CN107828667A - A kind of myrothecium verrucaria mutant strain T2901 and its application - Google Patents
A kind of myrothecium verrucaria mutant strain T2901 and its application Download PDFInfo
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Abstract
A kind of myrothecium verrucaria（Myrothecium verrucaria）Mutant strain T2901, its preserving number are CCTCC NO:M2017413.The T2901 bacterial strains that present invention separation mutagenesis obtains can be with the lignin component in efficient degradation maize straw.The T2901 bacterial strains of the present invention can secrete three kinds of laccase related to lignin degradation, lignin peroxidase and manganese peroxidase enzymes.And microorganisms pre-process the influence to maize straw structure and component, the loss of the carbohydrate such as T2901 bacterial strains cellulose-less of the invention and hemicellulose, high degree remains cellulose, there is provided improves the new way of stalk cellulose enzymolysis utilization rate.
Description
Technical field
The present invention relates to a kind of microorganism field, and in particular to a kind of application of myrothecium verrucaria in bioenergy.
Background technology
The shortage of chemical energy source and the environmental pollution of getting worse cause people constantly to seek reproducible green energy resource.Wood
Matter cellulose is as a kind of green energy resource of neutral carbon because the advantages of it has rich and easy to get and cost cheap is into preferably
Green candidate resource.Biorefinery technology based on sugared platform is the effective way using lignocellulosic.This process is main
That maize straw, straw and other lignocellulosic sources are converted into polysaccharide, then continued by way of biofermentation by
Polysaccharide conversion is other biological base product.Wherein, maize straw annual production occupies high, but utilization rate still has much room for improvement.Enzymatic hydrolysis
The cellulose in maize straw effectively can be converted into polysaccharide.But lignin and hemicellulose are combined in the form of covalent bond and incited somebody to action
Cellulose wraps up wherein, forms a kind of natural barrier and hinders contact of the cellulase with cellulosic molecule, therefore limits fibre
Tie up the decomposition of element.Lignin is the aromatics with three-dimensional structure formed by ehter bond and carbon-carbon bond by benzene-propane construction unit
Polymer, it is a kind of generally acknowledged cellulose hydrolyzation resisting substance.Meanwhile lignin absorbed portion cellulose to a certain extent
Enzyme, make it can not catalyzing cellulose hydrolysis reaction.Therefore, the removal of lignin is to improve the pass of cellulose enzymatic reaction efficiency
Key.
Pretreatment can destroy the structure of lignin, the accessibility of cellulase and cellulose be improved, so as to improve biomass
Utilization ratio.Physics, chemistry, biological method can make lignin degradation.Physics and chemical method can reduce wooden content
About 50%, but the cost of both approaches is higher, and secondary pollution can be brought.Microbial process is simple to operate with its, cost
It is cheap and environmentally safe have focus of the characteristics of good as research.
The microbe species of degradable lignin are various in nature, and the degradable of lignin is some bacteriums and fungi
Coefficient result, in this course, fungi are considered as playing a major role.The microorganism of degradable lignin has white rot true
Bacterium, trichobacteria, non-filamentous bacterium, brown rot fungus and soft-rot fungi etc..Wherein, can the microorganism of effectively lignin degrading be mainly
Whiterot fungi and other wood-rotting fungis.But some whiterot fungis can also cause portion of cellulose to drop while lignin degrading
Solution, and some whiterot fungis optionally lignin degrading.Therefore, select suitable fungi most important in preprocessing process.
Preferable preprocess method should farthest remove the lignin in maize straw, and cellulose is farthest protected
Stay.
Fungi relies on the enzyme of its secretion, as laccase, lignin peroxidase and manganese peroxidase carry out Partial digestion
Lignin.Three kinds of enzymes act synergistically, the lignin in common degradation biological matter.Lignin peroxidase and manganese peroxidase
By the H in lignin decomposable process2O2H caused by enzyme2O2.And laccase can be catalyzed the quinone of phenols dimer methoxy substitution,
Quinone is H2O2O needed for production2Source, three kinds of lignin-degrading enzymes synergy.Although not every whiterot fungi all may be used
Three kinds of enzymes are produced simultaneously, and only secreting the whiterot fungi of two kinds of enzymes can also have the function that to decompose lignin, but laccase is dropping
Played an important role in solution preocess.
But most lignin-degrading bacterias can only secrete one or both of three kinds of lignin-degrading enzymes, such as lignin degrading
Type strain Phanerochaete chrysosporium can only secrete lignin peroxidase and manganese peroxidase during the fermentation.
