CN105950591A - Neutral low-temperature xylanase CaXyn10A, and gene and application thereof - Google Patents
Neutral low-temperature xylanase CaXyn10A, and gene and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/248—Xylanases
Abstract
The invention relates to the field of genetic engineering, in particular to a neutral low-temperature xylanase CaXyn10A, and a gene and application thereof. The amino acid sequence of the neutral low-temperature xylanase CaXyn10A is shown as SEQ ID NO. 1. The xylanase is characterized in that the optimal pH is 6.0-6.5, the optimal temperature is 40 DEG C, over 20% of enzyme activity is kept at the temperature of 0 DEG C, and the specific activity is 453U/mg; the xylanase has activities of xylanase, glucanase and cellulose; industrialized fermentation production is facilitated. The neutral low-temperature xylanase CaXyn10A serving as a novel broad-spectrum preparation can be widely applied to aquatic feed, food, papermaking, energy industry and the like.
Description
Technical field
The present invention relates to genetic engineering field, in particular it relates to a kind of neutral low-temperature xylanase CaXyn10A
And gene and application.
Background technology
Xylan (xylan) is the main component of plant biomass, and the content in nature is only second to cellulose (Lynd
Et al.Microbiology and Molecular Biology Review 2002,66:506 577), in bioenergy side
There is potential value in face.Existing biotechnology can be with the cellulose components in efficient degradation plant and change into fermentable
Glucose, the latter generates bio-ethanol under yeast effect.The xylan of high component not only limits cellulase and cellulose
Association reaction, suppress catalytic reaction, and enter nature as side-product and can cause nutrition accumulation thus destroy ecology
System (Polizeli et al.Applied Microbiology and Biotechnology.2005,67:577-591).
Therefore in biomass bioconversion procedure, xylanase to be added, on the one hand remove the physical barriers of xylan, promote
Cellulase and combination (Hu et al.Biotechnology for Biofuels, the 2011,4:14 of cellulose；Qing and
Wyman CE.Biotechnology for Biofuels, 2011,4:18), the xylooligosaccharide on the other hand generated can also enter one
Step be degraded into fermentable xylose, for bio-ethanol produce provide 10-25% raw material (Ho et al.1998,64:
1852–1859；Jin et al.Applied and Environmental Microbiology,2004,70:6816–
6825)。
Based on aminoacid sequence and the structure of catalytic domain, major part xylanase (EC 3.2.1.8) is divided in glycoside hydrolysis
Enzyme the 10th and 11 family.Compared with the 11st family's xylanase of single-minded degradation of xylan, the xylanase of the 10th family has
Wide in range substrate specificity, in addition to acting on xylan, also can the degraded of the multiple substrate such as catalysis fibre element, glucosan, because of
This has wider application prospect in industrial circle.
Filamentous fungi is prone to cultivate, can secrete xylanase in a large number and become the ideal source (Tanaka of xylanase
et al.Journal of Bioscience and Bioengineering,2005,100:623–630).Most of thread very
The xylanase in bacterium source is all middle temperature or high temperature enzyme, loses enzyme and live under ambient temperature or cryogenic conditions.Existing biological second
Alcohol processing technique includes that the acid-base pretreatment of lignocellulose, long-time high temperature enzyme are reacted, thus consumes substantial amounts of energy and acid
Alkali chemical reagent, causes significantly promoting and serious environmental pollution of cost.Therefore the lignocellulose of the neutral low temperature of exploitation
Degradation enzyme system is being saved the energy, is being protected environment, is prone to the aspects such as catalytic reaction and has great importance (Ji et
al.Biotechnology for Biofuels,2014,7:130).So far, only one of which low-temperature xylanase system is reported
Road (Del-Cid A et al.Applied Biochemistry and Biotechnology, 2014,172:524 532),
There is no the report of low temperature the 10th family's xylanase of originated from fungus.
