CN101701214B - Xylanase XYNA4 with wide pH applicability and gene and application thereof - Google Patents
Xylanase XYNA4 with wide pH applicability and gene and application thereof Download PDFInfo
- Publication number
- CN101701214B CN101701214B CN2009102359431A CN200910235943A CN101701214B CN 101701214 B CN101701214 B CN 101701214B CN 2009102359431 A CN2009102359431 A CN 2009102359431A CN 200910235943 A CN200910235943 A CN 200910235943A CN 101701214 B CN101701214 B CN 101701214B
- Authority
- CN
- China
- Prior art keywords
- xylanase
- xyna4
- gene
- wide
- adaptive
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to the gene engineering field, in particular to a xylanase XYNA4 and gene and application thereof. The invention provides a xylanase XYNA4 derived from alicyclic acid bacillus Alicyclobacillus hesperidumA4 (CGMCC No. 3147), amino acid sequence thereof is shown as SEQ ID NO. 1, and the invention also provides gene xynA4 coding the xylanase. The xylanase of the invention has the following properties: optimal pH is 7.0, optimal temperature is 55 DEG C, and specific activity is 4.20.2U/mg; and the xylanase has pH stability with extreme range, higher pH adaptability and better thermostability. The xylanase is used as a novel enzymic preparation can be widely applied to industries of animal feed, food, papermaking and energy sources.
Description
Technical field
The present invention relates to the genetically engineered field, particularly, the present invention relates to a kind of wide pH adaptability Xylanase XYNA 4 and gene and application.
Background technology
Xylan (xylan) is the main component of plant hemicellulose, extensively is present in occurring in nature, its content be only second to Mierocrystalline cellulose be the second abundant saccharan (Collins et al.FEMS Microbiology Reviews.2005,29:3-23.).Zytase is the general name that xylan degrading can be become the class of enzymes of oligose and wood sugar.At present zytase is at feed, bread, and fields such as beer, papermaking, weaving, medicine and the energy have obtained increasingly extensive application.In paper industry, zytase can be applied to bio-pulping, association with pulp bleaching, deinking processing etc., particularly it is in pulp bio-bleaching, zytase cooperates with traditional chlorine bleaching, impel the degraded of residual lignin in the paper pulp and extracting of solvability xylogen, not only can improve the whiteness and the retention of whiteness of paper pulp, improve the drainability and the Papermaking Performance of fiber, and can reduce the follow-up consumption that floats the preface chemical substance, reduce the discharging of being rich in muriatic trade effluent, thereby the pollution that the reduction association with pulp bleaching produces environment (Polizeli et al.AppliedMicrobiology and Biotechnology.2005,67:577-591.).
Alicyclic acid genus bacillus (Alicyclobacillus) is the thermophilic aciduric bacteria of a class.Because alicyclic acid genus bacillus (A.acidocaldarius) etc. can cause the corruption of pasteurization fruit juice, produce the smell that is difficult to accept, add the viability that it is unique.Caused at present the very big concern of global foodstuffs industry and academia.Also there is not relevant report about xylanase gene and the cloning and expression thereof that derives from alicyclic acid genus bacillus source at present.
Because in feed and paper industry, bigger fluctuation appears in the pH value of animal gastrointestinal tract and bleached pulp.Therefore, obtaining novel research with zytase of good pH stability and suitability is significant.The clone with separates have the stable and adaptive zytase of high pH can better application in feed and paper industry, and can reduce production costs, it is necessary all to be that zytase is applied to suitability for industrialized production.
Summary of the invention
The wide pH adaptability zytase that the purpose of this invention is to provide a kind of energy efficient application.
A further object of the present invention provides the gene of the above-mentioned wide pH adaptability zytase of coding.
Another object of the present invention provides the recombinant vectors that comprises said gene.
Another object of the present invention provides the recombinant bacterial strain that comprises said gene.
Another object of the present invention provides a kind of gene engineering method for preparing above-mentioned wide pH adaptability body acidic xylanase.
Another object of the present invention provides the application of above-mentioned wide pH adaptability zytase.
Another object of the present invention provides a kind of alicyclic acid genus bacillus.
The present invention is from alicyclic acid genus bacillus Alicyclobacillus hesperidum A4, (be stored in (Datun Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms common micro-organisms center, Institute of Microorganism, Academia Sinica, 100101), its preserving number is: CGMCC No.3147, preservation date: separate obtaining a kind of new wide pH applicability Xylanase XYNA 4 on June 29th, 2009).
