CN105621630B - A kind of decoloration 'beta '-mannase and application thereof - Google Patents
A kind of decoloration 'beta '-mannase and application thereof Download PDFInfo
- Publication number
- CN105621630B CN105621630B CN201610031200.2A CN201610031200A CN105621630B CN 105621630 B CN105621630 B CN 105621630B CN 201610031200 A CN201610031200 A CN 201610031200A CN 105621630 B CN105621630 B CN 105621630B
- Authority
- CN
- China
- Prior art keywords
- beta
- mannase
- dyestuff
- enzyme
- bacillus licheniformis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
-
- 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)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/2488—Mannanases
- C12N9/2491—Beta-mannosidase (3.2.1.25), i.e. mannanase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01025—Beta-mannosidase (3.2.1.25), i.e. mannanase
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/308—Dyes; Colorants; Fluorescent agents
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Hydrology & Water Resources (AREA)
- Environmental & Geological Engineering (AREA)
- Water Supply & Treatment (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The present invention relates to a kind of 'beta '-mannases to be used for dye decolored purposes, and the dyestuff is azo dyes, triarylmethane compound dyestuff, wherein the azo dyes are Congo red, methyl orange, titan yellow and eriochrome black T；The triarylmethane compound dyestuff is water-tough treatment, malachite green, crystal violet and phenol red.The 'beta '-mannase is by bacillus licheniformis, preferably by bacillus licheniformis HDGLJT-01 output, specifically by following methods preparation: the bacillus licheniformis HDGLJT-01 seed liquor being incubated overnight is forwarded in konjac glucomannan enzymatic production culture medium, 37 DEG C, 160rpm shaken cultivation 48h, take 40mL fermentation liquid, 4500r/min, 4 DEG C of centrifugation 20min, obtained supernatant, i.e. crude enzyme liquid；Crude enzyme liquid is purified using acetone precipitation, then is further purified using ion-exchange chromatography and gel permeation chromatography.The 'beta '-mannase good decolorizing effect of product of the invention than commercialization.
Description
Technical field
The present invention relates to a kind of purposes of mannase, and in particular to the decoloration purposes of the enzyme.
Background technique
Dyestuff is widely used in the industries such as food, pharmacy, cosmetics, weaving and leather, and dyestuff reported at present is more than 10,000
Kind, the dyestuff produced every year is more than 70,000,000 tons.In DYE PRODUCTION and use process, a large amount of waste water from dyestuff is directly discharged to
In environment, the transparency and dissolved oxygen amount of water body are not only influenced, but also the carcinogen such as benzidine one kind, therefore dyestuff can be generated
The processing of waste water has been to be concerned by more and more people.Traditional physics and chemical method is since its is at high cost, and energy consumption is high, and is easy
Toxic by-product is formed, is subject to certain restrictions in practical applications.In this case, there is an urgent need to a kind of cost and energy
Processing method low, without secondary pollution, environmental-friendly, easy to operate is consumed, sight is gone to bio-enzyme degradation method by researcher.The party
Method is that dye molecule is aoxidized or restored by the related enzyme systems of microorganism, cracks its chromophoric group or unsaturated bond, is led to
The biochemical reaction for crossing some column, is degraded to small molecule compound.In recent years discovery 'beta '-mannase is useless in processing dyestuff
Most potential biological enzyme in water, substrate specificity is extensive, can a variety of different structures of catalytic degradation organic matter.Since it has
Quite extensive Substratspezifitaet, will not generate noxious material, and can under temperate condition at room temperature and atmospheric ionization pollution degradation
Object, saves equipment and energy consumption, thus in terms of have very huge application potential.
Summary of the invention
Present invention relates particularly to a kind of 'beta '-mannases to be used for dye decolored purposes.
Further, wherein the dyestuff is azo dyes, triarylmethane compound dyestuff.
Further, wherein the azo dyes are Congo red, methyl orange, titan yellow, eriochrome black T；The triaryl methane
Class dyestuff is water-tough treatment, malachite green, crystal violet or phenol red.
Further, the 'beta '-mannase is by bacillus licheniformis, preferably by bacillus licheniformis
HDGLJT-01 output.
Further, the 'beta '-mannase is prepared by following methods:
The bacillus licheniformis HDGLJT-01 seed liquor being incubated overnight is forwarded in konjac glucomannan enzymatic production culture medium,
37 DEG C, 160rpm shaken cultivation 48h, take 40mL fermentation liquid, 4500r/min, 4 DEG C of centrifugation 20min, obtained supernatant, i.e., slightly
Enzyme solution；Crude enzyme liquid is purified using acetone precipitation, then is further purified using ion-exchange chromatography and gel permeation chromatography.
