CN102643842A - Heat-resisting xylanase gene derived from actinomycetes (streptomyces rameus L2001) and product xylanase (Xyn A) thereof - Google Patents

Abstract

The invention discloses a gene of xylanase (Xyn A) derived from actinomycetes (streptomyces rameus L2001), which comprises nucleic acid and protein/peptide sequence thereof. The optimum reaction pH value of the gene coding xylanase (Xyn A) is 5.3; the stability range of the pH value is 2.2-11.3 at 50 DEG C; and the stability range of the pH value is 4.3-6.7 at 70 DEG C. The optimum reaction temperature of the Xyn A is 70 DEG C; and even the Xyn A is processed for 30 minutes at 70 DEG C, more than 60 percent of enzyme activity can be kept. As the novel xylanase, the Xyn A and products thereof have application values in paper industry, biological energy source, food processing, feed industry, cosmetics, medicines, health care products, brewing, crop production and various bioreactors.

Description

A kind of heat resistant xylanase gene and product zytase Xyn A thereof that derives from actinomycetes (Streptomyces rameus L2001)

Technical field

The present invention relates to a kind of gene of the heat resistant xylanase that derives from actinomycetes (Streptomyces rameus L2001) and the application of product zytase XynA thereof.

Technical background

Xylan (xylan) mainly is through β-1 by the D-pyranose form wood sugar (xylopryanose) of five carbon; The heterogeneity polysaccharide that the 4-glycosidic link forms with different polymerization degree; Being the plant hemicellulose staple, is the important composition composition that constitutes plant cell wall, accounts for the 15%-30% of angiosperm cell walls dry weight; Account for the 7%-12% of gymnosperm cell walls dry weight; Xylan extensively is present in roots of plants, stem, leaf, flower and the fruit, accounts for the 20%-40% of the total living weight of plant, is that occurring in nature is only second to the abundantest cellulosic renewable organic resource.

Xylan except containing the different wood sugar of the polymerization degree, also with other polysaccharide with multiple glycosidic link covalent cross-linking, also have a large amount of substitution in side chain bases simultaneously; Degraded needs a series of enzyme synergy fully, and that wherein play a major role is β-1, the 4-endo-xylanase; Destroy the glycosidic link of xylan backbone with the mode of inscribe, discharge oligomeric xylose and a spot of wood sugar monomer, next needs the β xylosidase; The reducing end of effect oligomeric xylose; Discharge the wood sugar monomer, the α-L-furans arabinofuranosidase/xylosidase (α-L-arabinofuranoside, EC 3.2.1.55) of the side chain of also need degrading in addition; Alpha-D-glucose aldehydic acid enzyme (α-D-glucuronidase; EC 3.2.1.139), the participation of acetyl xylan esterase (acetyl xylan esterase, EC 3.1.1.72) and phenolic acid esterase (phenolic acid esterase) zytases such as (comprising feruloyl esterase and p-PHCA esterase).

Zytase is in the news at present and extensively is present in the animals and plants, comprises fungi, bacterium; Algae, crustacean, snail; The sharp terrestrial plant of algae, filamentous fungus wherein, bacterium and actinomycetes are main producers; Comprise: the Trichoderma of filamentous fungus (Trichoderma sp.), the detritus enzyme belongs to (Humicola sp.), neurospora (Neurospora sp.); Aspergillus (Aspergillus sp.), fusarium (Fusarium sp.), penicillium (Penicillium sp.) and the mould genus of glue (Gliocladium sp.).The actinomycetes of bacterium comprise S.actuosus, S.cyaneus, S.halstedii, S.matensis, S.olivaceoviridis, S.roseiscleroticus, S.thermoviolaceus, S.viridosporus.

Zytase is with a wide range of applications and prospect, and it is mainly in papermaking, and feed and foodstuffs industry are used widely, and in fields such as weaving, bioenergy, makeup, Medicines and Health Products good prospects for application are arranged also.

