CN105713915A - Cloning method of culture-independent xylanase genes derived from soil macro-gene resource and product xylanase X1-19 - Google Patents

Abstract

The invention discloses a gene of culture-independent xylanase X1-19 derived from the soil metagenome, comprising the nucleic acid and protein/polypeptide sequences. The overall length of the encoded gene of the xylanase is 1047bp, the GC content is 47.7%, the code is 348aa, the protein molecular weight is 36.2KDa, and the enzyme has the typical structure of F/11 glucoside hydrolase, and has no other binding domains. The gene of X1-19 is used for constructing a recombinant expression vector pPIC9K-X-19. The enzyme content of the recombinase measured through a DNS method is 79.0+/-2.2 U/mL. The optimal pH of the recombinase is 4.8, the optimal reaction temperature is 55 DEG C, when oat spelt xylan, water-soluble corncob xylan and bran xylan as taken as the substrates, the affinity is strong, and the relative enzyme activities are 386%, 334% and 321% of that of the activity of birchwood xylan respectively. As the culture-independent acid xylanase with the novel gene sequence, the enzyme and an action product of the enzyme have application values in the paper manufacturing industry, the textile industry, the food processing industry, the biological energy source industry, the feed industry, the cosmetic industry, the medicine industry, the health care product industry, the wine making industry and the crop production industry and in various bioreactors.

Description

A kind of non-cultivation xylanase gene cloning process deriving from the grand genetic resources of soil and product xylanase X1-19 thereof

Technical field

The present invention relates to the character of a kind of non-cultivation xylanase gene cloning process deriving from the grand genetic resources of soil and DNA recombinant expression product xylanase X1-19 thereof and application.

Background technology

Xylan (xylan) is a kind of heterogeneous polysaccharide (heteropolysaeeharides), is that β-D-pyranoid form xylose units (xylopyranoseunits) connects formation by β-Isosorbide-5-Nitrae-glycosidic bond.It is widely present in nature to be the important component part of plant hemicellulose, accounts for the 15～35% of plant cell dry weight.Can it be a kind of important Renewable resource, effectively utilize xylan to have enormous industrial and be worth.The degradation of xylan that art dextranase energy is single-minded, and safe and efficient, utilizing xylanase degradation of xylan is the effective means currently with this resource, is paid attention to widely, becomes the study hotspot of researcher in recent years.Xylanase being widely used food, feedstuff, paper pulp help in the industries such as drift, medicine, weaving, the energy, and play a significant role.At present, xylan is applied primarily to the fields such as baking food, brewing industry, xylooligosaccharides production in food.

Xylanase most at present is derived from the fermentation of all kinds of original strain and produces, and the xylanase that original strain produces is originated less due to strain excellent and yielded poorly and causes that the application of its heavy industrialization exists restriction.Therefore genetic engineering means obtain xylanase research get more and more, xylanase gene is at escherichia coli, bacillus cereus, saccharomyces cerevisiae, Pichia sp.^]Express Deng host all has acquisition, xylanase especially from Situation of Microorganism Under Extremity Environment is originated, it is difficult to artificial culture under normal conditions and makes it produce enzyme, but we can obtain genes of interest and carry out heterogenous expression by engineered means, on producing, there is very big value, but the xylanase expressed by genetic engineering bacterium is comparatively single, and the homology of the xylanase produced with known bacterium is higher, be difficult to obtain differ relatively greatly with conventional properties of xylanase, the good xylanase of novelty.And utilize the method for metagenomics can obtain the genomic DNA of all microorganisms in a certain environment, then structure genomic library and library is screened, substantially increase the probability finding and finding new xylanase gene.

