CN114107093A - Cellulose degrading bacterium for high yield of cellulase and application thereof - Google Patents
Cellulose degrading bacterium for high yield of cellulase and application thereof Download PDFInfo
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- CN114107093A CN114107093A CN202111296618.3A CN202111296618A CN114107093A CN 114107093 A CN114107093 A CN 114107093A CN 202111296618 A CN202111296618 A CN 202111296618A CN 114107093 A CN114107093 A CN 114107093A
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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
- C12N9/244—Endo-1,3(4)-beta-glucanase (3.2.1.6)
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Abstract
The invention discloses a cellulose degrading bacterium for highly producing cellulase and application thereof. The cellulose degrading bacteria is named as Coniocaeta hoffmanii, and has a strain number ZJ2, and the preservation number is CGMCC No. 22458. The cellulose degrading bacteria are named as Coniochaeta hoffmanii, the strain number ZJ2, the preservation number is CGMCC No.22458, the cellulose degrading bacteria have strong cellulase producing capability at 28 ℃, the produced endo-beta-glucanase has strong capability of treating leaf vegetables, the final degradation rate can reach 98.5 percent, a high-efficiency degradation means is provided for treating cellulose, and the cellulose degrading bacteria have good industrial application prospect.
Description
Technical Field
The invention relates to the technical field of biology, in particular to cellulose degrading bacteria for high yield of cellulase and application thereof.
Background
Cellulose belongs to a linear glucan natural high molecular compound, is formed by connecting glucose molecules through beta-1, 4-glycosidic bonds, the basic repeating unit is cellobiose, residues are connected through the beta-1, 4 glycosidic bonds, and the cellobiose is formed by connecting two molecules of beta-D-glucopyranose through the beta-1, 4 glycosidic bonds, and is the most abundant biopolymer on the earth. Hydrogen bonds exist between cellulose, and cellulose chains form cellulose bundles through association of the hydrogen bonds. In this continuous structure of cellulose, where the molecular density is high, the molecules are arranged in parallel and well oriented to form crystalline regions of cellulose. As the degree of densification becomes smaller, the degree of bonding becomes weaker, intermolecular voids become larger, and the orientation becomes worse, forming amorphous regions of cellulose. In cellulose, hydrogen bonds exist in the molecule in addition to those between cellulose chains. Hydrogen bonds are high energy bonds and the presence of such bonds presents great difficulties in the hydrolysis of cellulose. Most cellulose resources are discarded or directly burned, which causes serious problems of resource waste and environmental pollution. Therefore, the effective development and utilization of cellulose resources have become one of the hot spots of current energy and environmental research.
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.
For example, the invention with the publication number of CN111893065A discloses a low-temperature cellulose degrading bacterium, and the bacterial strain MG-1 is Ochrobactrum sp, and the preservation number is CGMCC No. 18079. The MG-1 strain provided by the invention can well grow at the temperature of 4-35 ℃ and secrete cellulase such as filter paper enzyme, endo-beta-1, 4-glucanase, exo-beta-1, 4-glucanase, beta-glucosidase and the like. The strain MG-1 can be cultured for 30 days at 16 ℃, the degradation rate of the strain MG-1 to the corn straws reaches 22.30 percent, and the strain MG-1 can be used as a strain resource for the research and development of the corn straw returning decay promoting microbial inoculum in low-temperature areas.
For another example, the invention with the publication number of CN112195134A discloses a cellulose degradation complex microbial inoculum and a preparation method and application thereof, wherein the cellulose degradation complex microbial inoculum comprises Serratia marcescens (Serratia marcescens) with the preservation number of CGMCC NO.20557 and Acinetobacter calcoaceticus (Acinetobacter calcoaceticus) with the preservation number of CGMCC NO. 20558. The cellulase secreted by the two strains provided by the invention is used as a main active component, and the mushroom residue is used as a degradation substance, so that the cellulase is applied to mushroom residue compost, and has the characteristics of quick temperature rise, good mushroom residue degradation effect, no secondary pollution and the like. The microbial inoculum is cultured in a sodium carboxymethylcellulose CMC-NA culture medium, has a relatively obvious hydrolysis ring, has a remarkable degradation effect on mushroom residues in a mushroom residue fermentation culture medium, and obviously improves the germination rate and the maturity of compost seeds.