Although the xylanase of originated from fungus is widely used to different industrial circles, it is thus achieved that novel have good characteristic
Neutral low-temperature xylanase still there is great Research Significance and using value.Clone and heterogenous expression low-temperature xylanase,
There is provided economical and effective, eco-friendly candidate enzyme for biodegradation, feedstuff, food and paper industry, be that xylanase is applied to
Necessary to industrialized production.
Summary of the invention
It is an object of the invention to provide the neutral low-temperature xylanase of a kind of energy efficient application.
Another object of the present invention is to provide the gene of the above-mentioned neutral low-temperature xylanase of coding.
It is a further object of the present invention to provide the recombinant vector comprising said gene.
It is a further object of the present invention to provide the recombinant bacterial strain comprising said gene.
It is a further object of the present invention to provide a kind of gene engineering method preparing above-mentioned neutral low-temperature xylanase.
Another object of the present invention provides the application of above-mentioned neutral low-temperature xylanase.
The present invention a kind of new neutrality of isolated from cladosporium (Cladosporium acalyphae SL-16) is low
Temperature xylanase CaXyn10A.
The invention provides a kind of neutral low-temperature xylanase CaXyn10A, its aminoacid sequence such as SEQ ID NO.1 institute
Show.
SEQ ID NO.1:
MLISNAIAALSVLSLASAAAIPESHIEARAAKTVDAGFKAKGKKYWGTASDQGILSQSNINNAAKANFG
QITPENSMKFDATEPSRGNFNFNGADWLVNWAQTNGKLVRGHTLVWHSQVPAWVTNINDKNTLISVMQNHIKTVVGR
YKGKIYAWDVTNEIFNEDGTLRQSHWMKVIGEDYVRIAFETARAADPSAKLYINDYNLDVANYGKTQGMIKYVKKWR
AAGVPIDGIGSQMHLSNGNGWPTATDVVNAMKALCAAAPECAVTELDIQGASSADYLKAMHSCLDVQNCVGITSWGI
TDNTSWRADKTPLLFNSQAQPKAAYTALLNAL
Wherein, 332 aminoacid of this enzyme gene code, 18 aminoacid of N end are its signal peptide sequence
“mlisnaiaalsvlslasa”(SEQ ID NO.3)。
Therefore, the theoretical molecular of ripe neutral low-temperature xylanase CaXyn10A is 34.5kDa, its aminoacid sequence
As shown in SEQ ID NO.2:
AAIPESHIEARAAKTVDAGFKAKGKKYWGTASDQGILSQSNINNAAKANFGQITPENSMKFDATEPSRG
NFNFNGADWLVNWAQTNGKLVRGHTLVWHSQVPAWVTNINDKNTLISVMQNHIKTVVGRYKGKIYAWDVTNEIFNED
GTLRQSHWMKVIGEDYVRIAFETARAADPSAKLYINDYNLDVANYGKTQGMIKYVKKWRAAGVPIDGIGSQMHLSNG
NGWPTATDVVNAMKALCAAAPECAVTELDIQGASSADYLKAMHSCLDVQNCVGITSWGITDNTSWRADKTPLLFNSQ
AQPKAAYTALLNAL
The xylanase CaXyn10A of the present invention is respectively provided with in the range of neutral (pH 6.0-6.5) and middle low temperature (20-45)
Highly active characteristic.Xylanase CaXyn10A produced by Cladosporium acalyphae SL-16, its optimum pH
It is 6.5, in the range of pH6.0～7.2, maintains the enzymatic activity of more than 80%；Optimum temperature is 40 DEG C, all has between 20-45 DEG C
There is the enzyme activity of more than 70%；20% enzyme is also had to live under the conditions of 0 DEG C；And multiple xylan, glucosan, cellulose are had fall
Solution effect.The most unprecedented report of neutral low-temperature xylanase of this character.
