CN110358755A

CN110358755A - Acid high temperature resistant recombinant fiber element enzyme and its application

Info

Publication number: CN110358755A
Application number: CN201910686471.5A
Authority: CN
Inventors: 李相前; 夏继林; 朱晓燕; 时号
Original assignee: Huaiyin Institute of Technology
Current assignee: Huaiyin Institute of Technology
Priority date: 2019-07-26
Filing date: 2019-07-26
Publication date: 2019-10-22
Anticipated expiration: 2039-07-26
Also published as: CN110358755B

Abstract

The invention discloses three kinds of acid high temperature resistant recombinant fiber element enzymes and its applications, and the amino acid sequence of three kinds of recombinant fiber element enzymes is respectively as shown in SEQIDNO:1-3.Three kinds of recombinant fiber element enzymes of the invention are up to 1.540U/mg to CMC-Na enzyme activity under the conditions of being 4.0~5.0,60~80 DEG C in pH, are up to 0.167U/mg to Avicel vigor.Three kinds of recombinant fiber element enzymes of the invention are at 75 DEG C, and heat preservation 2h enzyme activity is held essentially constant under the conditions of pH is 4.0~4.5.Three kinds of recombinant fiber element enzymes can directly degrade CMC-Na, avicel cellulose, filter paper and other natural wooden fiber's element such as rice straws and wheat stalk, be up to 0.106U/mg to the vigor of rice straw.Three kinds of recombinant fiber element enzymes of the invention have bigger superiority than existing cellulase, and can effectively degrade natural wooden fiber's element, have potential industrial application value.

Description

Acid high temperature resistant recombinant fiber element enzyme and its application

Technical field

The invention belongs to technique for gene engineering and biorefinery technical fields, and in particular to three kinds of acid high temperature resistant recombinations Cellulase and its application.

Background technique

Lignocellulosic can be used for producing bio-fuel or other high value added products as a kind of renewable resource.However Cellulose in lignocellulosic is wrapped up by hemicellulose and lignin, and to be covalently keyed and the fento of cellulose itself Dimension crosslinking is so that it is difficult to be degraded by cellulase, therefore is difficult to be translated into fermentable sugars.Lignocellulosic is carried out Pretreatment can destroy hydrogen bond therein, remove lignin and hemicellulose, the accessibility of reinforcing fiber element enzyme.However conventional acid Alkali, the pretreatments such as ionic liquid not only increase production cost, and easily lead to serious environmental pollution.In addition, artificial synthesized Substrate such as: the ingredient of filter paper, avicel cellulose and CMC-Na is single, and structure is simple, only by different number of glucose molecule Composition.Therefore, by filter paper, avicel cellulose and CMC-Na is degradable only needs inside and outside to cut dextranase for glucose molecule With the synergistic effect of beta-glucosidase.However, need to a variety of glycoside hydrolases and non-to the degradable of natural wooden fiber's element The protein-bonded synergistic effect of enzyme, wherein endoglucanase, carbohydrate binding domain (CBM) and exoglucanase Play the role of in the process vital.The single catalytic domain cellulase to dissociate in nature often carries CBM structural domain, Catalyst structure domain can be securely adsorbed on substrate by CBM, improve catalytic efficiency.And endoglucanase cut at random first it is non- Crystal region discharges oligonucleotide chain, and secondly exoglucanase gradually cuts release fibre along crystal region or the end of noncrystalline domain oligonucleotide chain Tie up disaccharides.However, there is the invalid suction of individual inefficient enzymes during a variety of free cellulose enzyme mixed degradation lignocellulosics Attached problem.In addition, the endoglucanase in the source Thermophilic Bacteria Caldicellulosiruptor saccharolyticus (Endo5-CBM28) it can directly degrade Avicel with exoglucanase (CBM3b-Exo5), lignocellulosic can be saved Pre-treatment step saves cost and protection environment.However Endo5-CBM28 is 8.94U/mg to CMC-Na vigor, however it is right The vigor of Avicel and FP only has 0.0113U/mg and 0.0518U/mg respectively.In addition, Endo5 is to Avicel debility.

Summary of the invention

Goal of the invention: the problem of existing in view of the deficiencies of the prior art, to give full play to lignocellulolytic enzymes and CBM Intramolecular synergistic effect, in Endo5, CBM and Exo5 realize fusion, construct three kinds of fusion enzymes.The present invention provides three kinds entirely New acid high temperature resistant recombinant fiber element enzyme Endo5-2CBM-Exo5, Endo5-CBM3b-Exo5 and Endo5-CBM28-Exo5, Three kinds of recombinant fiber element enzymes of the invention all have acid-resistant and anti-high-temperature, and activity is high, and can directly degrade CMC-Na, avicel cellulose (Avicel), filter paper (FP) and other natural wooden fibers element.

The present invention also provides the nucleotide sequence of three kinds of encoding acidic high temperature resistant recombinant fiber element enzymes and three kinds of acidity are resistance to The application of high temperature recombinant fiber element.

Technical solution: in order to achieve the above-mentioned object of the invention, acidity high temperature resistant recombinant fiber element enzyme, described as described herein Acid high temperature resistant recombinant fiber element enzyme includes three kinds of acid high temperature resistant recombinant fiber element enzymes, and amino acid sequence is respectively such as Shown in SEQIDNO.1-3.It is respectively designated as Endo5-2CBM-Exo5, Endo5-CBM3b-Exo5 and Endo5-CBM28-Exo5.

Further, described three kinds acid high temperature resistant recombinant fiber element enzymes, DNA sequence dna is respectively such as SEQIDNO.4-6 institute Show.Respectively endo5-2cbm-exo5, endo5-cbm3b-exo5 and endo5-cbm28-exo5.

Wherein, the DNA sequence dna SEQIDNO.4-6 is expanded by following primer (SEQ ID NO.11-16) respectively Splicing obtains afterwards:

SEQ ID NO.11

Endo-F:5 '-AACGATCCATGGCTAATACTGCGTATGAAAAGGATA-3 '；

SEQ ID NO.12

Endocbm-R:5 '-AACGATGAGCTCTGTAAATCTTACATTGTCTAAG-3 '；

SEQ ID NO.13

Endo-R:5 '-AACGATGAGCTCTTTCTGGTCGCTGCCAGG-3 '；

SEQ ID NO.14

CbmExo-F:5 '-AACGATGAGCTCGGGGTAACAACATCATCTC-3 '；

SEQ ID NO.15

Exo-R:5 '-AACGATCTCGAGTTTTGAAGCTGGAACTGGCTC-3 '；

SEQ ID NO.16

Exo-F:5 '-AACGATGAGCTCTTTAAGAGTGGAGCAGGGCA-3 '.

Recombinant vector of the present invention, the recombinant vector clone includes any one DNA in SEQ ID NO.4-6 Sequence.

Recombinant bacterium of the present invention, the recombinant bacterium include the recombinant vector in claim 3.

Acidity high temperature resistant recombinant fiber element enzyme of the present invention is fine in enzymatic hydrolysis CMC-Na, filter paper, Avicel and natural wood Application in dimension element.

Preferably, natural wooden fiber's element includes rice straw and wheat stalk.

Further, the enzymolysis temperature is 60~80 DEG C, and pH is 4.0~4.5, reaction time 4h.

Recombinant vector of the present invention answering in enzymatic hydrolysis CMC-Na, filter paper, Avicel and natural wooden fiber's element With.

Three kinds of the present invention acid high temperature-resisting cellulases, the sequence of amino acid sequence SEQIDNO.1,2 and 3, by each Structural domain forms the fusion protein with difunctional cellulase by different amalgamation modes.

The characteristics of present invention is according to existing enzyme Endo5-CBM28, Endo5, CBM3b-Exo5 and Exo5 and Binding Capacity (another characteristic that can also add some points analysis, we synthesize new template again), devise completely new gene endo5-cbm28, endo5, Cbm3b-exo5 and exo5 gene (SEQ ID NO.7-10), and design completely new primer pair (SEQIDNO.11-16), primer and Newly-designed gene template carries out careful comparison, effectively prevents non-specific combination, then synthesized, then with completely new gene Based on endo5-cbm28, endo5, cbm3b-exo5 and exo5 gene, completely new recombination endo5- is dexterously constructed 2cbm-exo5, endo5-cbm3b-exo5 and endo5-cbm28-exo5 (SEQ ID NO.4-6), finally obtain of the invention Three kinds of completely new acid high temperature resistant recombinant fiber element enzyme Endo5-2CBM-Exo5, Endo5-CBM3b-Exo5 and Endo5- CBM28-Exo5.The three kinds of enzymes of designed completely new gene and formation completely newly are for substrate such as CMC-Na, filter paper, Avicel and day Right cellulosic activity is more preferable, and hydrolysis result is more excellent.

The utility model has the advantages that compared with prior art, the present invention has the advantage that

Three kinds provided by the invention completely new recombinant fiber element enzymes have acid-resistant and anti-high-temperature, pH be 4.0~5.0,60~ 80 DEG C of conditions are 1.540U/mg to CMC-Na enzyme activity highest；It is up to 0.167U/mg to Avicel enzyme activity；At 70~80 DEG C, PH shows high vigor under the conditions of being 4.0~6.0.Three kinds of recombinant fiber element enzymes are at 75 DEG C, under the conditions of pH is 4.0~4.5 Heat preservation 2h enzyme activity is held essentially constant.Avicel, FP in addition, three kinds of recombinant fiber element enzymes can directly degrade) and it is other natural Lignocellulosic such as rice straw (RS) and wheat stalk (WS), are up to 0.106U/mg to the vigor of rice straw.It is above-mentioned Characteristic makes the obtained three kinds of recombinant fiber element enzymes of the present invention have bigger superiority and wider array of than existing cellulase Substrate spectrum is 4.0~5.0,60~80 DEG C or so degradation natural wooden fiber elements in pH, and activity and degradation effect are substantially better than existing There are the enzyme Endo5-CBM28 and Endo5 and other cellulases in the prior art of endo5-cbm28, endo5 gene expression, And recombinant fiber element enzyme of the invention is easy to express, and can stablize acquisition, has potential industrial application value.

Detailed description of the invention

Fig. 1 is that three kinds of double catalytic domain cellulases construct mode figure.

Fig. 2 is three kinds of double catalytic domain cellulase Endo5-2CBM-Exo5, Endo5-CBM3b-Exo5 and Endo5- The SDS-PAGE protein electrophoresis figure of CBM28-Exo5；

Fig. 3 is the change of Endo5-2CBM-Exo5, Endo5-CBM3b-Exo5 and Endo5-CBM28-Exo5 activity with temperature Change result figure；

Fig. 4 is variation of Endo5-2CBM-Exo5, Endo5-CBM3b-Exo5 and Endo5-CBM28-Exo5 activity with pH Result figure；

Fig. 5 is Endo5-2CBM-Exo5, Endo5-CBM3b-Exo5 and Endo5-CBM28-Exo5 thermostabilization result of variations Figure；

Fig. 6 is Endo5-2CBM-Exo5, Endo5-CBM3b-Exo5 and Endo5-CBM28-Exo5pH steady change result Figure；

Fig. 7 is Endo5-2CBM-Exo5, Endo5-CBM3b-Exo5 and Endo5-CBM28-Exo5 degradation FP, Avicel And natural wooden fiber element TLC figure.

Specific embodiment

Below with reference to embodiment and attached drawing, the present invention is described further.

Material used in following embodiment, reagent etc. commercially obtain unless otherwise specified.

Embodiment 1

The base of endoglucanase (Endo5), carbohydrate binding domain (CBM) and exoglucanase (Exo5) Because of preparation.

