CN108251319A - A kind of fermentation of cutinase and cotton fabric enzyme refinery practice - Google Patents
A kind of fermentation of cutinase and cotton fabric enzyme refinery practice Download PDFInfo
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- CN108251319A CN108251319A CN201810145969.6A CN201810145969A CN108251319A CN 108251319 A CN108251319 A CN 108251319A CN 201810145969 A CN201810145969 A CN 201810145969A CN 108251319 A CN108251319 A CN 108251319A
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- cutinase
- enzyme
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- sorbierite
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01074—Cutinase (3.1.1.74)
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- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M16/00—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic
- D06M16/003—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic with enzymes or microorganisms
-
- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M2101/00—Chemical constitution of the fibres, threads, yarns, fabrics or fibrous goods made from such materials, to be treated
- D06M2101/02—Natural fibres, other than mineral fibres
- D06M2101/04—Vegetal fibres
- D06M2101/06—Vegetal fibres cellulosic
Abstract
The invention discloses a kind of fermentations of cutinase and cotton fabric enzyme refinery practice, belong to micro-organism enzyme preparation field.The present invention on the basis of keeping production of enzyme constant, the time required to reducing expression, was reduced to 100 hours or so by using the mode induced expression cutinase of compounding sorbierite from 150 hours or so；Present invention utilization is enzymatically treated cotton fabric and compares chemical Treatment, and step simple reaction mild condition is environmental-friendly, the demand of highly basic is avoided, required processing time reduces by 2 hours or more compared with chemical method and enzyme process, while refining effect gets a promotion, rate of dyeing rises, wettability enhancing.
Description
Technical field
The present invention relates to a kind of fermentations of cutinase and cotton fabric enzyme refinery practice, belong to micro-organism enzyme preparation field.
Background technology
Cotton fiber has excellent wearability, is a kind of important natural textile fiber, product include cotton fabrics,
Cotton fabrics.But in the growth course of cotton fiber, there are the substance and bio-metabolic process of a certain amount of protective effect
The substance of middle generation is grown altogether with cellulose symbiosis, these cellulose commensals mainly have pectin substance, nitrogen substance, wax
Matter, natural pigment etc., the cotton seed hulls also carried secretly when stripping cotton fiber.These impurity are known as the natural impurity on cotton fabric.
These commensals on cotton fiber can influence the dyeing and finishings such as the wearability such as the feel, appearance, whiteness of cotton fabric and water suction, dyeing and add
Therefore work performance, needs to remove in the preceding processing of dyeing and finishing processing, with the needs for meeting dyeing and finishing processing with taking.This removal
The process of commensal is known as the refining of cotton fabric on cotton fiber.
Traditional method of refining of cotton fabric (including yarn) is to carry out high-temperature process using alkali and surfactant, utilizes alkali
To the hydrolysis and surface-active of fatty acid ester substance, nitrogen substance (predominantly protein substance) in pectin, cotton wax
The emulsification of agent, peptizaiton remove all natural impuritys on cotton fiber in addition to natural pigment and cotton seed hulls, improve cotton fabric
Water absorbing properties.The traditional method of refining technical maturity of cotton fabric, high treating effect.But there is also fiber (fabric) is damaged
Greatly, processing chroma in waste water is deep, pH value is high, COD value big (commonly reaching thousands of to up to ten thousand mg/L), the problems such as slurry amount is big,
The quality of cotton fabric is seriously affected, while the waste water discharged also has seriously polluted the environment.And enzyme process carry out refining can be to avoid this
A little problems have the characteristics that the environmentally friendly, energy saving of biological treatment, however enzyme process still remains difficulty among commercial Application, pre-
In processing procedure, temperature can still reach 100 degrees Celsius, need to cool down after pretreatment, and secondly enzyme concentration is still higher.
