Aspergillus niger strain with high xylanase yield as well as breeding method and application of aspergillus niger strain

Abstract

The invention discloses an aspergillus niger mutant strain with high xylanase yield as well as a breeding method and application of the aspergillus niger mutant strain. An original starting strain Aspergillus niger is treated by adopting an ultraviolet (UV) mutagenesis technology, and an Aspergillus niger mutant strain with high xylanase yield is screened out. The strain is named as Aspergillus niger G63, and is preserved in the China General Microbiological Culture Collection Center (Institute of Microbiology, Chinese Academy of Sciences, 100101, No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing) on October 28, 2024, and the preservation number of the strain is CGMCC NO.41556. The invention also discloses a preparation method of the strain. The activity of xylanase produced by the strain is higher than that of an original strain, high enzyme activity can be shown under the solid-state fermentation condition, production benefits are improved, and in addition, the xylanase has good hydrolysis performance and has extremely wide application prospects in the papermaking industry, the feed industry, the food industry and the wine brewing industry.

Description

Technical Field

[0001] The invention relates to the technical field of microbial breeding, in particular to an Aspergillus niger strain with high xylanase production, a breeding method and application thereof. Background Art

[0002] Xylanase is mainly composed of β-1,4-D xylan endo-enzyme and β-1,4-D xylan exo-enzyme. In addition, there are some debranching enzymes that can degrade side chains composed of methyl ether, arabinose and other groups. Its main degradation product is oligoxylan, which has great potential in papermaking industry, feed industry, food industry, brewing industry, energy conversion and other aspects due to its good economic benefits.

[0003] The production of xylanase mainly adopts two methods: solid-state fermentation and submerged fermentation, and is controlled by various physical and nutritional parameters. In order to achieve successful industrial applications, such as biofuel production, pulp and paper making, baking and brewing, food and feed, waste paper deinking and other biotechnology fields, people use various suitable technologies to produce xylanase at commercial level to meet specific economic feasibility and industrial needs. Therefore, it is urgent to modify the properties of xylanase through physical and chemical methods or genetic engineering technology, so as to screen out xylanase with higher efficiency.

[0004] In order to solve the problem of low xylanase activity and low yield, the present invention aims to obtain a mutagenized strain of Aspergillus niger with high xylanase yield, so as to improve the application potential of the enzyme in industrial production. Summary of the invention

[0005] In view of the limitations of the prior art in the production of xylanase, the purpose of the present invention is to provide a strain of Aspergillus niger with high xylanase production, a breeding method and application thereof. The original starting strain Aspergillus niger is treated with ultraviolet (UV) mutagenesis technology to screen out a mutagenized strain of Aspergillus niger with high xylanase production. The xylanase activity produced by the strain is higher than that of the original strain, and it can show higher enzyme activity under solid-state fermentation conditions, thereby improving production efficiency. In addition, it has good hydrolysis performance and has extremely broad application prospects in the papermaking industry, feed industry, food industry and brewing industry.

[0006] The present invention first provides a high-yield xylanase Aspergillus niger strain, which is named Aspergillus sp. G63. The strain has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration (No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101) on October 28, 2024, and its deposit number is CGMCC NO.41556.

[0007] The invention also provides a method for breeding a high-yield xylanase-producing Aspergillus niger mutant strain, using Aspergillus niger preserved in a laboratory Aspergillus sp. CM3 (accession number CGMCC NO.23060) is the starting strain, and the breeding method includes the following steps: Step 1: After activating the Aspergillus sp. CM3 strain preserved in the laboratory on PDA solid medium, a spore suspension was prepared and diluted with sterile saline to achieve a spore suspension containing 1.0×10 6 The predetermined concentration of spores.

[0008] Step 2: Place the spore suspension prepared in step 1 under a 15-watt ultraviolet lamp, keep a distance of 25 cm, and irradiate it under stirring, and the irradiation time is controlled to be 1 to 10 minutes. Then, under red light illumination, the spore suspension is continuously diluted in a gradient manner and evenly coated on a plate of the primary screening culture medium. Finally, the coated culture medium plate is placed in a constant temperature incubator at 28°C for light-proof culture for 48 hours.

