CN105368804B - High-temperature-resistant and strong-alkali-resistant xylanase and application thereof in waste paper deinking - Google Patents
High-temperature-resistant and strong-alkali-resistant xylanase and application thereof in waste paper deinking Download PDFInfo
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- CN105368804B CN105368804B CN201510947137.2A CN201510947137A CN105368804B CN 105368804 B CN105368804 B CN 105368804B CN 201510947137 A CN201510947137 A CN 201510947137A CN 105368804 B CN105368804 B CN 105368804B
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- high temperature
- xylanase
- resistance
- resistant
- zytase
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- C—CHEMISTRY; METALLURGY
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- D21C5/025—De-inking
-
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- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
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Abstract
The invention provides high temperature resistant and strong base resistant xylanase and application thereof in waste paper deinking, wherein the amino acid sequence of the high temperature resistant and strong base resistant xylanase is shown as SEQ ID NO. 1; the nucleotide sequence of the gene for coding the high temperature and strong alkali resistant xylanase is shown as SEQ ID NO. 2. A recombinant plasmid comprising said thermophilic xylanase gene. A recombinant bacterium comprising said recombinant plasmid. The high temperature and strong alkali resistant xylanase is applied to waste paper deinking. Putting the waste paper to be deinked into a buffer solution containing 0.6-1.2U/mg of high-temperature-resistant and strong-base-resistant xylanase, and carrying out deinking reaction at the temperature of 50-90 ℃ and the pH value of 5.0-12.0. The xylanase gene provided by the invention realizes high expression in escherichia coli by using a codon optimization technology; the expressed xylanase has no cellulase activity and has the properties of high temperature resistance and high alkali resistance; the xylanase can be applied to waste paper deinking, has higher efficiency than commercial enzymes, and shows good industrial application value.
Description
Technical field
The present invention relates to genetic engineerings and microorganisms technical field, and in particular to a kind of high temperature resistant zytase of resistance to highly basic and
Its application in deinking.
Background technology
Hemicellulose is the second abundant renewable resource for being only second to cellulose, and efficient application solves to work as the mankind
The problems such as preceding crisis of resource, energy crisis, environmental pollution, is extremely important.Hemicellulose structure and composition are very multiple
It is miscellaneous, including the various ingredients such as xylan, mannosan, araban, arabogalactan and glucan.Wherein xylan
It is most representative hemicellulose in cell, it can be utilized as regenerated resources by people.Xylan is a kind of complicated
Poly pentose is mainly got up by β-Isosorbide-5-Nitrae-D- xylosides key connection, and band, there are many substituent group, its degradable needs are more
The synergistic effect of kind hydrolase.Wherein most important glycoside hydrolase is inscribe-β-Isosorbide-5-Nitraes-zytase, it can degrade wooden poly-
β -1,4- the glycosidic bonds of sugar backbone.Zytase has obtained increasingly wide in industries such as papermaking, food, feed, bioconversions at present
General application.In paper industry, zytase can be applied to bio-pulping, association with pulp bleaching, deinking processing etc., especially
In deinking, compared with traditional Chemical Deinking method, deinking is carried out using zytase, does not need a large amount of chemical drugs
Product, thus the influence to environment is smaller, the BOD and COD of deinking wastewater are relatively low, and the effect of Enzymatic Deinking and chemical method phase
Closely, but the physical property of fiber is then better than chemical deinking, therefore has good development prospect.
Before with zytase deinking, paper pulp is in about high temperature (55-70 DEG C) and high alkalinity pH (pH9-11)
Under.High temperature and high alkalinity state propose strict requirements to xylanase preparation.Many commercially available wild-type xylanases
The shortcomings that be that acid Optimal pH and about 55 DEG C of optimum temperature is presented in these enzymes, thus can largely effect on the effect of deinking.
Therefore the novel research with high temperature resistant and overbasic zytase is obtained with important value.
Planomicrobium glaciei CHR43 is a kind of extreme psychrophile, due to its unique survival ability, I
Speculate that the zytase for coming from this bacterium is likely to have resistance to high/low temperature and high pH adaptability.At present about fromPlanomicrobium glaciei There are no relevant reports with application for the xylanase gene of CHR43 and its expression.
Invention content
The deficiency of industrial requirement cannot be met in order to make up existing zytase temperature and pH, the present invention provides a kind of resistance to height
The warm zytase of resistance to highly basic and its application in deinking.
