CN103695393A - Method for producing cellulase by using beta-glucosidase and application of cellulase - Google Patents
Method for producing cellulase by using beta-glucosidase and application of cellulase Download PDFInfo
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- CN103695393A CN103695393A CN201310692426.3A CN201310692426A CN103695393A CN 103695393 A CN103695393 A CN 103695393A CN 201310692426 A CN201310692426 A CN 201310692426A CN 103695393 A CN103695393 A CN 103695393A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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Abstract
The invention discloses a method for producing acidity cellulase by using beta-glucosidase, and belongs to the field of an enzyme preparation. The method for producing the cellulase by using the beta-glucosidase is mainly characterized in that after trichoderma viride is cultured, 0.05-0.2% of glucose, 0.001-0.005% of microcrystalline cellulose, 0.5-2% of cellobiose and 0.001-0.005% of beta-glucosidase are subjected to fed-batch in a fermentation treatment process every two to four hours according to the weight of initial fermentation slurry, and after the fermentation is ended, residues in the fermentation slurry are filtered out, thus the acidity liquid cellulase is obtained. Compared with a conventional production technology and an existing technology, the method for producing the acidity liquid cellulase, provided by the invention, has the advantages that the yield of the cellulase is increased by more than two times, meanwhile, the fermentation period is shortened, the production efficiency is improved, and the production cost is reduced. The method also has the advantages of being simple in production technology, simple and convenient to operate and wide in sources of raw materials, having no pollution to environments, suitability for large-scale production and the like, and the technology is easy to master.
Description
Technical field:
The present invention relates to zymin field, relate in particular to a kind of method of utilizing beta-glucosidase to produce acidic cellulase.
Background technology:
1837, Liebig and Wohler found beta-glucosidase first in Semen Armeniacae Amarum juice.The English name of beta-glucosidase (EC3.2.1.21) is β-glucosidase, belongs to hydrolase, claims again β-D-Glucose glycosides lytic enzyme, another name gentiobiase, cellobiase and amygdalase.Beta-glucosidase hydrolyzable is incorporated into β-D-glycosidic link of end irreducibility, discharges β-D-Glucose and corresponding aglucon.It is found that in recent years beta-glucosidase also has transglycosylation, the aglucon that hydrolytic activity can be discharged and glucose, with β-1,6 glycosidic link forms are transferred on other sugared substrates, and adopt turning glycosides activity and can producing oligomeric dragon gallbladder sugar of this enzyme.
Cellulase is a kind of important prozyme product, mainly circumscribed beta-glucanase, Endo-β-glucanase and beta-glucosidase etc., consists of, and also has the Xylanase activity of very high vigor.In recent years, because cellulase has huge market potential in fields such as feed, alcohol, weaving and food, by insider is good both at home and abroad, so cellulase is a new growth point in Enzymes Industry.But in actual production process, yield of cellulase is very low, and production cost is high, and its major cause is the following aspects: 1, inductor raw material as lactose, glucose consumption is large and market price is high; 2, the production cycle long, up to 170h; 3, cellulose enzyme activity is on the low side; 4, rear extraction difficulty, enzyme pick-up rate is on the low side.
Summary of the invention:
Technical problem solved by the invention is to overcome production cycle length, the difficult defect of low, the rear extraction of cellulose enzyme vigor in existing cellulase production technology, a kind of method of utilizing beta-glucosidase production of cellulose enzyme is provided, the method production technique is simple, easy and simple to handle, shorten fermentation period simultaneously, enhance productivity, raw material sources are extensive, environmentally safe, are applicable to very much scale operation.
In order to achieve the above object, the present invention is by the following technical solutions:
A method of utilizing beta-glucosidase production of cellulose enzyme, comprises the steps:
(1) bacterial classification is chosen: viride is as producing bacterial strain, and saves backup on inclined-plane bacterium platform;
(2) spawn culture: the bacterial strain on inclined-plane bacterium platform is put into culturing bottle cultivate 1-3 days under the temperature condition of 20-40 ℃, then put into the seeding tank stir culture 12-40 hour that ventilates;
(3) fermentative processing: the cultivation bacterial classification of getting by weight percentage 5-15%, the Microcrystalline Cellulose of 2-5%, the glucose of 1-3%, the corn steep liquor of 1-2%, the water of 70-95% is mixed into fermentation slurry, fermentation slurry is put into fermentor tank to ventilate and stirs fermentation, temperature is controlled at 25-35 ℃ of fermentation and reduces the temperature between 5-9 ℃ after 25 hours, keep 0.5-1 hour, improve temperature and continue fermentation 45-85 hour to 25-35 ℃, in this fermenting process, every 2-4 hour, by initial fermentation slurry weight stream, add 0.05-0.2% glucose, the Microcrystalline Cellulose of 0.001-0.005%, 0.5-2% cellobiose, the beta-glucosidase of 0.001-0.005%, residue in fermentation ends elimination fermentation slurry, make acidic liquid cellulase.
