CN105950591B - A kind of neutrality low-temperature xylanase CaXyn10A and its gene and application - Google Patents
A kind of neutrality low-temperature xylanase CaXyn10A and its gene and application Download PDFInfo
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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 present invention relates to genetic engineering fields, in particular it relates to a kind of neutrality low-temperature xylanase CaXyn10A and its gene and application.For its amino acid sequence as shown in SEQ ID NO.1, zytase of the invention has the property that optimal pH 6.0-6.5,40 DEG C of optimum temperature, 20% or more enzyme activity is kept at 0 DEG C, than living for 453U/mg；Activity with zytase, dextranase, cellulase；And it is easy to industrial fermentation production.As a kind of novel wide spectrum enzyme preparation, aquatic feeds, food, papermaking, energy industry etc. can be widely used for.
Description
Technical field
The present invention relates to genetic engineering fields, in particular it relates to a kind of neutrality low-temperature xylanase CaXyn10A
And its gene and application.
Background technique
Xylan (xylan) is the main component of plant biomass, and 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 utility value in face.Existing biotechnology with the cellulose components in efficient degradation plant and can be converted to fermentable
Glucose, the latter generate bio-ethanol under yeast effect.The xylan of high component not only limits cellulase and cellulose
Association reaction, inhibit catalysis reaction, and being discharged into nature as byproduct can cause nutrition accumulationization to destroying ecology
System (Polizeli et al.Applied Microbiology and Biotechnology.2005,67:577-591).
Therefore in biomass bioconversion procedure, zytase is usually added, the physical barriers of xylan are on the one hand removed, is promoted
Combination (Hu et al.Biotechnology for Biofuels, 2011,4:14 of cellulase and cellulose；Qing and
Wyman CE.Biotechnology for Biofuels, 2011,4:18), the wood oligose on the other hand generated can also be into one
Step is degraded into fermentable xylose, for bio-ethanol production provide 10-25% raw material (Ho et al.1998,64:
1852–1859；Jin et al.Applied and Environmental Microbiology,2004,70:6816–
6825)。
Structure based on amino acid sequence and catalytic domain, most of zytase (EC 3.2.1.8) are divided in glycoside hydrolysis
Enzyme the 10th and 11 families.Compared with the 11st family's zytase of single-minded degradation of xylan, the zytase of the 10th family has
Wide in range substrate specificity, other than acting on xylan, can also a variety of substrates such as catalytic cellulose, glucan degradation, because
This has wider application prospect in industrial circle.
Ideal source (the Tanaka that filamentous fungi is easy to cultivate, can largely secrete zytase and become zytase
et al.Journal of Bioscience and Bioengineering,2005,100:623–630).It is most of Filamentous true
The zytase in bacterium source is all medium temperature or high temperature enzyme, loses enzyme activity under environment temperature or cryogenic conditions.Existing biology second
Alcohol processing technology includes the acid-base pretreatment of lignocellulosic, long-time high temperature enzyme reaction, to consume a large amount of energy and acid
Alkali chemical reagent causes the significantly promotion and serious environmental pollution of cost.Therefore the lignocellulosic of neutral low temperature is developed
Degradation enzyme system energy saving, protection environment, be easy to be catalyzed in terms of have great importance (Ji et
al.Biotechnology for Biofuels,2014,7:130).So far, only one 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 the 10th family's zytase of low temperature of originated from fungus.
Although the zytase of originated from fungus is widely used to different industrial circles, obtain novel with good characteristic
Neutral low-temperature xylanase still there is great research significance and application value.Clone and heterogenous expression low-temperature xylanase,
Economical and effective, environmental-friendly candidate enzyme are provided for biodegrade, feed, food and paper industry, is that zytase is applied to
Necessary to industrialized production.
Summary of the invention
The object of the present invention is to provide a kind of neutral low-temperature xylanases of energy efficient application.
Another object of the present invention is to provide the gene for encoding above-mentioned neutral low-temperature xylanase.
It is a further object of the present invention to provide the recombinant vectors comprising said gene.
It is a further object of the present invention to provide the recombinant bacterial strains comprising said gene.
