CN109370909A - The method of the fungi and its High Cellulase Production of High Cellulase Production - Google Patents

The method of the fungi and its High Cellulase Production of High Cellulase Production Download PDF

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CN109370909A
CN109370909A CN201810492142.2A CN201810492142A CN109370909A CN 109370909 A CN109370909 A CN 109370909A CN 201810492142 A CN201810492142 A CN 201810492142A CN 109370909 A CN109370909 A CN 109370909A
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fungi
cellulase production
high cellulase
cellulase
gorgeous
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戴玉成
司静
崔宝凯
员瑗
马鸿飞
吴怡
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Beijing Forestry University
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Beijing Forestry University
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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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • 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)
    • 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)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase

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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention provides a kind of method of the fungi and its High Cellulase Production of High Cellulase Production, select the gorgeous pore fungi Laetiporus ailaoshanensis of phosphorus pools as producing enzyme fungi, the enzyme activity range of gained cellulase is at least between 53.28 (U/mL) to 77.68 (U/mL) (including head and the tail).The gorgeous pore fungi of the phosphorus pools has the genetic fragment of GenBank Serial No. MG672515, belongs to the peculiar fungus resource in China, has abundant development prospect.

Description

The method of the fungi and its High Cellulase Production of High Cellulase Production
Technical field
The present invention relates to microorganism fields, belong to the innovation and creation of the microorganism of cellulase-producing.
Background technique
Cellulose is that distribution on global is most wide, the most abundant renewable organic resources.Cellulase is known a kind of energy Enough catalyzing hydrolysis celluloses generate the general name of glucose and biologically active enzyme.After cellulose hydrolyzation, cellulose quilt It is converted into soluble single molecules of glucose, this solubility single molecules of glucose has the potential quality for preparing bio-fuel, has wide Wealthy development prospect.
Unfortunately, most of cellulose is not yet utilized so far, to find out its cause, the bottleneck of one side is cellulase Production efficiency and quality also be not enough to support cellulose application landing be industry.Such reality, for as cellulose this For the widely distributed renewable resource of sample, undoubtedly a kind of serious waste；Moreover, for example, discarded building plank Also create certain environmental pollution.
Cellulase can be divided into according to property and function: endoglucanase (endo-1,4-D-glucanase, EC 3.2.1.4), exoglucanase (exo-1,4- β-D-glucanase, EC 3.2.1.91) and beta-glucosidase (3-D- Glucosidase, EC 3.2.1.21).Usually come using Filter paper Cellulase (Fi lter paper cel lulase, FPA) Characterize the total saccharification capability of cellulase system.Multiple-microorganism in nature can eccrine fiber element enzyme effect in cellulose, There were significant differences in composition for the cellulase of different microorganisms secretion, and also different to the hydrolysis ability of cellulose.It is long Since phase, the microorganism of High Cellulase Production has been found from nature and its method is the dream of several generations researcher.Especially It is to find that hope will be brought to the industrialization of cellulose application by improving production efficiency and the cellulase of fermentative activity.
The present invention is based on what such background proposed.Fungi provided by the invention is compared and existing cellulase-producing Fungi has higher producing enzyme efficiency and enzymatic activity.The present invention will play a key effect to the industrialization that cellulose is applied.
Summary of the invention
The present invention provides a kind of gorgeous pore fungi Laet iporus ai laoshanens is of phosphorus pools, be under the jurisdiction of mycota, Basidiomycota, agaric guiding principle, Aphyllophorales, quasi- shelf fungus section Fomitops idaceae；It has GenBank Serial No. The genetic fragment of MG672515.After measured, its enzyme activity range is at least between 53.28 (U/mL) to 77.68 (U/mL) (packet Include head and the tail), colonial morphology is shown in Fig. 1, Fig. 2.Bacterium colony shown in Fig. 1, Fig. 2 picks up from Yunnan Province Yongde County Daxueshan Nature Reserve The fructification culture of the gorgeous pore fungi of phosphorus pools on evergreen chinquapin stub forms.The incubation of the bacterium colony records: with the blade sterilized It cuts the fructification that surface has sterilized and is inoculated into plate culture close to the meat bacteria organization (about 0.5cm × 0.5cm × 0.5cm) of base portion On base, in 28 ± 2 DEG C of stationary cultures of constant incubator, the growing state of mycelia is during which observed under stereomicroscope daily, directly Bacterium colony is grown into culture medium.Mycelium inoculation under aseptic condition on picking plating medium far from miscellaneous bacteria bacterium colony is to new culture On base, in 28 ± 2 DEG C of stationary cultures of constant incubator, the growing state of mycelia is during which observed under stereomicroscope daily.Again After secondary discovery miscellaneous bacteria bacterium colony, again on the picking mycelium inoculation far from miscellaneous bacteria bacterium colony to new culture medium, in constant incubator 28 ± 2 DEG C of stationary cultures, until being the gorgeous pore fungi bacterium colony of single pure phosphorus pools in culture medium.Wherein, the component of plating medium Are as follows: peeled potatoes 300g/L, glucose 20g/L, agar powder 20g/L, peptone 5g/L, KH2PO4 1g/L, MgSO4 7H2O 0.5g/L, ZnSO47H2O 0.05g/L, vitamin B1 0.01g/L, pH 5.The present invention provides High Cellulase Production Method, prepared enzyme solution can be applied to bio-fuel manufacture, food processing, weaving papermaking etc. fields；This method apparatus There is the gorgeous pore fungi of phosphorus pools of the genetic fragment of GenBank Serial No. MG672515 as enzyme bacterium, cellulase-producing in this method Culture medium be made of carbon source fibrous raw material, inorganic nitrogen-sourced, liquid microelement.Wherein, the parts by weight of microelement are MnSO4· H2O(0.5-1.5)、MgSO4·7H2O(0.5-1.5)、CoCl2·6H2O(0.005-0.015)、FeSO4·7H2O(0.03- 0.07)、ZnSO4·7H2O (0.05-0.15), vitamin B1 (0.03-0.07) composition；PH is more than or equal to 3 and is less than or equal to 7.
