CN103571801A - Fermentation production method of fungal laccase and application of laccase - Google Patents
Fermentation production method of fungal laccase and application of laccase Download PDFInfo
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- CN103571801A CN103571801A CN201210270321.4A CN201210270321A CN103571801A CN 103571801 A CN103571801 A CN 103571801A CN 201210270321 A CN201210270321 A CN 201210270321A CN 103571801 A CN103571801 A CN 103571801A
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- 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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- 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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- D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
- D21C9/00—After-treatment of cellulose pulp, e.g. of wood pulp, or cotton linters ; Treatment of dilute or dewatered pulp or process improvement taking place after obtaining the raw cellulosic material and not provided for elsewhere
- D21C9/10—Bleaching ; Apparatus therefor
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Abstract
The invention discloses a fermentation production method of fungal laccase and an application of the laccase. The method comprises the following steps: preparing Panus conchatus suspension; inoculating the suspension into a fermentation tank containing 3-5L of fermentation medium and culturing for 10-15 days, thus obtaining fermentation liquor; and finally, filtering or centrifuging the fermentation liquor, thus obtaining a laccase preparation. The method of the invention is simple and convenient in subsequent treatment of the fermentation liquor and low in production cost, and the activity of the Panus conchatus laccase obtained through fermentation is over 200IU/mL. Compared with other domestic and overseas laccase production methods in a fermentation tank scale, the fermentation production method of the fungal laccase, which is disclosed by the invention, has the advantages that the total laccase yield and the laccase activity reach a relatively high level, and the prepared laccase preparation has a good pulp bleaching effect.
Description
Technical field
The present invention relates to fungi fermentation technical field, the preparation method who particularly relates to a kind of industrial laccase is exactly that a kind of white-rot fungi cultivation and fermentation in 5L scale fermentor tank is produced the method for laccase and laccase preparation prepared by the described method application in association with pulp bleaching.
Background technology
Laccase (Laccase, EC.1.10.3.2) is a kind of polyphenoloxidase of cupric, belongs to covellite oxydase family with the Vitamin C oxidase in plant, the copper-protein in mammalian plasma.In recent ten years, fungal laccase, especially the function and application of White-rot Fungi has caused increasing investigator's concern, because it has higher redox-potential, effect substrate scope is very extensive, the energy multiple phenol of catalyzed oxidation and aromatic amine compounds, under the condition existing at some small molecules redox mediators, non-phenol type structure in can also oxidative lignin, so there are huge researching value and industrial applications potentiality in the field such as the association with pulp bleaching of White-rot Fungi in pulp and paper industry and wastewater treatment.But the successful Application of White-rot Fungi in above-mentioned field, needs the critical problem solving is how under the prerequisite of controlling cost, to improve the output of laccase at present.Optimization of fermentation conditions with improve whiterot fungi enzymatic productivity and reduce produce and application cost be a kind of be substantially the most also the most frequently used Research Thinking.The scholar's research such as Borras whiterot fungi Trametes versicolor in wort semisynthetic medium, the cost control problem that matching stain orchid is received in free and easy grey decolorization, the expense of discovery culture medium raw material accounts for the ratio of decolouring total cost up to 95%, and pass through optimum culture condition, select at a low price without wort synthetic medium, can be by decolouring cost 96%, and constant (the Borras E of decolorizing effect, Blanquez P, Sarra M, et al.Trametes versicolor pellets production:low-cost medium and scale-up.Biochemical Engineering Journal, 2008, 42:61-66).The another kind of thinking of White-rot Fungi High-efficient Production is to find the heterogenous expression that suitable carrier carries out laccase gene, but because the existence of glycosyl in White-rot Fungi can impel the hydrolysis of laccase albumen, therefore with respect to some other, realized the oxydo-reductase of suitability for industrialized production, as the glucose oxidase of the gene recombination production by filamentous fungus, the high efficient expression of the allos of White-rot Fungi on active host is also comparatively difficult, also in the exploratory stage, be difficult to meet the requirement of laccase suitability for industrialized production and application generally.Therefore the current research about laccase production mainly still concentrates on the fermentation condition of optimizing whiterot fungi.