The invention provides a kind of wide pH adaptability Xylanase XYNA 4, its aminoacid sequence is shown in SEQ ID NO.1.
SEQ ID NO.1：
MTDTYRNIPSLSERYRPYFRIGAAVNAKSLNTHRDLLVTHFNSVTAENEMKWEEIHPEQDRYEFAKADALVNFAREHGMFVRGHTLVWHNQTPAAVFLDD LGQTATAAVVERRLEEHVATVLGRYHNDIYDWDVANEAVVDAGTGFLRDSRWLQTLGDDYIAKAFRIAHQAAPDALLFYNDYNETKPDKSERIYKLVAG L LDEGVPIHGIGMQGHWMLDDPALDEIERAIDRYASLGVHLHITELDVCVYGNVHGTGGQSQEVLPYDDELAKRLAERYRSLFSSLRARKDVIESVTFW
338 amino acid of this enzyme genes encoding, therefore the theoretical molecular of sophisticated Xylanase XYNA 4 is 38.6kDa.
Xylanase XYNA 4 of the present invention all has greater activity in neutrality and acid-basicity scope.The present invention screens the zytase that a kind of alicyclic acid genus bacillus Alicyclobacillus hesperidum A4 (CGMCCNo.3147) is produced, it is 7.0 in colibacillary recombinase optimum pH, keeps the enzymic activity more than 40% in the scope of pH3.8～9.4; In the scope of pH6.2～9.4, keep the enzymic activity more than 60%; 37 ℃ are incubated 60 minutes in the scope of pH2.6～12.0, can keep the enzymic activity more than 90%.Optimum temperuture is 55 ℃, all has the enzyme activity more than 60% between 45 ℃-65 ℃; 60 ℃ of insulations 60 minutes, residual enzyme work reached more than 90%.The also unprecedented report of the zytase of this character.
The invention provides the gene of the above-mentioned wide pH adaptability Xylanase XYNA 4 of coding.Particularly, the gene order of this gene is shown in SEQ ID NO.2:
SEQ ID NO.2：
ATGACTGACACTTATCGGAATATTCCTTCCTTGAGTGAGCGCTACCGCCCGTACTTTCGCATTGGTGCTGCCGTCAATGCCAAGTCCCTGAATACCCACCGTGACTTGTTGGTCACGCATTTTAACAGCGTGACCGCAGAGAACGAAATGAAGTGGGAAGAGATTCATCCAGAACAGGATCGATACGAGTTCGCAAAGGCGGATGCACTCGTGAATTTTGCCCGCGAGCACGGTATGTTTGTCCGCGGCCACACGTTGGTCTGGCACAATCAAACGCCAGCCGCAGTGTTTCTGGACGATCTCGGTCAAACAGCGACAGCGGCCGTTGTCGAGCGTCGACTCGAAGAGCATGTGGCAACAGTGCTTGGTCGATACCACAACGACATCTACGACTGGGATGTTGCCAACGAGGCAGTCGTCGATGCGGGTACAGGATTTTTACGGGACAGTCGCTGGCTGCAGACCCTTGGCGATGATTACATTGCGAAGGCTTTTCGCATCGCACATCAAGCGGCACCAGACGCACTGCTCTTCTACAACGACTACAATGAAACCAAACCCGATAAATCGGAGCGCATTTACAAACTTGTTGCAGGTCTGCTCGATGAAGGTGTCCCCATCCATGGGATTGGTATGCAGGGGCACTGGATGTTGGACGATCCGGCGCTGGACGAAATTGAACGAGCTATCGACCGCTACGCGTCACTCGGTGTGCACCTTCACATCACAGAACTCGACGTGTGTGTTTATGGCAATGTCCACGGAACGGGCGGCCAATCCCAAGAAGTTCTACCCTACGACGATGAACTTGCCAAACGTTTGGCCGAGCGTTATCGTTCTCTGTTTTCATCGTTGCGCGCTCGCAAGGATGTCATCGAAAGCGTCACGTTCTGGGGGGTTGCTGATGATGATACCTGGCGGGACAACTTCCCTGTTCGCGGACGCAAAGACTGGCCACTTCTGTTTGACGTGAACCACGGGCCGAAACAGGCGTTTTGGAGCGTGGTTGAATTTTGA
The method separating clone of the present invention by PCR xylanase gene XYNA4, the DNA complete sequence analysis is the result show, Xylanase XYNA 4 structure gene XYNA4 total length 1017bp contains a terminator TGA.The maturation protein theoretical molecular of Xylanase XYNA 4 is 38.6kDa.Xylanase gene XYNA4 sequence and the aminoacid sequence derived are carried out the BLAST comparison in GenBank.This gene is 62% with zytase (ACV59335.1) consensus amino acid sequence that derives from Alicyclobacillusacidocaldarius.With known activity zytase (ABI49937.2) consensus amino acid sequence that derives from Geobacillus stearothermophilus be 53%, illustrate that XYNA4 is a kind of new zytase.
