CN104975039A - Recombinant plasmid and application of recombinant plasmid to degrading cellulose raw material - Google Patents
Recombinant plasmid and application of recombinant plasmid to degrading cellulose raw material Download PDFInfo
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Abstract
The invention relates to a recombinant plasmid and application of the recombinant plasmid to degrading a cellulose raw material. A pUG6 plasmid is cut into linear plasmids through a NotI enzyme; then a trichoderma reesei xylanolytic enzyme II promoter (XYN2upstream), a beta-glucosaccharase encoding gene (Glucosidase), a beta-glucosaccharase terminator (Terminator), a hygromycin phosphotransferase coding gene segment (HPH) and a trichoderma reesei xylanolytic enzyme II terminator (XYN2downstream) are obtained through PCR amplification and are connected with the linear plasmids in sequence; and the recombinant plasmid PUG6-XYN2-GLU-HPH is obtained. The recombinant plasmid capable of expressing the high-enzyme-activity beta-glucosaccharase is constructed for the first time; and the high-enzyme-activity beta-glucosaccharase expressed by the recombinant plasmid can improve the efficiency of the cellulase for degrading lignocellulose.
Description
Technical field
The present invention relates to a kind of recombinant plasmid and the application in degraded cellulose raw material thereof, belong to technical field of biotechnology.
Background technology
At present, due to the exhaustion day by day of oil, the research and development that the agricultural wastes such as maize straw, straw and bagasse change into the alternative liquid fuel of crude oil (particularly bio-ethanol) production technology are paid much attention to.This technique, first the physico-chemical process Treating straws such as hydrothermal treatment consists method, diluted acid method, SPORL method are adopted, then the pretreated raw material of cellulase preparation enzymolysis is used, change into fermentability monose, then add the fermentable such as yeast, clostridium and become the liquid fuel/Chemicals such as ethanol, butanols.
But commercially available zymin major part derives from Trichodermareesei fermentation and obtains, and in enzyme system composition, beta-glucosidase activity is lower, causes enzymolysis process to produce more cellobiose, hinders cellulosic effective degraded, cause monose transformation efficiency lower.Meanwhile, yeast can not utilize cellobiose, therefore causes final ethanol fermentation yield also not high.
Chinese patent literature CN102787104A (application number 201210260079.2) discloses a kind of high-activity composite cellulase and preparation thereof and the application method in wood fibre enzymatic saccharification.Utilize Java penicillium ZN-205 to carry out shake flask fermentation and produce beta-glucosidase, best condition of enzyme production: initial pH is 6.0, peptone concentration is 0.75%, Microcrystalline Cellulose concentration is 2.5%, the addition of tween-80 is 0.05%, and culture temperature is 28 DEG C, and 250ml triangular flask liquid amount is 100ml, shaking speed is 175r/min, and inoculum size is 5%; The highest beta-glucosidase is lived as 2.312IU/ml.The cellulase that the beta-glucosidase produce Java penicillium ZN-205 and Trichodermareesei Rut C-30 produce carries out enzyme system compound, and obtaining optimum beta-glucosidase work/filter paper enzyme activity ratio is 1.4.This application needs the different microorganism of employing two kinds to carry out production enzyme liquid, then carries out enzyme system compound.Be unfavorable for suitability for industrialized production.
Chinese patent literature CN102286446A (application number 201110162149.6) discloses a kind of prozyme preparing monose for corn cob residue conversion, it comprises: cellulase 10 ~ 48FPU/g substrate, polygalacturonase 0 ~ 17.2IU/g substrate, tensio-active agent 0 ~ 1% (v/v).This invention utilizes the composite of different sources cellulase and the synergy with tensio-active agent thereof, reduces to use enzyme cost in production, substantially increases the conversion coefficient of corn cob residue, up to 74.8%.