Beneficial effect:
The method of production acidic liquid cellulase provided by the present invention, the output of the cellulase that the technology of producing than routine and prior art are produced improves more than 2 times, shortens fermentation period simultaneously, enhances productivity, and reduces production costs; The inventive method adopts alternating temperature control method, finds that this method effectively raises output and the enzyme activity of enzyme in production process; The method also has the advantages such as production technique is simple, easy and simple to handle, technology is easily grasped, raw material sources extensive, environmentally safe, applicable scale operation.The acidic liquid cellulase that the present invention produces can be widely used in feed, weaving, oil, garden stuff processing, paper-making industry, Chinese patent medicine processing, particularly biomass ethanol and the field such as produce.
Beta-glucosidase provided by the invention has transglycosylation, under transglycosylation, can make the oligosaccharides such as cellobiose generate the inductors such as oligomeric dragon gallbladder sugar, then the composition permease system on cytolemma enter in cell, active cell inner cellulose enzyme synthetic.So can utilize beta-glucosidase to catalyze and synthesize the production that oligomeric dragon gallbladder sugar is induced cellulase, there is huge commercial value and realistic meaning.
Made cellulase activity force rate prior art is produced has improved respectively 20-30%, and product pick-up rate has improved respectively 10% and 20%.
Embodiment
Below by specific embodiment narration the present invention.Unless stated otherwise, in the present invention, technique means used is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, but not limits the scope of the invention, and the spirit and scope of the invention are only limited by claims.To those skilled in the art, do not deviating under the prerequisite of essence of the present invention and scope various changes that the material component in these embodiment and consumption are carried out or change and also belong to protection scope of the present invention.
Embodiment 1
A method of utilizing beta-glucosidase production of cellulose enzyme, comprises the steps:
(1) bacterial classification is chosen: using viride CICC13001 as producing bacterial strain, and save backup on inclined-plane bacterium platform;
(2) spawn culture: the bacterial strain on inclined-plane bacterium platform is put into culturing bottle and cultivate under the temperature condition of 30 ℃ 2 days, then put into ventilate stir culture 30 hours of seeding tank;
(3) fermentative processing: get by weight percentage 10% cultivation bacterial classification, 4% Microcrystalline Cellulose, 2% glucose, 1% corn steep liquor, 83% water is mixed into fermentation slurry, fermentation slurry is put into fermentor tank to ventilate and stirs fermentation, temperature is controlled at 30 ℃ of fermentations and reduces the temperature between 6 ℃ after 25 hours, keep 0.6 hour, improve temperature to 30 ℃ and continue fermentation 60 hours, in this fermenting process, every 3 hours, by initial fermentation slurry weight stream, add 0.1% glucose, 0.003% Microcrystalline Cellulose, 1% cellobiose, 0.003% beta-glucosidase, residue in fermentation ends elimination fermentation slurry, make acidic liquid cellulase.
Adopt after the inventive method, the raising that the non-alternating temperature of made cellulase activity force rate is produced 21%, product pick-up rate has improved 13%.
Claims (6)
1. a method of utilizing beta-glucosidase production of cellulose enzyme, comprises the steps:
(1) bacterial classification is chosen: viride is as producing bacterial strain, and saves backup on inclined-plane bacterium platform;
(2) spawn culture: the bacterial strain on inclined-plane bacterium platform is put into culturing bottle cultivate 1-3 days under the temperature condition of 20-40 ℃, then put into the seeding tank stir culture 12-40 hour that ventilates;
(3) fermentative processing: the cultivation bacterial classification of getting by weight percentage 5-15%, the Microcrystalline Cellulose of 2-5%, the glucose of 1-3%, the corn steep liquor of 1-2%, the water of 70-95% is mixed into fermentation slurry, putting into fermentor tank ventilates and stirs fermentation, temperature is controlled at 25-35 ℃, ferment and reduce the temperature between 5-9 ℃ after 25 hours, keep 0.5-1 hour, improve temperature and continue fermentation 45-85 hour to 25-35 ℃, in this fermenting process, every 2-4 hour, by initial fermentation slurry weight stream, add 0.05-0.2% glucose, the Microcrystalline Cellulose of 0.001-0.005%, 0.5-2% cellobiose, the beta-glucosidase of 0.001-0.005%.
