CN113403207B - Aspergillus niger strain for high yield of beta-glucosidase and application thereof - Google Patents
Aspergillus niger strain for high yield of beta-glucosidase and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biomass material degradation and application of biomass material degradation to single-cell protein production, and particularly discloses a high-yield beta-glucosidase aspergillus niger strain and application thereof. The preservation number of the Aspergillus niger strain provided by the invention is as follows: CGMCC No.22465 is a compound cellulase preparation which is produced by a fermentation method and has the activity of beta-glucosidase as high as 88 IU/mL, and is simply compounded with a Trichoderma reesei cellulase preparation to form the compound cellulase preparation, the enzyme system of the compound cellulase preparation is more balanced, the release amount of glucose is improved by more than 35 percent under the condition of biomass high-solid enzymolysis, the conversion rate of cellulose in a biomass material is more than 90 percent, and the application is completed in straw single-cell protein to realize the protein content of the straw single-cell protein product to be more than 25 percent.
Description
Technical Field
The invention belongs to the technical field of biomass material degradation and application of biomass material degradation to single-cell protein production, and relates to a high-yield beta-glucosidase aspergillus niger strain and application thereof.
Background
At present, the enzymolysis efficiency of the lignocellulose hydrolase is low, the cost of enzyme is still one of the bottlenecks restricting the utilization of biomass resources, and the problem of how to improve the enzymolysis efficiency of the cellulase is the focus of research in the industry. Lignocellulose hydrolysis is the result of the synergistic action of multiple enzyme systems, wherein β -glucosidase is an important component of the cellulase system, which is responsible for the hydrolysis of cellooligosaccharides and cellobiose released by the synergistic action of endo-and exo-cellulolytic enzymes to glucose. Therefore, by improving the activity of the beta-glucosidase, the total enzyme activity of the cellulase can be effectively improved, and the inhibition effect of the cellulase on endo-cellulase and exo-cellulase can be reduced by degrading cellooligosaccharide and cellobiose, so that the hydrolysis efficiency of the cellulase system is improved.
In addition to playing a role in lignocellulose hydrolysis, β -glucosidase plays an important role in the synthesis of cellulase inducers and the rapid response of the cells to cellulose. On one hand, the beta-glucosidase has the function of transglycosylation, can convert a small amount of glucose into disaccharides such as sophorose and the like, and the disaccharides are efficient inducers in the production process of producing enzymes by cellulase fermentation. On the other hand, some beta-glucosidase has stronger beta-xylosidase activity or beta-galactosidase activity, and can produce xylooligosaccharide or lactose to be used as an inducer for synthesizing cellulase and hemicellulase, thereby playing an important role in improving the induced synthesis of the cellulase and the hemicellulase.
Therefore, from the viewpoint of improving the activity and hydrolysis efficiency of the cellulase system itself, or from the viewpoint of improving the inducible expression of cellulase by synthesizing an inducer, it was revealed that β -glucosidase plays a crucial role in the yield and hydrolysis efficiency of the cellulase system. However, the beta-glucosidase content and the enzyme activity of the strains such as trichoderma reesei and the like which are widely applied industrially are low at present, which becomes one of the main limiting factors that the hydrolysis efficiency of the cellulase system is not high. The additional compounding of beta-glucosidase is an effective method for improving the enzyme activity and the hydrolysis efficiency of cellulase.
Disclosure of Invention
Aspergillus niger (A) according to the present inventionAspergillus niger) Mutant 60B-3DW from Aspergillus nigerAspergillus niger) 3.316 (CGMCC number 3.316, preserved in the general strain preservation center of institute of microbiology of Chinese academy of sciences) as an original strain, and a mutant strain with greatly improved beta-glucosidase activity is bred by ARTP mutagenesis and a droplet microfluidic high-throughput screening technology.
