CN1506461A - A kind of thermostable xylanase and gene encoding the enzyme - Google Patents
A kind of thermostable xylanase and gene encoding the enzyme Download PDFInfo
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Abstract
本发明公开了由海栖热袍菌MSB8(Thermotogamaritima MSB8)基因组DNA借助PCR扩增而得到的木聚糖酶B基因(xylanase B gene，xynB)，和构建表达载体在大肠杆菌中表达出该基因编码的重组木聚糖酶。该酶的最适反应温度为90℃，在中性和偏碱性环境中热稳定性好，木聚糖酶酶活力是纤维素酶活力的1100倍以上。该酶作为助漂剂应用于制浆造纸行业表现出良好的应用前景。水解木聚糖的主要产物以木二糖为主，适合分解半纤维素中的木聚糖生产功能性低聚木糖。The invention discloses a xylanase B gene (xylanase B gene, xynB) obtained from the genomic DNA of Thermotogamaritima MSB8 (Thermotogamaritima MSB8) by means of PCR amplification, and constructing an expression vector to express the gene in Escherichia coli Encoded recombinant xylanase. The optimal reaction temperature of the enzyme is 90°C, and it has good thermal stability in neutral and alkaline environments, and the enzyme activity of xylanase is more than 1100 times that of cellulase. The enzyme has a good application prospect in the pulp and paper industry as a bleaching aid. The main product of hydrolyzed xylan is xylobiose, which is suitable for decomposing xylan in hemicellulose to produce functional xylooligosaccharides.
Description
Invention field
The present invention relates to a kind of zytase, the gene of this enzyme of encoding and contain this expression carrier.
Background of invention
Hemicellulose is to be only second to abundant the utilized natural resources of content after the Mierocrystalline cellulose second, and its main component is an xylan.β-1,4-endo-xylanase (EC. 3.2.1.8) can act on wood oligose and a spot of wood sugar that xylan backbone produces different lengths with internal-cutting way, is the enzyme of most critical in the xylan degrading enzyme.Zytase can be widely used in industries such as bio-transformation, food, feed, medicine, the energy, papermaking, weaving.Zytase can be by multiple microorganisms, but because the zytase that general Institute of Micro-biology produces is middle low temperature or slant acidity, has been subjected to considerable restraint on industrial application.
Heat-resisting or fire resistant xylanase has many good qualities in industrial application, and is therefore more about the patent documentation report of this respect abroad in recent years.For example, USP5,395,765, USP5,437,992, USP5,489,526, USP5,738,384, USP5,888,802, USP5,871,730, USP5,916,795, USP6,083,733, USP6,132,716, the source, recombinase and the application thereof that disclose fire resistant xylanase.The open CN1074935 of Chinese patent discloses that a kind of Xylanase for biobleaching, CN1126784 disclose a kind of technology of utilizing zytase to improve straw pulp performance, CN1170429 discloses a kind of cleaning combination that contains zytase.In addition, CN1143387 discloses a kind of thermostable xylanases, and the zytase of introduction is having activity more than 80 ℃.CN1266903A discloses a kind of recombined xylanase, its preparation method and application, introduction be the recombined xylanase that obtains by the xylanase gene in the pcr amplification Pseudomonas fluorescens (Psmdomonas fhomem); CN00814240.8 discloses the stability of improving G/11 family zytase and the method for expanding its pH scope, the Trichodermareesei of introduction (Trichoderma reesei) zytase XYNII gene.
Summary of the invention
The present invention is based on the present inventor's following discovery and finishes: owing to use in many commercial runs of zytase, normally carry out under hot conditions, need zytase to have good thermostability.Thermoduric bacteria is often cultivated difficulty, and what have also need cultivate under the high temperature anaerobic condition, and yield of enzyme is low and enzyme system is complicated, is difficult to obtain pure enzyme.Proved that the enzyme that Thermotoga maritima MSB8 produces is a more complicated, belonged to have 8 kinds of Mierocrystalline cellulose and hemicellulose degradation enzyme system.The enzyme that the Institute of Micro-biology of hydrolyzed xylan produces is a more complicated, and microorganism all produces more than one zytase mostly.These microorganisms itself of discovering of molecular genetics all contain more than one xylanase gene.This also is the multifarious major cause of zytase.The extreme thermophile bacteria T.maritima sp.strain FjSS3-B.1 that thermobacillus belongs to (Thermotoga) of dwelling has 3 different xylanase genes with T.neapolitana.So far, the research about Thermotoga maritima MSB8 zytase all only limits to first zytase (gene is xynA).For example, the xynA gene has been cloned and has been expressed in the intestinal bacteria, and clone's enzyme of purifying shows very high thermostability, and optimum temperuture and pH value are respectively 90 ℃ and pH6.2.The heat-resisting mechanism and the multi-functional zone that also have some report research zytase XynA.Do not see the report of second zytase of Thermotoga maritima MSB8 as yet.Based on above situation, is template in the present invention with the genomic dna of Thermotoga maritima MSB8, obtain second xylanase gene of Thermotoga maritima MSB8 by pcr amplification, this product is named as xynB, subsequently this dna fragmentation is inserted in plasmid, carry out that the xynB gene is cloned, the preparation of expression and recombinase.
