CN101845426B - Odontotermes formosanus beta-glucosidase, coding gene, vector and application thereof - Google Patents

Abstract

The invention provides a dontotermes formosanus beta-glucosidase, a coding gene, a vector and the application thereof. The beta-glucosidase has an amino acid sequence having more than 95% homology with a polypeptide shown by SEQ ID No.2. The invention discovers the gene for coding dontotermes formosanus beta-glucosidase, which can abundantly express in host cells to produce the beta-glucosidase for degrading cellulose, and the activity of the beta-glucosidase is detected to reach 117U. The beta-glucosidase provided by the invention and the coding gene thereof can be applied to the cellulose degradation.

Description

Blackwing subterranean beta-glucosidase, encoding sox, carrier and application

(1) technical field

The present invention relates to a kind of blackwing subterranean beta-glucosidase, encoding sox, carrier and application.

(2) background technology

The energy, resource and environmental problem are the 21 century severe challenges that faced of survival and development of mankind, also are the principal elements of restriction Chinese society Economic development.In the long run, petroleum resources will move towards exhausted edge rapidly in the first half in this century, and the task of the alternate resources of exploitation sustainable use is very urgent.China has become net import of oil state from beginning in 1993, and CNPC's import interdependency was near 52% in 2008.Undue dependence on import crude oil has constituted potential threat to China's energy and resource provisioning strategic security.Simultaneously, the burning of fossil oil causes CO2 emissions constantly to increase, and causes global warming.The CO2 emissions of China will continue to increase in the recent period, and this has just increased in the world the pressure of China's carbon dioxide discharge-reduction greatly, requires us to seek the alleviation approach as early as possible.

According to estimates, the biomass total amount that the annual photosynthesis of whole world green plants produces reaches 1,730 hundred million tons, and the energy that closes is 2X10 ²¹Joule, be equivalent to annual catabiotic 10 times of the whole world (Li Zhongren. the development and utilization of bioenergy. Yanbian University's agronomy journal, 2003,25 (3): 225-227).Biomass are renewable energy source, for the mankind provide energy; Also be renewable resources, produce required raw material for the mankind provide materiality; And its production and use do not pollute environment.Cellulose substances is the main part of biomass resource, and only the annual agriculture and forestry organic waste material that produces of China is just had an appointment 1,000,000,000 tons, and is cheap, in liberal supply, and fully developed.

At present, biomass energy and chemical industry use grain as raw material mostly.In China, because population is huge, grain security is extremely important, and it will be prerequisite with " do not strive grain, do not strive ground with grain, do not destroy ecotope with the people " that country explicitly calls for development of technologies.Along with the variation of China's life in the countryside energy structure and the development of intensification production, progressively bavin is burnt in heating to stalk and feed has developed into " waste " by traditional being used for.At harvest season on-site incineration usually, cause serious environmental to pollute, be badly in need of seeking the road of new efficient utilization.One of the most promising scheme is to utilize the high-performance bio catalyzer to come the Degradation and Transformation stalk, produces human liquid fuel and the Chemicals of being badly in need of.

Cellulosic enzyme liberating needs the participation of cellulase, and cellulase is not a kind of simple enzyme, but the enzyme system of a complicacy of forming by the some kinds of enzymes that are mutually related; In general, cellulase is mainly formed by three types: (1) endo-beta-1,4-glucanase (Endo--β-1; 4-glucanase, EC 3.2.1.4), mainly act on the inner noncrystalline domain of cellulosic molecule; Random hydrolysis β-1; 4 glycosidic links with the brachymemma of long chain cellulose molecular chain, produce the small molecules Mierocrystalline cellulose and the soluble fiber oligosaccharide that have non reducing end in a large number; (2) circumscribed-β-1,4-LSD (Exo-β-1,4-glucanase, EC 3.2.1.91) mainly acts on the non-reduced end of cellulose molecular chain, and hydrolysis β-1,4 glycosidic link produces cellobiose; (3) beta-glucosidase (β-glucosidase; EC 3.2.1.21); Cellobiose and other oligose that the above-mentioned two kinds of enzymes of hydrolysis produce generate glucose (Coughlin M P, Ljungdahl J G.Comparative biochemistry of fungal and bacterial cellulolyticenzyme systems.In Biochemistry and Genetics of Cellulose Degradation.New York Academic; 1988,11-30; Goyal A, et al.Characteristics of fungalcellulases.Bioresource Technology, 1991,36 (1): 37-50; Wang Lanfen. mechanism of action of cellulase and Application and Development. brewing science and technology, 1997,6:16-17; Yan Yan, Zhang Quanfu. the character of cellulase, application and Significance for Environment thereof. agricultural environment and development, 1997,1:17-20).