Meanwhile meeting degraded cellulose and hemicellulose while some white rot fungus degrading lignin, cause carbon hydrate
The loss of thing, such as after Phanerochaete chrysosporium is fermented, the content of lignin in maize straw have dropped 30.04%, but and this
The content of cellulose also reduced 17.02% simultaneously.Therefore, suitable bacterial strain should be screened to pre-process lignocellulosic,
Farthest to remove the lignin in biomass, and farthest retain cellulose.
The content of the invention
It is an object of the invention to provide a kind of myrothecium verrucaria to lignin with stronger degradation
(Myrothecium verrucaria) mutant strain T2901, wood of the bacterial strain that the present invention screens in maize straw is removed
There is positive role during cellulose conversion ratio in quality and raising stalk.And microorganisms pretreatment is to maize straw knot
The influence of structure and component, its degradation capability to lignin in maize straw of system research, propose to improve stalk cellulose enzymolysis
The new way of utilization rate.
The purpose of the present invention is achieved by the following technical solution：
A kind of myrothecium verrucaria (Myrothecium verrucaria) mutant strain T2901, it is characterised in that：
Myrothecium verrucaria (Myrothecium verrucaria) mutant strain T2901 of the present invention is gathered from Changbai Mountain
Separation screening and mutation obtain in pedotheque, and it is in China typical culture collection center (CCTCC) preservation, address
For：Wuhan, China Wuhan University, preservation date are on July 5th, 2017, and preserving number is CCTCC NO:M2017413.
The myrothecium verrucaria of the present invention is seeded on PDA solid mediums, and originally white flock is presented in bacterial strain mycelia, outwards
Week diverging growth, bacterium colony approximation irregular cycle, has sporodochia appearance, conidium is in initially after growing 5d on flat board
Existing blackish green, after cultivating 8d, conidium color continues to deepen, and Jiao Dot groups occurs；After cultivating 10d, bacterium colony is in concentric wheel stripe
Black is presented in shape, conidium Jiao Dot groups, and filbert emitting shape fold occurs in the bacterium colony back side.
The technical scheme is that bacterial strain → separation screening bacterial strain by collection Mt. Changbai Nature Reserve Area Forest soil
Classification position → atmospheric pressure at room plasma mutagenesis → screening of → identification bacterial strain obtains high yield laccase mutant strain → detection and analysis
The steps such as Lignin degradation rate, the myrothecium verrucaria (Myrothecium for the plant height lignin degradation effect that repeated screening goes out
Verrucaria) bacterial strain T2901.
The beneficial effect that the present invention obtains：1st, the myrothecium verrucaria (Myrothecium that present invention separation mutagenesis obtains
Verrucaria) T2901 can be with the lignin component in efficient degradation stalk.Because most lignin-degrading bacterias can only be secreted
One or both of three kinds of lignin-degrading enzymes, but the strain being related in the present invention can secrete the paint related to lignin degradation
Three kinds of enzyme, lignin peroxidase and manganese peroxidase enzymes.2nd, the myrothecium verrucaria that present invention separation mutagenesis obtains
The loss of (Myrothecium verrucaria) carbohydrate such as T2901 cellulose-less and hemicellulose, high degree remain
Cellulose.
Brief description of the drawings
Fig. 1：The phylogenetic tree of bacterial strain of the present invention.
Fig. 2：Lignin canonical plotting.
Embodiment
The separation of the bacterial strain of embodiment 1
Mt. Changbai Nature Reserve Area Forest soil is gathered, takes 1g pedotheques in the 250mL triangles for filling 99mL sterilized waters
In bottle, in vibrating 45min on 30 DEG C, 120r/min constant temperature oscillator.Supernatant is taken after standing, and is diluted by gradient multiple,
Then dilution spread flat band method is used by dilution factor 10-6~10-8Supernatant be forwarded to PDA plate, each dilution factor is made 3 times
Repeat, cultivated in 29 DEG C of incubators.After flora is grown, line separation repeatedly, is observed under the microscope on PDA plate
After being defined as pure single bacterium colony, it is inoculated on storage medium and is preserved.
The screening and identification of the bacterial strain of embodiment 2
(1) Morphological Identification and screening
The myrothecium verrucaria strain being separated to is seeded on PDA solid mediums, originally white flock is presented in bacterial strain mycelia,
Dissipate and grow to periphery, bacterium colony approximation irregular cycle, have sporodochia appearance after growing 5d on flat board, conidium is most
Just present it is blackish green, cultivate 8d after, conidium color continues to deepen, and Jiao Dot groups occurs；After cultivating 10d, bacterium colony is in concentric
Line shape is taken turns, black is presented in conidium Jiao Dot groups, and filbert emitting shape fold occurs in the bacterium colony back side.