Summary of the invention

The non-many nucleotide sequences of cultivation xylanase gene deriving from the grand genetic resources of soil that the present invention obtains are such as shown in SEQ-1:

ATGTTCAATCTTAAGAGAGTGGCGGCGCTCCTGCGCGTCGCAGGCCTGGGCATGTCTGCCGCAAATGCGCAGACCTGCCTCACGTCGAGCCAAACCGGCAACAACAACGGCTTC TACTATTCATTCTGGAAAGACAATCCGGGCACGGTGAATTTCTGCCTGCAGTCCGGCGGGCGCTACACATCGAACTGGAGCGGCATCAACAACTGGGTGGGCGGCAAGGGATGGCAGACCGGTTCGCGCCGGAATATCACGTACTCGGGCAGTTTCAGCTCGCCGGGCAACGGCTACCTGGCGGTGTATGGATGGACCACCAATCCACTCGTCGAGTACTACGTCGTCGAAAGCTGGGGCAACTGGCGTCCGCCGGGATCGGACGGCACGTTCCTGGGAACGGTCAACAGCGATGGCGGGACGTACGACATCTACCGCGCGCAGCGGGTCAACGCACCGTCGACCATCGGCAATGCCACGTTCTACCAATACTGGAGCGTCCGTCAGTCGAAGCGCGTGGGTGGAACGATCACCACCGGCAACCACTTCGATGCGTGGGCCAGCGTCGGCTTGAACCTGGGTACTCACAACTACCAGGTCATGGCGACCGAGGGCTATCAAAGCAGCGGCAGCTCCGACATCACGGTGAGTGAAGGCGGCGGGAGCAGCAGCAGCGGTGGAGGCAGCAGCACCAGCAGCAGCGGCGGCGGTGGTGGAAACAAGAGCTTCACCGTGCGGGCACGCGGCACCACGGGTGGTGAGTCCATCACGCCTCGCGTGAACAACCAGAACGTGCAGACCTGGACGCTCGGCACCGGCATGACGAACTACACGGCGTCGACGTCCCTGAGCGGCGGCATCACCGTGGCGTACACGAATGACAGTGGGAACCGGGACGTGCAGGTGGACTACATCGTCGTGAACGGCCAGACGCGGCAGTCCGAGTCCCAGAGCTGCAACACGGGGCTCTACGCCAATGGACGTTGCGGTGGTGGATCCAACAGCGAGTGGATGCATTGCAACGGCGCCATCGGCTACGGAAATACACCGTAG

The non-cultivation xylanase gene peptide sequence deriving from the grand genetic resources of soil that the present invention obtains is such as shown in SEQ-2:

MFNLKRVAALLRVAGLGMSAANAQTCLTSSQTGNNNGFYYSFWKDNPGTVNFCLQSGGRYTSNWSGINNWVGGKGWQTGSRRNITYSGSFSSPGNGYLAVYGWTTNPLVEYYVVESWGNWRPPGSDGTFLGTVNSDGGTYDIYRAQRVNAPSTIGNATFYQYWSVRQSKRVGGTITTGNHFDAWASVGLNLGTHNYQVMATEGYQSSGSSDITVSEGGGSSSSGGGSSTSSSGGGGGNKSFTVRARGTTGGESITPRVNNQNVQTWTLGTGMTNYTASTSLSGGITVAYTNDSGNRDVQVDYIVVNGQTRQSESQSCNTGLYANGRCGGGSNSEWMHCNGAIGYGNTP*

The non-cultivation xylanase gene signal peptide sequence deriving from the grand genetic resources of soil that the present invention obtains is such as shown in SEQ-3:

MFNLKRVAALLRVAGLGMSAANA

Accompanying drawing explanation

Fig. 1 is the agarose gel electrophoresis figure of the grand gene DNA of soil.Wherein: M1:1kbDNAMarker；1: the soil DNA that RNA isolation kit obtains；2: the soil DNA that physical-chemical process obtains；3: the soil DNA that enzyme solution obtains；M2: λ-HindIIIdigestedMarker

Fig. 2 is the electrophoretogram of non-cultivation xylanase gene core sequence.Wherein: M:DL500DNAMarker；1: xylanase core sequence.

Fig. 3 is that non-cultivation xylanase gene X1-19 conservative functional areas are analyzed.

Fig. 4 is the recombinant expression carrier pPIC9K-X1-19 built.(a) recombinant expression carrier pPIC9K collection of illustrative plates (b) BglII linearisation pPIC9K-X1-19.

Fig. 5 is Pichia yeast engineering fermented supernatant fluid SDS-PAGE.Wherein: M:marker；Recombined xylanase X1-19 after 1:DEAE process；2: the X1-19 that deglycosylation processes；3:EndoH enzyme；Arrow show recombined xylanase X1-19.

