CN110184244A - A kind of Sparassis crispa produces the preparation method of laccase - Google Patents
A kind of Sparassis crispa produces the preparation method of laccase Download PDFInfo
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- CN110184244A CN110184244A CN201910536882.6A CN201910536882A CN110184244A CN 110184244 A CN110184244 A CN 110184244A CN 201910536882 A CN201910536882 A CN 201910536882A CN 110184244 A CN110184244 A CN 110184244A
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- laccase
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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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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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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- C—CHEMISTRY; METALLURGY
- 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)
Abstract
A kind of Sparassis crispa disclosed by the invention produces the preparation method of laccase, comprising the following steps: (1) takes mixing pericarp as fermentation substrate, mixed active charcoal carries out homogeneous, and filtering after sterilizing, obtains pericarp solid medium；(2) it is inoculated with the silk ball bacteria strain activated in pericarp solid medium, carries out one time fermentation culture；(3) laccase activity monitoring is carried out to one time fermentation product, gained Sparassis crispa hypha body in picking step (2), is inoculated in the fluid nutrient medium containing Cu2+ and inducer, is transferred to fermentor after shaking flask producing enzyme after enzyme activity reaches peak value；(4) amino-oligosacchride and biofermentation defoaming agent are added in the fermenter, carries out secondary submerged fermentation, and tunning is that Sparassis crispa produces laccase, and laccase activity is high, and Sparassis crispa fermentation cell age is suitable for fermentation strain purity is high, and yield is big.
Description
Technical field
The present invention relates to Sparassis crispa fields, and in particular to a kind of Sparassis crispa produces the preparation method of laccase.
Background technique
Lignin and pollutant similar with lignin front body structure are one of the important sources of environmental pollution, sharp in recent years
Have become the hot spot of Study on Environmental Protection with fungal laccase this pollutant of degrading, especially paper pulp bleaching, it is dye decolored,
Environmental pollutants detoxification and degradation etc. laccase show biggish researching value and application potential.
Laccase is a kind of polyphenol oxidase of cupric, with ascorbic acid oxidase, mammalian plasma ceruloplasmin and gallbladder
Red pigment oxidizing ferment etc. belongs to blue blue multicopper oxidase family, and Sparassis crispa can secretion laccase, manganese peroxidase or lignin
The several frequently seen ectoenzyme such as peroxidase, these lignin-degrading enzymes be distributed in it is extracellular, since ectoenzyme is without will be thin
Born of the same parents destroy and can directly acquire, and keep the utilization of the degradation enzyme system easier.
There is the method using fungus fermentation lacquer producing enzyme in the prior art, it is common such as liquid deep layer fermenting or solid fermentation,
And liquid deep layer fermenting is easy to produce circulating oxygen difference and the problem of be unfavorable for the growth and development of aerobic Sparassis crispa, simultaneously as
Sparassis crispa is huge fungi, and mycelium is easily wrapped on power-equipment, blocking pipeline, and the chaotic expansive of mycelia can also improve
The viscosity of fermentation liquid seriously affects uniformly transferring and spreading for material, oxygen and heat；And the solid fermentation solid fermentation period
Length, yield of enzyme be not high, easily when bacterial strain is transferred to reproductive growth, mutually twists together between mycelia, so that the extracellular producing enzyme of bacterial strain is inhibited,
And opened since solid fermentation space relative liquid ferments, it is mixed into other miscellaneous bacterias easily to reduce producing enzyme efficiency.
Summary of the invention
To solve the above problems, the present invention provides a kind of preparation method of Sparassis crispa production laccase, laccase activity is high, Sparassis crispa
Cell age of fermenting is suitable for fermentation strain purity is high, and yield is big.
The technical scheme is that a kind of Sparassis crispa produces the preparation method of laccase, comprising the following steps: (1) take mixing
For pericarp as fermentation substrate, mixed active charcoal carries out homogeneous, and filtering after sterilizing, obtains pericarp solid medium；(2) solid in pericarp
The silk ball bacteria strain that state inoculation of medium has activated carries out one time fermentation culture；(3) laccase enzyme is carried out to one time fermentation product
Monitoring living, gained Sparassis crispa hypha body in picking step (2), is inoculated in containing Cu after enzyme activity reaches peak value2+And the liquid of inducer
In body culture medium, fermentor is transferred to after shaking flask producing enzyme；(4) amino-oligosacchride and biofermentation defoaming agent are added in the fermenter,
Secondary submerged fermentation is carried out, tunning is that Sparassis crispa produces laccase.
