CN112725194A - Fungus Flavodon sp.x10 for high yield of cellulase and application thereof - Google Patents
Fungus Flavodon sp.x10 for high yield of cellulase and application thereof Download PDFInfo
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- CN112725194A CN112725194A CN202110086125.0A CN202110086125A CN112725194A CN 112725194 A CN112725194 A CN 112725194A CN 202110086125 A CN202110086125 A CN 202110086125A CN 112725194 A CN112725194 A CN 112725194A
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- 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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Abstract
The invention discloses a fungus Flavodon sp.x10 for high yield of cellulase and application thereof, belonging to the technical field of biology. The fungus Flavodon sp.x10 for high yield of cellulase is preserved in China center for type culture Collection with the preservation number of CCTCC No: m2020961; the preservation date is as follows: 12/23/2020; and (4) storage address: wuhan university in Wuhan, China. The fungus Flavodon sp.x10 for efficiently degrading cellulose is obtained by separation in coastal areas, has stronger capability of producing cellulase at 30 ℃, produces endo-beta-glucanase, exo-beta-glucanase and beta-glucosidase with high activity, provides a means for efficiently degrading cellulose, and has good industrial application prospect.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a fungus Flavodon sp.x10 capable of highly producing cellulase and application thereof in producing cellulase and efficiently decomposing cellulose in a normal temperature environment.
Background
Cellulose is a macromolecular polysaccharide formed by connecting glucose by beta-1, 4-glycosidic bonds, is an important component of plant cell walls, accounts for about 35-50% of the dry weight of plants, and is the most abundant carbohydrate in nature. Meanwhile, cellulose is also an important renewable resource, and can be used as a raw material for energy, food and chemical industries, such as ethanol, lactic acid, single-cell protein and the like. However, since cellulose contains a large amount of high-energy hydrogen bonds, the cellulose is not easy to degrade and has low utilization rate, and most of cellulose resources are abandoned or directly burned, the problems of resource waste and environmental pollution are very serious. Therefore, the effective development and utilization of cellulose resources have become one of the hot spots of current energy and environmental research.
At present, the country strongly pushes the straws to return to the field, and a large amount of straws are crushed and directly applied to the land. However, straw spoilage is a lengthy process during which the growing microorganisms compete with the next crop for moisture and nutrients. The application of a proper microbial inoculum in the field after returning the straws to the field is beneficial to promoting the decomposition of the straws, reducing the use of part of chemical fertilizers and leading farmers to obtain higher economic benefits.
The cellulose-decomposing fungi with high efficiency have wide application prospect in industrial production and agricultural waste treatment. The way to treat and utilize cellulose, which is the largest polymer carbohydrate in the world, is a hotspot and difficulty in the field of research and utilization of plant bodies at present.
Disclosure of Invention
Aiming at the problems in the prior art, the first technical problem to be solved by the invention is to provide a fungus Flavodon sp.x10 for high yield of cellulase; the second technical problem to be solved by the invention is to provide the application of the fungus Flavodon sp.x10 for producing cellulase at high yield in the normal temperature environment and efficiently decomposing cellulose.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the invention separates a strain of white rot fungi from rotten fallen leaves and soil in a yellow sea forest park in Jiangsu east, and the specific separation method comprises the following steps: collecting rotten fallen leaves and lower layer humus soil in a east Taiwan yellow sea forest park of Jiangsu; putrefaction of the collected materialCrushing leaves and putting the soil into a centrifugal tube filled with sterile water for full oscillation, and taking the suspension; diluting the suspension in gradient to obtain 10-2To 10-7A solution of concentration; adding the solution to a CMC sodium culture medium plate for coating treatment, culturing in an incubator at 30 ℃, carrying out Congo red staining after culturing for 2-3 days, selecting the transparent ring with the largest size for purification culture, preserving, identifying as white rot fungi Flavodon, and naming as Flavodon sp.x10, wherein the ITS sequence is shown as SEQ ID NO. 1.
The colony of Flavodon sp.x10 on the PDA culture medium is white, and the aerial hyphae grow vigorously and have no spores. The hyphae on the CMC sodium culture medium are transparent, the growth is luxuriant, and no spores are produced.
10, Flavodon sp.x10, which is preserved in China center for type culture Collection with the preservation number of CCTCC No: m2020961; the preservation date is as follows: 12/23/2020; and (4) storage address: wuhan university in Wuhan, China.
Application of fungus Flavodon sp.x10 with high cellulase yield in cellulase production.
