CN109957571A - A kind of polysaccharide cracking monooxygenase encoding gene and enzyme and preparation and application - Google Patents
A kind of polysaccharide cracking monooxygenase encoding gene and enzyme and preparation and application Download PDFInfo
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- CN109957571A CN109957571A CN201711336932.3A CN201711336932A CN109957571A CN 109957571 A CN109957571 A CN 109957571A CN 201711336932 A CN201711336932 A CN 201711336932A CN 109957571 A CN109957571 A CN 109957571A
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- 102000008109 Mixed Function Oxygenases Human genes 0.000 title claims abstract description 71
- 238000005336 cracking Methods 0.000 title claims abstract description 49
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 20
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- 238000002360 preparation method Methods 0.000 title claims abstract description 10
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
- C12N9/0083—Miscellaneous (1.14.99)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
Abstract
The invention discloses preparation methods and application that a kind of polysaccharide from thermophilic fungus destroyed wire bacterium (Myceliophthora thermophila) cracks monooxygenase gene and its enzyme, utilize the technical method of genetic engineering, it will be in the gene cloning to yeast expression vector of polysaccharide cracking monooxygenase, obtain can the heterogenous expression enzyme Pichia pastoris recombinant bacterial strain, the polysaccharide of bacterial strain heterogenous expression preparation cracks monooxygenase, it can efficient degradation cellulose (oxicracking β-Isosorbide-5-Nitrae-glycosidic bond).Polysaccharide cracking monooxygenase provided by the invention can be widely applied to the energy, agricultural, food, feed addictive, medicine and other fields.
Description
Technical field
The present invention relates to a kind of gene order of polysaccharide cracking monooxygenase and its preparation method and application of enzyme.The present invention
Provide recombinant plasmid and recombination engineered strain and its application in terms of polysaccharide degradation of polysaccharide cracking monooxygenase.
Polysaccharide cracking monooxygenase provided by the invention can be widely applied to the energy, agricultural, food, feed addition, medicine and other fields.
Background technique
Plant cell wall is mainly made of polysaccharide and lignin.In the dry weight composition of plant cell wall, cellulose, half fiber
Dimension element and lignin account for 20-50%, 15-35% and 10-30% respectively.Wherein, cellulose is that distributed in nature is most extensive, is contained
Most biomass sources is measured, the effective use to cellulose can provide a large amount of chemicals and energy product for human society.It is fine
There are many methods for the utilization of dimension element, and wherein bioanalysis has the advantages such as environmental-friendly, mild condition, but bioanalysis utilizes cellulose
One bottleneck problem is that enzyme degradation efficiency is low, at high cost.This is because cellulose has the anti-degradation barrier of biomass.Traditional fibre
It ties up plain enzyme system and belongs to glycoside hydrolase Families, mainly include inscribe β-Isosorbide-5-Nitrae-dextranase, cellobiohydrolase and β -1,
4- glucuroide.Glycoside hydrolase is difficult to the crystal region degradation of cellulose, can only degrade, make in the noncrystalline domain of cellulose
Its higher cost in industrial application is obtained, the trans-utilization of biomass is unfavorable for.
2010, researcher had found a kind of metalloenzyme with oxidation activity, can be cracked by way of oxidation
Glycosidic bond in polysaccharide.This kind of polysaccharide cracking monooxygenase (LPMO) is a kind of oxidizing ferment for capableing of degradation of polysaccharide, is supplied in electronics
Under the conditions of body and oxygen are existing, oxicracking glycosidic bond generates the oligose fragment of the sugar-chain end containing oxidation.LPMO can and sugar
Glycosides hydrolase synergistic effect, improves the degradation efficiency of indissoluble substrate.Polysaccharide cracking monooxygenase is divided into bacterium and two kinds of fungi are come
Source, wherein the LPMO of bacterial origin belongs to AA (Auxiliary Activity) 10 families with auxiliary activity, principal degradation
Chitin, small part can be with degraded celluloses；The LPMO of originated from fungus points are AA9, AA11 and AA13 family, principal degradation substrate
Respectively cellulose, starch and chitin.Wherein, β-Isosorbide-5-Nitrae glycosidic bond in the LPMO degradation of substrates cellulose of AA9 family,
There is certain degradation to other substrates containing β -1,4-D- glucoside bond.The LPMO of AA9 family was also once classified as
Glycoside hydrolase GH (Glycoside Hydrolase) 61 families.The LPMO of AA9 family is divided into three according to oxidation site difference
Class, PMO1s and PMO2s are respectively C1 and C4 oxidations, and PMO3s is C1 and C4 simultaneous oxidation.The LPMO of AA9 family has height
The homology of degree and similar β sandwich structure, the histidine ring group that activated centre is conjugated by bivalent cupric ion and therewith
At.
