CN101225377B - Endoglucanase as well as encoding gene and use thereof - Google Patents

Abstract

The invention discloses an endoglucanase and a coding gene of the endoglucanase and an application of the endoglucanase and the coding gene. The endoglucanase is one type of the protein with the following amino acid residue sequence: 1) SEQ ID No.: 2 in the sequence table; 2) the amino acid residue sequence of the SEQ ID No.: 2 in the sequence table forms a protein with the endoglucanase activityby the substitution and/or the deletion and/or the addition of one or a plurality of amino acid residues. The endoglucanase and a coding gene of the endoglucanase has the advantages that: the endoglucanase has very high carboxymethyl cellulose enzyme activity (4029U/g), and the enzyme has a wide PH value range and a wide temperature range. The endoglucanase and the coding gene of the endoglucanase can be widely used in the degradation of cellulose.

Description

Endoglucanase and encoding gene thereof and application

Technical field

The present invention relates to a kind of endoglucanase and encoding gene thereof and application.

Background technology

Mierocrystalline cellulose mainly is that plant utilization carbonic acid gas and water are passing through the abundantest reproducible biomass (biomass) resource on the photosynthesis synthetic earth under the sun power effect.It is reported that the annual Mierocrystalline cellulose that produces by photosynthesis in the whole world is up to 1.55 * 10 ⁹Ton, wherein 89% as yet by human use (Dunlap C, Chiang GC.Utilization and recycle of agriculture wastes and residues.Shuler M L.Boca Raton, Florida.USA:CRC Press Inc.1980.19).Mierocrystalline cellulose be a plurality of glucosyl residues with β-1, the polymer that the 4-glycosidic link 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 carry out in good order folding arrangement regularly by cellulosic molecule to form.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, comprise that three fermentoids are inscribe-β-1,4-dextranase (endo-β-1,4-glucanase, EC 3.2.1.4), (exoglucanase is cellobiohydrolase cellobiohydrolase again to exoglucanase, EC3.2.1.91) and beta-glucosidase (β-glucosidase, EC3.2.1.21), these three kinds of enzyme synergies can become glucose with cellulose conversion.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 (Tomme P, WarrenR A J, Gilkes N is hydrolysis by bacteria and fungi.Adv.Microbiol.Physiol. R.1995.Cellulose, 37:1-81.1995; Bhat M K, Bhat be enzymes and their potential industrial applications.BiotechnologyAdvances S.1997.Cellulosedegrading, 15:583-620).Glucose can be used as important industrial raw material and produces Chemicals such as alcohol, acetone.Cellulosic utilization is significant for solving world energy sources crisis, grain and problems such as feed shortage, environmental pollution with conversion.Cellulase can be widely used in industries such as wine brewing, feed, food, weaving, papermaking.Can increase the digestibility of feed as cellulase as fodder additives, reduce excretory ight soil amount.Cellulase can replace " granite-wash " processing that float stone carries out jeans, also can handle other fibre-bearing fabric to reduce roughness and to increase light.Cellulase can add in the washing composition with the cleaning capacity that improves washing composition (Bhat MK.2000.Cellulases and related enzymes in biotechnology.BiotechnologyAdvances, 18:355-383).Need use cellulase of different nature owing to the extensive use of cellulase and at different purposes, make with the Mierocrystalline cellulose that to be that the cost of raw material production fuel alcohol is too high can't really realize industrialization to such an extent as to lower, the price of the efficient owing to cellulase is high, therefore, need new cellulase.

Cellulase belongs to glycosyl hydrolase enzyme (glycosyl hydrolases), many glycosyl hydrolases are by other functional domain such as carbohydrate-binding modules (carbohydrate-binding modules of a catalysis territory and one or more, CBMs) form, amino acid sequence similarity according to the catalysis territory, the glycosyl hydrolase enzyme is divided into different family (families), and (Davies G., Henrissat be and mechanisms of glycosyl hydrolases.Structure 3:853-859 B.1995.Structures; Henrissat is classification of glycosyl hydrolases based on amino-acidsequence similarities.Biochem.J.280:309-316 B.1991.A; Henrissat B., Bairoch be families in the classification of glycosyl hydrolases based on amino-acid sequence similarities.Biochem.J.293:781-788 A.1993New; Henrissat B., BairochA.1996.Updating the sequence-based classification of glycosyl hydrolases.Biochem.J.316:695-696).Go up the up-to-date inventory of listed glycosyl hydrolase enzyme according to Cazy server (server) (http://afmb.cnrs-mrs.fr/CAZY/), the glycosyl hydrolase enzyme has 108 families at present, and cellulase belongs to glycosyl hydrolase enzyme family 1,3,5,6,7,8,9,10,12,26,44,45,48,51,61,74.The cellulase of the unknown and known cellulase are done the sequence homology comparison can classify to it.

Human most of cellulose enzyme gene of being cloned all is to come from the microorganism of pure culture, but be not that all microorganisms of occurring in nature all are can be separated, cultivate, it is generally acknowledged that educable microbe species only accounts for 1% (Amann R I of occurring in nature microbe species, Ludwig W, Schleifer K is and in situ detection of individual microbial cells without cultivation.Microbiol.Rev.59:143-169 H.1995.Phylogeneticidentification), so remaining 99% can not contain a large amount of genetic resourceses in the cultured microorganism.In recent years from environmental sample not culturing micro-organisms extract genomic dna and make up then that to mix genome dna library be mature technology (Lorenz P, Schleper be source of enzyme discovery.Journal of Molecular Catalysis B:Enzymatic19-20:13-19 C.2002.Metagenome-achallenging) with isolated genes.Cloned at present the cellulose enzyme gene of the not culturing micro-organisms that obtains deriving from varying environment in the world, comprising (Healy F G such as Healy, Ray R M, Aldrich H C, Wilkie A C, Ingram L Oand Shanmugam K is isolation of functional genes encoding cellulases fromthe microbial consortia in a thermophilic T.1995.Direct, anaerobic digester maintained on lignocellulose.Appl Microbiol Biotechnol.43:667-674.) report in the wooden heap of anaerobic high temperature and be cloned into a cellulose enzyme gene; (Rees HC such as Rees, Grant S, Jones B, Grant WD, Heaphy is and esterase enzyme activities encoded by novel genes present in environmentalDNA libraries.Extremophiles.7 (5) S.2003.Detectingcellulase: 415-421) report is not cloned into 2 cellulose enzyme gene CRATCEL and HKCEL the culturing micro-organisms from lake water and lakebed settling, (Voget S such as Voget, Leggewie C, Uesbeck A, Raasch C, Jaeger KE, Streit WR.2003.Prospecting for novel biocatalysts in asoil metagenome.Appl Environ Microbiol., 69 (10): 6235-6242; Voget S, Steele H L, andStreit W be of a metagenome-derived halotolerant cellulase.J.Biotechnol.126:26-36 R.2006.Characterization) report and be not cloned into 3 cellulose enzyme gene gnuB, uvs080 and cel5A the culturing micro-organisms from soil; (Ferrer M such as Ferrer, Golyshina OV, Chernikova T N, Khachane A N, Reyes-Duarte D, Santos V A, Strompl C, Elborough K, Jarvis G, Neef A, Yakimov M M, Timmis K N, and Golyshin P be hydrolase diversity retrieved from ametagenomic library of bovine rumen microflora.Environ Microbiol.7:1996-2010 N.2005.Novel) do not identified 9 cellulose enzyme genes the culturing micro-organisms from bovine rumen.(Feng Y such as Feng, Duan CJ, Pang H, MoXC, Wu CF, Yu Y, Hu YL, Wei J, Tang JL, and Feng JX.2007.Cloning and identificationof novel cellulase genes from uncultured microorganisms in rabbit cecum andcharacterization of the expressed cellulases.Appl.Microbiol.Biotechnol.75:319-328) do not clone and identified 11 cellulose enzyme genes the culturing micro-organisms from the rabbit caecum.These derive from not the grand genomic cellulose enzyme gene of culturing micro-organisms all is new on sequence, illustrates that grand genome method is the good method that obtains new gene.The cud of ruminating animal is one of the most violent main place of occurring in nature cellulose degradation, and the plain class material of fibre of plant is by symbiotic cellulose degradation microbiological deterioration in the cud.Rumen microorganism includes fungi, bacterium, protozoon and archeobacteria.Research at present thinks that it is (the KrauseD O that does not cultivate that 85% microorganism is arranged in the cud, Denman S E, Mackie R I, Morrison M, Rae A L, Attwood G T, and McSweeney C is to improve fiber degradation in the rumen:microbiology S.2003.Opportunities, ecology, and genomics.FEMS Microbiol Rev 27:663-693), can infer that these necessarily do not contain a large amount of genetic resourceses such as cellulose enzyme gene resource in the culturing micro-organisms, by making up the not macro genome DNA library of culturing micro-organisms of buffalo cud, very likely therefrom screen the gene that obtains than the enzyme that known best cellulase also will be good at present.