[0009] Step 3. Select strains with good growth from the primary screening culture medium, and use the plate streak method to separate and purify the strains. The purified strains are re-inoculated onto the primary screening culture medium plate by the dot grafting method. After the strains grow, they are stained with Congo red solution and then eluted with sterile saline. During the elution process, observe whether a transparent circle is formed around the strain, which is an important indicator for screening strains. Measure the diameter of the strain itself (denoted as Dc) and the diameter of the transparent circle (denoted as Dp), and calculate the ratio of the two (Dp / Dc). Select the strain with the largest transparent circle ratio, transfer it to PDA solid culture medium, and culture it at 28°C for 3 days. After the culture is completed, use sterile saline to wash the spores and prepare a spore suspension with a concentration of 1.0×10 per milliliter. 6 spores. Then, a certain amount of spore suspension is inoculated into a solid fermentation medium for solid fermentation. After the culture is completed, the xylanase activity is measured to evaluate the enzyme production ability of the strain. According to the enzyme activity measurement results, the mutant strain with the highest enzyme activity is selected, which is the Aspergillus niger mutagenesis strain with high xylanase production, and its preservation number is CGMCCNO.41556.

[0010] The present invention further provides a method for producing xylanase by solid-state fermentation and separation and purification of the Aspergillus niger mutant strain obtained by mutagenesis rescreening.

[0011] Specifically, the selected Aspergillus niger mutant strain with the highest enzyme activity was activated and cultured on PDA solid medium for 3 days, the activated fresh strains were washed with sterile saline, and transferred to a conical flask containing glass beads, and the spores were fully shaken to ensure that the spores were evenly dispersed. Then, a sterile six-layer gauze was used for filtration to remove the hyphae to obtain a pure spore suspension. The number of spores was accurately calculated using a hemocytometer and a microscope for observation. Finally, appropriate dilution was performed with sterile saline to achieve a spore suspension containing 1.0×10 6 A predetermined concentration of spores is set, and then a certain amount of the spore suspension is inoculated into the solid fermentation medium for solid fermentation.

[0012] The fermentation wet base after solid-state fermentation is dried and ground to obtain a fermentation matrix; the fermentation matrix is ​​stirred and mixed with an acetic acid-sodium acetate buffer solution, and the volume is fixed to obtain a solution with a concentration of 10 mg / mL, which is stored in the dark at 4°C for 1h~2h. Centrifuge at 1000g for 3min~5min, take the supernatant, and then dilute it with a buffer solution to obtain a crude enzyme solution, extract the protein by ammonium sulfate precipitation, dialyze and concentrate, filter the filter membrane, and then load the sample on an anion exchange column for gradient elution to obtain purified xylanase.

[0013] The PDA solid culture medium of the present invention comprises the following components: 200 g / L potato, 20 g / L glucose and 20 g / L agar, and is sterilized at 115° C. for 30 min.

[0014] The components of the primary screening solid culture medium of the present invention are: KCl 0.5g / L, K2HPO4 1.0g / L, MgSO4·7H2O0.5g / L, FeSO4·7H2O 0.5g / L, agar 20g / L, water is added to make the volume to 1000mL, sterilized at 121°C for 20min, 8g of xylan is added after cooling, and the plate is poured after shaking.

[0015] The solid-state fermentation medium of the present invention comprises the following components: 23.4 g bran, 6.6 g soybean meal, 0.45 g (NH4)2SO4, 0.33 g MgCl2, 18.6 mL H2O, and is sterilized at 121°C for 20 min.

[0016] The composition of the acetic acid-sodium acetate buffer of the present invention is: 23.14 g of sodium acetate trihydrate, 1.7 mL of glacial acetic acid is added, water is added to dissolve, and the volume is fixed to 2000 mL. The pH of the solution is measured. If the pH deviates from 5.5, it is adjusted to 5.5 with acetic acid solution or sodium acetate solution.

[0017] The dialysis bag used in the present invention is immersed in boiling water and boiled for 20 minutes, taken out and immediately placed in distilled water for cooling, and after cooling to room temperature, stored in a 20% ethanol solution.

[0018] The fixed filler of the anion exchange column chromatography described in the present invention is DEAE Beads 6FF.

[0019] The present invention further provides the Aspergillus niger mutant strain with high xylanase production, and application of the xylanase produced by the strain in food and feed industries.

[0020] The enzymatic properties of xylanase produced by the isolated and purified Aspergillus niger mutant strain were studied. The enzymatic reaction was carried out in a phosphate system at different temperatures to determine the mutant strain. Aspergillus sp. The optimal temperature and thermal stability of xylanase produced by G63. The enzymatic reaction was carried out in phosphate system with different pH values ​​to determine the induced strain Aspergillus sp. The optimal pH and pH stability of G63 xylanase production. Different metal ions were added to the enzymatic reaction system to determine the optimal metal ion and concentration. The purified xylanase was reacted with the substrate under the optimal reaction conditions.