2. the method for utilizing beta-glucosidase production of cellulose enzyme as claimed in claim 1, is characterized in that, described viride is CICC13001.
3. the method for utilizing beta-glucosidase production of cellulose enzyme as claimed in claim 1, it is characterized in that, get by weight percentage 10% cultivation bacterial classification, 4% Microcrystalline Cellulose, 2% glucose, 1% corn steep liquor, 83% water and be mixed into fermentation slurry, putting into fermentor tank ventilates and stirs fermentation, temperature is controlled at 30 ℃ of fermentations and reduces the temperature between 6 ℃ after 25 hours, keep 0.6 hour, improve temperature to 30 ℃ and continue fermentation 60 hours.
4. the method for utilizing beta-glucosidase production of cellulose enzyme as claimed in claim 3, it is characterized in that, in fermenting process, every 3 hours, by initial fermentation slurry weight stream, add 0.1% glucose, 0.003% Microcrystalline Cellulose, 1% cellobiose, 0.003% beta-glucosidase.
5. the application of beta-glucosidase in production of cellulose enzyme, comprises the steps:
(1) bacterial classification is chosen: viride is as producing bacterial strain, and saves backup on inclined-plane bacterium platform;
(2) spawn culture: the bacterial strain on inclined-plane bacterium platform is put into culturing bottle cultivate 1-3 days under the temperature condition of 20-40 ℃, then put into the seeding tank stir culture 12-40 hour that ventilates;
(3) fermentative processing: the cultivation bacterial classification of getting by weight percentage 5-15%, the Microcrystalline Cellulose of 2-5%, the glucose of 1-3%, the corn steep liquor of 1-2%, the water of 70-95% is mixed into fermentation slurry, putting into fermentor tank ventilates and stirs fermentation, temperature is controlled at 25-35 ℃ of fermentation and reduces the temperature between 5-9 ℃ after 25 hours, keep 0.5-1 hour, improve temperature and continue fermentation 45-85 hour to 25-35 ℃, in this fermenting process, every 2-4 hour, by initial fermentation slurry weight stream, add 0.05-0.2% glucose, the Microcrystalline Cellulose of 0.001-0.005%, 0.5-2% cellobiose, the beta-glucosidase of 0.001-0.005%, residue in fermentation ends elimination fermentation slurry, make acidic liquid cellulase.
6. the application of beta-glucosidase as claimed in claim 5 in production of cellulose enzyme, comprises the steps:
(1) bacterial classification is chosen: using viride CICC13001 as producing bacterial strain, and save backup on inclined-plane bacterium platform;
(2) spawn culture: the bacterial strain on inclined-plane bacterium platform is put into culturing bottle and cultivate under the temperature condition of 30 ℃ 2 days, then put into ventilate stir culture 30 hours of seeding tank;
(3) fermentative processing: get by weight percentage 10% cultivation bacterial classification, 4% Microcrystalline Cellulose, 2% glucose, 1% corn steep liquor, 83% water is mixed into fermentation slurry, fermentation slurry is put into fermentor tank to ventilate and stirs fermentation, temperature is controlled at 30 ℃ of fermentations and reduces the temperature between 6 ℃ after 25 hours, keep 0.6 hour, improve temperature to 30 ℃ and continue fermentation 60 hours, in this fermenting process, every 3 hours, by initial fermentation slurry weight stream, add 0.1% glucose, 0.003% Microcrystalline Cellulose, 1% cellobiose, 0.003% beta-glucosidase, residue in fermentation ends elimination fermentation slurry, make acidic liquid cellulase.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105925551A (en) * | 2016-05-11 | 2016-09-07 | 上海交通大学 | Method for efficiently producing cellulose based on preparation of mixture through glucose glucoside conversion reaction |
| CN111593034A (en) * | 2020-06-24 | 2020-08-28 | 江南大学 | Method for preparing gentiooligosaccharide by using beta-1, 6-glucanase and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105925551A (en) * | 2016-05-11 | 2016-09-07 | 上海交通大学 | Method for efficiently producing cellulose based on preparation of mixture through glucose glucoside conversion reaction |
| CN111593034A (en) * | 2020-06-24 | 2020-08-28 | 江南大学 | Method for preparing gentiooligosaccharide by using beta-1, 6-glucanase and application thereof |
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