Summary of the invention

The purpose of this invention is to provide a kind of endoglucanase and encoding gene thereof and application.

Endoglucanase provided by the present invention, name is called Umcel5D, derives from not culturing bacterium of buffalo cud, is the protein with one of following amino acid residue sequences:

1) the N-terminal 20-552 amino acids residue sequence of the SEQ ID № .2 in sequence table;

2) with the N-terminal 20-552 amino acids residue sequence of the SEQ ID № .2 in sequence table through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have the protein of endoglucanase activity.

Wherein, the sequence in the sequence table 2 is made up of 552 amino-acid residues.N-terminal 1-19 amino acids from sequence 2 is signal peptide (signal peptide), is family's 5 glycosyl hydrolases (glycosyl hydrolase) functional domains from the N-terminal 39-336 amino acids of sequence 2.The homology of the endoglucanase of culturing micro-organisms (GenBank call number ABX76045) is the not highest with deriving from cud for Umcel5D, and both similaritys are 84%, homogeny is 74%.

The replacement of described one or several amino-acid residue and/or disappearance and/or interpolation are meant replacement and/or the disappearance and/or the interpolation of no more than ten amino-acid residues.

In order to make the Umcel5D in (a) be convenient to purifying, proteinic N end or C end that can the aminoacid sequence shown in the sequence 2 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 Umcel5D synthetic, also can synthesize its encoding gene earlier, carry out biology again and express and to obtain.The encoding gene of Umcel5D 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 ' end 295-1878 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 Umcel5D.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 19 amino acids residue in the sequence table.

Above-mentioned endoglucanase encoding gene (umcel5D) also belongs to protection scope of the present invention.

The genomic gene of above-mentioned endoglucanase can have one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 5 ' end 295-1878 position nucleotide sequence of 1;

2) SEQ ID № in the sequence table: 1 nucleotide sequence;

3) SEQ ID № in the code sequence tabulation: the polynucleotide of 2 protein sequences;

4) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 1 dna sequence dna hybridization that limits.

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.

Wherein, sequence 1 in the sequence table is made up of 2258 deoxynucleotides, from the 223rd to 1881 Nucleotide of 5 ' end of sequence 1 open reading frame (the Open Reading Frame that is umcel5D, ORF), 5 ' the 223-225 position Nucleotide of holding from sequence 1 is the initiator codon ATG of umcel5D gene, 5 ' the 1879-1881 position Nucleotide of holding from sequence 1 is the terminator codon TAA of umcel5D gene, the protein sequence of sequence 2 in the nucleotide coding sequence table of sequence 1 in the sequence table.The aminoterminal 25-552 amino acids residue sequence of the nucleotide sequence coded sequence 2 in sequence table in 5 of sequence 1 ' end 295-1878 position in sequence table.

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

The present invention is by making up buffalo cud not the macro genome DNA library of culturing micro-organisms and the dull and stereotyped sieve method that detects of endoglucanase activity of library clone, obtained new endo glucanase gene, this endo glucanase gene can be in host cell this gene of great expression to produce this endoglucanase, be used for cellulosic degraded, experimental results show that endoglucanase of the present invention has very high carboxymethylcelluloenzyme enzyme activity (reaching 4029u/g), and pH value scope and temperature range that this enzyme suits are very wide.Endoglucanase provided by the present invention and encoding gene thereof can be widely used in cellulosic degraded.

Description of drawings

Fig. 1 is the macro genome DNA of the not culturing micro-organisms extracted from buffalo rumen content sample.

Fig. 2 is not culturing micro-organisms gene library clone's restriction enzyme BamHI restriction analysis figure of buffalo cud.

Fig. 3 is that cud is not expressed the screening that endoglucanase activity is cloned, the lithograph at positive colony EPI100/pGXNC35 place in the culturing micro-organisms gene library.

Fig. 4 cuts banding pattern for the BamHI enzyme of the library clone plasmid pGXNC35 of the carboxymethyl cellulose of degrading.

The transformant that obtains behind the recombinant plasmid pGXNC35 transformed into escherichia coli of Fig. 5 for the primary dcreening operation acquisition is to carboxymethyl cellulose degraded hydrolysis circle photo.

Fig. 6 is that expression plasmid pET-umcel5D recombinant plasmid is through EcoRI and XhoI double digestion rear electrophoresis figure.

Fig. 7 is recombination bacillus coli Rosetta ^TM(DE3)/pET-umcel5D and intestinal bacteria Rosetta ^TM(DE3)/pET-30a carboxymethyl cellulose degraded detection.

The purifying of Fig. 8 umcel5D expression of gene, expression product Umcel5D and activity gel detect.

The relative vigor curve of the enzyme of Fig. 9 Umcel5D under different pH condition.

The relative vigor curve of the enzyme of Figure 10 Umcel5D under condition of different temperatures.

