CN109280672A

CN109280672A - Recombinant fiber element incision enzyme gene and its albumen and protein preparation method

Info

Publication number: CN109280672A
Application number: CN201811240711.0A
Authority: CN
Inventors: 李洪波; 胡兴; 江素珍; 米丹; 张赛名
Original assignee: Huaihua University
Current assignee: HUNAN BUSKY PHARMACEUTICAL Co.,Ltd.
Priority date: 2018-10-23
Filing date: 2018-10-23
Publication date: 2019-01-29
Anticipated expiration: 2038-10-23
Also published as: CN109280672B

Abstract

The present invention relates to a kind of recombinant fiber element incision enzyme gene, the gene nucleotide series are as shown in SEQ ID NO.1, and encoded albumen is as shown in SEQ ID NO.2.The present invention also provides one kind can be with high efficient expression and the method for purifying the recombinant protein, not only purity of protein after purification is up to 95%, protein concentration is also higher, and the enzymatic activity of glucose is generated with hydrocellulose, this has the industries such as the production of bio-fuel ethyl alcohol, food, feed important industrial value.

Description

Recombinant fiber element incision enzyme gene and its albumen and protein preparation method

Technical field

The invention belongs to biological gene engineering fields, and in particular to recombinant fiber element incision enzyme gene further relates to the gene Encoded albumen and protein preparation method.

Background technique

Cellulose is the polysaccharide being made of glucose with β-Isosorbide-5-Nitrae glycosidic bond, is the important component for constituting plant cell wall, It is the most abundant reproducibility resource of content on the current earth.Currently, the utilization rate of cellulose is also very low, how fiber is improved The utilization rate of element is still a world-class project.The efficient of cellulose utilizes the too busy to get away relevant enzyme of degraded cellulose.It is fine It ties up plain enzyme and belongs to glycoside hydrolases, be β in special catalyzing hydrolysis cellulose chain-Isosorbide-5-Nitrae-glycosidic bond class of enzymes general name, be A kind of high-activity biological catalyst, being capable of decomposition of cellulose generation glucose.It is different according to the structure of cellulase, it can be fiber Plain enzyme is divided into two classes: cellulase complex and non-composite body cellulase.Cellulase complex is a kind of supramolecular structure Multienzyme protein complexes, be made of multiple subunits.Non-composite body cellulase by endoglucanase, exoglucanase and Beta glucan glycosides enzyme composition.Non-composite body cellulase is mainly generated by aerobic filamentous fungi, they are decomposition of cellulose Most important enzyme source.

With the increasingly exhaustion of oil and coal resource, this nature of cellulose how is more effectively converted and utilized It is distributed most wide, most abundant also most cheap reproducibility organic resource and polysaccharide material, is the important research field of China's concern. So fuel, food and chemicals are converted it into using cellulose degraded cellulose, and such as sugar, ethyl alcohol, forage protein Deng to alleviating energy crisis, food and feed resource anxiety have great economic significance, and cellulase will be in food, feed, ring It plays a significant role in the fields such as border protection, the energy and development of resources.Although the biota kind of discovery cellulase-producing is very at present It is more, such as bacterium, fungi, actinomyces and insect, mollusk, but with cellulase the fields such as work, agriculture, poultry and doctor all There is extensive demand, demand just increasingly increases, and supply falls short of demand for cellulase preparation, and prospect is very wide.And the fibre in China Tie up that plain enzyme is industrially prepared also in the research and development stage, that there is also cellulase activities is lower for the production of cellulase, is produced into The problems such as this is higher, and the production cycle is longer is restricted the application of cellulase, and therefore, it is necessary to overcome cellulase a large amount of The bottleneck of production.The mechanism of action of cellulase is furtherd investigate, reinforces that the molecular biology research of cellulase is important to fill Divide the application using DNA gene recombination technology, produces the recombinant protein with high enzyme activity.Cellulase system includes restriction endonuclease, outer 3 kinds of enzymes of enzyme cutting and glucoside transferase.Wherein, cellulose restriction endonuclease plays an important role to the decomposition of cellulose.Presently commercially available fiber Plain enzyme is all the mixture of various enzymes, few to be obtained in big highest purity and the cellulose of high activity using genetic engineering means The Related product and technology of enzyme cutting.