Fig. 6 is the Congo red flat board transparent circle of recombined xylanase X1-19 hydrolyzed xylan substrate.

Fig. 7 is the pH specificity analysis of recombined xylanase X1-19.

Fig. 8 is the reaction temperature analysis of recombined xylanase X1-19.

Fig. 9 is recombined xylanase X1-19 substrate specificity.

Detailed description of the invention

The present invention is described further below in conjunction with instantiation, but is not the restriction present invention.Below in conjunction with accompanying drawing, the present invention is further described.The experimental technique of unreceipted actual conditions in embodiment, generally can bar routinely, such as the condition described in " the Molecular Cloning: A Laboratory guide third edition " write such as J. Pehanorm Brooker (Sambrook), or according to kit manufacturer it is proposed that condition carry out.Those skill in the art related can be more fully understood that by embodiment and grasp the present invention.

General reagent: agarose (Spain Biowest)；Tris (U.S. AMRESCO)；Congo red；Kanamycin (U.S. AMRESCO)；Ampicillin (U.S. AMRESCO)；Birch xylan, Zelkova schneideriana Hand.-Mazz. xylan, Herba bromi japonici xylan, chitosan (U.S. Sigma)；LB solid medium；Xylan-Congo red screening flat board；Peptone (Tryptone), yeast extract (YeastExtract) (Oxford company of Britain)；NaCl eluent: 1M.

Biochemical reagents: soil DNA extracts test kit (U.S. MOBIOLaboratories)；DNA agarose gel reclaims test kit (U.S. OMEGA)；Plasmid extraction kit (U.S. OMEGA)；Glue reclaims test kit (OMEGA, the U.S.)；LaTaqDNA polymerase (withGCbuffer)；Restricted enzyme EcoRI, NotI, BglII, T4DNA ligase.

Carrier: carrier T: pMD18-T is purchased from TAKARA company；Pichia vector: pPIC9K is purchased from Invitrogen company

Culture medium: YPD fluid medium: yeast extract 1%, tryptone 2%, glucose 2%, 4 DEG C of preservations.

MD:YNB1.34%, biotin 0.05%, glucose 2%, agar 1.5%, 4 DEG C of preservations.

MM:13.4g/LYNB, 4x10-4g/L biotin, 5ml/L methanol, 15g/L agar

RDB culture medium: 1M sorbitol, 1%YNB, 2% glucose, 0.02% biotin, 0.05% aminoacid.

Carrier T sequencing primer:

M13+ sequence: 5 '-GTTTTCCCAGTCACGAC-3 '

M13-sequence: 5 '-CAGGAAACAGCTATGAC-3 '

Pichia vector sequencing primer:

5 ' sequences: AOX1:5 '-GACTGGTTCCAATTGACAAGC-3 '；

Alpha-Factor-F:5 '-TACTATTGCCAGCATTGCTGC-3 '

3 ' sequences: AOX1:5 '-GCAAATGGCATTCTGACATCC-3 '

Xylanase the 10th family (GH10) and the 11st family (GH11) core sequence degenerate primer sequence:

X10-F:CTACGACTGGGAYGTNIBSAAYGA

X10-R:GTGACTCTGGAWRCCIABNCCRT

X11-F:AACTGCTACCTGKCNITNTAYGGNTGG

X11-R:CCGCACGGACCAGTAYTGNKIRAANGT

(note: the degenerate region that region is primer of underscore mark；Y represents C or T；R represents A or G；S represents C or G；W represents A or T；B represents C, G or T；N represents A, C, G or T；I: hypoxanthine, it is possible to combine with A, G, C or T pairing, for reducing the degeneracy of primer.)