The present invention also provides the recombinant vectors that comprises above-mentioned xylanase gene XYNA4, is preferably carrier pET-22b (+).Xylanase gene of the present invention is inserted between the suitable restriction enzyme site of expression vector, makes that its nucleotide sequence is exercisable to be connected with expression regulation sequence.As the most preferred embodiment of the present invention, be preferably xylanase gene of the present invention is inserted between the EcoR I and Hind III restriction enzyme site on the plasmid pET-22b (+), make this nucleotide sequence be positioned at the downstream of promotor and regulated and control by it, large intestine expression plasmid pET-22b (+)-XYNA4 obtains recombinating.
The present invention also provides the recombinant bacterial strain that comprises the adaptive xylanase gene XYNA4 of above-mentioned wide pH, and preferred described bacterial strain is intestinal bacteria, yeast, genus bacillus or lactobacillus, is preferably recombinant bacterial strain e. coli bl21 (DE3).
The present invention also provides a kind of method for preparing the adaptive Xylanase XYNA 4 of wide pH, may further comprise the steps:
1), gets recombinant bacterial strain with above-mentioned recombinant vectors transformed host cell;
2) cultivate recombinant bacterial strain, induce recombined xylanase to express; And
3) reclaim the also expressed Xylanase XYNA 4 of purifying.
Wherein, preferred described host cell is Bacillus coli cells, cerevisiae, pichia spp cell or many types of inferior yeast cell, preferably, obtain recombinant bacterial strain BL21 (DE3)/XYNA4 with recombinant expression plasmid transformed into escherichia coli cell (Escherichia coli) BL21 (DE3).
The present invention also provides above-mentioned wide pH the application of adaptive Xylanase XYNA 4.
The present invention's technical problem at first to be solved is to overcome the deficiencies in the prior art, provide a kind of character good, be suitable at feed, use new zytase in food and the paper industry.Zytase optimal pH of the present invention is 7.0, at pH 6.2～9.4 higher enzymic activity (more than 60%) is arranged all; The pH good stability; Than vigor is 420.2U/mg; Ability with better heat-resisting.Its superior heat resistance energy can effectively be reduced in the loss of enzyme activity in the feed course of processing, reduces the application cost of zytase.Higher pH subject range improves the transformation efficiency of non-starchiness polysaccharide in the feed, reduces formulation cost, reduces environmental pollution; Improve the exterior quality of the bread course of processing; Can be applicable to brewing industry, reduce the viscosity of wort, improve filtration efficiency, increase the wort productive rate; Can also be used for bioenergy, as the xylan in paper industry waste material and the agricultural wastes being converted into D-wood sugar monomer, and then, change into valuable fuel by the most of microbe metabolism.Hydrolysate (wood sugar and xylo-oligosaccharide) can be applicable to the protective foods industry; Xylan is used in combination with other material in pharmaceutical industry, can delay the release of pharmaceutical cpd.
Description of drawings
Fig. 1 analyzes at the SDS-PAGE of the recombined xylanase of expression in escherichia coli, wherein, and 1: low molecular weight protein Marker; 2: the recombined xylanase of purifying.
The optimal pH of Fig. 2 recombined xylanase.
The pH stability of Fig. 3 recombined xylanase.
The optimum temperuture of Fig. 4 recombined xylanase.
The thermostability of Fig. 5 recombined xylanase.
Alicyclic acid genus bacillus Alicyclobacillus hesperidum A4CGMCC3147, be stored in (Datun Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms common micro-organisms center, Institute of Microorganism, Academia Sinica, 100101), its preserving number is: CGMCC No.3147, preservation date: 2009 06 month No. 29.
