CN105062909A - Double-lipase cell surface co-display engineering bacterium, and construction method and application thereof - Google Patents
Double-lipase cell surface co-display engineering bacterium, and construction method and application thereof Download PDFInfo
- Publication number
- CN105062909A CN105062909A CN201510600813.9A CN201510600813A CN105062909A CN 105062909 A CN105062909 A CN 105062909A CN 201510600813 A CN201510600813 A CN 201510600813A CN 105062909 A CN105062909 A CN 105062909A
- Authority
- CN
- China
- Prior art keywords
- lipase
- cell
- rhizopus oryzae
- altogether
- sed1p
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Landscapes
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a double-lipase cell surface co-display engineering bacterium, and a construction method and application thereof. The engineering bacterium is co-displayed on the Pichia pastoris cell surface by using Saccharomyces cerevisiae cell wall protein Sed1p as ankyrin and Rhizopus oryzae lipase and Candida antarctica lipase B as double-lipase target proteins, thereby constructing the double-lipase cell surface co-display recombinant yeast engineering bacterium. The Rhizopus oryzae lipase and Candida antarctica lipase B are co-displayed on the Pichia pastoris cell wall surface to prepare the Rhizopus oryzae lipase/Candida antarctica lipase B cell surface co-display complete cell catalyst. The maximum enzyme activity of the surface co-display complete cell catalyst is 686U/g-cell dry weight, which is respectively 3 times or 2 times of the enzyme activity of the independently displayed Rhizopus oryzae lipase or Candida antarctica lipase B; and the methyl ester yield of the catalytic grease esterification reaction is up to 96%.
Description
Technical field
The invention belongs to the technical fields such as molecular biology, biomass energy and genetically engineered, be specifically related to a kind of two lipase cell surface and show engineering bacteria and construction process thereof and application altogether.
Background technology
Lipase (Lipase, EC3.1.1.3) is and the hydrolysis of triglyceride level, synthesis and transesterification relevant enzyme, is extensively present in animal and plant and microorganism, its most outstanding feature be can on oil-water interface catalyzed reaction.Lipase has been successfully applied to fats and oils processing, organic synthesis, makeup and medicine and other fields as a kind of green catalyst, is also used widely in recent years in the research of preparation biofuel.In the lipase taking fungi as source, candida antarctica lipase B (CandidaantarcticalipaseB, CALB) has chemo-selective and enantioselectivity, has very strong catalytic activity to water-insoluble and water-soluble substances.Rhizopus oryzae lipase (Rhizopusoryzaelipase, ROL) as a kind of 1,3-position specific lipase, in the aqueous phase that can well exist at organic solvent-free, the transesterification of catalysis grease, is one of primary fat enzyme of full cell method and Production by Enzymes biofuel.In the effect that catalysis grease is transesterification, candida antarctica lipase B catalytic activity is high, and the reaction times is short, but its aqueous catalysis less effective, Rhizopus oryzae lipase has 1,3-position specificity and well can play katalysis in aqueous phase.
In recent years, in the research of biofuel, be that the biological catalysis of catalyzer has the advantages such as, alcohol consumption little, aftertreatment simple, non-pollutant discharge gentleer than chemical method reaction conditions because it and more and more comes into one's own with lipase, especially immobilized enzyme catalysis is adopted, make reaction terminate rear catalyst easily to reclaim, can recycled for multiple times, greatly reduce production cost, demonstrate good application prospect.But, enzymatic process also exists the extraction and isolation of enzyme and purge process is complicated and high cost, enzyme easily produce the problems such as loss of activity in preparation process, thus directly in recent years produce with lipase Whole Cell Biocatalysis method that bacterial strain or cell are catalyzer and become an important developing direction, the microbial cell surface display technique grown up especially is in recent years that whole-cell catalytic and enzyme immobilizatio provide a kind of biological method based on gene recombination technology newly.
It is import microbial host cell after target protein gene sequence and specific carrier peptide gene sequence being merged that microbial cell surface is shown, target protein is expressed and is positioned microbial cell surface under the guiding of carrier proteins, keeps relatively independent space conformation and original biologic activity.Utilize surface display technologies that the enzyme with catalytic activity is showed in the microbial cell surface such as yeast and just define whole-cell catalyst.Different with external secretion enzyme from traditional desmo enzyme, the enzyme of surface display is fixed on cell outer surface with covalently or non-covalently key form, thus has many excellent characteristics, as temperature, organic solvent stability, can reuse.At present, antimicrobial surface display systems has developed phage display system, bacterium surface exhibiting system, yeast surface display system etc., is widely used in biological study and industrial many fields.
Summary of the invention
Goal of the invention: for the deficiencies in the prior art, the object of this invention is to provide a kind of two lipase cell surface and show engineering bacteria altogether, with yeast saccharomyces cerevisiae be source cell wall protein Sed1p be anchorin, Rhizopus oryzae lipase and candida antarctica lipase B are showed in Pichia pastoris GS115 cell wall altogether, and realize high expression.Another object of the present invention is to provide the construction process that a kind of above-mentioned pair of lipase cell surface shows engineering bacteria altogether.The application that the present invention also has an object to be to provide a kind of above-mentioned pair of lipase cell surface to show engineering bacteria altogether.
Technical scheme: in order to realize foregoing invention object, the technical solution used in the present invention as follows:
A kind of two lipase cell surface shows engineering bacteria altogether, for using brewing yeast cell wall-held protein Sed1p as anchorin, be two lipase target proteins with Rhizopus oryzae lipase and candida antarctica lipase B, jointly be illustrated in Pichia pastoris surface, the two lipase cell surfaces constructed show recombination yeast engineering bacteria altogether.