Pichia pastoris Pichia pastoris have been the most frequently used protein expression system for being only second to Escherichia coli at present, extensively
General protein preparation, characterization and structure elucidation applied to laboratory scale etc..It is induced using methanol complete red
Yeast has following several advantages：With alcohol oxidase AOX1 gene promoters, this is most strong at present, and Regulation Mechanism is most stringent of
One of promoter；Expression efficiency is high, and the foreign protein of expression can account for summary table reaches albumen more than 90%, be conducive to destination protein
Isolate and purify；High Density Cultivation can be achieved in simple synthetic media；Expression plasmid can genome specific site with
The form stable of single copy or multicopy is integrated；Since the yeast can be and most using methanol as sole carbon source and the energy
Microorganism can not can reduce pollution using methanol as carbon source.However there is also as fermentation period it is long；Methanol is inflammable and explosive to be had
Poison, there are certain danger；The shortcomings that culture medium and condition of culture are immature etc.Simultaneously as in Yeast fermentation process
There are carbon source consumption processes, there is certain injury to thalline, reduce the enzymatic productivity of yeast strain.
Invention content
The present invention first is designed to provide a kind of recombinant bacterium for producing cutinase, the recombinant bacterium using Pichia pastoris as
Host, expression is from the cutinase of Humicola insolens.
In one embodiment of the present invention, the amino acid sequence of the cutinase from Humicola insolen
As shown in SEQ ID NO.1.
In one embodiment of the present invention, the construction method of the recombinant bacterium is：It is SEQ ID by amino acid sequence
The gene of NO.1 cutinases is connected on expression vector pPICZ α A, and construction recombination plasmid pPICZ α A-hic will recombinate matter
Grain pPICZ α A-hic obtain recombinant bacterium after being transferred to Pichia pastoris.
Second purpose of the invention is to provide a kind of fermentation process for producing cutinase, and the specific steps of the method are such as
Under：
(1) the Spawn incubation stage：Recombinant bacterium is inoculated into fermentation medium, the stir culture 15- at 25~31 DEG C
36h；
(2) the fermentation inducement stage：Derivant is added during the fermentation, 96~216h of Fiber differentiation at 25~33 DEG C,
The derivant is methanol and sorbierite.
In one embodiment of the invention, the sorbierite containing 5~15g/L in the fermentation medium.
In one embodiment of the invention, the volume fraction of the middle methanol and sorbierite is 1：1~4, the first
The final concentration of 0.8-1.5% of volume of alcohol.
In one embodiment of the invention, the volume fraction of methanol and sorbierite is 1 in the derivant：1~2,
The final concentration of 0.8-1.5% of volume of the methanol.
In one embodiment of the invention, the volume fraction of methanol and sorbierite is 1 in the derivant：1~4,
The final concentration of 0.8-1.5% of volume of the methanol.
In one embodiment of the invention, the inducing temperature in the step (2) is 28~33 DEG C.
In one embodiment of the invention, the component of the fermentation medium is：0~20g/L potassium sulfates, 12~
18g/ L bitter salts, 0.5~1.5g/L calcium sulphate dihydrates, 3~6g/L potassium hydroxide, 20~30mL/L phosphoric acid, 20
~40g/L glycerine.
Third purpose of the present invention is to provide a kind of method that biorefining is carried out with cutinase, and the method includes following
Step：
(1) pretreatment includes：Cotton fabrics after desizing are first pre-processed in 70-100 DEG C of hot water, the time is
2-10min, 1: 10~50 (fabric weight of bath raio：Water volume)；
(2) enzymatic treatment includes：Add in cutinase, enzyme concentration：100~2000U/g, the temperature for the treatment of fluid is room temperature to 80
DEG C, the time is 30~120min.
In one embodiment of the invention, the step (2) the specific steps are in pretreated fabric every 10
~15min adds in the cutinase enzyme solution in Humicola insolen sources, and enzyme concentration is 100~2000U/g, amounts to processing 50-
60min。
In one embodiment of the invention, the cutinase in Humicola insolen sources in the step (2)
Enzyme concentration is 150~350U/g.
In one embodiment of the invention, the step (2) the specific steps are in fabric after the pre-treatment first
The cutinase in 200-400U Humicola insolen sources is added in, is handled 4-10 minutes, is subsequently placed into 40-60 DEG C of hot water
In handled, and add in the cutinase in Thermobifida fusca sources, enzyme concentration be 100~2000U/g, handle 40-
60min。
In one embodiment of the invention, the cutinase in step (2) the Thermobifida fusca sources
Enzyme concentration is 300~500U/g.