[0021] The enzymatic reaction of the present invention comprises a hydrolysis reaction system containing substrate xylan, xylanase, a phosphate buffer solution and metal ions.

[0022] The temperature of the hydrolysis reaction is 40-65°C, preferably 50-55°C, and most preferably 55°C.

[0023] The pH of the hydrolysis reaction is 4.5-7.5, preferably 4.5-6.5, most preferably 5.5.

[0024] The metal ion is any one of calcium ion, sodium ion, potassium ion and magnesium ion, preferably sodium ion; the final concentration of the metal ion is 2-10mmol / L, preferably 2mmol / L.

[0025] In some specific embodiments of the present invention, the xylanase obtained by fermentation and purification is used to catalyze the hydrolysis reaction of xylan, and by analyzing the reaction product, it is found that the main component thereof is xylose.

[0026] Compared with the prior art, the invention has the following beneficial effects: the Aspergillus niger mutagenesis strain with high xylanase production, its breeding method and application, the invention successfully screens out an efficient Aspergillus niger mutagenesis strain by applying ultraviolet mutagenesis technology combined with Congo red staining method, the strain shows excellent performance in the production of xylanase, and has significant industrial application potential. The use of ultraviolet mutagenesis technology significantly improves the mutation rate, while effectively reducing the production cost, providing an economically feasible solution for the large-scale production of xylanase. In addition, the invention adopts solid-state fermentation culture technology, which not only simplifies the culture process, but also reduces energy consumption. The strain cultured by solid-state fermentation can produce a wide variety of xylanases with high activity, and has the advantages of simple operation, rapid microbial growth and low pollution risk. These characteristics make the invention have important application value in the field of biotechnology, and provide a new efficient and environmentally friendly way for the industrial production of xylanase. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Other features, objects and advantages of the present application will become more apparent by reading the detailed description of non-limiting embodiments made with reference to the following drawings: Figure 1 is the UV-induced lethality curve; Figure 2 The strains that produce transparent circles after Congo red staining Aspergillus sp. G63 schematic diagram; Figure 3 Schematic diagram of SDS-PAGE of the original strain and the mutagenized strain; Figure 4 Schematic diagram of NATIVE-PAGE of the original strain and the mutagenized strain; Figure 5 The comparison chart of enzyme activity between the induced strain and the original strain is shown in Figure 5 In the figure, the abscissa is the mutagenic strain, and the ordinate is the xylanase activity; Figure 6 The xylanase activity in the supernatant and precipitate during the graded salting out of (NH4)2SO4; Figure 7 is the purified xylanase SDS-PAGE; lane 10 is the target band; Figure 8 is the optimal reaction temperature of xylanase; Fig. 9 is the thermostability of xylanase; Fig.10 is the optimal reaction pH of xylanase; Fig.11 is the pH stability of xylanase; Fig.12The effect of metal ions at a concentration of 2mmol / L on the activity of xylanase; Fig.13 The effect of metal ions at a concentration of 10mmol / L on the activity of xylanase; Fig.14 The original strain Aspergillus sp. CM3 and mutagenic strains Aspergillus sp. Lineweave-Burk curve of xylanase produced by G63. DETAILED DESCRIPTION

[0028] The present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It is to be understood that the specific embodiments described herein are only used to explain the relevant invention, rather than to limit the invention. It is also necessary to explain that, for ease of description, only the parts related to the invention are shown in the accompanying drawings. Example 1

[0029] Breeding of Aspergillus niger mutant strain with high xylanase production (1) Take out the Aspergillus niger strain stored in the laboratory from the low-temperature storage at -80℃ Aspergillus sp. CM3, and activated culture was performed on PDA solid medium for 3 days. Afterwards, these activated fresh strains were washed with an appropriate amount of sterile saline and transferred to a sterilized conical flask containing glass beads. By fully shaking, ensure that the spores are evenly dispersed. Next, filter with sterile six layers of gauze to remove the hyphae to obtain a pure spore suspension. Use a hemocytometer and a microscope to observe and accurately count the number of spores. Finally, dilute appropriately with sterile saline to achieve 1.0×10 per milliliter of spore suspension. 6 The predetermined concentration of spores.

