CN105779301A - Trichoderma reesei as well as culture method thereof and application thereof - Google Patents
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Abstract
The invention discloses trichoderma reesei as well as a culture method thereof and application thereof. Trichoderma reesei T1 has been preserved in China Center for Type Culture Collection at Luojiashan, Wuchang, Wuhan on December 30, 2015 with a preservation number as CCTCC NO:M2015804. The invention further relates to a culture method and application of the bacterial strain. When the bacterial strain is fermented, a fungal cellulose mixture containing a great deal of cellulose complex can be produced; and the content of the cellulose complex contained in the fungal cellulose mixture obtained by fermentation is remarkably higher than that of the cellulose complex contained in the fungal cellulose mixture obtained by fermentation of original trichoderma reesei QM6a, and therefore, the trichoderma reesei has a wide application prospect in the industry of industrially producing cellulose.
Description
Technical field
The present invention relates to a strain trichoderma reesei and cultural method thereof and application, particularly to the trichoderma reesei QM6a of a plant mutant and cultural method thereof and the application in producing cellulase complex, belong to technical field of biotechnology.
Background technology
The energy, Environmental and resource issue are the Main Bottlenecks of 21 century restriction Sustainable Socioeconomic Development.The whole world fossil feedstock resource based on oil reduces rapidly, and demand and the interdependency of oil are improved constantly by China's economic.Simultaneously as a large amount of discharges of the gases such as the carbon dioxide consuming fossil energy in a large number and causing also make " greenhouse effect " to aggravate rapidly.The atmospheric pollution in one line city is carrying out the conversion to automobile type of the bituminous coal type.Therefore, alcohol fuel receives much attention as the regenerative resource that can replace oil.But, the production of first generation alcohol fuel with starch based grain and sucrose etc. can food crop for raw material, cause the social problem of " striving grain with people ".Cellulose accounts for the 35～50% of plant dry weight, is be distributed the carbohydrate the widest, content is the abundantest on the earth.Utilize modern biotechnology that the ligocellulose degradation of non-grain is converted into the liquid fuels such as ethanol and bulk chemical; can effectively alleviate that energy resources are in short supply, open up new growth engines, the mode of accelerating economic development changes, the post that creates more jobs, promote agriculture-countryside-farmer's development, preserve the ecological environment that the sustainable development realizing China's economy is had highly important strategic importance.
But, cellulosic ethanol production technology also exists many bottleneck problems and not yet solves, cause great obstacle to the development of lignocellulose ethanol industry.Lignocellulose in sponge is mainly made up of cellulose, hemicellulose and lignin, and these macromolecules be combined with each other, and forms the natural cover for defense being difficult to be degraded.Therefore, firstly the need of adopting effective physical chemistry pretreatment to break this barrier in lignocellulose alcohol production, then utilize cellulase that pretreated ligocellulose degradation is become fermentability monosaccharide, finally fermentability monosaccharide is changed into the liquid fuels such as ethanol.Cellulase used in degradation process is relatively costly, and cellulase production speed is relatively slow, and this is an important bottleneck of restriction cellulosic ethanol large-scale industrial production.For this, USDOE puts into the fund of billions of dollars, is used for improving yield of cellulase, reduces the relevant R&D work of cellulase cost.
Additionally, cellulase is all used widely in food, weaving, papermaking, feed industry, and remarkable in economical benefits, the yield of cellulase preparation increases year by year, and the current global gross output value, more than 100,000,000 dollars, becomes a big enzyme in Enzymes Industry.
Producing in the microorganism of cellulase, filamentous fungi has plurality of advantages: cellulase produced by it is exoenzyme, it is simple to the separation of enzyme and extraction；Produce enzyme efficiency higher, and the enzyme architecture producing cellulase is relatively reasonable；Many hemicellulases, pectase, amylase etc. can be produced simultaneously.Therefore from cellulase preparation of industrialization and application angle, filamentous fungi, the lignocellulolyticenzymes superior strain trichoderma reesei especially generally acknowledged, there is high researching value.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, it is provided that a strain trichoderma reesei and cultural method thereof and application.