Further, the fungus Flavodon sp.x10 can produce cellulase under the normal temperature environment, and the cellulase comprises endo-beta-glucanase, exo-beta-glucanase and beta-glucosidase.
The Flavodon sp.x10 of the invention produces cellulase at 30 ℃. Under the temperature condition, the filter paper enzyme, the endo beta-glucanase, the exo beta-glucanase and the beta-glucosidase have higher enzyme activity which respectively reach 0.1388U/mL, 0.3592U/mL, 0.1359U/mL and 0.1222U/mL.
Fermentation broth of the fungus Flavodon sp.x10 containing high-yielding cellulase also belongs to the protection scope of the invention.
Further, the preparation process of the fermentation liquor comprises the following steps: inoculating Flavodon sp.x10 into a CMC sodium liquid culture medium, and shaking for 5 days at 30 ℃ in a shaking table to obtain a fermentation liquid of the Flavodon sp.x10, wherein the shaking rotation speed of the shaking table is 150 r/min.
Further, the formula of the CMC sodium liquid culture medium is as follows: 10g/L of sodium carboxymethylcellulose, 3g/L of peptone, 0.2g/L of yeast extract, 2g/L of ammonium sulfate, 4g/L of monopotassium phosphate and 0.3g/L of magnesium sulfate heptahydrate, wherein the pH of the liquid culture medium is natural, and the liquid culture medium is sterilized by high-pressure steam at 121 ℃ for 15 min.
The cellulase consists of a cellulose with a preservation number of CCTCC No: m2020961 Flavodon sp.x10, the preparation method comprises the following steps: inoculating Flavodon sp.x10 strain into seed culture medium, culturing at 30 deg.C and 150r/min for 24h, and inoculating into CMC sodium culture medium at 30 deg.C and 150r/min for 5d with 2% inoculum size; filtering the thallus and taking the supernatant as the crude enzyme liquid.
Further, the formula of the seed culture medium is as follows: 10g/L of sodium carboxymethylcellulose, 3g/L of peptone, 4g/L of potassium dihydrogen phosphate, 0.2g/L of yeast extract and 0.3g/L of magnesium sulfate heptahydrate.
The preservation number is CCTCC No: cellulase prepared by Flavodon sp.x10 fermentation of M2020961 is used for efficient degradation of cellulosic biomass.
Further, cellulosic biomass includes straw.
Has the advantages that: compared with the prior art, the invention has the advantages that:
the fungus Flavodon sp.x10 for efficiently degrading cellulose is obtained by separation in coastal areas, has stronger capability of producing cellulase at 30 ℃, produces endo-beta-glucanase, exo-beta-glucanase and beta-glucosidase with high activity, provides a means for efficiently degrading cellulose, and has good industrial application prospect.
Drawings
Fig. 1 is a flow chart of preparation of Flavodon sp.x 10;
FIG. 2 is a colony morphology of Flavodon sp.x10 in PDA medium (PDA, 30 ℃, 3 days);
FIG. 3 is a colony morphology of Flavodon sp.x10 in CMC sodium medium (CMC-Na, 30 ℃, 3 days);
fig. 4 is a schematic diagram of the hypha structure of Flavodon sp.x10 (20-fold objective lens);
fig. 5 is a schematic diagram of the hypha structure of Flavodon sp.x10 (100-fold objective lens);
FIG. 6 is a phylogenetic tree diagram constructed from the ITS sequence of Flavodon sp.x10;
FIG. 7 is a graph showing the hydrolysis effect of Flavodon sp.x10 on a Congo red sodium carboxymethyl cellulose flat plate (30 ℃ C., 3 days);
FIG. 8 is a graph showing the results of comparison of cellulase production by Flavodon sp.x10 and Trichoderma reesei which is a model strain;
fig. 9 is a graph showing the results of cellulase production assay by Flavodon sp.x 10.
Detailed Description
The invention is further described with reference to specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. In the following examples, unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
The media and formulations used in the following examples are as follows:
the formula of the PDA culture medium is as follows: 6g of potato extract powder, 20g of agar, 20g of glucose and 1000mL of water, wherein the pH is natural, and the potato extract powder is sterilized by high-pressure steam at 121 ℃ for 30 min.
The formula of the CMC sodium culture medium is as follows: 10g/L of sodium carboxymethylcellulose, 3g/L of peptone, 0.2g/L of yeast extract, 2g/L of ammonium sulfate, 4g/L of monopotassium phosphate, 0.3g/L of magnesium sulfate heptahydrate and 20g/L of agar. The medium was autoclaved at 121 ℃ for 20 min.