Artificial synthesized mode can be used and prepare original in the prior art endo5-cbm28, endo5, cbm3b-exo5 With exo5 gene, (U.S.'s culture presevation can also be purchased from the total DNA of Caldicellulosiruptor saccharolyticus Center) it is that template is obtained by PCR mode.

Completely new endo5-cbm28, endo5, cbm3b-exo5 and exo5 gene of the present embodiment is according to enzyme Endo5- CBM28, Endo5, CBM3b-Exo5 and Exo5 and the characteristics of Binding Capacity by sequence alignment, predict, devise new sequence, And new sequence is sent to the raw work full genome synthesis in Shanghai.Designed completely new gene for substrate such as CMC-Na, filter paper, Avicel, Plain active more preferable with natural wooden fiber, hydrolysis result is more excellent.

Using the new sequence of full genome synthesis as template, with following completely new primer pair (SEQ ID NO.11-16), expand respectively Increase endo5-cbm28, endo5, cbm3b-exo5 and exo5 gene.

Endo-F:5 '-AACGATCCATGGCTAATACTGCGTATGAAAAGGATA-3'；

Endocbm-R:5 '-AACGATGAGCTCTGTAAATCTTACATTGTCTAAG-3'；

Endo-R:5 '-AACGATGAGCTCTTTCTGGTCGCTGCCAGG-3'；

CbmExo-F:5 '-AACGATGAGCTCGGGGTAACAACATCATCTC-3'；

Exo-R:5 '-AACGATCTCGAGTTTTGAAGCTGGAACTGGCTC-3'；

Exo-F:5 '-AACGATGAGCTCTTTAAGAGTGGAGCAGGGCA-3’。

When above-mentioned primer synthesizes, Endo-F introduces NcoI restriction enzyme site, Endocbm-R, Endo-R, cbmExo-F and Exo- F introduces SacI restriction enzyme site respectively, and Exo-R introduces XhoI restriction enzyme site.

PCR reaction system: the C.saccharolyticus genomic DNA of 1 μ L, Endo-F, Endocbm-R, Endo-R, CbmExo-F, Exo-F and Exo-R add 1 μ L, 25 μ LBiouniquer2 × PCRMasterMix, 22 μ L ultrapure waters respectively.

PCR reaction condition: 94 DEG C of denaturation 5min；94 DEG C of denaturation 30sec, 50~55 DEG C of annealing 30sec, 72 DEG C extend 30~ 120sec, 30Cycles；72 DEG C of extension 10min；4 DEG C of heat preservations.

PCR product detects yield with 1% agarose gel electrophoresis and specificity, blend compounds QIAquick Gel Extraction Kit are purified (Trans, Nanjing).

Endo5-cbm28 gene is obtained by primer pair amplifies of Endo-F and Endocbm-R；It is with Endo-F and Endo-R Primer pair amplifies obtain endo5 gene；Cbm3b-exo5 gene is obtained by primer pair amplifies of cbmExo-F and Exo-R；With Exo-F and Exo-R is that primer pair amplifies obtain exo5 gene.Finally obtain newly-designed gene endo5-cbm28, endo5, Cbm3b-exo5 and exo5 gene order is respectively such as SEQ ID NO.7-10.SEQ ID NO.7 (endo5-cbm28), SEQ ID NO.8 (endo5), SEQ ID NO.9 (cbm3b-exo5), SEQ ID NO.10 (exo5).

Embodiment 2

Recombinant clone, expression vector pET28a-endo5-2cbm-exo5, pET28a-endo5-cbm3b-exo5 and The building and verifying of pET28a-endo5-cbm28-exo5

By PCR product endo5-cbm28, endo5 (preparation of embodiment 1) and pET28a (Novagen) difference after purification With NcoI and SacI double digestion, agarose gel electrophoresis recycles digestion PCR fragment and carrier large fragment.Purpose after being tapped and recovered Segment and carrier, 8 μ L sterile waters of concentrated addition are resuspended.1 μ L10 × LigaseBuffer and 1 μ LLigase is added, in 16 DEG C Connection overnight.Connection product converts Escherichia coli (E.coliDH5a), is then coated on containing 50 μ g/mL kanamycins (Kan) LB solid medium, 37 DEG C of 13~15h of culture.

From the multiple single colonies of picking on conversion plate, plasmid is extracted using the raw work plasmid extraction kit in Shanghai.To extraction Plasmid carry out double digestion verifying after carry out sequence verification.Sequencing result is shown, is inserted and is cloned respectively in pET28a carrier Target fragment (endo5-cbm28 and endo5 size is respectively 1629bp and 966bp), respectively recombinant vector pET28a- Endo5-cbm28 and pET28a-endo5.It is inoculated with positive recombinant, plasmid is extracted using the raw work plasmid extraction kit in Shanghai. Again by after purification PCR product cbm3b-exo5, exo5 (preparation of embodiment 1) and pET28a-endo5-cbm28, pET28a- Endo5 (preparation of embodiment 2) uses SacI and XhoI double digestion respectively, and agarose gel electrophoresis recycles digestion PCR fragment and carrier Large fragment.Target fragment cbm3b-exo5 and exo5 (preparation of embodiment 1) after being tapped and recovered are inserted into pET28a-endo5- respectively The downstream of cbm28；The downstream of target fragment cbm3b-exo5 insertion pET28a-endo5.8 μ L of each concentrated addition of segment are sterile Water is resuspended.1 μ L10 × LigaseBuffer and 1 μ LLigase is added, in 16 DEG C of connections overnight.Connection product converts Escherichia coli (E.coliDH5a), the LB solid medium containing 50 μ g/mL kanamycins (Kan), 37 DEG C of 13~15h of culture are then coated on. From the multiple single colonies of picking on conversion plate, plasmid is extracted using the raw work plasmid extraction kit in Shanghai.To the plasmid of extraction into Sequence verification is carried out after the verifying of row double digestion.Sequencing result is shown, inserts institute in pET28a-endo5-cbm28 carrier respectively The target fragment of clone (cbm3b-exo5 and exo5 size is respectively 1998bp and 1599bp)；It is inserted in pET28a-endo5 carrier The target fragment cloned has been entered (cbm3b-exo5 size is not 1998bp).And then respectively obtain recombinant expression carrier PET28a-endo5-2cbm-exo5, pET28a-endo5-cbm3b-exo5 and pET28a-endo5-cbm28-exo5.Wherein The DNA sequence dna of endo5-2cbm-exo5, endo5-cbm3b-exo5 and endo5-cbm28-exo5 such as SEQ ID NO.4,5 and 6 Shown, the amino acid sequence of the albumen (acid high temperature resistant recombinant fiber element enzyme) of translation is as shown in SEQ ID NO.1,2 and 3. These types of albumen is respectively designated as Endo5-2CBM-Exo5, Endo5-CBM3b-Exo5 and Endo5-CBM28-Exo5.Three kinds Double catalytic domain cellulases construct as shown in Figure 1.

SEQ ID NO.1 (Endo5-2CBM-Exo5), SEQ ID NO.2 (Endo5-CBM3b-Exo5), SEQ ID NO.3 (Endo5-CBM28-Exo5), SEQ ID NO.4 (endo5-2cbm-exo5), SEQ ID NO.5 (endo5-cbm3b- Exo5), SEQ ID NO.6 (endo5-cbm28-exo5).

Embodiment 3

The expression of recombinant fiber element enzyme Endo5-2CBM-Exo5, Endo5-CBM3b-Exo5 and Endo5-CBM28-Exo5 With purifying.

By recombinant clone, expression vector pET28a-endo5-2cbm-exo5, pET28a-endo5-cbm3b-exo5 and PET28a-endo5-cbm28-exo5 (preparation of embodiment 2) chemical conversion is to expressive host E.coliBL21 (DE3) (Novagen), the recombinant bacterium containing recombinant plasmid is obtained.Inoculation positive recombinant contains the Luria- of 50 μ g/mLKan in 5mL Bertanibroth (LB) fluid nutrient medium, is incubated overnight under the conditions of 200rpm by 37 DEG C.Transfer 4mL bacterium solution in contain 50 μ g/ The 500mLLB fluid nutrient medium of mLKan, cultivates cell concentration (OD by 37 DEG C under the conditions of 200rpm₆₀₀) to 0.6~0.8.It is added eventually Concentration is the IPTG of 0.5mM, at 25 DEG C, 12~14h of inducing expression under the conditions of 200rpm.By culture solution in high speed freezing centrifuge In with 4 DEG C, 10000rpm, centrifugation 8min collect thallus.The sterile water washing thalline of 40mL is added, again with 4 DEG C, 10000rpm is centrifuged 8min, discards supernatant.1 × bindingbuffer (NaCl of 0.5M, 20mM of 20mL are added into precipitating Tris-HCl, the imidazoles of 5mM, pH7.9), in ice-water bath pass through ultrasonic cell disruption instrument ultrasonication.Clasmatosis liquid At 4 DEG C, 15000rpm, it is centrifuged 15min, obtains the crude enzyme liquid containing acid high temperature resistant recombinant fiber element enzyme.

Crude enzyme liquid is first through 70 DEG C of heat pre-treatment 15min to remove the poor foreign protein of thermal stability, after Ni-NTA parent (operating procedure is shown in His-BindKits, Novagen) is purified with chromatographic column.The purity and molecular weight identification of enzyme are adopted after purification It is carried out with SDS-PAGE, as a result sees that Fig. 2,1,2 and No. 3 swimming lane are Endo5-2CBM-Exo5, Endo5- after purifying respectively CBM28-Exo5 and Endo5-CBM3b-Exo5, molecular weight are respectively 129kDa, 114kDa and 111kDa, close with theoretical value.

Embodiment 4

The zymetology of recombinant fiber element enzyme Endo5-2CBM-Exo5, Endo5-CBM3b-Exo5 and Endo5-CBM28-Exo5 Specificity analysis.

Enzyme activity definition: under pH value 4.5 and 80 DEG C reaction condition, CMC-Na substrate of degrading per minute releases 1 μm of Portugal ol The required enzyme amount of grape sugar.

(1) optimum temperature

The imidazoles for the 100mM for being 4.5 using pH value-Potassium Hydrogen Phthalate buffer dilutes what embodiment 3 was prepared Pure enzyme solution (20 times of dilution), carries out vitality test using the pure enzyme solution after dilution.The reaction system of enzyme activity determination is 150 μ L, packet 1%CMC-Na substrate containing 50 μ L, imidazoles-Potassium Hydrogen Phthalate buffer of the 100mM of 50 μ L are pure after the dilution of 50 μ L Enzyme solution, the pH of reaction system are 4.5.After reaction system is kept the temperature 30min in 40~90 DEG C of water-baths, it is immediately inserted into ice bath termination Reaction.The DNS reagent of 150 μ L is added, after boiling water bath boils 5min, is immediately inserted into ice bath and terminates reaction.Moisturizing is measured to 1mL Absorbance value at 520nm, wherein not enzyme reaction tube is as blank control.Experiment sets 3 repetitions, calculates enzyme activity, makes even Mean value mapping.As a result as shown in figure 3, result of study shows Endo5-2CBM-Exo5, Endo5-CBM3b-Exo5 and Endo5- The most suitable enzyme activity temperature of CBM28-Exo5 is respectively 80 DEG C, 60 DEG C and 80 DEG C, and wherein Endo5-2CBM-Exo5 is under the conditions of 80 DEG C 1.247U/mg is up to CMC-Na enzyme activity；Endo5-CBM28-Exo5 is up to 0.636U/ to CMC-Na enzyme activity under the conditions of 80 DEG C mg；Endo5-CBM3b-Exo5 is up to 0.320U/mg to CMC-Na enzyme activity under the conditions of 80 DEG C；By Endo5-2CBM-Exo5, The enzyme activity of Endo5-CBM28-Exo5, Endo5-CBM3b-Exo5 under the conditions of 80 DEG C is set as relative activity 100%, Qi Tawen The vigor that reaction system institute measured data under degree is calculated is using highest enzyme activity as relative activity.Under the conditions of 60~80 DEG C Enzyme activity is high.