The pH tolerance detected result of Figure 11 Umcel5D.

The temperature tolerance detected result of Figure 12 Umcel5D.

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); Expression strain Rosetta ^TM(DE3) (available from Novagen company); Coemid carrier pWEB::TNC (available from Epicentre company); The library prepares test kit (available from Epicentre company, pWEB::TNC cosmid cloning kit, catalog number (Cat.No.) WEBC931); Expression vector pET-30a (available from Novagen company); (Carboxymethylcellulose CMC) waits reagent available from Promega, TAKARA, SIGMA or MBI for restriction enzyme, modifying enzyme, polysaccharase and carboxymethyl cellulose.

The acquisition of embodiment 1, endoglucanase Umcel5D and encoding gene thereof

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

1, the not grand genomic extraction of culturing micro-organisms of buffalo cud

(source of sample is the cud of the buffalo just butchered of Nanning City meat processing combine to get 50g buffalo rumen content, the sample of gathering is preserved with liquid nitrogen immediately), be suspended in the 0.18M potassium phosphate buffer (pH6.5) of 200ml, light shaking is even, placed 10 minutes, allow the cellulose grain in the rumen content precipitate automatically, abandon supernatant liquor, the 0.18M potassium phosphate buffer (pH6.5) that adds 200ml is again washed precipitation piece twice, and last precipitation piece extracts the macro genome DNA of the microorganism that is adsorbed in cellulose grain with direct extraction method.In the precipitation piece, add 100ml and extract damping fluid (100mM sodium phosphate pH8.0; 100mM Tirs-HCl pH8.0; 100mM EDTA pH8.0; 1.5M NaCl; 1%CTAB; 2%SDS) mix, in liquid nitrogen and 65 ℃ of water-baths behind the multigelation three times, 37 ℃ of water-baths of N,O-Diacetylmuramidase (20mg/ml) solution (the N,O-Diacetylmuramidase final concentration is 0.4mg/ml) of adding 2ml 1 hour, put upside down mixing once in per during this time 10 minutes, the 37 ℃ of water-baths of Proteinase K (50mg/ml) solution (the Proteinase K final concentration is 1mg/ml) 1 hour that add 2ml then, during put upside down mixing once in per 10 minutes.Add 40ml PVPP (polyvinylpolypyrrolidone, polyvinylpolypyrrolidone) solution is (available from Sigma company, catalog number (Cat.No.) P-6755) (PVPP solution: every 100mg PVPP and 1ml 0.18M potassium phosphate buffer (pH7.2) mixing) vibrated 30 seconds, added 2ml 3M CaCl again ₂Solution vibrated after 30 seconds, in Beckman Coulter Avanti J-E whizzer (available from Beckman Coulter company, catalog number (Cat.No.) 369003) JA-10 rotary head with 8, centrifugal 20 minutes of 000g room temperature, supernatant liquor is transferred to another clean centrifuge tube.In throw out, add 100ml again and extract damping fluid, mix, placed 60 minutes at 65 ℃, during put upside down mixing once in per 10 minutes, 8, centrifugal 20 minutes of 000g merges twice supernatant liquor.The chloroform that in supernatant liquor, adds equivalent, the centrifuge tube mixing turns upside down, 8, centrifugal 10 minutes of 000g gets supernatant and goes into another centrifuge tube, the Virahol that adds 0.6 times of volume, fully see promptly behind the mixing that the DNA flocks separates out, cotton-shaped DNA, wash twice with 70% ethanol with the Tip choicest of sterilizing, in room temperature, dry, with 5ml TE dissolving.

2, the buffalo cud not the grand genomic purifying of culturing micro-organisms obtain the above DNA of 30kb

The DNA crude extract is added to Sephadex G200 (available from Pharmacia company, catalog number (Cat.No.) 17-0080-01) chromatography column (200mm * 10mm, contain 2%PVPP (available from Sigma company, catalog number (Cat.No.) P-6755) on, use the TE buffer solution elution, by every component 1ml Fractional Collections elutriant, each component adds 3M sodium acetate soln (pH5.2) and the 1ml isopropanol precipitating DNA of 100 μ l, throw out is dissolved among the TE, merge the gained dna solution, downcut the gel that contains the above DNA of 30kb behind 0.7% agarose gel electrophoresis, reclaim purify DNA with electroelution method, the DNA electrophorogram that reclaims purifying as shown in Figure 1, wherein swimming lane 1 is λ DNA (48.5kb); Swimming lane 2 for λ DNA with the EcoRI enzyme cut (clip size is followed successively by from big to small: 21.2kb, 7.4kb, 5.8kb, 5.6kb, 4.9kb, 3.5kb); Swimming lane 3 is the DNA crude extract that extracts from the buffalo rumen content; Swimming lane 4 is the DNA through SephadexG-200 gel preliminary purification; Swimming lane 5 is for reclaiming the above DNA of 30kb that obtains through electroelution method.

3, purifying obtains the not grand genomic end-filling of culturing micro-organisms of the above buffalo cud of 30kb

For the DNA that reclaims purifying with above-mentioned electroelution method makes gene library, at first these DNA are carried out end-filling, (library available from Epicentre company prepares test kit pWEB::TNC cosmid cloning kit to prepare test kit to produce blunt end with the library, the pWEB::TNC carrier of the same tool blunt end of having handled well catalog number (Cat.No.) WEBC931) links to each other, concrete grammar is: add in the Eppendorf tube of a new bacterium of going out on ice successively: 6 μ l 10 * terminal repair buffer liquid (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 dnNTP2.5mM), 6 μ l 10mM ATP, the DNA that 10ug 30kb is above, the terminal repairase mixture of 2 μ l (T ₄Archaeal dna polymerase and T ₄Polynueleotide kinase) (library available from Epicentre company prepares test kit pWEB::TNC cosmidcloning kit, catalog number (Cat.No.) WEBC931), the deionized water that adds sterilization adds to cumulative volume 60ul.Placed 45 minutes down for 25 ℃, transfer to 70 ℃ of water-baths again and place 10 minutes to stop enzyme reaction, the gel that downcuts the DNA that contains 30kb-45kb after the 1.0% low melting-point agarose gel electrophoresis carries out DNA and reclaims.

4, the not ligation of the grand genome of culturing micro-organisms and pWEB::TNC of the buffalo cud behind the end-filling

Above-mentioned end is mended the dna fragmentation and the library of reclaiming flat back prepare the pWEB::TNC carrier of the tool blunt end of having handled well in the test kit at T ₄Couple together under the effect of dna ligase, concrete grammar is: add in the Eppendorf tube of a new bacterium of going out on ice successively: 12 μ l sterilized waters, 2 μ l connect damping fluid (10 * Fast-Link Ligation Buffer) for 10 times fast, 1 μ l 10mM ATP, 1 μ l pWEB::TNC carrier (0.5 μ g), the DNA (0.1 μ g/ μ l) of the 30kb-45kb that 3 μ l low melting-point agarose gels reclaim, 1 μ l connects dna ligase (Fast-Link DNA Ligase fast, 2 units/μ l) (library available from Epicentre company prepares test kit pWEB::TNC cosmid cloning kit, catalog number (Cat.No.) WEBC931), placed 2 hours down at 25 ℃ behind the mixing, place 10 minutes to stop enzyme reaction at 70 ℃ again.

