CN104450649B - The application of 6 albumen of F52 and its encoding gene and hydrolyzed xylan - Google Patents

The application of 6 albumen of F52 and its encoding gene and hydrolyzed xylan Download PDF

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CN104450649B
CN104450649B CN201410654103.XA CN201410654103A CN104450649B CN 104450649 B CN104450649 B CN 104450649B CN 201410654103 A CN201410654103 A CN 201410654103A CN 104450649 B CN104450649 B CN 104450649B
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albumen
sequence
dna molecular
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recombinant vector
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CN104450649A (en
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吴晓磊
王蒙
来国莉
聂勇
耿爽
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Peking University
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/248—Xylanases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2445—Beta-glucosidase (3.2.1.21)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01021—Beta-glucosidase (3.2.1.21)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Abstract

The invention discloses the application of 6 albumen of F52 and its encoding gene and hydrolyzed xylan.The invention provides a kind of albumen, is following protein 1) or 2)：1) protein that the amino acid residue shown in sequence in sequence table 2 is constituted；2) by the amino acid sequence residue sequence of sequence in sequence table 2 through the replacement of one or several amino acid residues and/or disappearance and/or add and with identical function by derived from 1) protein.The experiment proves that, present invention discover that new zytase F52 6, with xylan as substrate, under the conditions of pH 5.0, optimum temperature is 75 DEG C, after 70 DEG C of incubations 2 hours, remains to retain 82.3% vigor；At 4 DEG C, after being incubated 24 hours in different pH buffer solutions, can retain the residual activity more than 80% in the range of pH 4.0 12.0.

Description

The application of F52-6 albumen and its encoding gene and hydrolyzed xylan
Technical field
The present invention relates to biological technical field, more particularly to F52-6 albumen and its encoding gene and hydrolyzed xylan should With.
Background technology
Xylan (Xylan) is the important component of hemicellulose in plant cell wall, accounts for the 35% of dry cell weight, is certainly The renewable resource for so enriching in boundary, is only second to cellulose.Zytase (Xylananse) is that xylan hydrolysis can be by a class The enzyme of xylo-oligosaccharide and wood sugar, many nature microorganisms contain zytase.As zytase can be widely applied to The industry such as weaving, papermaking, food and bio-fuel, have attracted the research interest of more and more scholars in recent years.In order that enzyme reaches To higher heat resistance and heat endurance, many scholars screen new enzyme from thermophilic microorganism, but they can seldom be suitable for In industrial condition.In bio-fuel, the i.e. industry of hydrolyzing biomass production bio-ethanol, seldom there is enzyme efficiently can exist Good stability is kept under 50 DEG C of industrial condition.Which limits the high-efficient development of bio-fuel industry, and restriction life Thing fuel reduces the key factor of production cost.
In nature, uncultured microorganisms account for the 99% of all microorganisms.The research of most of screening zytase Still confine in educable 1% microorganism.Over nearly 10 years, with sequencing technologies and the development of High Throughput Screening Assay, People gradually begin one's study the resource contained in uncultured microorganisms, use technique of metagenome ripe day by day, and The enzyme with outstanding property in a large number is found that, illustrates that technique of metagenome excavates the bright prospects of new enzyme.
In the research that technique of metagenome screens new enzyme, screening efficiency from natural environments such as soil to animal alimentary canal, The reactor for arriving domestication again is incremented by successively.This is related to the intensity and selection pressure of domestication.In natural environment, than Such as forest soil, the several years are needed, microorganism could be by biomass degradation, in animal alimentary canal, and this reaction needs some My god, but hydrolysis efficiency is not still high, here it is the reason for herbivore is heavy；And for the reactor of domestication, three It can reach 90% conversion ratio.This research screens zytase from domestication's reactor, it is ensured that its high efficiency.
The content of the invention
It is an object of the present invention to provide a kind of albumen.
The albumen that the present invention is provided, F52-6 is following protein 1) or 2)：
1) protein that the amino acid residue shown in sequence in sequence table 2 is constituted；
2) by the amino acid residue sequence of the protein shown in 1) through the replacement of one or several amino acid residues and/or Disappearance and/or add and with identical function by derived from 1) protein.
Above-mentioned replacement and/or disappearance through one or several amino acid residues and/or it is added to less than 10 amino The replacement of sour residue and/or disappearance and/or addition.