(2) optimal pH

Enzyme activity determination reaction system is 150 μ L, the 1%CMC-Na substrate comprising 50 μ L, the 100mM of 50 LpH4.0~8.0 μ Imidazoles-Potassium Hydrogen Phthalate buffer, the pure enzyme solution (ibid) after the dilution of 50 μ L.Reaction system is respectively placed in most suitable After keeping the temperature 30min in enzyme activity temperature 60 or 80 DEG C of water-baths, it is immediately inserted into ice bath and terminates reaction.The DNS reagent of 150 μ L is added, boils After 5min is boiled in water-bath, it is immediately inserted into ice bath and terminates reaction.Moisturizing measures the absorbance value at 520nm, wherein not adding to 1mL The reaction tube of enzyme is as blank control.Experiment sets 3 repetitions, calculates enzyme activity, is averaged mapping.As a result as shown in figure 4, research The result shows that the optimum pH of Endo5-2CBM-Exo5, Endo5-CBM3b-Exo5 and Endo5-CBM28-Exo5 are respectively 4.0,4.0 and 4.5, wherein Endo5-2CBM-Exo5 is up to 1.540U/mg to CMC-Na enzyme activity under the conditions of pH4.0 at 80 DEG C； Endo5-CBM28-Exo5 is up to 0.785U/mg to CMC-Na enzyme activity under the conditions of pH4.5 at 80 DEG C；Endo5-CBM3b-Exo5 At 80 DEG C, 0.357U/mg is up to CMC-Na enzyme activity under the conditions of pH4.0.By Endo5-2CBM-Exo5 at 80 DEG C, pH4.0 condition Under enzyme activity be set as relative activity 100%, the vigor that the reaction system institute measured data under the conditions of other pH is calculated is with most High enzyme activity is as relative activity.Enzyme activity is high under the conditions of pH4.0~4.5.

(3) thermal stability

The 4 pure enzymes being prepared are implemented in the imidazoles for the 100mM for being 4.5 with pH value-Potassium Hydrogen Phthalate buffer dilution Liquid carries out vitality test using the pure enzyme solution after dilution.By the enzyme solution after dilution at 70 DEG C, 75 DEG C, 80 DEG C, 85 DEG C, 90 DEG C of water 30min, 60min, 90min, 120min, measurement residual enzyme activity are kept the temperature in bath.Enzyme activity determination reaction is 4.0 or 4.5 in pH value, Measuring temperature is 60 or 80 DEG C.Experiment sets 3 repetitions, calculates enzyme activity, is averaged mapping.As a result as shown in figure 5, result of study It has been shown that, recombinase are heat-treated 2h under the conditions of 75 DEG C, have no apparent vigor decline, it is seen that recombinase has under the conditions of 75 DEG C Good thermal stability.

(4) pH stability

The 4 pure enzymes being prepared are implemented in the imidazoles for the 100mM for being 4.5 with pH value-Potassium Hydrogen Phthalate buffer dilution Liquid carries out vitality test using the pure enzyme solution after dilution.Enzyme solution after dilution is kept the temperature into 2h under the conditions of pH4.0~8.0,55 DEG C Afterwards, measurement residual enzyme activity.Enzyme activity determination is reacted at 60 or 80 DEG C.Experiment sets 3 repetitions, calculates enzyme activity, is averaged mapping. As a result as shown in fig. 6, result of study is shown, for recombinase under the conditions of pH4.0~6.0,55 DEG C of processing 2h have no apparent vigor Decline, it is seen that the stability that recombinase has had under the conditions of pH4.0~6.0.

Embodiment 5

Acid high temperature resistant recombinant fiber element enzyme Endo5-2CBM-Exo5, Endo5-CBM3b-Exo5 and Endo5-CBM28- The application study of Exo5.

Recombinant fiber element enzyme Endo5-2CBM-Exo5, Endo5-CBM3b-Exo5 and Endo5-CBM28-Exo5 to FP, The Study on degradation of the rice straw (SPRS) of Avicel, RS, WS and steam blasting, it is described that specific step is as follows:

(1) it prepares cellobiose and glucose solution, concentration is 1%；FP, Avicel, RS, WS of 50mg are weighed respectively Be placed in reaction tube with SPRS, be added 1mL pH value be 4.0 or 4.5 100mM imidazoles-Potassium Hydrogen Phthalate buffer and 1mL is by appropriate diluted pure enzyme solution (20 times of dilution, the pure enzyme solution that embodiment 3 is prepared).Reaction system is at 60 or 80 DEG C In shaking bath, 160rpm shakes 4h.

(2) hydrolyzate collected respectively in above-mentioned each reaction tube saves in -20 DEG C of refrigerator.

(3) above-mentioned hydrolyzate TLC solvent and color developing agent are prepared, solvent ingredient and proportion are n-butanol: acetic acid: water= 2:1:1；Color developing agent ingredient and proportion are sulfuric acid: methanol=1:9.

(4) a small amount of 1% cellobiose, glucose and above-mentioned sample are drawn with capillary, point sample is in width respectively 10cm, highly on the glass silica gel plate of 20cm.After point sample, after drying up glass silica gel plate with hair dryer, it is placed in containing above-mentioned In the chromatography cylinder of solvent.

(5) glass silica gel plate is taken out after 6h, is dried in 105 DEG C of baking oven, is uniformly sprayed at color developing agent in draught cupboard Glass silica gel plate is then placed again and is dried in 105 DEG C of baking oven.

As a result as shown in fig. 7, G1 and G2 in No. 1 swimming lane respectively represent glucose and cellobiose, 2,3,4 and 5 difference Indicate no enzyme (blank control), Endo5-2CBM-Exo5, Endo5-CBM3b-Exo5 and Endo5-CBM28-Exo5 hydrolysis supernatant Liquid.Result of study shows that FP, Avicel, RS, WS and SPRS effectively can be hydrolyzed to cellobiose and Portugal by recombinant fiber element enzyme Grape sugar.Therefore, these three recombinant fiber element enzymes are suitable for the glucose and cellobiose of the raw materials such as rice straw and wheat stalk Production.

(6) living to the degradation of CMC-Na, FP, Avicel, RS, WS and SPRS substrate for further three kinds of fusion enzymes of measurement Power takes the 300 μ L of hydrolyzate in each reaction tube of (2) step.300 μ LDNS are added, after boiling water bath boils 5min, are immediately inserted into Ice bath, moisturizing to 2mL measure the absorbance value at 520nm, wherein not enzyme reaction tube is as blank control.Experiment is set 3 times It repeats, calculates enzyme activity.

Three kinds of fusion enzymes show special Sexual potency to CMC-Na, FP, Avicel, RS, WS and SPRS.Wherein Endo5- 2CBM-Exo5, to the special Sexual potency highest of CMC-Na under the conditions of pH4.0, reaches 1.540 ± 0.013U/mg at 80 DEG C；Endo5- CBM3b-Exo5, to the special Sexual potency highest of FP under the conditions of pH4.0, reaches 0.074 ± 0.006U/mg at 60 DEG C；Endo5- 2CBM-Exo5, to the special Sexual potency highest of Avicel under the conditions of pH4.0, reaches 0.167 ± 0.008U/mg at 80 DEG C；Endo5- 2CBM-Exo5, to the special Sexual potency highest of SPRS under the conditions of pH4.0, reaches 0.075 ± 0.006U/mg at 80 DEG C；Endo5- CBM3b-Exo5, to the special Sexual potency highest of RS under the conditions of pH4.0, reaches 0.106 ± 0.002U/mg at 60 DEG C；Endo5- CBM3b-Exo5, to the special Sexual potency highest of WS under the conditions of pH4.0, reaches 0.078 ± 0.008U/mg at 60 DEG C.

Sequence table

<110>Huaiyingong College

<120>acid high temperature resistant recombinant fiber element enzyme and its application

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1221

<212> PRT

<213>artificial sequence (Endo5-2CBM-Exo5Artificial Sequence)