Described two lipase cell surfaces show the construction process of engineering bacteria altogether, take Sed1p as anchorin, make it hold with Rhizopus oryzae lipase N to be connected, tumor-necrosis factor glycoproteins is held by the N of this anchorin, with covalent, Rhizopus oryzae lipase ROL is fixed on Pichia pastoris surface, realizes abduction delivering, shown separately the recombination yeast of Rhizopus oryzae lipase; Again this is shown separately that the recombination yeast of Rhizopus oryzae lipase is prepared into competent cell, be converted in the recombination yeast competent cell showing separately ROL by the candida antarctica lipase B expression plasmid pGAPZ α A-CALB-Flag-Sed1p of structure electricity, screening obtains the recombination yeast positive transformant that ROL and CALB is shown on surface altogether.
Described two lipase cell surfaces show the construction process of engineering bacteria altogether, comprise the following steps:
(1) gene order of target protein Rhizopus oryzae lipase ROL is cloned in expression vector, obtains surface display recombinant plasmid vector; Again anchorin gene Sed1p is cloned in surface display recombinant plasmid vector, brewing yeast cell wall-held protein gene and target protein Rhizopus oryzae lipase gene is made to form fusion gene, the Rhizopus oryzae lipase cell surface display expression plasmid that to obtain with brewing yeast cell wall-held protein Sed1p be anchorin;
(2) Rhizopus oryzae lipase cell surface display expression plasmid electricity is converted into pichia pastoris phaff, is shown separately the recombinant yeast pichia pastoris positive transformant of Rhizopus oryzae lipase according to the selection markers screening on expression vector;
(3) will show that separately the recombination yeast of Rhizopus oryzae lipase ROL is prepared into competent cell.
(4) method identical with step (1) is adopted to build the cell surface display expression plasmid pGAPZ α A-CALB-Flag-Sed1p of candida antarctica lipase B, pGAPZ α A-CALB-Flag-Sed1p electricity is converted in the recombination yeast competent cell showing separately Rhizopus oryzae lipase, obtains by positive transformant screening the recombinant yeast cell that Rhizopus oryzae lipase ROL and candida antarctica lipase B cell surface show altogether.
Described two lipase cell surfaces show that engineering bacteria is as the application of display type whole-cell catalyst in the transesterification preparation biofuel of catalysis grease altogether of two lipase cell surface altogether.
Described application, the glyceride stock of the transesterification preparation biofuel of catalysis is soybean oil, rapeseed oil, Jatropha curcas oil or jatropha curcas seed oil, rubber seed wet goods Vegetable oil lipoprotein, and also can be microalgae grease, also can be used edible oil.
Rhizopus oryzae lipase of the present invention and candida antarctica lipase B cell surface display type whole-cell catalytic altogether, the advantages such as the catalytic efficiency in aqueous phase that can possess in conjunction with two kinds of enzymes high and catalyzed reaction time is short, than existing immobilized enzyme catalysis, non-surface display type whole-cell catalytic and single fat enzyme surface display type whole-cell catalytic, there is more significant advantage, the recent development trend of the transesterification preparation field of biodiesel oil of grease catalysis will be become.Therefore, it is extremely necessary for being total to display type whole-cell catalyst in conjunction with genetic engineering technique and cellular degeneration necrosis technical study lipase cell surface.
Beneficial effect: compared with prior art, Rhizopus oryzae lipase and candida antarctica lipase B are showed in Pichia pastoris wall surface by the present invention altogether, Rhizopus oryzae lipase and candida antarctica lipase B cell surface display type whole-cell catalyst are altogether prepared, this surface altogether the highest enzyme of display type whole-cell catalyst is lived as 686U/g-dry cell weight, be 3 times and 2 times of the enzyme activity showing separately Rhizopus oryzae lipase or candida antarctica lipase B respectively, catalysis grease transesterification methyl esters yield reaches 96%.
Accompanying drawing explanation
Fig. 1 is construction of recombinant plasmid figure;
Fig. 2 is that recombinant bacterial strain figure is shown on the surface of plate screening high yield lipase altogether; Wherein, (a) caprylin is dull and stereotyped, and (b) sweet oil-MMH-RB is dull and stereotyped;
Fig. 3 is the fluorometric result figure that recombination engineering bacteria is shown on surface altogether; Wherein, 1. white light, 2.488nm, 3.555nm;
Fig. 4 is Westernblot analytical results figure; Wherein, lane2, (a); Lane1, (b) is for showing recombinant bacterium GS115/pGAPZA-RCS cell wall protein altogether in surface; Lane3, a () is ROL surface display recombinant bacterium GS115/pPICZ α A-ROL-Sed1p cell wall protein, lane2, and (b) is CALB surface display recombinant bacterium GS115/pGAPZ α A-CALB-Sed1p cell wall protein, lane1, (a); Lane3, (b) is Pichia pastoris GS115 cell wall protein;
Fig. 5 is the enzyme activity result figure that surface is total to display lipase; Wherein,
the lipase activity of recombinant bacterium GS115/pGAPZ α A-RCS is shown on surface altogether;
the lipase activity of ROL surface display recombinant bacterium GS115/pGAPZ α A-CALB-Sed1p;
the lipase activity of CALB surface display recombinant bacterium GS115/pPICZ α A-ROL-Sed1p;
Fig. 6 is the methyl esters yield figure that surface is total to display type whole-cell catalyst catalysis soybean oil transesterification;
Fig. 7 is the repeat performance figure that surface is total to display type whole-cell catalyst.
Embodiment
The substratum that following examples use and related reagent as follows:
10 × YNB (1L): dissolve 134gYNB (liquid containing ammonium sulfate is not containing amino acid) in 1LddH
2in O, dissolve below 50 DEG C.
BMGY (1L): dissolve 10g yeast extract, 20g peptone in 700mLddH
2in O, 121 DEG C of sterilizing 20min, are chilled to room temperature, add 100mL1mol/L potassium phosphate buffer (pH6.0), 100mL10 × YNB, 2mL500 × B, 100mL10 × GY.
BMMY (1L): dissolve 10g yeast extract, 20g peptone in 700mLddH
2in O, 121 DEG C of sterilizing 20min, are chilled to room temperature, add 100mL1mol/L potassium phosphate buffer (pH6.0), 100mL10 × YNB, 2mL500 × B, 100mL10 × M.