To achieve the goals above, the technical solution adopted by the present invention is：
A kind of high temperature resistant zytase of resistance to highly basic, amino acid sequence is as shown in SEQ ID NO.1.
The gene xyn1 of the high temperature resistant zytase of resistance to highly basic is encoded, nucleotide sequence is as shown in SEQ ID NO.2.
A kind of recombinant plasmid, it includes the high temperature resistant xylanase gene of resistance to highly basic.
A kind of recombinant bacterium, it contains the recombinant plasmid.
The preparation method of the high temperature resistant zytase of resistance to highly basic cultivates the recombinant bacterium in nutrient medium, collects tool
There is the polypeptide of the high temperature resistant xylanase activity of resistance to highly basic.
Application of the high temperature resistant zytase of resistance to highly basic in deinking.To wait for the waste paper of deinking be put into containing 0.6 ~
In the buffer solution of the 1.2 U/mg high temperature resistants zytases of resistance to highly basic, deinking is carried out under the conditions of 50-90 DEG C, pH5.0-12.0
Reaction.
Further preferably, it will wait for that the waste paper of deinking is put into the buffering containing the 1 U/mg high temperature resistants zytase of resistance to highly basic
In solution, at 80 DEG C, deinking reaction is carried out under the conditions of pH9.0.
It should be appreciated that those skilled in the art can be according to amino acid sequence disclosed by the invention, not influencing, its is active
Under the premise of, replace, lack and/or increase one or several amino acid, obtains the mutant nucleotide sequence of the albumen.Therefore, of the invention
The high temperature resistant zytase of resistance to highly basic further include being substituted, lack or adding in the amino acid sequence shown in SEQ ID No.1
One or several amino acid and the protein with the same active protein derived shown in SEQ ID No.1.Such as it is logical
It crosses and adds sequence label, the derivative protein such as His-tag or Strep-tag in end.
Preferably, shown in the amino acid sequence of the derived protein of the high temperature resistant zytase of resistance to highly basic and SEQ ID No.1
The homology of amino acid sequence can be 70% or more, preferably 80% or more, more preferable 90% or more.
The present invention also provides carrier, cell line and the host strain containing the gene or gene cluster belong to the present invention's
Protection domain.
In the present invention, the high temperature resistant xylanase gene of resistance to highly basic obtains by the following method：It is given birth to using American National
The database of object technology information centre (NCBI), analysis, are found screeningPlanomicrobium glaciei Have one in CHR43
Duan Xulie withBacillus Sp. the zytase in Strain NG-27 (No. Genbank：AAB70918) sequence has higher
Homology, it is more likely that be the gene order of encoding xylanase.The sequence is screened, is optimized（Optimization includes part position
Mutation, the insertion etc. of point）, obtain the nucleotide sequence with SEQ ID NO.2.The high temperature resistant zytase of resistance to highly basic xyn1 genes
By Jin Sirui companies according to SEQ ID NO.2 nucleotide sequences carry out it is artificial synthesized, sequence both ends add respectively BamH I and
Xho I restriction enzyme sites are connected between BamH I and Xho the I restriction enzyme sites on pETDuet-1 plasmid vectors, obtain recombination matter
Grain pETDuet-1-xyn1.
Bacillus coli DH 5 alpha competent cell is converted, screen and positive recombinant plasmid, extraction recombination matter are identified by sequencing
Grain converts e. coli bl21（DE3）Competent cell obtains the recombination engineering Escherichia coli containing recombinant plasmidEscherichia coliBL21（DE3）.
It will be appreciated by those skilled in the art that as degenerate as a result, many different polynucleotides energy
Enough encode identical polypeptide.It is taken in addition, it will be appreciated that those skilled in the art can use conventional technique to carry out nucleotide
Generation, the substitution do not interfere with the polypeptide sequence of the polynucleotide encoding used in the present invention.Furthermore it is also possible to use ability
Known method in domain modifies polynucleotides, to enhance polynucleotides of the present invention activity in vivo or survival
Phase.
In the present invention, the known various carriers in this field, such as plasmid, clay, bacteriophage and retrovirus etc. can be selected.
Recombinant expression carrier can be imported with method well known in the art in host cell, these methods include：Calcium chloride
Heat shock method, electrotransformation, PEG mediated methods, particle bombardment etc..