Thermophilic fungus destroyed wire (Myceliophthora thermophile ATCC 42464, after be renamed as
Thermothelomyces thermophila) there is stronger cellulose degradation ability, have during cellulose utilization latent
In application value.International enzyme preparation giant Novi letter using thermophilic fungus destroyed wire as promotion cellulose degradation ability valuable source it
One, using the so-called substrate of Barley straw lignocellulosic, the thick enzyme of thermophilic fungus destroyed wire and cellulase are used in conjunction the said firm's discovery can
Greatly improve degradation capability.2011, thermophilic fungus destroyed wire genome was sequenced, so that adding to polysaccharide cracking is single in thermophilic fungus destroyed wire
The research of oxygenase is possibly realized.Currently, the report of only three LPMO about thermophilic fungus destroyed wire bacterium source.2015, discovery
MtLPMO9A has oxidation vigor to cellulose and xylan, and to the synergistic function of cellulase, but there is no correlation
Mechanism Study.2016, the influence of substrate and reducing agent to the LPMO degradation process in thermophilic fungus destroyed wire source is had studied, is had studied
Its degradation of substrates mode, and predict its structure.2017, by taking MtPMO3 as an example, to the secondary in copper ion activated centre in LPMO
Conjugated structure is studied.The polysaccharide cracking monooxygenase in thermophilic fungus destroyed wire bacterium source is mainly Mechanism Study at present, for
The research such as substrate specificity, synergy other than zymologic property, cellulose is less.
Clonal expression and purifying are carried out to polysaccharide cracking monooxygenase MtLPMO9E in the art of this patent return, and right
It its zymologic property, substrate specificity, enzymatic activity and its is characterized with glycoside hydrolase cooperative ability.Polysaccharide cracking is single to be added
Oxygenase MtLPMO9E has the extensive pH scope of application, under acid and alkaline condition, in energy the efficient oxidation cracking cellulose
β-Isosorbide-5-Nitrae-glycosidic bond illustrates that polysaccharide cracking monooxygenase can be applied to a variety of cellulose bottoms with different pretreatments
Object.The Tm value that polysaccharide cracks monooxygenase MtLPMO9E is 52 DEG C, and temperature capacity with higher can satisfy bioanalysis degradation
Utilize the temperature condition of cellulose.Polysaccharide crack monooxygenase MtLPMO9E except can β-Isosorbide-5-Nitrae-glycosidic bond in degraded cellulose,
To other simultaneously containing glucose and β-Isosorbide-5-Nitrae-glycosidic bond substrate, such as xyloglucan, also there is the energy of stronger oxidation and combination
Power, it is contemplated that also have certain degradation to hemicellulose in practical applications.Polysaccharide cracks monooxygenase MtLPMO9E can
β-Isosorbide-5-Nitrae-glycosidic bond in oxicracking cellulose, while hydrolysate and oxidation product are generated, and can be with cellulolytic enzyme
Mixed enzyme Cellulast 1.5L collaboration, effectively improves degradation efficiency.
Sequencing and analyzing described in this patent has no document or patent report.
Summary of the invention
Novel thermophilic fungus destroyed wire bacterium (Myceliophthora is derived from the first purpose of the invention is to provide a kind of
Thermophile ATCC 42464) polysaccharide cracking monooxygenase MtLPMO9E and its encoding gene.
