CN101402963A - Fire resistant xylanase XynA1, gene for encoding the enzyme and uses thereof - Google Patents
Fire resistant xylanase XynA1, gene for encoding the enzyme and uses thereof Download PDFInfo
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- CN101402963A CN101402963A CNA2008101530762A CN200810153076A CN101402963A CN 101402963 A CN101402963 A CN 101402963A CN A2008101530762 A CNA2008101530762 A CN A2008101530762A CN 200810153076 A CN200810153076 A CN 200810153076A CN 101402963 A CN101402963 A CN 101402963A
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Abstract
The invention discloses a xylanase XynA1 which is obtained by the PCR amplification of Geobacillus thermodenitrificans NG80-2 genome DNA and a recombinant xylanase for constructing an expression vector to express the genetic code in Escherichia coil. The optimal reaction temperature of the xylanase is 70 DEG C, the optimum reaction pH is 7.6, and the thermal stability is good in neutral and weak alkaline environments. The xylanase is applicable to decomposing xylan in hemicellulose to produce functional xylooligosaccharides.
Description
Technical field
The present invention relates to a kind of zytase, particularly in thermophilic denitrifying bacillocin (Geobacillusthermodenitrificans) NG80-2, the xylanase XynA 1 gene is carried out clonal expression, Function Identification and makes up the fire resistant xylanase of escherichia coli high-level expression and the gene and the application of this enzyme of encoding.
Background technology
Along with the develop rapidly of current society, population growth and resource consumption make the development and utilization of renewable resources become the common problem of paying close attention to of international community and academia.Xylan is the main composition of hemicellulose, is distributed widely in the cell walls of higher plant, is a kind of huge renewable biological source of occurring in nature, also is a class hemicellulose of the easiest extraction, degraded and utilization.Zytase and xylobiase are the topmost enzymes of degradation of xylan.In energy industry, the xylan in the agriculture and industry waste can be converted into wood sugar by zytase and xylosidase, and wood sugar can be changed into valuable fuel such as alcohol by bacterium and fungi; In the association with pulp bleaching of paper industry, xylobiase and zytase synergy can effectively improve bleachability; In pharmaceutical industries, zytase and xylosidase hydrolysis specific substrates can be created in the middle converted product that pharmaceutical industries has significant application value.Therefore the research for zytase and xylosidase has extensive and important purposes.
Hemicellulose is to be only second to the utilized natural resources that content of cellulose second enriches, and the xylan in the hemicellulose (xylan) is different according to the source, branch degree difference, and its main chain has multiple different substituted radical with side chain.Because the complicacy of xylan structure, its degraded fully needs the participation acting in conjunction of multiple lytic enzyme to finish.β wherein-1,4-endo-xylanase (EC.3.2.1.8) is the enzyme of most critical in the xylan degrading enzyme.This enzyme acts on the β-1 of xylan backbone inside with internal-cutting way, 4-wood sugar glycosidic bond, and making xylan degrading is the xylo-oligosaccharide of short chain, and has a small amount of wood sugar to generate; And β-D-xylosidase (β-xylosidase EC.3.2.1.37.) then acts on the xylo-oligosaccharide of short chain, and the end by the catalytic oligomerization wood sugar discharges xylose residues.
Zytase all has very big application potential and value in that bio-transformation, pulp and paper industry, foodstuffs industry, fodder industry are first-class.It extensively distributes at nature, all exists in ocean and land bacterium, marine algae, fungi (comprising yeast), cud and ruminating animal bacterium, snail, crustacean, land plant tissue and the various invertebrates.Because it is microbe-derived xylan degrading enzyme is prevalent in nature and Application Areas of a great variety is extensive, therefore a lot of for microbial xylanase research report.At present, people research and to use at most be the zytase in bacterium and mould source.Zytase source is different, and structure and character are not quite similar, and its pH scope of application and the suitableeest scope are also different.In general, the zytase of originated from fungus is many to have maximum vigor under acidic conditions, and the suitableeest scope of pH is 4.0～6.0, belongs to acidic xylanase; Bacterium and actinomycetes zytase belong to neutrality or alkalescent xylanase, and its optimal pH is between 6.0～8.0.Simultaneously, the thermotolerance of zytase is also different and different with its source with optimum temperature.Generally speaking, the zytase optimum temperature of originated from fungus is about 50 ℃, and the optimal reactive temperature of most bacterium and zytase that actinomycetes produce is between 50～60 ℃, and thermotolerance is more a little better than fungi zytase.
Aspect zytase research, the thermophilic bacterial classification that relates to has: whiterot fungi (Ceriporiopsis subvermispora), (Bacillus thermantarcticus), sporotrichum thermophile (Sporotrichum thermophile), short stalk mould (Aureobasidium pullulans ATCC 20524), blue streptomycete (Streptomyces cyaneus SN32), dredge the thermophilic hyphomycete of cotton shape (Thermomyces lanuginosus CAU44), small single-cell bacteria (Cellulosimicrobiumsp.HY-12), Cellulomonas (Cellulomonas cavigena), chaetomium thermophilum (Chaetomiumthermophilum), enterobacteria (Enterobacter sp.MTCC 5112), clostridium acetobutylicum (Clostridiumacetobutylicum ATCC 824), thermomonospora fusca (Thermomonospora sp.), (Caldibacilluscellulovorans), Clostridium thermocellum (clostridium thermocellum), genus bacillus (Bacillus sp.NG-27), subtilis (Bacillus subtilis C-01), thermophilic molten gemma spore bacillus (Geobacillus sp.MT-1), bacillus firmus (Bacillus firmus), heat-resistant bacillus (Thermoactinomyces thalpophilus).