Aminoacid sequence according to many glycosidic link lytic enzyme catalysis regions is formed and the hydrophobic cluster analysis, can be divided into many families.Known all zytases all belong to F/10 or G/11 family at present, different zytase molecules differ greatly on the number that its amino acid is formed, but their catalytic domain is relatively more consistent in size, has higher homology with the zytase catalysis region in the gang.The fire resistant xylanase B that the present invention relates to is a novel zytase.The reorganization xynB gene order of measuring is (SEQ ID NO:1) as shown in Figure 2, and by 1074 pairs of based compositions, 358 amino acid (SEQ ID NO:2) as shown in Figure 3 of encoding calculate molecular weight and are about 41,938kDa.By fire resistant xylanase B aminoacid sequence comparative analysis to the present invention relates to, homology investigation according to known zytase gal4 amino acid, find the homology maximum of the XynA of XynB and T.sp.strain FjSS3-B.1, reach 85% (85%identity, accession No.U33060), taking second place with the XynB homology of T.neapolitana, is 82% (82%identity, accession No.Z49961).With the homology of other zytase less than 43%.In addition, compare with other xylanase gene sequence and coded amino acid, it can also be seen that this enzyme belongs to F/10 family zytase, and only contain a simple function district from xynB gene order and coded amino acid.
The performance of the fire resistant xylanase B that the present invention relates to is different from known zytase, the optimal reactive temperature of this enzyme is 90 ℃, Heat stability is good in neutral and slight alkali environment, xylanase activity power is more than 1100 times of cellulase activity, is the highest in the fire resistant xylanase of finding so far.The primary product of hydrolyzed xylan is based on xylo-bioses, and the xylan that is fit to decompose in the hemicellulose is produced functional low polyxylose.
An object of the present invention is to provide a kind of high temperature resistant recombined xylanase.
Another object of the present invention provides a kind of gene of the zytase of the present invention of encoding.
An object of the present invention is to provide a kind of expression carrier of the present invention that contains.
The invention provides a kind of gene of the zytase of the present invention of encoding, this gene has the nucleotide sequence of the group of being selected from down:
(4) nucleotide sequence shown in the SEQ ID NO:1;
(5) because the degeneracy of genetic code is different from SEQ ID NO:1 but the amino acid sequence coded aminoacid sequence identical nucleotide sequence coded with SEQ ID NO:1;
(6) under rigorous hybridization conditions still can with the sequence hybridization in above-mentioned (1) or (2), but coding has the nucleotide sequence of active zytase simultaneously.
Rigorous hybridization conditions is meant, Hybond membrane is placed prehybridization solution (0.25mol/L
-Sodium phosphate buffer, pH7.2,7%SDS) in, 65 ℃ of prehybridization 30min; Abandon prehybridization solution, add hybridization solution (0.25mol/L sodium phosphate buffer, pH7.2,7%SDS, isotope-labeled nucleotide fragments), 65 ℃ of hybridization 12hr; Abandon hybridization solution, (20mmol/L sodium phosphate buffer, pH7.2 5%SDS), wash film 2 times for 65 ℃, each 30min to add film washing liquid I; (20mmol/L sodium phosphate buffer, pH7.2 1%SDS), wash film 30min for 65 ℃ to add film washing liquid II.
The present invention also provides a kind of zytase, and this enzyme has above-mentioned nucleotide sequence coded aminoacid sequence, perhaps preferably has the aminoacid sequence shown in the SEQ ID NO:2.
The present invention also provides the recombinant expression plasmid that contains aforesaid dna sequence dna, and described recombinant expression plasmid is the recombinant expression plasmid pET28a/xynB that contains above-described dna sequence dna.
Accompanying drawing is briefly described
Fig. 1 is the expression pattern of fire resistant xylanase recombination of the present invention in intestinal bacteria;
Fig. 2 is the dna sequence dna of high temperature resistant recombined xylanase B gene of the present invention;
Fig. 3 is the aminoacid sequence of high temperature resistant recombined xylanase B of the present invention;
Fig. 4 is the purification of the present invention SDS-PAGE in each step, wherein, and Lanes M, 10kDa gradient standard protein; Lane 0, after the thermal treatment; Lane 1, Ni-NTA affinity chromatography vigor peak; Lane 2, Q-sepharose ion exchange chromatography vigor peak; Lanes 3 and 4, Mono-Q ion exchange chromatography vigor peak;
Fig. 5 is the pH stability (70 ℃ (...) and 100 ℃ (-)) of high temperature resistant recombined xylanase B of the present invention, wherein, uses different buffer systems respectively at 70 ℃ (...) and 100 ℃ (-) insulation 30min, measures the residual enzyme vigor again.Used buffer system is respectively: Citrate (◆), Acetate (■), MES (Δ), MOPS (*), Phosphate (*), HEPES (●), Tris-HCl (zero), CHES (-) and Piperdine (+);
Fig. 6 is the hydrolysis properties of high temperature resistant recombined xylanase B of the present invention to natural substrate, and wherein, lane X is wood sugar, xylo-bioses, the mixture of xylotriose; Min. or the numeral among the h be hydrolysis time minute or hour.