Occurring in nature, cellulase extensively are present in the body of bacterium, fungi, mollusk, protozoon, crustacean and some insects such as cockroach, longicorn, termite etc.Wherein, people study at most to bacterium, the intravital cellulase of fungi, then study lessly to the intravital cellulase of insect.Research about insect body inner cellulose enzyme at present mainly concentrates on some kinds of longicorn and termite.

It is the ancient biology in main food source with lignocellulose that termite is one type, in earth physics circulation and flow of energy, plays crucial effect.In the evolution of long period of time process, termite has formed the cellulase system of degraded cellulose very efficiently in order to adapt to the digestion of cellulose family food in the body.The result of study of nearly decades shows that the intravital cellulase of termite mainly is made up of endo-beta-1,4-glucanase and beta-glucosidase, and under certain condition, these two kinds of enzymes also have circumscribed-β-1, the 4-dextranase activity.Blackwing subterranean Odontotermes formosanus is distributed in the wide geographic area on the south 35 ℃ of the north latitude in China; External then mainly be distributed in the ground such as Okinawa of Burma, Thailand and Japan; Be a kind of termite in the soil (Li Dong that fungus garden is cultivated fungi that sets up; Deng. the origin cause of formation and improvement thereof that the termite pipe leaks. insect knowledge, 2001,38 (3): 182-185; Yellow demutation waits and writes. Chinese fauna Insecta the 17 volume Isoptera. and Beijing: the .2000 of Science Press).The early stage result of study in contriver laboratory shows; Compare with Pingyang heap sand termite with eastern subterranean termite, thin jaw reticulitermes flavipe, Taiwan formosanes; It is high that the activity of beta-glucosidase of blackwing subterranean head is obviously wanted; And enzyme source research shows that the intravital cellulase of blackwing subterranean is by himself secretion (Mo Jianchu, et al.Cellulaseactivity in five species of important termites in China.Applied Entomologyand Zoology; 2004,39 (4): 635-641).

Utilize in the cellulosic production technique of cellulose degraded current; Perplex most people be enzymolysis efficiency that how to improve cellulase and the production cost that reduces cellulase (Wangjinglin. the progress of cellulase immobilization. life science .1997; 9 (3): 116-118,135).Seek highly active cellulase and utilize Protocols in Molecular Biology to transform cellulose enzyme gene from occurring in nature; And then the active height of exploitation, Heat stability is good, the substrate scope is wide, alcohol-tolerant ability is strong cellulase bioengineered strain, be the effective way that addresses this problem.

(3) summary of the invention

The purpose of this invention is to provide a kind of beta-glucosidase and encoding sox thereof, carrier and application.

The technical scheme that the present invention adopts is:

A kind of beta-glucosidase has and the aminoacid sequence of peptides homologous more than 95% shown in the SEQ ID No.2.

Because the singularity of aminoacid sequence; Anyly contain peptide protein homology with aminoacid sequence shown in the SEQ ID NO.2 at the fragment more than 95% or its variant; Like its examples of conservative variations, bioactive fragment or verivate, all belong to the row of protection domain of the present invention.Concrete change can comprise amino acid whose disappearance, insertion or replacement in the aminoacid sequence; Wherein, for the conservative property change of variant, the amino acid of being replaced has structure similar with original acid or chemical property, and as replacing Isoleucine with leucine, variant also can have non-conservation and change, as replacing glycocoll with tryptophane.