It is a further object of the present invention to provide a kind of gene engineering methods for preparing above-mentioned neutral low-temperature xylanase.
Another object of the present invention provides the application of above-mentioned neutral low-temperature xylanase.
A kind of present invention isolated new neutrality from cladosporium (Cladosporium acalyphae SL-16) is low
Warm zytase CaXyn10A.
The present invention provides a kind of neutral low-temperature xylanase CaXyn10A, amino acid sequence such as SEQ ID NO.1 institutes
Show.
SEQ ID NO.1:
MLISNAIAALSVLSLASAAAIPESHIEARAAKTVDAGFKAKGKKYWGTASDQGILSQSNINNAAKANFG
QITPENSMKFDATEPSRGNFNFNGADWLVNWAQTNGKLVRGHTLVWHSQVPAWVTNINDKNTLISVMQNHIKTVVGR
YKGKIYAWDVTNEIFNEDGTLRQSHWMKVIGEDYVRIAFETARAADPSAKLYINDYNLDVANYGKTQGMIKYVKKWR
AAGVPIDGIGSQMHLSNGNGWPTATDVVNAMKALCAAAPECAVTELDIQGASSADYLKAMHSCLDVQNCVGITSWGI
TDNTSWRADKTPLLFNSQAQPKAAYTALLNAL
Wherein, which encodes 332 amino acid, and 18 amino acid of N-terminal are its signal peptide sequence
“mlisnaiaalsvlslasa”(SEQ ID NO.3)。
Therefore, the theoretical molecular weight of mature neutral low-temperature xylanase CaXyn10A is 34.5kDa, amino acid sequence
As shown in SEQ ID NO.2:
AAIPESHIEARAAKTVDAGFKAKGKKYWGTASDQGILSQSNINNAAKANFGQITPENSMKFDATEPSRG
NFNFNGADWLVNWAQTNGKLVRGHTLVWHSQVPAWVTNINDKNTLISVMQNHIKTVVGRYKGKIYAWDVTNEIFNED
GTLRQSHWMKVIGEDYVRIAFETARAADPSAKLYINDYNLDVANYGKTQGMIKYVKKWRAAGVPIDGIGSQMHLSNG
NGWPTATDVVNAMKALCAAAPECAVTELDIQGASSADYLKAMHSCLDVQNCVGITSWGITDNTSWRADKTPLLFNSQ
AQPKAAYTALLNAL
Zytase CaXyn10A of the invention all has in neutral (pH 6.0-6.5) and middle low temperature (20-45) range
The characteristic of high activity.Zytase CaXyn10A caused by Cladosporium acalyphae SL-16, optimum pH
It is 6.5,80% or more enzymatic activity is maintained in the range of pH6.0~7.2；Optimum temperature is 40 DEG C, is had between 20-45 DEG C
There is 70% or more enzyme activity；There are also 20% enzyme activity under the conditions of 0 DEG C；And there is drop to a variety of xylans, glucan, cellulose
Solution effect.The also unprecedented report of the neutral low-temperature xylanase of this property.