5, the not acquisition and the quality test of the grand genomic library of culturing micro-organisms of buffalo cud

The ligation product is packed with the λ packaging protein, concrete grammar is: (library available from Epicentre company prepares test kit pWEB::TNC cosmid cloning kit to the λ packaging extract that will just dissolve on ice, catalog number (Cat.No.) WEBC931) a component, 50ul/ pipe altogether) 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 other 25 μ l dissolve, fully mixing be placed on 30 ℃ 90 minutes, to wherein adding 500 μ l phage dilution buffer liquid (10mM Tris-HCl (pH8.3), 100mM NaCl, 10mM MgCl ₂), obtain 560 μ l packing reaction product.

Again the above-mentioned 560 μ l packing reaction product that obtains is joined the OD of 5.6mL ₆₀₀(substratum is that (every liter contains Tryptones (Oxoid), 10g to LB to=1.0 host e. coli EPI100 nutrient solution; Yeast extract powder (Difco), 5g; NaCl, 5g; PH7.0)+10mM MgSO ₄) in, 25 ℃ times placements allowed the lambda particles phage of the above-mentioned packing that obtains adsorb in 20 minutes and infect host cell E.coli EPI100, (every liter contains Tryptones (Oxoid), 10g at the LA flat board that contains penbritin (final concentration is 100 μ g/mL) and paraxin (final concentration is 12ug/ul); Yeast extract powder (Difco), 5g; NaCl, 5g; Agar powder, 15g pH7.0) goes up the screening transduttant.The result obtains about 15 altogether, 000 transduttant, extract 14 clones' plasmid DNA arbitrarily, restriction enzyme BamHI enzyme is cut the back and is carried out electrophoretic analysis with 0.7% sepharose, all plasmids are except that the carrier segments that a 5.8kb is all arranged as a result, all contain the insertion fragment, and do not have to find that having two plasmids to have identical enzyme cuts banding pattern, enzyme is cut the result as shown in Figure 2, and wherein (clip size is followed successively by swimming lane 1 from big to small: 21.2kb, 7.4kb with the EcoRI endonuclease bamhi for λ DNA, 5.8kb, 5.6kb, 4.9kb, 3.5kb); Swimming lane 2 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); Other swimming lane is respectively the library clone plasmid that uses restriction enzyme BamHI enzyme to cut.The result shows that the library contains insertion dna fragmentation very at random, and that insert the fragment maximum be 42kb, minimum be 22kb, mean size is 35kb.Clone's capacity that the library is described also is sizable, and the quality in library is fairly good.

Two, the acquisition of endoglucanase and encoding gene thereof (umcel5D)

1, from the buffalo cud not the grand genomic library of culturing micro-organisms screening express the clone of endoglucanase activity

To xerox respectively and contain 0.5% carboxymethyl cellulose (carboxylmethylcellulose containing the library (about about 200 bacterium colonies of every flat board) that obtains on the LA flat board of penbritin and paraxin in the step 1 with dull and stereotyped photolithography, CMC) (available from Sigma company, catalog number (Cat.No.) C-5678), on the LA flat board of penbritin (final concentration 100 μ g/mL) and paraxin (final concentration 12ug/ml), flat board was inverted in 37 ℃ of incubators cultivations after 36 hours, is 0.5% Congo red solution-dyed 15 minutes with the cultured LA flat board that contains carboxymethyl cellulose with the quality percentage composition, NaCl solution with 1M decoloured 15 minutes, detected periphery of bacterial colonies then and had or not the hydrolysis circle.

The result as shown in Figure 3, the bacterium colony of arrow indication for around the clone of hydrolysis circle is arranged.The result shows and screens the clone (EPI100/pGXNC35) that 1 periphery of bacterial colonies has the hydrolysis circle, further extracts this clone's plasmid DNA and with its called after pGXNC35, behind restriction enzyme BamHI complete degestion pGXNC35, carry out 0.7% agarose gel electrophoresis analysis, the result as shown in Figure 4, pGXNC35 also has other 6 BamHI endonuclease bamhis except that the carrier segments that a 5.8kb is arranged, size is respectively 23.0kb, 3.8kb, 2.5kb, 1.5kb, 1.3kb, and 1kb.The result shows that pGXNC35 contains the insertion fragment of 33.1kb.Wherein, among Fig. 4, swimming lane 1 for the fragment of λ after cutting with the EcoRI enzyme (clip size is followed successively by from big to small: 21.2kb, 7.4kb, 5.8kb, 5.6kb, 4.9kb, 3.5kb); Swimming lane 2 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, 1kb); Swimming lane 3 is cut product (being respectively 23.0kb from top to bottom, 5.8kb, 3.8kb, 2.5kb, 1.5kb, 1.3kb, and 1kb) for the BamHI enzyme of pGXNC35.

For the insertion fragment that confirms pGXNC35 contains endo glucanase gene really, with pGXNC35 plasmid DNA and empty carrier pWEB::TNC difference Transformed E .coli EPI100, on the LA flat board that contains penbritin (100 μ g/mL), screen transformant, picking transforms 10 transformant points that obtain by each plasmid and receives and contain on the LA flat board that the quality percentage composition is 0.5% carboxymethyl cellulose at random, cultivate after 24 hours for 37 ℃, with 0.5% Congo red solution-dyed 15 minutes, NaCl solution decolouring with 1M, observe periphery of bacterial colonies then and have or not the hydrolysis circle, the result shows that all 10 transformants that obtained by empty carrier pWEB::TNC conversion all do not have the hydrolysis circle on every side, all 10 transformants that obtained by the pGXNC35 conversion all have the hydrolysis circle on every side, and the detected result of one of them transformant as shown in Figure 5.The result shows on the insertion fragment of recombinant plasmid pGXNC35 and contains endo glucanase gene really.Among Fig. 5, the bacterium colony on the right is the transformant (carboxymethyl cellulose of degrading) that obtains behind the recombinant plasmid pGXNC35 transformed into escherichia coli of primary dcreening operation acquisition, and the bacterium colony on the left side is the transformant (carboxymethyl cellulose of can not degrading) that obtains behind the empty carrier pWEB::TNC transformed into escherichia coli.

