CN103451162A - Method for Aspergillus oryzae secretion preparation of manganese peroxidase - Google Patents
Method for Aspergillus oryzae secretion preparation of manganese peroxidase Download PDFInfo
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Abstract
Relating to the field of bioengineering, the invention relates to a method for Aspergillus oryzae secretion preparation of manganese peroxidase. The method uses straw as a main raw material to produce manganese peroxidase by Aspergillus oryzae fermentation. The production process includes: subjecting the straw to crushing, pre-wetting and other pretreatment technologies, subjecting Aspergillus oryzae to liquid shaking flask culture, first class seed culture, and mixing with the straw to undergo solid fermentation, thus obtaining a manganese peroxidase-containing straw fermentation product with enzyme activity of 3-1000U/g dry fermentation product, and subjecting the fermentation product to water extraction, molecular interception and freeze drying, thus obtaining an enzyme preparation crude product with enzyme activity of 1000-10000U/g dry crude product. The fermentation process is simple, has no pollution to the environment, and also causes no resource waste. The manganese peroxidase prepared by the method can be used to degrade a lignin macromolecular structure and release small molecule substances, and can improve the digestion utilization rate of lignin-containing straw, thereby being conducive to improving the feeding value of straw.
Description
Technical field
The present invention relates to bioengineering field, the stalk substratum refered in particular to without high temperature, autoclaving prepares the manganese peroxidase enzyme preparation through fermentation, this goods water extraction, and extracting solution is through molecular retention, and lyophilize obtains the crude product of manganese peroxidase.
Background technology
China is large agricultural country, and annual agricultural crop straw annual production exceedes 600,000,000 tons, and except being used as on a small quantity weaving, paper industry, but most of form impouring environment to pile up, to burn had both been wasted resource and also to environment, caused greatly harm.
Stalk contains the energy over half that photosynthesis of plant accumulates, be rich in raw sugar, crude protein etc. and be conducive to the required composition of domestic animals and fowls, as noncompetitive feed resource, as long as can carry out reasonably processing modulation, be used for the livestock and poultry of feeding, particularly be used for the ruminant domestic animals such as ox, sheep of feeding, likely become the feed resource of high-quality.Simultaneously, China is again the big country of a feed (especially protein feed) critical shortage, needs every year a large amount of fish meal of import to make up the deficiency of protein feed.So stalk fibre can be converted into to the protein feed that animal can utilize and be of high nutritive value, to solve the wretched insufficiency of feed demand, just seem particularly important.
The stalk of plant mainly contains xylogen and Mierocrystalline cellulose forms.Xylogen is the natural polymer with aromatic series characteristic that a class is formed by connecting by ehter bond and carbon-carbon bond etc. by phenol and non-phenol structure unit, its diversity aspect component kind and connecting key type, the heterogeneity and the irregularity that mean its structure, also determined xylogen is carried out to biodegradable complicacy and singularity, and its in plant cell wall after hemicellulose is combined with the covalent linkage form, cellulosic molecule is embedded in wherein, form the firm natural cover for defense, make general microorganism be difficult to enter wherein decomposition of cellulose, therefore, can the degraded of xylogen be also the key factor that effectively utilize stalk resource.
The representational 5 kinds of methods such as steam-explosion pre-treatment, sulfur acid pretreatment, sodium hydroxide pre-treatment, pretreatment with agueous Ammonia and Biological Pretreatment that comprise in the main method of current degrading straw.Steam-explosion is with high pressure steam infiltrated fiber inside, mode with air-flow discharges from the space of sealing, certain mechanical breaking occurs in fiber, make most of hemicellulose and xylogen, cellulose degradation stripping on a small quantity in raw material, the method composition is higher, need large-scale equipment, xylogen also needs to process; Acidic treatment is to utilize sulphuric acid hydrolysis glycosidic link, ester bond etc., make the reticulated structure of corn stalk fiber loose, but the method consumes a large amount of organic acids or mineral acid, and xylogen still needs further processing, is not suitable with large-scale industrial production.Alkaline purification and ammonia treatment are to utilize xylogen to be dissolved in basic solution, equally exist problem of environmental pollution the same as acid treatment.Generally all there is the more difficult grasp of reaction conditions in these methods, temperature of reaction is high, the treatment time is long, take up room large, treatment effect is undesirable, can not remove the problems such as lignocellulose, the stalk of carrying out a biological disposal upon in recent years more and more is subject to people's attention, and the key of Biodegradation of Lignin is the enzyme that microorganism can secrete lignin degrading: manganese peroxidase, lignin peroxidase and laccase.