The present invention provides the method for preparing above-mentioned the high temperature resistant zytase of resistance to highly basic expression and purifying.By the recombination work of acquisition
Journey bacterium Escherichia coliEscherichia coliBL21（DE3）Switching culture, IPTG induce enzyme protein expression.Pass through ultrasound
Wave smudge cells, high speed centrifugation and the purifying of Ni-NTA sepharose4B affinity protein purifications；Then pass through SDS-PAGE
Electroresis appraisal protein molecular size；It finally concentrates, measures albumen concentration, analyze the zymologic property of albumen.It is provided by the invention resistance to
The high temperature zytase of resistance to highly basic keeps greater activity, optimal pH 9.0 within the scope of pH5.0-12.0；At 50-90 DEG C keep compared with
High activity is 80 DEG C suitable for reaction temperature.
Advantageous effect：
The present invention is from extreme psychrophilePlanomicrobium glaciei A kind of xylanase gene is obtained in CHR43,
It screened using codon-optimization techniques, optimize to obtain the high temperature resistant xylanase gene of resistance to highly basic, and phase is obtained by expression
Close zymoprotein.High expression in Escherichia coli, the zytase cellulose-less enzymatic activity of expression have high temperature resistant, resistance to high-alkali
Property；The zytase can be applied to deinking, and have efficiency more higher than commercial enzyme, show good commercial Application
Value.
The optimal pH of zytase provided by the invention is 9.0, and most suitable reaction temperature is 80 DEG C, and is had preferable
Heat-resisting quantity and high alkalinity.Meanwhile the zytase has good Deinking Efficiency to waste paper, and it is purchased from Shanghai source leaf biology section
The commercial enzyme of skill Co., Ltd（Model：MFCD00132594）（157%）It compares, has reached 298%, and higher than at present having reported
Come fromBacillusSp. the zytase of CKBx1D（200%）（Maity et al. Applied Biochemistry
and Biotechnology. 2012, 167: 1208-1219.）.
Description of the drawings
Fig. 1 high temperature resistants zytase of resistance to highly basic is expressed and SDS-PAGE analyses after purification, wherein：1 is resistance to for high temperature resistant
Band after highly basic zytase expression and purification；M is Protein Marker.
Influences and its pH stability of Fig. 2 pH to the high temperature resistant xylanase activity of resistance to highly basic.
Influence of Fig. 3 temperature to the high temperature resistant xylanase activity of resistance to highly basic.
The thermal stability of Fig. 4 high temperature resistants zytase of resistance to highly basic.
Waste paper configuration of surface after Fig. 5 Enzymatic Deinkings, wherein：A is control group；B is poly- for the high temperature resistant wood of resistance to highly basic of the present invention
Carbohydrase group；C is commercial enzyme group.
Specific implementation mode
The implementation of the present invention will be using the fields such as chemistry, molecular biology within the limit of power of those skilled in the art
Traditional technology.In addition, unless otherwise indicated, herein, nucleic acid is write from left to right with 5 ' to 3 ' direction, amino acid sequence
Row are then write with the direction of aminoterminal to c-terminus from left to right.In the following examples, the experimental methods for specific conditions are not specified,
It operates usually according to normal condition, such as《Molecular Cloning:A Laboratory guide》Or the condition proposed by manufacturer.
Test material and reagent
1, bacterial strain：Coli strainEscherichia coli DH5α、Escherichia coliBL21（DE3）
It is purchased from Novagen companies.
2, biochemical reagents：Xylan is purchased from Sigma companies；Other is all domestic reagent（It can be from common biochemistry
Reagent Company is commercially available）.
3, culture medium：LB culture mediums：Peptone 10g, Yeast extract 5g, NaCl 10g, add steaming
For distilled water to 1000ml, pH is natural（About 7）.Solid medium adds 2.0% on this basis（w/v）Agar.
