CN105567664A - Preparation method of enzymic preparation for degrading lignin and cellulose - Google Patents
Preparation method of enzymic preparation for degrading lignin and cellulose Download PDFInfo
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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
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
- C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
- C12N9/0061—Laccase (1.10.3.2)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/248—Xylanases
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
- C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
- C12Y110/03002—Laccase (1.10.3.2)
Abstract
The invention discloses a preparation method of an enzymic preparation for degrading lignin and cellulose. The preparation method comprises the following steps: (1) taking a waste fungous material, adding water and carrying out vibration extraction after smashing the waste fungous material, wherein a weight ratio of the waste fungous material and the water is 1:12, the rotating speed is 180 r/min, the extraction temperature is 25 DEG C, and the extraction time is 20 minutes; then centrifuging, thus obtaining a crude enzyme solution; (2) carrying out spray drying, wherein spray drying parameters are set as follows: the spray drying parameter of a draught fan is 26.0, the spray drying parameter of a nozzle cleaner is 5.0, the spray drying parameter of a peristaltic pump is 55.0, the hot air flow is 1.5 to 3.5 m<3>/min, the air inlet temperature is 95 to 115 DEG C, and the material inlet speed is 10 to 35 mL/min. According to the preparation method of the enzymic preparation for degrading the lignin and the cellulose, disclosed by the invention, the source of raw materials is wide, a fermentation step does not need to be carried out, the prepared enzymic preparation can be used for degrading the lignin, the cellulose and hemicellulose in a cultivation material, the utilization rate of fiber raw materials is increased, the production time is shortened, and quick development of second-generation biomass fuel-ethyl alcohol which uses lignocellulose as the raw material is promoted.
Description
Technical field
The invention belongs to zymin and preparation method thereof field, be specifically related to the preparation method of a kind of lignin degrading, cellulase preparation.
Background technology
The alcohol fuel overwhelming majority of current suitability for industrialized production take food crop as raw material, as corn, wheat and rice etc., has scale restriction and unsustainable property in the long run.Therefore, to take lignocellulose as the s-generation biomass fuel ethanol of raw material be determine following can the key of petroleum replacing on a large scale.The basic material of s-generation alcohol fuel is biomass, i.e. the sponge of bagasse, discarded maize straw and other types, and these raw materials are converted into sugar through cellulase hydrolysis, and then generate ethanol by fermentation.Mierocrystalline cellulose production alcohol fuel is utilized to represent the direction of future biological matter fuel development.Some enterprises of US and European have accelerated the research steps to s-generation biomass fuel ethanol technology.But the front-end investment of production of cellulosic ethanol is larger, according to the measuring and calculating of some research institutions of the U.S., under the condition that industrial scale is identical, the investment that cellulose fuel ethanol needs is 7 ~ 8 times of Corn Fuel Ethanol investment, its key reason is the preferential degradation of xylogen in lignocellulose pre-treatment is the key that constraints on fiber element utilizes, lignocellulose forms primarily of plant cell wall, the basal component mainly Mierocrystalline cellulose of cell walls, hemicellulose and xylogen, Mierocrystalline cellulose and hemicellulose are wrapped up layer by layer by xylogen, barrier action is play in hydrocellulose process, solve the degradation problem of xylogen, just can improve the degradation property of lignocellulose to a great extent, therefore the important method that effective preconditioning technique is a kind of Appropriate application lignocellulose is researched and developed.
Sugarcane is the C4 crop that photosynthetic capacity is the strongest, is also the field crop that biomass that the mankind are cultivated so far is the highest.Bagasse is the main waste of sugar industry, sugar refinery of China often produces 1 ton of sucrose and will produce 2-3 ton bagasse, bagasse is the main raw material of s-generation alcohol fuel, therefore, how to realize high-level efficiency, the enzymic degradation of baggasse fiber of low cost is the key point realizing cellulose fuel ethanol industrialization, and bagasse preconditioning technique is the vital the first step efficiently.
