CN103740678B - Cellulase and preparation thereof - Google Patents
Cellulase and preparation thereof Download PDFInfo
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- CN103740678B CN103740678B CN201310716678.5A CN201310716678A CN103740678B CN 103740678 B CN103740678 B CN 103740678B CN 201310716678 A CN201310716678 A CN 201310716678A CN 103740678 B CN103740678 B CN 103740678B
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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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- 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
- C12N9/2445—Beta-glucosidase (3.2.1.21)
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- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
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- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01091—Cellulose 1,4-beta-cellobiosidase (3.2.1.91)
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Abstract
The open cellulase of the present invention and preparation thereof, the invention belongs to field of microbial fermentation, utilizes trichoderma reesei (Trichoderma reesei) strain fermentation that a strain deposit number is CCTCC M2013540 to produce cellulase.The optimal pH 3.0 6.0 of described bacterial strain cellulase-producing, optimum temperature is 23～35 DEG C.The seed liquor of described bacterial strain is inoculated in fermentation medium, cultivating 104 hours for 25 DEG C, circumscribed beta glucan enzyme, Endocellulase, β glucuroide and the filter paper enzyme activity of zymotic fluid cellulase respectively reach 680U/mL, 1389U/mL, 486U/mL and 792U/mL.
Description
Technical field
The invention belongs to field of microbial fermentation, particularly to a kind of cellulase produced by fungi fermentation.
Background technology:
Cellulase is widely present in the organism of nature.Bacterium, fungi, cellulase can be produced in animal body etc..
The cellulase being generally used for producing comes from fungi, has wooden enzyme to belong to (Trichodema), aspergillus than more typical
And Penicillium (Penicillium) (Aspergillus).Cellulase is all widely used in food service industry and environmental industry.
When carrying out alcoholic fermentation, the interpolation of cellulase can increase the utilization rate of raw material, and has promoted vinosity.
Cellulase is of a great variety, originates the widest.Its 26S Proteasome Structure and Function of the cellulase of separate sources differs greatly.Due to fungi
Yield of cellulase is high, activity is big, therefore the cellulase of application is mainly fungal cellulase in animal husbandry and feed industry.
Cellulase can be divided into endoglucanase (1,4-β-D-glucan according to the difference of its catalytic reaction function
Glucanohydrolase or endo-1,4-β-D-glucanase, EC3.2.1.4), from the abbreviation EG of fungi, from carefully
The abbreviation Cen of bacterium, exoglucanase (1,4-β-D-glucan cellobilhydrolase or exo-1,4-β
-D-glucannase, EC.3.2.1.91), from the abbreviation CBH of fungi, from the abbreviation Cex of bacterium) and beta glucan glycosides
Enzyme (β-Isosorbide-5-Nitrae-glucosidase, EC.3.2.1.21) is called for short BG.In endoglucanase random cutting fibre element polysaccharide chain
The unformed area in portion, produces oligosaccharides and the end of new chain of different length.Exoglucanase acts on these reproducibilities and non-reduced
Property the end of fibrination sugar chain, release glucose or cellobiose.Beta-glucosidase hydrolysis fiber disaccharides produces two molecules
Glucose.Fungin production of enzyme is high, activity is big, the main fiber applying originated from fungus in animal husbandry and feed work
Element enzyme.
Cellulase reaction and general enzyme reaction are different, and its topmost difference is that cellulase is multicomponent enzyme system, and the end
Thing structure is extremely complex.Due to the water-insoluble of substrate, the suction-operated of cellulase instead of the ES of enzyme-to-substrate formation and is combined
Thing process.Cellulase first specifically adsorbs on substrate cellulose, then by cellulose under the synergy of several components
Resolve into glucose.
Nineteen fifty, Reese etc. proposes C1-Cx hypothesis, and this hypothesis is thought must be with different enzyme synergies, could be by fibre
Dimension element is hydrolyzed to glucose thoroughly.Synergy is commonly considered as the non-knot of endoglucanase (C1 enzyme) first attack cellulose
Crystalline region, forms the new free-end needed for Cx, is then cut fiber two by CX enzyme from reducing end or the non-reducing end of polysaccharide chain
Sugar unit, is finally hydrolyzed into two glucose by beta glucan glycosides enzyme by cellobiose.But, the synergy of cellulase is suitable
Sequence is not absolute, finds in research subsequently, and C1-Cx and beta glucan glycosides enzyme must exist simultaneously could hydrolyze natural fiber
Element.If first with C1 enzyme effect avicel cellulose, then removing C1 enzyme, adding Cx enzyme, such sequential action but can not will be tied
Crystalline cellulose hydrolyzes.