Chinese patent literature CN102071223A (application number 200910172724.3) discloses a kind of complex enzyme hydrolysis method of stalk raw materials, stalk raw materials is first through dilute acid hydrolysis lignin degrading, then zytase, cellulase, amylase, polygalacturonase and beta-glucanase is added, adjusting mixture pH with acidic substance or alkaline matter is 4.3 ~ 5.5, then at 40 ~ 55 DEG C, carries out enzymolysis 24 ~ 72h.The present invention adopts the method for prozyme to make stalk enzymolysis be converted into reducing sugar, select suitable enzyme ratio, make to produce between enzyme to interact, make hydrolysis reach maximization, the concentration of reduced sugar that enzymolysis produces is high, obtains sugared rate high, enzymatic hydrolysis condition is gentle, have no adverse effects to later stage fermentation, can not suppress the growth of later stage fermentation, ethanol production during fermentation is high.But it is composite that technique scheme needs several zymin zytase, cellulase, amylase, polygalacturonase and beta-glucanase to carry out enzyme system.
At present, prepare cellulase and hemicellulase often utilizes filamentous fungus to produce, and transform filamentous fungus industrial strain and improve the method that cellulase and hemicellulase enzyme live and have mutation breeding technologies, Protoplast Fusion Technique, gene rearrangement technology etc.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of recombinant plasmid and the application in degraded cellulose raw material thereof are provided.By transformation filamentous fungus industrial strain, improve beta-glucuroide enzymic activity in enzyme system, improve the composition of enzyme system, the method for monose and ethanol production thus the preparation of raising lignocellulose can be fermented.
Technical solution of the present invention is as follows:
A kind of recombinant plasmid PUG6-XYN2-GLU-HPH, it is characterized in that, utilize NotI enzyme that pUG6 plasmid is cut into linear plasmid, then by Trichodermareesei xylan solution enzyme II promotor (XYN2upstream) of pcr amplification acquisition, the encoding gene (Glucosidase) of beta-glucosidase and terminator (Terminator), the encoding gene segment (HPH) of hygromix phosphotransferase is connected with above-mentioned linear plasmid successively with Trichodermareesei xylan solution enzyme II terminator (XYN2downstream), obtained recombinant plasmid PUG6-XYN2-GLU-HPH (as shown in Figure 1),
The encoding gene nucleotide sequence (Glucosidase) of described beta-glucosidase is as shown in SEQ ID NO.1, and its terminator (Terminator) is as shown in SEQ ID NO.5; The nucleotide sequence (XYN2upstream) of Trichodermareesei xylan solution enzyme II promotor is as shown in SEQ ID NO.2; The nucleotide sequence (XYN2downstream) of Trichodermareesei xylan solution enzyme II terminator is as shown in SEQ ID NO.3; The nucleotide sequence (HPH) of the encoding gene of hygromix phosphotransferase is as shown in SEQ ID NO.4.
Preferred according to the present invention, described connection adopts the In-Fusion PCR Cloning test kit of Clotech company to carry out.
Preferred according to the present invention, described recombinant plasmid PUG6-XYN2-GLU-HPH nucleotide sequence is as shown in SEQ IDNO.6.
One strain recombinant bacterial strain, makes a variation bacterial classification for original bacteria with Trichodermareesei (Trichoderma reesei) QM9414 or its, obtained after transforming above-mentioned recombinant plasmid PUG6-XYN2-GLU-HPH.
Preferred according to the present invention, described Trichodermareesei (Trichoderma reesei) QM9414 derives from American Type Culture collection warehousing (ATCC), culture presevation ATCC 26921.
The application of above-mentioned recombinant bacterial strain in degraded cellulose raw material.
Preferred according to the present invention, described being degraded to utilizes above-mentioned recombinant bacterial strain to prepare cellulase hydrolyte after cultivating, and then utilizes cellulase hydrolyte process cellulosic material.
Preferred further according to the present invention, described cellulase hydrolyte, prepare as follows:
(1) get above-mentioned recombinant bacterial strain, be inoculated in seed culture medium, after seed culture, obtained seed liquor;
(2) seed liquor that step (1) is obtained is inoculated in culture medium, through fermentation culture, obtained fermented liquid;
Described culture medium, component is as follows, is all weight percentage:
Corn cob meal 1.0 ~ 3.0%, peptone 0.5 ~ 2.0%, wheat bran 1.0 ~ 4.0%, Microcrystalline Cellulose 0 ~ 1.0%, SODIUMNITRATE 0 ~ 1.0%, ammonium sulfate 0.1 ~ 0.5%, potassium primary phosphate 0.1 ~ 0.5%, magnesium sulfate 0.04 ~ 0.1%, urea 0 ~ 0.4%, tween 80 0 ~ 0.4%, excess water;
(3) by the fermentation liquor solid-liquid separation that step (2) is obtained, supernatant is got, obtained cellulase hydrolyte.