In the present invention, term " beta-glucosidase ", " blackwing subterranean beta-glucosidase " or " enzyme OfBG " all interchangeable use, all refer to have the beta-glucosidase amino acid residue sequence albumen or the polypeptide of (SEQ IDNo.2).

In the present invention, term " beta-glucosidase " refers to have the polypeptide of full length sequence (1-472 position) of the SEQ IDNo.2 of activity of beta-glucosidase.This term also comprises the variant form of the SEQ ID No.2 sequence with activity of beta-glucosidase.These variant forms comprise (but being not limited to): several amino acid whose disappearances, insertion and/or replacement, and at C-terminal and/or N-terminal interpolation one or several amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises the active fragments and the verivate of beta-glucosidase.

The variant form of this polypeptide comprises: homologous sequence, allelic variant, natural mutation, the induced mutation body, under high or low rigorous degree condition can with the coded albumen of DNA of beta-glucoside enzyme dna hybridization.The present invention also provides other polypeptide, as comprises beta-glucosidase or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of beta-glucosidase.Usually, this fragment have the beta-glucoside enzyme sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, about best 70 continuous amino acids.

Invention also provides the analogue of beta-glucosidase or polypeptide, and the difference of these analogues and natural beta-glucosidase can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain through various technology, as through radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L (like D-amino acid), and has non-natural analogue that exist or synthetic amino acid (like β, γ amino acid).Should be understood that polypeptide of the present invention is not limited to above-mentioned representational polypeptide of giving an example.

(the not changing primary structure usually) form of modification comprises; The chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, in the synthetic and processing of polypeptide or further, carries out glycosylation modified and polypeptide that produce in the procedure of processing like those.This modification can be carried out glycosylated enzyme (like mammiferous glycosylation or deglycosylating enzyme) and accomplishes through polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (like Tyrosine O-phosphate, Serine O-phosphate, phosphothreonine).Thereby also comprise and modified the polypeptide that has improved its anti-proteolyze performance or optimized solubility property.

Preferably, said beta-glucosidase has the aminoacid sequence shown in the SEQ ID No.2.

The invention still further relates to the application of described beta-glucosidase in cellulose degradation.

The invention still further relates to a kind of gene of the said beta-glucosidase of claim 1 of encoding.

Concrete, said gene has the nucleotide sequence shown in the SEQ ID NO.1.Perhaps, said gene has the nucleotide sequence shown in the SEQ ID NO.3.

In the present invention, term " polynucleotide of encoding beta-glucosidase " refer to the encode nucleotide sequence of polypeptide with activity of beta-glucosidase is like the 70th～1488 (SEQID No.3) nucleotide sequence and degenerate sequence thereof among the SEQ ID No.1.This degenerate sequence is meant the 70th～1488 (the SEQ ID No.3) Nucleotide of encoder block that is arranged in SEQ ID No.1 sequence, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, thus with SEQ ID No.1 in the 70th～1488 nucleotide sequence homology be low to moderate about 70%.The degenerate sequence described sequence of SEQ ID No.2 of also encoding out.This term also comprises can be under the moderate stringent condition, more preferably under the height stringent condition, with among the SEQ ID No.1 from the nucleotide sequence of the nucleotide sequence hybridization of the 70th～1488 in Nucleotide (SEQ ID No.3).This term also comprise with SEQ ID No.1 in from the homology of nucleotide sequence at least 70% of the 70th～1488 in Nucleotide (SEQ ID No.3), preferably at least 80%, at least 90% nucleotide sequence more preferably.

This term also comprises encoding to have the variant form with the proteic SEQ IDNo.2 sequence of beta-glucosidase identical function.These variant forms comprise (but being not limited to): the disappearance of several Nucleotide, insertion or replacement, and at 5 ' and/or 3 ' several Nucleotide of end interpolation.

