CN101870966B

CN101870966B - Cellulose degrading enzyme with glucosidase/xylosidase dual functions and preparation method and application thereof

Info

Publication number: CN101870966B
Application number: CN2009101717167A
Authority: CN
Inventors: 吕红; 周峻岗; 包蕾
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2009-04-27
Filing date: 2009-08-26
Publication date: 2012-08-01
Anticipated expiration: 2029-08-26
Also published as: CN101870966A

Abstract

The invention belongs to the biological engineering field, relating to a cellulose degrading enzyme RuBGX1 with glucosidase/xylosidase dual functions, a recombinant carrier, strains, a preparation method and application thereof. The invention is characterized in that the RuBGX1 can hydrolyze substrates such as glucoside, galactoside, xyloside, cellobiose, cellotriose and the like; proper amounts of the RuBGX1 added into xylanase can obviously increase the reduction yields of xylan; the RuBGX1 can be used in the fields such as cellulose biotransformation, chemical engineering, textile, food, bioenergy, feed ingredients, pharmaceutical industry application and the like; the RuBGX1 of the invention can degrade lignocellulose and reduce the categories of added enzymes, thereby simplifying the enzymolysis process; and the RuBGX1 of the invention can be used as feed additives and can improve digestion degree of animals on crude fibers, improve digestive tract environment of monogastric animals, increase utilization rate of feeds and promote good digestion of the feeds.

Description

Glucuroide/xylosidase dual functions cellulose degrading enzyme

Technical field

The invention belongs to bioengineering field, relate to a kind of glucuroide/xylosidase dual functions cellulose degrading enzyme.The present invention also provides the recombinant plasmid and the recombination engineering strain of this glucuroide/xylosidase.

Background technology

The plant lignocellulose is the main renewable resources of nature, and its staple is Mierocrystalline cellulose, semicellulose and xylogen, utilizes the cellulase bio-transformation to produce the most important developing direction that biofuel has become countries in the world new forms of energy strategy.Mierocrystalline cellulose in the lignocellulose is a kind ofly 100～1000 β-D-glucopyranose to be arranged with β-1, the straight-chain polysaccharide that the 4-glycosidic link connects, and a plurality of molecule parllels closely are arranged in thread insoluble micro fiber.The branched chain polymer that semicellulose then is made up of multiple polysaccharide molecule accounts for 35% of plant dry weight, is only second to Mierocrystalline cellulose at occurring in nature content.The cellulosic complicacy of the structure of semicellulose and ratio of components, it comprises various ingredients such as xylan, mannosans, ARABINANS, arabogalactan and xyloglucan.Make full use of semicellulose according to estimates and can make the production cost of cellulose ethanol reduce by 25%, this obviously should give the credit to the D-wood sugar.Wood sugar is only second to glucose at occurring in nature content, and the recycling from the waste paper in refuse tip and rubbish of the annual only U.S. just can be produced 400,000,000 tons of ethanol.The lignocellulose production alcohol fuel that sets out, must biomacromolecule be degraded to can be by the available small molecules carbohydrate of mikrobe material, and the degradation technique of lignocellulose has acid hydrolysis and two kinds of technological lines of enzymic hydrolysis usually.Acid hydrolyzation needs high temperature, high pressure, and inhibition is many, the fermentation difficulty, also has environmental issue.The enzymolysis process conversion condition is gentle, only need hydrolysis under normal condition, and its selectivity is high, inhibition is few, and the purity of sugar is also high.

In fodder industry, cellulase is with a wide range of applications at the nutritive value that improves silage equally.Add cellulase and can improve animal, improve the monogastric animal digestive tract environment, activate stomach en-coarse-fibred utilization ratio; The synergy of cellulase, hemicellulase and polygalacturonase can be destroyed plant cell wall, and entocyte is dissolved out, and has improved the digestion of nutrient, has also increased the digestibility of non-starch polysaccharide and the utilization ratio of feedstuff raw material simultaneously; Cellulase can also be eliminated the ANFs in the wood fibre.For example, pectin, semicellulose, beta-glucan and piperylene can be partly dissolved in the water, increase animal gastrointestinal tract content viscosity; Influence nutrition absorption rate utilization in the feed; Add cellulase and can reduce viscosity, improve digestive ferment and feed effect probability, promote the good digestion of feed.

Compare with fungal cellulase; The cellulase of Production by Bacteria all is superior to fungi in thermotolerance and pH tolerance; Excavate bacteria cellulose enzyme gene, both be beneficial to the development of new cellulose enzyme gene, also be beneficial to the variety of research cellulase; For the protein engineering transformation of cellulase provides abundant genetic resources, obtain novel cellulase preparation product.The cud of ruminating animal is the fine source of various bacterial origin cellulose degrading enzymes.The zytase that at present has a high yield in suitability for industrialized production derives from rumen microorganism Neocallinastix partricaiarum exactly.(isoperibol of acid, height anaerobism, high osmotic pressure) only has the rumen microorganism of less than 20% to obtain separating so far because the singularity of ruminal environment, the rumen microorganism more than 80% with and the genetic resources contained be not exploited as yet.Patent retrieval both domestic and external has also shown the exploitation famine of rumen microorganism resource, only has the rumen microorganism cellulose degrading enzyme of minority to obtain clone's evaluation, and all from educable rumen microorganism, obtains.

Cellulose degradation in the wood fibre is the multienzyme mechanism of a complicacy, so the complicated enzyme that cellulase is made up of multiple lytic enzyme is.According to enzyme function difference property wherein; Be divided into following three major types: (1) endoglucanase (1.4-β-D-glucanhydrolase or endo-1.4-β-D-glucanase E.C 3.2.1.4); This fermentoid acts on the inner noncrystalline domain of Mierocrystalline cellulose; Random hydrolysis β-1.4-glycosidic link produces a large amount of small molecules Mierocrystalline celluloses.(2) VISOSE excision enzyme (1.4-β-D-glucan cellobiohydrolase or exo-1.4-β-D-glucanase; E.C3.2.1.91); It is terminal that this fermentoid acts on the Mierocrystalline cellulose linear molecule, hydrolysis β-1,4-glycosidic link; Its hydrolysate is a cellobiose, so be called cellobiohydrolase (cellobiohydrolase) again.(3) (β-1.4-glucosidase, E.C3.2.1.21), this fermentoid is that cellobiose is hydrolyzed into glucose molecule to β-1.4-VISOSE glycosides enzyme.In cellulosic degradation process; Cellobiose has the product retarding effect to the degraded of cellulase; And thereby beta-glucosidase ability degradation of fibers disaccharides is eliminated the retarding effect of cellobiose, and therefore, beta-glucosidase plays important effect in cellulosic degradation process.