Whiterot fungi Panus conchatus is the basidiomycete of a strain secretion laccase, its lignocellulolytic enzymes cording has higher delignification selectivity, wherein laccase is its topmost lignin-degrading enzymes, in shaking flask liquid fermenting, show higher laccase output, there is higher research and application potential.The successful Application of this White-rot Fungi in the fields such as association with pulp bleaching and wastewater treatment, not only to improve the output of laccase, reduce its production cost, also will depend on ripe industrial fermentation technology, and wherein fermentor tank scale production is this White-rot Fungi key link and the only way which must be passed from laboratory study to suitability for industrialized production transition.The microbial liquid of ferment tank scale is cultivated and is commonly referred to submerged fermentation (Submerged fermentation, SmF), mechanical agitation type fermentor tank (Stirred tank reactor, STR) be current the most frequently used submerged fermentation equipment, form standardized general-purpose equipment, be suitable for the cultivation and fermentation process of most of microbe.It is simple in structure, do not need specific installation, utilize the effect of mechanical stirrer, thalline, fermented liquid, air and heat etc. are fully mixed, promote the dissolving with oxygen that is uniformly distributed of material, to guarantee to supply with microorganism growth breeding and required nutrition, temperature and the dissolved oxygen etc. of metabolism.When but this fermentor tank is used for the cultivation and fermentation of whiterot fungi, also there are some unfavorable factors, be mainly manifested in tank body the growth of Dead White Rot Fungus wayward, the unordered expansion of mycelia can improve the viscosity of fermented liquid, and be wrapped on paddle wheel, blocking pipeline, has a strong impact on the even transmission of material, heat and oxygen.Although can improve the problems referred to above by improving mixing speed, too high rotating speed can cause the shearing force that mycelia is subject to increase, and suppresses the secretion of cell proliferation metabolism and laccase, even causes cell rupture, and then causes that laccase yields poorly, enzyme work is low.
At present, about the report of the large scale fermentation technique of White-rot Fungi maturation also seldom, and do not solve all the time production cost problem, this is mainly that the production of whiterot fungi is wayward because exist the transmission characteristic with the diverse power of shake flask fermentation, heat, material and oxygen etc. in fermentor tank.Therefore, exploitation adapts to the whiterot fungi bacterial classification of laccase scale operation, the existing fermentor tank production technique of improvement and development of new fermentation equipment, simplify zymotechnique, reduce production costs, with the production of the White-rot Fungi of magnifying, become the active study hotspot in laccase production field.
Summary of the invention
The technical problem that the present invention mainly solves is to provide a kind of fermentation generation method of fungal laccase and the application of laccase, not only fermented liquid subsequent disposal is easy, low production cost, and the White-rot Fungi vigor that fermentation obtains is more than 200IU/mL, compare with the laccase production method of domestic and international other fermentor tank scales, laccase ultimate production and enzyme activity have all reached higher level, and the laccase preparation making has good association with pulp bleaching effect.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is: a kind of fermentation method for producing of fungal laccase is provided, said method comprising the steps of:
1) fungi is inoculated on seed culture medium and cultivates 2～4 days, the mycelium that nutrient solution obtains after sterile gauze filters is cleaned with sterilized water, mycelium is broken up at a high speed after approximately 10 seconds again and be collected in sterilized water and make bacterial classification suspension, described fungi is whiterot fungi Panus conchatus;
2) described bacterial classification suspension is inoculated in containing in the fermentor tank of 3～5L fermention medium and cultivates after 10～15 days, obtain fermented liquid;
3) by described filtering fermentation liquor or centrifugal rear acquisition laccase preparation.
Wherein, every liter of described seed culture medium is containing 20%～30% potato leach liquor, 10～50g glucose, 1～10g peptone, 3gKH
2pO
4, 3gMgSO
47H
2o.
Wherein, described fermention medium is in every liter of seed culture medium, to add 5～20g natural substrate and 1～5mmol inductor.
Wherein, described natural substrate is one or more in tealeaves, straw, wheat straw, Pericarppium arachidis hypogaeae, wheat bran, dregs of beans, bagasse, tangerine peel, milled powders of cornstalk.
Preferably, described natural substrate is wheat bran.
Wherein, described inductor is one or more in Li Lu alcohol, methyl catechol, xylidene(s), forulic acid, syringic aldehyde, Weibull, vanillic acid, coumaric acid, copper sulfate.
Preferably, described inductor is copper sulfate.
Wherein, the culture temperature described step 1) is that 28 ℃, rotating speed are 120～180rpm.
Wherein, described step 2) culture temperature in is 28 ℃～40 ℃, and stirring velocity is 150～300rpm.
In the present invention, with 2,2-azine-bis-, (3-ethyl benzothiazole-6-sulfonic acid (ABTS) is substrate (ε to the mensuration of laccase activity
420=3.6 * 10
4m
-1cm
-1), with isopyknic 0.5mmol/LABTS solution and crude enzyme liquid reaction, the increasing amount of 420nm place light absorption value in 3min before assaying reaction, it is 1 laccase activity unit (IU) that definition per minute makes 1 μ mol ABTS transform needed enzyme amount.
The invention still further relates to the application in association with pulp bleaching of the laccase preparation that obtains by described method.
