CN102747067B

CN102747067B - Application of TrPK protein in cellulase yield adjustment

Info

Publication number: CN102747067B
Application number: CN201210247297.2A
Authority: CN
Inventors: 董志扬; 陈秀珍
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2012-07-17
Filing date: 2012-07-17
Publication date: 2014-01-01
Anticipated expiration: 2032-07-17
Also published as: CN102747067A

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

The application of TrPK albumen in regulating yield of cellulase

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; find new renewable energy source and become the hot issue that current social is paid close attention to; the product that cellulose biomass absorbs sun power and forms by photosynthesis as plant 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, in lignocellulose resource conversion process, plays key effect, 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 by the mould fermentation of wood.Over 50 years, people make the ability of Trichodermareesei production of cellulose enzyme obtain significantly raising by methods such as optimum 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.Further improve yield of cellulase by traditional method very difficult.

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 builds engineering bacteria, comprise the steps: to suppress the expression of the mould middle TrPK gene of wood, obtain engineering bacteria; Described TrPK genes encoding following (a) or (b) described TrPK albumen:

(a) protein that the aminoacid sequence shown in sequence 1 forms in sequence table;

(b) by 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 to cellulase during wood the is mould protein derivative by sequence 1.

Described 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.

Described stringent condition is at 0.1 * SSPE(or 0.1 * SSC), in the solution of 0.1%SDS, hybridize under 65 ℃ of conditions and wash film.

The implementation method of described expression of mould middle TrPK gene " suppress wood " is as follows: by homologous recombination, knock out the described TrPK gene of wood in mould.

The implementation method of described " by homologous recombination, knocking out the described TrPK gene of wood in mould " is as follows: the recombinant plasmid that will contain the DNA fragmentation first import described wood mould in; Described DNA fragmentation first comprises the upstream homology arm of described TrPK gene and the downstream homology arm of described TrPK gene successively to downstream from upstream.

In described DNA fragmentation first, between described upstream homology arm and described downstream homology arm, also there is the selection markers gene.Described selection markers gene specifically can be the pyr4 gene; The gene of the pyr4 albumen shown in the sequence 5 that described pyr4 gene is the code sequence list.The reverse complementary sequence of described pyr4 gene specifically can be as the sequence 4 of sequence table from as shown in the 3000th to 4145 Nucleotide of 5 ' end.Described DNA fragmentation first specifically can comprise reverse complementary sequence and the described downstream homology arm of the expression cassette of described upstream homology arm, described pyr4 gene successively.Described upstream homology arm specifically can be as the sequence 4 of sequence table from as shown in the 9th to 1994 Nucleotide of 5 ' end.Described downstream homology arm specifically can be as the sequence 4 of sequence table from as shown in the 5511st to 7506 Nucleotide of 5 ' end.The reverse complementary sequence of the expression cassette of described pyr4 gene specifically can be as the sequence 4 of sequence table from as shown in the 2001st to 5504 Nucleotide of 5 ' end.Described DNA fragmentation first specifically can be the sequence 4 of sequence table from the double chain DNA molecule shown in the 9th to 7506 Nucleotide of 5 ' end.

The described recombinant plasmid that contains the DNA fragmentation first specifically can be the recombinant plasmid that the multiple clone site of described DNA fragmentation first importing plasmid pBluescript SK (+) is obtained.

The mould Trichodermareesei that can be of described wood, specifically can be TU6 △ tku70 bacterial strain, TU6 bacterial strain or QM9414 bacterial strain.

The engineering bacteria that above arbitrary described method obtains all belongs to protection scope of the present invention.

The present invention also protects a kind of method of production of cellulose enzyme, and the described engineering bacteria that comprises the steps: to ferment, obtain cellulase.The actual conditions of described fermentation is, described engineering bacteria is inoculated in to fermention medium, and 28 ℃, 48-144 hour is cultivated in the 200rpm concussion.

The present invention also protects the application of material in promoting wooden mould production of cellulose enzyme that suppresses described TrPK genetic expression.The material of the described TrPK genetic expression of described inhibition is the recombinant plasmid that contains above arbitrary described DNA fragmentation first.The material of the described TrPK genetic expression of described inhibition specifically can be the recombinant plasmid that the multiple clone site of described DNA fragmentation first importing plasmid pBluescript SK (+) is obtained.