Amplification primer used in flanking sequence

1. core sequence 1-19xynA-core5 ' end holds three nested specific primer sequences with 3 ':

5 ' end amplimer SP5-1CACGTCCCGCCATCGCTGTT

SP5-2CAGTTGCCCCAGCTTTCGACGAC

SP5-3CGTGGATTGGTGGTCCATCCATACACC

3 ' end amplimer SP3-1CTGCTACCTGGCGGTGTATGGATGG

SP3-2CGCTTCCTGGGAACGGTCAACAG

SP3-3TCGACCATCGGCAATGCCACG

2. expand 4 degenerate primers (LAD) of flank and 1 random primer (AC1) sequence:

LAD1-1ACGATGGACTCCAGAGCGGCCGCVNVNNNGGAA

LAD1-2ACGATGGACTCCAGAGCGGCCGCBNBNNNGGTT

LAD1-3ACGATGGACTCCAGAGCGGCCGCVVNVNNNCCAA

LAD1-4ACGATGGACTCCAGAGCGGCCGCBDNBNNNCGGT

AC1ACGATGGACTCCAGAG

Note: R=A/G, S=C/G, W=A/T, B=C/G/T, N=A/C/G/T

1-19xynA sequence is containing the primer sequence of EcoRI, NotI restriction enzyme site:

1-19xynF5′-CCGAATTCATGTTCAATCTTAAGAGAGTGGC-3′EcoRI

1-19xynR5′-ATAAGAATGCGGCCGCCTACGGTGTATTT-3′NotI

Embodiment 1: the not cultured xylanase gene clone of the grand GENE SOURCES of soil

The extraction of 1.1 pedotheque STb gene and purification

Pedotheque picks up from the throughout the year moist forest in the areas such as Shandong, Hunan, Heilungkiang or farmland.Depth selection is from soil surface 5～20cm, in the preservation of 4 DEG C of refrigerator after mixing.

1.1.1 RNA isolation kit

The method adopting SoilDNAisolationkit (MOBIOLaboratories, Inc., USA), operating procedure by specification.Collect DNA sample be stored in-20 or-80 DEG C standby.

1.1.2 physical-chemical process

Method in conjunction with physical grinding and chemical cracking.Isopropanol precipitating, centrifugal after abandon supernatant, take 5mLTEbuffer dissolution precipitation, add equal-volume phenol: chloroform: isoamyl alcohol (25: 24: 1), inhale and play mixing, 16000g is centrifuged 20min；Taking supernatant, add isopyknic chloroform: isoamyl alcohol (24: 1), inhale and play mixing, 16000g is centrifuged 20min；Taking supernatant, the isopropanol adding 0.7 times of volume precipitates again.

1.1.3 enzyme solution

Weigh 10g soil in 50mL sterile centrifugation tube, add the PVPP (polyvinylpolyp) of 15mLTENC buffer solution and 2% pickling, 3000rpm centrifuge washing；Abandoning supernatant, add 15mLDNA extraction buffer and 1.5mL lysozyme soln (50mg/mL), mix homogeneously, 37 DEG C, 225r/min vibrates 1h；Add 1.5mL20% (w/v) SDS and 90uL E.C. 3.4.21.64 (20mg/mL), 65 DEG C of water-bath 1h；The follow-up same physical-chemical process of DNA extraction step.

The DNA sample slightly carried is electrophoresis on 1% agarose gel, and EB dyes 15min, cuts glue and reclaims macro genome DNA band, be stored in after kits-20 DEG C standby.RNA isolation kit contains the purification step removing humic acid, phenol impurity, and the soil DNA impurities that therefore the method obtains is less, it is not necessary to purification again, can be directly used for pcr amplification.The soil DNA electrophoresis detection on 1% agarose gel obtained with RNA isolation kit after the DNA purification that first two method obtains, result is as shown in Figure 1.

1.2 sequence screenings obtain xylanase core sequence

1.2.1 the design of degenerate primer

By xylanase the 10th family (GH10) and the 11st family (GH11) a plurality of gene order are analyzed, obtain 2 conservative regions respectively, adopting CODE-HOPprimer design of primers principle, design is for expanding the degenerate primer of the tenth family and the 11st family's Xylanase coding gene.

1.2.2 degenerate pcr amplification xylanase core sequence

With the soil microorganism STb gene after purification for template, the degenerate primer of design is amplimer, expands GH10 and GH11 xylanase core sequence by degenerate pcr program.DNA fragmentation is connected with cloning vehicle PMD18-T after reclaiming, and builds xylanase core sequence library random picked clones order-checking.Degenerate primer X11-F and X11-R is utilized to obtain the 11st family (GH11) the xylanase core sequence that clip size is about 200bp, 1% agarose gel electrophoresis is as shown in Figure 2, show in pedotheque containing the microorganism producing the 11st family's xylanase gene, and after the extraction purification of soil DNA, corresponding gene order still has part to exist.