Strain improvement is the basic work of cellulase production, and the most many experts have carried out numerous studies, in order to produce height
The cellulase product of quality, Wang Jialin etc. (1996), on the basis of absorbing domestic and foreign experience, has successively introduced Trichoderma viride
Wood 10, Trichoderma viride Sn-91014, koning trichoderma NT-15, aspergillus niger XX-15A, on this basis, have employed ultraviolet,
Physics, the method for mutagenesis of chemistry such as specific electromagnetic wave radiates, linear accelerator, nitrosoguanidine, it is thus achieved that superior strain NT15-H,
NT15-H1、XT-15H、XT-15H1.Wooden mould NT-15H solid culture vigor is through food quality supervision inspection center of China National Light Industrial Products Department
The detection of station, Nanjing shows, filter paper vigor is 3670u/g, C1-enzyme activity 24460u/g, and Cx-enzyme activity 1800u/g reaches
International most advanced level.The stable performance in factorial praluction of this bacterial classification.Zhang Linghua etc. (1998) use koning trichoderma W-925,
J-931, after over-richness is 2% dithyl sulfate and ultraviolet (15W, 30cm, 2min) complex mutation, has obtained product enzyme and has lived
The Wu-932 bacterial classification that property is high, this bacterial classification CMC saccharifying power reaches 2975, and filter paper anase activity is 531, divides than the bacterium W-925 that sets out
Do not improve 100% and 81%.Feed additive technology service centre of the Ministry of Chemical Industry king Cheng Shu etc. (1997) use the Richter scale at this center
Wood is after mould A3 first carries out ultraviolet and nitrosoguanidine complex mutation, by the spore inoculating that processed on fiber double-layer plate, and 30
DEG C cultivate 5-8 days, place 7-10 days for 15 DEG C, select transparent circle diameter and the bigger single bacterium colony of colony diameter to carry out triangular flask solid
State fermentation is screened again, has obtained the trichoderma reesei 91-3 bacterial strain that cellulase-producing vigor is the highest.
The production technology of cellulase mainly has two kinds, i.e. solid fermentation and liquid fermentation, and its technique is as follows:
1, affect the factor of yield of enzyme and vigor: the factor affecting yield of cellulase and vigor is a lot, outside degerming kind, also have training
Support temperature, pH, moisture, matrix, incubation time etc..These factors are not isolated, but connect each other.Zhang Zhongliang etc.
(1997) use uniform Design Cl12 (1210), with Trichoderma viride (T.ViriclePers.expr) as bacterial classification, have studied shadow
Ring cellulase-producing five big factors to yield of enzyme and the effect of vigor, it is believed that matrix crude fiber content is 40%, initial pH7.5,
Add water 4 times, under the conditions of 26-31 DEG C, cultivate 45h can obtain maximum yield of enzyme 26mg/g and CMC enzyme activity 20mg/g h.
Wang Chenghua etc. (1997) also studied the condition of enzyme production of the trichoderma reesei 91-3 of its mutagenesis screening, and result shows that this bacterial classification is with 7:
The powder of straw of 3 and wheat bran, another interpolation 4% ammonium sulfate, 0.4% potassium dihydrogen phosphate, 0.1% magnesium sulfate are optimal medium, 28-32
DEG C being suitable cultivation temperature, 30 DEG C is optimum temperature, and 4% is optimum inoculation amount, and 96h arrives fermentation peak.Zhang Linghua etc. (1998)
Have studied with koning trichoderma W-925 for the bacterium that sets out, the optimal conditions of fermentation of the Wu-932 High-Cellulase-Yielding bacterium obtained after mutagenesis.
Result shows, with the wheat bran of 1:2 and rice straw powder as culture medium, the inoculum concentration of 5%, straw pulverizes average length 3-5mm, initially
PH4-5, temperature is at 28-35 DEG C, and fermentation time 72h is optimal conditions of fermentation.
Number of patent application is 201010040047, applicant Zhejiang University, the invention provides a kind of method producing cellulase,
Comprise the following steps: by trichoderma reesei (TriCh.Derma reesei) it is seeded in fermentation medium, start stream after fermentation
Adding cultivation, stream adds in incubation, and when zymotic fluid l, H value is higher than 4.8, fed-batch medium, when zymotic fluid pH value
During less than 4.5, stopping fed-batch medium, total fermentation time reaches 192 1 240 hours, stops fermentation.Side of the present invention
Insoluble carbon source and solubility carbon source are organically combined by method, the expression of co-induction cellulose enzyme gene.Add cultivation by stream and make bacterium
Kind the forming process of growth and purpose metabolite reach coordinated balance, solve merely with cellulose or soluble sugar for induction
Thing produces problem present in the technique of cellulase, is effectively increased the fermentation level of cellulase, and the cellulose obtained
Enzyme is stronger to the degradation property of cellulosic substrate.
