CN105385667B - A method of improving bolt bacterium producing enzyme using lignosulfonates as inducer - Google Patents
A method of improving bolt bacterium producing enzyme using lignosulfonates as inducer Download PDFInfo
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- CN105385667B CN105385667B CN201510961997.1A CN201510961997A CN105385667B CN 105385667 B CN105385667 B CN 105385667B CN 201510961997 A CN201510961997 A CN 201510961997A CN 105385667 B CN105385667 B CN 105385667B
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- 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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- 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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- 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 method that the invention discloses a kind of to improve bolt bacterium producing enzyme using lignosulfonates as inducer, belongs to technical field of microbial fermentation.The method of the present invention effectively increases the yield of enzyme of Trametessp.LEF01 bacterial strain by adding lignosulfonates in PDA culture medium as inducer.Method of the invention can be such that laccase, cellulase, hemicellulase, pectase enzyme activity is improved simultaneously, and wherein laccase activity can be improved to 4-7 times, cellulose enzyme activity to improve to 17-21 times, hemicellulase enzyme activity to improve to 4-6 times, pectase enzyme activity and improve to 3-4 times.
Description
Technical field
The method that the present invention relates to a kind of to improve bolt bacterium producing enzyme using lignosulfonates as inducer belongs to microorganism hair
Ferment technical field.
Background technique
Microbial fermentation technology is usually one enzyme preparation of production list, will usually be answered after several enzymes compounding again in practical application
With, but the compounding of various enzymes is likely to affect mutual zymologic property, and therefore, system of the fermentation liquid containing a variety of enzymes can obtain more
Good application.
Chinese patent publication No. CN102268379A, date of publication on December 07th, 2011, a kind of entitled " Trametes trogii
And its method of cellulase-producing ", the method for disclosure of the invention Trametes trogii fermentation cellulase-producing successfully measures cellulase
Enzyme activity, which, which is disadvantageous in that in crude enzyme liquid, contains only a kind of enzyme and is not added with inducer.
Chinese patent publication No. CN1023509764A, date of publication on 01 15th, 2014, a kind of entitled " application
In the culture medium of production of laccase from solid fermentation of tramete ", a kind of culture medium of production of laccase from solid fermentation of tramete of the disclosure of the invention, the hair
It is bright to be disadvantageous in that main production laccase, it is not directed to multi-enzyme system, and be not added with inducer.
Therefore, screening, which obtains one plant, can produce the problem of bacterial strain of a variety of enzymes is urgent need to resolve.
The present invention screens one plant of bacterial strain that can produce laccase, cellulase, hemicellulase, pectase simultaneously, but sends out
Ferment crude enzyme liquid enzyme activity need to be improved, in order to industrial applications.Display has been reported, induction is added in biofermentation technique
Agent can reinforce the enzymatic productivity of microorganism, improve crude enzyme liquid enzyme activity, veratryl alcohol, guaiacol and benzyl alcohol to the secretion of laccase all
With inducing action.How in same fermentation system, while improving the production of laccase, cellulase, hemicellulase, pectase
The problem of amount and urgent need to resolve.
Summary of the invention
To solve the above-mentioned problems, the present invention provides a kind of method for improving bolt bacterium producing enzyme, and the method is with lignin
Sulfonate is to add lignosulfonates in the culture medium of bolt bacterium as inducer.
In one embodiment of the invention, the bolt bacterium was Trametes sp.LEF01, on May 18th, 2015
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC, deposit number is CGMCC No.10489.
In one embodiment of the invention, the bolt bacterium CGMCC No.10489 is isolated from soil.
In one embodiment of the invention, the bolt bacterium CGMCC No.10489 can produce laccase, cellulase, half fiber
Plain enzyme, pectase are tieed up, is carrying out oscillation training using potato extracting solution and glucose as primary raw material (potato fluid nutrient medium)
Enzymatic production is supported, enzymatic production enzyme activity is reachable: laccase activity 630.6U/L, cellulose enzyme activity 1108.9U/L, hemicellulase
Enzyme activity 3844.9U/L, pectase enzyme activity 520.7U/L.
In one embodiment of the invention, the lignosulfonates are sodium lignin sulfonate.
In one embodiment of the invention, the additive amount of the lignosulfonates is 0.5-4g/L.
In one embodiment of the invention, the additive amount of the lignosulfonates is 0.8-1g/L.
In one embodiment of the invention, the culture medium is potato fluid nutrient medium.
In one embodiment of the invention, the method is by after bolt bacterium CGMCC No.10489 activation, and access contains
Have in the potato fluid nutrient medium of lignosulfonates, the shaken cultivation 6 days under conditions of 28 DEG C, 150rpm, in centrifuging and taking
Clear crude enzyme liquid to obtain the final product.