Embodiment
The extraction of embodiment 1 Thermotoga maritima genomic dna
Adopt Thermotoga maritima (Thermotoga maritima MSB8, Germany DSMZ, DSM3109) fresh wet thallus 20g is suspended from the 10mL 50mMTris damping fluid (pH8.0), add a small amount of N,O-Diacetylmuramidase and 8mL 0.25mM EDTA (PH8.0), place 20min at 37 ℃ behind the mixing, add 2mL 10%SDS then, place 5min for 55 ℃, use equal-volume phenol, each extracting of chloroform respectively once, get last supernatant liquor, add 2 times of volume ethanol, reclaim DNA.Again respectively with 70% and dehydrated alcohol wash, precipitation is dissolved in 0.5mL TE damping fluid (pH8.0,10mMTris, 1mM EDTA), add 10mg/mL RNase3 μ L, 37 ℃ of insulation 1h use equal-volume phenol, each extracting of chloroform respectively once, and supernatant liquor adds 2 times of volume ethanol, reclaim DNA, wash with 70% ethanol and dehydrated alcohol respectively, vacuum lyophilization is dissolved with ultrapure water.
The amplification of embodiment 2 xylanase gene xynB
Design a pair of primer, introduce the Nco I-Hind III restriction enzyme site that can insert pET28a (+) plasmid (Novagen) on the primer.The sequence of primer TM-XynB-Nco I-FWD and the TM-XynB-Hind-His-REV of using is as follows respectively:
TM-XynB-Nco?I-FWD：5′-CCATGGAAA?TATTACCTTCTGTGTGAT
TM-XynB-Hind-His-REV：5′-AAGCTTT?CTTTCTTCTATCTTTTTCTC?CA
When design TM-XynB-Nco I-FWD forward primer, consider that the C-end is with 6 * His label in expression vector, the translation back becomes E from second amino acid of Nco I restriction enzyme site by K like this.Guarantee that according to this sudden change of design of primers goal gene clones in open reading frame, have ATG initiator codon and 6 * His sequence label simultaneously, and the open reading frame drift can not take place.
Genomic dna with Thermotoga maritima is a template, adopts polysaccharase to carry out pcr amplification, and the pcr amplification reaction mixture is: Template DNA, 1 μ l; 10 * KOD-Plus buffer, 2.5 μ l; 2mM dNTP, 2.5 μ l; 25mM MgSO
4, 1.5 μ l; 100pM Forward primer, 0.5 μ l; 100pM Reverse primer, 0.5 μ l; 1Unit/ μ l KOD-Plus, 0.5 μ l; H
2O, 16 μ l; The reaction mixture cumulative volume is 25 μ l.
The condition of pcr amplification reaction is: loop parameter is 98 ℃, 5min; 98 ℃, 30 seconds, 62 ℃, 1min and 68 ℃, 1min, 22 circulations are extended 10min. for back 68 ℃, are cooled to 4 ℃ then, after reaction is finished, carry out agarose gel electrophoresis.Under ultraviolet, shine, downcut target dna, adopt QIAquick Gel Extraction kit (QIAGEN company) from sepharose, to reclaim DNA then, be the xynB gene DNA fragment that amplifies.
The clone and the determined dna sequence of embodiment 3PCR product
After the pcr amplification reaction, the present invention adopts topological TA to clone the xynB gene fragment clone to pCR -2.1Topo plasmid vector, has the bacterium colony of recon with the colony polymerase chain reaction (PCR) method screening.Then, the standard primer sequence on use pCR -2.1Topo plasmid vector is as the dna sequence dna of primer survey xynB gene, and the plasmid that will contain the xynB gene order is purified for further subclone and expression usefulness.
The present invention adopts terminal cessation method (capillary electrophoresis fluorescent method) order-checking, the order-checking agents useful for same be the Dye Terminor Cycle sequencing kit (Perkin-Elmer, USA).The sequencing reaction mixture consists of: Terminator Ready Reaction Mix, 4 μ l; Plasmid Solution, 3 μ l; Forward or Reverse primer, 1.5 μ l; H
2O, 1.5 μ l; The reaction mixture cumulative volume is 10 μ l.The circulating reaction that checks order earlier, the loop parameter of reaction is 98 ℃, 5min; 98 ℃, 15Seconds; 50 ℃, 5 Seconds and 60 ℃, 4min, 25 circulation postcooling to 4 ℃.
Reaction is added 2.0 μ l 3M NaAc (pH4.6), 50 μ l, 95% ethanol and 12.5 μ l water after finishing, and mixes.At room temperature leave standstill 15min., with 15, the centrifugal 20min. of 000rpm, abandoning supernatant adds 70% washing with alcohol precipitation again, with 15, the centrifugal 5min. of 000rpm, abandoning supernatant.Vacuum-drying precipitation adds 12 μ l TSR (Template SuppressionReagent) mixings then, puts 95～96 ℃ of insulation 2min. down, puts into frozen water cooling number minute rapidly, is transferred in the special-purpose tubule of order-checking.With Applied Biosystems 310 Genetic Analyser order-checking, GENETIX program software is used in data analysis at last.