A second object of the present invention is to provide a kind of methods for preparing novel polysaccharide cracking monooxygenase MtLPMO9E.
Third object of the present invention is to provide crack monooxygenase MtLPMO9E genetic recombination table containing the polysaccharide
Up to plasmid and recombination engineered strain.
Fourth object of the present invention is to provide a kind of novel polysaccharide cracking monooxygenase MtLPMO9E in β -1,4- glucosides
Application in key degradation.
Polysaccharide provided by the present invention cracks monooxygenase MtLPMO9E, and what is isolated and purified in soil thermophilic ruins silk
Mould, polysaccharide cracking monooxygenase MtLPMO9E encoding gene (being named as MtLPMO9E) therefrom amplified, has following cores
One of nucleotide sequence feature or two kinds or more:
1) in sequence table SEQ ID NO.1 DNA (DNA) sequence；
2) in polynucleotide SEQ ID NO.2 amino acid sequence DNA (DNA) sequence；
3) homology with SEQ ID NO.1 DNA (DNA) sequence limited reaches 80% or more, and energy
DNA (DNA) sequence of the protein of coding degradation beta-1,4-glucan；
4) one or several nucleotide are carried out to DNA (DNA) sequence of SEQ ID NO.1 in sequence table to take
Coding obtained from generation, missing or addition has the nucleotide sequence of polysaccharide cracking monooxygenase activity.
The present invention also provides the amino acid sequences of polysaccharide cracking monooxygenase MtLPMO9E, with one in following feature
Kind or two kinds or more:
1) 1-229 amino acids residue sequence of the SEQ ID NO.2 since aminoterminal in sequence table, wherein 1-
229 is with the polysaccharide cracking active amino acid sequences of monooxygenase LPMO9E；
2) 1-229 amino acids residue by the SEQ ID NO.2 in sequence table since aminoterminal carry out one or
More than two amino acid substitutions, deletions, or additions and formed with the constant amino acid sequence of polysaccharide cracking monooxygenase activity.
The method of preparation and reorganization enzyme MtLPMO9E is that polysaccharide cracking monooxygenase gene is cloned into recombinant expression carrier,
Or designed according to polysaccharide cracking monooxygenase amino acid sequence and the gene order of host cell is suitble to be cloned into expression vector, it imports
Host cell obtains the polysaccharide cracking monooxygenase of recombinant expression.
Above-mentioned polysaccharide cracks monooxygenase gene, and nucleotide sequence has one of following feature or two kinds or more:
1) DNA (DNA) sequence with SEQ ID NO.1 in sequence table；
2) DNA (DNA) sequence of SEQ ID NO.2 amino acid sequence is encoded；
3) one or more nucleosides is carried out to DNA (DNA) sequence of SEQ ID NO.1 in sequence table
Coding obtained from acid replaces, misses or adds has the nucleotide sequence of polysaccharide cracking monooxygenase activity；
The expression vector of the recombinant expression polysaccharide cracking monooxygenase MtLPMO9E can be Bacillus coli expression load
Body, Yeast expression carrier, hay bacillus expression vector, lactic acid bacteria expression vectors, streptomyces expression vector, phage vector, silk
Shape fungus expression vector, plant expression vector, insect expression vector or mammalian cell expression vector etc..
For recombinantly expressing the recombinant bacterium or transgenic cell line of polysaccharide cracking monooxygenase MtLPMO9E, large intestine can be
Bacillus host cell (such as Escherichia coli BL21, Escherichia coli JM109, Escherichia coli
DH5 α etc.), yeast host cells (such as Saccharomyces cerevisiae, Pichia pastoris, Kluyvero
Myceslactis etc.), hay bacillus host cell (such as Bacillus subtilis R25, Bacillus subtilis9920
Deng), lactic acid bacteria host cell (such as Lactic acid bacteria COCC101), actinomyces host cell (such as
Streptomyces spp. etc.), filamentous fungal host cell (such as Trichoderma viride, Trichoderma
Reesei, Aspergillus niger, Aspergillus nidulans etc.), insect cell (such as Bombyxmori,
Antharaea eucalypti etc.) or mammalian cell (such as Chinese hamster ovary cell CHO, baby hamster kidney cell
BHK, CHL cells CHL etc.).