The present invention provides encode above-mentioned neutral low-temperature xylanase Caxyn10A.Specifically, the genome sequence of the gene
Column are as shown in SEQ ID NO.4:
atgctgatttccaacgccattgccgcactctctgtgctgtcgcttgcttccgcagcggccattcctga
gtctcacatcgaggctcgcgctgctaagactgtcgatgccggcttcaaggccaagggcaagaagtcagtcaaaccc
taagctctaacacgcatttgggctttgctaatatccttataggtactggggcactgccagtgatcagggcattctt
tcccagagcaacatcaacaatgctgccaaggccaacttcggccagatcacccccgagaactccatgaagttcgatg
ccactgagccttcccgtggcaacttcaacttcaacggtgccgactggctcgtcaactgggctcagaccaacggcaa
gctcgtccgtggtcacactcttgtctggcacagccaggtccccgcctgggtcaccaacatcaacgacaagaacacc
ttgatctctgtcatgcagaaccacatcaagactgtcgttggccgctacaagggcaagatctacgcatgggtcagta
gagctggataccgctttgatcgaaccctatactgatgattgtaggacgtcaccaacgagatcttcaacgaggacgg
tactctcaggcagagccactggatgaaggtcatcggcgaggactacgtccgcattgccttcgagaccgctcgcgcc
gctgacccgtcggccaagctctacatcaacgattacaaccttgatgttgccaactacggcaagactcagggtatga
tcaagtacgtcaagaagtggcgcgccgccggcgttcccattgacggaattggcagccagatggtaagttcaacgat
caatctctttggaaagttgccaggatgctaacgtttcacagcacctttccaacggtaacggctggcccaccgccac
cgatgtcgttaacgccatgaaggccctctgtgccgccgctcctgagtgcgccgtcactgagctcgacattcagggc
gcatcgagcgctgactaccttaaggcgatgcactcttgccttgacgtccagaactgtgttggtatcacctcctggg
gtattaccgacaacaccgtaagttttgacatttttcacccaaaaatgacccaaaaaccgacactaaccatatcacc
tacagtcgtggcgcgccgacaagactcccctgctcttcaactcccaggctcagcccaaggccgcctacactgctct
cctcaacgctttgtaa
The present invention has cloned xylanase gene Caxyn10A, DNA complete sequence analysis result table by the method separation of PCR
Bright, zytase CaXyn10A structural gene Caxyn10A overall length 1224bp contains 4 intrones, cDNA long 999bp, and+132
~+186bp ,+518~+568bp ,+815~+869bp and+1074~+1137bp are the introne sequence of 55,51,55 and 64bp
Column, cDNA sequence is as shown in SEQ ID NO.5:
atgctgatttccaacgccattgccgcactctctgtgctgtcgcttgcttccgcagcggccattcctga
gtctcacatcgaggctcgcgctgctaagactgtcgatgccggcttcaaggccaagggcaagaagtactggggcact
gccagtgatcagggcattctttcccagagcaacatcaacaatgctgccaaggccaacttcggccagatcacccccg
agaactccatgaagttcgatgccactgagccttcccgtggcaacttcaacttcaacggtgccgactggctcgtcaa
ctgggctcagaccaacggcaagctcgtccgtggtcacactcttgtctggcacagccaggtccccgcctgggtcacc
aacatcaacgacaagaacaccttgatctctgtcatgcagaaccacatcaagactgtcgttggccgctacaagggca
agatctacgcatgggacgtcaccaacgagatcttcaacgaggacggtactctcaggcagagccactggatgaaggt
catcggcgaggactacgtccgcattgccttcgagaccgctcgcgccgctgacccgtcggccaagctctacatcaac
gattacaaccttgatgttgccaactacggcaagactcagggtatgatcaagtacgtcaagaagtggcgcgccgccg
gcgttcccattgacggaattggcagccagatgcacctttccaacggtaacggctggcccaccgccaccgatgtcgt
taacgccatgaaggccctctgtgccgccgctcctgagtgcgccgtcactgagctcgacattcagggcgcatcgagc
gctgactaccttaaggcgatgcactcttgccttgacgtccagaactgtgttggtatcacctcctggggtattaccg
acaacacctcgtggcgcgccgacaagactcccctgctcttcaactcccaggctcagcccaaggccgcctacactgc
tctcctcaacgctttgtaa
Wherein, the base sequence of signal peptide are as follows: ATGCTGATTTCCAACGCCATTGCCGCACTCTCTGTGCTGTCGCT
TGCTTCCGCA(SEQ ID NO.6)。
Maturation protein theoretical molecular weight is 34.5kDa.By xylanase gene Caxyn10A cDNA sequence and derive
Amino acid sequence carries out BLAST comparison in GenBank.The gene and the wood from Aspergillus fumigatus are poly-
Carbohydrase Amino acid sequence identity is 63%.Illustrate that CaXyn10A is a kind of new zytase.
