CN109929821A - A kind of cutin enzyme mutant and its application in plain boiled water processing - Google Patents
A kind of cutin enzyme mutant and its application in plain boiled water processing Download PDFInfo
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Abstract
The invention discloses a kind of cutin enzyme mutant and its applications in plain boiled water processing, belong to technical field of enzyme engineering.The vigor height of the resinous matters such as cutin enzyme mutant hydrolysed polyvinyl acetate of the invention, plain boiled water high treating effect, the vigor of its hydrolysed polyvinyl acetate can improve 54% compared with wild type compared with 1.72 times of wild type raising, plain boiled water treatment effect, there is high application prospect in plain boiled water process field.
Description
Technical field
The present invention relates to a kind of cutin enzyme mutant and its applications in plain boiled water processing, belong to technical field of enzyme engineering.
Background technique
Paper waste mainly includes black liquor, middle section water and plain boiled water.Wherein, plain boiled water be sheet forming during, from paper machine
Wet end and the water of press section abjection.Contain a large amount of fiber fines, inorganic filler and various chemical assistants in plain boiled water, therefore,
Industry can not only save the dosage of clear water usually by white water recycling in the preparation of paper making pulp in this way, reduce fiber and chemicals
Loss, saving resource and reduces cost, can also reduce environmental pollution, be good for the environment.
But if plain boiled water is without processing direct reuse, with the raising of white water system degree, having in plain boiled water
Evil substance also can be accumulated gradually, and the increase of System Charges amount, polyelectrolyte is caused to increase, the final effect effect for influencing chemical assistant
There are the quality problems such as holes at paper so that the retention rate and filler retention of the paper as obtained by the slurry are deteriorated in fruit.
In these harmful substances, wet end chemistry is influenced maximum, most intractable to be dissolution substance in plain boiled water
With colloidal substance (DCS).DCS can be derived from from soluble wood extractive (such as small molecule lignin, starch, 7 cap classes)
The natural materials such as resin, the various organic and inorganic additive that can be added in water and production process can also be derived from and be answered
Chemicals.
Wherein, resinous matter is some hydrophobic substances for being dissolved in neutral organic solvent, this moieties is in papermaking
Cheng Zhonghui is deposited on equipment surface in a variety of forms, and therefore, resinous matter can not only make papery quality decline, can also be to papermaking
Equipment itself causes damages, and is most urgent problem to be solved in plain boiled water processing.
Currently, resinous matter can not be decomposed by existing chemical reagent in plain boiled water, it is main to pass through the chemistry such as addition flocculant
The method of auxiliary agent solves, and the method can not fundamentally handle resinous matter.
Fortunately, Southern Yangtze University Wu Jing professor et al. has found the resinaes such as cutinase hydrolyzable polyvinyl acetate
Substance, and hydrolytic process has stable and green non-pollution advantage, therefore, cutinase has high application in plain boiled water processing
Prospect.
But, during using cutin enzymatic treatment plain boiled water, cutin enzyme hydrolysis vigor deficiency, plain boiled water processing are still remained
The problem of less effective, this problem hinder application of the cutinase in plain boiled water processing significantly.
Summary of the invention
[technical problem]
It is high, white the technical problem to be solved by the present invention is to obtain a kind of resinous matters such as hydrolysed polyvinyl acetate vigor
The good cutinase of water treatment efficiency.
[technical solution]
To solve the above problems, the present invention provides a kind of cutin enzyme mutant, the mutant is by by amino acid
The 66th leucine and/or the 169th isoleucine of sequence cutinase as shown in SEQ ID No.1 are mutated to obtain
's.
In one embodiment of the invention, the mutant is by by amino acid sequence such as SEQ ID No.1 institute
66th leucine of the cutinase shown sports what isoleucine obtained；
The mutant is by the way that the 66th leucine of amino acid sequence cutinase as shown in SEQ ID No.1 is dashed forward
Become what alanine obtained；
The mutant is by the way that the 66th leucine of amino acid sequence cutinase as shown in SEQ ID No.1 is dashed forward
Become what valine obtained；
The mutant is by by the 169th different bright ammonia of amino acid sequence cutinase as shown in SEQ ID No.1
Acid mutation is what leucine obtained；
The mutant is by by the 169th different bright ammonia of amino acid sequence cutinase as shown in SEQ ID No.1
Acid mutation is what alanine obtained；
The mutant is by by the 169th different bright ammonia of amino acid sequence cutinase as shown in SEQ ID No.1
Acid mutation is what valine obtained；
The mutant is by the way that the 66th leucine of amino acid sequence cutinase as shown in SEQ ID No.1 is dashed forward
Becoming alanine and the 169th isoleucine mutation is what leucine obtained；
The mutant is by the way that the 66th leucine of amino acid sequence cutinase as shown in SEQ ID No.1 is dashed forward
Becoming alanine and the 169th isoleucine mutation is what alanine obtained.