Therefore, the invention firstly provides an aspergillus niger strain 60B-3DW, which is named in classification: aspergillus nigerAspergillus nigerAspergillus niger strainAspergillus nigerThe 60B-3DW is preserved in the China general microbiological culture Collection center with the preservation number: CGMCC No.22465, the preservation time is as follows: on 2021, 07/05, the address of the depository is: xilu No.1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.
Further, the present invention provides a method for producing β -glucosidase, comprising the steps of:
1) inoculating Aspergillus niger strain 60B-3DW in a culture medium for culture;
2) harvesting the beta-glucosidase from the culture medium in the form of a fermentation broth of the Aspergillus niger strain 60B-3DW grown in step 1).
Preferably, in the method for producing β -glucosidase, the medium comprises the following components: 45-55 g/L of corncob, 4-6 g/L of yeast powder, 4-6 g/L of malt extract, 2.6-3 g/L of ammonium sulfate, 3-5 g/L of monopotassium phosphate, 0.8-1.0 g/L of crystalline magnesium sulfate, 0.8-1.0 g/L of crystalline calcium chloride and initial pH of the mixture is adjusted to 6.0.
Preferably, in the production method of the beta-glucosidase, in the step 1), aspergillus niger spores are cultured for 144-182 hours at the temperature of 26-30 ℃ and the rotating speed of 140-200 r/min;
in the step 2), the fermentation liquor obtained by the culture in the step 1) is subjected to solid-liquid separation, and the supernatant is collected to obtain a crude enzyme solution of the beta-glucosidase.
The invention also provides a compound cellulase preparation, which comprises the beta-glucosidase prepared by the method and cellulase. More preferably, the cellulase is derived from trichoderma reesei. Specifically, fermentation liquor of the two is used as crude enzyme liquid for mixing, preferably trichoderma reesei cellulase liquid: the Aspergillus niger beta-glucosidase fermentation liquor is compounded according to the volume ratio of 7-10:1, most preferably 8-9:1, the enzyme activity of the Trichoderma reesei cellulase filter paper is 30-40 IU/ml, and the enzyme activity of the Aspergillus niger beta-glucosidase is 80-100 IU/ml. )
Further, the invention provides the function of the compound cellulase preparation in degradation of biomass materials. Preferably, the biomass material is crop straw or distillers grains, such as rice straw, wheat straw, sorghum straw, corn stover (e.g., delignified corn stover, steam exploded corn stover), or cassava distillers grains, or the like.
According to the invention, by optimizing the conditions of Aspergillus niger liquid drop generation, a separation chip, liquid drop size and the like, the defect of cross metabolite pollution between liquid drops caused by package liquid drop puncture in the Aspergillus niger hypha growth process is overcome, the combined fluorescent substrate is screened and marked to achieve the purpose of separating Aspergillus niger micro liquid drops, the micro-fluidic high-flux screening technology of the Aspergillus niger beta-glucosidase liquid drops is established for the first time, the screening flux can reach 10000/h, the screening period of the traditional method is shortened from 7-15 days to 24 h, the technical bottleneck of the traditional filamentous fungus screening method is broken through, the beta-glucosidase activity of the Aspergillus niger strain is accurately and directionally improved, and the efficiency and success rate of beta-glucosidase strain breeding are greatly improved. Finally obtaining the aspergillus niger strain 60B-3DW with high yield of beta-glucosidase.
The Aspergillus niger strain 60B-3DW provided by the invention has the advantages that the activity of beta-glucosidase produced by a fermentation method is up to 88 IU/mL under the shake flask fermentation scale, the beta-glucosidase is simply compounded with a Trichoderma reesei cellulase preparation to form a composite cellulase preparation, the enzyme system of the composite cellulase preparation is more balanced, the glucose release amount is improved by more than 35% under the condition of biomass high-solid enzymolysis, the cellulose conversion rate in a biomass material is more than 90%, the application is completed in straw single-cell protein, and the protein content in a straw single-cell protein product is more than 25%.