The DNA molecular for encoding above-mentioned albumen is also the scope of protection of the invention.
Above-mentioned DNA molecular is following 1) -5) in arbitrary described DNA molecular：
1) code area is sequence 1 in sequence table；
2) code area is sequence 1 in sequence table from 5 ' end 14-1066 positions nucleotides；
3) code area is sequence 1 in sequence table from 5 ' end 11-1066 positions nucleotides；
4) under strict conditions with 1) or 2) or 3) hybridize and encode the DNA molecular with identical function albumen；
5) and 1) or 2) or 3) with more than 90% homology and encode the DNA molecular with identical function albumen.
Above-mentioned stringent condition can be that with 0.1 × SSPE (or 0.1 × SSC), the solution of 0.1%SDS is miscellaneous in DNA or RNA Hybridize at handing over 65 DEG C in testing and wash film.
Expression cassette containing above-mentioned DNA molecular, recombinant vector, recombinant bacterium, transgenic cell line or recombinant bacterium are also this The scope of bright protection.
In said method, the expression vector be pET-28a carriers, the recombinant vector embodiments of the invention be by Nde I and HindIII restriction enzyme site of the sequence 1 from 5 ' end 14-1066 positions nucleotides inserted pET-28a carriers in sequence table Between the carrier that obtains；
Above-mentioned recombinant vector is that above-mentioned DNA molecular is inserted the recombinant vector that expression vector is obtained.
Above-mentioned recombinant bacterium is that the recombinant vector is imported the recombinant bacterium obtained in purpose bacterium.
The purpose bacterium be Escherichia coli, specially Rosetta DE3.
Above-mentioned albumen as zytase or xylosidase or beta-glucosidase or the application in cellulase is being also The scope of protection of the invention.
The zymetology feature of the zytase is specially：It is 75 DEG C that optimum pH is 5.0, optimum temperature.
Above-mentioned albumen or above-mentioned DNA molecular or above-mentioned expression cassette, recombinant vector, recombinant bacterium, transgenic cell line or Recombinant bacterium is also protection of the present invention zytase or xylosidase or beta-glucosidase or the application in cellulase is prepared Scope.
It is a further object to provide one kind prepares zytase or xylosidase or beta-glucosidase or fibre The method of the plain enzyme of dimension.
The method of the present invention, is the above-mentioned recombinant bacterium of fermentation, that is, obtains zytase or xylosidase or beta-glucosidase Enzyme or cellulase.
In said method, the fermentation is to induce bottom fermentation culture in IPTG.
Application of the above-mentioned albumen in degradation biological matter is also the scope of protection of the invention, and the biomass are corn Core.
The experiment proves that, present invention discover that new zytase F52-6, with xylan as substrate, pH 5.0 Under the conditions of, optimum temperature is 75 DEG C, after incubating 2 hours at 70 DEG C, remains to retain 82.3% vigor；At 4 DEG C, different pH delay Rush after being incubated 24 hours in liquid, can retain the residual activity more than 80% in the range of pH 4.0-12.0.
Description of the drawings
Fig. 1 is the restructuring pET-28a-F52-6 expression vectors for building
Fig. 2 is the prediction to F52-6 three-dimensional structures and avtive spot
Fig. 3 is F52-6 abduction delivering SDS-PAGE collection of illustrative plates
Fig. 4 is impact of the temperature to F52-6 activity
Temperature stabilities of the Fig. 5 for F52-6
Optimal pH and pH stability of the Fig. 6 for F52-6
Fig. 7 is that F52-6 and F52-2 is studied to the hydrolysate of xylan
Fig. 8 is F52-6 and F52-2 and hydrolysate curves of the F52-6 to corncob.
Specific embodiment
Experimental technique used in following embodiments if no special instructions, is conventional method.
In following embodiments, material used, reagent etc., if no special instructions, commercially obtain.
The clone of embodiment 1, F52-6 genes
In early-stage Study, the grand genomic library of dry fermentation sludge system using Fosmid vector constructions.Using firm Arnotto-xylan flat board, screening have obtained a clone of the escherichia coli host containing xylanase activity.Contain in Fosmid Insert Fragment be 38176bp, ORF is predicted using softberry, and with NCBI and Pfam database annotations.Obtain F52-6 bases Cause, the nucleotides sequence of the gene be classified as sequence table in sequence 1 from 5 ' end 14-1063 positions nucleotides, the albumen life of its coding Entitled F52-6, its amino acid sequence are sequence 2 in sequence table from N ' ends 1-351 amino acids.Through comparing, F52-6 eggs There is 56% similitude of highest in vain with the zytase from Caldicoprobacter oshimai, predict which is xylan Enzyme.