Summary of the invention

In view of this, the purpose of the present invention is to provide a kind of recombinant fiber element incision enzyme gene and this gene institutes The albumen of coding, the present invention also provides a kind of recombinant vector of the building containing SEQ ID NO.1 gene order, more provide a kind of high Method effect expression and purify the recombinant protein.

In order to achieve the above objectives, the invention provides the following technical scheme:

1. recombinant fiber element inscribe enzyme coding gene, the gene at least DNA piece containing one of following nucleotide sequences Section:

1) in sequence table SEQ ID NO.1 nucleotide sequence；

2) there is 95% or more homology and the identical biological function of coding with nucleotide sequence shown in SEQ ID NO.1 The nucleotide sequence of protein；

3) nucleotides sequence of biological function protein identical as nucleotide sequence hybridization shown in SEQ ID NO.1 and coding Column.

Further, with nucleotide sequence hybridization condition shown in SEQ ID NO.1 are as follows: 50 DEG C, in 7% lauryl sodium sulfate (SDS), hybridize in the mixed solution of 0.5M Na3PO4 and 1mM EDTA, rinsed in 50 DEG C, 2 × SSC, 0.1%SDS；

It can be with hybridization conditions for 50 DEG C, in 7%SDS, 0.5M Na₃PO₄Hybridize in the mixed solution of 1mM EDTA, It 50 DEG C, rinses in 1 × SSC, 0.1%SDS；

It can be with hybridization conditions for 50 DEG C, in 7%SDS, 0.5M Na₃PO₄Hybridize in the mixed solution of 1mM EDTA, It 50 DEG C, rinses in 0.5 × SSC, 0.1%SDS；

It can be with hybridization conditions for 50 DEG C, in 7%SDS, 0.5M Na₃PO₄Hybridize in the mixed solution of 1mM EDTA, It 50 DEG C, rinses in 0.1 × SSC, 0.1%SDS；

It can be with hybridization conditions for 50 DEG C, in 7%SDS, 0.5M Na₃PO₄Hybridize in the mixed solution of 1mM EDTA, It 65 DEG C, rinses in 0.1 × SSC, 0.1%SDS；

It can be to hybridize at 65 DEG C in 6 × SSC, the solution of 0.5%SDS with hybridization conditions, then with 2 × SSC, It is primary that 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film.

2. wherein nucleotide sequence shown in SEQ ID NO.1 is made of 1020 deoxynucleotides, this sequence is cellulose The full-length cDNA reading frame of restriction endonuclease (EG), the albumen of amino acid sequence shown in SEQ ID NO.2 in polynucleotide include 340 amino acid residues.

1) in sequence table SEQ ID NO.1 nucleotide sequence；

2) there is 90% or more homology and the identical biological function of coding with nucleotide sequence shown in SEQ ID NO.1 The nucleotide sequence of protein；

Or there is 95% or more homology and the identical biological function of coding with nucleotide sequence shown in SEQ ID NO.1 The nucleotide sequence of protein；

Or there is 98% or more homology and the identical biological function of coding with nucleotide sequence shown in SEQ ID NO.1 The nucleotide sequence of protein；

The cellulose restriction endonuclease that any gene of three of the above encodes belongs to the scope of protection of the present invention.

3. recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing SEQ ID NO.1 gene order.

Further, recombinant vector is made of empty carrier with the target gene for being inserted into the empty carrier, and the target gene is power Benefit require 1 described in gene.

Further, the empty carrier is pET28 carrier.

4. a kind of preparation method of cellulose restriction endonuclease, which comprises the following steps:

1) by genetic recombination described in technical solution 1 to being building up in pET28 carrier；It is transformed into coli strain again In, obtain expression strain；

2) step 1) expression strain is cultivated in LB liquid medium, and the IPTG induction of 0.1~0.5mM, fermentation is added It finishes, ultrasonication, centrifuging and taking supernatant, obtains the cellulose restriction endonuclease of soluble recombination.

Further, further include protein purification steps: being purified with DEAE chromatographic column to the supernatant that step 2) obtains, first Column is crossed with equilibration buffer chromatographic column, then by supernatant, with Na containing 20mM₂HPO₄, 20~30mM NaCl, pH7.0 buffering Liquid prewashing pillar, then with containing 100mM~200mMNaCl, 20mM Na₂HPO₄, pH7.0 buffer soln gets off albumen wash-out ?.

