CN101100659A - Beta-glucosidase and its coding gene and application - Google Patents

Abstract

A beta-glucosidese, its encoding gene and use are disclosed. It consists of (a) or (b) protein, (a) protein is made of amino-acid sequence in sequence 3; (b) protein is derived by (a) and substituted and/or lost and/or added by one or several amino acid in sequence 3 and has beta-glucosidese. The process is carried out by: taking cellulose as material and synchronized mashing and common fermenting. It has better enzyme activity and can be used to produce alcohol.

Description

A kind of beta-glucosidase and encoding gene thereof and application

Technical field

The present invention relates to from not new beta-glucosidase and the encoding gene and the application of culturing micro-organisms of buffalo cud.

Background technology

Mierocrystalline cellulose mainly is that plant is passing through the abundantest reproducible biomass (biomass) resource on the photosynthesis synthetic earth with carbonic acid gas and water under the sun power effect.Mierocrystalline cellulose be a plurality of glucosyl residues with β-1, the polymer that the 4-glycosidic linkage is formed by connecting, its basic repeating unit is a cellobiose.The basic structure of natural cellulose is to be formed by the microfiber bundle set that protofibril constitutes.Protofibril is made up of the cellulosic molecule long-chain that crystallizing field and noncrystalline domain constitute the 15-40 root.Cellulosic crystallising part is to plan in good order that by cellulosic molecule ground folding arrangement forms.In natural cellulose, xylogen and hemicellulose form the mortise layer, closely surround Mierocrystalline cellulose.Cellulase is the general name that cellulose conversion can be become a series of enzymes of glucose, mainly comprise endoglucanase (endo-β-1,4-glucanase, EC 3.2.1.4), exoglucanase (exoglucanase, be cellobiohydrolase cellobiohydrolase again, EC3.2.1.91) and beta-glucosidase (β-glucosidase, EC3.2.1.21).Endoglucanase acts on the inside of cellulose long-chain molecule with the macrofiber cutting short-forming fiber, exoglucanase acts on an end of cellulosic molecule, with two glucosyl residues is that unit cuts the generation cellobiose, beta-glucosidase cutting fibre disaccharides generates glucose, and hydrolysis some fibre trisaccharide or cell-oligosaccharide (Lynd LR, Weimer PJ, Willem H Z, Pretorius IS.2002.Microbial cellulose utilization:fundamentals and biotechnology.MicrobiolMol Biol Rev, 66:506-577; Zhang YH, Lynd LR.2004.Toward an aggregatedunderstanding of enzymatic hydrolysis of cellulose:noncomplexed cellulasesystems.Biotechnology and Bioengineering, 88:797-824).

With the lignocellulose is that raw material, usefulness cellulase hydrolysis Mierocrystalline cellulose are produced alcohol, operational path the earliest is first saccharification secondary fermentation (Separate Hydrolysis and Fermentation, SHF) technology, elder generation's preprocessing lignocellulose, the plain enzymic hydrolysis Mierocrystalline cellulose of reprocess fibre produces sugar, then produce alcohol with yeast or other microbial fermentation again, the main drawback of this technology is: under the situation that the glucose that hydrolysis produces exists, beta-glucosidase stops the hydrolysis fiber disaccharides, and the accumulation of cellobiose causes the termination to cellulose degradation again.This technology was improved to simultaneous saccharification and fermentation (Simultaneous Saccharification and Fermentation afterwards, SSF) technology, in this technology, cellulase carries out in same container cellulosic hydrolysis and fermentation, enzymolysis produces sugar on one side, and organism of fermentation just changes into alcohol with sugar on one side.Not only reduced reactor, the more important thing is and avoided product to suppress problem.Now, the SSF process quilt is modified into synchronous saccharification ferment altogether (SimultaneousSaccharification and Cofermentation, SSCF) technology.SSCF technology is exactly that multiple sugared substrate ferments on the limit simultaneously for limit saccharification, organism of fermentation.The subject matter that this technology exists at present is: the enzyme work of cellulase is low, production cost is high; The operative temperature of cellulase and the saccharomycetic leavening temperature of organism of fermentation commonly used do not match.One of way that solves is that the high vigor cellulase of low temperature comprises beta-glucosidase (http://www1.eere.energy.gov/biomass/process-description.html in the research and development acidity;  hgren K, Bura R, Lesnicki G, Saddler J, Zacchi be comparison betweensimultaneous saccharification and fermentation and separate hydrolysis andfermentation using steam-pretreated corn stover.Process Biochemistry.42:834-839 G.2007.A).

A lot of microorganisms comprise that bacterium, actinomycetes and fungi etc. can both produce beta-glucosidase (Bhat MK, BhatS.1997.Cellulose degrading enzymes and their potential industrialapplications.Biotechnology Advances, 15:583-620).But the beta-glucosidase that is separated at present mainly obtains from the pure culture microorganism, and account for the genetic resources that is richly stored with in the not culturing micro-organisms (uncultured microorganisms) of occurring in nature microbe species more than 99% (AmannRI, Ludwig W, Schleifer KH.1995.Phylogenetic identification and in situdetection of individual microbial cells without cultivation.Microbiol Rev, 59:143-169), there has been more bibliographical information to obtain new gene by the grand genomic library of not culturing micro-organisms that makes up and screen environmental sample, as the lipase of encoding, proteolytic enzyme, chitinase, amylase, gene (the Streit WR of microbiotic and cellulase isoreactivity material, Daniel R, Jaeger KE.2004.Prospecting for biocatalysts and drugs in the genomes of non-culturedmicroorganisms.Curr Opin Biotechn, 15:285-290).Aspect culturing micro-organisms cellulase not, people such as Rees are not cloned into 2 cellulose enzyme genes the culturing micro-organisms from lake water and lakebed settling, the Genebank accession number is AJ537595 and AJ537596 (Rees H C, Grant S, Jones B, GrantWD, Heaphy is cellulase and esterase enzyme activitiesencoded by novel genes present in environmental DNA libraries.Extremophiles S.2003.Detecting, 7:415-421.).Voget etc. are not cloned into 2 cellulose enzyme gene gnuB and uvs080 (Voget S the culturing micro-organisms from soil, Leggewie C, Uesbeck A, Raasch C, Jaeger KE, Streit WR.2003.Prospecting for novel biocatalysts in a soil metagenome.Appl EnvironMicrobiol, 69:6235-6242).People such as Walter make up the not BAC gene library of culturing micro-organisms of mouse large intestine, and be cloned into a beta-glucosidase gene (Walter J, Mangold M, Tannock GW.2005.Construction, analysis, and beta-glucosidase screening of a bacterialartificial chromosome library from the large-bowel microbiota of mice.ApplEnviron Microbiol, 71:2347-2354).People such as Ferrer have made up the grand genomic library of the rumen content of milk cow, therefrom be cloned into and comprise 9 inscribe-β-1,4-glucanase gene and 1 beta-glucosidase gene (Ferrer M, Golyshina OV, Chernikova TN, Khachane AN, Reyes-DuarteD, Santos VA.2005.Novel hydrolase diversity retrieved from a metagenomiclibrary of bovine rumen microflora.Environ Microbiol, 7:1996-2010).2006, people such as Voget have not cloned an endo glucanase gene cel5A (VogetS the culturing micro-organisms from soil again, Steele HL, Streit WR.2006.Characterization of a metagenome-derivedhalotolerant cellulase.J Biotechnol, 126:26-36); 4 endoglucanase and 7 beta-glucosidase gene (Feng Y are not cloned and identified to people such as Feng report in 2007 the culturing micro-organisms from the rabbit caecum, Duan CJ, Pang H, Mo XC, Wu CF, Yu Y, Wei J, Tang JL, Feng JX.2007.Cloning and identification of novel cellulase genes from unculturedmicroorganisms in rabbit cecum and characterization of the expressedcellulases.Appl Microbiol Biotechnol, 75:319-328).

