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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- 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
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (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 of molecular biology, biomass energy, genetic engineering and the like, and particularly relates to a double-lipase cell surface co-display engineering bacterium and a construction method and application thereof.
Background
Lipases (EC 3.1.1.3) are enzymes involved in the hydrolysis, synthesis and transesterification of triglycerides, widely present in animals, plants and microorganisms, and are most notably characterized by the ability to catalyze reactions at the oil-water interface. Lipase as a green catalyst has been successfully applied in the fields of oil processing, organic synthesis, cosmetics, medicines and the like, and has been widely applied in the research of preparing biodiesel in recent years. Among lipases derived from fungi, Candida Antarctica Lipase B (CALB) has chemical selectivity and enantioselectivity, and has strong catalytic activity on water-insoluble and water-soluble substances. Rhizopus Oryzae Lipase (ROL) as a 1, 3-site specific lipase can well catalyze the transesterification of grease in an aqueous phase without an organic solvent, and is one of the main lipases for producing biodiesel by a whole cell method and an enzyme method. In the aspect of the effect of catalyzing transesterification of grease, the candida antarctica lipase B has high catalytic activity and short reaction time, but the water phase catalytic effect is not good enough, and the rhizopus oryzae lipase has 1, 3-site specificity and can well play a catalytic role in the water phase.
In recent years, in the research of biodiesel, the biocatalysis method using lipase as a catalyst is more and more emphasized because of the advantages of mild reaction conditions, small alcohol dosage, simple post-treatment, no pollutant emission and the like compared with the chemical method, and particularly, the catalyst is easy to recycle after the reaction is finished by adopting immobilized enzyme catalysis, so that the production cost is greatly reduced, and good application prospects are shown. However, the enzyme catalysis method has the problems of complex processes of extraction, separation and purification of enzyme, high cost, easy activity loss of enzyme in the preparation process and the like, so that the whole-cell biocatalysis method directly taking lipase production strains or cells as catalysts becomes an important development direction in recent years, and particularly, the microbial cell surface display technology developed in recent years provides a new biological method based on gene recombination technology for whole-cell catalysis and enzyme immobilization.
The microbial cell surface display is that target protein gene sequence is fused with specific carrier protein gene sequence and introduced into microbial host cell, and the target protein is expressed and located on the surface of microbial cell under the guide of carrier protein and maintained in independent spatial conformation and original biological activity. The whole-cell catalyst is formed by displaying enzyme with catalytic activity on the cell surface of microorganism such as yeast by using a surface display technology. Unlike conventional intracellular and exocrine enzymes, the surface-displayed enzyme is immobilized on the outer surface of a cell in a covalent or non-covalent bond, and thus has many excellent characteristics such as temperature, organic solvent stability, recyclability, etc. At present, phage display systems, bacterial display systems, yeast display systems, and the like have been developed for use in many fields of biological research and industrial production.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the defects in the prior art, the invention aims to provide a double-lipase cell surface co-display engineering bacterium, which takes cell wall protein Sed1p from saccharomyces cerevisiae as an anchor protein, co-displays rhizopus oryzae lipase and candida antarctica lipase B on the cell wall surface of pichia pastoris GS115, and realizes high-efficiency expression. The invention also aims to provide a construction method of the double-lipase cell surface co-display engineering bacterium. The invention also aims to provide application of the double-lipase cell surface co-display engineering bacterium.
The technical scheme is as follows: in order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
a double-lipase cell surface co-display engineering bacterium is constructed by using saccharomyces cerevisiae cell wall protein Sed1p as an anchor protein, using rhizopus oryzae lipase and candida antarctica lipase B as double-lipase target proteins and co-displaying the target proteins on the surface of a pichia pastoris cell.
The construction method of the double-lipase cell surface co-display engineering bacterium comprises the steps of taking Sed1p as an anchoring protein, connecting the anchoring protein with the N end of rhizopus oryzae lipase, fixing rhizopus oryzae lipase ROL on the surface of a pichia pastoris cell in a covalent bond mode through an N end repetitive sequence of the anchoring protein, realizing induced expression, and obtaining recombinant yeast for independently displaying rhizopus oryzae lipase; and then preparing the recombinant yeast which independently displays rhizopus oryzae lipase into competent cells, electrically transforming the constructed candida antarctica lipase B expression plasmid pGAPZ alpha A-CALB-Flag-set 1p into the recombinant yeast competent cells which independently display ROL, and screening to obtain the recombinant yeast positive transformant which co-displays ROL and CALB on the surface.
