CN105155167B - A kind of application of mesophilic protein in the bath process of polishing and dyeing one - Google Patents
A kind of application of mesophilic protein in the bath process of polishing and dyeing one Download PDFInfo
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- CN105155167B CN105155167B CN201510503973.1A CN201510503973A CN105155167B CN 105155167 B CN105155167 B CN 105155167B CN 201510503973 A CN201510503973 A CN 201510503973A CN 105155167 B CN105155167 B CN 105155167B
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Abstract
The invention discloses a kind of application of mesophilic protein in the bath process of polishing and dyeing one, the mesophilic protein uses trichoderma reesei or Pichia anomala expression.The mesophilic protein is the neutral cellulase with halophagia energy, the activity of the salt centering cellulase of high concentration is obviously improved effect, the optimum temperature of the neutral cellulase is 57~63 DEG C, so polish temperature is consistent with dyeing temperature, while polishing process, dyestuff can abundant level dyeing, ensure even dyeing.
Description
Technical field
The present invention relates to biological enzyme formulation field, and in particular to a kind of new application of mesophilic protein, especially
It is related to a kind of application of mesophilic protein in the bath process of polishing and dyeing one.
Background technology
In traditional cotton fabrics polishing and dyeing technique, what polishing was separately carried out with dyeing, general technology flow
It is to use acidic cellulase earliest, pH value general control is 4.5~5.0 or so as shown in figure 1, in above-mentioned technique.With
The progress of the development of enzyme preparation, particularly neutral cellulase technology, it can effectively remove fabric table in neutral conditions
The filoplume in face, and the adsorption process for dyeing early stage is typically also to carry out in neutral conditions.
Recent years, as people are attempted glossing dyeing the pay attention to day by day of environmental protection, many colourists
It is combined into one, i.e. the bath process of polishing and dyeing one.Glossing and dyeing are closed two by the bath process of cotton fabrics polishing and dyeing one
For one, the consumption that greatly reduces water and the energy, the discharge for reducing sewage, shorten the process time, improve production efficiency.The technique one
Through releasing, just paid attention to by many printing and dyeing enterprises and numerous colourists.Many factories have been entered using the technique at present
Row production, its technological process are typically as shown in Figure 2.
Compared with polishing and dyeing two-step method, the bath process of polishing and dyeing one can save the consumption and sewage discharge of 3/4 water, can
To reduce by 1/3 energy expenditure, it can save for 1/3 time.
In the process, the enzyme used is usually neutral cellulase, but the optimum temperature of most of enzyme is at present
50~55 DEG C, therefore polishing process temperature (and the upper dyer's sequence of dyeing starting) is typically chosen in 50~55 DEG C.General cotton-padded clothes
The dyeing temperature of dyestuff is 60 DEG C, therefore, adds alkali to carry out reaction color fixing temperature selection and is carried out under the conditions of 60 DEG C.
In the process, in order to promote to contaminate, substantial amounts of salt (sodium chloride or sodium sulphate) need to be typically added, even if dye light color, salt
Concentration be typically also not less than 15g/L.Dyeing is deeper, it is necessary to which the salt amount added is bigger, and salinity is higher.When contaminating black, salt
Concentration even can reach 90g/L.
Problems with actual production be present in above-mentioned technique：
1. the deactivation of salt centering cellulase.In above-mentioned concentration (15~90g/L) and temperature (50~55 DEG C) bar
Under part, salt has obvious deactivation to cellulase, and concentration is higher, and deactivation is bigger.Therefore, to ensure polishing effect,
Need to increase the dosage of starting neutral cellulase or add neutral cellulase later.