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 ZJ2 with high cellulase yield; the second technical problem to be solved by the invention is to provide the application of the fungus ZJ2 for producing cellulase with high yield in the normal temperature environment and decomposing cellulose with high efficiency.
A cellulose degrading bacterium with high cellulase yield is named as Coniochaeta hoffmanii, and has a strain number ZJ2, and the preservation number is CGMCC No. 22458.
The invention also provides application of the cellulose degrading bacteria in producing cellulase. The cellulase is endo-beta-glucanase. During the specific preparation, the cellulose degrading bacteria are inoculated into a liquid culture medium for fermentation culture, and the crude enzyme liquid containing the cellulase is obtained by filtering the bacteria and taking the supernatant.
Preferably, the liquid medium used in the fermentation culture is a CMC sodium liquid medium, and the formula of the CMC sodium liquid medium is as follows: 10g/L of sodium carboxymethylcellulose, 10g/L of peptone, 5g/L of beef extract, 4g/L of ammonium sulfate, 2g/L of monopotassium phosphate and 0.5g/L of magnesium sulfate heptahydrate, wherein the fermentation culture temperature is 25-35 ℃, and the fermentation culture time is 4-7 days. Preferably, the fermentation culture is carried out at 30 ℃ for 5 days.
The invention also provides application of the cellulose degrading bacteria in degrading cellulose.
Preferably, the application comprises inoculating the cellulose degrading bacteria into a liquid culture medium for fermentation culture to obtain a fermentation culture solution, and adding the fermentation culture solution into biomass containing cellulose for cellulose degradation.
Preferably, the temperature for degradation is 20-37 ℃. Most preferably, the temperature at which degradation occurs is 28 ℃.
Compared with the prior art, the invention has the advantages that:
the cellulose degrading bacteria are named as Coniochaeta hoffmanii, the strain number ZJ2, the preservation number is CGMCC No.22458, the cellulose degrading bacteria have stronger cellulase producing capability at 28 ℃, the activity of the produced endo-beta-glucanase can reach 0.1728U/mL, the cellulose degrading bacteria are greatly improved compared with Flavodon sp.x10 strain (0.1222U/mL) (the invention application with the publication number of CN 112725194A), the capability of degrading leaf vegetables is stronger, the final degradation rate can reach 98.5 percent, a high-efficiency degradation means is provided for treating cellulose, and the cellulose degrading bacteria have good industrial application prospect.
Drawings
FIG. 1 is a flow chart of screening and identification of ZJ 2.
FIG. 2 is a colony morphology of ZJ2 in PDA medium (PDA, 28 ℃, 6 days).
FIG. 3 is a colony morphology of ZJ2 in CMC sodium medium (CMC-Na, 28 ℃, 6 days).
FIG. 4 is a schematic drawing of the hyphal structure of ZJ2 (20-fold objective lens).
FIG. 5 is a schematic drawing of the hyphal structure of ZJ2 (100-fold objective lens).
FIG. 6 is a graph showing the effect of ZJ2 on hydrolysis of Congo red sodium carboxymethylcellulose on the flat plate (30 ℃ C., 5 days).
FIG. 7 is a graph showing the results of measurement of ZJ2 on degraded leaf vegetables.
Detailed Description
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 20 min.
The formula of the CMC sodium culture medium is as follows: 10g/L of sodium carboxymethylcellulose, 10g/L of peptone, 5g/L of beef extract, 4g/L of ammonium sulfate, 2g/L of monopotassium phosphate and 16g/L of agar, wherein the agar is 0.5g/L of magnesium sulfate heptahydrate. The medium was autoclaved at 121 ℃ for 20 min.