Drawings
FIG. 1 is a graph showing a linear relationship between a fluorescence value released by a droplet microfluidic substrate and enzyme activity of beta-glucosidase in an embodiment of the invention.
FIG. 2 is a graph showing the observation of fluorescence signals at different times in droplet culture in the example of the present invention.
FIG. 3 is a flow chart of droplet sorting of β -glucosidase in an embodiment of the invention.
FIG. 4 is a graph showing the genetic stability of Aspergillus niger strains according to the examples of the present invention.
FIG. 5 is a graph showing the comparison of glucose release when a complex cellulase system hydrolyzes different biomass materials in the example of the present invention.
Detailed Description
The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text. But are not to be construed as limiting the invention.
Example one Aspergillus niger 60B-3DW strain obtained by high-throughput screening method of beta-glucosidase strain
Aspergillus niger (A) toAspergillus niger) 3.316 as the basis, the inventor carries out mutagenesis by ARTP, optimizes conditions such as Aspergillus niger liquid drop generation, substrate sorting, liquid drop size and the like by means of a liquid drop microfluidic high-flux platform, after Aspergillus niger spores are embedded and pre-cultured for 16 hours through liquid drops, the diameter of the liquid drops is 80-90 mu m, Aspergillus niger hyphae do not excessively grow to puncture the liquid drops, meanwhile, the Aspergillus niger hyphae secrete enough beta-glucosidase to interact with a fluorogenic substrate, and the enzyme activity in the liquid drops is distinguished through a fluorescence value released by the substrate. The method overcomes the defect that cross metabolite pollution among liquid drops is caused by puncture of wrapped liquid drops in the growth process of aspergillus niger hypha, simultaneously screens and marks combined fluorescent substrates to achieve the aim of sorting aspergillus niger micro-droplets, firstly creates a microfluidic high-flux screening technology of aspergillus niger beta-glucosidase liquid drops, the screening flux can reach 10000/h, the screening period of the traditional method is completed within 24 h from 7-15 days, the technical bottleneck of the traditional filamentous fungus screening method is broken through, the activity of beta-glucosidase enzyme of the strain with the independent intellectual property right is accurately and directionally improved, and the efficiency and the success rate of breeding of the beta-glucosidase strain are greatly improved.
1. The aspergillus niger culture medium for producing beta-glucosidase and the formula of the fermentation culture solution are as follows:
50 g/L of corncob, 5 g/L of yeast powder, 5 g/L of malt extract, 2.8 g/L of ammonium sulfate, 4 g/L of monopotassium phosphate, 0.9 g/L of crystalline magnesium sulfate, 0.9 g/L of crystalline calcium chloride and initial pH adjustment to 6.0.
2. The culture method for producing the beta-glucosidase by the aspergillus niger comprises the following steps:
after the spore slope of the Aspergillus niger grows well, a loop is scraped from the slope by an inoculating loop and inoculated into a 500 mL shake flask filled with 50 mL fermentation culture solution, the mixture is cultured for 168 +/-24 hours under the conditions that the rotating speed is 170 revolutions per minute and the culture temperature is 28 +/-2 ℃, the fermentation solution is centrifuged for 10 minutes at 10000 revolutions per minute, and the supernatant is collected to be crude enzyme solution.
3. The formula of the fermentation medium for producing the cellulase preparation by the trichoderma reesei comprises the following components:
25 g/L glucose, 4 g/L corn steep liquor dry powder, 1.66 g/L KOH, (NH)4)2SO42.8 g/L and MgSO40.6 g/L, initial pH 5.5.
4. The culture method for producing the cellulase preparation by the trichoderma reesei comprises the following steps:
5. Yeast seed liquid culture medium:
YPD medium: 1% glucose, 2% peptone and 1% yeast powder.
