CN109182150B - Aspergillus melleus for high yield of cellulase and fermentation method and application thereof - Google Patents
Aspergillus melleus for high yield of cellulase and fermentation method and application thereof Download PDFInfo
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Abstract
The invention discloses aspergillus serrulatum Sartorya Vuill W-10 with high yield of cellulase, and the preservation number is CGMCC No. 11991. According to the invention, high-yield cellulose crude enzyme liquid can be obtained by fermenting aspergillus melleus (Sartorya Vuill W-10), wherein the filter paper enzyme activity of the cellulose can reach 1.26U/ml; the cellulase obtained by fermentation is applied to the preparation of bioethanol, so that the enzymolysis rate of straw residues and the yield of ethanol can be effectively improved, and the cellulase has certain application value. The invention has low production cost and high ethanol yield, is beneficial to the commercial application and implementation of the bioethanol, and provides a way for the comprehensive utilization of waste organisms.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation engineering, and relates to aspergillus melleus capable of realizing high yield of cellulase and a method for producing the cellulase by fermenting the aspergillus melleus.
Background
The resource utilization of the lignocellulose waste such as the straw and the like can not only reduce the negative influence on the environment, but also become an effective source of renewable and sustainable energy. For second generation bioethanol production, lignocellulose has become the most viable technology choice (Belal, E.B.Bioethanol production from edge strain solids. Braz JMicrobiol.2013,44(1): 225-. In the process of alcoholizing lignocellulose, hydrolyzing the lignocellulose by using cellulase, and fermenting the hydrolysate to prepare bioethanol; among them, the production of cellulase directly affects the hydrolysis of lignocellulose and the subsequent fermentation process, and is a major limitation for large-scale application of the ethanolation technology (Novy V, Longus K, Nidetzky b.2015).
Cellulose is polymerized from about one thousand glucose molecules, and cellulase can hydrolyze cellulose to form small molecular glucose (Zhouqiang Qiuhouping Hanyun et al, screening of cellulose-degrading bacteria and their enzyme-producing properties, environmental engineering report 2010 (03): 705-. At present, two major types of strains for producing cellulase mainly comprise fungi and bacteria, wherein the cellulase produced by the fungi has higher catalytic efficiency (Schering, Wurun, research progress of cellulase, grass science, 2004,21(4):72-76), and the difference from the bacteria is that the cellulase produced by most fungi is extracellular enzyme, which makes low-cost separation of active liquid enzyme possible (Korean, Yichenhui Qi, et al, research on enzyme production conditions of Bacillus subtilis C-36, university of Sichuan agriculture, 2006 (02): 178:181). Cellulase Production by fungi is being widely used in industrial Production of sugars and bioethanol by fermentation (F. Talebnia, D. Karakashev, I. Angelidaki, Production of biochemical from straw stream: an overview on pretreatment, hydrolysis and transfer. Bioresource Technol,2010,101: 4744-. Various cellulase-producing fungal species which have been isolated and screened include Mortierella, Chaetomium, Trichoderma viride, Trichoderma koningii, Penicillium, Cereus, Fusarium, Rhizopus, etc. (Murashima K, Nishimura T, Nakamura Y, et al, 2002Purification and characterization of new endo-1, 4-beta-d-glucanases from Rhizopus oryzae. enzymeMicrorob. Teh 30: 319-326; Lanchen Korea Li Hui, research on the breeding and enzyme production conditions of cellulase-producing strain TP1202, Biotechnology 2003,13(2): 12-13; Kovacs K, Marcellis S, Szakc G et al, 2009 enzymic hydrolysics of pretreated fungal species, Biotechnology 2009, Biotechnology 2. Biotechnology 1).
However, most of the fungi for producing cellulase mainly use composite microbial inoculum, and cellulase is still added in the production process to improve the enzymolysis rate of lignocellulose so as to improve the final ethanol yield, which inevitably increases the production cost and affects the economic benefit of enterprises.