The acquisition of the 1 high temperature resistant xylanase gene of resistance to highly basic xyn1 of embodiment, the structure of recombinant plasmid and recombination engineering
The acquisition of the high temperature resistant xylanase gene of resistance to highly basic xyn1
Using the database of National Center for Biotechnology Information (NCBI), analysis, is found screeningPlanomicrobium glaciei Have in CHR43 one section of sequence withBacillus Sp. the xylan in Strain NG-27
Enzyme (No. Genbank：AAB70918) sequence has compared with high homology, it is more likely that is the gene order of encoding xylanase.It is right
The sequence is screened, is optimized（Optimization includes mutation, insertion of moiety site etc.）, obtain the nucleosides with SEQ ID NO.2
Acid sequence.Gene is artificial synthesized according to the progress of SEQ ID NO.2 nucleotide sequences by Jin Sirui companies, distinguishes at sequence both ends
Add BamH I and Xho I restriction enzyme sites, be connected to BamH I and Xho I restriction enzyme sites on pETDuet-1 plasmid vectors it
Between, obtain recombinant plasmid pETDuet-1-xyn1.
The recombinant plasmid containing xylanase gene xyn1 of synthesis is imported into 100 μ l DH5 α competent cells,
Ice bath 30min, 42 DEG C of heat shock 60s, then ice bath 3min immediately, addition 1ml LB fluid nutrient mediums, 37 DEG C, shake culture
1h.Low-speed centrifugal bacterium solution discards part supernatant, and remaining thalline is resuspended, and is coated on containing 100 μ g/ml ampicillins
On LB tablets, the overnight incubation in 37 DEG C of incubators.Picking single bacterium colony carries out bacterium solution PCR identifications, is sequenced and determines positive gram
It is grand, extract the positive colony plasmid with the small extraction reagent kit of plasmid.
Recombinant plasmid transformed E.coli BL21（DE3）Obtain recombination engineering：
By recombinant plasmid transformed e. coli bl21（DE3）In competent cell, in green containing 100 μ g/ml ammonia benzyls
37 DEG C of overnight incubations on the LB tablets of mycin.Picking single bacterium is cloned in the training of the LB liquid containing 100 μ g/ml ampicillins
It supports and is cultivated in base, preserve strain with 20% glycerine, obtain with E.coli BL21（DE3）For the high temperature wood of host strain structure
Dextranase xyn1 recombinant bacterial strains.
The expression and purifying expression of 2 zytase xyn1 of embodiment：
Recombinant bacterium is first inoculated in respectively in LB fluid nutrient mediums of the 10ml containing 100 μ g/ml ampicillins, and 37
DEG C, 220rpm shaken cultivations stay overnight.Then with 1%（v/v）Amount be transferred to 500ml containing 100 μ g/ml ampicillins
LB liquid medium, 37 DEG C, 220rpm shaken cultivations to OD600Reach 0.6 or so, is added IPTG to final concentration 1mM, 37
DEG C, 220rpm shaken cultivations 6 hours.Thalline were collected by centrifugation.
Purifying：
Appropriate pH7.0 PBS buffer are added to thalline（137mmol/L NaCl, 2.7mmol/L KCl,
4.3mmol/L Na2HPO4, 1.4mmol/L KH2PO4, the pH value of solution is adjusted to 7.0 with HCl）It is resuspended, ultrasonication weight
Group bacterium, high speed centrifugation collect supernatant.Supernatant is incubated into half an hour in 65 DEG C, precipitable a large amount of nonrefractory host's mycoprotein, then
High speed centrifugation collects supernatant.Supernatant will be collected and cross Ni-NTA affinity columns, first with the banding buffer of 6 column volumes
（20mM Tris-HCl pH7.5,300mMNaCl, 40mM imidazoles）Wash column, then with a small amount of Elution Buffer（50mM
Tris-HCl pH7.5,300mMNaCl, 500mM imidazoles）Elution, SDS-PAGE electrophoresis detection eluents find to obtain height
The destination protein of purity.This result is as shown in Fig. 1.Then the protein liquid of collection is added to super filter tube, centrifugal concentrating obtains
The protein liquid of purifying.
The zymologic property of the 3 high temperature resistant zytase of resistance to highly basic of embodiment measures：
Xylanase activity is measured using DNS methods：It is added 200 μ L enzymes in 2mL reaction systems, concentration of substrate 0.9%,
10min is reacted under conditions of temperature 50 C, pH4.8, is added after 2mL DNS terminations are reacted and boiling water bath 10min.It is cooled to room
It is added water to after temperature after 15mL in its absorption value of 540nm wavelength detectings, reference standard curve obtains content of reducing sugar, enzyme activity list
Position（U）It is defined as the enzyme amount of release 1mM reduced sugars per minute.The high temperature resistant zytase of resistance to highly basic is measured in pH according to the method
Activity in 3.0 to 12.0 range of value.The results are shown in Figure 2, and as can be seen from the figure the high temperature resistant zytase of resistance to highly basic is most
Suitable pH is 9.0, and greater activity is kept within the scope of pH5.0-12.0.The high temperature resistant zytase of resistance to highly basic is also measured to arrive at 30 DEG C
Activity within the scope of 90 DEG C, xyn1 are 80 DEG C suitable for reaction temperature, and greater activity is kept at 50-90 DEG C.