25% polyvinyl alcohol sweet oil emulsion (1L): take 3g polyvinyl alcohol, adding distil water 150mL, heated and stirred is dissolved into 2% solution.Add 50mL sweet oil wherein, stir 3 ~ 4 times with high-speed tissue mashing machine, each 10s.
Sweet oil-MMH-RB flat board (1L): sweet oil emulsion 800mL, adds 15g agar, 121 DEG C of sterilizing 20min, when being chilled to 60 DEG C, add 100mL10 × YNB, 2mL500 × B, 100mL10 × M, 100mL10 × RB, 10mL100 × H, is down flat plate after mixing.
Finite concentration caprylin polyvinyl alcohol emulsion: take 3g polyvinyl alcohol, adding distil water 150mL, heated and stirred is dissolved into 2% solution.With 2% polyvinyl alcohol solution: caprylin volume ratio is 3:1 configuration, use miniature high-speed refiner to stir 4-5 time, each 20s, until there is white emulsion, if uneven or layering, can heat a little or increase and stir number of times and time.
Caprylin flat board (1L): with 10g yeast extract, 20g peptone in 900mLddH
2dissolve in O, add certain density caprylin polyvinyl alcohol emulsion, 1L (needing to add 10 × D then without the need to constant volume) is settled to deionized water, mixing, adds the agar powder of 1.5% (w/v), 121 DEG C of sterilizing 20min, as required, add 10 × D after sterilizing, be down flat plate, can 4 DEG C of preservations.
Embodiment 1
The genomic extraction of Saccharomyces Cerevisiae in S .cerevisiaeEBY100, step is as follows:
(1) S.cerevisiaeEBY100 is purchased from Invitrogen.First activated strains on YPD flat board, picking list bacterium colony in YPD shaking flask, 30 DEG C of incubated overnight.
(2) the centrifugal 5min of thalline 5000rpm/min taken the logarithm vegetative period, thalline sterile distilled water is washed once, removes supernatant.
(3) 5mLDNA broken wall damping fluid (100mmol/LTris-HCl, pH8.0,10mmol/LEDTA, 1%SDS) is added, mixing, 65 DEG C of insulation 1h.
(4) the centrifugal 15min of 10000rpm, gets supernatant.
(5) phenol chloroform isoamyl alcohol (25:24:1) extracting 2 times are added, the centrifugal 7min of each 5000rpm.
(6) get upper strata extract, add 6 μ LRNaseA (10mg/mL), spend the night (adding 0.3mol/L sodium-acetate, pH5.2) with 3 times of alcohol settling.The centrifugal 30min of 12000rpm.
(7) 75% ethanol wash 2 times, air-dry.
(8) add the aseptic ultrapure water of 50 μ L to dissolve.
Embodiment 2
The structure of Rhizopus oryzae lipase (ROL) Pichia pastoris surface display system, step is as follows:
A. surface display recombinant plasmid vector pPICZ α A-ROL builds
Clone's Rhizopus oryzae lipase gene ROL, merges with plasmid pPICZ α A.Design primer according to the plasmid pPIC9K-ROL containing Rhizopus oryzae lipase gene order, primer is synthesized by Shanghai Sheng Gong biotechnology company limited.
R1 (5 '-3 ', lower with): CCGGAATTCATGGTTCCTGTTTCTGGTAAATCTG, before add EcoR I restriction enzyme site.
R2:TCCCCGCGG
aTGATGATGATGATGATGcAAACAGCTTCCTTCGTTGATATCA, before add Sac II restriction enzyme site and His-tag label protein (underscore part).
Pcr amplification is carried out, PCR reaction system: 1 μ L plasmid pPIC9K-ROL, 1 μ LR1 primer, 1 μ LR2 primer, 25 μ LPremixExtaq, 22 μ LddH with the primer of synthesis
2o.Amplification condition is: sex change 94 DEG C, 2min; Sex change 94 DEG C, 30s; Anneal 55 DEG C, 1min; Extend 72 DEG C, 1min; Recirculation 30 times; Extend 72 DEG C, 10min; Reaction stops, 4 DEG C of insulations.Reclaim test kit by gel and purifying is carried out to PCR primer, obtain the Rhizopus oryzae lipase ROL gene with His-tag label protein gene.
Rhizopus oryzae lipase ROL gene and pPICZ α A EcoR I and Sac II enzyme to be cut and purifying reclaims.Connect with T4DNA ligase enzyme, build surface display recombinant plasmid pPICZ alpha A-ROL.
B. the structure of Yeast expression carrier pPICZ α A-ROL-Histag-Sed1p
(1) the anchorin gene Sed1p of pcr amplification maturation
Design primer according to the gene order (GeneID:851649) of known Saccharomyces Cerevisiae in S ed1p albumen, primer is synthesized by Shanghai Jierui Biology Engineering Co., Ltd.
S1:TCCCCGCGGCAATTTTCCAACAGTACATCTGCTTCT, before add Sac II restriction enzyme site.
S2:CTAGTCTAGATTATAAGAATAACATAGCAACACCAGC, before add Xba I restriction enzyme site.
With the yeast saccharomyces cerevisiae EBY100 genomic dna extracted for template, carry out pcr amplification with the primer of above-mentioned synthesis.PCR reaction system: 1 μ L yeast saccharomyces cerevisiae EBY100 genomic dna, 1 μ LS1 primer, 1 μ LS2 primer, 25 μ LPremixExtaq, 22 μ LddH
2o.Amplification condition is: sex change 94 DEG C, 5min; Sex change 94 DEG C, 30s; Anneal 56 DEG C, 30s; Extend 72 DEG C, 1min; Recirculation 30 times; Extend 72 DEG C, 10min; Reaction stops, 4 DEG C of insulations.Reclaim test kit by gel and purifying is carried out to PCR primer, obtain ripe anchorin gene Sed1p.