The fungus block for taking a diameter of 1cm is beaten with card punch, is inoculated on aniline blue Selective agar medium, 30 in aerobic incubator
Cultivate 10d under the conditions of DEG C, observe aniline blue discoloration, select that fade rates are fast, the strong bacterial strain of ability is candidate strain.
Simultaneously for the bacterial strain of directionality screening lignin degrading, liquid of the design culture medium using lignin as sole carbon source
Fermentation medium, by the restriction of single carbon source, the clearance of lignin is determined, screening has the true of degradation capability to lignin
Bacterium.Single bacterium colony is inoculated in so that in lignin liquid culture medium, 96h is cultivated under the conditions of 220rmp, 29 DEG C using 24 deep-well plates,
Determine the clearance of lignin.Selection rising trend in lignin culture medium is good, and to the wooden bacterium for being known as certain removal ability
Strain carries out the experimental pretreatment of follow-up maize straw.
The drafting of lignin standard curve：10g/L lignin liquor is prepared, is scanned and selects to determine wavelength.So
Afterwards again by dilute prepare concentration be 10,30,50,70,90mg/L lignin liquor.Each concentration is determined under selected wavelength
The absorbance of lignin, then according to lignin and absorbance relation, canonical plotting is drawn out, as shown in Figure 2.
The calculating of lignin removing rate：Each strain fermentating liquid lignin absorbance is determined with multi-function microplate reader, further according to
The lignin standard curve drawn out calculates the content of lignin in nutrient solution, finally calculates bacterial strain alkali wood according to below equation
Quality degradation rate.Calculation formula is as follows：
Lignin degradation rate=(C1-C2)/C1*100%
Wherein：C1 is the content for the lignin liquor for being not added with bacterium solution
C2 is the content of lignin liquor after strains for degrading
(2) ITS sequence amplification, sequencing and the molecular classification of bacterial strain
It is determined that bacterial strain is delivered into Sangon Biotech's sequencing after character is stable.Sequencing is tied
Shown in fruit SEQ ID NO.1, with carrying out similarity system design point with known ITS sequence in blast program and GenBank databases
Analysis, and it is as shown in Figure 1 with ClustalX1.83 and MEGA4 Neighbor-Joining method phylogenetic tree constructions.Identification knot
Such a bacterial strain of fruit is myrothecium verrucaria F2901, and the strain is laccase high yield fungus, and its laccase activity is in the myrothecium verrucaria reported
In the level of having the advantage reach 27921.39U/L, meanwhile, the bacterium can secrete lignin peroxidase and manganese peroxidase enzyme activity
Power reaches 71.97U/L and 335.4U/L.The degraded of Spruce lignin can be subsequently applied to.
The mutagenesis and screening of the bacterial strain of embodiment 3
The preparation of mutant strain is mainly as follows using the mutagenesis of atmospheric pressure at room plasma (ARTP) method, idiographic flow：
3-5 bead is positioned over bacterium colony surface, 1mL sterilized waters is added, prepares spore suspension.Then carry out dilute
Release, adjustment spore concentration is 107/ mL, the bacteria suspension for taking 10 μ L to dilute are dripped on ARTP slide glass, carry out mutagenesis.
Penetrated with ARTP mutation breeding systems 4mm away from irradiation, irradiate respectively 0s, 15s, 30s, 45s, 60s, 75s, 90s,
120s, slide glass after treatment is put in the centrifuge tube equipped with 1mL sterilized waters, 10 are diluted after shaken well-2、10-3、 10-4、10-5、10-6、10-76 gradients, each gradient coating 3 is parallel, it is determined that optimal mutation time is 75s.
By on the bacteria suspension even spread of dilution to guaiacol selective medium (concentration 0.4%), in aerobic training
Support and cultivate 10d in case under the conditions of 30 DEG C, observation becomes chromosphere situation, selects the big bacterial strain for becoming chromosphere as candidate strain.Forward direction is prominent
Mutant T2901 laccase activities are compared with wild-type strain lifting about 50%, and through multiple Secondary Culture, bacterial strain T2901 can stablize heredity.
Embodiment 4 tests and analyzes lignin removing rate
(1) drafting of lignin standard curve
Certain density lignin liquor is prepared, is scanned and selects to determine wavelength.Compound concentration is the wooden of 10g/L
Plain solution, then again by dilute prepare concentration be 10,30,50,70,90mg/L lignin liquor.Under selected wavelength
The absorbance of each concentration lignin is determined, then according to lignin and absorbance relation, draws out standard curve as shown in Fig. 2.
