A method for producing xylanase

Abstract

The present invention belongs to the field of bioengineering technology, and particularly relates to a method for producing xylanase. The production strain used in the method is specifically Trichoderma viride ( Trichoderma viride ) TP-1002, deposited with CGMCC No. 40415. The xylanase produced by this strain is heat-resistant, acid- and alkali-resistant, with high enzyme activity and stable performance. The present invention also optimizes the corresponding fermentation mechanism, resulting in higher fermentation activity, higher extraction yield, and lower manufacturing costs, with an average fermentation enzyme activity exceeding 29,000 U / mL. Furthermore, a xylanase coating and granulation method has been developed, addressing the inconvenient storage and transportation issues of existing xylanase, improving enzyme activity stability and ease of application, and significantly enhancing the granules' moisture and heat resistance, promising broad application prospects.

Description

Technical field:

[0002] The invention belongs to the technical field of biochemical engineering, relates to strain breeding for microbial fermentation, and particularly relates to a method for producing xylanase. Background technology:

[0004] The earliest research on xylanase began in 1955, and it was initially named pentasaccharidase. In 1961, the International Union of Biochemistry and Molecular Biology officially named it xylanase. Its official name is endo-1,4-β-xylanase, but other common names include xylanase and endo-xylanase, which are essentially the same.

[0005] Xylanases are an important class of xylanose hydrolases and are also induced enzymes. They primarily comprise a complex enzyme system consisting of multiple enzymes, including 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, and esterase. The synergistic action of these enzymes in this complex system degrades xylan into xylo-oligosaccharides and xylose. Xylanases are widely available, including marine and terrestrial bacteria, marine algae, fungi, the rumen of ruminants, snails, crustaceans, terrestrial plant tissues, and various invertebrates. However, due to the low enzyme content in animals and plants, the complex extraction and purification processes often fail to meet research needs. Therefore, a large number of xylanases are derived from microorganisms. Enzyme-producing microorganisms include Aspergillus, Trichoderma, Penicillium, yeast, Schizophyllum, and Bacillus.

[0006] The application of xylanase in the food industry is primarily reflected in the numerous physiological benefits of the xylooligosaccharides it produces, which can be used in the development of health foods. In winemaking, xylanase can significantly improve the efficiency and yield of alcohol fermentation. In baked goods, xylanase can also improve dough stability and fluffiness, enhancing gluten elasticity and the texture of pasta. In the feed industry, the addition of xylanase and other complex enzyme preparations can fully convert feed into easily digestible carbohydrates, improving utilization. In the papermaking industry, xylanase is added to pre-bleach pulp, improving bleaching efficiency and effectively reducing the use of chemical reagents in the papermaking industry. In the pharmaceutical industry, xylanase can improve intestinal microbial balance, enhance immunity, and prevent and treat diarrhea, constipation, and tumors. Xylanase has also been shown to have important physiological functions in plant tissues, potentially involved in fruit softening, seed germination, and plant defense mechanisms. Especially in the process of studying microbial xylanases, some scholars were surprised to find that microbial xylanases can induce ethylene biosynthesis in plant cells and thus act as a trigger for the plant defense system. Therefore, current research mainly focuses on xylanases produced by microorganisms.

[0007] For decades, the potential applications of xylanase have captured the attention of numerous researchers. Currently, industrial production of xylanase faces the following challenges: low enzyme activity; immature industrial production processes; inconvenient transportation; and insufficient enzyme stability. These issues contribute to high production costs. Research is focused on identifying strains that produce high xylanase yields, improving xylanase stability, and producing enzymes that are resistant to inactivation and have a broad activity range. Summary of the invention:

[0009] To solve the above problems, the present invention aims to provide a green Trichoderma capable of producing xylanase, specifically Trichoderma ( Trichoderma viride TP-1002, obtained through fast neutron irradiation and diethyl sulfate mutagenesis, significantly increases xylanase production, laying the foundation for low-cost, large-scale xylanase production. This strain was deposited on November 8, 2022, at the General Microbiology Center of the China Culture Collection Administration (CMC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, under the accession number CGMCC No. 40415.