In paper industry; Traditional chemical bleaching method adopts muriate under alkaline condition, to remove xylogen, produces a large amount of strong carcinogenic compounds of poisonous profit on the one hand, and the 2nd, in last handling process, consume a large amount of energy again; The application of zytase can strengthen bleaching effect and reduce follow-up muriatic consumption; The actual use not only greatly reduces environmental pollution, also improved the performance of paper, improved the quality of paper.On the working foundation of the zytase that separates nature, the researchist is also transforming zytase through biotechnology and engineered method, attempts further to obtain to be more suitable for paper industry and produces used zytase.

In fodder industry, owing to lack digestion such as zytase hemicellulase, can't utilize in the non-ruminant animal digestive tube semicellulose material abundant in the feed; Cause food utilization efficiency low, animal alimentary canal diseases increases and adverse consequences such as excrement and urine polluting environment, in feed, adds the enzyme that zytase etc. decomposes semicellulose; Can improve the utilising efficiency of animal to feed; Reduce the intestinal tract disease of animal, improve surviving rate, reduce fecal pollution.

In foodstuffs industry, the sugariness of wood sugar is 40% of a sucrose, in human body, can not cause the level of glucose to raise, and therefore can be used as diabetic subject and obese patient's sweeting agent; In addition, as difficult fermented type sugar, being not easy to cause carious tooth, also is the sweeting agent of infant foods therefore; On bread is produced, add the xylan syrup, can improve the bread color and luster; Tart up, keep effects such as bread moisture, these all are that zytase is in the Application in Food Industry direction.

In textile industry, zytase plays an important role in China grass degumming, and as good textile raw material, ramie has certain status in China's weaving industry; In process of production, an important step is to come unstuck, and traditional method adopts high temperature strong acid and strong base method, contaminate environment; And destroy fiber and reduce quality, China scientific research personnel is used zytase and polygalacturonase, creates the enzymatic degumming method; Improve work situation, improved product quality, and alleviated environmental pollution greatly.

In energy industry,, in the production of biomass energy, possessed potential application foreground because xylan is to be only second to cellulosic second largest renewable organism on the earth.

At makeup, zytase effect xylan, the few polymeric xylan of generation has good water conservation effect, can be used for makeup and keeps moisture of skin.

In preparation medicine and health care kind industry; Zytase effect xylan product separately or with other oligosaccharide (like Polylevulosan) mixing take; The few polymeric xylan that produces can improve the colony balance of human intestinal, helps the breeding of optimum bacterium, improves people's digestive function.

Summary of the invention

The present invention obtains xylanase gene multinuclear glycosides sequence shown in SEQIDNO1 from the heat resistant xylanase that derives from actinomycetes (Streptomyces rameusL2001):

CCATAGGACGCGCTCAGCGTCTCGGCGATCTCCTCGTCGGCCTGCGCCACGGTGCGGCGGATCGTACGGTGCCGAGCAAGGGCCTGTCGGCGGGGTCGCTCTCGAAGGGGCAGCGCCGGACATGGCCGGGCCCGTAGGCCTCGAGTCCGGCCGTCGCCACGCGGCGCAGCTCCTGTGTCGTCGAAACATTCGAAACCCAGTGTGGGGACTTCGGCGTCTCCATGGAGTGTTCCGGGATGTTCCTAGAGGGATCTCCTCACTCATGCCGATTCATCAGCGGACACGTAGCGACGGTCGCGCTAGGACGCAGACGAATGTTTCGAAATATCGACCGAAAGTGTTGACAGATGACATGGCCAGGCCAACACTCCCCATCACGTCACACCCCACCCGTCGCACAAGGAGGAAGTACGACATGAATCCGCTCGACCATGCGACGAGCCGCAGGGCCGCCTGCGCGCTGCTGCTCGGCACCGCGGCCGGACTGGCCCTGCCCGGCACCGCCCGTGCCGCCACGGTCGTCACCACGAACCAGACCGGCACCGACAACGGCTTCTACTACTCGTTCTGGACCGACGCGCAGGGCACGGTCTCGATGACCCTGGGCTCCGGTGGCAACTACAGCACCAGCTGGCGCAACACCGGCAACTTCGTGGCCGGCAAGGGCTGGAGCACCGGCGCCCGCAGGAACGTGACCTACTCCGGCAGCTTCAACCCGTCCGGCAACGGCTACCTGTCGCTCTACGGCTGGACGTCGAACCCGCTCGTGGAGTACTACATCGTCGACAACTGGGGCACCTACCGGCCCACGGGGACGTACAAGGGCAGTGTCACCAGTGACGGCGGAACGTACGACATCTACCAGACGACGCGGTACAACGCCCCGTCCGTCGAGGGCACCAGGACCTTCAACCAGTACTGGAGCGTGCGGCAGTCGAAGCGCACCGGCGGCACCATCACCACCGGCAACCACTTCGACGCCTGGGCCCGCGCCGGGATGCCCCTCGGCAGCTTCGCGTACTACATGATCCTCGCGACCGAGGGGTACCAGAGCAGCGGCAACTCCAATATCACGGTGTCGTCGTGAGGACCGCACGACAGCTCGCGTTACGTCCGGCGGTGACCGCCGCCGTCGCCGCACCGGCCCTCGCCGCTACGCTCATCGTCGGAGCCGCCCCTCGCACGCGCACCCTTCCC