Protection scope of the present invention is also belonged to according to the albumen after purification that the above-mentioned method for preparing albumen is prepared.

5. recombinant vector described in the albumen that gene described in above-mentioned technical proposal 1, technical solution 2 or 4 obtain, technical solution 3, The application of expression cassette, transgenic cell line or recombinant bacterium in the production of bio-fuel ethyl alcohol, food, raising and field of printing and dyeing It belongs to the scope of protection of the present invention.

The beneficial effects of the present invention are: the present invention will provide it is a kind of new through artificial reconstructed cellulose restriction endonuclease base Cause, and E. coli expression strains are reconnected after the gene constructed carrier to pET28, it realizes with cellulose endonuclease activity Expression product solubility expression and high efficient expression；It is additionally provided in the simple and effective purifying reconstituted protein cellulose of one kind simultaneously The method of enzyme cutting makes purity of protein after purification be up to 95%, this provides a kind of simple height for the preparation of cellulase preparation Technical foundation is established in the preparation method of effect, the extensive use for the enzyme in fields such as work, agriculture, poultry and doctors.

Additional aspect and advantage of the invention will be set forth in part in the description, and will partially become from the following description Obviously, or practice through the invention is recognized.

Detailed description of the invention

In order to keep the purpose of the present invention, technical scheme and beneficial effects clearer, the present invention provides following attached drawing and carries out Illustrate:

Fig. 1 is the pET28/EG vector construction schematic diagram in the embodiment of the present invention.

Fig. 2 is the SDS-PAGE testing result of the pET28/EG recombinant vector expression target protein in the embodiment of the present invention Figure.

Fig. 3 is elution curve of the recombinant fiber element restriction endonuclease through DEAE column chromatography in the embodiment of the present invention.

Fig. 4 is the SDS-PAGE testing result figure of the element restriction endonuclease of recombinant fiber after purification in the embodiment of the present invention.

Fig. 5 is the SDS-PAGE testing result figure of the recombinant fiber element restriction endonuclease of the concentration in the embodiment of the present invention.

Fig. 6 is the SDS-PAGE testing result figure that target protein is expressed in comparative example.

Fig. 7 is the HPLC result figure of Enzyme activity assay in the embodiment of the present invention.

Specific embodiment

Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other Embodiment shall fall within the protection scope of the present invention.Test method without specific conditions in embodiment, usually according to conventional strip Part or according to the normal condition proposed by manufacturer.Material used in embodiment, reagent etc. unless otherwise specified can be from quotient Industry approach obtains.% in embodiment is unless otherwise specified mass percentage.Quantitative test in following embodiment, Three repeated experiments are respectively provided with, data are the average value or mean+SD of three repeated experiments.

The present invention selects Bacillus coli expression bacterium, vector amplification bacterial strain TOP10 and expression vector pET28 to be purchased from the U.S. Invritrogen company.

Used medium formula is as follows:

1) LB liquid medium: NaCl 10g, peptone 10g, yeast extract 5g, distilled water 1L, high pressure sterilization, room temperature It saves；

2) LB/Kan plate: NaCl 10g, peptone 10g, yeast extract 5g, distilled water 1L, agar powder 15g, high pressure After sterilizing, 70 DEG C are cooled to hereinafter, the kanamycins (Kan) that 1mL concentration is 100mg/ml is added, is sufficiently mixed rear a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices, 4 DEG C It is kept in dark place；

3) LB/Kan culture medium: NaCl 10g, peptone 10g, yeast extract 5g, distilled water 1L are cold after high pressure sterilization But to 70 DEG C hereinafter, addition 1mLKan (100mg/ml), is sufficiently mixed, 4 DEG C are saved；LB liquid medium: NaCl10g, albumen Peptone 10g, yeast extract 5g, distilled water 1L, high pressure sterilization, room temperature preservation.

