CN102787078B - Trichoderma koningiopsis strain and application of trichoderma koningiopsis to preparation of cellulase - Google Patents

Trichoderma koningiopsis strain and application of trichoderma koningiopsis to preparation of cellulase Download PDF

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CN102787078B
CN102787078B CN201210276891.4A CN201210276891A CN102787078B CN 102787078 B CN102787078 B CN 102787078B CN 201210276891 A CN201210276891 A CN 201210276891A CN 102787078 B CN102787078 B CN 102787078B
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cellulase
fcd3
mould
wood
trichoderma
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CN102787078A (en
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冯家勋
张政
刘君梁
蓝健益
马庆生
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Guangxi University
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Abstract

The invention discloses a trichoderma koningiopsis strain and application of the trichoderma koningiopsis to the preparation of cellulase. The invention provides the trichoderma koningiopsis strain FCD3-1 capable of being used for preparing the cellulase, and the preservation number of the trichoderma koningiopsis strain FCD3-1 is CGMCC (China General Microbiological Culture Collection Center) No. 6050. The invention also provides a method for preparing the cellulase. The method comprises the following steps that the trichoderma koningiopsis strain FCD3-1 is subjected to liquid fermentation, fermentation products are collected, thalli are removed through centrifugation, obtained fermentation supernatant can be used as a cellulase preparation, the cellulase preparation can be used for efficiently hydrolyzing and crystallizing the cellulose, in addition, the final hydrolysis product is glucose, the cellulase preparation contains high-activity beta-glucosidase, the most proper pH value of the beta-glucosidase is 4.5, the most proper temperature is 60 DEG C, and in addition, better pH tolerance and temperature stability are realized. The cellulase preparation can be used for the lignocellulose hydrolysis.

Description

One strain trichoderma strain and the application in preparing cellulase thereof
Technical field
The present invention relates to biological technical field, relate in particular to a strain trichoderma strain and the application in preparing cellulase thereof.
Background technology
Plant biomass is the renewable biomass resource of maximum on the earth, and is considered to can play an important role like that by image-stone oil in future.Mierocrystalline cellulose and hemicellulose are the main ingredients of plant biomass, by cellulase to cellulosic hydrolysis, Mierocrystalline cellulose can be converted to glucose, glucose can further be changed into ethanol or other useful chemical (Lynd LR, Weimer PJ, van Zyl WH, et al.Microbial cellulose utilization:fundamentals and biotechnology.Microbiol Mol Biol Rev.2002,66:506 – 577).
First-generation alcohol fuel is mainly with sucrose or W-Gum or the production of other fermented grains.In in the past more than 30 year, Brazil, the U.S. and some other country be the blending ingredients using alcohol fuel as motor spirit or be widely used (Martin MA.First generation biofuels compete.N Biotechnol.2010,27:596 – 608) as fuel completely separately.S-generation alcohol fuel, mainly refer to and take ethanol (the Sims RE that lignocellulose is raw material production, Mabee W, Saddler JN, et al.An overview of second generation biofuel technologies.Bioresour Technol.2010,101:1570 – 1580).Since 20 century 70s, for take lignocellulose, carried out a large amount of research work as raw material production alcohol fuel.In the process of this bio-transformation, the stage of most critical is, by cellulase catalysis, cellulose hydrolysis is become to glucose.Compare with Starch Hydrolysis process in first-generation fuel ethanol production, due to the extremely strong crystallographic property of Mierocrystalline cellulose, much bigger (the Merino ST of the diastatic consumption of the amount ratio of cellulase during hydrolysis, Cherry J.Progress and challenges in enzyme development for biomass utilization.Adv Biochem Eng Biotechnol.2007,108:95-120).
Cellulase is the general name that a kind of polycomponent enzyme is, by its catalysis, the cellulase enzyme component that can be divided three classes, they are respectively inscribe-1, 4-callose enzyme (endo-1, 4-β-D-glucanase, EC3.2.1.4), circumscribed-1, 4-callose enzyme (exo-1, 4-β-D-glucanase) or cellobiohydrolase (cellobiohydrolase) (EC3.2.1.91) and beta-glucosidase (β-glucosidase, EC3.2.1.21) (Zhang PYH, Himmel ME, Mielenz JR.Outlook for cellulase improvement:screening and selection strategies.Biotechnol Advan.2006, 24:452-481).Inscribe-Isosorbide-5-Nitrae-callose enzyme (hereinafter to be referred as endoglucanase) acts on cellulosic molecule inside, and the β-Isosorbide-5-Nitrae-glycosidic link in random hydrolysis cellulosic molecule produces short cellulose chain, exposes the Mierocrystalline cellulose end making new advances.Circumscribed-Isosorbide-5-Nitrae-callose enzyme (hereinafter to be referred as exoglucanase), with orderly fashion (processive manner), along Mierocrystalline cellulose end (reducing end or non-reducing end) ecto-entad hydrolysis β-Isosorbide-5-Nitrae-glycosidic link, discharges cellobiose; In addition, exoglucanase also acts on crystalline cellulose, cellulose long-chain is peeled off out from crystallizing field (Teeri TT.Crystalline cellulose degradation:new insight into the function of cellobiohydrolase.Trends Biotechnol.1997,15:160-167).Beta-glucosidase is hydrolyzed into glucose molecule by the cell-oligosaccharide of cellobiose or other solubility, can eliminate the feedback inhibition of cellobiose to endoglucanase and exoglucanase.Under the synergy of this three fermentoid, Mierocrystalline cellulose is finally degraded to glucose (Lynd LR, Weimer PJ, van Zyl WH, et al.Microbial cellulose utilization:fundamentals and biotechnology.Microbiol Mol Biol Rev.2002,66:506 – 577).