2. the optimum temperature of cellulase is generally 50~55 DEG C, when temperature is higher than 60 DEG C, the vigor decline of enzyme is bright
It is aobvious.Therefore, polishing process from staining reaction process is carried out under different temperature conditionss.After 60 DEG C of heating, if protected
Warm time length, it is easy to cause larger strength damage, soaking time is shorter, and Dye Adsorption is uneven, adds alkali to carry out fixation
Reaction, it is easy to cause to dye dyeing defect.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of new application of mesophilic protein, i.e., is contaminated in polishing
Application in the bath process of color one.The mesophilic protein is the neutral cellulase with halophagia energy, high concentration
The activity of salt centering cellulase is obviously improved effect, and the optimum temperature of the neutral cellulase is 57~63
DEG C, such polish temperature is consistent with dyeing temperature, while polishing process, dyestuff can abundant level dyeing, ensure even dyeing.
Mesophilic protein of the present invention is applicant in identified patent applications number on July 5th, 2013
For 201310280267.6, the invention of entitled " a kind of mesophilic protein and its preparation method and application " is special
Profit application.
In order to solve the above technical problems, the present invention adopts the following technical scheme that：
In one aspect of the invention, there is provided a kind of mesophilic protein answering in the bath process of polishing and dyeing one
With the mesophilic protein is made as follows：
1) synthesis of mesophilic protein gene, the sequence of the mesophilic protein gene of synthesis is such as
Shown in SEQ ID NO.2, the gene code of synthesis more conforms to the gene code of trichoderma reesei high efficient expression；
2) structure of mesophilic protein expression plasmid；
3) conversion of trichoderma reesei and the screening of mesophilic protein superior strain, obtained chaetomium thermophilum are fine
The amino acid sequence of plain enzyme is tieed up as shown in SEQ ID NO.3, it is SEQ ID to be secreted into extracellular mesophilic protein
The 22-314 amino acids sequences of sequence shown in NO.3；Step 3) is specially：First, with EcoRI degradation steps 2) structure
Plasmid forms plasmid fragments, and the plasmid fragments are transferred into trichoderma reesei RUT C-30 host cells using chemical conversion process；So
Cultivated afterwards in the PDA culture medium containing hygromycin B, then eugonic colony inoculation is put in PDA culture medium, pass through growth
And germination, spore is then transferred on fresh PDA culture medium, to isolate and purify the transformant for turning generation stabilization, by these transformants
Spore inoculating in trichoderma cellulase induction broth, after culture, sampling centrifugation, take supernatant to carry out SDS-PAGE electrophoresis point
Analysis；The pH value of the trichoderma cellulase induction broth is 4.8, and inducer is used as by the use of lactose.
As currently preferred technical scheme, the mesophilic protein is that the neutrality with halophagia energy is fine
Plain enzyme is tieed up, the salt in 10g/L~90g/L concentration ranges (is preferably NaCl, Na2SO4In one or more) it is fine to the neutrality
The activity for tieing up plain enzyme is obviously improved effect.
As currently preferred technical scheme, the mesophilic protein is most suitable in the bath of polishing and dyeing one
Operative temperature is 57~63 DEG C (being preferably 60 DEG C).
In another aspect of this invention, there is provided a kind of mesophilic protein answering in the bath process of polishing and dyeing one
With the mesophilic protein is made as follows：
1) synthesis of mesophilic protein gene, the sequence of the mesophilic protein gene of synthesis is such as
Shown in SEQ ID NO.4；
2) structure of mesophilic protein expression plasmid；
3) conversion of Pichia pastoris and the screening of mesophilic protein superior strain, obtained chaetomium thermophilum are fine
The amino acid sequence of plain enzyme is tieed up as shown in SEQ ID NO.5；Step 3) is specially：First, will be walked with Spe I restriction enzymes
After the expression plasmid of rapid 2) structure is cut, Pichia yeast is directly converted；Multiple white Pichia pastoris bacterium colonies, training are selected immediately
Support, after induction, thalline is separated with nutrient solution, nutrient solution passes through SDS-PAGE electrophoretic analysis.