Preferably, the inducer includes one or more of lignanoid, cumarin, chalcone, O-VANILLIN.
Preferably, the pericarp such as is at the mixture of the horseshoe skins of quality, pineapple peel, shaddock ped, orange peel, longan skin.
Preferably, the additive amount of the active carbon is the 1/10-1/5 for mixing pericarp quality.
Preferably, the temperature of the one time fermentation culture is 30 DEG C -35 DEG C, is fermented under illumination.
It preferably, is the 6-8 days after one time fermentation at the beginning of the laccase activity monitoring.
Preferably, the Liquid Culture based component includes 1 part of inducer, potato leachate, 20 parts of glucose, 1 portion of egg
White peptone, 3 parts of KH2PO4, 3 parts of MgSO4·7H2O, 3 parts of (NH4) 2SO4, 10 portions of corn flour, 1 part of CuSO4·5H2O, 1 part of natural bottom
Object is in terms of 250 parts by potato quality.
Preferably, the natural substrate is tealeaves, straw, wheat straw, peanut shell, dregs of beans, bagasse, tangerine peel, cornstalk, wheat bran
One or more of.
Preferably, the shaking flask producing enzyme inoculum concentration 20%, shaking table 150rpm, 28 DEG C of temperature.
Preferably, the inoculum concentration of the secondary submerged fermentation is 10%, ventilatory capacity 0.5-1.0vvm, temperature 30-40
DEG C, speed of agitator 200-300rpm.
Most of fungal laccases are a kind of acid monomer albumen containing sugar, usually by polypeptide chain, polysaccharide and copper atom three
Part forms, and has many general character in physical and chemical properties, but between the laccase of different strain, there is also different
The difference of degree, such as the different laccase in source, its function is different, therefore prepares laccase by source of Sparassis crispa, need to be according to Sparassis crispa
Special fermentation character adjustment growth fermentation condition, if Sparassis crispa needs dark moist environment different from other saprophytic bacterias,
Fructification needs illumination 100 hours or more just be grown to silk ball shape, therefore fermentation condition is also provided with illumination.
In the present solution, first with the bacterial strain that solid medium culture activates, after strain fermentation producing enzyme to peak value, then picking
Sparassis crispa hypha body is inoculated in shaking flask producing enzyme in fluid nutrient medium, finally carries out fermentor submerged fermentation again, and expanding production is compared
In merely with for liquid fermentation or solid fermentation, two kinds of yeastings be used alternatingly in addition to overcome simple liquid fermentation or
Outside solid fermentation bring adverse effect, by first solid culture, Sparassis crispa mycelia is grown on solid medium, is easier to differentiate
With picking Sparassis crispa mycelia, and selectivity non-Sparassis crispa mycelia is stayed in solid medium, avoid being mixed into for miscellaneous bacteria so that
When depth is fermented, strain is purer, and fermentation efficiency is higher, also avoids being mixed into for other non-targeted property tunnings, and solid
In body fermentation process, strain enzyme-producing reaches maximum value when mycelia is covered with, however enzyme activity but sharply declines in former base formation, this
It is itself to expand numerous support to obtain since silk ball bacteria strain needs continuous secretion laccase to decompose substrate during nutrient growth
Material, and mutually twist together when bacterial strain is transferred to reproductive growth, between mycelia, to inhibit the extracellular producing enzyme of bacterial strain and produce laccase；If
The submerged fermentation for directly carrying out fermentor is equivalent to bacterial strain and is directly proliferated secretion laccase simultaneously in the fermenter, and as cell
Or for bacterial strain proliferation, when incipient cell quantity is few, the sizeable culture environment of relative growth environment is selected to be more conducive to bacterium
The proliferation and division of strain, for the fungies such as Sparassis crispa, cell age is too short often to occur that early growth is slow, and fermentation period prolongs
It is long, the time retardation that mycelium initially forms；Cell age is too long, then causes the too early self-dissolving of mycelium, production capacity is caused to decline, because
This enters back into fermentation tank culture after solid culture, so that bacterial strain expands culture in the suitable situation of cell age, deep layer hair
The increase of ferment secretion laccase, biomass increases the enzyme activity level in culture solution, is main lignin additionally, due to laccase
One of biodegradable enzyme, it can accelerate the decomposition of wooden aromatic polymer compound, provide battalion required for growth for mycelia
It supports, laccase activity is higher in fermentation liquid, and bacterial strain capacity of decomposition is stronger, and mycelial growth rate is also faster.