<400> 1

Met Ala Asn Thr Ala Tyr Glu Lys Asp Lys Tyr Pro His Leu Ile Gly

1 5 10 15

Asn Ser Leu Val Lys Lys Pro Ser Val Gly Ala Arg Ile Gln Leu Leu

20 25 30

Lys Asn Gln Gly Arg Lys Ile Ile Ala Asp Gln Asn Gly Glu Pro Ile

35 40 45

Gln Leu Arg Ala Met Thr Ser His Ala Leu Asn Trp Tyr Pro Asn Ile

50 55 60

Leu Asn Gln Asn Ala Tyr Ala Gly Leu Ala Gln Asp Gln Gly Leu Asn

65 70 75 80

Val Ile Arg Ile Ala Met Tyr Leu Gly Gln Gly Gly Tyr Gly Thr Gln

85 90 95

Pro Asn Val Arg Asp Lys Val Ile Glu Gly Ile Lys Ile Ala Leu Gln

100 105 110

Gln Asp Met Trp Val Leu Val Glu Trp His Ala Leu Asn Pro Ala Asp

115 120 125

Phe Asn Gly Glu Leu Tyr Lys Gly Gly Lys Asn Phe Pro Lys Glu Leu

130 135 140

Ala Asn Lys Phe Phe Asn Gln Phe His Leu Leu Tyr Glu Leu Cys Asn

145 150 155 160

Gln Pro Asp Pro Ser Asp Phe Gly Leu Thr Gln Asp Glu Gly Gly Trp

165 170 175

Arg Lys Val Arg Ala Trp Ala Gln Pro Leu Ile Arg Met Ile Arg Asn

180 185 190

Met Ala Asn Gln Asn Leu Ile Ile Ile Gly Ser Pro Asn Trp Ser Gln

195 200 205

Arg Pro Asp Phe Ala Ile Lys Asp Pro Ile Ala Asp Asp Lys Val Met

210 215 220

Tyr Ser Val His Phe Tyr Thr Gly Thr His Lys Val Asp Gly Tyr Val

225 230 235 240

Phe Glu Asn Met Lys Met Ala Ile Glu Ala Gly Val Pro Val Phe Val

245 250 255

Thr Glu Trp Gly Thr Ser Glu Ala Ser Gly Asp Gly Gly Pro Tyr Leu

260 265 270

Asp Glu Ala Asp Lys Trp Leu Glu Tyr Leu Asn Ala Asn Asn Ile Ser

275 280 285

Trp Val Asn Trp Ser Leu Thr Asn Lys Asn Glu Thr Ser Gly Ala Phe

290 295 300

Val Pro Tyr Ile Ser Gly Val Ser Gln Ala Thr Asp Leu Asp Pro Gly

305 310 315 320

Ser Asp Gln Lys Trp Asp Ile Ser Glu Leu Ser Ile Ser Gly Glu Tyr

325 330 335

Val Arg Ser Arg Ile Lys Gly Ile Pro Tyr Gln Pro Ile Glu Arg Thr

340 345 350

Leu Lys Ile Ser Gln Asp Gln Val Ala Cys Ala Pro Ile Gly Gln Pro

355 360 365

Ile Leu Pro Ser Asp Phe Glu Asp Gly Thr Arg Gln Gly Trp Asp Trp

370 375 380

Asp Gly Pro Ser Gly Val Lys Gly Ala Leu Thr Ile Glu Glu Ala Asn

385 390 395 400

Gly Ser Asn Ala Leu Ser Trp Glu Val Glu Tyr Pro Glu Lys Lys Leu

405 410 415

Gln Asp Gly Trp Ala Ser Ala Pro Arg Leu Ile Leu Arg Asn Ile Asn

420 425 430

Thr Thr Arg Gly Asp Cys Lys Tyr Leu Cys Phe Asp Phe Tyr Leu Lys

435 440 445

Pro Lys Gln Ala Thr Lys Gly Glu Leu Ala Ile Phe Leu Ala Phe Ala

450 455 460

Pro Pro Ser Leu Asn Tyr Trp Ala Gln Ala Glu Asp Ser Phe Asn Ile

465 470 475 480

Asp Leu Thr Asn Leu Ser Thr Leu Lys Lys Thr Pro Asp Asp Leu Tyr

485 490 495

Ser Phe Lys Ile Ser Phe Asp Leu Asp Lys Ile Lys Glu Gly Lys Ile

500 505 510

Ile Gly Pro Asp Thr His Leu Arg Asp Ile Ile Ile Val Val Ala Asp

515 520 525

Val Asn Ser Asp Phe Lys Gly Arg Met Tyr Leu Asp Asn Val Arg Phe

530 535 540

Thr Glu Leu Gly Val Thr Thr Ser Ser Pro Thr Pro Thr Pro Thr Pro

545 550 555 560

Thr Val Thr Val Thr Pro Thr Pro Thr Pro Thr Pro Thr Pro Thr Val

565 570 575

Thr Ala Thr Pro Thr Pro Thr Pro Thr Pro Val Ser Thr Pro Ala Thr

580 585 590

Gly Gly Gln Ile Lys Val Leu Tyr Ala Asn Lys Glu Thr Asn Ser Thr

595 600 605

Thr Asn Thr Ile Arg Pro Trp Leu Lys Val Val Asn Ser Gly Ser Ser

610 615 620

Ser Ile Asp Leu Ser Arg Val Thr Ile Arg Tyr Trp Tyr Thr Val Asp

625 630 635 640

Gly Glu Arg Ala Gln Ser Ala Val Ser Asp Trp Ala Gln Ile Gly Ala

645 650 655

Ser Asn Val Thr Phe Lys Phe Val Lys Leu Ser Ser Ser Val Ser Gly

660 665 670

Ala Asp Tyr Tyr Leu Glu Ile Gly Phe Lys Ser Gly Ala Gly Gln Leu

675 680 685

Gln Pro Gly Lys Asp Thr Gly Glu Ile Gln Ile Arg Phe Asn Lys Ser

690 695 700

Asp Trp Ser Asn Tyr Asn Gln Gly Asn Asp Trp Ser Trp Leu Gln Ser

705 710 715 720

Met Thr Ser Tyr Gly Glu Asn Glu Lys Val Thr Ala Tyr Ile Asp Gly

725 730 735

Val Leu Val Trp Gly Gln Glu Pro Ser Gly Ala Thr Pro Ala Pro Thr

740 745 750

Met Thr Val Ala Pro Thr Ala Thr Pro Thr Pro Thr Leu Ser Pro Thr

755 760 765

Val Thr Pro Thr Pro Ala Pro Thr Gln Thr Ala Ile Pro Thr Pro Thr

770 775 780

Leu Thr Pro Asn Pro Thr Pro Thr Ser Ser Ile Pro Asp Asp Thr Asn

785 790 795 800

Asp Asp Trp Leu Tyr Val Ser Gly Asn Lys Ile Val Asp Lys Asp Ala

805 810 815

Arg Phe Val Tyr Leu Thr Ala Ile Asn Tyr Phe Ala Tyr Asp Thr Ala

820 825 830

Thr Asn Leu Phe Asn Gly Ala Trp Ser Cys Asn Leu Lys Asp Thr Leu

835 840 845

Ala Glu Ile Gly Asn Lys Gly Tyr Asn Ile Ile Arg Ala Pro Leu Ser

850 855 860

Ala Glu Ile Ile Ile Asn Tyr Ser Asn Gly Leu Tyr Pro Lys Pro Asn

865 870 875 880

Ile Asn Tyr Tyr Val Asn Pro Glu Leu Glu Gly Lys Asn Ser Leu Glu

885 890 895

Val Phe Asp Ile Val Val Gln Thr Cys Lys Glu Val Gly Leu Lys Ile

900 905 910

Met Leu Asp Ile His Ser Ile Lys Thr Asp Ala Met Ala His Leu Tyr

915 920 925

Phe Val Trp Trp Asp Glu Arg Phe Ser Pro Gln Asp Pro Tyr Arg Ala

930 935 940

Cys Gln Trp Ile Thr Asn Arg Tyr Lys Asn Asp Asp Thr Ile Ile Ala

945 950 955 960

Phe Asp Leu Lys Asn Glu Pro His Gly Lys Pro Trp Gln Asp Thr Thr

965 970 975

Phe Ala Lys Trp Asp Asn Ser Thr Asp Ile Asn Asn Trp Lys Tyr Ala

980 985 990

Ala Glu Thr Cys Ala Lys Arg Ile Leu Asn Ile Asn Pro Asn Leu Leu

995 1000 1005

Ile Val Leu Glu Ala Ile Glu Gly Tyr Phe Lys Asn Asp Leu Thr Trp

1010 1015 1020

Ser Ser Lys Thr Thr Thr Asp Trp Tyr Thr Thr Trp Trp Gly Gly Asn

1025 1030 1035 1040

Leu Arg Gly Val Arg Lys Tyr Pro Ile Asn Leu Gly Lys Tyr Gln Asn

1045 1050 1055

Lys Leu Val Trp Ser Phe His Phe Tyr Ala Pro Ser Leu Tyr Glu Glu

1060 1065 1070

Pro Tyr Phe Trp Pro Gly Phe Thr Lys Glu Ser Leu Leu Gln Asp Cys

1075 1080 1085

Trp Arg Pro Asn Trp Ala Tyr Ile Met Glu Glu Asn Ile Ala Pro Leu

1090 1095 1100

Leu Ile Gly Glu Trp Gly Gly His Leu Asp Gly Ala Asp Asn Glu Lys

1105 1110 1115 1120

Trp Met Lys Tyr Leu Arg Asn Tyr Leu Leu Glu Asp His Leu His His

1125 1130 1135

Ser Phe Tyr Cys Pro Asn Ala Asn Ser Gly Asp Thr Gly Gly Leu Val

1140 1145 1150

Gly Tyr Asp Phe Thr Thr Trp Asp Glu Lys Lys Tyr Ser Phe Leu Lys

1155 1160 1165

Pro Ala Leu Trp Gln Asp Ser Gln Gly Arg Phe Val Gly Leu Asp His

1170 1175 1180

Lys Arg Pro Leu Gly Thr Asn Gly Lys Asn Ile Asn Ile Thr Thr Tyr

1185 1190 1195 1200

Tyr Asn Asn Asn Glu Pro Glu Pro Val Pro Ala Ser Lys Leu Glu His

1205 1210 1215

His His His His His

1220

<210> 2

<211> 1000

<212> PRT

<213>artificial sequence (Endo5-CBM3b-Exo5Artificial Sequence)