Therefore, it is of interest to develop a single fungal strain that can achieve high cellulase production.
Disclosure of Invention
The invention aims to provide aspergillus melleus Sartorya sp.Vuill W-10 capable of realizing high cellulase yield aiming at the current technical situation that a single cellulase strain is lack of.
The Aspergillus mansoni has been submitted to preservation in 2016, 1 month and 7 days, the preservation unit is China General Microbiological Culture Collection center (CGMCC), the preservation address is the institute of microbiology, China academy of sciences, 3, of northern Xilu 1 of the sunny district, Beijing, the preservation number is CGMCC No.11991, and the classification is named as Aspergillus mansoni Sartorya sp.
The species of A.meliloti can be determined by a method conventionally used in the art.
The study shows that the aspergillus melemingi grows fast on a seed (PDA, Potato Dextrose Agar) culture medium, and is cultured for 2-3 days at a constant temperature of 30 ℃, and the diameter of a colony reaches 3-4 cm. The mycelium layer is thin, the primary mycelium is white, the later-stage bacterial colony is grey brown, and the back of the bacterial colony is colorless; the Aspergillus melleus has conidium head with double-layer peduncle, conidium is in long chain shape, ascocarp is in sclerotium shape, ascospore is in oblate sphere or sphere shape, and ascospore is colorless.
The preparation process of the seed culture medium adopted by the invention is as follows: preparing a seed culture medium premix by using peptone 20g, glucose 20g, yeast extract 10g, sodium chloride 0.5g, ferrous sulfate heptahydrate 0.005g, calcium chloride 0.3g, magnesium sulfate 0.5g and dipotassium hydrogen phosphate 2g in a ratio, fixing the volume to 1000mL by using deionized water, adjusting the pH value of the seed culture medium premix to 7 by using 1mol/L hydrochloric acid and 1mol/L sodium hydroxide solution, sterilizing at 121 ℃ for 20min, and cooling to obtain a seed culture medium; the obtained seed culture medium can be stored at 4 ℃ for later use.
The invention also aims to provide a method for producing cellulase by adopting the aspergillus melleus fermentation, which comprises the following specific steps:
(1) preparing a culture medium: preparing a culture medium according to the proportion of 4g of potassium nitrate, 4g of lactose or glucose, 0.2g of urea, 1.5g of dipotassium hydrogen phosphate, 2-6 mL of Mandel's nutrient solution, 4-12 g of bran, 0.5g of magnesium sulfate, 0.3g of calcium chloride and 30g of straw powder pretreated by sodium hydroxide, fixing the volume to 1000mL by using deionized water, and adjusting the pH value of the culture medium to 2.5-3.5 by using inorganic acid;
(2) inoculating aspergillus melleus seed liquid: inoculating the aspergillus melleus seed liquid into the culture medium prepared in the step (1) according to 8-15% of the volume of the culture medium;
(3) preparing cellulase: and fermenting the culture medium inoculated with aspergillus melleus for 6-8 days at 45-55 ℃ under the condition of 180r/m to obtain a crude enzyme solution containing cellulase.
According to the method for producing the cellulase by adopting aspergillus melleus fermentation, potassium nitrate and urea are used as nitrogen sources, lactose or glucose, bran and straw powder are used as carbon sources, monopotassium phosphate is used as a phosphorus source, and monopotassium phosphate, magnesium sulfate and calcium chloride are used as inorganic salt sources.
The method for producing cellulase by fermenting aspergillus melleus can obtain Mandel's nutrient solution by adopting conventional means disclosed in the field (the study on the induction of Trichoderma koningii to produce cellulase by molasses alcohol waste liquor in the reference, Linyuan mountain, Guangxi university, Master academic thesis, 2004).
The method for producing cellulase by fermentation using Aspergillus melleus described above can obtain Straw powder after sodium hydroxide pretreatment by conventional means as disclosed in the art (references Y Liu, Z Qiu, G Wang optimized AlkalinePerformance Technology of Rice Straw for Ethanol Production [ J ] Advances in engineering Research2015, 1169 + 1173).
