CN102787104A - High-activity composite cellulase and preparation thereof, and application method for same in enzymatic saccharification of wood fiber - Google Patents

High-activity composite cellulase and preparation thereof, and application method for same in enzymatic saccharification of wood fiber Download PDF

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CN102787104A
CN102787104A CN2012102600792A CN201210260079A CN102787104A CN 102787104 A CN102787104 A CN 102787104A CN 2012102600792 A CN2012102600792 A CN 2012102600792A CN 201210260079 A CN201210260079 A CN 201210260079A CN 102787104 A CN102787104 A CN 102787104A
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beta
glucosidase
enzyme
cellulase
enzymatic saccharification
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陈介南
詹鹏
张�林
王义强
陈石兰
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CHANGSHA LUEN MEI BIOLOGICAL TECHNOLOGY Co Ltd
Central South University of Forestry and Technology
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Central South University of Forestry and Technology
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Abstract

The invention discloses a high-activity composite cellulase and preparation thereof, and an application method for the same in enzymatic saccharification of wood fiber. According to the invention, Eupenicillium javanicum ZN-205 is subjected to shake-flask fermentation so as to produce beta-glucosidase; best enzyme production conditions are that an initial pH value is 6, the concentration of peptone is 0.75%, the concentration of microcrystalline cellulose is 2.5%, the addition amount of tween-80 is 0.05%, culture temperature is 28 DEG C, the volume of liquid filled in a 250 ml triangular flask is 100 ml, a revolving speed of a shaker is 175 r/min, inoculum amount is 5% and the highest activity of beta-glucosidase is 2.312 IU/ml; beta-glucosidase prepared from Eupenicillium javanicum ZN-205 and cellulase prepared from Trichoderma reesei Rut C-30 are compounded, and an obtained optimal ratio of beta-glucosidase activity to filter paper activity of 1.4. Utilization of the compounded enzyme for enzymatic saccharification of wood fiber enables high-efficiency and low-cost enzymatic saccharification effects to be achieved.

Description

A kind of high reactivity complex cellulase and preparation thereof and the application method in the wood fibre enzymatic saccharification
Technical field
The present invention relates to prozyme of the high cellulase-producing preparation of a kind of fungi and preparation method thereof, and its application in the wood fibre enzymatic saccharification.
Background technology
Owing to can alleviate energy shortage, reduce environmental pollution, promote rural development, avoid vital role such as " striving grain with the people; strive ground " with farming; Utilize the abundant lignocellulosic plants resource of nature to produce concern and the support that alcohol fuel more and more receives society, cellulose fuel ethanol has become the important component part of renewable energy source.Generally include to the alcoholic acid biotransformation from lignocellulose: 1. lignocellulosic material pre-treatment; 2. the production of cellulase; 3. cellulosic hydrolysis and saccharification; 4. the fermentation of cellulose hydrolysis product (hexose) and the 5. fermentation of hydrolysis of hemicellulose product (pentose).
In the cellulosic ethanol production process, the cost of cellulase has accounted for very big ratio, even has reached the over half of whole cost.Cellulase is to utilize the bacterial strain of cellulase-producing to extract the refining liquid compound enzymic preparation that forms by fermentation, comprises inscribe beta-glucanase, circumscribed beta-glucanase and three kinds of enzyme systems of beta-glucosidase.At present, Trichodermareesei is the bacterial classification of a kind of outstanding cellulase-producing of generally acknowledging, but the activity of beta-glucosidase in the cellulase of its generation is lower, causes the cellobiose accumulation, has influenced enzymatic saccharification efficient, and then has reduced the alcoholic acid yield.Carry out the application that high active cellulase is produced and enzyme system optimizes in the wood fibre enzymatic saccharification wood fibre fuel ethanol production is had important more practical value.
