditions were initial pH 5.0, temperature 34°C, tank pressure 0.06Mpa, air volume: 0.25vvm for the 0th to 24th hour, 0.5vvm for the 25th to 48th hour, 1.0vvm for the 49th hour to tank release, rotation speed 600rpm, when the pH was lower than 4.5 during the fermentation period, ammonia was supplemented to control the pH at 4.5.

[0046] 3. Fed-batch culture Feed medium: glucose 60%, ammonium sulfate 0.5%, KH2PO4 0.4%, corn steep liquor 4%, the rest is water, pH 5.0.

[0047] Sterilization process of feed tank: Glucose is sterilized separately at 121-123°C and 0.11-0.12MPa for 30 min; the remaining raw materials are sterilized together at 121-123°C and 0.11-0.12MPa for 30 min, and then cooled to 40°C and mixed evenly.

[0048] After 10 h of the culture cycle, feed was added to control the dissolved oxygen at 20% and the culture was continued until the end of the fermentation.

[0049] 4. End of fermentation When the enzyme activity increases slowly and the bacteria autolyze seriously, the fermentation is terminated and the fermentation cycle is 150-160h. Prepare for tank extraction.

[0050] Table 5 Fermentation results of 50L fermenter

[0051] From the fermentation experiment results in Table 5, it can be seen that the enzyme activity of the fermentation broth is stable, and the average fermentation enzyme activity reaches more than 29000U / mL.

[0052] 5. Extraction and purification of xylanase (1) The pH of the fermentation liquid was adjusted to 7.0 with 1 mol / L sodium hydroxide, and 3% perlite filter aid was added to perform plate and frame filter pressing; (2) Add 2% diatomaceous earth to the filtrate for fine filtration and clarification; (3) ultrafiltration and concentration of the fine filtrate using a 20KDa ultrafiltration membrane; (4) The ultrafiltration concentrate is obtained, and then filtered and sterilized using a sterile membrane to obtain a liquid xylan finished enzyme preparation.

[0053] 6. Xylanase coating and granulation The activity of enzyme preparations is affected by many factors. In order to reduce the loss of enzyme activity and improve the convenience of use, the enzyme preparations are granulated.

[0054] (1) Accurately measure liquid xylanase, add 50% corn starch, 15% talcum powder, and 5% maltodextrin to the weight of the liquid xylanase, and mix the above materials thoroughly in a mixer.

[0055] (2) Place the material into a spinning extruder and extrude it into long strips. The diameter of the screen hole is 0.6 mm.

[0056] (3) Pour the extruded long strips into the spheronizer and adjust the spheronizer to a suitable speed to fully cut the material so that the enzyme particles are spherical.

[0057] (4) The spherical enzyme preparation granules are placed in a vertical air dryer for air drying at a temperature of 50°C and a material temperature of 35°C. The spherical enzyme preparation granules are dried for 30 minutes to obtain the finished xylanase granules.

[0058] The advantages of the finished granule product are: easy to preserve, convenient to transport, good enzyme activity stability and easy to use, which greatly enhances the moisture and heat capacity of the granules and avoids many problems of liquid enzyme preparations.

[0059] Example 4 Optimal reaction temperature of xylanase The liquid xylanase product enzyme preparation prepared in batch 1 of Example 3 was taken, and the xylanase activity was measured at 50, 55, 60, 65, 70, 75, 80, 85, and 90°C under normal conditions of pH 5.5. The enzyme activity at 65°C was taken as 100%, and the relative enzyme activity was calculated. The results are shown in FIG. Figure 1 As shown, the optimum reaction temperature is 65°C, and the activity can be maintained at more than 85% at 80°C. The experimental results show that the optimum reaction temperature of the xylanase produced by the mutant strain is significantly higher than that of xylanase from other sources in the prior art, and it has great market application value and good application value in industrial production.

[0060] Example 5 Optimal pH for xylanase reaction The liquid xylanase finished enzyme preparation prepared in batch 1 of Example 3 was taken, and the xylanase activity at pH values ​​of 3.0, 4.0, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 9.0, and 10.0 was measured at a temperature of 65°C, and the relative enzyme activity was calculated with the enzyme activity at pH 6.0 as 100%. The measurement results are as follows: Figure 2 As shown, the enzymatic activity of xylanase is highest when the pH is around 6.0.

