CN106754987A - A kind of polysaccharide cracks monooxygenase LPMO M1 encoding genes and its enzyme and preparation method and application - Google Patents
A kind of polysaccharide cracks monooxygenase LPMO M1 encoding genes and its enzyme and preparation method and application Download PDFInfo
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- CN106754987A CN106754987A CN201510830172.6A CN201510830172A CN106754987A CN 106754987 A CN106754987 A CN 106754987A CN 201510830172 A CN201510830172 A CN 201510830172A CN 106754987 A CN106754987 A CN 106754987A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
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Abstract
Monooxygenase LPMO M1 encoding genes and preparation method and application are cracked the invention discloses a kind of polysaccharide from Pyricularia oryzae.Polysaccharide cracking monooxygenase LPMO M1 involved in the present invention derive from paddy rice pathogenic bacteria Pyricularia oryzae (Magnaporthegrisea 70-15).The method that the novel polysaccharide cracks monooxygenase is prepared present invention also offers a kind of, i.e. using the technical method of genetic engineering, by in the gene cloning of the new polysaccharide cracking monooxygenase to coli expression carrier, obtain can the heterogenous expression enzyme E. coli recombinant stain, the polysaccharide prepared with the bacterial strain heterogenous expression cracks monooxygenase LPMO M1 and can degrade PASC (microcrystalline cellulose treated with phosphoric acid) generation cell-oligosaccharides and cell-oligosaccharide acid.The polysaccharide cracking monooxygenase LPMO M1 that the present invention is provided can be widely applied to chemical industry, agricultural, feed addition and medicine and other fields, with larger productive potentialities and economic worth.
Description
Technical field
Gene order the present invention relates to a kind of polysaccharide cracking monooxygenase and its preparation method and application.The present invention
Additionally provide recombinant plasmid and recombination engineered strain that the polysaccharide cracks monooxygenase.Polysaccharide of the invention splits
Solution monooxygenase LPMO M1 can be widely applied to agricultural, food, feed addition and medicine and other fields.
Background technology
With growth, the reduction of deposit and the aggravation of Global Greenhouse Effect of energy demand, seek alternativeization
The regenerative resource of stone fuel can not urgently be treated.In addition to the clear energy sources such as solar energy and wind energy, the life of rich content
The material energy turns into focus of concern.Lignocellulosic is to be distributed in nature extremely extensively and quantity is extremely more
Biomass resource, the yield of the annual cellulose in the whole world is up to about 8 × 1013Kg, reasonably using this kind of resource
The alleviation of energy crisis will be significant.The indissoluble polysaccharide such as efficient at present, environmental protection utilization cellulose
It is a big bottleneck of the biolobic material development and utilization, the utilization of cellulose mainly passes through chemical method and enzyme process, with
Chemical method has that equipment is simple, reaction condition gentle and advantages of environment protection compared to enzyme process.Enzyme process is mainly logical
Cross the enzyme system degradation of fibers being made up of endoglucanase, cellobiohydrolase and β -1,4-D- glucuroides
Element.Because traditional cellulase system belongs to glycoside hydrolase Families, their degradeds for substrate crystal region
Less efficient and relatively costly, this causes that traditional cellulase system is difficult to meet the demand of commercial Application, limit
The efficient utilization of the biomass such as cellulose is made.
It is that a class exists that polysaccharide cracks monooxygenase (lytic polysaccharide monooxygenases, LPMO)
The enzyme of Oxidative demage indissoluble polysaccharide crystal structure under conditions of bivalent metal ion and reducing agent presence.Its appearance
Improve the degradation efficiency of indissoluble polysaccharide so that the Efficient Conversion of biomass is possibly realized.The single oxygenation of polysaccharide cracking
The source of enzyme very enrich, at present from bacterium (such as Streptomyces coelicolorA3 (2)), fungi (such as
Neurosporacrassa OR74A) and viral (such as Anomalacupreaentomopoxvirus CV6M) in
It was found that the fermentoid.At present it has been reported that LPMO have 27, there are 14, AA10 in wherein AA9 families
Family has 12, and AA11 families have 1；Polysaccharide cracking monooxygenase LPMO M1 sources in the present invention
It is a new LPMO in Pyricularia oryzae, the clonal expression of the enzyme is that the plain efficient degradation of subsequent fiber is established
Fixed basis.