The present invention also comprises beta-glucosidase polypeptid coding sequence and segmental antisense sequences thereof, and this antisense sequences can be used for suppressing the expression of beta-glucosidase in the cell.

The invention still further relates to a kind of recombinant vectors that contains said gene, and the host cell that utilizes said recombinant vectors conversion, transduction or transfection to obtain.In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.The example of prokaryotic host cell commonly used comprises intestinal bacteria, Bacillus subtilus etc.Eukaryotic host cell commonly used comprises yeast cell, insect cell and mammalian cell, and preferably, this host cell is an eukaryotic cell, like Tn cell, Chinese hamster ovary celI, COS cell etc.

The invention still further relates to the application of described gene in preparation reorganization beta-glucosidase.

The present invention has found a kind of gene of the blackwing subterranean beta-glucosidase of encoding, and this gene can great expression be used for cellulosic degraded to produce this beta-glucosidase in host cell, reaches 117U through detecting beta-glucosidase enzyme of the present invention work.Beta-glucosidase provided by the present invention and encoding sox thereof can be applicable to cellulosic degraded.

(4) embodiment

Below in conjunction with specific embodiment the present invention is described further, but protection scope of the present invention is not limited in this:

Embodiment 1: the clone of blackwing subterranean beta-glucosidase gene (OfBG)

1. gene clone

Under condition of ice bath, dissect the sialisterium of blackwing subterranean (picking up from Yuhang District east, Hangzhou broom small stream embankment); And with the body of gland that obtains rapidly as in Trizol (Invitrogen) solution; Rapid its total RNA of extracting, and be primer reverse transcription generation cDNA with oligo (dT) 18.CDNA with reverse transcription is a template; With pair of degenerate primers (Primer 1:5 '-taycaytgggayytnccncarga-3 '; Primer2:5 '-tcraarttrtccatnarnswcca-3 ') carries out pcr amplification; The fragment that increases is carried out T carrier (Promega) clone, and it is carried out sequencing (Invitrogen).

Consequence devised Auele Specific Primer (the Primer 3:5 '-ttgaaccgataattgcctccaacgaaga-3 ' that obtains according to order-checking; Primer 4:5 '-ggtacaacctgtagccacgtagaagaac-3 '), be respectively applied for 3 ' and 5 ' end of amplification coding OfBG full-length cDNA.With total RNA is template, utilizes 3 ' and 5 ' RACE test kit (Takara-Clontech), reference reagent box specification sheets, 3 ' end and 5 ' terminal sequence of this gene that increases respectively.Respectively the fragment that increases is carried out T carrier (Promega) clone, and it is carried out sequencing (Invitrogen).The sequence that order-checking obtains is carried out sequence assembly with software, finally obtain full-length cDNA.

2. sequence alignment

Utilize DNAStar software that the full length cDNA sequence of coding OfBG is carried out correlation analysis, find the long 1962bp of this sequence (SEQ ID No.1), GC content is 41.7%.Wherein (open reading frame ORF) is 1419bp (SEQ ID No.3) to ORFs, 472 the derivation amino-acid residues (SEQ ID No.2) of encoding.

Utilize the protein molecular weight of this genes encoding of online tool prediction of http://au.expasy.org/ website to be 54.3KDa, iso-electric point is 5.75.Use the functional domain of online SMART instrument (http://smart.embl-heidelberg.de/) predicted protein matter, find that this albumen contains typical Glyco hydro 1 structural domain.

This gene ORF is read cDNA sequence that frame comprised in ncbi database, carry out the BLASTn compare of analysis, the result shows the beta-glucosidase gene height homology of itself and various insects.

Embodiment 2: the expression of blackwing subterranean beta-glucosidase gene (OfBG) in intestinal bacteria

Gene coding region (SEQ IDNo.3) with the blackwing subterranean beta-glucosidase (OfBG) that obtains; Behind Sac I and Hind III double digestion; Connect among the colibacillus expression plasmid pET-28a (Novagen) that same enzyme cuts; Transformed into escherichia coli DH5 α obtains expression plasmid pET-28a-OfBG after order-checking is identified correctly.Plasmid is converted in the e. coli bl21, screening positive clone, and it is carried out abduction delivering.