6. The yeast seed liquid culture method comprises the following steps:
a loopful of yeast colonies was scraped from the yeast plate by an inoculating loop and inoculated into YPD medium, and cultured at 30 ℃ and 200 rpm for 24 hours.
7. The method for measuring the activity of the beta-glucosidase comprises the following steps:
the amount of enzyme required to convert 1. mu. mol of substrate (15 mM cellobiose) per minute was one enzyme activity unit (IU) at 4.8 deg.C of C, pH.
The method for measuring the activity of the beta-glucosidase comprises the following steps:
add 1 ml of enzyme solution diluted appropriately to a5 ml centrifuge tube. Each sample should include at least 2 dilutions of enzyme solution, one dilution releasing slightly less than 1.0 mg of glucose under the reaction conditions and the other dilution releasing slightly more than 1.0 mg of glucose. Simultaneously making substrate blank and enzyme solution blank controls, and subtracting the glucose amount in the substrate blank and the enzyme solution blank during calculation;
heating the enzyme solution to 50 ℃, adding 1.0 ml of substrate solution, and uniformly mixing;
③ reacting for 30 min in a water bath at 50 ℃;
fourthly, boiling in water bath for 5.0 min, and stopping the reaction;
cooling, and measuring the content of the released glucose in the reaction solution by a biological sensing analyzer SBA-40C.
8. Sorting:
mutagenizing strain spores, performing high-throughput sorting by utilizing droplet microfluidics, and breeding the strain with high enzyme activity of the beta-glucosidase.
Firstly, the establishment of the high-throughput screening method of Aspergillus niger beta-glucosidase droplet microfluidics
A. Beta-glucosidase droplet microfluidic high-throughput screening substrate determination
Respectively taking beta-glucosidase with different enzyme activities and 2 mu M fluoroescein Di-beta-D-Glucopyranoside reaction liquid as water phases to simultaneously generate water-in-oil droplets, carrying out fluorescence detection on the droplets by using a droplet microfluidic screening system, and displaying that a clear linear relation exists between a fluorescence value released by a substrate fluoroescein Di-beta-D-Glucopyranoside and the enzyme activity of the beta-glucosidase by using a detection result, thereby showing that the liquid microfluidic system can use the fluorescence value released by fluoroescein Di-beta-D-Glucopyranoside as a basis for screening the beta-glucosidase droplets with high enzyme activity, as shown in figure 1.
The droplet microfluidic screening system is used for sorting the beta-glucosidase-embedded droplets, the sorting flux reaches 200/s, and the accuracy reaches 96%, as shown in figure 3. The liquid drop microfluidic high-throughput screening system is proved to be feasible in sorting the beta-glucosidase with high enzyme activity.
B. Determination of beta-glucosidase droplet microfluidic high-throughput screening and sorting time
Aspergillus niger spores, a culture medium and a reaction substrate fluoroscein Di-beta-D-Glucopyranoside are embedded in the liquid drops, and the liquid drops are embedded and cultured according to the optimized culture conditions to detect the fluorescence of the liquid drops. As can be seen from FIG. 2, the droplets started to produce weak fluorescence after 10 h of incubation, and the droplets emitted strong fluorescence after 16 h of incubation. The result proves that the beta-glucosidase secreted by the aspergillus niger hypha can react with the labeled substrate to emit fluorescence, and the fluorescence intensity is increased along with the prolonging of the culture time. The difference of fluorescence values released by the reaction of the liquid drop and the substrate after the liquid drop is cultured for 16 h is obvious, and in order to improve the sorting efficiency of the liquid drop, the liquid drop can be sorted after the liquid drop is cultured for 16 h.
