CN106047737A - Pichia pastoris engineered strain for expressing AA9 family polysaccharide monooxygenase genes TLAA9-2 - Google Patents
Pichia pastoris engineered strain for expressing AA9 family polysaccharide monooxygenase genes TLAA9-2 Download PDFInfo
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Abstract
The invention relates to a pichia pastoris engineered strain for expressing AA9 family polysaccharide monooxygenase genes TLAA9-2. Glucoside hydrolase genes TLAA9-2 are obtained from thermomyces lanuginosus by an RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method, are cloned and are inserted into pichia pastoris integrative expression vectors pPICZ[alpha]A; then, the obtained glucoside hydrolase gene TLAA9-2 expression vectors pPICZ[alpha] A/TLAA9-2 are transferred into the pichia pastoris GS115; and then, engineered yeast GS-CT-9-1 for expressing the glucoside hydrolase genes TLAA9-2 are screened out. The glucoside hydrolase TLAA9-2 expressed by the engineered strain has the obvious promoting effect on the activity of the endo-cellulase; and the cellulose degradation activity of mixed enzymes is improved by 1.2 times through being compared with that of original incision enzymes N24.
Description
(1) technical field
The present invention relates to biological engineering, specifically a kind of expression is Thermomyces lanuginosus Thermomyces
The pichia pastoris engineered strain Pichia pastoris of lanuginosusAA9 family polysaccharide monooxygenase gene TLAA9-2
GS-CT-9-2。
(2) background technology
Polysaccharide monooxygenase (English: Polysaccharide monooxygenases), also known as polysaccharide monooxygenase) it is one
Kind of special oxidation cracking glycosidic bond, and produce the ferment of multiple cell-oligosaccharide molecule, is also one of common ferment in nature.
This proteinoid has been applied in the industry of the mankind, such as, be used for being degraded into the biomass energies such as cellulose and hemicellulose
Available little molecule.Polysaccharide monooxygenase has multiple family.Thermomyces lanuginosus Thermomyces lanuginosus
It it is a kind of thermophilic fungal being distributed widely in compost.This bacterium is 50 DEG C of conditions in the culture medium that microcrystalline Cellulose is sole carbon source
Under can produce heat-staple cellulase and AA9 family polysaccharide monooxygenase.
The AA9 family polysaccharide monooxygenase TLAA9-2 that Thermomyces lanuginosus produces has oxicracking cellulose
Act on, and the enzymatic activity of the endo cellulase N24 producing this bacterial strain has obvious facilitation, and its activity can be made to carry
High 1.2 times.Polysaccharide monooxygenase plays its activity needs the existence of electron donor (ascorbic acid or CDH), through thin layer chromatography
(TLC) detection produces multiple cell-oligosaccharide.This enzyme can produce hydrogen peroxide in the case of lacking substrate, and through flight matter
It is cell-oligosaccharide that spectrum identifies that the cracking of this oxydasis interrupts cellulose long-chain primary product.
(3) summary of the invention
The present invention obtains polysaccharide monooxygenase cDNA sequence from Thermomyces lanuginosus Thermomyces lanuginosus
Row total length, named TLAA9-2, TLAA9-2 gene DNA total length 825bp, including signal peptide sequence, start codon and termination
Codon, intronless, encode 252 aminoacid.There is signal peptide sequence (1-22aaMKGSTTASLLLPLLASVTRTSA)
And 30 O glycosylation sites, there is a potential N glycosylation site.The aminoacid sequence encoded by TLAA9-2 is at international gene
Storehouse is retrieved, finds to belong to polysaccharide monooxygenase AA9 family.
Build recombinant expression plasmid carrier pPICZ α A/TLAA9-2, utilize electroporation to carry out electroporated
Pichiapastoris GS115, screens in Zeocin culture medium, and through PCR evaluation and screening positive transformant, high concentration is rich next
Mycin screening multicopy transformant, then carries out methanol induction expression, it is thus achieved that pichia pastoris engineered strain GS-CT-9-2.This work
Journey inoculation is in containing in BMGY culture medium, after 6d cultivated by 28 DEG C of 200rpm/min shaking tables, and the expression of polysaccharide monooxygenase
For 3.21mg/ml, molecular weight is that 45kDa, TLAA9-2 optimum temperature is 50-55 DEG C, is stable at 60 DEG C, processes 60min
After still have more than 90% enzymatic activity；The activity of 68% is still kept after 70 DEG C of process 30min；80 DEG C process holding after 30min
The activity of 23%；90 DEG C process 30min activity and almost completely lose.
