CN106497963A - Cell surface shows the recombinant yeast pichia pastoris of PET catabolic enzymes and hydrophobin altogether - Google Patents
Cell surface shows the recombinant yeast pichia pastoris of PET catabolic enzymes and hydrophobin altogether Download PDFInfo
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Abstract
The invention discloses cell surface shows the recombinant yeast pichia pastoris of PET catabolic enzymes and hydrophobin altogether, it is configured to：(1) anchorin gene is cloned from Pichia sp. genome；Chemosynthesis hydrophobin genes and PETase genes；(2) anchorin gene and PETase genes connect to obtain fusion sequence 1；Anchorin gene and hydrophobin genes connect to obtain fusion sequence 2；(3) fusion sequence 1 is connected on yeast expression vector, obtains fusion expression vector 1；Fusion sequence 2 is connected on yeast expression vector, fusion expression vector 2 is obtained；(4) fusion expression vector 1,2 is proceeded in expressive host Pichia sp., obtains the recombinant yeast pichia pastoris of the present invention.The present invention makes enzyme set be positioned at cell surface.With stability height, easily recovery, Reusability, low cost compared with resolvase, hydrophobin shows the stability of enhancing recombinant yeast pichia pastoris in Pichia sp. altogether with PETase.
Description
Technical field
The invention belongs to the gene engineering technology field of enzyme, it is related to a kind of cell surface and shows PET catabolic enzymes and hydrophobic altogether
The recombinant yeast pichia pastoris and construction method of albumen and application.
Background technology
White pollution is a kind of environmental pollution that global all cities are all present, be primarily referred to as people in daily life with
The destruction that the plastic garbage that meaning is abandoned is caused to environment, including common plastic cup, disposable lunch box, plastic bag etc..These modelings
Material class product is made using macromolecular compounds such as polrvinyl chloride, polystyrene, polypropylene, it is difficult to carry out with oil as raw material
Degraded, along with life in plastics package mostly white, by people using and arbitrarily discarding after can present cut a sorry figure, dirty, random,
The vision of the characteristics of miscellaneous etc. out of order, uncoordinated, interference and stimulation people, allows people to produce and is sick of psychology, so being referred to as white
Pollution.And white pollution thing is greatly PET (polyethylene terephthalate) product.At present, PET in the world
Annual production has reached more than 2,700 ten thousand tons, although PET directly will not work the mischief to environment, but its garbage is to air and microorganism
Repellence very strong, presence cycle in natural environment is 16-48, and the fast development with PET industries, and which is discarded
The quantity of thing is extremely huge, from environmental behaviour and Ecological Effect it is contemplated that PET garbages to have become global environmental pollution organic
Thing.
The method of degraded PET mainly has two big class of chemical degradation and biodegradation at present, in actual applications the chemistry of PET
Decomposition method mainly has Hydrolyze method and alcoholysis method, in addition with ammonolysis, amine solution and pyrolysis etc..But these method typically costs compared with
High, less efficient and not environmentally, it is to avoid not bring secondary pollution to environment after degraded PET；And biodegradation method is relative
Chemical degradation technique is simpler, more energy efficient environmental protection and do not result in secondary pollution.Therefore biodegradation will fundamentally be solved
The best method of waste PET pollution.
Polyethylene terephthalate catabolic enzyme (PETase) is degrading for currently the only one discovery in antibacterial
The enzyme of PET, molecular weight are about 32KD, PET can be degraded to list (2- ethoxys) p-phthalic acid or p-phthalic acid, and which is maximum
Advantage be, compared with enzyme LCC, TfH, the FsC in the funguses that PET can be degraded with other, its enzyme in low-temperature zone (20～40 DEG C)
Work is significantly higher than other enzymes, it means that be easier to reach suitable reaction condition in actual applications, prospects for commercial application compared with
Good.
Hydrophobin HGF1/HFB1 is small-molecular-weight (12KD or so) protein that a kind of filamentous fungis are produced, main
One of function exactly can independently be mounted in any parent/hydrophobic interfaces and form one layer of amphipathic film to change the property at interface,
And the albumen has stronger high temperature resistant, acid and alkali-resistance and the characteristics of compared with low immunogenicity etc..But not yet there is cell surface to open up altogether at present
Show the report of the recombinant bacterium of PET catabolic enzymes and hydrophobin.
List of references：
[1]Shosuke Yoshida,Kazumi Hiraga,Toshihiko Takehana,Ikuo Taniguchi,
Hironao Yamaji,Yasuhito Maeda,Kiyotsuna Toyohara,Kenji Miyamoto,Yoshiharu
Kimura,Kohei Oda.A bacterium that degrades and assimilates poly(ethylene
Terephthalate) [J] .science, 2016 (351)：1196-1199.
[2]Li Xiuhua,Wang Ziying.Research Progress on Degradation of PET[J].
Plastics science and technology, 2011 (4):110-114.
Content of the invention
The purpose of the present invention is to overcome the deficiencies in the prior art, there is provided a kind of cell surface shows PET catabolic enzymes altogether and dredges
The recombinant yeast pichia pastoris of water albumen.
Second object of the present invention is to provide a kind of cell surface and shows that the restructuring of PET catabolic enzymes and hydrophobin is finished altogether
The structure of red yeast.
Third object of the present invention is to provide a kind of cell surface and shows that the restructuring of PET catabolic enzymes and hydrophobin is finished altogether
The application of red yeast.
Technical scheme is summarized as follows：
Cell surface shows the structure of the recombinant yeast pichia pastoris of PET catabolic enzymes and hydrophobin altogether, comprises the steps：
(1) anchorin gene is cloned from Pichia pastoris GS115 genome；Chemosynthesis hydrophobin genes and such as
PETase genes shown in SEQ ID NO.1；
(2) the nucleotide sequence connection of the nucleotide sequence of anchorin gene and SEQ ID NO.1 is obtained using PCR
Fusion sequence 1；The nucleotide sequence of anchorin gene and hydrophobin genes connection are obtained fusion sequence 2 using PCR；
(3) fusion sequence 1 is connected on yeast expression vector, obtains fusion expression vector 1；By fusion sequence 2
It is connected on yeast expression vector, obtains fusion expression vector 2；
(4) fusion expression vector 1 and fusion expression vector 2 are proceeded in expressive host Pichia sp., obtains cell surface
Show the recombinant yeast pichia pastoris of PET catabolic enzymes and hydrophobin altogether.
Anchorin gene is preferred：The GCW61 shown in GCW51, SEQ ID No.4 or SEQ shown in SEQ ID No.3
GCW21 shown in ID No.8.
HFB1 genes shown in HGF1 genes, SEQ ID NO.9 shown in the preferred SEQ ID NO.2 of hydrophobin genes,
The HFB2 genes shown in SC3 genes or SEQ ID NO.11 shown in SEQ ID NO.10.
Yeast expression vector is preferred：ppic9、ppic9k、ppiczaA、ppiczaB、ppiczaC.
The cell surface that above-mentioned construction method builds shows the recombinant yeast pichia pastoris of PET catabolic enzymes and hydrophobin altogether.
Above-mentioned cell surface shows that PET catabolic enzymes and the recombinant yeast pichia pastoris of hydrophobin decompose in whole-cell catalytic altogether
The purposes of PET.
Beneficial effects of the present invention：
Application integrating carrier of the present invention expressed, it is to avoid selection pressure and carrier are not allowed easy to lose.When PETase exists
During cell surface expression, equivalent to enzyme immobilizatio has been done, its set is positioned at cell surface.Enzyme and trip after through immobilization
From enzyme compare have the advantages that stability high, reclaim convenient, easily controllable, can Reusability, with low cost, it is to avoid albumen is pure
That changed is loaded down with trivial details.And carry out common displaying in Pichia sp. using hydrophobin and PETase, due to hydrophobin have amphiphilic,
Spontaneous can be combined with yeast cell surface with hydrophilic surface, the hydrophobic stability for facing outwardly, strengthening recombinant yeast pichia pastoris.
Description of the drawings
Fig. 1 is recombinant yeast pichia pastoris P.pastoris PETase-GCW51-HGF1/P.pastoris PETase-
The Western Blot figures of GCW51-HFB1；Wherein：
Figure 1A is schemed for the Western Blot of recombinant yeast pichia pastoris P.pastoris PETase-GCW51-HGF1；
Figure 1B is schemed for the Western Blot of recombinant yeast pichia pastoris P.pastoris PETase-GCW51-HFB1.