The invention provides the above-mentioned neutral low-temperature xylanase Caxyn10A of coding.Specifically, the genome sequence of this gene
Row are as shown in SEQ ID NO.4:
atgctgatttccaacgccattgccgcactctctgtgctgtcgcttgcttccgcagcggccattcctgagtctcacat
cgaggctcgcgctgctaagactgtcgatgccggcttcaaggccaagggcaagaagtcagtcaaaccctaagctctaa
cacgcatttgggctttgctaatatccttataggtactggggcactgccagtgatcagggcattctttcccagagcaa
catcaacaatgctgccaaggccaacttcggccagatcacccccgagaactccatgaagttcgatgccactgagcctt
cccgtggcaacttcaacttcaacggtgccgactggctcgtcaactgggctcagaccaacggcaagctcgtccgtggt
cacactcttgtctggcacagccaggtccccgcctgggtcaccaacatcaacgacaagaacaccttgatctctgtcat
gcagaaccacatcaagactgtcgttggccgctacaagggcaagatctacgcatgggtcagtagagctggataccgct
ttgatcgaaccctatactgatgattgtaggacgtcaccaacgagatcttcaacgaggacggtactctcaggcagagc
cactggatgaaggtcatcggcgaggactacgtccgcattgccttcgagaccgctcgcgccgctgacccgtcggccaa
gctctacatcaacgattacaaccttgatgttgccaactacggcaagactcagggtatgatcaagtacgtcaagaagt
ggcgcgccgccggcgttcccattgacggaattggcagccagatggtaagttcaacgatcaatctctttggaaagttg
ccaggatgctaacgtttcacagcacctttccaacggtaacggctggcccaccgccaccgatgtcgttaacgccatga
aggccctctgtgccgccgctcctgagtgcgccgtcactgagctcgacattcagggcgcatcgagcgctgactacctt
aaggcgatgcactcttgccttgacgtccagaactgtgttggtatcacctcctggggtattaccgacaacaccgtaag
ttttgacatttttcacccaaaaatgacccaaaaaccgacactaaccatatcacctacagtcgtggcgcgccgacaag
actcccctgctcttcaactcccaggctcagcccaaggccgcctacactgctctcctcaacgctttgtaa
The present invention passes through the method separating clone of PCR xylanase gene Caxyn10A, DNA complete sequence analysis result table
Bright, xylanase CaXyn10A structural gene Caxyn10A total length 1224bp, containing 4 introns, the long 999bp of cDNA ,+132
～+186bp ,+518～+568bp ,+815～+869bp and+1074～+1137bp is 55,51,55 and the intron sequence of 64bp
Row, its cDNA sequence is as shown in SEQ ID NO.5:
atgctgatttccaacgccattgccgcactctctgtgctgtcgcttgcttccgcagcggccattcctgagtctcacat
cgaggctcgcgctgctaagactgtcgatgccggcttcaaggccaagggcaagaagtactggggcactgccagtgatc
agggcattctttcccagagcaacatcaacaatgctgccaaggccaacttcggccagatcacccccgagaactccatg
aagttcgatgccactgagccttcccgtggcaacttcaacttcaacggtgccgactggctcgtcaactgggctcagac
caacggcaagctcgtccgtggtcacactcttgtctggcacagccaggtccccgcctgggtcaccaacatcaacgaca
agaacaccttgatctctgtcatgcagaaccacatcaagactgtcgttggccgctacaagggcaagatctacgcatgg
gacgtcaccaacgagatcttcaacgaggacggtactctcaggcagagccactggatgaaggtcatcggcgaggacta
cgtccgcattgccttcgagaccgctcgcgccgctgacccgtcggccaagctctacatcaacgattacaaccttgatg
ttgccaactacggcaagactcagggtatgatcaagtacgtcaagaagtggcgcgccgccggcgttcccattgacgga
attggcagccagatgcacctttccaacggtaacggctggcccaccgccaccgatgtcgttaacgccatgaaggccct
ctgtgccgccgctcctgagtgcgccgtcactgagctcgacattcagggcgcatcgagcgctgactaccttaaggcga
tgcactcttgccttgacgtccagaactgtgttggtatcacctcctggggtattaccgacaacacctcgtggcgcgcc
gacaagactcccctgctcttcaactcccaggctcagcccaaggccgcctacactgctctcctcaacgctttgtaa
Wherein, the base sequence of signal peptide is: ATGCTGATTTCCAACGCCATTGCCGCACTCTCTGTGCTGTCGCTT
GCTTCCGCA(SEQ ID NO.6)。
Maturation protein theoretical molecular is 34.5kDa.By xylanase gene Caxyn10A cDNA sequence and derive
Aminoacid sequence carries out BLAST comparison in GenBank.This gene and the wood deriving from Aspergillus fumigatus gather
Carbohydrase Amino acid sequence identity is 63%.Illustrate that CaXyn10A is a kind of new xylanase.