In one embodiment of the invention, the amino acid sequence of the mutant is SEQ ID No.2, SEQ ID
No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 or SEQ ID
No.9。
In one embodiment of the invention, the cutinase derives from Humicola insolens (Humicola
insolens)。
The present invention also provides the genes for encoding above-mentioned cutin enzyme mutant.
The present invention also provides the recombinant plasmids for carrying said gene.
In one embodiment of the invention, the carrier of the recombinant plasmid is that pUC carrier, pET carrier or pGEX are carried
Body.
In one embodiment of the invention, the carrier of the recombinant plasmid is pET20b (+) carrier.
The present invention also provides the host cells for carrying said gene or above-mentioned recombinant plasmid.
In one embodiment of the invention, the host cell is bacterium or fungi.
In one embodiment of the invention, the host cell is Escherichia coli.
The present invention also provides the preparation method of above-mentioned cutin enzyme mutant, the method is to use above-mentioned host cell,
Above-mentioned host cell is seeded in fermentation medium and is fermented.
In one embodiment of the invention, the fermentation medium is LB culture medium or TB culture medium.
The present invention also provides the cutin enzyme mutants being prepared using the above method.
The present invention also provides above-mentioned cutin enzyme mutants or the above-mentioned cutin enzyme mutant being prepared in hydrolysis poly-vinegar
Application in terms of vinyl acetate.
The present invention also provides a kind of method for handling plain boiled water, the method is by above-mentioned cutin enzyme mutant or above-mentioned system
Standby obtained cutin enzyme mutant, which is added into plain boiled water, to be digested.
In one embodiment of the invention, additive amount of the cutin enzyme mutant in plain boiled water is 2.5~5U/g.
[beneficial effect]
Vigor height, the plain boiled water treatment effect of the resinous matters such as cutin enzyme mutant hydrolysed polyvinyl acetate of the invention
Good, the vigor of hydrolysed polyvinyl acetate can be improved compared with 1.72 times of wild type raising, plain boiled water treatment effect compared with wild type
54%, there is high application prospect in plain boiled water process field.
Specific embodiment
Combined with specific embodiments below, the present invention will be further elaborated.
E. coli jm109 involved in following embodiments, e. coli bl21 (DE3) be purchased from raw work bioengineering (on
Sea) limited liability company, pET-20b (+) plasmid is purchased from Novagen company, and paper mill secondary cycle residue plain boiled water is from Zhejiang
River paper mill.
Culture medium involved in following embodiments is as follows:
LB culture medium: tryptone 10gL-1, yeast powder 5gL-1, sodium chloride 10gL-1；
TB culture medium: tryptone 12gL-1, yeast powder 24gL-1, glycerol 5gL-1, KH2PO4 2.31g·L-1,
K2HPO4·3H2O 16.43g·L-1, glycine 7.5gL-1。
Detection method involved in following embodiments is as follows:
The enzyme activity determination method of cutinase: at 37 DEG C, enzyme activity is measured using continuous spectrophotometry.
Reaction total volume is 1.5mL, including 30 μ L fermentation crude enzyme liquid and the 1470 μ L NaTDC of sulphur containing 50mmol/L
It is recorded at wavelength 405nm to nitre with the Tris-HCl buffer (pH 8.0) of 50mmol/L p-nitrophenyl butyrate (pNPB)
The generating rate of base phenol；
Enzyme activity is defined as: at 37 DEG C, p-nitrophenyl butyrate catalyzing hydrolysis is generated into 1 μm of ol paranitrophenol institute per minute
The enzyme amount needed is an enzyme activity unit (1U).
The detection method of turbidity: it refers to national standard GB 13200-91 " measurement of water turbidity ".
Embodiment 1: the expression of wild type cutinase
Specific step is as follows:
(1) according to the gene order (core for the cutinase for deriving from Humicola insolens (Humicola insolens) in NCBI
Nucleotide sequence is as shown in SEQ ID No.10), obtain recombinant plasmid HIcut/pET20b (+) (recombinant plasmid HIcut/pET20b
The construction method of (+) can refer to document: Sun Yirong, Wu Jing, table of the tinkling of pieces of jade proper Humicola insolens cutinase in place in Escherichia coli
Up to fermentation optimization [J] food and mechanical, 2018 (4))；
(2) recombinant plasmid HIcut/pET20b (+) is transformed into e. coli bl21 (DE3), obtains HIcut/
pET20b(+)/BL21(DE3)；
(3) inoculation HIcut/pET20b (+)/BL21 (DE3) is in LB liquid medium (ampicillin containing 100mg/L)
8h is grown, seed is accessed into TB liquid fermentation medium (ampicillin containing 100mg/L) by 5% inoculum concentration；Escherichia coli exist
After 25 DEG C of shaking table culture fermentation 48h, certain volume fermentation liquid is centrifuged 15min in 4 DEG C, 12000rpm, takes fermentation supernatant, as
Wild enzyme fermentation crude enzyme liquid.