The beta-glucosidase of various bacteria and fungi has obtained isolation identification at present, like fungi Aspergillus (FEMS Microbiol Lett 97:149-154,1992); Humicola (FEMSMicrobiol Lett.146:291-295,1997), Fusarium (Eur.J.Biochem223:397-385; 1994), Volvariella (Appl Environ Microbiol.65:553-559,1999); Candida (Appl Environ Microbiol 61:518-525,1995).

About more existing patents of beta-glucosidase gene and bibliographical information.As: Dion etc. have reported the beta-glucosidase gene (glycoconjugate J, 16:27-37,1999) of Thermus thermophilus; Gonzalez etc. have reported the beta-glucosidase gene (Biochem Biophys Res Commun, 194:1359-1364,1993) of sporeformer Bacillus polymyxa; Wrightdd etc. have reported beta-glucosidase gene (Appl EnvironMicrobiol, 63 of Microbispora bispoera; 3902-3910,1997); Lieb etc. have reported the beta-glucosidase gene (Mol Gen Genet, 242:111-115,1994) of Thermotoga mariti; Tonouchi etc. have reported the beta-glucosidase gene (U.S. Pat P6316251, November 31 calendar year 2001) of Acetobacter xylinum.

Xylan (xylan) is a kind of heterozygosis poly five-carbon sugar, and main chain is linked to each other with β-1,4 wood sugar glycosidic bond by xylopyranosyl.The group of the multiple different sizes of ining succession on the side chain.The degrading enzyme of xylan mainly is: (1) inscribe β-1, and (EC3.2.1.8), this enzyme acts on inner β-1, the 4 wood sugar glycosidic bond of xylan backbone with internal-cutting way to the 4-zytase for endo-1,4-β-D-xylanase, and its main hydrolysate is oligomeric wood oligose; (2) β-1, (β-xylosidase, EC3.2.1.37), this enzyme comes hydrolysis to discharge wood sugar through the end of the oligomeric wood oligose of hydrolysis to the 4-xylosidase.The xylosidase major part of mikrobe is an intracellular enzyme, and the minority fungi then can secrete the outer xylobiase of born of the same parents.

Utilizing semicellulose to produce in the alcohol fuel, xylan is degraded into wood sugar through xylosidase and xylosidase, and the wood sugar of generation can be produced ethanol through bacterium or fungi fermentation, thereby the performance of the application potential in bioenergy receives much attention.And at present domestic about xylosidase patent also seldom, 200710063139.0) and the Danisco of Denmark (Chinese patent number: CN96194959.7) two Nankai University's (Chinese patent number: is only arranged.And the separation and purification and the gene clone of aspergillus (Aspergillus) xylobiase are also only arranged with the research of xylosidase report.

And has the bifunctional enzyme of beta-glucosidase and xylosidase degradation function; One piece of bibliographical information is only arranged both at home and abroad; Beta-glucosidase/xylosidase that Brunner etc. (Phytochemistry, 59 (2002) 689-696) are cloned into from phytophthora infestans (Phytophthora infestans).

Summary of the invention

The purpose of this invention is to provide a kind of novel glycoside hydrolase with beta-glucosidase/xylosidase dual functions.

Another object of the present invention provides the preparation method of this glucuroide/xylosidase dual functions cellulose degrading enzyme.

A purpose more of the present invention provides the application of this glucuroide/xylosidase dual functions cellulose degrading enzyme.

The invention provides a kind of glucuroide/xylosidase dual functions cellulose degrading enzyme, it has the aminoacid sequence shown in the SEQ ID NO.2.

Among the present invention, glucuroide/xylosidase dual functions cellulose degrading enzyme be meant possess simultaneously glucuroide and xylosidase activity, the polypeptide of aminoacid sequence shown in SEQ ID NO.2, abbreviate RuBGX1 as.This term also comprises possessing variant form glucuroide and xylosidase activity, SEQ IDNO.2 sequence simultaneously.These variant forms comprise (but being not limited to): several (are generally 1-50; Preferably 1-30; 1-20 more preferably, 1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20; Preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term comprises that also the homology with the aminoacid sequence of SEQ ID NO.2 reaches 80% and above sequence.

RuBGX1 of the present invention has the characteristic of the dual enzyme of beta-glucosidase/xylosidase, optimal reaction pH5.8,50 ℃ of optimal reactive temperatures, molecular weight 82,002Da.

Glucuroide of the present invention/xylosidase dual functions cellulose degrading enzyme, its nucleotide coding sequence can be shown in SEQ ID NO.1.

Glucuroide of the present invention/the nucleotide coding sequence of xylosidase dual functions cellulose degrading enzyme possesses the nucleotide sequence of the polypeptide of glucuroide and xylosidase activity, for example the nucleotide sequence of SEQ ID NO.1 and degenerate sequence thereof.This degenerate sequence is meant, in the SEQ ID NO.1 sequence, having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, so be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.2 of also encoding out with SEQ ID NO.1 homology.This term also comprises the homology of nucleotide sequence at least 70% with SEQ ID NO.1, preferably at least 80%, and at least 90% nucleotide sequence more preferably.

The present invention also provides the recombinant vectors of the nucleotide coding sequence that contains above-mentioned glucuroide/xylosidase dual functions cellulose degrading enzyme, and this recombinant vectors can be escherichia coli vector or yeast vector.

The RuBGX1 recombinant vectors can be coli expression carriers such as pET21a, pET28a among the present invention.Accordingly, the recombinant bacterial strain that can be used for expressing RuBGX1 is a kind of among Escherichia coli BL21, E.coliJM109, E.coli DH5 α or the E.coli top10 etc.