The present invention also protects described TrPK albumen regulating the application of the cellulase of wood in mould in synthetic.

The present invention's discovery, after knocking out the TrPK gene, the ability of wooden mould production of cellulose enzyme significantly increases.The present invention has great value for the production of cellulase, for solving energy dilemma and ecocrisis, has potential impact.

The accompanying drawing explanation

Fig. 1 is that the PCR in embodiment 1 identifies electrophorogram.

Fig. 2 is crude enzyme liquid SDS-PAGE analytical electrophoresis figure in embodiment 3.

The cellulose enzyme activity that Fig. 3 is crude enzyme liquid in embodiment 3.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, be ordinary method.Test materials used in following embodiment, if no special instructions, be and purchase available from routine biochemistry reagent shop.Quantitative test in following examples, all arrange 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 derivative by the QM9414 bacterial strain, the pyr4 gene function is lost, uridylic auxotrophy bacterial strain, and ATCC is numbered MYA-256.

Microcrystalline Cellulose (Avicel ^rpH-101): purchased from fluka company, catalog number 11365.

Plasmid pBluescript SK (+): purchased from Stratagene company, catalog number 212205.

Trichodermareesei (Trichoderma reesei) TU6 △ tku70 bacterial strain is derivative by the TU6 bacterial strain, knocks out the bacterial strain that the tku70 gene of the non-homogeneous restructuring of mediation obtains, 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 to the potato chopping, add 90mL water, boil 30min, double gauze filters and collects filtrate, 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, the 5-dinitrosalicylic acid is dissolved in 4L water, constantly stirs, slowly add 80g sodium hydroxide, make it to dissolve fully, continue to stir, the 1500g Seignette salt is divided and adds for several times, 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, then brown bottle room temperature storage.

TrPK albumen (claiming again trpk albumen), as shown in the sequence 1 of sequence table, is comprised of 1166 amino-acid residues, theoretical molecular 128.98kDa, iso-electric point (PI) 5.205.The open reading frame of the encoding gene of TrPK albumen (claiming again TrPK gene or trpk gene) is as shown in the sequence 2 of sequence table, and its full-length cDNA is as shown in the sequence 3 of sequence table.

The preparation of embodiment 1, restructuring fungus beetle

One, the structure of recombinant plasmid

1, the acquisition of trpk upstream region of gene homology arm (Ptrpk, about 1987bp)

The genomic dna of TU6 △ tku70 bacterial strain of take is template, and the primer pair formed with Fup and Rup carries out pcr amplification, obtains pcr amplification product.

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(purchased from being King Company entirely).

Pcr amplification program: 95 ℃ of denaturation 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)

The genomic dna of TU6 △ tku70 bacterial strain of take is template, and the primer pair formed with Fdown and Rdown carries out pcr amplification, obtains pcr amplification product.

Fdown:5 '- aAGCTTcGAGGTAAGTCTGATATATCGAG-3 ' (underscore mark HindIII restriction endonuclease recognition sequence);

Rdown:5 '- gGTACCgTCTGTTCTATTTCTGGTCCTTTC-3 ' (underscore mark KpnI restriction endonuclease recognition sequence).

Pcr amplification program: 95 ℃ of denaturation 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)

The genomic dna of QM9414 bacterial strain of take is template, and the primer pair formed with Fpyr4 and Rpyr4 carries out pcr amplification, obtains pcr amplification product.

Fpyr4:5 '- gGATCCgGTTGATTGTTGCCGTCCGTTTCG-3 ' (underscore mark BamHI restriction endonuclease recognition sequence).

Rpyr4:5 '- aAGCTTtCGACTAGACTGACCCCCCCG-3 ' (underscore mark HindIII restriction endonuclease recognition sequence).

Pcr amplification program: 95 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 55 ℃ of annealing 40s, 72 ℃ prolong 2min, 30 circulations; 3min is extended in 72 ℃ of expansions.

4, with the pcr amplification product of restriction enzyme NotI and BamHI double digestion step 1, reclaim enzyme and cut product.