As currently preferred technical scheme, the mesophilic protein is that the neutrality with halophagia energy is fine
Plain enzyme is tieed up, the salt in 10g/L~90g/L concentration ranges (is preferably NaCl, Na2SO4In one or more) it is fine to the neutrality
The activity for tieing up plain enzyme is obviously improved effect.
As currently preferred technical scheme, the mesophilic protein is most suitable in the bath of polishing and dyeing one
Operative temperature is 57~63 DEG C (being preferably 60 DEG C).
Compared with prior art, the beneficial effects of the present invention are：
1st, for salinity in the range of 10g/L~90g/L, centering cellulase has certain activation, can be lifted
The polishing effect of neutral cellulase.Therefore, the dosage of cellulase reduces, so as to reduce process costs.
2nd, the optimum temperature of neutral cellulase rises to 60 DEG C, consistent with cotton reactive dye dyeing temperature, can
To be polished the bath of dyeing one under the conditions of constant temperature (60 DEG C).Polishing is not only improved, is advantageous to dye again, ensures dyeing quality.
Brief description of the drawings
Fig. 1 is the process chart that traditional cotton fabrics polishing is separately carried out with dyeing.
Fig. 2 is the process chart of the existing bath process of polishing and dyeing one.
Fig. 3 is in the embodiment of the present invention 3 under the conditions of 60 DEG C, under the conditions of pH value 7.0, with 1.0g/L chaetomium thermophilum fibers
Plain enzyme polishes 60 minutes to fabric, the weight-loss ratio comparison diagram under the conditions of different sodium sulfate concentrations.
Fig. 4 is in the embodiment of the present invention 3 under the conditions of 60 DEG C, under the conditions of pH value 6.0, with 1.0g/L chaetomium thermophilum fibers
Plain enzyme polishes 60 minutes to fabric, the weight-loss ratio comparison diagram under the conditions of different sodium sulfate concentrations.
Fig. 5 is in the embodiment of the present invention 3 under the conditions of 50 DEG C, under the conditions of pH value 6.0, is collected with 1.0g/L in the markets big
The neutral cellulase of spore circle mould (Staphylotrichum coccosporum) the STCE1 gene codes of spore is to fabric polishing 60
Minute, the weight-loss ratio comparison diagram under the conditions of different sodium sulfate concentrations.
Fig. 6 is the analysis of Temperature Characteristics schematic diagram of mesophilic protein in the embodiment of the present invention 4.
Fig. 7 is the mould (Staphylotrichum of big spore circle spore that in the market is collected in the embodiment of the present invention 4
Coccosporum) the analysis of Temperature Characteristics schematic diagram of the neutral cellulase of STCE1 gene codes.
Fig. 8 is the process chart in the embodiment of the present invention 5.
Fig. 9 is the process chart in the embodiment of the present invention 6.
Figure 10 is the process chart in the embodiment of the present invention 7.
Figure 11 is the process chart in the embodiment of the present invention 8.
Embodiment
Following examples are only illustrative of the invention and is not intended to limit the scope of the invention.It is unreceipted specific in embodiment
The experimental method of condition, generally according to normal condition, such as Sambrook et al., molecular cloning:Laboratory manual (New
York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or built according to manufacturer
The condition of view.
Embodiment 1 use the mesophilic protein of trichoderma reesei expression preparation (referring to number of patent application for
| 0 | 5g/L | 10g/L | 20g/L | 40g/L | 60g/L | 80g/L | 90g/L | |
| Weight-loss ratio | 1.67% | 1.93% | 1.98% | 1.84% | 1.83% | 1.74% | 1.69% | 1.68% |
201310280267.6 application for a patent for invention disclosure CN103343111A specification embodiment 1).
Embodiment 2 use the mesophilic protein of Pichia anomala expression preparation (referring to number of patent application for
201310280267.6 application for a patent for invention disclosure CN103343111A specification embodiment 3).