(2) calculating of lignin removing rate
Each strain fermentating liquid lignin absorbance is determined with multi-function microplate reader, it is bent further according to the lignin standard drawn out
Line converses the content of lignin in nutrient solution, finally calculates bacterial strain Lignin degradation rate according to below equation.Calculate public
Formula is as follows：
Lignin degradation rate=(C1-C2)/C1*100%
Wherein：C1 is the absorbance for the lignin liquor for being not added with bacterium solution
C2 is the absorbance of lignin liquor after strains for degrading
The strain being related in this research carries out the solid state fermentation of maize straw in the form of solid state fermentation, to remove corn
Lignin in stalk.After measured, the three kind enzymes related to lignin degradation can be secreted simultaneously in solid ferment process, it is painted
The vigor of enzyme, lignin peroxidase and manganese peroxidase is respectively 6.61U/g, 0.78U/g and 1.31U/g biomass.
Content of lignin after fermentation in maize straw have dropped 42.30%, and the loss of the carbohydrate such as cellulose-less and hemicellulose.Through
The more untreated maize straw of the conversion ratio of cellulose improves 123.84% in pretreated maize straw；Concrete outcome is joined
It see the table below 1.
The weight-loss ratio and lignocellulosic relative amount of maize straw after table 1 pre-processes
| Sample | Weight-loss ratio (%) | Cellulose (%) | Hemicellulose (%) | Lignin (%) |
| Original stalk | ---- | 38.34±1.05 | 18.95±0.67 | 22.57±2.13 |
| Myrothecium verrucaria pre-processes | 23.67±0.52 | 48.79±0.38 | 24.60±2.79 | 16.99±0.49 |
| Phanerochaete chrysosporium pre-processes | 20.35±1.22 | 39.94±0.66 | 21.92±0.43 | 19.83±0.21 |
It is calculated according to the relative amount of the weight-loss ratio of sample after pretreatment and each material, after myrothecium verrucaria pretreatment
Corn stalk fiber cellulose content declines 2.48%, and hemicellulose level declines 0.53%, and content of lignin declines 42.30%；It is yellow
Corn stalk fiber cellulose content declines 17.02% after the flat lead fungi pretreatment of archespore hair, and hemicellulose level decline 7.62% is wooden
Cellulose content declines 30.04%.
Test result indicates that the bacterial strain screened has strong lignin degradation ability, through the pretreated corn stalk of bacterial strain
The industrialized utilization efficiency of stalk is obviously improved.This research provides a kind of new for the enzymatic saccharification of lignocellulosic in maize straw
Pretreatment approach.
Sequence table
<110>Jilin Agriculture University
<120>A kind of myrothecium verrucaria mutant strain T2901 and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 556
<212> DNA
<213>Myrothecium verrucaria (Myrothecium verrucaria)
<400> 1
ttgatatgct taagttcagc gggtattcct acctgatccg aggtcaactt tcagaagtgg 60
ggtgtttaac ggcatggcca ccgccgcgtt ccaactgcga ggttgtgcta ctacgcagag 120
gaggactaca gcgagaccgc cactagattt cggggccggc ggccttgcga gaaggctggg 180
ccgatcccca acaccaggca ctgggggcct gagggttgaa atgacgctcg aacaggcatg 240
cccgccagaa tactggcggg cgcaatgtgc gttcaaagat tcgatgattc actgaattct 300
gcaattcaca ttacttatcg catttcgctg cgttcttcat cgatgccaga accaagagat 360
ccgttgttga aagtttttat ttatttgttt atgtgtcact cagaggagaa aaccactaaa 420
gacataagag tttggggcct ccggcgggcg cctggttccg ggcccgaagg cgccggggcg 480
gtcccgccga agcaacaata tggtaaggtt cacaaagggt ttgggagttt gtaaactcgg 540
taatgatccc tccgca 556
Claims (3)
- A kind of 1. myrothecium verrucaria（Myrothecium verrucaria）Mutant strain T2901, preserving number are CCTCC NO: M2017413。
- 2. application of the bacterial strain in lignin degrading preparation is prepared as described in claim 1.