4) 50 × TAE agarose gel electrophoresis buffer: Tris alkali 121g, glacial acetic acid 28.6mL, 0.5mol/L EDTA (pH8.0) 50mL adds distilled water to be settled to 500mL, room temperature preservation；

5) 50mg/mL kanamycins saves liquid: kanamycins 0.5g adds distilled water to dissolve and is settled to 10mL, after packing It is saved in -20 DEG C；

6) 5 × SDS-PAGE sample-loading buffer: 1M Tris-HCl (pH 6.8) 1.25mL, SDS 0.5g, BPB 25mg, Glycerol 2.5mL is settled to 5mL after adding deionized water dissolving, in room temperature preservation after packing (about 500 every part of μ L), is added using every part Enter the mixing of 25 μ L beta -mercaptoethanols；

7) 5 × SDS-PAGE electrophoretic buffer: Tris 15.1g, glycine 94g, SDS 5.0g, be added about 800mL go from Sub- water is settled to 1L, room temperature preservation after dissolution is sufficiently stirred；

8) coomassie brilliant blue R_250 dyeing liquor: the ice second of 225mL methanol, 46mL is added in coomassie brilliant blue R_250 0.25g Acid, 225mL deionized water simultaneously stir evenly, after filter paper removes particulate matter, room temperature preservation；

9) Coomassie brilliant blue destainer: glacial acetic acid 50mL, methanol 150mL, deionized water 300mL, after being sufficiently mixed, room temperature It saves.

Embodiment 1

Present embodiments provide a kind of recombinant fiber element incision enzyme gene EG of the artificial chemistry synthesis of optimization, particular sequence As shown in the SEQ ID NO.1 in sequence table, protein sequence corresponding to the gene such as the SEQ ID NO.2 institute in sequence table Show.DNA sequence dna of the sequence of synthesis without obvious similitude in ncbi database, it is according to Escherichia coli and expression vector The characteristics of pET28, a kind of DNA molecular for optimizing and synthesizing, this is numerous Preferences that e. coli codon is utilized, base Data-optimized obtained one of the DNA molecular such as influence, G/C content because of higher structure to transcription, though remove the molecule hereinafter, The DNA molecular of right other optimizations is also able to achieve the expression in Escherichia coli but expression quantity will be far below DNA provided by the invention Molecule.

Gene after above-mentioned optimization is connected in coli expression carrier pET28 and is obtained recombinant vector, the above warp It is flat to be coated with corresponding resistance LB for the heat-shock transformed competent cell to E. coli expression strains of the recombinant vector of sequence verification Plate is cultivated 12 hours in 37 DEG C of constant incubators, and transformant is screened, wherein pET28/EG vector construction as shown in FIG. 1, FIG. 1 is PET28/EG vector construction schematic diagram in the embodiment of the present invention.

PET28 recombinant vector using optimized native cellulose incision enzyme gene sequence is expression vector, is corresponded to IPTG of the expression bacterium transformant through 0.1-0.5mM the expression for detecting target protein, the total egg of thallus are induced at 18 DEG C White SDS-PAGE result is as shown in Fig. 2, recombinant fiber element inscribe enzyme molecular weight is 40kDa or so, the target protein of expression such as arrow Shown in head.

Embodiment 2

The present embodiment provides a kind of methods for preparing albumen, specifically comprise the following steps:

S1: optimization gene, building prokaryotic expression carrier and conversion: artificial chemistry synthesizes optimized mature fibers element inscribe Enzyme gene is connected to pUC universal support and obtains pUC/EG, using BamHI and HindIII double digestion pUC/EG, the EG piece that will be obtained Section is subcloned again into expression vector pET28, obtains recombinant expression carrier pET28/EG, vector construction is as shown in Figure 1.pET28/ The key step of EG vector construction is as follows:

(1) BamH I and Hind III double digestion recombinant vector pUC/EG are used, purpose segment EG is obtained, reaction system is as follows (restriction endonuclease and buffer used are purchased from Dalian TAKARA company):

(2) BamH I and Hind III double digestion pET28 are used, carrier segment, the following (restriction endonuclease used of reaction system are obtained And buffer is purchased from Dalian TAKARA company):

(3) the purpose segment and carrier segment DNA gel obtained step (1) and (2) withdraws kit recycling, the examination Agent box is purchased from Dalian TAKARA company, and concrete operations are carried out by kit specification.