2, the acquisition of endoglucanase and encoding gene thereof (umcel5D)

In order to measure the dna sequence dna that recombinant plasmid pGXNC35 goes up endo glucanase gene, adopt the method for subclone that this gene is positioned.Use restriction enzyme EcoRI and HindIII that recombinant plasmid pGXNC35 has been carried out subclone successively, the step of subclone: add ligase enzyme one hour (behind the pGXNC35 complete degestion, the part of the 3.0kb of the carrier pWEB::TNC that this recombinant plasmid contains can be used to clone each external source EcoRI segment) of 25 ℃ of connections after getting plasmid pGXNC35 usefulness EcoRI complete degestion.Connect product with chemical method transformed into escherichia coli XL1-Blue, containing the activated clone of screening on the LA culture medium flat plate that the quality percentage composition is 0.5% carboxymethyl cellulose.Selecting 24 active clones of expression carboxymethylcelluloenzyme enzyme arbitrarily, extract plasmid and do the EcoRI restriction analysis, one of them plasmid clone has minimum external source fragment, with this plasmid called after pGXNC35E-9.The Hind III restriction analysis of plasmid pGXNC35E-9 shows that the pGXNC35E-9 clone has the external source fragment of 25.5kb.Plasmid pGXNC35E-9 with Hind III enzyme complete degestion after, add ligase enzyme, connect product transformed into escherichia coli XL1-Blue, express the active clone of carboxymethylcelluloenzyme enzyme is arranged containing on the LA culture medium flat plate that the quality percentage composition is 0.5% carboxymethyl cellulose screening once more, extract plasmid and do Hind III restriction analysis, find that a clone has minimum external source fragment.At last goal gene will be positioned on the Hind III segment of 4.2kb (will contain the segmental recombinant plasmid called after of this 4.2kb pGXNEH42).

This recombinant plasmid pGXNEH42 is sent Dalian Bao Bio-Engineering Company adopts the dideoxyribonucleoside acid system that this subclone is carried out two-way double-stranded order-checking.Sequencing result software DNAStar (DNASTAR company, version 5) and NCBI (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov) software on is analyzed dna sequence dna, as Blast (http://www.ncbi.nlm.nih.gov/BLAST), obtain the encoding gene of endoglucanase, this gene has the nucleotide sequence of sequence 1 in the sequence table, called after umcel5D.Open reading frame (the Open Reading Frame that the 223rd to 1881 Nucleotide of 5 of sequence 1 ' end is umcel5D in sequence table, ORF), form by 1659 Nucleotide, 223-225 position Nucleotide from 5 of sequence 1 ' end is the initiator codon ATG of umcel5D gene, and Nucleotide is the terminator codon TAA of umcel5D gene from the 1879-1881 position of 5 of sequence 1 ' end.。

One of endo glucanase gene umcel5D coding contains 552 amino acid whose protein Umcel5D, amino acid residue sequence with sequence 2 in the sequence table, with this proteinic theoretical molecular size of DNAStar software prediction is 61702.41 dalton, and iso-electric point pI is 5.923.With simple assemblies structural research instrument (Simple ModularArchitecture Research Tool, SMART, http://smart.embl-heidelberg.de) unit construction of analysis endoglucanase Umcel5D, the result is that the N-terminal 1-19 amino acids from sequence 2 is signal peptide (signal peptide), is family's 5 glycosyl hydrolases (glycosyl hydrolase) functional domains from the N-terminal 39-336 amino acids of sequence 2.The homology of the endoglucanase of culturing micro-organisms (GenBank call number ABX76045) is the not highest with deriving from cud for Umcel5D, and both similaritys are 84%, homogeny is 74%.

Embodiment 2, the umcel5D expression in intestinal bacteria

1, can express the structure of the recombinant vectors (pET-umcel5D) of umcel5D

After expressing in vivo, the gene that has a coded signal peptide probably expression product is secreted into the extracellular.For avoiding expression product to be secreted into the extracellular, only need artificial synthesized sequence 1 from 5 ' the 295th at end is to 1878 bit bases (being the dna sequence dna except that coded signal peptide functional domain and terminator codon in the umcel5D gene), 1585bp, estimate the molecular weight of synthetic fragment proteins encoded be 59.1202KDa, iso-electric point pI is 5.92.

The full sequence (promptly from 5 of sequence 1 ' end 295-1878 position nucleotide sequence) of synthetic umcel5D gene except signal peptide and terminator codon, synthetic umcel5D gene two ends have EcoRI and XhoI restriction enzyme site, after also order-checking shows that correct umcel5D gene fragment is cut with EcoRI and XhoI enzyme with synthetic back, insert between the EcoRI and XhoI site of carrier pET-30a (+) (available from Novagen company), obtain recombinant expression vector, this recombinant vectors is carried out the enzyme evaluation of cutting and check order, and enzyme is cut and is checked order and shows the correct recombinant plasmid called after pET-umcel5D that contains the umcel5D gene coded sequence.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 respectively for the N end and the C end of expression product.Wherein, the restriction enzyme digestion and electrophoresis collection of illustrative plates of pET-umcel5D recombinant plasmid can be seen recombinant plasmid carrier band and fragment with synthetic umcel5D gene onesize 1.585kb after EcoRI and XhoI enzyme are cut through discharging a 5.3kb behind EcoRI and the XhoI double digestion as shown in Figure 6 among Fig. 6.Among Fig. 6, 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 recombinant plasmid result behind EcoRI and XhoI double digestion.

2, the umcel5D gene is at E.coli Rosetta ^TM(DE3) expression in

PET-umcel5D is converted into E.coli Rosetta ^TM(DE3) obtain containing the transformant Rosetta of pET-umcel5D in ^TM(DE3)/and pET-umcel5D, picking Rosetta ^TM(DE3)/the single bacterium colony of pET-umcel5D (contains paraxin 34 μ g/mL, kantlex 25 μ g/ml, IPTG 1.0mmol/L) in containing on the LA flat board that the quality percentage composition is 0.5% carboxymethyl cellulose, uses Rosetta simultaneously ^TM(DE3)/pET-30a (changes the Rosetta of pET-30a over to ^TM(DE3)), cultivated 24 hours for 37 ℃ as blank.Thalline is 0.5% Congo red solution-dyed 15 minute with mass percentage concentration at 37 ℃ after cultivating 2 hours behind the chloroform broken wall again, decolours with the NaCl solution of 1mol/L, observes periphery of bacterial colonies then and has or not the hydrolysis circle.

The result as shown in Figure 7, the result shows reorganization bacterium Rosetta ^TM(DE3)/pET-umcel5D has the hydrolysis circle on every side, and blank Rosetta ^TM(DE3)/and there is not the hydrolysis circle around the pET-30a, illustrate that target gene umcel5D is at E.coliRosetta ^TM(DE3) efficiently expressed out the protein that the carboxymethyl cellulose degrading activity is arranged in.The bacterium colony on the right is recombination bacillus coli Rosetta among Fig. 7 ^TM(DE3)/and pET-umcel5D (carboxymethyl cellulose of degrading), the bacterium colony on the left side is the intestinal bacteria Rosetta that contains empty carrier ^TM(DE3)/pET-30a (carboxymethyl cellulose of can not degrading).