The formula of the seed culture medium is as follows: 8g/L of potato extract powder and 20g/L of glucose.
Example 1: isolation and characterization of ZJ2
The process of strain screening and identification is shown in FIG. 1.
The invention is separated from rotten fallen leaves and soil in national forest park of Torreya grandis (Zhu & City, Zhejiang province), and the specific separation method comprises the following steps:
in national forest parks of torreya grandis in Zhu and City of Zhejiang province, the growth cone method is adopted and the trunk diameter at breast height is combined to calculate the tree age of torreya grandis, branches of torreya grandis with different tree ages are collected, the branches are sealed and stored in an aseptic sealing bag and taken back to a laboratory, the branches are sealed and stored in a refrigerator at 4 ℃ for 2-4 days, and the branches are quickly separated.
Cleaning the collected separated sample with tap water, wiping the sample with clean gauze, and cutting the separated tissue. A stem section of about 0.5cm was cut from the middle of each sample and placed in a sterilized petri dish. Soaking the tissue in 70% alcohol for 60s in a superclean bench; then treating for 3-6 min by using a 3.5% sodium hypochlorite solution; soaking in 70% ethanol for 30s, and cleaning the tissue block with sterilized surface with sterile water for 5min for 3 times; placing the cleaned tissue on a PDA culture medium with the diameter of 9cm, culturing for 3-4 days at 25 ℃, observing the generation of sterile colonies, if the generation of the sterile colonies in a dish is found, proving that the surface of the material is thoroughly sterilized, otherwise, the material cannot be used; and then inoculating the sterile processed material on a water agar culture medium with the diameter of 9cm (4-6 blocks of tissue are placed on each plate), culturing at 25 ℃ for 7 days, recording the number of colonies, picking hyphae at the edges of the colonies, placing on a PDA culture medium, purifying for 1-2 times, and moving onto the PDA slant culture medium for storage after the hyphae grow out. And then selecting a well-grown bacterial colony, performing liquid activation for 48 hours, then dibbling the bacterial colony onto a CMC sodium culture medium plate, putting the plate into a 28 ℃ incubator for culture, performing Congo red staining after culturing for 3-4 days, selecting the bacterial colony with the largest transparent circle for purification culture, and setting the strain number of the bacterial strain as ZJ 2.
1. Morphological identification
The strain ZJ2 was inoculated on PDA solid medium and CMC sodium solid medium, then the plate was inverted, cultured at 28 ℃ for 5 days, and the growth of colonies on the plate was observed and recorded. The colony morphology of ZJ2 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. Then, a proper amount of cultured ZJ2 hyphae is clamped by sterile forceps to be arranged at the center of the glass slide, the hyphae is stirred to be evenly scattered, 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 the hyphae are dense, the branches are produced more, and the macroscopic morphology is adapted to the microscopic hyphae 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
Extracting DNA in the strain and carrying out ITS sequencing. A50. mu.L reaction system included: genomic DNA (20 ng/. mu.L) 1.0. mu.L, 10 XBuffer (containing 2.5mM Mg)2+) 5.0. mu.L, 1.0. mu.L of Taq polymerase (5U/. mu.L), 1.0. mu.L of dNTP (10mM), 1.5. mu.L of ITS1 primer (10. mu.M), 1.5. mu.L of ITS4 primer (10. mu.M), ddH2O39.0 μ L, reaction conditions were: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 1min, and 35 cycles; final extension at 72 ℃ for 7 min. After the reaction was completed, 3. mu.L of the PCR product was subjected to 1% agarose gel electrophoresis. The PCR amplified fragment was confirmed.
The sequence of the sequencing ITS of the strain is shown as SEQ ID No.1, the obtained sequence is subjected to BLAST similarity comparison in NCBI, the strain can be determined to be Coniocaeta hoffmanii, is named as ZJ2 and is preserved in China general microbiological culture Collection center (CGMCC) of China micro-organism culture Collection management Committee No.1 Hospital 3 of North Chen West Lu No.1 located in the sunward area of Beijing, the preservation number is CGMCC No.22458, and the preservation date is 2021, 7 and 13 days.