(2) Sed1p and pPICZ α A-ROL Sac II, Xba I enzyme cut and reclaimed with kits, connecting with T4DNA ligase enzyme, obtain Recombinant Pichia pastoris surface display expression plasmid pPICZ α A-ROL-Histag-Sed1p.By the pPICZ α A-ROL-Histag-Sed1p plasmid transformation escherichia coli host TOP10F obtained.With containing 50 μ g/mlZeocinLLB plate screening transformants, the transformant of the picking Zeocin resistance positive, extract plasmid and also check order, result shows that wall-held protein sed1p gene order is correctly inserted.Construction process is shown in shown in Fig. 1 (a).
C. recombinant plasmid pPICZ alpha A-ROL-Histag-Sed1p expresses in pichia spp
Adopt restriction enzyme SacI linearization plasmid pPICZ α A-ROL-Histag-Sed1p.Electricity transforming Pichia pastoris GS115, at the YPD grow on plates 72h containing 100 μ g/mLZeocin, picking resistance positive transformant, namely the transformant of fusion gene sequence is integrated with, point is dull and stereotyped to the high resistance YPD containing 400 μ g/mLZeocin, select larger bacterial strain, namely containing the transformant compared with multiple copied.
Embodiment 3
The structure of candida antarctica lipase B (CALB) pichia spp surface display system, step is as follows:
A. surface display recombinant plasmid vector pGAPZ α A-CALB builds
Clone's candida antarctica lipase B gene C ALB, merges with pGAPZ α A.According to candida antarctica lipase B gene order design primer, primer is synthesized by Shanghai Sheng Gong biotechnology company limited.
C1:CCGGAATTCATGAAGCTACTCTCTCTGACCGGTG, before add EcoR I restriction enzyme site.
C2:AAGGAAAAAAGCGGCCGCTGGGGGTGACGATGCCGGAGC, before add Not I restriction enzyme site.
With antarctic candida genomic dna for template, carry out pcr amplification with the primer of synthesis).PCR reaction system: 1 μ L antarctic candida genomic dna, 1 μ LC1 primer, 1 μ LC2 primer, 25 μ LPremixExtaq, 22 μ LddH
2o.Amplification condition is: sex change 94 DEG C, 2min; Sex change 94 DEG C, 30s; Anneal 55 DEG C, 1min; Extend 72 DEG C, 1min; Recirculation 30 times; Extend 72 DEG C, 10min; Reaction stops, 4 DEG C of insulations.Reclaim test kit by gel and purifying is carried out to PCR primer, obtain candida antarctica lipase B gene.
Candida antarctica lipase B gene and pGAPZ α A EcoR I and Not I enzyme to be cut and purifying reclaims.Connect with T4DNA ligase enzyme, build surface display recombinant plasmid pGAPZ α A-CALB.
B. the structure of Yeast expression carrier pGAPZ α A-CALB-Flag-Sed1p
(1) the anchorin gene Sed1p of pcr amplification maturation
According to the gene order design primer of known Saccharomyces Cerevisiae in S ed1p albumen, primer is synthesized by Shanghai Jierui Biology Engineering Co., Ltd.
SC1:AAGGAAAAAAGCGGCCGC
gATTACAAGGATGACGACGATAAGaTGAAATTATCAACTGTCCTATTATCT, before add Not I restriction enzyme site and FLAG label protein (underscore part).
SC2:CTAGTCTAGATTATAAGAATAACATAGCAACACCAG, before add Xba I restriction enzyme site.
With the yeast saccharomyces cerevisiae EBY100 genomic dna extracted for template, carry out pcr amplification with the primer of above-mentioned synthesis.PCR reaction system: 1 μ L yeast saccharomyces cerevisiae EBY100 genomic dna, 1 μ LSC1 primer, 1 μ LSC2 primer, 25 μ LPremixExtaq, 22 μ LddH
2o.Amplification condition is: sex change 94 DEG C, 5min; Sex change 94 DEG C, 30s; Anneal 56 DEG C, 30s; Extend 72 DEG C, 1min; Recirculation 30 times; Extend 72 DEG C, 10min; Reaction stops, 4 DEG C of insulations.Reclaim test kit by gel and purifying is carried out to PCR primer, obtain ripe anchorin gene Sed1p.
(2) Sed1p and pGAPZ α A-CALB Not I and Xba I enzyme cut and reclaim with kits, connecting with T4DNA ligase enzyme, obtain Recombinant Pichia pastoris surface display expression plasmid pGAPZ α A-CALB-Flag-Sed1p.By the pGAPZ α A-CALB-Flag-Sed1p plasmid transformation escherichia coli host TOP10F obtained.With containing 50 μ g/mlZeocinLLB plate screening transformants, the transformant of the picking Zeocin resistance positive, extract plasmid and also check order, result shows that wall-held protein Sed1p gene order is correctly inserted.Construction process is shown in Fig. 1 (b).
C. recombinant plasmid pGAPZ α A-CALB-Flag-Sed1p expresses in pichia spp
Adopt restriction enzyme SacI linearization plasmid pGAPZ α A-CALB-Flag-Sed1p.Electricity transforming Pichia pastoris GS115, at the YPD grow on plates 72h containing 100 μ g/mLZeocin, picking resistance positive transformant, namely the transformant of fusion gene sequence is integrated with, point is dull and stereotyped to the high resistance YPD containing 400 μ g/mLZeocin, select larger bacterial strain, namely containing the transformant compared with multiple copied.
Embodiment 4
The Construction and identification of recombination engineering bacteria is shown on Rhizopus oryzae lipase and candida antarctica lipase B surface altogether, and step is as follows:
A. the structure of recombination engineering bacteria GS115/pGAPZ α A-RCS is shown on surface altogether
The recombinant yeast pichia pastoris GS115/pPICZ α A-ROL-Sed1p of surface display Rhizopus oryzae lipase (ROL) is prepared competent cell.
