CN105462940B - A method of promote microorganism to produce pva-degrading enzyme - Google Patents
A method of promote microorganism to produce pva-degrading enzyme Download PDFInfo
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- CN105462940B CN105462940B CN201510961865.9A CN201510961865A CN105462940B CN 105462940 B CN105462940 B CN 105462940B CN 201510961865 A CN201510961865 A CN 201510961865A CN 105462940 B CN105462940 B CN 105462940B
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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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Abstract
The invention discloses a kind of methods that promotion microorganism produces pva-degrading enzyme, belong to textile technology field.The present invention is by using addition small molecule polyol as inducer, significantly improve the PVA degradation enzyme activity of mixed bacterial crude enzyme produced by the liquid, effectively reduce the required crude enzyme liquid dosage in degrading polyvinyl alcohol slurry, the incubation time for preparing crude enzyme liquid, toxigenic capacity have been saved, industrial application is suitble to.The problem of flocculation caused by polyvinyl alcohol inducer inventive process avoids high polymerization degree and PVA degrading enzyme separation difficulty.
Description
Technical field
The present invention relates to a kind of methods that promotion microorganism produces pva-degrading enzyme, belong to textile technology field.
Background technique
Polyvinyl alcohol (PVA) is a kind of artificial synthesized water soluble polymer.Because it is with pH stabilization, compatibility
Good and emulsifying capacity waits by force good characteristics, while having preferable adhesiveness, film forming to natural fiber and general synthetic fibers
Property and wearability, are the main slurries in current polyester-cotton fabric warp yarn sizing.Using the fabric of PVA starching, in pretreatment of spinning
Desizing treatment is had to pass through in the process just can increase its water imbibition.Traditional desizing method is the hot water using 70~90 DEG C to cotton
Fabric is eluted, and the PVA under eluting is discharged with waste water.So not only water consume, energy consumption is high, but also be easy cotton fiber is caused to damage
Wound；More seriously since PVA chemical oxygen consumption (COC) is big, biodegradability is poor, causes serious pollution to water body.
Concern with people to textile industry clean manufacturing, in desizing realize to PVA biodegrade by
The attention of researcher.PVA degradation enzyme desizing mild condition, can not only greatly reduce the discharge of PVA waste water, and can be avoided
The damage of cotton fiber caused by traditional desizing processes high temperature and oxidation.
It realizes and the biodegradable key of PVA is to screen the microorganism for capableing of efficient degradation PVA, but PVA drops
It is upper few in type and distribution to solve bacterium, cultivation cycle is long, and enzyme activity is low, and extraction is not easy, these, which all become, hinders PVA degrading enzyme to exist
An important factor for being applied in actual production.
At present produce pva-degrading enzyme mostly using polyvinyl alcohol of high degree of polymerization, glucose as carbon source and inducer, and mostly with
Based on single bacterium.And when using polyvinyl alcohol of high degree of polymerization as inducer, there are polyvinyl alcohol of high degree of polymerization easily to flocculate, meeting
The shortcomings that influencing microorganism to the degradation of polyvinyl alcohol and then yield of enzyme is caused to reduce.
Summary of the invention
To solve the above-mentioned problems, the present invention provides a kind of method that raising microorganism produces pva-degrading enzyme, it is
Using small molecule polyol as inducer, it is promoted to generate endocellular enzyme.
In one embodiment of the invention, the small molecule polyol includes any of the following or a variety of mixed
It closes: propylene glycol, 1,3 butylene glycol, 1,2,4- butantriol.
In one embodiment of the invention, the small molecule polyol is the 1,3-BDO of 4~6g/L.
In one embodiment of the invention, the inducer additive amount is 2~6g/L.
In one embodiment of the invention, the microorganism is mixed bacterial, mainly includes following several Pseudomonas:
Stenotrophomonas sp.、Pseudomonas sp.、Sphingopyxis sp.、Ochrobactrum sp.、
Shinella sp.、Castellaniella sp.、Microbacterium sp.。
In one embodiment of the invention, the mixed bacterial, in the weaving plant aeration tank polluted by PVA
Sludge.
In one embodiment of the invention, the Stenotrophomonas sp. in the mixed bacterial can be with
Descend any one: CGMCC No 6393, CGMCC No 9046；Pseudomonas sp. can be it is following any one:
CGMCC No10601, CGMCC No 6446；Sphingopyxis sp. can be it is following any one: CGMCC No
10900, CGMCC No 9098；Ochrobactrum sp. can be it is following any one: CGMCC No 10564, CGMCC
No 10477；Shinella sp. can be it is following any one: CGMCC No 6838；Castellaniella sp. can be with
Be it is following any one: CGMCC No 10715, CGMCC No 10720；Microbacterium sp. can be following any
It is a kind of: CGMCC No6777, CGMCC No 10251.