(4) the purpose segment and carrier T4DNA ligase obtained step (3) recycling (purchased from Dalian TAKARA company) It is attached reaction, target gene is properly inserted in expression vector reading frame, and reaction system is as follows:

Recombinant vector pET28/EG is transformed into Escherichia coli TOP10 bacterial strain, then extracts recombinant vector from TOP10 pET28/EG；Recombinant vector pET28/EG is transferred in host cell E. coli expression bacterial strain with heat shock method, with containing Kan The LB plate screening of resistance obtain include recombinant vector pET28/EG E. coli expression strains transformant.

S2: synthetic gene after optimization the expression and extraction of soluble recombinant fiber element restriction endonuclease: will be included It is 0.5-0.7 that Escherichia coli recombinant conversion of pET28/EG carrier, which is cultivated in 37 DEG C of LB liquid medium to OD600, so It is separately added into the IPTG that concentration is 0mM, 0.1mM, 0.2mM and 0.5mM afterwards, is induced 24 hours at 18 DEG C, the bacterium collected after induction Body ultrasonication is crushed power 300W, is crushed 2s, gap 6s, and after recycling 90 times, centrifuging and taking supernatant obtains recombinant fiber element Restriction endonuclease, SDS-PAGE result are as shown in Figure 2.

S3: the purifying of recombinant fiber element restriction endonuclease: being expanded culture and induces 20-24 small with 0.1mM IPTG at 18 DEG C When, thallus is resuspended in the buffer solution A of 40ml (containing 20mM by the thallus of the expression bacterium after collecting after IPTG inducing expression Na₂HPO₄It in 1mM protease inhibitors PMSF, pH 7.0), is then crushed with Ultrasonic Cell Disruptor, is crushed power 300W, Broken 2s, gap 6s, recycle 90 times；By broken bacterium solution at 4 DEG C, 30000g is centrifuged 15min；It will be centrifuged on obtained Clear liquid is added in the DEAE chromatographic column pre-equilibrated through buffer solution A；With 100ml buffer solution B (Na containing 20mM₂HPO₄, 20~ 30mMNaCl, pH7.0) rinsing protein purification pillar after, be added NaCl be 100~200mM buffer C (contain 20mM Na₂HPO₄, pH 7.0), albumen wash-out is got off, elution curve as shown in figure 3, collect figure in target arrow peak protein sample, SDS-PAGE analysis concrete outcome is carried out to sample protein as shown in figure 4, what 100~200mMNaCl was eluted using SDS-PAGE Albumen is recombinant fiber element restriction endonuclease of the purity 95% or more.

S4: the concentration of recombinant fiber element restriction endonuclease: by protein example pH4.0 20mM NaH₂PO₄It is slow with citric acid It dialyses under fliud flushing, after dialysis, be concentrated by ultrafiltration using the super filter tube that molecular cut off is 15kDa can be obtained purity and exist 95% or more high concentration recombinant fiber element restriction endonuclease, the albumen after concentration detect its purity using SDS-PAGE, As a result as shown in figure 5, obtained albumen is the recombinant fiber element endonuclease protein of high-purity.It is scanned and is combined using glue Bradford method detects the concentration of target protein, and table 1 is that soluble recombination is fine in thallus that 100ml is induced through IPTG Tie up yield and purity result of the plain endonuclease protein through each purification step.

1 protein purification result of table

It should be noted that SDS-PAGE sample buffer is added in the supernatant that step S2 is obtained, to its solvable egg It is white to be analyzed.When the concentration of IPTG is 0.1,0.2 and 0.5mM at a temperature of 18 DEG C, soluble recombinant fiber can be obtained Plain restriction endonuclease.For save the cost, shorten the production cycle, present invention preferably employs 18 DEG C of inducing temperature, the IPTG of 0.1mM is lured Lead expression.