Summary of the invention
The technical problem that the present invention will solve is the deficiency of customer service prior art; High reactivity complex cellulase of a kind of beta-glucosidase preparation by Java penicillium high yield and preparation method thereof is provided, and to carry out prozyme be its application in the enzymatic saccharification wood fibre of optimization implementation.
The objective of the invention is to realize in the following manner:
A kind of high reactivity complex cellulase: described complex cellulase be the beta-glucosidase produced by Java penicillium ZN-205 CCTCC NO:M208204 with the cellulase of Trichodermareesei Rut C-30 ATCC 56765 productions (method of Trichodermareesei Rut C-30 ATCC 56765 production of cellulose enzymes see reference document 1) through concentration; Enzyme liquid after concentrating mixed obtaining, the beta-glucosidase works/PFA enzyme work optimum ratio of the complex cellulase that obtains is 1.4;
The process that described Java penicillium ZN-205 CCTCC NO:M208204 produces beta-glucosidase is following:
Picking one ring Java penicillium ZN-205 CCTCC NO:M208204 spore inoculating shakes in the bottle in seed culture medium, cultivates on the shaking table as inoculum; By shaking 5% of bottled liquid measure volume, above-mentioned inoculum is inoculated in the 250ml that 100ml product enzyme substratum is housed shakes in the bottle, 28 ℃, cultivated 5 days under the 175rpm condition; Centrifugal, supernatant is beta-glucosidase;
Described Java penicillium ZN-205 CCTCC NO:M208204 produces the enzyme substratum: initial pH is 6.0; The Microcrystalline Cellulose mass concentration is 2.5%, peptone mass concentration 0.75%, KH 2PO 42g/L, CaCl 22H 2O 0.4g/L, MgSO 47H 2O 0.02g/L, Mandels microelement concentrate 1ml/L, 1mol/L citrate buffer solution 50ml/L; The interpolation mass percent of tween-80 is 0.05%, adds water and is mixed with 1L;
Wherein, Mandels microelement concentrate: FeSO 47H 2O 5g/L, MnSO 4H 2O 1.6g/L, ZnSO 47H 2O 1.4g/L, CoCl 26H 2O 3.7g/l adds water and is mixed with 1L;
1mol/l citrate buffer solution: Hydrocerol A (C 6H 8O 7H 2O 210g), H 2O 750ml adds NaOH 78g, and it is 4.2 that pH is surveyed in the cooling back, adds water to 1000ml, and pH is 4.45.
Described Java penicillium ZN-205 CCTCC NO:M208204 seed culture medium: glucose 20g/L; Peptone 1g/L, Mandels nutritive salt liquid concentrator 100ml/L, Mandels microelement concentrate 1ml/L; 1M citrate buffer solution 50ml/L adds water and is mixed with 1L;
Wherein, Mandels nutritive salt liquid concentrator: (NH 4) 2SO 414g/L, (NH 4) 2CO 33g/L, KH 2PO 420g/L, CaCl 22H 2O 4g/L, MgSO 47H 2O 0.2g/L adds water and is mixed with 1L;
Mandels microelement concentrate: FeSO 47H 2O 5g/L, MnSO 4H 2O 1.6g/L, ZnSO 47H 2O 1.4g/L, CoCl 26H 2O 3.7g/L adds water and is mixed with 1L;
1mol/l citrate buffer solution: Hydrocerol A (C 6H 8O 7H 2O) 210g, H 2O 750ml adds NaOH 78g, and it is 4.2 that pH is surveyed in the cooling back, adds water to 1000ml, and pH is 4.45.
The above-mentioned Java penicillium ZN-205 CCTCC NO:M208204 spore inoculating that utilizes is following in the detailed process that seed culture medium is cultivated: picking one ring spore inoculating shakes in the bottle in the 250ml of dress liquid 50ml seed culture medium from the Java penicillium ZN-205 CCTCC NO:M208204 solid plate substratum of preserving; 28 ℃, cultivate 2.5 ~ 3 days on the 200rpm shaking table as inoculum.