In one embodiment of the invention, the preparation method of the potato fluid nutrient medium: the formula of every L takes
Peeled potatoes 200g, it stripping and slicing, boils, filter, then complementing to 1L, and 20.0g glucose is added.
In one embodiment of the invention, the method is specifically: peeled potatoes 200g is weighed, is cut into small pieces,
Water 1.0L is added to boil 20~30min, 4 layers of filtered through gauze filter off potato ball, and filtrate is complemented to 1.0L, 20.0g grape is added
1g lignosulfonates are added in sugar；Cooling after packing after 121 DEG C of 20~30min of high pressure sterilization, sterile working is accessed in PDA
The bacteria suspension that 5~6 days CGMCC No.10489 bacterial strains have been cultivated on slant medium, 28~30 in constant-temperature shaking incubator
Shaken cultivation 6 days under the conditions of DEG C, revolving speed 150rpm, fermentation ends, fermentation liquid 4000rpm are centrifuged 30min, obtained supernatant
For crude enzyme liquid.
The present invention be also claimed the crude enzyme liquid obtained according to the method and its field of textiles application.
Beneficial effects of the present invention:
(1) bolt bacterium culture medium of the invention is potato, glucose, lignosulfonates, from a wealth of sources, hair using raw material
Zymotic fluid no pollution to the environment；
(2) by the present invention in that with bolt bacterium CGMCC No.10489, make in fermentation liquid containing there are many enzyme preparations.
(3) by adding inducer lignosulfonates in the medium, laccase, cellulase, hemicellulose can be made
Enzyme, pectase enzyme activity be significantly improved simultaneously, laccase activity can be improved to 4-7 times, cellulose enzyme activity improve to 2 times,
Hemicellulase enzyme activity is improved to 4-6 times, pectase enzyme activity and is improved to 32-38 times.
(4) by the method for the present invention, laccase activity 4100.8U/L, cellulose enzyme activity 2310U/L in obtained crude enzyme liquid,
Hemicellulase enzyme activity 21271.8U/L, pectase enzyme activity 19800U/L.
Biomaterial preservation:
A kind of bolt bacterium, taxology are named as bolt bacterium Trametes sp., are preserved in China Microbiological on May 18th, 2015
Culture presevation administration committee common micro-organisms center CGMCC, deposit number are CGMCC No.10489, and preservation address is Beijing
The institute 3 of Chaoyang District North Star West Road 1.
Specific embodiment
Enzyme activity determination method:
(1) laccase activity
Definition: enzyme amount needed for 1min internal oxidition ABTS generates 1 μm of ol ABTS free radical is 1 enzyme-activity unit.
Measuring method: being substrate measurement paint with ABTS (2,2'- phenodiazine-bis- (3- ethyl-benzothiazole -6- sulfonic acid) ammonium salt)
Enzyme enzyme activity, that is, taking 2mL 0.5mmol/L ABTS solution, (with pH5.0, concentration is the Acetic acid-sodium acetate buffer of 0.05mol/L
Prepare), blank sample is substituted with equivalent buffer solution, and 37 DEG C of preheatings are added 1mL crude enzyme liquid, measure absorbance change at 420nm
Rate.Laccase activity=absorbance change rate × reaction solution volume × 1000 × extension rate/molar absorptivity number
(2) cellulose enzyme activity
Definition: enzyme amount needed for generating 1 μm of ol glucose in 1min is 1 enzyme-activity unit.
Measuring method: cellulose enzyme activity is measured using DNS method (3,5- dinitrosalicylic acid system), that is, takes the thick enzyme of 0.2mL
Liquid, it (is 4.8,0.2mol/L acetic acid-with pH that 0.625% sodium carboxymethylcellulose of 1.8mL mass fraction (CMC-Na) solution, which is added,
Sodium acetate buffer is prepared), equivalent NaAc_HAc buffer solution is added in blank sample, adds immediately after 50 DEG C of reaction 30min
Enter 2mL DNS reagent, boiling water bath 10min colour developing, cooling, dilution is settled to certain multiple, mixes well, and measures and inhales at 550nm
Luminosity.Cellulose enzyme activity=concentration of glucose × reaction solution volume × 1000 × extension rate/reaction time
(3) hemicellulase enzyme activity
Definition: enzyme amount needed for generating 1 μm of ol xylose in 1min is 1 enzyme-activity unit.