Among the present invention the RuB6X1 recombinant vectors also can be Yeast expression carrier pPIC9, pPIC9K, pKLAC1, pYES wherein any one.Accordingly, the recombinant bacterial strain that can be used for expressing RuBGX1 is a kind of among Saccharomyces cerevisiae, Pichia pastoris, the Kluyveromyces lactis.

Above-mentioned recombinant vectors and engineering strain can adopt conventional technique means in this area and working method preparation.

The present invention also provides the method for a kind of RuBGX1 of preparation, may further comprise the steps successively:

(1) collects Chinese yak rumen microorganism thalline, extracting DNA;

(2) screening has the positive colony of activity of beta-glucosidase;

(3) primer amplified obtains the nucleotide coding sequence of RuBGX1;

(4) recombinant expressed RuBGX1.

Steps such as above-mentioned separated and collected thalline, extracting DNA, screening and cloning, amplification gene, expressing protein can adopt this area conventional technique means and working method.

The present invention adopts grand genome-based technologies method clone to derive from the β-1 of yak rumen microorganism cellulose degrading enzyme, and the 4-glucuroide has higher β-1 simultaneously, the enzymic activity of 4-xylosidase.

Grand genome (Metagenome) (is also claimed microbial environment genomics MicrobialEnvironmental Genomics; Grand genomics, first genomics Metagenomics; Ecological genomics Ecogenomics) is the new term that proposed in 1998 by Handelsman etc.; It is defined as " the genomes of the total microbiota found in nature ", i.e. the summation of whole tiny organism genetic material in the habitat.It has comprised gene educable and cannot not cultured microorganism, bacterium in the sample of at present main finger ring border and the genome summation of fungi.And so-called grand genomics (metagenomics) be exactly a kind of be research object with the microbial population genome in the environmental sample; Is research means with functional gene screening with sequencing analysis, with microbial diversity, population structure, evolutionary relationship, functional gene, each other cooperation relation and and environment between relation serve as to study the new microbe research method of purpose.

RuBGX1 nucleotide coding sequence of the present invention also can obtain according to the RuBGX1 nucleotide coding sequence synthetic of prediction.

The present invention utilizes first genome-based technologies and molecular biology method, and other recipient bacteriums are arrived in the enzyme gene clone that the present invention relates to, or produces glucuroide/xylosidase dual functions enzyme involved in the present invention by other bacterial strains or in other training methods.

The present invention also provides the application of RuBGX1 aspect preparation biofuel or fodder additives.

RuBGX1 of the present invention can be applicable to other commercial runs such as degraded and feed interpolation of Mierocrystalline cellulose, the semicellulose of lignocellulose.

The present invention also provides a kind of method that improves xylan reduction output, is about to RuBGX1 and zytase and unites use.

Experiment shows, in zytase, adds an amount of RuBGX1, under equal conditions can obviously increase the output of wood oligose.

The invention provides a kind of glucuroide/xylosidase dual functions cellulose degrading enzyme RuBGX1; This enzyme possesses the difunctional characteristic of glucuroide and xylosidase simultaneously; Can the hydrolyzation of glucose glycosides, galactoside, xyloside, cellobiose and trisaccharide etc., especially β type substrate.Add an amount of metals ion, for example Mg ²⁺, Co ²⁺Can improve the RuBGX1 activity.

Novel glucose glycosides enzyme provided by the invention/xylosidase dual functions albumen can be used for fields such as the biological conversion of Mierocrystalline cellulose, chemical industry, weaving, food, bioenergy, feed interpolation, the application of medicine industry aspect.

RuBGX1 of the present invention can be applicable to other commercial runs such as degraded and feed interpolation of Mierocrystalline cellulose, the semicellulose of lignocellulose.Utilize RuBGX1 degraded wood fibre of the present invention, mild condition, inhibition is few, and product purity is high, output is high, hydrolysis rate is fast.Be used for fodder additives, RuBGX1 of the present invention can improve animal to coarse-fibred utilization ratio, improves the monogastric animal digestive tract environment, activates stomach en-; Improve the digestible of nutrient, also increased the digestibility of non-starch polysaccharide and the utilization ratio of feedstuff raw material simultaneously; Cellulase can also be eliminated the ANFs in the wood fibre.Add cellulase and can reduce viscosity, improve digestive ferment and feed effect probability, promote the good digestion of feed.

Description of drawings

Fig. 1: the agarose gel electrophoresis figure of the mikrobe macro genome DNA that extracts in the yak rumen content, wherein, swimming lane M1::Lambda/Hind III Marker; Swimming lane 1: the rumen microorganism genomic dna of extraction.

Fig. 2: the BamH I atlas analysis in the grand genome cosmid of yak rumen microorganism library, wherein, swimming lane M1:Lambda/HindIII Marker; Swimming lane 11-20: rumen microorganism genome cosmid cloned plasmids DNA BamH I product; Swimming lane M2:1Kb DNA ladder Marker.

Fig. 3: the EcoR I of recombinant expression vector pET21a-RuBGX1 and Hind III restriction enzyme digestion and electrophoresis figure, wherein, swimming lane M2:1Kb DNA ladder Marker; Swimming lane 31,33: reorganization pET21a-RuBGX1 plasmid; Swimming lane 32,34: reorganization pET21a-RuBGX1 plasmid EcoR I and Hind III double digestion product; Swimming lane 35:pET21a plasmid.

Fig. 4: recombinase RuBGX1 expresses, purified proteins SDS-PAGE electrophorogram, and the sample that each swimming lane adds is respectively: swimming lane 41: with the Bacillus coli cells albumen of plasmid; Swimming lane 42-47: the imidazoles wash-out that is 300mmol, 150mmol, 100mmol, 80mmol, 60mmol, 40mmol is respectively collected liquid; Swimming lane 48: supernatant after the fragmentation of intestinal bacteria pET21a-RuBGX1 reorganization bacterium; Swimming lane 49: the broken postprecipitation of intestinal bacteria pET21a-RuBGX1 reorganization bacterium; Swimming lane 50: the intestinal bacteria of band pET21a empty plasmid.

Fig. 5: the optimal reaction pH value of recombinase RuBGX1.

Fig. 6: the optimal reactive temperature of recombinase RuBGX1.