Summary of the invention
The present invention passes through physico-chemical process mutation trichoderma reesei QM6a (ATCC), obtain the mutant T1 containing a large amount of born of the same parents' outer fiber element combined enzyme agents, and provide a kind of use mixing carbohydrate cultivation above-mentioned bacterial strains, to produce the fermentation process of the cellulase mixture containing a large amount of cellulase complexs.The cellulase mixture obtained comprises a large amount of cellulase complex, complex has played important function in ligocellulose degradation, the enzyme system low with complex content of enzyme system containing a large amount of cellulase complexs compares, and has the soluble sugar yield such as higher ligocellulose degradation's efficiency and glucose, cellobiose, xylose and 1,4-.beta.-Xylobiose.
Detailed Description Of The Invention
Technical solution of the present invention is as follows:
One strain trichoderma reesei (Trichodermareesei) T1, is preserved in China typical culture collection center (CCTCC), Luo Jia Shan, wuchang, wuhan, address, culture presevation numbering CCTCCNO:M2015804 on the 30th in December in 2015.
This bacterial strain is trichoderma reesei wild strain QM6a after repeatedly mutation improves, the modified form bacterial strain that protein secretion and ligocellulose degradation's efficiency are greatly promoted, and biology and genetics characteristics are stable.
The cultural method of above-mentioned trichoderma reesei (Trichodermareesei) T1, step is as follows:
(1) trichoderma reesei (Trichodermareesei) T1 is inoculated in plating medium, activation culture 68～76h at 28～32 DEG C, prepare activated strains；
(2) activated strains that step (1) prepares is inoculated in fluid medium, at 28～32 DEG C, cultivates 68～76h under 150～250rpm shaking table condition of culture, prepare liquid seeds；
(3) by step (2) prepare liquid seeds by 1:(4～6) volume ratio be inoculated in fluid medium, at 28～32 DEG C, cultivate 68～76h under 150～250rpm shaking table condition of culture, prepare trichoderma reesei (Trichodermareesei) T1 bacterium solution.
According to currently preferred, the plating medium in described step (1) is wheat bran plating medium, and preparation method is as follows:
Every 10g wheat bran adds in 500mL distilled water, boils 30min, and after 8 layers of filtered through gauze, adding mass percent in filtrate is the agar powder of 2%, 121 DEG C of high pressure steam sterilization 30min.
According to currently preferred, in described step (2) and (3), every liter of fluid medium component is as follows:
15～25g wheat bran, 15～25g microcrystalline Cellulose, 8～12g (NH3)2SO4, 12～18gKH2PO4, 0.4～0.8gMgSO4, 0.6～1.0gCaCl2, 0.004～0.006gFeSO4·7H2O, 0.0015～0.0017gMnSO4·H2O, 0.0013～0.0015gZnSO4·7H2O。
Above-mentioned trichoderma reesei (Trichodermareesei) T1 application in producing cellulase complex, step is as follows:
I trichoderma reesei (Trichodermareesei) T1 bacterium solution is inoculated in fermentation medium by (), at 28～32 DEG C, cultivate 68～76h, prepare fermentation liquid under 150～250rpm shaking table condition of culture；
(ii) by fermentation liquor solid-liquid separation prepared for step (i), taking supernatant, then rotary evaporation concentration volume is to 10%, and secondary solid-liquid separation takes supernatant, concentrates volume to 1% through super filter tube, prepares cellulase complex.
According to currently preferred, in described step (i), every liter of fermentation medium component is as follows:
15～25g wheat bran, 15～25g microcrystalline Cellulose, 8～12g (NH3)2SO4, 12～18gKH2PO4, 0.4～0.8gMgSO4, 0.6～1.0gCaCl2, 0.004～0.006gFeSO4·7H2O, 0.0015～0.0017gMnSO4·H2O, 0.0013～0.0015gZnSO4·7H2O。
According to currently preferred, in described step (ii), solid-liquid separation is the centrifugal 10～15min of 8000～10000rpm.
According to currently preferred, in described step (ii), secondary solid-liquid separation is the centrifugal 25～35min of 35000～40000rpm.
According to currently preferred, in described step (ii), super filter tube concentration is the super filter tube concentration 550～650min of 3kDa.
