CN103820422B - A kind of method improving mould cellulase-producing - Google Patents

A kind of method improving mould cellulase-producing Download PDF

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CN103820422B
CN103820422B CN201410066355.0A CN201410066355A CN103820422B CN 103820422 B CN103820422 B CN 103820422B CN 201410066355 A CN201410066355 A CN 201410066355A CN 103820422 B CN103820422 B CN 103820422B
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mould
buckwheat
extracting solution
order seed
cellulase
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CN103820422A (en
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曾柏全
冯金儒
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Central South University of Forestry and Technology
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Central South University of Forestry and Technology
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    • 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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    • 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
    • C12N9/2445—Beta-glucosidase (3.2.1.21)
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    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01021—Beta-glucosidase (3.2.1.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01091—Cellulose 1,4-beta-cellobiosidase (3.2.1.91)

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Abstract

The invention discloses a kind of method improving mould cellulase-producing.The method is cultivated in mould seed culture medium by the mould spore inoculating of rejuvenation in PDA substratum, obtains first order seed nutrient solution; And then first order seed nutrient solution is inoculated into the addition of buckwheat extracting solution fermention medium in cultivate.The present invention utilizes buckwheat extracting solution as the additive of mould fermention medium, and significantly improve mould cellulase-producing enzyme and live, method is simple, relative inexpensiveness, respond well.

