CN105462940A - Method for promoting microorganisms to produce polyvinyl alcohol degrading enzyme - Google Patents
Method for promoting microorganisms to produce polyvinyl alcohol degrading enzyme Download PDFInfo
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- CN105462940A CN105462940A CN201510961865.9A CN201510961865A CN105462940A CN 105462940 A CN105462940 A CN 105462940A CN 201510961865 A CN201510961865 A CN 201510961865A CN 105462940 A CN105462940 A CN 105462940A
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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)
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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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
- C12Y101/0303—Polyvinyl-alcohol oxidase (1.1.3.30)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y307/00—Hydrolases acting on carbon-carbon bonds (3.7)
- C12Y307/01—Hydrolases acting on carbon-carbon bonds (3.7) in ketonic substances (3.7.1)
- C12Y307/01007—Beta-diketone hydrolase (3.7.1.7)
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- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06L—DRY-CLEANING, WASHING OR BLEACHING FIBRES, FILAMENTS, THREADS, YARNS, FABRICS, FEATHERS OR MADE-UP FIBROUS GOODS; BLEACHING LEATHER OR FURS
- D06L1/00—Dry-cleaning or washing fibres, filaments, threads, yarns, fabrics, feathers or made-up fibrous goods
- D06L1/12—Dry-cleaning or washing fibres, filaments, threads, yarns, fabrics, feathers or made-up fibrous goods using aqueous solvents
- D06L1/14—De-sizing
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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
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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
- 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
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- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
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- Zoology (AREA)
- Wood Science & Technology (AREA)
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- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Textile Engineering (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for promoting microorganisms to produce polyvinyl alcohol (PVA) degrading enzyme, belonging to the technical field of spinning. By taking micromolecular polyhydric alcohol as an inducer, the enzyme activity of PVA degrading enzyme of crude enzyme produced by a mixed flora is remarkably improved, the use amount of crude enzyme needed when PVA slurry is degraded is effectively reduced, culture time and culture cost for preparing the crude enzyme are saved and lowered, and the method is suitable for being applied to industry. The method avoids the problems of flocculation caused by a high-polymerization-degree PVA inducer and PVA degrading enzyme separation difficulty.
Description
Technical field
The present invention relates to a kind of method promoting production by biological pva-degrading enzyme, belong to textile technology field.
Background technology
Polyvinyl alcohol (PVA) is a kind of high molecular weight water soluble polymer of synthetic.The good characteristic such as have that pH is stable, compatibility is good because of it and emulsifying capacity is strong, have good adhesivity, film-forming properties and wear resistance to natural fiber and general synthon, be the main slurry in current polyester-cotton fabric warp sizing simultaneously.Adopt the fabric of PVA starching, in pretreatment of spinning process, have to pass through destarch process could increase its water-absorbent.Traditional desizing method is that the hot water of use 70 ~ 90 DEG C carries out wash-out to cotton fabric, and the PVA under wash-out discharges with waste water.So not only water consumption, energy consumption greatly, and easily cause damage to cotton fibre; More seriously because PVA chemical oxygen demand is large, biodegradability is poor, causes serious pollution to water body.
Along with people are to the concern of textile industry cleaner production, in desizing, realize the attention biological degradation of PVA being subject to investigator.PVA degrading enzyme destarch mild condition, can not only greatly reduce the discharge of PVA waste water, and can avoid the damage of the cotton fibre that high mild oxidation causes in traditional desizing processes.
Realizing being to screen to the biodegradable key of PVA can the microorganism of efficient degradation PVA, but PVA degradation bacteria is few in kind and distribution, and culture cycle is long, and enzyme is lived low, extract not easily, these all become the important factor hindering PVA degrading enzyme to apply in actual production.
Current product pva-degrading enzyme is carbon source and inductor mainly with polyvinyl alcohol of high degree of polymerization, glucose, and is main mainly with single bacterium.And when adopting polyvinyl alcohol of high degree of polymerization as inductor, there is polyvinyl alcohol of high degree of polymerization and very easily flocculate, microorganism can be affected to the degraded of polyvinyl alcohol and then the shortcoming that causes yield of enzyme to reduce.