3, the expression of Umcel5D and purifying

1) RosettaTM (DE3)/pET-umcel5D thalline fermentation

Inoculation RosettaTM (DE3)/pET-umcel5D contains in 34 μ g/ml paraxin (CAM) and the 25 μ g/ml kantlex LB nutrient solutions to 10ml, 37 ℃ of 200rpm overnight incubation.Get the 5ml overnight culture and contain to 100ml in the LB nutrient solution of 34 μ g/ml paraxin (CAM) and 25 μ g/ml kantlex, cultivate 200rpm at 37 ℃ and be cultured to OD ₆₀₀Be 0.6.Adding IPTG is 1mM to final concentration, and continuation was cultivated 5 hours, 5, and the centrifugal collection thalline of 000g.Make 2 bottles of samples altogether, collect the thalline of the nutrient solution of 200ml.

2) extraction and purification of Umcel5D

Collect in the thalline that obtains in step 1), add the lysis buffer (50mMNaH of 5ml by every gram thalline weight ₂PO ₄, 300mM NaCl, 10mM imidazole (imidazoles), 1mM PMSF (phenylmethylsulfonyl fluoride), pH8.0), the suspension thalline adds N,O-Diacetylmuramidase to final concentration 1mg/ml, puts 30 minutes on ice.Use the ultrasonic disruption cell, 400w effect 20 times, each action time 10s, each effect is 12s at interval.12, centrifugal 20 minutes of 000g collects supernatant and obtains the Umcel5D crude enzyme liquid.Get 5ul and detect proteinic purity with SDS-PAGE.

The purifying of Umcel5D uses the The QIAexpressionist kit test kit (method reference reagent box specification sheets) of Qiagen company.Adding 1ml quality percentage composition by the cracking supernatant liquor of the above-mentioned acquisition of every 4ml is 50% NI-NTA colloid, shakes 60 minutes with 200rpm at 4 ℃, and mixture is filled into the incidental pillar of The QIAexpressionist kit test kit.Add 4ml lavation buffer solution (50mM NaH ₂PO ₄, 300mM NaCl, 20mMimidazole, 1mM PMSF pH8.0) in pillar, slowly stirs, and repeats to wash 6 times.Elution buffer (the 50mM NaH that adds 0.5ml ₂PO ₄, 300mM NaCl, 250mM imidazole, 1mM PMSF pH8.0), collects effluent, repeats wash-out 8 times.Merge 8 tubulin matter, be the Umcel5D behind the ni-sepharose purification, get 5ul and detect proteinic purity with SDS-PAGE.

Select Sephadex G-100 that Umcel5D is carried out further purifying, get 5g Sephadex G-100 in the 100ml deionized water, room temperature was placed 72 hours or boiling water boils 5 hours, and is standby; Get a long 40cm of being, internal diameter is the chromatography column of 1cm, cleans, and the pillar lower end is blocked with the sponge ball with filtering function, the pH of 1/4 volume of packing in post is citric acid-Sodium phosphate dibasic damping fluid of 6.0, and then that swelling is good Sephadex G-100 packs in the post.With pH Sephadex G-100 in citric acid-Sodium phosphate dibasic damping fluid balance columns of 6.0; Getting 1ml and be added in Jiao Mianshang through the Umcel5D of ni-sepharose purification along tube wall, is citric acid-Sodium phosphate dibasic buffer solution elution of 6.0 with pH, collects Umcel5D after elutriant promptly obtains molecular sieve purification by the every pipe of 0.5ml.Every pipe elutriant is got 5ul and is detected lipidated protein.

The result as shown in Figure 8, the result shows that it is pure that Umcel5D has reached the SDS-PAGE wall scroll behind molecular sieve (Sephadex G-100) purifying.

For whether the Umcel5D that detects purifying also has endoglucanase activity, detect with the activity gel staining, process is as described below: at first Umcel5D carries out protein electrophorese (SDS-PAGE) on polyacrylamide sex change glue, then to behind the electrophoresis in sex change glue the Umcel5D of sex change carry out renaturation and handle.Renaturation is handled: the sex change glue behind the electrophoresis is immersed in the (preparation of renaturation buffer: Tris 3.025g in the renaturation buffer of 50ml, 0.5M EDTA (pH8.0) 5ml, beta-mercaptoethanol 0.173ml, add water to cumulative volume 500ml), 4 ℃ were soaked 30 minutes down, change renaturation buffer, triplicate, the Virahol of preceding twice interpolation 0.5ml in renaturation buffer.Wash glue twice with the potassium phosphate buffer of 10mM pH7.0 again.The renaturation sticker containing on the LA culture dish that the quality percentage composition is 0.5% carboxymethyl cellulose, is wrapped up culture dish with plastics bag, and insulation is 1 hour in 37 ℃.Taking out dull and stereotyped is 0.5% Congo red solution-dyed 15 minutes with mass percentage concentration, with the NaCl solution decolouring of 1mol/L.The Umcel5D that activity gel detects purifying has CMCase (endoglucanase) activity, illustrates that umcel5D expresses by correct reading frame.1 is molecular weight standard (marker) among Fig. 8; 2 is the crude enzyme liquid of umcel5D expression of gene; 3 is the Umcel5D behind the ni-sepharose purification; 4 is the Umcel5D of molecular sieve purification; 5 is the activity gel detection of Umcel5D.

4, the research of Umcel5D enzymatic property

The enzyme activity determination of endoglucanase adopts the DNS method, and concrete operations are as follows:

1) preparation DNS reagent: take by weighing the about 400ml ddH of 10 gram NaOH ₂The O dissolving takes by weighing 10 gram dinitrosalicylic acids, 2 gram phenol, 0.5 gram sodium sulphite anhydrous 99.3,200 gram Rochelle salts again, and it is dissolved in about 300ml ddH ₂Among the O, two kinds of solution mix, and constant volume to 1 liter keeps in Dark Place.

2) drafting of glucose typical curve

Get 9 thin-walled test tubes, according to the form below adds solution

Reacted 5 minutes in boiling water behind the mixing, the room temperature cooling is surveyed A with microplate reader ₅₃₀

3) enzyme activity determination

Get the step 2 in the 10 μ l steps 3) the middle Umcel5D enzyme liquid (Umcel5D concentration is 0.203mg/ml) that obtains, add in 0.1M citric acid-0.2M Sodium phosphate dibasic damping fluid of 190 μ l pH4.5, adding 300 μ l quality percentage compositions again is 1%CMC (carboxymethyl cellulose) (solvent is the identical 0.1M citric acid of pH value-0.2M Sodium phosphate dibasic damping fluid) solution reaction, reaction is 10 minutes under 40 ℃ of temperature, the DNS reagent that question response finishes the preparation of back adding 1ml step 1) placed the boiling water water-bath 5 minutes, the room temperature cooling is surveyed A with microplate reader ₅₃₀

Enzyme (IU) alive is defined as: 1U produces the required enzyme amount of 1 μ mol reducing sugar for per minute catalysis.