Description

A kind of method improving mould cellulase-producing
Technical field
The present invention relates to the method improving enzyme and live, a kind of in particular method improving mould cellulase-producing.
Background technology
Along with traditional energy lays in day by day exhaustion, energy dilemma becomes the important factor of restriction various countries high speed development, and finding and greatly developing sustainable renewable resource becomes one of global hot issue.Mierocrystalline cellulose is the maximum renewable resources of global quantity, is mainly present in the cell walls of plant, and the phytomass that annual global photosynthesis produces is up to 1.145 × 10 12ton, and cellulose resource takes up an area 60% ~ 80% of ball total biomass.Mierocrystalline cellulose production new cleaning fuel and other biological chemical by-product is utilized to be the current focuses researched and developed.
China has abundant cellulose resource, and only stalk cot one, can reach more than 700,000,000 ton every year, and wherein corn stalk (35%), Wheat Straw (21%) and straw (19%) are the three large stalks of China.Calendar year 2001, China comes into effect alcohol fuel plan, has become the third-largest fuel ethanol production state being only second to Brazil and the U.S. in the world so far.2007, point out in " planning of renewable energy source Long-and Medium-term Development " that Chinese Government promulgates, the raw materials for production that to greatly develop with non-grain material be alcohol fuel, the year utilization expecting the year two thousand twenty biofuel ethanol was 1,000 ten thousand tons.
The general name of cellulase to be a class can be by the cellulose degradation polycomponent enzyme system of glucose, their synergy decomposition of cellulose produce oligosaccharides and cellobiose, are finally hydrolyzed to glucose.The multiply anchor-pile of cellulase mainly contains 3 compositions, endoglucanase (Isosorbide-5-Nitrae-β-D-glucangiucanohydrolase or endo-l, 4-β-D-glucanase, E.C3.2.1.4 are called for short EG from fungi); (Isosorbide-5-Nitrae-β-D-glucancellobiohydrolase or exo-1,4-β-D-glucanase, E.C3.2.1.91, be called for short CBH from fungi to exoglucanase; Cex is called for short) from bacterium.Beta-glucosidase (β-Isosorbide-5-Nitrae-glucosi-dase, E.C3.2.1.21 is called for short BG).Endoglucanase and the main dissolving cellulos of 1,4-BETA-D-glucancellobio-hydrolase, cellobiose, procellose mainly transform and generate glucose by beta-glucosidase.Through years of researches, cellulase is widely used in industry-by-industry, as food, weaving, wine brewing, feed, petroleum prospecting, traditional Chinese medicine ingredients extraction etc.Particularly in industries such as new forms of energy, washing, papermaking, weavings in occupation of very important status.
Cellulase wide material sources, bacterium, actinomycetes, filamentous fungus etc. all can produce cellulase.Wherein the fungi cellulose enzyme architecture of producing is comparatively complete, and mostly is extracellular enzyme, and extraction purification is easier to, and yield of enzyme is higher, advantage of lower cost, is the main bacteria seed of industry now.Studying more has Trichoderma, Aspergillus, Penicillium, Rhizopus and the mould genus of paint spot.
Rich in proteins, starch, mineral substance, VITAMIN and abundant flavonoid compound in buckwheat, these materials can stimulate the growth of Mierocrystalline cellulose producing strains and the generation of cellulase as somatomedin.At present, the main method that raising cellulase-producing bacterium enzyme is lived is selection by mutation and utilizes genetically engineered to build high yield cellulose-degrading bacteria, and these method stepss are loaded down with trivial details, cost is higher.
Summary of the invention
Technical problem to be solved by this invention is for prior art Problems existing in raising cellulase bacterium enzyme is lived, and provides a kind of method improving mould cellulase-producing.
Technical scheme of the present invention is as follows:
Improve a method for mould cellulase-producing, its step is as follows:
(1) preparation of buckwheat extracting solution
Added by buckwheat after soaking 1h in distilled water, be heated to boiling with electric furnace, keep micro-30min that boils, be cooled to room temperature, after sonicator process, centrifugal 15 minutes of 7000r/min, gets supernatant liquor, namely obtains buckwheat extracting solution;
(2) first order seed is cultivated
In gnotobasis, the mould spore inoculating rejuvenation in a ring PDA substratum, in 100ml mould seed culture medium, at 28 DEG C, is cultivated 48h under 190r/min condition, is obtained first order seed nutrient solution; Wherein, the composition of mould seed culture medium is: containing peptone 0.5g, glucose 1.0g in every 100mL distilled water, yeast leaches cream 0.4g, magnesium sulfate 0.05g, potassium primary phosphate 0.2g, dipotassium hydrogen phosphate 0.4g;
(3) fermentation culture
In gnotobasis, first order seed nutrient solution obtained in (2) is seeded in fermention medium, at 28 DEG C, under 185r/min condition, cultivates 6d; Wherein, the composition of fermention medium is: containing peptone 0.4g in every 100ml distilled water, yeast leaches cream 0.05g, ammonium sulfate 0.2g, potassium primary phosphate 0.4g, starch 3.0g, magnesium sulfate 0.03g, Microcrystalline Cellulose 3.6g, calcium chloride 0.03g, buckwheat extracting solution 10ml.
Described method, in step (1), the mass volume ratio of buckwheat and distilled water is 1:10.
Described method, in step (1), the processing parameter of ultrasonication is: total time 30min, sonication times 5s, intermittent time 3s, power 50%.
Described method, in step (3), the volume ratio of first order seed nutrient solution and fermention medium is 1:11.
The compositions such as beneficial effect of the present invention is: enlarged volume after buckwheat infusion, and seed coat breaks, and is conducive to ultrasonic disruption, and after ultrasonication, Nutrient Value of Buckwheat major part is dissolved in the water, centrifugal segregation cell walls organoid, obtain purer buckwheat extracting solution.Material of the present invention is cheap and easy to get, and method is simple, and the enzyme that buckwheat extracting solution effectively can improve mould cellulase-producing as the additive of mould fermention medium is lived.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
The preparation of embodiment 1 buckwheat extracting solution
Take 20g buckwheat, after adding 200ml distilled water immersion 1h, heating by electric cooker is to boiling, and adjustment Heating temperature, keep slight boiling condition 30min, now buckwheat has expanded and bleached.Buckwheat carries out ultrasonic disruption after just extracting solution is cooled to room temperature, and sonicator setup parameter is power 50%, total time 30min, sonication times 5s, intermittent time 3s.Finally, under 7000r/min, centrifugal 15min, gets supernatant, namely obtains buckwheat extracting solution.
Embodiment 2 first order seed is cultivated
Precise peptone 0.5g, yeast extract paste 1.0g, magnesium sulfate 0.05g, potassium primary phosphate 0.2g, potassium primary phosphate 0.4g also dissolve with 80ml distilled water in 250ml Erlenmeyer flask, take glucose 1.0g, 20ml distilled water dissolves, after both sterilizing coolings, in Bechtop, glucose solution is poured into 250ml Erlenmeyer flask, and the mould spore inoculated on a ring PDA substratum, at 28 DEG C, under 190r/min condition, cultivate 48h, obtain first order seed nutrient solution.
Embodiment 3 fermentation culture
Precise peptone 0.2g, yeast leaches cream 0.025g, ammonium sulfate 0.1g, potassium primary phosphate 0.2g, starch 1.5g, magnesium sulfate 0.015g, calcium chloride 0.015g, distilled water 50ml, Microcrystalline Cellulose 1.8g, buckwheat extracting solution 5ml, as in 250ml Erlenmeyer flask, do three repetitions according to this.After sterilizing cooling, in gnotobasis, the primary seed solution that inoculation 5ml embodiment 2 is obtained, as in shaking table, 28 DEG C, cultivates 6d under 185r/min condition.After collecting crude enzyme liquid, measure CMC enzyme and live as 156.65U/ml, filter paper enzyme activity is 24.78U/ml.
Embodiment 4 fermentation culture
Implementation step, as embodiment 3, does not just add buckwheat extracting solution in substratum, and measure CMC enzyme and live as 58.48U/ml, filter paper enzyme activity is 9.66U/ml.
Should be understood that, for those of ordinary skills, can be improved according to the above description or convert, and all these improve and convert the protection domain that all should belong to claims of the present invention.