The present invention obtains the xylanase gene peptide sequence shown in SEQ IDNO2 from the heat resistant xylanase that derives from actinomycetes (Streptomyces rameus L2001):

MAGPVGLESGRRHAAQLLCRRNIRNPVWGLRRLHGVFRDVPRGISSLMPIHQRTRSDGRARTQTNVSKYRPKVLTDDMARPTLPITSHPTRRTRRKYDMNPLDHATSRRAACALLLGTAAGLALPGTARA ATVVTTNQTGTDNGFYYSFWTDAQGTVSMTLGSGGNYSTSWRNTGNFVAGKGWSTGARRNVTYSGSFNPSGNGYLSLYGWTSNPLVEYYIVDNWGTYRPTGTYKGSVTSDGGTYDIYQTTRYNAPSVEGTRTFNQYWSVRQSKRTGGTITTGNHFDAWARAGMPLGSFAYYMILATEGYQSSGNSNITVSS

The present invention obtains the xylanase gene peptide sequence shown in SEQ IDNO3 from the heat resistant xylanase that derives from actinomycetes (Streptomyces rameusL2001):

MNPLDHATSRRAACALLLGTAAGLALPGTARA ATVVTTNQTGTDNGFYYS?FWTDAQGTVSMTLGSGGNYSTSWRNTGNFVAGKGWSTGARRNVTYSGSFNPSGNGYLSLYGWTSNPLVEYYIVDNWGTYRPTGTYKGSVTSDGGTYDIYQTTRYNAPSVEGTRTFNQYWSVRQSKRTGGTITTGNHFDAWARAGMPLGSFAYYMILATEGYQSSGNSNITVSS

The present invention obtains xylanase gene signal peptide sequence shown in SEQ ID NO4 from the heat resistant xylanase that derives from actinomycetes (Streptomyces rameus L2001):

MNPLDHATSRRAACALLLGTAAGLALPGTARA

Description of drawings

Fig. 1 is a branch streptomycete L2001 genome dna electrophoresis photo.

Fig. 2 is the encode conserved sequence of zytase of Fig. 2.

Fig. 3 Tail-PCR process schematic drawing.Sp1 wherein, Sp2 is a nested primers, according to the core sequence design, LAD is a degenerate primer.

Fig. 4-1 is 5 ' distolateral wing sequence amplification electrophoresis photo.

The third round Tail-PCR amplification that swimming lane 1,2,3 carries out for the combination between Sp5 ' and the Different L AD primer.

Fig. 4-2 is 3 ' distolateral wing sequence amplification electrophoresis photo.

The third round Tail-PCR amplification that swimming lane 1,2,3 carries out for the combination between Sp3 ' and the Different L AD primer.