The expression strain that will contain pET-28a-OfBG is inoculated in the LB nutrient solution; 37 ℃ of overnight cultures obtain seed liquor and are inoculated in the big volume LB liquid nutrient medium by 1: 100 volume ratio, and 37 ℃ are cultured to OD value 0.6; Add IPTG to final concentration be 1mmol/L, induced 4 hours for 28 ℃.With the centrifugal collection thalline of inducing culture thing; Solution with containing 20mM Tris-HCl pH8.0,50mM NaCl, 0.5mM EDTA is washed thalline twice; Be resuspended in the solution that contains 20mM Tris-HCl pH8.0,50mM NaCl, 0.5mM EDTA, 0.5mM PMSF, 0.5mg/ml N,O-Diacetylmuramidase; The supersound process lysing cell is got supernatant survey enzyme and is lived, and does not have activity.

Embodiment 3: the expression of blackwing subterranean beta-glucosidase gene (OfBG) in eukaryotic cell (Tn cell strain, i.e. trichoplusia ni cell strain)

Gene coding region (SEQ IDNo.3) with the blackwing subterranean beta-glucosidase (OfBG) that obtains; Behind Not I and Hind III double digestion; Connect among the Tn fibrocyte expression vector pBacFastHTa (Invitrogen) that same enzyme cuts; Transformed into escherichia coli DH5 α obtains expression plasmid pBacFastHTa-OfBG after order-checking is identified correctly.Plasmid is converted among the intestinal bacteria DH10Bac screening positive clone, extracting plasmid.

Recombinant plasmid is at Lipofectin (GiBco Life) mediation transfection Tn cell down, and transfection is after 48 hours, collecting cell and cell conditioned medium, and the cell conditioned medium that contains the recombinant virus particle is used for the next round infection.The TC-100 continuous passages in 2～3 weeks of warp are cultivated, and collecting cell with ultrasonic degradation method smudge cells, is got supernatant survey enzyme and lived, and enzyme size alive is 117U.

Embodiment 4: the enzyme activity determination of blackwing subterranean beta-glucosidase

The enzyme activity determination of beta-glucosidase adopts Somogyi-Nelson micromethod (Somogyi M; Areagent for the copper-iodometric determination of very small amounts ofsugar.Journal of Biological Chemistry; 1937,117 (2): 771-776; Somogyi M, Anew reagent for the determination of sugars.Journal of Biological Chemistry, 1945,160 (1): 61-68; Somogyi M, Notes on sugar determination.Journal ofBiological Chemistry, 1952,195 (1): 19-23; Nelson N, A photometricadaptation of the Somogyi method for the determination of glucose.Journalof Biological Chemistry, 1951,193 (1): 375-380), concrete operations are following:

1. reagent preparation

Somogyi I: the 288g SODIUM SULPHATE ANHYDROUS 99PCT is dissolved in the zero(ppm) water that 1L boils, and then adds the 24g Rochelle salt crystal successively, 48g yellow soda ash, the 32g sodium hydrogencarbonate, the dissolving back with distilled water diluting to 1.6L, 27 ℃ of preservations.

Somogyi II: the 72g SODIUM SULPHATE ANHYDROUS 99PCT is dissolved in the zero(ppm) water that 300ml boils, and then adds 8g copper sulfate, with the distilled water diluting that boils to 400ml, 27 ℃ of preservations.

Nelson Reagent: the 100g ammonium molybdate is dissolved in the 1.8L zero(ppm) water; Add the 84ml98% vitriol oil then; Add 100ml liquor sodii arsenatis (the 12g sodium arseniate is dissolved in the 100ml zero(ppm) water) again; Solution is kept in the brown bottle and at 37 ℃ and stores 24～48h down then, preserves at room temperature at last.