Detect the cutinase enzyme activity in wild enzyme fermentation crude enzyme liquid, testing result 85U/mL.
Embodiment 2: the preparation and expression of cutinase single mutant
Specific step is as follows:
(1) preparation of single mutant
According to the gene order (nucleosides for the cutinase for deriving from Humicola insolens (Humicola insolens) in NCBI
Acid sequence is as shown in SEQ ID No.10), the primer for introducing mutation is separately designed and synthesizes, and utilize fast PCR technology, with reality
The expression vector HIcut/pET-20b (+) for applying the gene of the carrying encoding wild type cutinase in example 1 is template, to cutinase
Gene HIcut carries out rite-directed mutagenesis, obtains cutin enzyme mutant；Whether just sequencing confirms the encoding gene of enzyme mutant respectively
Really, it and will be expressed in the vector introduction Escherichia coli for carrying mutant gene, obtain mutation cutinase；
Wherein, the rite-directed mutagenesis primer of L66I mutation is introduced are as follows:
Nucleotide sequence forward primer as shown in SEQ ID No.11: 5 '-CATACGATGCCGCCTATGCTACTAA -
3 ' (underscore is mutating alkali yl)；
Nucleotide sequence reverse primer as shown in SEQ ID No.12: 5 '-GAAAAGTTAGTAGCATAGGCGGCAT -
3 ' (underscore is mutating alkali yl)；
Introduce the rite-directed mutagenesis primer of L66A mutation are as follows:
Nucleotide sequence forward primer as shown in SEQ ID No.13: 5 '-CATACGATGCCGCCGCAGCTACTAA -
3 ' (underscore is mutating alkali yl)；
Nucleotide sequence reverse primer as shown in SEQ ID No.14: 5 '-GAAAAGTTAGTAGCTGCGGCGGCAT -
3 ' (underscore is mutating alkali yl)；
Introduce the rite-directed mutagenesis primer of L66V mutation are as follows:
Nucleotide sequence forward primer as shown in SEQ ID No.15: 5 '-CATACGATGCCGCCGTAGCTACTAA -
3 ' (underscore is mutating alkali yl)；
Nucleotide sequence reverse primer as shown in SEQ ID No.16: 5 '-GAAAAGTTAGTAGCTACGGCGGCAT -
3 ' (underscore is mutating alkali yl)；
Introduce the rite-directed mutagenesis primer of I169L mutation are as follows:
Nucleotide sequence forward primer as shown in SEQ ID No.17: 5 '-ACCGGAACTTTAATTTATACTCCT-3’
(underscore is mutating alkali yl)；
Nucleotide sequence reverse primer as shown in SEQ ID No.18: 5 '-
AGATGAGCAGGAGTAATAATTAAAGT-3 ' (underscore is mutating alkali yl)；
Introduce the rite-directed mutagenesis primer of I169A mutation are as follows:
Nucleotide sequence forward primer as shown in SEQ ID No.19: 5 '-ACCGGAACTTTAATTGCTACTCCT -
3 ' (underscore is mutating alkali yl)；
Nucleotide sequence reverse primer as shown in SEQ ID No.20: 5 '-
AGATGAGCAGGAGTAGCAATTAAAGT-3 ' (underscore is mutating alkali yl)；
Introduce the rite-directed mutagenesis primer of I169V mutation are as follows:
Nucleotide sequence forward primer as shown in SEQ ID No.21: 5 '-ACCGGAACTTTAATTGTAACTCCT -
3 ' (underscore is mutating alkali yl)；
Nucleotide sequence reverse primer as shown in SEQ ID No.22: 5 '-
AGATGAGCAGGAGTACATATTAAAGT-3 ' (underscore is mutating alkali yl)；
Introduce the rite-directed mutagenesis primer of I169H mutation are as follows:
Nucleotide sequence forward primer as shown in SEQ ID No.23: 5 '-ACCGGAACTTTAATTCATACTCCT -
3 ' (underscore is mutating alkali yl)；
Nucleotide sequence reverse primer as shown in SEQ ID No.24: 5 '-
AGATGAGCAGGAGTAATGATTAAAGT-3 ' underscore is mutating alkali yl)；
PCR reaction system are as follows: 20 μM of forward primers and each 4 μ L, 5 × PS Buffer of 0.5 μ L, dNTPMix of reverse primer
The 0.5 μ L of PrimeStar polymerase of 10 μ L, 2.5U/ μ L, 0.5 μ L of template add 50 μ L of distilled water polishing；
PCR condition are as follows: 94 DEG C of initial denaturation 4min；Then carry out 25 circulations (94 DEG C of 10s, 55 DEG C of 5s, 72 DEG C of 7min
50s) 72 DEG C of extension 10min；Last 4 DEG C of heat preservations, PCR product are detected with 1% agarose gel electrophoresis；
The correct PCR product of above-mentioned verifying is subjected to Dpn I digestion, escherichia coli jm109 competent cell is transferred to, converts
Product is coated on the LB plate of the ampicillin containing 100mg/L, is incubated overnight through 37 DEG C, and 2 single colonies of picking, connect on plate
Enter LB liquid medium, plasmid is extracted after 8h and be sequenced, obtain sequencing correctly carry respectively mutant L66I, L66A,
The recombinant plasmid of L66V, I169L, I169A, I169V or I169H.