Pericarp can be used as a kind of fermentation substrate, be rich in carbohydrate and protein matter, can mention for the growth and metabolism of thallus
Contain a certain amount of lignin, cellulose or half fiber for nutriment, especially horseshoe skin, pineapple peel, shaddock ped, orange peel, longan skin
Dimension element is outer also to contain certain colloid, and the inducer that can be used as Sparassis crispa secretion laccase is microorganism, makees mushroom training using pericarp
Waste utilization can be realized by supporting matrix production laccase, economized on resources, be of great significance to the sustainable development of environment, this programme
In be mixed into active carbon in the fermentation substrate of pericarp, one side active carbon can be used as carbon source, and another aspect active carbon is porous
There is structure stronger adsorption capacity and electrostatic attraction mycelia is adsorbed onto surface and is fixed, is made in solid medium culture
Mycelia ordering growth is unlikely to tangle.
The mechanism of action that inducer is added is that it can be combined with certain substances for checking target protein synthesis, makes its hair
Raw allosteric effect reduces these substances and checks effect to what zymoprotein synthesized, promotes genetic transcription, translation to generate corresponding enzyme
Albumen, it is mostly some low molecule aromatic compounds similar with lignin structure that whiterot fungi can be induced, which to produce the substance of laccase,
With the fragment compound of lignin degradation, they are largely the substrate specificity of laccase, lignanoid, cumarin, chalcone, adjacent perfume
Lan Su is the aromatic cycle compound of hydroxyl or amino in structure, can reach inducing effect.
Metallic element, such as Cu, Mn, Zn, Fe are usually contained in the active group of zymoprotein, are Microbe synthesis enzyme eggs
Nutrient necessary to white, certain metallic elements are also the active activator of zymoprotein, and the influence to enzyme activity can not be ignored,
Therefore addition metallic element is necessary in fluid nutrient medium, it is worth mentioning at this point that, a certain amount of Cu is added in the medium2+
Ion, since laccase is a kind of polyphenol oxidase of cupric, mostly containing there are four copper ion in each zymoprotein molecule, therefore phase
The presence of elemental copper is needed when correlation gene transcription synthesis laccase, and under the condition of culture of limit copper or scarce copper, the synthesis of laccase is then
It can be impacted.
There is the natural quality adhered to solid matter surface using filamentous fungi, silk ball is controlled using immobilization technology
The free growth of bacterium, advantage are that somatic cells can be easily isolated from fluid nutrient medium, simplify feed supplement and subsequent
Operation, to realize producing enzyme of continuously fermenting, immobilized cell technique can also reduce the viscosity of fermentation liquid, make the rheology of fermentation liquid
Characteristic is more advantageous to the supply of oxygen and the transmitting of material, in addition, this technology can also protect hyphal cell from shearing force
It acts on, the interference to hyphal cell growth and producing enzyme of the external environments such as reduction pH value, temperature, noxious material, in this programme
Using amino-oligosacchride as fixative.
This programme has the beneficial effect that:
(1) in the present solution, first with the bacterial strain that solid medium culture activates, after strain fermentation producing enzyme to peak value, then picking
Sparassis crispa hypha body is inoculated in shaking flask producing enzyme in fluid nutrient medium, finally carries out fermentor submerged fermentation again, and bacterial strain is purer, fermentation
When, cell age is suitable, and it is higher to produce laccase activity；
(2) mycelia is adsorbed onto surface and fixed by pericarp mixed active charcoal as solid medium, makes mycelia ordering growth, unlikely
In entanglement, it is conducive to mycelia and grows.
(3) inducer and Cu is added2+, accelerate the synthesis of laccase.
(4) using amino-oligosacchride as the fixed bacterial strain of fixative, fermentation efficiency is improved.
Detailed description of the invention
Fig. 1 is laccase activity detection data；
Fig. 2 is that hypha form observes result.
Specific embodiment
In order to make content of the present invention easily facilitate understanding, With reference to embodiment to of the present invention
Technical solution is described further, but the present invention is not limited only to this.