Comparative example

The formation of Poria cocos is parasitized on pine in heaven under appropriate conditions by Poria mycelium, is constantly decomposed in pine Nutrition, and by after bacteriumization extra substance accumulation expand rapidly, the vegetative storage organ and suspend mode organ of formation are sclerotium, It is commonly called as loose Poria cocos.The main component of timber is cellulose.Therefore, it there will more than likely be the secretion fiber type of high activity in Poria cocos mycelia Plain enzyme.Inventor carries out analysis using express spectra of the transcript profile technology to the cellulase catabolic enzyme of Poria cocos and has found the high abundance Cellulose decomposition enzyme gene.The data obtained using Poria cocos transcript profile, design primer, simultaneously through RT-PCR amplification target gene It is connected to cloning vector, the target gene sequence of amplification is as shown in SEQ ID NO.3 in sequence table, by the Poria cocos recombinant fiber element Incision enzyme gene BamH I and Hind III double digestion, and be connected to equally through BamH I and Hind III double digestion PET28 expression vector.Recombinant vector is coated with corresponding resistance through the heat-shock transformed competent cell to E. coli expression strains LB plate is cultivated 12 hours in 37 DEG C of constant incubators, and transformant is screened.The pET28/EG carrier of gene before optimizing will be included Escherichia coli recombinant conversion is cultivated in 37 DEG C of LB liquid medium to OD₆₀₀Be 0.4, be then respectively adding concentration be 0, 0.1, the IPTG of 0.2,0.5mM is induced 24 hours, the thallus ultrasonication collected after induction at 18 DEG C, is crushed power 300W, is broken Broken 2s, gap 6s, after recycling 90 times, centrifuging and taking supernatant does not obtain soluble recombinant fiber element restriction endonuclease, SDS-PAGE result As shown in Figure 6.Comparative example result explanation, the Poria cocos recombinant fiber element incision enzyme gene ability after artificial optimization only of the present invention Soluble expression is realized in Escherichia coli.

Embodiment 3

The present invention is generated using high performance liquid chromatography detection sodium carboxymethylcellulose (CMC-Na) by inscribe enzyme hydrolysis a small amount of The ability of glucose determines enzyme activity.The specific method is as follows: the 200 μ g of recombinant fiber element restriction endonuclease of purifying is added to containing for pH4 In 1% CMC-Na solution, reacted 8 hours at 40 DEG C；After reaction, by sample 0.22 μm of filtering with microporous membrane to sample Bottle, for liquid-phase chromatographic analysis.The method of liquid phase is as follows: chromatographic column: Aglient nh 2 column, 250 × 4.6mm, and 5 μm；Mobile phase: Acetonitrile: water=70:30 (volume ratio), flow velocity: 1.0mL/min, sample volume: 10uL, column temperature: 35 DEG C, detector: differential refraction inspection Survey device.HPLC result is as shown in fig. 7, wherein upper figure a is that CMC-Na is not examined in the case where the recombinant fiber element restriction endonuclease is not added Glucose peaks are measured, following figure b is after CMC-Na is added the enzyme reaction of cellulose inscribe 8 hours prepared by the present invention, to detect bright Aobvious glucose peaks, the retention time at the peak are 5.581 minutes, and concentration of glucose is about 56ng/ μ L, illustrates the restriction endonuclease really The ability of glucose is generated with hydrocellulose.

Therefore, according to result above, according to the present invention the provided building of the gene order as shown in SEQ ID NO.1 Recombinant vector, further it is expressed go out new cellulose restriction endonuclease be that a kind of sodium carboxymethylcellulose that can decompose generates glucose Novel recombinant fiber element restriction endonuclease.

Finally, it is stated that preferred embodiment above is only used to illustrate the technical scheme of the present invention and not to limit it, although logical It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be Various changes are made to it in form and in details, without departing from claims of the present invention limited range.

Sequence table

<110>Huaihua College

<120>preparation method of recombinant fiber element incision enzyme gene and its albumen and albumen