<400> 2

Met Ala Asn Thr Ala Tyr Glu Lys Asp Lys Tyr Pro His Leu Ile Gly

1 5 10 15

Asn Ser Leu Val Lys Lys Pro Ser Val Gly Ala Arg Ile Gln Leu Leu

20 25 30

Lys Asn Gln Gly Arg Lys Ile Ile Ala Asp Gln Asn Gly Glu Pro Ile

35 40 45

Gln Leu Arg Ala Met Thr Ser His Ala Leu Asn Trp Tyr Pro Asn Ile

50 55 60

Leu Asn Gln Asn Ala Tyr Ala Gly Leu Ala Gln Asp Gln Gly Leu Asn

65 70 75 80

Val Ile Arg Ile Ala Met Tyr Leu Gly Gln Gly Gly Tyr Gly Thr Gln

85 90 95

Pro Asn Val Arg Asp Lys Val Ile Glu Gly Ile Lys Ile Ala Leu Gln

100 105 110

Gln Asp Met Trp Val Leu Val Glu Trp His Ala Leu Asn Pro Ala Asp

115 120 125

Phe Asn Gly Glu Leu Tyr Lys Gly Gly Lys Asn Phe Pro Lys Glu Leu

130 135 140

Ala Asn Lys Phe Phe Asn Gln Phe His Leu Leu Tyr Glu Leu Cys Asn

145 150 155 160

Gln Pro Asp Pro Ser Asp Phe Gly Leu Thr Gln Asp Glu Gly Gly Trp

165 170 175

Arg Lys Val Arg Ala Trp Ala Gln Pro Leu Ile Arg Met Ile Arg Asn

180 185 190

Met Ala Asn Gln Asn Leu Ile Ile Ile Gly Ser Pro Asn Trp Ser Gln

195 200 205

Arg Pro Asp Phe Ala Ile Lys Asp Pro Ile Ala Asp Asp Lys Val Met

210 215 220

Tyr Ser Val His Phe Tyr Thr Gly Thr His Lys Val Asp Gly Tyr Val

225 230 235 240

Phe Glu Asn Met Lys Met Ala Ile Glu Ala Gly Val Pro Val Phe Val

245 250 255

Thr Glu Trp Gly Thr Ser Glu Ala Ser Gly Asp Gly Gly Pro Tyr Leu

260 265 270

Asp Glu Ala Asp Lys Trp Leu Glu Tyr Leu Asn Ala Asn Asn Ile Ser

275 280 285

Trp Val Asn Trp Ser Leu Thr Asn Lys Asn Glu Thr Ser Gly Ala Phe

290 295 300

Val Pro Tyr Ile Ser Gly Val Ser Gln Ala Thr Asp Leu Asp Pro Gly

305 310 315 320

Ser Asp Gln Lys Glu Leu Gly Val Thr Thr Ser Ser Pro Thr Pro Thr

325 330 335

Pro Thr Pro Thr Val Thr Val Thr Pro Thr Pro Thr Pro Thr Pro Thr

340 345 350

Pro Thr Val Thr Ala Thr Pro Thr Pro Thr Pro Thr Pro Val Ser Thr

355 360 365

Pro Ala Thr Gly Gly Gln Ile Lys Val Leu Tyr Ala Asn Lys Glu Thr

370 375 380

Asn Ser Thr Thr Asn Thr Ile Arg Pro Trp Leu Lys Val Val Asn Ser

385 390 395 400

Gly Ser Ser Ser Ile Asp Leu Ser Arg Val Thr Ile Arg Tyr Trp Tyr

405 410 415

Thr Val Asp Gly Glu Arg Ala Gln Ser Ala Val Ser Asp Trp Ala Gln

420 425 430

Ile Gly Ala Ser Asn Val Thr Phe Lys Phe Val Lys Leu Ser Ser Ser

435 440 445

Val Ser Gly Ala Asp Tyr Tyr Leu Glu Ile Gly Phe Lys Ser Gly Ala

450 455 460

Gly Gln Leu Gln Pro Gly Lys Asp Thr Gly Glu Ile Gln Ile Arg Phe

465 470 475 480

Asn Lys Ser Asp Trp Ser Asn Tyr Asn Gln Gly Asn Asp Trp Ser Trp

485 490 495

Leu Gln Ser Met Thr Ser Tyr Gly Glu Asn Glu Lys Val Thr Ala Tyr

500 505 510

Ile Asp Gly Val Leu Val Trp Gly Gln Glu Pro Ser Gly Ala Thr Pro

515 520 525

Ala Pro Thr Met Thr Val Ala Pro Thr Ala Thr Pro Thr Pro Thr Leu

530 535 540

Ser Pro Thr Val Thr Pro Thr Pro Ala Pro Thr Gln Thr Ala Ile Pro

545 550 555 560

Thr Pro Thr Leu Thr Pro Asn Pro Thr Pro Thr Ser Ser Ile Pro Asp

565 570 575

Asp Thr Asn Asp Asp Trp Leu Tyr Val Ser Gly Asn Lys Ile Val Asp

580 585 590

Lys Asp Ala Arg Phe Val Tyr Leu Thr Ala Ile Asn Tyr Phe Ala Tyr

595 600 605

Asp Thr Ala Thr Asn Leu Phe Asn Gly Ala Trp Ser Cys Asn Leu Lys

610 615 620

Asp Thr Leu Ala Glu Ile Gly Asn Lys Gly Tyr Asn Ile Ile Arg Ala

625 630 635 640

Pro Leu Ser Ala Glu Ile Ile Ile Asn Tyr Ser Asn Gly Leu Tyr Pro

645 650 655

Lys Pro Asn Ile Asn Tyr Tyr Val Asn Pro Glu Leu Glu Gly Lys Asn

660 665 670

Ser Leu Glu Val Phe Asp Ile Val Val Gln Thr Cys Lys Glu Val Gly

675 680 685

Leu Lys Ile Met Leu Asp Ile His Ser Ile Lys Thr Asp Ala Met Ala

690 695 700

His Leu Tyr Phe Val Trp Trp Asp Glu Arg Phe Ser Pro Gln Asp Pro

705 710 715 720

Tyr Arg Ala Cys Gln Trp Ile Thr Asn Arg Tyr Lys Asn Asp Asp Thr

725 730 735

Ile Ile Ala Phe Asp Leu Lys Asn Glu Pro His Gly Lys Pro Trp Gln

740 745 750

Asp Thr Thr Phe Ala Lys Trp Asp Asn Ser Thr Asp Ile Asn Asn Trp

755 760 765

Lys Tyr Ala Ala Glu Thr Cys Ala Lys Arg Ile Leu Asn Ile Asn Pro

770 775 780

Asn Leu Leu Ile Val Leu Glu Ala Ile Glu Gly Tyr Phe Lys Asn Asp

785 790 795 800

Leu Thr Trp Ser Ser Lys Thr Thr Thr Asp Trp Tyr Thr Thr Trp Trp

805 810 815

Gly Gly Asn Leu Arg Gly Val Arg Lys Tyr Pro Ile Asn Leu Gly Lys

820 825 830

Tyr Gln Asn Lys Leu Val Trp Ser Phe His Phe Tyr Ala Pro Ser Leu

835 840 845

Tyr Glu Glu Pro Tyr Phe Trp Pro Gly Phe Thr Lys Glu Ser Leu Leu

850 855 860

Gln Asp Cys Trp Arg Pro Asn Trp Ala Tyr Ile Met Glu Glu Asn Ile

865 870 875 880

Ala Pro Leu Leu Ile Gly Glu Trp Gly Gly His Leu Asp Gly Ala Asp

885 890 895

Asn Glu Lys Trp Met Lys Tyr Leu Arg Asn Tyr Leu Leu Glu Asp His

900 905 910

Leu His His Ser Phe Tyr Cys Pro Asn Ala Asn Ser Gly Asp Thr Gly

915 920 925

Gly Leu Val Gly Tyr Asp Phe Thr Thr Trp Asp Glu Lys Lys Tyr Ser

930 935 940

Phe Leu Lys Pro Ala Leu Trp Gln Asp Ser Gln Gly Arg Phe Val Gly

945 950 955 960

Leu Asp His Lys Arg Pro Leu Gly Thr Asn Gly Lys Asn Ile Asn Ile

965 970 975

Thr Thr Tyr Tyr Asn Asn Asn Glu Pro Glu Pro Val Pro Ala Ser Lys

980 985 990

Leu Glu His His His His His His

995 1000

<210> 3

<211> 1088

<212> PRT

<213>artificial sequence (Endo5-CBM28-Exo5Artificial Sequence)