The gene order of polysaccharide cracking monooxygenase MtLPMO9E of the invention can be from thermophilic fungus destroyed wire bacterium
It clones and obtains in the cDNA library of (Myceliophthora thermophile ATCC 42464).Polysaccharide cracks monooxygenase
The amino acid sequence of MtLPMO9E is expressed by the yeast expression vector constructed.The long 1071bp in the gene coding region belongs to
In auxiliary activity enzyme AA (Auxiliary Activity) 9 families, original is classified as glycoside hydrolase GH (glycoside
Hydrolase) 61 family.
Polysaccharide cracking monooxygenase provided by the invention can be applied in lignocellulose degradation, including in applying below
One kind or two kinds:
1) in fracture β-Isosorbide-5-Nitrae-glucan, β-Isosorbide-5-Nitrae-glycosidic bond of xyloglucan, the application in monosaccharide or oligosaccharides is obtained；
2) in the β-Isosorbide-5-Nitrae-glycosidic bond being broken in other polysaccharide, the application in monosaccharide or oligosaccharides is obtained；
3) it after being mixed with other cellulases, is applied in terms of β-Isosorbide-5-Nitrae-glycosidic bond in collaboration fracture polysaccharide.
The present invention cracks monooxygenase MtLPMO9E from the polysaccharide that Pichia pastoris recombinant expression obtains, can be with efficient degradation fibre
β -1,4- glycosidic bond in dimension element and xyloglucan.LPMO and cellulase synergistic solve existing lignocellulosic conversion and produce
Problem at high cost has important practical value, can be applied to large-scale industrial production.
Polysaccharide cracking monooxygenase MtLPMO9E of the invention can be widely applied to the energy, agricultural, food, feed addition,
Medicine and other fields.
Detailed description of the invention
Fig. 1: polysaccharide cracks the recombinant plasmid after monooxygenase gene recombinant plasmid pPICZ alpha A-MtLPMO9E and linearisation
Agarose gel electrophoresis detection.Swimming lane M is BM5000DNA Marker；Swimming lane 1 is recombinant plasmid pPICZ alpha A-MtLPMO9E；Swimming
Road two is the recombinant plasmid after linearisation
Fig. 2: polysaccharide cracks the SDS-PAGE figure of monooxygenase MtLPMO9E expression and purifying.Swimming lane M is albumen in left figure
Marker, swimming lane 1,2 are the thick enzyme of MtLPMO9E secreting, expressing sample, Pichia anomala expression；Swimming lane M is albumen in right figure
Marker, 1 sample of swimming lane are sugar cracking monooxygenase MtLPMO9E after purification.
Fig. 3: polysaccharide cracks the Binding Capacity lab diagram of monooxygenase MtLPMO9E and cellulose, and swimming lane 1 is precipitating, swimming lane
2 be supernatant, and swimming lane 3 is no Substrate controls.
Fig. 4: polysaccharide cracks monooxygenase MtLPMO9E, and (swimming lane 2,3 is chitin pearl, swimming lane with chitin class substrate respectively
4,5 be α-chitin, and swimming lane 6,7 is β-chitin) it combines, swimming lane 1 is control, and swimming lane 2/4/6 is precipitating, and swimming lane 3/5/7 is
Supernatant.
Fig. 5: polysaccharide cracks monooxygenase MtLPMO9E ITC testing result figure in conjunction with xyloglucan.
Fig. 6: polysaccharide cracks monooxygenase MtLPMO9E to the MALDI-TOF-MS map of cellulose degradation product.
Fig. 7: polysaccharide cracks monooxygenase MtLPMO9E to the MALDI-TOF-MS map of xyloglucan catabolite.
Fig. 8: polysaccharide cracks monooxygenase MtLPMO9E and cellulase synergistic.
