CN102747067A - Application of TrPK protein to cellulase yield adjustment - Google Patents
Application of TrPK protein to cellulase yield adjustment Download PDFInfo
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Abstract
The invention discloses application of TrPK protein to cellulase yield adjustment. An engineering bacterium constructing method includes the steps: inhibiting expression of TrPK genes in trichoderma so as to obtain engineering bacteria; and enabling TrPK protein to be (a) or (b): (a) protein composed of amino acid sequences shown in sequence No.1 in a sequence table; and (b) protein obtained by subjecting the amino acid sequences in the sequence No.1 to substitution and/or deletion and/or addition of one or more amino acid residues, relevant to cellulase synthesis in the trichoderma and derivative from the sequence No.1. The invention further provides application of the TrPK protein to cellulase synthesis in trichoderma adjustment. According to the application, after knockout of the TrPK genes, capability of the trichoderma in cellulase production is remarkably improved. The application is of great significance to cellulase production and has potential influences on solution to energy crisis and environmental crisis.
Description
Technical field
The present invention relates to the application of a kind of TrPK albumen in regulating yield of cellulase.
Background technology
Along with going from bad to worse of global energy and ecocrisis; Seek new renewable energy source and become the hot issue that current social is paid close attention to; Cellulose biomass is as plant absorbing sun power and pass through the product that photosynthesis forms, and is the abundantest, the most feasible renewable new forms of energy material on the earth.But how becoming the available energy substance of modern industry to have problems Wood Adhesives from Biomass, wherein the most critical issue is exactly to realize the efficient degradation of natural biomass.Cellulase is that occurring in nature exists the most extensively, the most effective biomass degrading enzymes, plays key effect in lignocellulose resource conversion process, exploitation, utilization that its output, active height direct relation new biomass energy.
Filamentous fungus Trichodermareesei (Trichoderma reesei) is main cellulase production bacterial strain, and the whole world about 90% above cellulase product generates through the mould fermentation of wood.Over 50 years, people significantly improve the ability acquisition of Trichodermareesei production of cellulose enzyme through optimizing methods such as culture condition, transgenation and bacterial strain mutagenesis.But production of cellulose enzyme cost is high, yielding poorly is still the bottleneck that the restriction biomass energy can not mass-producing.It is very difficult further to improve yield of cellulase through traditional method.
Summary of the invention
The purpose of this invention is to provide the application of a kind of TrPK albumen in regulating yield of cellulase.
The invention provides a kind of method that makes up engineering bacteria, comprise the steps: to suppress the mould middle TrPK expression of gene of wood, obtain engineering bacteria; Said TrPK genes encoding is (a) or (b) described TrPK albumen as follows:
(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;
(b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and synthetic relevant with cellulase during wood is mould by sequence 1 deutero-protein.
Said TrPK gene is following 1) to 4) in arbitrary described dna molecular:
1) dna molecular shown in the sequence 2 of sequence table;
2) dna molecular shown in the sequence 3 of sequence table;
3) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of encoding said proteins;
4) with 1) or 2) dna molecular with 90% above homology and encoding said proteins of the dna sequence dna that limits.
Said stringent condition be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, hybridization and wash film under 65 ℃ of conditions.
The implementation method of said " suppress wood mould in TrPK expression of gene " is following: knock out the said TrPK gene of wood in mould through homologous recombination.
The implementation method of said " knocking out the said TrPK gene of wood in mould through homologous recombination " is following: the recombinant plasmid that will contain the dna fragmentation first import said wood mould in; Said dna fragmentation first comprises the upper reaches homology arm of said TrPK gene and the downstream homology arm of said TrPK gene successively to downstream from the upper reaches.
In the said dna fragmentation first, between said upper reaches homology arm and said downstream homology arm, also has the selection markers gene.Said selection markers gene specifically can be the pyr4 gene; Said pyr4 gene is the proteic gene of pyr4 shown in the sequence 5 of code sequence tabulation.The reverse complementary sequence of said pyr4 gene specifically can be like the sequence 4 of sequence table from shown in 5 ' terminal the 3000th to 4145 Nucleotide.Said dna fragmentation first specifically can comprise the reverse complementary sequence and the said downstream homology arm of said upper reaches homology arm, said pyr4 expression of gene box successively.Said upper reaches homology arm specifically can be like the sequence 4 of sequence table from shown in 5 ' terminal the 9th to 1994 Nucleotide.Said downstream homology arm specifically can be like the sequence 4 of sequence table from shown in 5 ' terminal the 5511st to 7506 Nucleotide.The reverse complementary sequence of said pyr4 expression of gene box specifically can be like the sequence 4 of sequence table from shown in 5 ' terminal the 2001st to 5504 Nucleotide.Said dna fragmentation first specifically can be the sequence 4 of sequence table from the double chain DNA molecule shown in 5 ' terminal the 9th to 7506 Nucleotide.