The subclone of embodiment 4 xylanase gene xynB and the expression in intestinal bacteria
Employed expressing gene carrier is pET28a (+) in specific embodiment, is one of expression vector of using always, and target gene is cloned on pET28a (+) carrier, make its expression place T7 phage strong promoter transcribe and translation signals control under.Fig. 1 is the clone of xynB gene and the expression pattern in intestinal bacteria; Fig. 2 is the recombinant plasmid mode chart.Because the gene that Thermotoga belongs to is difficult at expression in escherichia coli, research and design makes the C-terminal of xynB gene have 6 * His label, the target protein that gives expression to like this has 6 * His label, utilize the affinity of 6 * His label and Ni-NTA, the recombinant protein that gives expression to can be carried out affinity chromatography and purify, the enzyme of purifying expression so quickly and easily.The coding of Nco I-Hind III restriction enzyme site pET28a (+) carrier carboxyl terminal His label, can remove the His label with carboxypeptidase, but need control enzyme tangent condition rationally.In fact, connect 6 * His purification tag on protein molecule, this modification does not all have influence to protein expression, folding and biologic activity.
The plasmid pCR2.1-Topo 10/xynB that extraction contains the Topo clone of xynB gene order cuts with a pair of restriction enzyme Nco I-HindIII enzyme respectively, under ultraviolet, shines, and the sepharose behind the observation electrophoresis, and downcut target dna rapidly.Adopt QIAquick Gel Extractionkit (QIAGEN company) to reclaim DNA then from sepharose, the dna fragmentation of recovery is the xynB gene that will insert.Adopt same enzyme blanking method to prepare pET28a (+) carrier, xynB gene fragment and pET28a (+) carrier for preparing mixed by a certain percentage, and add ligationHigh T
4DNA ligase kit (TOYOBO company) connects 4 hours down at 16 ℃.Change competent cell E.coli BL21 over to electroporation then, 100 μ l bacterium liquid are coated on the LB flat board that contains kalamycin resistance, in 37 ℃ of incubated overnight.With the single bacterium colony label that grows, adopt the bacterium colony of bacterium colony PCR screening band recombinant plasmid.Extract the plasmid of several positive bacterium colonies, carry out the double digestion electrophoresis.3 methods of being introduced are carried out determined dna sequence (using pET28a (+) carrier primer) among the employing embodiment, finally determine the complete sequence of expressed gene.Determine the positive colony bacterium colony simultaneously, behind inducing culture, carry out the expression experiment of enzyme.Preserve standby as bacterial classification entirely true and satisfactory bacterium colony.
Expression, purifying and the characteristic of embodiment 5 recombined xylanase B
Prepared fresh 100ml contains the E coli BL21 of recombinant plasmid, is inoculated in Luria-Bertani broth (LB) substratum that 1000ml contains 50 μ g/ml kantlex, cultivates in 30 ℃ shaking table, and shaking speed is 150rpm.Measure the variation of the absorbancy of nutrient solution under 600nm, when absorbancy reached 0.5～0.6, the 1M IPTG that adds 1ml induced, and continued to cultivate 16 hours again.With high speed freezing centrifuge with nutrient solution under 4 ℃ with the centrifugal 10min of 8000rpm, the results bacterial precipitation, (50mM pH8.0) suspends with the 50ml phosphate buffer solution.In ice-water bath, with ultrasonication crusher machine bacterial cell, with broken liquid under 4 ℃ with the centrifugal 10min of 16000rpm, the gained supernatant liquor is crude enzyme liquid.
Crude enzyme liquid through the thermal treatment of 70 ℃ of heating 10min, is removed heat labile foreign protein and other impurity earlier.Next can remove the foreign protein that does not have 6 * His tag with the Ni-NTA affinity column chromatography.Use two step ion-exchange chromatographies (Q-sepharose and Mono-Q) at last enzyme is finally purified, obtained electrophoretically pure zytase XynB.The mensuration of the evaluation of enzyme purity and molecular weight adopts SDS-PAGE (SDS-polyacrylamide gel electrophoresis) method to carry out in the purge process, sees Fig. 4.Press Laemmli (U.K.Laemmli et al, Nature 227,1970) method and carry out the electrophoresis of protein example, resolving gel concentration 10% concentrates gum concentration 3%, uses PAGEL NPU-12.5L electrophoresis chamber (ATTO, company).Sample solution mixes with the Tris/HCl sample-loading buffer earlier, boiling water bath heating 3 minutes.Separation gel behind the electrophoresis dyes with Xylene Brilliant Cyanine G R-250.Protein standard 10kDaprotein ladder.SDS-PAGE with standard protein schemes the relative migration value R of working sample and standard protein per sample
fValue is with the R of standard protein
fValue is made typical curve to the logarithm of molecular weight.R by pure enzyme sample
fValue can be tried to achieve its molecular weight MW (Molecular Weight).SDS-PAGE shows the relative molecular weight 42kDa of zytase XynB, with the molecular weight of theoretical calculate (42,067Da) match.