Summary of the invention
In order to solve the problem, the invention provides a kind of method improving production by biological pva-degrading enzyme, is using small molecule polyol as inductor, impels it to produce intracellular enzyme.
In one embodiment of the invention, described small molecule polyol comprises following any one or multiple mixing: propylene glycol, 1,3 butylene glycol, BT.
In one embodiment of the invention, described small molecule polyol is the 1,3 butylene glycol of 4 ~ 6g/L.
In one embodiment of the invention, described inductor addition is 2 ~ 6g/L.
In one embodiment of the invention, described microorganism is mixed bacterial, mainly comprises following several Pseudomonas: Stenotrophomonassp., Pseudomonassp., Sphingopyxissp., Ochrobactrumsp., Shinellasp., Castellaniellasp., Microbacteriumsp..
In one embodiment of the invention, described mixed bacterial, from the mud in the textile mills aeration tank of polluting by PVA.
In one embodiment of the invention, the Stenotrophomonassp. in described mixed bacterial can be following any one: CGMCCNo6393, CGMCCNo9046; Pseudomonassp. can be following any one: CGMCCNo10601, CGMCCNo6446; Sphingopyxissp. can be following any one: CGMCCNo10900, CGMCCNo9098; Ochrobactrumsp. can be following any one: CGMCCNo10564, CGMCCNo10477; Shinellasp. can be following any one: CGMCCNo6838; Castellaniellasp. can be following any one: CGMCCNo10715, CGMCCNo10720; Microbacteriumsp. can be following any one: CGMCCNo6777, CGMCCNo10251.
In one embodiment of the invention, can also containing bacterial classifications such as Boseasp., Delftiasp., Phenylobacteriumsp., Devosiasp., Rhizobiumsp., Pseudoxanthobactersp., Leucobactersp. in described mixed bacterial, the existence of these bacterial classifications can not have a negative impact in PVA degraded to produced crude enzyme liquid.
In one embodiment of the invention, described method: to cultivate mixed bacterial containing the broth culture of small molecule polyol as inductor, impels it to produce intracellular enzyme.
In one embodiment of the invention, described method, that (by bacterium mass ratio, Stenotrophomonassp:Pseudomonassp.:Sphingopyxissp.:Ochrob actrumsp.:Shinellasp.:Castellaniellasp:Microbacteriumsp. is about 5817:1568:24:39:36:7:31 by mixed bacterial; Or also containing other bacterial strains a small amount of, Microbacteriumsp., Boseasp:Delftiasp., Phenylobacteriumsp., Devosiasp:Rhizobiumsp, Pseudoxanthobactersp., Leucobactersp.) on broth culture, cultivate preparation seed liquor, then be inoculated in the broth culture of the small molecule polyol containing 4 ~ 6g/L, in 30 DEG C of fermentations 3 days, then collecting cell, broken wall, centrifuging and taking supernatant liquor is crude enzyme liquid.
In one embodiment of the invention, described method also comprises the cell after producing enzyme to induction and carries out broken wall treatment, and after broken wall completes, centrifuging and taking supernatant is the crude enzyme liquid of produced intracellular enzyme.Containing PVA oxydase (a kind of secondary-alcohol oxidase), PVA lytic enzyme (a kind of β-diketone lytic enzyme) in this crude enzyme liquid.
In one embodiment of the invention, described broth culture, the small molecule polyol containing peptone 10.0 ~ 15.0g/L, beef powder 3.0 ~ 5.0g/L, 2 ~ 6g/L, sodium-chlor 5.0 ~ 6.0g/L, pH7.2 ± 0.2.
Beneficial effect of the present invention:
(1) adopt interpolation small molecule polyol as inductor, significantly improve mixed bacterial produce the PVA degradation enzyme activity of crude enzyme liquid, effectively reduce the crude enzyme liquid consumption required when degrading polyvinyl alcohol slurry, save the incubation time preparing crude enzyme liquid, toxigenic capacity, be applicable to industrial application.
(2) for the microorganism culturing of producing pva-degrading enzyme, general method is for carbon source and inductor with the polyvinyl alcohol of the polymerization degree about 1700, but the polyvinyl alcohol of high-polymerization degree is degraded not exclusively in the medium, in enzyme extraction process, meeting and zymoprotein coprecipitation produce undissolvable throw out, cause can not get PVA degrading enzyme product.The present invention adds small molecule polyol using broth culture and cultivates microorganism as inductor, has both avoided the appearance of flocculation, solves again the problem of PVA degrading enzyme separation difficulty.