Ruminating animal can efficiently utilize fibering food, can secrete with microorganism in its cud to produce the cellulosic cellulase of efficient degradation and be undivided.Rumen microorganism comprises fungi, bacterium, protozoon and archeobacteria.People are by discovering rumen microorganism, microorganism in the cud has kind more than 8000, wherein is (Krause DO, the Denman SE that does not cultivate more than 85%, Mackie RI, Morrison M, Rae AL, Attwood GT, McSweeney CS.2003.Opportunities to improve fiber degradation in the rumen:microbiology, ecology, and genomics.FEMS Microbiol Rev, 27:663-693; KamraDN.2005.Rumen microbial ecosystem.Curr Sci 89:124-135.), in these microorganisms, may contain a large amount of genetic resourceses that comprises cellulose enzyme gene.

Summary of the invention

The purpose of this invention is to provide a kind of beta-glucosidase new, that high enzyme is lived.

Beta-glucosidase provided by the present invention, name is called Umcel3G, derives from not culturing micro-organisms of buffalo cud, is following (a) or protein (b):

(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 3;

(b) with the aminoacid sequence of sequence in the sequence table 3 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have activity of beta-glucosidase by (a) deutero-protein.

Wherein, the sequence in the sequence table 3 is made up of 717 amino-acid residues.From N-terminal (N end) 98-387 amino acids residue is the catalysis territory (glycosyl hydrolase 3domain) of glycosyl hydrolase family 3, is the structural domain (glycosyl hydrolase 3 C terminal domain) of glycosyl hydrolase family 3C from N-terminal (N end) 462-705 amino acids residue.

The replacement of described one or several amino-acid residue and/or disappearance and/or interpolation are meant and replace outside above-mentioned two structural domains of sequence 3 and/or lack and/or add.

In order to make the Umcel3G in (a) be convenient to purifying, proteinic N end or C end that can the aminoacid sequence shown in the sequence 3 is formed in by sequence table connect label as shown in table 1.

The sequence of table 1. label

Label	Residue	Sequence
			Poly-Arg	5-6 (being generally 5)	RRRRR
Poly-His	2-10 (being generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

Above-mentioned (b) but in the Umcel3G synthetic, also can synthesize its encoding gene earlier, carry out biology again and express and to obtain.The encoding gene of Umcel3G in above-mentioned (b) can pass through SEQ ID № in the sequence table: 1 the codon that lacks one or several amino-acid residue in the dna sequence dna shown in 5 ends the 661st to 2814 bit base, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.

For the ease of proteic secreting, expressing, signal peptide sequence on also can adding at the N-terminal of described Umcel3G.As adding, obtain the albumen of forming by the aminoacid sequence shown in 2 in the sequence table by 2 the polypeptide of forming from N-terminal the 1st to 23 amino acids residue in the sequence table.

Sequence 2 in the sequence table is made up of 740 amino-acid residues.From N-terminal (N end) 1-23 amino acids residue is signal peptide (signal peptide) sequence, from N-terminal (N end) 121-410 amino acids residue is the catalysis territory (glycosyl hydrolase 3 domain) of glycosyl hydrolase family 3, is the structural domain (glycosyl hydrolase 3 C terminal domain) of glycosyl hydrolase family 3C from N-terminal (N end) 485-728 amino acids residue.

The Umcel3G shown in the sequence 2 is with to derive from beta-glucosidase (the Genbank call number BAA36161) homology of Bacillus sp. the highest in the sequence table, and their amino acid sequence similarity is 73%, consistence is 60%.

The encoding gene of above-mentioned beta-glucosidase also belongs to protection scope of the present invention.

The encoding gene of described beta-glucosidase specifically can be following 1) or 2) or 3) gene:

1) its nucleotide sequence be in the sequence table sequence 1 from the dna molecular shown in the 5 end 661-2814 position deoxyribonucleotides;

2) its nucleotide sequence is the dna molecular shown in the sequence 1 in the sequence table;

3) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of the described beta-glucosidase of encoding.

The rigorous condition of above-mentioned height can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, under 65 ℃, hybridize and wash film.

Sequence 1 in the sequence table is made up of 3334 deoxyribonucleotides, from the 592nd to 2814 Nucleotide of 5 ' end open reading frame (the Open Reading Frame that is umcel3G, ORF), 592-594 position Nucleotide from 5 ' end is the initiator codon ATG of umcel3G, is the terminator codon TAG of umcel3G from 5 ' the 2812-2814 position Nucleotide of holding.

The recombinant expression vector, transgenic cell line and the reorganization bacterium that contain gene of the present invention all belong to protection scope of the present invention.

Another object of the present invention provides a kind of method of expressing above-mentioned beta-glucosidase.

The method of the above-mentioned beta-glucosidase of expression provided by the present invention is that the recombinant expression vector that will contain above-mentioned beta-glucoside enzyme coding gene imports host cell, expresses obtaining beta-glucosidase.

Described host can be yeast, intestinal bacteria, mammalian cell, insect cell or Bacillus subtilus etc., is preferably intestinal bacteria.

Described yeast specifically can be pichia pastoris (Pichia pastoris).Wherein, described pichia pastoris is preferably pichia pastoris GS115, KM71 (can available from American I nvitrogen company) or SMD1168 (can available from American I nvitrogen company).

Described intestinal bacteria specifically can be BL21 (DE3) pLysS, E.coli JM109, E.coliHB101 or E.coli Top10 etc.

When described host is yeast, the carrier that sets out that is used for making up described recombinant expression vector can be existing expression vector at above-mentioned yeast expression alien gene, as pPIC9, pPIC3, pHIL-D1, pA0804, pA0815, pPSC3K or the pPIC9K that can express in pichia pastoris (Pichia pastoris).

When described host is pichia pastoris (Pichia pastoris), need to carry out abduction delivering with methyl alcohol, the final concentration of methyl alcohol can be 0.5% (volumn concentration).

When described host was intestinal bacteria, the carrier that sets out that is used to make up described recombinant expression vector can be existing at above-mentioned expression in escherichia coli expression of exogenous gene carrier,

Above-mentioned recombinant expression vector all can make up according to ordinary method.

Cultivation contains the substratum and the culture condition of the host cell of beta-glucoside enzyme coding gene of the present invention, all can be substratum and the culture condition of cultivating the host that sets out.

The optimal pH that experiment showed, the Umcel3G enzymatic reaction is 6.0-6.5 (Fig. 9 A), and this enzyme has 65% relative enzyme work when pH4.5; The optimum temperuture of Umcel3G enzymatic reaction is 45 ℃ (Fig. 9 B), and this enzyme has 80% relative enzyme work in the time of 35 ℃.Under optimal pH and optimum temperuture, the recombinase of purifying is 35.4IU/mg to the ratio vigor of substrate pNPG.The Ca of 5mmol/L ²⁺Can improve 40% to the activity of Umcel3G, reach 52.5IU/mg.In addition, the Ca of 5mmol/L is being arranged ²⁺Under existence, pH4.5 and the 35 ℃ of conditions, the ratio vigor that records this enzyme is 22.8IU/mg.Therefore under the organism of fermentation yeast the most adaptable method in the common zymotechnique of the synchronous saccharification that with the Mierocrystalline cellulose is the raw material production fuel alcohol, this enzyme has higher enzyme lives, and can be applied in this technology to produce fuel alcohol.

The present invention by make up the buffalo cud not culturing micro-organisms the macro genome DNA library and library clone carried out screening active ingredients, obtained a new beta-glucosidase gene.Can in host cell, be used for cellulosic degraded to produce this cellulase by this gene of great expression.New beta-glucosidase provided by the present invention and encoding gene thereof have purposes widely in cellulosic degraded.