Fig. 2 is recombinant yeast pichia pastoris P.pastoris PETase-GCW51-HGF1/P.pastoris PETase-
The relative enzyme activity of GCW51-HFB1.
Fig. 3 is recombinant yeast pichia pastoris P.pastoris PETase-GCW51-HGF1/P.pastoris PETase-
The Immunofluorescence test result figure of GCW51-HFB1；Wherein：
Immunofluorescence test result figures of the Fig. 3-1 for recombinant yeast pichia pastoris P.pastoris PETase-GCW51-HGF1；
Immunofluorescence test result figures of the Fig. 3-2 for recombinant yeast pichia pastoris P.pastoris PETase-GCW51-HFB1；
Specific embodiment
With reference to specific embodiment, the present invention is further illustrated.
Anchorin GCW21 (NCBI Reference Sequence:XM_002491407)；
Anchorin GCW51 (NCBI Reference Sequence:XP_002493782)；
Anchorin GCW61 (NCBI Reference Sequence:XP_002494322) sequence.
Pichia pastoris GS115 (pichia pastoris GS115) is commercially available.Can also be applied to other Pichia sp.
The present invention.
Ppic9, ppic9k, ppiczaA, ppiczaB, ppiczaC, are commercially available.
Escherichia coli (E.coli) DH5 α, commercially available.
1 cell surface of embodiment shows the recombinant yeast pichia pastoris (P.pastoris of PET catabolic enzymes and hydrophobin altogether
PETase-GCW51-HGF1 preparation) and application：
First, clone from Pichia pastoris GS115 genome GCW51 anchorin genes as shown in SEQ ID No.3,
GCW61 anchorin genes as shown in SEQ ID No.4；Comprise the following steps that：
Pichia pastoris GS115 is inoculated in YPD culture medium, and incubated overnight is collected by centrifugation cell, using carrying gene group reagent
Box is extracted and obtains GS115 yeast genomic DNAs.
According to the GCW51 sequences as shown in SEQ ID No.3, the GCW61 sequences as shown in SEQ ID No.4, using upper
Trip primer 51-F/61-F and downstream primer 51-R/61-R, wherein dashed part are restriction enzyme site, with P.pastoris GS115
Genomic DNA is template, expands GCW51, GCW61 gene.
51-F：GGTGGTGGAGGTAGTGGAGGAGGTGGTAGTGATGACGATGACTCATTAC
51-R：CCGGAATTCCTAGATCAATAGGGCAATGG
61-F：GGTGGTGGAGGTAGTGGAGGAGGTGGTAGTAACAACCTATCAAACGAGAGT
61-R：ATAGTTTAGCGGCCGCTTAAATCAATAGAGCAACACCGGC
PCR product carries out 1% agarose gel electrophoresiies, reclaims genes of interest fragment, obtains GCW51 genes, GCW61 bases
Cause.
2nd, hydrophobin HGF1 gene of the chemosynthesis as shown in SEQ ID NO.2 and as shown in SEQ ID NO.1
PETase genes.
3rd, PCR is utilized by the nucleotide sequence and SEQ ID of the GCW51 anchorin genes as shown in SEQ ID No.3
The nucleotide sequence connection of NO.1 obtains fusion sequence 1；Using PCR by the GCW61 anchorin bases as shown in SEQ ID No.4
The nucleotide sequence connection of the nucleotide sequence of cause and hydrophobin genes HGF1 as shown in SEQ ID NO.2 obtains merging sequence
Row 2, comprise the following steps that：
Hydrophobin genes HGF1 according to chemosynthesis as shown in SEQ ID NO.2 and as shown in SEQ ID NO.1
PETase genes, design forward primer P-F/hg-F and downstream primer P-R/hg-R, and wherein underscore part is enzyme
Enzyme site, with composition sequence as template amplification PETase genes and HGF1 genes.
P-F:CCGCTCGAGAAAAGAGATTACAAGGATGACGACGATAAGCAAACCAATCCTTATGCCCGTG
P-R:ACTACCACCTCCTCCACTACCTCCACCACCGCTACAGTTGGCGGTACGAA
hg-F:CCGGAATTCCAACAGTGCACCACTGG
hg-R:ACTACCACCTCCTCCACTACCTCCACCACCGACGTTAACCGGAACACATC
PCR product carries out 1% agarose gel electrophoresiies, reclaims genes of interest fragment, obtains PETase genes, HGF1 bases
Cause.
Gained PETase and GCW51 is carried out overlap with the linker as shown in SEQ ID No.5 as overlay region
pcr；Gained HGF1 and GCW61 is carried out overlap pcr with the linker as shown in SEQ ID No.5 as overlay region.
PCR product carries out 1% agarose gel electrophoresiies, reclaims genes of interest fragment, obtains as shown in SEQ ID No.6
PETase-linker-GCW51 genes (fusion sequence 1) and the HGF1-linker-GCW61 bases as shown in SEQ ID No.7
Because of (fusion sequence 2).
4th, fusion sequence 1 is connected on yeast expression vector ppic9, obtains fusion expression vector 1；Will fusion
Sequence 2 is connected on yeast expression vector ppiczaA, is obtained fusion expression vector 2, is comprised the following steps that：
PETase-linker-GCW51 genes and ppic9 carriers are used after Xho I and EcoR I double digestions simultaneously, will
To enzyme action PETase-linker-GCW51 genetic fragments be connected on the ppic9 carriers after double digestion；By HGF1-linker-
After GCW61 genes and ppiczaA carriers use EcoR I and Not I double digestions, by the enzyme action HGF1-linker- for obtaining simultaneously
GCW61 genetic fragments are connected on the ppiczaA carriers after double digestion；Above two connection product is transformed into competence respectively
In cell E.coli DH5 α, and picking positive colony, plasmid is extracted after expanding on LB fluid mediums, must be connected with
The fusion expression vector p9P51 (fusion expression vector 1) of PETase-linker-GCW51 and it is connected with HGF1-linker-
The fusion expression vector pAHG61 (fusion expression vector 2) of GCW61 genes.
With Xho I and EcoR I double digestion plasmid p9P51, and verified with 1% agarose gel electrophoresiies；With EcoR I and
Not I double digestion plasmid pAHG61 are simultaneously verified with 1% agarose gel electrophoresiies；
In above-mentioned steps, described genes of interest fragment is connected with the T4DNA for being connected by thermo companies of carrier
Enzyme is attached.
In above-mentioned steps, the colibacillary conversion of described connection product is carried out as follows：
(1) pipe competent cell E.coli DH5 α are taken in slow mechanism dissolved on ice, adds plasmid to be converted or connection to produce
Thing (10 μ L), light mixed rear ice bath 30min；
After (2) 42 DEG C of heat shock 90s, rapid ice bath 5min；
(3) 900 μ L, the LB culture medium of 37 DEG C of temperature baths, 37 DEG C of shaking table cultures 1h are added；
(4) 200 microlitres of LB coated containing ampicillin (100 μ g/mL) are taken flat board is selected (for p9P51 carriers
Speech) and the LB containing zeocin (25 μ g/mL) select on flat board (for pAHG61 carriers), 37 DEG C are inverted culture 12-16h；
(5) picking monoclonal, carries out follow-up plasmid extraction and confirmatory experiment.
In above-mentioned steps, described connection product positive colony is verified, is carried out as follows：
(1) the picking p9P51 transformants from screening flat board, access in 5mL LB fluid mediums (containing 100 μ g/mL ammonia benzyls
Penicillin), 37 DEG C of shaking table cultures 12-16h；The picking pAHG61 transformants from screening flat board, access 5mL less salt LB liquid cultures
In base (sodium chloride concentration is common LB culture medium half) (containing 25 μ g/mLzeocin), 37 DEG C, lucifuge shaking table culture 12-16h
(2) 5mL bacterium solutions are taken, and plasmid are extracted with small amount plasmid extraction kit；
(3) taking 1 μ L plasmid extractions liquid carries out Nanodrop detections, the plasmid concentration of Detection and Extraction；
(4) plasmid of extraction is carried out double digestion, verifies the connection effect of genetic fragment and carrier；
(5) correct for digestion verification recombiant plasmid is sequenced or is carried out other experiments, the only intelligence life of sequencing trust money
Thing company completes.