[0010] The present invention also provides the strain Trichoderma viride ( Trichoderma viride ) Applications of TP-1002, particularly in the production of xylanase;

[0011] Furthermore, the present invention provides a method of using Trichoderma viride ( Trichoderma viride ) TP-1002 completed the liquid microbial fermentation production method of xylanase with high fermentation enzyme activity, high extraction yield and low manufacturing cost, as follows:

[0012] Fermentation tank culture: The seed liquid is transferred into the fermentation tank at a ratio of 8-12% (v / v) of the transplant volume. The culture conditions are: initial pH 4.5-5.5, temperature 30-34°C, air volume: 0.1-0.25 vvm for the 0-24th hour, 0.3-0.6 vvm for the 25th-48th hour, 0.6-1.0 vvm for the 49th hour until the tank is discharged, rotation speed 200-800 rpm. If the pH is lower than 4.5 during the fermentation period, ammonia is added to control the pH at 4.5-4.7.

[0013] Furthermore, after 10 h of fermentation, the fed-batch culture medium was fed to control the dissolved oxygen at 15-30%;

[0014] Furthermore, the fermentation cycle is 150-160 hours, during which time the enzyme activity in the fermentation tank increases slowly and the bacteria undergo severe autolysis. At the end of fermentation, the xylanase activity in the fermentation broth can reach more than 29,000 U / ml.

[0015] Furthermore, the fermentation enzyme production medium (w / v) comprises: 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.

[0016] Fermentation tank sterilization process: 121-123℃, 0.11-0.12MPa, sterilization for 30-35min.

[0017] Furthermore, the feed medium (w / v) comprises: glucose 40%-70%, ammonium sulfate 0.4%-0.6%, KH2PO4 0.3%-0.5%, corn steep liquor 2%-5%, and the remainder water, pH 5.0.

[0018] Sterilization process of feeding tank: Sterilize glucose alone at 121-123℃ and 0.11-0.12MPa for 30-35min; sterilize the remaining raw materials together at 121-123℃ and 0.11-0.12MPa for 30-35min, cool to 40℃ and then mix.

[0019] Furthermore, the extraction and purification method is as follows:

[0020] The pH of the fermentation liquid is adjusted to 7.0 with alkaline solution, and perlite filter aid is added for plate and frame filtration. Diatomaceous earth is then added for fine filtration. The fine filtrate is ultrafiltered and concentrated using an ultrafiltration membrane. The concentrate is filtered and sterilized using a sterile membrane (stabilizers and preservatives may be added) to obtain a liquid finished enzyme preparation.

[0021] Furthermore, the coating and granulation method of xylanase is as follows:

[0022] Accurately measure the required finished liquid xylanase preparation, add 30%-50% corn starch, 10%-20% talc, and 2%-7% maltodextrin by weight of the liquid xylanase, and thoroughly mix the above materials in a mixer; put the mixture 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 to an appropriate speed to fully cut the material so that the enzyme granules are spherical; and subject the spherical enzyme preparation granules to air drying at a blast temperature of 45-55°C and a material temperature of 30-38°C for 20-35 minutes to obtain finished xylanase granules.

[0023] The xylanase obtained by the present invention has the following enzymatic properties:

[0024] (1) The optimal 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.

[0025] (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%.

[0026] Beneficial effects:

[0027] The present invention provides a Trichoderma viride strain suitable for industrial production of xylanase and an optimized fermentation mechanism. This fermentation mechanism offers higher fermentation activity, lower manufacturing costs, and an average fermentation enzyme activity exceeding 29,000 U / mL.

[0028] 2. The present invention provides a xylanase product that is heat-resistant, acid- and alkali-resistant, has high enzyme activity, stable performance, and low production cost. This 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:

[0030] Figure 1 Optimum action temperature curve;

[0031] Figure 2 Optimal pH curve;

[0032] Figure 3 Temperature stability curve;

[0033] Figure 4 pH stability curve. Specific implementation method:

[0035] In order to make the purpose, technical solutions and advantages of this patent more clear, the following is a further detailed description of this patent in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain this patent and are not intended to limit the present invention.

[0036] Example 1 Mutation breeding of strains

[0037] 1. Culture medium

[0038] Slant activation medium: PDA medium.