Embodiment
Test materials and reagent
1, bacterial strain and carrier: alicyclic acid genus bacillus Alicyclobacillus hesperidum A4CGMCC3147, be stored in (Datun Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms common micro-organisms center, Institute of Microorganism, Academia Sinica, 100101), its preserving number is: CGMCC No.3147.Coli expression carrier pET-22b (+) and bacterial strain BL21 (DE3) are available from Merch company.
2, enzyme and other biochemical reagents: restriction endonuclease is available from TaKaRa company, and ligase enzyme is available from Invitrogen company.The oat xylan is available from Sigma company, and other all is domestic reagent (all can buy from common biochemical reagents company and obtain).
3, substratum:
(1) alicyclic acid genus bacillus Alicyclobacillus hesperidum A4CGMCC3147 substratum consists of:
Growth medium: 0.2% peptone, 0.1% yeast extract, 0.2% glucose, pH3.0.
Produce the enzyme substratum: 0.2% peptone, 0.1% yeast extract, 0.5% xylan, pH3.0.
(2) intestinal bacteria substratum LB (1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0).
Illustrate: make the experimental methods of molecular biology specify in following examples, all carry out, perhaps carry out according to test kit and product description with reference to listed concrete grammar in " molecular cloning experiment guide " (third edition) J. Sa nurse Brooker one book.
The separation and purification of embodiment 1 alicyclic acid genus bacillus Alicyclobacillus hesperidum A4 (CGMCC No.3147) and product enzyme characteristic thereof
Pick up from Yunnan hyperthermal spring trickling thing soil sample and be transferred to enrichment medium (yeast extract 1.0g, Tryptones 2.0g transfer pH2.0 with hydrochloric acid), cultivate after 72 hours for 60 ℃, the dilution coated plate is to isolation medium (yeast extract 1.0g, Tryptones 2.0g, oat xylan 5.0g, agar 30, Congo red 0.5g, transfer pH3.0 with hydrochloric acid) 60 ℃ cultivate after 72 hours, according to transparent circle have or not with big or small picking to a strain bacterium producing multi enzyme preparation, be numbered A4, be accredited as alicyclic acid genus bacillus Alicyclobacillus hesperidum A4 according to 16SRNA and strain characteristic.With the alicyclic acid genus bacillus Alicyclobacillus hesperidum A4 growth medium (yeast extract 1.0g, Tryptones 2.0g transfer pH3.0 with hydrochloric acid) of transferring then, test its optimum growth temperature and pH value.The result shows that its optimum growth temperature is 60 ℃ and pH3.5.With alicyclic acid genus bacillus Alicyclobacillus hesperidum A4 multiparity enzyme substratum (yeast extract 1.0g, Tryptones 2.0g oat xylan 5.0g, transfer pH3.0 with hydrochloric acid) 60 ℃ cultivate after 48 hours, measure the xylanase activity of supernatant liquor.Prove that it has xylanase activity.
The clone of embodiment 2 alicyclic acid genus bacillus Alicyclobacillus hesperidum A4 (CGMCC No.3147) Xylanase coding gene XYNA4
Extract alicyclic acid genus bacillus Alicyclobacillus hesperidum A4 (CGMCC No.3147) genomic dna:
With 2 days bacterium liquid centrifuging and taking thalline of liquid culture, add the 1mL N,O-Diacetylmuramidase, handle 60min for 37 ℃, add lysate again, 65 ℃ of water-bath cracking 30min, every the 10min mixing once, at 4 ℃ of centrifugal 5min of following 10000rpm.Get supernatant extrct foreigh protein removing in phenol/chloroform, get supernatant again and add the equal-volume Virahol, after room temperature leaves standstill 5min, 4 ℃ of centrifugal 10min of following 10000rpm.Abandon supernatant, precipitation is with 70% washing with alcohol twice, and vacuum-drying adds an amount of TE and dissolves, place-20 ℃ standby.
Conservative (YDWDV and HGIGM) sequences Design according to the tenth family's xylanase gene has been synthesized degenerated primer XYN10F, XYN10R such as table 1
With the total DNA of alicyclic acid genus bacillus Alicyclobacillus hesperidum A4 (CGMCC No.3147) is that template is carried out pcr amplification.The PCR reaction parameter is: 94 ℃ of sex change 5min; Preceding 10 circulations, 94 ℃ of sex change 30sec, 48 ℃ of-53 ℃ of touchdown (0.5 ℃/circulation) annealing 30sec, 72 ℃ are extended 1min, and 25 cycling conditions are back 94 ℃ of sex change 30sec then, 53 ℃ of annealing 30sec, 72 ℃ are extended 1min.Last 72 ℃ of insulation 10min.Obtain an about 258bp fragment, this fragment recovery back is linked to each other with the pEASY-T3 carrier send the order-checking of three rich Bioisystech Co., Ltd.