1.2.3 the comparison analysis of xylanase core sequence

With DNAman software, DNA sequence is carried out multisequencing sequence analysis, then the homology sequence less than 99% is carried out on NCBI nucleic acid homology comparison, the nucleotide sequence that screening is higher with the xylanase gene homology of Anticipated transient without scram.Wherein having 11 sequences Homologous gene sequences on NCBI less, and homologous genes is almost entirely from Anticipated transient without scram, therefore, these 11 sequences, very likely from the xylanase gene of Anticipated transient without scram, have good researching value.Embodiment selects with the xylanase gene not cultivating bacterium microbe of synthetic for dominant clone, with 1-19 for researching and analysing object, design nested primers, utilize HiTail-PCR to expand flanking sequence, obtain the full-length gene of encoding xylanase, and it is carried out bioinformatic analysis.

1.3HiTail-PCR expands flanking sequence

With soil microorganism STb gene for template, adopt HiTail-PCR that the flanking sequence of bacterial origin sequence 1-19xynA-core is expanded.At the high nested special primer taking off fire temperature of core sequence indoor design three, with degenerate primer LAD, random primer AC carries out nest-type PRC, obtains the fragment that specificity is stronger after three-wheel amplified reaction.The sequence spliced being searched open reading frame (ORF) on NCBI, obtains the full length gene SEQ-1 of encoding xylanase 1-19xynA, the accession number on NCBI is KP209314.1.

1.3.1xynA bioinformatic analysis

With DNAman software, full length gene SEQ-1 is carried out bioinformatic analysis；NCBI website carries out (www.ncbi.nlm.nih.gov) conservative functional areas with DomainSearch instrument analyze；It is analyzed predicting to signal peptide by online website (http://www.cbs.dtu.dk/services/SignalP/).Software analysis shows SEQ-1 full length gene 1047bp, G/C content 47.7%, encodes 348aa, it was predicted that the protein molecular weight of coding is 36.2Kda (SEQ-2), encoding one section 23 amino acid whose signal peptides (SEQ-3), after removing signal peptide, molecular weight of albumen is 33.8KDa.

1.3.2 conservative functional areas are analyzed

Data in contrast conserved sequence data base (CDD), result is as shown in Figure 3, it was shown that this albumen is the 11st family's glycoside hydrolase, without other binding domain.

Embodiment 2: grand GENE SOURCES xylanase gene is recombinant expressed and recombiant protein character

The pichia yeast expression system of 2.1 xylanase X1-19 builds

2.1.1PCR the gene xynA of amplified band restriction enzyme site

Consider the restriction enzyme sites in the multiple clone site (MCS) of pPIC9K carrier and X1-19 sequence, select two restriction enzyme sites of EcoRI and NotI.Design primer, introduces EcoRI in upstream, and downstream introduces NotI.PCR reacts amplification, cuts glue and reclaims the target DNA fragment that fragment is bigger.

2.1.2 the structure of recombiant plasmid pPIC9K-xynA

Respectively with restricted enzyme EcoRI and NotI enzyme action carrier pPIC9K and the genetic fragment X1-19 containing EcoRI, NotI restriction enzyme site, 37 DEG C of reaction 16h.Reaction terminate after on 1% agarose gel electrophoresis detection, cut glue reclaim purpose fragment.Connecting genetic fragment 1-19xynA and linearizing carrier pPIC9K (mol ratio 3: 1) with sticky end with T4DNA ligase, 16 DEG C connect 12h.Recombiant plasmid pPIC9K plasmid map such as Fig. 4 (a), the recombinant expression carrier pPIC9K-X1-19 agarose gel electrophoresis after BglII linearisation detects such as Fig. 4 (b).Result shows: occur two bands varied in size after BglII enzyme action, according to plasmid map it can be seen that the band that size is about 8kb is the part containing genes of interest xynA.Cutting glue and reclaim purpose band, electric shock proceeds to Pichia sp..