Most of edible mushrooms have very strong capacity of decomposition to natural wood, the degraded of edible mushrooms to xylogen is the result of mycelia secretion extracellular enzyme effect, mainly comprises lignin peroxidase, depends on the peroxidase of manganese and the important lignin-degrading enzymes of laccase three kinds.Wherein, main body oxydase is the Extracellular laccase of thalline secretion, and other enzyme mainly plays synergy.
The method preparing lignin degradation zymin, cellulase preparation, xylanase preparation etc. is at present liquid fermenting thalline, then extraction and isolation enzyme from fermented liquid, this process also needs to experience fermenting experiment, period also can be subject to the impact of fermentation condition, and it is not extensive to have material source, the shortcomings such as preparation time is long, complicated.
Summary of the invention
The present invention is directed to above-mentioned weak point and the preparation method of a kind of lignin degrading provided, cellulase preparation, obtain high purity zymin by raw material pulverizing-lixiviate-filtration-spraying dry-Powdered zymin crude product-dissolving-ammonium sulfate precipitation-dialysis-DEAE ion exchange chromatography-lyophilize; In the present invention with waste edible fungus bacterium bag for raw material, its material source is extensive, do not need to experience fermentation step, also useless bacterium bag is utilized again simultaneously, more economic worth can be increased, resource have also been obtained effective utilization, can not cause waste, also can not directly abandoning and burning and cause environmental pollution because of useless bacterium bag.
For achieving the above object, the technical solution adopted for the present invention to solve the technical problems is:
A preparation method for lignin degrading, cellulase preparation, comprises the following steps:
(1) will give up bacterium bag by following condition lixiviate: get waste mushroom, after pulverizing, add water concussion lixiviate, and material water weight ratio is 1:12, and rotating speed is 180r/min, and extraction temperature is 25 DEG C, and extraction time is 20min, then centrifugal, obtains crude enzyme liquid;
(2) spraying dry crude zyme preparation: get the crude zyme preparation of crude enzyme liquid through spraying dry; Spray drying parameters is blower fan setting 26.0, cleansing pin setting 5.0, peristaltic pump setting 55.0, hot air flow 1.5-3.5m
3/ min, inlet temperature 95-115 DEG C, input speed 10-35mL/min;
Because laccase content is higher in crude zyme preparation, is further advanced by following methods and purifying carried out to laccase:
(1) ammonium sulfate precipitation: crude zyme preparation is dissolved, add ammonium sulfate adjustment saturation ratio to 40%, 25 DEG C, after 150r/min shaking table 2-3h, 10000r/min, 25 DEG C of centrifugal 20min, get supernatant liquor and then adjust ammonium sulfate saturation ratio to 60%, 25 DEG C, after 150r/min shaking table 2-3h, 10000r/min, 25 DEG C of centrifugal 20min remove supernatant liquors, use distilled water to suspend precipitation to dissolve, dialysis is desalted;
(2) DEAE ion exchange chromatography: the enzymes soln that above-mentioned steps obtains is crossed DEAE ion exchange chromatography, collect elutriant, lyophilize obtains high purity zymin, and wherein, applied sample amount is 4mL; Pillar specification is 50cm × 2cm, column volume 100mL; Elutriant is pH value is 6.0, and concentration is the sodium phosphate buffer of 0.02mol/L, and working concentration is the NaCl solution gradient elution of 0-0.5mol/L, and flow velocity is 1.5mL/min.
Further, described hot air flow 3.5m
3/ min, inlet temperature 95 DEG C, input speed 20mL/min.
The preparation method of a kind of lignin degrading provided by the invention, cellulase preparation, has following beneficial effect:
(1) material source is extensive, does not need to experience fermentation step.
(2) again utilize useless bacterium bag, can increase more economic worth, resource have also been obtained effective utilization, can not cause waste, also can not directly abandoning and burning and cause environmental pollution because of useless bacterium bag.