<400> 3

Met Ala Asn Thr Ala Tyr Glu Lys Asp Lys Tyr Pro His Leu Ile Gly

1 5 10 15

Asn Ser Leu Val Lys Lys Pro Ser Val Gly Ala Arg Ile Gln Leu Leu

20 25 30

Lys Asn Gln Gly Arg Lys Ile Ile Ala Asp Gln Asn Gly Glu Pro Ile

35 40 45

Gln Leu Arg Ala Met Thr Ser His Ala Leu Asn Trp Tyr Pro Asn Ile

50 55 60

Leu Asn Gln Asn Ala Tyr Ala Gly Leu Ala Gln Asp Gln Gly Leu Asn

65 70 75 80

Val Ile Arg Ile Ala Met Tyr Leu Gly Gln Gly Gly Tyr Gly Thr Gln

85 90 95

Pro Asn Val Arg Asp Lys Val Ile Glu Gly Ile Lys Ile Ala Leu Gln

100 105 110

Gln Asp Met Trp Val Leu Val Glu Trp His Ala Leu Asn Pro Ala Asp

115 120 125

Phe Asn Gly Glu Leu Tyr Lys Gly Gly Lys Asn Phe Pro Lys Glu Leu

130 135 140

Ala Asn Lys Phe Phe Asn Gln Phe His Leu Leu Tyr Glu Leu Cys Asn

145 150 155 160

Gln Pro Asp Pro Ser Asp Phe Gly Leu Thr Gln Asp Glu Gly Gly Trp

165 170 175

Arg Lys Val Arg Ala Trp Ala Gln Pro Leu Ile Arg Met Ile Arg Asn

180 185 190

Met Ala Asn Gln Asn Leu Ile Ile Ile Gly Ser Pro Asn Trp Ser Gln

195 200 205

Arg Pro Asp Phe Ala Ile Lys Asp Pro Ile Ala Asp Asp Lys Val Met

210 215 220

Tyr Ser Val His Phe Tyr Thr Gly Thr His Lys Val Asp Gly Tyr Val

225 230 235 240

Phe Glu Asn Met Lys Met Ala Ile Glu Ala Gly Val Pro Val Phe Val

245 250 255

Thr Glu Trp Gly Thr Ser Glu Ala Ser Gly Asp Gly Gly Pro Tyr Leu

260 265 270

Asp Glu Ala Asp Lys Trp Leu Glu Tyr Leu Asn Ala Asn Asn Ile Ser

275 280 285

Trp Val Asn Trp Ser Leu Thr Asn Lys Asn Glu Thr Ser Gly Ala Phe

290 295 300

Val Pro Tyr Ile Ser Gly Val Ser Gln Ala Thr Asp Leu Asp Pro Gly

305 310 315 320

Ser Asp Gln Lys Trp Asp Ile Ser Glu Leu Ser Ile Ser Gly Glu Tyr

325 330 335

Val Arg Ser Arg Ile Lys Gly Ile Pro Tyr Gln Pro Ile Glu Arg Thr

340 345 350

Leu Lys Ile Ser Gln Asp Gln Val Ala Cys Ala Pro Ile Gly Gln Pro

355 360 365

Ile Leu Pro Ser Asp Phe Glu Asp Gly Thr Arg Gln Gly Trp Asp Trp

370 375 380

Asp Gly Pro Ser Gly Val Lys Gly Ala Leu Thr Ile Glu Glu Ala Asn

385 390 395 400

Gly Ser Asn Ala Leu Ser Trp Glu Val Glu Tyr Pro Glu Lys Lys Leu

405 410 415

Gln Asp Gly Trp Ala Ser Ala Pro Arg Leu Ile Leu Arg Asn Ile Asn

420 425 430

Thr Thr Arg Gly Asp Cys Lys Tyr Leu Cys Phe Asp Phe Tyr Leu Lys

435 440 445

Pro Lys Gln Ala Thr Lys Gly Glu Leu Ala Ile Phe Leu Ala Phe Ala

450 455 460

Pro Pro Ser Leu Asn Tyr Trp Ala Gln Ala Glu Asp Ser Phe Asn Ile

465 470 475 480

Asp Leu Thr Asn Leu Ser Thr Leu Lys Lys Thr Pro Asp Asp Leu Tyr

485 490 495

Ser Phe Lys Ile Ser Phe Asp Leu Asp Lys Ile Lys Glu Gly Lys Ile

500 505 510

Ile Gly Pro Asp Thr His Leu Arg Asp Ile Ile Ile Val Val Ala Asp

515 520 525

Val Asn Ser Asp Phe Lys Gly Arg Met Tyr Leu Asp Asn Val Arg Phe

530 535 540

Thr Glu Leu Phe Lys Ser Gly Ala Gly Gln Leu Gln Pro Gly Lys Asp

545 550 555 560

Thr Gly Glu Ile Gln Ile Arg Phe Asn Lys Ser Asp Trp Ser Asn Tyr

565 570 575

Asn Gln Gly Asn Asp Trp Ser Trp Leu Gln Ser Met Thr Ser Tyr Gly

580 585 590

Glu Asn Glu Lys Val Thr Ala Tyr Ile Asp Gly Val Leu Val Trp Gly

595 600 605

Gln Glu Pro Ser Gly Ala Thr Pro Ala Pro Thr Met Thr Val Ala Pro

610 615 620

Thr Ala Thr Pro Thr Pro Thr Leu Ser Pro Thr Val Thr Pro Thr Pro

625 630 635 640

Ala Pro Thr Gln Thr Ala Ile Pro Thr Pro Thr Leu Thr Pro Asn Pro

645 650 655

Thr Pro Thr Ser Ser Ile Pro Asp Asp Thr Asn Asp Asp Trp Leu Tyr

660 665 670

Val Ser Gly Asn Lys Ile Val Asp Lys Asp Ala Arg Phe Val Tyr Leu

675 680 685

Thr Ala Ile Asn Tyr Phe Ala Tyr Asp Thr Ala Thr Asn Leu Phe Asn

690 695 700

Gly Ala Trp Ser Cys Asn Leu Lys Asp Thr Leu Ala Glu Ile Gly Asn

705 710 715 720

Lys Gly Tyr Asn Ile Ile Arg Ala Pro Leu Ser Ala Glu Ile Ile Ile

725 730 735

Asn Tyr Ser Asn Gly Leu Tyr Pro Lys Pro Asn Ile Asn Tyr Tyr Val

740 745 750

Asn Pro Glu Leu Glu Gly Lys Asn Ser Leu Glu Val Phe Asp Ile Val

755 760 765

Val Gln Thr Cys Lys Glu Val Gly Leu Lys Ile Met Leu Asp Ile His

770 775 780

Ser Ile Lys Thr Asp Ala Met Ala His Leu Tyr Phe Val Trp Trp Asp

785 790 795 800

Glu Arg Phe Ser Pro Gln Asp Pro Tyr Arg Ala Cys Gln Trp Ile Thr

805 810 815

Asn Arg Tyr Lys Asn Asp Asp Thr Ile Ile Ala Phe Asp Leu Lys Asn

820 825 830

Glu Pro His Gly Lys Pro Trp Gln Asp Thr Thr Phe Ala Lys Trp Asp

835 840 845

Asn Ser Thr Asp Ile Asn Asn Trp Lys Tyr Ala Ala Glu Thr Cys Ala

850 855 860

Lys Arg Ile Leu Asn Ile Asn Pro Asn Leu Leu Ile Val Leu Glu Ala

865 870 875 880

Ile Glu Gly Tyr Phe Lys Asn Asp Leu Thr Trp Ser Ser Lys Thr Thr

885 890 895

Thr Asp Trp Tyr Thr Thr Trp Trp Gly Gly Asn Leu Arg Gly Val Arg

900 905 910

Lys Tyr Pro Ile Asn Leu Gly Lys Tyr Gln Asn Lys Leu Val Trp Ser

915 920 925

Phe His Phe Tyr Ala Pro Ser Leu Tyr Glu Glu Pro Tyr Phe Trp Pro

930 935 940

Gly Phe Thr Lys Glu Ser Leu Leu Gln Asp Cys Trp Arg Pro Asn Trp

945 950 955 960

Ala Tyr Ile Met Glu Glu Asn Ile Ala Pro Leu Leu Ile Gly Glu Trp

965 970 975

Gly Gly His Leu Asp Gly Ala Asp Asn Glu Lys Trp Met Lys Tyr Leu

980 985 990

Arg Asn Tyr Leu Leu Glu Asp His Leu His His Ser Phe Tyr Cys Pro

995 1000 1005

Asn Ala Asn Ser Gly Asp Thr Gly Gly Leu Val Gly Tyr Asp Phe Thr

1010 1015 1020

Thr Trp Asp Glu Lys Lys Tyr Ser Phe Leu Lys Pro Ala Leu Trp Gln

1025 1030 1035 1040

Asp Ser Gln Gly Arg Phe Val Gly Leu Asp His Lys Arg Pro Leu Gly

1045 1050 1055

Thr Asn Gly Lys Asn Ile Asn Ile Thr Thr Tyr Tyr Asn Asn Asn Glu

1060 1065 1070

Pro Glu Pro Val Pro Ala Ser Lys Leu Glu His His His His His His

1075 1080 1085

<210> 4

<211> 3663

<212> DNA

<213>artificial sequence (endo5-2cbm-exo5Artificial Sequence)

<400> 4

atggctaata ctgcgtatga aaaggataag tatccacacc tgattggcaa cagcttggta 60

aaaaaacctt ctgtgggtgc tagaatccaa ctcctaaaaa atcagggaag aaaaattatt 120

gccgaccaaa atggagagcc tattcagctt cgtgcaatga cttctcacgc actgaattgg 180

tacccaaata tactcaacca gaatgcgtac gctggtcttg cacaggacca aggtcttaat 240

gtaattcgca ttgcaatgta tcttggacag ggtggatatg gaacacagcc taatgtaagg 300

gacaaggtca ttgaaggtat taaaattgca ctacaacagg acatgtgggt acttgtagag 360

tggcatgccc tcaatcccgc tgactttaat ggtgaactat ataaaggggg taaaaatttt 420

cctaaggagc ttgccaacaa atttttcaac cagtttcatc ttctttatga gttgtgtaat 480

caaccagatc caagtgactt tggtcttaca caagatgaag gtgggtggag gaaggtaagg 540

gcttgggctc agccactcat acgtatgatc cgtaatatgg ctaatcagaa cttaataatt 600

ataggttcgc ctaactggag ccaaagacca gattttgcga taaaagaccc aattgcagat 660

gacaaggtca tgtattctgt tcatttctat acaggtacac acaaggtaga tggatatgtg 720

tttgaaaata tgaagatggc aattgaagca ggcgttccag tatttgtaac agaatggggg 780

acaagtgagg caagcggtga tggtggacca tatcttgatg aggctgacaa atggcttgag 840

tacctcaacg caaacaatat aagctgggta aactggtcct tgacaaataa aaatgagact 900

tctggtgctt ttgtacccta cattagcggt gtgtctcaag caacagattt ggaccctggc 960

agcgaccaga aatgggatat ttcagaactt agcatctctg gtgaatatgt acgatcaaga 1020

ataaagggta ttccatatca gcctattgag agaacattga aaatttctca agatcaagtc 1080

gcgtgtgccc caattggtca acctattttg ccatctgatt ttgaagatgg tacacggcaa 1140

ggttgggact gggatggacc atctggtgtt aaaggtgctt taacaataga agaggcaaat 1200

ggttcaaatg cactttcatg ggaggttgag taccctgaaa aaaagcttca agatggttgg 1260

gcttctgctc caaggctaat tttgaggaat ataaatacaa caagaggtga ttgtaagtat 1320

ctctgctttg acttttacct aaaaccaaaa caggcaacaa aaggtgagct tgcaatcttc 1380

ctggcgtttg caccaccttc tcttaactat tgggcacagg cagaggatag ctttaatata 1440

gatttgacca atttatctac actgaagaaa acaccagatg acctttattc tttcaaaatt 1500

tcttttgatt tagataagat aaaagagggg aagattattg gacctgatac ccatctgcga 1560

gacataatca tagttgttgc agatgtaaac agtgatttta aaggtagaat gtacttagac 1620

aatgtaagat ttacagagct cggggtaaca acatcatctc cgacacctac acctacgccg 1680

acagtaacag taacaccaac tccaacaccg acaccgacac cgacagtaac agcgactcca 1740

acacctacac ctacacctgt tagcacacct gcgacaggtg ggcagataaa ggtactgtat 1800

gctaacaagg agacaaacag cacgacaaac acgataaggc catggttgaa ggtagtgaat 1860

agtggcagca gtagcataga tttgagcagg gtaacgataa ggtactggta cacggtagat 1920

ggtgaaaggg cacagagtgc ggtatcagac tgggcacaga taggagcaag caatgtaaca 1980

ttcaagtttg tgaagctgag cagtagtgta agtggagcgg attattactt ggagatagga 2040

tttaagagtg gagcagggca gttacagcct gggaaggaca caggagagat acagataagg 2100

tttaacaaga gtgactggag caattacaat caggggaatg actggtcatg gttacagagc 2160

atgacgagtt atggagagaa tgagaaggta acagcgtata tagatggagt gctggtatgg 2220

ggacaggagc cgagtggagc gacgcctgcg ccgacgatga cggttgcacc gacagcgacc 2280

ccgacaccaa ctctgagtcc gacggtaacg ccgacgccgg caccgacgca gacagcaata 2340

ccaacgccaa cattaactcc aaatccaacg ccaacatcga gtattcctga tgatacaaat 2400

gatgattggc tttatgtaag tggtaataaa atagttgata aagacgccag attcgtatac 2460

ttaacagcca ttaactactt tgcctatgac actgccacaa atctctttaa cggtgcctgg 2520

agttgtaatc tgaaagatac tcttgctgaa ataggcaata agggctacaa catcatcaga 2580

gccccactct ctgcagagat aataataaac tactcgaatg gtctatatcc aaaaccaaat 2640

ataaactact acgtaaatcc agagcttgaa ggcaaaaaca gcttggaagt atttgacata 2700

gtagtgcaaa catgtaaaga agttggtttg aaaattatgt tggatattca cagtataaaa 2760

acagacgcga tggcccatct atatttcgta tggtgggatg aacgttttag tccacaagat 2820

ccatatcgtg cgtgccagtg gattacaaat agatataaaa atgatgacac tattatagct 2880

tttgacctaa agaatgagcc acatggaaaa ccgtggcaag atacaacatt tgcaaaatgg 2940

gataattcaa cagatattaa taattggaaa tatgcagctg aaacatgtgc aaaacgtata 3000

ctgaatataa atccaaacct tctaattgta ctagaagcca ttgaaggtta tttcaagaat 3060

gacctaacat ggagttcaaa aactactact gactggtata ctacatggtg gggcggtaac 3120

ttgcgaggtg ttagaaagta tcctattaat ctgggtaaat atcaaaataa actagtatgg 3180

tcgttccatt tctacgcccc atctctatac gaagaaccgt atttttggcc agggttcaca 3240

aaagagtctt tactacaaga ttgttggcgt ccgaattggg cgtacattat ggaagaaaac 3300

attgcgccac ttctgatagg tgagtggggt ggtcatctgg atggagccga taacgaaaag 3360

tggatgaagt atttacgaaa ctatctacta gaagaccatc tacatcacag tttttattgc 3420

ccaaatgcta actcgggtga cactggagga ttggttggat atgattttac gacatgggat 3480

gagaaaaaat actcattttt gaaaccggct ctatggcaag acagtcaagg taggtttgtt 3540

ggattagatc acaaaagacc cttaggtaca aatgggaaaa acattaatat tacaacatat 3600

tacaacaata atgagcctga gccagttcca gcttcaaaac tcgagcacca ccaccaccac 3660

cac 3663

<210> 5

<211> 3000

<212> DNA

<213>artificial sequence (endo5-cbm3b-exo5Artificial Sequence)