The content of the invention
First purpose of the invention is to provide a kind of new from Pyricularia oryzae
(Magnaporthegrisea70-15) polysaccharide cracking monooxygenase LPMO M1 and its encoding gene.
Second object of the present invention is to provide a kind of novel polysaccharide for preparing and cracks monooxygenase LPMO M1's
Method.
Third object of the present invention is to provide and cracks monooxygenase lpmo M1 gene weights containing above-mentioned polysaccharide
Group expression plasmid and recombination engineered strain.
Dropped in cellulose it is a further object to provide novel polysaccharide cracking monooxygenase LPMO M1
Application in solution.
Polysaccharide provided by the present invention cracks monooxygenase LPMO M1, from paddy rice pathogenic bacteria Pyricularia oryzae
(Magnaporthgrisea 70-15), its amino acid sequence has at least one of following feature：
1) 1-272 or 22-272 amino acids residue sequences of the SEQ ID NO.2 since aminoterminal in sequence table,
Wherein 1-21 is signal peptide, and 22-272 is active polysaccharide cracking monooxygenase LPMO M1
Amino acid sequence.
2) 1-272 the or 22-272 amino acids residues by the SEQ ID NO.2 in sequence table since aminoterminal
Carry out one or more 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, missing or addition and formed and crack monooxygenase with polysaccharide
The constant amino acid sequence of activity.
Present invention also offers the encoding gene (being named as lpmo M1) of polysaccharide cracking monooxygenase LPMO M1,
With at least one of following nucleotide sequence feature：
1) in sequence table SEQ ID NO.1 DNA (DNA) sequence；
2) in polynucleotide SEQ ID NO.2 amino acid sequences DNA (DNA) sequence；
3) with SEQ ID NO.1 limit DNA (DNA) sequence homology reach 80% and
More than, and DNA (DNA) sequence of the protein of degraded cellulose can be encoded.
4) DNA (DNA) sequence to SEQ ID NO.1 in sequence table carries out one or two
The substitution of above nucleotides, missing or coding obtained from addition crack the nucleotides of monooxygenase activity with polysaccharide
Sequence.
The amino acid sequence and its nucleotide coding sequence of polysaccharide cracking monooxygenase LPMO M1 of the invention
Can also be according to the amino acid sequence of the LPMO M1 of prediction and its artificial synthesized acquisition of nucleotide coding sequence.
The method of Prepare restructuring enzyme LPMO M1, is that encoding gene lpmo M1 are cloned into recombinant expression carrier,
Host cell is imported, the polysaccharide cracking monooxygenase of recombination expression is obtained.
The expression vector of described recombination expression polysaccharide cracking monooxygenase LPMO M1 can be Escherichia coli
Expression vector, Yeast expression carrier, hay bacillus expression vector, lactic acid bacteria expression vectors, streptomycete expression are carried
Body, phage vector, filamentous fungi expression vector, plant expression vector, insect expression vector or lactation are moved
Thing fibrocyte expression vector etc..
The recombinant bacterium or transgenic cell line of the polysaccharide cracking monooxygenase LPMO M1 for recombinantly expressing, can
Be e. coli host cell (such as Escherichia coli BL21, Escherichia coli JM109,
Escherichia coli DH5 α etc.), yeast host cells (such as Saccharomyces cerevisiae,
Pichiapastoris, Kluyveromyceslactis etc.), hay bacillus host cell (such as Bacillus subtilis
R25, Bacillus subtilis 9920 etc.), lactic acid bacteria host cell (such as Lactic acid bacteria COCC101
Deng), actinomyces host cell (such as Streptomyces spp.), filamentous fungal host cell (such as
Trichodermaviride, Trichodermareesei, Aspergillusniger, Aspergillusnidulans etc.),
Insect cell (such as Bombyxmori, Antharaea eucalypti etc.) or mammalian cell are (such as Chinese storehouse
Mouse gonad cell CHO, baby hamster kidney cell BHK, CHL cells CHL etc.).