In one embodiment of the invention, the preparation reference of the cutinase in the Thermobifida fusca sources
Document Lingqia Su, Ruoyu Hong, Jing Wu*.Enhanced extracellular expression of
gene-optimized Thermobifida fusca cutinase in Escherichia coli by
optimization of induction strategy.Process Biochemistry.2015.50(7):1039-1046。
In one embodiment of the invention, the cutinase comes from thermophilic sporangium Thermobifida fusca
Or the homology of amino acid sequence and the cutinase at least 60% derived from Thermobifida fusca.
In one embodiment of the invention, the cutinase comes from Humicola insolens Humicola insolens
Or the homology of amino acid sequence and the cutinase at least 60% derived from Humicola insolens.
Beneficial effects of the present invention：
(1) the method for the invention induced expression cutinase by the way of sorbierite is compounded is cancelled in conventional procedure for the first time
The process of carbon source starvation, the time required to reducing expression, on the basis of keeping production of enzyme constant, from original induction 180 hours
Left and right is reduced to 100 hours or so, is reduced methanol consumption amount, is carried high production intensity.
(2) chemical Treatment is compared using being enzymatically treated cotton fabric, step simple reaction mild condition is environmental-friendly, keeps away
Exempt from the demand of highly basic, required processing time reduces by 2 hours or more compared with chemical method and enzyme process, while refining effect gets a promotion,
Rate of dyeing rises, wettability enhancing.Cutinase production needed for reaction is simple, and enzyme concentration is low.These advantages are in environmental protection
The today's society being paid more and more attention, it appears particularly important.
Specific embodiment
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 enzyme solutions
It is buffered with the Tris-HCl of 1470 μ L sulphur containing 50mmol/L NaTDC and 50mmol/L p-nitrophenyls butyrate (pNPB)
Liquid (pH 8.0) at wavelength 405nm, records the generating rate of paranitrophenol.The definition of enzyme activity is：It is per minute to incite somebody to action at 37 DEG C
The enzyme amount that p-nitrophenyl butyrate catalyzing hydrolysis generates 1 μm of ol paranitrophenol is an enzyme activity unit.
Rate of dyeing computational methods：
The maximum absorption wavelength of standard dyes is obtained with the scanning of continuous spectrophotometer first.At this wavelength, dye is measured
Dye liquor absorbance value before and after color calculates dye uptake.Using the time as abscissa, dye uptake makes dye speed for ordinate
Rate curve.
Embodiment 1：The structure of genetic engineering bacterium pichia methanolica KM71/pPICZ α A-HIC
(1) it is connect, formed as masterplate, and with pPICZ α A using the gene that amino acid sequence is SEQ ID NO.1 cutinases
Recombinant plasmid pPICZ α A-HIC.
(2) recombinant plasmid is linearized through Sac I restriction endonucleases, and electric shocking method is transferred in Pichia pastoris KM71 competence.
(3) the above-mentioned bacterium solution handled well is uniformly coated in 1mgmL-1Zeocin resistant panels, in 30 DEG C of constant temperature items
It is cultivated under part, until growing single bacterium colony, about 2-3 days, then by shaking flask culture, carries out expression verification, obtain genetic engineering bacterium
Pichia methanolica KM71/pPICZ α A-HIC.
(4) shake flask fermentation, the specific steps are：
Monoclonal on picking YPD/G418 (YPD/Zeocin) concentration gradient tablet in 10mL YPD culture mediums, in
The switching above-mentioned seed liquors of 2.5mL are to 50mL BMGY culture mediums after 30 DEG C of culture 20-24h in 200 rpm constant-temperature tables, in 200
30 DEG C of culture 20-24h in rpm constant-temperature tables, whole thalline 25mL BMMY culture mediums are resuspended, add in derivant methanol extremely
Final concentration of 11gL-1, induction to the HIC enzyme activity that 30 DEG C of cultures carry out recombination HIC reaches peak, and regular (for 24 hours) is to culture
Base adds methanol.
Shake flask results show highest enzyme activity in 200U/mL or so, and OD is 60 or so.