Example 2: ZJ2 determination of cellulose degradation ability
The method for measuring the cellulose degrading capacity of the strain by adopting a Congo red dyeing method comprises the following specific implementation steps: covering a Congo red solution with the mass concentration of 1mg/mL on a culture medium with a colony, pouring the Congo red solution after 10-15 min, adding a NaCl solution with the mass concentration of 1mol/L, and pouring the NaCl solution after 15min, wherein a transparent ring appears around the colony generating cellulase. Fig. 6 is a schematic diagram of hydrolysis effect of the congo red sodium carboxymethyl cellulose flat plate of ZJ2 (the other three strains FH8, 508 and 530 are other strains screened from the same batch), and it can be seen that, at 28 ℃, after inoculating the CMC sodium flat plate of ZJ2 for 4 days, congo red staining shows an obvious hydrolysis ring, the ratio of the transparent ring to the colony is the maximum, which is about 2.33, indicating that the strain ZJ2 has the strongest capacity of degrading cellulose. The higher efficiency of producing cellulase can be seen from the colony growth size and speed and the hydrolysis circle.
Example 3: determination of cellulase production capability of ZJ2
1. Preparation of crude enzyme solution
The screened ZJ2 strain is inoculated into a seed culture medium, cultured for 48h at 25 ℃ and 180r/min, and then inoculated into a CMC sodium culture medium at 28 ℃ and 180r/min in an inoculum size of 10 percent for culture for 6 d. Filtering the thallus and taking the supernatant as the crude enzyme liquid.
2. Preparation of glucose Standard Curve
Taking 0-1.2 mL (0.2 mL difference of each tube) of 1mg/mL glucose standard solution into a 7-branch test tube, adding distilled water to complement to 2mL, then adding 2mL DNS, uniformly mixing, carrying out boiling water bath for 5min, taking out, cooling to room temperature, then adding 6mL distilled water into each test tube, fully and uniformly mixing, and measuring OD540 by using a spectrophotometer. The optical density value is used as the abscissa and the glucose content (mg/mL) 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
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 substrate solution into 1mL of the prepared crude enzyme solution, reacting in a water bath kettle at 50 ℃ for 20min, adding 2mL of DNS reagent after stopping the reaction, shaking all tubes uniformly, carrying out boiling water bath for 10min, 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. The determination method can prove the enzyme activity obtained by the corresponding determination method, namely the enzyme activity of the endo-beta-glucanase, and the determination method introduces a reference (Shenxueliang, Xiahizing, screening of cellulase-producing bacteria and research of enzymological characteristics [ J ]. forest chemical and industry, 2002(01): 47-51.).
The enzyme activity of ZJ2 for producing enzyme is measured by the method, and the measured activity of endo-beta-glucanase is the highest at 28 ℃, 200rpm and 72h of fermentation, and is 0.1728U/mL.
4. Determination of degraded cellulosic Biomass
And (3) determination of degraded leaf vegetables:
the ZJ2 strain is inoculated into a seed culture medium, cultured for 48h at 25 ℃ and 180r/min, and then inoculated into a CMC sodium culture medium at 28 ℃ and 180r/min for 6d in an inoculum size of 10%. Adding the fermentation liquor obtained by culturing into a small kitchen garbage treatment machine, uniformly mixing with 500g of sawdust, adding 250g of leaf vegetable vegetables every day, wherein the degradation period is 7d, measuring the weight of the residual residues, and comparing with the weight of the added leaf vegetable vegetables, wherein the final degradation rate can reach 98.5%. The degradation scheme is shown in FIG. 7.