Filamentous fungus is the main source of commercial fibres element enzyme, the filamentous fungus that comprises Trichoderma (Trichoderma sp.), fungus Trichoderma viride (Trichoderma viride), long shoot wood mould (Trichoderma longibrachiatum), it is the highest that Rui Shi wood mould (Trichoderma reesei) etc. is considered to yield of cellulase, to one of best fungi of crystalline cellulose hydrolysis effect (Gusakov AV.Alternatives to Trichoderma reesei in biofuel production.Trends Biotechnol.2011, 29:419-425).
Mandels and Sternberg have assessed and have surpassed the fungal bacterial strain that 14000 strains are collected and screened, measured their cellulase activity, found that producing the highest bacterial strain of cellulase activity is mould (the Mandels M of Rui Shi wood, Sternberg D.Recent advances in cellulase technology.Ferment Technol.1976,54:267 – 286).According to this discovery, developed subsequently the Rui Shi trichoderma strain of different High Cellulase Productions, its Zhong Ruishi trichoderma strain Rut-C30 is one of the most distinctive bacterial strain, in the Rui Shi of High-efficient Production cellulase trichoderma strain, bacterial strain Rut-C30 becomes reference strain (Le Crom S, Schackwitz W, Pennacchio L, et al.Tracking the roots of cellulase hyperproduction by the fungus Trichoderma reesei using massively parallel DNA sequencing.Proc Natl Acad Sci USA.2009, 106:16151 – 16156).
At present, the commercial fibres element zymin that many companies carry out industrial-scale production is in the world to use the mould mutant strain of Rui Shi wood to produce.But the beta-glucosidase activity level of Rui Shi trichoderma strain is very low, can not fast and fully the cellobiose of cellulose hydrolysis generation be all hydrolyzed into glucose.Therefore, when preparing cellulase preparation with the mould mutant strain of Rui Shi wood, the beta-glucosidase of other microorganisms of having to augment wherein so that cellulosic substrate be more effectively hydrolyzed into completely glucose (
Figure BDA00001978229000021
margeot A, Dolla A, et al.Comparative secretome analyses of two Trichoderma reesei RUT-C30 and CL847hypersecretory strains.Biotechnol Biofuels.2008,1:18).
Summary of the invention
An object of the present invention is to provide a strain wood mould (Trichoderma koningiopsis) bacterial strain FCD3-1.
Wood provided by the invention mould (Trichoderma koningiopsis) bacterial strain FCD3-1, its deposit number is CGMCC No.6050.
The application of mould (Trichoderma koningiopsis) the bacterial strain FCD3-1 of above-mentioned wood in preparing cellulase or cellulase preparation is also the scope of protection of the invention.
Second object of the present invention is to provide a kind of method of production of cellulose enzyme.
Method provided by the invention, the above-mentioned wood that comprises the steps: to ferment mould (Trichoderma koningiopsis) FCD3-1, collects tunning, obtains cellulase.
In aforesaid method, the condition of described fermentation is to cultivate 5-7 days at 26-32 ℃, 180rpm.In an embodiment of the present invention, the condition of fermentation is specially at 28 ℃, 180rpm and cultivates 5 days;
In aforesaid method, the component of the fermention medium that described fermentation adopts is as follows: described in every L, fermention medium is by 2.0g KH 2pO 4, 1.4g (NH 4) 2sO 4, 0.3g urea, 0.3g MgSO 47H 2o, 0.3g CaCl 2, 5.0mgFeSO 47H 2o, 1.56mg MnSO 4h 2o, 1.4mg ZnSO 47H 2o, 2.0mg CoCl 2, 2mlTween-80,1.0g peptone, 20g wheat bran, 10g crystalline cellulose and water forms, water is supplied volume;
The pH value of described fermention medium is pH4.0-6.0; In embodiments of the invention, the pH value of fermention medium is specially 5.0.
In aforesaid method, above-mentioned fermentation is cultivated for the spore solution of above-mentioned wood mould (Trichoderma koningiopsis) FCD3-1 is seeded in described fermention medium;
Above-mentioned spore solution is for to be suspended in by the spore of above-mentioned wood mould (Trichoderma koningiopsis) FCD3-1 the solution obtaining in sterilized water.
In aforesaid method, after described collection tunning, also comprise the steps: described tunning centrifugally, collect supernatant liquor, obtain cellulase; Above-mentionedly centrifugally be specially 4 ℃, 12000rpm, centrifugal 5min, centrifugal radius is 7cm;
The cellulase being prepared by above-mentioned method is also the scope of protection of the invention.
Above-mentioned cellulase is the mixture that contains beta-glucosidase.
The 3rd object of the present invention is to provide a kind of cellulase preparation.
Cellulase preparation provided by the invention, its activeconstituents is above-mentioned mould (Trichoderma koningiopsis) the bacterial strain FCD3-1 of wood or above-mentioned cellulase.