Fig. 7: the TLC analysis of RuBGX1 degradation of fibers disaccharides and MU-cellobiose, wherein, Marker: glucose+cellobiose; Cellobiose+: cellobiose+RuBGX1: cellobiose-: do not add enzyme liquid in the cellobiose; The MU-cellobiose+: MU-cellobiose+RuBGX1:MU-cellobiose-: do not add RuBGX1 enzyme liquid in the MU-cellobiose.

The synergistic effect of Fig. 8: RuBGX1 and zytase degradation of xylan.

Fig. 9: zytase degradation of xylan 1h after product high performance anion exchange chromatography (HPAEC) analysis.

Figure 10: the common degradation of xylan 1h of RuBGX1 zytase after product HPAEC analysis.

Figure 11: zytase degradation of xylan 16h after product HPAEC analysis.

Figure 12: the common degradation of xylan 16h of RuBGX1 zytase after product HPAEC analysis.

Embodiment

The extraction of embodiment 1 Chinese yak rumen microorganism macro genome DNA

Gather Chinese Qinghai yak cud sample, adopt 3 layers of filtered through gauze, the centrifugal collection rumen microorganism thalline of filtrating is got 100-200ul thalline sample, and PBS washes 2～3 times, adds 650uL DNA extraction buffer (Tris-HCl, 100mM pH8.0; Na ₂EDTA 100mM pH8.0; Na ₃PO ₄Buffer 100mMpH8.0; NaCl 1.5M; CTAB 1%; PH8.0), behind the mixing, put in-80 ℃, be placed on then in 65 ℃ of water-baths and melt triplicate; Cooling back adding 3-4 μ L N,O-Diacetylmuramidase (100mg/L) level vibration in shaking table (37 ℃, 225rpm) about 30min; Add the about 30min of 2-3 μ L Proteinase K (20mg/mL) continued vibration; Add 50-70uL SDS (20%), behind the mixing, 65 ℃ of insulation 1-2h are whenever at a distance from 10～20min centrifuge tube mixing that turns upside down; 12, the centrifugal 10min of 000rpm room temperature collects supernatant, adds the phenol of 400-500ul: chloroform: twice of primary isoamyl alcohol (25: 24: 1) extracting; Chloroform: primary isoamyl alcohol (24: 1) extracting is the Virahol of 0.6 times of volume of back adding once, after room temperature is placed 15-20min, and the centrifugal 15min of 12000rpm; Deposition is with 70% ethanol rinsing, and dry back adds 1 μ LRNAase and removes RNA with 60-100 μ L TE (pH8.0) dissolving.0.8% agarose electrophoresis detected result is as shown in Figure 1, and the grand genome size of extractive cud meets the grand genome cosmid library requirement of structure fully about 30～40kb.

The structure in the grand genome cosmid of embodiment 2 yak rumen microorganisms library and the screening active ingredients of glucuroide

Cud not the construction process of the grand genomic library of culturing bacterium with reference to the pWEB::TNC Cosmid Cloning Kit of Epicentre company test kit product description.Extractive macro genome DNA is through End-Repair Enzyme Mix end-filling, is connected with dephosphorylation carrier pWEB::TNC in the test kit, connects product and uses MaxPlax ^TMLambda Packaging Extracts packs the back, infects host bacterium E.coli EP I100, infects product and is coated with flat board, cultivates 12～16h for 37 ℃ and grows bacterium colony, is the grand genomic library of these lot sample article.Transfer 10 cosmid clones at random and extract DNA, with BamH I restriction analysis (like Fig. 2), the result shows that the library quality of structure is better, the about 30～40kb of genomic fragment size of insertion.

The screening active ingredients of beta-glucosidase adopts fluorogenic substrate 4-methyl umbelliferone β-D-1, and the 4-glucoside is as substrate.With the 1mg/ml 4-methyl umbelliferone β-D-1 that adds 20ul after the 96 orifice plates trace cultured cells freeze thawing fragmentation, the substrate of 4-glucoside (50mmol citrate-phosphate pH4.8), 37 ℃ of reactions add 30ul 1M Na after 30 minutes ₂CO ₃, present fluorescence under the ultraviolet and be positive colony with activity of beta-glucosidase.Through screening, obtain 50 altogether and have β-D-1, the positive colony of 4-glucosidase activity 5000 cosmid library clones.

The clone and the analysis of the sequence of the RuBGX1 gene in embodiment 3 rumen microorganisms source

With the positive library clone of the cosmid that screens, carry out in pGEM11z that RuBGX1 gene, its promotor/adjusting that the specific oligonucleotide of subclone, functional screening and use measures the rumen microorganism source as the sequencing reaction primer are gone, the structure division of gene and the sequence of terminator.

For measuring nucleotide sequence; Isolated part Sau3A I enzyme section disconnected (2-3kb); Then it is cloned among the carrier pGEM11z; Utilize 4-methyl umbelliferone β-D-1, the 4-glucoside is the functional screening that substrate carries out the beta-glucosidase of Ya Wenku, and the positive inferior library clone of acquisition is measured nucleotide sequence through T7 sequencing primer and the SP6 sequencing primer of carrier pGEM11z.Operational analysis software DNAMAN carries out the coding region sequence analysis.In SEQ ID NO 1, provided the gene ORF sequence of measuring.

What obtain has a β-1; The unnamed gene of 4-glucosidase activity is RuBGX1; 854 amino-acid residues of RuBGX1 genes encoding are formed (SEQ ID NO.2), and have 82, the deduction molecular weight of 002Da; Front end from the N terminal amino acid sequence is the hydrophobicity signal peptide sequence of one 17 amino acid long; The 73-292 amino acids is the catalysis territory (glycosylhydrolase 3domain) of glycosyl hydrolase family 3, glycosyl hydrolase family 3C end domain (glycosylhydrolase 3C domain) during 358-616, through NCBI ( Http:// www.ncbi.nlm.nih.gov/) the blastx software analysis, the homology of RuBGX1 and Parabacteroides distasonis ATCC 8503 β-glucosidase is the highest, its aminoacid sequence similarity 58%.Analytical results shows that the RuBGX1 of acquisition is a novel cellulose degrading enzyme gene.