AA9 family polysaccharide monooxygenase TLAA9-2 can be by the presence of electron donor (ascorbic acid and CDH) and copper ion
Cellulose long-chain oxicracking is different cell-oligosaccharide, and can produce hydrogen peroxide in the case of lacking substrate, right
The enzymatic activity of endo cellulase N24 has obvious facilitation, the ability of the mixed enzyme degraded cellulose than protoenzyme N24
Improve 1.2 times.First histidine of polysaccharide monooxygenase has important function, and front end is connected with other aminoacid can affect its work
Property.Different polysaccharide monooxygenase oxicracking cellulose long-chains occurs, at different company's key positions, to identify through flight mass spectrum
TLAA9-2 oxicracking cellulose long-chain primary product is cell-oligosaccharide.
(4) accompanying drawing explanation:
The SDS-PAGE of Fig. 1 AA9 family polysaccharide monooxygenase TLAA9-2 analyzes
Swimming lane 1: low molecular weight protein (LMWP) standard
Swimming lane 2: the AA9 family polysaccharide monooxygenase TLAA9-2 of purification
The optimum temperature of Fig. 2 AA9 family polysaccharide monooxygenase TLAA9-2
Fig. 3 AA9 family polysaccharide monooxygenase TLAA9-2 facilitation to endo cellulase N24 activity
Fig. 4 AA9 family's polysaccharide monooxygenase TLAA9-1 oxicracking cellulose thin-layer chromatography detection and electron donor are (anti-
Bad hematic acid) on impact active for AA9 family polysaccharide monooxygenase TLAA9-1
Swimming lane 1: standard fibers oligosaccharide molecular (M)
Swimming lane 2: do not add polysaccharide monooxygenase TLAA9-1 (E) of ascorbic acid
Swimming lane 3: add polysaccharide monooxygenase TLAA9-1 (E+Vc) of ascorbic acid
Fig. 5 AA9 family polysaccharide monooxygenase TLAA9-1 enzymatic hydrolysate flight mass spectrum is analyzed
(5) detailed description of the invention
Embodiment 1: the isolation identification of Thermomyces lanuginosus Thermomyces lanuginosusermophilum
(1) collection of specimens: gather from compost.
(2) separation and Culture: collect specimen is taken 0.5 gram be placed on PDA plate 50 DEG C cultivate 3 days after, carry out separating pure
Change.Operating procedure is with reference to Cooney and Emerson (1964) document.
(3) identify: with reference to Cooney and Emerson (1964) and LaTouche (1950) document.
Embodiment 2: the clone of polysaccharide monooxygenase gene TLAA9-2
(1) extraction of Thermomyces lanuginosus Thermomyces lanuginosusermophilum total serum IgE: reference
Trizol test kit explanation.
(2) synthesis of cDNA Article 1 chain: try according to TaKaRa RNA PCR kit (AMV) Ver3.0 of Takara company
Agent box description is carried out: takes 1～2 μ g total serum IgE, adds RNase Free ddH2O to 9.5 μ L, by RNA sample 75 DEG C of degeneration
5min, cools down 5min immediately in ice bath, the most centrifugal once, ice bath is sequentially added into following various composition:
10mmol/L dNTP Mixture 2 μ L, 10 × RT Buffer (Mg2+) 2 μ L, 25mmol/L MgCl24 μ L, Oligo d (T)-
Adaptor Primer 1 μ L, RNase Inhibiter 0.5 μ L, AMV Reverse Transcriptase 1 μ L (Final
Volume 20 μ L), after reactant liquor is mixed, ambient temperatare 10min, then 42 DEG C of incubation 60min, then boil 5min with inactivation
Reverse transcription.Add the ddH that 180 μ L DEPC process2O, is diluted to 200 μ L, mixing, the most centrifugal, is stored in-20 DEG C, standby
With.