The application in hydrolysis begasse pulp of above-mentioned wood mould (Trichoderma koningiopsis) bacterial strain FCD3-1, above-mentioned cellulase or above-mentioned cellulase preparation is also the scope of protection of the invention; In this application, the pH value of described hydrolysis is 4.0-5.0, and temperature is 45-55 ℃; The pH value of described hydrolysis is specially 4.0, and temperature is specially 55 ℃;
Above-mentioned cellulase or above-mentioned cellulase preparation are also the scope of protection of the invention at hydrolysis of lignocellulose or the application prepared in glucose.
The 4th object of the present invention is to provide a kind of method of producing glucose.
The method providing of the present invention, comprises the steps: take that begasse pulp is as substrate, adds above-mentioned cellulase or above-mentioned cellulase preparation, in pH4.0,55 ℃ of Water Under solutions, obtains glucose.
Above-mentioned bacterial strains FCD3-1 has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (has been called for short CGMCC on 04 24th, 2012, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.6050, and Classification And Nomenclature is for intending the mould Trichoderma koningiopsis of healthy and free from worry wood.
Of the present invention experimental results show that, the present invention has found a strain trichoderma strain FCD3-1, it is carried out to fermentation culture, fermented liquid can be used as cellulase, this cellulase can be used for Production of Alcohol from Lignocellulose, can be hydrolyzed efficiently crystalline cellulose, and hydrolysis end product is mainly glucose, do not need additionally to add beta-glucosidase in cellulase, can reduce cost.In addition, this cellulase is higher to the enzyme activity of begasse pulp, is 2.74U/ml, in to the trans-utilization of bagasse, has application potential.
Accompanying drawing explanation
Fig. 1 is the colonial morphology of wooden mould FCD3-1 on MEA flat board
Fig. 2 is wooden mould FCD3-1 conidiophore, bottle stalk and conidial microscopic morphology
Fig. 3 is the result of the ITS sequence of wooden mould FCD3-1 and the ITS sequence alignment of Trichoderma koningiopsis bacterial strain DMC 795b
Fig. 4 is that wooden mould FCD3-1 fermented liquid is as the suitableeest action pH curve of cellulase
Fig. 5 is that wooden mould FCD3-1 fermented liquid is as the optimum temperature curve of cellulase
Fig. 6 is that wooden mould FCD3-1 fermented liquid is as silica gel thin-layer chromatography (TLC) collection of illustrative plates of the product of the hydrolysis crystalline cellulose (Avicel) of cellulase
M: glucose sugar and cellobiose; Swimming lane 1-5: corresponding A vicel suspension reacts the reaction mixture supernatant liquor after 0h, 2h, 4h, 8h and 16h with wooden mould FCD3-1 fermented liquid under pH4.0,50 ℃ of conditions respectively; The direction of silica gel thin-layer chromatography is bottom-up
Fig. 7 is that the mould Rut-C30 fermented liquid of Rui Shi wood is as silica gel thin-layer chromatography (TLC) collection of illustrative plates of the product of the hydrolysis crystalline cellulose (Avicel) of cellulase
M: glucose sugar and cellobiose; Swimming lane 1-5: corresponding A vicel suspension reacts the reaction mixture supernatant liquor after 0h, 2h, 4h, 8h and 16h with the mould Rut-C30 fermented liquid of Rui Shi wood under pH4.0,50 ℃ of conditions respectively;
The direction of silica gel thin-layer chromatography is bottom-up
Fig. 8 is that wooden mould FCD3-1 fermented liquid is as the suitableeest action pH curve of beta-glucosidase
Fig. 9 is that wooden mould FCD3-1 fermented liquid is as the optimum temperature curve of beta-glucosidase
Figure 10 is that wooden mould FCD3-1 fermented liquid is as the pH tolerability curves of beta-glucosidase
Figure 11 is that wooden mould FCD3-1 fermented liquid is as the temperature tolerance curve of beta-glucosidase
Figure 12 is that wooden mould FCD3-1 fermented liquid is as the tolerability curves under simultaneous saccharification and fermentation condition of beta-glucosidase
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.% in following embodiment, if no special instructions, is quality percentage composition.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.
Minimum medium formula: KH 2pO 42.0g, (NH 4) 2sO 41.4g, urea 0.3g, MgSO 47H 2o 0.3g, CaCl 20.3g, FeSO 47H 2o 5.0mg, MnSO 4h 2o 1.56mg, ZnSO 47H 2o 1.4mg, CoCl 22.0mg, Tween-802ml, peptone 1.0g, wheat bran 20g, Avicel(crystalline cellulose, Sigma PH101) 10g; Use 800ml deionized water dissolving, then regulate pH value to the 1M HCl aqueous solution or the adjusting of the 1M NaOH aqueous solution for 5.0(); With deionized water, be settled to 1L; 121 ℃ of moist heat sterilization 20min.
Citric acid-Sodium phosphate dibasic damping fluid preparation of pH4.0: by 7.71 milliliters of 0.2M Na 2hPO 4the aqueous solution and 12.29 milliliters of 0.1M aqueous citric acid solutions mix.