The preparation method of 2,013,102,687,288 1 kinds of low-temperature neutral cellulases of application number, described low-temperature neutral cellulase is with inner
Family name's wood is mould to be prepared for bacterial strain, and this preparation method includes: activated spawn, ultraviolet mutagenesis, cultivates the most at low temperatures, purifies, and
Purifying bacterial strain is prepared seed liquor in 10 1 15 DEG C of bottom fermentations, seed liquor is inoculated in culture medium, 10 1 15 DEG C
Lower cultivation 96 1 144 hours, just obtains low-temperature neutral cellulase；Present invention also offers a kind of described low-temperature neutral fiber
The preparation method of element enzyme.The invention have the benefit that simple to operate, fermentation period is short, it is possible to decrease the application temperature of enzyme, from
And reduce the power consumption of commercial Application so that it is and more environmentally friendly, have broad application prospects；Additionally, present invention also offers a kind of being somebody's turn to do
The preparation method of cellulase, it can be widely used for the modification of paper-making fibre, reduces the cost of paper industry fibre modification.
Summary of the invention
The technical problem to be solved is to provide a kind of high activity cellulase produced by fungi fermentation, can be by with lower section
Prepared by method.
The seed liquor of described bacterial strain is inoculated in fermentation medium, cultivates 104 hours for 25 DEG C, zymotic fluid cellulase circumscribed
1,4 beta-glucanase, Endo-β-glucanase, beta-glucosidase and filter paper enzyme activity respectively reach 680U/mL, 1389U/mL,
486U/mL and 792U/mL.
The production bacterial classification of described cellulase is trichoderma reesei (Trichodema reesei) 601-17, and this bacterial strain was in 2013
November 3 was preserved in China typical culture collection center, and deposit number is CCTCC NO:M2013540, and Classification And Nomenclature is
Trichoderma reesei 601-17Trichodema reesei601-17, preservation address: Luo Jia Shan Wuhan University of wuchang, wuhan district is raw
Life science institute, postcode 430072.
The optimal pH 3.0-6.0 of described bacterial strain cellulase-producing；Optimum temperature is 23～35 DEG C.
Described bacterial strain physiological and biochemical property:
This bacterial strain at PDA cultured on solid medium, the colony characteristics of formation be bacterium colony be flocculence, bacterium colony is light green, bacterium
Fall flat, high 0.1-0.75mm, colony edge white, neatly；Fast growth, 48h colony diameter reaches 1.0-8.5mm, 72h
Reach 30-50mm；White mycelium, has barrier film, and mycelia wall is smooth, and diameter is at 2-5 ū m.Conidiophore is from the short lateral branch of mycelia
Occur, to life on side shoot.Conidiophore is ampuliform, and uprightly, colourless, spore is spherical in shape, green, diameter 20-100 ū m.
This bacterial strain can grow on wheat bran, and main metabolites is cellulase (endo cellulase, exocellulase and Portugal
Polyglycoside enzyme).According to " An Introduction industrial mycology " (George Smith1954),
" Fungal identification handbook " (Wei Jing surpasses 1982), " common and conventional fungi " (institute of microbiology of the Chinese Academy of Sciences 1973),
Identify that this bacterial strain is: trichoderma reesei.
Utilize Li's Trichoderma strains, by ultraviolet mutagenesis, cultivation, fermenting and producing cellulase method as follows.
Li's Trichoderma strains is inoculated on slant activation culture medium, activated spawn；Cultivate the bacterial classification after activation, picking list bacterium colony system
Standby spore suspension, and irradiate spore suspension with ultraviolet, mutagenesis obtains spore bacterium colony.
Spore bacterium colony after mutagenic and breeding is coated in primary dcreening operation culture medium, cultivates 3-5 days, select primary dcreening operation bacterial strain at 20-35 DEG C
And purify this bacterial strain；Purify bacterial strain to be inoculated in fermentation medium by the inoculum concentration of 5%, expand step by step and cultivate preparation seed liquor,
Incubation time is 72-96 hour；Seed liquor is inoculated in fermentation medium by the inoculum concentration of fermentating liquid volume 5-10%, 20-35
DEG C cultivate 96-144 hour, i.e. trichoderma reesei fermenting and producing cellulase terminates；Zymotic fluid is centrifuged at 4000-600orpm,
Collecting gained liquid is crude enzyme liquid；The crude enzyme liquid obtained carries out hyperconcetration filtration, obtains concentrating enzyme liquid.