About the up-to-date patent report of xylanase gene.Domestic patent has: Zhan Zhichun has reported a kind of zytase and gene (No. 200510018452.3, date of application 2005.03.25) thereof; People such as Yuan Jianguo have reported bacillus pumilus (Bacillu spumilus) zytase and gene (No. 200510042557.2, date of application 2005.03.10) thereof; People such as Dong Zhiyang have reported the zytase of bacillus pumilus BP19 and gene thereof respectively (02131439.X number, date of application 2002.10.14) and pichia pastoris (Pichia Pastoris) PGX722 zytase and gene (No. 200310103246.3, date of application 2003.11.03) thereof; People such as Jiang Zhengqiang have reported Thermotoga maritima (Thermotogamaritima) MSB8 zytase and gene (No. 02156022.6, date of application 2002.12.11) thereof; People such as Li Ying have reported a kind of zytase and gene (No. 200310113562.9, date of application 2003.11.17) thereof; People such as Tu Jun have reported yeast gene engineering bacteria GS115/HB705 zytase and gene (No. 200510018574.2, date of application 2005.04.19) thereof; Song Yonglin has reported Trichodermareesei (Trichoderma reesei) zytase and gene (No. 02823195.3, date of application 2002.11.20) thereof; People such as Zhang Guimin have reported a kind of zytase and gene (No. 200610020049.9, date of application 2006.08.29) thereof; People such as V lattice Robbie that have reported a kind of from anaerobic thermophilic bacterium acquisition thermostable xylanases and gene (No. 95190553.8, date of application 1995.06.14) thereof.International monopoly has: people such as De Buyl have reported a kind of zytase and gene (the 09/909th, No. 207, date of application 2001.7.19) thereof that obtains from genus bacillus (Bacillus sp.) 720/1; People such as Sung have reported a kind of thermostable xylanases and gene (the 09/856th, No. 025, date of application 1999.11.16) thereof; People such as Bhosle have reported a kind of thermophilic alkali resistant xylanase and gene (the 10/223rd, No. 852, date of application 2002.8.20) thereof that obtains from Pseudomonas stutzeri (Pseudomonas stutzeri); People such as Bentzien have reported a kind of thermophilic alkali resistant xylanase and gene (the 09/570th, No. 856, date of application 2000.5.12) thereof; People such as Dunlop have reported a kind of thermophilic alkali resistant xylanase and gene (the 09/639th, No. 354, date of application 2000.4.16) thereof that obtains from subtilis (Bacillussubtilis); People such as De Buyl have reported a kind of zytase and gene (the 08/470th, No. 953, date of application 1995.6.6) thereof of the wide pH scope that obtains from genus bacillus (Bacillus sp.) 720/1; People such as Williams have reported a kind of thermophilic alkali resistant xylanase that obtains and gene (the 08/501st, No. 126, date of application 1995.12.29) thereof from the basophilia bacterium; People such as Morgan have reported a kind of thermophilic alkali resistant xylanase and gene (the 08/732nd, No. 242, date of application 1997.4.7) thereof that obtains from gentle bent micropolysporaceae (Microtetraspora flexuosa); People such as Gronberg have reported a kind of thermophilic zytase and gene (the 08/591st, No. 685, date of application 1997.2.5) thereof; People such as Outtrup have reported a kind of alkali resistant xylanase and gene (the 08/470th, No. 398, date of application 1995.6.6) thereof that obtains from basophilia genus bacillus (Bacillus sp.) AC13; People such as Vehmaanpera have reported a kind of thermostable xylanases and gene (the 08/468th, No. 812, date of application 1995.6.6) thereof that obtains from actinomycetes (Actinomadura spp.); People such as Rele have reported alkalescent xylanase and the gene (the 08/294th, No. 068, date of application 1994.8.22) thereof that obtains in a kind of from the beginning born of the same parents bacterium (Ccphalosporium); People such as Casimir-Schenkel have reported a kind of thermostable xylanases and gene (the 08/292nd, No. 147, date of application 1994.4.17) thereof that obtains from brown high temperature Zymomonas mobilis (Thermomonospora fusca); People such as Rosenberg have reported a kind of thermophilic alkali resistant xylanase and gene (the 08/063rd, No. 551, date of application 1993.5.18) thereof that obtains from bacstearothermophilus (Bacillusstearothermophilus) NCIMB 40221; People such as Campbell have reported a kind of thermostable xylanases and gene (the 08/044th, No. 621, date of application 1993.4.8) thereof that obtains from Bacillus circulans (Bacillus circulans); People such as Yu have reported a kind of thermostable xylanases and gene (the 07/340th, No. 307, date of application 1989.4.19) thereof or the like.Although the patent report about xylanase gene has many pieces, the gene that obtains thermostable xylanases and coding thereof from thermophilic denitrifying bacillocin is not seen bibliographical information as yet.
The inventor discloses first in thermophilic Crude Oil-degrading Bacteria, adopts thermophilic denitrifying bacillocin (Geobacillusthermodenitrificans) NG80-2 that xylanase gene is carried out clonal expression, Function Identification and makes up the escherichia coli high-level expression engineering strain.By aminoacid sequence comparison and analysis, prove the albumen of the poly-enzyme XynA1 genes encoding of wood of the present invention and gene (the GenBank accession no.P40943) similarity the highest (consensus amino acid sequence is 85%) of the coding zytase among known bacstearothermophilus (Geobacillus stearothermophilus) T6, with other known zytase similarity less than 58%.This enzyme belongs to GH10 family, and the N end has the signal peptide of 28 amino-acid residues.The 30th of the aminoacid sequence of xylanase XynA 1 of the present invention, 216,364,377 is Threonine, the 31st, 32,195,274,315,394 is L-glutamic acid, the 39th, 109,143 is glutamine, the 55th is Serine, the 68th is leucine, the 93rd, 383,396 is l-asparagine, the 105th is L-Ala, the 44th, 117,118 is Histidine, the 116th, 223,373 is Methionin, the 269th, 285,323,392 is aspartic acid, the 281st is Isoleucine, this aminoacid sequence with the zytase of Geobacillus stearothermophilus T6 different (seeing Figure 11) therefore shows better thermostability.This XynA1 gene has been carried out further Function Identification, and compare with xylanase activity among the Geobacillus stearothermophilus T-6, experimental result shows: xylanase XynA 1 of the present invention is a kind of novel enzyme, this kind of enzyme is not only high temperature resistant, and good heat stability, thereby finished work of the present invention.