Description of drawings

Fig. 1 is the gel electrophoresis figure of the mixing genomic dna of the not culturing micro-organisms extracted from the buffalo rumen content.

Fig. 2 is not culturing micro-organisms gene library cloned plasmids BamHI restriction analysis figure of buffalo cud.

Fig. 3 for the buffalo cud not culturing micro-organisms gene library be cloned in screening active ingredients on the esculin medium, the lithograph at positive colony EPI100/pGLU64 place.

Fig. 4 cuts banding pattern for the BamHI enzyme of the cloned plasmids pGLU64 of activity of beta-glucosidase.

The transformant (right side) that obtains behind the recombinant plasmid pGLU64 transformed into escherichia coli that Fig. 5 obtains for primary dcreening operation has activity of beta-glucosidase, and the intestinal bacteria that contain empty carrier pWEB::TNC do not have activity (left side).

Fig. 6 is the electrophorogram of pET30-umcel3G recombinant plasmid behind KpnI and XhoI double digestion.

Fig. 7 has activity (right side) for reorganization e. coli bl21 (DE3) pLys/pumcel3G on the Vitamin C2 flat board, and e. coli bl21 (DE3) pLys that contains empty carrier pET30a (+) does not have activity (left side) on the Vitamin C2 flat board.

The electrophorogram of Fig. 8 Umcel3G expression, purifying and renaturation product.

Fig. 9 is the optimum reaction conditions of the beta-glucosidase of this work acquisition.

Embodiment

Experimental technique among the following embodiment if no special instructions, is ordinary method.

Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.

Used in an embodiment of the present invention material comprises: intestinal bacteria (Escherichia coli) strain is EPI100 (available from an Epicentre company); Coemid carrier pWEB::TNC (available from Epicentre company); The library prepares test kit (pWEB::TNC cosmid cloning kit, catalog number (Cat.No.) WEBC931 is available from Epicentre company); Expression vector pET30a (+) (available from Novagen company); Expression strain BL21 (DE3) pLys (available from Novagen company); Restriction enzyme, modifying enzyme, polysaccharase, Vitamin C2 (Esculin hydrate), citric acid high ferro ammonium reagent such as (Ammonium iron (III) citrate) is available from Promega, Stratagene, SIGMA.

The acquisition of embodiment 1, beta-glucosidase Umcel3G and encoding gene thereof

One, the buffalo cud structure of the grand genomic library of culturing micro-organisms not

The fresh of 5 buffalos and exsiccant buffalo rumen content are relatively gathered in slaughterhouse from the Nanning City, and-20 ℃ of preservations are stand-by in the laboratory.

From each sample, take by weighing buffalo rumen content 10g respectively and mix, be suspended in the 0.18M potassium phosphate buffer (pH7.2) of 100ml, fully in Beckman Coulter Avanti J-E whizzer JA-10 rotary head, use centrifugal 10 minutes of 2000g centrifugal force behind the mixing, remove supernatant liquor, throw out extracts damping fluid (100mMsodium phosphate pH8.0 with 100ml; 100mM Tirs-HCl pH8.0; 100mM EDTA pH8.0; 1.5M NaCl; 1%CTAB; 2%SDS) suspend, fully blend sample, and behind liquid nitrogen and 65 ℃ of water-bath multigelation samples three times, add N,O-Diacetylmuramidase (20mg/ml is dissolved in TE solution), 37 ℃ of effects 30 minutes down to final concentration 2mg/ml with stirrer.Add Proteinase K then to final concentration 0.1mg/ml, act on 30 minutes down at 50 ℃.Add SDS again to final concentration 1.0%, act on 10 minutes down at 75 ℃.Centrifugal 10 minutes of 8000g centrifugal force is got supernatant liquor, adds the dehydrated alcohol of two volumes, fully see promptly behind the mixing that the DNA flocks separates out, choose the DNA flocks, wash DNA 2 times, after the drying DNA is dissolved in 500 μ lTE solution and promptly gets the DNA crude extract with 70% ethanol.

The DNA crude extract is added to contains Sephadex G200 and 2%PVPP (polyvinylpolypyrrolidone, polyvinylpolypyrrol idone) chromatography column is (on the 200mm * 10mm), use the TE buffer solution elution, by every component 1ml fraction collection elutriant, each component adds 3M sodium acetate soln (pH5.2) and the 1ml isopropanol precipitating DNA of 100 μ l.Merge the DNA that is settled out, be dissolved in the dna solution that obtains purifying among the TE, then with the DNA more than the electroelution method recovery 30kb.

(library available from Epicentre company prepares test kit pWEB::TNCcosmid cloning kit owing to the library construction test kit, catalog number (Cat.No.) WEBC931) carrier that provides (pWEB::TNC) is a blunt end, therefore needing at first the dna fragmentation that reclaims to be carried out end repairs to produce flat terminal, concrete grammar is: add in the Eppendorf tube of the new bacterium of going out on ice successively: 6 μ l 10 * terminal repair enzyme damping fluid (330mM Tris-acetic acid [pH7.8], the 660mM Potassium ethanoate, the 100mM magnesium acetate, 5mM DTT), 6 μ l 2.5mM dNTP mixtures (every kind of 2.5mM), 6 μ l 10mM ATP, 40 μ l DNA (0.2 μ g/ μ l), the terminal repair enzyme mixture (T4 archaeal dna polymerase and T4 polynueleotide kinase) of 2 μ l, repair reaction under 25 ℃, transfer to 10 minutes termination reactions of 70 ℃ of water-baths after 45 minutes, reclaim the dna fragmentation of mending the 30kb-50kb after putting down with 1.0% low melting-point agarose gel.Recovery electrophorogram after the extraction of DNA, purifying and terminal the reparation is (swimming lane M: λ DNA (48.5kb) as shown in Figure 1; Swimming lane 1: slightly carry total DNA; Swimming lane 2: through the dna fragmentation of Sephadex G200 gel column purifying and electrodialysis recovery; Swimming lane 3: through the dna fragmentation of end reparation and the recovery of low melting point glue).

The DNA (0.1 μ g/ μ l) that adds the 25kb-45kb of 12 μ l sterilized waters, 10 times of quick ligase enzyme damping fluids of 2 μ l (10 * Fast-Link Ligation Buffer), 1 μ l 10mM ATP, 1 μ lpWEB::TNC carrier (0.5 μ g), the recovery of 3 μ l low melting-point agarose gels successively on ice in the Eppendorf tube of a new bacterium of going out is connected dna ligase (Fast-Link DNA Ligase fast with 1 μ l, 2 units/μ l), under 25 ℃, carry out ligation 2 hours behind the mixing, place 10 minutes to stop enzyme reaction at 70 ℃ again.

The λ packaging extract that will just dissolve on ice (the library construction test kit provides) 25 μ l transfer in the Eppendorf tube of a new bacterium of going out immediately and place fast on ice, again toward wherein adding 10 μ l ligation products, fully mixing was placed on 30 ℃ after 90 minutes, again toward wherein adding the λ packaging extract that remaining 25 μ l dissolve, fully mixing be placed on 30 ℃ 90 minutes, to wherein add 500 μ l phage dilution buffer liquid (10mMTris-HCl[pH 8.3], 100mM NaCl, 10mM MgCl ₂).

560 μ l packing reaction product is joined the OD of 5.6ml ₆₀₀(substratum is that every liter of LB[contains Tryptones (Oxoid), 10g to=1.0 host e. coli EPI100 nutrient solution; Yeast extract powder (Difco), 5g; NaCl, 5g; PH7.0]+10mM MgSO ₄) in, place down for 25 ℃ and allowed lambda particles phage absorption in 20 minutes and infect host cell E.coliEPI100, go up at the LA flat board (above-mentioned LB substratum+1.5% agar powder) that contains penbritin (100 μ g/mL) and paraxin (12 μ g/ul) and screen transformant.