5th, fusion expression vector 1 and fusion expression vector 2 are proceeded in expressive host Pichia sp., obtains cell surface
Show the recombinant yeast pichia pastoris P.pastoris PETase-GCW51-HGF1 of PET catabolic enzymes and hydrophobin, concrete steps altogether
As follows：
Prepared by electric shocking method conversion P.pastoris GS115 cells competence
(1) from picking single bacterium colony on fresh Pichia pastoris GS115 (His-) flat board, 5mL YPD Tube propagations are forwarded to
In base, 30 DEG C, 250rpm overnight incubations；
0.1-0.5ml overnight cultures are taken from tube culture within (2) second days, be seeded to containing the fresh YPD cultures of 500ml
The 2L shaking flasks of base, overnight growth are about 1.3-1.5 to OD600；
(3) yeast culture is taken from shaking table, 4 DEG C, 1500g is centrifuged 5 minutes to collect cell, afterwards in 100mLYPD-
HEPES-DTT (adds the HEPES buffer solution of 20ml pH 8.0, adds the freshly prepared 1M's of 2.5ml in 100ml YPD
DTT) suspension cell again in culture medium, 30 DEG C are cultivated 15 minutes on shaking table；Cell, ice bath half an hour is removed from shaking table；
(4) cell, ice bath half an hour are removed from shaking table；4 DEG C, 1500g is centrifuged 5 minutes to collect cell, then uses 250ml
The aquesterilisa suspension cell of pre-cooling；；
(5) as above it is centrifuged, then the 1M Sorbitol suspension cells with 20ml pre-coolings；
(6) as above it is centrifuged, with the 1M Sorbitol suspension cells of 1ml pre-coolings, to final volume about 1.5ml；As above it is centrifuged, uses
The cold ultra-pure waters of 500 μ L suspension cell again, standby in ice bath.
Electric shocking method conversion is verified with transformant
(1) the above-mentioned cells of 80 μ L p9P51 carriers linearizing with 5-20ug are taken and pAHG61 carrier DNAs mixes, proceeded to pre-
In cold 0.2cm electricity revolving cups, 5min is placed on ice；
(2) electric revolving cup outer wall water droplet is dried, is placed on electroporation；
(3) yeast parameter " fungi " is set, and " pic " clicks on Pulse；
(4) electric revolving cup is taken out, 600 microlitres of L ice pre-cooling 1mol/L Sorbitol is added immediately, is gently blown and beaten with rifle, gentle
It is transferred in 1.5mL centrifuge tubes, after 30 DEG C of quiescent culture 1h, adds 600 microlitres of YPD culture medium, 30 DEG C, 200rpm shaking tables is distinguished
It is divided into 200-600 μ L aliquots after culture 1h, 3h, is applied on the YPDS flat boards containing 75 mcg/mls zeocin and (arranges in competence
It is added without the matched group of carrier)；After 30 DEG C of lucifuge culture 48h, picking single bacterium colony is screened on MD flat boards, positive bacterium colony bacterium
Strain shows the recombinant yeast pichia pastoris of PET catabolic enzymes and hydrophobin altogether for cell surface, is named as P.pastoris PETase-
GCW51-HGF1；
(5) genomic DNA of transformant (P.pastoris PETase-GCW51-HGF1) is extracted, enters performing PCR checking, than
Whether identical with positive control compared with electrophoretic band position, the primer that PCR is adopted is for AOX upstream and downstream primers, P-51-F/R and hg-
61-F/R, carries out glycerol and protects bacterium to positive bacteria.
P-51-F：CCGCTCGAGAAAAGAGATT
P-51-R：CCGGAATTCCTAGATCAATAGGGCAATGG
hg-61-F：CCGGAATTCCAACAGTGCA
hg-61-R：ATAGTTTAGCGGCCGCTTAAATC
6th, cell surface shows the recombinant yeast pichia pastoris P.pastoris of PET catabolic enzymes and hydrophobin altogether
The application of PETase-GCW51-HGF1, comprises the following steps that：
Western Blot are tested
First, the SDS-PAGE electrophoresis of P.pastoris PETase-GCW51-HGF1
2nd, transferring film
(1) turn the nitrocellulose filter that a film need to prepare the filter paper and 1 7.3～8.6cm of 6 7.0～8.3cm.Cut
Glove must be worn when filter paper and film.
(2) clip of transferring film, two pieces of foam-rubber cushions, filter paper and films are put in the enamel tray added with transfer liquid.
(3) opening clip makes black one side holding level.In one foam-rubber cushion of pad above, three metafiltration of pad on mat
Paper.
(4) glass plate sled to be fallen ability peelable glue first, action is light when sled, be on two sides gently repeatedly
Sled.Glass plate just starts to loosen sled a little while, until sled goes glass plate.After removing little glass plate, concentration glue is gently scraped off (concentration
Glue affects operation), separation gel will be avoided to scratch.Carefully peel separation gel to be placed on filter paper, which is made with filter paper pair with hand adjustment is whole
Together, bubble is gently rolled.By membrane cover on glue, completely whole glue (can not move again after under membrane cover) bubble removing to be covered.On film
3 filter paper of lid simultaneously remove bubble.Another foam-rubber cushion is finally covered, clip is closed.Whole operation is carried out in transfer liquid.
(5) clip is put in transfer groove groove, the black flour of the black flour of folder to be made to groove, the red face of the flour of folder to groove.Electricity
During transfer can heat production, put an ice cube to lower the temperature in groove.General 100V shifts 1h.
3rd, immunoreation
(1) by film transfer to the box containing confining liquid, shake on decolorization swinging table under room temperature after closing 1h, with PBST in room
Wash three times on the lower decolorization swinging table of temperature, each 10min.
(2) an anti-PBST is diluted to debita spissitudo, after 4 DEG C of night incubation, with PBST at room temperature decolorization swinging table
Wash three times, each 10min.
(3) two anti-PBST are diluted to debita spissitudo, under room temperature after incubation 1h, with PBST at room temperature decolorization swinging table
Wash three times, each 10min.
4th, chemiluminescence, development are fixed
The result explanation PETase and HGF1 of Figure 1A is in recombinant yeast pichia pastoris P.pastoris PETase-GCW51-HGF1
Successful expression.
HPLC surveys enzyme activity experiment
A series of used Buffer PBSs of configuration enzyme activity reaction：8g NaCl are dissolved with 800ml distilled waters,
0.2g KCl, 1.44g Na2HPO4,0.24g KH2PO4, adjust the pH value of solution to 7.4 with HCl, add water to 1L, and sucking filtration is removed
Bacterium.
Glycine/NaOH Buffer：0.2M Glycine solution 50ml；0.2M NaOH solutions 8.8ml
Constant volume 200ml is mixed, pH to 9.0 is adjusted mutually
Monophosphate monophosphate sodium dihydrogen pH of buffer=2.50：0.02M H3PO4 1L；0.02M NaH2PO4 1L,
First Jia 200 in 1L beakers～300ml phosphoric acid, then convert sodium dihydrogen phosphate inward, until pH=2.50
Methanol：One bottle of methanol is 500ml, directly by the big bottle of two bottles of sucking filtration a to 1L, organic filter paper.
Terminate liquid：90mL PBS+10mL DMSO
1. sample preparation (arrange 1 experimental group and 1 matched group).
A. take appropriate P.pastoris PETase-GCW51-HGF1 bacterium solutions to manage in EP, 3000g, 4 DEG C of centrifugation 5min go
Clearly.
B. appropriate glycine sodium hydrate buffer solution (pH=9.0) resuspended bacterium solution, centrifugation is added to remove supernatant.
C. it is repeated once.
D. arrange difference P.pastoris PETase-GCW51-HGF1 bacterium amount gradients (2ml, 1ml, 0.5ml, 0.1ml,
0.01ml).
E. 100 microlitres of glycine sodium hydrate buffer solution resuspended bacterium solutions are added.
2. experimental group adds the good PET film of advanced processing, matched group to be not added with PET film
3. 40 DEG C in constant-temperature table, 18h, 220rpm incubation reaction.
4. bacterium is frozen：Remaining thalline PBS is washed twice, resuspended with 20% glycerol, frozen.
5., after reaction terminates, 4 DEG C of 5000g are centrifuged 10min
6. supernatant is taken, terminate liquid is added
7.85 DEG C of inactivation 10min
8.12000rpm, is centrifuged 10min, takes 100 microlitres in new EP pipes.
