CN104694513A - Method for producing ligninase by using edible mushroom stick - Google Patents
Method for producing ligninase by using edible mushroom stick Download PDFInfo
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- CN104694513A CN104694513A CN201410719177.7A CN201410719177A CN104694513A CN 104694513 A CN104694513 A CN 104694513A CN 201410719177 A CN201410719177 A CN 201410719177A CN 104694513 A CN104694513 A CN 104694513A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
- C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
- C12N9/0061—Laccase (1.10.3.2)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
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- C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
- C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
- C12Y110/03002—Laccase (1.10.3.2)
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- C12Y111/01—Peroxidases (1.11.1)
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- C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
- C12Y111/01—Peroxidases (1.11.1)
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- C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
- C12Y111/01—Peroxidases (1.11.1)
- C12Y111/01014—Lignin peroxidase (1.11.1.14)
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Abstract
The invention discloses a method for preparing ligninase from mushroom residue. The method comprises the specific steps: crushing the mushroom stick to obtain mushroom residue, adding enzyme-production induced liquid, cultivating under proper condition, recycling crude enzyme and repeatedly adding the enzyme-production induced liquid; the enzyme activity of obtained paint enzyme is 1-10U, the manganese peroxidase is 1-10U, and the lignin peroxidase is 10-30U. compared with other prior art related to the ligninase production method, the method saves the mushroom growth process, saves the energy source and the material, the waste is utilized, the environment is improved, and the cost for preparing the ligninase is effectively reduced.
Description
Technical field
The present invention relates to the production method of lignoenzyme, particularly relating to a kind of take edible fungus stick as the method for raw material production lignoenzyme.
Technical background
Lignoenzyme (Lignin-degrading enzymes), be can the general name of one group of enzyme of Catalytic lignin oxidative degradation, comprise laccase (being called for short Lac), manganese peroxidase (being called for short Mnp) and lignin peroxidase (abbreviation Lip).Laccase and system are called this diphenol oxydo-reductase, act on adjacent keto-alcohol with to keto-alcohol, the oxidizing reaction of amino-phenol and this diamines; Manganese peroxidase and system are called bivalent manganese hydrogen peroxide redox enzyme, and catalysis bivalent manganese is oxidized to manganic reaction; Lignin peroxidase and system are called xylogen hydrogen peroxide redox enzyme, the oxidizing reaction of Catalytic lignin.Research finds, they have stronger degradation capability to many compound such as pollutents such as polycyclic aromatic hydrocarbons, phenols, chlorinated aromatic compound, dyestuff, agricultural chemicals containing aromatic structure, and this is because edible mushrooms secretes the non-specific decision of the Ligninolytic Enzymes substrate produced.And degradation enzyme system is exocytosis, thus is more prone to contact these and treats degradation product.This special degradation mechanism presents huge application prospect in environment protection, fodder industry, chemical industry and coal chemistry, has caused the extensive concern of academia and industry member.
The production of current lignoenzyme adopts liquid submerged fermentation to produce mostly, and technical requirements is harsh, and energy consumption is high, and enzyme is lived low and can be produced a large amount of factory effluents, produces detrimentally affect to environment.In recent years, various countries researchist starts to produce lignoenzyme with solid-state fermentation technology gradually, and it can imitate the process of growth of natural microbial, produce the product that people need, but because the microorganism producing lignoenzyme is all generally filamentous fungus, the speed of growth is comparatively slow, therefore the production cycle is longer.Also studies have reported that and directly from edible fungi residue, extract lignoenzyme, but produce lignoenzyme enzyme in bacterium slag that enzyme induction liquid cultivates live lower owing to not adding, need very high separation and purification cost, economic benefit is lower, is unfavorable for large-scale application.
Up to the present, there is not yet any technology taking edible fungus stick as raw material and add that product enzyme induction liquid carries out second incubation production lignoenzyme.
Summary of the invention
The object of the present invention is to provide a kind of edible fungus stick to produce the method for lignoenzyme, utilize the bacterium rod containing hypha of edible fungus to be raw material, make the bacterium rod in agriculture production obtain high-valued recycling, environmental protect, reduce the cost that lignoenzyme is produced.
Bacterium rod refers to cotton seed hulls, wood chip, straw, corn cob, bagasse and multiple kinds of crops stalk, industrial waste for main raw material, is used for the bar-shaped solid mixt producing fruit body of edible fungi or sporophore of gathering is later remaining after inoculation hypha of edible fungus cultivates for some time.When hypha of edible fungus grows in the medium, need to produce a large amount of lignoenzyme to decompose lignocellulose in matrix to obtain nutrition.Therefore, results or not yet gather in the crops sporophore bacterium rod in containing a large amount of hypha of edible fungus and lignocellulose.The concrete composition of bacterium rod can not as the restriction to lignoenzyme preparation method of the present invention.