Above-mentioned polysaccharide cracking monooxygenase can be applied in cellulose degradation, including the one kind in following application
Or two kinds；
1) in fracture cellulose glycosidic bonds, the application in cell-oligosaccharide is obtained；
2) in biomass such as oxidative degradation lignin, the application in cell-oligosaccharide and cell-oligosaccharide acid is obtained；
After polysaccharide cracking monooxygenase LPMO M1 mix with cellulase system, Synergistic degradation cellulose,
Preparing bio-ethanol aspect can apply.
The gene order of polysaccharide cracking monooxygenase LPMO M1 of the invention is by unrestricted clone (RF clones)
Technology the clone from Pyricularia oryzae (Magnaporthegrisea 70-15) obtain.Gene code head of district 819bp,
272 amino acid are encoded, molecular weight about 28.85KDa belongs to the family of glycoside hydrolase 61 (now known as AA9
Family).The LPMO M1 that Recombinant protein expression is obtained, can be with oxygen under conditions of 37 DEG C, ph=6.5
Change the microcrystalline cellulose (PASC) after degraded is processed through peroxophosphoric acid.Polysaccharide of the invention cracks monooxygenase
LPMO M1 with same family it has been reported that LPMO carry out sequence alignment, as a result find homology reach as high as
41%, and the enzyme is first polysaccharide cracking monooxygenase of successful clone expression in the bacterial strain, can extensive use
In agricultural, food, feed addition and medicine and other fields.
Brief description of the drawings
Fig. 1:Polysaccharide cracks the electrophoresis detection of monooxygenase lpmo M1 genes；
The sample that each swimming lane is added is respectively：Swimming lane M-BM5000DNA Marker；Swimming lane 1-lpmo M1PCR are produced
Thing
Fig. 2:The polyacrylamide gel electrophoresis figure of restructuring polysaccharide cracking monooxygenase LPMO M1 expression
(SDS-PAGE).The sample that each swimming lane is added is respectively：Swimming lane M- pre-dyed Protein Markers；Swimming lane
Full bacterium before 1-E.coli BL21 (DE3)/pET22b-lpmo M1 inductions；Swimming lane 2- induces the full bacterium of 24h；Swimming
Albumen supernatant after road 3- osmotic shocks method treatment；
Fig. 3：The product MALDI-TOF analysis of spectra of LPMO M1 degradeds PASC.
Specific embodiment
The polysaccharide of embodiment 1 cracking monooxygenase full-length gene clone
Pyricularia oryzae bacterial strain is extracted by fungal rna extracts kit (giving birth to work, SK8659 in Shanghai) operating procedure
The RNA of Magnaporthegrisea 70-15, by cDNA the first chain synthetic agents of TaKaRa biotech firms
Box (CodeNO.:Operating procedure synthesis cDNA 6210A).Nucleotide sequence design according to genes of interest
Primer, sense primer 5 '
- CTGCCCAGCCGGCGATGGCCCACTACAACTTCGAGTCCCTC-3 ', anti-sense primer：
5′-TTGTTAGCAGCCGGATCTCAGTAGTGTGCGGAGCGGCGGC-3′.To synthesize
CDNA for template enter performing PCR amplification.PCR reaction conditions are：98 DEG C of predegeneration 3min, 98 DEG C
Denaturation 10s, 55 DEG C of annealing 5s, 72 DEG C of extension 2min, totally 30 circulations, 72 DEG C of extension 5min.PCR
After product is analyzed through agarose gel electrophoresis, gel extraction purpose fragment, by (RF grams of unrestricted PCR cloning PCR
It is grand) genes of interest is connected to after expression vector pET-22b it is sequenced.
The polysaccharide of embodiment 2 cracks monooxygenase gene sequence analysis
The result of sequencing is using the Basic Local Alignment Search Tool in GenBank databases
(BLAST) analyze, the softwares of Vector NTI Suite 8.0 carry out Multiple Sequence Alignment, analyze its homology.Using
The domain of Simple Modular Architecture Research Tool (SMART) online tool analytical sequence.