The invention has the beneficial effects as follows: the situation that White-rot Fungi yields poorly, enzyme activity is low, production cost is high that is different from existing ferment tank fermentative production, the present invention expands the production of White-rot Fungi to 5L fermentor tank by shaking flask, industrial scale expands approximately 100 times, not only fermented liquid subsequent disposal is easy, low production cost, and the White-rot Fungi vigor that fermentation obtains is more than 200IU/mL, compare with the laccase production method of domestic and international other fermentor tank scales, laccase ultimate production and enzyme activity have all reached higher level.In addition the laccase preparation making, has good bleaching effect during for association with pulp bleaching.
Embodiment
Below in conjunction with embodiment, the present invention is described in detail.
In the present invention, whiterot fungi Panus conchatus is provided by Pulp and Paper Engineering National Key Laboratory of South China Science & Engineering University.
Embodiment 1
The preparation of potato leach liquor: take 300g fresh potato, boil 30min after peeling section in 1000mL deionized water, cooling rear by filtered through gauze, filtrate is settled to 1000mL with deionized water.
PDA substratum: add dextrose anhydrous 20g in every liter of potato leach liquor, agar 20g.
Seed culture medium: add dextrose anhydrous 20g in every liter of potato leach liquor, peptone 1g, KH
2pO
43g, MgSO
47H
2o 3g.
Fermention medium: add the wheat bran 10g through pulverizing in every liter of seed culture medium, fermentation dedicated defoamer 0.2mL, copper sulfate 3mmol.
1, the preparation of bacterial classification suspension
Whiterot fungi Panus conchatus is transferred and is equipped with in the 300mL triangular flask of 50mL seed culture medium by PDA slant medium, in shaking table, 150rpm, 28 ℃ of concussions are cultivated after 3d, nutrient solution filters through sterile gauze, mycelium is cleaned with sterilized water and break up about 10s in being smashed cup at a high speed, is collected in that in sterilized water, to make bacterial classification suspension standby.
2, fermentor tank produces enzyme
The bacterial classification suspension preparing is inoculated in the fermentor tank that fermention medium is housed under aseptic condition to fermentor tank liquid amount 3L, inoculum size 500mL.The fermentor tank that the present invention adopts is 5L full-automatic mechanical stirred-tank fermenter, and its every operating parameter can regulate by the digital controller being connected with it.After inoculation, controlling mechanical mixing speed is 150rpm, and after fermentation for some time, due to mycelium amount reproduction, fermented liquid viscosity enlarges markedly, and therefore mixing speed is increased to 300rpm to promote being uniformly distributed of material.Culture-liquid temp maintains 28 ℃ by circulating condensing water, passes into air to maintain the oxygen dissolved of fermented liquid in fermentor tank simultaneously, and air flow is 1.0Lpm, and total fermentation time is 15 days.The biological fermentation dedicated defoamer adding in fermention medium can be avoided the obstruction to pipeline of foam that bacterial metabolism and mechanical stirring produce.
After fermentation, measure enzyme and live, laccase activity is up to more than 200IU/mL.
3, the acquisition of laccase preparation
Fermented liquid after gauze removes by filter mycelium and wheat bran residue, 5000r/min frozen centrifugation 15min at 4 ℃, fine particulate material is removed in separation, collects supernatant liquor and obtains industrial finish zymin.
Laccase preparation is for association with pulp bleaching
1, bleaching conditions:
Laccase treatment (L): paper pulp (starching dense 10%) is depressed at 0.5MPa oxygen, in the buffered soln of 50 ℃ and pH5.0, add the laccase (to oven dry stock) of 2.0IU/g the present embodiment acquisition and 1% nitro humus acid (nitrohumic acid, NHA) (amboceptor), in the autoclave with electric stirring, process 2h, clean.
Oxygen delignification (O): oxygen delignification is to carry out in the autoclave with electric stirring.Condition is: starch dense 10%, MgSO
4consumption 0.5%, NaOH consumption 2.0%, oxygen is pressed as 0.7MPa, time 60min.
Chelation treatment (Q): starch dense 10%, 1.0%EDTA, pH3.0, temperature 70 C, time 60min.
Hydrogen peroxide bleaching (P): starch dense 10%, H
2o
22.0%, NaOH2.0%, MgSO
40.05%., 90 ℃ of temperature, time 4h.
2, bleaching effect: obtain reed pulp through the laccase treatment (L) of consumption 2.0IU/g (to oven dry stock), Kappa value reduces by 17.8%, whiteness decline 3.4%ISO, viscosity changes less, after QP bleaching, whiteness reaches 72.5%ISO again, compared with QP pulp brightness, increases 3.3%ISO, viscosity 1250ml/g, than the high 113mL/g of reed reed QP slurry viscosity.Reed reed OLQP slurry, whiteness reaches 83.2%ISO, than OQP pulp brightness rising 8.2%ISO.Kappa value significantly declines, and viscosity degradation is few.