Present invention also offers the recombinant vector comprising above-mentioned neutral low-temperature xylanase gene C axyn10A, be preferably
pET30-Caxyn10A.The xylanase gene of the present invention is inserted between the suitable restriction enzyme site of expression vector,
Make that its nucleotide sequence is exercisable to be connected with expression regulation sequence.As the most preferred embodiment of the present invention,
Be preferably NdeI and EcoRI restriction enzyme site that the xylanase gene of the present invention is inserted on plasmid pET30 (a) it
Between, make this nucleotide sequence be positioned at the downstream of T7 promoter and be regulated and controled by it, obtain expression of recombinant e. coli plasmid pET30-
Caxyn10A。
Present invention also offers the recombinant bacterial strain comprising above-mentioned neutral low-temperature xylanase gene C axyn10A, preferably described
Bacterial strain is escherichia coli, yeast, bacillus cereus, lactobacillus, aspergillosis or Trichoderma spp., preferably recombinant bacterial strain pET30/
Caxyn10A。
Present invention also offers a kind of method preparing neutral low-temperature xylanase CaXyn10A, comprise the following steps:
1) with above-mentioned recombinant vector transformed host cell, recombinant bacterial strain is obtained；
2) cultivating recombinant bacterial strain, induction recombined xylanase is expressed；And
3) the xylanase CaXyn10A also expressed by purification is reclaimed.
Wherein, the most described host cell is escherichia coli, preferably by expression of recombinant e. coli Plastid transformation large intestine bar
Bacterium cell (Escherichia coli) BL21, obtains recombinant bacterial strain pET30/Caxyn10A.
Present invention also offers the application of above-mentioned neutral low-temperature xylanase CaXyn10A.
The most to be solved technical problem is that of the present invention overcomes the deficiencies in the prior art, it is thus achieved that the bacterium of low-temperature xylanase
Goods and materials source, it is provided that a kind of good properties, be suitable in feedstuff, food, industry, apply new neutral low-temperature xylanase.This
The xylanase optimum pH of invention is 6.0, has higher enzymatic activity at pH6.0～7.2；PH good stability；Rate activity is
453U/mg；There is wide in range substrate specificity.The feature of its neutral low temperature, it is possible to decrease xylanase energy in commercial production
Source cost.PH subject range improves aquatic feeds digestible energy and metabolizable energy, reduces formulation cost, reduces environmental pollution.It is wide in range
Substrate specificity can improve the nutritive value of grain processing co-product, promote feed product quality.This xylanase can be answered
For brewing industry, reduce material viscosity, diastatic action can be conducive in stratum granulosum, improve starch utilization ratio, increase wine
The productivity of essence.Under normal temperature condition, the xylan in paper industry waste material and agricultural wastes can also can be converted into wood
Oligosaccharide, and xylooligosaccharide can be become valuable fuel by antibacterial, yeast and fungal transformation.Therefore, this xylanase is in energy work
Application in industry also shows that its huge potentiality.Hydrolyzate (xylose and oligomeric xylose) can be applicable to food service industry, as
Thickening agent, fat sub, prebiotic oligosaccharide and freeze proof food additive；In pharmaceuticals industry, xylan is combined with other material and makes
With, the release of ingredient can be delayed.The hydrolyzate of xylan can also be further converted to liquid fuel, single cell protein
In vain, solvent and low calorie sweetener.
