CN105385667A - Method for improving trametes enzyme production with lignosulfonate as inductive agent - Google Patents
Method for improving trametes enzyme production with lignosulfonate as inductive agent Download PDFInfo
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- CN105385667A CN105385667A CN201510961997.1A CN201510961997A CN105385667A CN 105385667 A CN105385667 A CN 105385667A CN 201510961997 A CN201510961997 A CN 201510961997A CN 105385667 A CN105385667 A CN 105385667A
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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.)
- C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
- C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
- C12N9/0061—Laccase (1.10.3.2)
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- 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)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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- 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
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
- C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
- C12Y110/03002—Laccase (1.10.3.2)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
- C12Y402/02006—Oligogalacturonide lyase (4.2.2.6)
Abstract
The invention discloses a method for improving trametes enzyme production with lignosulfonate as an inductive agent, and belongs to the technical field of microbial fermentation. The method effectively increases the enzyme yield of Trametessp.LEF01 strains by adding lignosulfonate into a PDA culture medium to serve as the inductive agent. Enzyme activity of laccase, enzyme activity of cellulase, enzyme activity of hemicellulase and enzyme activity of pectinase are improved by 4-7 times, 17-21 times, 4-6 times and 3-4 times respectively at the same time.
Description
Technical field
The present invention relates to a kind of method improving bolt bacterium product enzyme using sulfonated lignin as inductor, belong to technical field of microbial fermentation.
Background technology
Microbial fermentation technology normally produces single zymin, again usually by composite for several enzyme rear application in practical application, but the composite zymologic property that likely can affect each other of various enzyme, therefore, fermented liquid can better be applied containing the system of multiple enzyme.
Chinese patent publication No. CN102268379A, date of publication on December 07th, 2011, denomination of invention is " a kind of method of Trametes trogii and cellulase-producing thereof ", the method of this disclosure of the invention Trametes trogii fermentation cellulase-producing, successfully record cellulose enzyme activity, the weak point of this invention is only contain a kind of enzyme in crude enzyme liquid and do not add inductor.
Chinese patent publication No. CN1023509764A, date of publication on 01 15th, 2014, denomination of invention is " a kind of substratum being applied to bolt bacterium Produced by Solid-state Fermentation laccase ", a kind of substratum of bolt bacterium Produced by Solid-state Fermentation laccase of this disclosure of the invention, the weak point of this invention is mainly to produce laccase, do not relate to multi-enzyme system, and do not add inductor.
Therefore, screening obtains the bacterial strain that a strain can produce multiple enzyme is the problem needing solution badly.
The present invention screens the bacterial strain that a strain can produce laccase, cellulase, hemicellulase, polygalacturonase simultaneously, but alive the needing of fermentation crude enzyme liquid enzyme is improved, so that industrial applications.Have been reported display, add the enzymatic productivity that inductor can strengthen microorganism in biofermentation technique, improve crude enzyme liquid enzyme and live, the secretion to laccase of veratryl alcohol, methyl catechol and phenylcarbinol all has inducing action.How in same fermentation system, improve the output of laccase, cellulase, hemicellulase, polygalacturonase, be also the problem needing solution badly simultaneously.
Summary of the invention
In order to solve the problem, the invention provides a kind of method improving bolt bacterium product enzyme, described method is using sulfonated lignin as inductor, is to add sulfonated lignin in the substratum of bolt bacterium.
In one embodiment of the invention, described bolt bacterium is Trametessp.LEF01, and be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC on May 18th, 2015, preserving number is CGMCCNo.10489.
In one embodiment of the invention, described bolt bacterium CGMCCNo.10489 is separated and obtains from soil.
In one embodiment of the invention, described bolt bacterium CGMCCNo.10489 can produce laccase, cellulase, hemicellulase, polygalacturonase, with potato extracting solution and glucose for main raw material (potato liquid nutrient medium) carries out shaking culture enzymatic production, the work of enzymatic production enzyme can reach: laccase activity 630.6U/L, cellulose enzyme activity 1108.9U/L, hemicellulase enzyme 3844.9U/L alive, polygalacturonase enzyme 520.7U/L alive.
In one embodiment of the invention, described sulfonated lignin are sodium lignosulfonate.
In one embodiment of the invention, the addition of described sulfonated lignin is 0.5-4g/L.
In one embodiment of the invention, the addition of described sulfonated lignin is 0.8-1g/L.
In one embodiment of the invention, described substratum is potato liquid nutrient medium.
In one embodiment of the invention, described method is after being activated by bolt bacterium CGMCCNo.10489, and access is containing in the potato liquid nutrient medium of sulfonated lignin, and 28 DEG C, shaking culture 6 days under the condition of 150rpm, namely centrifuging and taking supernatant obtains crude enzyme liquid.