Summary of the invention
Purpose of the present invention is to disclose a kind of high temperature resistant recombined xylanase XynA1.
Another purpose of the present invention is the gene that discloses a kind of xylanase XynA 1 of the present invention of encoding.
Still a further object of the present invention is the recombinant plasmid that discloses a kind of expressed xylanase, wherein comprises the encoding gene of above-mentioned xylanase XynA 1 at least.
A present invention also purpose is to disclose a kind of above-mentioned xylanase XynA 1 encoding gene that imported, and produces the reorganization bacterium of zytase, for example contains the recombinant bacterial strain H1739 of recombinant plasmid.
Technical scheme of the present invention is as follows:
A kind of gene of the xylanase XynA 1 of encoding, this gene have the nucleotide sequence of the group of being selected from down:
A) nucleotide sequence shown in the SEQ ID NO:1; Or b) is different from SEQ ID NO:1 but the amino acid sequence coded aminoacid sequence identical nucleotide sequence coded with SEQ ID NO:1; Or c) under stringent hybridization condition with described a) or b) in sequence hybridization, and coding has an active zytase nucleotide sequence.
Should be pointed out that the above-mentioned term of mentioning " stringent condition " implication in this manual is meant has formed so-called specific hybridization and has not formed non-specific hybridization under this condition.For example, this stringent condition can be, homology each other is not less than can hybridize between 70% the DNA and be lower than between the DNA of above-mentioned numerical value and can not hybridize, and preferably homology is no less than between 90% the DNA and can hybridizes.For common wash conditions in the Southern hybridization, can for example be following hybridization conditions: with Hybond membrane place prehybridization solution (the 0.25mol/L sodium phosphate buffer, pH7.0,7%SDS) in, 50 ℃ of prehybridization 30min; Abandon prehybridization solution, add hybridization solution (0.25mol/L sodium phosphate buffer, pH7.0,7%SDS, isotope-labeled nucleotide fragments), 50 ℃ of hybridization 12hr; Abandon hybridization solution, add film washing liquid I (2 * SSC and 0.1%SDS), wash film 2 times for 50 ℃, each 30min; Add film washing liquid II (0.5 * SSC and 0.1%SDS), wash film 30min for 50 ℃.
The present invention further discloses xylanase XynA 1, this enzyme has nucleotide sequence coded aminoacid sequence.Perhaps preferably has the aminoacid sequence shown in the SEQ ID NO:2; Or disappearance in above-mentioned, replace or insert behind one or more amino acid and have the aminoacid sequence of described xylanase activity.
The present invention further discloses a kind of recombinant expression plasmid and reorganization bacterium again, and it contains the gene of the nucleotide sequence shown in the SEQ ID NO:1.The carrier of described recombinant plasmid is pET-28a (+), and recombinant plasmid is pLW1380.Contain the recombinant bacterial strain H1739 of recombinant plasmid, these all belong to protection scope of the present invention.
Any segmental primer is right in the amplification xylanase XynA 1, also within protection scope of the present invention.
In the preferred embodiment of the present invention, the suitableeest substrate of above-mentioned xylanase XynA 1 is the beech wood glycan;
In the preferred embodiment of the present invention, the temperature of reaction of above-mentioned zytase is 35 ℃-95 ℃, preferred 70 ℃.
In the preferred embodiment of the present invention, above-mentioned zytase pH value in reaction is 4.0-10.6, and preferred optimal reaction pH value is 7.6.
The recombinant plasmid of expressed xylanase provided by the invention is pLW1380, comprises the encoding gene of above-mentioned xylanase XynA 1 at least.
In the preferred embodiment of the present invention, the carrier of above-mentioned recombinant plasmid is pET-28a (+).
In the preferred embodiment of the present invention, imported the encoding gene of xylanase XynA 1 in the reorganization bacterium of zytase.
Described reorganization bacterium is intestinal bacteria.Be preferably e. coli strain bl21.
The person of ordinary skill in the field will be appreciated that, the encode dna sequence dna of xylanase XynA 1 of the present invention comprises that also coding carries out one or more amino acid to the aminoacid sequence of the expressed enzyme molecule of nucleotide sequence shown in the SEQ ID NO:1 and replaces, inserts or disappearance and the proteinic nucleotide sequence that still has this enzymic activity.
In addition, the amino acid of the expressed enzyme molecule of xylanase gene of the present invention is carried out one or more amino acid replace, insert or lack resulting protein, also can reach purpose of the present invention.Thereby the present invention also comprises with the aminoacid sequence shown in the SEQ IDNO:2 having at least 70% homology, preferably has at least 90% homology, but has the protein of xylanase activity simultaneously.The term that uses above " a plurality of " can be the number less than 100, is preferably the number less than 10.
XynA1 gene of the present invention, it is to be template with isolating xylanase XynA 1 from thermophilic denitrifying bacillocin NG80-2 (GTNG_1761 genes encoding) genomic dna, according to XynA1 gene complete sequence, design two pairs of primers, amplify the XynA1 gene fragment effectively and carried out topology, clone and determined dna sequence, further experiment shows: xylanase XynA 1 interrupts the long-chain of xylan and is the oligomerization xylan, then by xylosidase with oligomerization xylan thoroughly degrade (as Fig. 1).