Fig. 5 is xylanase activity analysis in the transgenic zytase A yeast.

Fig. 6 is the SDS-PAGE protein electrophoresis figure of escherichia coli expression zytase A.

1:Marker, 2-3: contain empty carrier escherichia coli expression albumen 4-7: the escherichia coli expression albumen that contains the XylA expression vector.

Fig. 7 is the SDS-PAGE protein electrophoresis figure than red yeast expression zytase A.1:Marker, 2-5: positive pichia spp pPIC9K-XylA expressing protein.

Embodiment

The present invention is described further below in conjunction with specific examples, but is not restriction the present invention.

Bacterial strain: bacillus coli DH 5 alpha and Transtetta (DE3) competent cell is available from full formula King Company; Pichi strain GS115 is available from invitrogen company.

Carrier: T carrier pEASY-blunt simple and pEASY-simple T1 are available from full formula King Company; PET-28a (+) and pET-32a are available from Merk company, and yeast expression vector is available from pPIC9K Invitrogen company.

Biochemical reagents: rTaq, ExTaq, EcoRI, EcoRV, AvrII, XhoI and T4ligase etc. are available from Takara company, and KDO-plus is available from Toyobo company.N,O-Diacetylmuramidase is available from Amresco company.Plasmid extracts test kit in a small amount, and amount is extracted test kit in the plasmid, and glue reclaims test kit available from Omiga company.Bacterial genomes DNA extraction test kit is available from sky root biochemical technology ltd.

General chemistry reagent: chloroform, methyl alcohol, ethanol, Virahol, sodium-chlor; Hydrochloric acid, Glacial acetic acid min. 99.5 etc. are available from Beijing chemical reagent factory, beta-mercaptoethanol, Tris, SDS; Mountain plough alcohol, glycerine, DMSO, kantlex, acillin; Xylene Brilliant Cyanine G, ammonium persulphate, acrylic amide, bisacrylamides etc. are available from Amresco company.Birch xylan is available from Sigma company.The saturated phenol of Tris-, agar powder is available from ancient cooking vessel state biotech firm.Agarose is available from Spain Biowest company.Tryptones and yeast extract are available from Oxford company.Congo red, imidazoles-Potassium Hydrogen Phthalate is available from rare your the reagent chemical plant of Tianjin Wonder.

Primer:

The used degenerate primer of amplification zytase core sequence:

Forward primer Xyn-p1 sequence: 5 '-STBSTACTCSTTCTGGACSGA-3 '

Reverse primer Xyn-p2 sequence: 5 '-CCASGCGTCGAAGTGRTT-3 '

(annotate: R=A/G, S=C/G, W=A/T, B=C/G/T, N=A/C/G/T).

The T carrier is used sequencing primer:

The M13+ sequence: 5 '-GTTTTCCCAGTCACGAC-3 '

The M13-sequence: 5 '-CAGGAAACAGCTATGAC-3 '

The full-length gene amplimer:

xynA-p1：5′-CCATAGGACGCGCTCAG-3′

xynA-p2：5′-GGGAAGGGTG?CGCGTG-3′

The Tail-PCR the primer:

Upper reaches amplimer: sp5-1:5 '-CGATTCATCAGCGGACACGTAGC-3 '

sp5-2：5′-GATGTTCCTAGAGGGATCTCCTCACTCAT-3′

sp5-3：5′-GTCGAAACATTCGAAACCCAGTGTGG-3′

Downstream amplimer: sp3-1:5 '-CCACACTGGGTTTCGAATGTTTCGAC-3 '

sp3-2：5′-CATGAGTGAGGAGATCCCTCTAGGAACAT-3′

sp3-3：5′-GCTACGTGTCCGCTGATGAA?TCG-3′

The coli expression carrier primer:

Forward long segment primer xynA-eL1:5 '-CAGAATTCATGAATCCGCTCGACCATG-3 '

Forward short segments primer xynA-eS1:5 '-CA GAATTCACGGTCGTCACCACGAA-3 '