Seed culture medium in described step (1), component is as follows, is all weight percentage:
Glucose 0.5 ~ 2.0%, peptone 0.5 ~ 2.0%, wheat bran 1.0 ~ 4.0%, SODIUMNITRATE 0 ~ 1.0%, ammonium sulfate 0.1 ~ 0.5%, potassium primary phosphate 0.1 ~ 0.5%, magnesium sulfate 0.04 ~ 0.1%, urea 0 ~ 0.4%, excess water.
In described step (1), seed culture condition is: under the condition of 25 ~ 32 DEG C, cultivates 20 ~ 30 hours.
In described step (2), by volume the ratio of per-cent 5 ~ 10% is inoculated.
In described step (2), fermentation culture conditions is: under the condition of 25 ~ 30 DEG C, cultivates 5 ~ 7 days.
In described step (3), solid-liquid separation is centrifugal, and condition is the centrifugal 12 ~ 18min of 10000 ~ 15000r/min.
Preferred further according to the present invention, the described step of cellulase hydrolyte process cellulosic material that utilizes is as follows:
I () carries out pre-treatment to the biomass material of cellulose, by 15 ~ 25% (mass percent) solid substance final concentration, the biomass material after pre-treatment and water are mixed, then adjust ph scope to 5.0 ~ 6.0, cellulase hydrolyte is added again in the ratio of every gram of over dry biomass material interpolation 8 ~ 15FPU, oscillatory reaction 32 ~ 40h at 45 ~ 50 DEG C of temperature, through solid-liquid separation, obtained hydrolyzed solution;
(ii) get the hydrolyzed solution that step (i) is obtained, according to the ratio inoculation thermotolerant yeast of mass percent 1 ~ 5%, carry out anaerobically fermenting at 28 ~ 32 DEG C and cultivate 45 ~ 50 hours, purified separation, obtained ethanol.
In described step (i), pre-treatment is acid treatment, alkaline purification, hydrothermal treatment consists, subcritical water process, Crushing of Ultrafine process, boiling process, drying treatment, sulfiting, hydrothermal treatment consists; Preferably, sulfiting, hydrothermal treatment consists, dilute sulphuric acid process.
In described step (i), solid-liquid separation is centrifugation, and centrifugation condition is the centrifugal 30min of 8000rpm.
In described step (ii), purifies and separates is fractionation by distillation.
FPU refers to filter paper enzyme activity unit of force.50mg filter paper (with glucose production standard curve) is added, 1.5ml acetate buffer solution (50mM, pH4.8) in test tube, add 0.5ml enzyme liquid, 50 DEG C of water-bath 60min, after add 2.5ml DNS termination reaction, after boiling 10 minutes, constant volume, to 25ml, shakes up rear OD
540measure absorbance.Intercept the reducing sugar (transformation efficiency 4%) calculating filter paper enzyme activity (FPA) discharging 2.0mg glucose equivalent at 60min, unit represents with FPU.
Beneficial effect
1, the present invention constructs the recombinant plasmid can expressing the beta-glucuroide that high enzyme is lived first, and the high enzyme beta-glucuroide alive of this expression of recombinant plasmid can improve the efficiency of cellulose degraded lignocellulose.
The recombinant plasmid of the beta-glucuroide of 2, being lived by the high enzyme that the present invention builds imports in filamentous fungus T. reesei (Trichoderma reesei) QM9414 (ATCC26921), effectively can improve beta-glucuroide enzymic activity, improve the composition of enzyme system, improve efficiency and the ethanol production of cellulose degraded lignocellulose.
Accompanying drawing explanation
Fig. 1 is the design of graphics of Trichodermareesei recombinant expression vector PUG6-XYN2-GLU-HPH;
Wherein: XYN2upstream is promotor; Glucosidase is the gene of the beta-glucoside expression of enzymes deriving from aspergillus niger; Terminator and XYN2downstream is terminator; HPH is hygromycin gene selection markers box; BamHI, HindIII, BglII and NotI are restriction enzyme site;
Embodiment
Below by embodiment, technical scheme of the present invention is further elaborated, should be noted that protection scope of the present invention is not limited only to this.