2. the making of glucose typical curve

In 8 test tubes, be respectively charged into 200 μ g/ml standard glucose 0,0.2,0.4,0.6,0.8,1.0,1.2,1.4ml.Add zero(ppm) water 2.0,1.8,1.6,1.4,1.2,1.0,0.8,0.6ml more successively, be made into every milliliter of glucose solution that contains 0,20,40,60,80,100,120,140 μ g.Add Tong Shiji 2ml (Somogyi I 1.6ml in every test tube; Somogyi II 0.4ml), heats 15min behind the mixing in the boiling water bath, immediately cooling; Add 2ml arsenic molybdic acid reagent (NelsonReagent) again, vibrate and under 520nm, measure light absorption value with spectrophotometer after two minutes.(μ g/ml) is X-coordinate with glucose content, and light absorption value is an ordinate zou under the 520nm, the drawing standard curve.

3. enzyme activity determination

(solvent is 0.1M acetic acid-sodium-acetate buffer, pH5.6) is substrate with the salicin of 0.6% (w/v).Get 950 μ l substrates and 50 μ l enzyme liquid thorough mixing, at 37 ℃ of following water-bath 30min, the reducing sugar that hydrolysis produces adopts the Somogyi-Nelson micromethod to measure.Under 520nm, measure light absorption value at last.An enzyme activity unit is defined as micromole's number of the every gram reducing sugar that protein produces of PM, is expressed as U.

Through measuring, the enzyme work of beta-glucosidase of the present invention reaches 117U.

SEQUENCE LISTING

< 110>Zhejiang University

< 120>blackwing subterranean beta-glucosidase, encoding sox, carrier and application

<130>

<160>7

<170>PatentIn version 3.4

<210>1

<211>1962

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cgaaacgaca cttgttactg tgtgttaatt cgtcacagac agctgtacca caaacgcagt 60

tgtagcttca tggctggtca ggcgttcccc gctggatttc tgtttggaac agcatcgtct 120

tcttatcaag taaaaggcgg ttggaacgaa aatggtaaag gcgagagtat atgggacaga 180

ctgacccacg atcatccgga gatcatcaaa gataaatcaa ctggtgatgt ggcctgtaat 240

tcctatcatt tgtataagga aaacgtccga atgctaaagg aattgggggt tcatttctac 300

cggttctctg tgtcgtggcc gcgaattcta ccaactggac atgacaacgt agtgaatgaa 360

gctggaattg cctattataa taatctgata aacgaactga tagccaatgg aatacagcct 420

atgataacga tgtaccactg ggatctgcca caacctctgc aagatcttgg tggatggacc 480

aatcctgctc tagcgaacta cttcgaggat tatgctcgtg tgctgtacgc taactttgga 540

gacagggtaa aatggtggaa cactatcaac gaaccacaaa acattgctgt aggatatagc 600

tcaccgtttg gagttgcacc taacattctt acacctggtc acggggacta cctggctatg 660

catacgattc tgttatcaca cgccagggcg tacagactgt atgagcggga atttaaggat 720

aaacaggaag ggaaagtatc tattgcggct tcgtgcgtct ggattgaacc gataattgac 780

tccaacgaag aagaagaatc ggcttcgaga gtacggcaaa tgcatattgg atgggtgtta 840

catcccatct acagtgcaac tggagattat ccgactgtaa tgaaggagtg gatagctaaa 900

aaaagcaagg aggaaggtta cagcaggtcg agactgccga gattcaccaa ggaggagatt 960

gaaatggtga aaggtacttg ggactacctg ggaattaacc actacaccac attcttcact 1020

taccggagtg aaagtgaatc acttctactc ttgggtaccg gagttgctaa tattgcaaat 1080

gaaaaatatg caactggttc ttctacgtgg ctacaggttg taccgtgggg tttcagaaag 1140

ttgctgaact ggatagccaa agagtacaac aatccacctg tcttggtaac agagaatggg 1200

ttctctgatt atggagaact taacgacaga gatagaattg attaccacat aaagtacatg 1260

tgggaattac tgaaggccat gaaggaagat ggatgcaacg taattggtta taccgcgtgg 1320

agtctgatgg acgactttga atgggcatct ggatatacag agaaatttgg cctgtttcac 1380

gtagatttca atgaccctga tcggaaaaga acagccaaga agtcagcaga agtcttctct 1440

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cgcaatgtca cagtttgcag ctataaattc accacattga tttgtttgta catttgtcag 1560