Industrial waste is mainly selected from vinegar grain, vinasse, paper mill effluent.
Edible mushrooms is can be biological edible mushroom under artificial culture or wild environment.Lignoenzyme can be secreted to decompose lignocellulose in matrix to obtain nutrition in its process of growth.Described edible mushrooms is mainly selected from mushroom (Agaricus campestris), mushroom (Lentinula edodes), tea tree mushroom (Agrocybe aegerita), Hericium erinaceus (Bull. Ex Fr.) Pers. (Hericium erinaceum), Pleurotus sajor-caju (Pleurotus sajor-caju), Twospore Mushroom (Agaricus bisporus), auricularia auriculajudae (Auricularia auricula), Cordyceps sinensis (Cordyceps sinensis), glossy ganoderma (Ganoderma lucidum), pycnoporus samguineus (Pycnoporus sanguineus), gold ear (Tremella aurantialba), Pleurotus abalonus (Pleurotus abalonus), flat mushroom (Pleurotus ostreatus), needle mushroom (Flammulinavelutipes), Pleurotus eryngii (Pleurotus eryngii), Pleurotus geesteranus (Pleurotus geesteranus), Coprinus comatus (Coprinus comatus), Pleurotus nebrodensis (Pleurotus nebrodensis), lung shape picks up the ears (Pleurotus pulmonarius), Agaricus blazei Murrill (Agaricus blazei) etc.
The invention provides a kind of method utilizing edible fungus stick to produce lignoenzyme, specifically comprise the steps: that the break process of bacterium rod obtains bacterium chaff; Add and produce enzyme induction liquid; Cultivate under suitable condition; Crude enzyme liquid reclaims; Repeatedly add induced liquid and produce enzyme.
1. broken bacterium rod obtains bacterium chaff
After the bacterium of sporophore of or not yet gathering rod removing plastics bag, utilize pulverizer that the fragmentation of bacterium rod is obtained uniform bacterium chaff.
2. add induction and produce enzyme liquid
The inductor that enzyme liquid contains lignoenzyme is produced in induction used, and other substances provide nitrogen element and trace element for hypha of edible fungus, can also adjust carbon-nitrogen ratio (C/N) and the pH value of system in addition, stimulates mycelia diauxic growth.Induction is produced enzyme liquid and is obtained by the prolonged and repeated test in this laboratory.
A kind of concrete embodiment, the usual add-on that enzyme liquid is produced in every 10kg bacterium chaff induction is 10-50L, 10-30L can be selected further, the diauxic growth that more effectively can stimulate hypha of edible fungus in bacterium chaff when 12,13,15,16,17,19,20,23,25 or 28L is selected, the high expression of induction lignoenzyme and secretion when concrete.
3. cultivate under suitable condition
Suitable extraneous culture condition can change the physiological status of hypha of edible fungus, better expression and secretion ligninase.
A concrete embodiment, culture temperature is generally 17-35 DEG C, can be chosen as 22-30 DEG C further, or is specifically chosen as 22,24,25,27,28,30 DEG C.The time of cultivating under above temperature condition is 24-192 hour, or selects 24,72,96,120 or 144 hours further, can obtain the bacterium chaff that lignoenzyme content is higher.
4. the recovery of crude enzyme liquid
What obtain to step 3 contains the distilled water immersion adding 1-20 times of volume in the fermentation maturation system of a large amount of lignoenzyme, and vibration is filtered.Can select 1-10 further, the lignoenzyme that mycelia can be made when being specifically chosen as 1,3,5,7,9 times to secrete is dissolved in solution as much as possible and does not hurt mycelia again.The lignoenzyme total amount obtained when distilled water volume is very few will be very few, when distilled water volume is excessive, will in elution process, make mycelia suffer damage.
5. add for many times and produce enzyme induction liquid
The bacterium chaff obtained to step 3 replace repeating step 2 add produce enzyme induction liquid, step 3 suitable condition under cultivate, the recovery of step 4 crude enzyme liquid, amount to 1-6 cycle, the object done so more efficiently utilizes bacterium chaff.Multiplicity too much after aging due to mycelia, enzymatic productivity declines.