Embodiment 1
It configures slant medium and activates silk ball bacteria strain, weigh 200g potato, 20g glucose, 18g agar powder, 3g
KH2PO4、1.5gMgSO4, vitamin B1,1L distilled water that 1mL concentration is 0.05g/L, configure slant medium, 30 DEG C of cultures embroideries
Meningitidis strains 2 days, silk ball bacteria strain is enable to activate；
Horseshoe skin, pineapple peel, shaddock ped, orange peel, longan skin, each 5g, active carbon are separately added into the culture dish that diameter is 9cm
0.5g, distilled water 25mL after cooling, access silk ball bacteria strain, are placed in 30 DEG C of illumination cultivations, observe in 121 DEG C of sterilizing 30min
Mycelium growth vigor is sampled for measuring laccase activity, until enzyme activity no longer increases for the 6th, 10,15,20 day in culture.
Weigh 1g lignanoid, 1L potato leachate, 20g glucose, 1g peptone, 3gKH2PO4、3gMgSO4•7H2O、
3g(NH4) 2SO4, 10g corn flour, 1gCuSO4•5H2O, 1gFeSO4, 1gMnSO4, 1g tealeaves stirs evenly, and sterilizes and liquid is made
Body culture medium, wherein potato leachate is equipped with the preparation of 1L distilled water with 250g potato, connects in the above-mentioned solid medium of picking
The Sparassis crispa hypha body of kind amount 20%, rejects miscellaneous bacteria, is seeded in fluid nutrient medium, shaking flask culture, shaking table 150rpm, temperature 28
DEG C, illumination cultivation 7 days；
According to the above ratio, expand configuration fluid nutrient medium to 5L, and add 2g amino-oligosacchride as fixative, 1g in fermentor
Biofermentation defoaming agent, the biofermentation defoaming agent are commercial product, and by 10% Sparassis crispa of volume inoculum concentration, ventilatory capacity is
0.5vvm, temperature are 30 DEG C, speed of agitator 200rpm, carry out submerged fermentation, fermentation liquid is laccase crude liquid.
Embodiment 2
It configures slant medium and activates silk ball bacteria strain, weigh 200g potato, 20g glucose, 18g agar powder, 3g
KH2PO4、1.5gMgSO4, vitamin B1,1L distilled water that 1mL concentration is 0.05g/L, configure slant medium, 30 DEG C of cultures embroideries
Meningitidis strains 2 days, silk ball bacteria strain is enable to activate；
Horseshoe skin, pineapple peel, shaddock ped, orange peel, longan skin, each 5g, active carbon are separately added into the culture dish that diameter is 9cm
1g, distilled water 25mL after cooling, access silk ball bacteria strain, are placed in 35 DEG C of illumination cultivations, observe bacterium in 121 DEG C of sterilizing 30min
Filament length gesture is sampled for measuring laccase activity, until enzyme activity no longer increases for the 8th, 12,17,22 day in culture.
Weigh 1g cumarin, 1L potato leachate, 20g glucose, 1g peptone, 3gKH2PO4、3gMgSO4•7H2O、
3g(NH4) 2SO4, 10g corn flour, 1gCuSO4•5H2O, 1gFeSO4, 1gMnSO4, 1g straw stirs evenly, and sterilizes and liquid is made
Body culture medium, wherein potato leachate is equipped with the preparation of 1L distilled water with 250g potato, connects in the above-mentioned solid medium of picking
The Sparassis crispa hypha body of kind amount 20%, rejects miscellaneous bacteria, is seeded in fluid nutrient medium, shaking flask culture, shaking table 150rpm, temperature 28
DEG C, illumination cultivation 7 days；
According to the above ratio, expand configuration fluid nutrient medium to 5L, and add 2g amino-oligosacchride as fixative, 1g in fermentor
Biofermentation defoaming agent, the biofermentation defoaming agent are commercial product, and by 10% Sparassis crispa of volume inoculum concentration, ventilatory capacity is
1.0vvm, temperature are 40 DEG C, speed of agitator 300rpm, carry out submerged fermentation, fermentation liquid is laccase crude liquid.
Embodiment 3
It configures slant medium and activates silk ball bacteria strain, weigh 200g potato, 20g glucose, 18g agar powder, 3g
KH2PO4、1.5gMgSO4, vitamin B1,1L distilled water that 1mL concentration is 0.05g/L, configure slant medium, 30 DEG C of cultures embroideries
Meningitidis strains 2 days, silk ball bacteria strain is enable to activate；
Horseshoe skin, pineapple peel, shaddock ped, orange peel, longan skin, each 5g, active carbon are separately added into the culture dish that diameter is 9cm
0.8g, distilled water 25mL after cooling, access silk ball bacteria strain, are placed in 32 DEG C of illumination cultivations, observe in 121 DEG C of sterilizing 30min
Mycelium growth vigor is sampled for measuring laccase activity, until enzyme activity no longer increases for the 7th, 11,16,21 day in culture.