The characteristic of embodiment 6 recombined xylanase B
The standard method that the present invention measures enzyme activity is: (RemazolBrilliant Blue-R-D-xylan, SIGMA Germany) are substrate to the RBB-xylan with 1.15%, and 50 ℃ are incubated 20min. down in 50mM pH6.14 MES damping fluid.The enzyme solution 25 μ l that standard reaction mixture (100 μ l) consists of 1.15% RBB-xylan 50 μ l, 200mM MES damping fluid 25 μ l and rationally dilutes.Add 100% ethanol, 200 μ l and stop enzyme reaction, precipitate residual substrate simultaneously.At room temperature leave standstill 15min. then at least, mixture is with 15, and the centrifugal 5min. of 000rpm gets supernatant liquor.With the absorbance under the spectrophotometric determination 595nm (Abs) OD
595The barren operation just replaces the enzyme solution of dilution rationally with 25 μ l water with above-mentioned the same.1 enzyme activity unit (unit) is defined as: under above-mentioned experiment condition, per minute causes Δ OD
595=1 required enzyme amount is defined as 1 enzyme activity unit.
The optimal pH of recombined xylanase B is 6.14, can keep 50% of the highest vigor at pH 5.14～8.20.The suitableeest enzyme reaction temperature under optimal pH 6.14 is 90 ℃.Recombined xylanase B shows very high pH stability and thermostability, and more stable in alkaline range internal ratio acid range.Consider the surge capability and the influence of buffered soln itself, the buffer preparation reaction system of different pH is adopted in test.Most of zytases belong to acidic xylanase, and not only optimal pH is in acid range, and the pH stable range is also narrower.Under differing temps the pH stability of (70 and 100 ℃) XynB as shown in Figure 5, interesting is that this enzyme shows very high pH stability, and more stable in alkaline range internal ratio acid range.Under 70 ℃, pH 5.5～11.5, even under 100 ℃, also all very stable in pH 6.5～8.5 scopes.Thisly help industrial application in neutral meta-alkalescence scope stable properties.
The kinetic parameter of embodiment 7 recombined xylanase B
Method according to introductions such as Lawson et al, adopt synthetic to contain p-nitrophenyl (pNP, p-nitrophenyl) substrate, pNP-β-D-xylobioside, pNP-β-D-xylopyranoside, pNP-β-D-cellobioside, pNP-α-L-arabinopyranoside and pNP-β-D-fucopyranoside with different concns, concentration range between 0.5～2.0Km, is measured the speed of enzyme reaction as far as possible in the 50mM of 30 ℃ of pH6.14 MES damping fluid.With absorbance (Abs) OD under Beckman spectrophotometer (ModelDU640, USA, colorimetric troughed belt constant temperature water bath device) monitoring in the per 15 seconds 405nm
405With Line Weaver-Burk graphing method, use " Grafit " analysis software to try to achieve Michaelis-Menton constant Km, maximum reaction velocity V
MaxWith catalytic constant kcat and standard deviation thereof.Here, 1 enzyme activity unit (unit) is defined as: under above-mentioned experiment condition, per minute forms the required enzyme amount of 1 μ mole p-nitrophenyl and is defined as 1 enzyme activity unit.
Adopt aforesaid method mensuration and calculate the kinetic parameter of this enzyme, list in table 1 its substrate.As can be seen, the Km value of pNP-β-D-xylobioside is very little, and only 0.0095 ± 0.0006 mM illustrates that the avidity of enzyme and this substrate is very big.The Km value of pNP-β-D-xylopyranoside, pNP-β-D-cellobioside, pNP-α-L-arabinopyranoside and pNP-β-D-fucopyranoside is respectively 10.4 ± 0.6,3.6 ± 0.25,10.2 ± 0.9 and 12.9 ± 1.2mM.What deserves to be mentioned is the catalytic constant (k of XynB hydrolysis pNP-β-D-xylobioside
Cat/ K
m) be 1730mM
-1.s
-1, more much bigger than other substrate, be the maximum value of finding report under the present similarity condition, be the catalytic constant (1.56mM of hydrolysis pNP-β-D-cellobioside
-1.s
-1) 1109 times.Though this illustrates that also XynB effect substrate is extensive, and is little to the glycosidic link effect in the Mierocrystalline cellulose, can be applied to the pulping and paper-making industry fully.
The kinetic parameter of table 1 XynB
Substrate K
m(mM) k
Cat(s
-1) k
Cat/ K
m(mM
-1S
-1)
pNP-β-xylobioside 0.0095±0.0006 16.4±1.1 1730±100
pNP-β-D-xylopyranoside 10.4±0.6 2.67±0.08 0.26±0.008
pNP-β-D-cellobioside 3.6±0.3 5.65±0.17 1.56±0.06
pNP-α-L- 10.2±0.9 1.80±0.08 0.14±0.005
arabinopyranoside
pNP-β-D-fucopyranoside 12.9±1.2 0.36±0.02 0.028±0.002
Comprise in the reaction mixture: 1% birch xylan (Sigama), 50mM MES damping fluid (pH 6.14) and the pure enzyme that rationally dilutes.At 90 ℃ of insulation reaction 12h, sampling regularly removes ion wherein, uses the thin layer chromatography analysis hydrolysate then.Standard comprises wood sugar, xylo-bioses and xylotriose for the wood oligose mixture.Thin layer chromatography (TLC) is adopted in the analysis of hydrolysate, and silica-gel plate use Merck Silica Gel 60F 254 (E.Merck, Darmstadt, Germany).Thin-layer chromatography adopts upper strata method exhibition layer, exhibition layer 2～4 times, and developing agent is an acetonitrile: water (85: 15, volume ratio).The good silica-gel plate dipping in 5% sulfuric acid methanol solution of exhibition layer once put into baking oven then and baked several minutes, and different carbohydrates just are presented on the silica-gel plate.As shown in Figure 6, the primary product of hydrolyzed xylan reaches as high as about 70% based on xylo-bioses, and the xylan that is fit to decompose in the hemicellulose is produced functional low polyxylose.