The said recombinant plasmid that contains the dna fragmentation first specifically can be said dna fragmentation first is imported the recombinant plasmid that the MCS of plasmid pBluescript SK (+) obtains.
The mould Trichodermareesei that can be of said wood specifically can be TU6 △ tku70 bacterial strain, TU6 bacterial strain or QM9414 bacterial strain.
More than the engineering bacteria that obtains of arbitrary said method all belong to protection scope of the present invention.
The present invention also protects a kind of method of production of cellulose enzyme, and the said engineering bacteria that comprises the steps: to ferment obtains cellulase.The actual conditions of said fermentation does, said engineering bacteria is inoculated in fermention medium, and 28 ℃, the 200rpm concussion was cultivated 48-144 hour.
The present invention also protects the application of material in promoting wooden mould production of cellulose enzyme that suppresses said TrPK genetic expression.The material of the said TrPK genetic expression of said inhibition is the recombinant plasmid that contains above arbitrary said dna fragmentation first.The material of the said TrPK genetic expression of said inhibition specifically can be said dna fragmentation first is imported the recombinant plasmid that the MCS of plasmid pBluescript SK (+) obtains.
The mould Trichodermareesei that can be of said wood specifically can be TU6 △ tku70 bacterial strain, TU6 bacterial strain or QM9414 bacterial strain.
The present invention also protects said TrPK albumen regulating the application of the cellulase of wood in mould in synthetic.
The mould Trichodermareesei that can be of said wood specifically can be TU6 △ tku70 bacterial strain, TU6 bacterial strain or QM9414 bacterial strain.
The present invention finds, knock out the TrPK gene after, the ability of wooden mould production of cellulose enzyme significantly increases.The present invention has great value for the production of cellulase, has potential impact for solving energy dilemma and ecocrisis.
Description of drawings
Fig. 1 is that the PCR among the embodiment 1 identifies electrophorogram.
Fig. 2 is crude enzyme liquid SDS-PAGE analytical electrophoresis figure among the embodiment 3.
Fig. 3 is the cellulose enzyme activity of crude enzyme liquid among the embodiment 3.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.
Trichodermareesei (Trichoderma reesei) QM9414 bacterial strain: US mode culture collection warehousing (being called for short ATCC, network address www.atcc.org/), ATCC is numbered 26921.
Trichodermareesei (Trichoderma reesei) TU6 bacterial strain is by QM9414 bacterial strain deutero-, the pyr4 gene function is lost, uridylic auxotrophy bacterial strain, and ATCC is numbered MYA-256.
Microcrystalline Cellulose (Avicel
RPH-101): available from fluka company, catalog number 11365.
Plasmid pBluescript SK (+): available from Stratagene company, catalog number 212205.
Trichodermareesei (Trichoderma reesei) TU6 △ tku70 bacterial strain is by TU6 bacterial strain deutero-, knocks out the bacterial strain that obtains of tku70 gene of the non-homogeneous reorganization of mediation, and the public can obtain from Institute of Microorganism, Academia Sinica; Mention the document of TU6 △ tku70 bacterial strain (T.reesi KU70): Zhang Guangtao, Lukas Hartl, Andre Schuster; Stefan Polak, Monika Schmoll, Tianhong Wang; Verena Seidl; Bernhard Seiboth.Gene targeting in a nonhomologous end joining deficient Hypocrea jecorina.Journal of biotechnology, 2009,139 (2): 146-151..
Potato substratum (PDA solid medium): 20g is removed the peel the yam chopping, add 90mL water, boil 30min, double gauze filters and collects filtrating, adds 2g glucose and 1.8g agar powder, and water is settled to 100mL.
MM liquid nutrient medium (pH is 5.2+0.1): (NH
4)
2SO
40.5g/100mL, KH
2PO
41.5g/100mL, MgSO
40.06g/100mL, CaCl
20.06g/100mL, FeSO
47H
2O 0.0005g/100mL, MnSO
4H
2O0.00016g/100mL, ZnSO
47H
2O 0.00014g/100mL, CoCl
20.0002g/100mL all the other are water.