Weigh 1g chalcone, 1L potato leachate, 20g glucose, 1g peptone, 3gKH2PO4、3gMgSO4•7H2O、
3g(NH4) 2SO4, 10g corn flour, 1gCuSO4•5H2O, 1gFeSO4, 1gMnSO4, 1g straw stirs evenly, and sterilizes and liquid is made
Body culture medium, wherein potato leachate is equipped with the preparation of 1L distilled water with 250g potato, connects in the above-mentioned solid medium of picking
The Sparassis crispa hypha body of kind amount 20%, rejects miscellaneous bacteria, is seeded in fluid nutrient medium, shaking flask culture, shaking table 150rpm, temperature 28
DEG C, illumination cultivation 7 days；
According to the above ratio, expand configuration fluid nutrient medium to 5L, and add 2g amino-oligosacchride as fixative, 1g in fermentor
Biofermentation defoaming agent, the biofermentation defoaming agent are commercial product, and by 10% Sparassis crispa of volume inoculum concentration, ventilatory capacity is
0.8vvm, temperature are 35 DEG C, speed of agitator 250rpm, carry out submerged fermentation, fermentation liquid is laccase crude liquid.
Embodiment 4
It configures slant medium and activates silk ball bacteria strain, weigh 200g potato, 20g glucose, 18g agar powder, 3g
KH2PO4、1.5gMgSO4, vitamin B1,1L distilled water that 1mL concentration is 0.05g/L, configure slant medium, 30 DEG C of cultures embroideries
Meningitidis strains 2 days, silk ball bacteria strain is enable to activate；
Horseshoe skin, pineapple peel, shaddock ped, orange peel, longan skin, each 5g, active carbon are separately added into the culture dish that diameter is 9cm
0.8g, distilled water 25mL after cooling, access silk ball bacteria strain, are placed in 32 DEG C of illumination cultivations, observe in 121 DEG C of sterilizing 30min
Mycelium growth vigor is sampled for measuring laccase activity, until enzyme activity no longer increases for the 7th, 11,16,21 day in culture.
Weigh 1g O-VANILLIN, 1L potato leachate, 20g glucose, 1g peptone, 3gKH2PO4、3gMgSO4•
7H2O、3g(NH4) 2SO4, 10g corn flour, 1gCuSO4•5H2O, 1gFeSO4, 1gMnSO4, 1g straw stirs evenly, sterilizing system
Fluid nutrient medium is obtained, wherein potato leachate is equipped with the preparation of 1L distilled water, the above-mentioned solid medium of picking with 250g potato
The Sparassis crispa hypha body of middle inoculum concentration 20% is rejected miscellaneous bacteria, is seeded in fluid nutrient medium, shaking flask culture, shaking table 150rpm, temperature
28 DEG C, illumination cultivation 7 days of degree；
According to the above ratio, expand configuration fluid nutrient medium to 5L, and add 2g amino-oligosacchride as fixative, 1g in fermentor
Biofermentation defoaming agent, the biofermentation defoaming agent are commercial product, and by 10% Sparassis crispa of volume inoculum concentration, ventilatory capacity is
0.8vvm, temperature are 35 DEG C, speed of agitator 250rpm, carry out submerged fermentation, fermentation liquid is laccase crude liquid.
Embodiment 5
It configures slant medium and activates silk ball bacteria strain, weigh 200g potato, 20g glucose, 18g agar powder, 3g
KH2PO4、1.5gMgSO4, vitamin B1,1L distilled water that 1mL concentration is 0.05g/L, configure slant medium, 30 DEG C of cultures embroideries
Meningitidis strains 2 days, silk ball bacteria strain is enable to activate；
Weigh 1g lignanoid, 1L potato leachate, 20g glucose, 1g peptone, 3gKH2PO4、3gMgSO4•7H2O、3g
(NH4) 2SO4, 10g corn flour, 1gCuSO4•5H2O, 1gFeSO4, 1gMnSO4, 1g tealeaves stirs evenly, and sterilizes and liquid is made
Culture medium, wherein potato leachate is equipped with the preparation of 1L distilled water with 250g potato, is inoculated with Sparassis crispa hypha body, is seeded to liquid
In body culture medium, shaking flask culture, shaking table 150rpm, 28 DEG C of temperature, illumination cultivation 7 days；
According to the above ratio, expand configuration fluid nutrient medium to 5L, and add 2g amino-oligosacchride as fixative, 1g in fermentor
Biofermentation defoaming agent, the biofermentation defoaming agent are commercial product, and by 10% Sparassis crispa of volume inoculum concentration, ventilatory capacity is
0.5vvm, temperature are 30 DEG C, speed of agitator 200rpm, carry out submerged fermentation, fermentation liquid is laccase crude liquid.