The XynA1 that the present invention proposes compares with known zytase, and the XynA1 zytase that the present invention proposes belongs to novel enzyme, and is not only high temperature resistant, and good heat stability.Long the having of zytase thermostability introduced in the article of having published at present: the zytase among bacstearothermophilus (Geobacillus stearothermophilus) T6 can be preserved 10 hours (Khasin in 65 ℃, A., et al, Purification and characterization of athermostable xylanase from Bacillus stearothermophilus T-6.Appl.Environ.Microbiol.1993.59:1725-30.); Zytase among actinomycetes (Streptomyces cyaneus) SN32 can be preserved 1 hour (Ninawe in 60 ℃, S., et al, Purification and characterization of extracellular xylanasefrom Streptomyces cyaneus SN32, Bioresour.Technol.2007.); Zytase in the cellulomonas cartae (Cellulomonasxavigena) can be preserved 1 hour (Santiago-Hern á ndez in 55 or 60 ℃, A., et al, Purification and characterization of two sugarcane bagasse-absorbable thermophilicxylanases from the mesophilic Cellulomonas Xavigena, J.Ind.Microbiol.Biotechnol.2007.34:331-338); Zytase among enterobacter cloacae (Enterobacter sp.) MTCC 5112 can be preserved 50 hours (Khandeparkar in 50 ℃, R., et al, Purification and characterization ofthermoalkalophilic xylanase isolated from the Enterobacter sp.MTCC 5112, Res.Microbiol.2006.157:315-325); Zytase among Aureobasidium pullulans (Aureobasidium pullulans) ATCC 20524 can be preserved half an hour (Tanaka in 65 ℃, H., et al, Purification and properties ofa family-10 xylanase from Aureobasidium pullulans ATCC 20524 and characterization ofthe encoding gene, Appl.Microbiol.Biotechnol.2006.70:202-211); Zytase in the bacillus firmus (Bacillus firmus) can be preserved 16 hours (Chang in 62 ℃, P., et al, Cloning andcharacterization of two thermostable xylanases from an alkaliphilic Bacillus firmus.Biochem.Bioph.Res.Co.2004.319:1017-1025); Zytase in the bacillus acidocldarius (Bacillusthermantarcticus) can be preserved 24 hours (Lama in 60 ℃, L., et al, Purificationand characterization of thermostable xylanase and β-xylosidase by the thermophilicbacterium Bacillus thermantarcticus, Res.Microbiol.2004.155:283-289); Zytase among clostridium acetobutylicum (Clostridium acetobutylicum) ATCC82 can be preserved 1 hour (Ali in 60 ℃, M.K.et al, Thermostable xylanase 10B from Clostridium acetobutylicum ATCC824, J.Ind.Microbiol.Biotechnol.2004.31:229-234); Zytase in the thermomonospora fusca (Thermomonospora sp.) can be preserved 8 hours (Geore in 60 ℃, S.P., et al, A novel thermostable xylanase fromThermomonospora sp.:influence of additives on thermostability, Bioresour.Technol.2001.78:221-224); Zytase in the thermophilic actinomycete (Thermoactinomyces thalophilus) can be preserved 2 hours (Kohli in 65 ℃, U.et al, Thermostable, alkalophilic and cellulase free xylanaseproduction by Thermoactinomyces thalophilus subgroup C, Enzyme Microb.Technol.2001.28:606-610).
Xylanase XynA 1 of the present invention beneficial effect compared with prior art is: compare the thermostability height of this enzyme with known zytase, and still have the activity more than 50% after hatching 24 hours under 55 ℃, can preserve 4 hours 75 ℃ of following activity.
Xylanase XynA 1 performance of the present invention is different from the zytase of having reported, and this xylanase XynA 1 has the highly active feature of maintenance under high temperature, high pH condition.The xylanase XynA 1 optimal reactive temperature is 70 ℃; Optimal reaction pH 7.6.Heat stability is good in neutral and meta-alkalescence.The xylan that is fit to decompose in the hemicellulose is produced functional low polyxylose.
Description of drawings
Fig. 1 is a zytase degradation of xylan effect synoptic diagram of the present invention;
Fig. 2 is the xylanase gene construction of recombinant plasmid mode chart in the embodiment of the invention
Fig. 3 represents zytase SDS-PAGE electrophorogram of the present invention.
Fig. 4 represents the relative vigor of zytase of the present invention to different substrates.
Fig. 5 represents the relative vigor of zytase of the present invention under differing temps.
Fig. 6 represents the relative vigor of zytase of the present invention under different pH values.
Fig. 7 represents the thermostability under 55 ℃ of zytase of the present invention.
Fig. 8 represents the thermostability under 75 ℃ of zytase of the present invention.
Fig. 9 represents the aminoacid sequence shown in the zytase SEQ ID NO:2 of the present invention.
Figure 10 represents the nucleotide sequence shown in the zytase SEQ ID NO:1 of the present invention.
Figure 11 represents the comparison of the zytase aminoacid sequence among xylanase XynA 1 of the present invention and the Geobacillus stearothermophilus T6.
Embodiment
For above and other objects of the present invention, feature and advantage can be become apparent, preferred embodiment cited below particularly, and conjunction with figs. elaborate.Following specific embodiment is only used for explanation rather than restriction the present invention.
Embodiment one
1. the extraction of the total DNA of thermophilic denitrifying bacillocin NG80-2 (CGMCC No.1228)
The extraction of the total DNA of thermophilic denitrifying bacillocin NG80-2 (CGMCC No.1228) studies have shown that: the gene that can be isolated the coding alcoholdehydrogenase by thermophilic denitrifying bacillocin (Geobacillus thermodenitrificans) NG80-2 genome.Therefore, in the present embodiment, (its preserving number at China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCC No.1228 to the thermophilic denitrifying bacillocin NG80-2 that employing obtains from Chinese Tianjin Dagang Oilfield official 69-8 block oil-well strata water sepn, preservation date is on October 09th, 2004, patent of invention and obtaining the authorization in the applicant country, denomination of invention: thermophilic denitrifying bacillocin and screening thereof and application, the patent No.: ZL200410072759.7), get the fresh culture thing 3ml of its incubated overnight, centrifugal collection thalline, thalline is suspended from the 250 μ l50mM Tris damping fluids (pH8.0), add 10 μ l 0.4M EDTA (pH8.0), 37 ℃ of insulation 20min behind the mixing, add 30 μ l 20mg/ml N,O-Diacetylmuramidases afterwards, 37 ℃ are incubated 20min again behind the mixing, add 5 μ l 20mg/ml Proteinase Ks again, behind the gentle mixing, add 20 μ l 10%SDS again, 50 ℃ are incubated to solution and clarify, use equal-volume phenol respectively: chloroform: primary isoamyl alcohol extracting twice, chloroform: the primary isoamyl alcohol extracting once, last supernatant solution, the dehydrated alcohol that adds 2.5 times of volume precoolings, reclaim DNA, wash with 70% ethanol, precipitation is dissolved in 100 μ l TE damping fluid (pH8.0,10mMTris, 1mMEDTA), add 10mg/ml RNase 2 μ l, 65 ℃ of insulation 30min, use phenol respectively: chloroform: primary isoamyl alcohol, chloroform: each extracting of primary isoamyl alcohol once, supernatant liquor adds the dehydrated alcohol of 2.5 times of volume precoolings, reclaims DNA, washes with 70% ethanol, vacuum-drying, precipitation is dissolved in 50 μ l TE damping fluids.The ultraviolet spectrophotometer measurement result of dna solution is A
260/ A
280=1.95, A
260=0.73.