In embodiment 1 and embodiment 2, cellulase activity adopts 3 in measuring, 5-dinitrosalicylic acid (3,5-dinitrosalicylate, DNS) method is measured the reducing sugar (Miller producing, GL.Use of dinitrosalicyclic acid reagent for determination of reducing sugar.Analytical Chemistry 1959,31:426-428), concrete steps are as follows:
1, with the glucose reference liquid of deionized water preparation 2mg/ml, carry out 8 gradient dilutions; Each extent of dilution is got 500 μ l Glucose Liquids and is added 1ml DNS reagent, and boiling water bath 5min colour developing, is cooled to room temperature, gets 200 μ l and adds 96 hole enzyme plates, and 540nm place measures absorbance value; Obtain the typical curve of absorbance value and glucose content, the functional expression of typical curve is y=0.006x-0.030 (R 2=0.999), y is absorbance value (OD 540), x is glucose content (μ g).
2, by 1g crystalline cellulose Avicel(Sigma PH101) be suspended from citric acid-Sodium phosphate dibasic damping fluid of 100ml pH4.0, obtain the suspension of crystalline cellulose; In 2ml EP pipe, add 450 μ l crystalline cellulose suspensions, be placed in the water-bath of 50 ℃; Add again 50 μ l liquid cellulase preparations, 50 ℃ insulation 60min, during constantly vibration, enzyme-to-substrate is fully contacted; Then add 1ml DNS reagent, boiling water bath 5min colour developing, is cooled to room temperature, gets 200 μ l clear liquors and adds 96 hole enzyme plates, measures absorbance value in 540nm place, according to glucose typical curve and absorbance value, calculates enzyme activity.Cellulase activity definition: under certain condition, enzymic hydrolysis crystalline cellulose (Avicel), it is an enzyme activity unit (U) that every min discharges the required enzyme amount of 1 μ mol reducing sugar (glucose that is equivalent to equivalent).
The isolation identification of embodiment 1, trichoderma strain FCD3-1
One, the acquisition of bacterial strain
1, the collection of pedotheque
Be collected in March, 2009 near Guangxi China Fangchenggang City starch factory; Remove earth's surface overburden soil, 50 grams, soil getting about 5-20cm place under earth's surface is a sample; Totally 13 parts, distributed collection sample.
2, the separation screening of cellulase producing strain
(1) with deionized water, prepare isolation medium; In every liter of isolation medium, contain: KH 2pO 42.0g, (NH 4) 2sO 41.4g, urea 0.3g, MgSO 47H 2o 0.3g, CaCl 20.3g, FeSO 47H 2o 5.0mg, MnSO 4h 2o 1.56mg, ZnSO 47H 2o 1.4mg, CoCl 22.0mg, Avicel(Sigma PH101) 10g, agar 15g; PH5.0; 121 ℃ of moist heat sterilization 20min; While being cooled to 50 ℃ after sterilizing, be down flat plate.
(2) get 10g soil sample and put into 250ml Erlenmeyer flask, add 90ml sterilized water and add appropriate granulated glass sphere, on magnetic stirring apparatus, stir 30min, soil sample is fully mixed with water, cell is disperseed.Get 1ml soil supension and add in the finger-type bottle that fills 9ml sterilized water and fully mix, then therefrom get 1ml and fill in the finger-type bottle of 9ml sterilized water to another, mix, make by that analogy 10 -1, 10 -2, 10 -3the different dilution soil solution, respectively gets 100 μ l and is coated on isolation medium flat board, 28 ℃ of cultivations.
After (3) 5 days, observe fungal colony growing state, select single bacterium colony, be transferred on new isolation medium, line separation and purification.
(4) minimum medium (liquid) of preparation pH 5.0.
(5) fungal bacterial strain step (3) being obtained is seeded in minimum medium (liquid), 28 ℃, 180rpm are cultivated 7 days, get supernatant liquor, take crystalline cellulose (Avicel) as substrate carries out cellulase activity mensuration, therefrom filter out the bacterial strain FCD3-1 that cellulase-producing vigor is the highest.
Two, the evaluation of bacterial strain
The colonial morphology of bacterial strain FCD3-1 on wort agar (MEA) flat board is shown in Fig. 1, and this bacterial strain is the dark colony radius of cultivating 72h on MEA culture medium flat plate: 42-44mm in the time of 20 ℃, 63-65mm in the time of 25 ℃, 59-60mm in the time of 30 ℃, 17-18mm in the time of 35 ℃.The bacterium colony of the upper 25 ℃ of dark 72h of cultivation of MEA forms 3 and takes turns concentric ring, produces closeer white hypha on concentric ring, and bacterium colony is cotton-shaped quality, the thick 2-3mm of flora; On the concentric ring of bacterium colony central authorities, produce closeer green conidium, form the ring of an about 10mm; The nondiscoloration of the bacterium colony back side; Bacterium colony is without obvious smell, bacterium colony surface drying, and without transudate, chromogenesis not.
The form of the mycelia of bacterial strain FCD3-1 under bright-field microscope is shown in Fig. 2.The main shaft of conidiophore is obvious, width approximately 3 μ m; Can educate branch and produce successively along major axes orientation, the angle of life, branch and main shaft is slightly less than to 90 degree; Longer branch is positioned at main shaft base portion; Main shaft top branch is few, and branch spacing is larger; One-level branch is branch or directly produce bottle stalk again, and second branch is inclined to life; All branch tops of educating are the bottle stalk that whirlpool shape is arranged.Bottle stalk is ampoule shape straight or slight curvature, and obviously expand middle part, the base portion contracting of slightly hanging, and several bottles of stalks produce from same point.Conidium is oval.