Biological material source
Trichodermareesei (Trichoderma reesei) QM6a purchased from American standard biological product preservation center, culture presevation ATCCNo.13631;
Trichodermareesei (Trichoderma reesei) QM9414 purchased from American standard biological product preservation center, culture presevation ATCCNo.26921
PET-32A plasmid vector is purchased from Novagen company.
Embodiment 1
Fungal cellulase enzyme regulatory gene alive knocks out the acquisition of box:
(1) extraction of Trichodermareesei QM6a and aspergillus niger total serum IgE:
Trichodermareesei QM6a and Aspergillus niger strain are cultivated 2 days in the MM substratum being added with 2wt% Microcrystalline Cellulose, collect mycelia with filter paper filtering.The mortar that the mycelia of collection puts into precooling is ground, wherein adds liquid nitrogen during grinding.Moved in 1.5ml centrifuge tube by the mycelia powder ground to form, and add 1ml RNAiso (purchased from Sheng Gong biotechnology company limited B6402-1) and shake in vibrator evenly, room temperature puts 5min.Then the centrifugal 10min of 12000rpm.Then supernatant is drawn onto in clean 1.5ml centrifuge tube.Then add 160 μ l chloroforms, concussion 15s mixing, room temperature places 5min, 12000rpm, 4 degree of centrifugal 5min.Then suct clearly to new 1.5ml centrifuge tube.And then add 800 μ l Virahols, turn upside down 5 times.Room temperature places 10min, 12000rpm, and 4 degree of centrifugal 10min, abandon supernatant.Add 75% ethanol purge RNA of 1ml precooling, the centrifugal 5min of 7500rpm after concussion.Add 50 μ l DEPC treated waters, dissolve RNA.
MM nutrient media components is as follows: ammonium sulfate 3g, potassium primary phosphate 4.5g, magnesium sulfate 0.18g, Calcium dichloride dihydrate 0.24g urea 1.5g, 1000 × trace element (green vitriol 5g/L, manganese sulfate monohydrate 1.6g/L, Zinc Sulphate Heptahydrate 1.4g/L, cobalt chloride 2g/L) 30 μ l, supply 300ml with water.
(2) clone of encoding gene total serum IgE:
With Trichodermareesei QM6a and aspergillus niger total serum IgE for template, reverse transcription is utilized to synthesize cDNA (purchased from takara Reverse Transcription box BK1201):
1. the removal reaction of genomic dna
By following proportions reaction solution:
Above-mentioned reaction solution is reacted 2min under 42 degree of conditions, obtained RNA solution.
2. reverse transcription reaction:
By following proportions reaction solution:
By above-mentioned reaction solution at 37 DEG C of reaction 15min, then at 85 DEG C of reaction 5s.Amplify bgl gene.
PCR reaction is carried out in 50 μ l systems: 2 × PCR Buffer 25 μ l, 2mM dNTPs 10 μ l, primer 1.5 μ l, template DNA 1 μ l, KOD FX polysaccharase 1 μ l, adds distilled water and supply 50 μ l.
The primer sequence is as follows:
The primer of SEQ ID NO.1:
Upstream primer: ccgctcgagatgaggttcactttgatcga
Downstream primer: cccaagcttttagtgaacagtaggcagag
The primer of SEQ ID NO.2:
Upstream primer: ccagtacttgctcgtgtcaatctcc
Downstream primer: aagcggccgcttggatcccggttgatgtcttcttgcttcagctagtagg
The primer of SEQ ID NO.3:
Upstream primer: tgacactatagaacgcgcactctttgccttgtcctgtttgaca
Downstream primer: cggcagatccgcggccacagatgccaggttcgatcctcacca
The primer of SEQ ID NO.4:
Upstream primer: ggaagatctggccgcgacgttaactgatattgaagg
Downstream primer: aaagggcccaacccaggggctggtgacg
The primer of SEQ ID NO.5:
Upstream primer: cccaagcttgatgccgaccggatcgatccact
Downstream primer: ggaagatctaacccaggggctggtgacg
PCR response procedures is as follows:
94 DEG C of denaturation 2min; 98 DEG C of sex change 10s, 60 DEG C of annealing 30s, 68 DEG C extend 90s, 35 circulations; 68 DEG C extend 10min.