C. Aspergillus niger strain culture and enzyme activity verification after high-throughput screening
Spore micro-liquid obtained after aspergillus niger mutagenesis by utilizing droplet microfluidic high-throughput screening systemThe drops were sorted with a sorting throughput of 10000/h. And (3) separating out droplets with strong fluorescence signals (the microfluidic separation speed of the droplets is 20 mu l/h, and each sample is collected for 20 min), and coating the droplets on a PDA (personal digital assistant) plate for subsequent culture. And respectively inoculating the grown single colonies into a fermentation culture medium for culture, measuring the enzyme activity of the beta-glucosidase in the fermentation liquid after 7 days of culture, and selecting a transformant with the highest enzyme activity from the measured enzyme activity to obtain a mutant strain with greatly improved enzyme activity of the beta-glucosidase, wherein the mutant strain is named as Aspergillus niger 60B-3 DW. Aspergillus nigerAspergillus niger) The beta-glucosidase activity of the mutant strain 60B-3DW can reach about 88 IU/mL, compared with the original strain Aspergillus niger (A), (B), (C) and (D)Aspergillus niger) 3.316, the enzyme activity of the beta-glucosidase enzyme secreted by the enzyme is 17.16 IU/mL, which is improved by 412.8 percent.
EXAMPLE two genetic stability testing of Aspergillus niger 60B-3DW Strain
To verify Aspergillus nigerAspergillus niger) And (3) the genetic stability of the mutant strain 60B-3DW, after the mutant strain is subjected to passage 6, fermentation culture is carried out under the same fermentation condition, and the change condition of the enzyme activity is compared. The average value of the enzyme activity was plotted against the number of passages, and the results are shown in FIG. 4.
The enzyme activities of the mutant strain 60B-3DW generation strains are respectively as follows: 88, 85.8, 87.6, 88.8, 88.5 and 86.3 IU/mL, and the variation range of the enzyme activity is small after the passage of the number of times, so that the mutant strain 60B-3DW is shown to have good genetic stability and can be used as an experimental strain for subsequent fermentation.
Example III hydrolysis of Biomass efficiency by Aspergillus niger 60B-3DW fermented beta-glucosidase and Trichoderma reesei cellulase
And (3) compounding the Trichoderma reesei cellulase liquid and the Aspergillus niger beta-glucosidase fermentation liquid obtained by the culture method according to different proportions (9.5: 1, 8.5:1, 8.0:1, 7.5:1 and 7: 1) to form a composite cellulase preparation, and measuring the enzyme activity.
1. Filter paper enzyme activity assay
The amount of enzyme required to release 1. mu. mol of reducing sugar by degradation from a substrate (50 mg Whatman No.1 filter paper) per minute at 4.8 deg.C of 50 ° C, pH was one enzyme activity unit (IU).
The filter paper enzyme activity determination comprises the following three parts of parallel and same treatment of test tubes: blank control, glucose standard curve and fermentation liquor detection.
Adding 1.0 ml of 0.05M citric acid buffer solution with pH4.8 into a test tube, and adding 0.5 ml of Trichoderma reesei diluted enzyme solution (the amount of glucose released by the diluted enzyme solution under the reaction conditions is about 2.0 mg);
② adding Whatman No.1 filter paper strips with the specification of 1cm multiplied by 6 cm (50 +/-1 mg) for mixing;
placing the mixture in a water bath at 50 ℃ to react for 60 min;
adding 3.0 ml DNS solution, boiling water bath for 5 min to terminate the reaction, and cooling water bath to room temperature;
fifthly, adding 170 mu l of the mixed solution into 1 ml of water for dilution;
sixthly, taking 200 mu l of mixed solution to read in a 96-well plate to determine A540, and converting the glucose content of the sample from a glucose DNS standard curve. Wherein the filter paper enzyme activity calculation formula is as follows: x (IU/ml) =
The experimental result data are as follows:
| trichoderma reesei | Trichoderma reesei: aspergillus niger =9:1 | Trichoderma reesei: aspergillus niger =8.5:1 | Trichoderma reesei: aspergillus niger =8:1 | Trichoderma reesei: aspergillus niger =7.5:1 | Trichoderma reesei: aspergillus niger =7:1 | |
| OD540nm | 0.96 | 0.98 | 1.14 | 1.11 | 1.03 | 0.95 |
The results of calculating the filter paper enzyme activity by using a formula are as follows:
| trichoderma reesei | Trichoderma reesei: aspergillus niger =9:1 | Trichoderma reesei: aspergillus niger =8.5:1 | Trichoderma reesei: aspergillus niger =8:1 | Trichoderma reesei: aspergillus niger =7.5:1 | Trichoderma reesei: aspergillus niger =7:1 | |
| FPU/mL | 29.51 | 30.34 | 35.06 | 34.20 | 31.79 | 29.26 |
The results show that the filter paper enzyme activity of the composite cellulase preparation is improved to different degrees compared with the Trichoderma reesei cellulase preparation. Especially when the Trichoderma reesei cellulase: when the Aspergillus niger beta-glucosidase fermentation liquor =8.5:1, the filter paper enzyme activity of the composite cellulase preparation is improved to 35.06 FPU/mL, which is 18.81% higher than that of 29.51 FPU/mL of the Trichoderma reesei cellulase filter paper enzyme.