Accompanying drawing explanation
The SDS-PAGE of the recombined xylanase that Fig. 1 expresses in coliform analyzes, wherein, and 1: the Scrimber polysaccharide of purification
Enzyme；M: low molecular weight protein Marker.
The optimum pH of Fig. 2 recombined xylanase.
The pH stability of Fig. 3 recombined xylanase.
The optimum temperature of Fig. 4 recombined xylanase.
The heat stability of Fig. 5 recombined xylanase.
Detailed description of the invention
Test material and reagent
1, bacterial strain and carrier: the present invention, from cladosporium Cladosporium acalyphae SL-16, is stored in China's agriculture
Agricultural DSMZ of industry academy of science (No.12 ,zhongguancun south street,Haidian District, Beijing, Chinese Academy of Agricultural Sciences's agricultural strain
Preservation center, 100081), its preserving number is: ACCC32700.Coli expression carrier pET30 (a) and bacterial strain BL21 are by this
Laboratories Accession.
2, enzyme and other biochemical reagents: restriction endonuclease is purchased from TaKaRa company, ligase is purchased from Invitrogen company.Beech
Wood xylan is purchased from Sigma company, and other is all domestic reagent (all can be commercially available from common biochemical Reagent Company).
3, culture medium:
(1) Cladosporium acalyphae SL-16 culture medium is potato juice culture medium: 1000mL potato juice,
10g glucose, 25g agar, pH5.0.
(2) Escherichia coli culture medium LB (1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0).
Illustrate: following example are not made the experimental methods of molecular biology illustrated, all with reference to " Molecular Cloning: A Laboratory
Guide " concrete grammar listed in (third edition) J. Pehanorm Brooker one book carries out, or according to test kit and product description
Carry out.
Embodiment 1 cladosporium Cladosporium acalyphae SL-16 Enzymatic characteristic
By cladosporium Cladosporium acalyphae SL-16 after potato juice culture medium culturing, make spore and hang
Liquid, be inoculated in wheat bran fluid medium (Luo et al.Enzyme Microbial Technology, 2009,45:126
133), cultivating 7d for 15 DEG C, the Zelkova schneideriana Hand.-Mazz. xylan with 1%, as substrate, reacts 10min, with DNS method under the conditions of pH 6.0 and 30 DEG C
(Miller 1959) determines that it has xylanase activity.
Embodiment 2 cladosporium Cladosporium acalyphae SL-16 Xylanase coding gene Caxyn10A gram
Grand
Extraction cladosporium Cladosporium acalyphae SL-16 genomic DNA:
Being filtered by the liquid culture mycelium aseptic filter paper of 3 days puts in mortar, adds 2mL extracting solution, grinds 5min,
Then lapping liquid is placed in 50mL centrifuge tube, 65 DEG C of water-bath cracking 20min, mixes once every 10min, at 4 DEG C
10000rpm is centrifuged 5min.Take supernatant extrct foreigh protein removing in phenol/chloroform, then take supernatant addition equal-volume isopropanol, in
After room temperature stands 5min, at 4 DEG C, 10000rpm is centrifuged 10min.Abandon supernatant, precipitation with 70% washing with alcohol twice, vacuum is dry
Dry, add appropriate TE dissolve, be placed in-20 DEG C standby.