The result obtains about 14000 transformants altogether, extract 15 clones' plasmid DNA arbitrarily, cut back 0.7% agarose gel electrophoresis analysis with restriction enzyme BamHI enzyme, all plasmids are except that the carrier segments that a 5.8kb is all arranged as a result, all contain the insertion fragment, that insert the fragment maximum is 48kb, and that minimum is 20.5kb, mean size is 38.2kb, and the total volume in library is 5.35 * 10 ⁸Bp, and do not have to find that have two plasmids to have identical enzyme cuts banding pattern illustrates that the library contains insertion dna fragmentation very at random, and the storage capacity in library also reaches requirement substantially.Fig. 2 is not culturing micro-organisms gene library cloned plasmids BamHI restriction analysis figure of buffalo cud.M1: λ/EcoRI (clip size is followed successively by from big to small: 21.2kb, and 7.4kb, 5.8kb, 5.6kb, 4.9kb, 3.5kb); M2:1kb ladder (clip size is followed successively by from big to small: 10.0kb, and 8.0kb, 6.0kb, 5.0kb, 4.0kb, 3.5kb, 3.0kb, 2.5kb, 2.0kb, 1.5kb, 1.0kb, 0.75kb); Other swimming lane is respectively 15 library clone plasmids cutting through BamH I enzyme.

Two, the buffalo cud is not expressed the screening that activity of beta-glucosidase is cloned in the grand genomic library of culturing micro-organisms

Clone on the original flat board in library (about about 200 bacterium colonies of every flat board) is xeroxed on the active LA flat board (being called for short the Vitamin C2 flat board) that contains penbritin (100 μ g/mL), 0.1% Vitamin C2 (esculin hydrate) and 0.2% ferric ammonium citrate (ferric ammonium citrate) with dull and stereotyped photolithography, flat board is inverted in after 37 ℃ of incubators cultivate 24 hours, periphery of bacterial colonies occur black hydrolysis circle for the positive colony (being called EPI100/pGLU64) of expressing activity of beta-glucosidase (Fig. 3).The present invention only relates to one of them positive colony, further extract this clone's plasmid DNA and with its called after pGLU64, behind restriction enzyme BamHI complete degestion pGLU64, carry out electrophoretic analysis (Fig. 4), pGLU64 is except that the carrier segments that a 5.8kb is arranged as a result, give other 6 BamHI fragments, size is about 9kb, 5.5kb, 5kb, 2.3kb, 2kb and 1.3kb, illustrate pGLU64 contain the insertion fragment of 25.3kb (swimming lane M: λ/EcoRI, clip size is followed successively by from big to small: 21.2kb, 7.4kb, 5.8kb, 5.6kb, 4.9kb, 3.5kb; Swimming lane 1:1kbladder, clip size is followed successively by from big to small: 10.0kb, 8.0kb, 6.0kb, 5.0kb, 4.0kb, 3.5kb, 3.0kb, 2.5kb, 2.0kb, 1.5kb, 1.0kb, 0.75kb, the BamHI enzyme of 0.5kb swimming lane 2:pGLU64 is cut banding pattern).With pGLU64 plasmid DNA and empty carrier pWEB::TNC difference Transformed E .coliEPI100, on active Vitamin C2 flat board, verify transformant, the transformant of empty carrier pWEB::TNC does not have hydrolysis circle (Fig. 5 left side) as a result, black hydrolysis circle (Fig. 5 right side) is arranged around the transformant of pGLU64, thereby contain alpha-glucosidase gene (Fig. 5) really on the insertion fragment of proof recombinant plasmid pGLU64.

The location of embodiment 2, beta-glucosidase gene

Adopt the method for subclone that the beta-glucosidase gene on the pGLU64 is positioned.Earlier cut pGLU64 with restriction enzyme XhoI enzyme, again from connect, transform, active detection, plasmid analysis, goal gene is positioned on the XhoI fragment of 14kb.Use the BamHI enzyme to cut then, goal gene is positioned on the BamHI fragment of 5.0kb the most at last, this BamHI fragment is sent Dalian Bao Bio-Engineering Company adopt the dideoxyribonucleoside acid system that this gene is carried out two-way double-stranded order-checking.

With software DNAStar (DNASTAR company, version 5) sequence is spliced, with NCBI (NationalCenter for Biotechnology Information, http://www.ncbi.nlm.nih.gov) the software Blast on (http://www.ncbi.nlm.nih.gov/BLAST) analyzes sequence, obtain cellulosic encoding gene, this gene has the dna sequence dna of sequence table 1, called after umcel3G.Open reading frame (the Open Reading Frame that the DNA of sequence 1 is umcel3G from 5 ' the 592nd to 2814 Nucleotide of holding in the sequence table, ORF), form by 2223 Nucleotide, 592-594 position Nucleotide from 5 ' end is the initiator codon ATG of umcel3G, is the terminator codon TAG of umcel3G from 5 ' the 2812-2814 position Nucleotide of holding.

One of cellulose enzyme gene umcel3G coding contains 740 amino acid whose protein Umcel3G, is 80997.08 dalton with this proteinic theoretical molecular size of DNAStar software prediction, and iso-electric point pI is 4.77.Analyzed the unit construction of beta-glucosidase Umcel3G with SMART (http://smart.embl-heidelberg.de), the result is signal peptide (signal peptide) for 1-23 amino acid from the N end, the 121-410 amino acids is glycosyl hydrolase family 3 catalysis territories (glycosyl hydrolase 3 domain), and the 485-728 amino acids is glycosyl hydrolase family 3C end functional domain (glycosyl hydrolase 3 Cterminal domain).With Umcel3G catalysis territory homology the highest be the beta-glucosidase (Genebank accession number BAA36161) that derives from Bacillus sp., their amino acid sequence similarity is 73%, consistence is 60%.

Embodiment 3, the umcel3G expression in intestinal bacteria

1, the structure of umcel3G gene expression plasmid

In order in the pET system, to express umcel3G, the full sequence (promptly from 5 of sequence 1 ' end 661-2811 position) of synthetic umcel3G gene except signal peptide and terminator codon, insert between the KpnI and XhoI site of carrier pET-30a (+) (available from Novagen company), obtain recombinant expression vector pET30-umcel3G.Initiator codon and terminator codon are provided by expression vector pET30a (+).A His label that is provided by expression vector (6 * His Tag) is provided the N end of expression product.

PET30-umcel3G is transformed among the host E.coli EPI100.Extract the plasmid of transformant, with KpnI and XhoI restriction analysis, the result shows that recombinant plasmid pET30-umcel3G discharges the carrier band of a 5.4kb and the external source insertion fragment that conforms to the expection size behind KpnI and XhoI double digestion.Fig. 6 is the restriction enzyme digestion and electrophoresis collection of illustrative plates of recombinant plasmid pET30-umcel3G, swimming lane 1 for 1kb ladder (clip size is followed successively by from big to small: 10.0kb, 8.0kb, 6.0kb, 5.0kb, 4.0kb, 3.5kb, 3.0kb, 2.5kb, 2.0kb, 1.5kb); Swimming lane 2 is the electrophorogram of recombinant plasmid behind KpnI and XhoI double digestion.And with dna sequence analysis test kit (U.S. USB company product) recombinant vectors pET30-umcel3G is checked order, the result show the umcel3G gene order in this recombinant vectors be in the sequence table sequence 1 from the 661st to 2811 deoxynucleotides of 5 ' end.