(3) crude enzyme liquid extracted from Pleurotus geesteranus gives up bacterium bag contains laccase, cellulase, zytase, after spraying dry, this mixing enzyme preparation is used for lignin degrading in planting material, Mierocrystalline cellulose and hemicellulose, thus accelerate the speed of growth of edible mushrooms, strengthen raw material availability, increase economic worth.
(4) the crude zyme preparation pre-treatment bagasse containing Ligninolytic Enzymes is adopted, change baggasse fiber structure, remove xylogen, explore enzyme process pre-treatment approach, realize cellulose fuel ethanol industrialization, improve the utilization ratio of fibrous material, shorten the production time, promote the fast development of the s-generation biomass fuel ethanol taking lignocellulose as raw material.
(5) preparation method's simple possible, is applicable to suitability for industrialized production.
Accompanying drawing explanation
The dye decolored result of Fig. 1 zymin.
Embodiment
Embodiment 1 prepares crude zyme preparation and purifying
A kind of lignin degrading, cellulase preparation high efficiency preparation method, comprise the following steps:
(1) discarded Pleurotus geesteranus is given up bacterium bag by following condition lixiviate: get waste mushroom, after pulverizing, add water concussion lixiviate, and material water weight ratio is 1:12, and rotating speed is 180r/min, and extraction temperature is 25 DEG C, and extraction time is 20min, then centrifugal, obtains crude enzyme liquid;
(2) spraying dry crude zyme preparation: spray drying parameters is blower fan setting 26.0, cleansing pin setting 5.0, peristaltic pump setting 55.0, hot air flow 1.5-3.5m
3/ min, inlet temperature 95-115 DEG C, input speed 10-35mL/min;
(3) ammonium sulfate precipitation: the crude zyme preparation of step (2) is dissolved, add ammonium sulfate adjustment saturation ratio to 40%, 25 DEG C, after 150r/min shaking table 2-3h, 10000r/min, 25 DEG C of centrifugal 20min, get supernatant liquor and then adjust ammonium sulfate saturation ratio to 60%, 25 DEG C, after 150r/min shaking table 2-3h, 10000r/min, 25 DEG C of centrifugal 20min remove supernatant liquors, use distilled water to suspend precipitation to dissolve, dialysis is desalted.
Crude zyme preparation comprises cellulase, zytase, laccase, and before and after its purifying, enzyme lives change in table 1:
Before and after table 1 crude zyme preparation purifying, enzyme is lived and is changed
Can find that crude zyme preparation, laccase has higher activity from measurement result, from crude zyme preparation, thus extract laccase has good economic benefits and using value.
Embodiment 2 input speed is on the impact of laccase activity
A kind of lignin degrading, cellulase preparation high efficiency preparation method, comprise the following steps:
(1) discarded Pleurotus geesteranus is given up bacterium bag by following condition lixiviate: get waste mushroom, after pulverizing, add water concussion lixiviate, and material water weight ratio is 1:12, and rotating speed is 180r/min, and extraction temperature is 25 DEG C, and extraction time is 20min, then centrifugal, obtains crude enzyme liquid;
(2) spraying dry painting enzyme crude product: carry out spraying dry to crude enzyme liquid and obtain Powdered laccase and laccase crude product, spray drying parameters is: blower fan setting 26.0, cleansing pin setting 5.0, peristaltic pump setting 55.0, hot air flow 2.6m
3/ min, inlet temperature 105 DEG C, input speed 10-35mL/min, the impact of input speed on laccase activity the results are shown in Table 2.
Table 2 input speed is on the impact of laccase activity
As shown in Table 2, when input speed is 15mL/min, laccase activity is the highest.