Embodiment 6
It configures slant medium and activates silk ball bacteria strain, weigh 200g potato, 20g glucose, 18g agar powder, 3g
KH2PO4、1.5gMgSO4, vitamin B1,1L distilled water that 1mL concentration is 0.05g/L, configure slant medium, 30 DEG C of cultures embroideries
Meningitidis strains 2 days, silk ball bacteria strain is enable to activate；
Horseshoe skin, pineapple peel, shaddock ped, orange peel, longan skin, each 5g, distilled water are separately added into the culture dish that diameter is 9cm
25mL after cooling, accesses silk ball bacteria strain, is placed in 30 DEG C of illumination cultivations, observe mycelium growth vigor in 121 DEG C of sterilizing 30min,
Sampling in 6th, 10,15,20 day of culture is for measuring laccase activity, until enzyme activity no longer increases.
Weigh 1g lignanoid, 1L potato leachate, 20g glucose, 1g peptone, 3gKH2PO4、3gMgSO4•7H2O、
3g(NH4) 2SO4, 10g corn flour, 1gCuSO4•5H2O, 1gFeSO4, 1gMnSO4, 1g tealeaves stirs evenly, and sterilizes and liquid is made
Body culture medium, wherein potato leachate is equipped with the preparation of 1L distilled water with 250g potato, connects in the above-mentioned solid medium of picking
The Sparassis crispa hypha body of kind amount 20%, rejects miscellaneous bacteria, is seeded in fluid nutrient medium, shaking flask culture, shaking table 150rpm, temperature 28
DEG C, illumination cultivation 7 days；
According to the above ratio, expand configuration fluid nutrient medium to 5L, and add 2g amino-oligosacchride as fixative, 1g in fermentor
Biofermentation defoaming agent, the biofermentation defoaming agent are commercial product, and by 10% Sparassis crispa of volume inoculum concentration, ventilatory capacity is
0.5vvm, temperature are 30 DEG C, speed of agitator 200rpm, carry out submerged fermentation, fermentation liquid is laccase crude liquid.
Embodiment 7
It configures slant medium and activates silk ball bacteria strain, weigh 200g potato, 20g glucose, 18g agar powder, 3g
KH2PO4、1.5gMgSO4, vitamin B1,1L distilled water that 1mL concentration is 0.05g/L, configure slant medium, 30 DEG C of cultures embroideries
Meningitidis strains 2 days, silk ball bacteria strain is enable to activate；
Horseshoe skin, pineapple peel, shaddock ped, orange peel, longan skin, each 5g, distilled water are separately added into the culture dish that diameter is 9cm
25mL after cooling, accesses silk ball bacteria strain, is placed in 30 DEG C of illumination cultivations, observe mycelium growth vigor in 121 DEG C of sterilizing 30min,
Sampling in 6th, 10,15,20 day of culture is for measuring laccase activity, until enzyme activity no longer increases.
Weigh 1L potato leachate, 20g glucose, 1g peptone, 3gKH2PO4、3gMgSO4•7H2O、3g(NH4)2SO4, 10g corn flour, 1gFeSO4, 1gMnSO4, 1g tealeaves stirs evenly, and sterilizes and fluid nutrient medium is made, and wherein potato is soaked
Liquid is equipped with the preparation of 1L distilled water with 250g potato out, the Sparassis crispa hypha body of inoculum concentration 20% in the above-mentioned solid medium of picking,
Miscellaneous bacteria is rejected, is seeded in fluid nutrient medium, shaking flask culture, shaking table 150rpm, 28 DEG C of temperature, illumination cultivation 7 days；
According to the above ratio, expand configuration fluid nutrient medium to 5L, and add 2g amino-oligosacchride as fixative, 1g in fermentor
Biofermentation defoaming agent, the biofermentation defoaming agent are commercial product, and by 10% Sparassis crispa of volume inoculum concentration, ventilatory capacity is
0.5vvm, temperature are 30 DEG C, speed of agitator 200rpm, carry out submerged fermentation, fermentation liquid is laccase crude liquid.