2.umcel3G expression, the purifying of gene in intestinal bacteria

Expressive host BL21 (DE3) pLysS that contains recombinant expression plasmid pET30-umcel3G is seeded on the above-mentioned Vitamin C2 flat board that contains 34 μ g/mL paraxin and 25 μ g/ml kantlex, host bacterium BL21 (DE3) pLysS that inoculation simultaneously only contains empty carrier pET-30a (+) in contrast, cultivate after 12 hours for 37 ℃, the IPTG that adds 10 μ l 1.0mM on two kinds of bacterium colonies respectively induces the expression of foreign protein.Continuation was cultivated 6 hours down at 37 ℃, thalline was cultivated 1 hour through 37 ℃ behind the stifling broken wall of chloroform again, can see that the host's periphery of bacterial colonies that contains recombinant expression plasmid pET30-umcel3G has black hydrolysis circle, and the contrast periphery of bacterial colonies does not have the hydrolysis circle, the results are shown in accompanying drawing 7: expressive host BL21 (DE3) pLysS that contains recombinant expression plasmid pET30-umcel3G has activity of beta-glucosidase (right side), and e. coli bl21 (DE3) pLysS that contains empty carrier pET-30a (+) does not have activity of beta-glucosidase (left side).Illustrate that gene umcel3G has given expression to the protein with activity of beta-glucosidase in E.coli.This experiment is carried out 3 times altogether and is repeated.Expressive host BL21 (DE3) pLysS that will contain pET30-umcel3G is called BL21 (DE3) pLys/pUmcel3G.

In order to obtain expression product Umcel3G, promptly contain BL21 (DE3) pLys of empty carrier pET30a (+) with IPTG processing expression strain BL21 (DE3) pLys/pUmcel3G and control strain.Compare with the protein banding pattern (Fig. 8 swimming lane 1) that the BL21 that contains empty carrier pET30a (+) (DE3) pLys provides, expression strain BL21 (DE3) pLys/pUmcel3G obviously provides the protein belt (Fig. 8 swimming lane 2) of extra about 83 kD, and it is similar to the molecular mass (83902.11 Daltons) that expression product is estimated; Only demonstrate a main protein belt (Fig. 8 swimming lane 3) through the product behind the Ni-NTA column purification on SDS-PAGE, the purified product that obtains can be used for carrying out the analysis of enzymatic property; After the expression product renaturation of the purifying on the gel, on the Vitamin C2 flat board, demonstrate activity of beta-glucosidase (Fig. 8 swimming lane 4).

The determination of activity of embodiment 4, Umcel3G

With right-nitrophenols β-D-glucuronide (p-nitrophenyl-β-D-glucopyranoside, pNPG) measure the activity of beta-glucosidase for substrate, with per minute catalysis pNPG generate 1 μ mol right-the required enzyme amount of nitrophenols (p-nitrophenol) is the enzyme unit (IU) that lives.Optimal pH is measured used damping fluid: pH3.0～7.0: citric acid-Sodium phosphate dibasic damping fluid (citrate-phosphate buffer); PH6.0～8.0:0.1mol/L sodium-phosphate (sodium phosphate buffer); PH7.0～9.0:0.1mol/L Tris-HCl damping fluid.Reaction system is: the pNPG of 14 μ l 25mmol/L, and the enzyme liquid of 10 μ l proper concns, the damping fluid of the different pH of 116 μ l, 37 ℃ were reacted 5 minutes, added the Na of 70 μ l 0.4mol/L ₂CO ₃Solution is with termination reaction, and the light absorption value (A of 410nm is surveyed in the reaction back with microplate reader ₄₁₀) (with add 100 ℃ of water-baths after 10 minutes the enzyme liquid of inactivation be blank reaction).Calculate contained enzyme amount in the enzyme liquid according to made protein standard opisometer, again according to made p-NP curve (y=3.6094x+0.05595, x represents the concentration of p-NP behind the reaction terminating, on behalf of microplate reader, y survey the light absorption value of 410nm) calculate the concentration of p-NP behind the reaction terminating, calculate the enzyme unit that lives according to the enzyme unit definition of living at last.Identical with above-mentioned reaction system and method, in the damping fluid of optimal pH (6.0), measure the relative enzyme of this enzyme under differing temps and live.Relative enzyme work among Fig. 9 A is meant the ratio that enzyme is lived and the enzyme under pH6.0 is lived under other pH; Relative enzyme work among Fig. 9 B is meant the ratio that enzyme is lived and the enzyme under 45 ℃ is lived under other temperature.

(pH6.0,45 ℃) measures metal ion to the active influence of Umcel3G under the optimum condition of enzyme reaction.Reaction system is: the pNPG of 14 μ l 25mmol/L, the enzyme liquid of 5 μ l proper concns, (metal ion is all from its muriate for the metal ion solution of 7 μ l 0.1mol/L, the reaction final concentration is 5mmol/L), the damping fluid of 114 μ lpH6.0,45 ℃ were reacted 5 minutes, added the Na of 70 μ l 0.4mol/L ₂CO ₃Solution is with termination reaction, and the light absorption value (A of 410nm is surveyed in the reaction back with microplate reader ₄₁₀).The result shows that final concentration is the Ca of 5mmol/L ²⁺Enzyme work to this enzyme has significant promoter action.

In above-mentioned all mensuration, each sample is all done three times and is repeated.

Light absorption value and enzyme activity during Umcel3G enzyme activity determination under table 2, the different pH

pH	A 410				Enzyme activity (IU/mg)
									Repeat 1	Repeat 2	Repeat 3	Mean value	Repeat	1	Repeat 2	Repeat 3	Mean value
	3	0.104	0.098	0.124	0.109	0.7	0.6	1.0										0.8
3.5									0.185	0.162	0.170	0.172	1.9	1.6	1.7	1.7
	4	0.224	0.321	0.249	0.265	2.5	2.9	2.9									2.8
4.5									0.474	0.580	0.499	0.518	6.3	7.9	6.7	7.0
	5	0.862	0.884	0.891	0.877	12.2	12.6	12.7									12.5
5.5									1.123	1.248	1.084	1.152	16.2	18.0	15.6	16.6
	6	1.387	1.428	1.582	1.466	20.2	20.8	23.1									21.4
6.5									1.207	1.184	1.417	1.269	17.5	17.1	20.6	18.4
	7	1.020	1.007	1.010	1.012	14.6	14.4	14.5									14.5
7.5									0.827	1.087	0.999	0.971	11.7	15.6	14.3	13.9
	8	0.752	0.893	0.759	0.801	10.6	12.7	10.7									11.3
8.5									0.555	0.674	0.657	0.629	7.6	9.4	9.1	8.7
	9	0.461	0.577	0.465	0.521	6.1	7.9	6.2									6.7

Light absorption value and enzyme activity during Umcel3G enzyme activity determination under table 3, the differing temps

Temperature (℃)	A 410				Enzyme activity (IU/mg)
									Repeat 1	Repeat 2	Repeat 3	Mean value	Repeat l	Repeat	2	Repeat 3	Mean value
	25	0.496	0.445	0.448	0.463	6.7	6.0	5.9										6.2
30									0.582	0.581	0.578	0.580	8.0	7.9	7.9	7.9
	35	0.903	0.876	0.90	0.893	12.8	12.4	12.8									12.6
40									0.902	0.942	1.046	0.964	12.8	13.4	15.0	13.7
	45	1.162	1.165	1.164	1.130	16.8	16.7	16.7									16.7
50									0.929	0.905	0.876	0.903	13.2	12.9	12.4	12.8
	55	0.229	0.232	0.176	0.212	2.6	2.7	1.8									2.4
60									0.114	0.114	0.091	0.103	0.9	0.9	0.6	0.7
	65	0.087	0.087	0.075	0.081	0.5	0.5	0.3									0.4