Under 4000rpm, centrifugal 10min obtains supernatant after the described Java penicillium ZN-205 CCTCC NO:M208204 process product enzyme culture medium culturing.
A kind of high reactivity complex cellulase is to be prepared by above-mentioned method.
Above-mentioned high reactivity complex cellulase is used for the enzymatic saccharification lignocellulose; Specifically can the pretreated poplar of enzymolysis steam explosion.
The mass concentration that the pretreated poplar of described steam explosion accounts for described complex cellulase liquid is 5%,, enzymatic saccharification uses the enzyme amount to be the 15FPU/g poplar, and the enzymatic saccharification time is 48h.
The pretreated poplar of described steam explosion is in the explosion reactor drum of steam blasting device, to load the poplar sheet; Open vapour generator, steam can feed the explosion reactor drum after arriving 210 ℃ of preset temps; Keep pressure under the 3MPa vapor pressure 20 minutes, and utilized the chuck on the reactor drum to heat simultaneously; Abrupt release pressure, explosion is accomplished, and is then that the oven dry of explosion liquid is subsequent use to anhydrous collection.
Compared with prior art; The invention has the advantages that: produce the beta-glucosidase working condition through optimizing Java penicillium ZN-205; Can make the beta-glucosidase that activity is high, cost is low; It mixes with the cellulase that Trichodermareesei Ru-C30 produces again, optimizes the optimum enzyme that obtains both proportioning alive, is used for efficient enzymolysis lignocellulose.Though the beta-glucosidase enzyme is lived low in the cellulase that Trichodermareesei Ru-C30 produces; But it also comprises inscribe beta-glucanase and the circumscribed beta-glucanase that plays an important role, so the active high beta-glucoside endonuclease capable that Java penicillium ZN-205 makes remedies the defective of the cellulase of Trichodermareesei Ru-C30 production, both are complementary.
Application of the present invention possibly open up new efficiently, enzymolysis lignocellulose technology cheaply, produce alcohol fuel for the lignocellulose fermentation technical guarantee be provided.
The used Java of the present invention penicillium (Eupenicillium javanicum) ZN-205; Be preserved in the preservation strain at Chinese typical culture collection center for autonomous seed selection and on November 6th, 2008; Preserving number is: CCTCC NO:M208204, and this bacterial strain of patent applied for protection (license notification number: CN101463327B).
Trichodermareesei Ru-C30 buys from China National Academy of Food & Fermentation Industries, is numbered ATCC56765 (it is the product of agency's U.S. ATCC (Biological resources center)).
Description of drawings
Poplar before and after Fig. 1 steam explosion is handled;
Fig. 2 complex cellulase is an enzymatic saccharification poplar effect;
Produce enzyme activity in Fig. 3 fermentor tank;
Fig. 4 complex cellulase is the experiment of enzymatic saccharification wood fibre fermentor tank.
Embodiment
Be intended to further specify the present invention below in conjunction with embodiment, and unrestricted the present invention.
Embodiment 1
Picking one ring spore inoculating shakes in the bottle in the 250ml of dress liquid 50ml seed culture medium from the Java penicillium ZN-205 potato solid plate substratum that preserve in the laboratory, 28 ℃, cultivates 2.5 ~ 3 days on the 200rpm shaking table as inoculum.By 5% (v/v) inoculum size, above-mentioned culture is inoculated in the 250ml that 50ml product enzyme substratum is housed shakes in the bottle, 28 ℃, cultivated 5 days under the 200rpm condition.Under the 4000rpm, centrifugal 10min, supernatant is crude enzyme liquid.Measure its beta-glucosidase 1.521IU/ml of being alive.
Java penicillium ZN-205 seed culture medium: glucose 20g/L, peptone 1g/L, Mandels nutritive salt liquid concentrator 100ml/L, Mandels microelement concentrate 1ml/L, 1M citrate buffer solution 50ml/L adds water to 1L.