[0030] The components of PDA solid culture medium are: 200 g / L potato, 20 g / L glucose, 20 g / L agar, sterilized at 115°C for 30 min.

[0031] (2) Place the spore suspension obtained in step (1) under a 15W ultraviolet lamp, 25 cm away from the light source, and irradiate it under stirring for 1 to 10 minutes. Under red light illumination, take 1 mL of sample from the spore suspension and add it to test tubes containing sterile saline for gradient dilution. Subsequently, take 100 μL of the diluted spore suspension and evenly spread it on a plate of the primary screening culture medium, and place it in an incubator at 28°C for 48 hours in the dark.

[0032] The components of the primary screening solid culture medium are: KCl 0.5g / L, K2HPO4 1.0g / L, MgSO4·7H2O 0.5g / L, FeSO4·7H2O 0.5g / L, agar 20g / L, add water to make up to 1000mL, sterilize at 121℃ for 20min, add 8g of xylan after cooling, shake well and pour into the plate.

[0033] By observing the culture results, the lethality of the spores is calculated, and a lethality curve is drawn based on the obtained data, such as Figure 1 shown. Figure 1 The analysis results show that when the ultraviolet light irradiation time is set to 7 minutes, the mortality rate can reach 81.63%, that is, the positive mutation rate is higher when the ultraviolet light is irradiated for more than 7 minutes.

[0034] (3) The strains with a lethality rate of 80% on the primary screening medium in step (2) were separated by the plate streak method to purify the strains. The purified strains were re-inoculated onto the primary screening medium plate by the spot grafting method. After the strains grew, they were stained with Congo red solution and then eluted with sterile saline. During the elution process, it was observed whether a transparent circle was formed around the strain, which was an important indicator for screening strains. The diameter of the strain itself (denoted as Dc) and the diameter of the transparent circle (denoted as Dp) were measured, and the ratio of the two was calculated (Dp / Dc). The strain with the largest transparent circle ratio was selected, transferred to PDA solid medium, and cultured at 28°C for 3 days. After the culture was completed, the spores were washed with sterile saline and poured into a sterile conical flask containing glass beads and shaken thoroughly to disperse the spores. The mycelium was removed by filtering through a sterile six-layer gauze to obtain a pure spore suspension. Count the spores in the spore suspension using a hemocytometer and microscope, and adjust the concentration of the suspension to 1.0×10 per ml. 6 spores. Then, a certain amount of spore suspension was inoculated into the solid fermentation medium. The components of the solid fermentation medium were: 23.4g bran, 6.6g soybean meal, 0.45g (NH4)2SO4, 0.33g MgCl2, 18.6mL H2O, sterilized at 121℃ for 20min. At the same time, the initial strain that had not been induced by mutation was used as a control group for solid fermentation. After the culture was completed, the enzyme activity of xylanase was measured to evaluate the enzyme production ability of the strain. According to the results of the enzyme activity determination, the mutant strain with the highest enzyme activity was selected for subsequent experimental research.

[0035] The Congo red staining method produces transparent circles. Figure 2 As shown, an obvious transparent circle was formed around the mutagenic strain.

[0036] Solid-state fermentation was performed on each Aspergillus niger mutant strain obtained by mutagenesis, and its enzyme activity was determined. The mutant strain with the highest enzyme activity relative to the original strain was selected as the final target strain. Aspergillus sp.G63. Figure 3 It can be seen that in the case of protein quantification, the SDS-PAGE result of the mutagenized strain (G63) is darker in color. Figure 4 It can be seen that in the case of protein quantification, the clearing zone produced by NATIVE-PAGE of the mutagenic strain (G63) is larger.

[0037] (4) Determination of xylanase activity Extraction of crude enzyme solution: Weigh 1.00 g of dried and crushed fermentation substrate. Add 40 mL of acetic acid-sodium acetate buffer solution. Stir magnetically for 30 minutes, then dilute to 100 mL with buffer solution, and store at 4°C in the dark for 1 hour to 2 hours. Centrifuge at 1000g for 3 minutes to 5 minutes, take 1.0 mL of supernatant, and dilute to 50 mL with buffer solution.

[0038] Determination steps: a. Take 4.0 mL of acetic acid-sodium acetate buffer solution, add 5.0 mL of DNS reagent, and heat in a boiling water bath for 5 minutes. Cool to room temperature with tap water, and make up to 25.0 mL with water to prepare a standard blank sample.