Claims (1)

1. improve a method for mould cellulase-producing, it is characterized in that, its step is as follows:
(1) preparation of buckwheat extracting solution
Buckwheat is added after soaking 1h in distilled water, is heated to boiling with electric furnace, keeps micro-30min that boils,
Be cooled to room temperature, after sonicator process, centrifugal 15 minutes of 7000r/min, gets supernatant liquor,
Namely buckwheat extracting solution is obtained;
(2) first order seed is cultivated
In gnotobasis, inoculate the mould spore of rejuvenation in a ring PDA substratum to 100ml mould kind
In sub-substratum, at 28 DEG C, under 190r/min condition, cultivate 48h, obtain first order seed nutrient solution; Its
In, the composition of mould seed culture medium is: containing peptone 0.5g in every 100mL distilled water, glucose 1.0
G, yeast leaches cream 0.4g, magnesium sulfate 0.05g, potassium primary phosphate 0.2g, dipotassium hydrogen phosphate 0.4g;
(3) fermentation culture
In gnotobasis, first order seed nutrient solution obtained in (2) is seeded in fermention medium,
28 DEG C, under 185r/min condition, cultivate 6d; Wherein, the composition of fermention medium is: every 100ml steams
Heat up in a steamer containing peptone 0.4g in water, yeast leaches cream 0.05g, ammonium sulfate 0.2g, potassium primary phosphate 0.4g,
Starch 3.0g, magnesium sulfate 0.03g, Microcrystalline Cellulose 3.6g, calcium chloride 0.03g, buckwheat extracting solution 10
ml；
In step (1), the mass volume ratio of buckwheat and distilled water is 1:10;
In step (1), the processing parameter of ultrasonication is: total time 30min, sonication times 5s,
Intermittent time 3s, power 50%;
In step (3), the volume ratio of first order seed nutrient solution and fermention medium is 1:11.
CN201410066355.0A 2014-02-26 2014-02-26 A kind of method improving mould cellulase-producing Expired - Fee Related CN103820422B (en)

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CN104844354B (en) * 2015-04-29 2017-11-14 天津农学院 A kind of high density pleurotus eryngii liquid strain fermentation medium

Citations (3)

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CN1657613A (en) * 2005-01-31 2005-08-24 湖南大学 Method for raising green trichoderma cellulase active by biological surfae active agent
CN1262327C (en) * 2003-04-25 2006-07-05 谢福胜 Spiral fractionation tower
CN102108347B (en) * 2009-12-25 2012-10-31 中国科学院生态环境研究中心 Method for increasing enzyme activity of cellulase produced from fungal

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CN1149282C (en) * 2000-01-26 2004-05-12 广西大学 Process for increasing cellulase activity by means of waste alcohol liquid in sugar refinery

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CN1262327C (en) * 2003-04-25 2006-07-05 谢福胜 Spiral fractionation tower
CN1657613A (en) * 2005-01-31 2005-08-24 湖南大学 Method for raising green trichoderma cellulase active by biological surfae active agent
CN102108347B (en) * 2009-12-25 2012-10-31 中国科学院生态环境研究中心 Method for increasing enzyme activity of cellulase produced from fungal

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