Java penicillium ZN-205 produces the enzyme substratum: Microcrystalline Cellulose 25g/L, peptone 5g/L, KH 2PO 42g/L, CaCl 22H 2O 0.4g/L, MgSO 47H 2O 0.02g/L, Mandels microelement concentrate 1ml/L, 1mol/L citrate buffer solution 50ml/l adds water to 1L.
Wherein, Mandels nutritive salt liquid concentrator: (NH 4) 2SO 414g/L, (NH 4) 2CO 33g/L, KH 2PO 420g/L, CaCl 22H 2O 4g/L, MgSO 47H 2O 0.2g/L;
Mandels microelement concentrate: FeSO 47H 2O 5g/L, MnSO 4H 2O 1.6g/L, ZnSO 47H 2O 1.4g/L, CoCl 26H 2O 3.7g/L;
1mol/L citrate buffer solution: Hydrocerol A (C 6H 8O 7H 2O) 210g, H 2O 750ml adds NaOH 78g, and the cooling back is surveyed pH and is about 4.2, adds water to 1000ml, and pH is about 4.45.
The invention provides the measuring method of beta-glucoside enzyme activity, specific as follows:
Get the saligenin (being dissolved in the 0.05mol/L pH=4.8 citrate buffer solution) of 1.0ml 1.0%; Add the suitably enzyme liquid of dilution of 0.5ml, 50 ℃ of water bath heat preservation 30min add 3ml DNS solution; In boiling water, boil 10min; The cooling back is settled to 25ml with adding distil water, shakes up, and be to measure absorbancy under the 540nm at the spectrophotometer wavelength.Press same method as blank with the enzyme liquid of heat treated.The definition of enzyme activity: under these conditions, 1ml enzyme liquid makes substrate produce the reducing sugar amount of 1 μ mol glucose in 1min, is 1 enzyme activity unit, representes with IU/ml.
Figure BDA00001910322500041
Wherein, m is glucose content (mg), and n is the extension rate of crude enzyme liquid.
Embodiment 2
The present invention adopts the experiment of single factor design that penicillium ZN-205 in Java among the embodiment 1 is produced the beta-glucoside enzymatic process and proceeds to optimize, and is specific as follows.
Produce enzyme substratum single factor experiment: keep producing other components unchanged in the enzyme liquid nutrient medium; Change carbon source, nitrogenous source, pH value and these four factors (table 1) of tensio-active agent (tween-80) respectively, filter out the single factor that Java penicillium ZN-205 produces the best product enzyme substratum of beta-glucosidase that influences.Each factor to the size order of the influence of activity of beta-glucosidase is: carbon source＞nitrogenous source＞tensio-active agent＞initial pH.Draw optimum combination from test-results (table 2): initial pH is 6.0, and the nitrogenous source mass concentration is 0.75%, and the carbon source mass concentration is 2.5%, and the addition of tween-80 is 0.05wt%.
Table 1 produces enzyme substratum orthogonal experimental design
Figure BDA00001910322500042
Table 2 produces enzyme substratum orthogonal experimental design result
Figure BDA00001910322500052
Produce enzyme external conditions single factor experiment: keep producing enzyme liquid nutrient medium components unchanged; Change culture temperature, inoculum size, liquid amount and shaking speed (table 3) respectively, filtering out influences the best product enzyme external conditions that Java penicillium ZN-205 produces beta-glucosidase.Each factor to the size order of the influence of activity of beta-glucosidase is: culture temperature＞inoculum size＞liquid amount＞shaking speed (table 4).
Table 3 produces enzyme external conditions orthogonal experimental design
Figure BDA00001910322500053
Table 4 produces enzyme external conditions orthogonal experimental design result
Figure BDA00001910322500054
Figure BDA00001910322500061
Draw optimum combination from product enzyme substratum orthogonal test, external conditions orthogonal test: initial pH is 6.0; The peptone mass concentration is 0.75%, and the Microcrystalline Cellulose mass concentration is 2.5%, and the addition of tween-80 is 0.05wt%; Culture temperature is 28 ℃; 250ml triangular flask liquid amount is 100ml, and shaking speed is 175r/min, and inoculum size is 5%.