The polysaccharide of acquisition cracks monooxygenase gene (being named as lpmo M1) coding head of district 819bp, its nucleosides
Acid sequence is as shown in SEQ ID NO 1.Lpmo M1 encode 271 amino acid and 1 terminator codon,
Its amino acid sequence as shown in SEQ ID NO 2, protein theoretical molecular be 28.85kDa, prediction etc. electricity
Point is 7.66.SMART analysis shows, 1-21 is signal peptide in the amino acid sequence of LPMO M1.LPMO
The domains characteristic of M1 is increasingly similar with AA9 family members, is indicated above LPMO M1 for AA9 families
A newcomer.With the homologous Modeling Server (http of SWISS-MODEL://swissmodel.expasy.org)
Protein three-dimensional structure to polysaccharide cracking monooxygenase LPMO M1 carries out homologous modeling, as a result shows structure
LPMO M1 structural models the QMEAN score value of evaluation model quality (QMEAN for) it is relatively low, say
The bright LPMO M1 3 d structure models for obtaining are insincere.
SEQ ID NO.1
ATGCACTGCAACACCCTGGCCACCATCGTCGCCGCCGTGGGCCTCATCCCC
ACGGCGCTGGCGCACTACAACTTCGAGTCCCTCGTGGTCAACGGCAAGTC
GACCGGGCCGTACGAGTACGTGCGACGGACGACCAACTCCAACTCGCCCA
TCGAGGACGTCAAGTCGCAAAACATGGTGTGCAACCAGGGCGGGCTCGAC
GCCTCCATCATGGCCGCCACCAAGACCTACACGGTGGCGCCGGGCGACGA
GGTCGGATTCCTCGTCAACTCGAACCTGGGACACCCGGGCCCGCAGGCCG
TGTACCTCTCGCGCGCTCCCGACGGCGTCGCCGCCAACCAGTACAAGGGC
GACGGCGACTGGTTCAAGGTCTACTCGCTGACCAGCGGCAAGATCGACGC
CAACACGGGCATCGACTGGGCCACCTTCCCCGGCAGCCAGGGCGCCAGCA
GCTTCACCTTCAAGCTGCCGCAGAACCTGCCGCCGGGCGACTACCTCATGC
GCGCCGAGCACATTGGCCTGCACGGCGCCGGCACCTTTGGCGGCGCGCAG
TTCTACATTGGCTGCGCCCAGCTCAAGGTCACGGGCAGCGGGAGCGGCAA
GCCCGGCCCCACCGTCAAGTTCCCCGGCGCCTACACCGGCAACGAGCCCG
GCATCAAGATCAACATCTACTGGCCGCCGCTGACCAGCTACACCCCGCCCG
GCCCCGTCACCTGGCCCAACGCCTGCGAGGACCACACCACAAACCTCTTT
GGTAAGCCTAGCGATGGCGACTGCACTCCGCTCCGCAGCTCTCGCCGCCGC
TCCGCACACTACTAA
SEQ ID NO.2
MHCNTLATIVAAVGLIPTALAHYNFESLVVNGKSTGPYEYVRRTTNSNSPIEDV
KSQNMVCNQGGLDASIMAATKTYTVAPGDEVGFLVNSNLGHPGPQAVYLSR
APDGVAANQYKGDGDWFKVYSLTSGKIDANTGIDWATFPGSQGASSFTFKLP
QNLPPGDYLMRAEHIGLHGAGTFGGAQFYIGCAQLKVTGSGSGKPGPTVKFG
AYTGNEPGIKINIYWPPLTSYTPPGPVTWPNACEDHTTNLFGKPSDGDCTPLRS
SRRRSAHY
Recombination expression of the embodiment 3lpmo M1 genes in Escherichia coli
Genomic DNA with Pyricularia oryzae cDNA as template, using design sense primer (5 '-
CTGCCCAGCCGGCGATGGCCCACTACAACTTCGAGTCCCTC-3 ') and downstream draw
Thing (5 '-TTGTTAGCAGCCGGATCTCAGTAGTGTGCGGAGCGGCGGC-3 ') is pressed
The gene order of the maturation protein of the Program amplification coding polysaccharide of embodiment 1 cracking monooxygenase is not (including signal
Peptide gene).Pcr amplification product and expression vector pET22b (Novagen companies of the U.S.) pass through unrestricted gram
Grand method (RF clones) connection, Escherichia coli TOP10 competent cells are converted by connection product, are coated on and are contained
On the Luria-Bertani culture medium solid plates of 100 μ g/mL ampicillins, 37 DEG C of culture 12-16h choose
Monoclonal is taken, bacterium colony PCR checkings is carried out with upstream and downstream primer, as a result obtain the correct amplified production of size,；
Cultivated during correct monoclonal is accessed into the liquid Luria-Bertani culture mediums containing 100 μ g/mL ampicillins,