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1020

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

atgttgaaca tcaaggcagc ctctctcgtg ctactggcag cggcagcgcc ggtggtactt 60

ggtcatggtc aagtccactc tgtcatcatc gcttcccctt ctgcgacgtt cccagctgca 120

gacgcctacg ctgctgctga tcccactagc cctctgcgca aactaaacac ctacggacct 180

gctgccaact ttactggccc tgacattacg tgtggaccag gagggaatat ccccatcacg 240

ccattggccc ctgtagatgc tggaagtttg gtcacatttg attggcagag ttggaactcc 300

gttcaccctg ggcctgtcat gacgtatatc gccaaatgtc cggatggctg cgctaacttt 360

aagggcgacg aaggcaacgt ttgggttaaa attgaccagg accaatacaa tcccgatcgt 420

ggttctgatc tcgcttgggg tgaagagttg ctgcgtttga agacaaactc aaagtatagc 480

gtcaacgttc ctgcaggttt agaaaacggg gagtatatcc ttcgtcacga aattcttggt 540

ctccatgttg ctggcaccgt catgggtgcc caattctacc cgaactgctt gcaaatcaag 600

gttcaaaacg gtggaagtgt tactctaccc gaaggtatcc ctcttccagg gtcctatgac 660

ccatatgatc cgggtatcct tgttcagctc tggcaaatta ctcttcagaa ccccaactac 720

accgctcctg gtggccctgt gcttcttcct ggtggttcag gcgactgggc cgaggctaac 780

tacggatcga aggccgtcag ttcacctgca gcccctccaa caacgacagc atctcagtcg 840

gcgcacccga gcaccacaac ggctgctcct actaccatca actcatcacc cgcgtcgtcc 900

cctactggcg ttacggtcca gaagtacggg caatgcggag gtcagggata ttcgggagcg 960

acactttgtg catcgggaac gacttgcacg gctttaaacg attactactc tcaatgcctc 1020

<210> 2

<211> 340

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 2

Met Leu Asn Ile Lys Ala Ala Ser Leu Val Leu Leu Ala Ala Ala Ala

1 5 10 15

Pro Val Val Leu Gly His Gly Gln Val His Ser Val Ile Ile Ala Ser

20 25 30

Pro Ser Ala Thr Phe Pro Ala Ala Asp Ala Tyr Ala Ala Ala Asp Pro

35 40 45

Thr Ser Pro Leu Arg Lys Leu Asn Thr Tyr Gly Pro Ala Ala Asn Phe

50 55 60

Thr Gly Pro Asp Ile Thr Cys Gly Pro Gly Gly Asn Ile Pro Ile Thr

65 70 75 80

Pro Leu Ala Pro Val Asp Ala Gly Ser Leu Val Thr Phe Asp Trp Gln

85 90 95

Ser Trp Asn Ser Val His Pro Gly Pro Val Met Thr Tyr Ile Ala Lys

100 105 110

Cys Pro Asp Gly Cys Ala Asn Phe Lys Gly Asp Glu Gly Asn Val Trp

115 120 125

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

130 135 140

Ala Trp Gly Glu Glu Leu Leu Arg Leu Lys Thr Asn Ser Lys Tyr Ser

145 150 155 160

Val Asn Val Pro Ala Gly Leu Glu Asn Gly Glu Tyr Ile Leu Arg His

165 170 175

Glu Ile Leu Gly Leu His Val Ala Gly Thr Val Met Gly Ala Gln Phe

180 185 190

Tyr Pro Asn Cys Leu Gln Ile Lys Val Gln Asn Gly Gly Ser Val Thr

195 200 205

Leu Pro Glu Gly Ile Pro Leu Pro Gly Ser Tyr Asp Pro Tyr Asp Pro

210 215 220

Gly Ile Leu Val Gln Leu Trp Gln Ile Thr Leu Gln Asn Pro Asn Tyr

225 230 235 240

Thr Ala Pro Gly Gly Pro Val Leu Leu Pro Gly Gly Ser Gly Asp Trp

245 250 255

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

260 265 270

Pro Thr Thr Thr Ala Ser Gln Ser Ala His Pro Ser Thr Thr Thr Ala

275 280 285

Ala Pro Thr Thr Ile Asn Ser Ser Pro Ala Ser Ser Pro Thr Gly Val

290 295 300

Thr Val Gln Lys Tyr Gly Gln Cys Gly Gly Gln Gly Tyr Ser Gly Ala

305 310 315 320

Thr Leu Cys Ala Ser Gly Thr Thr Cys Thr Ala Leu Asn Asp Tyr Tyr

325 330 335

Ser Gln Cys Leu

340

<210> 3

<211> 1020

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

atgttaaata ttaaggcagc ctcgctggtg ctactcgcag cggcagcgcc cgtcgtactg 60

ggacacgggc aggtgcattc ggtcatcatc gcttcccctt cggcgacgtt cccggctgca 120

gacgcctacg ctgctgctga tccgactagc cctttgcgca aactaaacac ctacggacct 180

gctgccaact ttactggacc ggacattacg tgtggaccag gcggcaatat cccgatcacg 240

ccattggccc cagtagatgc tggaagtttg gtcacatttg attggcagag ttggaactca 300

gttcaccctg gacctgtcat gacgtatatc gccaaatgtc cggatggctg cgctaacttt 360

aagggcgacg aagggaacgt ttgggttaaa attgaccagg accaatacaa tccagatcgt 420