The invention provides a kind of method utilizing edible fungus stick to produce lignoenzyme.It is a kind of new way of bacterium chaff higher value application, it is a kind of brand-new lignoenzyme production method, compared with current all relevant lignoenzyme production methods reported, present invention eliminates edible fungi bulk-growth process, shorten and produce the enzyme time, having saved the energy and material, is that in all relevant lignoenzyme production methods reported, cost is minimum.Therefore, utilize waste mushroom leftover to prepare lignoenzyme, both can accomplish utilization of waste material environmental protect, the production cost of lignoenzyme can be reduced again, there is good application prospect.
Embodiment
Technical scheme of the present invention is described in detail below in conjunction with example.Embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although with reference to preferred embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that, can modify to the technical scheme of invention or equivalent replacement, and not departing from the spirit and scope of technical solution of the present invention, it all should be encompassed in right of the present invention.
Embodiment 1 lignoenzyme enzyme activity determination method
Get the crude enzyme liquid 10mi that step 4 obtains, be divided in the centrifuge tube of 10 1ml, under 12000r/min centrifugal 10 minutes, get supernatant liquor and measure lignoenzyme enzyme and live.
Manganese peroxidase (Mnp) enzyme activity determination method: at 30 DEG C, containing 50mmol in reaction system, the tartrate one sodium tartrate damping fluid 2.0mL of pH5.0, the manganese sulfate solution 0.8mL of 1.6mmol/L, enzyme liquid 0.1mL, the DMP25 μ L of 20mmol/L, the hydrogen peroxide 75 μ L adding 1.6mmol/L starts reaction.The absorbancy change at assaying reaction initial 1 minute inherent 470nm place.1 enzyme activity unit is defined as per minute and produces enzyme amount required for 1 μm of ol product.
Lignin peroxidase (Lip) enzyme activity determination method: with Li Lu alcohol for substrate, the Li Lu alcohol 100 μ L of the tartrate of pH 3.5-sodium tartrate damping fluid 2.3mL, 10mmol/L, the hydrogen peroxidase 10 .3mL of 0.4mmol/L, enzyme liquid is appropriate.The absorbancy change at 310nm place is detected at 30 DEG C.1 enzyme activity unit is defined as per minute and produces enzyme amount required for 1 μm of ol product.
Laccase activity (Lacase) measuring method: reaction system is 3mL, comprising the phosphate buffered (pH3.5) of 2.4mL 0.1mol μ L, the enzyme liquid of 10mmol/L DMP and 0.1mL of 0.5mL, reaction system is incubated 5min adding at first 50 DEG C of enzyme liquid, adds enzyme liquid afterwards and surveys absorbancy change at 470nm place.1 enzyme activity unit is defined as per minute and produces enzyme amount required for 1 μm of ol product.
Embodiment 2
Get 10kg flat mushroom bacterium rod, fragmentation adds the product enzyme induction liquid of 15L after obtaining bacterium chaff, and cultivate 3 days at 28 DEG C, the system obtained adds 150L distilled water immersion, and vibration is filtered and obtained crude enzyme liquid and bacterium chaff.The product enzyme induction liquid adding 15L is continued in residual bacterium chaff, cultivate 3 days at 28 DEG C, the system obtained adds 150L distilled water immersion, vibration, filters and obtains crude enzyme liquid and bacterium chaff, so circulation 3 times, obtain the crude enzyme liquid of 450L, recording lignoenzyme enzyme according to embodiment 1 lives as follows: manganese peroxidase enzyme 4U alive, lignin peroxidase enzyme 3U, laccase activity 22U alive.
Embodiment 3
Get 10kg agaricus bisporus mushroom rod, fragmentation adds the product enzyme induction liquid of 25L after obtaining bacterium chaff, and cultivate 4 days at 22 DEG C, the system obtained adds 150L distilled water immersion, and vibration is filtered and obtained crude enzyme liquid and bacterium chaff.The product enzyme induction liquid adding 25L is continued in residual bacterium chaff, cultivate 4 days at 22 DEG C, the system obtained adds 150L distilled water immersion, vibration, filters and obtains crude enzyme liquid and bacterium chaff, so circulation 3 times, obtain the crude enzyme liquid of 450L, recording lignoenzyme enzyme according to embodiment 1 lives as follows: manganese peroxidase enzyme 2U alive, lignin peroxidase enzyme 1U, laccase activity 17U alive.