Sequence table
＜110〉China Agricultural University
＜120〉gene of a kind of fire resistant xylanase and this enzyme of coding
<130>I020532
<160>4
<170>PatentIn?version?3.1
<210>1
<211>1074
<212>DNA
<213>Thermotoga?maritima
<220>
<221>CDS
<222>(1)..(1074)
<223>
<400>1
atg?gaa?ata?tta?cct?tct?gtg?ttg?atc?ctt?ttg?ttg?gga?tgt?gtt?cca 48
Met?Glu?Ile?Leu?Pro?Ser?Val?Leu?Ile?Leu?Leu?Leu?Gly?Cys?Val?Pro
1 5 10 15
gtt?ttc?agc?tct?cag?aat?gta?tct?ctg?aga?gaa?ctc?gca?gaa?aag?ctg 96
Val?Phe?Ser?Ser?Gln?Asn?Val?Ser?Leu?Arg?Glu?Leu?Ala?Glu?Lys?Leu
20 25 30
aac?atc?tat?att?ggt?ttt?gcc?gca?atc?aac?aac?ttt?tgg?tct?ctt?tcc 144
Asn?Ile?Tyr?Ile?Gly?Phe?Ala?Ala?Ile?Asn?Asn?Phe?Trp?Ser?Leu?Ser
35 40 45
gac?gca?gaa?aag?tac?atg?gaa?gtt?gca?aga?aga?gaa?ttc?aac?atc?ctg 192
Asp?Ala?Glu?Lys?Tyr?Met?Glu?Val?Ala?Arg?Arg?Glu?Phe?Asn?Ile?Leu
50 55 60
acc?cct?gag?aac?cgg?atg?aag?tgg?gat?acg?att?cat?cca?gaa?aga?gac 240
Thr?Pro?Glu?Asn?Arg?Met?Lys?Trp?Asp?Thr?Ile?His?Pro?Glu?Arg?Asp
65 70 75 80
aga?tac?aat?ttc?act?ccc?gca?gaa?aaa?cac?gtt?gag?ttt?gca?gaa?gaa 288
Arg?Tyr?Asn?Phe?Thr?Pro?Ala?Glu?Lys?His?Val?Glu?Phe?Ala?Glu?Glu
85 90 95
aac?gac?atg?atc?gtg?cat?gga?cac?act?ctt?gtc?tgg?cac?aac?cag?ctt 336
Asn?Asp?Met?Ile?Val?His?Gly?His?Thr?Leu?Val?Trp?His?Asn?Gln?Leu
100 105 110
cct?gga?tgg?atc?act?ggt?aga?gaa?tgg?aca?aag?gaa?gaa?ctt?ttg?aac 384
Pro?Gly?Trp?Ile?Thr?Gly?Arg?Glu?Trp?Thr?Lys?Glu?Glu?Leu?Leu?Asn
115 120 125
gtt?ctt?gaa?gac?cac?ata?aaa?acg?gtg?gtg?tct?cat?ttc?aaa?ggt?aga 432
Val?Leu?Glu?Asp?His?Ile?Lys?Thr?Val?Val?Ser?His?Phe?Lys?Gly?Arg
130 135 140
gtg?aag?atc?tgg?gat?gtg?gtg?aac?gaa?gcg?gtg?agc?gat?tct?gga?acc 480
Val?Lys?Ile?Trp?Asp?Val?Val?Asn?Glu?Ala?Val?Ser?Asp?Ser?Gly?Thr
145 150 155 160
tac?agg?gaa?agc?gtg?tgg?tac?aag?acg?atc?ggt?cct?gaa?tac?att?gaa 528
Tyr?Arg?Glu?Ser?Val?Trp?Tyr?Lys?Thr?Ile?Gly?Pro?Glu?Tyr?Ile?Glu
165 170 175
aaa?gcg?ttc?aga?tgg?gcg?aaa?gaa?gcc?gat?cca?gat?gcg?att?ctc?atc 576
Lys?Ala?Phe?Arg?Trp?Ala?Lys?Glu?Ala?Asp?Pro?Asp?Ala?Ile?Leu?Ile
180 185 190
tac?aac?gac?tac?agc?ata?gaa?gaa?atc?aac?gca?aaa?tcg?aac?ttc?gtc 624
Tyr?Asn?Asp?Tyr?Ser?Ile?Glu?Glu?Ile?Asn?Ala?Lys?Ser?Asn?Phe?Val
195 200 205
tac?aac?atg?ata?aaa?gag?ctg?aaa?gaa?aag?gga?gta?cct?gtt?gat?gga 672
Tyr?Asn?Met?Ile?Lys?Glu?Leu?Lys?Glu?Lys?Gly?Val?Pro?Val?Asp?Gly
210 215 220
ata?gga?ttt?cag?atg?cac?ata?gac?tac?aga?ggg?ctc?aat?tat?gac?agt 720
Ile?Gly?Phe?Gln?Met?His?Ile?Asp?Tyr?Arg?Gly?Leu?Asn?Tyr?Asp?Ser
225 230 235 240
ttc?aga?agg?aat?ttg?gag?aga?ttt?gcg?aaa?ctc?ggt?ctt?caa?ata?tac 768
Phe?Arg?Arg?Asn?Leu?Glu?Arg?Phe?Ala?Lys?Leu?Gly?Leu?Gln?Ile?Tyr
245 250 255
atc?aca?gag?atg?gat?gtg?aga?att?cct?ctc?agt?ggt?tcg?gag?gag?tat 816
Ile?Thr?Glu?Met?Asp?Val?Arg?Ile?Pro?Leu?Ser?Gly?Ser?Glu?Glu?Tyr
260 265 270
tat?ttg?aaa?aaa?cag?gct?gaa?gtt?tgt?gcg?aag?atc?ttc?gat?ata?tgc 864
Tyr?Leu?Lys?Lys?Gln?Ala?Glu?Val?Cys?Ala?Lys?Ile?Phe?Asp?Ile?Cys
275 280 285
ttg?gac?aac?cct?gca?gtt?aaa?gcg?atc?cag?ttt?tgg?gga?ttc?aca?gac 912
Leu?Asp?Asn?Pro?Ala?Val?Lys?Ala?Ile?Gln?Phe?Trp?Gly?Phe?Thr?Asp
290 295 300
aaa?tac?tcc?tgg?gtt?ccc?ggc?ttt?ttc?aaa?ggg?tac?ggg?aaa?gcg?ttg 960