(3) PCR reaction (25 μ L): cDNA2 μ L, 10 × Buffer 2.5 μ L, 10mmol/L dNTP 2 μ L, 25mmol/
LMgCl22 μ L, each 2 μ L of upstream and downstream primer, Taq archaeal dna polymerase 0.5 μ L (5U/ μ L), ddH2O 12 μ L.Reaction condition is 94
DEG C 5min denaturation；94 DEG C of 40s, 56 DEG C of 40s, 72 DEG C of 1min, totally 32 circulations, 72 DEG C extend 10min, 4 DEG C of preservations.
Forward primer: 5 '-AGGGGTATCTCTCGAGAAAAGACACGGGTTTGTCTCCAACCT-3 '
Downstream primer: 5 '-GAGTTTTTGTTCTAGACCCGCCGCCGCCGGCGTCG-3 '
(4) gene clone: take 0.5 μ l PCR primer and be attached with pMD18-T carrier, operating procedure is public according to TAKARA
Department's product description is carried out.Then connect product and convert e.colistraindh5α, scribble penbritin (100 μ g/ on surface
ML) grow overnight on LB flat board.Picking white colony, overnight incubation in LB fluid medium.
(5) extraction of plasmid DNA: alkalinity extraction plasmid DNA.
(6) sequencing: DNA double deoxidation method measures nucleotide sequence, and biological engineering company limited is carried out in Shanghai.Sequence
Primer is M13 promoter primer.Thermomyces lanuginosus TLAA9-2 gene cDNA total length 825bp, comprise start codon and
Termination codon, intronless, encode 252 aminoacid.This aminoacid sequence is retrieved in international gene bank, finds
Belong to polysaccharide monooxygenase AA9 family.This sequence is as follows:
(A) information of SEQ ID NO 1
(a) sequence signature: * length: 825 base pairs；* type: nucleic acid；* chain: double-strand；* topological structure: linear
(b) molecule type: DNA
C () is assumed: no
(d) antisense: no
E () is initially originated: Thermomyces lanuginosus Thermomyces lanuginosus
(f) sequence description:
(B) information of SEQ ID NO 2
(a) sequence signature: * length: 252 aminoacid；* type: aminoacid；* chain: strand；* topological structure: linear
(b) molecule type: protein
(c) sequence description:
Embodiment 3: the structure of expression vector
(1) genes of interest CDS sequence is carried out signal peptide analyses and prediction, remove signal peptide, draw according to carrier information design
Thing, introduces Xhol I and Xba I restriction enzyme site, and completion sequence is to the Kex2 protein signal cleavage site of Ppicz α A carrier, reversely
Primer 3 ' end is removed termination codon and adds two bases to ensure that sequence is normally translated to 6 × His label.
Forward primer: 5 '-AGGGGTATCTCTCGAGAAAAGACACGGGTTTGTCTCCAACCT-3 '
Downstream primer: 5 '-GAGTTTTTGTTCTAGACCCGCCGCCGCCGGCGTCG-3 '
(2) extraction of Thermomyces lanuginosus total serum IgE: utilize Trizol reagent to extract.
(3) reverse transcription synthesis cDNA Article 1 chain: take 2 μ g total serum IgE, add 5 × reaction buffer 4 μ L, 10mM dNTP 2
μ L, ribonuclease inhibitor (40-200u/ μ L) 0.5 μ L primer oligodT (1 μ g/ μ L) 1 μ L, reverse transcription (10u/ μ L) 2
μ L, 42 DEG C of reaction 60min, then 85 DEG C of 10min terminate reaction, are diluted to 200 μ L.
(4) PCR reaction (25 μ L): cDNA 2 μ L, 10 × Buffer 2.5 μ L, 10mmol/L dNTP2 μ L, 25mmol/
LMgCl22 μ L, each 2 μ L of upstream and downstream primer, Taq archaeal dna polymerase 0.5 μ L (5U/ μ L), ddH2O 12μL.Reaction condition is 94 DEG C
5min denaturation；94 DEG C of 40s, 56 DEG C of 40s, 72 DEG C of 1min, totally 32 circulations, 72 DEG C extend 10min, 4 DEG C of preservations.