The application in degraded cellulose of the above-mentioned cellulase complex, step is as follows:
Compound concentration is the cellulose solution of 1～200g/L, then according to the ratio of every gram of cellulose interpolation 0.1～100mg albumen adds cellulase complex fermentation liquid, react 4～120 hours when temperature 30 DEG C～60 DEG C, pH3.5～7.0, obtain catabolite.
Beneficial effect
First passage of the present invention sudden change obtains trichoderma reesei (Trichodermareesei) T1 bacterial strain, this bacterial strain, when fermenting the culture medium containing hemicellulose source carbohydrate (HDC) and the carbohydrate source (such as wheat bran and microcrystalline Cellulose) of the carbohydrate (CIC) of cellulase induction, can produce the fungin enzymatic mixture containing a large amount of cellulase complexs；The content of the cellulase mixture cellulase complex that trichoderma reesei (Trichodermareesei) T1 strain fermentation obtains is significantly higher than the content of the cellulase mixture cellulase complex that original trichoderma reesei (Trichodermareesei) QM6a fermentation obtains, in industrialized production cellulase industry, have broad application prospects.
Accompanying drawing explanation
The electrophoretogram of extracellular protein composition detection after trichoderma reesei QM6a and trichoderma reesei T1 under Fig. 1, same culture conditions；
Wherein: represent respectively in trichoderma reesei in born of the same parents' outer fiber element combined enzyme agent three kinds of I, II, III are dissimilar；
The electrophoretogram of extracellular protein composition detection after Fig. 2, employing distinct methods cultivation trichoderma reesei T1；
Fig. 3, four kinds of enzyme liquid that the method for comparative example obtains are adopted to carry out the block diagram of saccharifying measuring for four kinds of substrates；
Four kinds of enzyme liquid that Fig. 4, the present invention obtain carry out the block diagram of saccharifying measuring for four kinds of substrates；
The block diagram that Fig. 5, trichoderma reesei QM6a contrast with trichoderma reesei T1 extracellular protein yield and filter paper enzyme activity (FPA)；
Detailed description of the invention
By the examples below technical scheme being further elaborated, it should explanation, protection scope of the present invention is not limited only to this.
Trichoderma reesei (trichodermareesei) QM6a purchased from American standard biological product preservation center, culture presevation ATCCNo.13631；
Detection method
Trichoderma reesei biomass estimation method
Preparatory work of experiment: prepare 10% trichloroacetic acid (TCA) solution and at room temperature store.
1. from the culture fluid of mix homogeneously, draw the bacteria suspension of 1ml in the centrifuge tube of 15ml, add the pure water of 4ml；
Centrifugal 10 minutes of 2.40C, 10000rpm, remove supernatant；
3. in centrifuge tube, add the TCA solution of 1ml10%, fully mix with agitator, then ice bath 3 minutes；
Centrifugal 10 minutes of 4.10000rpm, removes supernatant, then respectively adds the TCA solution of 1ml10%, and agitator fully mixes, ice bath 3 minutes；
5., with the TCA of 10% for comparison, water-bath 30 minutes in boiling water put into by comparison and sample；
6.10000rpm takes supernatant after centrifugal 10 minutes；
7. supernatant surveys OD after suitable dilution (5-10 times)260。
The estimating and measuring method (the existence method of proof of extracellular protein complex) of trichoderma reesei exoenzyme liquid protein molecular weight
One. preparation
A) acrylamide concentrated solution (Acrylamide), buffering Buffer, distilled water, tetramethylethylenediamine (TEMED), 75% glycerol are taken out from refrigerator；
B) use distilled water that gradient mixer is rinsed, and side opening adds rotor to the right；
C) offset plate is installed, clip with clip；
D) Ammonium persulfate. (APS) (weighing in 1.5mL centrifuge tube) weighing 0.05g adds the dissolving of 1mL distilled water.