Lys?Tyr?Ser?Trp?Val?Pro?Gly?Phe?Phe?Lys?Gly?Tyr?Gly?Lys?Ala?Leu
305 310 315 320
ctc?ttc?gat?gag?aat?tac?aac?ccc?aag?cct?tgt?tat?tac?gcg?ata?aaa 1008
Leu?Phe?Asp?Glu?Asn?Tyr?Asn?Pro?Lys?Pro?Cys?Tyr?Tyr?Ala?Ile?Lys
325 330 335
gag?gtg?ctg?gag?aaa?aag?ata?gaa?aga?aag?ctt?gcg?gcc?gca?ctc?gag 1056
Glu?Val?Leu?Glu?Lys?Lys?Ile?Glu?Arg?Lys?Leu?Ala?Ala?Ala?Leu?Glu
340 345 350
cac?cac?cac?cac?cac?cac 1074
His?His?His?His?His?His
355
<210>2
<211>358
<212>PRT
<213>Thermotoga?maritima
<400>2
Met?Glu?Ile?Leu?Pro?Ser?Val?Leu?Ile?Leu?Leu?Leu?Gly?Cys?Val?Pro
1 5 10 15
Val?Phe?Ser?Ser?Gln?Asn?Val?Ser?Leu?Arg?Glu?Leu?Ala?Glu?Lys?Leu
20 25 30
Asn?Ile?Tyr?Ile?Gly?Phe?Ala?Ala?Ile?Asn?Asn?Phe?Trp?Ser?Leu?Ser
35 40 45
Asp?Ala?Glu?Lys?Tyr?Met?Glu?Val?Ala?Arg?Arg?Glu?Phe?Asn?Ile?Leu
50 55 60
Thr?Pro?Glu?Asn?Arg?Met?Lys?Trp?Asp?Thr?Ile?His?Pro?Glu?Arg?Asp
65 70 75 80
Arg?Tyr?Asn?Phe?Thr?Pro?Ala?Glu?Lys?His?Val?Glu?Phe?Ala?Glu?Glu
85 90 95
Asn?Asp?Met?Ile?Val?His?Gly?His?Thr?Leu?Val?Trp?His?Asn?Gln?Leu
100 105 110
Pro?Gly?Trp?Ile?Thr?Gly?Arg?Glu?Trp?Thr?Lys?Glu?Glu?Leu?Leu?Asn
115 120 125
Val?Leu?Glu?Asp?His?Ile?Lys?Thr?Val?Val?Ser?His?Phe?Lys?Gly?Arg
130 135 140
Val?Lys?Ile?Trp?Asp?Val?Val?Asn?Glu?Ala?Val?Ser?Asp?Ser?Gly?Thr
145 150 155 160
Tyr?Arg?Glu?Ser?Val?Trp?Tyr?Lys?Thr?Ile?Gly?Pro?Glu?Tyr?Ile?Glu
165 170 175
Lys?Ala?Phe?Arg?Trp?Ala?Lys?Glu?Ala?Asp?Pro?Asp?Ala?Ile?Leu?Ile
180 185 190
Tyr?Asn?Asp?Tyr?Ser?Ile?Glu?Glu?Ile?Asn?Ala?Lys?Ser?Asn?Phe?Val
195 200 205
Tyr?Asn?Met?Ile?Lys?Glu?Leu?Lys?Glu?Lys?Gly?Val?Pro?Val?Asp?Gly
210 215 220
Ile?Gly?Phe?Gln?Met?His?Ile?Asp?Tyr?Arg?Gly?Leu?Asn?Tyr?Asp?Ser
225 230 235 240
Phe?Arg?Arg?Asn?Leu?Glu?Arg?Phe?Ala?Lys?Leu?Gly?Leu?Gln?Ile?Tyr
245 250 255
Ile?Thr?Glu?Met?Asp?Val?Arg?Ile?Pro?Leu?Ser?Gly?Ser?Glu?Glu?Tyr
260 265 270
Tyr?Leu?Lys?Lys?Gln?Ala?Glu?Val?Cys?Ala?Lys?Ile?Phe?Asp?Ile?Cys
275 280 285
Leu?Asp?Asn?Pro?Ala?Val?Lys?Ala?Ile?Gln?Phe?Trp?Gly?Phe?Thr?Asp
290 295 300
Lys?Tyr?Ser?Trp?Val?Pro?Gly?Phe?Phe?Lys?Gly?Tyr?Gly?Lys?Ala?Leu
305 310 315 320
Leu?Phe?Asp?Glu?Asn?Tyr?Asn?Pro?Lys?Pro?Cys?Tyr?Tyr?Ala?Ile?Lys
325 330 335
Glu?Val?Leu?Glu?Lys?Lys?Ile?Glu?Arg?Lys?Leu?Ala?Ala?Ala?Leu?Glu
340 345 350
His?His?His?His?His?His
355
<210>3
<211>27
<212>DNA
＜213〉artificial sequence
<400>3
ccatggaaat?attaccttct?gtgtgat 27
<210>4
<211>29
<212>DNA
＜213〉artificial sequence
<400>4
aagctttctt?tcttctatct?ttttctcca 29
Claims (5)
1. the gene of the zytase of encoding, this gene have the nucleotide sequence of the group of being selected from down:
(1) nucleotide sequence shown in the SEQ ID NO:1;
(2) because the degeneracy of genetic code is different from SEQ ID NO:1 but the amino acid sequence coded aminoacid sequence identical nucleotide sequence coded with SEQ ID NO:1;
(3) under rigorous hybridization conditions still can with the sequence hybridization in above-mentioned (1) or (2), but coding has the nucleotide sequence of active zytase simultaneously.
2. zytase, this enzyme has the described nucleotide sequence coded aminoacid sequence of claim 1.