2. Enzyme activity assay for degradation of different substrates
Delignified corn straws, steam-exploded corn straws and cassava vinasse are respectively used as substrates, the concentration of the substrates is 25%, and the reaction volume is 20 mL. Trichoderma reesei cellulase liquor: aspergillus niger beta-glucosidase fermentation liquor is compounded according to different proportions (9: 1, 8.5:1, 8.0:1, 7.5:1, 7: 1), and the enzyme dosage of the composite cellulase is kept at 10 FPU/g substrate and reacts for 72 hours in a water bath shaker at 50 ℃. After 72 hours of hydrolysis, the samples were centrifuged at high speed and the hydrolyzed supernatant was analyzed for changes in glucose concentration by HPLC-87P column. The results are given in the following table:
| enzyme system | The delignified straws release the glucose amount (g/L) | Cassava jellyAmount of glucose (g/L) | Glucose amount (g/L) released by steam explosion of straw |
| Trichoderma reesei | 17.41 | 7.34 | 10.3 |
| Trichoderma reesei: aspergillus niger =9:1 | 17.89 | 9.12 | 12.39 |
| Trichoderma reesei: aspergillus niger =8.5:1 | 24.14 | 9.9 | 14.4 |
| Trichoderma reesei: aspergillus niger =8:1 | 18.94 | 9.7 | 13.45 |
| Trichoderma reesei: aspergillus niger =7.5:1 | 18.55 | 9.3 | 11.98 |
| Trichoderma reesei: aspergillus niger =7:1 | 18.46 | 8.11 | 11.12 |
As shown in FIG. 5, the filter paper enzyme activity of the composite cellulase preparation is improved to a different extent than that of the Trichoderma reesei cellulase preparation in the release amount of glucose after hydrolysis. Especially when the Trichoderma reesei cellulase: when the aspergillus niger beta-glucosidase fermentation liquor is =8.5:1, compared with the saccharification result of the trichoderma reesei enzyme system, the glucose amount released by delignified corn straws, cassava vinasse and steam exploded straws is respectively increased by 38.68%, 34.88% and 39.81%, and the cellulose conversion rate in the biomass material is more than 90% under the biomass high-solid enzymolysis condition.
Example four application of Aspergillus niger 60B-3DW and Trichoderma reesei in production of straw single-cell protein
Trichoderma reesei/aspergillus niger spore liquid: respectively streaking PDA plate with Aspergillus niger 60B-3DW and Trichoderma reesei A2H, culturing at 30 deg.C for 5d, eluting spore with sterile water, filtering to remove mycelium with cell filter, counting with blood counting plate, and adjusting spore concentration of Aspergillus niger and Trichoderma reesei to 1.2 × 108one/mL.