(2) preparation of double-mutant
Recombinant plasmid to carry mutant L66A separately designs and synthesizes the primer for introducing mutation as template, and
Using fast PCR technology, rite-directed mutagenesis is carried out to cutinase gene HIcut again, obtains cutin enzyme mutant；Sequencing is true respectively
Whether the encoding gene for recognizing enzyme mutant is correct, and will express in the vector introduction Escherichia coli for carrying mutant gene,
Obtain mutation cutinase；
Wherein, the rite-directed mutagenesis primer of L66A/I169L mutation is introduced are as follows:
Nucleotide sequence forward primer as shown in SEQ ID No.25: 5 '-ACCGGAACTTTAATTTATACTCCT-3’
(underscore is mutating alkali yl)；
Nucleotide sequence reverse primer as shown in SEQ ID No.26: 5 '-
AGATGAGCAGGAGTAATAATTAAAGT-3 ' (underscore is mutating alkali yl)；
Introduce the rite-directed mutagenesis primer of L66A/I169A mutation are as follows:
Nucleotide sequence forward primer as shown in SEQ ID No.27: 5 '-ACCGGAACTTTAATTGCTACTCCT -
3 ' (underscore is mutating alkali yl)；
Nucleotide sequence reverse primer as shown in SEQ ID No.28: 5 '-
AGATGAGCAGGAGTAGCAATTAAAGT-3 ' (underscore is mutating alkali yl)；
Introduce the rite-directed mutagenesis primer of L66A/I169H mutation are as follows:
Nucleotide sequence forward primer as shown in SEQ ID No.29: 5 '-ACCGGAACTTTAATTCATACTCCT -
3 ' (underscore is mutating alkali yl)；
Nucleotide sequence reverse primer as shown in SEQ ID No.30: 5 '-
AGATGAGCAGGAGTAATGATTAAAGT-3 ' (underscore is mutating alkali yl)；
PCR reaction system are as follows: 20 μM of forward primers and each 4 μ L, 5 × PS Buffer of 0.5 μ L, dNTPMix of reverse primer
The 0.5 μ L of PrimeStar polymerase of 10 μ L, 2.5U/ μ L, 0.5 μ L of template add 50 μ L of distilled water polishing；
PCR condition are as follows: 94 DEG C of initial denaturation 4min；Then carry out 25 circulations (94 DEG C of 10s, 55 DEG C of 5s, 72 DEG C of 7min
50s) 72 DEG C of extension 10min；Last 4 DEG C of heat preservations, PCR product are detected with 1% agarose gel electrophoresis；
The correct PCR product of above-mentioned verifying is subjected to Dpn I digestion, escherichia coli jm109 competent cell is transferred to, converts
Product is coated on the LB plate of the ampicillin containing 100mg/L, is incubated overnight through 37 DEG C, and 2 single colonies of picking, connect on plate
Enter LB liquid medium, plasmid is extracted after 8h and be sequenced, obtain sequencing correctly carry respectively mutant L66A/I169L,
The recombinant plasmid of L66A/I169A or L66A/I169H.
(3) expression of mutant
It is inoculated with the correct recombinant plasmid of above-mentioned sequencing and grows 8h in LB liquid medium (ampicillin containing 100mg/L),
Seed is accessed into TB liquid fermentation medium (ampicillin containing 100mg/L) by 5% inoculum concentration；Escherichia coli are in 25 DEG C of shaking tables
After cultivation and fermentation 48h, certain volume fermentation liquid is centrifuged 15min in 4 DEG C, 12000rpm, takes fermentation supernatant, as mutant
Ferment crude enzyme liquid.