Embodiment 2：The influence that derivant additive amount ferments to Humicola insolens 3L tanks
(1) prepare bacterial strain and culture medium
Bacterial strain：Genetic engineering bacterium pichia methanolica KM71/pPICZ α A-HIC；
Fermentation medium：18.2g/L potassium sulfates, 14.9g/L bitter salts, 0.93g/L calcium sulphate dihydrates,
4.13g/L potassium hydroxide, 26.7mL/L phosphoric acid, 30g/L glycerine, 10g/L sorbierites；
(2) genetic engineering bacterium grown cultures
Taken from solid slope the strain activated on a small quantity pichia methanolica PMAD16/pPICZ α A-HIC be seeded in through
In the fermentation medium of sterilizing, liquid amount 30%, 30 DEG C of cultivation temperature, stir culture to thalline OD is 100；
(3) Fiber differentiation of genetic engineering bacterium
Derivant continuously is added, the addition manner of derivant is as shown in table 1,30 DEG C of Fiber differentiation temperature, induction time
108h is separated by filtration to obtain cutinase (crude preparation).
According to table 1 it is found that in sorbierite and methanol additional proportion 1:In the case of 1, enzyme activity and production intensity reach most
It is high.
The influence of 1 derivant ratio of table and additive amount to producing enzyme
Embodiment 3：The influence that sorbierite amount ferments to Humicola insolens 3L tanks in fermentation medium
(1) prepare bacterial strain and culture medium
Bacterial strain：Genetic engineering bacterium pichia methanolica KM71/pPICZ α A-HIC；
Fermentation medium：18.2g/L potassium sulfates, 14.9g/L bitter salts, 0.93g/L calcium sulphate dihydrates,
4.13g/L potassium hydroxide, 26.7mL/L phosphoric acid, 30g/L glycerine.Sorbierite additive amount is as shown in table 2；
(2) genetic engineering bacterium grown cultures
Taken from solid slope the strain activated on a small quantity pichia methanolica PMAD16/pPICZ α A-HIC be seeded in through
In the fermentation medium of sterilizing, liquid amount 30%, 30 DEG C of cultivation temperature, stir culture to thalline OD is 100；
(3) Fiber differentiation of genetic engineering bacterium
Continuously add methanol and sorbierite volume ratio 1：1 derivant maintains methanol final volume a concentration of 1%, induction training
30 DEG C, induction time 108h of temperature is supported, is separated by filtration to obtain cutinase (crude preparation).
According to table 2 it is found that in sorbierite and methanol additional proportion 1:In the case of 1, enzyme activity and production intensity reach most
It is high.
Influence of the 2 sorbierite additive amount of table to producing enzyme
Embodiment 4：The influence that fermentation parameter ferments to Humicola insolens 3L tanks
(1) prepare bacterial strain and culture medium
Bacterial strain：Genetic engineering bacterium pichia methanolica KM71/pPICZ α A-HIC；
Fermentation medium：18.2g/L potassium sulfates, 14.9g/L bitter salts, 0.93g/L calcium sulphate dihydrates,
4.13g/L potassium hydroxide, 26.7mL/L phosphoric acid, 30g/L glycerine, 10g/L sorbierites；
(2) genetic engineering bacterium grown cultures
Taken from solid slope the strain activated on a small quantity pichia methanolica PMAD16/pPICZ α A-HIC be seeded in through
In the fermentation medium of sterilizing, liquid amount 30%, 30 DEG C of cultivation temperature, stir culture to thalline OD is 100；
(3) Fiber differentiation of genetic engineering bacterium
Continuously add methanol and sorbierite volume ratio 1：1 derivant maintains methanol final volume a concentration of 1%, certain
At a temperature of Fiber differentiation, be separated by filtration to obtain cutinase (crude preparation), Fiber differentiation temperature, induction time is as shown in table 2.
According to table 3 it is found that in the case where inducing temperature is 28 DEG C, enzyme activity and production intensity reach highest.
Influence of 3 induction parameters of table to producing enzyme
Embodiment 5：Biorefining is carried out using cutinase
Sample：Desizing pure cotton fabric
A. it pre-processes：Pure cotton after desizing is first pre-processed in 80 DEG C of hot water, pH=8.5, time 5min, bath
Than 1: 10.