In the method for producing the cellulase by fermenting the aspergillus melleus, the inorganic acid is hydrochloric acid, nitric acid or sulfuric acid, and the concentration of the inorganic acid is 1-5 mol/L.
According to the method for producing the cellulase by adopting aspergillus melleus fermentation, in the step (1), the prepared culture medium is subpackaged into fermentation containers according to the bottling amount of 40-60 ml/250 ml.
In the step (2), a conventional method disclosed in the art can be used to obtain a seed solution of aspergillus melleus (ref. wangmi; shijing; Tan deshui, etc., a property study on beta-mannase produced by bacillus subtilis MSJ-5 [ J ] Fujian agricultural science, 2015, 03).
In the method for producing cellulase by fermentation of aspergillus melleus, in the step (3), the product obtained by fermentation can be further centrifuged to remove solids; the centrifugal speed is 3500-4000 r/min, and the centrifugal time is 5-10 min.
The invention further provides the application of the cellulase prepared by the method in preparing bioethanol, and further illustrates the preparation process of bioethanol by adopting the cellulase, wherein crude enzyme liquids containing cellulase and cellobiase are respectively obtained by fermenting Aspergillus melleus (Sartoyasp. Vuill W-10) and Aspergillus niger (Aspergillus niger); then carrying out enzymolysis on the straws pretreated by 2% of sodium hydroxide by using cellulase and cellobiase together; simultaneously, the saccharomyces cerevisiae is used for carrying out synchronous saccharification and fermentation on the straws, and the bioethanol can be prepared. The preparation process of the bioethanol comprises the following steps: adding a substrate and crude enzyme liquid containing cellulase into a fermentation container by taking straw residues as the substrate to obtain mixed liquid with the substrate concentration of 40-100 g/L, adjusting the pH value of the mixed liquid to 5-6 by using inorganic acid (the same as the above) and sodium hydroxide solution, adding a Tween-80 nonionic surfactant, the crude enzyme liquid containing cellobiase and saccharomyces cerevisiae under the conditions of 30-50 ℃ and 120-180 r/min, and performing synchronous saccharification and fermentation for 3-4 days to obtain bioethanol; the final concentration of the Tween-80 is 1-10 g/L; the paper filter enzyme activity of cellulase in each gram of substrate is 5-15 FPU, and the cellobiase enzyme activity is 5-15 CBU; the concentration of the saccharomyces cerevisiae is 8 g/L.
In the above process for preparing bioethanol, the sodium hydroxide-treated Straw powder (i.e. Straw residue) is obtained by treating Straw by conventional means as disclosed in the art (references Y Liu, Z Qiu, G Wang optimized alkaline advanced Technology of Rice Straw for Ethanol Production [ J ] Advances in Engineering Research2015, 1169-. The processing method adopted by the invention comprises the following steps: crushing straws, sieving with a 20-mesh sieve, air-drying the sieved product at 60 ℃ to constant weight, pretreating with 2% NaOH solution at 100 ℃ for 2h, and separating out residues.
In the preparation process of the bioethanol, the crude enzyme solution containing cellobiase is obtained by fermenting aspergillus niger, and the fermentation process can adopt the conventional means disclosed in the field (reference documents of brave, royal loyalty and zephyr, bearable research on the fermentation conditions of cellobiase produced by aspergillus niger, university of Sichuan (Nature edition), polysaccharide I2002, 39 (5); 938-942).
The straw adopted by the invention is rice straw or/and wheat straw.