(5) gene clone: take 2 μ L PCR primer and be attached with pMD18-T carrier, it is thus achieved that recombiant plasmid pMD18T/
TLAA9-2 operating procedure is carried out by TAKARA Products description.Then connect product and convert bacillus coli DH 5 alpha, scribbling
Grow overnight on the LB flat board of AMP.Picking white colony, overnight incubation in LB fluid medium.
(6) extraction of plasmid DNA: alkalinity extraction plasmid DNA.
(7) with XholI and XbaI recombiant plasmid pMD18-T/TLAA9-2 product carried out double digestion, simultaneously with XholI and
XbaI double digestion expression plasmid of yeast pPICZ α A, DNA glue reclaims test kit and reclaims purification TLAA9-2 gene and carrier pPICZ α
A segment.Then Ligation in vitro is carried out, it is thus achieved that recombinant plasmid pPICZ alpha A/TLAA9-2, recombinant plasmid transformed e. coli jm109,
Converting picking individual colonies on flat board at the LB containing Amp, extract plasmid DNA, carry out PCR and enzyme action is identified, order-checking confirms restructuring matter
The reading frame of grain is correct.
Embodiment 4. expresses structure and the screening of the Yeast engineering bacterium strain of polysaccharide monooxygenase gene TLAA9-2
(1) linearisation of recombinant expression plasmid: by recombinant expression plasmid pPICZ α A/TLAA9-2 restricted enzyme
Sac I linearisation.
(2) convert: electroporated yeast strain Pichia pastoris GS115 competent yeast cells, method for transformation
See Invitrogen company Pichia sp. workbook.
(3) screening: with sterilizing toothpick picking transformant correspondence dibbling on Zeocin flat board, cultivates 2-4d, picking for 28 DEG C
On Zeocin flat board, the most well-grown transformant is positive transformant.The dibbling respectively of picking positive transformant is won in high concentration
Multicopy transformant is screened on bleomycin flat board.
Embodiment 5: the abduction delivering of Pichia sp. Pichia pastoris GS115 and Activity determination
(1) being inoculated in by positive transformant containing in 25mL BMGY culture medium, 24h cultivated by 30 DEG C of 200rpm/min shaking tables
(OD600 valuesReach 1.8), centrifugal collection thalline, cell is resuspended in the BMMY culture medium of proper volume, to OD600Value is 1.0,
28 DEG C of 200rpm/min continue to cultivate, and every 24h supplements methanol extremely final concentration of 0.5%, and every 24h samples, room temperature 10, and 000g is centrifuged
5min, takes supernatant and carries out SDS-PAGE analysis.
(2) Enzyme assay: enzyme assay uses enzymatic hydrolysate thin layer chromatography, and reaction system and reaction condition are
The enzyme liquid of 100 μ L, adds the 0.5% phosphoric acid expansion cellulose of 200 μ L, the acetate buffer solution (0.2mol/L, pH 5.0) of 100 μ L
50 DEG C of reaction 48h, centrifugal after take supernatant 3ul to chromatoplate, spray nitrite ion, 85 degree of 20min observed results after exhibition layer.Albumen
Assay uses Bradford method.
(3) optimal reactive temperature of restructuring polysaccharide monooxygenase TLAA9-2: the restructuring polysaccharide monooxygenase warp of abduction delivering
Cross the HisTrap FF crude metal-ligand affinity chromatography column purification of GE company with the recombinant protein of histidine mark, obtain
Obtain the purifying protein that electrophoresis is homogeneous, measure its character with the restructuring polysaccharide monooxygenase of purification.Different at identical PH (PH=5)
Temperature conditions under (30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C) polysaccharide monooxygenase reaction 36h, then distinguish
Adding cellulase, react half an hour at 50 DEG C, DNS method measures the amount of the reducing sugar produced.Numerical measuring is averaged for three times,
The highest enzyme activity is defined as 100%.