B. in fabric after the pre-treatment certain unit H.i sources cutinase enzyme solution coprocessing is once added in per 15min
60min。
Each enzyme concentration is as shown in table 3.
C. it after being disposed, is first washed 1 time with 95 DEG C of hot water, time about 10s, room temperature is washed 3 times later, is finally dried
It is dry.After the above method is handled, according to table 3 it is found that when each enzyme concentration is 250U/ml, the wetting time of pure cotton by
It is more than that 20min is promoted to 5s before reason.Rate of dyeing is promoted to 92.2% from 52.5%.
Influence of the table 4HIC enzyme concentrations to biorefining
Embodiment 6：Biorefining is carried out using cutinase
Sample：Desizing pure cotton fabric
A. it pre-processes：Pure cotton after desizing is first pre-processed in 80 DEG C of hot water, pH=8.5, time 5min, bath
Than 1: 10.
B. 200U H.i sources cutin enzymatic treatment is first added in fabric after the pre-treatment 5 minutes, is subsequently placed into 50 DEG C
It is handled in hot water, and adds in (the cutinase preparation method reference in Thermobifida fusca sources of T.f sources cutinase
Document： Lingqia Su,Ruoyu Hong,Jing Wu*.Enhanced extracellular expression of
gene-optimized Thermobifida fusca cutinase in Escherichia coli by
optimization of induction strategy.Process Biochemistry.2015.50(7):1039-
1046).It is handled, specific enzyme concentration and processing time are as shown in table 4.
C. it after being disposed, is first washed 1 time with 95 DEG C of hot water, time about 10s, room temperature is washed 3 times later, is finally dried
It is dry.According to table 4 it is found that when T.f cutinases enzyme concentration is 300U/mL, after the above method is handled, the wetting time of pure cotton
It is more than that 20min is promoted to 2.5s by before processing.Rate of dyeing is promoted to 94.2% from 52.5%
Influence of the table 5T.f cutinases enzyme concentration to biorefining
Embodiment 7：It is refined using NaOH
Sample：Desizing pure cotton fabric
A. it pre-processes：Pure cotton after desizing is first pre-processed in 100 DEG C of hot water, time 5min, bath raio 1: 10.
B. 100mmol/L NaOH are first added in fabric after the pre-treatment to handle 180 minutes, are subsequently placed into 50 DEG C of heat
It is handled in water.
C. it after being disposed, is first washed 1 time with 95 DEG C of hot water, time about 10s, room temperature is washed 3 times later, is finally dried
It is dry.After the above method is handled, the wetting time of pure cotton is more than that 20min is promoted to 25s by before processing.Rate of dyeing from
52.5% is promoted to 89.2%.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not limited to the present invention, any to be familiar with this skill
The people of art without departing from the spirit and scope of the present invention, can do various change and modification, therefore the protection model of the present invention
Enclosing be subject to what claims were defined.
Sequence table
<110>A kind of fermentation of cutinase and cotton fabric enzyme refinery practice
<120>Southern Yangtze University
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 194
<212> PRT
<213>It is artificial synthesized
<400> 1
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 Ile 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
Claims (10)
1. a kind of recombinant bacterium for producing cutinase, which is characterized in that using Pichia pastoris as host, expression derives from the recombinant bacterium
The cutinase of Humicola insolens.
2. recombinant bacterium according to claim 1, which is characterized in that the cutin from Humicola insolens
The amino acid sequence of enzyme has more than 60% similitude as shown in SEQ ID NO.1 or with SEQ ID NO.1.
3. recombinant bacterium according to claim 1 or 2, which is characterized in that the construction method of the recombinant bacterium is：By amino acid
Sequence is connected to for the gene of SEQ ID NO.1 cutinases on expression vector pPICZ α A, construction recombination plasmid pPICZ α A-hic,
Recombinant bacterium is obtained after recombinant plasmid pPICZ alpha A-hic is transferred to Pichia pastoris.