Adopt above-mentioned five pairs of primers to increase respectively and obtain PCR fragment, carry out cutting glue and reclaim, obtain the DNA fragmentation reclaimed; After testing, nucleotide sequence is respectively as shown in SEQ ID NO.1 ~ 5;
(2) gene order (SEQID NO.2) of Trichodermareesei xylan solution enzyme II promotor (xyn2) comprising secretion peptide sequence will be obtained; The gene order (SEQ ID NO.3) of Trichodermareesei xylan solution enzyme II terminator (xyn2); The gene order (SEQ ID NO.4) of hygromix phosphotransferase; The gene order (SEQ IDNO.1) of the beta-glucosidase of Aspergillus niger origin, its terminator (Terminator) is as shown in SEQ ID NO.5.
Utilize NotI enzyme that pUG6 plasmid is cut into linear plasmid, then by Trichodermareesei xylan solution enzyme II promotor (XYN2upstream) of pcr amplification acquisition, the encoding gene (Glucosidase) of beta-glucosidase and terminator (Terminator), encoding gene segment (HPH) and Trichodermareesei xylan solution enzyme II terminator (XYN2downstream) of hygromix phosphotransferase adopt the In-Fusion PCR Cloning test kit of Clotech company to be connected with above-mentioned linear plasmid, obtained recombinant plasmid PUG6-XYN2-GLU-HPH (as shown in Figure 1), nucleotide sequence is as shown in SEQ ID NO.6,
Described PCR increases expansion, digestion with restriction enzyme is all this area Normal practice.
(3) transforming Trichoderma reesei bacterial strain
Li's Trichoderma strains is cultivated three days on bran mass, utilizes physiological saline, spore under wash-out, at wheat bran bran mass upper cover one glassine paper, and add 100 μ l spore suspensions in the above, coating evenly, cultivate about 25h for 30 DEG C, the obtained glassine paper with sprouting spore.
Add 0.1g lyase (purchased from sigma company, trade name sigma#L-1412) to 20ml solution 1 (1.2M sorbyl alcohol, 0.1M KH
2pO
4) in, shake all gently; Draw 2 ~ 3ml enzyme liquid in sterile petri dish, add one deck with the glassine paper sprouting spore, then add 2 ~ 3ml enzyme liquid, stack 10 layers successively.Culture dish holding is in 30 DEG C of incubators.After enzymolysis is about 90min, with tweezers (aseptic) picking glassine paper, mycelium residual on glassine paper is washed out by liquid-transfering gun draw solution, protoplast suspension is filtered to the 50ml centrifuge tube be placed on ice with glass wool funnel, then glass wool is rinsed with number ml soln 1, then 2000rpm, 4 DEG C of centrifugal 10min, remove supernatant liquor and use 4ml solution 2 (1M sorbyl alcohol, 50mmol/LCaCl
2, 10mmol/L TrisHCl) and resuspended protoplastis, 2000rpm, 4 DEG C of centrifugal 10min, remove supernatant liquor, with 0.5 ~ 1.0ml solution 2 (4 DEG C) resuspended protoplastis, places protoplastis on ice, obtained protoplast suspension.
Transformation system (200 μ l protoplast suspension, 10 μ l purifying pE, 50 μ l molecular weight polyethylene glycols (5000-7000)) is placed 20min on ice, after add 2ml PEG (room temperature), mix gently, 20 DEG C place 5min, add 4ml solution 2, mixing; Draw in the upper strata substratum of 0.2 ~ 1ml to 4ml pre-incubation, mix gently, be poured on down be covered with lower floor's substratum flat board on, after culture medium solidifying, put 30 DEG C of cultivations.After screening culture medium cultivates 3 ~ 4d, with inoculating needle picking transformant to Selective agar medium, cultivate raw spore.
(cultivate 3 days) after passing for two generations, proceeded to by spore in minimum medium, extraction karyomit(e) obtains dissociant after verifying.
Embodiment 2
The preparation of cellulase and hemicellulase and mensuration:
Get Trichodermareesei (Trichoderma reesei) QM9414 and dissociant, be inoculated in seed culture medium, cultivate 1 day under the condition of 30 DEG C, then transfer in culture medium by the volume ratio of 10%, 28 DEG C, the condition bottom fermentation of 180rpm cultivates 6 days, obtained microbial culture medium.By centrifugal for nutrient solution 12000r/min 15min, Aspirate supernatant is crude enzyme liquid.