Embodiment 2
The preparation of potato leach liquor: take 300g fresh potato, boil 30min after peeling section in 1000mL deionized water, cooling rear by filtered through gauze, filtrate is settled to 1000mL with deionized water.
PDA substratum: add dextrose anhydrous 20g in every liter of potato leach liquor, agar 20g.
Seed culture medium: add dextrose anhydrous 10g in every liter of potato leach liquor, peptone 10g, KH
2pO
43g, MgSO
47H
2o3g.
Fermention medium: add the Pericarppium arachidis hypogaeae 20g through pulverizing in every liter of seed culture medium, fermentation dedicated defoamer 0.2mL, Li Lu alcohol 5mmol.
1, the preparation of bacterial classification suspension
Whiterot fungi Panus conchatus is transferred and is equipped with in the 300mL triangular flask of 50mL seed culture medium by PDA slant medium, in shaking table, 150rpm, 28 ℃ of concussions are cultivated after 3d, nutrient solution filters through sterile gauze, mycelium is cleaned with sterilized water and break up about 10s in being smashed cup at a high speed, is collected in that in sterilized water, to make bacterial classification suspension standby.
2, fermentor tank produces enzyme
The bacterial classification suspension preparing is inoculated in the fermentor tank that fermention medium is housed under aseptic condition to fermentor tank liquid amount 3L, inoculum size 500mL.The fermentor tank that the present invention adopts is 5L full-automatic mechanical stirred-tank fermenter, and its every operating parameter can regulate by the digital controller being connected with it.After inoculation, controlling mechanical mixing speed is 150rpm, and after fermentation for some time, due to mycelium amount reproduction, fermented liquid viscosity enlarges markedly, and therefore mixing speed is increased to 300rpm to promote being uniformly distributed of material.Culture-liquid temp maintains 28 ℃ by circulating condensing water, passes into air to maintain the oxygen dissolved of fermented liquid in fermentor tank simultaneously, and air flow is 1.0Lpm, and total fermentation time is 10 days.The biological fermentation dedicated defoamer adding in fermention medium can be avoided the obstruction to pipeline of foam that bacterial metabolism and mechanical stirring produce.
After fermentation, measure enzyme and live, laccase activity is up to more than 200IU/mL.
3, the acquisition of laccase preparation
Fermented liquid after gauze removes by filter mycelium and Pericarppium arachidis hypogaeae residue, 5000r/min frozen centrifugation 15min at 4 ℃, fine particulate material is removed in separation, collects supernatant liquor and obtains industrial finish zymin.
Laccase preparation is for association with pulp bleaching
1, bleaching conditions:
Laccase treatment (L): paper pulp (starching dense 10%) is depressed at 0.5MPa oxygen, in the buffered soln of 50 ℃ and pH5.0, add the laccase (to oven dry stock) of 2.0IU/g the present embodiment acquisition and 1% nitro humus acid (nitrohumic acid, NHA) (amboceptor), in the autoclave with electric stirring, process 2h, clean.
Oxygen delignification (O): oxygen delignification is to carry out in the autoclave with electric stirring.Condition is: starch dense 10%, MgSO
4consumption 0.5%, NaOH consumption 2.0%, oxygen is pressed as 0.7MPa, time 60min.
Chelation treatment (Q): starch dense 10%, 1.0%EDTA, pH3.0, temperature 70 C, time 60min.
Hydrogen peroxide bleaching (P): starch dense 10%, H
2o
22.0%, NaOH 2.0%, MgSO
40.05%., 90 ℃ of temperature, time 4h.
2, bleaching effect: Straw Pulp is through the laccase treatment (L) of consumption 2.0IU/g, and Kappa value reduces by 8.7%, whiteness decline 1%ISO, viscosity changes less, then after QP bleaching, whiteness 74.6%ISO, compared with QP pulp brightness, increase 1.9%ISO, viscosity ratio QP slurry declines a bit.Oxygen delignification Straw Pulp is through the laccase treatment of consumption 2.0IU/g, Kappa value, viscosity degradation.OLQP slurry, whiteness reaches 81.8%ISO, than OQP pulp brightness rising 2.0%ISO.
Embodiment 3
The preparation of Ma Lingzhu leach liquor: take 200g fresh potato, boil 30min after peeling section in 1000mL deionized water, cooling rear by filtered through gauze, filtrate is settled to 1000mL with deionized water.
PDA substratum: add dextrose anhydrous 20g in every liter of potato leach liquor, agar 20g.
Seed culture medium: add dextrose anhydrous 50g in every liter of potato leach liquor, peptone 1g, KH
2pO
43g, MgSO
47H
2o3g.
Fermention medium: add the dregs of beans 5g through pulverizing in every liter of seed culture medium, fermentation dedicated defoamer 0.2mL, methyl catechol 1mmol.