The embodiment 6:AA9 family polysaccharide monooxygenase TLAA9-2 facilitation to endo cellulase N24 activity
(1) the AA9 family polysaccharide monooxygenase TLAA9-2 facilitation to endo cellulase N24 activity
Using DNS method, polysaccharide monooxygenase, at PH=5, temperature 50 C, under conditions of 200RPM, arranges two groups of polysaccharide lists
Oxygenase and fibrin reaction 36h, one group adds cellulase, and one group is not added with cellulase, reacts 30min, reaction at 50 DEG C
System is: the 0.5% phosphoric acid expansion cellulose of TLAA9-250 μ L, N2450 μ L, 200 μ L, the acetate buffer solution of 100 μ L
(0.2mol/L, pH 5.0), adds 400 μ L DNS reagent, boils 10min, measures absorbance value under 540nm wavelength.With not
The enzymatic activity added in the reaction system of polysaccharide monooxygenase TLAA9-2 is defined as 100%, calculates polysaccharide monooxygenase respectively
TLAA9-2, endo cellulase N24 and the relative activity of mixed enzyme.In triplicate, average.
(2) electron donor (ascorbic acid) impact on polysaccharide monooxygenase TLAA9-2 cellulase activity
Arranging two groups of reaction systems, one group adds ascorbic acid so that it is final concentration of 10mM, another group is not added with Vitamin C
Acid, measures its oxicracking cellulase activity under polysaccharide monooxygenase TLAA9-2 optimum reaction conditions, after reaction 48h, and silicon
Glue thin layer chromatography analysis product, measures activity.
Embodiment 7:AA9 family polysaccharide monooxygenase enzymatic hydrolysate flight mass spectrum is analyzed
AA9 family polysaccharide monooxygenase TLAA9-2 is pacified buffer with the acetic acid that phosphoric acid expansion cellulose is blended in PH=5
In, under the conditions of optimum temperature, react 48h, take supernatant and do flight mass spectrum.AA9 family polysaccharide monooxygenase TLAA9-2 oxidation is split
Solve the polysaccharide produced and have various ways, mainly have C1 and C4 to aoxidize, it is also possible to have C6 to aoxidize.C1 oxidation C6 oxidation C4 water and product
Molecular weight all increase by 16, and C4 oxidized molecules quantitative change turns to 2.What flight mass spectrum was surveyed molecular weight deducts the molecule of corresponding oligosaccharide
It is the molecular weight of product after oxidation with the quantity combining sodium ion that amount deducts the quantity of the carbon atom that oxidation reduces again.
Embodiment 8: express the preservation of the pichia pastoris engineered strain GS-CT-9-2 of TLAA9-2 gene
The depositary institution of the pichia pastoris engineered strain GS-CT-TLAA9-2 of expression TLAA9-2 gene: China Microbiological bacterium
Plant preservation administration committee common micro-organisms center (CGMCC)；Address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, China
Institute of microbiology of academy of science；Preservation date: on April 21st, 2016；Yeast engineering bacterium strain is numbered: CGMCC No.12384；
The Classification And Nomenclature of pichia pastoris engineered strain is: pichia pastoris Pichiapastoris.
Claims (1)
1. express Thermomyces lanuginosus Thermomyces lanuginosusAA9 family polysaccharide monooxygenase gene for one kind
The Yeast engineering bacterium strain of TLAA9-2, it is characterised in that this bacterium is a kind of Pichia sp., thermophilic from thin continuous shape by RT-PCR method
Hyphomycete Thermomyces lanuginosus obtains thermally-stabilised AA9 family glycoside hydrolase gene TLAA9-2, by this gene
It is cloned into Pichia sp. secreted expression carrier pPICZ α A, obtains recombinant expression pPICZ α A/TLAA9-2, convert complete red
Yeast GS115, therefrom filters out the pichia pastoris engineered strain GS-CT-expressing thermally-stabilised polysaccharide monooxygenase gene TLAA9-2
9-2, this glycoside hydrolase TLAA9-2 have obvious facilitation to the activity of endo cellulase, and can be by phosphoric acid
Expansion cellulose oxicracking is cell-oligosaccharide, and flight mass spectrum result identifies that cracking cellulose long-chain primary product is that fiber is few
Sugar.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103384678A (en) * | 2011-02-23 | 2013-11-06 | 诺维信股份有限公司 | Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same |
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| CN103384678A (en) * | 2011-02-23 | 2013-11-06 | 诺维信股份有限公司 | Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same |
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| Title |
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| 张煜等: "里氏木霉纤维二糖水解酶II在毕赤酵母中的高效表达", 《菌物学报》 * |
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