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aggttgaacc ttcaaaaatt ctacattgtt ttcgcttcga gttgagtgtt ttgtagggat 1680

ctcagaacag acagtgaatt ttgccttata catcattgac tgattggttt ttataaccgt 1740

ggtggaaagt gtttacagcg cggtacgaac tgggtcttta aagaaagctg tctgcgcttc 1800

gtatttaaaa ggttaacttg gacattgctt gaagtcactt tatatattat tgtactactt 1860

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Ser Ser Tyr Gln Val Lys Gly Gly Trp Asn Glu Asn Gly Lys Gly Glu

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Ser Ile Trp Asp Arg Leu Thr His Asp His Pro Glu Ile Ile Lys Asp

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Lys Ser Thr Gly Asp Val Ala Cys Asn Ser Tyr His Leu Tyr Lys Glu

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Asn Val Arg Met Leu Lys Glu Leu Gly Val His Phe Tyr Arg Phe Ser

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Val Ser Trp Pro Arg Ile Leu Pro Thr Gly His Asp Asn Val Val Asn

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Asn Gly Ile Gln Pro Met Ile Thr Met Tyr His Trp Asp Leu Pro Gln

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Pro Leu Gln Asp Leu Gly Gly Trp Thr Asn Pro Ala Leu Ala Asn Tyr

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Phe Glu Asp Tyr Ala Arg Val Leu Tyr Ala Asn Phe Gly Asp Arg Val

145 150 155 160

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

165 170 175

Ser Ser Pro Phe Gly Val Ala Pro Asn Ile Leu Thr Pro Gly His Gly

180 185 190

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

195 200 205

Arg Leu Tyr Glu Arg Glu Phe Lys Asp Lys Gln Glu Gly Lys Val Ser

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Ile Ala Ala Ser Cys Val Trp Ile Glu Pro Ile Ile Asp Ser Asn Glu

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Glu Glu Glu Ser Ala Ser Arg Val Arg Gln Met His Ile Gly Trp Val

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Leu His Pro Ile Tyr Ser Ala Thr Gly Asp Tyr Pro Thr Val Met Lys

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Glu Trp Ile Ala Lys Lys Ser Lys Glu Glu Gly Tyr Ser Arg Ser Arg

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Leu Pro Arg Phe Thr Lys Glu Glu Ile Glu Met Val Lys Gly Thr Trp

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Asp Tyr Leu Gly Ile Asn His Tyr Thr Thr Phe Phe Thr Tyr Arg Ser

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Glu Ser Glu Ser Leu Leu Leu Leu Gly Thr Gly Val Ala Asn Ile Ala

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

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Trp Gly Phe Arg Lys Leu Leu Asn Trp Ile Ala Lys Glu Tyr Asn Asn

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Pro Pro Val Leu Val Thr Glu Asn Gly Phe Ser Asp Tyr Gly Glu Leu

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

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Leu Lys Ala Met Lys Glu Asp Gly Cys Asn Val Ile Gly Tyr Thr Ala

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

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Phe Gly Leu Phe His Val Asp Phe Asn Asp Pro Asp Arg Lys Arg Thr

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Ala Lys Lys Ser Ala Glu Val Phe Ser Glu Ile Ile Lys Ser Asn Lys

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Ile Pro Val Glu Trp Leu Lys Leu