According to above optimum combination, all the other conditions are carried out the test of beta-glucosidase shake flask fermentation with operation with embodiment 1, and the vigor of the beta-glucosidase of the 5d that must ferment is 2.312IU/ml.
The positive blue or green enzyme ZN-205 in Java after optimizing and other bacterial strains produce the comparison of beta-glucosidase.
Figure BDA00001910322500062
Embodiment 3
The preparation method of complex cellulase of the present invention, specific as follows:
Utilize the Java penicillium ZN-205 of autonomous seed selection under the optimal conditions of the foregoing description 2, to carry out shake flask fermentation and produce beta-glucosidase, the Trichodermareesei Ru-C30 of purchase (ATCC 56765) prepares cellulase (its method reference 1).Measure beta-glucosidase method alive and adopt method provided by the invention, the method for measuring filter paper enzyme activity (FPA) adopts the method (document 2 sees reference) of Ghose recommendation.The filter paper enzyme activity (FPA) that obtains Trichodermareesei Ru-C30 crude enzyme liquid is 6.212IU/ml, and beta-glucosidase is lived and is 1.352IU/ml; The filter paper enzyme activity (FPA) of enzyme liquid is 46.912IU/ml after the ultrafiltration and concentration appearance (UOF 48) of Tianjin Motimo Membrane Technology Co., Ltd. concentrates 20 minutes; Beta-glucosidase is lived and is 10.662IU/ml, beta-glucosidase work/filter paper enzyme activity (FPA)=0.227.The filter paper enzyme activity (FPA) of Java penicillium ZN-205 crude enzyme liquid is 0.342IU/ml, and beta-glucosidase is lived and is 2.312IU/ml; The filter paper enzyme activity (FPA) of enzyme liquid is 3.245IU/ml after the ultrafiltration and concentration appearance (UOF48) of Tianjin Motimo Membrane Technology Co., Ltd. concentrates 20 minutes; Beta-glucosidase is lived and is 21.598IU/ml, beta-glucosidase work/filter paper enzyme activity (FPA)=6.656.These two kinds of spissated enzymes are carried out the compound proportioning that forms different beta-glucosidase work/filter paper enzyme activities (FPA).
Embodiment 4
The present invention utilizes steam explosion for pretreatment process the enzymatic saccharification substrate to be provided, and is specific as follows: in the explosion reactor drum of steam blasting device (manufacturing of Jiangsu Province Haian Huada Petroleum Instrument Co., Ltd.), load the poplar sheet; Open vapour generator, steam can feed the explosion reactor drum after arriving 210 ℃ of preset temps; 3MPa vapor pressure dimension was down pressed 20 minutes, can utilize the chuck on the reactor drum to heat simultaneously; Abrupt release pressure, explosion is accomplished, and is then that the oven dry of explosion liquid is subsequent use to anhydrous collection.Poplar before and after steam explosion is handled is seen Fig. 1.
Utilize poplar after the complex cellulase enzymatic saccharification steam explosion of the present invention pre-treatment, specific as follows:
Poplar behind the complex cellulase enzymatic saccharification steam explosion of the present invention (mass concentration that poplar accounts for complex cellulase enzyme liquid is 5%).When the enzyme amount is the 15FPU/g poplar; When the ratio of the beta-glucosidase work/filter paper enzyme activity of complex cellulase is respectively 0.3,0.4,0.5,0.6,0.7,0.8,0.9,1.0,1.1,1.2,1.3,1.4,1.5 and 1.6; Enzymolysis 48h measures its reducing sugar amount.Its result such as Fig. 2 when utilizing Trichodermareesei to concentrate enzyme liquid enzymolysis poplar separately, can produce the reducing sugar of 24.90g/l; After adding Java penicillium enzyme liquid in the enzyme liquid at Trichodermareesei, beta-glucosidase work/filter paper enzyme activity ratio increases, and the reducing sugar content that enzymolysis produces increases gradually; But work as ratio and be higher than 1.4, reducing sugar content descends gradually.Therefore the best beta-glucosidase work/PFA enzyme of enzymolysis ratio alive is 1.4.