In one embodiment of the invention, the preparation method of described potato liquid nutrient medium: the formula of every L, gets peeled potatoes 200g, stripping and slicing, boils, filters, then complement to 1L, and add 20.0g glucose.
In one embodiment of the invention, described method specifically: take peeled potatoes 200g, be cut into small pieces, the 1.0L that adds water boils 20 ~ 30min, and 4 layers of filtered through gauze elimination potato ball, complement to 1.0L by filtrate, add 20.0g glucose, add 1g sulfonated lignin; After packing after 121 DEG C of autoclaving 20 ~ 30min, cooling, aseptic technique, the bacteria suspension of the CGMCCNo.10489 bacterial strain of 5 ~ 6 days has been cultivated in access on PDA slant medium, shaking culture 6 days under 28 ~ 30 DEG C of conditions in constant-temperature shaking incubator, rotating speed is 150rpm, fermentation ends, the centrifugal 30min of fermented liquid 4000rpm, the supernatant liquor obtained is crude enzyme liquid.
The present invention is the claimed crude enzyme liquid obtained by described method also, and it is in the application of field of textiles.
Beneficial effect of the present invention:
(1) bolt bacterium culture medium of the present invention uses that raw material is potato, glucose, sulfonated lignin, wide material sources, fermented liquid environmentally safe;
(2) the present invention is by using bolt bacterium CGMCCNo.10489, makes in fermented liquid containing multiple zymin.
(3) by adding inductor sulfonated lignin in the medium, the enzyme of laccase, cellulase, hemicellulase, polygalacturonase can be made to live be significantly improved simultaneously, laccase activity can be increased to 4-7 doubly, cellulose enzyme activity be increased to 2 times, hemicellulase enzyme live be increased to 4-6 doubly, polygalacturonase enzyme lives and is increased to 32-38 doubly.
(4) by the inventive method, laccase activity 4100.8U/L in the crude enzyme liquid obtained, cellulose enzyme activity 2310U/L, hemicellulase enzyme 21271.8U/L alive, polygalacturonase enzyme 19800U/L alive.
Biomaterial preservation:
A kind of bolt bacterium, taxonomy called after bolt bacterium Trametessp., be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC on May 18th, 2015, preserving number is CGMCCNo.10489, and preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
Embodiment
Enzyme activity determination method:
(1) laccase activity
Definition: it is 1 Ge Meihuo unit that 1min internal oxidition ABTS generates enzyme amount needed for 1 μm of olABTS free radical.
Measuring method: with ABTS (2,2'-phenodiazine-bis-(3-ethyl-benzothiazole-6-sulfonic acid) ammonium salt) be substrate mensuration laccase activity, namely 2mL0.5mmol/LABTS solution is got (with pH5.0, concentration is the Acetic acid-sodium acetate buffer of 0.05mol/L), blank sample equivalent buffered soln substitutes, 37 DEG C of preheatings, add 1mL crude enzyme liquid, measure 420nm place absorbancy velocity of variation.Laccase activity=absorbancy velocity of variation × reaction solution volume × 1000 × extension rate/molar absorptivity number
(2) cellulose enzyme activity
In definition: 1min, the enzyme amount produced needed for 1 μm of ol glucose is 1 Ge Meihuo unit.
Measuring method: utilize DNS method (3,5-dinitrosalicylic acid system) measure cellulose enzyme activity, namely 0.2mL crude enzyme liquid is got, adding 1.8mL massfraction 0.625% Xylo-Mucine (CMC-Na) solution (is 4.8 with pH, 0.2mol/L NaAc_HAc buffer solution is prepared), blank sample adds equivalent NaAc_HAc buffer solution, 2mLDNS reagent is added immediately after 50 DEG C of reaction 30min, boiling water bath 10min develops the color, cooling, dilution is settled to certain multiple, fully mixes, and measures 550nm place absorbancy.Cellulose enzyme activity=glucose concn × reaction solution volume × 1000 × extension rate/reaction times
(3) hemicellulase enzyme is lived
In definition: 1min, the enzyme amount produced needed for 1 μm of ol wood sugar is 1 Ge Meihuo unit.
Measuring method: utilize DNS method to measure hemicellulase enzyme and live, namely 1mL crude enzyme liquid is got, adding 2mL massfraction is that the xylan suspension of 0.5% is (with pH4.8, the NaAc_HAc buffer solution preparation of concentration 0.05mol/L), blank sample adds equivalent NaAc_HAc buffer solution, 2mLDNS reagent termination reaction is added immediately process 30min at 60 DEG C after, boiling water bath 10min develops the color, be cooled to room temperature, dilution is settled to proper volume, mixes rear mensuration 540nm place absorbancy.Hemicellulase enzyme method of calculation alive are: obtain xylose concentration according to xylose standard curve, hemicellulase enzyme work=xylose concentration × reaction solution volume × 1000 × extension rate/reaction times
(4) polygalacturonase enzyme is lived
Definition: make polygalacturonic acid-catalyzed cleavage produce the enzyme amount of 1 μm of unsaturated galacturonic acid of ol in 1min.