In one embodiment of the invention, Bosea sp., Delftia can also be contained in the mixed bacterial
sp.、Phenylobacterium sp.、Devosia sp.、Rhizobium sp.、Pseudoxanthobacter sp.、
The presence of the strains such as Leucobacter sp., these strains will not have an adverse effect in PVA degradation to crude enzyme produced by the liquid.
In one embodiment of the invention, the method: to contain small molecule polyol as the meat soup of inducer
Culture medium cultivates mixed bacterial, it is promoted to generate endocellular enzyme.
In one embodiment of the invention, the method, be by mixed bacterial (by bacterium mass ratio,
Stenotrophomonas sp:Pseudomonas sp.:Sphingopyxis sp.:Ochrobactrum sp.:
Shinella sp.:Castellaniella sp:Microbacterium sp. is about 5817:1568:24:39:36:7:31；
Or also containing other a small amount of bacterial strains, Microbacterium sp., Bosea sp:Delftia sp.,
Phenylobacterium sp., Devosia sp:Rhizobium sp, Pseudoxanthobacter sp.,
Leucobacter sp.) in culture preparation seed liquor on broth bouillon, it is more to be then inoculated in the small molecule containing 4~6g/L
It in the broth bouillon of first alcohol, ferments 3 days in 30 DEG C, then collects cell, broken wall, centrifuging and taking supernatant is crude enzyme liquid.
In one embodiment of the invention, the method also includes carrying out at broken wall to the cell after induction producing enzyme
It manages, centrifuging and taking supernatant is the crude enzyme liquid of produced endocellular enzyme after the completion of broken wall.It is (a kind of secondary containing PVA oxidizing ferment in the crude enzyme liquid
Alcohol oxidase), PVA hydrolase (a kind of β-diketone hydrolase).
In one embodiment of the invention, the broth bouillon contains 10.0~15.0g/L of peptone, beef
3.0~5.0g/L of powder, the small molecule polyol of 2~6g/L, sodium chloride 5.0~6.0g/L, pH 7.2 ± 0.2.
Beneficial effects of the present invention:
(1) the PVA drop of mixed bacterial crude enzyme produced by the liquid is significantly improved as inducer using addition small molecule polyol
Enzyme enzyme activity is solved, the required crude enzyme liquid dosage in degrading polyvinyl alcohol slurry is effectively reduced, has saved and prepared crude enzyme liquid
Incubation time, toxigenic capacity are suitble to industrial application.
(2) for the microculture of production pva-degrading enzyme, universal method is with the poly- of the degree of polymerization 1700 or so
Vinyl alcohol is carbon source and inducer, but the polyvinyl alcohol of high polymerization degree is degraded in the medium not exclusively, in enzyme extraction process
Undissolvable flocculate can be generated with zymoprotein coprecipitation, cause to cannot get PVA degradation enzyme product.The present invention is with broth cultivation
Feeding base adds small molecule polyol to cultivate as inducer microorganism, has not only avoided flocculated appearance, but also solves PVA drop
Solve the difficult problem of enzyme separation.
(3) contain a variety of associations such as PVA oxidizing ferment (secondary-alcohol oxidase), PVA hydrolase (β-diketone hydrolase) in crude enzyme liquid
With the enzyme system of degradation PVA, which can be used to the polyvinyl alcohol of effectively degradation high polymerization degree, and the crude enzyme liquid of acquisition is to PVA1799
Degradation enzyme activity reach 1.985U/mL.Glucose induction does not go out enzyme.
Specific embodiment
Enzyme activity determination method:
Enzyme activity test is carried out by substrate of the PVA1799 of 1g/L.
Enzyme activity determination: the PVA solution of 1mL crude enzyme liquid and 1mL 1g/L is added in 25mL colorimetric cylinder, by this mixed system
6h is reacted in 30 DEG C of shaking bath pot, respectively the absorbance of PVA content corresponding to the mixed liquor of measurement reaction front and back.Every point
Clock absorbance decline 0.001 is defined as an enzyme-activity unit.
Ultrasonic cell disintegration:
The thallus that culture is completed is centrifuged (10000r/min), removes supernatant, precipitating is washed with deionized, sufficiently after oscillation
It is centrifuged (10000r/min), removes supernatant, repeat above operation repeatedly, sediment is suspended in phosphate buffer solution (pH
7.2,20mmol/L), mass ratio is about 1:5, with ultrasonication (4 DEG C, 400W, work 3s, gap 2s, 20min) and is centrifuged receipts
Collect supernatant, as produced endocellular enzyme crude enzyme liquid.
Embodiment 1: mixed bacterial produces crude enzyme liquid (not inducing)
By mixed bacterial in culture preparation seed liquor on broth bouillon, then it is inoculated in broth bouillon, in 30
DEG C fermentation 3 days；Then cell, broken wall are collected, centrifuging and taking supernatant is crude enzyme liquid.