2. the clone of xylanase gene XynA1 and screening
Get the total dna solution 0.5 μ l (about 10ng) of thermophilic denitrifying bacillocin NG80-2 (CGMCC No.1228) as template, as primer, carry out pcr amplification with following oligonucleotide sequence.
(50ul) is as follows for reaction mixture:
Dd H
2O, 34ul; 10 * PCR Buffer, 5ul; MgCl
2Buffer, 5ul; DNTP Mix, 2ul; Upstream primer, 1ul; Downstream primer, 1ul; The NG80-2 genomic dna, 1ul; Taq DNA polymerase, 1ul.
Primer sequence is as follows:
Upstream primer is the nucleotide sequence shown in the SEQ ID NO:3, and downstream primer is the nucleotide sequence shown in the SEQ ID NO:4
The PCR loop parameter of setting is as follows:
95℃，5min；95℃，30s；50℃，45s；72℃，2min；72℃，10min；4℃，2hr。Carry out 25 cycle P CR by the PCR loop parameter of above-mentioned setting.
Above-mentioned PCR product EcoRI and XhoI double digestion, the agarose gel electrophoresis through 0.8% is cut glue recovery enzyme and is cut the product segment.With through same restricted type restriction endonuclease enzymolysis and cut the plasmid pET-28a (+) that glue reclaims and be connected, behind transformed competence colibacillus intestinal bacteria (E.coli) the DH5 α, be applied on the LB solid medium that contains 50 μ g/ml Kan (kantlex).Cultivated 12 hours for 37 ℃, the picking mono-clonal is transferred in the 20ml LB substratum cultivates (50 μ g/ml Kan), cultivate after 12 hours for 37 ℃, the extraction plasmid is identified, pET-28a (+) plasmid that is inserted with the dna sequence dna of SEQ ID NO:1 is a recombinant plasmid pLW1380 (see figure 2), (this bacterial strain can be ordered to the thick general biotechnology in development area, Tianjin development corporation, Ltd. to change recombinant plasmid pLW1380 over to the expressive host e. coli bl21, article No. is 69387-3) in, the recombinant bacterial strain that obtains containing this recombinant plasmid is H1739, but this recombinant plasmid pLW1380 high frequency transformed into escherichia coli BL21 expressed xylanase is active and anti-kantlex performance.
Enzyme wherein cut and ligation all with reference to table 1.
Table 1 enzyme is cut system and linked system
3.XynA1DNA segmental mensuration
Adopt the Sanger method that this dna fragmentation is checked order, sequencing result shows that the insertion fragment among the pLW1380 contains the open reading frame of long 1224bp (SEQ ID NO:1), the protein that coding is made up of the individual amino acid of 407 (SEQ ID NO:2).
Embodiment two
The expression and purification of recombined xylanase and characteristic:
Above-mentioned reorganization bacterium H1739 mono-clonal is inserted 20ml to be contained in the LB substratum of 50 μ g/ml Kan, 37 ℃, 180rpm cultivated 12 hours, then culture was inserted the LB substratum that 200ml contains 50 μ g/ml Kan by 1% (v/v) inoculum size, 37 ℃, 220rpm is cultured to OD
600Be 0.6 o'clock, add IPTG to final concentration be 0.05mM, induced 3 hours for 45 ℃.The centrifugal 5min of 5000rpm collects thalline, is suspended to contain in 50mM Tris-HCl (pH8.0) damping fluid, utilizes the ultrasonic disruption cell, and the centrifugal 20min of 14000g, supernatant liquor are the crude extract of alcoholdehydrogenase.This supernatant liquor is through chelating sepharose (Chelating Sepharose) nickel affinity column chromatography purification, and the zymin that obtains shows a band (see figure 3) on SDS-PAGE.Utilize the standard method of known protein materialization to measure the fundamental characteristics of this enzyme system.The molecular weight of the recombinase that records with SDS-PAGE is 48kDa, and is similar to the molecular weight of calculating in theory (47399Da); The iso-electric point pI of the recombinase that isoelectric point precipitation records is 5.83.
Embodiment three
1. the ratio vigor when mensuration zytase of the present invention acts on different sources xylan substrate:
Zytase is made an addition in animal-feed or the paper making pulp, should consider its activity and the influence of specificity to using to the xylan of different sources, the oat xylan is as wheat crops (wheat, rye and triticale) xylan pattern substrate, and beech wood glycan and birchwood glycan are investigated its ratio vigor to the different sources xylan as timber source xylan pattern substrate.