Further, ion-exchange chromatography and gel permeation chromatography refer to: enzyme solution will be concentrated, the DEAE- balanced is added
In sepharose FF ion exchange column, ladder is carried out with the Tris-HCl that the NaCl of 0.1mol/L and 0.005mol/L, pH are 7.0
Degree elution, collects eluent, detects enzyme activity and protein content；The enzyme solution of 5mL ion-exchange purification is added to have balanced
In sepadexG-75 pillar, use pure water as eluent, elution flow rate 0.5mol/min collects eluent.
It is produced using bacillus licheniformis (Bacillus licheniformis) HDGLJT-01 for possessing independent intellectual property right
Raw 'beta '-mannase carries out decolorization to the dyestuff of different structure, is applied to dye decolored process field for the enzyme and establishes
Theory and technology basis (HDGLJT-01 is disclosed in patent CN102191235A before excessively, and has carried out preservation,
It is preserved in China typical culture collection center, deposit number CCTCC M209288).
Detailed description of the invention
Fig. 1 is the SDS-PAGE electrophoresis of 'beta '-mannase.
Fig. 2 is the optimum temperature of 'beta '-mannase.
Fig. 3 is influence of the temperature to beta-mannase enzyme stability.
Fig. 4 is the optimal pH of 'beta '-mannase.
Fig. 5 is influence of the pH to beta-mannase enzyme stability.
Fig. 6 is decolorizing effect of the 'beta '-mannase to dyestuff.
Fig. 7 is purifying and the 'beta '-mannase being commercialized to dye decolored comparison.
Specific embodiment
The acquisition of 1. 'beta '-mannase of embodiment
The B.licheniformis HDGLJT-01 seed liquor being incubated overnight is forwarded to konjac glucomannan enzymatic production culture medium
In, 37 DEG C, 160rpm shaken cultivation 48h take 40mL fermentation liquid, 4500r/min, 4 DEG C of centrifugation 20min, obtained supernatant, i.e.,
Crude enzyme liquid.Crude enzyme liquid is purified using acetone precipitation, then is further purified using ion-exchange chromatography and gel permeation chromatography.Tool
Body will be concentrated enzyme solution and be added in the DEAE-sepharose FF ion exchange column balanced, with the NaCl of 0.1mol/L with
The Tris-HCl that 0.005mol/L, pH are 7.0 carries out gradient elution, collects eluent, detects enzyme activity and protein content.By 5mL
The enzyme solution of ion-exchange purification is added in the sepadexG-75 pillar to have balanced, and uses pure water as eluent, elution flow rate is
0.5mol/min collects eluent, detects enzyme activity and protein content.Purity of protein and molecular size range are detected using SDS-PAGE
(Fig. 1).
The analysis of 2. 'beta '-mannase some properties of embodiment
Activity analysis is carried out to 'beta '-mannase of the invention, optimum temperature and thermal stability and enzyme including enzyme are most
Suitable pH and pH stability.
(1) optimum temperature and thermal stability of 'beta '-mannase
It takes the Tris-HCl buffer (0.1mol/L, pH 8.0) of 2mL to mix with the pure enzyme solution of 0.5mL, keeps the temperature 30min
Afterwards, enzyme activity is measured immediately, and as compareing, (this enzyme activity is protoenzyme to the pure enzyme enzyme activity being dissolved in Tris-HCl buffer using at 4 DEG C
Living is 100%), to calculate the enzyme activity retention rate under different temperatures, determine the optimum temperature of 'beta '-mannase.The result shows that (figure
2), which is 60 DEG C, illustrates that this enzyme is more heat-resisting.
The pure enzyme solution of the Tris-HCl buffer and 0.5mL that take 2mL respectively at 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C and
It is mixed at 80 DEG C, and measures enzyme activity in 6,12,24,48 and 72h, to be stored in the pure enzyme enzyme activity of Tris-HCl buffer at 4 DEG C
As control (this enzyme activity is original enzyme activity i.e. 100%), different temperatures and the enzyme activity retention rate under the time are calculated, determines the enzyme enzyme
Equilibrium temperature range living and stable time.The result shows that (Fig. 3), which handles 72h at 60 DEG C, and remaining enzyme activity exists
27.81% or more.