The MM solid medium: on the basis of MM liquid nutrient medium, every liter is added the 20g agar powder.
LB substratum (natural pH value): 1g/100mL peptone, 1g/100mL sodium-chlor, 0.5g/100mL yeast extract, all the other are water.
DNS reagent: 50g 3,5-dinitrosalicylic acid are dissolved in the 4L water, constantly stir, and slowly add 80g sodium hydroxide; Make it to dissolve fully, continue to stir, the 1500g Seignette salt is divided for several times add; And careful heating, the solution top temperature is no more than 45 ℃, is settled to 5L after being cooled to room temperature; If solution is not clarified, with the Whatman1 filter paper filtering, brown bottle room temperature storage then.
TrPK albumen (claiming trpk albumen again) is made up of 1166 amino-acid residues shown in the sequence 1 of sequence table, theoretical molecular 128.98kDa, iso-electric point (PI) 5.205.The ORFs of the proteic encoding sox of TrPK (claiming TrPK gene or trpk gene again) is shown in the sequence 2 of sequence table, and its full-length cDNA is shown in the sequence 3 of sequence table.
The preparation of embodiment 1, reorganization fungus beetle
One, construction of recombinant plasmid
1, the acquisition of trpk upstream region of gene homology arm (Ptrpk, about 1987bp)
Genomic dna with TU6 △ tku70 bacterial strain is a template, and the primer of forming with Fup and Rup obtains pcr amplification product to carrying out pcr amplification.
Fup:5 '-
GCGGCCGCCTTGCCCGTTTGCTACCTATGATG-3 ' (underscore mark NotI restriction endonuclease recognition sequence);
Rup:5 '-
GGATCCGTGGGAGGGGGAGATAGTTG-3 ' (underscore mark BamHI restriction endonuclease recognition sequence).
The Taq enzyme that pcr amplification adopts is high-fidelity fastpfu (available from being King Company entirely).
Pcr amplification program: 95 ℃ of preparatory sex change 5min; 94 ℃ of sex change 30s, 55 ℃ of annealing 40s, 72 ℃ prolong 1min, 30 circulations; 3min is extended in 72 ℃ of expansions.
2, the acquisition of trpk gene downstream homology arm (Ttrpk, about 1996bp)
Genomic dna with TU6 △ tku70 bacterial strain is a template, and the primer of forming with Fdown and Rdown obtains pcr amplification product to carrying out pcr amplification.
Fdown:5 '-
AAGCTTCGAGGTAAGTCTGATATATCGAG-3 ' (underscore mark HindIII restriction endonuclease recognition sequence);
Rdown:5 '-
GGTACCGTCTGTTCTATTTCTGGTCCTTTC-3 ' (underscore mark KpnI restriction endonuclease recognition sequence).
The Taq enzyme that pcr amplification adopts is high-fidelity fastpfu (available from being King Company entirely).
Pcr amplification program: 95 ℃ of preparatory sex change 5min; 94 ℃ of sex change 30s, 52 ℃ of annealing 40s, 72 ℃ prolong 1min, 30 circulations; 3min is extended in 72 ℃ of expansions.
3, the acquisition of pyr4 expression casette (about 3504bp)
Genomic dna with the QM9414 bacterial strain is a template, and the primer of forming with Fpyr4 and Rpyr4 obtains pcr amplification product to carrying out pcr amplification.
Fpyr4:5 '-
GGATCCGGTTGATTGTTGCCGTCCGTTTCG-3 ' (underscore mark BamHI restriction endonuclease recognition sequence).
Rpyr4:5 '-
AAGCTTTCGACTAGACTGACCCCCCCG-3 ' (underscore mark HindIII restriction endonuclease recognition sequence).
The Taq enzyme that pcr amplification adopts is high-fidelity fastpfu (available from being King Company entirely).
Pcr amplification program: 95 ℃ of preparatory sex change 5min; 94 ℃ of sex change 30s, 55 ℃ of annealing 40s, 72 ℃ prolong 2min, 30 circulations; 3min is extended in 72 ℃ of expansions.
4,, reclaim enzyme and cut product with the pcr amplification product of restriction enzyme NotI and BamHI double digestion step 1.