Fig. 9: polysaccharide cracks monooxygenase MtLPMO9E three dimensional structure diagram.
Specific embodiment
Sequence table
The information of SEQ ID No.1
(a) sequence signature
Length: 696 nucleotide
Type: nucleotide
Chain: single-stranded
(b) molecule type: DNA
Sequence description: SEQ ID NO.1
Overall length 696bp
CACGCTATCTTCCAGAAGGTTTCAGTTAACGGAGCTGATCAAGGATCTTTGACAGGTTTAAGAGCCCCA
AACAACAACAACCCAGTTCAAAACGTCAACTCCCAAGATATGATTTGCGGTCAATCCGGTTCTACTTCCAACACCAT
TATCGAAGTTAAGGCAGGAGATAGAATTGGAGCTTGGTATCAACACGTTATTGGAGGTGCTCAATTTCCTAACGATC
CAGATAACCCAATCGCTAAGTCTCATAAGGGTCCAGTTATGGCTTACTTGGCTAAAGTTGATAACGCAGCTACAGCT
TCTAAGACCGGTCTTAAGTGGTTCAAGATTTGGGAAGACACTTTCAACCCATCTACTAAGACTTGGGGAGTTGATAA
CCTGATCAACAACAACGGATGGGTTTACTTCAATTTGCCTCAGTGCATTGCAGACGGAAACTACCTTTTGAGAGTGG
AAGTTTTGGCTTTGCATTCAGCTTACTCTCAAGGTCAAGCTCAATTCTACCAGTCTTGTGCTCAAATTAACGTTTCA
GGAGGAGGATCTTTCACTCCAGCTTCTACCGTTTCTTTCCCAGGAGCTTATTCAGCTTCAGATCCAGGAATCTTGAT
CAACATCTACGGTGCTACCGGTCAACCAGATAATAACGGTCAACCATACACCGCTCCAGGTCCAGCTCCAATTTCTT
GTTAATCTAGA
The information of SEQ ID No.2
(a) sequence signature
Length: 229 amino acid
Type: amino acid
Chain: single-stranded
(b) molecule type: albumen
Sequence description: SEQ ID NO.2
229 amino acid of overall length, 1-229 is with the polysaccharide cracking active amino acid sequences of monooxygenase LPMO9E.
HAIFQKVSVNGADQGSLTGLRAPNNNNPVQNVNSQDMICGQSGSTSNTIIEVKAGDRIGAWYQHVIGGA
QFPNDPDNPIAKSHKGPVMAYLAKVDNAATASKTGLKWFKIWEDTFNPSTKTWGVDNLINNNGWVYFNLPQCIADGN
YLLRVEVLALHSAYSQGQAQFYQSCAQINVSGGGSFTPASTVSFPGAYSASDPGILINIYGATGQPDNNGQPYTAPG
PAPISC
The building of 1 polysaccharide of embodiment cracking monooxygenase MtLPMO9E eukaryotic expression bacterial strain
By polysaccharide in The National Center for Biotechnology Information (NCBI) database
After cracking the progress Multiple Sequence Alignment analysis of monooxygenase gene sequence, the middle polysaccharide for preparing synthesis thermophilic fungus destroyed wire bacterium cracks single add
Oxygenase.Target gene MtLPMO9E encodes head of district 741bp, nucleotide sequence such as SEQ ID NO in thermophilic fungus destroyed wire genome
Shown in 1,246 amino acid and a terminator codon are encoded, amino acid sequence is as shown in SEQ ID NO 2, Theoretical molecular
Amount is 26.04KDa.Prediction MtLPMO9E signal peptide is 1-17 amino acid, and predicted molecular weight is after removing signal peptide
24.29KDa, prediction isoelectric point are 6.03.The amino acid of MtLPMO9E coding includes AA9 (GH61) family structure domain.It is logical
Codon optimization is crossed, which is building up on pPICZ α A carrier for expression of eukaryon, is expanded in Escherichia coli Top10.