[0039] Wash and peel the potatoes, weigh 200g of potatoes and cut them into small pieces, add water and boil for 20-30 minutes, filter through 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 bandage, and sterilize at 121℃ for 20 minutes.

[0040] Seed activation medium: bran 2%, glucose 1%, (NH4)2SO40.4%, MgSO40.2%, K2HPO40.1%, KCl0.05%, FeSO40.01%, and the rest is water, pH5.0-5.2.

[0041] 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.

[0042] 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.

[0043] 2. Preparation of bacterial suspension:

[0044] The green mold spores were washed off from the laboratory-preserved slant culture medium with physiological saline, placed in a triangular flask and added 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.

[0045] 3. Mutagenic lethality and positive mutation rates

[0046] 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%

[0047] Positive mutation rate (%) = number of mutants with a hydrolysis zone diameter larger than that of the control group / total number of colonies × 100%

[0048] 4. Fast neutron irradiation mutagenesis

[0049] 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 to 10 -6 , 0.2 ml of each 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.

[0050] Table 1 Lethality rate and positive mutation rate of strains induced by fast neutron irradiation

[0051]

[0052] As shown in the table, the lethality rate gradually increases with increasing radiation dose. Positive mutations first rise and then decrease. At a dose of 1 Gy, the positive mutation rate reaches a peak of 45.2%, and the lethality rate is 39.6%. The radiation dose of 1 Gy was determined.

[0053] 5. Diethyl sulfate (DES) mutagenesis

[0054] Take 1 ml of diethyl sulfate and add it to 9 ml of 95% ethanol to prepare diethyl sulfate dilution solution. Mix the diluted diethyl sulfate solution with the bacterial suspension in a certain ratio (diethyl sulfate dilution solution: 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 , 0.2 mL of each was taken and spread on the primary screening culture medium, and cultured in a 34°C incubator. The bacterial solution that had not been mutagenized with diethyl sulfate was diluted and spread on the plate as a control.

[0055] Table 2 Lethality and positive mutation rate of strains induced by diethyl sulfate

[0056]

[0057] As shown in the table, the highest positive mutation rate was 43.3% at a DES solution / bacterial suspension ratio of 1:250 and a mutagenesis time of 20 minutes. The optimal conditions for diethyl sulfate mutagenesis were determined: a DES solution / bacterial suspension ratio of 1:250 and a mutagenesis time of 20 minutes.

[0058] 6. Compound mutagenesis

[0059] After the above two mutagenesis methods, the mutagenesis methods 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.

[0060] Table 3 Xylanase activity of 7 mutant strains

[0061]

[0062] After composite mutagenesis, the mutant seed No. 3 was selected, and the enzyme activity reached 4258U / mL, which was 3.4 times higher than that of the original strain.

[0063] 7. Genetic stability

[0064] The strain was used as the research object to determine its genetic stability, and the enzyme activity experiment was carried out by shake 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 the 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 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.

[0065] Table 4 Genetic stability results of strains

[0066]

[0067] Example 2 Determination and definition of xylanase activity of the present invention

[0068] 1. Definition of xylanase activity unit

[0069] 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.

[0070] 2. Enzyme activity determination method

[0071] Take 2ml of diluted enzyme solution (equilibrated at 50℃ for 10min), add it to a graduated test tube, then add 5ml of DNS reagent and oscillate electromagnetically for 3s-5s. Then add 2ml of 1% xylan substrate (prepared with pH 5.5 acetic acid-sodium acetate buffer), keep warm at 50℃ for 30min, and heat in a boiling water bath for 5min. Cool to room temperature with tap water, add distilled water to make up to 25ml, and oscillate electromagnetically for 3s-5s. Use a standard blank sample (4.0ml of acetic acid-sodium acetate buffer solution, 5.0ml of DNS reagent, heat in a boiling water bath for 5min, cool, and make up to 25ml) as a blank control, and measure the absorbance A at 540nm. B .