5,, reclaim enzyme and cut product with the pcr amplification product of restriction enzyme HindIII and KpnI double digestion step 2.
6,, reclaim enzyme and cut product with the pcr amplification product of restriction enzyme BamHI and HindIII double digestion step 3.
7,, reclaim carrier framework (about 2961bp) with restriction enzyme NotI and KpnI double digestion plasmid pBluescript SK (+).
8, the enzyme that the enzyme that the enzyme of the enzyme of step 4 being cut product, step 6 is cut product, step 5 is cut product and step 7 is cut product and is connected, and obtains recombinant plasmid pSK-Ptrpk-pyr4-Ttrpk (trpk gene knockout carrier).According to sequencing result, it is following that recombinant plasmid pSK-Ptrpk-pyr4-Ttrpk is carried out structrual description: between the NotI of plasmid pBluescript SK (+) and KpnI restriction enzyme site, inserted the double chain DNA molecule shown in the sequence 4 of sequence table.
In the sequence 4 of sequence table; From 5 ' terminal the 1st to 8 Nucleotide is the NotI restriction endonuclease recognition sequence; The the 9th to 1994 Nucleotide is trpk upstream region of gene homology arm; The the 1995th to 2000 Nucleotide is the BamHI restriction endonuclease recognition sequence; The reverse complementary sequence that the 2001st to 5504 Nucleotide is the pyr4 expression casette (wherein the 2001st to 2999 Nucleotide reverse complementary sequence that is terminator, the 3000th to 4145 Nucleotide are the pyr4 gene reverse complementary sequence, the 4146th to 5504 Nucleotide are the reverse complementary sequence of promotor); The the 5505th to 5510 Nucleotide is the HindIII restriction endonuclease recognition sequence, and the 5511st to 7506 Nucleotide is trpk gene downstream homology arms, and the 7507th to 7512 Nucleotide is the KpnI restriction endonuclease recognition sequence.
Two, the acquisition of reorganization fungus beetle
1, Tu6 △ tku70 bacterial strain protoplastis preparation
(1) gets the spore of Tu6 △ tku70 bacterial strain; Wash spore and process spore suspension with an amount of sterilized water; Remove by filter remaining mycelia with 200 mesh; Spore suspension after filtering is seeded in the 500mL triangular flask that 100mL MM liquid nutrient medium is housed, and 28 ℃ are cultured to mycelia and stretch (13h-14h).
(2) culture system that step (1) is obtained filters through 200 mesh, collects thalline, with sterilized water washing 2-3 time, uses 1.2M MgSO then
4Solution washing once.
(3) thalline that step (2) is obtained adds that (lysate is to contain the lyase of 150mg and the 1.2M MgSO of 15mg cellulase in the triangular flask that the 15mL lysate is housed
4The aqueous solution), 30 ℃, 80rpm oscillatory reaction 1.5h, microscopically is observed the situation that protoplastis produces, and every separated 10min sampling is observed once behind the reaction 1h.
(4) in the step (3); When protoplastis produces in a large number and still have a large amount of mycelia to exist, add equal-volume 0.6M sorbitol aqueous solution termination reaction, remove by filter remaining mycelia through 200 mesh; The centrifugal 10min of room temperature 3000rpm collects the protoplastis deposition.
(5) the protoplastis deposition of step (4) is resuspended with the 1.0M sorbitol aqueous solution, the centrifugal 10min of room temperature 3000rpm collects the protoplastis deposition.
(6) the protoplastis deposition of step (5) is resuspended with the 1.0M sorbitol aqueous solution, the centrifugal 10min of room temperature 3000rpm collects the protoplastis deposition and is suspended in the 200 μ L 1.0M sorbitol aqueous solutions observation of blood cell plate telltale and counting (10
8Individual protoplastis/mL).
2, the acquisition of reorganization fungus beetle
50%PEG4000 solution: contain 50g/100g PEG4000,50mM CaCl
210mM Tris-HCL (pH8.0) damping fluid.
(1) recombinant plasmid pSK-Ptrpk-pyr4-Ttrpk is added in the protoplastis of step 1 preparation, mixing adds 50 μ L, 50% PEG4000 solution more gently, and mixing is placed 30min on ice once more.
(2) in the culture system of step (1), add 1mL 50% PEG4000 solution, mixing, room temperature is placed 20min.