465 470

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atggctggtc aggcgttccc cgctggattt ctgtttggaa cagcatcgtc ttcttatcaa 60

gtaaaaggcg gttggaacga aaatggtaaa ggcgagagta tatgggacag actgacccac 120

gatcatccgg agatcatcaa agataaatca actggtgatg tggcctgtaa ttcctatcat 180

ttgtataagg aaaacgtccg aatgctaaag gaattggggg ttcatttcta ccggttctct 240

gtgtcgtggc cgcgaattct accaactgga catgacaacg tagtgaatga agctggaatt 300

gcctattata ataatctgat aaacgaactg atagccaatg gaatacagcc tatgataacg 360

atgtaccact gggatctgcc acaacctctg caagatcttg gtggatggac caatcctgct 420

ctagcgaact acttcgagga ttatgctcgt gtgctgtacg ctaactttgg agacagggta 480

aaatggtgga acactatcaa cgaaccacaa aacattgctg taggatatag ctcaccgttt 540

ggagttgcac ctaacattct tacacctggt cacggggact acctggctat gcatacgatt 600

ctgttatcac acgccagggc gtacagactg tatgagcggg aatttaagga taaacaggaa 660

gggaaagtat ctattgcggc ttcgtgcgtc tggattgaac cgataattga ctccaacgaa 720

gaagaagaat cggcttcgag agtacggcaa atgcatattg gatgggtgtt acatcccatc 780

tacagtgcaa ctggagatta tccgactgta atgaaggagt ggatagctaa aaaaagcaag 840

gaggaaggtt acagcaggtc gagactgccg agattcacca aggaggagat tgaaatggtg 900

aaaggtactt gggactacct gggaattaac cactacacca cattcttcac ttaccggagt 960

gaaagtgaat cacttctact cttgggtacc ggagttgcta atattgcaaa tgaaaaatat 1020

gcaactggtt cttctacgtg gctacaggtt gtaccgtggg gtttcagaaa gttgctgaac 1080

tggatagcca aagagtacaa caatccacct gtcttggtaa cagagaatgg gttctctgat 1140

tatggagaac ttaacgacag agatagaatt gattaccaca taaagtacat gtgggaatta 1200

ctgaaggcca tgaaggaaga tggatgcaac gtaattggtt ataccgcgtg gagtctgatg 1260

gacgactttg aatgggcatc tggatataca gagaaatttg gcctgtttca cgtagatttc 1320

aatgaccctg atcggaaaag aacagccaag aagtcagcag aagtcttctc tgagataatt 1380

aaaagcaaca agattcctgt agaatggctg aagttatag 1419

<210>4

<211>23

<212>DNA

<213>Unknown

<220>

< 223>artificial sequence

<220>

<221>misc_feature

<222>(15)..(15)

<223>n is a，c，g，or t

<220>

<221>misc_feature

<222>(18)..(18)

<223>n is a，c，g，or t

<400>4

taycaytggg ayytnccnca rga 23

<210>5

<211>23

<212>DNA

<213>Unknown

<220>

< 223>artificial sequence

<220>

<221>misc_feature

<222>(15)..(15)

<223>n is a，c，g，or t

<220>

<221>misc_feature

<222>(18)..(18)

<223>n is a，c，g，or t

<400>5

tcraarttrt ccatnarnsw cca 23

<210>6

<211>28

<212>DNA

<213>Unknown

<220>

< 223>artificial sequence

<400>6

ttgaaccgat aattgcctcc aacgaaga 28

<210>7

<211>28

<212>DNA

<213>Unknown

<220>

< 223>artificial sequence

<400>7

ggtacaacct gtagccacgt agaagaac 28

Claims (8)

1. beta-glucosidase, its aminoacid sequence is shown in SEQ ID No.2.

2. the application of beta-glucosidase as claimed in claim 1 in cellulose degradation.

3. gene of the said beta-glucosidase of claim 1 of encoding.

4. gene as claimed in claim 3 is characterized in that said gene nucleotide series is shown in SEQ IDNO.1.

5. gene as claimed in claim 3 is characterized in that said gene nucleotide series is shown in SEQ IDNO.3.

6. recombinant vectors that contains the said gene of one of claim 3～5.

7. host cell that utilizes the said recombinant vectors conversion of claim 6, transduction or transfection to obtain.

8. the application of the described gene of one of claim 3～5 in preparation reorganization beta-glucosidase.