[0072] Take 2ml of diluted enzyme solution (equilibrated at 50℃ for 10min), add it to a graduated test tube, then add 2ml of 1% xylan substrate (prepared with pH 5.5 acetic acid-sodium acetate buffer), electromagnetically oscillate for 3s-5s, and keep warm at 50℃ for 30min. Add 5ml of DNS reagent and electromagnetically oscillate for 3s-5s to terminate the enzymatic reaction. Heat in a boiling water bath for 5min. Cool to room temperature with tap water, add distilled water to make up to 25ml, and electromagnetically oscillate for 3s-5s. Use a standard blank sample (4.0ml of acetic acid-sodium acetate buffer solution, 5.0ml of DNS reagent, heat in a boiling water bath for 5min, cool, and make up to 25ml) as a blank control, and measure the absorbance A at 540nm. E .

[0073] Enzyme activity calculation formula:

[0074]

[0075] 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, 1 mmol = 1000 μmol.

[0076] Example 3: Production of xylanase by submerged fermentation of Trichoderma viride TP-1002 and product processing

[0077] 1. Seed tank culture

[0078] Seed tank culture medium: glucose 1.5%, bran 2.5%, (NH4)2SO41%, MgSO40.03%, CaCl20.1%, pH5.0, and the rest is water.

[0079] Seed tank sterilization process: 121-123℃, 0.11-0.12MPa, sterilize for 30min.

[0080] 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 0.06 MPa, air volume 0.3 vvm, and rotation speed 400 rpm. When the pH dropped to 4.4 during the culture process, ammonia was added to control the pH to 4.5. The culture time was 24 hours, and more bacteria were observed under the microscope.

[0081] 2. Fermentation tank culture

[0082] 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.

[0083] Fermentation tank sterilization process: 121-123℃, 0.11-0.12MPa, sterilization for 35min.

[0084] 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 0.06 MPa, air volume: 0.25 vvm for the 0th to 24th hour, 0.5 vvm for the 25th to 48th hour, 1.0 vvm for the 49th hour to tank release, rotation speed 600 rpm. When the pH was lower than 4.5 during the fermentation period, ammonia was added to control the pH at 4.5.

[0085] 3. Fed-batch culture

[0086] Feed medium: glucose 60%, ammonium sulfate 0.5%, KH2PO4 0.4%, corn steep liquor 4%, the rest is water, pH 5.0.

[0087] Sterilization process of feeding tank: Glucose is sterilized separately at 121-123℃ and 0.11-0.12MPa for 30min; the rest of the raw materials are sterilized together at 121-123℃ and 0.11-0.12MPa for 30min, and then cooled to 40℃ and mixed evenly.

[0088] 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.

[0089] 4. Fermentation ends

[0090] When the enzyme activity increases slowly and the bacteria autolyze seriously, the fermentation is terminated. The fermentation cycle is 150-160 hours. Prepare for tank extraction.

[0091] Table 5 Fermentation test results in 50L fermenter

[0092]

[0093] From the fermentation test 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.

[0094] 5. Extraction and purification of xylanase

[0095] (1) Adjust the pH of the fermentation liquid to 7.0 with 1 mol / L sodium hydroxide, add 3% perlite filter aid, and perform plate and frame filter pressing;

[0096] (2) Add 2% diatomaceous earth to the filtrate for fine filtration and clarification;

[0097] (3) Ultrafiltration and concentration of the seminal filtrate using a 20 kDa ultrafiltration membrane;

[0098] (4) Obtain the ultrafiltration concentrate, which is then filtered and sterilized using a sterile membrane to obtain a liquid xylan finished enzyme preparation.

[0099] 6. Xylanase coating and granulation

[0100] 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.

[0101] (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 blender.

[0102] (2) Place the material into a spinning extruder and extrude it into long strips. The diameter of the screen hole is 0.6 mm.

[0103] (3) Pour the extruded long strip material into the spheronizer and adjust the spheronizer to a suitable speed to fully cut it so that the enzyme particles appear spherical.

[0104] (4) Place the spherical enzyme preparation granules in a vertical air dryer for air drying at an air temperature of 50°C and a material temperature of 35°C. Air dry for 30 minutes to obtain the finished xylanase granules.

[0105] 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.