(3) in the culture system of step (2), add 1mL 1.0M sorbitol aqueous solution; Branch four times and MM solid medium (below 58 ℃) each and a 4ml thawing are mixed behind the mixing; Be laid on immediately on the MM solid medium flat board that contains the 1.0M sorbyl alcohol, cultivate 4-7d for 30 ℃.
(4) after son to be transformed grows; It is transferred on the PDA flat board, cultivates 3-5 days (having spore to generate) for 28 ℃, with sterilized water the spore on the flat board is washed and be prepared into spore suspension; After doing gradient dilution; It is coated on the MM solid medium flat board that contains 0.1% (volume ratio) Triton-X100 divides monospore, treat that mycelia grows after, the extracting genomic dna carries out PCR to be identified.
PCR identifies that used primer is following:
Frup:5 '-CAACTATCTCCCCCTCCCAC-3 '; It is terminal to be positioned at upper reaches homology arm
Rfdown:5 '-CTCGATATATCAGACTTACCTCG-3 '; Be positioned at downstream homology arm front end
Tu6 △ tku70 bacterial strain can only obtain the fragment of about 3930bp, can obtain the segmental bacterial strain of about 3559bp and be the reorganization fungus beetle.
Qualification result is seen Fig. 1 (arrow mark purpose band), and wherein M is 1kb DNA ladder marker, and 1 is the reorganization fungus beetle, and 2 is Tu6 △ tku70 bacterial strain.
The acquisition of embodiment 2, reorganization bacterium second
Replace recombinant plasmid pSK-Ptrpk-pyr4-Ttrpk to carry out the operation of the step 2 of embodiment 1 plasmid pBluescript SK (+), bacterium second obtains recombinating.
The fermentation of embodiment 3, reorganization bacterium and the cellulase activity of crude enzyme liquid are identified
The reorganization fungus beetle of embodiment 1 acquisition, reorganization bacterium second or the bacterium that sets out (TU6 △ tku70 bacterial strain) that embodiment 2 obtains are carried out following steps respectively:
One, the fermentation of reorganization bacterium
1, bacterial strain is gone on the PDA solid medium flat board produces spore, process the spore suspension 4 * 10 of high density then with sterilized water washing spore and with sterilized water
7-5 * 10
7Individual spore/mL.
2, the 50mL that the 0.5mL spore suspension is inoculated in the 250mL triangular flask contains in the MM liquid nutrient medium of 2g/100mL glucose, and 28 ℃, 200rpm cultivates 48h in advance, and four layers of filtered through gauze are also collected thalline.
3, take by weighing 1.8 gram thalline (weight in wet base) in the 50mL fermention medium, 28 ℃, 200rpm shakes cultivation, takes a sample after 48 hours, 72 hours, 96 hours, 120 hours and 144 hours respectively at shaking culture.
Fermention medium (pH5.2 ± 0.1): the MM liquid nutrient medium that contains the 1g/100mL Microcrystalline Cellulose.
4, the centrifugal 5min of sample 12000r/min that step 3 is obtained collects supernatant.
Reorganization fungus beetle shaking culture was taken a sample and carried out the supernatant called after reorganization fungus beetle crude enzyme liquid that step 4 obtains in 48 hours
48 hoursReorganization fungus beetle shaking culture was taken a sample and carried out the supernatant called after reorganization fungus beetle crude enzyme liquid that step 4 obtains in 72 hours
72 hoursReorganization fungus beetle shaking culture was taken a sample and carried out the supernatant called after reorganization fungus beetle crude enzyme liquid that step 4 obtains in 96 hours
96 hoursReorganization fungus beetle shaking culture was taken a sample and carried out the supernatant called after reorganization fungus beetle crude enzyme liquid that step 4 obtains in 120 hours
120 hoursReorganization fungus beetle shaking culture was taken a sample and carried out the supernatant called after reorganization fungus beetle crude enzyme liquid that step 4 obtains in 144 hours
144 hours
Reorganization bacterium second shaking culture was taken a sample and carried out the supernatant called after reorganization bacterium second crude enzyme liquid that step 4 obtains in 48 hours
48 hoursReorganization bacterium second shaking culture was taken a sample and carried out the supernatant called after reorganization bacterium second crude enzyme liquid that step 4 obtains in 72 hours
72 hoursReorganization bacterium second shaking culture was taken a sample and carried out the supernatant called after reorganization bacterium second crude enzyme liquid that step 4 obtains in 96 hours