The present invention also provides the recombinant vectors comprising above-mentioned neutral low-temperature xylanase gene C axyn10A, preferably
pET30-Caxyn10A.Xylanase gene of the invention is inserted between suitable restriction enzyme cleavage sites of the expression vector,
Make its nucleotide sequence is operable to be linked to the expression control sequence.As the most preferred embodiment of the invention,
Preferably by xylanase gene of the invention be inserted into NdeI the and EcoRI restriction enzyme site on plasmid pET30 (a) it
Between, so that the nucleotide sequence is located at the downstream of T7 promoter and regulated and controled by it, obtains expression of recombinant e. coli plasmid pET30-
Caxyn10A。
It is preferably described the present invention also provides the recombinant bacterial strain comprising above-mentioned neutral low-temperature xylanase gene C axyn10A
Bacterial strain is Escherichia coli, saccharomycete, bacillus, Bacillus acidi lactici, aspergillus or trichoderma, preferably recombinant bacterial strain pET30/
Caxyn10A。
The present invention also provides a kind of methods for preparing neutral low-temperature xylanase CaXyn10A, comprising the following steps:
1) host cell is converted with above-mentioned recombinant vector, obtains recombinant bacterial strain；
2) recombinant bacterial strain, induction recombined xylanase expression are cultivated；And
3) it recycles and purifies expressed zytase CaXyn10A.
Wherein, the preferably described host cell is Escherichia coli, and expression of recombinant e. coli plasmid is preferably converted large intestine bar
Bacterium cell (Escherichia coli) BL21, obtains recombinant bacterial strain pET30/Caxyn10A.
The present invention also provides the applications of above-mentioned neutral low-temperature xylanase CaXyn10A.
The technical problem to be solved is that overcome the deficiencies in the prior art first by the present invention, obtain the bacterium of low-temperature xylanase
Goods and materials source, provide a kind of good properties, be suitable in feed, food, industry using new neutral low-temperature xylanase.This
The zytase optimal pH of invention is 6.0, has higher enzymatic activity in pH6.0~7.2；PH stability is good；Rate activity is
453U/mg；With wide in range substrate specificity.The characteristics of its neutral low temperature, energy of the zytase in industrial production can be reduced
Source cost.PH adaptation range improves aquatic feeds digestible energy and metabolic energy, reduces formulation cost, reduces environmental pollution.Its is wide in range
Substrate specificity the nutritive value of grain processing co-product can be improved, promote feed product quality.This zytase can answer
For brewing industry, material viscosity is reduced, diastatic action can be conducive in stratum granulosum, starch utilization ratio is improved, increase wine
The yield of essence.The xylan in paper industry waste material and agricultural wastes can also be converted into wood under normal temperature conditions
Oligosaccharides, and wood oligose can be by bacterium, yeast and fungal transformation at valuable fuel.Therefore, this zytase is in energy work
Application in industry also shows that its huge potentiality.Hydrolysate (xylose and xylo-oligosaccharide) can be applicable to food service industry, as
Thickener, fatty sub, prebiotic oligosaccharides and freeze proof food additives；Xylan makes in conjunction with other materials in pharmaceuticals industry
With the release of drug ingedient can be delayed.The hydrolysate of xylan can also be further converted to liquid fuel, single cell protein
White, solvent and low calorie sweetener.
Detailed description of the invention
The SDS-PAGE for the recombined xylanase that Fig. 1 is expressed in coliform is analyzed, wherein 1: the recombination xylan of purifying
Enzyme；M: low molecular weight protein Marker.
The optimal pH of Fig. 2 recombined xylanase.
The pH stability of Fig. 3 recombined xylanase.
The optimum temperature of Fig. 4 recombined xylanase.
The thermal stability of Fig. 5 recombined xylanase.
Specific embodiment
Test material and reagent
1, bacterial strain and carrier: the present invention is stored in Chinese agriculture from cladosporium Cladosporium acalyphae SL-16
Industry academy of sciences agricultural Culture Collection Center (No.12 ,zhongguancun south street,Haidian District, Beijing, Chinese Academy of Agricultural Sciences's agricultural strain
Collection, 100081), deposit number are as follows: ACCC32700.Coli expression carrier pET30 (a) and bacterial strain BL21 is by this
Laboratory preservation.