The nucleotide sequence that obtains according to order-checking, each three TAIL-PCR specificity nested primers of design upstream and downstream: design direction is for needing the zone of ignorance direction of amplification, and with they difference called after usp1, usp2, usp3 (upstream Auele Specific Primer), dsp1, dsp2, dsp3 (downstream Auele Specific Primer) sees Table 1.
Table 1. Xylanase XYNA 4 TAIL-PCR Auele Specific Primer
N represents A, G, C or T; M represents A or C; S represents C or G; W represents A or T; D represents A, G or T; Y represents C or T
Obtain the flanking sequence of known sequence by TAIL-PCR, amplification obtains product and reclaims the back order-checking.By this full length gene of discovery 1017bp behind the genome sequence that compares zytase, encode 338 amino acid and a terminator codon, the N end does not have the signal peptide sequence of prediction.The nucleotide sequence of measured gene XYNA4 and the xylanase gene sequence on the GeneBank are carried out homology relatively, and the highest consistence is 65%, and the highest consistence of aminoacid sequence is 62%.With known activity zytase consensus amino acid sequence be 53%, illustrate that XYNA4 is a kind of new zytase, show that the gene of the coding zytase that separating clone obtains from Alicyclobacillus hesperidumA4 (CGMCC No.3147) is new gene.
The preparation of embodiment 3 recombined xylanases.
Expression vector PET-22B (+) is carried out double digestion (EcoRI+Hind III), to encode the simultaneously gene XYNA4 double digestion (EcoRI+Hind III) of zytase, the gene fragment that cuts out the encoding mature zytase is connected with expression vector pPET-22B (+), acquisition contains recombinant plasmid PET-22b (+)-XYNA4 and the transformed into escherichia coli BL21 (DE3) of Alicyclobacillus hesperidum A4 (CGMCC No.3147) xylanase gene XYNA4, obtains recombinant escherichia coli strain BL21 (DE3)/XYNA4.
Get BL21 (DE3) bacterial strain that contains recombinant plasmid, be inoculated in (1000ml triangular flask) in the 200mL LB nutrient solution, behind 37 ℃ of 250rpm shaking culture 3～4h, add IPTG and induce.Behind 30 ℃ of 250rpm shaking culture 10～12h, centrifugal collection supernatant.Measure the vigor of zytase.The expression amount of recombined xylanase is 0.38U/mL.Expressed zytase is through after the ni-sepharose purification, and through the SDS-PAGE electrophoretic analysis, result (Fig. 1) shows that recombined xylanase has obtained expression in intestinal bacteria.
The activation analysis of embodiment 4 recombined xylanases
The DNS method: concrete grammar is as follows: at pH7.0 (0.1M SODIUM PHOSPHATE, MONOBASIC-citric acid), under 55 ℃ of conditions, the reaction system of 1mL comprises 100 μ L suitable dilution enzyme liquid and 900 μ L (1%, w/v) substrate, reaction 10min adds 1.5mL DNS termination reaction, and boiling water boils 5min.Cooling back 540nm measures the OD value.1 enzyme unit (U) that lives is defined as the enzyme amount that under given condition per minute discharges 1 μ mol reducing sugar.
The property testing of embodiment 5 recombined xylanase XYNA4
1, the measuring method of the optimal pH of recombined xylanase XYNA4 and pH stability is as follows:
The recombined xylanase of embodiment 3 purifying is carried out enzymatic reaction to measure its optimal pH under different pH.The substrate xylan is with damping fluid (the 0.1M glycine-hydrochloric acid pH2.2-5.4 of different pH; 0.1mol/L citric acid-Sodium phosphate dibasic pH5.4-7.8; 0.1M Tris-HCl pH7.4-8.6; 0.1M glycine-sodium hydroxide pH8.6-12.0), carrying out Xylanase activity under 55 ℃ measures.Result (Fig. 2) shows that the optimal pH of XYNA4 is 7.0, and in the scope of pH6.2～9.4, enzymic activity all maintains more than 60% of maximum enzyme activity.Zytase is 37 ℃ of processing 60min in the damping fluid of above-mentioned various different pH, measure enzymic activity again under 55 ℃ in the pH7.0 buffer solution system, with the pH patience of research enzyme.Result (Fig. 3) shows that zytase is all very stable between pH2.6-12.0, and the residual enzyme activity is more than 90% behind the processing 60min in this pH scope, and this illustrates that this enzyme has pH stability preferably.