Measuring method: measuring hemicellulase enzyme activity using DNS method, that is, take 1mL crude enzyme liquid, and 2mL mass fraction is added and is
0.5% xylan suspension (using pH4.8, the NaAc_HAc buffer solution of concentration 0.05mol/L is prepared), blank sample adds
Enter equivalent NaAc_HAc buffer solution, the termination reaction of 2mLDNS reagent, boiling water are added immediately after 30min is handled at 60 DEG C
10min colour developing is bathed, is cooled to room temperature, dilution is settled to proper volume, measures absorbance at 540nm after mixing.Hemicellulose
Plain enzyme enzyme activity calculation method are as follows: xylose concentration, hemicellulase enzyme activity=xylose concentration × anti-are obtained according to xylose standard curve
Answer liquid product × 1000 × extension rate/reaction time
(4) pectase enzyme activity
Definition: polygalacturonic acid-catalyzed cleavage is made to generate the enzyme amount of 1 μm of ol unsaturation galacturonic acid in 1min.
Measuring method: pectase enzyme activity is measured by substrate of pectin, that is, takes 1mL crude enzyme liquid, 0.2% pectin of 2mL is added
Glycine-NaOH buffer (pH9.4), the Glycine-NaOH buffer of blank sample addition equivalent pH9.4,45 DEG C
15min is reacted in water-bath, the phosphoric acid 3mL of 0.03mol/L is added, and measures light absorption value at 235nm.Pectase enzyme activity calculation method
Are as follows: pectase enzyme activity=enzyme activity determination value × reaction solution volume × 1000 × extension rate/reaction time
Embodiment 1: bolt bacterium enzymatic production
Peeled potatoes 200g is weighed, is cut into small pieces, water 1.0L is added to boil 30min, 4 layers of filtered through gauze filter off potato
Filtrate is complemented to 1.0L, 20.0g glucose is added by block, is added 0.8g lignosulfonates, is sufficiently dissolved.121 DEG C after packing
Cooling after high pressure sterilization 20min, 5~6 days Trametes have been cultivated in sterile working, access on PDA slant medium
The bacteria suspension of sp.LEF01 bacterial strain, shaken cultivation 6 days under the conditions of 28 DEG C, revolving speed 150rpm in constant-temperature shaking incubator.Hair
Ferment terminates, and fermentation liquid 4000rpm is centrifuged 30min, and supernatant is taken to survey enzyme activity, laccase activity 3080.8U/L, cellulose enzyme activity
1910U/L, hemicellulase enzyme activity 16271.8U/L, pectase enzyme activity 16800U/L.
Wherein, it compares as follows: not adding lignosulfonates in culture medium, other steps and embodiment 2 are consistent.As a result it shows
Show, in fermented supernatant fluid, laccase activity 630.6U/L, cellulose enzyme activity 1108.9U/L, hemicellulase enzyme activity 3844.9U/
L, pectase enzyme activity 520.7U/L.
Compared with the control, using the method for embodiment 1, the enzyme of laccase in fermentation liquid, hemicellulase, pectase can be made
Work is all significantly improved, and wherein laccase activity is improved to 4.89 times, hemicellulase enzyme activity and improved to 4.23 times, pectase enzyme
It is living to improve to 32.3 times.
Embodiment 2: bolt bacterium enzymatic production
Peeled potatoes 200g is weighed, is cut into small pieces, water 1.0L is added to boil 30min, 4 layers of filtered through gauze filter off potato
Filtrate is complemented to 1.0L, 20.0g glucose is added by block, is added 1g lignosulfonates, is sufficiently dissolved.121 DEG C of height after packing
After pressure sterilizing 20min, cooling, 5~6 days Trametes have been cultivated in sterile working, access on PDA slant medium
The bacteria suspension of sp.LEF01 bacterial strain, shaken cultivation 6 days under the conditions of 28 DEG C, revolving speed 150rpm in constant-temperature shaking incubator.Hair
Ferment terminates, and fermentation liquid 4000rpm is centrifuged 30min, and supernatant is taken to survey enzyme activity, laccase activity 4100.8U/L, cellulose enzyme activity
2310U/L, hemicellulase enzyme activity 21271.8U/L, pectase enzyme activity 19800U/L.
Wherein, it compares as follows: not adding lignosulfonates in culture medium, other steps and embodiment 2 are consistent.As a result it shows
Show, in fermented supernatant fluid, laccase activity 630.6U/L, cellulose enzyme activity 1108.9U/L, hemicellulase enzyme activity 3844.9U/
L, pectase enzyme activity 520.7U/L.
Compared with the control, using the method for embodiment 2, can make laccase in fermentation liquid, cellulase, hemicellulase,
The enzyme activity of pectase is all significantly improved, wherein laccase activity improve to 6.5 times, cellulose enzyme activity improve to 2.08 times,
Hemicellulase enzyme activity is improved to 5.53 times, pectase enzyme activity and is improved to 38.01 times.