2, enzyme and other biochemical reagents: restriction endonuclease is purchased from TaKaRa company, and ligase is purchased from Invitrogen company.Beech
The wooden xylan is purchased from Sigma company, other all (to be commercially available from common biochemical Reagent Company) for domestic reagent.
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: not making the experimental methods of molecular biology illustrated in following embodiment, referring to " Molecular Cloning: A Laboratory
Guide " specific method listed in book of (third edition) J. Pehanorm Brooker one carries out, or according to kit and product description
It carries out.
1 cladosporium Cladosporium acalyphae SL-16 Enzymatic characteristic of embodiment
By cladosporium Cladosporium acalyphae SL-16 after potato juice culture medium culture, it is outstanding that spore is made
Liquid, be inoculated in wheat bran fluid nutrient medium (Luo et al.Enzyme Microbial Technology, 2009,45:126-
133), 15 DEG C of culture 7d react 10min under the conditions of pH 6.0 and 30 DEG C, with DNS method using 1% beech xylan as substrate
(Miller 1959) determines it with xylanase activity.
Gram of 2 cladosporium Cladosporium acalyphae SL-16 Xylanase coding gene Caxyn10A of embodiment
It is grand
Extract cladosporium Cladosporium acalyphae SL-16 genomic DNA:
Mycelium aseptic filter paper filtering in Liquid Culture 3 days is put into mortar, 2mL extracting solution is added, grinds 5min,
Then lapping liquid is placed in 50mL centrifuge tube, 65 DEG C of water-baths crack 20min, mix once, at 4 DEG C every 10min
10000rpm is centrifuged 5min.Supernatant extrct foreigh protein removing in phenol/chloroform is taken, then takes supernatant that isometric isopropanol is added, in
After being stored at room temperature 5min, 10000rpm is centrifuged 10min at 4 DEG C.Abandon supernatant, precipitating with 70% ethanol washing twice, vacuum do
It is dry, appropriate TE dissolution is added, be placed in -20 DEG C it is spare.
Design synthesis special primer Caxyn10A-F/Caxyn10A-F:
Caxyn10A-F:5'-atgctgatttccaacgccattgccg-3'；
Caxyn10A-R:5'-ttacaaagcgttgaggagagcagtgtaggc-3'；
PCR amplification is carried out by template of cladosporium Cladosporium acalyphae SL-16 total DNA.PCR reaction ginseng
Number are as follows: 94 DEG C of denaturation 5min；Then 94 DEG C of denaturation 30sec, 60 DEG C of annealing 30sec, 72 DEG C of extension 60sec, 72 after 30 circulations
DEG C heat preservation 10min.An about 1224bp segment is obtained, send three rich biotechnologys for being connected after segment recycling with pEASY-T3 carrier
Co., Ltd's sequencing.
The RT-PCR of 3 cladosporium Cladosporium acalyphae SL-16 xylanase gene of embodiment is analyzed
The total serum IgE for extracting cladosporium Cladosporium acalyphae SL-16, obtains cDNA's using reverse transcriptase
Then one chain expands the single-stranded cDNA with primer Caxyn10A-E-F/Caxyn10A-E-F, obtain the cDNA sequence of zytase
Column, amplification obtain the sequencing of product recycling Hou Songsanbo Bioisystech Co., Ltd.
Caxyn10A-E-F:5'-ccccatatgatgctgatttccaacgccattgcc-3'；
Caxyn10A-E-R:5'-cccgaattcttagtggtggtggtggtggtgcaaagcgttg aggagagcagtg-
3'；
By comparing finding that the gene has 4 intrones, cDNA long after the genome sequence and cDNA sequence of zytase
999bp, encodes 332 amino acid and a terminator codon, and 18 amino acid of N-terminal are its signal peptide sequence.Measured base
Because the xylanase sequence on the maturation protein part nucleotide sequence and GenBank of Caxyn10A carries out Homology search,
The gene for the encoding xylanase that separation clone obtains is new gene.
The preparation of 4 recombined xylanase of embodiment.