<400> 5

atggctaata ctgcgtatga aaaggataag tatccacacc tgattggcaa cagcttggta 60

aaaaaacctt ctgtgggtgc tagaatccaa ctcctaaaaa atcagggaag aaaaattatt 120

gccgaccaaa atggagagcc tattcagctt cgtgcaatga cttctcacgc actgaattgg 180

tacccaaata tactcaacca gaatgcgtac gctggtcttg cacaggacca aggtcttaat 240

gtaattcgca ttgcaatgta tcttggacag ggtggatatg gaacacagcc taatgtaagg 300

gacaaggtca ttgaaggtat taaaattgca ctacaacagg acatgtgggt acttgtagag 360

tggcatgccc tcaatcccgc tgactttaat ggtgaactat ataaaggggg taaaaatttt 420

cctaaggagc ttgccaacaa atttttcaac cagtttcatc ttctttatga gttgtgtaat 480

caaccagatc caagtgactt tggtcttaca caagatgaag gtgggtggag gaaggtaagg 540

gcttgggctc agccactcat acgtatgatc cgtaatatgg ctaatcagaa cttaataatt 600

ataggttcgc ctaactggag ccaaagacca gattttgcga taaaagaccc aattgcagat 660

gacaaggtca tgtattctgt tcatttctat acaggtacac acaaggtaga tggatatgtg 720

tttgaaaata tgaagatggc aattgaagca ggcgttccag tatttgtaac agaatggggg 780

acaagtgagg caagcggtga tggtggacca tatcttgatg aggctgacaa atggcttgag 840

tacctcaacg caaacaatat aagctgggta aactggtcct tgacaaataa aaatgagact 900

tctggtgctt ttgtacccta cattagcggt gtgtctcaag caacagattt ggaccctggc 960

agcgaccaga aagagctcgg ggtaacaaca tcatctccga cacctacacc tacgccgaca 1020

gtaacagtaa caccaactcc aacaccgaca ccgacaccga cagtaacagc gactccaaca 1080

cctacaccta cacctgttag cacacctgcg acaggtgggc agataaaggt actgtatgct 1140

aacaaggaga caaacagcac gacaaacacg ataaggccat ggttgaaggt agtgaatagt 1200

ggcagcagta gcatagattt gagcagggta acgataaggt actggtacac ggtagatggt 1260

gaaagggcac agagtgcggt atcagactgg gcacagatag gagcaagcaa tgtaacattc 1320

aagtttgtga agctgagcag tagtgtaagt ggagcggatt attacttgga gataggattt 1380

aagagtggag cagggcagtt acagcctggg aaggacacag gagagataca gataaggttt 1440

aacaagagtg actggagcaa ttacaatcag gggaatgact ggtcatggtt acagagcatg 1500

acgagttatg gagagaatga gaaggtaaca gcgtatatag atggagtgct ggtatgggga 1560

caggagccga gtggagcgac gcctgcgccg acgatgacgg ttgcaccgac agcgaccccg 1620

acaccaactc tgagtccgac ggtaacgccg acgccggcac cgacgcagac agcaatacca 1680

acgccaacat taactccaaa tccaacgcca acatcgagta ttcctgatga tacaaatgat 1740

gattggcttt atgtaagtgg taataaaata gttgataaag acgccagatt cgtatactta 1800

acagccatta actactttgc ctatgacact gccacaaatc tctttaacgg tgcctggagt 1860

tgtaatctga aagatactct tgctgaaata ggcaataagg gctacaacat catcagagcc 1920

ccactctctg cagagataat aataaactac tcgaatggtc tatatccaaa accaaatata 1980

aactactacg taaatccaga gcttgaaggc aaaaacagct tggaagtatt tgacatagta 2040

gtgcaaacat gtaaagaagt tggtttgaaa attatgttgg atattcacag tataaaaaca 2100

gacgcgatgg cccatctata tttcgtatgg tgggatgaac gttttagtcc acaagatcca 2160

tatcgtgcgt gccagtggat tacaaataga tataaaaatg atgacactat tatagctttt 2220

gacctaaaga atgagccaca tggaaaaccg tggcaagata caacatttgc aaaatgggat 2280

aattcaacag atattaataa ttggaaatat gcagctgaaa catgtgcaaa acgtatactg 2340

aatataaatc caaaccttct aattgtacta gaagccattg aaggttattt caagaatgac 2400

ctaacatgga gttcaaaaac tactactgac tggtatacta catggtgggg cggtaacttg 2460

cgaggtgtta gaaagtatcc tattaatctg ggtaaatatc aaaataaact agtatggtcg 2520

ttccatttct acgccccatc tctatacgaa gaaccgtatt tttggccagg gttcacaaaa 2580

gagtctttac tacaagattg ttggcgtccg aattgggcgt acattatgga agaaaacatt 2640

gcgccacttc tgataggtga gtggggtggt catctggatg gagccgataa cgaaaagtgg 2700

atgaagtatt tacgaaacta tctactagaa gaccatctac atcacagttt ttattgccca 2760

aatgctaact cgggtgacac tggaggattg gttggatatg attttacgac atgggatgag 2820

aaaaaatact catttttgaa accggctcta tggcaagaca gtcaaggtag gtttgttgga 2880

ttagatcaca aaagaccctt aggtacaaat gggaaaaaca ttaatattac aacatattac 2940

aacaataatg agcctgagcc agttccagct tcaaaactcg agcaccacca ccaccaccac 3000

<210> 6

<211> 3264

<212> DNA

<213>artificial sequence (endo5-cbm28-exo5Artificial Sequence)

<400> 6

atggctaata ctgcgtatga aaaggataag tatccacacc tgattggcaa cagcttggta 60

aaaaaacctt ctgtgggtgc tagaatccaa ctcctaaaaa atcagggaag aaaaattatt 120

gccgaccaaa atggagagcc tattcagctt cgtgcaatga cttctcacgc actgaattgg 180

tacccaaata tactcaacca gaatgcgtac gctggtcttg cacaggacca aggtcttaat 240

gtaattcgca ttgcaatgta tcttggacag ggtggatatg gaacacagcc taatgtaagg 300

gacaaggtca ttgaaggtat taaaattgca ctacaacagg acatgtgggt acttgtagag 360

tggcatgccc tcaatcccgc tgactttaat ggtgaactat ataaaggggg taaaaatttt 420

cctaaggagc ttgccaacaa atttttcaac cagtttcatc ttctttatga gttgtgtaat 480

caaccagatc caagtgactt tggtcttaca caagatgaag gtgggtggag gaaggtaagg 540

gcttgggctc agccactcat acgtatgatc cgtaatatgg ctaatcagaa cttaataatt 600

ataggttcgc ctaactggag ccaaagacca gattttgcga taaaagaccc aattgcagat 660

gacaaggtca tgtattctgt tcatttctat acaggtacac acaaggtaga tggatatgtg 720

tttgaaaata tgaagatggc aattgaagca ggcgttccag tatttgtaac agaatggggg 780

acaagtgagg caagcggtga tggtggacca tatcttgatg aggctgacaa atggcttgag 840

tacctcaacg caaacaatat aagctgggta aactggtcct tgacaaataa aaatgagact 900

tctggtgctt ttgtacccta cattagcggt gtgtctcaag caacagattt ggaccctggc 960

agcgaccaga aatgggatat ttcagaactt agcatctctg gtgaatatgt acgatcaaga 1020

ataaagggta ttccatatca gcctattgag agaacattga aaatttctca agatcaagtc 1080

gcgtgtgccc caattggtca acctattttg ccatctgatt ttgaagatgg tacacggcaa 1140

ggttgggact gggatggacc atctggtgtt aaaggtgctt taacaataga agaggcaaat 1200

ggttcaaatg cactttcatg ggaggttgag taccctgaaa aaaagcttca agatggttgg 1260

gcttctgctc caaggctaat tttgaggaat ataaatacaa caagaggtga ttgtaagtat 1320

ctctgctttg acttttacct aaaaccaaaa caggcaacaa aaggtgagct tgcaatcttc 1380

ctggcgtttg caccaccttc tcttaactat tgggcacagg cagaggatag ctttaatata 1440

gatttgacca atttatctac actgaagaaa acaccagatg acctttattc tttcaaaatt 1500

tcttttgatt tagataagat aaaagagggg aagattattg gacctgatac ccatctgcga 1560

gacataatca tagttgttgc agatgtaaac agtgatttta aaggtagaat gtacttagac 1620

aatgtaagat ttacagagct ctttaagagt ggagcagggc agttacagcc tgggaaggac 1680

acaggagaga tacagataag gtttaacaag agtgactgga gcaattacaa tcaggggaat 1740

gactggtcat ggttacagag catgacgagt tatggagaga atgagaaggt aacagcgtat 1800

atagatggag tgctggtatg gggacaggag ccgagtggag cgacgcctgc gccgacgatg 1860

acggttgcac cgacagcgac cccgacacca actctgagtc cgacggtaac gccgacgccg 1920

gcaccgacgc agacagcaat accaacgcca acattaactc caaatccaac gccaacatcg 1980

agtattcctg atgatacaaa tgatgattgg ctttatgtaa gtggtaataa aatagttgat 2040

aaagacgcca gattcgtata cttaacagcc attaactact ttgcctatga cactgccaca 2100

aatctcttta acggtgcctg gagttgtaat ctgaaagata ctcttgctga aataggcaat 2160

aagggctaca acatcatcag agccccactc tctgcagaga taataataaa ctactcgaat 2220

ggtctatatc caaaaccaaa tataaactac tacgtaaatc cagagcttga aggcaaaaac 2280

agcttggaag tatttgacat agtagtgcaa acatgtaaag aagttggttt gaaaattatg 2340

ttggatattc acagtataaa aacagacgcg atggcccatc tatatttcgt atggtgggat 2400

gaacgtttta gtccacaaga tccatatcgt gcgtgccagt ggattacaaa tagatataaa 2460

aatgatgaca ctattatagc ttttgaccta aagaatgagc cacatggaaa accgtggcaa 2520

gatacaacat ttgcaaaatg ggataattca acagatatta ataattggaa atatgcagct 2580

gaaacatgtg caaaacgtat actgaatata aatccaaacc ttctaattgt actagaagcc 2640

attgaaggtt atttcaagaa tgacctaaca tggagttcaa aaactactac tgactggtat 2700

actacatggt ggggcggtaa cttgcgaggt gttagaaagt atcctattaa tctgggtaaa 2760

tatcaaaata aactagtatg gtcgttccat ttctacgccc catctctata cgaagaaccg 2820

tatttttggc cagggttcac aaaagagtct ttactacaag attgttggcg tccgaattgg 2880

gcgtacatta tggaagaaaa cattgcgcca cttctgatag gtgagtgggg tggtcatctg 2940

gatggagccg ataacgaaaa gtggatgaag tatttacgaa actatctact agaagaccat 3000

ctacatcaca gtttttattg cccaaatgct aactcgggtg acactggagg attggttgga 3060

tatgatttta cgacatggga tgagaaaaaa tactcatttt tgaaaccggc tctatggcaa 3120

gacagtcaag gtaggtttgt tggattagat cacaaaagac ccttaggtac aaatgggaaa 3180

aacattaata ttacaacata ttacaacaat aatgagcctg agccagttcc agcttcaaaa 3240

ctcgagcacc accaccacca ccac 3264

<210> 7

<211> 1629

<212> DNA

<213>artificial sequence (endo5-cbm28Artificial Sequence)

<400> 7

aatactgcgt atgaaaagga taagtatcca cacctgattg gcaacagctt ggtaaaaaaa 60

ccttctgtgg gtgctagaat ccaactccta aaaaatcagg gaagaaaaat tattgccgac 120

caaaatggag agcctattca gcttcgtgca atgacttctc acgcactgaa ttggtaccca 180

aatatactca accagaatgc gtacgctggt cttgcacagg accaaggtct taatgtaatt 240

cgcattgcaa tgtatcttgg acagggtgga tatggaacac agcctaatgt aagggacaag 300

gtcattgaag gtattaaaat tgcactacaa caggacatgt gggtacttgt agagtggcat 360

gccctcaatc ccgctgactt taatggtgaa ctatataaag ggggtaaaaa ttttcctaag 420

gagcttgcca acaaattttt caaccagttt catcttcttt atgagttgtg taatcaacca 480

gatccaagtg actttggtct tacacaagat gaaggtgggt ggaggaaggt aagggcttgg 540

gctcagccac tcatacgtat gatccgtaat atggctaatc agaacttaat aattataggt 600

tcgcctaact ggagccaaag accagatttt gcgataaaag acccaattgc agatgacaag 660

gtcatgtatt ctgttcattt ctatacaggt acacacaagg tagatggata tgtgtttgaa 720

aatatgaaga tggcaattga agcaggcgtt ccagtatttg taacagaatg ggggacaagt 780

gaggcaagcg gtgatggtgg accatatctt gatgaggctg acaaatggct tgagtacctc 840

aacgcaaaca atataagctg ggtaaactgg tccttgacaa ataaaaatga gacttctggt 900

gcttttgtac cctacattag cggtgtgtct caagcaacag atttggaccc tggcagcgac 960

cagaaatggg atatttcaga acttagcatc tctggtgaat atgtacgatc aagaataaag 1020

ggtattccat atcagcctat tgagagaaca ttgaaaattt ctcaagatca agtcgcgtgt 1080

gccccaattg gtcaacctat tttgccatct gattttgaag atggtacacg gcaaggttgg 1140

gactgggatg gaccatctgg tgttaaaggt gctttaacaa tagaagaggc aaatggttca 1200

aatgcacttt catgggaggt tgagtaccct gaaaaaaagc ttcaagatgg ttgggcttct 1260

gctccaaggc taattttgag gaatataaat acaacaagag gtgattgtaa gtatctctgc 1320

tttgactttt acctaaaacc aaaacaggca acaaaaggtg agcttgcaat cttcctggcg 1380

tttgcaccac cttctcttaa ctattgggca caggcagagg atagctttaa tatagatttg 1440

accaatttat ctacactgaa gaaaacacca gatgaccttt attctttcaa aatttctttt 1500

gatttagata agataaaaga ggggaagatt attggacctg atacccatct gcgagacata 1560

atcatagttg ttgcagatgt aaacagtgat tttaaaggta gaatgtactt agacaatgta 1620

agatttaca 1629

<210> 8

<211> 966

<212> DNA

<213>artificial sequence (endo5Artificial Sequence)