The active influence of different metal ion pair Umcel3G under table 4 optimum condition

Metal ion (5mM)	A 410				Enzyme (IU/mg) alive
									Repeat
	1	Repeat 1	Repeat 1	Mean value	Repeat	1	Repeat 1	Repeat 1		Mean value
Do not contain ion									0.996		1.353	1.347	1.232	28.5	39.3	39.1	35.4
	Zn2+	1.171	1.584	1.591	1.449	33.8	46.3	46.5		42.2
Mg2+									1.008		1.369	1.363	1.247	28.9	39.8	39.6	36.1
	Mn2+	1.514	1.120	1.521	1.385	44.2	32.3	44.4		40.3
Ca2+									1.819		1.814	1.727	1.787	53.5	53.3	50.7	52.5
	Na+	1.331	1.808	1.694	1.611	38.7	53.1	49.7		47.2
Fe3+									0.984		1.337	1.330	1.217	28.1	38.8	38.6	35.2
	Fe2+	0.918	1.247	1.242	1.136	26.1	36.1	35.9		32.7
Cu2+									0.896		1.256	0.947	1.03	25.5	36.4	27.0	29.6
	Co2+	0.256	0.274	0.340	0.29	6.1	8.6	6.6		7.1
K+									0.228		0.321	0.239	0.263	5.2	8.1	5.6	6.3

The result is shown in table 2, table 3 and table 4, and the optimal pH that records the Umcel3G enzymatic reaction is 6.0-6.5 (A among Fig. 9), and this enzyme has 65% relative enzyme work when pH4.5; The optimum temperuture of Umcel3G enzymatic reaction is 45 ℃ (B among Fig. 9), and this enzyme has 80% relative enzyme work in the time of 35 ℃.Under optimal pH and optimum temperuture, the recombinase Umcel3G of purifying is 35.4 IU/mg to the ratio vigor of substrate pNPG.The Ca of 5 mmol/L ²⁺Can improve about 40% to the activity of Umcel3G, reach 52.5IU/mg.In addition, the Ca of 5mmol/L is being arranged ²⁺Under existence, pH4.5 and the 35 ℃ of conditions, the ratio vigor that records this enzyme is 22.8IU/mg.As shown in table 5.

Table 5 is having the Ca of 5mmol/L ²⁺The ratio of recombinase Umcel3G under existence, pH4.5 and the 35 ℃ of conditions is lived

	A 410				Enzyme (IU/mg) alive
									Multiplicity	Repeat
1	Repeat 2	Repeat 3	Mean value	Repeat	1	Repeat 2	Repeat 3	Mean value
									For the first time	0.991	0.937	0.974	0.941	22.0	22.7	23.7	22.8
For the first time	0.970	0.960	0.927	0.952	23.6	23.3	22.4	23.1
									For the first time	0.883	0.914	0.981	0.926	21.3	22.1	23.8	22.4
Enzyme mean value (IU/mg) alive	(22.8+23.1+22.4)÷3＝22.8