Compared with the prior art, the invention has the following beneficial effects:
1. according to the invention, cellulase can be obtained through single strain fermentation of the provided aspergillus melleus (Sartorya sp. Vuill W-10), and the cellulase for improving the yield of the lignocellulose does not need to be additionally added, so that the production cost is reduced, and the economic benefit of an enterprise is improved;
2. the filter paper enzyme activity of the cellulase in the crude enzyme liquid obtained by fermenting aspergillus melleus (Sartorya sp. Vuill W-10) can reach 1.26U/ml, and the enzyme production efficiency is higher;
3. the cellulase obtained by fermenting aspergillus melleus (Sartorya sp. Vuill W-10) is applied to the preparation of bioethanol, so that the enzymolysis process is promoted, the proportion of glucose in reducing sugar is increased, the enzymolysis rate of straw residues can reach 87%, the ethanol yield can reach 0.43g/g DW, the enzymolysis rate of the straw residues is increased, the bioethanol yield is increased, and the cellulase has a certain application value;
4. the invention has low production cost and high ethanol yield, is beneficial to the commercial application and implementation of the bioethanol, and provides a way for the comprehensive utilization of waste organisms.
Drawings
FIG. 1 is a schematic view of the morphology of Aspergillus melleus; wherein (a) is the colony characteristic of aspergillus melleus, (b) is the microscopic form 1 cultured by aspergillus melleus, (c) is the microscopic form 2 cultured by aspergillus melleus, and (d) is aspergillus melleus spores.
Detailed Description
The present invention is further explained below with reference to examples.
Misinamine used in examples 1 to 3 belowThe preparation process of the aspergillus sporulata seed liquid comprises the following steps: inoculating Aspergillus candidus spore in 50ml seed culture medium, culturing at 30 deg.C and 180r/min for 36 hr to obtain Aspergillus candidus seed solution with OD concentration6002.0 (ref. Wangmei, SHIJING, Tanshu, etc.), Property study of beta-mannase produced by Bacillus subtilis MSJ-5 [ J]Fujian agricultural science, year 2015, 03 th). The preparation process of the adopted seed culture medium comprises the following steps: preparing 20g of peptone, 20g of glucose, 10g of yeast extract, 0.5g of sodium chloride, 0.005g of ferrous sulfate heptahydrate, 0.3g of calcium chloride, 0.5g of magnesium sulfate and 2g of dipotassium phosphate to obtain a seed culture medium premix, fixing the volume to 1000mL by using deionized water, adjusting the pH value of the seed culture medium premix to 7 by using 1mol/L hydrochloric acid and 1mol/L sodium hydroxide solution, sterilizing at 121 ℃ for 20min, and cooling to obtain a seed culture medium; the obtained seed culture medium can be stored at 4 ℃ for later use.
The Mandel's nutrient solution used in examples 1 to 3 was prepared according to the preparation process of the Mandel's nutrient solution in "research on the production of cellulase by Trichoderma koningii induced by molasses alcohol waste liquid" (Linyuanshan, Guangxi university, Master's academic thesis, 2004).
The following Straw powder after sodium hydroxide Pretreatment used in examples 1 to 3, that is, the substrate (Straw residue) in examples 4 to 18, was prepared in the same manner as in the preparation process according to the sodium hydroxide Pretreatment Straw powder in Optimized alkali Pretreatment Technology of rice strand for Ethanol Production (Y Liu, Z Qiu, G Wang, [ J ] advanced in engineering Research2015, 1169-.
The crude enzyme solutions containing cellobiase used in examples 8 to 18 below were prepared according to the procedures for preparing cellobiase in "study on fermentation conditions for producing cellobiase by Aspergillus niger (bore, Wangzui, Miaoyan, Waring, Nature's edition of university of Sichuan," A-Chi ", 2002,39 (5); 938 to 942).
The cellulase activity test method in the following examples 1-3 is as follows: adding 50mg Xinhua filter paper strips into a test tube, adding 1mL of citric acid buffer solution (pH4.8, the concentration of citric acid in the buffer solution is 0.05mol/L), then adding 0.5mL of appropriately diluted crude enzyme solution, reacting for 30min under the condition of 50 ℃ water bath, adding 2mL of a secondary sodium cyanide (LDNS) reagent after the reaction is finished, boiling the water bath for 5min, adding distilled water to dilute to 20mL, uniformly mixing, carrying out color comparison under the condition of 520nm, measuring an absorbance value, calculating the amount of reducing sugar after the reaction is finished by glucose standard, deducting the enzyme solution and substrate reducing sugar (which can be obtained by comparing with an enzyme blank and a substrate blank control experiment group), and calculating the enzyme activity.