2, the optimum temperuture of zytase and thermal stability determination method are as follows:
Enzymatic reaction is carried out in being determined as under citric acid-Sodium phosphate dibasic damping fluid (pH7.0) buffer solution system and differing temps of the optimum temperuture of zytase.Temperature tolerance is determined as zytase and handles different time under differing temps, carries out enzyme assay again under 55 ℃.Enzyme reaction optimum temperuture measurement result (Fig. 4) shows that its optimum temperuture is 55 ℃.The thermostability test of enzyme shows (Fig. 5), and recombinase stability in the time of 60 ℃ is very good.65 ℃ are incubated 60min down, and the residual enzyme activity is 31.6%.
3, the K of zytase
mValues determination method is as follows:
Xylan (4-O-Me-D-glucurono-D-xylan, Sigma From birchwood) with different concns is a substrate, in citric acid-Sodium phosphate dibasic damping fluid (pH7.0) buffer solution system, measures enzymic activity down, calculates its k under 50 ℃ for 55 ℃
mValue.After measured, this zytase is the k of substrate with the xylan under 55 ℃
mValue is 1.90mg/mL, maximum reaction velocity V
MaxBe 417.93 μ mol/minmg.
4, different metal ion chemistry reagent is as follows to XYLA4 enzyme influence mensuration alive:
Add the different metal ions and the chemical reagent of different concns in enzymatic reaction system, study its influence to enzymic activity, various material final concentrations are 1 and 10mmol/L.Under 55 ℃, pH7.0 condition, measure enzymic activity.Result's (table 2) shows that the vigor of recombined xylanase did not have considerable change when most of ions and chemical reagent were 1mmol in concentration.Zn
2+, Cu
2+, Ni
2+, Ag
+Can partly suppress its vigor, but Hg
2+With SDS its vigor of strongly inhibited almost.Zn when concentration is 10mmo
2+, Cu
2+, Ni
2+, Ag
+, Hg
2+And its vigor of the equal strongly inhibited of SDS.Beta-mercaptoethanol can make the recombinase vigor be increased to original 1.13 and 1.32 times when 1mmol and 10mmol respectively.
The various chemical reagent of table 2. are to the influence of Xylanase XYNA 4 vigor
Annotate: " ND " representative can't detect.
5, being analyzed as follows of zytase degraded oat xylan product:
The enzyme liquid (about 4.0U) that adds 200 μ L purifying in the xylan of 200 μ L2%, 37 ℃ are incubated 12h down.2500 chromatographic instruments are used with behind the ultra-filtration membrane high speed centrifugation of 3k in the centrifugal back of 12000rpm, utilize high performance anion exchange chromatography-pulse ampere (HPAEC-PAD) detection method, carry out the analysis of sugar type in the product.Analytical results shows: the product of Xylanase XYNA 4 degraded oat xylan mainly is a wood sugar, xylo-bioses and a small amount of xylotriose and Xylotetrose.Wood sugar content is 51.50% in the product, and xylobiose content is 34.30%, and the content of xylotriose is 7.53%, and Xylotetrose content is 6.65%.