Extract plasmid；Then the recombinant plasmid is sent into Hua Da gene (Beijing) sequencing, is as a result shown, in pET-22b
PelB signal peptides and his labels between insert lpmo M1 genes shown in SEQ ID NO 1, and insertion
It is in the right direction, so further proving that the recombinant plasmid for building is correct, the recombinant plasmid is named as
pET22b-lpmo M1。
By pET22b-lpmo M1 convert coli strain BL21 (DE3) (Novagen companies of the U.S.,
CB1493450), the operating procedure for then being provided according to the said firm carries out polysaccharide cracking monooxygenase LPMO M1
Induced expression.Detect that polysaccharide cracks the expression of monooxygenase with polyacrylamide gel electrophoresis, as a result such as
Shown in Fig. 2, polysaccharide cracks monooxygenase obvious expression under IPTG inductions.
The product analysis of the polysaccharide of embodiment 4 cracking monooxygenase LPMO M1 oxidative degradations PASC
5mgPASC is taken, adds polysaccharide to crack monooxygenase LPMO M1 (1 μM), vitamin C (1mM)
And CuSO4(1 μM), terminating reaction after 37 DEG C of reaction 48h, is centrifuged off insoluble substrate, takes supernatant, uses
Savage methods are according to product：Savage reagent=4:1 ratio vibration is centrifuged off in product after protein
Product is detected with MALDI-TOF.
Product as shown in Figure 3 is mainly the oligosaccharides of DP=5-10 and the saccharic acid of DP=6-9, therefore the enzyme can be with
It is used for the preparation of oligosaccharides and saccharic acid, and cellulose drop can be improved with cellulase system Synergistic degradation cellulose
Solution efficiency, for the degraded of cellulose provides new visual angle.
Claims (7)
1. a kind of polysaccharide cracks monooxygenase LPMO M1 encoding genes, it is characterised in that：With at least one of following features：
1) with DNA (DNA) sequence of SEQ ID NO.1 in sequence table；
2) DNA (DNA) sequence of SEQ ID NO.2 amino acid sequences is encoded；
3) DNA (DNA) sequence to SEQ ID NO.1 in sequence table carries out the nucleotide sequence of the enzyme of one or more nucleotides substitution, missing or coding obtained from addition with polysaccharide cracking monooxygenase activity；
4) homology of DNA (DNA) sequence limited with SEQ ID NO.1 reach 80% and more than, and DNA (DNA) sequence of the protein of degraded cellulose can be encoded.
2. a kind of polysaccharide of the polysaccharide cracking monooxygenase LPMO M1 encoding gene codings described in claim 1 cracks monooxygenase, it is characterised in that：Its amino acid sequence has at least one of following feature：
1) 1-272s or 22-272 amino acids residue sequence of the SEQ ID NO.2 since aminoterminal in sequence table；
2) amino acid sequence that monooxygenase activity is cracked with polysaccharide for one or more 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, missing or addition being carried out to the amino acid sequence in sequence table shown in SEQ ID NO.2 and being formed.
3. polysaccharide as claimed in claim 2 cracks monooxygenase, it is characterised in that the enzyme has oxidative degradation activity to cellulose family substrate PASC.