Embodiment 3 hot air flow is on the impact of laccase activity
A kind of lignin degrading, cellulase preparation high efficiency preparation method, comprise the following steps:
(1) discarded Pleurotus geesteranus is given up bacterium bag by following condition lixiviate: get waste mushroom, after pulverizing, add water concussion lixiviate, and material water weight ratio is 1:12, and rotating speed is 180r/min, and extraction temperature is 25 DEG C, and extraction time is 20min, then centrifugal, obtains crude enzyme liquid;
(2) spraying dry painting enzyme crude product: carry out spraying dry to crude enzyme liquid and obtain Powdered laccase and laccase crude product, spray drying parameters is: blower fan setting 26.0, cleansing pin setting 5.0, peristaltic pump setting 55.0, input speed 15mL/min, inlet temperature 105 DEG C, hot air flow 1.5-3.5m
3/ min, the impact of hot air flow on laccase activity the results are shown in Table 3.
Table 3 hot air flow is on the impact of laccase activity
As shown in Table 3, as hot air flow 3.0m
3during/min, laccase activity is the highest.
Embodiment 4 orthogonal experiment
A kind of lignin degrading, cellulase preparation high efficiency preparation method, comprise the following steps:
(1) discarded Pleurotus geesteranus is given up bacterium bag by following condition lixiviate: get waste mushroom, after pulverizing, add water concussion lixiviate, and material water weight ratio is 1:12, and rotating speed is 180r/min, and extraction temperature is 25 DEG C, and extraction time is 20min, then centrifugal, obtains crude enzyme liquid;
(2) spraying dry painting enzyme crude product: carry out spraying dry to crude enzyme liquid and obtain Powdered laccase, spray drying parameters is: blower fan setting 26.0, cleansing pin setting 5.0, peristaltic pump setting 55.0, input speed 10-20mL/min, inlet temperature 95-115 DEG C, hot air flow 2.5-3.5m
3/ min, with input speed, inlet temperature and hot air flow for orthogonal experiment, measures its impact on laccase activity, the results are shown in Table 4.
Table 4 Orthogonal experiment results
Can be obtained by table 4, when inlet temperature be 95 DEG C, input speed is 20mL/min, hot air flow is 3.5m
3during/min, laccase activity is the highest.
Embodiment 5
A kind of lignin degrading, cellulase preparation high efficiency preparation method, comprise the following steps:
(1) discarded Pleurotus geesteranus is given up bacterium bag by following condition lixiviate: get waste mushroom, after pulverizing, add water concussion lixiviate, and material water weight ratio is 1:12, and rotating speed is 180r/min, and extraction temperature is 25 DEG C, and extraction time is 20min, then centrifugal, obtains crude enzyme liquid;
(2) spraying dry crude zyme preparation: spraying dry is carried out to crude enzyme liquid and obtains Powdered laccase, spray drying parameters is: blower fan setting 26.0, cleansing pin setting 5.0, peristaltic pump setting 55.0, inlet temperature is 95 DEG C, input speed is 20mL/min, hot air flow is 3.5m
3/ min;
(3) ammonium sulfate precipitation:
By the laccase dissolving crude product that step (2) prepares, add ammonium sulfate adjustment saturation ratio to initial saturation (40%), 25 DEG C, after 150r/min shaking table 3h, 10000r/min, 25 DEG C of centrifugal 20min get supernatant, gather supernatant adjustment ammonium sulfate saturation ratio to stopping concentration (60%), 25 DEG C, after 150r/min shaking table 3h, 10000r/min, 25 DEG C of centrifugal 20min remove supernatant, use 25mL distilled water to suspend precipitation to dissolve, dialysis is desalted;
(4) DEAE ion exchange chromatography:
Applied sample amount: 4mL
Pillar specification: 50cm × 2cm, column volume 100mL
The sodium phosphate buffer (pH6.0) of elutriant: 0.02mol/L, NaCl(0-0.5mol/L) gradient elution, flow velocity 1.5mL/min
Often 4.5mL collected by pipe, measures the laccase activity that every root collects liquid.