Expression vector pET30 (a) is subjected to double digestion (NedI+EcoRI), while by the gene of encoding xylanase
Caxyn10A double digestion (NedI+EcoRI), the genetic fragment for cutting out encoding mature zytase and expression vector pET30 (a) are even
It connects, obtains the recombinant plasmid containing cladosporium Cladosporium acalyphae SL-16 xylanase gene Caxyn10A
PET30-Caxyn10A simultaneously converts e. coli bl21, obtains recombinant escherichia coli strain pET30/Caxyn10A.
Building in the same way includes the recombinant expression carrier of signal peptide sequence, and obtains recombinant bacterial strain.
The BL21 bacterial strain containing recombinant plasmid is taken, is inoculated in 100mL LB culture solution, 16 DEG C of 150rpm shaken cultivation 12h
Afterwards, 0.6%IPTG is added and induces 10h, collect cell, supernatant is collected by centrifugation in ultrasonic disruption.Measure the work of zytase
Power.The expression quantity of recombined xylanase is 1.54U/mL.SDS-PAGE result (Fig. 1) shows recombined xylanase in large intestine bar
It is expressed in bacterium.Expressed zytase after purifying, the content of protein reach the 15% of total protein with
On.
The property of 5 recombined xylanase CaXyn10A of embodiment measures
DNS method: the specific method is as follows: under the conditions of pH6.5,40 DEG C, the reaction system of 1mL includes 100 μ L appropriate dilute
Enzyme solution is released, 900 μ L substrates react 10min, and 1.5mL DNS is added and terminates reaction, boiling water boiling 5min.540nm measures OD after cooling
Value.1 enzyme-activity unit (U) is defined as releasing the enzyme amount of 1 μm of ol reduced sugar per minute under given conditions.
1, the measuring method of the optimal pH of recombined xylanase CaXyn10A and pH stability is as follows:
The recombined xylanase that embodiment 4 purifies is subjected to enzymatic reaction at different pH to measure its optimal pH.Bottom
Object xylan carries out Xylanase activity survey in the 0.1mol/L citrate-phosphate disodium hydrogen buffer of different pH 30 DEG C
It is fixed.As a result (Fig. 2) shows that the optimal pH of CaXyn10A is 6.0, and in the range of pH6.0~7.2, enzymatic activity is kept at most
80% or more of big enzymatic activity.Zytase 30 DEG C of processing 60min in the buffer of above-mentioned various difference pH, then in pH6.0
Enzymatic activity is measured at 40 DEG C in buffer solution system, with the pH patience of studying enzyme.As a result (Fig. 3) shows zytase in pH 4.0-
It is very stable between 10.0, remaining enzymatic activity is handled after 60min within the scope of this pH 70% or more, this illustrate this enzyme have compared with
Good pH stability.
2, the optimum temperature of zytase and thermal stability determination method are as follows:
The optimum temperature of zytase is measured as in citrate-phosphate disodium hydrogen buffer (pH6.0) buffer solution system
And enzymatic reaction is carried out under different temperatures.Temperature tolerance is measured as zytase and handles different time at different temperatures, then
PH6.0, enzyme assay is carried out at 40 DEG C.Enzyme reaction optimum temperature measurement result (Fig. 4) shows that its optimum temperature is 40 DEG C.Enzyme
Thermal stability experiments have shown that (Fig. 5), recombinase stability at 30 DEG C are very good.60min is kept the temperature at 40 DEG C, is lost completely
Enzyme activity.