Detect mutant L66I, L66A, L66V, I169L, I169A, I169V, I169H, L66A/I169L, L66A/
Cutinase enzyme activity in the fermentation crude enzyme liquid of I169A, L66A/I169H, testing result is respectively 82U/mL, 78U/mL, 75U/
mL、 113U/mL、128U/mL、125U/mL、120U/mL、116U/mL、112U/mL、95U/mL。
Embodiment 3: the application of cutinase and cutin enzyme mutant in terms of hydrolysed polyvinyl acetate
Specific step is as follows:
It takes 1mL polyvinyl acetate (about 1g/mL) lotion to be added in the Tris-HCl buffer of 99mL pH 8.0, mixes
Take 10mL that wild cutinase or cutin enzyme mutant that 200U embodiment 1-2 is obtained is added after closing uniformly, it is smart in 50 DEG C of water-baths
Really after reaction 120min, the ethyl alcohol for taking 5mL sample that 5mL 95% is added terminates reaction, then utilizes 0.05mol/L sodium hydroxide
Titer be titrated on potentiometric titrimeter pH 10.3 be terminal, according to titration consumption NaOH titer titration volume come
Judge cutinase to the decomposition situation of polyvinyl acetate (the results are shown in Table 1).
As shown in Table 1, mutant L66I, L66A, L66V, I169L, I169A, I169V, L66A/I169L, L66A/
The wilder cutinase of hydrolysed polyvinyl acetate ability of I169A is significantly improved, wherein mutant L66A/I169L,
Wilder enzyme has been respectively increased 1.52 and 1.72 times to L66A/I169A respectively；The hydrolysis of mutant I169H, L66A/I169H are poly-
The wilder cutinase of vinylacetate ability, which has, to be decreased obviously, and wilder enzyme reduces 16% and 4% respectively.
1 cutin enzyme mutant hydrolysed polyvinyl acetate of table
| Enzyme | Sodium hydroxide consumption (mL) |
| Wild enzyme | 5 |
| L66I | 5.4 |
| L66A | 6.8 |
| L66V | 5.9 |
| I169L | 6.2 |
| I169A | 6.8 |
| I169V | 5.3 |
| I169H | 4.2 |
| L66A/I169L | 7.6 |
| L66A/I169A | 8.6 |
| L66A/I169H | 4.8 |
Embodiment 4: the application in terms of cutinase and cutin enzyme mutant processing paper mill secondary cycle residue plain boiled water
1, water and water quality are handled:
Handle water: 1500m3
Influent quality: 50~400mg/L of COD, pH 7.0,200~1000NTU of turbidity.
2, treatment process:
Plain boiled water pH to 8.0 ± 0.2 is adjusted, the wilder cutinase of hydrolysed polyvinyl acetate ability in embodiment 3 is added and mentions
High cutin enzyme mutant L66I, L66A, L66V, I169L, I169A, I169V, L66A/I169L, L66A/I169A 50000U
(enzyme concentration about 2.5~5U/g) is handled 4 hours under room temperature (16~25 DEG C).
Measurement processing after water quality, the results are shown in Table 2, wild enzyme turbidity reduction amount be 108NTU, mutant L66I,
L66A, L66V, I169L, I169A, I169V, L66A/I169L, L66A/I169A respectively compared with wild type improve 25%, 32%,
12%, 27%, 38%, 16%, 45%, 54%.