(2) optimal pH of 'beta '-mannase and pH stability
Prepare the citric acid-sodium citrate buffer solution that pH is respectively 4.0,5.0,5.5,6.0 and 6.5；7.0,7.5,8.0,
8.5 and 9.0 Tris-HCI buffer and pH is respectively 9.5,10.0 and 11.0 Glycine-NaOH buffer.In room temperature
Under, it takes the above-mentioned 3 kinds of buffers of 2mL to mix with the pure enzyme solution of 0.5mL and stands 10min (enzyme solution concentration is 0.19mg/mL after mixing), with
Highest enzyme activity measures the enzyme activity of the 'beta '-mannase under different pH as control (this enzyme activity is set as 100%).It calculates other
Opposite enzyme activity under pH value, determines optimum pH.The result shows that (Fig. 4), the optimal pH of the enzyme is 8.0.
The pH above-mentioned buffer of 2mL for being 6.5,7.5,8.0,8.5 and 9.0 is mixed (after mixing with the pure enzyme solution of 0.5mL respectively
Enzyme solution concentration is 0.21mg/mL), it is placed at room temperature for 0,40,80,120,160 and 200min, using highest enzyme activity as control (this enzyme
It is living to be set as 100%), calculating the opposite enzyme activity under different time and pH, it determines the stability range of enzyme and stablizes the time.The result shows that
(Fig. 5), when pH is 8.0, the opposite enzyme activity of preceding 160min is held essentially constant；After 160min, enzyme activity declines, and opposite enzyme activity is
56.35%, enzyme activity 273.28U/mL.It can be seen that the enzyme stability is stronger when pH is 8.0.
The decolorizing effect of 3. 'beta '-mannase of embodiment
Dyestuff used in 1 Decolorant Test of table
The dyestuff (table 1) for choosing 8 kinds of different structures, detects the decoloring ability of 'beta '-mannase.A certain amount of β-is sweet
Reveal dextranase and dyestuff mixing, 37 DEG C, 160rpm oscillating reactions carry out full wavelength scanner, detect dyestuff in 0,6 and 12h
Residual condition.The result shows that (Fig. 7), 'beta '-mannase are best to the dye decolored effect of azo, wherein methyl orange, Congo red
Decolourize with titan yellow best, in 12h, percent of decolourization is up to almost to 100%, and eriochrome black T percent of decolourization reaches 50%, decoloration compared with
It is good.Preferable to the dye decolored effect of triarylmethane compound, wherein water-tough treatment, cardinal malachite green and phenol red percent of decolourization reach 90%
Left and right, effect are obvious；Crystal violet percent of decolourization reaches 50% or more, and decolorizing effect is preferable.
By 'beta '-mannase of the invention and the 'beta '-mannase of commercialization (4000U/mg) to azo and three virtues
The decolorizing effect of methane class dyestuff is compared.The result shows that (Fig. 7), to Congo red, methyl orange, titan yellow, eriochrome black T, water
Soluble aniline indigo plant, crystal violet, malachite green, phenol red decolourize in result, the 'beta '-mannase decoloration of the ratio commercialization of purifying
Effect is good.
Claims (1)
1. a kind of 'beta '-mannase is used for dye decolored purposes；
'beta '-mannase is by bacillus licheniformis HDGLJT-01 output；
The bacillus licheniformis HDGLJT-01 seed liquor being incubated overnight is forwarded in konjac glucomannan enzymatic production culture medium, 37 DEG C,
160rpm shaken cultivation 48h takes 40mL fermentation liquid, 4500r/min, 4 DEG C of centrifugation 20min, obtained supernatant, i.e. crude enzyme liquid；
Crude enzyme liquid is purified using acetone precipitation, then is further purified using ion-exchange chromatography and gel permeation chromatography；
The optimal pH of 'beta '-mannase is 8.0；
Ion-exchange chromatography and gel permeation chromatography refer to: enzyme solution will be concentrated, the DEAE-sepharose FF balanced is added
In ion exchange column, gradient elution is carried out with the Tris-HCl that the NaCl of 0.1mol/L and 0.005mol/L, pH are 7.0, is collected
Eluent detects enzyme activity and protein content；The enzyme solution of 5mL ion-exchange purification is added to the sepadexG-75 column to have balanced
In son, use pure water as eluent, elution flow rate 0.5mol/min collects eluent；
The dyestuff is azo dyes or triarylmethane compound dyestuff；
The azo dyes are methyl orange, Congo red and titan yellow；
The triarylmethane compound dyestuff is water-tough treatment, cardinal malachite green and phenol red.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610031200.2A CN105621630B (en) | 2016-01-18 | 2016-01-18 | A kind of decoloration 'beta '-mannase and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610031200.2A CN105621630B (en) | 2016-01-18 | 2016-01-18 | A kind of decoloration 'beta '-mannase and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105621630A CN105621630A (en) | 2016-06-01 |