3. according to the described zytase of claim 2, it has the aminoacid sequence shown in the SEQ ID NO:2.
4. recombinant expression plasmid, it contains the described gene of claim 1.
5. according to the described plasmid of claim 4, it is pET28a/xynB.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02156022 CN1219063C (en) | 2002-12-11 | 2002-12-11 | A kind of thermostable xylanase and gene encoding the enzyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02156022 CN1219063C (en) | 2002-12-11 | 2002-12-11 | A kind of thermostable xylanase and gene encoding the enzyme |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1506461A true CN1506461A (en) | 2004-06-23 |
| CN1219063C CN1219063C (en) | 2005-09-14 |
Family
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 02156022 Expired - Fee Related CN1219063C (en) | 2002-12-11 | 2002-12-11 | A kind of thermostable xylanase and gene encoding the enzyme |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1219063C (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100491533C (en) * | 2005-04-13 | 2009-05-27 | 中国农业科学院饲料研究所 | Improved high specific activity xylanase and gene thereof, expression vector comprising the gene, recombinant yeast cell and expression method |
| CN1916171B (en) * | 2006-09-11 | 2010-05-12 | 中国农业大学 | A method for producing xylobiose and its special immobilized enzyme |
| CN104357427A (en) * | 2014-11-11 | 2015-02-18 | 福州大学 | High-temperature-resistant alkaline salt-tolerant xylanase XynSL4 as well as gene and application thereof |
| US9012186B2 (en) | 2009-04-27 | 2015-04-21 | The Board Of Trustees Of The University Of Illinois | Hemicellulose-degrading enzymes |
| EP2775857B1 (en) | 2011-11-09 | 2020-01-01 | Puratos N.V. | A feed composition supplemented with a xylanase |
| CN112831486A (en) * | 2020-11-21 | 2021-05-25 | 中国林业科学研究院林产化学工业研究所 | A kind of endo-xylanidase and its application for preparing xylo-oligosaccharides |
-
2002
- 2002-12-11 CN CN 02156022 patent/CN1219063C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100491533C (en) * | 2005-04-13 | 2009-05-27 | 中国农业科学院饲料研究所 | Improved high specific activity xylanase and gene thereof, expression vector comprising the gene, recombinant yeast cell and expression method |
| CN1916171B (en) * | 2006-09-11 | 2010-05-12 | 中国农业大学 | A method for producing xylobiose and its special immobilized enzyme |
| US9012186B2 (en) | 2009-04-27 | 2015-04-21 | The Board Of Trustees Of The University Of Illinois | Hemicellulose-degrading enzymes |
| EP2775857B1 (en) | 2011-11-09 | 2020-01-01 | Puratos N.V. | A feed composition supplemented with a xylanase |
| CN104357427A (en) * | 2014-11-11 | 2015-02-18 | 福州大学 | High-temperature-resistant alkaline salt-tolerant xylanase XynSL4 as well as gene and application thereof |
| CN112831486A (en) * | 2020-11-21 | 2021-05-25 | 中国林业科学研究院林产化学工业研究所 | A kind of endo-xylanidase and its application for preparing xylo-oligosaccharides |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1219063C (en) | 2005-09-14 |
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