Application of the embodiment 2, F52-6 as zytase
1st, the structure of recombinant vector
With artificial synthesized sequence 1 as template, it is primer with F52-6F and F52-6R, using TAKARA companies PrimeStar high-fidelity enzymes enter performing PCR amplification, obtain the PCR primer of 1075bp, and its nucleotides sequence is classified as sequence 1.
F52-6F:5’-GGAATTCCATATGGAAGCTGGAGGTGTTCCCAT-3 ' includes Nde I restriction enzyme sites
F52-6R:5’-CCCAAGCTTTTAGCGATCCTGTGCAGTGCT-3 ' includes Hind III digestions site
The system of above-mentioned PCR amplifications is as follows：
PCR programs are as follows：
By above-mentioned PCR primer Nde I and Hind III double digestions, the digestion products for obtaining and through same digestion PET-28a carriers (Novagen pET-28a DNA Cat.No.69864-3) connect, and obtain recombinant vector.
Through sequencing, the recombinant vector is from 5 ' end 14-1066 positions nucleotides inserteds pET- by sequence in sequence table 1 The carrier obtained between the Nde I and Hind III digestions site of 28a carriers, the albumen shown in expressed sequence 2, by the recombinant vector It is named as pET-28a-F52-6 (Fig. 1).
In DNA molecular in sequence table shown in sequence 1,11-1066 positions nucleotides is F52-6 genes；
In sequence table, in albumen shown in sequence 2, from N ' ends, 1-351 amino acids are F52-6 albumen.
2nd, the expression and purification of albumen
1) express
Above-mentioned recombinant vector pET-28a-F52-6 is converted into Rosseta DE3 competent cells, containing 50 μ g/ 37 DEG C of overnight incubations in the LB flat boards of mL kanamycins and 12.5 μ g/mL chloramphenicol, obtain single bacterium colony, bacterium colony PCR identifications, primer For F52-6F and F52-6R, obtain 1075bp for positive restructuring bacterium, be named as DE3/pET-28a-F52-6.
Empty carrier pET-28a is proceeded to by Rosseta DE3 competent cells using same method, DE3/pET- is obtained 28a。
DE3/pET-28a-F52-6 single bacterium colonies are seeded to into 100mL and contain 50 μ g/mL kanamycins and 12.5 μ g/mL chlorine 37 DEG C of overnight incubations in the LB culture mediums of mycin, when OD600nm reaches 0.4, add the IPTG of final concentration of 1mM (Isopropyl β-D-1-Thiogalactopyranoside) is induced four hours.1mL bacterium solutions are taken, collected after centrifugation thalline hangs In 200 μ L 1 × sds gel sample loading buffers, 10min, 25 DEG C of high speed centrifugation 1min are boiled, take supernatant, 20 μ L of applied sample amount. With DE3/pET-28a as control.
10%SDS polyacrylamide gel electrophoresises, coomassie brilliant blue staining detection protein expression, as a result as shown in figure 3,1 DE3/pET-28a-F52-6 is for DE3/pET-28a, 2-5, it can be seen that DE3/pET-28a-F52-6 is through four The induction of hour, F52-6 success abduction deliverings, destination protein size is 40KD.
2), purify
Collect DE3/pET-28a-F52-6 thalline, with PBS (NaCl 137mM, KCl 2.7mM, Na2HPO410mM,KH2PO42mM, pH 7.4) washing thalline three times, resuspended, three broken walls of multigelation.Centrifugation, collects supernatant, Using nickel column packing purifying protein.Cleaning solution (NaH first2PO450mM,NaCl 300mM,imidazole 20mM,pH 8.0) Wash three times, then eluent (NaH2PO48.0) 50mM, NaCl 300mM, imidazole 250mM, pH elute three times, washing And elution requirement is 500g, 2min, the eluent for collecting three times column volume is purpose albumen F52-6.