[0061] Example 6 Xylanase Thermal Stability The liquid xylanase finished enzyme preparation prepared in batch 1 of Example 3 was taken, and the xylanase liquid was placed under 80°C for heat preservation treatment, and samples were taken at regular intervals to determine the enzyme activity. The experimental results were all expressed in relative enzyme activity, that is, the initial enzyme activity was set as 100% enzyme activity, and the enzyme activity was determined by the method described in Example 2 to determine the relative enzyme activity of the remaining xylanase after heat preservation for different times. The experimental results are as follows: Figure 3 When kept at 80℃ for 2.5h, the enzyme activity can still be kept above 85%, indicating good thermal stability.

[0062] Example 7 Acid and Alkali Resistance of Xylanase Take the liquid xylan finished enzyme preparation prepared in batch 1 of Example 3, adjust the pH of the xylanase to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0 with NaOH or HCl, respectively, and leave it at room temperature for 2 hours. After the end of the standing, the enzyme activity is determined by the method described in Example 2, and the relative enzyme activity is calculated with the enzyme activity before the acid or alkali treatment as 100%. The determination results are as follows Figure 4 As shown, after treatment at pH 3.0-10.0 for 2 h, the relative enzyme activity still remained above 80%.

[0063] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. A method for producing xylanase, characterized in that The method for producing xylanase by fermentation is as follows: green Trichoderma seed liquid is inoculated into a fermentation medium at a ratio of 8-12% of the transplanting amount, the culture conditions are initial pH 4.5-5.5, temperature 30-34°C, air volume: 0.1-0.25 vvm in the 0th to 24th hour, 0.3-0.6 vvm in the 25th to 48th hour, 0.6-1.0 vvm in the 49th hour to tank release, rotation speed 200-800 rpm, when pH is lower than 4.5 during fermentation, ammonia is supplemented to control pH 4.5-4.7; feeding is started after 10 hours of culture, dissolved oxygen is controlled at 15-30%, and culture is continued until enzyme activity increases slowly and fermentation is terminated when bacterial autolysis is serious, and the fermentation cycle is 150-160 hours; The green Trichoderma is specifically green Trichoderma ( Trichoderma viride )TP-1002, the deposit number is CGMCC No.40415.

2. The method according to claim 1, characterized in that The composition of the fermentation medium is as follows: corn cob powder 2%-5%, glucose 2%-3%, bran 0.5%-3%, MgSO4 0.02%-0.04%, (NH4)2SO4 0.4%-0.7%, K2HPO4 0.4%-0.6%, pH 5.0, and the rest is water.

3. The method according to claim 1, characterized in that The composition of the feed medium is as follows: glucose 40%-70%, ammonium sulfate 0.4%-0.6%, KH2PO4 0.3%-0.5%, corn steep liquor 2%-5%, and the rest is water, pH 5.

0.

4. The method according to claim 1, characterized in that The extraction and purification methods of xylanase are as follows: The pH value of the fermentation liquid was adjusted to 7.0 with alkaline solution, and 3% perlite filter aid was added to perform plate and frame filtration; 2% diatomaceous earth was added to the filtrate for fine filtration and clarification; the fine filtrate was ultrafiltered and concentrated with a 20KDa ultrafiltration membrane; the ultrafiltration concentrate was obtained, and then filtered and sterilized with a sterile membrane to obtain a liquid xylan finished enzyme preparation.

5. The method according to claim 4, characterized in that The coating and granulation method of xylanase is as follows: Accurately measure the required finished liquid xylanase preparation, add 30%-50% corn starch, 10%-20% talcum powder, and 2%-7% maltodextrin in the weight of the liquid xylanase, and fully mix the above materials in a mixer; put it into a spinning extruder to extrude a long strip material with a mesh diameter of 0.6 mm; pour the extruded long strip material into a spheronizer, adjust the spheronizer at a suitable speed to fully cut it, so that the enzyme particles are spherical; perform air drying on the spherical enzyme preparation particles, the air temperature is 45-55°C, the material temperature is 30-38°C, and the air drying is performed for 20-35 minutes to obtain a finished xylanase particle product.

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