Reverse universal primer xynA-eD:5 '-TA CTCGAGTCACGACGACACCGTGA-3 '

The yeast expression vector primer:

Forward long segment primer xynA-yL1:5 '-CAGAATTCATGAATCCGCTCGACCATG-3 '

Forward short segments primer xynA-yS1:5 '-CAGAATTCACGGTCGTCACCACGAA-3 '

Reverse universal primer xynA-yD:5 '-TACCTAGGTCACGACGACACCGTGA-3 '

The extraction of genomic dna:

The actinomycetes genome DNA is extracted and is used bacterial genomes DNA extraction test kit (day root biochemical technology ltd); Method is slightly done improvement; Thalline after the cracking fully; Adopt the saturated phenol of isopyknic Tris-/chloroform extracting 2 times, isopyknic chloroform extracting is once leant on again, can obtain pure and actinomycetes genome DNA (Fig. 1) that be not easy to degrade.

The design of degenerate primer:

Pure enzyme with the zytase after deriving from actinomycetes Streptomyces rameus fermented liquid and passing through purifying; Carrying out the N-terminal protein sequence measures; This enzyme N end 15 aminoacid sequence ATVVTTNQTGTDNGF (Ala-Thr-Val-Val-Thr-Thr-Asn-Gln-Thr-Gly-Thr-Asp-Asn-Gly-Phe) have been recorded; Use these 15 aminoacid sequences that the albumen database of NCBI is carried out the BLAST retrieval, obtain the protein sequence of 16 high zytases of similarity, these protein sequences are carried out the ALIGMENT comparison through software DNAman; Analysis obtains the conserved sequence district (Fig. 2) of such zytase, design degenerate primer Xyn-p1 and Xyn-p2.

Pcr amplification:

Genomic dna to extract is a template, uses the high-fidelity amplification enzyme KOD-plus of Toyobo company, and the pcr amplification condition is: 94 ℃ of sex change 3 minutes; 94 ℃ of sex change 30 seconds, 50 ℃ of annealing 30 seconds, 68 ℃ were extended totally 10 circulations 1 minute; 94 ℃ of sex change 30 seconds, 54 ℃ of annealing 30 seconds, 68 ℃ were extended totally 20 circulations 1 minute; 68 ℃ were extended 10 minutes.Obtain an about 450bp dna fragmentation.

The T-carrier connects:

Above-mentioned PCR product fragment is passed through 1% agarose electrophoresis; Use the glue of Omiga company to reclaim test kit; And the step that adopts specification sheets to provide reclaims the PCR fragment; To reclaim fragment subsequently and be connected PCR product and T carrier, by " molecular cloning " method heat shock transformed into escherichia coli DH5 α bacterial strain with T-carrier pEASY-blunt simple (full formula King Company) by specification.

Determined dna sequence:

Positive DH5 α clone after the above-mentioned conversion is inoculated in 1ml LB substratum, hands in sea living worker biotech company and carry out determined dna sequence, use universal sequencing primer thing M13+ and M13-, order-checking obtains following sequence:

GTGGTACTCGTTCTGGACCGACGCGCAGGGCACGGTCTCGATGACCCTGGGCTCCGGTGGCAACTACAGCACCAGCTGGCGCAACACCGGCAACTTCGTGGCCGGCAAGGGCTGGAGCACCGGCGCCCGCAGGAACGTGACCTACTCCGGCAGCTTCAACCCGTCCGGCAACGGCTACCTGTCGCTCTACGGCTGGACGTCGAACCCGCTCGTGGAGTACTACATCGTCGACAACTGGGGCACCTACCGGCCCACGGGGACGTACAAGGGCAGTGTCACCAGTGACGGCGGAACGTACGACATCTACCAGACGACGCGGTACAACGCCCCGTCCGTCGAGGGCACCAGGACCTTCAACCAGTACTGGAGCGTGCGGCAGTCGAAGCGCACCGGCGGCACCATCACCACCGGC AACCACTTCGACGCCTGGG