96 hoursReorganization bacterium second shaking culture was taken a sample and carried out the supernatant called after reorganization bacterium second crude enzyme liquid that step 4 obtains in 120 hours
120 hoursReorganization bacterium second shaking culture was taken a sample and carried out the supernatant called after reorganization bacterium second crude enzyme liquid that step 4 obtains in 144 hours
144 hours
The bacterium shaking culture of setting out was taken a sample and carried out supernatant called after that step 4 the obtains bacterium crude enzyme liquid that sets out in 48 hours
48 is little The timeThe bacterium shaking culture of setting out was taken a sample and carried out supernatant called after that step 4 the obtains bacterium crude enzyme liquid that sets out in 72 hours
72 hoursThe bacterium shaking culture of setting out was taken a sample and carried out supernatant called after that step 4 the obtains bacterium crude enzyme liquid that sets out in 96 hours
96 hoursThe bacterium shaking culture of setting out was taken a sample and carried out supernatant called after that step 4 the obtains bacterium crude enzyme liquid that sets out in 120 hours
120 hoursThe bacterium shaking culture of setting out was taken a sample and carried out supernatant called after that step 4 the obtains bacterium crude enzyme liquid that sets out in 144 hours
144 hours
Two, SDS-PAGE analyzes
Each crude enzyme liquid that step 1 is obtained carries out the SDS-PAGE analysis, and the result sees Fig. 2.Among Fig. 2,1 represents the bacterium that sets out, 2 representative reorganization fungus beetles, and 48h, 72h, 96h, 120h and 144h represent fermentation time.Because crude enzyme liquid is a fermented liquid supernatant; According to prior art and document record; Mould for wood, be that substrate carries out under the condition of fermentation culture with the Mierocrystalline cellulose earlier, being secreted into the outer albumen of born of the same parents all is the cellulose degradation involved enzyme basically; By observing among Fig. 2, each proteic expression amount all rises with fermentation time.
Three, the cellulase activity of crude enzyme liquid is identified
Each crude enzyme liquid that step 1 is obtained carries out cellulase activity mensuration with reference to the IUPAC standard method, and concrete grammar is following:
(1) gets the supernatant that 100 μ l step 1 obtain, add 10mL and contain in the citric acid-sodium citrate damping fluid (0.05M, pH4.8) of 0.05g/100mL Sodium Benzoate, obtain being diluted to 101 times enzyme liquid.
(2) Whatman1 filter paper is processed the shape that 6cm is long, 1cm is wide (about 50mg), be converted into 4 foldings, be the M type.
(3) packet transaction
Experimental group: the filter paper bar of step (2) is placed the test tube bottom, add citric acid-sodium citrate damping fluid (0.05M, the pH4.8) 1.5mL that contains the 0.05g/100mL Sodium Benzoate, 50 ℃ of water-baths 60 minutes; Add the enzyme liquid that 0.5mL step (1) obtains then, mix, make solution submergence filter paper in the pipe, 50 ℃ of water-baths 1 hour, cooling rapidly; In test tube, add 3mL DNS reagent, mix, in boiling water, boil 10min, cooling is settled to 25mL with zero(ppm) water rapidly.
Control group: the filter paper bar of step (2) is placed the test tube bottom; Add citric acid-sodium citrate damping fluid (0.05M, the pH4.8) 1.5mL contain the 0.05g/100mL Sodium Benzoate, 50 ℃ of water-baths 60 minutes make solution submergence filter paper in the pipe; 50 ℃ of water-baths 1 hour, cooling rapidly; In test tube, add 3mL DNS reagent, add the enzyme liquid that 0.5mL step (1) obtains then, mix, in boiling water, boil 10min, cooling is settled to 25mL with zero(ppm) water rapidly.
With the control group test tube is reference, measures the absorbancy (A of 540nm
540nm).
Under 50 ℃, the condition of pH4.8, PM hydrolysis filter paper substrate produces the needed enzyme amount of 1umol reducing sugar (with glucose meter) and is defined as an iu filter paper enzyme activity (1FPU).
Adopt glucose as standard substance, make A
540nmWith the typical curve of glucose concn, the typical curve equation is following: Y=0.6692X-0.0082, Y represent glucose concn (umol/mL), and X represents A
540nmNumerical value; R
2=0.9988.