Above-mentioned seed culture medium component is as follows, is all weight percentage:
Glucose 1%, peptone 1%, wheat bran 1%, SODIUMNITRATE 0.1%, ammonium sulfate 0.1%, potassium primary phosphate 0.1%, magnesium sulfate 0.04%, urea 0.15%, excess water.
Above-mentioned culture medium component is as follows, is all weight percentage:
Corn cob meal 3%, peptone 1%, wheat bran 3%, Microcrystalline Cellulose 0.4%, SODIUMNITRATE 0.1%, ammonium sulfate 0.1%, potassium primary phosphate 0.1%, magnesium sulfate 0.04%, urea 0.15%, tween 80 0.2%, excess water.
Extracellular protein content: add sample (with 0.1ml pure water in contrast, with bovine serum albumin production standard curve) after the dilution of 0.1ml in 5ml centrifuge tube.Add the 1mol/L NaOH solution of 0.2ml again.Add the first reagent (2%NaCO of 2ml
3: 0.5%CuSO
4: 1% Seignette salt (KNa
4h
2o)=50:1:1), at room temperature place about 10 minutes.After reaction in 10 minutes, add the second reagent (Folin phenol reagent+isopyknic pure water) of 0.3ml.At room temperature place about 30 minutes.Absorbancy is measured under 660nm wavelength.
Filter paper enzyme activity: add 50mg filter paper (with glucose production standard curve) in test tube, 1.5ml acetate buffer solution (50mM, pH4.8), add 0.5ml enzyme liquid, 50 DEG C of water-bath 60min, after add 2.5ml DNS termination reaction, after boiling 10 minutes, constant volume, to 25ml, shakes up rear OD
540measure absorbance.Intercept the reducing sugar (transformation efficiency 4%) calculating filter paper enzyme activity (FPA) discharging 2.0mg glucose equivalent at 60min, unit represents with FPU.
Other enzyme activity units in crude enzyme liquid are defined as 1 enzyme activity unit (IU) with the hydrolysis substrate enzyme amount produced needed for 1 μm of ol reducing sugar or p-NP in a minute.
Measuring method is as follows:
The oat xylan suspension of xylanase activity: 1mL 1%, the enzyme liquid after 0.5mL dilution, 50 DEG C of enzymolysis 30min.
Endoglucanase is lived: the CMC-Na solution of 1mL 1%, the enzyme liquid after 0.5mL dilution, 50 DEG C of enzymolysis 30min.
In above two kinds of enzyme activity determinations, all measure the reducing sugar amount in enzymolysis solution with DNS method.
Beta-glucuroide or exoglucanase enzyme are lived: the crude enzyme liquid after 0.5mL dilution, adds 50 μ L pNPG or pNPC (1mg/mL), 50 DEG C of insulation 30min; Add 150 μ L 10%Na
2cO
3termination reaction.
Experimental result is as shown in table 1.The change of extracellular protein content and filter paper enzyme activity is little, and endoglucanase and xylanase activity decline to some extent, but beta-glucuroide and the work of exoglucanase enzyme increase substantially.
The comparison that in table 1 embodiment spawn culture secondary fermentation liquid, various enzyme is lived
Embodiment 3
1) preparation method's (hydrothermal treatment consists) of pretreated maize straw
Maize straw (cellulose 10wt%) is immersed in water, stirs and carry out autoclave process in 120 minutes at 190 DEG C, after high-concentration paste mill mechanical treatment, obtain material previously treated through solid/liquid separation.
2) preparation method's (dilute acid pretreatment) of pretreated wheat stalk
Wheat stalk (cellulose 10wt%) is immersed in the sulphuric acid soln of 1wt%, stirs and carry out autoclave process in 20 minutes at 180 DEG C.
3) preparation method's (sulfiting) of pretreated rice straw
In sulfurous acid rice straw (cellulose 10wt%) being immersed in 2wt% or sulfurous acid ammonia sodium solution, stir while carry out autoclave process in 30 minutes at 160 DEG C, after high-concentration paste mill mechanical treatment, obtain material previously treated through solid/liquid separation.Above-mentioned process specifically can see " hydrosulfate pre-treatment is on the impact of Factor of Enzymolysis Corn Stalk " (Liu Yunyun, Wang Gaosheng, general spring just, Liu Zhiya, " chemistry of forest product and industry ", the 30th volume the 4th phase, 73-77 page).