Detailed description of the invention
Fig. 1 is that the gorgeous pore fungi colonial morphology of phosphorus pools of the present invention far shines；
Fig. 2 is the gorgeous pore fungi colonial morphology recent photograph of phosphorus pools of the present invention.
Specific embodiment
1. molecular biology identification
It will aseptically be put into mortar after the mycelia scraping on plating medium, be fully ground with liquid nitrogen to powder Shape is fitted into immediately in 1.5mL centrifuge tube, and the gene of the bacterial strain is extracted using CTAB plant genome DNA rapidly extracting kit Group DNA.
ITS1-5.8S-ITS2 (ITS) rDNA genetic fragment is expanded using ITS5 and ITS4.PCR reaction system (30 μ L): 15 μ L 2 × Easy Taq PCR SuperMix, 1 μ L, 10 μm of ol/L primer I TS5,1 μ L, 10 μm of ol/L primer I TS4,1 μ L base Because of group DNA and 12 μ L deionized waters.PCR response procedures: 95 DEG C of initial denaturation 3min, 94 DEG C of denaturation 40s, 54 DEG C of renaturation 45s, 72 DEG C Extend 1min, 72 DEG C of extension 10min after 35 circulations, 4 DEG C of heat preservations.PCR product is finally transferred into Beijing six directions Hua Da gene section Skill Co., Ltd is sequenced, and carries out Blast comparison to sequencing result.
It is completely the same with the sequence of the gorgeous pore fungi Laet iporus ai laoshanens is of phosphorus pools after comparing, GenBank Serial No. MG672515 is accredited as the gorgeous pore fungi Laet iporus ai laoshanens is of phosphorus pools.It is crucial Gene order is as follows:
2 seed cultures
It takes 250mL triangular flask to dispense 100mL fluid nutrient medium, 5 diameter 1cm bacteria cakes is inoculated with, in constant-temperature table 28 ± 2 DEG C, 150r/min shaken cultivation 7 days.Seed fermentation is made in culture with 5000r/min, 1min using interior cut type refiner to hang Liquid sufficiently vibrates spare.
Fluid nutrient medium: peeled potatoes 300g/L, glucose 20g/L, peptone 5g/L, KH2PO41g/L, MgSO4 7H2O 0.5g/L, ZnSO47H2O 0.05g/L, vitaminB10 .01g/L, pH 5.
3 cellulase-producing medium optimizations
It takes 500mL triangular flask to dispense cellulase-producing culture medium, 10mL seed fermentation suspension is inoculated with, in constant incubator 28 ± 2 DEG C stationary culture 10 days.
100mL 0.05mol/L citrate-phosphate disodium hydrogen buffering is added into the 500mL triangular flask of above-mentioned solid fermentation Liquid (pH 5) reacts 2h in 28 ± 2 DEG C of constant-temperature table, 150r/min, extract obtained to be centrifuged in 4 DEG C, 12000r/min 15min, supernatant are cellulase solution.
Cellulase-producing culture medium: 80-120 mesh cotton seed hulls 20g/L (5-35), 8-120 mesh corncob 10g/L (5-15), Microcrystalline cellulose 10g/L (5-15), peptone 5g/L (3-7), Tween 802mL/L (1-3), PEG40000.5g/L (0.3- 0.7), KH2PO41g/L (0.5-1.5), CaCl20.3g/L (0.1-0.5), liquid microelement 50mL/L.
Liquid microelement: MnSO4H2O 1g/L (0.5-1.5), MgSO47H2O 1g/L (0.5-1.5), CoCl2 6H2O 0.01g/L (0.005-0.015), FeSO47H2O 0.05g/L (0.03-0.07), ZnSO47H2O 0.1g/L (0.05-0.15), vitaminB10 .05g/L (0.03-0.07), pH 5 (3-7).
The measurement of 4 cellulase activities
DNS preparation of reagents: accurately weighing 5.3g 3, and 5- dinitrosalicylic acid is dissolved in 500mL deionized water, stirs 5s, Water-bath is to 45 DEG C.9.9g sodium hydroxide is added afterwards, is during which stirred continuously, until solution is as clear as crystal.153g tetra- is gradually added again Water sodium potassium tartrate tetrahydrate, 3.8mL phenol (50 DEG C of thawings) and 4.15g anhydrous sodium sulfite, 45 DEG C of heating water baths are added 208mL and are gone During which ionized water is stirred continuously, until the substance being added is completely dissolved, be cooled to 25 DEG C, spare after being kept in dark place 7 days, effectively Phase 6 months.
Standard curve making: 0.1,0.2,0.3,0.4,0.5,0.6,0.7mg/mL glucose standard are accurately prepared.Point Not Xi Qu the above-mentioned glucose standard of 0.5mL in 8 20mL plug test tube, be added 1.5mL DNS reagent, shake up boiling water 5min is bathed, 20mL is settled to deionized water after taking-up is cooling, mixes well.Light absorption value is measured at 540nm, if 3 repetitions, It averages.It is that ordinate makees standard curve using the concentration of dextrose standard sample in reaction system as abscissa, light absorption value, calculates Regression equation are as follows: y=0.0569x-0.0139, coefficient R 2=0.9997.
FPA measurement: drawing 0.5mL enzyme solution and 1.5mL 0.05mol/L citrate-phosphate salt buffer (pH 5) is added In 20mL plug test tube, after 50 DEG C of water-bath heat preservation 10min, it is added 50mg filter paper item (about 1cm × 6cm), 50 DEG C of water-baths are protected Warm 1h takes out and 1.5mL DNS reagent is added immediately, takes out cooling after boiling 5min, is diluted to 20mL with deionized water, in Light absorption value is measured at 540nm, if 3 parallel, is averaged.DNS is directly added into the test tube for having drawn enzyme solution and buffer Reagent is to be passivated enzyme activity, as blank control.Defining enzyme amount needed for 1 μm of ol glucose of catalysis generation in 1min is 1 enzyme activity Unit of force (U).
Cellulase activity of the gorgeous pore fungi of 1 phosphorus pools of table under the conditions of different culture medium
Above embodiments are completed in laboratory conditions, it has been verified that the effect of the invention mentioned, also to this The those of ordinary skill in field illustrates the required necessary means of the practice present invention, and the protection scope of the present invention should be respected, no In deliberately reducing, it is all done when industry application be adaptively adjusted or change after technical solution should recognize it is of the invention Spirit is implemented.