- 3. the bacterial strain as described in claim 1, it is characterised in that：The bacterial strain is obtained using atmospheric pressure at room plasma mutagenesis Arrive.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108753640A (en) * | 2018-05-04 | 2018-11-06 | 吉林农业大学 | A kind of complex microbial inoculum of efficient degradation straw lignin |
| CN109182163A (en) * | 2018-08-17 | 2019-01-11 | 吉林农业大学 | A kind of composite bacteria agent of efficient cryogenic degrading maize straws and its application |
| CN109868224A (en) * | 2019-02-01 | 2019-06-11 | 吉林农业大学 | A kind of myrothecium verrucaria of high yield laccase and application |
| CN110042063A (en) * | 2018-06-22 | 2019-07-23 | 北京农学院 | A kind of rouge and powder glues mould (Clonostachys rosea) the bacterial strain YZC3 of broom and its application |
| CN110835612A (en) * | 2018-08-17 | 2020-02-25 | 吉林农业大学 | Corn straw degradation composite microbial inoculum |
| CN110835610A (en) * | 2018-08-17 | 2020-02-25 | 吉林农业大学 | Composite microbial inoculum suitable for degrading straw and preparation method thereof |
| CN110835611A (en) * | 2018-08-17 | 2020-02-25 | 吉林农业大学 | Candida parapsilosis-containing straw degrading composite microbial inoculum and application thereof |
| CN110835613A (en) * | 2018-08-17 | 2020-02-25 | 吉林农业大学 | Composite microbial inoculum containing bacillus subtilis and application thereof in degrading straws at low temperature |
| CN110835607A (en) * | 2018-08-17 | 2020-02-25 | 吉林农业大学 | Low-temperature efficient degradation straw composite bacteria containing saccharomyces cerevisiae and application thereof |
| CN110857431A (en) * | 2018-08-17 | 2020-03-03 | 吉林农业大学 | Composite microbial agent containing bacillus licheniformis and used for degrading straw and application thereof |
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Cited By (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108753640A (en) * | 2018-05-04 | 2018-11-06 | 吉林农业大学 | A kind of complex microbial inoculum of efficient degradation straw lignin |
| CN110042063A (en) * | 2018-06-22 | 2019-07-23 | 北京农学院 | A kind of rouge and powder glues mould (Clonostachys rosea) the bacterial strain YZC3 of broom and its application |
| CN110042063B (en) * | 2018-06-22 | 2021-07-20 | 北京农学院 | Gliocladium roseum (Clinostacchys rosea) strain YZC3 and application thereof |
| CN110835612A (en) * | 2018-08-17 | 2020-02-25 | 吉林农业大学 | Corn straw degradation composite microbial inoculum |
| CN109182163B (en) * | 2018-08-17 | 2020-10-27 | 吉林农业大学 | Composite microbial inoculum for efficiently degrading corn straws at low temperature and application thereof |
| CN110835610A (en) * | 2018-08-17 | 2020-02-25 | 吉林农业大学 | Composite microbial inoculum suitable for degrading straw and preparation method thereof |
| CN110835611A (en) * | 2018-08-17 | 2020-02-25 | 吉林农业大学 | Candida parapsilosis-containing straw degrading composite microbial inoculum and application thereof |
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| CN110835607A (en) * | 2018-08-17 | 2020-02-25 | 吉林农业大学 | Low-temperature efficient degradation straw composite bacteria containing saccharomyces cerevisiae and application thereof |
| CN110857431A (en) * | 2018-08-17 | 2020-03-03 | 吉林农业大学 | Composite microbial agent containing bacillus licheniformis and used for degrading straw and application thereof |
| CN110835607B (en) * | 2018-08-17 | 2023-05-02 | 吉林农业大学 | Low-temperature efficient degradation straw composite bacteria containing saccharomyces cerevisiae and application thereof |
| CN110857431B (en) * | 2018-08-17 | 2022-09-13 | 吉林农业大学 | Composite microbial agent containing bacillus licheniformis and used for degrading straw and application thereof |
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| CN110835612B (en) * | 2018-08-17 | 2021-11-02 | 吉林农业大学 | Corn straw degradation composite microbial inoculum |
| CN110835613B (en) * | 2018-08-17 | 2022-08-09 | 吉林农业大学 | Composite microbial inoculum containing bacillus subtilis and application thereof in degrading straws at low temperature |
| CN110835610B (en) * | 2018-08-17 | 2022-08-26 | 吉林农业大学 | Composite microbial inoculum suitable for degrading straw and preparation method thereof |
| CN109868224B (en) * | 2019-02-01 | 2021-03-16 | 吉林农业大学 | Myrothecium verrucaria with high laccase yield and application thereof |
| CN109868224A (en) * | 2019-02-01 | 2019-06-11 | 吉林农业大学 | A kind of myrothecium verrucaria of high yield laccase and application |
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