By expression vector pGAPZ α A-CALB-Flag-Sed1p Restriction Enzyme SacI linearization for enzyme restriction, whether electrophoresis detection linearizing is complete.Expression vector, after total Linearization, is converted into the competent cell of recombinant yeast pichia pastoris GS115/pPICZ α A-ROL-Sed1p, coats containing the antibiotic YPDS of Zeocin dull and stereotyped.By obtaining the recombinant bacterium of high copy containing the antibiotic YPDS plate screening of Zeocin of different concns.Picking transformant, point is on certain density sweet oil-MMH-RB flat board and caprylin flat board, and larger bacterium (Fig. 2 (a), (b)) is enclosed in picking hydrolysis, surface is shown altogether bacterial strain is designated as GS115/pGAPZ α A-RCS, for follow-up cultivation.
B. the qualification of recombination engineering bacteria GS115/pGAPZ α A-RCS is shown on surface altogether
(1) fluorometric analysis of recombination engineering bacteria is shown on surface altogether
Positive transformant GS115/pGAPZ α A-RCS is inoculated in 50mLBMGY substratum, 30 DEG C, 180rpm shaking culture 16-20h to OD
600to 2-6.Collected by centrifugation thalline, then be suspended in BMMY substratum, be diluted to OD
600be 1, continue shaking culture, the methyl alcohol adding 0.5% every 24h in BMMY substratum carries out abduction delivering.After fermentation 72h, after carrying out immune response with anti-Histag and anti-Flag antibody respectively, adopt fluorescent microscope to GS115/pGAPZ α A-RCS, GS115/pPICZ α A-ROL-Sed1p, GS115/pGAPZ α A-CALB-Sed1p and control strain GS115 carries out com-parison and analysis.The detected result at ultraviolet 488nm and 555nm place is incubated in as Fig. 3 through the cultivation induction of the same terms and fluorescence antibody.Under 488nm is blue-light excited, green fluorescence is presented containing histidine-tagged lipase recombinant bacterium GS115/pPICZ α A-ROL-Sed1p and GS115/pGAPZ α A-RCS, and cannot not being combined with fluorescence antibody containing histidine-tagged blank bacterium GS115, therefore unstressed configuration display.Similarly, under 555nm green glow excites, red fluorescence is presented containing the lipase recombinant bacterium GS115/pGAPZ α A-CALB-Sed1p of Flag label and GS115/pGAPZ α A-RCS, and cannot not being combined with fluorescence antibody containing the blank bacterium GS115 of Flag label, therefore unstressed configuration display.This result proves that lipase ROL and CALB is all successful and is illustrated in Pichia pastoris surface by anchorin Sed1p grappling.
(2) analysis of recombinant yeast pichia pastoris cell wall protein is shown on surface altogether
The two lipase of shake-flask culture shows bacterial strain GS115/pGAPZ α A-RCS altogether, more respectively using blank bacterial strain Pichia pastoris GS115, single fat enzyme surface display bacterial strain GS115/pPICZ α A-ROL-Sed1p and GS115/pGAPZ α A-CALB-Sed1p as negative control.Extracted the cell wall protein of above-mentioned bacterial strains by laminarinase, and carry out WesternBlot analysis.As shown in Figure 4, Fig. 4 (a) is the Western-blot result adopting anti-His tag antibody immuning hybridization to result.Swimming lane 2,3 are respectively displaying the recombinant bacterium GS115/pGAPZ α A-RCS of ROL, the cell wall protein of GS115/pPICZ α A-ROL-Sed1p, there is an obvious band, size is about 75kDa, consistent with theoretical value, in addition the GS115 mycetocyte wall-held protein of swimming lane 1 is as negative control, is formed without band.Similarly, Fig. 4 (b) is the Western-blot result adopting anti-Flag tag antibody immuning hybridization.Swimming lane 1,2, for showing have the recombinant bacterium GS115/pGAPZ α A-RCS of CALB, the cell wall protein of GS115/pGAPZ α A-CALB-Sed1p, have an obvious band at about 75kDa equally, consistent with theoretical value, the GS115 mycetocyte wall-held protein of swimming lane 3, as negative control, is formed without band.This result again demonstrates surface and shows that recombinant yeast pichia pastoris successfully constructs altogether.
Embodiment 5
The mensuration of recombination engineering bacteria GS115/pGAPZ α A-RCS lipase activity is shown on surface altogether, and process is as follows:
The two lipase of the above-mentioned screening of shake-flask culture shows bacterial strain GS115/pGAPZ α A-RCS, investigates the best induction time of bacterial strain and most high enzymatic activity with the optimal reaction substrate pNPD (4-nitrophenyldodecanoate) of lipase.1000 μ L enzyme activity determination systems: 0.05mol/LTris-HCLbufferpH7.5850 μ L, dilute the pNPD50 μ L of certain density bacterium liquid 100 μ L, 13.5mmol/L.
After recombinant bacterial strain abduction delivering 144h, 6,000g room temperature collected by centrifugation thalline, with the PBS wash buffer thalline 3 times of 50mMpH7.5, and final resuspended thalline is in the PBS damping fluid of 50mMpH7.5.Adjust the OD of different sample respectively
600=50, preliminary acquisition is display lipase recombinant bacterium GS115/pGAPZ α A-RCS altogether, the recombinant bacterium GS115/pPICZ α A-ROL-Sed1p of independent display lipase and the thalline suspension of GS115/pGAPZ α A-CALB-Sed1p and control strain Pichia pastoris GS115, and at 45 DEG C, under pH8.0 and suitable reaction rotating speed, react 15min.Display lipase vitality test selects absorbance method.1 enzyme activity unit (IU) is defined as: the enzyme amount needed for l μm of ol p-NP that per minute hydrolysis substrate p-nitrophenyl phenolic ester pNPD generates.
As shown in Figure 5, the enzyme work being illustrated in the lipase on pichia spp surface increases along with the increase of induction time, when altogether display lipase GS115/pGAPZA-RCS is cultured to 144h on surface, vigor is the highest, the highest enzyme is lived and is reached 686U/g stem cell, and the lipase activity of surface display ROL and CALB is 217U/g stem cell and 379U/g stem cell respectively separately, visible surface is total to display lipase vigor apparently higher than the lipase activity showing separately ROL and CALB.