Design synthesis special primer Caxyn10A-F/Caxyn10A-F:
Caxyn10A-F:5'-atgctgatttccaacgccattgccg-3'；
Caxyn10A-R:5'-ttacaaagcgttgaggagagcagtgtaggc-3'；
PCR amplification is carried out for template with cladosporium Cladosporium acalyphae SL-16 STb gene.PCR reacts ginseng
Number is: 94 DEG C of degeneration 5min；Then 94 DEG C of degeneration 30sec, 60 DEG C annealing 30sec, 72 DEG C extend 60sec, 30 circulation after 72
DEG C insulation 10min.Obtain an about 1224bp fragment, be connected with pEASY-T3 carrier after this fragment is reclaimed and send three to win biotechnology
Company limited checks order.
The RT-PCR of embodiment 3 cladosporium Cladosporium acalyphae SL-16 xylanase gene analyzes
Extract the total serum IgE of cladosporium Cladosporium acalyphae SL-16, utilize reverse transcription to obtain cDNA's
Article one, chain, then expands this strand cDNA with primer Caxyn10A-E-F/Caxyn10A-E-F, it is thus achieved that the cDNA sequence of xylanase
Row, amplification obtains product and reclaims the order-checking of Hou Songsanbo Bioisystech Co., Ltd.
Caxyn10A-E-F:5'-ccccatatgatgctgatttccaacgccattgcc-3'；
Caxyn10A-E-R:5'-cccgaattcttagtggtggtggtggtggtgcaaagcgttg aggagagcagtg-
3'；
By finding that this gene has 4 introns, cDNA length after comparing the genome sequence of xylanase and cDNA sequence
999bp, encodes 332 aminoacid and a termination codon, and 18 aminoacid of N end are its signal peptide sequence.Measured base
Because the maturation protein part nucleotide sequence of Caxyn10A carries out Homology search with the xylanase sequence on GenBank,
The gene of the encoding xylanase that separating clone obtains is new gene.
The preparation of embodiment 4 recombined xylanase.
Expression vector pET30 (a) is carried out double digestion (NedI+EcoRI), simultaneously by the gene of encoding xylanase
Caxyn10A double digestion (NedI+EcoRI), cuts out the genetic fragment of encoding mature xylanase with expression vector pET30 (a) even
Connect, it is thus achieved that containing the recombiant plasmid of cladosporium Cladosporium acalyphae SL-16 xylanase gene Caxyn10A
PET30-Caxyn10A also converts e. coli bl21, it is thus achieved that recombinant escherichia coli strain pET30/Caxyn10A.
Build the recombinant expression carrier comprising signal peptide sequence in the same way, and obtain recombinant bacterial strain.
Take the BL21 bacterial strain containing recombiant plasmid, be inoculated in 100mL LB culture fluid, 16 DEG C of 150rpm shaken cultivation 12h
After, add 0.6%IPTG and induce 10h, collect cell, ultrasonic disruption, centrifugal collection supernatant.Measure the work of xylanase
Power.The expression of recombined xylanase is 1.54U/mL.SDS-PAGE result (Fig. 1) shows, recombined xylanase is at large intestine bar
Bacterium is expressed.Expressed xylanase after purification, the content of its protein reach the 15% of total protein with
On.
The property testing of embodiment 5 recombined xylanase CaXyn10A
DNS method: concrete grammar is as follows: at pH6.5, under the conditions of 40 DEG C, the reaction system of 1mL includes suitable dilute of 100 μ L
Release enzyme liquid, 900 μ L substrates, react 10min, add 1.5mL DNS and terminate reaction, boiling water boiling 5min.After cooling, 540nm measures OD
Value.1 enzyme unit (U) alive is defined as the enzyme amount discharging 1 μm ol reducing sugar the most per minute.