5, with the pcr amplification product of restriction enzyme HindIII and KpnI double digestion step 2, reclaim enzyme and cut product.

6, with the pcr amplification product of restriction enzyme BamHI and HindIII double digestion step 3, reclaim enzyme and cut product.

7, with restriction enzyme NotI and KpnI double digestion plasmid pBluescript SK (+), reclaim carrier framework (about 2961bp).

8, the enzyme that the enzyme that the enzyme of the enzyme of step 4 being cut to 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, recombinant plasmid pSK-Ptrpk-pyr4-Ttrpk is carried out to structrual description as follows: inserted the double chain DNA molecule shown in the sequence 4 of sequence table between the NotI of plasmid pBluescript SK (+) and KpnI restriction enzyme site.

In the sequence 4 of sequence table, from the 1st to 8 Nucleotide of 5 ' end, it is the NotI restriction endonuclease recognition sequence, the 9th to 1994 Nucleotide are trpk upstream region of gene homology arm, the 1995th to 2000 Nucleotide are the BamHI restriction endonuclease recognition sequence, the 2001st to 5504 reverse complementary sequences that Nucleotide is the pyr4 expression casette (the 2001st to 2999 reverse complementary sequences that Nucleotide is terminator wherein, the 3000th to 4145 reverse complementary sequences that Nucleotide is the pyr4 gene, the 4146th to 5504 reverse complementary sequences that Nucleotide is promotor), the 5505th to 5510 Nucleotide are the HindIII restriction endonuclease recognition sequence, the 5511st to 7506 Nucleotide are trpk gene downstream homology arm, the 7507th to 7512 Nucleotide are the KpnI restriction endonuclease recognition sequence.

Two, the acquisition of restructuring fungus beetle

1, Tu6 △ tku70 Strain Protoplast preparation

(1) get the spore of Tu6 △ tku70 bacterial strain, wash spore and make spore suspension with appropriate sterilized water, remove by filter remaining mycelia with 200 mesh sieve, 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 step (1) obtained filters through 200 mesh sieve, collects thalline, with sterilized water washing 2-3 time, then uses 1.2M MgSO ₄solution washing once.

(3) thalline step (2) obtained adds in the triangular flask that the 15mL lysate is housed that (lysate is the lyase that contains 150mg and the 1.2M MgSO of 15mg cellulase ₄the aqueous solution), 30 ℃, 80rpm oscillatory reaction 1.5h, the situation that micro-Microscopic observation protoplastis produces, observe once every the 10min sampling after reaction 1h.

(4) in 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 sieve, the centrifugal 10min of room temperature 3000rpm, collect the protoplastis precipitation.

(5) protoplastis of step (4) precipitation is resuspended with the 1.0M sorbitol aqueous solution, the centrifugal 10min of room temperature 3000rpm, collect the protoplastis precipitation.

(6) protoplastis of step (5) precipitation is resuspended with the 1.0M sorbitol aqueous solution, the centrifugal 10min of room temperature 3000rpm, collect protoplastis and precipitate and be suspended in 200 μ L 1.0M sorbitol aqueous solutions, and blood cell plate counter observes and count (10 ⁸individual protoplastis/mL).

2, the acquisition of restructuring fungus beetle

50%PEG4000 solution: contain 50g/100g PEG4000,50mM CaCl ₂10mM Tris-HCL (pH8.0) damping fluid.

(1) in the protoplastis that adds step 1 to prepare recombinant plasmid pSK-Ptrpk-pyr4-Ttrpk, mix gently, then add 50 μ L 50% PEG4000 solution, again mix, place 30min on ice.

(2) add 1mL 50% PEG4000 solution in the culture system of step (1), mix, room temperature is placed 20min.

(3) add 1mL 1.0M sorbitol aqueous solution in the culture system of step (2), after mixing, minutes four times and each MM solid medium (below 58 ℃) melted with a 4ml mix, be laid on immediately on the MM solid medium flat board containing the 1.0M sorbyl alcohol, cultivate 4-7d for 30 ℃.