Embodiment 4RuBGX1 gene recombinant expressed in intestinal bacteria

According to RuBGX1 gene order and coli expression carrier pET-21a sequence, synthetic one group of Oligonucleolide primers of design: forward primer RuBGX1F:TCC GAATTC

(SEQ ID NO 3) and reverse primer RuBGX1R:CGC AAGCTT

(SEQ ID NO 4).The restricted interior following marking of enzyme recognition sequence in the sequence, gene order represent that with italic the cloned genes sequence removes except 17 signal peptide amino acid of N end.Pcr amplification RuBGX1 gene product after agarose electrophoresis reclaims with EcoR I and Hind III after enzyme is cut respectively; Be connected with the carrier pET-21a (+) (available from Novagen company) that Hind III enzyme is cut with EcoR I; Connect product transformed into escherichia coli E.coliTop10, transformant utilizes primer RuBGX1F and RuBGX1R to carry out bacterium colony PCR screening and checking.Extract the plasmid of transformant; With EcoR I and Hind III restriction analysis (result such as Fig. 3), select to have 2.3kb and insert segmental recombinant vectors (called after pET21a-RuBGX1) transformed into escherichia coli expression strain E.coli BL21 (DE3) (available from Novagen company) and carry out recombinant expressed.

The technique means of above-mentioned preparation recombinant vectors also is applicable to preparation pET28a-RuBGX1, and the preparation thinking of said gene engineering strain is applicable to that also preparation contains Escherichia coliBL21 (DE3), E.coli JM109, E.coli DH5 α or the Bacillussubtilis engineering strain of pET28a-RuBGX1 or pET21a-RuBGX1.

The specificity analysis of embodiment 5RuBGX1

The substrate specificity type analysis utilizes following substrate: p-nitrophenyl-β-glucopyranside (p-nitrophenyl-β-glucopyranoside); P-nitrophenyl-α-glueopyranside (p-nitrophenyl-α-glucopyranoside), p-nitrophenyl-β-galactoside (p-nitrophenyl-beta galactose glycosides); P-nitrophenyl-α-galactoside (p-nitrophenyl-alpha galactosides); P-nitrophenyl-β-D-xylopyranoside (p-nitrophenyl-beta-D-xyloside), sucrose are analyzed the hydrolysis ability of RuBGX1 to these substrates.The result shows; RuBGX1 does not have activity to the glucosides substrate of α-type configuration; But RuBGX1 has certain broad spectrum to the substrate of beta comfiguration; Wherein p-nitrophenyl-β-glucopyranside and p-nitrophenyl-β-D-xylopy-ranoside's is active higher, is respectively 100% and 29.5%, and the relative reactivity less than 8.3% of the substrate p-nitrophenyl-β-galactoside of beta comfiguration of the same type.

Use spectrophotometer and detect the activity that product is measured enzyme at 410nm, generating the needed enzyme amount of 1umol p-NP (p-nitrophenol) with PM catalysis pNPG or pNPX is enzyme unit (IU) alive.The result shows that reorganization RuBGX1 is respectively 37.6IU/mg albumen, 11.1IU/mg albumen to the ratio work of pNPG and pNPX.The mensuration of the most suitable reaction conditions of enzyme is substrate with pNPG, measures the used damping fluid scope of ph optimum to be: pH 3.0-8.0,50mmol Hydrocerol A-Sodium phosphate, dibasic damping fluid (citrate-phosphate buffer).The result shows that (like Fig. 5) is the highest in the activity of pH5.8 enzyme.Under the righttest pH, measure the catalytic efficiency (of differing temps (33 ℃-62 ℃) enzyme, the result finds the activity of RuBGX1 under 50 ℃ the highest (like Fig. 6).

Embodiment 6 metals ions and monose are to the active influence of RuBGX1

(pH5.8,50 ℃) mensuration metals ion and monose are to the influence of RuBGX1 hydrolysis pNPG and pNPX under the most suitable condition of enzyme.Reaction system is: 50uL 1.25mmol pNPG/pNPX, and the metals ion of 5mmol or monose, the enzyme liquid of 5uL, 50 ℃ of reactions add the 1MNa of 2 times of volumes after 10 minutes ₂CO ₃Stopped reaction, 410nm spectrophotometry product pNP growing amount, result such as following table show proper metal ion such as Mg ²⁺, Co ²⁺It is active to improve 20% RuBGX1, and glucose has than the obvious suppression effect the glucosidase activity of RuBGX1, to the activity influence of RuBGX1 xylosidase and not obvious.

The β of embodiment 7 RuBGX1-1-4-glucuroide property analysis

The property analysis of the β of RuBGX1-1-4-glucuroide utilizes cellobiose and 4-methyl umbelliferone β-D-1, and the 4-cellobiose is a substrate, analyzes the product of generation with thin-layer chromatography (TLC).Thin-layer chromatography condition: developping agent: propyl carbinol: ethanol: water (5: 3: 2); Developing time 1 hour, developer: H ₂SO ₄: methanol (3: 7); Coloration method: 80-90 ℃ of baking 15-20min.The result is as shown in Figure 7, and can degrade disaccharides substrate cellobiose and trisaccharide substrate MU-cellobiose of RuBGX1 produces glucose.