Embodiment 6
Surface is the transesterification preparation biofuel of display type whole-cell catalyst catalysis grease altogether, and step is as follows:
A. the preparation of surface altogether display type whole-cell catalyst: surface shows that enzyme secretion that Pichia yeast engineering produces is outside born of the same parents altogether, and target product is thalline, the object of abduction delivering as much as possiblely obtains the high cell of enzyme work.Recombinant yeast pichia pastoris engineering bacteria derivational expression method is as follows:
(1) to screening the GS115/pGAPZ α A-RCS genetic engineering bacterium that obtains on YPD flat board, 2d are cultivated for 30 DEG C.Picking list colony inoculation is in the 500mL shaking flask containing 150mLBMGY substratum, and 28 DEG C, 200r/min is cultured to OD
600=2 ~ 6.
(2) room temperature, the centrifugal 5min of 3000 × g gathers in the crops thalline, removes supernatant, with the new resuspended thalline of BMMY substratum to OD
600approximate 1.0.
(3) join in 1L shaking flask by resuspended BMMY culture 400mL, with 4 layers of sterile gauze lid bottleneck, continue 28 DEG C, 200r/min shaking table is cultivated.
(4) every 24h adds maintenance induction in a methyl alcohol to shaking flask, and methyl alcohol addition is final concentration 0.5%.
(5) after BMMY substratum starts to induce 72h, 6000rpm collected by centrifugation thalline.
(6) thalline will collected, after buffer solution for cleaning 3 times, uses for the follow-up whole-cell catalyst that does.
(7) measure the lipase activity shown altogether on surface, method is shown in embodiment 5.
It is B. surperficial that display type whole-cell catalyst catalysis grease is transesterification altogether
In 50mL tool plug triangular flask, 1:1 adds 5g grease and 0.18g methyl alcohol in molar ratio, adding a certain amount of deionized water makes the moisture control of reaction system at 50-80%, taking total enzyme amount is that recombinant bacterium GS115/pGAPZA-RCS whole-cell catalyst is shown on the surface of 60-120U altogether, joins in triangular flask.This system is placed in the constant temperature culture oscillator of 35-50 DEG C, esterification reaction of organic acid is carried out under 200r/min, add the methyl alcohol of mol ratio 1:1 at interval of 12 hours in batches, get 100 μ L supernatants at regular intervals, centrifugally rear gas chromatographic analysis is carried out to sample, calculate the methyl esters yield of grease transesterification.
1) surface display type whole-cell catalyst catalysis soybean oil esterification altogether
In 50mL tool plug triangular flask, 1:1 adds the commercially available soybean oil of 5g and 0.18g methyl alcohol in molar ratio, and adding total enzyme amount is that recombinant bacterium GS115/pGAPZ α A-RCS whole-cell catalyst is shown on the surface of 80U altogether, adds water and regulates reaction system water ratio to be 70%.React under temperature of reaction 45 DEG C, shaking flask rotating speed 200r/min condition, and add methyl alcohol according to mol ratio 1:1 respectively at 12h, 24h, 48h in batches.Get 100 μ L supernatants at set intervals, centrifugally carry out gas chromatographic analysis to sample afterwards, the methyl esters yield of transesterification changes as shown in Figure 6 in time.During reaction 60h, methyl esters yield reaches as high as 96.0%.
2) surface display type whole-cell catalyst catalysis rapeseed methylester altogether
In 50mL tool plug triangular flask, 1:1 adds the commercially available rapeseed oil of 5g and 0.18g methyl alcohol in molar ratio, and adding total enzyme amount is that recombinant bacterium GS115/pGAPZ α A-RCS whole-cell catalyst is shown on the surface of 80U altogether, adds water and regulates reaction system water ratio to be 70%.React under temperature of reaction 45 DEG C, shaking flask rotating speed 200r/min condition, and add methyl alcohol respectively at 12h, 24h, 48h according to mol ratio 1:1 in batches.When reacting 60h under this condition, methyl esters yield is 94.8%.
3) surface display type whole-cell catalyst catalysis jatropha curcas seed oil (Jatropha curcas oil) esterification altogether
In 50mL tool plug triangular flask, add 5g through solvent extraction and the jatropha curcas seed of degumming process oil and 0.18g methyl alcohol, adding total enzyme amount is that recombinant bacterium GS115/pGAPZ α A-RCS whole-cell catalyst is shown on the surface of 100U altogether, adds water and regulates reaction system water ratio to be 60%.React under temperature of reaction 40 DEG C, shaking flask rotating speed 200r/min condition, and add methyl alcohol respectively at 12h, 24h, 48h according to mol ratio 1:1 in batches.When reacting 60h under this condition, methyl esters yield is 93.2%.
4) surface display type whole-cell catalyst catalysis used edible oil esterification altogether
In 50mL tool plug triangular flask, add 5g used edible oil after pretreatment and 0.18g methyl alcohol, adding total enzyme amount is that recombinant bacterium GS115/pGAPZ α A-RCS whole-cell catalyst is shown on the surface of 100U altogether, adds water and regulates reaction system water ratio to be 60%.React under temperature of reaction 40 DEG C, shaking flask rotating speed 200r/min condition, and add methyl alcohol respectively at 12h, 24h, 48h according to mol ratio 1:1 in batches.When reacting 60h under this condition, methyl esters yield is 94.0%.
5) surface display type whole-cell catalyst catalysis microalgae grease esterification altogether
In 50mL tool plug triangular flask, add 5g and extracted the microalgae grease and 0.18g methyl alcohol that obtain by chlorella, adding total enzyme amount is that recombinant bacterium GS115/pGAPZ α A-RCS whole-cell catalyst is shown on the surface of 120U altogether, adds water and regulates reaction system water ratio to be 60%.React under temperature of reaction 45 DEG C, shaking flask rotating speed 200r/min condition, and add methyl alcohol respectively at 12h, 24h, 48h according to mol ratio 1:1 in batches.When reacting 60h under this condition, methyl esters yield is 95.5%.