ggctcggatc tcgcttgggg cgaagagttg ctgcgtttga agacaaactc caagtatagc 480

gtcaacgttc cggcaggctt agaaaacggc gagtatatcc ttcgtcacga aattcttggc 540

ttgcatgttg ctggaaccgt catgggcgcc caattctacc ccaactgctt gcaaatcaag 600

gttcaaaacg gcggcagtgt tactctacct gaaggcatcc ctcttccagg ctcatatgac 660

ccatatgatc cgggcatcct ggttcagctc tggcaaatta ctcttcagaa ccccaactac 720

accgctccgg ggggccctgt gcttcttcct ggaggttcag gcgactgggc cgaggctaac 780

tacggctcca aggccgtcag ttcgccagca gccccgccca caacgacagc atcccagtcg 840

gcgcacccaa gcaccacaac ggctgctccc actaccatca actcgtcgcc ggcgtcctca 900

ccgactgggg ttacggtcca gaagtacggc caatgcggcg gacaggggta ttccggggcg 960

acactttgtg catcgggcac gacttgcacg gctttaaacg attactactc acaatgcctc 1020

Claims

1. recombinant fiber element incision enzyme gene, which is characterized in that the gene is at least containing the DNA of one of following nucleotide sequences Segment:

1) in sequence table SEQ ID NO.1 nucleotide sequence；

2) there is 95% or more homology and the identical biological function albumen of coding with nucleotide sequence shown in SEQ ID NO.1 The nucleotide sequence of matter；

3) nucleotide sequence of biological function protein identical as nucleotide sequence hybridization shown in SEQ ID NO.1 and coding.

2. the cellulose restriction endonuclease that gene according to claim 1 encodes.

3. cellulose restriction endonuclease according to claim 2, it is characterised in that: the cellulose restriction endonuclease be following (1) or (2) albumen:

(1) amino acid sequence of albumen is as shown in SEQ ID NO.2 in sequence table；

(2) amino acid sequence shown in SEQ ID NO.2 passes through the substitution of one or several amino acid residues, missing in sequence table And/or add and have the protein as derived from SEQ ID NO.2 of cellulose endonuclease activity.

4. recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing gene described in claim 1.

5. recombinant vector according to claim 4 is made of, feature empty carrier and the target gene for being inserted into the empty carrier It is, the target gene is gene described in claim 1.

6. recombinant vector according to claim 5, which is characterized in that the empty carrier is pET28 carrier.

7. a kind of preparation method of recombinant fiber element restriction endonuclease described in claim 2 or 3, which is characterized in that including following step It is rapid:

1) by genetic recombination described in claim 1 to being building up in pET28 carrier；It is transformed into coli strain, obtains again To expression strain；

2) step 1) expression strain to be cultivated in LB liquid medium, and the IPTG induction of 0.1~0.5mM is added, fermentation finishes, Ultrasonication, centrifuging and taking supernatant obtain the cellulose restriction endonuclease of soluble recombination.

8. preparation method according to claim 7, which is characterized in that further include protein purification steps: using DEAE chromatographic column The supernatant that step 2) obtains is purified, first uses equilibration buffer chromatographic column, then supernatant is crossed into column, with containing 20mM Na₂HPO₄, 20~30mM NaCl, pH7.0 buffer prewashing pillar, then with the NaCl containing 100mM~200mM, 20mM Na₂HPO₄, pH7.0 buffer soln gets off albumen wash-out.

9. the albumen obtained according to the preparation method of claim 7 or 8.

10. recombinant vector described in gene described in claim 1, claim 2,3 or 9 albumen, claim 4, expression cassette, The application of transgenic cell line or recombinant bacterium in the production of bio-fuel ethyl alcohol, food, raising and/or field of printing and dyeing.