One filter paper enzyme activity unit (FPU) is defined as: the amount of enzyme required to produce 1. mu. mol glucose per minute in the enzymatic reaction.
Wherein the dilution factor is the ratio of the volume of the crude enzyme solution obtained in examples 1 to 3 after dilution to the volume before dilution.
The invention provides aspergillus melemingiae Sartorya sp. The Aspergillus mansoni has been submitted to preservation in 2016, 1 month and 7 days, the preservation unit is China General Microbiological Culture Collection center (CGMCC), the preservation address is the institute of microbiology, China academy of sciences, 3, of northern Xilu 1 of the sunny district, Beijing, the preservation number is CGMCC No.11991, and the classification is named as Aspergillus mansoni Sartorya sp.
As shown in FIG. 1, the Aspergillus melleus has the following characteristics: the mycelium layer is thin, the primary mycelium is white, the later-stage bacterial colony is grey brown, and the back of the bacterial colony is colorless; the Aspergillus melleus has conidium head with double-layer peduncle, conidium is in long chain shape, ascocarp is in sclerotium shape, ascospore is in oblate sphere or sphere shape, and ascospore is colorless.
Example 1
The specific preparation process of cellulase prepared by Aspergillus melleus (Sartorya sp. Vuill W-10) is as follows:
(1) preparing a culture medium: preparing a culture medium according to the proportion of 4g of potassium nitrate, 4g of lactose, 0.2g of urea, 1.5g of dipotassium hydrogen phosphate, 3mL of Mandel's nutrient solution, 6g of bran, 0.5g of magnesium sulfate, 0.3g of calcium chloride and 30g of straw powder pretreated by sodium hydroxide, fixing the volume to 1000mL by using deionized water, and adjusting the pH value of the culture medium to 3 by using 1mol/L hydrochloric acid; then subpackaging the prepared culture medium into fermentation containers according to the bottling amount of 43mL/250 mL;
(2) inoculating aspergillus melleus seed liquid: inoculating the aspergillus melleus seed liquid into a fermentation container containing the culture medium according to the volume of 10 percent of the culture medium;
(3) preparing cellulase: fermenting the culture medium inoculated with aspergillus melleus for 6 days at 50 ℃ under the condition of 180r/m, and centrifuging the fermentation product for 5min at 4000r/min to obtain the crude enzyme solution containing the cellulase.
And performing cellulase activity test on the obtained crude enzyme solution to obtain the cellulase with the filter paper enzyme activity of 1.26 FPU/ml.
Example 2
The specific preparation process of cellulase prepared by Aspergillus melleus (Sartorya sp. Vuill W-10) is as follows:
(1) preparing a culture medium: preparing a culture medium according to the proportion of 4g of potassium nitrate, 4g of lactose, 0.2g of urea, 1.5g of dipotassium hydrogen phosphate, 6mL of Mandel's nutrient solution, 12g of bran, 0.5g of magnesium sulfate, 0.3g of calcium chloride and 30g of straw powder pretreated by sodium hydroxide, fixing the volume to 1000mL by using deionized water, and adjusting the pH value of the culture medium to 2.5 by using 1mol/L of hydrochloric acid; then subpackaging the prepared culture medium into fermentation containers according to the bottling amount of 60mL/250 mL;
(2) inoculating aspergillus melleus seed liquid: inoculating the aspergillus melleus seed liquid into a fermentation container containing the culture medium according to 8% of the volume of the culture medium;
(3) preparing cellulase: fermenting the culture medium inoculated with aspergillus melleus for 7 days at 45 ℃ under the condition of 180r/m, and centrifuging the fermentation product at 3500r/min for 10min to obtain crude enzyme liquid containing cellulase.