C. surface is total to the repeat performance of display type whole-cell catalyst
In 50mL tool plug triangular flask, 1:1 adds the commercially available soybean oil of 5g and 0.18g methyl alcohol in molar ratio, and adding total enzyme amount is that recombinant bacterium GS115/pGAPZ α A-RCS whole-cell catalyst is shown on the surface of 80U altogether, adds water and regulates reaction system water ratio to be 70%.React under temperature of reaction 45 DEG C, shaking flask rotating speed 200r/min condition, and add methyl alcohol respectively at 12h, 24h, 48h according to mol ratio 1:1 in batches.
After reaction 60h, in 6000rpm collected by centrifugation thalline, reuse with after deionized water wash thalline 3 times, the result of its catalysis soybean oil esterification as shown in Figure 7.After this whole-cell catalyst reuses 10 times, the methyl esters yield of catalysis grease transesterification still remains on more than 80%, shows that catalyzer has good repeat performance.
SEQUENCELISTING
<110> Nanjing Forestry University
The two lipase cell surface of <120> shows engineering bacteria and construction process thereof and application altogether
<130>100
<160>8
<170>PatentInversion3.3
<210>1
<211>34
<212>DNA
<213>Artificial
<220>
<223>R1 sequence
<400>1
ccggaattcatggttcctgtttctggtaaatctg34
<210>2
<211>51
<212>DNA
<213>Artificial
<220>
<223>R2 sequence
<400>2
tccccgcggatgatgatgatgatgatgcaaacagcttccttcgttgatatc51
<210>3
<211>36
<212>DNA
<213>Artificial
<220>
<223>S1 sequence
<400>3
tccccgcggcaattttccaacagtacatctgcttct36
<210>4
<211>37
<212>DNA
<213>Artificial
<220>
<223>S2 sequence
<400>4
ctagtctagattataagaataacatagcaacaccagc37
<210>5
<211>34
<212>DNA
<213>Artificial
<220>
<223>C1 sequence
<400>5
ccggaattcatgaagctactctctctgaccggtg34
<210>6
<211>39
<212>DNA
<213>Artificial
<220>
<223>C2 sequence
<400>6
aaggaaaaaagcggccgctgggggtgacgatgccggagc39
<210>7
<211>69
<212>DNA
<213>Artificial
<220>
<223>SC1 sequence
<400>7
aaggaaaaaagcggccgcgattacaaggatgacgacgataagatgaaattatcaactgtc60
ctattatct69
<210>8
<211>36
<212>DNA
<213>Artificial
<220>
<223>SC2 sequence
<400>8
ctagtctagattataagaataacatagcaacaccag36
Claims (5)
1. a two lipase cell surface shows engineering bacteria altogether, it is characterized in that: for using brewing yeast cell wall-held protein Sed1p as anchorin, be two lipase target proteins with Rhizopus oryzae lipase and candida antarctica lipase B, jointly be illustrated in Pichia pastoris surface, the two lipase cell surfaces constructed show recombination yeast engineering bacteria altogether.
2. the according to claim 1 pair of lipase cell surface shows the construction process of engineering bacteria altogether, it is characterized in that, take Sed1p as anchorin, make it hold with Rhizopus oryzae lipase N to be connected, hold tumor-necrosis factor glycoproteins by the N of this anchorin, with covalent, Rhizopus oryzae lipase ROL is fixed on Pichia pastoris surface, realize abduction delivering, shown separately the recombination yeast of Rhizopus oryzae lipase; Again this is shown separately that the recombination yeast of Rhizopus oryzae lipase is prepared into competent cell, be converted in the recombination yeast competent cell showing separately ROL by the candida antarctica lipase B expression plasmid pGAPZ α A-CALB-Flag-Sed1p of structure electricity, screening obtains the recombination yeast positive transformant that ROL and CALB is shown on surface altogether.
3. the according to claim 2 pair of lipase cell surface shows the construction process of engineering bacteria altogether, it is characterized in that, comprises the following steps:
(1) gene order of target protein Rhizopus oryzae lipase ROL is cloned in expression vector, obtains surface display recombinant plasmid vector; Again by anchorin gene
sed1pbe cloned in surface display recombinant plasmid vector, brewing yeast cell wall-held protein gene and target protein Rhizopus oryzae lipase gene is made to form fusion gene, the Rhizopus oryzae lipase cell surface display expression plasmid that to obtain with brewing yeast cell wall-held protein Sed1p be anchorin;
(2) Rhizopus oryzae lipase cell surface display expression plasmid electricity is converted into pichia pastoris phaff, is shown separately the recombinant yeast pichia pastoris positive transformant of Rhizopus oryzae lipase according to the selection markers screening on expression vector;
(3) will show that separately the recombination yeast of Rhizopus oryzae lipase ROL is prepared into competent cell;
(4) method identical with step (1) is adopted to build the cell surface display expression plasmid pGAPZ α A-CALB-Flag-Sed1p of candida antarctica lipase B, pGAPZ α A-CALB-Flag-Sed1p electricity is converted in the recombination yeast competent cell showing separately Rhizopus oryzae lipase, obtains by positive transformant screening the recombinant yeast cell that Rhizopus oryzae lipase ROL and candida antarctica lipase B cell surface show altogether.
4. according to claim 1 pair of lipase cell surface shows that engineering bacteria is as the application of display type whole-cell catalyst in the transesterification preparation biofuel of catalysis grease altogether of two lipase cell surface altogether.