The formula of the seed culture medium is as follows: 10g/L of sodium carboxymethylcellulose, 3g/L of peptone, 4g/L of potassium dihydrogen phosphate, 0.2g/L of yeast extract and 0.3g/L of magnesium sulfate heptahydrate.
Example 1 isolation and characterization of Flavodon sp.x10
As shown in fig. 1, the preparation steps of the fungus Flavodon sp.x10 capable of efficiently degrading cellulose provided in this embodiment are as follows: collecting rotten leaves and humus soil of a national forest park of east Tai of Jiangsu; shearing the collected humus soil and putrefactive leaves, putting the sheared humus soil and putrefactive leaves into a centrifugal tube filled with sterile water, and fully vibrating to obtain a suspension; diluting the suspension in gradient to obtain 10-2To 10-7A gradient dilution of concentration; adding the gradient dilution solution to a CMC sodium culture medium plateCoating, culturing in 30 deg.C incubator for 2-3 days, dyeing with Congo red, selecting large transparent colony, purifying, and preserving strain at low temperature; the strain with the largest hydrolysis circle was selected and named x-10.
1. Morphological identification
The strain x-10 was inoculated on PDA solid medium and CMC sodium solid medium, then the plate was inverted, cultured at 30 ℃ for 3 days, and the growth of colonies on the plate was observed and recorded. The colony morphology of x-10 in PDA medium is shown in FIG. 2, and it can be seen that: the bacterial colony grows gently, is white hypha all around, and central part hypha is comparatively transparent, and the hypha polarity grows stronger, and the bacterial colony diffusion is comparatively fast. The fungus did not have spores on PDA. Then, a proper amount of cultured x-10 hyphae are clamped by sterile tweezers to be arranged in the center of the glass slide, the hyphae are stirred to be evenly dispersed, a cover glass is covered, and the glass slide is placed under a microscope to observe the hyphae form. The hyphal structure of the strain is shown in detail in FIG. 4. From FIG. 4, it can be seen that x-10 hyphae are longer, branch production is less, polar growth is stronger, and macroscopic morphology is adapted to microscopic hyphal characteristics. When observed on CMC sodium medium, the growth rate of hyphae is higher, but the hyphae of the colony are sparse and the hyphae are more transparent (FIG. 3). The hyphae were observed to have a large number of cavities during growth by observation under a 100-fold objective microscope (FIG. 5).
2. ITS sequence analysis
DNA in the strain is extracted by a traditional method and subjected to ITS sequence analysis, and a 30-microliter PCR reaction system comprises 1uL template primer (ITS1/ITS4), 1uL template DNA, 15uL taq enzyme premix and 12uL sterile water. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 45 s; annealing at 55 ℃ for 45 s; extension at 72 deg.C for 1min, 30 cycles, and storage at 4 deg.C. The PCR product was sequenced by Oncorks Ltd, and the sequencing result is shown in SEQ ID NO. 1. BLAST similarity alignment of the obtained sequences in GenBank is carried out and MEGA7.0 software is used for constructing a phylogenetic tree of the strains. The development tree of this strain is shown in FIG. 6. By combining the morphological observation and ITS sequence analysis results, the present strain x-10 can be determined to be a member of the Flavodon genus and named Flavodon sp.x10. The strain is preserved in China center for type culture Collection (CCTCC for short, the preservation unit address: Wuhan, Wuhan university, and postal code: 430072) in 12 months and 23 days in 2020, and is named by classification as follows: flavodon sp.x10, with the preservation number of CCTCC NO: m2020961.
Example 2 Flavodon sp.x10 ability to degrade cellulose
Fig. 7 is a schematic view of hydrolysis effect of Flavodon sp.x10 congo red sodium carboxymethyl cellulose plate, and it can be seen that at 30 ℃, after inoculating Flavodon sp.x10 CMC sodium plate for 2 days, congo red staining appears obvious hydrolysis ring, and the growth size and speed of bacterial colony and hydrolysis ring show that the efficiency of producing cellulase is higher.
Example 3 measurement of cellulase producing ability of Flavodon sp.x10
1. Preparation of crude enzyme solution
The Flavodon sp.x10 strain provided by the embodiment 1 of the invention is inoculated into a seed culture medium, cultured for 24h at 30 ℃ and 150r/min, and then inoculated into a CMC sodium culture medium at 30 ℃ and 150r/min by the inoculation amount of 2% for culture for 5 d. Filtering the thallus and taking the supernatant as the crude enzyme liquid.