Embodiment 3: influence of the inducer type to bolt bacterium enzymatic production
Sodium lignin sulfonate is replaced with 2mmol guaiacol, other steps and embodiment 3 are consistent, as the result is shown fermentation liquid
In supernatant, fiber is not detected in laccase activity 730U/L, hemicellulase enzyme activity 3076U/L, pectase enzyme activity 1090U/L
Plain enzyme.Be not added compared with inducer, laccase activity is improved to 1.16 times, and hemicellulose enzyme activity does not improve, pectin enzyme activity improve
To 2.09 times.
Lignosulfonates are replaced with 2mmol alkali lignin, other steps and embodiment 3 are consistent, as the result is shown fermentation liquid
In supernatant, cellulose is not detected in laccase activity 873U/L, hemicellulase enzyme activity 3200U/L, pectase enzyme activity 490U/L
Enzyme.Be not added compared with inducer, laccase activity is improved to 1.38 times, and hemicellulose enzyme activity does not improve, and pectin enzyme activity does not improve.
Sodium lignin sulfonate is replaced with 2mmol pectin, other steps and embodiment 3 are consistent, as the result is shown fermented liquid supernatant
In liquid, cellulase is not detected in laccase activity 660U/L, hemicellulase enzyme activity 3237U/L, pectase enzyme activity 1090U/L.
Be not added compared with inducer, laccase activity does not improve, and hemicellulose enzyme activity does not improve, and pectin enzyme activity is improved to 2.09 times.
Embodiment 4: the application of bacterial strain
Existing cotton fabric enzymatic scouring technology either uses single enzyme or complex enzyme, on to cotton seed hulls removal effect
It is undesirable, (even if wetability obtains good effect) especially even more so to the higher fabric of cloth cover cotton seed hulls, many documents
Or patent report avoids this point (not mentioning cotton seed hulls treatment effect).However, cotton seed hulls naked eyes are as it can be seen that be in factory's actual production
One of most intuitive cotton scouring result.Bacterial strain crude enzyme produced by the liquid of the invention can solve these problems.
Bacterial strain of the present invention is used for the concise of cotton fabric according to the crude enzyme liquid that the method for embodiment 2 is fermented, method is such as
Under:
(1) it pads enzyme solution: using the bolt bacterium CGMCC No.10489 crude enzyme liquid obtained according to the method for embodiment 2, rolling enzyme
40 DEG C of temperature, enzyme dosage 1g/L, bleeding agent 5g/L roll fabric clot after enzyme；
(2) heat preservation is banked up: clot fabric is banked up 24 hours at 25 DEG C, and fabric slowly rotates during banking up；
(3) it washes: being washed after fabric uncoiling in 90 DEG C, to go to dezymotize the impurity of degradation, and have both to make enzyme deactivation.
Using method of the invention, scouring result is good, wetability: the time of dripping is less than 1s, 70% or more whiteness, strength damage
It loses less than 5%, COD value of waste water is the concise 10-20% of alkali；Cloth cover is remained without cotton seed hulls after subsequent conventional bleaching.
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 (6)
1. it is a kind of using lignosulfonates as inducer improve bolt bacterium producing enzyme method, which is characterized in that the method be
Lignosulfonates are added in the culture medium of bolt bacterium；The bolt bacterium is Trametes sp.LEF01, is protected on May 18th, 2015
It is hidden in China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC, deposit number is CGMCC No.10489；Institute
The additive amount for stating lignosulfonates is 0.8-1g/L.
2. the method according to claim 1, wherein the culture medium is potato fluid nutrient medium.
3. the method according to claim 1, wherein the method is to activate bolt bacterium CGMCC No.10489
Afterwards, it accesses in the potato fluid nutrient medium containing lignosulfonates, the shaken cultivation 6 days under conditions of 28 DEG C, 150rpm,
Centrifuging and taking supernatant is up to crude enzyme liquid.
4. the method according to claim 1, wherein the bolt bacterium is isolated from soil.
5. the method according to claim 1, wherein the method is specifically: weighing peeled potatoes 200g, cut
At fritter, water 1.0L is added to boil 20~30min, 4 layers of filtered through gauze filter off potato ball, filtrate is complemented to 1.0L, are added
1g lignosulfonates are added in 20.0g glucose；It is cooling after packing after 121 DEG C of 20~30min of high pressure sterilization, sterile working,
The bacteria suspension of 5~6 days CGMCC No.10489 bacterial strains has been cultivated in access on PDA slant medium, in constant-temperature shaking culture
Shaken cultivation 6 days under the conditions of 28~30 DEG C in case, revolving speed 150rpm, fermentation ends, fermentation liquid 4000rpm are centrifuged 30min, obtain
The supernatant arrived is crude enzyme liquid.
6. a kind of any method for improving bolt bacterium producing enzyme using lignosulfonates as inducer of claim 1-5 is being spun
Knit the application in field.
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