[0106] Example 4 Optimal reaction temperature of xylanase

[0107] 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 relative enzyme activity was calculated with the enzyme activity at 65°C as 100%. The results are shown in the figure. Figure 1 As shown, the optimal reaction temperature is 65°C, and the activity can still be maintained at more than 85% at 80°C. The experimental results show that the optimal reaction temperature of the xylanase produced by this mutant strain is significantly higher than that of xylanases from other sources in the prior art, which has great market application value and has good application value in industrial production.

[0108] Example 5 Optimal reaction pH of xylanase

[0109] The liquid xylanase product 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. The relative enzyme activity was calculated with the enzyme activity at pH 6.0 as 100%. The measurement results are shown in FIG. Figure 2 As shown in the figure, the enzymatic activity of xylanase is highest when the pH is around 6.0.

[0110] Example 6 Xylanase Thermal Stability

[0111] The liquid xylanase finished enzyme preparation prepared in batch 1 of Example 3 was taken and the xylanase liquid was kept at 80°C. Samples were taken at regular intervals to measure the enzyme activity. The experimental results were expressed as relative enzyme activity, that is, the initial enzyme activity was set as 100% enzyme activity, and the enzyme activity was measured using the method described in Example 2 to determine the relative enzyme activity of the xylanase remaining after different holding times. The experimental results are shown in Figure 2. Figure 3 When kept at 80℃ for 2.5h, the enzyme activity can still be maintained above 85%, indicating good thermal stability.

[0112] Example 7 Acid and Alkali Resistance of Xylanase

[0113] The liquid xylanase finished enzyme preparation prepared in batch 1 of Example 3 was taken, and the pH of the xylanase was adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0 with NaOH or HCl, respectively. The xylanase was allowed to stand at room temperature for 2 hours. After the standing period, the enzyme activity was determined using the method described in Example 2, and the relative enzyme activity was calculated with the enzyme activity before acid or alkali treatment as 100%. The determination results are shown in FIG. Figure 4 As shown, after treatment at pH 3.0-10.0 for 2 h, the relative enzyme activity still remained above 80%.

[0114] The above-described embodiments merely represent several implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that, without departing from the concept of this patent, a person skilled in the art would be able to make various variations, combinations, and improvements to the above-described embodiments, all of which fall within the scope of protection of this patent. Therefore, the scope of protection of this patent shall be determined by the claims.

Claims

1. A method for producing xylanase, characterized in that: The method for producing xylanase by fermentation is as follows: the green Trichoderma seed liquid is inoculated into the 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 0-24th hour, 0.3-0.6 vvm in the 25th to 48th hour, 0.6-1.0 vvm in the 49th hour until the tank is released, the rotation speed is 200-800 rpm, when the pH is lower than 4.5 during the fermentation period, ammonia is added to control the pH to 4.5-4.7; after 10 hours of culture, feeding is started, and the dissolved oxygen is controlled at 15-30%, and the culture is continued until the enzyme activity increases slowly and the fermentation is terminated when the bacteria are severely autolyzed. 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, wherein The fermentation medium is composed of 2%-5% corn cob meal, 2%-3% glucose, 0.5%-3% bran, 0.02%-0.04% MgSO4, 0.4%-0.7% (NH4)2SO4, 0.4%-0.6% K2HPO4, and the remainder water; pH 5.

0.

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

0.

4. The method according to claim 1, wherein The extraction and purification method of xylanase is as follows: adjust the pH of the fermentation liquid to 7.0 with alkaline solution, add 3% perlite filter aid, and perform plate and frame filtration; add 2% diatomaceous earth to the filtrate for fine filtration and clarification; use a 20KDa ultrafiltration membrane to ultrafilter and concentrate the fine filtrate; obtain the ultrafiltration concentrate, and then filter and sterilize it with a sterile membrane to obtain a liquid xylanase finished enzyme preparation.

5. The method according to claim 4, wherein: 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 thoroughly mix the above materials in a mixer; put the materials 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 an appropriate speed to fully cut it, so that the enzyme particles are spherical; subject the spherical enzyme preparation particles to air drying at a blast temperature of 45-55°C and a material temperature of 30-38°C for 20-35 minutes to obtain the finished xylanase granules.

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