Sequence table

<160>3

<210>1

<211>3334

<212>DNA

＜213〉the unknown

<220>

<223>

<400>1

tcgggggtga gcagcgagaa gacgttgcgc ggcgtgaagc cgtattcggc gagcagccgg 60

tcgtcgtcga gcgcgcggtg caccgtcgcg gaatccagca gcagctggct cgcgatcttg 120

gcggcgatgt tgcccttgat gcggagattg cccgtcaggg agaggcgcac ggggctctgc 180

cagccgttgt tgagcatccg ggccacgtac acgctcggat tgcccggcga aacggtatag 240

tgcccgggcg tgaggtactc cgccacctgc ttggcccgga agcagcgctc caggctcgcg 300

cggttgcgca cctgcgcctt ttccgcgatc tcctccagca ccttgttggc cgaggcgccc 360

ggatagacgt agatctccgc ctgcttgcgg aaattgggca gtttgttgtc ctgcagccag 420

ttgtagccga ggaacgccgc gaccgcaagg gccgccagcg tcaggatgat gaataccgct 480

ttcttcatag cggggcaaag ataccgattt ttgttcaata gtttatcccg atgtgcggga 540

cgcgcccgga ttcccgggaa aaagcttacc tttacggaaa accaattacc catgaaacga 600

ttgatccctt tctgcgcact cgtcctgctg gccgcctgcg gcccccggtg gacggaaacg 660

gaagccgacg gctaccggct catcacccag cgcaacggcg ccacgctcgg cgtcaccagc 720

gcgcccctgc tcgatctgaa cggccatata ttcaaggacc tcaaccgcaa cggccgcgtc 780

gatccctacg aggactggcg cctgcccgcg ctgacgcgcg cgcaggacct cgccgcgcag 840

ctcagcatcg aggaaatcgc cggactgatg ctctacagcg cgcatcagag cgtgccgacc 900

cccgagatca cggagcggca gaagaaattc ctcgaagagg acaacctccg tgcggtgctc 960

gtgaccacgg tcgggagccc ggagatcgcc gcccgctgga acaacaacgt gcaggccttc 1020

gtggaggcgc tgggccacgg catcccggcc aacaactctt ccgacccgcg caacgaatgt 1080

agcgccacgg ccgaattcaa cctcggctcg ggcggacaga tttccctctg gcccaccccg 1140

ctgggcctcg ccgcgacctt cgacccggcc ctcgtggagc agttcgggcg gatcgcctcg 1200

gcggaatacc gggccctcgg catcgccacg gccctcagcc cgcagatcga cctcgccacc 1260

gagccgcgct ggagccgctt caacggcacg ttcggcgagg atcccgagct cgacgtcgcc 1320

ctggcgcgcg cctacgtgga cggcttccag acgacggaag acgccccgga cggctggggc 1380

gcacagagcg tcaacgcgat ggtcaagcac tggccgtccg gcggcccgga agaaggcggc 1440

cgcgacgccc atttcaacta cggcaaatac gccgtctatc ccggcggcaa cttcgcgacg 1500

cacctgcgtc cgttcaccga aggcgccttc cgcctggacg gcggcacgaa gagcgcctcg 1560

gcggtcatgc cctactacac gatctcctac ggcgtcgatc cctccgggaa gaacgccggc 1620

aacagctaca acgaatacat catcggcgac ctgctgcgcg gggaatacgg cttcgacggc 1680

gtggtctgca ccgactgggg catcaccgcc gacaacgccg ctgtctcatc cttcgacggc 1740

aagtgctggg gaatggagga gctgagcgtc gcggagcgcc actacgcggt catcaaggcc 1800

ggcgtggacc agttcggcgg caacaacgac aagggccccg tcctggaggc ctacaagatg 1860

tgggtggcgg aattcggcga ggagagcgcc cgcgcgcgct tcgagcagtc cgccgtgcgc 1920

ctgctgatga attccttccg caccggactc ttcgagaatc cctataccga tccggcggcc 1980

gccgcggccg tcgtgggcaa tccggaatat atggaagcgg gcttccaggc gcagcgcaag 2040

tccatcgtga tgctgaagaa ccacggaggc gtcctgccga acgacagcgc cagggtctat 2100

gtcccgcagc ggctgtatcc ccagacgccc ggcatgttcg gcctgtcgat ggggccggcc 2160

gcgcactggg actaccccat cgacaaggaa ctggtcggaa agtacttcca gtggaccgag 2220

gacccggaag cggccgactt cgcgctcgtg atgatccagg agcccttccc gggcgccggc 2280

tatgacgtga acgaccgcaa acggggcggc aacggctacg tgcccatcag cctgcagtac 2340

cggccctaca aggccgaata cgcccgtccc gtgtccatcg cgggcggcga tccgaaagag 2400

acgttcacca accgctccta ccggggcaag aaggtcacga cctacaacga gagcgacctc 2460

gacctggtca tcgagacgaa gcgcaggatg ggcgacaagc ccgtcgtcgt ggtcatcggc 2520

gtgagccgac ccctcgtcct ggcggagctg gagccgtatg ccgacgccat cctcctcacc 2580

ttcggcgtcc agaaccaggc ggtgctggac atcctctccg gcgcagcgga gccgtccggc 2640

ctgctgccga tgcagctgcc ggccgatatg cgcaccgtcg aggagcaggc cgaggacgtg 2700

ccgcgcgata tgcgcgtcta tgtcgacgcc gacggccacg cctacgattt cgcctacggc 2760

ctcggctggg acggcgtcat caacgacgcc cgcgtgtcca tctaccgcag atagaaatta 2820

gcggaaaagc tgctgcgcga agaccgtcag gtacacgcgc cagttgcgcc agatgtgccc 2880

gccttcggat tcgtaatagg tgacgggata accgtgcgcg tcgcaataat ccttgagcgc 2940

cagcgaggtg acccgcacgc cgtcgtcctt gccgacgccg atccaccaga gtttcgggcg 3000

ggcggcgaag acggccgcga tcgcgtcctc attcccttcc ggcagcgccg cgccggagaa 3060

cattcccaca tagccgaagc gcttcggata gcgcagcgac agctgcgcgg actggcgacc 3120

gcccatcgac aggccggcca cggccgtgtt ggcggcgccc ttcgccacgc ggtagtgccc 3180

ctcgatgaac tgcatcacgt cggggaagct ttcttcgatc tcgaccgtgg actgggaacg 3240

gctgttctgc atcgtgggct ggaacatatt gacggcggcg ccgggcgcgg cccggttgaa 3300

atacacgccg ttcggcatca ccacgatcat cggg 3334

<210>2

<211>740

<212>PRT

＜213〉the unknown

<220>

<223>

<400>2

Met Lys Arg Leu Ile Pro Phe Cys Ala Leu Val Leu Leu Ala Ala Cys

1 5 10 15

Gly Pro Arg Trp Thr Glu Thr Glu Ala Asp Gly Tyr Arg Leu Ile Thr

20 25 30

Gln Arg Asn Gly Ala Thr Leu Gly Val Thr Ser Ala Pro Leu Leu Asp

35 40 45

Leu Asn Gly His Ile Phe Lys Asp Leu Asn Arg Asn Gly Arg Val Asp

50 55 60

Pro Tyr Glu Asp Trp Arg Leu Pro Ala Leu Thr Arg Ala Gln Asp Leu

65 70 75 80

Ala Ala Gln Leu Ser Ile Glu Glu Ile Ala Gly Leu Met Leu Tyr Ser

85 90 95

Ala His Gln Ser Val Pro Thr Pro Glu Ile Thr Glu Arg Gln Lys Lys

100 105 110

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

115 120 125

Ser Pro Glu Ile Ala Ala Arg Trp Asn Asn Asn Val Gln Ala Phe Val

130 135 140

Glu Ala Leu Gly His Gly Ile Pro Ala Asn Asn Ser Ser Asp Pro Arg

145 150 155 160

Asn Glu Cys Ser Ala Thr Ala Glu Phe Asn Leu Gly Ser Gly Gly Gln

165 170 175

Ile Ser Leu Trp Pro Thr Pro Leu Gly Leu Ala Ala Thr Phe Asp Pro

180 185 190

Ala Leu Val Glu Gln Phe Gly Arg Ile Ala Ser Ala Glu Tyr Arg Ala

195 200 205

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

210 215 220

Pro Arg Trp Ser Arg Phe Asn Gly Thr Phe Gly Glu Asp Pro Glu Leu

225 230 235 240

Asp Val Ala Leu Ala Arg Ala Tyr Val Asp Gly Phe Gln Thr Thr Glu

245 250 255

Asp Ala Pro Asp Gly Trp Gly Ala Gln Ser Val Asn Ala Met Val Lys

260 265 270

His Trp Pro Ser Gly Gly Pro Glu Glu Gly Gly Arg Asp Ala His Phe

275 280 285

Asn Tyr Gly Lys Tyr Ala Val Tyr Pro Gly Gly Asn Phe Ala Thr His

290 295 300

Leu Arg Pro Phe Thr Glu Gly Ala Phe Arg Leu Asp Gly Gly Thr Lys

305 310 315 320

Ser Ala Ser Ala Val Met Pro Tyr Tyr Thr Ile Ser Tyr Gly Val Asp

325 330 335

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

340 345 350

Asp Leu Leu Arg Gly Glu Tyr Gly Phe Asp Gly Val Val Cys Thr Asp

355 360 365

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

370 375 380

Cys Trp Gly Met Glu Glu Leu Ser Val Ala Glu Arg His Tyr Ala Val

385 390 395 400

Ile Lys Ala Gly Val Asp Gln Phe Gly Gly Asn Asn Asp Lys Gly Pro

405 410 415

Val Leu Glu Ala Tyr Lys Met Trp Val Ala Glu Phe Gly Glu Glu Ser

420 425 430

Ala Arg Ala Arg Phe Glu Gln Ser Ala Val Arg Leu Leu Met Asn Ser

435 440 445

Phe Arg Thr Gly Leu Phe Glu Asn Pro Tyr Thr Asp Pro Ala Ala Ala

450 455 460

Ala Ala Val Val Gly Asn Pro Glu Tyr Met Glu Ala Gly Phe Gln Ala

465 470 475 480

Gln Arg Lys Ser Ile Val Met Leu Lys Asn His Gly Gly Val Leu Pro

485 490 495

Asn Asp Ser Ala Arg Val Tyr Val Pro Gln Arg Leu Tyr Pro Gln Thr

500 505 510

Pro Gly Met Phe Gly Leu Ser Met Gly Pro Ala Ala His Trp Asp Tyr

515 520 525

Pro Ile Asp Lys Glu Leu Val Gly Lys Tyr Phe Gln Trp Thr Glu Asp

530 535 540

Pro Glu Ala Ala Asp Phe Ala Leu Val Met Ile Gln Glu Pro Phe Pro

545 550 555 560