Sequence listing
<110> Zhejiang Ningbo theory of technology, college
<120> cellulose degrading bacterium for high-yield cellulase and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 526
<212> DNA
<213> Coniochaeta hoffmannii
<400> 1
tactgatccg aggtcacctt ggttaagatg ggtctttaac ggcaggagca cgccgcaacc 60
tccctagcga gataatgaat tactacgctc ggagttgtag cgagcccgcc acttcctttc 120
agggcctacg gcagccgtag ggccccaaca ccaagcaggg cttgagggtt gaaatgacgc 180
tcgaacaggc atgcccggta gagtactacc gggcgcaatg tgcgttcaaa gattcgatga 240
ttcactgaat tctgcaattc acattactta tcgcatttcg ctgcgttctt catcgatgcc 300
agagccaaga gatccgttgt tgaaagtttt aacttatttg ctttacactc agagatgcca 360
ctataataca gagtttcgta cctccggcgg gcgccccgaa cctccggaga ggcagggctg 420
cggcgccccc gttagggggt gccgcgccgc cgaagcaact tggacgagtt cgcaatggtt 480
tgaagtagcc tttcggcttc ttgtaatgat ccctccgcag gtcacc 526
Claims (8)
1. The cellulose degrading bacterium for highly producing cellulase is named as Coniochaeta hoffmanii strain number ZJ2 with the preservation number of CGMCC No. 22458.
2. Use of the cellulose-degrading bacterium of claim 1 for producing cellulase.
3. The use according to claim 2, wherein the cellulase is an endo- β -glucanase.
4. The use of claim 2, wherein the cellulose-degrading bacteria are inoculated into a liquid culture medium for fermentation culture, and the crude enzyme solution containing the cellulase is obtained by filtering the bacteria and taking the supernatant.
5. The use according to claim 4, wherein the liquid medium used in the fermentation culture is a CMC sodium liquid medium, and the formula of the CMC sodium liquid medium is as follows: 10g/L of sodium carboxymethylcellulose, 10g/L of peptone, 5g/L of beef extract, 4g/L of ammonium sulfate, 2g/L of monopotassium phosphate and 0.5g/L of magnesium sulfate heptahydrate,
the fermentation culture temperature is 25-35 deg.C, and the fermentation culture time is 4-7 days.
6. Use of the cellulose-degrading bacterium of claim 1 for degrading cellulose.
7. The use of claim 6, wherein the cellulose-degrading bacteria are inoculated into a liquid medium and subjected to fermentation culture to obtain a fermentation culture solution, and the fermentation culture solution is added into biomass containing cellulose to degrade the cellulose.
8. The use according to claim 7, wherein the temperature at which degradation occurs is 20-37 ℃.
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| US7067303B1 (en) * | 2003-01-24 | 2006-06-27 | The United States Of America As Represented By The Secretary Of Agriculture | Culture containing biomass acid hydrolysate and Coniochaeta ligniaria fungus |
| CN101353629A (en) * | 2008-09-24 | 2009-01-28 | 首都师范大学 | In situ detoxication alcohol fermentation method of ligno-cellulose hydrolysate using single culture |
| CN112725194A (en) * | 2021-01-21 | 2021-04-30 | 南京林业大学 | Fungus Flavodon sp.x10 for high yield of cellulase and application thereof |
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| US7067303B1 (en) * | 2003-01-24 | 2006-06-27 | The United States Of America As Represented By The Secretary Of Agriculture | Culture containing biomass acid hydrolysate and Coniochaeta ligniaria fungus |
| CN101353629A (en) * | 2008-09-24 | 2009-01-28 | 首都师范大学 | In situ detoxication alcohol fermentation method of ligno-cellulose hydrolysate using single culture |
| CN112725194A (en) * | 2021-01-21 | 2021-04-30 | 南京林业大学 | Fungus Flavodon sp.x10 for high yield of cellulase and application thereof |
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| 曾思泉;凌娟;林丽云;MANZOOR AHMAD;张燕英;张颖;董俊德;: "1株红树林来源枝孢属真菌的分离鉴定及纤维素酶性质分析", 微生物学杂志, no. 02 * |
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