3, the K of zytasemValues determination method is as follows:
It is substrate with the beech xylan of various concentration, in citrate-phosphate disodium hydrogen buffer (pH6.0) buffering liquid
In system, enzymatic activity is measured at 20 DEG C and 40 DEG C, calculates its K at 20 DEG C and 40 DEG Cm、Vmax、kcatAnd kcat/KmValue.Through surveying
Fixed, this zytase is at 40 DEG C using xylan as the k of substratemValue is 1.87mg/mL, maximum reaction velocity VmaxFor 331.6 μ
Mol/minmg, kcatValue is 190.7/s and kcat/KmValue is 102.0ml/mgs；Using xylan as the k of substrate at 20 DEG Cm
Value is 12.23mg/mL, maximum reaction velocity VmaxFor 322.9 μm of ol/minmg, kcatValue is 185.7/s and kcat/KmValue is
15.2ml/mg·s
4, influence measurement of the different metal ions chemical reagent to CaXyn10A enzyme activity is as follows:
The different metal ions and chemical reagent of various concentration are added in enzymatic reaction system, study it to enzymatic activity
Influence, the various final concentration of 5mmol/L of substance.Enzymatic activity is measured under the conditions of 40 DEG C, pH6.0.As a result (table 1) shows greatly
Most ions and the chemical reagent vigor of recombined xylanase when concentration is 5mmol do not have significant change.But Ag+Strong suppression
Make its vigor, Pb2+, Zn2+, SDS and EDTA have partial inhibition.Ni2+And Co2+It can make to recombinate enzyme activity increase in 5mmol
To original 1.27 and 1.16 times.
Influence of the various chemical reagent of table 1. to zytase CaXyn10A vigor
5, the substrate specificity of CaXyn10A
Different substrates is added in enzymatic reaction system, studies the enzymatic activity of CaXyn10A.In 40 DEG C, pH6.0 condition
Lower measurement enzymatic activity.As a result (table 2) shows CaXyn10A while having the activity of zytase, cellulase and dextranase.
The substrate specificity of table 2.CaXyn10A
6, CaXyn10A zytase degradation beech xylan product is analyzed as follows:
The enzyme solution of 100 μ L purifying is added in the xylan of 500 μ L 1%, keeps the temperature 12h under optimum temperature.Use dehydrated alcohol
Zymoprotein is precipitated, 2500 chromatographs of supernatant, is examined using High Performance Anion Exchange Chromatography Coupled with Pulsed Amperometric (HPAEC-PAD)
Survey method carries out the analysis of sugar type in product.Analysis the result shows that: zytase CaXyn10A degradation beech xylan production
If owner's xylobiose, xylotriose and Xylotetrose.Xylobiose content is 50.1% in product, and xylotriose content is 45.9%, and
Xylotetrose content is 4.0%.
Claims (9)
1. a kind of neutrality low-temperature xylanase CaXyn10A, which is characterized in that its amino acid sequence such as SEQ ID NO.1 or SEQ
Shown in ID NO.2.
2. a kind of neutrality low-temperature xylanase gene C axyn10A, which is characterized in that coding neutral low temperature described in claim 1
Zytase CaXyn10A.
3. neutrality low-temperature xylanase gene C axyn10A as claimed in claim 2, which is characterized in that its base sequence is such as
Shown in SEQ ID NO.4 or 5.
4. the recombinant vector comprising neutrality low-temperature xylanase gene C axyn10A described in claim 2.
5. the recombinant vector pET30-Caxyn10A comprising neutrality low-temperature xylanase gene C axyn10A described in claim 2,
Wherein, by encoding amino acid sequence, the neutrality low-temperature xylanase gene C axyn10A as shown in SEQ ID NO.2 is inserted into matter
On grain pET30 (a), so that the gene is located at the downstream of T7 promoter and regulated and controled by it, obtain recombinant vector pET30-Caxyn10A.
6. the recombinant bacterial strain comprising neutrality low-temperature xylanase gene C axyn10A described in claim 2.
7. recombinant bacterial strain as claimed in claim 6, which is characterized in that the bacterial strain is Escherichia coli, saccharomycete, bacillus, cream
Acidfast bacilli, aspergillus or trichoderma.
8. a kind of method for preparing neutrality low-temperature xylanase CaXyn10A described in claim 1, which is characterized in that including following
Step:
1) host cell is converted with the recombinant vector of claim 4, obtains recombinant bacterial strain；
2) recombinant bacterial strain, induction recombined xylanase expression are cultivated；And
3) it recycles and purifies expressed neutral low-temperature xylanase CaXyn10A.
9. the application that neutrality low-temperature xylanase CaXyn10A described in claim 1 is used for hydrolyzed xylan.
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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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