A kind of 4. method of fermenting and producing cutinase, which is characterized in that include the following steps：
(1) the Spawn incubation stage：Any recombinant bacterium of claims 1 to 3 is inoculated into fermentation medium, at 25~31 DEG C
Lower stir culture 15-36h；
(2) the fermentation inducement stage：Derivant is added during the fermentation, and 96~216h of Fiber differentiation, described at 25~33 DEG C
Derivant is methanol and sorbierite.
5. according to the method described in claim 4, it is characterized in that, contain the sorbierite of 5~15g/L in the fermentation medium.
6. according to the method described in claim 4, it is characterized in that, the volume fraction of methanol and sorbierite is in the derivant
1：1~4, the additive amount of the methanol reaches the final concentration of 0.8-1.5% of volume.
A kind of 7. method that biorefining is carried out with cutinase, which is characterized in that include the following steps：
(1) pretreatment includes：Cotton fabrics after desizing are first pre-processed in 70-100 DEG C of hot water, time 2-
10min, 1: 10~50 (fabric weight of bath raio：Water volume)；
(2) enzymatic treatment includes：Add in cutinase, enzyme concentration：100~2000U/g, the temperature for the treatment of fluid are room temperature to 80 DEG C, when
Between be 30~120min.
8. the method according to the description of claim 7 is characterized in that the step (2) is knitted the specific steps are pretreated
The enzyme concentration that every 10~15min adds in the cutinase in Humicola insolen sources in object is 150~350U/g, and total is handled
50-60min。
9. method according to claim 7 or 8, which is characterized in that the step (2) the specific steps are after the pre-treatment
Fabric in first add in the cutinase in 200-400U Humicola insolen sources, handle 4-10 minutes, be subsequently placed into 40-
It is handled in 60 DEG C of hot water, and adds in the cutinase in Thermobifida fusca sources, enzyme concentration is 100~2000U/
G handles 40-60min.
10. according to application of the cutinase that any the methods of claim 4-6 obtain in dyeing and finishing processing.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110306346A (en) * | 2019-07-10 | 2019-10-08 | 广东湛丰精细化工有限公司 | A kind of refining dye enzyme and preparation method thereof promoting the anti-fuzz balls performance of cotton knitwear |
| CN110983849A (en) * | 2019-12-20 | 2020-04-10 | 江南大学 | Method for degrading adhesive by compounding multiple enzymes and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101792729A (en) * | 2009-12-18 | 2010-08-04 | 江南大学 | Genetically engineered bacteria for efficiently secreting, expressing and reconstructing cutinase and method for constructing same |
| CN102146633A (en) * | 2011-01-15 | 2011-08-10 | 江南大学 | Method for scouring cotton fabric by applying cutinase and CBD (cellulose-binding domain)-alkaline pectinase |
| KR20120039808A (en) * | 2010-10-18 | 2012-04-26 | 주식회사 에코웍스 | Natural dyeing method for ecotextiles |
| 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
- 2018-02-12 CN CN201810145969.6A patent/CN108251319A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101792729A (en) * | 2009-12-18 | 2010-08-04 | 江南大学 | Genetically engineered bacteria for efficiently secreting, expressing and reconstructing cutinase and method for constructing same |
| KR20120039808A (en) * | 2010-10-18 | 2012-04-26 | 주식회사 에코웍스 | Natural dyeing method for ecotextiles |
| CN102146633A (en) * | 2011-01-15 | 2011-08-10 | 江南大学 | Method for scouring cotton fabric by applying cutinase and CBD (cellulose-binding domain)-alkaline pectinase |
| 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 (3)
| Title |
|---|
| 吴建华: "《纤维素纤维织物的染整》", 31 May 2015, 中国纺织出版社 * |
| 李光磊等: "嗜麦芽寡养单胞菌角蛋白酶基因在毕赤酵母中的表达", 《生物技术通报》 * |
| 陈静奕等: "棉织物酶精练及检测方法研究", 《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110306346A (en) * | 2019-07-10 | 2019-10-08 | 广东湛丰精细化工有限公司 | A kind of refining dye enzyme and preparation method thereof promoting the anti-fuzz balls performance of cotton knitwear |
| CN110983849A (en) * | 2019-12-20 | 2020-04-10 | 江南大学 | Method for degrading adhesive by compounding multiple enzymes and application thereof |
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