And performing cellulase activity test on the obtained crude enzyme solution to obtain the cellulase with the filter paper enzyme activity of 0.92U/ml.
Example 3
The specific preparation process of cellulase prepared by Aspergillus melleus (Sartorya sp. Vuill W-10) is as follows:
(1) preparing a culture medium: preparing a culture medium according to the proportion of 4g of potassium nitrate, 4g of lactose, 0.2g of urea, 1.5g of dipotassium hydrogen phosphate, 2mL of Mandel's nutrient solution, 4g of bran, 0.5g of magnesium sulfate, 0.3g of calcium chloride and 30g of straw powder pretreated by sodium hydroxide, fixing the volume to 1000mL by using deionized water, and adjusting the pH value of the culture medium to 3.5 by using 3mol/L hydrochloric acid; then subpackaging the prepared culture medium into fermentation containers according to the bottling amount of 40mL/250 mL;
(2) inoculating aspergillus melleus seed liquid: inoculating the aspergillus melleus seed liquid into a fermentation container containing the culture medium according to 15% of the volume of the culture medium;
(3) preparing cellulase: fermenting the culture medium inoculated with aspergillus melleus for 8 days at 55 ℃ under the condition of 180r/m, and centrifuging the fermentation product for 5min at 4000r/min to obtain the crude enzyme solution containing the cellulase.
And performing cellulase activity test on the obtained crude enzyme solution to obtain the cellulase with the filter paper enzyme activity of 1.15U/ml.
Examples 4 to 7
The crude enzyme solution prepared in example 1 was added to four containers containing substrates for saccharification experiments at different substrate concentrations: enzymolysis is carried out for 72 hours at the speed of 120r/min in a shaking table at normal temperature. The substrate concentrations and experimental results are shown in table 1.
TABLE 1 results of saccharification experiments corresponding to different substrate concentrations in examples 4-7
As can be seen from Table 1, the enzyme yield is continuously decreased with the increase of the substrate concentration; in order to ensure the concentration of ethanol produced by subsequent fermentation, the enzymolysis liquid is required to have higher concentration of reducing sugar, so that the enzymolysis yield and the concentration of reducing sugar are comprehensively considered, and the concentration of 80g/L substrate is preferably selected when preparing the bioethanol.
Examples 8 to 12
Adding the crude enzyme solution prepared in example 1 into five containers containing substrates to prepare a mixed solution with the substrate concentration of 80g/L, and adding crude enzyme solution containing cellobiase (the dosage is calculated by the enzyme activity of the cellobiase) and tween-80 into the mixed solution to carry out enzymolysis experiments on the substrates respectively: enzymolysis is carried out for 72 hours at 120r/min in a shaking table at normal temperature. The proportions of the raw materials and the results are shown in Table 2.
TABLE 2 examples 8-12 raw material ratios and experimental results of enzymolysis experiments
Note: the concentration of the Tween-80 is the concentration of the Tween-80 in the mixed liquid in the container.
As can be seen from Table 2, the substrate enzymolysis rate increases with the increase of the concentration of Tween-80, and the enzymolysis yield of the straw stalk reaches the maximum value of 81.8 percent after 72 hours when the concentration of Tween-80 reaches 5 g/L; however, when the concentration of Tween-80 was increased to 10g/L, the enzyme digestion yield was 81%. Therefore, when preparing bioethanol, tween-80 is preferably added in an amount of 5 g/L.
Examples 13 to 15
Adding the crude enzyme solution prepared in the example 1 into three containers containing substrates to prepare 50ml of mixed solution with the substrate concentration of 80g/L, and adding crude enzyme solution containing cellobiase (the dosage is calculated by the enzyme activity of the cellobiase) and tween-80 into the 50ml of mixed solution to carry out enzymolysis experiments on the substrates respectively: enzymolysis is carried out for 72 hours at 120r/min in a shaking table at normal temperature. The proportions of the raw materials and the results are shown in Table 3.