The Tail-PCR primer design:

Dna sequence dna according to above acquisition; Design amplification xylanase gene upstream and downstream Tail-PCR is with each three of primers in this sequence: the amplification direction is the primer called after sp5-1 respectively of updrift side; Sp5-2 and sp5-3; Primer sp5-1 is in the primer sp5-2 outside, and primer sp5-2 surveys outside primer sp5-3; The primer amplification direction is the primer difference called after sp3-1 of downstream direction, sp3-2 and sp3-3, and primer sp3-1 is in the primer sp3-2 outside, and primer sp3-2 is in the primer sp3-3 outside (table).The overlap (Fig. 3) that has 200 bp between primer sp3-3 and the primer sp5-3 after the order-checking of assurance Tail-PCR product, can correctly be pieced together and save out complete gene order.

Tail-PCR：

The method of Tail-PCR is with reference to the method for Liu Yaoguang (document); Obtaining upstream region of gene direction PCR fragment respectively is 500bp (Fig. 4-1), and downstream direction is 400bp (Fig. 4-2).The amplification enzyme that uses that makes is the ExTaq enzyme of Takara company.

The T-carrier connects:

With upstream region of gene and downstream amplified fragments through 1% agarose electrophoresis after, reclaim test kit by the glue of Omiga company and reclaim the PCR fragment, connect heat shock transformed into escherichia coli DH5 α bacterial strain with T carrier pEASY-simple T1 (full formula King Company) then.

Determined dna sequence:

Positive DH5A after the above-mentioned conversion is inoculated in 1ml LB substratum, hands in sea living worker biotech company and carry out determined dna sequence, adopt universal sequencing primer thing M13+ and M13-.

The full-length gene pcr amplification:

According to recording upstream region of gene and downstream sequence; Carry out sequence assembly by software DNAman (), obtain full-length gene and get dna sequence dna, according to obtaining sequence; Design primer xynA-p1 and xynA-p2; The employing complete genome DNA is a template, high-fidelity amplification enzyme KOD-plus, and amplification condition is: 94 ℃ of sex change 3 minutes; 94 ℃ of sex change 30 seconds, 50 ℃ of annealing 30 seconds, 68 ℃ were extended totally 10 circulations 1 minute; 94 ℃ of sex change 30 seconds, 54 ℃ of annealing 30 seconds, 68 ℃ were extended totally 20 circulations 1 minute; 68 ℃ were extended 10 minutes.Obtain single about 1200bp dna fragmentation.

The T-carrier connects:

Above-mentioned PCR product fragment is passed through 1% agarose electrophoresis; The step that adopts the glue recovery test kit of Omiga company and adopt specification sheets to provide reclaims the PCR fragment; To reclaim fragment subsequently and be connected PCR product and T carrier, by " molecular cloning " method heat shock transformed into escherichia coli DH5 α bacterial strain with T-carrier pEASY-simple blunt (full formula King Company) by specification.

Determined dna sequence:

Positive DH5 α after the above-mentioned conversion is inoculated in 1ml LB substratum, hands in sea living worker biotech company and carry out determined dna sequence, adopt universal sequencing primer thing M13+ and M13-.Obtain the XylA full length protein encoding sequences that the ORF total length is 671 bases or 966 bases through the DNAman analysis.Through NCBI is carried out the blast sequence alignment, sequential analysis is a zytase, and the length of possibly encoding is 234 amino acid and 322 amino acid, two peptide species.

The structure of pET28a (+) expression vector:

According to two different peptide species of gene order possibility code length; Design gene forward primer xynA-eL1 and xynA-eS1 and reverse primer xynA-eD2 respectively; The employing complete genome DNA is a template, high-fidelity amplification enzyme KOD-plus, and amplification condition is: 94 ℃ of sex change 3 minutes; 94 ℃ of sex change 30 seconds, 50 ℃ of annealing 30 seconds, 68 ℃ were extended totally 10 circulations 1 minute; 94 ℃ of sex change 30 seconds, 54 ℃ of annealing 30 seconds, 68 ℃ were extended totally 20 circulations 1 minute; 68 ℃ were extended 10 minutes.Obtain a 680bp DNA sheet and a section 985bp (Fig. 6) dna fragmentation.