1, the preparation of bacterial classification suspension
Whiterot fungi Panus conchatus is transferred and is equipped with in the 300mL triangular flask of 50mL seed culture medium by PDA slant medium, in shaking table, 180rpm, 28 ℃ of concussions are cultivated after 2d, nutrient solution filters through sterile gauze, mycelium is cleaned with sterilized water and break up about 10s in being smashed cup at a high speed, is collected in that in sterilized water, to make bacterial classification suspension standby.
2, fermentor tank produces enzyme
The bacterial classification suspension preparing is inoculated in the fermentor tank that fermention medium is housed under aseptic condition to fermentor tank liquid amount 4L, inoculum size 500mL.The fermentor tank that the present invention adopts is 5L full-automatic mechanical stirred-tank fermenter, and its every operating parameter can regulate by the digital controller being connected with it.After inoculation, controlling mechanical mixing speed is 150rpm, and after fermentation for some time, due to mycelium amount reproduction, fermented liquid viscosity enlarges markedly, and therefore mixing speed is increased to 250rpm to promote being uniformly distributed of material.Culture-liquid temp maintains 30 ℃ by circulating condensing water, passes into air to maintain the oxygen dissolved of fermented liquid in fermentor tank simultaneously, and air flow is 1.0Lpm, and total fermentation time is 11 days.The biological fermentation dedicated defoamer adding in fermention medium can be avoided the obstruction to pipeline of foam that bacterial metabolism and mechanical stirring produce.
After fermentation, measure enzyme and live, laccase activity is up to more than 200IU/mL.
3, the acquisition of laccase preparation
Fermented liquid after gauze removes by filter mycelium and dregs of beans residue, 5000r/min frozen centrifugation 15min at 4 ℃, fine particulate material is removed in separation, collects supernatant liquor and obtains industrial finish zymin.
Laccase preparation is for association with pulp bleaching
1, bleaching conditions:
Laccase treatment (L): paper pulp (starching dense 10%) is depressed at 0.5MPa oxygen, in the buffered soln of 50 ℃ and pH5.0, add the laccase (to oven dry stock) of 2.0IU/g the present embodiment acquisition and 1% nitro humus acid (nitrohumic acid, NHA) (amboceptor), in the autoclave with electric stirring, process 2h, clean.
Oxygen delignification (O): oxygen delignification is to carry out in the autoclave with electric stirring.Condition is: starch dense 10%, MgSO
4consumption 0.5%, NaOH consumption 2.0%, oxygen is pressed as 0.7MPa, time 60min.
Chelation treatment (Q): starch dense 10%, 1.0%EDTA, pH3.0, temperature 70 C, time 60min.
Hydrogen peroxide bleaching (P): starch dense 10%, H
2o
22.0%, NaOH2.0%, MgSO
40.05%., 90 ℃ of temperature, time 4h.
2, bleaching effect: reed pulp is through the laccase treatment of consumption 2.0IU/g, and Kappa value reduces by 17.8%, whiteness decline 2.2%ISO, viscosity changes little, then after QP bleaching, whiteness reaches 74.7%ISO, compared with QP pulp brightness, increase 3.4%ISO, viscosity 1250ml/g is higher than QP slurry.Oxygen delignification reed pulp is through the laccase treatment of consumption 2.0IU/g, Kappa value, viscosity degradation.OLQP slurry, whiteness reaches 84.1%ISO, than the high 6.8%ISO of the whiteness of OQP bleached pulp, and the viscosity 65mL/g that only declines.
Embodiment 4
The preparation of potato leach liquor: take 280g fresh potato, boil 30min after peeling section in 1000mL deionized water, cooling rear by filtered through gauze, filtrate is settled to 1000mL with deionized water.
PDA substratum: add dextrose anhydrous 20g in every liter of potato leach liquor, agar 20g.
Seed culture medium: add dextrose anhydrous 20g in every liter of potato leach liquor, peptone 8g, KH
2pO
43g, MgSO
47H
2o3g.
Fermention medium: add the straw 15g through pulverizing in every liter of seed culture medium, fermentation dedicated defoamer 0.2mL, forulic acid 2mmol.
1, the preparation of bacterial classification suspension
Whiterot fungi Panus conchatus is transferred and is equipped with in the 300mL triangular flask of 50mL seed culture medium by PDA slant medium, in shaking table, 120rpm, 28 ℃ of concussions are cultivated after 4d, nutrient solution filters through sterile gauze, mycelium is cleaned with sterilized water and break up about 10s in being smashed cup at a high speed, is collected in that in sterilized water, to make bacterial classification suspension standby.