TABLE 3 examples 13-15 raw material ratios and experimental results of enzymolysis experiments
Note: the concentration of the Tween-80 is the concentration of the Tween-80 in the mixed liquid in the container.
As can be seen from Table 3, the substrate enzymatic hydrolysis rate increases with the increase of the cellobiase activity, the maximum increase reaches 2.3%, but when the cellobiase activity exceeds 10 CBU/g substrate, the increase of the enzymatic hydrolysis yield is not very obvious any more, so that when the bioethanol is prepared, the cost and the enzymatic hydrolysis yield are comprehensively considered, and the cellobiase with the enzyme activity of 10 CBU/g substrate is preferably adopted.
Examples 16 to 18
Adding the crude enzyme solution prepared in the embodiment 1 into three fermentation containers containing substrates to prepare 50ml of mixed solution with the substrate concentration of 80g/L, adjusting the pH value of the mixed solution to 5-6 by using 1mol/L hydrochloric acid and 1mol/L sodium hydroxide solution, adding Tween-80, crude enzyme solution containing cellobiase and saccharomyces cerevisiae under the conditions of 30-50 ℃ and 120-180 r/min, and performing synchronous saccharification and fermentation on the materials for 3-4 days to obtain the bioethanol. The ethanol concentration was measured by gas chromatography. The ratios of the raw materials and the test results are shown in Table 4.
TABLE 4 examples 16-18 bioethanol preparation test raw material ratios and test results
Note: (1) the concentration of the Tween-80 is measured by the concentration of the Tween-80 in the mixed liquid of the container;
(2) ethanol yield% DW refers to the yield of ethanol produced per 1g of dry straw.
As can be seen from Table 4, the cellulase obtained by fermentation of Aspergillus melleus provided by the invention is applied to the preparation of bioethanol, so that the enzymolysis process can be promoted, the proportion of glucose in reducing sugar can be increased, the production cost is low, and the prepared ethanol has high yield, thereby being beneficial to the commercial application and implementation of bioethanol.
It will be appreciated by those of ordinary skill in the art that the embodiments described herein are intended to assist the reader in understanding the principles of the invention and are to be construed as being without limitation to such specifically recited embodiments and examples. Those skilled in the art can make various other specific changes and combinations based on the teachings of the present invention without departing from the spirit of the invention, and these changes and combinations are within the scope of the invention.
Claims (4)
1. A high-yield cellulase of Aspergillus mixtureus Sartorya Vuill W-10 with the preservation number of CGMCC No. 11991.
2. A method for producing cellulase by fermentation of Aspergillus melleus according to claim 1, comprising the steps of:
(1) preparing a culture medium: preparing a culture medium according to the proportion of 4g of potassium nitrate, 4g of lactose or glucose, 0.2g of urea, 1.5g of dipotassium hydrogen phosphate, 2-6 mL of Mandel's nutrient solution, 4-12 g of bran, 0.5g of magnesium sulfate, 0.3g of calcium chloride and 30g of straw powder pretreated by sodium hydroxide, fixing the volume to 1000mL by using deionized water, and adjusting the pH value of the culture medium to 2.5-3.5 by using inorganic acid;
(2) inoculating aspergillus melleus seed liquid: inoculating the aspergillus melleus seed liquid into the culture medium prepared in the step (1) according to 8-15% of the volume of the culture medium;
(3) preparing cellulase: and fermenting the culture medium inoculated with the aspergillus melleus for 6-8 days at 45-55 ℃ under 180r/min to obtain a crude enzyme solution containing the cellulase.
3. The method for producing cellulase by fermentation of aspergillus melleus according to claim 2, wherein in the step (1), the inorganic acid is hydrochloric acid, nitric acid or sulfuric acid.
4. The method for producing cellulase by fermentation of aspergillus melleus according to claim 2, wherein in the step (1), the prepared culture medium is subpackaged into fermentation containers according to bottling amount of 40-60 ml/250 ml.
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