The broth bouillon, containing 10.0~15.0g/L of peptone, 3.0~5.0g/L of powdered beef, sodium chloride 5.0~
6.0g/L, pH 7.2 ± 0.2.
Enzyme activity is measured after collecting breaking-wall cell, PVA degrading enzymatic activity is not detected.
Embodiment 2: addition 1,3 butylene glycol inducer induces mixed bacterial producing enzyme
By mixed bacterial in culture preparation seed liquor on broth bouillon, it is then inoculated in 1,3- containing 4~6g/L
In the broth bouillon of butanediol induction, ferment 3 days in 30 DEG C；Then cell, broken wall are collected, centrifuging and taking supernatant is thick enzyme
Liquid.
The broth bouillon contains peptone 10.0g/L, powdered beef 5.0g/L, sodium chloride 5.0g/L, 1,3-BDO
4-6g/L, pH 7.2 ± 0.2.
Enzyme activity in crude enzyme liquid is measured, as the result is shown up to 1.985U/mL.
Embodiment 3: addition propylene glycol inducer induces mixed bacterial producing enzyme
By mixed bacterial in culture preparation seed liquor on broth bouillon, it is then inoculated in the third two containing 2~5g/L
It in the broth bouillon of alcohol, ferments 3 days in 30 DEG C, then collects cell, broken wall, centrifuging and taking supernatant is crude enzyme liquid.
The broth bouillon contains peptone 15.0g/L, powdered beef 3.0g/L, sodium chloride 6.0g/L, propylene glycol 2-
5g/L, pH 7.2 ± 0.2.Enzyme activity in crude enzyme liquid is measured, as the result is shown up to 1.32U/mL.
Embodiment 4: addition 1,2,4-butanetriol inducer induces mixed bacterial producing enzyme
By mixed bacterial in culture preparation seed liquor on broth bouillon, it is then inoculated in 1,2,4- containing 2-4g/L
In the broth bouillon of butantriol, ferment 3 days in 30 DEG C；Then cell, broken wall are collected, centrifuging and taking supernatant is crude enzyme liquid.
The broth bouillon, containing peptone 12.0g/L, 3.0~5.0g/L of powdered beef, 5.0~6.0g/L of sodium chloride,
1,2,4-butanetriol 2-4g/L, pH 7.2 ± 0.2.Enzyme activity in crude enzyme liquid is measured, as the result is shown up to 1.364U/mL.
Reference examples:
(1) PVA1799 that the 1,3-BDO in embodiment 2 is changed into 3g/L can also induce producing enzyme, but enzyme activity is only
1.02U/mL, and the polyvinyl alcohol of high polymerization degree is degraded not exclusively in the medium, it can be with zymoprotein one in enzyme extraction process
It plays precipitating and generates undissolvable flocculate, cause to cannot get PVA degradation enzyme product.
(2) 1,3-BDO in embodiment 2 is replaced using glucose, there is no PVA degradation enzyme in obtained fermentation liquid
It is living.
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.
Claims (5)
1. a kind of method for improving microorganism and producing pva-degrading enzyme, which is characterized in that the method is added with small molecule
Polyalcohol cultivates mixed bacterial as the broth bouillon of inducer, promote its generate film on and endocellular enzyme；It is described small
Molecular polylol includes any of the following or a variety of mixing: propylene glycol, 1,3 butylene glycol, 1,2,4- butantriol；It is described to lure
Leading agent additive amount is 4~6g/L.
2. the method according to claim 1, wherein the microorganism is mixed bacterial, including following several bacterium
Belong to:Stenotrophomonas sp.；Pseudomonas sp.；Sphingopyxis sp.；Ochrobactrum sp.；Shinella sp.；Castellaniella sp.；Microbacterium sp.。
3. the method according to claim 1, wherein the method: being by mixed bacterial on broth bouillon
Culture preparation seed liquor, is then inoculated in the broth bouillon of the small molecule polyol containing 4~6g/L, ferments in 30 DEG C
3 days.
4. the method according to claim 1, wherein the method also includes to the cell progress after induction producing enzyme
Broken wall treatment, centrifuging and taking supernatant is produced crude enzyme liquid after the completion of broken wall.
5. according to the method described in claim 3, it is characterized in that, the broth bouillon, containing peptone 10.0~
15.0g/L, 3.0~5.0g/L of powdered beef, 5.0~6.0g/L of sodium chloride, small molecule polyol 4-6g/L, pH 7.2 ± 0.2.
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| 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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| 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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| 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 |
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| 聚乙烯醇降解酶研究进展;张兴等;《中国生物工程杂志》;20030228;第23卷(第2期);第69-73页 |
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