1, the optimum pH of recombined xylanase CaXyn10A and the assay method of pH stability are as follows:
The recombined xylanase of embodiment 4 purification is carried out enzymatic reaction to measure its optimum pH under different pH.The end
The 0.1mol/L citrate-phosphate disodium hydrogen buffer of the different pH of thing xylan carries out Xylanase activity survey at 30 DEG C
Fixed.Result (Fig. 2) shows, the optimum pH of CaXyn10A is 6.0, and in the range of pH6.0～7.2, enzymatic activity is kept at
More than the 80% of big enzymatic activity.Xylanase is 30 DEG C of process 60min in the buffer of above-mentioned various different pH, then at pH6.0
Buffer solution system measures enzymatic activity, with the pH patience of studying enzyme at 40 DEG C.Result (Fig. 3) shows that xylanase is at pH 4.0-
Between 10.0 the most stable, in the range of this pH, process after 60min that residual enzyme activity is more than 70%, this illustrates that this enzyme has relatively
Good pH stability.
2, optimum temperature and the thermal stability determination method of xylanase are as follows:
Being determined as at citrate-phosphate disodium hydrogen buffer (pH6.0) buffer solution system of the optimum temperature of xylanase
And under different temperatures, carry out enzymatic reaction.Temperature tolerance is determined as xylanase and processes different time at different temperatures, then
PH6.0, carry out enzyme assay at 40 DEG C.Enzyme reaction optimum temperature measurement result (Fig. 4) shows that its optimum temperature is 40 DEG C.Enzyme
Heat stability test show (Fig. 5), recombinase stability when 30 DEG C is the best.It is incubated 60min at 40 DEG C, loses completely
Enzyme is lived.
3, the K of xylanasemValues determination method is as follows:
It is substrate with the Zelkova schneideriana Hand.-Mazz. xylan of variable concentrations, at citrate-phosphate disodium hydrogen buffer (pH6.0) buffering liquid
In system, measure enzymatic activity at 20 DEG C and 40 DEG C, calculate its K at 20 DEG C and 40 DEG Cm、Vmax、kcatAnd kcat/KmValue.Through surveying
Fixed, this xylanase k with xylan as substrate at 40 DEG CmValue is 1.87mg/mL, maximum reaction velocity VmaxIt is 331.6 μ
Mol/min mg, kcatValue is 190.7/s and kcat/KmValue is 102.0ml/mg s；K with xylan as substrate at 20 DEG Cm
Value is 12.23mg/mL, maximum reaction velocity VmaxIt is 322.9 μm ol/min mg, kcatValue is 185.7/s and kcat/KmValue is
15.2ml/mg·s
4, the impact that CaXyn10A enzyme is lived by different metal ion chemistry reagent is determined as follows:
In enzymatic reaction system, add different metal ions and the chemical reagent of variable concentrations, study it to enzymatic activity
Impact, the final concentration of 5mmol/L of various materials.40 DEG C, measure enzymatic activity under the conditions of pH6.0.Result (table 1) shows, greatly
Most ions and chemical reagent vigor of recombined xylanase when concentration is 5mmol do not have significant change.But Ag+Strongly press down
Make its vigor, Pb2+, Zn2+, SDS and EDTA has partial inhibition.Ni2+And Co2+Recombinase vigor can be made to increase when 5mmol
To original 1.27 and 1.16 times.
The impact on xylanase CaXyn10A vigor of the table 1. various chemical reagent
5, the substrate specificity of CaXyn10A
Different substrates, the enzymatic activity of research CaXyn10A is added in enzymatic reaction system.40 DEG C, pH6.0 condition
Lower mensuration enzymatic activity.Result (table 2) shows, CaXyn10A has the activity of xylanase, cellulase and glucanase simultaneously.