9. by the phosphoric acid/phosphate sodium dihydrogen buffer solution (pH=2.5) for preparing and methanol ultrasound 30min.
10. HPLC, 100 microlitres of loading, the good order of record are crossed.
The result explanation recombinant yeast pichia pastoris P.pastoris PETase-GCW51-HGF1 of Fig. 2 can degrade PET, and wilder
Raw type PETase enzyme activity is obviously improved.
Immunofluorescence test
(1) 200uLP.pastoris PETase-GCW51-HGF1 induction broths are taken in 4 DEG C, and 12000rpm is centrifuged
1min, retains thalline.
(2) PBS recombination yeast P.pastoris PETase-GCW51-HGF1 cells are carried out resuspended, in 4 DEG C,
12000rpm, is centrifuged 1min, abandons supernatant and stay thalline.
(3) using the resuspended thalline of PBS of the 3mg/mLBSA of 200uL, 1h is reacted at room temperature, period should make bacterium
Body is mixed up and down, and in 4 DEG C, 12000rpm is centrifuged 1min, retains thalline.
(4) the resuspended thalline of the PBS of 3mg/mLBSA of the utilization containing the anti-200uL of 1uL mono-, is overnight processed in 4 DEG C,
Period should be such that thalline mixes up and down.Next day, 12000rpm was centrifuged 1min, retains thalline in 4 DEG C.
(5) PBS recombination yeast P.pastoris PETase-GCW51-HGF1 cells are carried out resuspended, at room temperature
Reason 10min, in 4 DEG C, 12000rpm is centrifuged 1min, abandons supernatant and stay thalline.
(6) step (5) is repeated twice.
(7) using the resuspended thalline of the PBS for containing the anti-200uL of 1uL bis-, lucifuge room temperature treatment 1h, period should make bacterium
Body is mixed up and down, and in 4 DEG C, 12000rpm is centrifuged 1min, retains thalline.
(8) PBS carries out resuspended, lucifuge room to recombination yeast P.pastoris PETase-GCW51-HGF1 cells
Temperature processes 5min, and period should be such that thalline mixes up and down, and in 4 DEG C, 12000rpm is centrifuged 1min, abandons supernatant and stay thalline.
(9) step (8) is repeated once.
(10) PBS with 200uL is resuspended, takes 10uL every time in clean static load slide film-making.
The result explanation PETase genes successful presentation of Fig. 3-1 is in recombinant yeast pichia pastoris P.pastoris PETase-
GCW51-HGF1 surfaces.
2 cell surface of embodiment shows the recombinant yeast pichia pastoris P.pastoris of PET catabolic enzymes and hydrophobin altogether
The preparation and application of PETase-GCW51-HFB1, comprises the steps：
First, with 1 step one of embodiment.
2nd, hydrophobin genes HFB1 of the chemosynthesis as shown in SEQ ID NO.9 and as shown in SEQ ID NO.1
PETase genes.
3rd, PCR is utilized by the nucleotide sequence and SEQ ID of the GCW51 anchorin genes as shown in SEQ ID No.3
The nucleotide sequence connection of NO.1 obtains fusion sequence 1；Using PCR by the GCW61 anchorin bases as shown in SEQ ID No.4
The nucleotide sequence connection of the nucleotide sequence of cause and hydrophobin genes HFB1 as shown in SEQ ID NO.9 obtains merging sequence
Row 2, comprise the following steps that：
Hydrophobin genes HFB1 according to chemosynthesis as shown in SEQ ID NO.9 and as shown in SEQ ID NO.1
PETase genes, design forward primer P-F/hf-F and downstream primer P-R/hf-R, and wherein underscore part is restriction enzyme site, with
Composition sequence is template amplification PETase genes and HFB1 genes.
P-F:CCGCTCGAGAAAAGAGATTACAAGGATGACGACGATAAGCAAACCAATCCTTATGCCCGTG
P-R:ACTACCACCTCCTCCACTACCTCCACCACCGCTACAGTTGGCGGTACGAA
hf-F:CCGGAATTCAGCAACGGCAACGGCAATGT
hf-R:ACTACCACCTCCTCCACTACCTCCACCACCAGCACCGACGGCGGTCT
PCR product carries out 1% agarose gel electrophoresiies, reclaims genes of interest fragment, obtains PETase genes, HFB1 bases
Cause.
Gained PETase and GCW51 is carried out overlap with the linker as shown in SEQ ID No.5 as overlay region
pcr；Gained HFB1 and GCW61 is carried out overlap pcr with the linker as shown in SEQ ID No.5 as overlay region
PCR product carries out 1% agarose gel electrophoresiies, reclaims genes of interest fragment, obtains as shown in SEQ ID No.6
PETase-linker-GCW51 genes (fusion sequence 1) and the HFB1-linker-GCW61 bases as shown in SEQ ID No.12
Because of (fusion sequence 2).
4th, fusion sequence 1 is connected on yeast expression vector ppic9, obtains fusion expression vector 1；Will fusion
Sequence 2 is connected on yeast expression vector ppiczaA, obtains fusion expression vector 2, and concrete steps are shown in 1 step of embodiment
Four.
5th, fusion expression vector 1 and fusion expression vector 2 are proceeded in expressive host Pichia sp., obtains cell surface
Show the recombinant yeast pichia pastoris P.pastoris PETase-GCW51-HFB1 of PET catabolic enzymes and hydrophobin, concrete steps altogether
As follows：
Prepared by electric shocking method conversion P.pastoris GS115 cells competence
(1) from picking single bacterium colony on fresh Pichia pastoris GS115 (His-) flat board, 5mL YPD Tube propagations are forwarded to
In base, 30 DEG C, 250rpm overnight incubations；
0.1-0.5ml overnight cultures are taken from tube culture within (2) second days, be seeded to containing the fresh YPD cultures of 500ml
The 2L shaking flasks of base, overnight growth are about 1.3-1.5 to OD600；
(3) yeast culture is taken from shaking table, 4 DEG C, 1500g is centrifuged 5 minutes to collect cell, afterwards in 100mLYPD-
HEPES-DTT (adds the HEPES buffer solution of 20ml pH 8.0, adds the freshly prepared 1M's of 2.5ml in 100ml YPD
DTT) suspension cell again in culture medium, 30 DEG C are cultivated 15 minutes on shaking table；Cell, ice bath half an hour is removed from shaking table；
(4) cell, ice bath half an hour are removed from shaking table；4 DEG C, 1500g is centrifuged 5 minutes to collect cell, then uses 250ml
The aquesterilisa suspension cell of pre-cooling；；
(5) as above it is centrifuged, then the 1M Sorbitol suspension cells with 20ml pre-coolings；
(6) as above it is centrifuged, with the 1M Sorbitol suspension cells of 1ml pre-coolings, to final volume about 1.5ml；As above it is centrifuged, uses
The cold ultra-pure waters of 500 μ L suspension cell again, standby in ice bath.
Electric shocking method conversion is verified with transformant
(1) the above-mentioned cells of 80 μ L p9P51 carriers linearizing with 5-20ug are taken and pAHF61 carrier DNAs mixes, proceeded to pre-
In cold 0.2cm electricity revolving cups, 5min is placed on ice；
(2) electric revolving cup outer wall water droplet is dried, is placed on electroporation；
(3) yeast parameter " fungi " is set, and " pic " clicks on Pulse；
(4) electric revolving cup is taken out, 600 microlitres of L ice pre-cooling 1mol/L Sorbitol is added immediately, is gently blown and beaten with rifle, gentle
It is transferred in 1.5mL centrifuge tubes, after 30 DEG C of quiescent culture 1h, adds 600 microlitres of YPD culture medium, 30 DEG C, 200rpm shaking tables is distinguished
It is divided into 200-600 μ L aliquots after culture 1h, 3h, is applied on the YPDS flat boards containing 75 mcg/mls zeocin and (arranges in competence
It is added without the matched group of carrier)；After 30 DEG C of lucifuge culture 48h, picking single bacterium colony is screened on MD flat boards, positive bacterium colony bacterium
Strain cell surface shows the recombinant yeast pichia pastoris of PET catabolic enzymes and hydrophobin altogether, is named as P.pastoris PETase-
GCW51-HFB1；
(5) genomic DNA of transformant (P.pastoris PETase-GCW51-HFB1) is extracted, enters performing PCR checking, than
Whether identical with positive control compared with electrophoretic band position, the primer that PCR is adopted is for AOX upstream and downstream primers, P-51-F/R and hf-
61-F/R, carries out glycerol and protects bacterium to positive bacteria.