2, fermentor tank produces enzyme
The bacterial classification suspension preparing is inoculated in the fermentor tank that fermention medium is housed under aseptic condition to fermentor tank liquid amount 5L, inoculum size 500mL.The fermentor tank that the present invention adopts is 5L full-automatic mechanical stirred-tank fermenter, and its every operating parameter can regulate by the digital controller being connected with it.After inoculation, controlling mechanical mixing speed is 150rpm, and after fermentation for some time, due to mycelium amount reproduction, fermented liquid viscosity enlarges markedly, and therefore mixing speed is increased to 200rpm to promote being uniformly distributed of material.Culture-liquid temp maintains 35 ℃ by circulating condensing water, passes into air to maintain the oxygen dissolved of fermented liquid in fermentor tank simultaneously, and air flow is 1.0Lpm, and total fermentation time is 12 days.The biological fermentation dedicated defoamer adding in fermention medium can be avoided the obstruction to pipeline of foam that bacterial metabolism and mechanical stirring produce.
After fermentation, measure enzyme and live, laccase activity is up to more than 200IU/mL.
3, the acquisition of laccase preparation
Fermented liquid after gauze removes by filter mycelium and straw residue, 5000r/min frozen centrifugation 15min at 4 ℃, fine particulate material is removed in separation, collects supernatant liquor and obtains industrial finish zymin.
Laccase preparation is for association with pulp bleaching
1, bleaching conditions:
Laccase treatment (L): paper pulp (starching dense 10%) is depressed at 0.5MPa oxygen, in the buffered soln of 50 ℃ and pH5.0, add the laccase (to oven dry stock) of 2.0IU/g the present embodiment acquisition and 1% nitro humus acid (nitrohumic acid, NHA) (amboceptor), in the autoclave with electric stirring, process 2h, clean.
Oxygen delignification (O): oxygen delignification is to carry out in the autoclave with electric stirring.Condition is: starch dense 10%, MgSO
4consumption 0.5%, NaOH consumption 2.0%, oxygen is pressed as 0.7MPa, time 60min.
Chelation treatment (Q): starch dense 10%, 1.0%EDTA, pH3.0, temperature 70 C, time 60min.
Hydrogen peroxide bleaching (P): starch dense 10%, H
2o
22.0%, NaOH 2.0%, MgSO
40.05%., 90 ℃ of temperature, time 4h.
2, bleaching effect: Eucalyptus Kraft Pulp is through oxygen delignification (O), the laccase treatment (L) of consumption 2.0IU/g (to oven dry stock) then, then through QP bleaching, pulp brightness can reach 85%ISO, the viscosity degradation of paper pulp is seldom.
Embodiment 5
The preparation of potato leach liquor: take 250g fresh potato, boil 30min after peeling section in 1000mL deionized water, cooling rear by filtered through gauze, filtrate is settled to 1000mL with deionized water.
PDA substratum: add dextrose anhydrous 20g in every liter of potato leach liquor, agar 20g.
Seed culture medium: add dextrose anhydrous 30g in every liter of potato leach liquor, peptone 6g, KH
2pO
43g, MgSO
47H
2o3g.
Fermention medium: add the bagasse 10g through pulverizing in every liter of seed culture medium, fermentation dedicated defoamer 0.2mL, syringic aldehyde 3mmol.
1, the preparation of bacterial classification suspension
Whiterot fungi Panus conchatus is transferred and is equipped with in the 300mL triangular flask of 50mL seed culture medium by PDA slant medium, in shaking table, 140rpm, 28 ℃ of concussions are cultivated after 3d, nutrient solution filters through sterile gauze, mycelium is cleaned with sterilized water and break up about 10s in being smashed cup at a high speed, is collected in that in sterilized water, to make bacterial classification suspension standby.
2, fermentor tank produces enzyme
The bacterial classification suspension preparing is inoculated in the fermentor tank that fermention medium is housed under aseptic condition to fermentor tank liquid amount 3L, inoculum size 500mL.The fermentor tank that the present invention adopts is 5L full-automatic mechanical stirred-tank fermenter, and its every operating parameter can regulate by the digital controller being connected with it.After inoculation, controlling mechanical mixing speed is 150rpm, and after fermentation for some time, due to mycelium amount reproduction, fermented liquid viscosity enlarges markedly, and therefore mixing speed is increased to 300rpm to promote being uniformly distributed of material.Culture-liquid temp maintains 40 ℃ by circulating condensing water, passes into air to maintain the oxygen dissolved of fermented liquid in fermentor tank simultaneously, and air flow is 1.0Lpm, and total fermentation time is 13 days.The biological fermentation dedicated defoamer adding in fermention medium can be avoided the obstruction to pipeline of foam that bacterial metabolism and mechanical stirring produce.
After fermentation, measure enzyme and live, laccase activity is up to more than 200IU/mL.