At present, existing many patents relate to the microorganism secretion manganese peroxidase, as patent (application number/patent No.: 200810064332) introduced the strengthening white rot fungus to excrete manganese peroxidase method, this method has shifted to an earlier date three days than the method for existing fermented by white rot fungus manganese peroxidase, fermentation period has shortened half, and production of enzyme is higher than 900U/L; (application number: 200680022879) target is the output that increases the outer laccase of white rot basidiomycetes production born of the same parents and manganese peroxidase to patent, and reduces costs, and this method, with respect to existing culture technique, can make laccase and manganese peroxidase production of enzyme significantly increase; Patent (application number: 200910079781) introduced another kind of manganese peroxidase and produced bacterium---trichoderma harziarum, this bacterium (4-5 days) in a short time forms a large amount of conidiums, it forms conidial speed than the fast about week age of Phanerochaete chrysosporium, and sporiparous ability can reach 10 times of Phanerochaete chrysosporium, Phanerochaete chrysosporium is growth and the stronger bacterial classification of sporiparous ability in basidiomycetes.Aspergillus oryzae by united States food and drug administration (FDA) and U.S. feed public determine that association (AAFCO), China Ministry of Agriculture assert can the Direct-fed animal the feed level microbe additive bacterial classification, but the patent and the pertinent literature thereof that have no aspergillus oryzae secretion manganese peroxidase are reported.
The inventor is through deep research, finds out a kind ofly to take stalk as main raw material, obtains the method for manganese peroxidase by the aspergillus oryzae cell solid culture.The method and method difference in the past be used bacterial classification by united States food and drug administration (FDA) and U.S. feed public determine association (AAFCO), the identification of China Ministry of Agriculture can the Direct-fed animal the aspergillus oryzae bacterial classification of feed level microbe additive, adopt stalk as main raw material, obtain the fermented product goods that contain manganese peroxidase by solid state fermentation, these goods can be through further extracting and make the manganese peroxidase crude product.
Summary of the invention
Purpose of the present invention has been to provide a kind ofly take aspergillus oryzae as bacterial classification, and the stalk of take carries out the method for solid state fermentation as raw material, and the aspergillus oryzae fermented product prepared by the method, in this fermented product, contains manganese peroxidase.
A kind of aspergillus oryzae secretion of the present invention prepares the method for manganese peroxidase, according to following step, carry out: take aspergillus oryzae as starting strain, the agricultural crop straw that contains xylogen of take is raw material, carrying out test tube enlarged culturing, liquid shaking bottle cultivation, first order seed cultivation and solid fermentation cultivates, obtain the solid state fermentation thing that contains manganese peroxidase, then extract and obtain the Aspergillus oryzae solid culture that contains manganese peroxidase.
Wherein said bacterial classification is any bacterial classification of aspergillus oryzae, as the bacterial classification of Chinese common micro-organisms culture presevation administrative center (CCGMC), Chinese agriculture microbial strains preservation administrative center (ACCC), Chinese industrial microbial strains preservation administrative center (CICC), Chinese medicine microbial strains preservation administrative center (CMCC), national veterinary microorganism DSMZ (CVCC) and microbiotic bacterial classification preservation pipe reason center etc. preservation.
The wherein said agricultural crop straw raw material that contains xylogen is any one stalk, and as maize straw, rice straw, Wheat Straw, broomcorn straw, soybean stalk, cotton stalk etc., stalk, at first through pulverizing, is crossed 20～100 purpose sieves.
In wherein said test tube enlarged culturing technique, its enlarged culturing base is the potato sucrose substratum;
The substratum that wherein said liquid shaking bottle is cultivated and first order seed is cultivated is: glucose 5～30 grams, ammonium tartrate 0.1～5 gram, phenylcarbinol 0.2～3 gram, sal epsom 0.2～1 gram, tween 80 0.5～10 gram, potassium primary phosphate 2～9 grams, phthalic acid 3～18 grams, add water constant volume to 1 liter, adjust 5～8,120～140 ℃ of sterilizings of pH 20～40 minutes;
Wherein said shake-flask culture processing condition are, inoculate a ring aspergillus oryzae test tube slant spore in the triangular flask that volume ratio 16～48% substratum are housed, at rotating speed: 150 rev/mins, 25～35 ℃, cultivate 24～72 hours; Before carrying out shake-flask culture, at first will in the potato sucrose substratum of the new configuration of aspergillus oryzae bacterial classification access, be cultivated 25～35 ℃ of culture temperature, incubation time 48～144 hours; 4～10 ℃ save backup.
Wherein said first order seed culture process is, by 1～20%(volume ratio) inoculum size the shake-flask culture seed liquor is inoculated in the first order seed substratum, 25～35 ℃ of temperature, 50～200 rev/mins of mixing speed, air flow 0.2～2:1 volume/volume/per minute (ventilation gas volume/fermentating liquid volume/per minute), cultivate 18～72 hours;
In wherein said solid fermentation cultivation, substratum consists of: 10 kilograms of stalks, 0.5～3 kilogram of glucose, 0.5～2 kilogram of soybean cake powder; Copper sulfate 5～15 grams, ferrous sulfate 5～15 grams, manganous sulfate 5～20 grams, 20～60 kilograms, water, each composition of this substratum can amplify in this ratio;
Wherein said zymotechnique is: by 2～20%(seed liquor volume/fermention medium weight), culture material thickness is 0.1～0.4 meter, expect wide 0.5～2 meter, length is not limit, air flow is 0.3～1.5:1(gas volume/fermention medium weight)/per minute, incubation time is 3 days～23 days, and stirring is 1～2 time midway.
The technique of wherein said production manganese peroxidase crude product, the solid state fermentation thing obtained is pulverized, cross 20～100 mesh sieves, the fermenting culture of pulverizing mixes in 1:10～50 ratios with water, 50～300 rev/mins of stirrings, 5～10 ℃ are extracted 4～8 hours, suction filtration, filter residue mixes with the water of 10～50 times, 50～300 rev/mins of stirrings, 5～10 ℃ are extracted 4～8 hours, suction filtration, twice filtrate merges, and by the film of molecular weight 5000～10000, holds back concentrated 50～100 times,-20 ℃ of lyophilizes, obtain the zymin crude product.
The zymin crude product that technique obtains thus, chromaticness white is to brown, and the manganese peroxidase enzymic activity can reach the dry crude product of 1000～10000U/g.
Obtain the stalk fermentation thing by the present invention and contain a large amount of yellow-green colour aspergillus oryzae spores, the fermented product orange, to brown, contains distiller's yeast fragrance, and the manganese peroxidase enzymic activity reaches the dry fermented product of 3～1000U/g.
According to fermentating culturing process of the present invention, in conjunction with the ABC of this area, can be according to need of production, increase even level Four seeding tank of secondary, three grades, further enlarge the solid state fermentation industrial scale, to carry out suitability for industrialized production.Secondary, the three grades substratum that even the level Four seeding tank adopts are identical with the first order seed substratum with the shake-flask culture base, and inoculum size is 5～20%, and culture condition is same as the first order seed cultivation,
Adopt the advantage of this technique to adopt solid state fermentation, and, without high temperature high pressure process, can not give environment like this, also can not waste resource.
The accompanying drawing explanation
The process flow sheet that Fig. 1 is this patent.
Embodiment
According to the manganese peroxidase enzyme testing method of this process using, be: accurately take 1 gram fermented product or zymin crude product, be dissolved in 50mL water, room temperature vibration 2 hours, suction filtration, filtrate is settled to 50mL, is enzyme liquid.Contain Sodium.alpha.-hydroxypropionate damping fluid 3.4 mL of pH 4.5 50 mmol/L in 4 mL reaction systems, manganese sulfate solution 0.1 mL of 1.6 mmol/L, enzyme liquid X mL, when distilled water (0.4-X) mL is preheated to 37 ℃, add the H of 1.6 mmol/L
2o
2solution 0.1 mL starts reaction.At 240 nm UV-light places, OD value changing value in assaying reaction 4 min.In control group, replace original enzyme liquid to boil deactivation 15 min enzyme liquid, replace H with distilled water
2o
2solution, other reactants are constant.Per minute makes the Mn of 1 μ mol/L
2+be converted into Mn
3+required enzyme amount is 1 enzyme activity unit (U).
Here
x: enzyme liquid to be measured amasss (≤0.4mL)
In order further to set forth related material and the technique of technical scheme of the present invention, provided following examples, but these embodiment do not limit the scope of the invention in any form.
Embodiment 1
1, the making of test tube slant bacterial classification
Access one ring aspergillus oryzae bacterial classification (Aspergillus oryzae) in the potato sucrose substratum (Zhu Gejian, Wang Zhengxiang chief editor's " industrial microorganism experimental technique handbook ", 1994,367 pages) of new configuration, 26 ℃, incubation time 50 hours; 4 ℃ save backup.
2, liquid shaking bottle is cultivated the making of bacterial classification
Accurately take glucose 5 grams, ammonium tartrate 0.1 gram, phenylcarbinol 0.2 gram, sal epsom 0.2 gram, tween 80 0.5 gram, potassium primary phosphate 2 grams, O-phthalic acid buffer 3 grams, water 1000 mL, pH 5, packing 250mL triangular flask, every bottle of 50 grams, totally 20 bottles, 120 ℃ of sterilizings 40 minutes; After cooling, every bottle graft enters the aspergillus oryzae bacterial classification of 4 ℃ of preservations of a ring, at 25 ℃, 150 rev/mins, cultivates 72 hours;
3, the making of level liquid bacterial classification
Accurately take glucose 50 grams, ammonium tartrate 1 gram, phenylcarbinol 2 grams, sal epsom 2 grams, tween 80 5 grams, potassium primary phosphate 20 grams, phthalic acid 30 grams, water 10L, be loaded in the seeding tank of 15L, 120 ℃ of sterilizings 20 minutes; After sterilizing is cooling, 25 ℃ of temperature, 50 rev/mins of mixing speed, 0.2: 1 volume/volume/per minute (ventilation gas volume/fermentating liquid volume/per minute) of air flow, cultivate 72 hours;
4, solid state fermentation is produced manganese peroxidase
Rice straw is pulverized, and crosses 20 mesh sieves, takes 750 kilograms of this straw powder, 1500 kilograms, water, 37.5 kilograms of glucose, 37.5 kilograms of soybean cake powders; Copper sulfate 375 grams, ferrous sulfate 375 grams, manganous sulfate 375 grams, fully mix thoroughly, access above-mentioned first order seed nutrient solution, mix, making culture material thickness is 0.1 meter, expects wide 0.5 meter, the stockpile that length is 37.5 meters, control 25 ℃ of temperature and cultivate 23 days, and stirring is 2 times midway.
Obtain the stalk fermentation thing and contain a large amount of yellow-green colour aspergillus oryzae spores, the fermented product orange, to brown, has wine flavour.The manganese peroxidase enzymic activity is the dry fermented product of 30U/g.
5, the preparation of thick enzyme preparation
The solid state fermentation thing is pulverized, and crosses 20 mesh sieves, and the fermenting culture of pulverizing mixes in the 1:10 ratio with water, 60 rev/mins of stirrings, 10 ℃ are extracted 6 hours, suction filtration, filter residue mixes with the water of 10 times, 60 rev/mins of stirrings, 10 ℃ are extracted 6 hours, suction filtration, twice filtrate merges, hold back concentrated 50 times by the film of molecular weight 5000 ,-20 ℃ of lyophilizes, obtain the zymin crude product.The zymin crude product that technique obtains thus, chromaticness white is to brown, and the manganese peroxidase enzymic activity can reach the dry crude product of 2000U/g.
Embodiment 2
1, the making of test tube slant bacterial classification
Access one ring aspergillus oryzae bacterial classification (Aspergillus oryzae) in the potato sucrose substratum (Zhu Gejian, Wang Zhengxiang chief editor's " industrial microorganism experimental technique handbook ", 1994,367 pages) of new configuration,, 30 ℃, incubation time 100 hours; 7 ℃ save backup.
2, liquid shaking bottle is cultivated the making of bacterial classification
Accurately take glucose 150 grams, ammonium tartrate 250 grams, phenylcarbinol 15 grams, sal epsom 0.4 gram, tween 80 4 grams, potassium primary phosphate 5 grams, phthalic acid cushions 20 grams, water 10 L, pH 6, packing 250mL triangular flask, every bottle of 100 grams, totally 100 bottles, 130 ℃ of sterilizings 30 minutes; After cooling, every bottle graft enters the aspergillus oryzae bacterial classification of 7 ℃ of preservations of a ring, at 30 ℃, 150 rev/mins, cultivates 50 hours;
3, the making of level liquid bacterial classification
Accurately take glucose 2000 grams, ammonium tartrate 200 grams, phenylcarbinol 150 grams, sal epsom 60 grams, tween 80 60 grams, potassium primary phosphate 400 grams, phthalic acid 1000 grams, water 100L, be loaded in the seeding tank of 130L, 130 ℃ of sterilizings 30 minutes; After cooling, 30 ℃ of temperature, 125 rev/mins of mixing speed, air flow 1:1 volume/volume/per minute (ventilation gas volume/fermentating liquid volume/per minute), cultivate 50 hours;
4, solid state fermentation is produced manganese peroxidase
Corn straw smashing, cross 60 mesh sieves, takes 1000 kilograms of this straw powder, 3000 kilograms, water, 150 kilograms of glucose, soybean cake powder double centner; Copper sulfate 600 grams, ferrous sulfate 600 grams, manganous sulfate 1000 grams, fully mix thoroughly, access above-mentioned first order seed nutrient solution, mix, making culture material thickness is 0.2 meter, expects wide 1 meter, the stockpile that length is 225 meters, control 30 ℃ of temperature and cultivate 15 days, and stirring is 2 times midway.
Obtain the stalk fermentation thing and contain a large amount of yellow-green colour aspergillus oryzae spores, the fermented product tawny, have wine flavour.The manganese peroxidase enzymic activity is the dry fermented product of 100U/g.
5, the preparation of thick enzyme preparation
The solid state fermentation thing is pulverized, and crosses 50 mesh sieves, and the fermenting culture of pulverizing mixes in the 1:30 ratio with water, 150 rev/mins of stirrings, 10 ℃ are extracted 8 hours, suction filtration, filter residue mixes with the water of 30 times, 150 rev/mins of stirrings, 10 ℃ are extracted 8 hours, suction filtration, twice filtrate merges, hold back concentrated 75 times by the film of molecular weight 10000 ,-20 ℃ of lyophilizes, obtain the zymin crude product.The zymin crude product that technique obtains thus, chromaticness white is to brown, and the manganese peroxidase enzymic activity can reach the dry crude product of 7500U/g.
Embodiment 3
1, the making of test tube slant bacterial classification
Access one ring aspergillus oryzae bacterial classification (Aspergillus oryzae) in the potato sucrose substratum (Zhu Gejian, Wang Zhengxiang chief editor's " industrial microorganism experimental technique handbook ", 1994,367 pages) of new configuration,, 35 ℃, incubation time 144 hours; 10 ℃ save backup.
2, liquid shaking bottle is cultivated the making of bacterial classification
Accurately take glucose 300 grams, ammonium tartrate 50 grams, phenylcarbinol 30 grams, sal epsom 10 grams, tween 80 100 grams, potassium primary phosphate 90 grams, O-phthalic acid buffer 180 grams, water 10L, pH7.5, packing 250mL triangular flask, every bottle of 120 mL, totally 80 bottles, 140 ℃ of sterilizings 20 minutes; After cooling, every bottle graft enters the aspergillus oryzae bacterial classification of 10 ℃ of preservations of a ring, at 35 ℃, 150 rev/mins, cultivates 72 hours;
3, the making of level liquid bacterial classification
Accurately take glucose 1500 grams, ammonium tartrate 250 grams, phenylcarbinol 150 grams, sal epsom 50 grams, tween 80 500 grams, potassium primary phosphate 450 grams, O-phthalic acid buffer 900 grams, water 50L, be loaded in the first class seed pot of 70L, 140 ℃ of sterilizings 40 minutes; After sterilizing is cooling, 35 ℃ of temperature, 200 rev/mins of mixing speed, air flow 2:1 volume/volume/per minute (ventilation gas volume/fermentating liquid volume/per minute), cultivate 72 hours;
4, the making of second-class liquid isolate
Accurately take glucose 15000 grams, ammonium tartrate 2500 grams, phenylcarbinol 1500 grams, sal epsom 500 grams, tween 80 5000 grams, potassium primary phosphate 4500 grams, O-phthalic acid buffer 9000 grams, water 500L, be loaded in the first class seed pot of 700L, 140 ℃ of sterilizings 40 minutes; After sterilizing is cooling, 35 ℃ of temperature, 200 rev/mins of mixing speed, air flow 2:1 volume/volume/per minute (ventilation gas volume/fermentating liquid volume/per minute), cultivate 18 hours;
5, solid state fermentation is produced manganese peroxidase
Wheat straw waste is pulverized, and crosses 100 mesh sieves, takes 3500 kilograms of this straw powder, 21000 kilograms, water, 525 kilograms of glucose, 525 kilograms of soybean cake powders; Copper sulfate 5000 grams, ferrous sulfate 5000 grams, manganous sulfate 6000 grams, fully mix thoroughly, access above-mentioned secondary seed nutrient solution, mix, making culture material thickness is 0.3 meter, expects wide 2 meters, the stockpile that length is 300 meters, control 35 ℃ of temperature and cultivate 6 days, and stirring is 1 time midway.
Obtain the stalk fermentation thing and contain a large amount of yellow-green colour aspergillus oryzae spores, the fermented product brown, have wine flavour.The manganese peroxidase enzymic activity is the dry fermented product of 980U/g.
6, the preparation of thick enzyme preparation
The solid state fermentation thing is pulverized, and crosses 80 mesh sieves, and the fermenting culture of pulverizing mixes in the 1:50 ratio with water, 250 rev/mins of stirrings, 5 ℃ are extracted 4 hours, suction filtration, filter residue mixes with the water of 50 times, 250 rev/mins of stirrings, 5 ℃ are extracted 4 hours, suction filtration, twice filtrate merges, hold back concentrated 100 times by the film of molecular weight 7000 ,-20 ℃ of lyophilizes, obtain the zymin crude product.The zymin crude product that technique obtains thus, the chromaticness canescence, the manganese peroxidase enzymic activity can reach the dry crude product of 9500U/g.
Claims (10)
1. an aspergillus oryzae is secreted the method for preparing manganese peroxidase, it is characterized in that carrying out according to following step: take aspergillus oryzae as starting strain, the agricultural crop straw that contains xylogen of take is raw material, carrying out test tube enlarged culturing, liquid shaking bottle cultivation, first order seed cultivation and solid fermentation cultivates, obtain the solid state fermentation thing that contains manganese peroxidase, then extract and obtain the Aspergillus oryzae solid culture that contains manganese peroxidase.
2. a kind of aspergillus oryzae secretion according to claim 1 prepares the method for manganese peroxidase, it is characterized in that wherein said bacterial classification is any bacterial classification of aspergillus oryzae, as the bacterial classification of Chinese common micro-organisms culture presevation administrative center (CCGMC), Chinese agriculture microbial strains preservation administrative center (ACCC), Chinese industrial microbial strains preservation administrative center (CICC), Chinese medicine microbial strains preservation administrative center (CMCC), national veterinary microorganism DSMZ (CVCC) and microbiotic bacterial classification preservation pipe reason center etc. preservation.
3. a kind of aspergillus oryzae secretion according to claim 1 prepares the method for manganese peroxidase, it is characterized in that the wherein said agricultural crop straw raw material that contains xylogen is any one stalk, as maize straw, rice straw, Wheat Straw, broomcorn straw, soybean stalk, cotton stalk etc., stalk, at first through pulverizing, is crossed 20～100 purpose sieves.
4. a kind of aspergillus oryzae secretion according to claim 1 prepares the method for manganese peroxidase, it is characterized in that in wherein said test tube enlarged culturing technique, its enlarged culturing base is the potato sucrose substratum.
5. a kind of aspergillus oryzae secretion according to claim 1 prepares the method for manganese peroxidase, it is characterized in that the substratum that wherein said liquid shaking bottle is cultivated and first order seed is cultivated is: glucose 5～30 grams, ammonium tartrate 0.1～5 gram, phenylcarbinol 0.2～3 gram, sal epsom 0.2～1 gram, tween 80 0.5～10 gram, potassium primary phosphate 2～9 grams, phthalic acid 3～18 grams, add water constant volume to 1 liter, adjust 5～8,120～140 ℃ of sterilizings of pH 20～40 minutes.
6. a kind of aspergillus oryzae secretion according to claim 1 prepares the method for manganese peroxidase, it is characterized in that wherein said shake-flask culture processing condition are, inoculation one ring aspergillus oryzae test tube slant spore is in the triangular flask that volume ratio 16～48% substratum are housed, at rotating speed: 150 rev/mins, 25～35 ℃, cultivate 24～72 hours; Before carrying out shake-flask culture, at first will in the potato sucrose substratum of the new configuration of aspergillus oryzae bacterial classification access, be cultivated 25～35 ℃ of culture temperature, incubation time 48～144 hours; 4～10 ℃ save backup.
7. a kind of aspergillus oryzae secretion according to claim 1 prepares the method for manganese peroxidase, it is characterized in that wherein said first order seed culture process is, 1～20% inoculum size is inoculated into the shake-flask culture seed liquor in the first order seed substratum by volume, 25～35 ℃ of temperature, 50～200 rev/mins of mixing speed, air flow 0.2～2:1 volume/volume/per minute (ventilation gas volume/fermentating liquid volume/per minute), cultivate 18～72 hours.
8. a kind of aspergillus oryzae secretion according to claim 1 prepares the method for manganese peroxidase, it is characterized in that in wherein said solid fermentation cultivation, substratum consists of: 10 kilograms of stalks, 0.5～3 kilogram of glucose, 0.5～2 kilogram of soybean cake powder; Copper sulfate 5～15 grams, ferrous sulfate 5～15 grams, manganous sulfate 5～20 grams, 20～60 kilograms, water, each composition of this substratum can amplify in this ratio.
9. a kind of aspergillus oryzae secretion according to claim 1 prepares the method for manganese peroxidase, it is characterized in that wherein said zymotechnique is: by 2～20%(seed liquor volume/fermention medium weight), culture material thickness is 0.1～0.4 meter, expect wide 0.5～2 meter, length is not limit, air flow is 0.3～1.5:1(gas volume/fermention medium weight)/minute, incubation time is 3 days～23 days, stirring is 1～2 time midway.
10. a kind of aspergillus oryzae secretion according to claim 1 prepares the method for manganese peroxidase, the technique that it is characterized in that wherein said production manganese peroxidase crude product, the solid state fermentation thing obtained is pulverized, cross 20～100 mesh sieves, the fermenting culture of pulverizing mixes in 1:10～50 ratios with water, 50～300 rev/mins of stirrings, 5～10 ℃ are extracted 4～8 hours, suction filtration, filter residue mixes with the water of 10～50 times, 50～300 rev/mins of stirrings, 5～10 ℃ are extracted 4～8 hours, suction filtration, twice filtrate merges, hold back concentrated 50～100 times by the film of molecular weight 5000～10000,-20 ℃ of lyophilizes, obtain the zymin crude product.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104531640A (en) * | 2015-01-08 | 2015-04-22 | 北京绿科天成生物科技有限公司 | Aspergillus oryzae fermentation liquor, corn stalk sugar liquor prepared through same, and preparation method and application of corn stalk sugar liquor |
| CN105039286A (en) * | 2015-02-06 | 2015-11-11 | 张志才 | Aspergillus oryzae fermentation liquid, liquid glucose prepared therewith through degradation of straw powder, and preparation method and application of the liquid glucose |
| CN107012131A (en) * | 2017-01-12 | 2017-08-04 | 中国农业科学院饲料研究所 | A kind of manganese peroxidase and its gene and the application on detoxicating mycotoxin |
| CN114480146A (en) * | 2022-04-15 | 2022-05-13 | 中国科学院天津工业生物技术研究所 | Application of aspergillus oryzae strain in copper, iron, manganese and zinc enrichment |
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| WO2004113490A2 (en) * | 2003-06-24 | 2004-12-29 | HöFer Bioreact GmbH | Directed selective solid-phase culturing of stable microbial mixed populations for the continuous preparation of defined enzyme and metabolite mixture and bioreactor |
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| CN104531640A (en) * | 2015-01-08 | 2015-04-22 | 北京绿科天成生物科技有限公司 | Aspergillus oryzae fermentation liquor, corn stalk sugar liquor prepared through same, and preparation method and application of corn stalk sugar liquor |
| CN104531640B (en) * | 2015-01-08 | 2018-08-28 | 北京绿科天成生物科技有限公司 | A kind of aspergillus oryzae zymotic fluid, the maize straw liquid glucose prepared by the zymotic fluid and preparation method and purposes |
| CN105039286A (en) * | 2015-02-06 | 2015-11-11 | 张志才 | Aspergillus oryzae fermentation liquid, liquid glucose prepared therewith through degradation of straw powder, and preparation method and application of the liquid glucose |
| CN105039286B (en) * | 2015-02-06 | 2018-11-06 | 江苏一鸣生物股份有限公司 | Liquid glucose and preparation method and purposes prepared by aspergillus oryzae zymotic fluid, the zymotic fluid degradation of rice straw powder |
| CN107012131A (en) * | 2017-01-12 | 2017-08-04 | 中国农业科学院饲料研究所 | A kind of manganese peroxidase and its gene and the application on detoxicating mycotoxin |
| CN107012131B (en) * | 2017-01-12 | 2020-11-06 | 中国农业科学院北京畜牧兽医研究所 | Manganese peroxidase, gene thereof and application of manganese peroxidase in mycotoxin detoxification |
| CN114480146A (en) * | 2022-04-15 | 2022-05-13 | 中国科学院天津工业生物技术研究所 | Application of aspergillus oryzae strain in copper, iron, manganese and zinc enrichment |
| CN114480146B (en) * | 2022-04-15 | 2022-06-24 | 中国科学院天津工业生物技术研究所 | Application of aspergillus oryzae strain in copper, iron, manganese and zinc enrichment |
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