2. Preparation of glucose Standard Curve
Adding 0-1mL (0.2 mL per tube) of 1mg/mL glucose standard solution into a 7-branch test tube, adding distilled water to make up to 1mL, adding DNS 2mL, 2mL boric acid buffer solution with pH of 10, mixing, boiling in water bath for 30min, taking out, cooling to room temperature, and measuring OD with spectrophotometer540、OD530. The optical density value is used as the abscissa and the glucose content (mg) is used as the ordinate to make a glucose standard curve. The OD value of the sample was measured, and the amount of glucose produced was calculated from the standard curve.
3. Determination of enzyme Activity
(1) Determination of endo-beta-glucanase activity
Adding sodium carboxymethylcellulose into citric acid buffer solution with pH of 4.5 to prepare 1% substrate solution, adding 1mL of the crude enzyme solution into 2mL of the substrate solution, reacting in a water bath kettle at 50 ℃ for 30min, stopping the reaction, adding 2mL of DNS reagent and 2mL of boric acid buffer solution with pH of 10 into 1mL of the reaction solution, shaking the tubes uniformly, carrying out boiling water bath for 30min, taking out, cooling to room temperature, and measuring the OD value at 540 nm. The blank group was not subjected to a 50 ℃ water bath, and DNS was added first to inactivate the enzyme activity, and the others were the same as those in the test group. The enzyme activity unit is the glucose which generates 1 mu mol per minute.
(2) Determination of exo-beta-glucanase activity
Adding microcrystalline cellulose into citric acid buffer solution with pH of 4.5 to prepare 1% substrate solution, adding 1mL of the crude enzyme solution into 2mL of the substrate solution, reacting in a water bath kettle at 50 ℃ for 30min, stopping the reaction, adding 2mL of DNS reagent and 2mL of boric acid buffer solution with pH of 10 into 1mL of the reaction solution, shaking the tubes uniformly, carrying out boiling water bath for 30min, taking out, cooling to room temperature, and measuring the OD value at 540 nm. The blank group was not subjected to a 50 ℃ water bath, and DNS was added first to inactivate the enzyme activity, and the others were the same as those in the test group. The enzyme activity unit is the glucose which generates 1 mu mol per minute.
(3) Determination of beta-glucosidase Activity
Adding saligenin into citric acid buffer solution with pH of 4.5 to prepare 1% substrate solution, adding 1mL of the crude enzyme solution into 2mL of the substrate solution, reacting in a water bath kettle at 50 ℃ for 30min, stopping the reaction, adding 2mL of DNS reagent and 2mL of boric acid buffer solution with pH of 10 into 1mL of the reaction solution, shaking the tubes uniformly, carrying out boiling water bath for 30min, taking out, cooling to room temperature, and measuring the OD value at 540 nm. The blank group was not subjected to a 50 ℃ water bath, and DNS was added first to inactivate the enzyme activity, and the others were the same as those in the test group. The enzyme activity unit is the glucose which generates 1 mu mol per minute.
(4) Filter paper enzyme activity assay
Cutting 1g of filter paper into pieces, adding the cut pieces into 2mL of sterile water, adding 1mL of the crude enzyme solution prepared above, reacting in a water bath kettle at 50 ℃ for 30min, stopping the reaction, adding 2mL of DNS reagent and 2mL of boric acid buffer solution with pH of 10 into 1mL of reaction solution, shaking the tubes uniformly, carrying out boiling water bath for 30min, taking out, cooling to room temperature, and measuring the OD value at 540 nm. The blank group was not subjected to a 50 ℃ water bath, and DNS was added first to inactivate the enzyme activity, and the others were the same as those in the test group. The enzyme activity unit is the glucose which generates 1 mu mol per minute.
By making enzyme production activity contrast with cellulose enzyme production model strain trichoderma reesei ACCC30590 (China agricultural microbial culture collection management center) (figure 8), the enzyme activity of Flavodon sp.x10 is far greater than that of trichoderma reesei, which indicates that Flavodon sp.x10 has higher application potential and development prospect.