Two. join separation gel
A) preparation (APS and TEMED adds before encapsulating) of glue it is easily separated according to following table
| Resolving gel concentration | 6% | 12% | Concentration glue |
| Acrylamide(mL) | 0.42 | 0.85165 | 0.242 |
| Buffering Buffer (mL) | 1.165 | 1.165 | 1 |
| 75% glycerol (mL) | — | 0.933 | — |
| Distilled water (mL) | 1.9133 | 0.54833 | 1.72 |
| TEMED(μL) | 3.5 | 3.5 | 12 |
| APS(μL) | 17.5 | 17.5 | 64 |
| Cumulative volume (mL) | 3.5 | 3.5 | 3 |
Three. fill separation gel
A) close the central flowing valve of the gradient mixer after rinsing and constant flow pump speed is adjusted to 0；
B) side opening adds 6% separation gel to the left, and right ports adds 12% separation gel；
C) open flowing valve, it was observed that after adverse current, valve cuts out, then with 1mL rifle, left side circulation hole is mixed；
D) rotor rotates, and opens constant flow pump simultaneously, and speed is adjusted to 2.5, and end rifle point is inserted offset plate；
E) open flowing valve, make holes separation gel liquid level keep contour as far as possible by regulating spinner velocity；
F) carry out sealing (using 200 μ L rifles) with water after encapsulating and rinse gradient mixer with distilled water；
G) offset plate is put into 4 DEG C of refrigerators, overnight gel；
Four. preparation concentration glue and loading
A) being poured out by the water in offset plate, residual moisture filter paper blots；
B) preparation concentrates glue and uses blue electron gun point to be rapidly injected to offset plate, inserts comb；
C) after waiting 20 minutes, concentration gelling is solid, extracts comb, puts into electrophresis apparatus, adds support offset plate and regulates extruding dynamics；
D) pour negative electrode and anode buffer (negative electrode buffer dyestuff) into, observe offset plate from the side with or without leakage situation (blue decorative pattern)；
E) without leakage, suck negative electrode buffer with yellow rifle point and loading hole is softly rinsed；
F) carrying out loading, each hole can loading 5～15 μ L.
Five. leakage of electricity is swum
A), after loading, 4 DEG C of refrigerators put into by whole electrophoretic apparatus, are plugged electrode, open electrophresis apparatus；
B) electric current is not intended to, voltage is from 50V, electrophoresis time is 30min, every 30min increases 25V, with 5mL rifle sucking-off negative electrode buffer after 100V electrophoresis, and adds the negative electrode buffer without dyestuff, continue to increase voltage, until observing band after the 30min of 250V, if indicia band (bottom) is run to bottom, terminating electrophoresis, if do not run to bottom, 250V continues electrophoresis and arrives bottom to it.
Six. fluorescence staining
A) albumin glue being put into colouration box, add fluorescent dye and adjust liquid 100mL, shaking table shakes 30min, changes adjustment liquid and repeats this step once；
B) outwell adjustment liquid, add 60mL fluorescence dye liquor, incubator overnight；
C) fluorescent dye being poured special waste liquid bottle into, add 100mL and wash color liquid, shaking table shakes 30min；
D) washing color liquid to outwell, and add 100mL distilled water, shaking table shakes 30min.
Result is compared with albumen marker, when result occurring in that more than 440kDa the protein band less than 669kDa, then proves the existence of extracellular protein complex.Its brightness and complex concentration are proportionate, and therefore may be used for the content of the different enzyme liquid mesocomplex of comparison.
Embodiment 1
Trichoderma reesei (trichodermareesei) T1 is that wild-type T. reesei bacterial strain (trichodermareesei) QM6a accidentally obtains diverted via after too much time radioinduction improvement, the modified form bacterial strain that protein secretion and ligocellulose degradation's efficiency are greatly promoted, excellent results is shown in Fig. 5.
The biological property of trichoderma reesei (trichodermareesei) T1:
Filamentous fungus T. reesei (Trichodermareesei) is cellulous eukaryotic microorganisms, the phorozoon of Hypocrea jecorina (Hypocreajecorina), is under the jurisdiction of Moniliales (Moniliales) trichoderma (Penicillium).It for producing the enzyme decomposing different vegetable materials, including cellulase, hemicellulase, protease, amylase etc., has history for many years as industrial strain.
Trichoderma reesei T1 bacterium colony, in the flocculence of wide paving, is originally the smooth mycelia of white dense, and light green product spore Cong Shu district occurs in back edge, and reverse side is colourless.The short lateral branch of conidiophore mycelia, transparent, multi-branched；Stigma doleiform, middle bent；Conidium is oval or elongated, unicellular, transparent, colourless, and wall is smooth, green time in heaps.