(4) after son to be transformed grows, transfer them on the PDA flat board, cultivate 3-5 days (having spore to generate) for 28 ℃, with sterilized water, the spore on flat board is washed and got off to be prepared into spore suspension, after doing gradient dilution, it is coated on to the volume ratio containing 0.1%() the dull and stereotyped upper sheet spore of the MM solid medium of Triton-X100, after mycelia grows, the extracting genomic dna carries out the PCR evaluation.

PCR identifies that primer used is as follows:

Frup:5 '-CAACTATCTCCCCCTCCCAC-3 '; Be positioned at upstream homology arm end

Rfdown:5 '-CTCGATATATCAGACTTACCTCG-3 '; Be positioned at downstream homology arm front end

Tu6 △ tku70 bacterial strain can only obtain the fragment of about 3930bp, and the bacterial strain that can obtain about 3559bp fragment is the restructuring fungus beetle.

Qualification result is shown in Fig. 1 (arrow mark purpose band), and wherein M is 1kb DNA ladder marker, and 1 is the restructuring fungus beetle, and 2 is Tu6 △ tku70 bacterial strain.

The acquisition of embodiment 2, recombinant bacterium second

Replace recombinant plasmid pSK-Ptrpk-pyr4-Ttrpk to carry out the operation of the step 2 of embodiment 1 plasmid pBluescript SK (+), obtain recombinant bacterium second.

The fermentation of embodiment 3, recombinant bacterium and the cellulase activity of crude enzyme liquid are identified

Recombinant bacterium second or the bacterium that sets out (TU6 △ tku70 bacterial strain) that the restructuring fungus beetle that embodiment 1 is obtained, embodiment 2 obtain carry out respectively following steps:

One, the fermentation of recombinant bacterium

1, bacterial strain is gone on PDA solid medium flat board and produce spore, then with sterilized water washing spore and make the spore suspension 4 * 10 of high density with sterilized water ⁷-5 * 10 ⁷individual spore/mL.

2, the 50mL 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 preculture 48h, four layers of filtered through gauze are also collected thalline.

3, take 1.8 gram thalline (weight in wet base) in the 50mL fermention medium, 28 ℃, 200rpm shakes cultivation, respectively at shaking culture, after 48 hours, 72 hours, 96 hours, 120 hours and 144 hours, samples.

Fermention medium (pH5.2 ± 0.1): containing the MM liquid nutrient medium of 1g/100mL Microcrystalline Cellulose.

4, the centrifugal 5min of sample 12000r/min step 3 obtained, collect supernatant liquor.

Restructuring fungus beetle shaking culture is sampled and carries out the supernatant liquor called after restructuring fungus beetle crude enzyme liquid that step 4 obtains in 48 hours _{48 hours}.Restructuring fungus beetle shaking culture is sampled and carries out the supernatant liquor called after restructuring fungus beetle crude enzyme liquid that step 4 obtains in 72 hours _{72 hours}.Restructuring fungus beetle shaking culture is sampled and carries out the supernatant liquor called after restructuring fungus beetle crude enzyme liquid that step 4 obtains in 96 hours _{96 hours}.Restructuring fungus beetle shaking culture is sampled and carries out the supernatant liquor called after restructuring fungus beetle crude enzyme liquid that step 4 obtains in 120 hours _{120 hours}.Restructuring fungus beetle shaking culture is sampled and carries out the supernatant liquor called after restructuring fungus beetle crude enzyme liquid that step 4 obtains in 144 hours _{144 hours}.

Recombinant bacterium second shaking culture is sampled and carries out the supernatant liquor called after recombinant bacterium second crude enzyme liquid that step 4 obtains in 48 hours _{48 hours}.Recombinant bacterium second shaking culture is sampled and carries out the supernatant liquor called after recombinant bacterium second crude enzyme liquid that step 4 obtains in 72 hours _{72 hours}.Recombinant bacterium second shaking culture is sampled and carries out the supernatant liquor called after recombinant bacterium second crude enzyme liquid that step 4 obtains in 96 hours _{96 hours}.Recombinant bacterium second shaking culture is sampled and carries out the supernatant liquor called after recombinant bacterium second crude enzyme liquid that step 4 obtains in 120 hours _{120 hours}.Recombinant bacterium second shaking culture is sampled and carries out the supernatant liquor called after recombinant bacterium second crude enzyme liquid that step 4 obtains in 144 hours _{144 hours}.