Plasmid is extracted, is linearized (Fig. 1 is shown in agarose gel electrophoresis detection) with Pme I, glue recycling linearisation product,
It is electroporated into pichia pastoris X-33 competent cell, be coated on the solid YPDSZ culture medium containing 100 μ g/mL bleomycins
On, 30 DEG C are cultivated 5 days, picking monoclonal, carry out bacterium colony PCR verifying using universal primer.PCR reaction condition are as follows: 94 DEG C
2min, 1 circulation；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 2min, 30 circulations；72 DEG C of 5min, 1 circulation.Select verifying
Correct bacterial strain is ready for expressing.
2 polysaccharide of embodiment cracks monooxygenase MtLPMO9E heterogenous expression and purifying in Pichia pastoris
Polysaccharide cracking monooxygenase MtLPMO9E is subjected to inducing expression by Pichia anomala expression manual technique.Use polypropylene
Acrylamide gel electrophoresis detection polysaccharide cracks the expression of monooxygenase LPMO9E, as a result as shown in Figure 2.With BIO-PAD albumen
Purification system is according to the isoelectric point protein of interest of albumen, and polysaccharide cracking monooxygenase LPMO9E after purification is on running gel
In single band, and position matches with the molecular weight of prediction.
3 polysaccharide of embodiment cracks the analysis of monooxygenase MtLPMO9E substrate binding activity
Utilize the binding ability of affine incubation and SDS-PAGE technical research MtLPMO9E and insoluble substrate: MtLPMO9E exists
Under buffer condition, it is incubated for for 24 hours with by the substrate of pretreatment and cleaning, centrifugation obtains supernatant and precipitating, and precipitating need to wash twice,
Its binding ability is determined with MtLPMO9E protein content ratio in the detection upper layer SDS-PAGE and precipitating, and find: MtLPMO9E can
With with cellulose family Binding Capacity (see Fig. 3), MtLPMO9E and chitin class substrate are without combining vigor (see Fig. 4).
Utilize the binding ability of isothermal measuring ball method (ITC) research MtLPMO9E and dissolvable substrate: by MtLPMO9E
It is balanced in identical buffer with xyloglucan overnight, is titrated according to isothermal calorimeter operating process, as a result illustrated
MtLPMO9E and xyloglucan have in conjunction with active (see Fig. 5).
4 polysaccharide of embodiment cracks the oxidation activity measurement of monooxygenase MtLPMO9E
In 1mL system, polysaccharide cracks final concentration of 2 μM of monooxygenase, Final substrate concentrations 5mg/mL, and ascorbic acid is whole
Concentration is 1mM, and buffer solution is the Tris-HCl (pH 8.0) of 20mM.Centrifuge tube is placed in Eppendorf Thermomixer
Middle 100rpm, 40 DEG C, reaction is for 24 hours.After reaction, centrifuging and taking supernatant confirms product structure, discovery with MALDI-TOF-MS
MtLPMO9E has oxidation activity (see respectively Fig. 6,7) to PASC and xyloglucan.It can be generated when MtLPMO9E degradation PASC
A variety of degree of polymerization oxidation products, DP=3-15.Hex can be generated when MtLPMO9E degradation xyloglucan4-6Pen3- ox and Hex9- 11Pen6The oxidation product of-ox.
5 polysaccharide of embodiment cracks monooxygenase MtLPMO9E and cellulase synergistic function analysis
In 1mL system, polysaccharide cracks final concentration of 2 μM of monooxygenase, Final substrate concentrations 5mg/mL, and ascorbic acid is whole
Concentration is 1mM, and buffer solution is the Tris-HCl (pH 8.0) of 20mM, and 1 μ L Cellulast 1.5L is added.Centrifuge tube is set
The 1000rpm in Eppendorf Thermomixer, 40 DEG C of reactions, samples at 10,30,60min respectively.After reaction,
Centrifuging and taking supernatant, with HPAEC-PAD method to the product of the independent degraded cellulose of Cellulast 1.5L, and its with MtLPMO9E assist
Product with degraded cellulose is analyzed (see Fig. 8).By product analysis, catalytic site can analyze, it is different by reaction
The product of time is distributed, and the rule that product changes over time can be intuitively understood, to analyze the vigor of the enzyme.