Above-mentioned PCR product fragment is passed through 1% agarose electrophoresis; The step that adopts the glue recovery test kit of Omiga company and adopt specification sheets to provide reclaims the PCR fragment; Press the embodiment method and connect the T carrier; And carry out sequencing by embodiment, measure the structure that the right-on clone of result is used for expression vector.

Correct T-carrier bacterial strain is inoculated in to accompany in 37 ℃ of training bases of 5ml LB to support spends the night, press the Omiga kit method and extract plasmid, plasmid passes through EcoRI and the XhoI enzyme is cut; Cut product through the OMIGA PCR of company purification kit purifying enzyme; With the purifying after product in same through EcoRI with the XhoI enzyme is cut and through 1% agarose electrophoresis, the recovery carrier framework connection of adopting the glue of Omiga company to reclaim test kit, linked system is a 100ngPCR product endonuclease bamhi; 10ng pET28a (+) product fragment; 1 μ l T4 ligase enzyme damping fluid, 1 μ l T4DNA ligase enzyme replenishes aseptic ultrapure water to 10 μ l; Condition of contact spends the night for connecting at 16 ℃ of low temperature, by " molecular cloning " method heat shock transformed into escherichia coli DH5 α bacterial strain.The positive colony that conversion obtains is used for cultivating and zytase is expressed.

The structure of Yeast expression carrier:

According to two different peptide species of gene order possibility code length; Design gene forward primer xynA-yL1 and xynA-yS1 and reverse primer xynA-yD2 respectively; The employing complete genome DNA is a template, high-fidelity amplification enzyme KOD-plus, and amplification condition is: 94 ℃ of sex change 3 minutes; 94 ℃ of sex change 30 seconds, 50 ℃ of annealing 30 seconds, 68 ℃ were extended totally 15 circulations 1 minute; 94 ℃ of sex change 30 seconds, 54 ℃ of annealing 30 seconds, 68 ℃ were extended totally 15 circulations 1 minute; 68 ℃ were extended 10 minutes.Obtain a 680bp DNA sheet and a section 985bp (Fig. 7) dna fragmentation.

Above-mentioned PCR product fragment is passed through 1% agarose electrophoresis; The step that adopts the glue recovery test kit of Omiga company and adopt specification sheets to provide reclaims the PCR fragment; Press the embodiment method and connect the T carrier; And carry out sequencing by embodiment, measure the structure that the right-on clone of result is used for expression vector.

Correct T-carrier bacterial strain is inoculated in to accompany in 37 ℃ of training bases of 5ml LB to support spends the night, press the OMGA kit method and extract plasmid, plasmid passes through EcoRI and the AvrII enzyme is cut; Cut product through the Omiga PCR of company purification kit purifying enzyme; With the purifying after product in same through EcoRI with the AvrII enzyme is cut and through 1% agarose electrophoresis, the recovery carrier framework connection of adopting the glue of Omiga company to reclaim test kit, linked system is a 100ngPCR product endonuclease bamhi; 10ng pPIC9K product fragment; 1 μ l T4 ligase enzyme damping fluid, 1 μ l T4DNA ligase enzyme replenishes aseptic ultrapure water to 10 μ l; Condition of contact spends the night for connecting at 16 ℃ of low temperature, by " molecular cloning " method heat shock transformed into escherichia coli DH5 α bacterial strain.The pichia spp electricity swashs conversion:

The LB substratum that above-mentioned positive colony is inoculated in 500ml spends the night; Press the medium-sized extraction test kit extraction of Omiga company plasmid DNA after collecting thalline; After DNA is cut through the EcoRV enzyme; Reclaim test kit with Omiga company glue and reclaim dna fragmentation, be taken to the sharp pichia spp host bacterium GS115 that transforms of the linearizing DNA electricity of few 2 μ g, the RD substratum recovers to cultivate.