Sequence table
＜110〉Institute of Feeds,China Academy of Agriculture Sciences
＜120〉a kind of wide pH adaptability Xylanase XYNA 4 and gene and application
<160>2
<210>1
<211>338
<212>PRT
＜213〉alicyclic acid genus bacillus (Alicyclobacillus hesperidum A4)
<400>1
MTDTYRNIPS LSERYRPYFR IGAAVNAKSL NTHRDLLVTH FNSVTAENEM
RYEFAKADAL VNFAREHGMF VRGHTLVWHN QTPAAVFLDD LGQTATAAVV
ERRLEEHVAT 120
VLGRYHNDIY DWDVANEAVV DAGTGFLRDS RWLQTLGDDY IAKAFRIAHQ
AAPDALLFYN 180
DYNETKPDKS ERIYKLVAGL LDEGVPIHGI GMQGHWMLDD PALDEIERAI
DRYASLGVHL 240
HITELDVCVY GNVHGTGGQS QEVLPYDDEL AKRLAERYRS LFSSLRARKD
VIESVTFWGV 300
ADDDTWRDNF PVRGRKDWPL LFDVNHGPKQ AFWSVVEF 338
<210>2
<211>1017
<212>DNA
＜213〉alicyclic acid genus bacillus (Alicyclobacillus hesperidum A4)
<400>2
atgactgaca cttatcggaa tattccttcc ttgagtgagc gctaccgccc gtactttcgc 60
attggtgctg ccgtcaatgc caagtccctg aatacccacc gtgacttgtt ggtcacgcat 120
tttaacagcg tgaccgcaga gaacgaaatg aagtgggaag agattcatcc agaacaggat 180
cgatacgagt tcgcaaaggc ggatgcactc gtgaattttg cccgcgagca cggtatgttt 240
gtccgcggcc acacgttggt ctggcacaat caaacgccag ccgcagtgtt tctggacgat 300
ctcggtcaaa cagcgacagc ggccgttgtc gagcgtcgac tcgaagagca tgtggcaaca 360
gtgcttggtc gataccacaa cgacatctac gactgggatg ttgccaacga ggcagtcgtc 420
gatgcgggta caggattttt acgggacagt cgctggctgc agacccttgg cgatgattac 480
attgcgaagg cttttcgcat cgcacatcaa gcggcaccag acgcactgct cttctacaac 540
gactacaatg aaaccaaacc cgataaatcg gagcgcattt acaaacttgt tgcaggtctg 600
ctcgatgaag gtgtccccat ccatgggatt ggtatgcagg ggcactggat gttggacgat 660
ccggcgctgg acgaaattga acgagctatc gaccgctacg cgtcactcgg tgtgcacctt 720
cacatcacag aactcgacgt gtgtgtttat ggcaatgtcc acggaacggg cggccaatcc 780
caagaagttc taccctacga cgatgaactt gccaaacgtt tggccgagcg ttatcgttct 840
ctgttttcat cgttgcgcgc tcgcaaggat gtcatcgaaa gcgtcacgtt ctggggggtt 900
gctgatgatg atacctggcg ggacaacttc cctgttcgcg gacgcaaaga ctggccactt 960
ctgtttgacg tgaaccacgg gccgaaacag gcgttttgga gcgtggttga attttga 1017
Claims (10)
1. the adaptive Xylanase XYNA 4 of wide pH is characterized in that its aminoacid sequence is shown in SEQ IDNO.1.
2. the adaptive xylanase gene xynA4 of wide pH is characterized in that, described wide pH stability of coding claim 1 and adaptive Xylanase XYNA 4.
3. the adaptive xylanase gene xynA4 of wide pH as claimed in claim 2 is characterized in that its base sequence is shown in SEQ ID NO.2.
4. the recombinant vectors that comprises the adaptive xylanase gene xynA4 of the described wide pH of claim 3.
5. recombinant vectors according to claim 4, it is characterized in that, the preparation method of described recombinant vectors comprises: expression vector PET-22B is carried out EcoRI+Hind III double digestion, simultaneously with xylanase gene xynA4 through EcoRI+Hind III double digestion, cut out the gene fragment of encoding mature zytase and be connected with expression vector pPET-22B through EcoRI+Hind III double digestion.
6. the recombinant bacterial strain that comprises the adaptive xylanase gene xynA4 of the described wide pH of claim 3.
7. recombinant bacterial strain as claimed in claim 6 is characterized in that, described bacterial strain is intestinal bacteria, yeast, genus bacillus or lactobacillus.
8. a method for preparing Xylanase XYNA 4 is characterized in that, may further comprise the steps:
1) with the recombinant vectors transformed host cell of claim 4, gets recombinant bacterial strain;
2) cultivate recombinant bacterial strain, induce recombined xylanase to express; And
3) reclaim the also expressed Xylanase XYNA 4 of purifying.
The adaptive Xylanase XYNA 4 of the described wide pH of claim 1 at feed, bread, the application of beer, papermaking, weaving, pharmacy and energy field.