The invention provides a measurement result chart for efficiently decomposing cellulose Flavodon sp.x10 to produce cellulase, including endo-beta-glucanase, exo-beta-glucanase, beta-glucosidase and filter paper enzyme, and the result chart refers to FIG. 9. As can be seen from FIG. 9, the strain has stronger enzyme production capability at 30 ℃, and when the strain is cultured for 5 days by fermentation, the filter paper enzyme, the endo-beta-glucanase, the exo-beta-glucanase and the beta-glucosidase all have higher enzyme activities which respectively reach 0.1388U/mL, 0.3592U/mL, 0.1359U/mL and 0.1222U/mL, so that the strain has good industrial application prospect.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Nanjing university of forestry
<120> fungus Flavodon sp.x10 for highly producing cellulase and application thereof
<130> 1
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 555
<212> DNA
<213> Flavodon sp.x10 ITS sequence (artificial)
<400> 1
tgtctcggta agagacgatt ataagcacga actaataaat acttcaacac cacagcgcag 60
ataattatca cactgaaggc gatccgtaag attcacgcta atgcatttca gaggagtcga 120
ctagaagccg acacaacctc caagtccaag cccactaaac ttcattacaa aatttagggg 180
ttgagaattc catgagactc aaacaggcat actcctcgga ataccaagga gtgcaaggtg 240
cgttcaaaga ttcgatgatt cactgaattc tgcaattcac attacttatc gcatttcgct 300
gcgttcttca tcgatgcgag agccaagaga tccgttgctg aaagttgtat ataattgtgt 360
tatacacagt aaacattcta aaactgaagc gtttgtagta aacataagaa aggcttatta 420
ccaactaata aatagctggc ttacaccgtt tcttacataa agtgcacaga ggttgagagt 480
ggatgagcca ggcgtgcaca tacctcgtta aaggccagct acaacccgtt caaaactcga 540
taatgatcct tccgc 555
Claims (10)
1. A fungus Flavodon sp.x10 with high cellulase yield is preserved in China center for type culture Collection with the preservation number of CCTCC No: m2020961; the preservation date is as follows: 12/23/2020; and (4) storage address: wuhan university in Wuhan, China.
2. Use of the highly cellulase producing fungus Flavodon sp.x10 according to claim 1 for the production of cellulase.
3. The use according to claim 2, wherein the fungus Flavodon sp.x10 is capable of producing cellulases comprising endo- β -glucanases, exo- β -glucanases and β -glucosidases in a normothermic environment.
4. A fermentation broth of the fungus flavedo sp.x10 containing the high-yielding cellulase of claim 1, characterized in that the preparation process of the fermentation broth is as follows: inoculating Flavodon sp.x10 into a CMC sodium liquid culture medium, and shaking a shaking table to obtain a fermentation liquid of Flavodon sp.x10.
5. The fermentation broth of fungus Flavodon sp.x10 with high yield of cellulase in accordance with claim 4, wherein the preparation process of the fermentation broth is as follows: inoculating Flavodon sp.x10 into a CMC sodium liquid culture medium, and shaking for 5 days at 30 ℃ in a shaking table to obtain a fermentation liquid of the Flavodon sp.x10, wherein the shaking speed of the shaking table is 150 r/min.
6. The fermentation broth of fungus Flavodon sp.x10 with high yield of cellulase enzyme according to claim 4, wherein the formulation of CMC sodium liquid medium is: 10g/L of sodium carboxymethylcellulose, 3g/L of peptone, 0.2g/L of yeast extract, 2g/L of ammonium sulfate, 4g/L of monopotassium phosphate and 0.3g/L of magnesium sulfate heptahydrate.
7. The cellulase is characterized by comprising a cellulose with a preservation number of CCTCC No: m2020961, Flavodon sp.x10.
8. A method for preparing the cellulase of claim 7, comprising the steps of: inoculating Flavodon sp.x10 strain into seed culture medium, culturing at 30 deg.C and 150r/min for 24h, and inoculating into CMC sodium culture medium at 30 deg.C and 150r/min for 5d with 2% inoculum size; filtering the thallus and taking the supernatant as the crude enzyme liquid.
9. The method for preparing cellulase according to claim 8, wherein the formulation of the seed culture medium is: 10g/L of sodium carboxymethylcellulose, 3g/L of peptone, 4g/L of potassium dihydrogen phosphate, 0.2g/L of yeast extract and 0.3g/L of magnesium sulfate heptahydrate.
10. Use of the cellulase according to claim 7, characterised in that the cellulase is used for efficient degradation of cellulosic biomass.
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| CN103045484A (en) * | 2011-10-11 | 2013-04-17 | 济南圣泉集团股份有限公司 | Penicillium strain producing cellulase and application in cellulose enzymatic hydrolysis thereof |
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| CN114107093A (en) * | 2021-11-03 | 2022-03-01 | 浙大宁波理工学院 | Cellulose degrading bacterium for high yield of cellulase and application thereof |
| CN114107093B (en) * | 2021-11-03 | 2023-10-27 | 浙大宁波理工学院 | Cellulose degrading bacterium for high-yield cellulase and application thereof |
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