4. the polysaccharide described in a kind of claim 2 cracks the preparation method of monooxygenase, it is characterised in that：Polysaccharide cracking monooxygenase LPMO M1 encoding genes are cloned into recombinant expression carrier, host cell is imported, the polysaccharide cracking monooxygenase of recombination expression is obtained；
Above-mentioned polysaccharide cracking monooxygenase lpmo M1 encoding genes have at least one in following features：
1) with DNA (DNA) sequence of SEQ ID NO.1 in sequence table,
2) DNA (DNA) sequence of SEQ ID NO.2 amino acid sequences is encoded,
3) DNA (DNA) sequence to SEQ ID NO.1 in sequence table carries out the nucleotide sequence of the enzyme of one or more nucleotides substitution, missing or coding obtained from addition with polysaccharide cracking monooxygenase activity；
Described recombination expression polysaccharide cracks the expression vector of monooxygenase, refer to one or two or more kinds in coli expression carrier, Yeast expression carrier, hay bacillus expression vector, lactic acid bacteria expression vectors, streptomyces expression vector, phage vector, filamentous fungi expression vector, plant expression vector, insect expression vector or mammalian cell expression vector.
5. in accordance with the method for claim 4, it is characterised in that：The recombinant bacterium or transgenic cell line of monooxygenase are cracked for recombinantly expressing polysaccharide,Refer to e. coli host cell Escherichia coli BL21,Escherichia coli JM109,Escherichia coli DH5α,Yeast host cells Saccharomyces cerevisiae,Pichiapastoris,Kluyveromyceslactis,Hay bacillus host cell Bacillus subtilis R25,Bacillus subtilis 9920,Lactic acid bacteria host cell Lactic acid bacteria COCC101,Actinomyces host cell Streptomycesspp filamentous fungal host cells Trichodermaviride,Trichodermareesei,Aspergillusniger,Aspergillusnidulans,Insect cell Bombyxmori,Antharaea eucalypti,Mammalian cell Chinese hamster ovary cell CHO,One or two or more kinds in baby hamster kidney cell BHK or CHL cells CHL.
6. application of a kind of polysaccharide cracking monooxygenase as claimed in claim 2 in cellulose degradation, it is characterised in that：Including the one kind in following application or two kinds；
1) in fracture cellulose glycosidic bonds, the application in cell-oligosaccharide is obtained；
2) in biomass such as oxidative degradation lignin, the application in cell-oligosaccharide and cell-oligosaccharide acid is obtained.
7. application as claimed in claim 6, it is characterised in that：After polysaccharide cracking monooxygenase LPMO M1 mix with cellulase system, in Synergistic degradation cellulose, prepare bio-ethanol in terms of application.
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Cited By (4)
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| CN108753741A (en) * | 2018-03-28 | 2018-11-06 | 天津科技大学 | A kind of extracellular AA9 families polysaccharide monooxygenase AnLPMO15g and its application |
| CN109897859A (en) * | 2019-03-11 | 2019-06-18 | 大连大学 | Polysaccharide cracks monooxygenase gene PsLMPO10A and its coding albumen and function |
| CN109957571A (en) * | 2017-12-14 | 2019-07-02 | 中国科学院大连化学物理研究所 | A kind of polysaccharide cracking monooxygenase encoding gene and enzyme and preparation and application |
| CN114874334A (en) * | 2022-04-27 | 2022-08-09 | 首都师范大学 | Chimeric fibrosome and application thereof |
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2015
- 2015-11-25 CN CN201510830172.6A patent/CN106754987A/en active Pending
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109957571A (en) * | 2017-12-14 | 2019-07-02 | 中国科学院大连化学物理研究所 | A kind of polysaccharide cracking monooxygenase encoding gene and enzyme and preparation and application |
| CN108753741A (en) * | 2018-03-28 | 2018-11-06 | 天津科技大学 | A kind of extracellular AA9 families polysaccharide monooxygenase AnLPMO15g and its application |
| CN108753741B (en) * | 2018-03-28 | 2020-01-10 | 天津科技大学 | Extracellular AA9 family polysaccharide monooxygenase AnLPMO15g and application thereof |
| CN109897859A (en) * | 2019-03-11 | 2019-06-18 | 大连大学 | Polysaccharide cracks monooxygenase gene PsLMPO10A and its coding albumen and function |
| CN114874334A (en) * | 2022-04-27 | 2022-08-09 | 首都师范大学 | Chimeric fibrosome and application thereof |
| CN114874334B (en) * | 2022-04-27 | 2023-12-22 | 首都师范大学 | Chimeric fiber corpuscle and application thereof |
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Application publication date: 20170531 |