The bacterium shaking culture of setting out is sampled and carries out supernatant liquor called after that step 4 the obtains bacterium crude enzyme liquid that sets out in 48 hours _{48 is little} _{the time}.The bacterium shaking culture of setting out is sampled and carries out supernatant liquor called after that step 4 the obtains bacterium crude enzyme liquid that sets out in 72 hours _{72 hours}.The bacterium shaking culture of setting out is sampled and carries out supernatant liquor called after that step 4 the obtains bacterium crude enzyme liquid that sets out in 96 hours _{96 hours}.The bacterium shaking culture of setting out is sampled and carries out supernatant liquor called after that step 4 the obtains bacterium crude enzyme liquid that sets out in 120 hours _{120 hours}.The bacterium shaking culture of setting out is sampled and carries out supernatant liquor 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, the results are shown in Figure 2.In Fig. 2,1 represents the bacterium that sets out, 2 representative restructuring fungus beetles, and 48h, 72h, 96h, 120h and 144h represent fermentation time.Because crude enzyme liquid is fermented liquid supernatant, according to prior art and document record, mould for wood, the Mierocrystalline cellulose of first take carries out under the condition of fermentation culture as substrate, being secreted into the outer albumen of born of the same parents is all the cellulose degradation involved enzyme basically, in Fig. 2, can observe, the expression amount of each albumen 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 as follows:

(1) get the supernatant liquor that 100 μ l step 1 obtain, add 10mL to contain in the citric acid-sodium citrate damping fluid (0.05M, pH4.8) of 0.05g/100mL Sodium Benzoate, obtain being diluted to the enzyme liquid of 101 times.

(2) Whatman1 filter paper is made to the shape that 6cm is long, 1cm is wide (50mg left and right), be converted into 4 foldings, be the M type.

(3) packet transaction

Experimental group: the filter paper bar of step (2) is placed in to test tube bottom, adds citric acid-sodium citrate damping fluid (0.05M, the pH4.8) 1.5mL containing the 0.05g/100mL Sodium Benzoate, 50 ℃ of water-baths 60 minutes; Then the enzyme liquid that adds 0.5mL step (1) to obtain, mix, and makes to manage interior solution submergence filter paper, and 50 ℃ of water-baths 1 hour are cooling rapidly; Add 3mL DNS reagent in test tube, mix, boil 10min in boiling water, cooling rapidly, be settled to 25mL with distilled water.

Control group: the filter paper bar of step (2) is placed in to the test tube bottom, add citric acid-sodium citrate damping fluid (0.05M, the pH4.8) 1.5mL containing the 0.05g/100mL Sodium Benzoate, 50 ℃ of water-baths 60 minutes, make to manage interior solution submergence filter paper, 50 ℃ of water-baths 1 hour, cooling rapidly; Add 3mL DNS reagent in test tube, the enzyme liquid that then adds 0.5mL step (1) to obtain, mix, and boils 10min in boiling water, cooling rapidly, with distilled water, is settled to 25mL.

Take the control group test tube as reference, measure the absorbancy (A of 540nm _540nm).

Under 50 ℃, the condition of pH4.8, per minute hydrolysis filter paper substrate, produce the needed enzyme amount of 1umol reducing sugar (with glucose meter) and be defined as an international unit filter paper enzyme activity (1FPU).

Adopt glucose as standard substance, make A _540nmwith the typical curve of glucose concn, the typical curve equation is as follows: Y=0.6692X-0.0082, and Y represents glucose concn (umol/mL), X represents A _540nmnumerical value; R ²=0.9988.

Every milliliter of crude enzyme liquid enzyme biopsy is surveyed and be the results are shown in Figure 3.The cellulase activity of the crude enzyme liquid that the restructuring 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 recombinant bacterium second obtains is consistent with the crude enzyme liquid that the bacterium that sets out obtains.Result shows, after knocking out the TrPK gene, can make the ability of wooden mould production of cellulose enzyme significantly increase.

Claims

The application during 1.TrPK the cellulase of albumen in adjusting wood is mould is synthetic; Described TrPK albumen is the protein that the aminoacid sequence shown in sequence 1 forms in sequence table.