P-51-F：CCGCTCGAGAAAAGAGATT
P-51-R：CCGGAATTCCTAGATCAATAGGGCAATGG
hf-61-F：CCGGAATTCAGCAACGGCAAC
hf-61-R：ATAGTTTAGCGGCCGCTTAAATC
6th, cell surface shows the recombinant yeast pichia pastoris P.pastoris PETase- of PET catabolic enzymes and hydrophobin altogether
The application of GCW51-HFB1, concrete steps are with 1 step 6 of embodiment.
The result explanation PETase and HFB1 of Figure 1B is in recombinant yeast pichia pastoris P.pastoris PETase-GCW51-HFB1
Middle successful expression.
The result explanation recombinant yeast pichia pastoris P.pastoris PETase-GCW51-HFB1 of Fig. 2 can degrade PET, and wilder
Raw type PETase enzyme activity is obviously improved.
The result explanation PETase genes successful presentation of Fig. 3-2 is in recombinant yeast pichia pastoris P.pastoris PETase-
GCW51-HFB1 surfaces.
With SC3 hydrophobins (SEQ ID NO.10) or HFB2 hydrophobins (SEQ ID NO.11), the present embodiment is substituted
Hydrophobin genes HFB1, other are prepared cell surface and show PET catabolic enzymes and hydrophobin altogether with reference to the present embodiment
Recombinant yeast pichia pastoris, are named as P.pastoris PETase-GCW51-SC3；Or cell surface shows PET catabolic enzymes altogether and dredges
The recombinant yeast pichia pastoris of water albumen, are named as P.pastoris PETase-GCW51-HFB2.
3 cell surface of embodiment shows the recombinant yeast pichia pastoris P.pastoris of PET catabolic enzymes and hydrophobin altogether
The preparation and application of PETase-GCW21-HGF1, comprises the steps：
First, clone from Pichia pastoris GS115 genome GCW21 anchorin genes as shown in SEQ ID No.8,
GCW61 anchorin genes as shown in SEQ ID No.4；Comprise the following steps that：
Pichia pastoris GS115 is inoculated in YPD culture medium, and incubated overnight is collected by centrifugation cell, using carrying gene group reagent
Box is extracted and obtains GS115 yeast genomic DNAs.
According to the GCW21 sequences as shown in SEQ ID No.8, the GCW61 sequences as shown in SEQ ID No.4, using upper
Trip primer 2 1-F/61-F and downstream primer 21-R/61-R, wherein dashed part are restriction enzyme site, with P.pastoris GS115
Genomic DNA is template, expands GCW21, GCW61 gene.
21-F:GGTGGTGGAGGTAGTGGAGGAGGTGGTAGTGACCAGCGAATCTCTGTCAC
21-R:CCGGAATTCTTATAACAAAGCAGCGGCGGCAATT
61-F：GGTGGTGGAGGTAGTGGAGGAGGTGGTAGTAACAACCTATCAAACGAGAGT
61-R：ATAGTTTAGCGGCCGCTTAAATCAATAGAGCAACACCGGC
PCR product carries out 1% agarose gel electrophoresiies, reclaims genes of interest fragment, obtains GCW21 genes, GCW61 bases
Cause.
2nd, hydrophobin HGF1 gene of the chemosynthesis as shown in SEQ ID NO.2 and as shown in SEQ ID NO.1
PETase genes.
3rd, PCR is utilized by the nucleotide sequence and SEQ ID of the GCW21 anchorin genes as shown in SEQ ID No.8
The nucleotide sequence connection of NO.1 obtains fusion sequence 1；Using PCR by the GCW61 anchorin bases as shown in SEQ ID No.4
The nucleotide sequence connection of the nucleotide sequence of cause and hydrophobin genes HGF1 as shown in SEQ ID NO.2 obtains merging sequence
Row 2, comprise the following steps that：
Hydrophobin genes HGF1 according to chemosynthesis as shown in SEQ ID NO.2 and as shown in SEQ ID NO.1
PETase genes, design forward primer P-F/hg-F and downstream primer P-R/hg-R, with composition sequence as template amplification PETase
Gene and HGF1 genes.
P-F:CCGCTCGAGAAAAGAGATTACAAGGATGACGACGATAAGCAAACCAATCCTTATGCCCGTG
P-R:ACTACCACCTCCTCCACTACCTCCACCACCGCTACAGTTGGCGGTACGAA
hg-F:CCGGAATTCCAACAGTGCACCACTGG
hg-R:ACTACCACCTCCTCCACTACCTCCACCACCGACGTTAACCGGAACACATC
PCR product carries out 1% agarose gel electrophoresiies, reclaims genes of interest fragment, obtains PETase genes, HGF1 bases
Cause.
Gained PETase and GCW21 is carried out overlap with the linker as shown in SEQ ID No.5 as overlay region
pcr；Gained HGF1 and GCW61 is carried out overlap pcr with the linker as shown in SEQ ID No.5 as overlay region
PCR product carries out 1% agarose gel electrophoresiies, reclaims genes of interest fragment, obtains such as SEQ ID No.28 institutes
The PETase-linker-GCW21 genes (fusion sequence 1) for showing and the HGF1-linker-GCW61 as shown in SEQ ID No.7
Gene (fusion sequence 2).
4th, fusion sequence 1 is connected on yeast expression vector ppic9, obtains fusion expression vector 1；Will fusion
Sequence 2 is connected on yeast expression vector ppiczaA, obtains fusion expression vector 2, and concrete steps are with 1 step of embodiment
Four.
5th, fusion expression vector 1 and fusion expression vector 2 are proceeded in expressive host Pichia sp., obtains cell surface
Show the recombinant yeast pichia pastoris P.pastoris PETase-GCW21-HGF1 of PET catabolic enzymes and hydrophobin, concrete steps altogether
As follows.
Prepared by electric shocking method conversion P.pastoris GS115 cells competence
(1) from picking single bacterium colony on fresh Pichia pastoris GS115 (His-) flat board, 5mL YPD Tube propagations are forwarded to
In base, 30 DEG C, 250rpm overnight incubations；
0.1-0.5ml overnight cultures are taken from tube culture within (2) second days, be seeded to containing the fresh YPD cultures of 500ml
The 2L shaking flasks of base, overnight growth are about 1.3-1.5 to OD600；
(3) yeast culture is taken from shaking table, 4 DEG C, 1500g is centrifuged 5 minutes to collect cell, afterwards in 100mLYPD-
HEPES-DTT (adds the HEPES buffer solution of 20ml pH 8.0, adds the freshly prepared 1M's of 2.5ml in 100ml YPD
DTT) suspension cell again in culture medium, 30 DEG C are cultivated 15 minutes on shaking table；Cell, ice bath half an hour is removed from shaking table；
(4) cell, ice bath half an hour are removed from shaking table；4 DEG C, 1500g is centrifuged 5 minutes to collect cell, then uses 250ml
The aquesterilisa suspension cell of pre-cooling；；
(5) as above it is centrifuged, then the 1M Sorbitol suspension cells with 20ml pre-coolings；
(6) as above it is centrifuged, with the 1M Sorbitol suspension cells of 1ml pre-coolings, to final volume about 1.5ml；As above it is centrifuged, uses
The cold ultra-pure waters of 500 μ L suspension cell again, standby in ice bath.
Electric shocking method conversion is verified with transformant
(1) the above-mentioned cells of 80 μ L p9P21 carriers linearizing with 5-20ug are taken and pAHG61 carrier DNAs mixes, proceeded to pre-
In cold 0.2cm electricity revolving cups, 5min is placed on ice；
(2) electric revolving cup outer wall water droplet is dried, is placed on electroporation；
(3) yeast parameter " fungi " is set, and " pic " clicks on Pulse；
(4) electric revolving cup is taken out, 600 microlitres of L ice pre-cooling 1mol/L Sorbitol is added immediately, is gently blown and beaten with rifle, gentle
It is transferred in 1.5mL centrifuge tubes, after 30 DEG C of quiescent culture 1h, adds 600 microlitres of YPD culture medium, 30 DEG C, 200rpm shaking tables is distinguished
It is divided into 200-600 μ L aliquots after culture 1h, 3h, is applied on the YPDS flat boards containing 75 mcg/mls zeocin and (arranges in competence
It is added without the matched group of carrier)；After 30 DEG C of lucifuge culture 48h, picking single bacterium colony is screened on MD flat boards, positive bacterium colony bacterium
Strain cell surface shows the recombinant yeast pichia pastoris of PET catabolic enzymes and hydrophobin altogether, is named as P.pastoris PETase-
GCW21-HGF1；
(5) genomic DNA of transformant (P.pastoris PETase-GCW21-HGF1) is extracted, enters performing PCR checking, than
Whether identical with positive control compared with electrophoretic band position, the primer that PCR is adopted is for AOX upstream and downstream primers, P-21-F/R and hg-
61-F/R, carries out glycerol and protects bacterium to positive bacteria.