Therefore, MtLPMO9E, which can be used for acting synergistically with cellulase system, improves the biomass economy of cellulose, Yi Jiyu
Research containing β-Isosorbide-5-Nitrae-D-Glucose glycosidic bond polysaccharide degradation related fields, including the energy, agricultural, food, feed addition, doctor
Medicine and containing β -1,4-D- glucoside bond oligosaccharide preparation etc. fields.
6 polysaccharide of embodiment cracks the analysis of monooxygenase MtLPMO9E tertiary structure
The primary sequence that monooxygenase MtLPMO9E is cracked according to polysaccharide, constructs its three-level knot by way of homologous modeling
Structure model (see Fig. 9).The enzyme is the oxidoreducing enzyme with copper activity center, and the tertiary structure of MtLPMO9E is one and includes 2
The sandwich structure of a alpha-helix and 8 beta sheets.Its end C- is a wedge angle, and the opposite of wedge angle is MtLPMO9E and substrate
Combination plane, an including copper ion activated centre.
Sequence table
<110>Dalian Inst of Chemicophysics, Chinese Academy of Sciences
<120>a kind of polysaccharide cracking monooxygenase encoding gene and enzyme and preparation and application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 696
<212> DNA
<213>thermophilic fungus destroyed wire bacterium (Myceliophthora thermophila)
<400> 1
cacgctatct tccagaaggt ttcagttaac ggagctgatc aaggatcttt gacaggttta 60
agagccccaa acaacaacaa cccagttcaa aacgtcaact cccaagatat gatttgcggt 120
caatccggtt ctacttccaa caccattatc gaagttaagg caggagatag aattggagct 180
tggtatcaac acgttattgg aggtgctcaa tttcctaacg atccagataa cccaatcgct 240
aagtctcata agggtccagt tatggcttac ttggctaaag ttgataacgc agctacagct 300
tctaagaccg gtcttaagtg gttcaagatt tgggaagaca ctttcaaccc atctactaag 360
acttggggag ttgataacct gatcaacaac aacggatggg tttacttcaa tttgcctcag 420
tgcattgcag acggaaacta ccttttgaga gtggaagttt tggctttgca ttcagcttac 480
tctcaaggtc aagctcaatt ctaccagtct tgtgctcaaa ttaacgtttc aggaggagga 540
tctttcactc cagcttctac cgtttctttc ccaggagctt attcagcttc agatccagga 600
atcttgatca acatctacgg tgctaccggt caaccagata ataacggtca accatacacc 660
gctccaggtc cagctccaat ttcttgttaa tctaga 696
Claims (10)
1. a kind of polysaccharide cracks monooxygenase gene, nucleotide sequence has one of following feature or two kinds or more:
1) DNA (DNA) sequence with SEQ ID NO.1 in sequence table；
2) DNA (DNA) sequence of SEQ ID NO.2 amino acid sequence is encoded；
3) one or more nucleotide is carried out to DNA (DNA) sequence of SEQ ID NO.1 in sequence table to take
Coding obtained from generation, missing or addition has the nucleotide sequence of polysaccharide cracking monooxygenase activity；
4) homology with SEQ ID NO.1 DNA (DNA) sequence limited reaches 80% or more, and can encode
DNA (DNA) sequence of the protein of degraded cellulose.
2. a kind of polysaccharide of polysaccharide cracking monooxygenase gene coding described in claim 1 cracks monooxygenase, feature exists
In: its amino acid sequence has one of following feature or two kinds:
1) 1-631 amino acids residue sequence of the SEQ ID NO.2 since aminoterminal in sequence table；
2) one or more amino acid substitution, missing are carried out to amino acid sequence shown in SEQ ID NO.2 in sequence table
Or the amino acid sequence with polysaccharide cracking monooxygenase activity of addition and formation.