The cutinase hydrolysed residual plain boiled water in 2 source Humicola Insolens of table
| Enzyme | Turbidity reduction amount (NTU) |
| Wild enzyme | 108 |
| L66I | 135 |
| L66A | 144 |
| L66V | 121 |
| I169L | 137 |
| I169A | 148 |
| I169V | 126 |
| L66A/I169L | 156 |
| L66A/I169A | 166 |
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Sequence table
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Asn Met Gly Ile Thr Val Gly Pro Ala Leu Ala Asn Gly Leu Glu Ser
35 40 45
His Ile Arg Asn Ile Trp Ile Gln Gly Val Gly Gly Pro Tyr Asp Ala
50 55 60
Ala Leu Ala Thr Asn Phe Leu Pro Arg Gly Thr Ser Gln Ala Asn Ile
65 70 75 80
Asp Glu Gly Lys Arg Leu Phe Ala Leu Ala Asn Gln Lys Cys Pro Asn
85 90 95
Thr Pro Val Val Ala Gly Gly Tyr Ser Gln Gly Ala Ala Leu Ile Ala
100 105 110
Ala Ala Val Ser Glu Leu Ser Gly Ala Val Lys Glu Gln Val Lys Gly
115 120 125
Val Ala Leu Phe Gly Tyr Thr Gln Asn Leu Gln Asn Arg Gly Gly Ile
130 135 140
Pro Asn Tyr Pro Arg Glu Arg Thr Lys Val Phe Cys Asn Val Gly Asp
145 150 155 160
Ala Val Cys Thr Gly Thr Leu Ile Ala Thr Pro Ala His Leu Ser Tyr
165 170 175
Thr Ile Glu Ala Arg Gly Glu Ala Ala Arg Phe Leu Arg Asp Arg Ile
180 185 190
Arg Ala
<210> 7
<211> 194
<212> PRT
<213>artificial sequence
<400> 7
Gln Leu Gly Ala Ile Glu Asn Gly Leu Glu Ser Gly Ser Ala Asn Ala
1 5 10 15
Cys Pro Asp Ala Ile Leu Ile Phe Ala Arg Gly Ser Thr Glu Pro Gly
20 25 30
Asn Met Gly Ile Thr Val Gly Pro Ala Leu Ala Asn Gly Leu Glu Ser
35 40 45
His Ile Arg Asn Ile Trp Ile Gln Gly Val Gly Gly Pro Tyr Asp Ala
50 55 60
Ala Leu Ala Thr Asn Phe Leu Pro Arg Gly Thr Ser Gln Ala Asn Ile
65 70 75 80
Asp Glu Gly Lys Arg Leu Phe Ala Leu Ala Asn Gln Lys Cys Pro Asn
85 90 95
Thr Pro Val Val Ala Gly Gly Tyr Ser Gln Gly Ala Ala Leu Ile Ala
100 105 110
Ala Ala Val Ser Glu Leu Ser Gly Ala Val Lys Glu Gln Val Lys Gly
115 120 125
Val Ala Leu Phe Gly Tyr Thr Gln Asn Leu Gln Asn Arg Gly Gly Ile
130 135 140
Pro Asn Tyr Pro Arg Glu Arg Thr Lys Val Phe Cys Asn Val Gly Asp
145 150 155 160
Ala Val Cys Thr Gly Thr Leu Ile Val Thr Pro Ala His Leu Ser Tyr
165 170 175
Thr Ile Glu Ala Arg Gly Glu Ala Ala Arg Phe Leu Arg Asp Arg Ile
180 185 190
Arg Ala
<210> 8
<211> 194
<212> PRT
<213>artificial sequence
<400> 8
Gln Leu Gly Ala Ile Glu Asn Gly Leu Glu Ser Gly Ser Ala Asn Ala
1 5 10 15
Cys Pro Asp Ala Ile Leu Ile Phe Ala Arg Gly Ser Thr Glu Pro Gly
20 25 30
Asn Met Gly Ile Thr Val Gly Pro Ala Leu Ala Asn Gly Leu Glu Ser
35 40 45
His Ile Arg Asn Ile Trp Ile Gln Gly Val Gly Gly Pro Tyr Asp Ala
50 55 60
Ala Ala Ala Thr Asn Phe Leu Pro Arg Gly Thr Ser Gln Ala Asn Ile
65 70 75 80
Asp Glu Gly Lys Arg Leu Phe Ala Leu Ala Asn Gln Lys Cys Pro Asn
85 90 95
Thr Pro Val Val Ala Gly Gly Tyr Ser Gln Gly Ala Ala Leu Ile Ala
100 105 110
Ala Ala Val Ser Glu Leu Ser Gly Ala Val Lys Glu Gln Val Lys Gly
115 120 125
Val Ala Leu Phe Gly Tyr Thr Gln Asn Leu Gln Asn Arg Gly Gly Ile
130 135 140
Pro Asn Tyr Pro Arg Glu Arg Thr Lys Val Phe Cys Asn Val Gly Asp
145 150 155 160
Ala Val Cys Thr Gly Thr Leu Ile Leu Thr Pro Ala His Leu Ser Tyr
165 170 175
Thr Ile Glu Ala Arg Gly Glu Ala Ala Arg Phe Leu Arg Asp Arg Ile
180 185 190
Arg Ala
<210> 9
<211> 194
<212> PRT
<213>artificial sequence
<400> 9
Gln Leu Gly Ala Ile Glu Asn Gly Leu Glu Ser Gly Ser Ala Asn Ala
1 5 10 15
Cys Pro Asp Ala Ile Leu Ile Phe Ala Arg Gly Ser Thr Glu Pro Gly
20 25 30
Asn Met Gly Ile Thr Val Gly Pro Ala Leu Ala Asn Gly Leu Glu Ser
35 40 45
His Ile Arg Asn Ile Trp Ile Gln Gly Val Gly Gly Pro Tyr Asp Ala