The β of embodiment 8RuBGX1-1-4-xylosidase property analysis

(pH5.8 in 2% the xylan; The preparation of 50mmol citrate/phosphate damping fluid) in; Add the zytase (xylanase) of 0.2V and the recombinant expressed RuBGX1 of intestinal bacteria of 0.3V respectively, be warming up to 45 ℃ of hydrolysis, divide in 20min, 40min, 60min sampling; With 3,5-dinitrosalicylic acid (DNS) method is measured the reducing sugar product of generation to analyze zytase and RuBGX1 Synergistic degradation xylan.Result such as Fig. 8; Add an amount of RuBGX1 and can improve the output of reducing sugar significantly; Reaction solution continues 45 ℃ of effects 16 hours; Utilize high-effect ionic exchange chromatography-pulse Amperometric Detection Coupled method (HPAEC-PAD) to analyze single enzymolysis and double enzymolysis product, ion-exchange chromatogram analysis condition: model: DX500 (U.S. Dionex company); Analytical column: CarboPac PA-1 (250mm * 4.6mm) (U.S. Dionex company); Elution requirement 0-100mM sodium-acetate/0.1M NaOH gradient elution 5min is then with 100-400mM sodium-acetate/0.1M NaOH gradient elution 35min.The qualitative employing standard addition method of wood sugar.The ion-exchange chromatogram analysis result shows that the single enzyme effect of xylanase 1 hour (like Fig. 9) or 16 hours (like Figure 11) does not all have the generation of wood sugar; And RuBGX1 and xylanase act on the accumulation (like Figure 10) that a spot of wood sugar was arranged in the product in 1 hour at 45 ℃; And under equal conditions; Two enzyme effects are higher than the amount of the wood oligose that single xylanase hydrolysis produces, the accumulation (like Figure 12) of a large amount of wood sugars after the reaction times extends to 16 hours.Have collaborative promotion degradation effect when presentation of results RuBGX1 and xylanase degradation of xylan, and can xylan degrading be become the fermentable D-wood sugar of mikrobe.

Glucuroide xylosidase dual functions cellulose degrading enzyme

SEQUENCE LISTING

< 110>Fudan University

< 120>glucuroide/xylosidase dual functions cellulose degrading enzyme

<210>1

<211>2298

<212>DNA

< 213>Chinese yak rumen microorganism

<220>

<221>CDS

<222>(1)..(2298)