Can the degrade xylan of different sources of the zytase that makes in the foregoing description two, the three kind xylan substrates of this experiment to buying at present: oat xylan (the Sigma article No. is XO627), beech wood glycan (the Sigma article No. is X4252), birchwood glycan (the Sigma article No. is X0502), carried out the enzyme activity detection.Concrete grammar is: preparation xylanase XynA 1 reaction system (100 μ l) is: add oat xylan, beech wood glycan, birchwood glycan substrate to final concentration 1% (w/v), certain density xylanase XynA 1 is mended to 100 μ l with the Tris-HCl damping fluid of 50mM pH7.6.Mixing, reaction is 15 minutes in 70 ℃ of water-baths, and after reaction finished, the ice bath termination reaction added 1%DNS reagent (3,5 dinitrosalicylic acids 1% of 150 μ l; Phenol 0.2%; Sodium sulfate 0.05%; Seignette salt 20%; Sodium hydroxide 1%.Lucifuge is placed the back use of two weeks.) boiling water bath 10 minutes, measure photoabsorption then at the 540nm place.With the wood sugar is typical curve, calculates per minute and generates the required enzyme amount of 1 μ mol reducing sugar, is defined as 1 enzyme activity unit.Measurement result is referring to Fig. 4, and as can be seen from the figure, zytase all can be degraded to the commercialization xylan of three kinds of different sourcess, and its suitableeest substrate is the beech wood glycan.
2. measure the ratio vigor of zytase of the present invention under differing temps:
The zytase that makes in the foregoing description two is carried out the mensuration of optimal reactive temperature, concrete grammar is: preparation xylanase XynA 1 reaction system (100 μ l) is: add oat xylan substrate to final concentration 1% (w/v), certain density xylanase XynA 1 is mended to 100 μ l with the Tris-HCl damping fluid of 50mM pH 7.6.Mixing, respectively at reaction in 25～100 ℃ of water-baths 15 minutes, after reaction finishes, the ice bath termination reaction.The DNS reagent boiling water bath 10 minutes that adds 150 μ l is measured photoabsorption then at the 540nm place.With the wood sugar is typical curve, calculates per minute and generates the required enzyme amount of 1 μ mol reducing sugar, is defined as 1 enzyme activity unit.Measurement result is referring to Fig. 5.As can be seen from the figure, the optimal reactive temperature of zytase is about 70 ℃.
3. measure the ratio vigor of zytase of the present invention under different pH:
The zytase that makes in the foregoing description two is carried out the mensuration of optimal reaction pH value, concrete grammar is: prepare 100 μ l reaction systems, the final concentration of substrate oat xylan is 1% (M/V), certain density xylanase XynA 1 uses the damping fluid (prescription sees Table 2) of 50mM pH3-11.9 to mend to 100 μ l respectively.Mixing, reaction is 15 minutes in 70 ℃ of water-baths, after reaction finishes, the ice bath termination reaction.The DNS reagent boiling water bath 10 minutes that adds 150 μ l is measured photoabsorption then at the 540nm place.With the wood sugar is typical curve, calculates per minute and generates the required enzyme amount of 1 μ mol reducing sugar, is defined as 1 enzyme activity unit.Measurement result is referring to Fig. 6, and as can be seen from the figure, the pH value of zytase is 4.0-10.6, and preferred optimal reaction pH value is 7.6.This in neutral meta-alkalescence scope stable properties help industrial application.
Used damping fluid during table 2 xylanase activity is measured
| The pH scope | Damping fluid |
| pH 3-6 | citrate-sodium citrate |
| pH 6-8 | K 2HPO 4-KH 2PO 4 |
| pH 8-9 | Tris-HCl |
| pH 8.6-10.6 | glycine-NaOH |
| pH 11-11.9 | Na 2HPO 4-NaOH |
4. measure the thermostability of zytase of the present invention:
The zytase that makes in the foregoing description two is carried out the mensuration of thermostability, and concrete grammar is: certain density xylanase XynA 1 is put into 55 and 75 ℃ of water-bath temperature bathe, took out a part of enzyme every one hour and do the enzyme reaction of living.
Preparation xylanase XynA 1 reaction system (100 μ l) is: add oat xylan substrate to final concentration 1% (w/v), certain density xylanase XynA 1 is mended to 100 μ l with the Tris-HCl damping fluid of 50mM pH 7.6.Mixing, reaction is 15 minutes in 70 ℃ of water-baths, after reaction finishes, the ice bath termination reaction.The DNS reagent boiling water bath 10 minutes that adds 150 μ l is measured photoabsorption then at the 540nm place.With the wood sugar is typical curve, calculates per minute and generates the required enzyme amount of 1 μ mol reducing sugar, is defined as 1 enzyme activity unit.Measurement result is referring to Fig. 7,8.As can be seen from the figure, xylanase XynA 1 still keeps the enzymic activity more than 50% after 55 ℃ of temperature are bathed 24 hours, bathe 4 hours inactivations 75 ℃ of temperature.
Embodiment four
1. the aminoacid sequence of xylanase XynA 1 of the present invention and the aminoacid sequence of Geobacillus stearothermophilus T6 zytase are compared, comparison result is referring to Figure 11.
2. it is different that xylanase XynA 1 of the present invention and the every zymetology parameter of zytase among the Geobacillus stearothermophilus T6 all have, and sees table 3 for details.
The comparison of the zytase zymetology parameter among table 3XynA1 and the Geobacillus stearothermophilus T6
| NG80-2 | T6 | |
| Optimal reactive temperature (℃) | 70 | 65 |
| Optimal reaction pH | 7.6 | 6.5 |
| Thermostability | 55 ℃ of temperature are bathed and are still had activity more than 50% after 24 hours, and 75 ℃ of following activity can be preserved 4 hours | Bathe 10 hours inactivations 65 ℃ of temperature |
| Than vigor (U/mg) | 404.85 | 288 |
From Figure 11 and table 3 as can be seen, because amino acid whose variation, the xylanase XynA 1 among the NG80-2 is higher than the xylanase activity among the Geobacillus stearothermophilus T6, and is more alkaline-resisting thermophilic, has more thermostability.
Though the present invention discloses as above with preferred embodiment, so it is not that any person of ordinary skill in the field not breaking away from change and the improvement that the spirit and scope of the present invention are done, will fall into protection scope of the present invention in order to qualification the present invention.