Cross post according to above-mentioned condition, reclaim the higher collection tube of enzyme activity according to laccase activity changing conditions and gather.The changing conditions that after crossing post, enzyme is lived is as table 5:
The changing conditions that table 5 enzyme is lived
More than test other edible mushroomss all can be taked discarded waste mushroom to prepare lignin degrading, cellulase preparation, be not limited to Pleurotus geesteranus waste mushroom.
The crude zyme preparation prepared is used for the decolouring of dyestuff by experimental example 1
Prepare the dyestuff 20mL of 0.5/L and the dry powder (22.6U) after adding 0.1g spraying dry in dyestuff, survey the concentration of each dye liquor, be placed in shaking table 40 DEG C, after 150r/min reacts one hour, survey the concentration of each dye liquor again, not add the solution being dissolved with laccase dry powder of dyestuff for blank, decolouring the results are shown in Table 6.
The decolouring result of table 6 different dyes
From Fig. 1, table 6, can observe out significantly, this zymin has good decolorizing effect to Methylene blue and aniline blue, and percent of decolourization reaches 46.4% and 38.3% respectively.
Experimental example 2 crude zyme preparation is applied to xylogen, cellulosic degraded
0.1g zymin dry powder (22.6U) adds 20mL to and contains in 0.5g bagasse powder solution, and when pretreatment time is 24h, temperature 30 DEG C, increasing sugared concentration is 0.45g/L, and Lignin degradation rate improves 12.6%, and cellulose degradation rate improves 16.12%.Increase sugared concentration and improve bagasse xylogen, cellulosic degraded with regard to representative.
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
Claims (3)
1. a preparation method for lignin degrading, cellulase preparation, is characterized in that, comprises the following steps:
(1) will give up bacterium bag by following condition lixiviate: get waste mushroom, after pulverizing, add water concussion lixiviate, and material water weight ratio is 1:12, and rotating speed is 180r/min, and extraction temperature is 25 DEG C, and extraction time is 20min, then centrifugal, obtains crude enzyme liquid;
(2) spraying dry crude zyme preparation: get the crude zyme preparation of crude enzyme liquid through spraying dry; Spray drying parameters is blower fan setting 26.0, cleansing pin setting 5.0, peristaltic pump setting 55.0, hot air flow 1.5-3.5m
3/ min, inlet temperature 95-115 DEG C, input speed 10-35mL/min.
2. the preparation method of a kind of lignin degrading according to claim 1, cellulase preparation, is characterized in that, further comprising the steps of:
(1) ammonium sulfate precipitation: crude zyme preparation is dissolved, add ammonium sulfate adjustment saturation ratio to 40%, 25 DEG C, after 150r/min shaking table 2-3h, 10000r/min, 25 DEG C of centrifugal 20min, get supernatant liquor and then adjust ammonium sulfate saturation ratio to 60%, 25 DEG C, after 150r/min shaking table 2-3h, 10000r/min, 25 DEG C of centrifugal 20min remove supernatant liquor, use distilled water to suspend precipitation to dissolve, dialysis is desalted, and obtains enzymes soln;
(2) DEAE ion exchange chromatography: the enzymes soln that above-mentioned steps obtains is crossed DEAE ion exchange chromatography, collect elutriant, lyophilize obtains high purity zymin, and wherein, applied sample amount is 4mL; Pillar specification is 50cm × 2cm, column volume 100mL; Elutriant is pH value is 6.0, and concentration is the sodium phosphate buffer of 0.02mol/L, and working concentration is the NaCl solution gradient elution of 0-0.5mol/L, and flow velocity is 1.5mL/min.
3. the preparation method of a kind of lignin degrading according to claim 1, cellulase preparation, is characterized in that, described hot air flow 3.5m
3/ min, inlet temperature 95 DEG C, input speed 20mL/min.
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Application publication date: 20160511 |