Taking steam-exploded pretreated straws, adding water according to the material-water ratio of 1:2.5, and taking the straws as a culture medium for solid state fermentation. The trichoderma reesei and aspergillus niger spore liquid are added on the surface of a culture medium for steam explosion pretreatment of straw solid state fermentation according to the inoculum size of 5% (namely the ratio of 1: 1), and cultured for 24-36 hours at the temperature of 30 ℃. After the straws are hydrolyzed by aspergillus niger and trichoderma reesei for 24 hours, candida utilis seed liquid is added into the straws according to the addition amount of 10 percent, and solid state fermentation is continued for 120 hours at the temperature of 30 ℃. The trichoderma reesei and the aspergillus niger secrete cellulase and beta-glucosidase to the outside of cells respectively in the straw growing process, and the two enzyme systems are mutually cooperated to efficiently degrade cellulose of corn straws and convert the cellulose into fermentable sugar which can be utilized by thalli. After yeast seed liquid is added, the yeast can efficiently utilize fermentable sugar to convert the fermentable sugar into mycoprotein; the addition of the yeast can efficiently reduce the concentration of fermentable sugar in the culture medium, and effectively reduce the feedback inhibition effect of glucose on the Aspergillus niger/Trichoderma reesei cellulase, thereby being beneficial to improving the secretion of the Aspergillus niger/Trichoderma reesei cellulase and accelerating the enzymolysis efficiency of the straw. And (4) drying the fermented straws after solid state fermentation for 3, 4, 5, 6 and 7 days respectively, and then measuring the total nitrogen content of the solid. The crude protein content of the fermented straw was calculated as crude protein = total nitrogen content of solid 6.25. The results showed that the crude protein content of the fermented straw was about 25.13% after 7 days of fermentation.
| Time (d) | 3 | 4 | 5 | 6 | 7 |
| Total nitrogen content (%) | 1.56 | 2.01 | 2.45 | 3.01 | 4.02 |
| Crude protein content (%) | 9.75 | 12.56 | 15.31 | 18.81 | 25.13 |
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
Claims (8)
1. Aspergillus niger strain (A)Aspergillus niger) The culture medium is characterized by being preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms with the preservation number as follows: CGMCC No.22465, the preservation time is as follows: year 2021, month 07, day 05.
2. A method for producing beta-glucosidase, which comprises the following steps:
1) inoculating the Aspergillus niger strain of claim 1 into a fermentation medium of Aspergillus niger for culture;
2) harvesting the beta-glucosidase from the fermentation medium as a fermentation broth of the Aspergillus niger strain grown in step 1).
3. The method for producing β -glucosidase of claim 2, wherein the fermentation medium of aspergillus niger comprises the following components: 45-55 g/L of corncob, 4-6 g/L of yeast powder, 4-6 g/L of malt extract, 2.6-3 g/L of ammonium sulfate, 3-5 g/L of monopotassium phosphate, 0.8-1.0 g/L of crystalline magnesium sulfate, 0.8-1.0 g/L of crystalline calcium chloride and initial pH of the mixture is adjusted to 6.0.
4. The method for producing beta-glucosidase as claimed in claim 2, wherein in step 1), Aspergillus niger spores are cultured for 144-182 hours at 26-30 ℃ and 140-200 rpm.
5. The method for producing β -glucosidase as defined in claim 2 wherein, in step 2), the fermentation liquid obtained by culturing in step 1) is subjected to solid-liquid separation, and the supernatant is collected to obtain a crude enzyme solution of β -glucosidase.
6. Use of an Aspergillus niger strain according to claim 1, a β -glucosidase prepared by a method according to any of claims 2 to 5 for the degradation of biomass material.
7. The use of claim 6, wherein the biomass material is crop straw or distillers grains.
8. The use of claim 7, wherein the crop straw is rice straw, wheat straw, sorghum straw, corn stover; the vinasse is cassava vinasse.
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