10. alicyclic acid genus bacillus Alicyclobacillus hesperidum A4, its deposit number is CGMCCNo.3147.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102359431A CN101701214B (en) | 2009-10-30 | 2009-10-30 | Xylanase XYNA4 with wide pH applicability and gene and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102359431A CN101701214B (en) | 2009-10-30 | 2009-10-30 | Xylanase XYNA4 with wide pH applicability and gene and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101701214A CN101701214A (en) | 2010-05-05 |
| CN101701214B true CN101701214B (en) | 2011-09-21 |
Family
ID=42156141
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009102359431A Active CN101701214B (en) | 2009-10-30 | 2009-10-30 | Xylanase XYNA4 with wide pH applicability and gene and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101701214B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101886064B (en) * | 2010-06-28 | 2012-06-06 | 中国农业科学院饲料研究所 | Acid amylase AMYA4 and gene and application thereof |
| CN102978188B (en) * | 2012-08-06 | 2014-10-22 | 江南大学 | Wide pH action range xylanase and applications thereof |
| CN103074317B (en) * | 2012-11-08 | 2014-06-11 | 中国农业科学院饲料研究所 | High temperature neutral xylanase XYNA used for brewing beer and gene and application thereof |
| CN103540573B (en) * | 2013-10-19 | 2015-10-07 | 沅江浣溪沙酶技术有限公司 | A kind of lignoenzyme and production method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1930285A (en) * | 2004-01-06 | 2007-03-14 | 诺和酶股份有限公司 | Polypeptides of alicyclibacillus |
-
2009
- 2009-10-30 CN CN2009102359431A patent/CN101701214B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1930285A (en) * | 2004-01-06 | 2007-03-14 | 诺和酶股份有限公司 | Polypeptides of alicyclibacillus |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101701214A (en) | 2010-05-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104357427A (en) | High-temperature-resistant alkaline salt-tolerant xylanase XynSL4 as well as gene and application thereof | |
| CN101457207A (en) | Eosinophil beta-mannanase MAN5A and gene and application thereof | |
| CN101457206B (en) | Acidic xylanase XYL10A and gene and application thereof | |
| CN107828806A (en) | A kind of β alpha-glucosidase genes of new resistance to glucose and its application | |
| CN101701214B (en) | Xylanase XYNA4 with wide pH applicability and gene and application thereof | |
| CN101701205B (en) | Alkali-resistant xylanase XynE2, genes thereof and application thereof | |
| CN104745612A (en) | Genes of high temperature resistant xylanase and high temperature resistant xylosidase and protein expression and application thereof | |
| CN101402964B (en) | Alkali-fast high-activity alkalescent xylanase and encoding gene thereof | |
| CN102220303A (en) | Xylanase XynAHJ3 with protease resistance and gene thereof | |
| CN102719417B (en) | High-temperature resistance arabinfuranosidease Abf51B8, as well as gene and application thereof | |
| CN102816728A (en) | Construction of beta-1,4-endoxylanase engineering bacterium, and application of enzyme of beta-1,4-endoxylanase engineering bacterium | |
| CN101838636B (en) | High-specific-activity xylanase XYN11F63 and genes and application thereof | |
| CN101724565A (en) | Acid xylanase XYNB, genes and application thereof | |
| CN101701213B (en) | Dual-function xylanase XYNBE18 and gene and application thereof | |
| CN101724613B (en) | Alkali proof intermediate temperature xylanase XYNAM6, gene thereof and application thereof | |
| CN101921739B (en) | Low-temperature xylanase XYNGR40 and gene and application thereof | |
| CN107236692B (en) | Paenibacillus cellulolyticus NP1 and xylanase PtXyn1 as well as encoding gene and application thereof | |
| CN103060290B (en) | Alkaline xylanase, its coding gene and application | |
| CN102242092A (en) | Isolation and identification for bombyx mori intestinal tract probiotics, and cloning and expression of enzyme gene produced by bombyx mori probiotics | |
| CN104560833A (en) | Basophilic micrococcus and alkaline xylanase produced from basophilic micrococcus and application | |
| CN102174494B (en) | Marine cryophilic endo beta-xylanase XynB as well as expressing gene xynB and application thereof | |
| CN103695397B (en) | Mesophilic acidic xylanase XYN10L1 and gene and application thereof | |
| CN101892208B (en) | High-temperature acidic xylanase XYN10J88, gene and application thereof | |
| CN103290039B (en) | Alpha-amylase derived from animal feces metagenome and gene of the alpha-amylase | |
| CN102952790B (en) | Multifunctional cellulose as well as expression gene and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20200902 Address after: 100193 Beijing Old Summer Palace West Road, Haidian District, No. 2 Patentee after: Beijing Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences Address before: 100086 No. 12 South Main Street, Haidian District, Beijing, Zhongguancun Patentee before: FEED Research Institute CHINESE ACADEMY OF AGRICULTURAL SCIENCES |
|
| TR01 | Transfer of patent right |