| CN105621630B true CN105621630B (en) | 2019-02-01 |
Family
ID=56037037
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610031200.2A Expired - Fee Related CN105621630B (en) | 2016-01-18 | 2016-01-18 | A kind of decoloration 'beta '-mannase and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105621630B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109825451A (en) * | 2019-01-25 | 2019-05-31 | 牡丹江医学院 | A kind of 'beta '-mannase superior strain and its application in dye decolored |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102071156B (en) * | 2009-12-22 | 2012-07-25 | 黑龙江大学 | Bacillus licheniformis capable of producing beta-mannanase |
| US8633148B2 (en) * | 2010-04-06 | 2014-01-21 | The Procter & Gamble Company | Encapsulates |
| CN101870968A (en) * | 2010-05-18 | 2010-10-27 | 南京大学 | Triphenylmethane dye decolorization enzyme and application thereof |
| CN103555629B (en) * | 2013-11-04 | 2015-11-11 | 赵静 | A kind of bacterial strain and application thereof of producing middle low temperature 'beta '-mannase |
-
2016
- 2016-01-18 CN CN201610031200.2A patent/CN105621630B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN105621630A (en) | 2016-06-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Massana et al. | Changes in marine bacterioplankton phylogenetic composition during incubations designed to measure biogeochemically significant parameters | |
| Tachibana et al. | Purification and characterization of an extremely thermostable cyclomaltodextrin glucanotransferase from a newly isolated hyperthermophilic archaeon, a Thermococcus sp | |
| Cabral et al. | Carotenoids production from a newly isolated Sporidiobolus pararoseus strain by submerged fermentation | |
| CN102965418A (en) | Extraction method for biological oat polypeptide and applications thereof | |
| Flores-Gallegos et al. | Comparative study of fungal strains for thermostable inulinase production | |
| CN109294950B (en) | High-activity gellan gum oligosaccharide producing strain and application thereof | |
| Yang et al. | Enhanced production of volatile fatty acids (VFAs) from sewage sludge by β-cyclodextrin | |
| Chi et al. | Production and characterization of a novel thermostable extracellular agarase from Pseudoalteromonas hodoensis newly isolated from the west sea of South Korea | |
| Verma et al. | Whole cell based miniaturized fiber optic biosensor to monitor L-asparagine | |
| CN101838616A (en) | Halomonas capable of degrading polyaromatic hydrocarbon and application thereof | |
| Balaraman et al. | Sustainable valorization of meat processing wastewater with synergetic eutectic mixture based purification of R-Phycoerythrin from porphyrium cruentium | |
| CN105621630B (en) | A kind of decoloration 'beta '-mannase and application thereof | |
| Wang et al. | The biodegradation of indigo carmine by Bacillus safensis HL3 spore and toxicity analysis of the degradation products | |
| Arnosti | Contrasting patterns of peptidase activities in seawater and sediments: an example from Arctic fjords of Svalbard | |
| Zambelli et al. | Sweet-and-salty biocatalysis: fructooligosaccharides production using Cladosporium cladosporioides in seawater | |
| CN110055233A (en) | The MTSase mutant and its application that a kind of thermal stability improves | |
| CN113215179A (en) | Fungal laccase, recombinant pichia pastoris engineering bacteria thereof and application | |
| CN105543128A (en) | Polar cold-adapted salt-tolerant alginate lyase and coding gene c3 and application thereof | |
| CN109161556B (en) | M1PDH gene in kelp, protein and application thereof | |
| CN106754851A (en) | TaGPI1mS543A protein and coding gene and application thereof | |
| CN103451123A (en) | Macrococcus caseolyticus and preparation method as well as application thereof | |
| Ruginescu et al. | Preliminary characterization of a cellulase producing bacterial strain isolated from a Romanian hypersaline lake | |
| CN102433290B (en) | Strain for producing citrulline and method for biologically synthesizing citrulline with same | |
| CN105733953A (en) | Separation and optimization method and application of laccase-producing fungus strain | |
| Chiu et al. | Production of pigments by Monascus purpureus using sugar-cane bagasse in roller bottle cultures |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190201 Termination date: 20200118 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |