CN105062984A - Method for preparing laccase by fungus fermentation - Google Patents
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- CN105062984A CN105062984A CN201510589399.6A CN201510589399A CN105062984A CN 105062984 A CN105062984 A CN 105062984A CN 201510589399 A CN201510589399 A CN 201510589399A CN 105062984 A CN105062984 A CN 105062984A
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- 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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Abstract
The invention relates to a method for preparing laccase by fungus fermentation. Bran, tealeaves, peptones, KH2PO4, MgSO4.7H2O, MnSO4.5H2O and CuSO4.5H2O are taken as main raw materials of a culture medium, and the method comprises the following steps of inoculating the main raw materials into eurotium cristatum spore suspension, and performing shake-flask cultivation for 3 to 4 days under the conditions of temperature of 26 to 28 DEG C and rotating speed of 120r/min to obtain crude enzyme liquid; performing ammonium sulfate fractional precipitation, DEAE (DiEthyl-AminoEthanol) DEAE-cellulose anion exchange column chromatography and glucan G-150 gel chromatography on the crude enzyme liquid to obtain purified laccase. According to the method, a fermental cultivation condition for laccase production is optimized, and under the optimized cultivation condition, the purification multiple of laccase reaches 8.98, the recovery rate is 24.88 percent, and the activity of laccase reaches 2,413U/L.
Description
Technical field
The present invention relates to a kind of method utilizing fungi fermentation to prepare laccase.
Technical background
Laccase (laccase, EC.1.10.3.1) polyphenoloxidase (Polyphenoloxidase is belonged to, PPO), it is the oxydo-reductase of a class cupric, be the class cuproprotein all extensively existed in bacterium, fungi, plant and mammalian body, and there is substrate specificity widely.Polyphenoloxidase the oxidation of catalysis polyphenolic compound can form corresponding quinones substance effectively, obtains good investigation and application in phenolic wastewater treatment, lignin degradation and the field such as dye decolored.Being rich in polyphenoloxidase in fresh tea leaf in its cell, catechins can being impelled to be oxidized and form theoflavin, thearubigins and other oxypolymers etc., produce volatile compound, is the biochemical basis forming black tea local flavor and speciality.Therefore, polyphenoloxidase all plays an important role in the process such as Physiological Metabolism of Tea Tree and Tea Processing.
Tea is one of three large nonalcoholic drinks.Large quantity research finds to drink tea except having the effects such as benefit is thought to sleep less, aid digestion, improving eyesight diuresis, prevents cardiovascular diseases, diabetes and the effect such as anticancer in addition.Along with the production of the development of tea industry, particularly instant tea and liquid tea drink, traditional tea production technique is only relied on to can not meet the needs of production.High added value tea product production technology, particularly enzyme engineering technology will be applied in Tea Processing gradually.At present, have the zymins such as tannase, cellulase, polyphenoloxidase and proteolytic enzyme and be applied to Tea Processing.
The invention is characterized in and utilize tealeaves (or tea) as culture medium mainly composition, produce a kind of laccase by fungi fermentation, this laccase has certain biotransformation to tea composition, is expected to be applied to tea comprehensive processing and produces.
Summary of the invention
The object of the present invention is to provide a kind of method utilizing fungi fermentation to prepare laccase.By inoculation in substratum, obtain the fermented liquid containing laccase after fermentation culture, this fermentation liquor is crossed separation and purification and namely obtains laccase.
Adopt technical scheme as follows for realizing object of the present invention:
1, take final volume as the fermention medium aqueous solution of 1L, the formula of its each component is:
2, concrete preparation process:
(1) in each 250mL triangular flask, load above-mentioned fermention medium, liquid amount is 60mL, according to a conventional method sterilizing.
(2) in each triangular flask, access coronoid process to fall apart capsule bacterium spore suspension 2mL, temperature 26 ~ 28 DEG C, under the condition of rotating speed 120r/min, shake-flask culture is after 3 ~ 4 days, gets fermentation liquor filtered through gauze, centrifugal acquisition supernatant liquor is crude enzyme liquid.
Described coronoid process falls apart capsule bacterium spore suspension, and its concentration is 1 ~ 2 × 10
8individual spore/mL.
(3) crude enzyme liquid is successively through ammonium sulfate precipitation, DEAE-cellulose anion exchange column chromatography and dextran G-150 gel chromatography, namely obtains the laccase of separation and purification.
Described ammonium sulfate precipitation, refers to and the crude enzyme liquid prepared is placed in ice-water bath, slowly adds ground ammonium sulfate to saturation ratio 50%, and leave standstill 2h, 10000r/min, 4 DEG C of centrifugal 10min, get supernatant liquor; Continue to add ammonium sulfate to saturation ratio 80% in supernatant liquor, leave standstill 12 ~ 18h, 10000r/min, 4 DEG C of centrifugal 10min, collecting precipitation; Precipitate with appropriate pH6.0,0.05mol/L Acetic acid-sodium acetate buffer solution, then dialysis tubing is placed in, at pH6.0,0.05mol/L Acetic acid-sodium acetate damping fluid dialysis desalting under 4 DEG C of conditions, exchange buffering liquid 2-3 time, after dialysis terminates, measure enzyme activity and protein content, freeze-drying concentrates.
Described DEAE-cellulose anion exchange column chromatography, referring to first is immersed in distilled water by the Mierocrystalline cellulose of dry powder, after fully swelling, remove impurity, with the HCl solution soaking 2h of 0.5mol/L, clean to pH neutral with ultrapure water, be immersed in 2h in the NaOH solution of 0.5mol/L again, after being washed to neutrality with ultrapure water, then balance with pH6.0,0.05mol/L Acetic acid-sodium acetate damping fluid, carry out dress post, after compacting, leave standstill 12 ~ 15h; Pillar and chromatograph are coupled together, flow velocity is 0.8mL/min, first use pH6.0,0.05mol/L Acetic acid-sodium acetate damping fluid balanced gel for some time, until baseline is steady, enzyme liquid after ammonium sulfate precipitation freeze-drying being concentrated carries out loading, wash-out is carried out with 1 ~ 1.5mol/LNaCl, pH6.0,0.05mol/L Acetic acid-sodium acetate damping fluid, collect with the pipe of 10mL, often 4mL collected by pipe, the enzyme detecting each pipe is lived, dialysis desalting, and freeze-drying concentrates, obtain the enzyme powder of freeze-drying, and save backup in-20 DEG C.
Described dextran G-150 gel chromatography, refers to the enzyme powder of freeze-drying, with pH6.0,0.05mol/L Acetic acid-sodium acetate buffer solution, and 10000r/min, 4 DEG C of centrifugal 10min, get supernatant liquor stand-by, get appropriate dextran G-150 powder, add in ultrapure water, swelling 5 ~ the 6h of boiling water bath, removing gel upper water and fine particle, with ultrapure water repetitive scrubbing 2 ~ 3 times, again with pH6.0, 0.05mol/L NaAc_HAc buffer solution washing 2-3 time, carry out dress post, spend the night after compacting, pillar and chromatograph are coupled together, flow velocity is 0.4mL/min, first use pH6.0, 0.05mol/L Acetic acid-sodium acetate damping fluid balanced gel for some time, until baseline balance, sample thief loading, buffer solution elution, collect with the pipe of 10mL, often 4mL collected by pipe, obtain laccase, the enzyme detecting each pipe laccase is lived, freeze-drying concentrates,-20 DEG C of preservations.
Coronoid process of the present invention falls apart capsule bacterium spore suspension, and its concentration is 1 ~ 2 × 10
8individual spore/mL, process for preparation is as follows: the inoculation of the capsule bacterium that fallen apart by coronoid process, on the flat board of fresh PDA substratum, is cultivated 5d and obtained ripe spore, wash lower spore with sterile distilled water under temperature 26 ~ 28 DEG C of conditions, put into the Erlenmeyer flask that sterile glass beads is housed, fully vibrate.After spore is fully broken up, survey the value of spore suspension OD600, and adjust its OD value to 2.0 by stroke-physiological saline solution, obtain spore suspension.
Coronoid process of the present invention falls apart capsule bacterium (Eurotiumcristatum), and purchased from China General Microbiological culture presevation administrative center (CGMCC), it is numbered: 3.07934.The front coronoid process capsule bacterium (Eurotiumcristatum) that falls apart is used to be kept on potato dextrose agar slant substratum under 4 DEG C of conditions.
Adopt method of the present invention to prepare in laccase process, through the laccase that dextran G-150 gel chromatography obtains, through SDS-PAGE detected through gel electrophoresis, laccase relative molecular mass is 70kDa.SDS-PAGE condition: resolving gel concentration is 12%, concentrated gum concentration is 5%.Voltage is at 80-120V electrophoresis 2h, and electrophoresis terminates rear gel coomassie brilliant blue R_250 staining reagent 1h, rear use 30% methyl alcohol and 10% Glacial acetic acid decolouring 2h.
Described SDS-PAGE preparation sees the following form:
Adopt method of the present invention, optimize the fermentation culture conditions of laccase production, under the culture condition optimized, the purification of laccase reaches 8.98, and the rate of recovery is 24.88%, and enzyme is lived and reached 2413U/L.
Innovation of the present invention is to utilize tealeaves as one of main component fermentative production of laccase, has developed the new way that tealeaves utilizes, has had good directive function to waste utilization in low-grade tealeaves and Tea Production process.
Accompanying drawing explanation
Fig. 1 is the bovine serum albumin canonical plotting measured in embodiment 1 used by crude enzyme liquid Rate activity.
Fig. 2 is the graphic representation utilizing ammonium sulfate precipitation determination ammonium sulfate saturation ratio in embodiment 1.
Fig. 3 be measure in embodiment 1 after ammonium sulfate precipitation used by enzyme liquor ratio vigor bovine serum albumin canonical plotting.
Fig. 4 is that the crude enzyme liquid for preparing of embodiment 1 is through DEAE-cellulose anion displacement chromatography collection of illustrative plates.
Fig. 5 is the collection of illustrative plates of crude enzyme liquid after dextran G-150 gel chromatography that embodiment 1 prepares.
The electrophoretogram that Fig. 6 is that embodiment 1 prepares, laccase enzyme liquid after separation and purification carries out SDS-PAGE.
Embodiment:
In order to better understand the present invention, existing the present invention is described further by way of example by reference to the accompanying drawings.
In Fig. 1, recording crude enzyme liquid laccase activity is 2470U/L; Measure crude enzyme liquid protein content with Coomassie Brilliant Blue, obtaining total protein in crude enzyme liquid is 47.44mg, and crude enzyme liquid Rate activity is 10.41U/mg.
In Fig. 2, the ammonium sulfate of crude enzyme liquid different saturation is carried out fractionation precipitation, after centrifugal to the supernatant liquor of enzyme liquid under each saturation ratio, carry out enzyme activity determination.As can be seen from Figure 2, ammonium sulfate saturation ratio is between 20% ~ 50%, and supernatant liquor enzyme is lived and substantially remained unchanged, and illustrates that the protein now settled down is foreign protein; Ammonium sulfate saturation ratio is by 50% to 80%, and in supernatant liquor, enzyme is lived and sharply declined, and enzyme is lived and only remained about 5%, illustrates that now most of laccase precipitates under ammonium sulfate effect.Therefore, 50%-80% ammonium sulfate saturation ratio is defined as the scope of laccase fractionation precipitation.
In Fig. 3, the thick liquid of enzyme first removes foreign protein with the ammonium sulfate of 50% saturation ratio, then adds ammonium sulfate, makes the saturation ratio of ammonium sulfate reach 80%.Collect the precipitation of 50% ~ 80% ammonium sulfate saturation ratio, with pH6.00.05mol/L Acetic acid-sodium acetate dissolution precipitation, 4 DEG C of refrigerator dialysed overnight, and exchange buffering liquid 3 times, use BaCl
2detect SO
4 2+whether dialyse completely, after having dialysed, and measure enzyme activity and protein content, laccase activity, measure crude enzyme liquid protein content with Coomassie Brilliant Blue, obtaining total protein in crude enzyme liquid is 6.76mg, and crude enzyme liquid Rate activity is 25.53U/mg if being 17.26U/mL.
In Fig. 4, instrument is tested with two protein peaks, and first protein peak is smaller, when NaCl concentration is 50%, occurs second protein peak.After Enzyme activity assay, laccase, in second protein peak, does not have enzyme to live, illustrates that first protein peak is foreign protein peak in first protein peak.Measure crude enzyme liquid protein content with Coomassie Brilliant Blue, obtaining total protein in crude enzyme liquid is 1.17mg, and crude enzyme liquid Rate activity is 100.43U/mg.
In Fig. 5, chromatograph detects an obvious elution peak, and recording laccase activity is 10.15U/mL, and measure crude enzyme liquid protein content with Coomassie Brilliant Blue, obtaining total protein in crude enzyme liquid is 0.6mg, and crude enzyme liquid Rate activity is 135.3U/mg.
In Fig. 6, laccase enzyme liquid separation and purification final step obtained carries out SDS-PAGE purity detecting, and obtain single protein band, its molecular size range is at about 70kDa.M in figure: albumen Marker; 1: dextran G-150 gel chromatography; 2: crude enzyme liquid after freeze-drying is concentrated.
The preparation of reagents related in an embodiment:
1) Coomassie brilliant G-250: take 100mg Coomassie brilliant G-250 and be dissolved in 50mL90% ethanol, add 85% (W/V) phosphatase 11 00mL, be finally settled to l000mL with ultrapure water, this solution can place one month at normal temperatures.
2) bovine serum albumin (BSA) preparation: get 0.01g bovine serum albumin, add in 20mL ultrapure water, constant volume is to 100mL.
3) 5 × Glycine-TrisBuffer: take Gly94g, Tris15.1g and SDS5g respectively, adds 800mLddH
2o dissolves, and is settled to 1L.
4) preparation of 10% (W/V) ammonium persulphate (APS): take 1g ammonium persulphate and add 8mLddH
2o, by thorough for pressed powder stirring and dissolving, is settled to 10mL and is stored in 4 DEG C.
5) 30% acrylamide soln: by 29g acrylamide (Acylamide) and 1gN, N '-methylene bisacrylamide (Bis) in cumulative volume be the ddH of 60mL
2in O.Be heated to 37 DEG C of dissolvings, mending and adding water to final volume is 100mL.With filter (0.45 μm of aperture) filtration sterilization, be placed in brown bottle 4 DEG C preservation, the pH value of this solution can not be greater than 7.0.
6) 5 × SDS-PAGE electrophoresis sample-loading buffer: take 1gSDS, 50mg tetrabromophenol sulfonphthalein and 3mL glycerine adds 2.5mL1mol/LTris-HCl (pH6.8) 50mmol/L, and use ddH
2o constant volume is to 10mL, and packing 1mL mono-manages, room temperature preservation.The beta-mercaptoethanol mixing of 50 μ L is added before using.
7) Coomassie brilliant blue staining fluid: Coomassie brilliant blue R2501.25g adds methyl alcohol 250mL and acetic acid 50mL and uses ddH
2o is settled to 500mL.
8) destainer (1L): ethanol 300mL, glacial acetic acid 80mL, ddH
2o620mL mixes.
Embodiment 1
1, take final volume as the fermention medium aqueous solution of 1L, its each component consumption is as follows:
After above-mentioned component adds, the constant volume that adds water is to 1L.
2, concrete preparation process:
(1) get 8 250mL triangular flasks and load above-mentioned fermention medium, each triangular flask liquid amount is 60mL, according to a conventional method sterilizing.
(2) in each triangular flask, access coronoid process to fall apart capsule bacterium spore suspension 2mL, its concentration is 1 ~ 2 × 10
8individual spore/mL, temperature 28 DEG C, under the condition of rotating speed 120r/min, shake-flask culture is after 3 days, gets fermentation liquor filtered through gauze, centrifugal acquisition supernatant liquor is crude enzyme liquid.
(3) crude enzyme liquid is successively through ammonium sulfate precipitation, DEAE-cellulose anion chromatography and dextran G-150 gel chromatography, namely obtains the laccase of separation and purification.
The ammonium sulfate precipitation related in the present embodiment, DEAE-cellulose anion chromatography and dextran G-150 gel chromatography are as described in technical scheme.
Coronoid process described in the present embodiment falls apart capsule bacterium (Eurotiumcristatum), and purchased from China General Microbiological culture presevation administrative center (CGMCC), it is numbered: 3.07934.The front coronoid process capsule bacterium (Eurotiumcristatum) that falls apart is used to be kept on potato dextrose agar slant substratum under 4 DEG C of conditions.
Adopt the laccase that the method described in the present embodiment prepares, its purification reaches 8.98, and the rate of recovery is 24.68%, and enzyme is lived and reached 2411U/L.
Embodiment 2
1, take final volume as the fermention medium aqueous solution of 1L, its each component consumption is as follows:
After above-mentioned component adds, the constant volume that adds water is to 1L.
2, concrete preparation process:
(1) get 10 250mL triangular flasks and load above-mentioned fermention medium, each triangular flask liquid amount is 60mL, according to a conventional method sterilizing.
(2) in each triangular flask, access coronoid process to fall apart capsule bacterium spore suspension 2mL, the concentration of spore suspension is 1 ~ 2 × 10
8individual spore/mL, temperature 26 DEG C, under the condition of rotating speed 120r/min, shake-flask culture is after 4 days, gets fermentation liquor filtered through gauze, centrifugal acquisition supernatant liquor is crude enzyme liquid.
(3) crude enzyme liquid is successively through ammonium sulfate precipitation, DEAE-cellulose anion chromatography and dextran G-150 gel chromatography, namely obtains the laccase of separation and purification.
The ammonium sulfate precipitation related in the present embodiment, DEAE-cellulose anion chromatography and dextran G-150 gel chromatography are as described in technical scheme.
Coronoid process described in the present embodiment falls apart capsule bacterium (Eurotiumcristatum), and purchased from China General Microbiological culture presevation administrative center (CGMCC), it is numbered: 3.07934.The front coronoid process capsule bacterium (Eurotiumcristatum) that falls apart is used to be kept on potato dextrose agar slant substratum under 4 DEG C of conditions.
Adopt the laccase that the method described in the present embodiment prepares, its purification reaches 8.96, and the rate of recovery is 24.88%, and enzyme is lived and reached 2413U/L.
Embodiment 3
1, take final volume as the fermention medium aqueous solution of 1L, the formula of its each component is:
After above-mentioned component adds, the constant volume that adds water is to 1L.
2, concrete preparation process:
(1) get in 10 250mL triangular flasks and load above-mentioned fermention medium, liquid amount is 60mL, according to a conventional method sterilizing.
(2) in each triangular flask, access coronoid process to fall apart capsule bacterium spore suspension 2mL, temperature 26 ~ 28 DEG C, under the condition of rotating speed 120r/min, shake-flask culture is after 3 ~ 4 days, gets fermentation liquor filtered through gauze, centrifugal acquisition supernatant liquor is crude enzyme liquid.
Described coronoid process falls apart capsule bacterium spore suspension, and its concentration is 1 ~ 2 × 10
8individual spore/mL.
(3) crude enzyme liquid is successively through ammonium sulfate precipitation, DEAE-cellulose anion exchange column chromatography and dextran G-150 gel chromatography, namely obtains the laccase of separation and purification.
The ammonium sulfate precipitation related in the present embodiment, DEAE-cellulose anion chromatography and dextran G-150 gel chromatography are as described in technical scheme.
Coronoid process described in the present embodiment falls apart capsule bacterium (Eurotiumcristatum), and purchased from China General Microbiological culture presevation administrative center (CGMCC), it is numbered: 3.07934.The front coronoid process capsule bacterium (Eurotiumcristatum) that falls apart is used to be kept on potato dextrose agar slant substratum under 4 DEG C of conditions.
Adopt the laccase that the method described in the present embodiment prepares, its purification reaches 8.86, and the rate of recovery is 25.02%, and enzyme is lived and reached 2415U/L.
Claims (5)
1. utilize fungi fermentation to prepare a method for laccase, it is characterized in that
1) take final volume as the fermention medium aqueous solution of 1L, its each component is:
Wheat bran 30.0 ~ 35.0g
Tealeaves 6 ~ 10g
Peptone 2.5 ~ 4g
KH
2PO
42.0～3.0g
MgSO
4·7H
2O0.2～0.5g
MnSO
4·5H
2O0.45～0.55g
CuSO
4·5H
2O0.01～0.02g
2) concrete preparation process:
(1) by above fermentative medium formula preparation substratum, in each 250mL triangular flask, load fermention medium, liquid measure is 60mL, according to a conventional method sterilizing;
(2) in each triangular flask, access coronoid process to fall apart capsule bacterium spore suspension 2mL, temperature 26 ~ 28 DEG C, under the condition of rotating speed 120r/min, shake-flask culture is after 3 ~ 4 days, gets fermentation liquor filtered through gauze, centrifugal acquisition supernatant liquor is crude enzyme liquid;
(3) crude enzyme liquid is successively through ammonium sulfate precipitation, DEAE-cellulose anion exchange column chromatography and dextran G-150 gel chromatography, namely obtains the laccase of separation and purification.
2. a kind of method utilizing fungi fermentation to prepare laccase according to claim 1, it is characterized in that described ammonium sulfate precipitation, refer to and the crude enzyme liquid prepared is placed in ice-water bath, slowly add ground ammonium sulfate to saturation ratio 50%, leave standstill 2h, 10000r/min, 4 DEG C of centrifugal 10min, get supernatant liquor; Continue to add ammonium sulfate to saturation ratio 80% in supernatant liquor, leave standstill 12 ~ 18h, 10000r/min, 4 DEG C of centrifugal 10min, collecting precipitation; Precipitate with appropriate pH6.0,0.05mol/L Acetic acid-sodium acetate buffer solution, then dialysis tubing is placed in, at pH6.0,0.05mol/L Acetic acid-sodium acetate damping fluid dialysis desalting under 4 DEG C of conditions, exchange buffering liquid 2-3 time, after dialysis terminates, measure enzyme activity and protein content, freeze-drying concentrates.
3. a kind of method utilizing fungi fermentation to prepare laccase according to claim 1, it is characterized in that described DEAE-cellulose anion exchange column chromatography, referring to first is immersed in distilled water by the Mierocrystalline cellulose of dry powder, after fully swelling, remove impurity, with the HCl solution soaking 2h of 0.5mol/L, clean to pH neutral with ultrapure water, be immersed in 2h in the NaOH solution of 0.5mol/L again, after being washed to neutrality with ultrapure water, again by pH6.0,0.05mol/L Acetic acid-sodium acetate damping fluid balance, carry out dress post, after compacting, leave standstill 12 ~ 15h; Pillar and chromatograph are coupled together, flow velocity is 0.8mL/min, first use pH6.0,0.05mol/L Acetic acid-sodium acetate damping fluid balanced gel for some time, until baseline is steady, enzyme liquid after ammonium sulfate precipitation freeze-drying being concentrated carries out loading, wash-out is carried out with 1 ~ 1.5mol/LNaCl, pH6.0,0.05mol/L Acetic acid-sodium acetate damping fluid, collect with the pipe of 10mL, often 4mL collected by pipe, the enzyme detecting each pipe is lived, dialysis desalting, and freeze-drying concentrates, obtain the enzyme powder of freeze-drying, and save backup in-20 DEG C.
4. a kind of method utilizing fungi fermentation to prepare laccase according to claim 1, it is characterized in that described dextran G-150 gel chromatography, refer to the enzyme powder of freeze-drying, with pH6.0,0.05mol/L Acetic acid-sodium acetate buffer solution, 10000r/min, 4 DEG C of centrifugal 10min, get supernatant liquor stand-by, get appropriate dextran G-150 powder, add in ultrapure water, swelling 5 ~ the 6h of boiling water bath, removing gel upper water and fine particle, with ultrapure water repetitive scrubbing 2 ~ 3 times, again with pH6.0, 0.05mol/L NaAc_HAc buffer solution washing 2-3 time, carry out dress post, spend the night after compacting, pillar and chromatograph are coupled together, flow velocity is 0.4mL/min, first use pH6.0, 0.05mol/L Acetic acid-sodium acetate damping fluid balanced gel for some time, until baseline balance, sample thief loading, buffer solution elution, collect with the pipe of 10mL, often 4mL collected by pipe, obtain laccase, the enzyme detecting each pipe laccase is lived, freeze-drying concentrates,-20 DEG C of preservations.
5. a kind of method utilizing fungi fermentation to prepare laccase according to claim 1, it is characterized in that described coronoid process falls apart capsule bacterium spore suspension, its concentration is 1 ~ 2 × 10
8individual spore/mL, process for preparation is as follows: the inoculation of the capsule bacterium that fallen apart by coronoid process is on the flat board of fresh PDA substratum, ripe spore is obtained cultivate 5d under temperature 28 DEG C of conditions after, wash lower spore with sterile distilled water, put into the Erlenmeyer flask that sterile glass beads is housed, fully vibrate, after spore is fully broken up, survey the value of spore suspension OD600, and adjust its OD value to 2.0 by stroke-physiological saline solution, obtain spore suspension.
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| CN104278018A (en) * | 2014-10-09 | 2015-01-14 | 福建农林大学 | Method for extracting cellulase from hypsizigus marmoreus culture waste |
| CN108220258A (en) * | 2016-12-15 | 2018-06-29 | 天津市林业果树研究所 | A kind of preparation method of the Stropharia rugoso-annulata activated protein with laccase activity |
| CN106497894A (en) * | 2017-01-11 | 2017-03-15 | 浙江农林大学 | The culture of the hair keyhole bacterium laccase of tool dyestuff Synergistic degradation effect prepares method and purposes |
| CN106497894B (en) * | 2017-01-11 | 2019-11-05 | 浙江农林大学 | Have the culture the preparation method and purposes of the hair keyhole bacterium laccase of dyestuff Synergistic degradation effect |
| CN109468289A (en) * | 2018-12-07 | 2019-03-15 | 广西师范大学 | A kind of process for separation and purification of tremella laccase |
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