Embodiment 4
Get 10kg pycnoporus samguineus bacterium rod, fragmentation adds the product enzyme induction liquid of 20L after obtaining bacterium chaff, and cultivate 3 days at 25 DEG C, the system obtained adds 250L distilled water immersion, and vibration is filtered and obtained crude enzyme liquid and bacterium chaff.The product enzyme induction liquid adding 20L is continued in residual bacterium chaff, cultivate 3 days at 25 DEG C, the system obtained adds 250L distilled water immersion, vibration, filters and obtains crude enzyme liquid and bacterium chaff, so circulation 4 times, obtain the crude enzyme liquid of 1000L, recording lignoenzyme enzyme according to embodiment 1 lives as follows: manganese peroxidase enzyme 6U alive, lignin peroxidase enzyme 4U, laccase activity 28U alive.
Embodiment 5
Get 10kg lentinus edodes strain stick, fragmentation adds the product enzyme induction liquid of 17L after obtaining bacterium chaff, and cultivate 3 days at 23 DEG C, the system obtained adds 150L distilled water immersion, and vibration is filtered and obtained crude enzyme liquid and bacterium chaff.The product enzyme induction liquid adding 17L is continued in residual bacterium chaff, cultivate 3 days at 23 DEG C, the system obtained adds 150L distilled water immersion, vibration, filters and obtains crude enzyme liquid and bacterium chaff, so circulation 4 times, obtain the crude enzyme liquid of 600L, recording lignoenzyme enzyme according to embodiment 1 lives as follows: manganese peroxidase enzyme 2U alive, lignin peroxidase enzyme 5U, laccase activity 26U alive.
Claims (9)
1. one kind utilizes the method for edible fungus stick fermentative production lignoenzyme.It is characterized in that: with the edible fungus stick of sporophore of not yet or gathering for raw material, add and produce enzyme induction liquid fermentative production lignoenzyme.
2., according to the method for producing lignoenzyme described in claim 1 with edible fungus stick, it is characterized in that described method is specially:
1) fragmentation of bacterium rod obtains bacterium chaff: by the edible fungus stick of sporophore of or not yet gathering by obtaining bacterium chaff after physical means fragmentation;
2) product enzyme induction liquid is added: add according to the ratio of solid-liquid mass volume ratio 1: 1-4 in the bacterium chaff obtained by step 1 and produce enzyme induction liquid, obtain after mixing producing enzyme system;
3) cultivate under suitable condition: product enzyme system step 2 obtained cultivates 24-192 hour under 17-35 DEG C of condition, carries out fermentative production lignocellulolyticenzymes;
4) crude enzyme liquid reclaims: the distilled water wash-out of fermentation maturation system 1-10 times of volume step 3 obtained obtains crude enzyme liquid;
5) repeatedly induced liquid is added: the bacterium chaff that step 4 stays can repeat to cultivate and the recovery of step 4 crude enzyme liquid under alternate steps 2 adds product enzyme induction liquid, step 3 suitable condition, amounts to 1-6 cycle.
3. a kind of method utilizing edible fungus stick to produce lignoenzyme according to claim 1, is characterized in that edible fungus stick needs after physics fragmentation, obtain even bacterium chaff.
4. a kind of method utilizing edible fungus stick fermentative production lignoenzyme according to claim 2, it is characterized in that described product enzyme induction liquid formula is: in mass, dextrin 1 ~ 3%, urea 0.1 ~ 0.3%, ammonium nitrate 0.2 ~ 0.4%, dipotassium hydrogen phosphate 0.035 ~ 0.07%, potassium primary phosphate 0.04 ~ 0.08%, copper sulfate 0.002 ~ 0.01%, magnesium sulfate 0.02 ~ 0.04%, wheat bran 4 ~ 7%, tween-80 0.01 ~ 0.08%, veratryl alcohol 0.0228 ~ 0.14%, water 89.0702 ~ 94.375%, wherein said each component sum is 100%.
5. a kind of method utilizing edible fungus stick to produce lignoenzyme according to claim 2, is characterized in that every 10kg bacterium rod needs the product enzyme induction liquid of 10L-40L.
6. a kind of method utilizing edible fungus stick fermentative production lignoenzyme according to claim 2, is characterized in that described culture temperature is 20-30 DEG C.
7. a kind of method utilizing edible fungus stick fermentative production lignoenzyme according to claim 2, is characterized in that the incubation time in an enzymatic production cycle is 24-168 hour.
8. a kind of method utilizing edible fungus stick to produce lignoenzyme according to claim 2, is characterized in that the mass volume ratio of enzymatic production system and each wash-out water is 1: 1-10.
9. a kind of method utilizing edible fungus stick fermentative production lignoenzyme according to claim 2, is characterized in that the described number of times repeatedly adding product enzyme induction liquid is 1-6.