3. a kind of preparation method of polysaccharide cracking monooxygenase as claimed in claim 2, it is characterised in that: add polysaccharide cracking list
Oxygenase gene cloning enters recombinant expression carrier, imports host cell, obtains the polysaccharide cracking monooxygenase of recombinant expression；
Above-mentioned polysaccharide cracks monooxygenase gene, and nucleotide sequence has one of following feature or two kinds or more:
1) DNA (DNA) sequence with SEQ ID NO.1 in sequence table；
2) DNA (DNA) sequence of SEQ ID NO.2 amino acid sequence is encoded；
3) one or more nucleotide is carried out to DNA (DNA) sequence of SEQ ID NO.1 in sequence table to take
Coding obtained from generation, missing or addition has the nucleotide sequence of polysaccharide cracking monooxygenase activity；
4) homology with SEQ ID NO.1 DNA (DNA) sequence limited reaches 80% or more, and can encode
DNA (DNA) sequence of the protein of degraded cellulose.
4. according to the method for claim 3, it is characterised in that: the expression of the recombinant expression polysaccharide cracking monooxygenase
Carrier refers to coli expression carrier, Yeast expression carrier, hay bacillus expression vector, lactic acid bacteria expression vectors, streptomycete
Expression vector, phage vector, filamentous fungi expression vector, plant expression vector, insect expression vector or mammalian cell
One of expression vector or two kinds or more.
5. according to the method for claim 3, it is characterised in that: the host cell, i.e., for recombinantly expressing polysaccharide cracking
The recombinant bacterium or transgenic cell line of monooxygenase, refer to e. coli host cell (such as Escherichia coli BL21,
Escherichia coli JM109, Escherichia coli DH5 α etc.), yeast host cells (such as
Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyceslactis etc.), hay bacillus host
Cell (such as Bacillus subtilis R25, Bacillus subtilis9920), lactic acid bacteria host cell (such as Lactic
Acid bacteria COCC101 etc.), actinomyces host cell (such as Streptomyces spp.), filamentous fungi host it is thin
Born of the same parents (such as Trichodermaviride, Trichodermareesei, Aspergillusniger, Aspergillusnidulans
Deng), insect cell (such as Bombyxmori, Antharaea eucalypti), mammalian cell (such as Chinese hamster ovary
One of cell CHO, baby hamster kidney cell BHK, CHL cells CHL etc.).
6. a kind of polysaccharide cracking monooxygenase as claimed in claim 2 is in polysaccharide of the degradation containing glucose and β -1,4- glycosidic bond
Application.
7. a kind of polysaccharide as claimed in claim 2 cracks monooxygenase, it is characterised in that enzyme temperature capacity with higher is used
It is 52 DEG C that DSC method, which detects its fusion temperature, and boiling 10min at 100 DEG C will not complete deactivation.
8. a kind of polysaccharide as claimed in claim 2 cracks monooxygenase, it is characterised in that the enzyme has extensive pH adaptability,
It is active in pH 4-9.
9. a kind of polysaccharide as claimed in claim 2 cracks monooxygenase, degradation is simultaneously containing glucose and β-Isosorbide-5-Nitrae-glycosidic bond
The C4 position oxidation product and hydrolysate for being 3-15 including but not limited to the degree of polymerization in polysaccharide product.
10. applying according to claim 6, it is characterised in that: including in applying below one kind or two kinds:
1) monosaccharide, oligosaccharides or derivatives thereof are obtained simultaneously containing glucose and β-Isosorbide-5-Nitrae-glycosidic bond polysaccharide substrate in fracture
In application；
2) after being mixed with other cellulases, in collaboration fracture simultaneously containing glucose and β-Isosorbide-5-Nitrae-glycosidic bond polysaccharide substrate
The application of aspect.
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| CN115011571A (en) * | 2022-06-17 | 2022-09-06 | 中国科学院天津工业生物技术研究所 | Lytic polysaccharide monooxygenase and application thereof |
| CN115992104A (en) * | 2022-07-22 | 2023-04-21 | 浙江大学 | Schizochy polysaccharide monooxygenase from bacillus subtilis and application thereof |
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Application publication date: 20190702 |