The Molecular Identification of bacterial strain FCD3-1 the results are shown in Figure 3.Extract total DNA of bacterial strain FCD3-1 and as template, utilize universal primer ITS1(5 ' TCCGTAGGTGAACCTGCGG 3 ') and ITS4(5 ' TCCTCCGCTTATTGATATG 3 '), pcr amplification obtains its ITS, obtains the nucleotide sequence of a 609bp through order-checking.Sequence analysis analysis shows: and the ITS of itself and Trichoderma koningiopsis bacterial strain DMC 795b (GenBank accession number: comparison score EU718083) is the highest, sequential covering degree is 98%, consistence is 99%.
According to the morphological specificity of this bacterial strain, with reference to < < fungi identification handbook > > (Wei Jingchao, Shanghai: Shanghai science tech publishing house, 1979), and binding molecule qualification result, bacterial strain FCD3-1 is initially identified as and intends healthy and free from worry wood mould (Trichoderma koningiopsis).
Above-mentioned bacterial strains FCD3-1 has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (has been called for short CGMCC on 04 24th, 2012, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.6050, and Classification And Nomenclature is for intending the mould Trichoderma koningiopsis of healthy and free from worry wood.
Embodiment 2, utilize trichoderma strain FCD3-1 to prepare cellulase
One, the acquisition of fermented liquid
1, the minimum medium (liquid) of preparation pH5.0
2, the preparation of spore liquid
(1) by 121 ℃ of sterilizing 20min of PDA substratum (solid).
(2) spore of the mould FCD3-1 of wood being obtained by embodiment 1 that activates 5 days going down to posterity on PDA flat board is with making spore suspension after aseptic washing, and spore concentration is 1 * 10 10individual/ml.
3, the acquisition of fermented liquid
(1) getting 100ml minimum medium is contained in 500ml Erlenmeyer flask.
(2) spore liquid of getting wooden mould FCD3-1 is seeded in minimum medium by 1% inoculum size (volumn concentration), and 28 ℃, 180rpm, cultivate 5 days, collects culture.
(3) centrifugal (4 ℃, centrifugal radius 7cm, 12000rpm, centrifugal 5min) culture is removed thalline, collects supernatant liquor and is wooden mould FCD3-1 fermented liquid.
Two, cellulase zymetology characteristic and the checking of fermented liquid
1, the Optimun pH of fermented liquid and optimum temperature
1), Optimun pH
The mould FCD3-1 fermented liquid of wood is detected the enzyme of crystalline cellulose Avicel is lived according to aforesaid DNS method, use respectively citric acid-Sodium phosphate dibasic damping fluid of the alternative pH4.0 of citric acid-Sodium phosphate dibasic damping fluid (pH 3.0,3.5,4.0,4.5,5.0,5.5,6.0) of different pH, temperature of reaction adopts 50 ℃, and cellulase activity is measured the DNS method that adopts; Enzyme activity definition: under 50 ℃ of conditions, enzymic hydrolysis Avicel, it is an enzyme activity unit (U) that every min discharges the required enzyme amount of 1 μ mol reducing sugar (glucose that is equivalent to equivalent).
The high enzymatic activity of take is 100%, and the enzyme activity of other pH value is enzyme activity with the ratio of high enzymatic activity, take pH value as X-coordinate, and enzyme activity is ordinate zou mapping, sees Fig. 4.Result shows, the Optimun pH of wooden mould FCD3-1 fermented liquid in the time of 50 ℃ is 4.0.
2), optimum temperature
The mould FCD3-1 fermented liquid of wood is detected the enzyme of crystalline cellulose Avicel is lived according to aforesaid DNS method, adopt respectively different temperature of reaction (30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃), reaction buffer adopts citric acid-Sodium phosphate dibasic of pH4.0, and cellulase activity is measured the DNS method that adopts; Enzyme activity definition: under pH4.0 condition, enzymic hydrolysis Avicel, it is an enzyme activity unit (U) that every min discharges the required enzyme amount of 1 μ mol reducing sugar (glucose that is equivalent to equivalent).
Using high enzymatic activity as 100%, and the enzyme activity of other temperature is enzyme activity with the ratio of high enzymatic activity, take temperature as X-coordinate, and enzyme activity is ordinate zou mapping, sees Fig. 5.Result shows, the optimum temperature of wooden mould FCD3-1 fermented liquid when pH4.0 is 55 ℃.
Therefore, wooden mould FCD3-1 fermented liquid is cellulase, also can be called cellulase preparation.
2, the enzyme activity of fermented liquid to crystalline cellulose
DNS method detects enzyme and lives, and method is the same, and wooden mould FCD3-1 fermented liquid (cellulase) (pH4.0,55 ℃) under optimal condition is 0.37 ± 0.007U/ml to the vigor of crystalline cellulose Avicel.