The cultural method of above-mentioned trichoderma reesei (Trichodermareesei) T1, step is as follows:
(1) trichoderma reesei (Trichodermareesei) T1 is inoculated in plating medium, activation culture 72h at 30 DEG C, prepare activated strains；
(2) activated strains that step (1) prepares is inoculated in fluid medium, activation culture 72h at 30 DEG C, prepare liquid seeds；
(3) ratio of the liquid seeds 1:5 by volume step (2) prepared is inoculated in fluid medium, activation culture 72h at 30 DEG C, prepares trichoderma reesei (Trichodermareesei) T1 bacterium solution.
In described step (2) (3), fluid medium component is as follows:
20g/L wheat bran, 20g/L microcrystalline Cellulose, 10g/L (NH3)2SO4, 15g/LKH2PO4, 0.6g/LMgSO4, 0.8g/LCaCl2, 0.005g/LFeSO4·7H2O, 0.0016g/LMnSO4·H2O, 0.0014g/LZnSO4·7H2O。
After testing, the cellular protein concentration of trichoderma reesei (Trichodermareesei) T1 bacterium solution is 0.750mg/mL.
Comparative example 1
(1) trichoderma reesei (Trichodermareesei) T1 is inoculated in plating medium, activation culture 72h at 30 DEG C, prepare activated strains；
(2) activated strains that step (1) prepares is inoculated in fluid medium, activation culture 72h at 30 DEG C, prepare liquid seeds；
(3) ratio of the liquid seeds 1:5 by volume step (2) prepared is inoculated in fluid medium, activation culture 72h at 30 DEG C, prepares trichoderma reesei (Trichodermareesei) T1 bacterium solution.
In described step (2) (3), fluid medium component is as follows:
Carbon source, 10g/L (NH3)2SO4, 15g/LKH2PO4, 0.6g/LMgSO4, 0.8g/LCaCl2, 0.005g/LFeSO4·7H2O, 0.0016g/LMnSO4·H2O, 0.0014g/LZnSO4·7H2O。
Above-mentioned carbon source first group is 20g/L cellobiose, and second group is 20g/L lactose.
After testing, first group of cellular protein concentration of trichoderma reesei (Trichodermareesei) T1 bacterium solution is 0.097mg/mL, and second group of cellular protein concentration is 0.332mg/mL.
Embodiment 2
Above-mentioned trichoderma reesei (Trichodermareesei) T1 application in producing cellulase complex, step is as follows:
I trichoderma reesei (Trichodermareesei) the T1 bacterium solution that embodiment 1 prepares is inoculated in fermentation medium by (), cultivate 72h at 30 DEG C of condition bottom fermentations, prepares fermentation liquid；
(ii) by fermentation liquor solid-liquid separation prepared for step (i), take supernatant, be then concentrated into percent by volume 10%, secondary solid-liquid separation, take supernatant, be concentrated into 1% through super filter tube, prepare cellulase complex.
According to currently preferred, in described step (i), every liter of fermentation medium component is as follows:
20g/L wheat bran, 20g/L microcrystalline Cellulose, 10g/L (NH3)2SO4、15g/LKH2PO4、0.6g/LMgSO4、0.8g/LCaCl2、0.005g/LFeSO4·7H2O、0.0016g/LMnSO4·H2O、0.0014g/LZnSO4·7H2O。
According to currently preferred, in described step (ii), solid-liquid separation is the centrifugal 10min of 8000rpm.
According to currently preferred, in described step (ii), secondary solid-liquid separation is the centrifugal 30min of 35000rpm.
According to currently preferred, in described step (ii), super filter tube concentration is the super filter tube concentration 600min of 3kDa.
After testing, the exoenzyme liquid protein content after concentration is 75.05mg/mL；The mass spectrometry results of cellulase complex is as shown in table 1 below (its ratio in complex of enzyme title digitized representation below):
Table 1
Comparative example 2
I trichoderma reesei (Trichodermareesei) T1 bacterium solution in comparative example 1 is inoculated in fermentation medium by (), cultivate 72h at 30 DEG C of condition bottom fermentations, prepares fermentation liquid；
(ii) by fermentation liquor solid-liquid separation prepared for step (i), take supernatant, be then concentrated into percent by volume 10%, secondary solid-liquid separation, take supernatant, be concentrated into 1% through super filter tube, prepare cellulase complex.