[0039] b. Pipette 10.0 mL of xylan solution and equilibrate at 37 °C for 20 min. Pipette 10.0 mL of appropriately diluted enzyme solution and equilibrate at 37 °C for 10 min. Pipette 2.00 mL of appropriately diluted enzyme solution (already equilibrated at 37 °C) and add it to a graduated test tube, then add 5 mL of DNS reagent and oscillate electromagnetically for 3s to 5s. Then add 2.0 mL of xylan solution, keep warm at 37 °C for 30 min, and heat in a boiling water bath for 5 min. Cool to room temperature with tap water, add water to make up to 25 ml, and oscillate electromagnetically for 3s to 5s. Use the standard blank sample as a blank control and measure the absorbance A at 540 nm. B .

[0040] c. Take 2.00 mL of appropriately diluted enzyme solution (equilibrated at 37°C) and add it to a graduated test tube. Then add 2.0 mL of xylan (equilibrated at 37°C), oscillate by electromagnetic field for 3s~5s, and keep the temperature at 37°C for 30 min. Add 5.0 mL of DNS reagent and oscillate by electromagnetic field 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 water to make up to 25 mL, and oscillate by electromagnetic field for 3s~5s. Use the standard blank sample as the blank control and measure the absorbance A at 540 nm. C Definition of enzyme activity unit: Under the conditions of 37°C and pH 5.50, the amount of enzyme required to degrade and release 1 μmol of reducing sugar from a 5 mg / mL xylan solution per minute is one enzyme activity unit U.

[0041] The enzyme activity of the high-yield xylanase-producing strain induced by UV mutagenesis is as follows Figure 5 As shown, the xylanase activity of the original strain (CM3) was 12605 U / g dry basis. Aspergillus sp. The enzyme activity of G63 is 15365U / g dry basis, which is 21.9% higher than the original one. Aspergillus sp. G63 enters the next step of the experiment. Example 2

[0042] Isolation, Purification and Enzymatic Reaction of Xylanase The target strain obtained by mutagenesis screening is separated and purified after solid-state fermentation. The mutant strain with the highest enzyme activity selected in step 3 is purified by Aspergillus sp.G63 was activated and cultured on PDA solid medium for 3 days. Afterwards, these activated fresh strains were washed with an appropriate amount of sterile saline and transferred to a sterilized conical flask containing glass beads. By fully shaking, ensure that the spores are evenly dispersed. Next, filter with sterile six layers of gauze to remove the hyphae to obtain a pure spore suspension. Use a hemocytometer and a microscope to observe and accurately count the number of spores. Finally, dilute appropriately with sterile saline to achieve a spore suspension containing 1.0×10 per milliliter. 6 The predetermined concentration of spores is set, and then a certain amount of spore suspension is inoculated into the solid-state fermentation medium and cultured for 5 days. Dry and grind the fermentation wet base after the solid-state fermentation, and weigh 1.00g of the fermentation matrix. Add acetic acid-sodium acetate buffer solution. Stir magnetically for 30 minutes, then make up to 100mL with buffer solution, and store in the dark at 4°C for 1h~2h. Centrifuge at 1000g for 3min~5min, take the supernatant, and then dilute it appropriately with buffer solution. Add (NH4)2SO4 powder gradually under ice bath conditions, control the generation of foam during the addition process, and perform graded gradient salting out. First, adjust the final concentration of (NH4)2SO4 to 50%, retain the supernatant, and gradually add (NH4)2SO4 powder to the supernatant until the final concentration reaches 100%. Then, place the solution at 4°C overnight, centrifuge to obtain the supernatant and precipitate, and use Figure 6It can be seen that when the final saturation concentration of ammonium sulfate is 100%, the relative enzyme activity of the precipitate reaches the maximum. After the precipitate is re-dissolved in the buffer solution, it is placed in the treated dialysis bag, sealed and soaked in a buffer solution with a pH of 3.5, and kept at 4°C overnight. Take a certain amount of the initially purified xylanase solution and put it into the treated dialysis bag, clamp the two ends and place it in a blank glass dish, and spread polyethylene glycol 2000. When the crude enzyme solution in the dialysis bag is about 1 / 4 of the original volume, take it out. Take a gravity column of appropriate specifications and rinse the column tube and gasket with deionized water. Pipette 6mL of the stationary phase filler DEAEBeads 6FF into the gravity column. After the protective solution in the filler flows out, rinse the filler with deionized water until there is no ethanol smell. Filter the above crude enzyme solution with a 0.22mm filter membrane before loading. The equilibrium buffer and eluent used are filtered with a 0.22mm filter membrane. Manual loading is used for column loading. First, balance the pre-installed filler and rinse 5 times the column volume with the balancing solution; slowly add 1 mL of the crude enzyme sample to the column along the tube wall, keep it for 2 minutes to allow full contact, collect the effluent, and repeat the loading to increase the binding rate; then perform gradient elution, and the eluent is a NaCl solution of different concentrations (0.1-0.5 mol / L). Each gradient uses about 5 times the column volume for elution, and one tube is collected for each column volume, and the enzyme activity and protein content are tested respectively. After purification, first wash the remaining retained protein with 1 mol / L NaCl solution, then rinse with buffer for at least 5 times the column volume, then rinse with deionized water, about 5 times the column volume, and finally rinse with 20% ethanol solution for 2-3 times the column volume, and store in a 4°C refrigerator. Figure 7 It can be seen that when the NaCl concentration is 0.5 mol / L, the eluted xylanase is relatively ideal. According to the relative mobility of the protein, combined with the protein marker, the molecular weight of the xylanase is calculated to be 28.0 KDa.