5. application according to claim 4, is characterized in that, the glyceride stock of the transesterification preparation biofuel of catalysis is soybean oil, rapeseed oil, Jatropha curcas oil or jatropha curcas seed oil, rubber seed oil, microalgae grease, used edible oil.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510600813.9A CN105062909A (en) | 2015-09-18 | 2015-09-18 | Double-lipase cell surface co-display engineering bacterium, and construction method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510600813.9A CN105062909A (en) | 2015-09-18 | 2015-09-18 | Double-lipase cell surface co-display engineering bacterium, and construction method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105062909A true CN105062909A (en) | 2015-11-18 |
Family
ID=54492430
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510600813.9A Pending CN105062909A (en) | 2015-09-18 | 2015-09-18 | Double-lipase cell surface co-display engineering bacterium, and construction method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105062909A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113088533A (en) * | 2021-04-15 | 2021-07-09 | 华中科技大学 | Yeast engineering bacterium for efficiently expressing barnacle viscose protein and preparation method thereof |
| CN114058526A (en) * | 2021-11-18 | 2022-02-18 | 广西科学院 | Saccharomyces cerevisiae engineering bacterium for displaying alkaline lipase on cell surface as well as preparation method and use method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1681942A (en) * | 2002-09-13 | 2005-10-12 | 韩国生命工学研究院 | Method for screening of a lipase having improved enzymatic activity using yeast surface display vector and the lipase |
| CN102180954A (en) * | 2011-03-01 | 2011-09-14 | 华南理工大学 | Pichia pastoris wall protein and surface display system constructed by same and construction method of surface display system |
| CN102876594A (en) * | 2012-09-11 | 2013-01-16 | 南京林业大学 | Surface displaying system for rhizopus oryzaelipase, and preparation method and application of surface displaying system |
| CN102911701A (en) * | 2012-11-02 | 2013-02-06 | 南京林业大学 | Preparation method of biodiesel and compound enzyme used by preparation method |
-
2015
- 2015-09-18 CN CN201510600813.9A patent/CN105062909A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1681942A (en) * | 2002-09-13 | 2005-10-12 | 韩国生命工学研究院 | Method for screening of a lipase having improved enzymatic activity using yeast surface display vector and the lipase |
| CN102180954A (en) * | 2011-03-01 | 2011-09-14 | 华南理工大学 | Pichia pastoris wall protein and surface display system constructed by same and construction method of surface display system |
| CN102876594A (en) * | 2012-09-11 | 2013-01-16 | 南京林业大学 | Surface displaying system for rhizopus oryzaelipase, and preparation method and application of surface displaying system |
| CN102911701A (en) * | 2012-11-02 | 2013-02-06 | 南京林业大学 | Preparation method of biodiesel and compound enzyme used by preparation method |
Non-Patent Citations (4)
| Title |
|---|
| 代敏: "具有协同效应的南极假丝酵母脂肪酶B和疏棉状嗜热丝孢菌脂肪酶在毕赤酵母中的共展示研究", 《中国优秀硕士学位论文全文数据库 基础科技辑》 * |
| 张燕: "在毕赤酵母表面共展示具有协同作用的米根霉脂肪酶和褶皱假丝酵母脂肪酶LIP1的研究", 《华中科技大学硕士论文》 * |
| 江逢春等: "利用Sed1p锚定蛋白在毕赤酵母表面展示米黑根毛霉脂肪酶及其应用", 《微生物学报》 * |
| 陈美龄: "全细胞脂肪酶生物催化剂的构建及其在生物转化中的应用", 《中国博士学位论文全文数据库 工业科技I辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113088533A (en) * | 2021-04-15 | 2021-07-09 | 华中科技大学 | Yeast engineering bacterium for efficiently expressing barnacle viscose protein and preparation method thereof |
| CN114058526A (en) * | 2021-11-18 | 2022-02-18 | 广西科学院 | Saccharomyces cerevisiae engineering bacterium for displaying alkaline lipase on cell surface as well as preparation method and use method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104651383A (en) | Recombinant pichia pastoris engineering bacteria and production method thereof | |
| CN103361327B (en) | The recombinant yeast pichia pastoris of allos high expression lipase | |
| Bader et al. | Efficient saccharification of microalgal biomass by Trichoderma harzianum enzymes for the production of ethanol | |
| JP2020501607A (en) | Genetically modified Candida utilis capable of decomposing and utilizing garbage and a method for constructing the same | |
| Hoover et al. | Bacterial production of free fatty acids from freshwater macroalgal cellulose | |
| CN104017786A (en) | Phospholipase A2 mutant and preparation method thereof | |
| Zhang et al. | Heterologous expression of an alkali and thermotolerant lipase from Talaromyces thermophilus in Trichoderma reesei | |
| CN104480083A (en) | Lipase, engineering bacterium and preparing methods of the lipase and the engineering bacterium | |
| CN105062909A (en) | Double-lipase cell surface co-display engineering bacterium, and construction method and application thereof | |
| CN102876594B (en) | Surface displaying system for rhizopus oryzaelipase, and preparation method and application of surface displaying system | |
| Liu et al. | High-level expression and characterization of Aspergillus niger ATCC 1015 xylanase B in Komagataella phaffii | |
| Bai et al. | Immobilized lipase of reconstructed oil bodies and its potential application in biodiesel production | |
| CN103981197A (en) | Novel leader peptide-containing rhizomucor mieheilipase gene and expression of rhizomucor mieheilipase gene in pichia pastoris | |
| CN104726477A (en) | Lipase coding gene and engineering strain thereof | |
| CN102586312A (en) | Method for expressing intracellular protein matrix and application thereof | |
| CN102911701A (en) | Preparation method of biodiesel and compound enzyme used by preparation method | |
| RU2701642C1 (en) | Yeast strain pichia pastoris - producer of xylanase | |
| CN104560833B (en) | A kind of basophilic micrococcus luteus and its alkalescent xylanase and the application of generation | |
| CN103305426A (en) | Mutant strains for producing cellulose, mutant strains capable of performing high-efficiency expression on target proteins and construction methods and application of mutant strains | |
| CN103834640B (en) | A kind of Chloroplast gene sequence to micro- method and correlation intended and foreign DNA is imported in ball algae chloroplast | |
| CN103122342A (en) | Heat-resisting glucamylase as well as coding gene and application thereof | |
| CN103088434B (en) | Construction method and application of Pichia stipitis large-fragment DNA (deoxyribonucleic acid) genome library | |
| CN111893107A (en) | Pichia pastoris engineering strain for heterologous expression of cellulase gene EG IV and application | |
| KR101350955B1 (en) | Novel exoglucanase and the Use thereof | |
| CN104371926A (en) | Microalgae culture solution treatment method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20151118 |
|
| RJ01 | Rejection of invention patent application after publication |