The mesophilic protein of embodiment 3 has qualitative test (the different sodium sulfate concentration centerings of halophagia energy
Cellulase polishing effect influences)
1st, under the conditions of 60 DEG C, under the conditions of pH value 7.0, the mesophilic protein pair made from 1.0g/L embodiments 1
Fabric polishes 60 minutes, the weight-loss ratio situation under the conditions of different sodium sulfate concentrations, and being shown in Table 1 and Fig. 3, (weight-loss ratio is higher, polishing effect
Fruit is relatively better).
Table 1
| 0 | 5g/L | 10g/L | 20g/L | 40g/L | 60g/L | 80g/L | 90g/L | |
| Weight-loss ratio | 1.34% | 1.57% | 1.76% | 1.51% | 1.52% | 1.43% | 1.36% | 1.35% |
2nd, under the conditions of 60 DEG C, under the conditions of pH value 6.0, the mesophilic protein pair made from 1.0g/L embodiments 2
Fabric polishes 60 minutes, the weight-loss ratio situation under the conditions of different sodium sulfate concentrations, and being shown in Table 2 and Fig. 4, (weight-loss ratio is higher, polishing effect
Fruit is relatively better).
Table 2
| 0 | 5g/L | 10g/L | 20g/L | 40g/L | 60g/L | 80g/L | 90g/L | |
| Weight-loss ratio | 1.67% | 1.93% | 1.98% | 1.84% | 1.83% | 1.74% | 1.69% | 1.68% |
It can be seen that the mesophilic protein as made from embodiment 1,2 has halophagia energy, i.e., the presence of salt is to fiber
Plain enzyme has certain activation, can lift the polishing action effect of cellulase.
3rd, under the conditions of 50 DEG C, under the conditions of pH value 6.0, the big spore circle spore collected with 1.0g/L in the markets is mould
The neutral cellulase of (Staphylotrichum coccosporum) STCE1 gene codes polishes 60 minutes to fabric, not
With weight-loss ratio situation under the conditions of sodium sulfate concentration, it is shown in Table 3 and Fig. 5 (weight-loss ratio is higher, and polishing effect is relatively better).
Table 3
| 0 | 5g/L | 10g/L | 20g/L | 40g/L | 60g/L | 80g/L | 90g/L | |
| Weight-loss ratio | 1.67% | 1.60% | 1.48% | 1.32% | 1.27% | 1.17% | 1.10% | 1.06% |
It can be seen that big spore circle mould (Staphylotrichum coccosporum) the STCE1 genes of spore that salt is collected in the market
The neutral cellulase of coding has obvious inhibitory action, and salinity is higher, and inhibitory action is stronger.
The FPA vigor analysis of Temperature Characteristics of the mesophilic protein of embodiment 4
Cellulase FPA vigor (filter paper vigor) method of testing：
Preheat in 1.5ml buffer solution+0.05g Xinhua's filter paper (1*6cm) → thermostat water bath and added in 5min → test specimens
Add 1.5mlDNS solution terminating reactions in every test tube after accurate response 30min in 1ml dilution enzyme liquid → thermostat water baths
1ml dilution enzyme liquid → all reaction tubes are added in → blank sample test tube 5min is boiled in 100 DEG C of water-baths, it is fixed after cooling
Hold to 5ml → 540nm, 10mm cuvettes measure light absorption value, enzyme activity is calculated according to light absorption value and standard curve.
Cellulase FPA vigor analysis of Temperature Characteristics methods：
By above-mentioned test cellulase FPA vigor methods, the accurate response 30min under conditions of different temperatures, test out
The FPA vigor of cellulase at different temperatures, analysis cellulase activity vary with temperature situation.
Concrete outcome is shown in that (in the market is collected by Fig. 6 (the analysis of Temperature Characteristics schematic diagram of mesophilic protein) and Fig. 7
Big spore circle mould (Staphylotrichum coccosporum) the STCE1 gene codes of spore neutral cellulase temperature it is special
Property analysis schematic diagram).As shown in fig. 6, mesophilic protein optimum temperature of the present invention is 60 DEG C, most suitable action temperature
It is 57~63 DEG C to spend scope, and when temperature is 70 DEG C, enzyme activity is kept still more than 80%, is well suited for using in dyeing.