<223>

<400>1

atg aag aaa atc att ctc ctc tcc gcc gct gcc ctg gcc atc gcc ggg 48

Met Lys Lys Ile Ile Leu Leu Ser Ala Ala Ala Leu Ala Ile Ala Gly

1 5 10 15

tgc aag ccg ccc cag ctc gga aaa gca tcc atc gac aag gtc ctc aag 96

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

20 25 30

gcc atg acc ctc gag gaa aag gtc cat ttc gtc atc ggc acc ggc atg 144

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

35 40 45

gcc ggc atg gat gac ggc gct tcc gcc acc gtg ggc gcc acg cag aag 192

Ala Gly Met Asp Asp Gly Ala Ser Ala Thr Val Gly Ala Thr Gln Lys

50 55 60

atc gtc ccg ggc gcc gcg ggc acc acg tat ccc atc gag cgg ctg ggc 240

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

65 70 75 80

att ccg tcc atc gtc ctc gcc gac ggc ccc gcc ggc ctc cgc atc gac 288

Ile Pro Ser Ile Val Leu Ala Asp Gly Pro Ala Gly Leu Arg Ile Asp

85 90 95

ccc atc cgc gaa ggc gac gag aac act tac tac tgc acg cac ttc ccc 336

Pro Ile Arg Glu Gly Asp Glu Asn Thr Tyr Tyr Cys Thr His Phe Pro

100 105 110

atc ggc acc ctg ctg gca tcg acc tgg aac cag gag ctg gtg gaa agc 384

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

115 120 125

Glucuroide xylosidase dual functions cellulose degrading enzyme

gtg ggc aag gcc atg ggc gaa gag gtc cat gag tac ggc gct gac gtc 432

Val Gly Lys Ala Met Gly Glu Glu Val His Glu Tyr Gly Ala Asp Val

130 135 140

tat ctc gct ccg gcc ctg aac atc cat cgc aac ccg ctg aac ggc cgt 480

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

145 150 155 160

aac ttc gag tat tat tcc gag gat ccg gtg gtc gcc ggc aag acc gcg 528

Asn Phe Glu Tyr Tyr Ser Glu Asp Pro Val Val Ala Gly Lys Th Ala

165 170 175

gcg gcc tat gtc cgc ggc gtc cag agc aac gac gtg ggc acc tcc atc 576

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

180 185 190

aag cat ttc gcc tac aac aac cag gag acg aac cgc acc ggc aac aac 624

Lys His Phe Ala Tyr Asn Asn Gln Glu Thr Asn Arg Thr Gly Asn Asn

195 200 205

gcc atc atc tcc ccg cgc gca cag cgc gaa atc tat ctg aag ggc ttc 672

Ala Ile Ile Ser Pro Arg Ala Gln Arg Glu Ile Tyr Leu Lys Gly Phe

210 215 220

gaa atc acc gtc aag gaa tcc gct ccc tgg acc gtg atg agc tcc tat 720

Glu Ile Thr Val Lys Glu Ser Ala Pro Trp Thr Val Met Ser Ser Tyr

225 230 235 240

aac aag atc aac ggc acc tat act tcc cag agc cgc gac ctc atc acc 768

Asn Lys Ile Asn Gly Thr Tyr Thr Ser Gln Ser Arg Asp Leu Ile Thr

245 250 255

acc gtc ctc cgc gac gag tgg ggc ttc aag ggc ctc gtg atg acc gac 816

Thr Val Leu Arg Asp Glu Trp Gly Phe Lys Gly Leu Val Met Thr Asp

260 265 270

tgg tac ggc ggt gac gac ggt gcc gcg cag atg gcc gcc ggc aac gac 864

Trp Tyr Gly Gly Asp Asp Gly Ala Ala Gln Met Ala Ala Gly Asn Asp

275 280 285

atg ctc cag ccg ggc acc caa ctg cag tac gac cag atc atg gcc gcc 912

Met Leu Gln Pro Gly Thr Gln Leu Gln Tyr Asp Gln Ile Met Ala Ala

290 295 300

ctg aac gcc gga acc ctc agc gaa gag gag ctg gac gtc tgc gtc cgc 960

Leu Asn Ala Gly Thr Leu Ser Glu Glu Glu Leu Asp Val Cys Val Arg

305 310 315 320

cgc tgc ctg gaa ctg gtg gcc cgc agc ccc aag gca aag aaa tat gcc 1008

Arg Cys Leu Glu Leu Val Ala Arg Ser Pro Lys Ala Lys Lys Tyr Ala

325 330 335

tat tcc aac aag cct gac ctg acg gcc cat gcc gcc gtc acc cgt cag 1056

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

340 345 350

Glucuroide xylosidase dual functions cellulose degrading enzyme

agc gcc ctg gaa ggc atg gtg ctc ctg gag aac aag ggt gtc ctg ccg 1104

Ser Ala Leu Glu Gly Met Val Leu Leu Glu Asn Lys Gly Val Leu Pro

355 360 365

ctc aag gac gtg aag aac gtg gcc gtg ttc ggc tgc acc tcc ttc gac 1152

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

370 375 380

ttc atc gcg ggc ggc acc ggc tcc ggc aac gtg aac cgc gcc tac acc 1200

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

385 390 395 400

gtg tcc ctg ctg gac ggc ctc aag aac gcc gga ttc aat gtg gac gag 1248

Val Ser Leu Leu Asp Gly Leu Lys Asn Ala Gly Phe Asn Val Asp Glu

405 410 415

cgc atg aaa gat gcg tgc ctg cag cac atc gcc gac gag gcg gcc gcc

1296

Arg Met Lys Asp Ala Cys Leu Gln His Ile Ala Asp Glu Ala Ala Ala

420 425 430

ttc aag gcc ggc ctg ccc gac gac ggt ctt tcc gcc ttc tat ccc gtg 1344

Phe Lys Ala Gly Leu Pro Asp Asp Gly Leu Ser Ala Phe Tyr Pro Val

435 440 445

ccg cgc ccg acc gag ctc atc ccg gcc ccc aag gac ctg gct gcc atg 1392

Pro Arg Pro Thr Glu Leu Ile Pro Ala Pro Lys Asp Leu Ala Ala Met

450 455 460

gcc cat gcc aat gac gtc gcc atc atc aca ttc ggc cgc aac tcc ggc 1440

Ala His Ala Asn Asp Val Ala Ile Ile Thr Phe Gly Arg Asn Ser Gly

465 470 475 480

gag ttc ttc gac cgc acc agc gcc gac ttc gcc ctc tcc gcc cag gag 1488

Glu Phe Phe Asp Arg Thr Ser Ala Asp Phe Ala Leu Ser Ala Gln Glu

485 490 495

cgc aag ctc ctt gtc aac gtc gcc aac gcc ttc cac gcc gtc cga aag 1536

Arg Lys Leu Leu Val Asn Val Ala Asn Ala Phe His Ala Val Arg Lys

500 505 510

aag gtc gtg gtc gtg ctg aac atc ggc ggc gtg atc gag acc gct tct 1584

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

515 520 525

tgg aag aac att ccg gac gcc atc ctc ctg gcc tgg cag gcc ggt cag 1632

Trp Lys Asn Ile Pro Asp Ala Ile Leu Leu Ala Trp Gln Ala Gly Gln

530 535 540

gaa ggc ggc aac tcc gtc acc gac atc cta acg ggc gcc aag agc ccg

1680

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

545 550 555 560

tcc ggc aag ctg ccg atg acc ttc ccg gtg aac ctg atg gac gcc ggc 1728

Glucuroide xylosidase dual functions cellulose degrading enzyme

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

565 570 575

tcc tcc gcg aac ttc ccg atc gac gcg acc aac gag gtg tac ttc ctg 1776

Ser Ser Ala Asn Phe Pro Ile Asp Ala Thr Asn Glu Val Tyr Phe Leu

580 585 590

aac aag cgt gag gac gtg ggc cag aag gat gtc gat gtc acc aag tac 1824

Asn Lys Arg Glu Asp Val Gly Gln Lys Asp Val Asp Val Thr Lys Tyr

595 600 605

gag gaa ggc atc tat gtg ggc tac cgc tgg ttc gac aag cag aac ctc 1872

Glu Glu Gly Ile Tyr Val Gly Tyr Arg Trp Phe Asp Lys Gln Asn Leu

610 615 620

aag gtc tcc tac ccg ttc ggc tac ggt ctc agc tac acc acc ttc gag 1920

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

625 630 635 640

tac agc gcg ccc gcc gtc gcc aat gac ggc acg acc gtc acg gcc aag

1968

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

645 650 655

gtt acc gtg aag aac acc ggt tcc gtg gcc ggc aag gaa gcc gtc caa 2016

Val Thr Val Lys Asn Thr Gly Ser Val Ala Gly Lys Glu Ala Val Gln

660 665 670

ctg tac gtg agc gcc ccc gcc ggc acc ctc gac aag ccc gtg aag gag

2064

Leu Tyr Val Ser Ala Pro Ala Gly Thr Leu Asp Lys Pro Val Lys Glu

675 680 685

ctg aag gcc tat gcc aag acg aag gaa ctg gcc cct ggc gag tcc cag 2112

Leu Lys Ala Tyr Ala Lys Thr Lys Glu Leu Ala Pro Gly Glu Ser Gln

690 695 700

gaa ctc acc ctc acc ttc ccg acc tca gag ctc gcc tcc ttc gac gag 2160

Glu Leu Thr Leu Thr Phe Pro Thr Ser Glu Leu Ala Ser Phe Asp Glu

705 710 715 720

gcc gcc tcg gcc tgg aag gtc gat gcc ggc acc tac acc ttc ctg ttc 2208

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

725 730 735

ggc gct tct tcc cgc gac atc cgc tgc acg gcc acg gcc gac gcg gac 2256

Gly Ala Ser Ser Arg Asp Ile Arg Cys Thr Ala Thr Ala Asp Ala Asp

740 745 750

gcc gcc gag acg ccc acc cac act gtc ctt ctg atg caa tag 2298

Ala Ala Glu Thr Pro Thr His Thr Val Leu Leu Met Gln

755 760 765

<210>2

Glucuroide xylosidase dual functions cellulose degrading enzyme

<211>765

<212>PRT

< 213>Chinese yak rumen microorganism

<400>2

Met Lys Lys Ile Ile Leu Leu Ser Ala Ala Ala Leu Ala Ile Ala Gly

1 5 10 15

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

20 25 30

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

35 40 45

Ala Gly Met Asp Asp Gly Ala Ser Ala Thr Val Gly Ala Thr Gln Lys

50 55 60

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

65 70 75 80

Ile Pro Ser Ile Val Leu Ala Asp Gly Pro Ala Gly Leu Arg Ile Asp

85 90 95

Pro Ile Arg Glu Gly Asp Glu Asn Thr Tyr Tyr Cys Thr His Phe Pro

100 105 110

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

115 120 125

Val Gly Lys Ala Met Gly Glu Glu Val His Glu Tyr Gly Ala Asp Val

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Glucuroide xylosidase dual functions cellulose degrading enzyme