Pichia spp produces the screening of zytase transformant:

The transformant of normal growth on the RD substratum is transferred in the MD substratum, behind the sub-purifying to be transformed, be inoculated in 3ml BMGY liquid nutrient medium enrichment culture 36h; 1ml BMMY liquid nutrient medium inducing culture 24h, 4 ℃, 5000rpm are gone in switching again; Centrifugal 5min obtains supernatant.5 μ l supernatants are put in the screening culture medium that contains 0.5% birch xylan substrate 50 ℃ of reaction 30min, 1% congo red staining 20min, 1M NaCl 20min that decolours.The positive transformant that the transparent circle phenomenon is arranged.

Xylanase activity is analyzed:

After above-mentioned positive transformant was inoculated in 15ml BMGY liquid nutrient medium enrichment culture 48h, 50ml BMMY liquid nutrient medium inducing culture 216h was gone in switching, and every 24h sampling 1ml adds the inductor methyl alcohol of 1mL final concentration 0.5% simultaneously.4 ℃, 5000rpm, centrifugal 5min handles fermentation broth sample, obtains supernatant, adopts the DNS method to measure Xylanase activity.Concrete operations are following: use 0.05M, the pH value is 5.3 citrate buffer solution dilution supernatant samples, gets 100 μ l samples and is added in the birch xylan substrate of 900 μ l 1%; In 55 ℃ of reaction 5min; Add 1ml DNS reaction terminating liquid, the 15min that in boiling water, develops the color, the potassium sodium tartrate solution of adding 1ml 40%; Measure absorbancy down in 540nm, calculate xylanase activity.Through above-mentioned xylanase activity power measuring method; Obtain 6 high yield enzymatic conversion; Be respectively 1-76,3-59,6-28,6-63,7-111,9-3, enzyme activity is respectively 403 ± 3U/mL, 464 ± 9U/mL, 447 ± 13U/mL, 479 ± 10U/mL, 768 ± 35U/mL, 473 ± 10U/mL.(Fig. 5)

The zytase recombinant protein is analyzed

Xylanase gene XylA is connected with carrier pEASY-simple blunt and changes in the bacillus coli DH 5 alpha; Fermented liquid is measured Xylanase activity behind broken wall be 163U/mL; Fig. 6 is its SDS-PAGE electrophorogram, can know that by analyzing the zytase molecular weight of albumen is 27KD.

Xylanase gene XylA is connected with pPIC9K, induces fermentation 196h.Fermented liquid is that 12.5% SDS-PAGE denaturing gel electrophoresis is handled through concentration, and after electrophoresis finished, with the coomassie brilliant blue staining liquid 20min that dyes, it was clear to protein band to decolour with 1M NaCl elutriant again, and the result is as shown in Figure 7, and zytase albumen size is 38kDa.

Claims (13)

1. the nucleotide sequence of a zytase, its sequence details is SEQ NO1.

2. at least 90% of this sequence homology.

3. the polypeptide of a zytase, its sequence details is SEQ NO2.

4. at least 90% of this sequence homology.

5. the polypeptide of a zytase, its sequence details is SEQ NO3.

6. at least 90% of this sequence homology.

7. the signal peptide of a zytase, its sequence details is SEQ NO4.

8. at least 90% of this sequence homology.

9. comprise right 1,2,3,4,5,6,7,8 recombinant vectors.

10. the colibacillary recombinant expression system of right 9.

11. the recombinant expression system of the pichia spp of right 9.

12. be similar to right 10,11 at lactobacillus spp, yeast saccharomyces cerevisiae, genus bacillus (like subtilis), the expression in the aspergillus (like black mold or aspergillus oryzae) is used.

13. right 1,2,3,4,5,6,7,8,9,10,11,12 in paper industry, food-processing, fodder industry, textile industry, bioenergy, makeup, Medicines and Health Product, and the application of various bio-reactors.

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