As shown in fig. 7, big spore circle mould (Staphylotrichum coccosporum) the STCE1 gene codes of spore that in the market is collected
The optimum temperature general range of neutral cellulase is 50~53 DEG C.When temperature is more than 60 DEG C, vigor drastically declines；Temperature
During more than 70 DEG C, vigor declines 50% or so, and this kind of cellulase is not suitable for using in dyeing.It can be seen that the present invention will be thermophilic
The optimum temperature of cupreum cellulase is improved to 57~63 DEG C, Ke Yi consistent with cotton reactive dye dyeing temperature
The bath of dyeing one is polished under the conditions of constant temperature (60 DEG C), polishing is not only improved, is advantageous to dye again, ensures dyeing quality.
Embodiment 5：Mesophilic protein in the bath process of polishing and dyeing one application (first it is enzyme-added, afterwards plus dyestuff,
Again plus salt technique)：
Cloth weight：180Kg fabrics：Contaminate colour of camel's hair bath raio to 20S pearls：1：10
Type：ECO-38-1T overflow dyeing machines (Hong Kong Fog's National Engineering Co., Ltd)
Technological process as shown in Figure 8 is dyed：Above-mentioned polishing and dyeing one is bathed, and fabric face filoplume removes bright during sampling
It is aobvious, contaminate color and require solid colour, dye than more uniform, every fastness index is qualified.
Embodiment 6：Application of the mesophilic protein in the bath process of polishing and dyeing one is (first plus salt, rear enzyme-added, again
Add dye technology)：
Cloth weight：711Kg fabrics：20S rib-loops contaminate navy bath raio：1：10
Type：ECO-38-3T overflow dyeing machines (Hong Kong Fog's National Engineering Co., Ltd)
Technological process as shown in Figure 9 is dyed：Above-mentioned polishing and dyeing one is bathed, and fabric face filoplume removes bright during sampling
It is aobvious, contaminate color and require solid colour, dye than more uniform, every fastness index is qualified.
Embodiment 7：Application of the mesophilic protein in the bath process of polishing and dyeing one：
Cloth weight：218Kg fabrics：The light blue bath raio of the two-sided Burans of 40S：1：12
Type：The pipe high temperature overflow dyeing machine of three skill two
Technological process as shown in Figure 10 is dyed：Above-mentioned polishing and dyeing one is bathed, and fabric face filoplume removes during sampling
Substantially, contaminate color and require solid colour, dye than more uniform, every fastness index is qualified.
Embodiment 8：Application of the mesophilic protein in the bath process of polishing and dyeing one：
Cloth weight：858Kg fabrics：26S plain weave Buran navy bath raioes：1：12
Type：LAN ' s dyeing machine LTO-6T overflow dyeing machines (Hong Kong Wei Xi dyeing and finishing machineries Co., Ltd)
Technological process as shown in Figure 11 is dyed：Above-mentioned polishing and dyeing one is bathed, and fabric face filoplume removes during sampling
Substantially, contaminate color and require solid colour, dye than more uniform, every fastness index is qualified.