Commercial strain Rui Shi wood mould (Trichoderma reesei) Rut-C30[of the method fermentative production cellulase according to above-mentioned is US mode culture collection warehousing (American type culture collection) purchased from ATCC, ATCC numbering: 56765], collect Rut-C30 fermented liquid (being cellulase), according to aforesaid DNS method, detecting Rut-C30 fermented liquid lives to the enzyme of crystalline cellulose Avicel, (pH4.5 under its optimal condition, 55 ℃) Rut-C30 fermented liquid (cellulase) is 0.39 ± 0.014U/ml to the vigor of crystalline cellulose Avicel, the two does not have significant difference.
3, fermented liquid hydrolysis crystalline cellulose product analysis
1), prepare crystalline cellulose suspension
Get 1.25g crystalline cellulose Avicel(Sigma PH101) be suspended from citric acid-Sodium phosphate dibasic damping fluid of 100ml pH4.0, obtain the suspension of crystalline cellulose.
2), product analysis
Prepare 5 aseptic finger-type bottles, the mould FCD3-1 fermented liquid of wood that adds respectively the Avicel suspension of the above-mentioned preparation of 4ml and the preparation that 1ml above-mentioned obtains, be placed in 50 ℃ of shaking tables, 150rpm vibration, correspondence is reacted 0h, 2h, 4h, 8h and 16h successively, get 1ml reaction mixture, boiling water boiling 5min deactivation cellulase, cooling; The centrifugal 5min of 12,000rpm, gets supernatant liquor, and silica gel thin-layer chromatography (TLC) detects the sugar (standard specimen is glucose, cellobiose) in supernatant liquor.
The results are shown in Figure 6, wooden mould FCD3-1 fermented liquid acts on crystalline cellulose Avicel, and the amount that just can discharge a large amount of glucose and glucose during 2h is on the increase with the prolongation in reaction times; In addition, it is important that the hydrolysate detecting only has glucose, does not have cellobiose.
Utilize the fermented liquid of commercial strain Rui Shi wood mould (Trichoderma reesei) Rut-C30 to do above-mentioned identical experiment, TLC detects the sugar in supernatant liquor, the results are shown in Figure 7,2h-16h hydrolysate and is mainly cellobiose, and glucose yield is less.
Illustrate that thus wooden mould FCD3-1 fermented liquid can be hydrolyzed into glucose by crystalline cellulose quickly and efficiently, further prove that wooden mould FCD3-1 fermented liquid is cellulase.
4, the substrate specificity of wooden mould FCD3-1 fermented liquid
Use respectively different substrate Whatman No.1 filter paper (the Whatman company of equal in quality, production code member: 1001-110), Xylo-Mucine (CMC-Na) (Sigma company, production code member: 126k0119), phosphoric acid expansion Avicel(utilizes Sigma PH101 self-control), begasse pulp (Pu Miao paper mill, Nanning City, Guangxi Zhuang Autonomous Region) replaces Avicel, temperature of reaction adopts 55 ℃, reaction buffer adopts citric acid-Sodium phosphate dibasic of pH4.0, and cellulase activity is measured the DNS method that adopts; Enzyme activity definition: under pH4.0,55 ℃ of conditions, the above-mentioned a kind of substrate of enzymic hydrolysis, it is an enzyme activity unit (U) that every min discharges the required enzyme amount of 1 μ mol reducing sugar (glucose that is equivalent to equivalent).
Liquid cellulase preparation to the cellulase activity measurement result of different substrates in Table 1.
The substrate specificity of the wooden mould FCD3-1 liquid cellulase preparation of table 1
Figure BDA00001978229000091
Using to Avicel enzyme activity as 100%, to the enzyme activity of other substrate with to the ratio of the enzyme activity of Avicel, be enzyme activity.Wood mould FCD3-1 fermented liquid to the enzyme activity of various substrates by weak order being by force: CMC-Na, phosphoric acid expansion Avicel, begasse pulp, Whatman No.1 filter paper, Avicel.The mould FCD3-1 liquid cellulase preparation of wood is stronger to the enzyme activity of begasse pulp, illustrates that wooden mould FCD3-1 fermented liquid has application potential in the trans-utilization of bagasse.
Three, the enzymatic property of beta-glucosidase and checking in fermented liquid
1, the beta-glucoside enzyme activity determination of fermented liquid
Beta-glucoside enzyme activity determination is with reference to the described methods such as Odoux (Odoux E, Escoute J, Verdeil JL, et al.Localization of β-D-glucosidase activity and glucovanillin in vanilla bean (Vanilla planifolia Andrews) .Ann Botany.2003,92:437-444), with 4-nitrophenyl-β-D-glucopyranoside (p-NPG, p-Nitrophenyl β-D-glucopyranoside) be substrate, concrete steps are as follows:
1), with 4-nitrophenols (p-NP, the p-Nitrophenol) reference liquid of deionized water preparation 10mM, carry out 5 gradient dilutions (10 -1-10 -5); Get each dilution p-NP solution of 140 μ l, add respectively the Na of 70 μ l 0.4M 2cO 3solution, beats and mixes with the careful suction of pipettor, avoids occurring bubble.Get 200 μ l mixed solutions, add 96 hole enzyme plates, 410nm place measures absorbance value; Obtain the typical curve of absorbance value and p-NP concentration, the functional expression of typical curve is y=6.851x+0.059 (R 2=0.997), y is absorbance value (OD 410), x is p-NP concentration (mM).