3, the acquisition of laccase preparation
Fermented liquid after gauze removes by filter mycelium and bagasse, 5000r/min frozen centrifugation 15min at 4 ℃, fine particulate material is removed in separation, collects supernatant liquor and obtains industrial finish zymin.
Laccase preparation is for association with pulp bleaching
1, bleaching conditions:
Laccase treatment (L): paper pulp (starching dense 10%) is depressed at 0.5MPa oxygen, in the buffered soln of 50 ℃ and pH5.0, add the laccase (to oven dry stock) of 2.0IU/g the present embodiment acquisition and 1% nitro humus acid (nitrohumic acid, NHA) (amboceptor), in the autoclave with electric stirring, process 2h, clean.
Oxygen delignification (O): oxygen delignification is to carry out in the autoclave with electric stirring.Condition is: starch dense 10%, MgSO
4consumption 0.5%, NaOH consumption 2.0%, oxygen is pressed as 0.7MPa, time 60min.
Chelation treatment (Q): starch dense 10%, 1.0%EDTA, pH3.0, temperature 70 C, time 60min.
Hydrogen peroxide bleaching (P): starch dense 10%, H
2o
22.0%, NaOH2.0%, MgSO
40.05%., 90 ℃ of temperature, time 4h.
2, bleaching effect: masson s pine pulp is through oxygen delignification (O), the laccase treatment (L) of consumption 2.0IU/g (to oven dry stock) then, then through QP bleaching, pulp brightness can reach 83%ISO, the viscosity degradation of paper pulp is seldom.
Embodiment 6
The preparation of potato leach liquor: take 220g fresh potato, boil 30min after peeling section in 1000mL deionized water, cooling rear by filtered through gauze, filtrate is settled to 1000mL with deionized water.
PDA substratum: add dextrose anhydrous 20g in every liter of potato leach liquor, agar 20g.
Seed culture medium: add dextrose anhydrous 40g in every liter of potato leach liquor, peptone 4g, KH
2pO
43g, MgSO
47H
2o3g.
Fermention medium: add the corn stalk 18g through pulverizing in every liter of seed culture medium, fermentation dedicated defoamer 0.2mL, vanillic acid 4mmol.
1, the preparation of bacterial classification suspension
Whiterot fungi Panus conchatus is transferred and is equipped with in the 300mL triangular flask of 50mL seed culture medium by PDA slant medium, in shaking table, 160rpm, 28 ℃ of concussions are cultivated after 3d, nutrient solution filters through sterile gauze, mycelium is cleaned with sterilized water and break up about 10s in being smashed cup at a high speed, is collected in that in sterilized water, to make bacterial classification suspension standby.
2, fermentor tank produces enzyme
The bacterial classification suspension preparing is inoculated in the fermentor tank that fermention medium is housed under aseptic condition to fermentor tank liquid amount 3L, inoculum size 500mL.The fermentor tank that the present invention adopts is 5L full-automatic mechanical stirred-tank fermenter, and its every operating parameter can regulate by the digital controller being connected with it.After inoculation, controlling mechanical mixing speed is 150rpm, and after fermentation for some time, due to mycelium amount reproduction, fermented liquid viscosity enlarges markedly, and therefore mixing speed is increased to 280rpm to promote being uniformly distributed of material.Culture-liquid temp maintains 37 ℃ by circulating condensing water, passes into air to maintain the oxygen dissolved of fermented liquid in fermentor tank simultaneously, and air flow is 1.0Lpm, and total fermentation time is 14 days.The biological fermentation dedicated defoamer adding in fermention medium can be avoided the obstruction to pipeline of foam that bacterial metabolism and mechanical stirring produce.
After fermentation, measure enzyme and live, laccase activity is up to more than 200IU/mL.
3, the acquisition of laccase preparation
Fermented liquid after gauze removes by filter mycelium and corn stalk residue, 5000r/min frozen centrifugation 15min at 4 ℃, fine particulate material is removed in separation, collects supernatant liquor and obtains industrial finish zymin.
Laccase preparation is for association with pulp bleaching
1, bleaching conditions:
Laccase treatment (L): paper pulp (starching dense 10%) is depressed at 0.5MPa oxygen, in the buffered soln of 50 ℃ and pH5.0, add the laccase (to oven dry stock) of 2.0IU/g the present embodiment acquisition and 1% nitro humus acid (nitrohumic acid, NHA) (amboceptor), in the autoclave with electric stirring, process 2h, clean.
Oxygen delignification (O): oxygen delignification is to carry out in the autoclave with electric stirring.Condition is: starch dense 10%, MgSO
4consumption 0.5%, NaOH consumption 2.0%, oxygen is pressed as 0.7MPa, time 60min.
Chelation treatment (Q): starch dense 10%, 1.0%EDTA, pH3.0, temperature 70 C, time 60min.