Claims (6)
1. application of a kind of mesophilic protein in the bath process of polishing and dyeing one, it is characterised in that be in salinity
10g/L~90g/L and temperature using the mesophilic protein and dyestuff are polished dye under the conditions of being 57~63 DEG C
Color one is bathed, and the mesophilic protein is made as follows：
1) synthesis of mesophilic protein gene, the sequence such as SEQ ID of the mesophilic protein gene of synthesis
Shown in NO.2, the gene code of synthesis more conforms to the gene code of trichoderma reesei high efficient expression；
2) structure of mesophilic protein expression plasmid；
3) conversion of trichoderma reesei and the screening of mesophilic protein superior strain, obtained Chaetomium thermophile fungin
For the amino acid sequence of enzyme as shown in SEQ ID NO.3, it is SEQ ID NO.3 to be secreted into extracellular mesophilic protein
The 22-314 amino acids sequences of shown sequence；Step 3) is specially：First, with EcoRI degradation steps 2) structure plasmid
Plasmid fragments are formed, the plasmid fragments are transferred to by trichoderma reesei RUT C-30 host cells using chemical conversion process；Then exist
Cultivated in PDA culture medium containing hygromycin B, then eugonic colony inoculation is put in PDA culture medium, by growing and sending out
Bud, spore is then transferred on fresh PDA culture medium, to isolate and purify the transformant for turning generation stabilization, by the spore of these transformants
Son is seeded in trichoderma cellulase induction broth, after culture, sampling centrifugation, takes supernatant to carry out SDS-PAGE electrophoretic analysis；
The pH value of the trichoderma cellulase induction broth is 4.8, and inducer is used as by the use of lactose.
2. application as claimed in claim 1, it is characterised in that the salt is NaCl, Na2SO4In one or more.
3. application as claimed in claim 1, it is characterised in that the mesophilic protein is in the bath of polishing and dyeing one
Optimum temperature be 60 DEG C.
4. application of a kind of mesophilic protein in the bath process of polishing and dyeing one, it is characterised in that be in salinity
10g/L~90g/L and temperature using the mesophilic protein and dyestuff are polished dye under the conditions of being 57~63 DEG C
Color one is bathed, and the mesophilic protein is made as follows：
1) synthesis of mesophilic protein gene, the sequence such as SEQ ID of the mesophilic protein gene of synthesis
Shown in NO.4；
2) structure of mesophilic protein expression plasmid；
3) conversion of Pichia pastoris and the screening of mesophilic protein superior strain, obtained Chaetomium thermophile fungin
The amino acid sequence of enzyme is as shown in SEQ ID NO.5；Step 3) is specially：First, with Spe I restriction enzymes by step 2)
After the expression plasmid of structure is cut, Pichia yeast is directly converted；Multiple white Pichia pastoris bacterium colonies are selected immediately, are cultivated, are lured
After leading, thalline is separated with nutrient solution, nutrient solution passes through SDS-PAGE electrophoretic analysis.
5. application as claimed in claim 4, it is characterised in that the salt is NaCl, Na2SO4In one or more.
6. application as claimed in claim 4, it is characterised in that the mesophilic protein is in the bath of polishing and dyeing one
Optimum temperature be 60 DEG C.
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| EP3390625B1 (en) * | 2015-12-18 | 2023-09-06 | Danisco US Inc. | Polypeptides with endoglucanase activity and uses thereof |
| CN105624140A (en) * | 2016-03-29 | 2016-06-01 | 常州大学 | Method for improving storage stability of hydrogen peroxide |
| CN108753492B (en) * | 2018-07-04 | 2020-06-05 | 湖南尤特尔生化有限公司 | Application of chaetomium thermophilum cellulase in preparation of detergent |
| CN109778407B (en) * | 2019-02-18 | 2020-08-11 | 宁波荣昌祥服饰股份有限公司 | Antibacterial fabric and preparation method thereof |
| EP3715445A1 (en) * | 2019-03-29 | 2020-09-30 | Clariant Produkte (Deutschland) GmbH | Cellulolytic enzyme comprising laundry product |
| CN112127173A (en) * | 2020-10-12 | 2020-12-25 | 浙江美欣达纺织印染科技有限公司 | Neutral enzyme-based cloth dyeing and polishing one-bath method |
| US11987824B2 (en) * | 2020-12-22 | 2024-05-21 | Fornia Biosolutions, Inc. | Additional endoglucanase variants and methods |
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| CN103343111A (en) * | 2013-07-05 | 2013-10-09 | 山东尤特尔生物科技有限公司 | Thermophilic chaetomium cellulase, as well as preparation method and application thereof |
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