[0043] The enzymatic properties of xylanase produced by the isolated and purified Aspergillus niger mutant strain were studied. In the enzymatic reaction system, the substrate was 2 mL of 10 mg / mL xylan solution, 2 mL of purified xylanase solution with a concentration of 100 U / mL, and phosphate buffer solution, with a total reaction volume of 25 mL. The enzymatic reaction was carried out in a phosphate system at different temperatures (30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, and 65°C), and it was determined that the optimal temperature for the mutagenic strain (G63) to produce xylanase was 50°C. Figure 8 At 50°C, the xylanase produced by the mutant strain (G63) was incubated for 0, 30, 60, 90, 120, and 150 min, respectively, and the xylanase activity was measured, the relative enzyme activity was calculated, and the thermal stability of the enzyme was determined, as shown in Figure 2. Fig. 9The enzymatic reaction was carried out in a phosphate system with different pH values ​​(2.5, 3.5, 4.5, 5.5, 6.5, 7.5), and the optimum pH value for the production of xylanase by the mutant strain (G63) was determined to be 5.5, as shown in Fig.10 As shown. Under the condition of pH 5.5, the xylanase produced by the mutant strain (G63) was incubated for 0, 30, 60, 90, 120, and 150 minutes respectively, and the xylanase activity was measured, the relative enzyme activity was calculated, and the pH stability of the enzyme was determined, as shown in Figure 2. Fig.11 As shown. Different types of metal ions (Ca + , Fe 3+ , Cu 2+ 、Na + , K + 、Co 2+ , Mn 2+ 、Zn + Mg 2+ ) were added into the enzymatic reaction of xylanase, so that the final concentration of metal ions was 2mmol / L and 10mmol / L respectively, the xylanase activity was measured, the relative enzyme activity was calculated, and the effect of different concentrations of metal ions on the production of xylanase by the mutagenic strain (G63) was determined. Fig.12 , Fig.13 As shown. Xylanase was incubated with substrates of different concentrations, and the enzymatic reaction was carried out under the optimal reaction conditions. The enzyme activity at different concentrations was measured. The Lineweaver-Burk double reciprocal plot was drawn, the Km and Vmax values ​​were calculated, and the kinetic parameters and substrate specificity were determined. The curve equation of the mutagenic strain (G63) was:

[0044] in K m =2.99 mg / mL, V max =3425.51 mg / (min×mL) .

[0045] The curve equation of strain (CM3) is:

[0046] in K m = 3.57 mg / mL,V max = 4082.52 mg / (min×mL) .

[0047] According to the meaning of each parameter of the Michaelis-Menten equation, the substrate concentration when the xylanase produced by strain (G63) reaches the maximum rate during the enzymatic reaction is 2.99 mg / mL; the substrate concentration when the xylanase produced by strain (CM3) reaches the maximum rate during the enzymatic reaction is 3.57 mg / mL. K m Smaller, V max / K m A higher value indicates that the conversion efficiency of the enzymatic degradation reaction product is improved and the enzyme has a stronger affinity for the substrate, e.g. Fig.14 shown.