(3) the enzyme system containing the multiple Synergistic degradation PVA such as PVA oxydase (secondary-alcohol oxidase), PVA lytic enzyme (β-diketone lytic enzyme) in crude enzyme liquid, this enzyme liquid can be used to effectively to degrade the polyvinyl alcohol of high-polymerization degree, and the degradation enzyme activity of crude enzyme liquid to PVA1799 of acquisition reaches 1.985U/mL.Glucose induction does not go out enzyme.
Embodiment
Enzyme activity determination method:
With the PVA1799 of 1g/L for substrate carries out enzyme test alive.
Enzyme activity determination: the PVA solution adding 1mL crude enzyme liquid and 1mL1g/L in 25mL colorimetric cylinder, reacts this mixed system to 6h in the shaking bath pot of 30 DEG C, the absorbancy of the PVA content respectively before and after assaying reaction corresponding to mixed solution.Per minute absorbancy decline 0.001 is defined as a Ge Meihuo unit.
Ultrasonic cell disintegration:
The thalline cultivated centrifugal (10000r/min), removes supernatant, precipitates with deionized water wash, centrifugal after abundant vibration (10000r/min), removes supernatant, repeats above operation repeatedly, throw out is suspended in phosphate buffer soln (pH7.2,20mmol/L), mass ratio is about 1:5, with ultrasonication (4 DEG C, 400W, work 3s, gap 2s, 20min) and collected by centrifugation supernatant liquor, produced intracellular enzyme crude enzyme liquid is.
Embodiment 1: mixed bacterial produces crude enzyme liquid (not inducing)
Mixed bacterial is cultivated on broth culture preparation seed liquor, and then be inoculated in broth culture, in 30 DEG C of fermentations 3 days; Then collecting cell, broken wall, centrifuging and taking supernatant liquor is crude enzyme liquid.
Described broth culture, containing peptone 10.0 ~ 15.0g/L, beef powder 3.0 ~ 5.0g/L, sodium-chlor 5.0 ~ 6.0g/L, pH7.2 ± 0.2.
Measure enzyme after collecting cell broken wall to live, do not detect PVA degrading enzymatic activity.
Embodiment 2: add 1,3 butylene glycol inductor induction mixed bacterial and produce enzyme
Mixed bacterial is cultivated on broth culture preparation seed liquor, and then be inoculated in broth culture that the 1,3 butylene glycol containing 4 ~ 6g/L induce, ferment 3 days in 30 DEG C; Then collecting cell, broken wall, centrifuging and taking supernatant liquor is crude enzyme liquid.
Described broth culture, containing peptone 10.0g/L, beef powder 5.0g/L, sodium-chlor 5.0g/L, 1,3 butylene glycol 4-6g/L, pH7.2 ± 0.2.
Measure enzyme in crude enzyme liquid to live, result display can reach 1.985U/mL.
Embodiment 3: add propylene glycol inductor induction mixed bacterial and produce enzyme
Mixed bacterial is cultivated on broth culture preparation seed liquor, and then be inoculated in the broth culture of the propylene glycol containing 2 ~ 5g/L, in 30 DEG C of fermentations 3 days, then collecting cell, broken wall, centrifuging and taking supernatant liquor was crude enzyme liquid.
Described broth culture, containing peptone 15.0g/L, beef powder 3.0g/L, sodium-chlor 6.0g/L, propylene glycol 2-5g/L, pH7.2 ± 0.2.Measure enzyme in crude enzyme liquid to live, result display can reach 1.32U/mL.
Embodiment 4: add BT inductor induction mixed bacterial and produce enzyme
Mixed bacterial is cultivated on broth culture preparation seed liquor, and then be inoculated in the broth culture of the BT containing 2-4g/L, in 30 DEG C of fermentations 3 days; Then collecting cell, broken wall, centrifuging and taking supernatant liquor is crude enzyme liquid.