Claims (6)

1. the fungi of High Cellulase Production, which is characterized in that it is the gorgeous pore fungi of phosphorus pools, it has GenBank Serial No. The genetic fragment of MG672515.
2. the fungi of High Cellulase Production according to claim 1, which is characterized in that its enzyme activity range is 53.28 (U/mL) to (including head and the tail) between 77.68 (U/mL).
3. the method for High Cellulase Production, which is characterized in that with the genetic fragment with GenBank Serial No. MG672515 The gorgeous pore fungi of phosphorus pools is as enzyme bacterium.
4. the method for High Cellulase Production according to claim 3, which is characterized in that the culture medium of its cellulase-producing by Carbon source fibrous raw material, inorganic nitrogen-sourced, liquid microelement composition.
5. the method for High Cellulase Production according to claim 4, which is characterized in that the parts by weight of the microelement are MnSO4·H2O(0.5-1.5)、MgSO4·7H2O(0.5-1.5)、CoCl2·6H2O(0.005-0.015)、FeSO4·7H2O (0.03-0.07)、ZnSO4·7H2O (0.05-0.15), vitamin B1 (0.03-0.07) composition.
6. the method for High Cellulase Production according to claim 3, which is characterized in that the culture medium of its cellulase-producing PH is more than or equal to 3 and is less than or equal to 7.
CN201810492142.2A 2018-05-22 2018-05-22 The method of the fungi and its High Cellulase Production of High Cellulase Production Pending CN109370909A (en)

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US20140220150A1 (en) * 2000-10-04 2014-08-07 Paul Edward Stamets Integrative fungal solutions for protecting bees and overcoming colony collapse disorder (CCD): methods and compositions
JP5565625B2 (en) * 2010-08-27 2014-08-06 学校法人武庫川学院 Worcester sauce manufacturing method and Worcester sauce obtained thereby
CN103961379A (en) * 2014-05-15 2014-08-06 神农架天润生物科技有限责任公司 Laetiporus sulphureus extract and application thereof
CN105238704A (en) * 2015-11-18 2016-01-13 中国农业科学院饲料研究所 Method for rapidly improving enzyme activity of Trichoderma reesei cellulase
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Title
DAI YC: ""Laetiporus ailaoshanensis voucher Dai 15655 18S ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and 28S ribosomal RNA gene, partial sequence", 《GENBANK DATABASE》 *
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