As the pre-treating process of stalk, be not limited to above-mentioned three kinds for the treatment of processs, can also adopt: acid treatment, alkaline purification, subcritical water process, Crushing of Ultrafine process, boiling process, drying treatment etc.; Wherein, sulfiting, hydrothermal treatment consists, dilute sulphuric acid process or alkaline purification, compared with additive method, enzyme glycolysis efficiency is excellent, enzyme usage quantity is less can complete, thus preferably sulfiting, hydrothermal treatment consists, dilute sulphuric acid process in the present invention.
Embodiment 4
I () gets 8 mass parts Microcrystalline Celluloses, pretreated maize straw, pretreated wheat stalk or pretreated rice straw, joining 89 parts of pH is 4.8, ionic strength is in the NaAc_HAc buffer solution of 50mmol/L, adds cellulase oscillatory reaction 36h at 45-50 DEG C of temperature of 3 mass parts.After reaction terminates, solid-liquid separation (8000rpm, 30min are centrifugal), obtains saccharide compound hydrolyzed solution, by hplc determination glucose content;
(ii) in the obtained hydrolyzed solution of step (i), access 0.01 mass parts thermotolerant yeast, at 30 DEG C of quiescent culture, carry out ethanol fermentation 48 hours, be then separated through distillation purifying, obtained ethanol, by hplc determination glucose content.
Experimental result is as shown in table 2 and table 3.With Microcrystalline Cellulose, pretreated maize straw, pretreated wheat stalk or pretreated rice straw for substrate, the glucose yield of genetic engineering modified rear acquisition improves 99%, 76% before comparatively transforming respectively.After adding yeast fermentation, alcohol getting rate improves 105%, 69% before comparatively transforming respectively.Although improved enzyme liquid xylanase activity declines (table 1) to some extent, the result from table 2 to table 7, the yield of wood sugar is not affected.
The comparison of table 2 hydrothermal treatment consists saccharification yield
The comparison of table 3 hydrothermal treatment consists saccharification secondary fermentation alcohol getting rate
The comparison of table 4 dilute acid pretreatment saccharification yield
The comparison of table 5 dilute acid pretreatment saccharification secondary fermentation alcohol getting rate
The comparison of table 6 sulfiting saccharification yield
The comparison of table 7 sulfiting saccharification secondary fermentation alcohol getting rate
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1. a recombinant plasmid PUG6-XYN2-GLU-HPH, it is characterized in that, utilize NotI enzyme that pUG6 plasmid is cut into linear plasmid, then by Trichodermareesei xylan solution enzyme II promotor (XYN2upstream) of pcr amplification acquisition, the encoding gene (Glucosidase) of beta-glucosidase and terminator (Terminator), the encoding gene segment (HPH) of hygromix phosphotransferase is connected with above-mentioned linear plasmid successively with Trichodermareesei xylan solution enzyme II terminator (XYN2downstream), obtained recombinant plasmid PUG6-XYN2-GLU-HPH,
The encoding gene nucleotide sequence (Glucosidase) of described beta-glucosidase is as shown in SEQ ID NO.1, and its terminator (Terminator) is as shown in SEQ ID NO.5; The nucleotide sequence (XYN2upstream) of Trichodermareesei xylan solution enzyme II promotor is as shown in SEQ ID NO.2; The nucleotide sequence (XYN2downstream) of Trichodermareesei xylan solution enzyme II terminator is as shown in SEQ ID NO.3; The nucleotide sequence (HPH) of the encoding gene of hygromix phosphotransferase is as shown in SEQ ID NO.4.
2. recombinant plasmid PUG6-XYN2-GLU-HPH as claimed in claim 1, it is characterized in that, the nucleotide sequence of recombinant plasmid PUG6-XYN2-GLU-HPH is as shown in SEQ ID NO.6.