Definition than vigor: the enzyme activity (U/g) that every g protein is contained.

The result shows, Umcel5D is 4029U/g to the ratio vigor of carboxymethyl cellulose under pH4.5,40 ℃ of temperature.

4) mensuration of the optimum pH of enzyme

Measure Umcel5D at 37 ℃, the pH scope is that the enzyme in 0.1M citric acid-0.2M Sodium phosphate dibasic damping fluid of 2.5～8.0 is lived, and gradient is 0.5.Concrete grammar is: get the step 2 in the 10 μ l steps 3 respectively) the middle Umcel5D enzyme liquid that obtains, add respectively in 0.1M citric acid-0.2M Sodium phosphate dibasic damping fluid that 190 μ l pH values are 2.5～8.0 (gradient is 0.5), add 300 μ l quality percentage compositions more respectively and be 1%CMC (carboxymethyl cellulose) (solvent for the 0.1M citric acid of identical pH value-0.2M Sodium phosphate dibasic damping fluid) before solution, 37 ℃ the reaction 10 minutes after, the DNS reagent that adds the preparation of 1ml step 1) immediately placed the boiling water water-bath 5 minutes, the room temperature cooling is surveyed A with microplate reader ₅₃₀Enzyme (IU) alive is defined as: 1U produces the required enzyme amount of 1 μ mol reducing sugar for per minute catalysis.Under 37 ℃ reaction conditions, the ratio vigor of Umcel5D in citric acid-disodium hydrogen phosphate buffer solution of pH 4.5 is the highest, as 100%, the relative enzyme activity under each pH value that converts, the result as shown in Figure 9, the result shows that the optimal pH of Umcel5D is 4.5.

5) mensuration of the optimum temperuture of enzyme

Live at the Umcel5D enzyme of measuring (20 ℃～70 ℃) under the differing temps under optimal pH 4.5 conditions.Concrete grammar is: get the step 2 in the 10 μ l steps 3 respectively) the middle Umcel5D enzyme liquid that obtains, add 190 μ l pH values and be in 0.1M citric acid-0.2M Sodium phosphate dibasic damping fluid of 4.5, adding 300 μ l quality percentage compositions again is 1%CMC (carboxymethyl cellulose) (solvent is that the pH value is 0.1M citric acid-0.2M Sodium phosphate dibasic damping fluid of 4.5) solution, after placing 20 ℃～70 ℃ (gradient is 5 ℃) to react 10 minutes respectively, the DNS reagent that adds the preparation of 1ml step 1) placed the boiling water water-bath 5 minutes, the room temperature cooling is surveyed A with microplate reader ₅₃₀Enzyme (IU) alive is defined as: 1U produces the required enzyme amount of 1 μ mol reducing sugar for per minute catalysis.In the pH value is in 0.1M citric acid-0.2M Sodium phosphate dibasic damping fluid of 4.5 under the reaction conditions, Umcel5D is higher than vigor in the time of 40 ℃, as 100%, and conversion relative enzyme activity at each temperature, the result as shown in figure 10, the result shows that the optimum temperuture of Umcel5D is 40 ℃

6) the pH tolerance of enzyme is measured

With the step 2 in the step 3) in the Umcel5D enzyme liquid that obtains be stored in the damping fluid of different pH values (pH 2.5-10.0, gradient is 0.5), 40 ℃ down measure enzyme alive (method same step 3)) in optimum pH 4.5 and optimum temperuture in 4 ℃ after placing 24 hours.Its contrast (i.e. 100% vigor) is the step 2 in the untreated step 3) the middle Umcel5D enzyme liquid that obtains is at pH4.5, and 40 ℃ are descended measured vigor.The result as shown in figure 11, the result shows that Umcel5D still has the vigor more than 80% after keeping 24 hours between pH3.5～10.0, this illustrates that this enzyme has pH tolerance preferably.

7) temperature tolerance of enzyme is measured

With the step 2 in the step 3) in the Umcel5D enzyme liquid that obtains be stored in the damping fluid of optimum pH, place (20 ℃～80 ℃, gradient is 5 ℃) under the differing temps to place after 1 hour under the optimum pH of this enzyme and optimum temperuture, to measure enzyme live (the same step 3) of method).Its contrast (i.e. 100% vigor) is the step 2 in the untreated step 3) the middle Umcel5D enzyme liquid that obtains is at pH4.5, and 40 ℃ are descended measured vigor.The result as shown in figure 12, the result shows Umcel5D at the 25-45 ℃ of relative vigor that can keep more than 60%, basically with regard to loss of activity, this shows that Umcel5D is not very wide to the tolerance range of temperature in the time of 50 ℃.