The substrate specificity of table 2.CaXyn10A
6, CaXyn10A xylanase degraded being analyzed as follows of Zelkova schneideriana Hand.-Mazz. xylan product:
In the xylan of 500 μ L 1%, add the enzyme liquid of 100 μ L purification, under optimum temperature, be incubated 12h.Use dehydrated alcohol
Pheron is precipitated, supernatant 2500 chromatographs, utilize High Performance Anion Exchange Chromatography Coupled with Pulsed Amperometric (HPAEC-PAD) to examine
Survey method, carries out the analysis of sugar type in product.Analysis result shows: the product of xylanase CaXyn10A degraded Zelkova schneideriana Hand.-Mazz. xylan
If owner's 1,4-.beta.-Xylobiose, xylotriose and Xylotetrose..In product, xylobiose content is 50.1%, and xylotriose content is 45.9%, and
Xylotetrose. content is 4.0%.
Claims (9)
1. a neutral low-temperature xylanase CaXyn10A, it is characterised in that its aminoacid sequence such as SEQ ID NO.1 or SEQ
Shown in ID NO.2.
2. neutral low-temperature xylanase gene C axyn10A, it is characterised in that the coding neutral low temperature described in claim 1
Xylanase CaXyn10A.
3. neutral low-temperature xylanase gene C axyn10A as claimed in claim 2, it is characterised in that its base sequence is such as
Shown in SEQ ID NO.4 or 5.
4. comprise the recombinant vector of neutral low-temperature xylanase gene C axyn10A described in claim 2.
5. comprise the recombinant vector pET30-Caxyn10A of neutral low-temperature xylanase gene C axyn10A described in claim 2.
6. comprise the recombinant bacterial strain of neutral low-temperature xylanase gene C axyn10A described in claim 2.
7. recombinant bacterial strain as claimed in claim 6, it is characterised in that described bacterial strain is escherichia coli, yeast, bacillus cereus, breast
Acidfast bacilli, aspergillosis or Trichoderma spp..
8. prepare the method for neutral low-temperature xylanase CaXyn10A described in claim 1 for one kind, it is characterised in that include following
Step:
1) with the recombinant vector transformed host cell of claim 4, recombinant bacterial strain is obtained；
2) cultivating recombinant bacterial strain, induction recombined xylanase is expressed；And
3) the neutral low-temperature xylanase CaXyn10A also expressed by purification is reclaimed.
9. the application of neutral low-temperature xylanase CaXyn10A described in claim 1.
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| CN108048506A (en) * | 2018-01-09 | 2018-05-18 | 山东大学 | The method with side chain wood oligose is prepared using thermophilic filamentous fungi fermentation one-step method |
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| CN101701213A (en) * | 2009-10-30 | 2010-05-05 | 中国农业科学院饲料研究所 | Dual-function xylanase XYNBE18 and gene and application thereof |
| CN102220304A (en) * | 2011-05-30 | 2011-10-19 | 云南师范大学 | Low-temperature xylanase XynAHJ2 and gene thereof |
| CN103898079A (en) * | 2014-04-16 | 2014-07-02 | 中国农业科学院饲料研究所 | Intermediate-temperature neutral xylanase XYN11B as well as gene and application thereof |
| WO2015114110A1 (en) * | 2014-01-31 | 2015-08-06 | Dupont Nutrition Biosciences Aps | Protein |
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| CN101701213A (en) * | 2009-10-30 | 2010-05-05 | 中国农业科学院饲料研究所 | Dual-function xylanase XYNBE18 and gene and application thereof |
| CN102220304A (en) * | 2011-05-30 | 2011-10-19 | 云南师范大学 | Low-temperature xylanase XynAHJ2 and gene thereof |
| WO2015114110A1 (en) * | 2014-01-31 | 2015-08-06 | Dupont Nutrition Biosciences Aps | Protein |
| CN103898079A (en) * | 2014-04-16 | 2014-07-02 | 中国农业科学院饲料研究所 | Intermediate-temperature neutral xylanase XYN11B as well as gene and application thereof |
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| CN108048506A (en) * | 2018-01-09 | 2018-05-18 | 山东大学 | The method with side chain wood oligose is prepared using thermophilic filamentous fungi fermentation one-step method |
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