Measuring method: be that substrate mensuration polygalacturonase enzyme is lived with pectin, namely 1mL crude enzyme liquid is got, add the Glycine-NaOH damping fluid (pH9.4) of 2mL0.2% pectin, blank sample adds the Glycine-NaOH damping fluid of equivalent pH9.4,15min is reacted in 45 DEG C of water-baths, add the phosphoric acid 3mL of 0.03mol/L, measure 235nm place light absorption value.Polygalacturonase enzyme method of calculation alive are: polygalacturonase enzyme work=enzyme activity determination value × reaction solution volume × 1000 × extension rate/reaction times
Embodiment 1: bolt bacterium enzymatic production
Take peeled potatoes 200g, be cut into small pieces, the 1.0L that adds water boils 30min, and 4 layers of filtered through gauze elimination potato ball, complement to 1.0L by filtrate, add 20.0g glucose, adds 0.8g sulfonated lignin, fully dissolves.After packing after 121 DEG C of autoclaving 20min, cooling, aseptic technique, accesses the bacteria suspension of the Trametessp.LEF01 bacterial strain having cultivated 5 ~ 6 days on PDA slant medium, and shaking culture 6 days under 28 DEG C of conditions in constant-temperature shaking incubator, rotating speed is 150rpm.Fermentation ends, the centrifugal 30min of fermented liquid 4000rpm, gets supernatant liquor survey enzyme and lives, laccase activity 3080.8U/L, cellulose enzyme activity 1910U/L, hemicellulase enzyme 16271.8U/L alive, polygalacturonase enzyme 16800U/L alive.
Wherein, contrast as follows: do not add sulfonated lignin in substratum, other steps are consistent with embodiment 2.Result shows, in fermented supernatant fluid, and laccase activity 630.6U/L, cellulose enzyme activity 1108.9U/L, hemicellulase enzyme 3844.9U/L alive, polygalacturonase enzyme 520.7U/L alive.
Compared with the control, adopt the method for embodiment 1, the work of the enzyme of laccase in fermented liquid, hemicellulase, polygalacturonase can be made all to be significantly improved, and wherein laccase activity is increased to 4.89 times, the work of hemicellulase enzyme is increased to 4.23 times, polygalacturonase enzyme is alive is increased to 32.3 times.
Embodiment 2: bolt bacterium enzymatic production
Take peeled potatoes 200g, be cut into small pieces, the 1.0L that adds water boils 30min, and 4 layers of filtered through gauze elimination potato ball, complement to 1.0L by filtrate, add 20.0g glucose, adds 1g sulfonated lignin, fully dissolves.After packing after 121 DEG C of autoclaving 20min, cooling, aseptic technique, accesses the bacteria suspension of the Trametessp.LEF01 bacterial strain having cultivated 5 ~ 6 days on PDA slant medium, and shaking culture 6 days under 28 DEG C of conditions in constant-temperature shaking incubator, rotating speed is 150rpm.Fermentation ends, the centrifugal 30min of fermented liquid 4000rpm, gets supernatant liquor survey enzyme and lives, laccase activity 4100.8U/L, cellulose enzyme activity 2310U/L, hemicellulase enzyme 21271.8U/L alive, polygalacturonase enzyme 19800U/L alive.
Wherein, contrast as follows: do not add sulfonated lignin in substratum, other steps are consistent with embodiment 2.Result shows, in fermented supernatant fluid, and laccase activity 630.6U/L, cellulose enzyme activity 1108.9U/L, hemicellulase enzyme 3844.9U/L alive, polygalacturonase enzyme 520.7U/L alive.
Compared with the control, adopt the method for embodiment 2, the work of the enzyme of laccase in fermented liquid, cellulase, hemicellulase, polygalacturonase can be made all to be significantly improved, and wherein laccase activity is increased to 6.5 times, cellulose enzyme activity is increased to 2.08 times, the work of hemicellulase enzyme is increased to 5.53 times, polygalacturonase enzyme is alive is increased to 38.01 times.
Embodiment 3: inductor kind is on the impact of bolt bacterium enzymatic production
Replace sodium lignosulfonate with 2mmol methyl catechol, other steps are consistent with embodiment 3, and in result display fermented liquid supernatant liquid, laccase activity 730U/L, hemicellulase enzyme 3076U/L alive, polygalacturonase enzyme 1090U/L alive, does not detect cellulase.With do not add compared with inductor, laccase activity is increased to 1.16 times, and hemicellulase is lived and do not improved, and polygalacturonase is lived and is increased to 2.09 times.