2.1.3 electroporated recombinant yeast pichia pastoris

Take 1～5 linear recombiant plasmid of μ g and 100 μ L Pichia sp. competent cells mix gently, proceed in the aseptic electric shock cup (0.2cm type) of pre-cooling.Light and slow knock electric shock cup, make mixture sink to bottom electric shock cup, stand 5min on ice.The electroporated operation of recombinant yeast pichia pastoris is carried out at electric shock instrument (BioRad).Adjust the configuration of electroporated instrument, voltage 1.5kV, electric capacity 25 μ F, resistance 400 Ω, electroporated Pichia sp. after charging complete.In electric shock cup, add rapidly the 1mol/L sorbitol of 1mL pre-cooling on ice, after mixing, coat RDB flat board immediately.30 DEG C of incubators are inverted and are cultivated, and occur to transformant.

2.1.4 the screening of high yield transformant

Linearisation recombiant plasmid pPIC9K-X1-19 electric shock is proceeded to Pichia pastoris GS115, coats cultivation 3d on RDB flat board, flat board occurs a large amount of His+ transformant bacterium colony.The copy number of transformant is relevant with the ability of anti-G418, can obtain high copy transformant by screening the strong yeast of anti-G418 ability.Select the transformant of growth on the flat board of the G418 containing 3.0mg/ml, the G418 flat board containing 4.0,6.0mg/ml is adopted to screen further more successively, finally from the YPD flat board of the G418 containing 6.0mg/ml, obtain 3 bacterium colonies, through MM, MD plate assay, selected bacterial strain is methanol and quickly utilizes type (Mut+).3 transformants obtained by primary dcreening operation are cultivated, and sampling is surveyed enzyme and lived, it has been found that during fermentation 216h, enzyme is lived the highest.Through multiple sieve, transformant induction product enzyme 216h enzyme is lived and is up to 79.0 ± 2.2U/mL.

2.2 recombined xylanase X1-19 character

2.2.1 the purification of recombined xylanase (X1-19)

Ice-water bath adds ammonium sulfate precipitation crude enzyme liquid, gradient of saltouing is 20%-80%, the centrifugal 20min of 12000 × g after mixing, with 0.05M, the resuspended precipitation of Tirs-HCL buffer of pH8.0, then with the buffer (Tirs-HCL buffer, 0.05M, pH8.0) of 100 times of sample volumes, dialyse 24h.DEAE chromatographic column (1 × 10cm) crossed by sample after dialysis, the Tirs-HCL buffer balance of chromatographic column pH8.0,0.05M.Remove the foreign protein not being combined with post material with identical buffer after loading.The NaCl of the albumen 0～0.2M being attached on post material (is dissolved in pH8.0, the Tirs-HCL buffer of 0.05M) gradient elution, flow velocity is 0.5mL/min, 280nm detects protein content, eluting peak 2mL centrifuge tube is collected, and survey enzyme is lived and protein content (Lowry method) respectively, SDS-PAGE electrophoresis detection lipidated protein.

Secretory protein can be carried out O-and N-and connect glycosylation modified by Pichia sp., therefore the recombined xylanase deglycosylation through DEAE purification is processed: the reaction system containing 20 μ g recombined xylanase X1-19 adds 1 μ L10 × glycoprotein denaturation buffer, adding sterilizing high purity water to cumulative volume is 10 μ L, mix homogeneously, reacts 10min in boiling water bath；Being subsequently adding 2 μ L10 × G5 buffer, 1 μ LEndoH enzyme, adding high purity water to total system is 20 μ L, 37 DEG C of reaction 1.5h, then with SDS-PAGE electrophoresis detection lipidated protein, as it is shown in figure 5, this molecular weight of albumen is sized to 36kDa, identical with the result of prediction.

2.2.2 Congo red transparent circle method detection recombined xylanase activity

Utilize the xylanase activity of Congo red transparent circle method detection recombiant protein.Xylanase can decompose the Zelkova schneideriana Hand.-Mazz. xylan on flat board and produce xylooligosaccharide, and the complexing power between congo red weakens, and by the NaCl solution eluting of 1M, can present transparent circle.Drawing the supernatant of 5 μ L transformants 1 and transformant 2 respectively and crude enzyme liquid carries out the detection of Congo red transparent circle method, as shown in Figure 6, all there is transparent circle transformant 1 position corresponding with the crude enzyme liquid of 2 to result.Comprehensive Congo red transparent circle method and xylanase activity measurement result are known: recombiant protein has xylanase activity.