Destination protein F52-6 is carried out into SDS-PAGE detections, it is 40KD to obtain size, illustrates that purifying obtains destination protein F52-6。
3rd, the functional verification of destination protein F52-6 and enzyme Characteristics Detection
The measure of xylanase activity：Reaction system：+ 100 μ L 100mM sodium acetate buffers of 100 μ L, 1% xylan solutions Liquid.During 5 μ L enzyme liquids add the reaction system of 50 DEG C of preheatings, 5min is incubated, 500 μ L DNS reagents are subsequently added.Boiling water bath 5min Afterwards, light absorption value is determined at 540nm.
The enzyme activity (U) of one unit is defined as the amount (μ that enzyme molecule hydrolyzed xylan per minute discharges reduced sugar mol)。
1) condition determination of optimal pH：
Buffer solution is replaced by into different pH, enzyme activity is determined.The preparation of buffer solution：Disodium hydrogen phosphate-citric acid (3.5- 8.0), Tris-HCl (8.0-10.0), disodium hydrogen phosphate-NaOH (10.0-12.0).
As a result as shown in fig. 6, destination protein F52-6 can degradation of xylan, be zytase, and its optimal pH be 5.0.
2) measure of optimum temperature：
Enzyme reaction system is preheated into 5min at 20-90 DEG C in advance, enzyme is added and is reacted.React for pH 5.0.
As a result as shown in figure 4, destination protein F52-6 can degradation of xylan, be zytase, and its optimum temperature be 75 ℃。
3) temperature stability condition determination：
Enzyme is incubated 20min, 40min, 60min, 80min, 100min and 120min at various temperatures respectively, subsequently At 75 DEG C, pH 5.0 determines remaining enzyme activity；
As a result as shown in figure 5, destination protein F52-6 can degradation of xylan, be zytase, and its 70 DEG C incubate 2 After hour, 82.3% activity is still remained with.
4) pH Stability Determinations：
Enzyme is added in the buffer solution of correspondence pH, 24h is placed in 4 DEG C, finally at 75 DEG C, determine remaining under the conditions of pH 5.0 Activity.
As a result as shown in fig. 6, destination protein F52-6 can degradation of xylan, be zytase, and which be at 4 DEG C, it is different After being incubated 24 hours in pH buffer solutions, can retain the residual activity more than 80% in the range of pH 4.0-12.0.
5) measure of Michaelis constant：
Prepare the xylan solution of 0-45mg/mL, determine the reactive kinetics parameters (Lineweaver-Burk of enzyme plot)。
Method of the research of enzymolysis product using thin-layer chromatography, after 50 DEG C with substrate reactions 4h, sample point is in silica gel for enzyme On plate, chromatography poststaining observation catabolite.
As a result as shown in fig. 7, mark product are wood sugar X1, xylobiose X2 and xylotriose X3, swimming lane 1：Negative control；Swimming lane 2： F52-2 results of hydrolysis；Swimming lane three：F52-6 results of hydrolysis；Swimming lane four：F52-2+F52-6 results of hydrolysis, as a result shows, works as F52- During 2 independent role xylan, hydrolysate is only a small amount of wood sugar；When F52-6 independent roles, product be wood sugar, xylobiose with And a large amount of oligosaccharides；When both merge use, the amount of xylobiose is significantly reduced, and the amount of wood sugar increases, and illustrates the common of two kinds of enzymes Effect can improve the yield of wood sugar.
4th, F52-6 is to corncob degradation experiment
The ratio of 200U/g biomass (corncob) adds the F52-6 of above-mentioned 2 purifying, 50 DEG C of incubation 72h, liquid phase measurement sugar Content.
Separation condition：
Analytical column：Shim-pack ISA-07(4.0mm×25cm)
Mobile phase：A liquid 0.1M boric acid (being adjusted to pH=8 with potassium hydroxide)
B liquid 0.4M boric acid (being adjusted to pH=9 with potassium hydroxide)
Gradient：A liquid 100%-B liquid 100%, 2%/min linear gradients wash-out
Flow：0.6mL/min
Temperature：65℃
Derivatization conditions：
Derivative liquid：1%L- arginine, 3% boric acid
Flow velocity：0.5mL/min
Reaction temperature：150℃
As a result see Fig. 8, the hydrolysis efficiency of the enzyme is 23.1%, and after adding the F52-2 of 50U/g biomass, hydrolysis efficiency increases Add to 79.3%.
5th, the simulation of three-dimensional structure
Most suitable template is searched in swiss-model, finally image is modified using pymol softwares.Using Cluster W carry out Multiple Sequence Alignment, obtain avtive spot, see Fig. 2.