Gly Ala Gly Tyr Asp Val Asn Asp Arg Lys Arg Gly Gly Asn Gly Tyr

565 570 575

Val Pro Ile Ser Leu Gln Tyr Arg Pro Tyr Lys Ala Glu Tyr Ala Arg

580 585 590

Pro Val Ser Ile Ala Gly Gly Asp Pro Lys Glu Thr Phe Thr Asn Arg

595 600 605

Ser Tyr Arg Gly Lys Lys Val Thr Thr Tyr Asn Glu Ser Asp Leu Asp

610 615 620

Leu Val Ile Glu Thr Lys Arg Arg Met Gly Asp Lys Pro Val Val Val

625 630 635 640

Val Ile Gly Val Ser Arg Pro Leu Val Leu Ala Glu Leu Glu Pro Tyr

645 650 655

Ala Asp Ala Ile Leu Leu Thr Phe Gly Val Gln Asn Gln Ala Val Leu

660 665 670

Asp Ile Leu Ser Gly Ala Ala Glu Pro Ser Gly Leu Leu Pro Met Gln

675 680 685

Leu Pro Ala Asp Met Arg Thr Val Glu Glu Gln Ala Glu Asp Val Pro

690 695 700

Arg Asp Met Arg Val Tyr Val Asp Ala Asp Gly His Ala Tyr Asp Phe

705 710 715 720

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

725 730 735

Ile Tyr Arg Arg

740

<210>3

<211>717

<212>PRT

<212>PRT

＜213〉the unknown

<220>

<223>

<400>3

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

1 5 10 15

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

20 25 30

Asp Leu Asn Arg Asn Gly Arg Val Asp Pro Tyr Glu Asp Trp Arg Leu

35 40 45

Pro Ala Leu Thr Arg Ala Gln Asp Leu Ala Ala Gln Leu Set Ile Glu

50 55 60

Glu Ile Ala Gly Leu Met Leu Tyr Ser Ala His Gln Ser Val Pro Thr

65 70 75 80

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

85 90 95

Arg Ala Val Leu Val Thr Thr Val Gly Ser Pro Glu Ile Ala Ala Arg

100 105 110

Trp Asn Asn Asn Val Gln Ala Phe Val Glu Ala Leu Gly His Gly Ile

115 120 125

Pro Ala Asn Asn Ser Ser Asp Pro Arg Asn Glu Cys Ser Ala Thr Ala

130 135 140

Glu Phe Asn Leu Gly Ser Gly Gly Gln Ile Ser Leu Trp Pro Thr Pro

145 150 155 160

Leu Gly Leu Ala Ala Thr Phe Asp Pro Ala Leu Val Glu Gln Phe Gly

165 170 175

Arg Ile Ala Ser Ala Glu Tyr Arg Ala Leu Gly Ile Ala Thr Ala Leu

180 185 190

Ser Pro Gln Ile Asp Leu Ala Thr Glu Pro Arg Trp Ser Arg Phe Asn

195 200 205

Gly Thr Phe Gly Glu Asp Pro Glu Leu Asp Val Ala Leu Ala Arg Ala

210 215 220

Tyr Val Asp Gly Phe Gln Thr Thr Glu Asp Ala Pro Asp Gly Trp Gly

225 230 235 240

Ala Gln Ser Val Asn Ala Met Val Lys His Trp Pro Ser Gly Gly Pro

245 250 255

Glu Glu Gly Gly Arg Asp Ala His Phe Asn Tyr Gly Lys Tyr Ala Val

260 265 270

Tyr Pro Gly Gly Asn Phe Ala Thr His Leu Arg Pro Phe Thr Glu Gly

275 280 285

Ala Phe Arg Leu Asp Gly Gly Thr Lys Ser Ala Ser Ala Val Met Pro

290 295 300

Tyr Tyr Thr Ile Ser Tyr Gly Val Asp Pro Ser Gly Lys Asn Ala Gly

305 310 315 320

Asn Ser Tyr Asn Glu Tyr Ile Ile Gly Asp Leu Leu Arg Gly Glu Tyr

325 330 335

Gly Phe Asp Gly Val Val Cys Thr Asp Trp Gly Ile Thr Ala Asp Asn

340 345 350

Ala Ala Val Ser Ser Phe Asp Gly Lys Cys Trp Gly Met Glu Glu Leu

355 360 365

Ser Val Ala Glu Arg His Tyr Ala Val Ile Lys Ala Gly Val Asp Gln

370 375 380

Phe Gly Gly Asn Asn Asp Lys Gly Pro Val Leu Glu Ala Tyr Lys Met

385 390 395 400

Trp Val Ala Glu Phe Gly Glu Glu Ser Ala Arg Ala Arg Phe Glu Gln

405 410 415

Ser Ala Val Arg Leu Leu Met Asn Ser Phe Arg Thr Gly Leu Phe Glu

420 425 430

Asn Pro Tyr Thr Asp Pro Ala Ala Ala Ala Ala Val Val Gly Asn Pro

435 440 445

Glu Tyr Met Glu Ala Gly Phe Gln Ala Gln Arg Lys Ser Ile Val Met

450 455 460

Leu Lys Asn His Gly Gly Val Leu Pro Asn Asp Ser Ala Arg Val Tyr

465 470 475 480

Val Pro Gln Arg Leu Tyr Pro Gln Thr Pro Gly Met Phe Gly Leu Ser

485 490 495

Met Gly Pro Ala Ala His Trp Asp Tyr Pro Ile Asp Lys Glu Leu Val

500 505 510

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

515 520 525

Leu Val Met Ile Gln Glu Pro Phe Pro Gly Ala Gly Tyr Asp Val Asn

530 535 540

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

545 550 555 560

Arg Pro Tyr Lys Ala Glu Tyr Ala Arg Pro Val Ser Ile Ala Gly Gly

565 570 575

Asp Pro Lys Glu Thr Phe Thr Asn Arg Ser Tyr Arg Gly Lys Lys Val

580 585 590

Thr Thr Tyr Asn Glu Ser Asp Leu Asp Leu Val Ile Glu Thr Lys Arg

595 600 605

Arg Met Gly Asp Lys Pro Val Val Val Val Ile Gly Val Ser Arg Pro

610 615 620

Leu Val Leu Ala Glu Leu Glu Pro Tyr Ala Asp Ala Ile Leu Leu Thr

625 630 635 640

Phe Gly Val Gln Asn Gln Ala Val Leu Asp Ile Leu Ser Gly Ala Ala

645 650 655

Glu Pro Ser Gly Leu Leu Pro Met Gln Leu Pro Ala Asp Met Arg Thr

660 665 670

Val Glu Glu Gln Ala Glu Asp Val Pro Arg Asp Met Arg Val Tyr Val

675 680 685

Asp Ala Asp Gly His Ala Tyr Asp Phe Ala Tyr Gly Leu Gly Trp Asp

690 695 700

Gly Val Ile Asn Asp Ala Arg Val Ser Ile Tyr Arg Arg

705 710 715

Claims (10)

1, a kind of beta-glucosidase is following (a) or protein (b):

(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 3;

(b) with the aminoacid sequence of sequence in the sequence table 3 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have activity of beta-glucosidase by (a) deutero-protein.

2, beta-glucosidase according to claim 1 is characterized in that: the replacement of described one or several amino-acid residue and/or disappearance and/or interpolation are meant replacing from N-terminal 98-387 amino acids residue with outside N-terminal 462-705 amino acids residue and/or lack and/or add in sequence 3.

3, beta-glucosidase according to claim 1 is characterized in that: the proteinic aminoacid sequence of described (b) is shown in the sequence in the sequence table 2.

4, according to the encoding gene of claim 1,2 or 3 described beta-glucosidases.

5, gene according to claim 4 is characterized in that: described beta-glucoside enzyme coding gene is following 1) or 2) or 3) gene:

1) its nucleotide sequence be in the sequence table sequence 1 from the dna molecular shown in the 5 end 661-2814 position deoxyribonucleotides;

2) its nucleotide sequence is the dna molecular shown in the sequence 1 in the sequence table;

3) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of the described beta-glucosidase of encoding.

6, the recombinant expression vector, transgenic cell line or the reorganization bacterium that contain claim 4 or 5 described genes.

7, a kind of method of expressing claim 1,2 or 3 described beta-glucosidases is that the recombinant expression vector that will contain claim 1,2 or 3 described beta-glucoside enzyme coding genes imports host cell, expresses obtaining beta-glucosidase.

8, method according to claim 7 is characterized in that: described host is intestinal bacteria; Described intestinal bacteria are preferably BL21 (DE3) pLysS; Described recombinant expression vector between the KpnI of pET30a (+) and XhoI restriction enzyme site, insert sequence 1 in the sequence table from the 661st to 2811 recombinant expression vector pET30-umcel3G that deoxynucleotide obtains of 5 ' end.

9, claim 1,2 or 3 described beta-glucosidases or claim 4 or 5 application of described beta-glucoside enzyme coding gene in cellulose degradation.

10, claim 1,2 or 3 described beta-glucosidases or claim 4 or 5 application of described beta-glucoside enzyme coding gene in Alcohol Production.

Images