Every milliliter of crude enzyme liquid enzyme biopsy is surveyed the result and is seen Fig. 3.The cellulase activity of the crude enzyme liquid that the reorganization fungus beetle obtains is significantly higher than the crude enzyme liquid that the bacterium that sets out obtains.The cellulase activity of the crude enzyme liquid that reorganization bacterium second obtains is consistent with the crude enzyme liquid that the bacterium that sets out obtains.The result shows, knock out the TrPK gene after, the ability of wooden mould production of cellulose enzyme is significantly increased.
Claims (10)
1. a method that makes up engineering bacteria comprises the steps: to suppress the mould middle TrPK expression of gene of wood, obtains engineering bacteria; Said TrPK genes encoding is (a) or (b) described TrPK albumen as follows:
(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;
(b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and synthetic relevant with cellulase during wood is mould by sequence 1 deutero-protein.
2. the method for claim 1, it is characterized in that: said TrPK gene is following 1) to 4) in arbitrary described dna molecular:
1) dna molecular shown in the sequence 2 of sequence table;
2) dna molecular shown in the sequence 3 of sequence table;
3) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of encoding said proteins;
4) with 1) or 2) dna molecular with 90% above homology and encoding said proteins of the dna sequence dna that limits.
3. according to claim 1 or claim 2 method is characterized in that: the implementation method of said " suppress wood mould in TrPK expression of gene " is following: knock out the said TrPK gene of wood in mould through homologous recombination.
4. method as claimed in claim 3 is characterized in that: the implementation method of said " knocking out the said TrPK gene of wood in mould through homologous recombination " is following: the recombinant plasmid that will contain the dna fragmentation first import said wood mould in; Said dna fragmentation first comprises the upper reaches homology arm of said TrPK gene and the downstream homology arm of said TrPK gene successively to downstream from the upper reaches.
5. method as claimed in claim 4 is characterized in that: in the said dna fragmentation first, between said upper reaches homology arm and said downstream homology arm, also have the selection markers gene.
6. like arbitrary described method in the claim 1 to 5, it is characterized in that: said wood is mould to be Trichodermareesei.
7. the engineering bacteria that arbitrary said method obtains in the claim 1 to 6.
8. the method for a production of cellulose enzyme, the said engineering bacteria of claim 7 that comprises the steps: to ferment obtains cellulase.
9. suppress the application of material in promoting wooden mould production of cellulose enzyme of TrPK genetic expression; Said TrPK genes encoding is (a) or (b) described TrPK albumen as follows:
(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;
(b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and synthetic relevant with cellulase during wood is mould by sequence 1 deutero-protein.
The application during 10.TrPK the cellulase of albumen in adjusting wood is mould is synthetic; Said TrPK albumen is (a) or (b) as follows:
(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;
(b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and synthetic relevant with cellulase during wood is mould by sequence 1 deutero-protein.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104130946A (en) * | 2014-05-16 | 2014-11-05 | 吉林大学 | Trichoderma reesei strain and preparation method thereof |
| CN114085782A (en) * | 2021-11-23 | 2022-02-25 | 深圳中科欣扬生物科技有限公司 | Ergothioneine synthetic gene derived from natural hot spring of Quzhuomu and development and application thereof |
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| CN101250485A (en) * | 2008-04-16 | 2008-08-27 | 中国石油化工股份有限公司 | Trichoderma reesei cultivation method for improving yield of cellulase |
| CN101735993A (en) * | 2010-01-19 | 2010-06-16 | 浙江大学 | Method for efficiently producing cellulase |
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| CN101250485A (en) * | 2008-04-16 | 2008-08-27 | 中国石油化工股份有限公司 | Trichoderma reesei cultivation method for improving yield of cellulase |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104130946A (en) * | 2014-05-16 | 2014-11-05 | 吉林大学 | Trichoderma reesei strain and preparation method thereof |
| CN104130946B (en) * | 2014-05-16 | 2017-04-12 | 吉林大学 | Trichoderma reesei strain and preparation method thereof |
| CN114085782A (en) * | 2021-11-23 | 2022-02-25 | 深圳中科欣扬生物科技有限公司 | Ergothioneine synthetic gene derived from natural hot spring of Quzhuomu and development and application thereof |
| CN114085782B (en) * | 2021-11-23 | 2023-10-24 | 深圳中科欣扬生物科技有限公司 | Ergothioneine synthetic gene from Qu Zhuomu natural hot spring and development and application thereof |
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