Embodiment 5
Adopt following condition to realize the positive blue or green enzyme strain fermentation jar high-yield beta-glucosidase in Java: dress liquid 5L in the 7.5L fermentor tank, substratum are the product enzyme substratum after embodiment 2 optimizes, 5% inoculum size; 28 ℃, 200rpm, the pH value keeps 6.0; Ventilation is than 1～0.2vvm; Keep dissolved oxygen 20%～30%, cultivated 5 ~ 6 days, every separated 12h sampling once.During 0～5d, along with the prolongation of fermentation time, enzyme is lived constantly to be increased; When fermenting to 5d, enzyme work is up to 2.201IU/ml; After crossing 5d, enzyme is lived and slowly descended again, and is as shown in Figure 3.Fermentation time is 5d, and the highest enzyme is lived and is 2.201IU/ml.
The beta-glucosidase of above-mentioned fermentor tank preparation mixes the fermentor tank experiment (complex cellulase beta-glucosidase work/filter paper enzyme activity ratio=1.39) of the complex cellulase enzymatic saccharification poplar that obtains with the cellulase of Trichodermareesei Ru-C30 (ATCC 56765) preparation among the embodiment 3.2.5L dress liquid 1.5L in the BioFlo110 fermentor tank, when the mass concentration that accounts for complex cellulase when the poplar behind the substrate steam explosion was 5%, enzyme dosage was the 15FPU/g poplar; Hydrolysis temperature is 50 ℃, and pH is 5.0, measures the variation of different time reducing sugar amount behind the enzymolysis.Can know that by Fig. 4 initial reducing sugar is 2.09g/l; Enzymolysis 48h can produce the reducing sugar of 25.54g/l, and conversion coefficient is 78.18%.
Reference:
1. Ai Bin insults, Wang Yiqiang, Chen Jienan. and wheat bran is to the promoter action [J] of Trichodermareesei RUT C30 cellulase-producing. biomass chemical engineering .2009,43 (2): 27-33.
2.Ghose?T?K.Measurement?of?cellulase?activities?[J].Pure?and?Appl.Chem.,1987,59(2):257-268.

Claims (9)

1. the preparation method of a high reactivity complex cellulase; It is characterized in that; Described complex cellulase be the beta-glucosidase produced by Java penicillium ZN-205 CCTCC NO:M208204 with the cellulase of Trichodermareesei Rut C-30 ATCC 56765 productions through concentration; Enzyme liquid after concentrating mixed obtaining, the beta-glucosidase works/PFA enzyme work ratio of the complex cellulase that obtains is 1.4;
The process that described Java penicillium ZN-205 CCTCC NO:M208204 produces beta-glucosidase is following:
Picking one ring Java penicillium ZN-205 CCTCC NO:M208204 spore inoculating shakes in the bottle in seed culture medium, cultivates on the shaking table as inoculum; By shaking 5% of bottled liquid measure volume, above-mentioned inoculum is inoculated in the 250ml that 100ml product enzyme substratum is housed shakes in the bottle, 28 ℃, cultivated 5 days under the 175rpm condition; Centrifugal, supernatant is beta-glucosidase;
Described Java penicillium ZN-205 CCTCC NO:M208204 produces the enzyme substratum: initial pH is 6.0; The Microcrystalline Cellulose mass concentration is 2.5%, peptone mass concentration 0.75%, KH 2PO 42g/L, CaCl 22H 2O 0.4g/L, MgSO 47H 2O 0.02g/L, Mandels microelement concentrate 1ml/L, 1mol/L citrate buffer solution 50ml/L; The interpolation mass percent of tween-80 is 0.05%, adds water and is mixed with 1L;
Wherein, Mandels microelement concentrate: FeSO 47H 2O 5g/L, MnSO 4H 2O 1.6g/L, ZnSO 47H 2O 1.4g/L, CoCl 26H 2O 3.7g/L adds water and is mixed with 1L;
1mol/L citrate buffer solution: C 6H 8O 7H 2O 210g, H 2O 750ml adds NaOH 78g, and it is 4.2 that pH is surveyed in the cooling back, adds water to 1000ml, and pH is 4.45.