Sequence table
＜110〉Nankai University
＜120〉gene and the application of fire resistant xylanase XynA 1 and this enzyme of coding
<160>4
<210>1
<211>1224
<212>DNA
＜213〉thermophilic denitrifying bacillocin (Geobacillus thermodenitrificans) NG80-2
<220>
<221>gene
<222>(1)..(1224)
<400>1
atgttgaaaa gatcgcgaaa agcgataata gttggattct catttatgct gttgctccct 60
ttagggatga cgaatgcatt ggcaaaaact gaacaatcat acgctaaaaa gcctcaaatc 120
agcgcattgc atgccccaca attggaccag cgctacaaag attcattcac tattggtgcg 180
gctgttgaac cttatcagct actaaacgag aaagacgcac aaatgctaaa acgccatttt 240
aacagcattg tcgctgagaa cgttatgaag ccgattaata ttcaaccgga agaaggaaaa 300
ttcaattttg ctgaggcgga tcaaatcgtt cggtttgcta aaaaacacca tatggatatc 360
cgtttccaca cgctcgtttg gcatagccaa gtacctcaat ggttctttct tgacaaggaa 420
ggccaaccaa tggtcaatga aacggaccct gtgaaacgcg aacaaaataa acagctgtta 480
ttaaaacgga tcgaaaccca tattaaaacg attgtcgagc ggtataaaga tgacatcaaa 540
tattgggacg ttgtaaatga ggtagtcggg gatgatggag aattgcgcga ttccccatgg 600
tatcaaatcg ctggcatcga ttatattaag gtagcgttcc aaacagcgag aaaatatggc 660
ggcaacaaga ttaaacttta cattaatgat tacaatacgg aagttgaacc aaagcgaagc 720
gctctttata acttggtgaa acaattaaaa gaagagggca ttcccattga tggtattggc 780
catcagtccc acatccaaat tgactggcct tctgaagagg aaatcgaaaa aacgattatc 840
atgtttgccg atctagggtt agacaaccaa attactgagc tggatgtgag catgtacggc 900
tggccgccgc gggcttacct gtcgtatgac gccattccgg agcaaaagtt tttggaccaa 960
gcggatcgct atgatcgatt gtttaagctg tatgaaaaac tcagcgataa aatcagtaac 1020
gtcaccttct ggggcatcgc cgacaaccat acgtggctcg acagtcgagc ggatgtttac 1080
tatgatactg atgggaatgt gattgtagac ccgaaagccc catatacgag agtggaaaaa 1140
gggaatggaa aagatgcgcc atttgtgttc gaccccgaat acaacgtaaa acctgcgtat 1200
tgggctatta tcgatcataa gtga 1224
<210>2
<211>407bp
<212>PRT
＜213〉thermophilic denitrifying bacillocin (Geobacillus thermodenitrificans) NG80-2
<220>
<221>CHAIN
<222>(1)..(407)
<400>2
Met Leu Lys Arg Ser Arg Lys Ala Ile Ile Val Gly Phe Ser Phe Met
1 5 10 15
Leu Leu Leu Pro Leu Gly Met Thr Asn Ala Leu Ala Lys Thr Glu Gln
20 25 30
Ser Tyr Ala Lys Lys Pro Gln Ile Ser Ala Leu His Ala Pro Gln Leu
35 40 45
Asp Gln Arg Tyr Lys Asp Ser Phe Thr Ile Gly Ala Ala Val Glu Pro
50 55 60
Tyr Gln Leu Leu Asn Glu Lys Asp Ala Gln Met Leu Lys Arg His Phe
65 70 75 80
Asn Ser Ile Val Ala Glu Asn Val Met Lys Pro Ile Asn Ile Gln Pro
85 90 95
Glu Glu Gly Lys Phe Asn Phe Ala Glu Ala Asp Gln Ile Val Arg Phe
100 105 110
Ala Lys Lys His His Met Asp Ile Arg Phe His Thr Leu Val Trp His
115 120 125
Ser Gln Val Pro Gln Trp Phe Phe Leu Asp Lys Glu Gly Gln Pro Met
130 135 140
Val Asn Glu Thr Asp Pro Val Lys Arg Glu Gln Asn Lys Gln Leu Leu
145 150 155 160
Leu Lys Arg Ile Glu Thr His Ile Lys Thr Ile Val Glu Arg Tyr Lys
165 170 175
Asp Asp Ile Lys Tyr Trp Asp Val Val Asn Glu Val Val Gly Asp Asp
180 185 190
Gly Glu Leu Arg Asp Ser Pro Trp Tyr Gln Ile Ala Gly Ile Asp Tyr
195 200 205
Ile Lys Val Ala Phe Gln Thr Ala Arg Lys Tyr Gly Gly Asn Lys Ile
210 215 220
Lys Leu Tyr Ile Asn Asp Tyr Asn Thr Glu Val Glu Pro Lys Arg Ser
225 230 235 240
Ala Leu Tyr Asn Leu Val Lys Gln Leu Lys Glu Glu Gly Ile Pro Ile
245 250 255
Asp Gly Ile Gly His Gln Ser His Ile Gln Ile Asp Trp Pro Ser Glu
260 265 270
Glu Glu Ile Glu Lys Thr Ile Ile Met Phe Ala Asp Leu Gly Leu Asp
275 280 285
Asn Gln Ile Thr Glu Leu Asp Val Ser Met Tyr Gly Trp Pro Pro Arg
290 295 300
Ala Tyr Leu Ser Tyr Asp Ala Ile Pro Glu Gln Lys Phe Leu Asp Gln
305 310 315 320
Ala Asp Arg Tyr Asp Arg Leu Phe Lys Leu Tyr Glu Lys Leu Ser Asp
325 330 335
Lys Ile Ser Asn Val Thr Phe Trp Gly Ile Ala Asp Asn His Thr Trp
340 345 350
Leu Asp Ser Arg Ala Asp Val Tyr Tyr Asp Thr Asp Gly Asn Val Ile
355 360 365
Val Asp Pro Lys Ala Pro Tyr Thr Arg Val Glu Lys Gly Asn Gly Lys
370 375 380
Asp Ala Pro Phe Val Phe Asp Pro Glu Tyr Asn Val Lys Pro Ala Tyr
385 390 395 400
Trp Ala Ile Ile Asp His Lys
405
<210>3
<211>29bp
<212>DNA
＜213〉artificial sequence
<400>3
ccggaattca tgttgaaaag atcgcgaaa 29
<210>4
<211>29bp
<212>DNA
＜213〉artificial sequence
<400>4
ccgctcgagt cacttatgat cgataatag 29
Claims (9)
1, a kind of gene of the xylanase XynA 1 of encoding, this gene have the nucleotide sequence of the group of being selected from down:
A) nucleotide sequence shown in the SEQ ID NO:1; Or
B) be different from SEQ ID NO:1 but the amino acid sequence coded aminoacid sequence identical nucleotide sequence coded with SEQ ID NO:1; Or
C) under stringent hybridization condition with described a) or b) in sequence hybridization, and coding has an active zytase nucleotide sequence.