P-21-F：CCGCTCGAGAAAAGAGATT
P-21-R：CCGGAATTCTTATAACAAAGCAGCG
hg-61-F：CCGGAATTCCAACAGTGCA
hg-61-R：ATAGTTTAGCGGCCGCTTAAATC
Six .HPLC survey enzyme activity experiment
Operating procedure surveys enzyme activity experiment with reference to the HPLC of embodiment 1, and experimental result illustrates recombinant yeast pichia pastoris
P.pastoris PETase-GCW21-HGF1 can degrade PET, and be obviously improved compared with wild type PETase enzyme activity.
4 cell surface of embodiment shows the recombinant yeast pichia pastoris P.pastoris of PET catabolic enzymes and hydrophobin altogether
The preparation and application of PETase-GCW21-HFB1, comprises the steps：
First, with 3 step one of embodiment；
2nd, hydrophobin genes HFB1 of the chemosynthesis as shown in SEQ ID NO.9 and as shown in SEQ ID NO.1
PETase genes.
3rd, PCR is utilized by the nucleotide sequence and SEQ ID of the GCW21 anchorin genes as shown in SEQ ID No.8
The nucleotide sequence connection of NO.1 obtains fusion sequence 1；Using PCR by the GCW61 anchorin bases as shown in SEQ ID No.4
The nucleotide sequence connection of the nucleotide sequence of cause and hydrophobin genes HFB1 as shown in SEQ ID NO.9 obtains merging sequence
Row 2, comprise the following steps that：
Hydrophobin genes HFB1 according to chemosynthesis as shown in SEQ ID NO.9 and as shown in SEQ ID NO.1
PETase genes, design forward primer P-F/hf-F and downstream primer P-R/hf-R, with composition sequence as template amplification PETase
Gene and HGF1 genes.
P-F:CCGCTCGAGAAAAGAGATTACAAGGATGACGACGATAAGCAAACCAATCCTTATGCCCGTG
P-R:ACTACCACCTCCTCCACTACCTCCACCACCGCTACAGTTGGCGGTACGAA
hf-F:CCGGAATTCAGCAACGGCAACGGCAATGT
hf-R:ACTACCACCTCCTCCACTACCTCCACCACCAGCACCGACGGCGGTCT
PCR product carries out 1% agarose gel electrophoresiies, reclaims genes of interest fragment, obtains PETase genes, HFB1 bases
Cause.
Gained PETase and GCW21 is carried out overlap with the linker as shown in SEQ ID No.5 as overlay region
pcr；Gained HFB1 and GCW61 is carried out overlap pcr with the linker as shown in SEQ ID No.5 as overlay region
PCR product carries out 1% agarose gel electrophoresiies, reclaims genes of interest fragment, obtains such as SEQ ID No.28 institutes
The PETase-linker-GCW21 genes (fusion sequence 1) for showing and the HFB1-linker-GCW61 as shown in SEQ ID No.12
Gene (fusion sequence 2).
4th, fusion sequence 1 is connected on yeast expression vector ppic9, obtains fusion expression vector 1；Will fusion
Sequence 2 is connected on yeast expression vector ppiczaA, obtains fusion expression vector 2, and concrete steps are with 1 step of embodiment
Four.
5th, fusion expression vector 1 and fusion expression vector 2 are proceeded in expressive host Pichia sp., obtains cell surface
Show the recombinant yeast pichia pastoris P.pastoris PETase-GCW21-HFB1 of PET catabolic enzymes and hydrophobin, concrete steps altogether
As follows.
Prepared by electric shocking method conversion P.pastoris GS115 cells competence
(1) from picking single bacterium colony on fresh Pichia pastoris GS115 (His-) flat board, 5mL YPD Tube propagations are forwarded to
In base, 30 DEG C, 250rpm overnight incubations；
0.1-0.5ml overnight cultures are taken from tube culture within (2) second days, be seeded to containing the fresh YPD cultures of 500ml
The 2L shaking flasks of base, overnight growth are about 1.3-1.5 to OD600；
(3) yeast culture is taken from shaking table, 4 DEG C, 1500g is centrifuged 5 minutes to collect cell, afterwards in 100mLYPD-
HEPES-DTT (adds the HEPES buffer solution of 20ml pH 8.0, adds the freshly prepared 1M's of 2.5ml in 100ml YPD
DTT) suspension cell again in culture medium, 30 DEG C are cultivated 15 minutes on shaking table；Cell, ice bath half an hour is removed from shaking table；
(4) cell, ice bath half an hour are removed from shaking table；4 DEG C, 1500g is centrifuged 5 minutes to collect cell, then uses 250ml
The aquesterilisa suspension cell of pre-cooling；；
(5) as above it is centrifuged, then the 1M Sorbitol suspension cells with 20ml pre-coolings；
(6) as above it is centrifuged, with the 1M Sorbitol suspension cells of 1ml pre-coolings, to final volume about 1.5ml；As above it is centrifuged, uses
The cold ultra-pure waters of 500 μ L suspension cell again, standby in ice bath.
Electric shocking method conversion is verified with transformant
(1) the above-mentioned cells of 80 μ L p9P21 carriers linearizing with 5-20ug are taken and pAHF61 carrier DNAs mixes, proceeded to pre-
In cold 0.2cm electricity revolving cups, 5min is placed on ice；
(2) electric revolving cup outer wall water droplet is dried, is placed on electroporation；
(3) yeast parameter " fungi " is set, and " pic " clicks on Pulse；
(4) electric revolving cup is taken out, 600 microlitres of L ice pre-cooling 1mol/L Sorbitol is added immediately, is gently blown and beaten with rifle, gentle
It is transferred in 1.5mL centrifuge tubes, after 30 DEG C of quiescent culture 1h, adds 600 microlitres of YPD culture medium, 30 DEG C, 200rpm shaking tables is distinguished
It is divided into 200-600 μ L aliquots after culture 1h, 3h, is applied on the YPDS flat boards containing 75 mcg/mls zeocin and (arranges in competence
It is added without the matched group of carrier)；After 30 DEG C of lucifuge culture 48h, picking single bacterium colony is screened on MD flat boards, positive bacterium colony bacterium
Strain cell surface shows the recombinant yeast pichia pastoris of PET catabolic enzymes and hydrophobin altogether, is named as P.pastoris PETase-
GCW21-HFB1；
(5) genomic DNA of transformant (P.pastoris PETase-GCW21-HFB1) is extracted, enters performing PCR checking, than
Whether identical with positive control compared with electrophoretic band position, the primer that PCR is adopted is for AOX upstream and downstream primers, P-21-F/R and hf-
61-F/R, carries out glycerol and protects bacterium to positive bacteria.
P-21-F：CCGCTCGAGAAAAGAGATT
P-21-R：CCGGAATTCTTATAACAAAGCAGCG
hf-61-F：CCGGAATTCAGCAACGGCAAC
hf-61-R：ATAGTTTAGCGGCCGCTTAAATC
Six .HPLC survey enzyme activity experiment
Operating procedure surveys enzyme activity experiment with reference to the HPLC of embodiment 1, and experimental result illustrates recombinant yeast pichia pastoris
P.pastoris PETase-GCW21-HFB1 can degrade PET, and be obviously improved compared with wild type PETase enzyme activity.
5 cell surface of embodiment shows the recombinant yeast pichia pastoris P.pastoris of PET catabolic enzymes and hydrophobin altogether
The preparation and application of PETase-GCW61-HGF1, comprises the steps：
First, GCW61 anchorin gene as shown in SEQ ID No.4 is cloned from Pichia pastoris GS115 genome；
Comprise the following steps that：
Pichia pastoris GS115 is inoculated in YPD culture medium, and incubated overnight is collected by centrifugation cell, using carrying gene group reagent
Box is extracted and obtains GS115 yeast genomic DNAs.