Described broth culture, containing peptone 12.0g/L, beef powder 3.0 ~ 5.0g/L, sodium-chlor 5.0 ~ 6.0g/L, BT 2-4g/L, pH7.2 ± 0.2.Measure enzyme in crude enzyme liquid to live, result display can reach 1.364U/mL.
Reference examples:
(1) by 1 in embodiment 2,3-butyleneglycol changes the PVA1799 of 3g/L into, also product enzyme can be induced, the only 1.02U/mL but enzyme is lived, and the polyvinyl alcohol of high-polymerization degree is degraded not exclusively in the medium, in enzyme extraction process, meeting and zymoprotein coprecipitation produce undissolvable throw out, cause can not get PVA degrading enzyme product.
(2) use the 1,3 butylene glycol in glucose replacement embodiment 2, in the fermented liquid obtained, there is no PVA degradation enzyme activity.
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.
Claims (10)
1. improve a method for production by biological pva-degrading enzyme, it is characterized in that, described method is using small molecule polyol as inductor, impels it to produce intracellular enzyme.
2. method according to claim 1, is characterized in that, described small molecule polyol comprises following any one or multiple mixing: propylene glycol, 1,3 butylene glycol, BT.
3. method according to claim 1, is characterized in that, described inductor addition is 4 ~ 6g/L.
4. method according to claim 1, is characterized in that, described microorganism is mixed bacterial, mainly comprises following several Pseudomonas: Stenotrophomonassp.; Pseudomonassp.; Sphingopyxissp.; Ochrobactrumsp.; Shinellasp.; Castellaniellasp.; Microbacteriumsp..
5. method according to claim 1, is characterized in that, described method is added with small molecule polyol to cultivate mixed bacterial as the broth culture of inductor, impels it to produce on film and intracellular enzyme.
6. method according to claim 1, is characterized in that, described method: be mixed bacterial is cultivated on broth culture preparation seed liquor, and then be inoculated in the broth culture of the small molecule polyol containing 4 ~ 6g/L, in 30 DEG C of fermentations 3 days.
7. method according to claim 1, is characterized in that, described method also comprises the cell after producing enzyme to induction and carries out broken wall treatment, and after broken wall completes, centrifuging and taking supernatant is produced crude enzyme liquid.
8. the method according to claim 5 or 6, is characterized in that, described broth culture, containing peptone 10.0 ~ 15.0g/L, beef powder 3.0 ~ 5.0g/L, sodium-chlor 5.0 ~ 6.0g/L, small molecule polyol 4-6g/L, and pH7.2 ± 0.2.
9. by the pva-degrading enzyme that the arbitrary described method of claim 1-7 obtains.
10. pva-degrading enzyme described in claim 9 is in the application of field of textiles.
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| CN105462940B CN105462940B (en) | 2019-01-04 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109553656A (en) * | 2018-10-31 | 2019-04-02 | 上海健康医学院 | Antimicrobial compound and preparation method thereof, pharmaceutical composition and purposes |
| CN109593667A (en) * | 2017-09-30 | 2019-04-09 | 天津科技大学 | The identification of Cr (VI) reducing bacteria and its cultural method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101724609A (en) * | 2009-12-18 | 2010-06-09 | 江南大学 | Method for increasing yield of PVA-degrading enzyme produced by fermentation method |
| CN101850167A (en) * | 2010-05-25 | 2010-10-06 | 江南大学 | Fermentation method for improving PVA degradation speed |
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- 2015-12-18 CN CN201510961865.9A patent/CN105462940B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101724609A (en) * | 2009-12-18 | 2010-06-09 | 江南大学 | Method for increasing yield of PVA-degrading enzyme produced by fermentation method |
| CN101850167A (en) * | 2010-05-25 | 2010-10-06 | 江南大学 | Fermentation method for improving PVA degradation speed |
Non-Patent Citations (1)
| Title |
|---|
| 张兴等: "聚乙烯醇降解酶研究进展", 《中国生物工程杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109593667A (en) * | 2017-09-30 | 2019-04-09 | 天津科技大学 | The identification of Cr (VI) reducing bacteria and its cultural method |
| CN109553656A (en) * | 2018-10-31 | 2019-04-02 | 上海健康医学院 | Antimicrobial compound and preparation method thereof, pharmaceutical composition and purposes |
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| CN105462940B (en) | 2019-01-04 |
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