Replace sulfonated lignin with 2mmol alkali lignin, other steps are consistent with embodiment 3, and in result display fermented liquid supernatant liquid, laccase activity 873U/L, hemicellulase enzyme 3200U/L alive, polygalacturonase enzyme 490U/L alive, does not detect cellulase.With do not add compared with inductor, laccase activity is increased to 1.38 times, and hemicellulase is lived and do not improved, and polygalacturonase is lived and do not improved.
Replace sodium lignosulfonate with 2mmol pectin, other steps are consistent with embodiment 3, and in result display fermented liquid supernatant liquid, laccase activity 660U/L, hemicellulase enzyme 3237U/L alive, polygalacturonase enzyme 1090U/L alive, does not detect cellulase.With do not add compared with inductor, laccase activity does not improve, and hemicellulase is lived and do not improved, and polygalacturonase is lived and is increased to 2.09 times.
Embodiment 4: the application of bacterial strain
No matter existing cotton fabric enzymatic scouring technology is adopt single enzyme or prozyme, to undesirable in cotton seed hulls removal effect, particularly to the fabric that cloth cover cotton seed hulls is higher (even if wettability obtains good effect) all the more so, a lot of document or patent report avoid this point (not carrying cotton seed hulls treatment effect).But cotton seed hulls naked eyes are visible, it is one of the most cotton scouring result in factory's actual production.Crude enzyme liquid that bacterial strain of the present invention produces can address these problems.
Bacterial strain of the present invention is used for the concise of cotton fabric according to the method for embodiment 2 crude enzyme liquid obtained that ferments, and method is as follows:
(1) pad enzyme liquid: adopt the bolt bacterium CGMCCNo.10489 crude enzyme liquid obtained according to the method for embodiment 2, roll enzyme temperature 40 DEG C, enzyme dosage 1g/L, permeate agent 5g/L, roll fabric clot after enzyme;
(2) insulation is banked up: clot fabric is banked up 24 hours at 25 DEG C, and in process of banking up, fabric slowly rotates;
(3) wash: in 90 DEG C of washings after fabric uncoiling, to remove the impurity of enzyme liberating, and have concurrently and make enzyme deactivation effect.
Adopt method of the present invention, scouring result is good, wettability: the time of dripping is less than 1s, whiteness more than 70%, and strength loss is less than 5%, and COD value of waste water is the concise 10-20% of alkali; After follow-up conventional bleaching, cloth cover remains without cotton seed hulls.
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 the method that bolt bacterium produces enzyme using sulfonated lignin as inductor, it is characterized in that, described method adds sulfonated lignin in the substratum of bolt bacterium.
2. method according to claim 1, is characterized in that, described bolt bacterium is Trametessp.LEF01, and be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC on May 18th, 2015, preserving number is CGMCCNo.10489.
3. method according to claim 1, is characterized in that, the addition of described sulfonated lignin is 0.5-4g/L.
4. method according to claim 1, is characterized in that, the addition of described sulfonated lignin is 0.8-1g/L.
5. method according to claim 1, is characterized in that, described substratum is potato liquid nutrient medium.
6. method according to claim 1, it is characterized in that, described method is that after being activated by bolt bacterium CGMCCNo.10489, access is containing in the potato liquid nutrient medium of sulfonated lignin, 28 DEG C, shaking culture 6 days under the condition of 150rpm, namely centrifuging and taking supernatant obtains crude enzyme liquid.
7. bolt bacterium according to claim 2, is characterized in that, described bolt bacterium is separated and obtains from soil.
8. method according to claim 1, it is characterized in that, described method is specifically: take peeled potatoes 200g, be cut into small pieces, the 1.0L that adds water boils 20 ~ 30min, and 4 layers of filtered through gauze elimination potato ball, complement to 1.0L by filtrate, add 20.0g glucose, add 1g sulfonated lignin; After packing after 121 DEG C of autoclaving 20 ~ 30min, cooling, aseptic technique, the bacteria suspension of the CGMCCNo.10489 bacterial strain of 5 ~ 6 days has been cultivated in access on PDA slant medium, shaking culture 6 days under 28 ~ 30 DEG C of conditions in constant-temperature shaking incubator, rotating speed is 150rpm, fermentation ends, the centrifugal 30min of fermented liquid 4000rpm, the supernatant liquor obtained is crude enzyme liquid.
9. according to the crude enzyme liquid that the arbitrary described method of claim 1-8 obtains.
10. crude enzyme liquid described in claim 9 is in the application of field of textiles.
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| CN112042472A (en) * | 2020-09-28 | 2020-12-08 | 上海市农业科学院 | Culture medium for slowing down aging of hypsizigus marmoreus plate culture |
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