2), with the p-NPG solution of deionized water preparation 25mM as substrate, use tubule packing, pipe external application aluminium foil wraps up lucifuge, is stored in-20 ℃ of refrigerators.
3), in 1.5ml EP pipe, add citric acid-Sodium phosphate dibasic damping fluid of 116 μ l pH4.0 and the p-NPG solution of 14 μ l 25mM, be positioned over temperature and be in the water-bath of 55 ℃; Add again 10 μ l mould FCD3-1 fermented liquid of a resulting wood in embodiment 2,55 ℃ of insulation 15min; Then the Na that adds 70 μ l 0.4M 2cO 3solution, to stop enzymatic reaction, is got 200 μ l mixed solutions, adds 96 hole enzyme plates, and 410nm place measures absorbance value; According to p-NP typical curve and absorbance value, calculate enzyme activity.The definition of beta-glucoside enzyme activity: under certain condition, enzymic hydrolysis p-NPG, it is an enzyme activity unit (U) that every min discharges the required enzyme amount of 1 μ mol p-NP.
Result is that fermented liquid beta-glucoside enzyme activity at pH4.0,55 ℃ of wooden mould FCD3-1 is 1.17 ± 0.022U/ml.
Commercial strain Rui Shi wood mould (Trichoderma reesei) Rut-C30 of production of cellulose enzyme of take is contrast, the Rut-C30 fermented liquid of preparing by same method under same culture condition, under pH4.5, the reaction conditions of 55 ℃, beta-glucoside enzyme activity is 0.07 ± 0.003U/ml.
The mould FCD3-1 fermented liquid of above-mentioned explanation wood is the beta-glucosidase in cellulase, and it is 16.7 times of the beta-glucosidase that produces of Rut-C30 as beta-glucoside enzyme activity.
2, the enzymatic property of the beta-glucosidase of fermented liquid
1), Optimun pH
The mould FCD3-1 fermented liquid of wood is carried out to the detection of beta-glucoside enzyme activity, adopt respectively citric acid-Sodium phosphate dibasic damping fluid (pH 3.0,3.5,4.0,4.5,5.0,5.5,6.0), Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution (pH6.5,7.0,7.5,8.0), glycine-sodium hydrate buffer solution (pH 8.5,9.0,9.5,10.0), temperature of reaction adopts 37 ℃, and beta-glucoside enzyme activity determination is with reference to described methods such as Odoux; Enzyme activity definition: under 37 ℃ of conditions, enzymic hydrolysis p-NPG, it is an enzyme activity unit (U) that every min discharges the required enzyme amount of 1 μ mol p-NP.
The high enzymatic activity of take is 100%, and the enzyme activity of other pH value is enzyme activity with the ratio of high enzymatic activity, take pH value as X-coordinate, and enzyme activity is ordinate zou mapping, sees Fig. 8.Result shows, the Optimun pH of the beta-glucoside enzyme activity of wooden mould FCD3-1 fermented liquid in the time of 37 ℃ is 4.5, pH6.0-7.0, and its enzyme activity declines rapidly, under alkaline pH condition, and its enzyme suppressed or inactivation of living.
2), optimum temperature
The mould FCD3-1 fermented liquid of wood is carried out to the detection of beta-glucoside enzyme activity, adopt respectively different temperature of reaction (30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃), reaction buffer adopts citric acid-Sodium phosphate dibasic damping fluid of pH4.5, and beta-glucoside enzyme activity determination is with reference to described methods such as Odoux; Enzyme activity definition: under pH4.5 condition, enzymic hydrolysis p-NPG, it is an enzyme activity unit (U) that every min discharges the required enzyme amount of 1 μ mol p-NP.
Using high enzymatic activity as 100%, and the enzyme activity of other temperature is enzyme activity with the ratio of high enzymatic activity, take temperature as X-coordinate, and enzyme activity is ordinate zou mapping, sees Fig. 9.Result shows, the optimum temperature of the beta-glucoside enzyme activity of wooden mould FCD3-1 fermented liquid when pH4.5 is 60 ℃.
3), pH tolerance
The mould FCD3-1 fermented liquid of wood is carried out to the detection of beta-glucoside enzyme activity, (pH 3.0 to adopt respectively citric acid-Sodium phosphate dibasic damping fluid, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0), Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution (pH6.5, 7.0, 7.5, 8.0), (pH 8.5 for glycine-sodium hydrate buffer solution, 9.0, 9.5, 10.0), with reference to Eckert and the described method of Schneider (Eckert K, Schneider E.A thermoacidophilic endoglucanase (CelB) from Alicyclobacillus acidocaldarius displays high sequence similarity to arabinofuranosidases belonging to family 51 of glycoside hydrolases.Eur J Biochem.2003, 270:3593-3602), enzyme is stored in the damping fluid of different pH values, in 4 ℃ of placements, after 24 hours, in optimum pH (pH4.5) and the lower enzyme of measuring of optimum temperuture (60 ℃), live.Beta-glucoside enzyme activity determination is with reference to described methods such as Odoux; Enzyme activity definition: under pH4.5,60 ℃ of conditions, enzymic hydrolysis p-NPG, it is an enzyme activity unit (U) that every min discharges the required enzyme amount of 1 μ molp-NP.