The invention have the benefit that the acidic cellulase having obtained a kind of high vigor, its preparation method is simple, fermentation period
Short, the power consumption of commercial Application can be substantially reduced so that it is more environmentally friendly, there is wide prospects for commercial application.
Detailed description of the invention:
Bacterial classification primary dcreening operation: original strain trichoderma reesei HYX01 picks up from the soil sample screening point that Jinshi City is protected in river levee domestic fungus cultivating base
Separate out.Li's Trichoderma strains is inoculated on slant activation culture medium, activated spawn；Cultivate the bacterial classification after activation, picking list bacterium colony
Preparing spore suspension, and use ultraviolet irradiation spore suspension, mutagenesis obtains spore bacterium colony；Spore concentration is regulated by suitably dilution
Being 103/mL, take last dilution bacterium solution 0.2mL, dilution spread is on cellulose-Congo red plate screening culture medium.?
Cultivate the bacterial strain 200 that picking transparent circle/colony diameter is bigger after 3 days for 30 DEG C.(described cellulose-Congo red plate screening is cultivated
Base composition is as follows: cellulose powder 10g, Congo red 0.2g, ammonium sulfate 5g, magnesium sulfate 0.25g, potassium dihydrogen phosphate 1g, chlorination
Sodium 0.1g, gelatin 2g, agar 20g, running water constant volume 1000mL, pH value 5-6,121 DEG C of sterilizing 20min).
Multiple sieve: with sterile toothpick, the 200 strain bacterium obtained are inoculated in slant medium respectively, 30 DEG C of cultivation to spores are paved with inclined-plane.
Respectively spore is fermented to be inoculated under aseptic washing in the 250mL triangular flask sieving culture medium equipped with 50mL again, inoculum concentration 10
%(v/v), 30 DEG C, 100r/min cultivate 96h, measure the cellulase activity of each bacterial strain respectively.(described sieve culture medium again
Form as follows: cellulose powder 50g, ammonium sulfate 5g, magnesium sulfate 0.25g, potassium dihydrogen phosphate 1g, sodium chloride 0.1g, running water are fixed
Hold 1000mL, pH value 5-6,121 DEG C of sterilizing 20min).Choose the highest bacterial strain of cellulose enzyme vigor and be amplified experiment.
Filtering out bacterial strain again is trichoderma reesei (Trichodema reesei) 601-17, and deposit number is CCTCC NO:M2013540.
Cultural characteristic: the optimal pH 3.0-6.0 of this bacterial strain cellulase-producing；Optimum temperature is 23～35 DEG C.
Genetic stability is tested: passed on for continuous ten times on inclined-plane by this bacterial strain, and the method detection sieved again by shaking flask is passed on every time
After fermentation situation.Experiment finds, passing on for continuous ten times on inclined-plane, this bacterial classification proterties does not has significant change, properties to refer to
Mark is all normal, illustrates that the genetic stability of this bacterial classification is stronger.
Scale-up
Seed culture: bacterial strain the highest for cellulose enzyme vigor is accessed in 500mL triangular flask, seed culture medium loading amount 100 milli
Rise, 30 DEG C, 150rpm shaking table cultivation 72-96h.
Seed tank culture: by seed liquor with 10%(v/v) inoculum concentration accesses equipped with in the 10L fermentation tank of 7.5L fermentation medium,
Control ph is constant is 5.0 ± 0.2, cultivation temperature 27 ± 0.1 DEG C, mixing speed 300rpm, ventilation (v/v) 1:0.8-1.2,
Incubation time 104h, dissolved oxygen 20-30%.Described fermentation medium preparation method is: cellulose powder 10%, ammonium sulfate 0.5%,
Magnesium sulfate 0.025%, potassium dihydrogen phosphate 0.1%, sodium chloride 0.01%, remaining be water, pH value 5-6,121 DEG C of sterilizing 20min.
After fermentation ends, take fermented supernatant fluid (crude enzyme liquid) and carry out enzyme activity detection, after measured, circumscribed 1,4 beta-glucanase, interior
Cut 1,4 beta-glucanase, beta-glucosidase and filter paper enzyme activity and respectively reach 680U/mL, 1389U/mL, 486U/mL and 792
U/mL。
Bacterial strain after trichoderma reesei (Trichodema reesei) mutagenesis used in the present invention can preserve 2 in 4 DEG C of environment
Month, in the sorbitol solution of 10-20%, can preserve for a long time at subzero 80 DEG C.