In described step (i), fermentation medium component is as follows:
Carbon source, 10g/L (NH3)2SO4, 15g/LKH2PO4, 0.6g/LMgSO4, 0.8g/LCaCl2, 0.005g/LFeSO4·7H2O, 0.0016g/LMnSO4·H2O, 0.0014g/LZnSO4·7H2O。
Above-mentioned carbon source first group is 20g/L cellobiose, and second group is 20g/L lactose.
After testing, after concentration, exoenzyme liquid protein content first group is 9.73mg/mL, and second group is 33.48mg/mL.The mass spectrometry results of first group of cellulase complex is as shown in table 2, and the mass spectrometry results of second group of cellulase complex is as shown in table 3:
Table 2
Table 3
Comparative example 3
I trichoderma reesei (Trichodermareesei) the QM6a bacterium solution that embodiment 1 prepares is inoculated in fermentation medium by (), cultivate 72h at 30 DEG C of condition bottom fermentations, prepares fermentation liquid；
(ii) by fermentation liquor solid-liquid separation prepared for step (i), take supernatant, be then concentrated into percent by volume 10%, secondary solid-liquid separation, take supernatant, be concentrated into 1% through super filter tube, prepare cellulase complex.
In described step (i), fermentation medium component is as follows:
20g/L wheat bran
20g/L microcrystalline Cellulose
10g/L(NH3)2SO4
15g/LKH2PO4
0.6g/LMgSO4
0.8g/LCaCl2
0.005g/LFeSO4·7H2O
0.0016g/LMnSO4·H2O
0.0014g/LZnSO4·7H2O。
After testing, exoenzyme liquid protein content respectively 33.23mg/mL, mass spectrometry results is as shown in table 4.
Table 4
Embodiment 3
The application in degraded cellulose of the above-mentioned cellulase complex, step is as follows:
Prepare the industrial substrates of following four rich cellulose:
Microcrystalline Cellulose, corn cob residue, ball milling corn cob, alkali process corn cob；
Wherein:
It is as follows that alkali processes corn cob preparation method:
Take 20g corn cob (smashing), add in the triangular flask of 5L, then pour the 1MNaOH of 1L into；Heating, to boiling, timing 1h, intermittent stirring, fills into 1MNaOH and is about 300ml, keep fluidized state in process；By 8 layers of filtered through gauze, continuous tap water about 12 hours is to pH close to the pH of tap water simultaneously；The corn cob being trapped on gauze is placed in 45 DEG C of baking ovens and dries (about 48 hours).The material of drying is placed in mortar, grinds a little and namely use.
Ball milling corn cob preparation method
Weigh the corn cob less than or equal to 14g (smashing) and be placed in grinding pot, add 15 mill balls；It is placed in ball mill, 200rpm positive and negative alternate 5min, rotate centrifugal 1h；Corn cob after ball milling is taken out and namely uses.
Compound concentration is the cellulose solution of 1～200g/L, then according to every gram of cellulose adds the cellulase complex fermentation liquid that the ratio addition embodiment 2 of 0.1～100mg albumen prepares, react 4～120 hours when temperature 30 DEG C～60 DEG C, pH3.5～7.0, obtain catabolite.
When waiting albumen, use the enzyme liquor ratio relatively saccharifying obtained in embodiment 2 and comparative example 2, comparative example 3 and result is compared.
1, according to etc. the method for albumen carry out saccharifying experiment, rotating and culturing under 45 DEG C of conditions；
2, to sample respectively at 6h, 12h, 24h, 48h point in time sampling, then sample is centrifuged, dilution；
3, the sample after saccharifying uses HPLC to measure the concentration of glucose etc., and measurement result asks for an interview Fig. 3, Fig. 4.
Every ligocellulose degradation relative specific enzyme live data of cellulase mixture is as shown in table 5:
Table 5
Relative analysis
By the Data Comparison of embodiment 1 and comparative example 1 it can be seen that the culture medium used in embodiment 1 can be effectively increased the extracellular protein yield of trichoderma reesei T1.
Be can be seen that with the Data Comparison with comparative example 2 by embodiment 2, the protein concentrate concentration that embodiment 2 obtains is higher than in comparative example 2 industrial method for culturing used, compared with comparative example 3, the extracellular protein yield of Li's Trichoderma strains T1 is higher than wild strain QM6a.