The high enzymatic activity of take is 100%, and the enzyme activity that other pH value is preserved liquid is enzyme activity with the ratio of high enzymatic activity, and the pH value of preserving liquid of take is X-coordinate, and enzyme activity is that ordinate zou is mapped, and sees Figure 10.Result shows, the enzyme work of the beta-glucosidase of wooden mould FCD3-1 fermented liquid has good pH tolerance within the scope of pH3.0-9.0, and under the preservation condition higher than pH9.0, enzyme is lived and declined rapidly.
4), temperature tolerance
The mould FCD3-1 fermented liquid of wood is carried out to the detection of beta-glucoside enzyme activity, adopt respectively (30 ℃ of different storage temperatures, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃), with reference to the described methods such as Inoue (Inoue T, Moriya S, Ohkuma M, et al.Molecular cloning and characterization of a cellulase gene from a symbiotic protist of the lower termite, Coptotermes formosanus.Gene.2005, 349:67-75), enzyme is stored under differing temps, place after 1 hour at optimum pH (pH4.5) and the lower enzyme activity of measuring of optimum temperuture (60 ℃).Beta-glucoside enzyme activity determination is with reference to described methods such as Odoux; Enzyme activity definition: under pH4.5,60 ℃ of conditions, enzymic hydrolysis p-NPG, it is an enzyme activity unit (U) that every min discharges the required enzyme amount of 1 μ mol p-NP.
The high enzymatic activity of take is 100%, and the enzyme activity of other storage temperature is enzyme activity with the ratio of high enzymatic activity, take storage temperature as X-coordinate, and enzyme activity is ordinate zou mapping, sees Figure 11.Result shows, the beta-glucosidase enzyme work of wooden mould FCD3-1 fermented liquid has satisfactory stability under 30-60 ℃ of condition.
5), the tolerance of beta-glucosidase under simultaneous saccharification and fermentation condition
It is under pH4.0,30 ℃ of conditions that the mould FCD3-1 fermented liquid of wood is stored in to simultaneous saccharification and fermentation condition, place some hours, at optimum pH (pH4.5) and the lower beta-glucoside enzyme activity of measuring of optimum temperuture (60 ℃), to observe the stability of this enzyme under simultaneous saccharification and fermentation condition.Beta-glucoside enzyme activity determination is with reference to described methods such as Odoux; Enzyme activity definition: under pH4.5,60 ℃ of conditions, enzymic hydrolysis p-NPG, it is an enzyme activity unit (U) that every min discharges the required enzyme amount of 1 μ mol p-NP.
The high enzymatic activity of take is 100%, and other enzyme activity is enzyme activity with the ratio of high enzymatic activity, take the shelf time as X-coordinate, and enzyme activity is ordinate zou mapping, sees Figure 12.Result shows, the beta-glucosidase enzyme work of wooden mould FCD3-1 fermented liquid is good at simultaneous saccharification and fermentation condition stability inferior, places and within 10 days, still can retain higher enzyme activity in 240 hours under pH4.0,30 ℃ of conditions.

Claims (10)

  1. Wood mould (Trichoderma koningiopsis) bacterial strain FCD3-1, its deposit number is CGMCC No.6050.
  2. 2. the application of mould (Trichoderma koningiopsis) the bacterial strain FCD3-1 of wood claimed in claim 1 in preparing cellulase or cellulase preparation.
  3. 3. a method for production of cellulose enzyme, is fermentation wood claimed in claim 1 mould (Trichoderma koningiopsis) bacterial strain FCD3-1, collects tunning, obtains cellulase.
  4. 4. method as claimed in claim 3, is characterized in that: the condition of described fermentation is for cultivating 5-7 days at 26-32 ℃, 180rpm.
  5. 5. method as claimed in claim 4, is characterized in that: the condition of described fermentation is for cultivating 5 days at 28 ℃, 180rpm.
  6. 6. as the method as described in arbitrary in claim 3-5, it is characterized in that:
    The component of the fermention medium that described fermentation adopts is as follows: described in every L, fermention medium is by 2.0g KH 2pO 4, 1.4g (NH 4) 2sO 4, 0.3g urea, 0.3g MgSO 47H 2o, 0.3g CaCl 2, 5.0mg FeSO 47H 2o, 1.56mg MnSO 4h 2o, 1.4mg ZnSO 47H 2o, 2.0mg CoCl 2, 2ml Tween-80,1.0g peptone, 20g wheat bran, 10g crystalline cellulose and water forms, water is supplied volume;
    The pH value of described fermention medium is pH4.0-6.0.
  7. 7. method as claimed in claim 6, is characterized in that:
    Described fermentation is cultivated for the spore solution of wood claimed in claim 1 mould (Trichoderma koningiopsis) FCD3-1 is seeded in described fermention medium;
    Described spore solution is for to be suspended in by the spore of wood claimed in claim 1 mould (Trichoderma koningiopsis) FCD3-1 the solution obtaining in aseptic deionized water.
  8. 8. method as claimed in claim 7, is characterized in that: after described collection tunning, also comprise the steps: described tunning centrifugally, collect supernatant liquor, obtain cellulase.
  9. 9. a cellulase preparation, its activeconstituents is the tunning of wood claimed in claim 1 mould (Trichoderma koningiopsis) bacterial strain FCD3-1.
  10. Described in claim 9 cellulase preparation at hydrolysis of lignocellulose or prepare the application in glucose.
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