50 55 60
Ala Ala Ala Thr Asn Phe Leu Pro Arg Gly Thr Ser Gln Ala Asn Ile
65 70 75 80
Asp Glu Gly Lys Arg Leu Phe Ala Leu Ala Asn Gln Lys Cys Pro Asn
85 90 95
Thr Pro Val Val Ala Gly Gly Tyr Ser Gln Gly Ala Ala Leu Ile Ala
100 105 110
Ala Ala Val Ser Glu Leu Ser Gly Ala Val Lys Glu Gln Val Lys Gly
115 120 125
Val Ala Leu Phe Gly Tyr Thr Gln Asn Leu Gln Asn Arg Gly Gly Ile
130 135 140
Pro Asn Tyr Pro Arg Glu Arg Thr Lys Val Phe Cys Asn Val Gly Asp
145 150 155 160
Ala Val Cys Thr Gly Thr Leu Ile Ala Thr Pro Ala His Leu Ser Tyr
165 170 175
Thr Ile Glu Ala Arg Gly Glu Ala Ala Arg Phe Leu Arg Asp Arg Ile
180 185 190
Arg Ala
<210> 10
<211> 585
<212> DNA
<213>artificial sequence
<400> 10
caattgggtg ctattgaaaa cggacttgaa tcaggatcag ctaacgcctg tcctgatgcc 60
attcttattt ttgccagagg ttcaactgaa cctggaaaca tgggaattac cgttggacca 120
gctttagcca acggtttaga atctcatatt cgtaacattt ggattcaagg tgttggaggt 180
ccatacgatg ccgccttagc tactaacttt cttcctcgtg gtacttcaca agccaacatt 240
gatgaaggaa agagattatt tgccttggcc aaccaaaagt gtccaaacac cccagttgtt 300
gcgggtggct actcacaagg ggccgcttta attgctgccg ccgtttccga attatccgga 360
gctgttaagg aacaagttaa gggagttgcc ttgtttggtt acactcaaaa cttgcaaaac 420
agaggtggta ttcctaacta ccctagagaa agaactaagg tattctgtaa cgttggtgac 480
gctgtttgta ccggaacttt aattattact cctgctcatc tttcatacac cattgaagcc 540
cgtggagaag ccgctagatt tcttcgtgat cgtattcgtg cttaa 585
<210> 11
<211> 25
<212> DNA
<213>artificial sequence
<400> 11
catacgatgc cgcctatgct actaa 25
<210> 12
<211> 25
<212> DNA
<213>artificial sequence
<400> 12
gaaaagttag tagcataggc ggcat 25
<210> 13
<211> 25
<212> DNA
<213>artificial sequence
<400> 13
catacgatgc cgccgcagct actaa 25
<210> 14
<211> 25
<212> DNA
<213>artificial sequence
<400> 14
gaaaagttag tagctgcggc ggcat 25
<210> 15
<211> 25
<212> DNA
<213>artificial sequence
<400> 15
catacgatgc cgccgtagct actaa 25
<210> 16
<211> 25
<212> DNA
<213>artificial sequence
<400> 16
gaaaagttag tagctacggc ggcat 25
<210> 17
<211> 24
<212> DNA
<213>artificial sequence
<400> 17
accggaactt taatttatac tcct 24
<210> 18
<211> 26
<212> DNA
<213>artificial sequence
<400> 18
agatgagcag gagtaataat taaagt 26
<210> 19
<211> 24
<212> DNA
<213>artificial sequence
<400> 19
accggaactt taattgctac tcct 24
<210> 20
<211> 26
<212> DNA
<213>artificial sequence
<400> 20
agatgagcag gagtagcaat taaagt 26
<210> 21
<211> 24
<212> DNA
<213>artificial sequence
<400> 21
accggaactt taattgtaac tcct 24
<210> 22
<211> 26
<212> DNA
<213>artificial sequence
<400> 22
agatgagcag gagtacatat taaagt 26
<210> 23
<211> 24
<212> DNA
<213>artificial sequence
<400> 23
accggaactt taattcatac tcct 24
<210> 24
<211> 26
<212> DNA
<213>artificial sequence
<400> 24
agatgagcag gagtaatgat taaagt 26
<210> 25
<211> 24
<212> DNA
<213>artificial sequence
<400> 25
accggaactt taatttatac tcct 24
<210> 26
<211> 26
<212> DNA
<213>artificial sequence
<400> 26
agatgagcag gagtaataat taaagt 26
<210> 27
<211> 24
<212> DNA
<213>artificial sequence
<400> 27
accggaactt taattgctac tcct 24
<210> 28
<211> 26
<212> DNA
<213>artificial sequence
<400> 28
agatgagcag gagtagcaat taaagt 26
<210> 29
<211> 24
<212> DNA
<213>artificial sequence
<400> 29
accggaactt taattcatac tcct 24
<210> 30
<211> 26
<212> DNA
<213>artificial sequence
<400> 30
agatgagcag gagtaatgat taaagt 26
Claims (10)
1. a kind of cutin enzyme mutant, which is characterized in that the mutant is by by amino acid sequence such as SEQ ID No.1 institute
What the 66th leucine and/or the 169th isoleucine of the cutinase shown were mutated.