Embodiment 8
Measure each stage enzyme activity in embodiment 1-7, measuring method are as follows: the ABTS in 4ml reaction system containing 0.5mmol/L is as anti-
Answer substrate, lemon acid acid-lemon acid sodium buffer (pH4.0) of 0.1mol/L, the diluted crude enzyme liquid of 1ml.35 DEG C of reactions 5min, Yu Bo
Absorbance is surveyed at long 420nm, takes the linear segment of absorbance change, measures protein content by Bradford method.Definition: every point
Enzyme amount needed for the 1 μm of olABST oxidation of clock catalysis oxidation is an enzyme activity unit, and unit is indicated with U/g, as shown in table 1.
The hypha form in embodiment 1-7 fermentor is observed, determines biomass, as described in Table 2.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
Claims (10)
1. the preparation method that a kind of Sparassis crispa produces laccase, which comprises the following steps: (1) take mixing pericarp as hair
Ferment matrix, mixed active charcoal carry out homogeneous, and filtering after sterilizing, obtains pericarp solid medium；(2) in pericarp solid medium
It is inoculated with the silk ball bacteria strain activated, carries out one time fermentation culture；(3) laccase activity monitoring is carried out to one time fermentation product, to
Enzyme activity reaches the middle gained Sparassis crispa hypha body of picking step (2) after peak value, is inoculated in containing Cu2+And the fluid nutrient medium of inducer
In, fermentor is transferred to after shaking flask producing enzyme；(4) amino-oligosacchride and biofermentation defoaming agent are added in the fermenter, are carried out secondary
Submerged fermentation, tunning are that Sparassis crispa produces laccase.
2. the preparation method that a kind of Sparassis crispa according to claim 1 produces laccase, which is characterized in that the inducer includes
One or more of lignanoid, cumarin, chalcone, O-VANILLIN.
3. the preparation method that a kind of Sparassis crispa according to claim 1 produces laccase, which is characterized in that the pericarp such as is at the matter
The horseshoe skin of amount, pineapple peel, shaddock ped, orange peel, longan skin mixture.
4. the preparation method that a kind of Sparassis crispa according to claim 1 produces laccase, which is characterized in that the active carbon adds
Dosage is the 1/10-1/5 for mixing pericarp quality.
5. the preparation method that a kind of Sparassis crispa according to claim 1 produces laccase, which is characterized in that the one time fermentation training
Feeding temperature is 30 DEG C -35 DEG C, is fermented under illumination.
6. the preparation method that a kind of Sparassis crispa according to claim 1 produces laccase, which is characterized in that the laccase activity prison
It is the 6-8 days after one time fermentation at the beginning of survey.
7. the preparation method that a kind of Sparassis crispa according to claim 1 produces laccase, which is characterized in that the fluid nutrient medium
Ingredient includes 1 part of inducer, potato leachate, 20 parts of glucose, 1 part of peptone, 3 parts of KH2PO4, 3 parts of MgSO4·7H2O、3
Part (NH4) 2SO4, 10 portions of corn flour, 1 part of CuSO4·5H2O, 1 part of FeSO4, 1 part of MnSO4, 1 part of natural substrate, with potato matter
Amount is 250 parts of meters.
8. the preparation method that a kind of Sparassis crispa according to claim 7 produces laccase, which is characterized in that the natural substrate is
One or more of tealeaves, straw, wheat straw, peanut shell, dregs of beans, bagasse, tangerine peel, cornstalk, wheat bran.
9. the preparation method that a kind of Sparassis crispa according to claim 1 produces laccase, which is characterized in that the shaking flask producing enzyme connects
It plants and measures 20%, shaking table 150rpm, 28 DEG C of temperature.
10. the preparation method that a kind of Sparassis crispa according to claim 1 produces laccase, which is characterized in that the secondary deep layer
The inoculum concentration of fermentation is 10%, ventilatory capacity 0.5-1.0vvm, and temperature is 30-40 DEG C, speed of agitator 200-300rpm.
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Application publication date: 20190830 |