The 4 high temperature resistant zytase of resistance to highly basic of embodiment is applied to deinking
One 100% surface area is cut into the small scraps of paper of 3x3 by the waste paper for coming from Hewlett-Packard's printer that ink covers,
By the scraps of paper be placed in 5ml respectively containing the 1 U/mg high temperature resistants zytase of resistance to highly basic, the pH7.0 of commercial enzyme PBS buffering in, in
It 80 DEG C, is reacted 12 hours under the conditions of pH9.0, the not enzyme reaction of setting is control group, uses uv-spectrophotometric after reaction
Meter measures light absorption value of the reaction solution at 596nm, while with micro- sem observation paper surface form（Fig. 5）.As a result high temperature is shown
The Deinking Efficiency of zytase xyn1 is 298%, higher than the commercial enzyme purchased from the Shanghai bio tech ltd Yuan Ye（Model：
MFCD00132594）（157%）, and come from higher than what is reported at presentBacillusSp. the zytase of CKBx1D
（200%）（Maity et al. Applied Biochemistry and Biotechnology. 2012, 167: 1208-
1219.）.
<110>Nanjing University of Technology
<120>A kind of high temperature resistant zytase of resistance to highly basic and its application in deinking
<130>
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 360
<212> PRT
<213>Artificial sequence
<400> 1
Ala Ala Ser Asn Ala Pro Val Ser Ala Leu Glu Ala Ala Pro Leu Asp
1 5 10 15
Glu Arg Tyr Glu Lys Ser Phe Thr Ile Gly Ala Ala Val Glu Thr Ser
20 25 30
Gln Leu Glu Gly Lys Asp Gly Glu Met Leu Lys Lys His Tyr Asn Ser
35 40 45
Leu Val Ala Glu Asn Ile Met Lys Pro Ile Asn Ile Gln Pro Glu Glu
50 55 60
Gly Lys Phe Asn Phe Lys Glu Ala Asp Lys Ile Val Lys Phe Ala Lys
65 70 75 80
Glu Asn Asp Met Asp Leu Arg Phe His Thr Leu Ile Trp His Ser Gln
85 90 95
Val Pro Glu Trp Phe Phe Leu Asp Lys Glu Gly Lys Lys Met Val Asp
100 105 110
Glu Thr Asp Pro Lys Gln Arg Glu Lys Asn Lys Lys Leu Leu Leu Lys
115 120 125
Arg Val Glu Thr His Val Lys Thr Ile Val Lys Arg Tyr Lys Asn Asp
130 135 140
Val Asp Ser Trp Asp Val Val Asn Glu Val Ile Asp Glu Tyr Ala Pro
145 150 155 160
Asn Asp Lys Gly Leu Arg Glu Ser Pro Trp Tyr Gln Ile Thr Gly Thr
165 170 175
Asp Tyr Ile Ala Val Ala Phe Glu Thr Ala Arg Arg Phe Ala Gly Lys
180 185 190
Asp Ala Lys Leu Tyr Ile Asn Asp Tyr Asn Thr Glu Val Glu Pro Lys
195 200 205
Arg Thr His Leu Tyr Asn Leu Val Lys Glu Leu Leu Glu Gln Gly Val
210 215 220
Pro Ile Asp Gly Val Gly His Gln Gly His Ile Gln Leu Gly Trp Pro
225 230 235 240
Ser Leu Gln Glu Thr Arg Asp Ser Ile Asn Met Phe Ala Ser Leu Gly
245 250 255
Leu Asp Asn Gln Ile Thr Glu Leu Asp Val Ser Leu Tyr Gly Trp Pro
260 265 270
Pro Arg Pro Ala Phe Lys Ser Tyr Glu Glu Ile Pro Asp Glu Val Phe
275 280 285
Gln Ala Gln Ala Glu Arg Tyr Asp Ala Leu Phe Lys Leu Tyr Glu Glu
290 295 300
Leu Asp Asp Lys Ile Ser Asn Val Thr Phe Trp Gly Ile Thr Asp Asn
305 310 315 320
His Thr Trp Leu Asp Asp Arg Ala Glu Glu Tyr Asn Asp Gly Val Gly
325 330 335
Lys Asp Ala Pro Phe Val Phe Asp Pro Tyr Tyr Asn Val Lys Pro Ala
340 345 350
Tyr Trp Ala Ile Ile Asp Leu Glu
355 360
<210> 2
<211> 1080
<212> DNA