[0048] Aspergillus niger mutant strain provided by the present invention Aspergillus sp. G63, which has been specially selected and bred, can be widely used in the industrial production of xylanase. The use of this strain can significantly reduce production costs and increase yields, thus providing economic and technical advantages for the widespread application of xylanase in industries such as food and feed.

[0049] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0050] The above description is only a preferred embodiment of the present application and an explanation of the technical principles used. Those skilled in the art should understand that the scope of the invention involved in the present application is not limited to the technical solution formed by a specific combination of the above technical features, but should also cover other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the inventive concept. For example, the above features are replaced with (but not limited to) technical features with similar functions disclosed in the present application.

Claims

1. A strain of Aspergillus niger, characterized in that: Classification and naming of the Aspergillus niger strain Aspergillus niger G63, It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 28, 2024, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC NO.41556.

2. The method for breeding the Aspergillus niger strain according to claim 1, characterized in that: The following steps are involved: Step 1: After activating the Aspergillus sp. CM3 strain preserved in the laboratory on PDA solid medium, a spore suspension was prepared and diluted with sterile saline to achieve a spore suspension containing 1.0×10 6 A predetermined concentration of spores; Step 2: placing the spore suspension prepared in step 1 under a 15-watt ultraviolet lamp, maintaining a distance of 25 cm, and irradiating it under stirring, and the irradiation time is controlled at 1 to 10 minutes. Then, under red light illumination, the spore suspension is continuously diluted in a gradient manner, and evenly spread on a plate of the primary screening culture medium, and cultured at a constant temperature of 28° C. in the dark for 48 hours; Step 3: Select the strains with good growth from the primary screening medium, separate and purify the strains by plate streak method; re-inoculate the purified strains on the primary screening medium plate by spot grafting method, stain with Congo red solution after the strains grow, elute with sterile saline, measure and calculate the ratio of the diameter of the transparent circle to the diameter of the strain itself, select the strain with the largest transparent circle ratio, transfer it to PDA solid medium, culture it at 28°C for 3 days, and prepare a spore suspension with a concentration of 1.0×10 per milliliter. 6 spores; inoculating the spore suspension into a solid fermentation medium for solid fermentation culture, determining the xylanase activity, and selecting the strain with the highest enzyme activity, which is the Aspergillus niger mutant strain with high xylanase production.

3. The method for breeding Aspergillus niger strain according to claim 2, characterized in that: The PDA solid culture medium comprises: 200 g / L potato, 20 g / L glucose, and 20 g / L agar; the primary screening solid culture medium comprises: 0.5 g / L KCl, 1.0 g / L K2HPO4, 0.5 g / L MgSO4·7H2O, 0.5 g / L FeSO4·7H2O, and 20 g / L agar. After sterilization and cooling, 8 g / L xylan is added, shaken, and poured into a plate; the solid fermentation culture medium comprises: 23.4 g bran, 6.6 g soybean meal, 0.45 g (NH4)2SO4, 0.33 g MgCl2, and 18.6 mL H2O.

4. The use of the Aspergillus niger strain according to claim 1, characterized in that: The invention relates to an application of the Aspergillus niger strain in producing xylanase.

5. The use of the Aspergillus niger strain according to claim 4, characterized in that: Aspergillus niger strain is used as the strain to prepare a spore suspension, which is inoculated into a solid fermentation medium to induce the production of xylanase; the fermentation wet base after the solid fermentation is dried and ground, the crude enzyme liquid is separated, and the xylanase is obtained after purification by anion exchange column chromatography.

6. The use of the Aspergillus niger strain according to claim 5, characterized in that: The fixed filler of the anion exchange column chromatography is DEAE Beads 6FF.

7. Use of the xylanase prepared by the Aspergillus niger strain according to claim 1 or any one of claims 4 to 5 in catalyzing xylan hydrolysis reaction.

8. The use according to claim 7, characterized in that: The hydrolysis reaction system comprises substrate xylan, xylanase, phosphate buffer solution and metal ions; the metal ions are any one of calcium ions, sodium ions, potassium ions and magnesium ions; and the final concentration of the metal ions is 2-10 mmol / L.

9. The use according to claim 8, characterized in that: The temperature of the hydrolysis reaction is 40-65° C., and the pH is 4.5-7.

5.

10. The use according to claim 8, characterized in that: In the hydrolysis reaction system, the metal ion is a sodium ion, and the final concentration of the metal ion is 2 mmol / L.

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