6th, impact of the metal ion (5mM) to enzymatic activity
Method：100 μ L, 1% xylan solutions (solvent is 100mM sodium-acetate buffers (pH 5.0))+100 μ L 100mM Sodium-acetate buffer (pH 5.0), wherein adding each metal ion species of final concentration of 5mM.The F52-6 of above-mentioned 2 purifying of 5 μ L adds Enter in the reaction system of 75 DEG C of preheatings, incubate 5min, be subsequently added 500 μ L DNS reagents.After boiling water bath 5min, at 540nm Determine light absorption value.
Table 1 is impact of the metal ion (5mM) to enzymatic activity
As a result it is as shown in table 1, it can be seen that most of metal ion is little to the activity influence of F52-6.Manganese ion has increasing The effect of strong enzyme activity, and mercury ion and nickel ion have to enzyme activity than stronger inhibitory action.
7th, the enzyme kinetics parameter of F52-6
Prepare the xylan solution of 0-45mg/mL ,+100 μ L 100mM sodium acetates of xylan solution of 100 μ L variable concentrations Buffer solution (pH 5.0).During the F52-6 of above-mentioned 2 purifying of 5 μ L adds the reaction system of 75 DEG C of preheatings, 5min is incubated, is subsequently added 500 μ L DNS reagents.After boiling water bath 5min, light absorption value is determined at 540nm.
As a result it is as shown in table 2, it can be seen that F52-6 is more affine in beech xylan, it is per second to convert 91 substrates point Son, and it is higher than the hydrolysis efficiency of oat xylan.
Enzyme kinetics parameter of the table 2 for F52-6
8th, the substrate specificity of F52-6
100 μ L different+100 μ L 100mM sodium-acetate buffers (pH 5.0) of substrate solution.Above-mentioned 2 purifying of 5 μ L During F52-6 adds the reaction system of 75 DEG C of preheatings, 30min is incubated, 500 μ L DNS reagents are subsequently added.After boiling water bath 5min, in Light absorption value is determined at 540nm.Substrate includes beech xylan, CMC (sodium carboxymethylcellulose), pNPX (p-nitrophenol wood sugars Glycosides), pNPG (p-nitrophenol glucoside), pNPA (p-nitrophenol Arabinoside), MIC (microcrystalline cellulose) and filter paper (50mg)。
As a result as shown in table 3, show that F52-6 has the extensive substrate-function spectrum of comparison, the active highest to xylan；In addition The activity of also certain xylosidase and beta-glucosidase, minimal amount of cellulase activity may also detect that.
Substrate specificity of the table 3 for F52-6

Claims (11)

1. a kind of albumen, the protein that the amino acid residue shown in sequence in sequence table 2 is constituted.
2. the DNA molecular of albumen described in claim 1 is encoded.
3. DNA molecular according to claim 2, it is characterised in that：The DNA molecular is following DNA molecular：Code area is In sequence table, sequence 1 is from 5 ' end 11-1066 positions nucleotides.
4. the recombinant vector containing DNA molecular described in Claims 2 or 3.
5. recombinant vector according to claim 4, it is characterised in that：The recombinant vector is by described in Claims 2 or 3 The recombinant vector that DNA molecular insertion expression vector is obtained.
6. the recombinant bacterium containing DNA molecular described in Claims 2 or 3.
7. recombinant bacterium according to claim 6, it is characterised in that：The recombinant bacterium is recombinant vector to be imported in purpose bacterium The recombinant bacterium for obtaining；
The recombinant vector is the recombinant vector for obtaining DNA molecular insertion expression vector described in Claims 2 or 3.
8. the albumen described in claim 1 is in as zytase or xylosidase or beta-glucosidase or cellulase Using；
The zymetology feature of the zytase is specially：It is 75 DEG C that optimum pH is 5.0, optimum temperature.
9. DNA molecular described in the albumen or Claims 2 or 3 described in claim 1 or the recombinant vector described in claim 4 or Recombinant bacterium described in claim 6 answering in zytase or xylosidase or beta-glucosidase or cellulase is prepared With.
10. a kind of method for preparing xyloglucanase enzymes or xylosidase or beta-glucosidase or cellulase, is fermentation right The recombinant bacterium described in 6 is required, that is, obtains xyloglucanase enzymes or xylosidase or beta-glucosidase or cellulase.
Application of the albumen described in 11. claims 1 in degradation biological matter, the biomass are corncob.
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