<400> 8

aatactgcgt atgaaaagga taagtatcca cacctgattg gcaacagctt ggtaaaaaaa 60

ccttctgtgg gtgctagaat ccaactccta aaaaatcagg gaagaaaaat tattgccgac 120

caaaatggag agcctattca gcttcgtgca atgacttctc acgcactgaa ttggtaccca 180

aatatactca accagaatgc gtacgctggt cttgcacagg accaaggtct taatgtaatt 240

cgcattgcaa tgtatcttgg acagggtgga tatggaacac agcctaatgt aagggacaag 300

gtcattgaag gtattaaaat tgcactacaa caggacatgt gggtacttgt agagtggcat 360

gccctcaatc ccgctgactt taatggtgaa ctatataaag ggggtaaaaa ttttcctaag 420

gagcttgcca acaaattttt caaccagttt catcttcttt atgagttgtg taatcaacca 480

gatccaagtg actttggtct tacacaagat gaaggtgggt ggaggaaggt aagggcttgg 540

gctcagccac tcatacgtat gatccgtaat atggctaatc agaacttaat aattataggt 600

tcgcctaact ggagccaaag accagatttt gcgataaaag acccaattgc agatgacaag 660

gtcatgtatt ctgttcattt ctatacaggt acacacaagg tagatggata tgtgtttgaa 720

aatatgaaga tggcaattga agcaggcgtt ccagtatttg taacagaatg ggggacaagt 780

gaggcaagcg gtgatggtgg accatatctt gatgaggctg acaaatggct tgagtacctc 840

aacgcaaaca atataagctg ggtaaactgg tccttgacaa ataaaaatga gacttctggt 900

gcttttgtac cctacattag cggtgtgtct caagcaacag atttggaccc tggcagcgac 960

cagaaa 966

<210> 9

<211> 1998

<212> DNA

<213>artificial sequence (cbm3b-exo5Artificial Sequence)

<400> 9

ggggtaacaa catcatctcc gacacctaca cctacgccga cagtaacagt aacaccaact 60

ccaacaccga caccgacacc gacagtaaca gcgactccaa cacctacacc tacacctgtt 120

agcacacctg cgacaggtgg gcagataaag gtactgtatg ctaacaagga gacaaacagc 180

acgacaaaca cgataaggcc atggttgaag gtagtgaata gtggcagcag tagcatagat 240

ttgagcaggg taacgataag gtactggtac acggtagatg gtgaaagggc acagagtgcg 300

gtatcagact gggcacagat aggagcaagc aatgtaacat tcaagtttgt gaagctgagc 360

agtagtgtaa gtggagcgga ttattacttg gagataggat ttaagagtgg agcagggcag 420

ttacagcctg ggaaggacac aggagagata cagataaggt ttaacaagag tgactggagc 480

aattacaatc aggggaatga ctggtcatgg ttacagagca tgacgagtta tggagagaat 540

gagaaggtaa cagcgtatat agatggagtg ctggtatggg gacaggagcc gagtggagcg 600

acgcctgcgc cgacgatgac ggttgcaccg acagcgaccc cgacaccaac tctgagtccg 660

acggtaacgc cgacgccggc accgacgcag acagcaatac caacgccaac attaactcca 720

aatccaacgc caacatcgag tattcctgat gatacaaatg atgattggct ttatgtaagt 780

ggtaataaaa tagttgataa agacgccaga ttcgtatact taacagccat taactacttt 840

gcctatgaca ctgccacaaa tctctttaac ggtgcctgga gttgtaatct gaaagatact 900

cttgctgaaa taggcaataa gggctacaac atcatcagag ccccactctc tgcagagata 960

ataataaact actcgaatgg tctatatcca aaaccaaata taaactacta cgtaaatcca 1020

gagcttgaag gcaaaaacag cttggaagta tttgacatag tagtgcaaac atgtaaagaa 1080

gttggtttga aaattatgtt ggatattcac agtataaaaa cagacgcgat ggcccatcta 1140

tatttcgtat ggtgggatga acgttttagt ccacaagatc catatcgtgc gtgccagtgg 1200

attacaaata gatataaaaa tgatgacact attatagctt ttgacctaaa gaatgagcca 1260

catggaaaac cgtggcaaga tacaacattt gcaaaatggg ataattcaac agatattaat 1320

aattggaaat atgcagctga aacatgtgca aaacgtatac tgaatataaa tccaaacctt 1380

ctaattgtac tagaagccat tgaaggttat ttcaagaatg acctaacatg gagttcaaaa 1440

actactactg actggtatac tacatggtgg ggcggtaact tgcgaggtgt tagaaagtat 1500

cctattaatc tgggtaaata tcaaaataaa ctagtatggt cgttccattt ctacgcccca 1560

tctctatacg aagaaccgta tttttggcca gggttcacaa aagagtcttt actacaagat 1620

tgttggcgtc cgaattgggc gtacattatg gaagaaaaca ttgcgccact tctgataggt 1680

gagtggggtg gtcatctgga tggagccgat aacgaaaagt ggatgaagta tttacgaaac 1740

tatctactag aagaccatct acatcacagt ttttattgcc caaatgctaa ctcgggtgac 1800

actggaggat tggttggata tgattttacg acatgggatg agaaaaaata ctcatttttg 1860

aaaccggctc tatggcaaga cagtcaaggt aggtttgttg gattagatca caaaagaccc 1920

ttaggtacaa atgggaaaaa cattaatatt acaacatatt acaacaataa tgagcctgag 1980

ccagttccag cttcaaaa 1998

<210> 10

<211> 1599

<212> DNA

<213>artificial sequence (exo5Artificial Sequence)

<400> 10

tttaagagtg gagcagggca gttacagcct gggaaggaca caggagagat acagataagg 60

tttaacaaga gtgactggag caattacaat caggggaatg actggtcatg gttacagagc 120

atgacgagtt atggagagaa tgagaaggta acagcgtata tagatggagt gctggtatgg 180

ggacaggagc cgagtggagc gacgcctgcg ccgacgatga cggttgcacc gacagcgacc 240

ccgacaccaa ctctgagtcc gacggtaacg ccgacgccgg caccgacgca gacagcaata 300

ccaacgccaa cattaactcc aaatccaacg ccaacatcga gtattcctga tgatacaaat 360

gatgattggc tttatgtaag tggtaataaa atagttgata aagacgccag attcgtatac 420

ttaacagcca ttaactactt tgcctatgac actgccacaa atctctttaa cggtgcctgg 480

agttgtaatc tgaaagatac tcttgctgaa ataggcaata agggctacaa catcatcaga 540

gccccactct ctgcagagat aataataaac tactcgaatg gtctatatcc aaaaccaaat 600

ataaactact acgtaaatcc agagcttgaa ggcaaaaaca gcttggaagt atttgacata 660

gtagtgcaaa catgtaaaga agttggtttg aaaattatgt tggatattca cagtataaaa 720

acagacgcga tggcccatct atatttcgta tggtgggatg aacgttttag tccacaagat 780

ccatatcgtg cgtgccagtg gattacaaat agatataaaa atgatgacac tattatagct 840

tttgacctaa agaatgagcc acatggaaaa ccgtggcaag atacaacatt tgcaaaatgg 900

gataattcaa cagatattaa taattggaaa tatgcagctg aaacatgtgc aaaacgtata 960

ctgaatataa atccaaacct tctaattgta ctagaagcca ttgaaggtta tttcaagaat 1020

gacctaacat ggagttcaaa aactactact gactggtata ctacatggtg gggcggtaac 1080

ttgcgaggtg ttagaaagta tcctattaat ctgggtaaat atcaaaataa actagtatgg 1140

tcgttccatt tctacgcccc atctctatac gaagaaccgt atttttggcc agggttcaca 1200

aaagagtctt tactacaaga ttgttggcgt ccgaattggg cgtacattat ggaagaaaac 1260

attgcgccac ttctgatagg tgagtggggt ggtcatctgg atggagccga taacgaaaag 1320

tggatgaagt atttacgaaa ctatctacta gaagaccatc tacatcacag tttttattgc 1380

ccaaatgcta actcgggtga cactggagga ttggttggat atgattttac gacatgggat 1440

gagaaaaaat actcattttt gaaaccggct ctatggcaag acagtcaagg taggtttgtt 1500

ggattagatc acaaaagacc cttaggtaca aatgggaaaa acattaatat tacaacatat 1560

tacaacaata atgagcctga gccagttcca gcttcaaaa 1599

<210> 11

<211> 36

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

aacgatccat ggctaatact gcgtatgaaa aggata 36

<210> 12

<211> 34

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 12

aacgatgagc tctgtaaatc ttacattgtc taag 34

<210> 13

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 13

aacgatgagc tctttctggt cgctgccagg 30

<210> 14

<211> 31

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 14

aacgatgagc tcggggtaac aacatcatct c 31

<210> 15

<211> 33

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 15

aacgatctcg agttttgaag ctggaactgg ctc 33

<210> 16

<211> 32

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 16

aacgatgagc tctttaagag tggagcaggg ca 32

Claims

1. acid high temperature resistant recombinant fiber element enzyme, which is characterized in that the acidity high temperature resistant recombinant fiber element enzyme includes three kinds of acid Property high temperature resistant recombinant fiber element enzyme, amino acid sequence is respectively as shown in SEQ ID NO.1-3.

2. acid high temperature resistant recombinant fiber element enzyme, which is characterized in that the acidity high temperature resistant recombinant fiber element enzyme includes three kinds of acid Property high temperature resistant recombinant fiber element enzyme, DNA sequence dna is respectively as shown in SEQIDNO.4-6.

3. acidity high temperature resistant recombinant fiber element enzyme according to claim 2, which is characterized in that the DNA sequence dna SEQ ID NO.4-6 splices after being expanded respectively by following primer SEQ ID NO.11-16 and obtains:

Endo-F:5 '-AACGATCCATGGCTAATACTGCGTATGAAAAGGATA-3 '；

Endocbm-R:5 '-AACGATGAGCTCTGTAAATCTTACATTGTCTAAG-3 '；

Endo-R:5 '-AACGATGAGCTCTTTCTGGTCGCTGCCAGG-3 '；

CbmExo-F:5 '-AACGATGAGCTCGGGGTAACAACATCATCTC-3 '；

Exo-R:5 '-AACGATCTCGAGTTTTGAAGCTGGAACTGGCTC-3 '；

Exo-F:5 '-AACGATGAGCTCTTTAAGAGTGGAGCAGGGCA-3 '.

4. a kind of recombinant vector, which is characterized in that recombinant vector clone include in SEQ ID NO.4-6 institute any one DNA sequence dna.

5. a kind of recombinant bacterium, which is characterized in that the recombinant bacterium includes the recombinant vector in claim 3.

6. acid high temperature resistant recombinant fiber element enzyme answering in enzymatic hydrolysis CMC-Na, filter paper, Avicel and natural wooden fiber's element With.

7. application according to claim 6, which is characterized in that natural wooden fiber's element includes rice straw and wheat Stalk.

8. application according to claim 6, which is characterized in that the enzymolysis temperature is preferably 60~80 DEG C, and pH is 4.0~4.5, reaction time 4h.

9. recombinant vector as claimed in claim 4 answering in enzymatic hydrolysis CMC-Na, filter paper, Avicel and natural wooden fiber's element With.

10. the answering in enzymatic hydrolysis CMC-Na, filter paper, Avicel and natural wooden fiber's element of recombinant bacterium described in claim 5 With.