2. preparation method according to claim 1; It is characterized in that described Java penicillium ZN-205 CCTCCNO:M208204 seed culture medium: glucose 20g/L, peptone 1g/L; Mandels nutritive salt liquid concentrator 100ml/L; Mandels microelement concentrate 1ml/L, 1M citrate buffer solution 50ml/L adds water and is mixed with 1L;
Wherein, Mandels nutritive salt liquid concentrator: (NH 4) 2SO 414g/L, (NH 4) 2CO 33g/L, KH 2PO 420g/L, CaCl 22H 2O 4g/L, MgSO 47H 2O 0.2g/L adds water and is mixed with 1L;
Mandels microelement concentrate: FeSO 47H 2O 5g/L, MnSO 4H 2O 1.6g/L, ZnSO 47H 2O 1.4g/L, CoCl 26H 2O 3.7g/L adds water and is mixed with 1L;
1mol/L citrate buffer solution: C 6H 8O 7H 2O 210g, H 2O 750ml adds NaOH 78g, and it is 4.2 that pH is surveyed in the cooling back, adds water to 1000ml, and pH is 4.45.
3. preparation method according to claim 1; It is characterized in that; Picking one ring spore inoculating shakes in the bottle in the 250ml of dress liquid 50ml seed culture medium from the Java penicillium ZN-205 CCTCC NO:M208204 solid plate substratum of preserving; 28 ℃, cultivate 2.5 ~ 3 days on the 200rpm shaking table as inoculum.
4. preparation method according to claim 1 is characterized in that, under 4000rpm, centrifugal 10min obtains supernatant after the described Java penicillium ZN-205 CCTCC NO:M208204 process product enzyme culture medium culturing.
5. a high reactivity complex cellulase is characterized in that, is prepared by each described method of claim 1-4.
6. the application method of the described high reactivity complex cellulase of claim 5 is characterized in that, adopts described complex cellulase enzymatic saccharification lignocellulose.
7. application method according to claim 6 is characterized in that, adopts the pretreated poplar of described complex cellulase enzymolysis steam explosion.
8. application method according to claim 7 is characterized in that, the mass concentration that the pretreated poplar of described steam explosion accounts for described complex cellulase liquid is 5%, and it is 15 FPU/g poplars that enzymatic saccharification uses the enzyme amount, and the enzymatic saccharification time is 48h.
9. application method according to claim 7 is characterized in that, the pretreated poplar of described steam explosion is in the explosion reactor drum of steam blasting device, to load the poplar sheet; Open vapour generator, steam can feed the explosion reactor drum after arriving 210 ℃ of preset temps; Kept pressure under the 3MPa vapor pressure 20 minutes, abrupt release pressure, explosion is accomplished, and is then that the oven dry of explosion liquid is subsequent use to anhydrous collection.
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CN104293861A (en) * 2014-09-10 2015-01-21 中南林业科技大学 Method for improving enzymatic saccharification of wood fibre by combination of dilute phosphoric acid and steam explosion pretreatment
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CN114032227A (en) * 2021-09-16 2022-02-11 厦门大学 Composite cellulase for saccharification of lignocellulose and preparation method thereof

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