3. a strain recombinant bacterial strain, is characterized in that, makes a variation bacterial classification for original bacteria with Trichodermareesei (Trichoderma reesei) QM9414 or its, obtained after transforming recombinant plasmid PUG6-XYN2-GLU-HPH described in claim 1;
Preferably, described Trichodermareesei (Trichoderma reesei) QM9414 derives from American Type Culture collection warehousing (ATCC), culture presevation ATCC 26921.
4. the application of recombinant bacterial strain described in claim 3 in degraded cellulose raw material.
5. apply as claimed in claim 4, it is characterized in that, described being degraded to utilizes above-mentioned recombinant bacterial strain to prepare cellulase hydrolyte after cultivating, and then utilizes cellulase hydrolyte process cellulosic material.
6. apply as claimed in claim 5, it is characterized in that, described cellulase hydrolyte, prepare as follows:
(1) get above-mentioned recombinant bacterial strain, be inoculated in seed culture medium, after seed culture, obtained seed liquor;
(2) seed liquor that step (1) is obtained is inoculated in culture medium, through fermentation culture, obtained fermented liquid;
Described culture medium, component is as follows, is all weight percentage:
Corn cob meal 1.0 ~ 3.0%, peptone 0.5 ~ 2.0%, wheat bran 1.0 ~ 4.0%, Microcrystalline Cellulose 0 ~ 1.0%, SODIUMNITRATE 0 ~ 1.0%, ammonium sulfate 0.1 ~ 0.5%, potassium primary phosphate 0.1 ~ 0.5%, magnesium sulfate 0.04 ~ 0.1%, urea 0 ~ 0.4%, tween 80 0 ~ 0.4%, excess water;
(3) by the fermentation liquor solid-liquid separation that step (2) is obtained, supernatant is got, obtained cellulase hydrolyte.
7. apply as claimed in claim 6, it is characterized in that, the seed culture medium in described step (1), component is as follows, is all weight percentage:
Glucose 0.5 ~ 2.0%, peptone 0.5 ~ 2.0%, wheat bran 1.0 ~ 4.0%, SODIUMNITRATE 0 ~ 1.0%, ammonium sulfate 0.1 ~ 0.5%, potassium primary phosphate 0.1 ~ 0.5%, magnesium sulfate 0.04 ~ 0.1%, urea 0 ~ 0.4%, excess water.
8. apply as claimed in claim 6, it is characterized in that, in described step (1), seed culture condition is: under the condition of 25 ~ 32 DEG C, cultivates 20 ~ 30 hours;
Preferably, in described step (2), by volume the ratio of per-cent 5 ~ 10% is inoculated;
Preferably, in described step (2), fermentation culture conditions is: under the condition of 25 ~ 30 DEG C, cultivates 5 ~ 7 days;
Preferably, in described step (3), solid-liquid separation is centrifugal, and condition is the centrifugal 12 ~ 18min of 10000 ~ 15000r/min.
9. apply as claimed in claim 5, it is characterized in that, the described step of cellulase hydrolyte process cellulosic material that utilizes is as follows:
I () carries out pre-treatment to the biomass material of cellulose, by 15 ~ 25% (mass percent) solid substance final concentration, the biomass material after pre-treatment and water are mixed, then adjust ph scope to 5.0 ~ 6.0, cellulase hydrolyte is added again in the ratio of every gram of over dry biomass material interpolation 8 ~ 15FPU, oscillatory reaction 32 ~ 40h at 45 ~ 50 DEG C of temperature, through solid-liquid separation, obtained hydrolyzed solution;
(ii) get the hydrolyzed solution that step (i) is obtained, according to the ratio inoculation thermotolerant yeast of mass percent 1 ~ 5%, carry out anaerobically fermenting at 28 ~ 32 DEG C and cultivate 45 ~ 50 hours, purified separation, obtained ethanol.
10. apply as claimed in claim 9, it is characterized in that, in described step (i), pre-treatment is acid treatment, alkaline purification, hydrothermal treatment consists, subcritical water process, Crushing of Ultrafine process, boiling process, drying treatment, sulfiting, hydrothermal treatment consists; Preferably, sulfiting, hydrothermal treatment consists, dilute sulphuric acid process;
Preferably, in described step (i), solid-liquid separation is centrifugation, and centrifugation condition is the centrifugal 30min of 8000rpm.
Preferably, in described step (ii), purifies and separates is fractionation by distillation.
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