Sequence table

<160>2

<210>1

<211>2258

<212>DNA

＜213〉the unknown

<220>

<223>

<400>1

tctatcatca gatcatgaag cgtctgattg tttgtgcaat ataagcactg tttgggatca 60

ctttgcatat tcgttttgtt ttaagatttg attttagtgg tgcaaaatta aacaaattct 120

tccgattctc aaaatctttc caaatatatt tgtctacttc acaaataaat tgtacttttg 180

cagaagaaat ctataattac aaatattatt aaactaaaat ttatgaagaa aattctactc 240

ttagtatgca gcgcgatgct atgtgcatcc attcaggctg ccgattttga aacggcaaaa 300

gatgccgtca agaacatggg cgtaggttgg aacctgggaa acacccttga tgctgctgat 360

gcttctaaaa cctggaccac cacagcacag catgaaacct gctggggaca gcccgtcacc 420

aagcccgaac tcatgaagat gatgaaggag gcaggattcg gtgccatccg tgtgccaata 480

acctggtatc aagaaatgga caaggacggt aaggtaaacg atgcatggat gaagcgtgtg 540

aaagaagtcg ttgactatgt gattgacaat ggtatgtact gtatcatcaa cgtgcatcac 600

gatacaggtg acggcacaca atggttgcat gccagctcta ccaactacaa tacgaaccag 660

gcgaagtatg aaggtttatg gaaacagata gccgagaaat tcaaggacta tgatcagaag 720

ctgctctttg aggcctacaa cgagatgctc gacgataaga acacctggaa cgaacccctt 780

agtgacgatg gctacaaggc catcaacagc tatgccaaga gtttcgtcac aaccgttcgt 840

aatacgggag gcaacaacaa ggatcgcaac ctgatagtca atacctactc tgccagcagt 900

actgctaacg cgatgaaagc gctggatttg ccagaagaat cagggcacat tattttccag 960

ctgcacagct atcctaactg gaagagcgaa agtaacgcca aaagtgaaat cgacaacctg 1020

atcaacaata tcaagacgaa tctgctgaac agggccccag tgatcatcgg agaatatgcc 1080

acatttacca cttggcctgc taatctcgac tattacgcaa ctgacaagaa agtagccttc 1140

tatgcgatgg actatctgat caagcagacc aaggagaacg gtatcggcac cttctactgg 1200

atgggactct cggatggtga atcacgctca ttgcccgcct tcaatcaggc agaccttgca 1260

cagacgctta tcaaggccta ctacggcagt actgacggct ataagtttcc cacagctgac 1320

gactttcaaa taacatatac cgtgaaatat acagatgaat ggtcggaggc ctttctcttt 1380

ggtgactggg gtcgtacagc ccagaaactg agtgactata aaggtatccg tctggagatg 1440

gacaatgact attctggtaa gttgcagttt aaaatctatg gcgacaagac aggagaaaag 1500

aatactgacg gcagcgataa attcaaagag caatatactg ggttgacggc tggctcgaat 1560

acctcagagg ttacctttga cacttccatt cttggctcaa ccttttgggg cgtcacacta 1620

cagacccttt caggagccct gactgccaaa gttaagaagg caacactcat caaggctgac 1680

ggtactgagg tttctctgac cgttacgaaa gcttggggat gtgaagtgag ctcagaatca 1740

gcagcgcctt ctggtatccg ttccatcagt gcgatgccca acaagggtga aagtcagatc 1800

tacaatctga atggccagcg catatcaaca ccgcataaag gcatctatat cctaaatggc 1860

aaaaaatatg ttatgaaata agtaccgctt attgaacaat aagggaccct tatcgacaaa 1920

taagggaccc ttatttgaga ataagtaccc cataaagcaa gagagccgca ggataaaaat 1980

cttgcggctc tcttcttatg atcgatagat ttatttgttt acctggaact tggtgtcgcc 2040

atccttgaag aaggcattga tctgcttggc agcagcgata ccagcgttga tgttggcttc 2100

agctgtctga gcacccatct tcttaggagt agagaagtaa cggccctcga acttcaggaa 2160

ctcgtcgttg gcatcaggca tgatgtcagt aacgaacttc aaatcctcgc gctctgccat 2220

cagcttaatc agctcaggct cgttaatgac ctccttgc 2258

<210>2

<211>552

<212>PRT

＜213〉the unknown

<220>

<223>

<400>2

Met Lys Lys Ile Leu Leu Leu Val Cys Ser Ala Met Leu Cys Ala Ser

1 5 10 15

Ile Gln Ala Ala Asp Phe Glu Thr Ala Lys Asp Ala Val Lys Asn Met

20 25 30

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

35 40 45

Lys Thr Trp Thr Thr Thr Ala Gln His Glu Thr Cys Trp Gly Gln Pro

50 55 60

Val Thr Lys Pro Glu Leu Met Lys Met Met Lys Glu Ala Gly Phe Gly

65 70 75 80

Ala Ile Arg Val Pro Ile Thr Trp Tyr Gln Glu Met Asp Lys Asp Gly

85 90 95

Lys Val Asn Asp Ala Trp Met Lys Arg Val Lys Glu Val Val Asp Tyr

100 105 110

Val Ile Asp Asn Gly Met Tyr Cys Ile Ile Asn Val His His Asp Thr

115 120 125

Gly Asp Gly Thr Gln Trp Leu His Ala Ser Ser Thr Asn Tyr Asn Thr

130 135 140

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

145 150 155 160

Lys Asp Tyr Asp Gln Lys Leu Leu Phe Glu Ala Tyr Asn Glu Met Leu

165 170 175

Asp Asp Lys Asn Thr Trp Asn Glu Pro Leu Ser Asp Asp Gly Tyr Lys

180 185 190

Ala Ile Asn Ser Tyr Ala Lys Ser Phe Val Thr Thr Val Arg Asn Thr

195 200 205

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

210 215 220

Ser Ser Thr Ala Asn Ala Met Lys Ala Leu Asp Leu Pro Glu Glu Ser

225 230 235 240

Gly His Ile Ile Phe Gln Leu His Ser Tyr Pro Asn Trp Lys Ser Glu

245 250 255

Ser Asn Ala Lys Ser Glu Ile Asp Asn Leu Ile Asn Asn Ile Lys Thr

260 265 270

Asn Leu Leu Asn Arg Ala Pro Val Ile Ile Gly Glu Tyr Ala Thr Phe

275 280 285

Thr Thr Trp Pro Ala Asn Leu Asp Tyr Tyr Ala Thr Asp Lys Lys Val

290 295 300

Ala Phe Tyr Ala Met Asp Tyr Leu Ile Lys Gln Thr Lys Glu Asn Gly

305 310 315 320

Ile Gly Thr Phe Tyr Trp Met Gly Leu Ser Asp Gly Glu Ser Arg Ser

325 330 335

Leu Pro Ala Phe Asn Gln Ala Asp Leu Ala Gln Thr Leu Ile Lys Ala

340 345 350

Tyr Tyr Gly Ser Thr Asp Gly Tyr Lys Phe Pro Thr Ala Asp Asp Phe

355 360 365

Gln Ile Thr Tyr Thr Val Lys Tyr Thr Asp Glu Trp Ser Glu Ala Phe

370 375 380

Leu Phe Gly Asp Trp Gly Arg Thr Ala Gln Lys Leu Ser Asp Tyr Lys

385 390 395 400

Gly Ile Arg Leu Glu Met Asp Asn Asp Tyr Ser Gly Lys Leu Gln Phe

405 410 415

Lys Ile Tyr Gly Asp Lys Thr Gly Glu Lys Asn Thr Asp Gly Ser Asp

420 425 430

Lys Phe Lys Glu Gln Tyr Thr Gly Leu Thr Ala Gly Ser Asn Thr Ser

435 440 445

Glu Val Thr Phe Asp Thr Ser Ile Leu Gly Ser Thr Phe Trp Gly Val

450 455 460

Thr Leu Gln Thr Leu Ser Gly Ala Leu Thr Ala Lys Val Lys Lys Ala

465 470 475 480

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

485 490 495

Ala Trp Gly Cys Glu Val Ser Ser Glu Ser Ala Ala Pro Ser Gly Ile

500 505 510

Arg Ser Ile Ser Ala Met Pro Asn Lys Gly Glu Ser Gln Ile Tyr Asn

515 520 525

Leu Asn Gly Gln Arg Ile Ser Thr Pro His Lys Gly Ile Tyr Ile Leu

530 535 540

Asn Gly Lys Lys Tyr Val Met Lys

545 550

Claims (5)

1. the encoding gene of endoglucanase is characterized in that: the encoding gene of described endoglucanase is the 5 ' end 295-1878 position nucleotide sequence of SEQ ID NO:1 in the sequence table.

2. the recombinant expression vector that contains the described endoglucanase encoding gene of claim 1.

3. the transgenic cell line that contains the described endoglucanase encoding gene of claim 1.

4. the host bacterium that contains the described endoglucanase encoding gene of claim 1.

5. the application of the described endoglucanase encoding gene of claim 1 in cellulose degradation.

Images