According to the GCW61 sequences as shown in SEQ ID No.4, using forward primer 61-F '/61-F and downstream primer 61-
R '/61-R, wherein dashed part are restriction enzyme site, with P.pastoris GS115 genomic DNAs template, expand GCW61 bases
Cause.
61-F’：GGTGGTGGAGGTAGTGGAGGAGGTGGTAGTAACAACCTATCAAACGAGAG
61-R’：CCGGAATTCTTAAATCAATAGAGCAACACCG
61-F：GGTGGTGGAGGTAGTGGAGGAGGTGGTAGTAACAACCTATCAAACGAGAGT
61-R：ATAGTTTAGCGGCCGCTTAAATCAATAGAGCAACACCGGC
PCR product carries out 1% agarose gel electrophoresiies, reclaims genes of interest fragment, obtains GCW61 genes.
2nd, hydrophobin HGF1 gene of the chemosynthesis as shown in SEQ ID NO.2 and as shown in SEQ ID NO.1
PETase genes.
3rd, PCR is utilized by the nucleotide sequence and SEQ ID of the GCW61 anchorin genes as shown in SEQ ID No.4
The nucleotide sequence connection of NO.1 obtains fusion sequence 1；Using PCR by the GCW61 anchorin bases as shown in SEQ ID No.4
The nucleotide sequence connection of the nucleotide sequence of cause and hydrophobin genes HGF1 as shown in SEQ ID NO.2 obtains merging sequence
Row 2, comprise the following steps that：
Hydrophobin genes HGF1 according to chemosynthesis as shown in SEQ ID NO.2 and as shown in SEQ ID NO.1
PETase genes, design forward primer P-F/hg-F and downstream primer P-R/hg-R, with composition sequence as template amplification PETase
Gene and HGF1 genes.
P-F:CCGCTCGAGAAAAGAGATTACAAGGATGACGACGATAAGCAAACCAATCCTTATGCCCGTG
P-R:ACTACCACCTCCTCCACTACCTCCACCACCGCTACAGTTGGCGGTACGAA
hg-F:CCGGAATTCCAACAGTGCACCACTGG
hg-R:ACTACCACCTCCTCCACTACCTCCACCACCGACGTTAACCGGAACACATC
PCR product carries out 1% agarose gel electrophoresiies, reclaims genes of interest fragment, obtains PETase genes, HGF1 bases
Cause.
Gained PETase and GCW61 is carried out overlap with the linker as shown in SEQ ID No.5 as overlay region
pcr；Gained HGF1 and GCW61 is carried out overlap pcr with the linker as shown in SEQ ID No.5 as overlay region
PCR product carries out 1% agarose gel electrophoresiies, reclaims genes of interest fragment, obtains such as SEQ ID No.29 institutes
The PETase-linker-GCW61 genes (fusion sequence 1) for showing and the HGF1-linker-GCW61 as shown in SEQ ID No.7
Gene (fusion sequence 2).
4th, fusion sequence 1 is connected on yeast expression vector ppic9, obtains fusion expression vector 1；Will fusion
Sequence 2 is connected on yeast expression vector ppiczaA, obtains fusion expression vector 2, and concrete steps are with 1 step of embodiment
Four.
5th, fusion expression vector 1 and fusion expression vector 2 are proceeded in expressive host Pichia sp., obtains cell surface
Show the recombinant yeast pichia pastoris P.pastoris PETase-GCW61-HGF1 of PET catabolic enzymes and hydrophobin, concrete steps altogether
As follows.
Prepared by electric shocking method conversion P.pastoris GS115 cells competence
(1) from picking single bacterium colony on fresh Pichia pastoris GS115 (His-) flat board, 5mL YPD Tube propagations are forwarded to
In base, 30 DEG C, 250rpm overnight incubations；
0.1-0.5ml overnight cultures are taken from tube culture within (2) second days, be seeded to containing the fresh YPD cultures of 500ml
The 2L shaking flasks of base, overnight growth are about 1.3-1.5 to OD600；
(3) yeast culture is taken from shaking table, 4 DEG C, 1500g is centrifuged 5 minutes to collect cell, afterwards in 100mLYPD-
HEPES-DTT (adds the HEPES buffer solution of 20ml pH 8.0, adds the freshly prepared 1M's of 2.5ml in 100ml YPD
DTT) suspension cell again in culture medium, 30 DEG C are cultivated 15 minutes on shaking table；Cell, ice bath half an hour is removed from shaking table；
(4) cell, ice bath half an hour are removed from shaking table；4 DEG C, 1500g is centrifuged 5 minutes to collect cell, then uses 250ml
The aquesterilisa suspension cell of pre-cooling；；
(5) as above it is centrifuged, then the 1M Sorbitol suspension cells with 20ml pre-coolings；
(6) as above it is centrifuged, with the 1M Sorbitol suspension cells of 1ml pre-coolings, to final volume about 1.5ml；As above it is centrifuged, uses
The cold ultra-pure waters of 500 μ L suspension cell again, standby in ice bath.
Electric shocking method conversion is verified with transformant
(1) the above-mentioned cells of 80 μ L p9P61 carriers linearizing with 5-20ug are taken and pAHG61 carrier DNAs mixes, proceeded to pre-
In cold 0.2cm electricity revolving cups, 5min is placed on ice；
(2) electric revolving cup outer wall water droplet is dried, is placed on electroporation；
(3) yeast parameter " fungi " is set, and " pic " clicks on Pulse；
(4) electric revolving cup is taken out, 600 microlitres of L ice pre-cooling 1mol/L Sorbitol is added immediately, is gently blown and beaten with rifle, gentle
It is transferred in 1.5mL centrifuge tubes, after 30 DEG C of quiescent culture 1h, adds 600 microlitres of YPD culture medium, 30 DEG C, 200rpm shaking tables is distinguished
It is divided into 200-600 μ L aliquots after culture 1h, 3h, is applied on the YPDS flat boards containing 75 mcg/mls zeocin and (arranges in competence
It is added without the matched group of carrier)；After 30 DEG C of lucifuge culture 48h, picking single bacterium colony is screened on MD flat boards, positive bacterium colony bacterium
Strain cell surface shows the recombinant yeast pichia pastoris of PET catabolic enzymes and hydrophobin altogether, is named as P.pastoris PETase-
GCW61-HGF1；
(5) genomic DNA of transformant (P.pastoris PETase-GCW61-HGF1) is extracted, enters performing PCR checking, than
Whether identical with positive control compared with electrophoretic band position, the primer that PCR is adopted is for AOX upstream and downstream primers, P-61-F/R and hg-
61-F/R, carries out glycerol and protects bacterium to positive bacteria.
P-61-F：CCGCTCGAGAAAAGAGATT
P-61-R：CCGGAATTCTTAAATCAATAGAGCAACACCG
hg-61-F：CCGGAATTCCAACAGTGCA
hg-61-R：ATAGTTTAGCGGCCGCTTAAATC
Six .HPLC survey enzyme activity experiment
Operating procedure surveys enzyme activity experiment with reference to the HPLC of embodiment 1, and experimental result illustrates recombinant yeast pichia pastoris
P.pastoris PETase-GCW61-HGF1 can degrade PET, and be obviously improved compared with wild type PETase enzyme activity.
6 cell surface of embodiment shows the recombinant yeast pichia pastoris P.pastoris of PET catabolic enzymes and hydrophobin altogether
The preparation and application of PETase-GCW61-HFB1, comprises the steps：
First, with 5 step one of embodiment.
2nd, hydrophobin genes HFB1 of the chemosynthesis as shown in SEQ ID NO.9 and as shown in SEQ ID NO.1
PETase genes.
3rd, PCR is utilized by the nucleotide sequence and SEQ ID of the GCW61 anchorin genes as shown in SEQ ID No.4
The nucleotide sequence connection of NO.1 obtains fusion sequence 1；Using PCR by the GCW61 anchorin bases as shown in SEQ ID No.4
The nucleotide sequence connection of the nucleotide sequence of cause and hydrophobin genes HFB1 as shown in SEQ ID NO.9 obtains merging sequence
Row 2, comprise the following steps that：
Hydrophobin genes HFB1 of the chemosynthesis as shown in SEQ ID NO.9 and as shown in SEQ ID NO.1
PETase genes, design forward primer P-F/hf-F and downstream primer P-R/hf-R, with composition sequence as template amplification PETase
Gene and HFB1 genes.