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105013813A (en) * | 2015-07-01 | 2015-11-04 | 江南大学 | Method for biologically remediating crude oil contaminated soil by using edible fungi residues |
| CN105016800A (en) * | 2015-07-01 | 2015-11-04 | 江南大学 | Method for using fungus bran to produce organic fertilizer |
| CN105010725A (en) * | 2015-07-01 | 2015-11-04 | 江南大学 | Feed production method by adding enzyme producing inducer into edible fungus brans |
| CN105032923A (en) * | 2015-07-01 | 2015-11-11 | 江南大学 | Method used for bioremediation of PAHs polluted soil with edible mushroom residue |
| CN105080961A (en) * | 2015-07-01 | 2015-11-25 | 江南大学 | Method for remedying soil contaminated by phenols through residues of mushroom media |
| CN105124170A (en) * | 2015-07-01 | 2015-12-09 | 江南大学 | Method for producing forage by using edible fungus chaff |
| CN105146053A (en) * | 2015-07-01 | 2015-12-16 | 江南大学 | Method for production of high protein feed from edible fungus chaff |
| CN105567664A (en) * | 2016-03-11 | 2016-05-11 | 福建农林大学 | Preparation method of enzymic preparation for degrading lignin and cellulose |
| CN107736185A (en) * | 2017-11-07 | 2018-02-27 | 王逸帆 | Utilize the method for lentinus edodes strain stick waste material cultivation line Radix Codonopsis |
| CN112210547A (en) * | 2019-07-12 | 2021-01-12 | 湖南农业大学 | Mushroom bran crude enzyme preparation and preparation method thereof |
| CN112831480A (en) * | 2021-04-14 | 2021-05-25 | 成都信息工程大学 | Method for producing laccase by solid-state fermentation of pseudo-ginseng residue by lucid ganoderma |
| CN114084967A (en) * | 2021-11-12 | 2022-02-25 | 河南省科学院生物研究所有限责任公司 | Ganoderma lucidum fermented Shuanghuanglian mushroom residue for repairing water environment polluted by polycyclic aromatic hydrocarbon benzo [ a ] pyrene and application thereof |
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105013813A (en) * | 2015-07-01 | 2015-11-04 | 江南大学 | Method for biologically remediating crude oil contaminated soil by using edible fungi residues |
| CN105016800A (en) * | 2015-07-01 | 2015-11-04 | 江南大学 | Method for using fungus bran to produce organic fertilizer |
| CN105010725A (en) * | 2015-07-01 | 2015-11-04 | 江南大学 | Feed production method by adding enzyme producing inducer into edible fungus brans |
| CN105032923A (en) * | 2015-07-01 | 2015-11-11 | 江南大学 | Method used for bioremediation of PAHs polluted soil with edible mushroom residue |
| CN105080961A (en) * | 2015-07-01 | 2015-11-25 | 江南大学 | Method for remedying soil contaminated by phenols through residues of mushroom media |
| CN105124170A (en) * | 2015-07-01 | 2015-12-09 | 江南大学 | Method for producing forage by using edible fungus chaff |
| CN105146053A (en) * | 2015-07-01 | 2015-12-16 | 江南大学 | Method for production of high protein feed from edible fungus chaff |
| CN105567664A (en) * | 2016-03-11 | 2016-05-11 | 福建农林大学 | Preparation method of enzymic preparation for degrading lignin and cellulose |
| CN107736185A (en) * | 2017-11-07 | 2018-02-27 | 王逸帆 | Utilize the method for lentinus edodes strain stick waste material cultivation line Radix Codonopsis |
| CN112210547A (en) * | 2019-07-12 | 2021-01-12 | 湖南农业大学 | Mushroom bran crude enzyme preparation and preparation method thereof |
| CN112831480A (en) * | 2021-04-14 | 2021-05-25 | 成都信息工程大学 | Method for producing laccase by solid-state fermentation of pseudo-ginseng residue by lucid ganoderma |
| CN114084967A (en) * | 2021-11-12 | 2022-02-25 | 河南省科学院生物研究所有限责任公司 | Ganoderma lucidum fermented Shuanghuanglian mushroom residue for repairing water environment polluted by polycyclic aromatic hydrocarbon benzo [ a ] pyrene and application thereof |
| CN114084967B (en) * | 2021-11-12 | 2024-01-23 | 河南省科学院生物研究所有限责任公司 | Lucid ganoderma fermented Shuanghuanglian mushroom dreg for repairing polycyclic aromatic hydrocarbon benzo [ a ] pyrene polluted water environment and application thereof |
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