<213>Artificial sequence
<400> 2
gctgcctcta atgcacctgt ttccgcgtta gaagctgccc ccttggatga gcgttatgaa 60
aaatcattta ctattggtgc agcggtcgag acctcgcaac ttgaaggcaa ggacggagag 120
atgctcaaaa agcattacaa cagtctagta gctgaaaata tcatgaaacc aataaacatt 180
cagccggagg aagggaagtt caattttaaa gaggccgata agatcgtgaa attcgcaaag 240
gaaaacgaca tggatctgcg ctttcacaca ttaatatggc atagccaagt tcctgagtgg 300
ttctttttgg acaaagaagg taagaaaatg gtcgatgaga cggaccccaa gcagcgagaa 360
aaaaataaga aacttctcct aaagcgggta gagactcacg tgaaaaccat tgttaagaga 420
tataaaaacg atgtcgactc ttgggatgta gtgaatgaag ttatcgacga gtacgcgcca 480
aacgataagg gcctgaggga atccccgtgg tatcaaataa caggaacgga ctacattgct 540
gtcgccttcg agactgcacg tcgctttgcg gggaaagatg ctaagttata tatcaatgac 600
tacaacaccg aagtagagcc taaacgaaca catttgtata atcttgtgaa ggaactccta 660
gagcagggtg ttcccataga tggcgtcgga caccaagggc atattcagct gggttggcca 720
tcattacaag aaacgcggga ctcgatcaac atgttcgcca gtttgggcct tgataatcag 780
ataactgagc tcgacgtaag cctatacgga tggccgccta gacccgcatt taaatcttat 840
gaagagattc cagatgaagt gttccaagcg caggctgaga ggtacgacgc cctgtttaag 900
ttatatgaag agttggatga caaaatctcc aacgttacct tctgggggat aacagataat 960
cacacgtggc ttgacgatcg tgcagaagag tacaacgacg gtgtcggcaa ggatgcgccg 1020
tttgtattcg acccttatta caatgtgaaa cccgcttatt gggccattat cgatctcgaa 1080
Claims (8)
1. a kind of high temperature resistant zytase of resistance to highly basic, as shown in SEQ ID NO. 1, the zytase exists amino acid sequence
50-90 DEG C, greater activity is kept within the scope of pH5.0-12.0, there is high temperature resistant, resistance to high-alkali property can be applied to deinking.
2. a kind of gene of the coding high temperature resistant zytase of resistance to highly basic described in claim 1, nucleotide sequence such as SEQ ID
NO. shown in 2.
3. a kind of recombinant plasmid, it includes the high-temperature xylanase gene described in claim 2.
4. a kind of recombinant bacterium, it contains the recombinant plasmid described in claim 3.
5. the preparation method of the high temperature resistant zytase of resistance to highly basic, which is characterized in that cultivate claim 4 institute in nutrient medium
The recombinant bacterium stated collects the polypeptide with the high temperature resistant xylanase activity of resistance to highly basic.
6. application of the high temperature resistant zytase of resistance to highly basic described in claim 1 in deinking.
7. application according to claim 6, which is characterized in that will wait for that the waste paper of deinking is put into containing 0.6 ~ 1.2 U/mg
In the buffer solution of the high temperature resistant zytase of resistance to highly basic, deinking reaction is carried out under the conditions of 50-90 DEG C, pH5.0-12.0.
8. application according to claim 7, which is characterized in that will wait for that the waste paper of deinking is put into containing 1 U/mg high temperature resistants
In the buffer solution for the zytase of resistance to highly basic, at 80 DEG C, deinking reaction is carried out under the conditions of pH9.0.
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