P-F:CCGCTCGAGAAAAGAGATTACAAGGATGACGACGATAAGCAAACCAATCCTTATGCCCGTG
P-R:ACTACCACCTCCTCCACTACCTCCACCACCGCTACAGTTGGCGGTACGAA
hf-F:CCGGAATTCAGCAACGGCAACGGCAATGT
hf-R:ACTACCACCTCCTCCACTACCTCCACCACCAGCACCGACGGCGGTCT
PCR product carries out 1% agarose gel electrophoresiies, reclaims genes of interest fragment, obtains PETase genes, HFB1 bases
Cause.
Gained PETase and GCW61 is carried out overlap with the linker as shown in SEQ ID No.5 as overlay region
pcr；Gained HFB1 and GCW61 is carried out overlap pcr with the linker as shown in SEQ ID No.5 as overlay region
PCR product carries out 1% agarose gel electrophoresiies, reclaims genes of interest fragment, obtains such as SEQ ID No.29 institutes
The PETase-linker-GCW61 genes (fusion sequence 1) for showing and the HFB1-linker-GCW61 as shown in SEQ ID No.12
Gene (fusion sequence 2).
4th, fusion sequence 1 is connected on yeast expression vector ppic9, obtains fusion expression vector 1；Will fusion
Sequence 2 is connected on yeast expression vector ppiczaA, obtains fusion expression vector 2, and concrete steps are with 1 step of embodiment
Four.
5th, fusion expression vector 1 and fusion expression vector 2 are proceeded in expressive host Pichia sp., obtains cell surface
Show the recombinant yeast pichia pastoris P.pastoris PETase-GCW61-HFB1 of PET catabolic enzymes and hydrophobin, concrete steps altogether
As follows.
Prepared by electric shocking method conversion P.pastoris GS115 cells competence
(1) from picking single bacterium colony on fresh Pichia pastoris GS115 (His-) flat board, 5mL YPD Tube propagations are forwarded to
In base, 30 DEG C, 250rpm overnight incubations；
0.1-0.5ml overnight cultures are taken from tube culture within (2) second days, be seeded to containing the fresh YPD cultures of 500ml
The 2L shaking flasks of base, overnight growth are about 1.3-1.5 to OD600；
(3) yeast culture is taken from shaking table, 4 DEG C, 1500g is centrifuged 5 minutes to collect cell, afterwards in 100mLYPD-
HEPES-DTT (adds the HEPES buffer solution of 20ml pH 8.0, adds the freshly prepared 1M's of 2.5ml in 100ml YPD
DTT) suspension cell again in culture medium, 30 DEG C are cultivated 15 minutes on shaking table；Cell, ice bath half an hour is removed from shaking table；
(4) cell, ice bath half an hour are removed from shaking table；4 DEG C, 1500g is centrifuged 5 minutes to collect cell, then uses 250ml
The aquesterilisa suspension cell of pre-cooling；；
(5) as above it is centrifuged, then the 1M Sorbitol suspension cells with 20ml pre-coolings；
(6) as above it is centrifuged, with the 1M Sorbitol suspension cells of 1ml pre-coolings, to final volume about 1.5ml；As above it is centrifuged, uses
The cold ultra-pure waters of 500 μ L suspension cell again, standby in ice bath.
Electric shocking method conversion is verified with transformant
(1) the above-mentioned cells of 80 μ L p9P61 carriers linearizing with 5-20ug are taken and pAHF61 carrier DNAs mixes, proceeded to pre-
In cold 0.2cm electricity revolving cups, 5min is placed on ice；
(2) electric revolving cup outer wall water droplet is dried, is placed on electroporation；
(3) yeast parameter " fungi " is set, and " pic " clicks on Pulse；
(4) electric revolving cup is taken out, 600 microlitres of L ice pre-cooling 1mol/L Sorbitol is added immediately, is gently blown and beaten with rifle, gentle
It is transferred in 1.5mL centrifuge tubes, after 30 DEG C of quiescent culture 1h, adds 600 microlitres of YPD culture medium, 30 DEG C, 200rpm shaking tables is distinguished
It is divided into 200-600 μ L aliquots after culture 1h, 3h, is applied on the YPDS flat boards containing 75 mcg/mls zeocin and (arranges in competence
It is added without the matched group of carrier)；After 30 DEG C of lucifuge culture 48h, picking single bacterium colony is screened on MD flat boards, positive bacterium colony bacterium
Strain cell surface shows the recombinant yeast pichia pastoris of PET catabolic enzymes and hydrophobin altogether, is named as P.pastoris PETase-
GCW61-HFB1；
(5) genomic DNA of transformant (P.pastoris PETase-GCW61-HFB1) is extracted, enters performing PCR checking, than
Whether identical with positive control compared with electrophoretic band position, the primer that PCR is adopted is for AOX upstream and downstream primers, P-61-F/R and hf-
61-F/R, carries out glycerol and protects bacterium to positive bacteria.
P-61-F：CCGCTCGAGAAAAGAGATT
P-61-R：CCGGAATTCTTAAATCAATAGAGCAACACCG
hf-61-F：CCGGAATTCAGCAACGGCAAC
hf-61-R：ATAGTTTAGCGGCCGCTTAAATC
Claims (6)
1. cell surface shows the structure of the recombinant yeast pichia pastoris of PET catabolic enzymes and hydrophobin altogether, and its feature includes following step
Suddenly：
(1) anchorin gene is cloned from Pichia pastoris GS115 genome；Chemosynthesis hydrophobin genes and such as SEQ
PETase genes shown in ID NO.1；
(2) the nucleotide sequence connection of the nucleotide sequence of anchorin gene and SEQ ID NO.1 is merged using PCR
Sequence 1；The nucleotide sequence of anchorin gene and hydrophobin genes connection are obtained fusion sequence 2 using PCR；
(3) fusion sequence 1 is connected on yeast expression vector, obtains fusion expression vector 1；Fusion sequence 2 is connected
To on yeast expression vector, fusion expression vector 2 is obtained；
(4) fusion expression vector 1 and fusion expression vector 2 are proceeded in expressive host Pichia sp., obtains cell surface and open up altogether
Show the recombinant yeast pichia pastoris of PET catabolic enzymes and hydrophobin.
2. cell surface according to claim 1 shows the structure of the recombinant yeast pichia pastoris of PET catabolic enzymes and hydrophobin altogether
Build, anchorin gene described in its feature is the GCW61 or SEQ shown in GCW51, SEQ ID No.4 shown in SEQ ID No.3
GCW21 shown in ID No.8.
3. cell surface according to claim 1 shows the structure of the recombinant yeast pichia pastoris of PET catabolic enzymes and hydrophobin altogether
Build, hydrophobin genes described in its feature are the HGF1 genes shown in SEQ ID NO.2, the HFB1 bases shown in SEQ ID NO.9
SC3 genes or the HFB2 genes shown in SEQ ID NO.11 shown in cause, SEQ ID NO.10.
4. cell surface according to claim 1 shows the structure of the recombinant yeast pichia pastoris of PET catabolic enzymes and hydrophobin altogether
Build, yeast expression vector described in its feature is ppic9, ppic9k, ppiczaA, ppiczaB, ppiczaC.
5. the cell surface that one of claim 1-4 builds shows the recombinant yeast pichia pastoris of PET catabolic enzymes and hydrophobin altogether.
6. the cell surface of claim 5 shows the recombinant yeast pichia pastoris of PET catabolic enzymes and hydrophobin in whole-cell catalytic altogether
The purposes of decomposed P ET.
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| CN108004254A (en) * | 2017-12-13 | 2018-05-08 | 天津大学 | The albumen and application of hydrophobin mHGFI genes and expression |
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| CN113584057A (en) * | 2021-07-26 | 2021-11-02 | 天津大学 | ICCG expression element, expression vector, bacillus subtilis recombinant strain and method for degrading PET or monomer thereof |
| CN113584057B (en) * | 2021-07-26 | 2023-08-15 | 天津大学 | ICCG expression element, expression vector, bacillus subtilis recombinant strain and method for degrading PET or monomer thereof |
| CN114941004A (en) * | 2022-04-15 | 2022-08-26 | 大连理工大学 | Engineering enzyme compound for efficiently degrading PET (polyethylene terephthalate) plastic as well as preparation method and application thereof |
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