2. cutin enzyme mutant as described in claim 1, which is characterized in that the mutant be by by amino acid sequence such as
66th leucine of cutinase shown in SEQ ID No.1 sports what isoleucine obtained；
Or the mutant is by the way that the 66th leucine of amino acid sequence cutinase as shown in SEQ ID No.1 to be mutated
It is obtained for alanine；
Or the mutant is by the way that the 66th leucine of amino acid sequence cutinase as shown in SEQ ID No.1 to be mutated
It is obtained for valine；
Or the mutant is by by the 169th isoleucine of amino acid sequence cutinase as shown in SEQ ID No.1
Sport what leucine obtained；
Or the mutant is by by the 169th isoleucine of amino acid sequence cutinase as shown in SEQ ID No.1
Sport what alanine obtained；
Or the mutant is by by the 169th isoleucine of amino acid sequence cutinase as shown in SEQ ID No.1
Sport what valine obtained；
Or the mutant is by the way that the 66th leucine of amino acid sequence cutinase as shown in SEQ ID No.1 to be mutated
It is what leucine obtained for alanine and the 169th isoleucine mutation；
Or the mutant is by the way that the 66th leucine of amino acid sequence cutinase as shown in SEQ ID No.1 to be mutated
It is what alanine obtained for alanine and the 169th isoleucine mutation.
3. cutin enzyme mutant as claimed in claim 1 or 2, which is characterized in that the amino acid sequence of the mutant is SEQ
ID No.2、SEQ ID No.3、SEQ ID No.4、SEQ ID No.5、SEQ ID No.6、SEQ ID No.7、SEQ ID
No.8 or SEQ ID No.9.
4. encoding the gene of any cutin enzyme mutant of claim 1-3.
5. carrying the recombinant plasmid of gene described in claim 4.
6. carrying the host cell of recombinant plasmid described in gene described in claim 4 or claim 5.
7. the preparation method of any cutin enzyme mutant of claim 1-3, which is characterized in that the method is to use right
It is required that host cell described in 6, host cell as claimed in claim 6 is seeded in fermentation medium and is fermented.
8. the cutin enzyme mutant being prepared using claim 7 the method.
9. claim 1-3 any the cutin enzyme mutant or the cutin enzyme mutant according to any one of claims 8 being prepared
Application of the body in terms of hydrolysed polyvinyl acetate.
10. a kind of method for handling plain boiled water, which is characterized in that the method is by any cutinase of claim 1-3
Mutant or the cutin enzyme mutant according to any one of claims 8 being prepared are added into plain boiled water and are digested.
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|---|---|---|---|
| CN201811612801.8A CN109929821A (en) | 2018-12-27 | 2018-12-27 | A kind of cutin enzyme mutant and its application in plain boiled water processing |
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| CN109929821A true CN109929821A (en) | 2019-06-25 |
Family
ID=66984817
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060194303A1 (en) * | 2004-12-21 | 2006-08-31 | Novozymes Biologicals, Inc. | Wastewater treatment compositions |
| CN101423824A (en) * | 2000-06-02 | 2009-05-06 | 诺维信公司 | Cutinase variants |
| CN104271737A (en) * | 2012-05-14 | 2015-01-07 | 诺维信公司 | Cutinase variants and polynucleotides encoding same |
| CN106480771A (en) * | 2016-12-23 | 2017-03-08 | 江南大学 | A kind of method of utilization cutin ferment treatment mthod of white water from paper making |
-
2018
- 2018-12-27 CN CN201811612801.8A patent/CN109929821A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101423824A (en) * | 2000-06-02 | 2009-05-06 | 诺维信公司 | Cutinase variants |
| US20060194303A1 (en) * | 2004-12-21 | 2006-08-31 | Novozymes Biologicals, Inc. | Wastewater treatment compositions |
| CN104271737A (en) * | 2012-05-14 | 2015-01-07 | 诺维信公司 | Cutinase variants and polynucleotides encoding same |
| CN106480771A (en) * | 2016-12-23 | 2017-03-08 | 江南大学 | A kind of method of utilization cutin ferment treatment mthod of white water from paper making |
Non-Patent Citations (1)
| Title |
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| 郝玥: "造纸废水处理方法概述", 《科技展望》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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