By the experimental result of embodiment 3 it can be seen that embodiment 2 and other cultural methods and use compared with other bacterial strains that identical cultural method cultivates, the protein concentrate obtained in embodiment 2 has higher cellulase specific enzyme activity and higher saccharification efficiency.
Claims (10)
1. strain trichoderma reesei (Trichodermareesei) T1, is preserved in China typical culture collection center (CCTCC), Luo Jia Shan, wuchang, wuhan, address, culture presevation numbering CCTCCNO:M2015804 on the 30th in December in 2015.
2. the cultural method of trichoderma reesei (Trichodermareesei) T1 as claimed in claim 1, it is characterised in that step is as follows:
(1) trichoderma reesei (Trichodermareesei) T1 is inoculated in plating medium, activation culture 68～76h at 28～32 DEG C, prepare activated strains；
(2) activated strains that step (1) prepares is inoculated in fluid medium, at 28～32 DEG C, cultivates 68～76h under 150～250rpm shaking table condition of culture, prepare liquid seeds；
(3) by step (2) prepare liquid seeds by 1:(4～6) volume ratio be inoculated in fluid medium, at 28～32 DEG C, cultivate 68～76h under 150～250rpm shaking table condition of culture, prepare trichoderma reesei (Trichodermareesei) T1 bacterium solution.
3. cultural method as claimed in claim 2, it is characterised in that the plating medium in described step (1) is wheat bran plating medium, and preparation method is as follows:
Every 10g wheat bran adds in 500mL distilled water, boils 30min, and after 8 layers of filtered through gauze, adding mass percent in filtrate is the agar powder of 2%, 121 DEG C of high pressure steam sterilization 30min.
4. cultural method as claimed in claim 2, it is characterised in that in described step (2) and (3), every liter of fluid medium component is as follows:
15～25g wheat bran, 15～25g microcrystalline Cellulose, 8～12g (NH3)2SO4, 12～18gKH2PO4, 0.4～0.8gMgSO4, 0.6～1.0gCaCl2, 0.004～0.006gFeSO4·7H2O, 0.0015～0.0017gMnSO4·H2O, 0.0013～0.0015gZnSO4·7H2O。
5. trichoderma reesei described in claim 1 (Trichodermareesei) T1 application in producing cellulase complex, it is characterised in that step is as follows:
I trichoderma reesei (Trichodermareesei) T1 bacterium solution is inoculated in fermentation medium by (), at 28～32 DEG C, cultivate 68～76h, prepare fermentation liquid under 150～250rpm shaking table condition of culture；
(ii) by fermentation liquor solid-liquid separation prepared for step (i), taking supernatant, then rotary evaporation concentration volume is to 10%, and secondary solid-liquid separation takes supernatant, concentrates volume to 1% through super filter tube, prepares cellulase complex.
6. applying as claimed in claim 5, it is characterised in that in described step (i), every liter of fermentation medium component is as follows:
15～25g wheat bran, 15～25g microcrystalline Cellulose, 8～12g (NH3)2SO4, 12～18gKH2PO4, 0.4～0.8gMgSO4, 0.6～1.0gCaCl2, 0.004～0.006gFeSO4·7H2O, 0.0015～0.0017gMnSO4·H2O, 0.0013～0.0015gZnSO4·7H2O。
7. applying as claimed in claim 5, it is characterised in that in described step (ii), solid-liquid separation is the centrifugal 10～15min of 8000～10000rpm.
8. applying as claimed in claim 5, it is characterised in that in described step (ii), secondary solid-liquid separation is the centrifugal 25～35min of 35000～40000rpm.
9. applying as claimed in claim 5, it is characterised in that in described step (ii), super filter tube concentration is the super filter tube concentration 550～650min of 3kDa.
10. cellulase complex application in degraded cellulose described in claim 5, it is characterised in that step is as follows:
Compound concentration is the cellulose solution of 1～200g/L, then according to the ratio of every gram of cellulose interpolation 0.1～100mg albumen adds cellulase complex fermentation liquid, react 4～120 hours when temperature 30 DEG C～60 DEG C, pH3.5～7.0, obtain catabolite.
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