2, a kind of xylanase XynA 1, it has the aminoacid sequence shown in the SEQ ID NO:2;
3, a kind of recombinant expression plasmid, it contains the described xylanase XynA 1 encoding gene of claim 1.
4, the described recombinant expression plasmid of claim 3, the carrier of described recombinant plasmid is pET-28a (+).
5, the described xylanase XynA 1 encoding gene of claim 1 is characterized in that described zytase separates to obtain in thermophilic denitrifying bacillocin NG80-2.
6, the described xylanase XynA 1 encoding gene of claim 1 is characterized in that the suitableeest substrate of described zytase is the beech wood glycan; Optimal reactive temperature is 70 ℃.
7, the described xylanase XynA 1 encoding gene of claim 1 is characterized in that described zytase pH value is 7.6.
8, a kind of reorganization bacterium that produces zytase is characterized in that it contains the described xylanase XynA 1 encoding gene of claim 1.
9, the described xylanase XynA 1 reorganization of claim 8 bacterium is characterized in that described reorganization bacterium is an e. coli strain bl21.
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|---|---|---|---|
| CNA2008101530762A CN101402963A (en) | 2008-11-14 | 2008-11-14 | Fire resistant xylanase XynA1, gene for encoding the enzyme and uses thereof |
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|---|---|---|---|
| CNA2008101530762A CN101402963A (en) | 2008-11-14 | 2008-11-14 | Fire resistant xylanase XynA1, gene for encoding the enzyme and uses thereof |
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| CN101402963A true CN101402963A (en) | 2009-04-08 |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101701205B (en) * | 2009-10-30 | 2011-08-17 | 中国农业科学院饲料研究所 | Alkali-resistant xylanase XynE2, genes thereof and application thereof |
| CN101724613B (en) * | 2009-12-15 | 2012-07-04 | 中国农业科学院饲料研究所 | Alkali proof intermediate temperature xylanase XYNAM6, gene thereof and application thereof |
| CN102643842A (en) * | 2012-02-27 | 2012-08-22 | 北京工商大学 | Heat-resisting xylanase gene derived from actinomycetes (streptomyces rameus L2001) and product xylanase (Xyn A) thereof |
| CN102676464A (en) * | 2012-05-18 | 2012-09-19 | 南开大学 | Thermophilic long-chain alkyl aldehyde dehydrogenase and crystal structure thereof |
| CN102712932A (en) * | 2010-02-10 | 2012-10-03 | 永丰馀造纸股份有限公司 | Modified nucleotide molecules of xylanase and application thereof |
| CN103184154A (en) * | 2013-04-02 | 2013-07-03 | 南开大学 | Biotechnology capable of producing No. 380 admiralty fuel oil and application thereof |
| CN104031860A (en) * | 2014-05-27 | 2014-09-10 | 南京工业大学 | Bacillus sphaericus with high yield and high-temperature xylanase resistance and application thereof |
| CN104946614A (en) * | 2015-06-18 | 2015-09-30 | 江南大学 | Preparation of recombinant xylanase and method for applying recombinant xylanase to cassava residue degradation |
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- 2008-11-14 CN CNA2008101530762A patent/CN101402963A/en active Pending
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101701205B (en) * | 2009-10-30 | 2011-08-17 | 中国农业科学院饲料研究所 | Alkali-resistant xylanase XynE2, genes thereof and application thereof |
| CN101724613B (en) * | 2009-12-15 | 2012-07-04 | 中国农业科学院饲料研究所 | Alkali proof intermediate temperature xylanase XYNAM6, gene thereof and application thereof |
| CN102712932A (en) * | 2010-02-10 | 2012-10-03 | 永丰馀造纸股份有限公司 | Modified nucleotide molecules of xylanase and application thereof |
| CN102643842A (en) * | 2012-02-27 | 2012-08-22 | 北京工商大学 | Heat-resisting xylanase gene derived from actinomycetes (streptomyces rameus L2001) and product xylanase (Xyn A) thereof |
| CN102676464A (en) * | 2012-05-18 | 2012-09-19 | 南开大学 | Thermophilic long-chain alkyl aldehyde dehydrogenase and crystal structure thereof |
| CN102676464B (en) * | 2012-05-18 | 2014-03-05 | 南开大学 | Thermophilic long-chain alkyl aldehyde dehydrogenase and crystal structure thereof |
| CN103184154A (en) * | 2013-04-02 | 2013-07-03 | 南开大学 | Biotechnology capable of producing No. 380 admiralty fuel oil and application thereof |
| CN104031860A (en) * | 2014-05-27 | 2014-09-10 | 南京工业大学 | Bacillus sphaericus with high yield and high-temperature xylanase resistance and application thereof |
| CN104031860B (en) * | 2014-05-27 | 2016-05-18 | 南京工业大学 | A kind of spherical bacillus of high-yield thermostable zytase and application thereof |
| CN104946614A (en) * | 2015-06-18 | 2015-09-30 | 江南大学 | Preparation of recombinant xylanase and method for applying recombinant xylanase to cassava residue degradation |
| CN104946614B (en) * | 2015-06-18 | 2018-11-02 | 江南大学 | A kind of method of recombined xylanase prepared and its degrade applied to manioc waste |
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