Slant medium: potato 20%, glucose 1%, agar 2%, remaining is water, and pH is natural, temperature 28 DEG C.
Cellulase is prepared by following methods:
The trichoderma reesei seed liquor obtained will be cultivated with 10%(v/v) inoculum concentration accesses equipped with the 10L fermentation tank of 7.5L fermentation medium
In, control ph is constant is 5.0 ± 0.2, cultivation temperature 27 ± 0.1 DEG C, mixing speed 300rpm, ventilation (v/v) 1:1.2,
Incubation time 104h, dissolved oxygen 25%.After fermentation ends, take fermented supernatant fluid (crude enzyme liquid) and carry out enzyme activity detection, after measured,
Circumscribed 1,4 beta-glucanase, Endo-β-glucanase, beta-glucosidase and filter paper enzyme activity respectively reach 680U/mL,
1389U/mL, 486U/mL and 792U/mL.
Described fermentation medium preparation method is: cellulose powder 10%, ammonium sulfate 0.5%, magnesium sulfate 0.025%, potassium dihydrogen phosphate
0.1%, sodium chloride 0.01%, remaining be water, pH value 5-6,121 DEG C of sterilizing 20min.
Claims (3)
1. a cellulase, it is characterised in that described cellulase is by the trichoderma reesei of deposit number CCTCC NO:M 2013540
Prepared by strain fermentation, method is as follows: CTCC M 2013540 purifies bacterial strain and is inoculated in fermentation medium by the inoculum concentration of 5%,
Expanding step by step and cultivate preparation seed liquor, incubation time is 72-96 hour；Seed liquor is connect by the inoculum concentration of fermentating liquid volume 5-10%
Plant in fermentation medium, cultivate 96-144 hour for 20-35 DEG C, i.e. trichoderma reesei fermenting and producing cellulase terminates；To send out
Ferment liquid is centrifuged at 4000-6000rpm, and collecting gained liquid is crude enzyme liquid；The crude enzyme liquid obtained carries out hyperconcetration filtration, obtains
Concentrate enzyme liquid；
Described fermentation medium preparation method is: cellulose powder 10%, ammonium sulfate 0.5%, magnesium sulfate 0.025%, potassium dihydrogen phosphate
0.1%, sodium chloride 0.01%, remaining be water, pH value 5-6,121 DEG C of sterilizing 20min.
Cellulase the most according to claim 1, it is characterised in that the circumscribed beta glucan of described crude enzyme liquid cellulase
Enzyme, Endo-β-glucanase, beta-glucosidase and filter paper enzyme activity respectively reach 680U/mL, 1389U/mL, 486U/mL
And 792U/mL.
The preparation method of cellulase the most according to claim 1, comprises the steps: that described cellulase is by deposit number
The trichoderma reesei bacterial classification fermentation preparation of CCTCC NO:M 2013540, trichoderma reesei seed liquor is inoculated in fermentation medium, 20-35 DEG C
Cultivate 96-144 hour, i.e. trichoderma reesei fermenting and producing cellulase terminates；Zymotic fluid is centrifuged at 4000-6000rpm, receives
Integrate gained liquid as crude enzyme liquid；The crude enzyme liquid obtained carries out hyperconcetration filtration, obtains concentrating enzyme liquid.
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| CN201310716678.5A CN103740678B (en) | 2013-12-23 | 2013-12-23 | Cellulase and preparation thereof |
| US14/578,408 US9605247B2 (en) | 2013-12-23 | 2014-12-20 | Strain and a method to produce cellulase and its use |
| US15/360,076 US10053680B2 (en) | 2013-12-23 | 2016-11-23 | Strain and a method to produce cellulase and its use |
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| CN101280330A (en) * | 2008-05-08 | 2008-10-08 | 南昌航空大学 | Method for preparing chitosan oligosaccharide with trichoderma reesei cellulase |
| CN101735993A (en) * | 2010-01-19 | 2010-06-16 | 浙江大学 | Method for efficiently producing cellulase |
| CN101418289B (en) * | 2008-11-28 | 2011-05-04 | 天津科技大学 | Trichoderma reesei liquid submerged fermentation cellulase and enzymatic beating process thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101280330A (en) * | 2008-05-08 | 2008-10-08 | 南昌航空大学 | Method for preparing chitosan oligosaccharide with trichoderma reesei cellulase |
| CN101418289B (en) * | 2008-11-28 | 2011-05-04 | 天津科技大学 | Trichoderma reesei liquid submerged fermentation cellulase and enzymatic beating process thereof |
| CN101735993A (en) * | 2010-01-19 | 2010-06-16 | 浙江大学 | Method for efficiently producing cellulase |
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