Hydrogen peroxide bleaching (P): starch dense 10%, H
2o
22.0%, NaOH2.0%, MgSO
40.05%., 90 ℃ of temperature, time 4h.
2, bleaching effect: bamboo pulp is through oxygen delignification (O), the laccase treatment (L) of consumption 2.0IU/g (to oven dry stock) then, then through QP bleaching, pulp brightness can reach 82%ISO, the viscosity degradation of paper pulp.
Those skilled in the art do not depart from essence of the present invention and spirit, can have various deformation scheme to realize the present invention, the foregoing is only the better feasible embodiment of the present invention, not thereby limit to interest field of the present invention.In addition, should be appreciated that those skilled in the art can make various changes or modifications the present invention after having read the content of the present invention's instruction, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. a fermentation method for producing for fungal laccase, is characterized in that, said method comprising the steps of:
1) fungi is inoculated on seed culture medium and cultivates 2～4 days, the mycelium that nutrient solution obtains after sterile gauze filters is cleaned with sterilized water, mycelium is broken up at a high speed after approximately 10 seconds again and be collected in sterilized water and make bacterial classification suspension, described fungi is whiterot fungi Panus conchatus;
2) described bacterial classification suspension is inoculated in containing in the fermentor tank of 3～5L fermention medium and cultivates 10～15 days, obtain fermented liquid;
3) by described filtering fermentation liquor or centrifugal rear acquisition laccase preparation.
2. the method for claim 1, is characterized in that, every liter of described seed culture medium is containing 20%～30% potato leach liquor, 10～50g dextrose anhydrous, 1～10g peptone, 3gKH
2pO
4, 3gMgSO
47H
2o.
3. method as claimed in claim 2, is characterized in that, described fermention medium is in every liter of seed culture medium, to add 5～20g natural substrate and 1～5mmol inductor.
4. method as claimed in claim 3, is characterized in that, described natural substrate is one or more in tealeaves, straw, wheat straw, Pericarppium arachidis hypogaeae, wheat bran, dregs of beans, bagasse, tangerine peel, milled powders of cornstalk.
5. method as claimed in claim 4, is characterized in that, described natural substrate is wheat bran.
6. method as claimed in claim 3, is characterized in that, described inductor is one or more in Li Lu alcohol, methyl catechol, xylidene(s), forulic acid, syringic aldehyde, Weibull, vanillic acid, coumaric acid, copper sulfate.
7. method as claimed in claim 6, is characterized in that, described inductor is copper sulfate.
8. the method for claim 1, is characterized in that, described step 1) in culture temperature be 28 ℃, rotating speed is 120～180rpm.
9. the method for claim 1, is characterized in that, described step 2) in culture temperature be 28 ℃～40 ℃, stirring velocity is 150～300rpm.
10. the application of the laccase preparation that the method for claim 1 obtains in association with pulp bleaching.
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| CN105542551A (en) * | 2015-12-15 | 2016-05-04 | 林康艺 | Composition for deinking waste paper |
| CN105624133A (en) * | 2016-01-20 | 2016-06-01 | 徐向群 | Technique for producing Inonotus obliquus lignocellulose catabolic enzyme by liquid submerged fermentation |
| CN110184244A (en) * | 2019-06-20 | 2019-08-30 | 福清市火麒麟食用菌技术开发有限公司 | A kind of Sparassis crispa produces the preparation method of laccase |
| CN110358647A (en) * | 2019-07-01 | 2019-10-22 | 福清市火麒麟食用菌技术开发有限公司 | A kind of preparation method of Sparassis crispa barley wine |
| CN110477234A (en) * | 2019-08-20 | 2019-11-22 | 广东省生物工程研究所（广州甘蔗糖业研究所） | A kind of synchronous method and device thereof for being coupled fruit juice and taking away the puckery taste of fungi fermentation |
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| CN105542551A (en) * | 2015-12-15 | 2016-05-04 | 林康艺 | Composition for deinking waste paper |
| CN105624133A (en) * | 2016-01-20 | 2016-06-01 | 徐向群 | Technique for producing Inonotus obliquus lignocellulose catabolic enzyme by liquid submerged fermentation |
| CN110184244A (en) * | 2019-06-20 | 2019-08-30 | 福清市火麒麟食用菌技术开发有限公司 | A kind of Sparassis crispa produces the preparation method of laccase |
| CN110358647A (en) * | 2019-07-01 | 2019-10-22 | 福清市火麒麟食用菌技术开发有限公司 | A kind of preparation method of Sparassis crispa barley wine |
| CN110477234A (en) * | 2019-08-20 | 2019-11-22 | 广东省生物工程研究所（广州甘蔗糖业研究所） | A kind of synchronous method and device thereof for being coupled fruit juice and taking away the puckery taste of fungi fermentation |
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