Lys His Phe Ala Tyr Asn Asn Gln Glu Thr Asn Arg Thr Gly Asn Asn

195 200 205

Ala Ile Ile Ser Pro Arg Ala Gln Arg Glu Ile Tyr Leu Lys Gly Phe

210 215 220

Glu Ile Thr Val Lys Glu Ser Ala Pro Trp Thr Val Met Ser Ser Tyr

225 230 235 240

Asn Lys Ile Asn Gly Thr Tyr Thr Ser Gln Ser Arg Asp Leu Ile Thr

245 250 255

Thr Val Leu Arg Asp Glu Trp Gly Phe Lys Gly Leu Val Met Thr Asp

260 265 270

Trp Tyr Gly Gly Asp Asp Gly Ala Ala Gln Met Ala Ala Gly Asn Asp

275 280 285

Met Leu Gln Pro Gly Thr Gln Leu Gln Tyr Asp Gln Ile Met Ala Ala

290 295 300

Leu Asn Ala Gly Thr Leu Ser Glu Glu Glu Leu Asp Val Cys Val Arg

305 310 315 320

Arg Cys Leu Glu Leu Val Ala Arg Ser Pro Lys Ala Lys Lys Tyr Ala

325 330 335

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

340 345 350

Ser Ala Leu Glu Gly Met Val Leu Leu Glu Asn Lys Gly Val Leu Pro

355 360 365

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

370 375 380

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

385 390 395 400

Val Ser Leu Leu Asp Gly Leu Lys Asn Ala Gly Phe Asn Val Asp Glu

405 410 415

Glucuroide xylosidase dual functions cellulose degrading enzyme

Arg Met Lys Asp Ala Cys Leu Gln His Ile Ala Asp Glu Ala Ala Ala

420 425 430

Phe Lys Ala Gly Leu Pro Asp Asp Gly Leu Ser Ala Phe Tyr Pro Val

435 440 445

Pro Arg Pro Thr Glu Leu Ile Pro Ala Pro Lys Asp Leu Ala Ala Met

450 455 460

Ala His Ala Asn Asp Val Ala Ile Ile Thr Phe Gly Arg Asn Ser Gly

465 470 475 480

Glu Phe Phe Asp Arg Thr Ser Ala Asp Phe Ala Leu Ser Ala Gln Glu

485 490 495

Arg Lys Leu Leu Val Asn Val Ala Asn Ala Phe His Ala Val Arg Lys

500 505 510

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

515 520 525

Trp Lys Asn Ile Pro Asp Ala Ile Leu Leu Ala Trp Gln Ala Gly Gln

530 535 540

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

545 550 555 560

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

565 570 575

Ser Ser Ala Asn Phe Pro Ile Asp Ala Thr Asn Glu Val Tyr Phe Leu

580 585 590

Asn Lys Arg Glu Asp Val Gly Gln Lys Asp Val Asp Val Thr Lys Tyr

595 600 605

Glu Glu Gly Ile Tyr Val Gly Tyr Arg Trp Phe Asp Lys Gln Asn Leu

610 615 620

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

625 630 635 640

Glucuroide xylosidase dual functions cellulose degrading enzyme

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

645 650 655

Val Thr Val Lys Asn Thr Gly Ser Val Ala Gly Lys Glu Ala Val Gln

660 665 670

Leu Tyr Val Ser Ala Pro Ala Gly Thr Leu Asp Lys Pro Val Lys Glu

675 680 685

Leu Lys Ala Tyr Ala Lys Thr Lys Glu Leu Ala Pro Gly Glu Ser Gln

690 695 700

Glu Leu Thr Leu Thr Phe Pro Thr Ser Glu Leu Ala Ser Phe Asp Glu

705 710 715 720

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

725 730 735

Gly Ala Ser Ser Arg Asp Ile Arg Cys Thr Ala Thr Ala Asp Ala Asp

740 745 750

Ala Ala Glu Thr Pro Thr His Thr Val Leu Leu Met Gln

755 760 765

<210>3

<211>33

<212>DNA

< 213>synthetic

<400>3

tccgaattca agccgcccca gctcggaaaa gca 33

<210>4

<211>33

<212>DNA

< 213>synthetic

<400>4

Glucuroide xylosidase dual functions cellulose degrading enzyme

cgcaagcttt tgcatcagaa ggacagtgtg ggt 33

1

Claims

1. glucuroide/xylosidase dual functions cellulose degrading enzyme is characterized in that, it is the glycoside hydrolase of beta-glucosidase/xylosidase dual functions, and its aminoacid sequence is shown in SEQ ID NO.2.

2. glucuroide as claimed in claim 1/xylosidase dual functions cellulose degrading enzyme, the nucleotide coding sequence that it is characterized in that it is shown in SEQ ID NO.1.

3. recombinant vectors that contains the nucleotide coding sequence of the described glucuroide of claim 2/xylosidase dual functions cellulose degrading enzyme, this recombinant vectors is escherichia coli vector or yeast vector.

4. recombinant vectors as claimed in claim 3 is characterized in that this recombinant vectors is escherichia coli vector expression vector pET21a or pET28a.

5. recombinant bacterial strain that contains the described recombinant vectors of claim 4, this bacterial strain is Escherichia coliBL21, E.coli JM109, E.coli DH5 α.

6. recombinant vectors as claimed in claim 3, it is characterized in that this recombinant vectors be Yeast expression carrier pPIC9, pPIC9K, pKLAC1, pYES wherein any one.

7. recombinant bacterial strain that contains the described recombinant vectors of claim 6, this bacterial strain are a kind of among Saccharomyces cerevisiae, Pichia pastoris, the Kluyveromyces lactis.

8. the purposes of the glucuroide of claim 1/xylosidase dual functions cellulose degrading enzyme in preparation biofuel or fodder additives.

9. a method that improves wood oligose output is characterized in that the described glucuroide of claim 1/xylosidase dual functions cellulose degrading enzyme and zytase are united use.