2.2.3 the zymologic property research of recombined xylanase

2.2.3.1 recombined xylanase optimum pH

The mensuration of optimum pH carries out as shown in Figure 7 at 50 DEG C.Adopting 7 kinds of buffer systems to configure a series of concentration is the buffer of 0.05mol/L, and pH value range is between 2.2～11.3.Buffer solution system and pH value range thereof be citric acid-sodium citrate buffer (pH2.20～4.20) respectively, Acetic acid-sodium acetate buffer (pH3.80～5.80), MES buffer (pH5.20～7.20), MOPS buffer (pH6.20～8.20), Tris-HCl buffer (pH7.00～9.00), CHES buffer (pH8.20～10.20), CAPS buffer (pH9.30～11.30).Every kind of buffer system has 5 different pH value, totally 35 pH value.When measuring Xylanase activity, the buffer in DNS assay method replacing with the buffer of above-mentioned different pH value, the enzyme activity of mensuration is with maximum for 100%.When the acetate buffer solution that reaction buffer is pH4.8, enzyme is lived the highest and relatively stable.When pH is at 4.3-8.5, enzyme activity, all more than 70%, has certain acid resistance and alkali resistance.

2.3.3.2 recombined xylanase optimum temperature

The mensuration of optimum temperature is as shown in Figure 8: with optimum pH buffer, enzyme liquid is suitably diluted, then respectively under the different temperatures of 40～80 DEG C (between adjacent temperature 5 DEG C of interval), reaction 10min, measures Xylanase activity, with maximum for 100%.Its optimum temperature is 55 DEG C.The enzyme activity of xylanase is all more than 70% between 47-65 DEG C for temperature, and remnant enzyme activity sharply declines when temperature reaches 65 DEG C.

2.3.3.3 recombined xylanase substrate specificity

With the Xylanase activity under the natural substrate of separate sources and synthetic substrate bioassay standard method, thus analyzing the substrate specificity of xylanase.Substrate is changed into different xylan substrate (1%, W/V), and according to standard method under 50 DEG C of conditions, reaction 10min measures enzyme activity.Enzyme activity during using birch xylan as enzyme reaction substrate is as 100%.Natural substrate includes birch xylan, Zelkova schneideriana Hand.-Mazz. xylan, Herba bromi japonici xylan, corn cob Water soluble pentosan, insoluble corncob xylan, bagasse xylan, bean stalk xylan, cotton seed hull xylan, wheat bran xylan.Synthetic substrate includes soluble starch, carboxymethyl cellulose, chitosan, dextrin.From embodiment Fig. 9 interpretation of result, when recombinase X1-19 is with Herba bromi japonici xylan, water solublity Corncob Xylan, wheat bran xylan for substrate, enzyme activity is of a relatively high, 386,334, the 321% of enzyme activity respectively birch, differs greatly with the substrate specificity that water solublity Corncob Xylan enzyme activity is higher with common xylanase majority.Synthetic substrate soluble starch, CMC, chitosan and dextrin are substantially free of effect.

Claims (10)

1. deriving from a non-cultivation xylanase gene nucleotide sequence for the grand genetic resources of soil, its sequence details is SEQ-1.

2. the homology of at least the 90% of this sequence.

3. a polypeptide for xylanase, its sequence details is SEQ-2.

4. the homology of at least the 90% of this sequence.

5. a signal peptide for xylanase, its sequence details is SEQ-3.

6. the homology of at least the 90% of this sequence.

7. comprise the recombinant vector of right 1,2,3,4,5,6.

8. the recombinant expression system of the Pichia sp. of right 7.

9. it is similar to right 8 at lactobacillus, saccharomyces cerevisiae, bacillus cereus (such as bacillus subtilis), the Expression and Application in aspergillosis (such as aspergillus niger or aspergillus oryzae).

10. right 1,2,3,4,5,6,7,8,9 is in paper industry, food processing, textile industry, feed industry, bioenergy, cosmetics, Medicines and Health Product, and the application of various bioreactor.