The construction method of the double-lipase cell surface co-display engineering bacteria comprises the following steps:
(1) cloning a gene sequence of a target protein rhizopus oryzae lipase ROL into an expression vector to obtain a surface display recombinant plasmid vector; cloning the anchoring protein gene Sed1p into a surface display recombinant plasmid vector to form a fusion gene between a saccharomyces cerevisiae cell wall protein gene and a target protein rhizopus oryzae lipase gene, so as to obtain a rhizopus oryzae lipase cell surface display expression plasmid taking the saccharomyces cerevisiae cell wall protein Sed1p as anchoring protein;
(2) electrically converting the rhizopus oryzae lipase cell surface display expression plasmid into pichia pastoris, and screening according to a screening marker on the expression vector to obtain a recombinant pichia pastoris positive transformant for independently displaying rhizopus oryzae lipase;
(3) recombinant yeast displaying rhizopus oryzae lipase ROL alone was prepared as competent cells.
(4) And (2) constructing a cell surface display expression plasmid pGAPZ alpha A-CALB-Flag-Sed1p of the Candida antarctica lipase B by adopting the same method as the step (1), electrically converting the pGAPZ alpha A-CALB-Flag-Sed1p into a recombinant yeast competent cell which independently displays the Rhizopus oryzae lipase, and screening positive transformants to obtain the recombinant yeast cell which co-displays the Rhizopus oryzae lipase ROL and the Candida antarctica lipase B cell surface.
The double-lipase cell surface co-display engineering bacteria is used as a double-lipase cell surface co-display type whole-cell catalyst in the application of catalyzing grease transesterification to prepare biodiesel.
The grease raw materials for preparing the biodiesel by catalytic transesterification are vegetable grease such as soybean oil, rapeseed oil, jatropha curcas oil or jatropha curcas oil, rubber seed oil and the like, and can also be microalgae grease and waste catering grease.
The rhizopus oryzae lipase and candida antarctica lipase B cell surface co-display type whole cell catalysis can combine the advantages of high catalytic efficiency and short catalytic reaction time of two enzymes in a water phase, has more remarkable advantages than the existing immobilized enzyme catalysis, non-surface display type whole cell catalysis and single lipase surface display type whole cell catalysis, and is certainly the latest development trend in the field of biodiesel preparation by grease catalytic transesterification. Therefore, it is very necessary to research the lipase cell surface co-display type whole cell catalyst by combining genetic engineering technology and cell surface engineering technology.
Has the advantages that: compared with the prior art, the rhizopus oryzae lipase and candida antarctica lipase B are displayed on the cell wall surface of the pichia pastoris together, the surface co-display type whole-cell catalyst for the rhizopus oryzae lipase and candida antarctica lipase B is prepared, the highest enzyme activity of the surface co-display type whole-cell catalyst is 686U/g-dry cell weight, the highest enzyme activity is 3 times and 2 times that of the surface co-display type whole-cell catalyst for the rhizopus oryzae lipase or candida antarctica lipase B which is independently displayed, and the yield of catalytic grease transesterification methyl ester reaches 96%.
Drawings
FIG. 1 is a diagram of recombinant plasmid construction;
FIG. 2 is a diagram of a surface co-display recombinant strain for plate screening of high lipase production; wherein (a) a tricaprylin plate, (b) an olive oil-MMH-RB plate;
FIG. 3 is a graph showing the results of fluorescence analysis of the surface co-displaying recombinant genetically engineered bacteria; wherein, 1, white light, 2.488nm, 3.555 nm;
FIG. 4 is a graph of the results of a Westernblot analysis; wherein, lane2, (a); lane1, (b) is surface co-display recombinant bacteria GS115/pGAPZA-RCS cell wall protein; lane3, (a) is ROL surface display recombinant bacterium GS115/pPICZ alpha A-ROL-Sed1p cell wall protein, lane2, (b) is CALB surface display recombinant bacterium GS115/pGAPZ alpha A-CALB-Sed1p cell wall protein, lane1, (a); lane3, (b) is pichia pastoris GS115 cell wall protein;
FIG. 5 is a graph showing the results of enzyme activities of surface-co-displayed lipases; wherein,the surface co-displays the lipase activity of the recombinant strain GS115/pGAPZ alpha A-RCS;displaying the lipase activity of the recombinant strain GS115/pGAPZ alpha A-CALB-set 1p on the surface of the ROL;the CALB surface displays the lipase activity of the recombinant strain GS115/pPICZ alpha A-ROL-Sed1 p;
FIG. 6 is a graph of methyl ester yield from transesterification of soybean oil catalyzed by a surface co-display whole-cell catalyst;
FIG. 7 is a graph of the reusability of surface co-displayed whole cell catalysts.
Detailed Description
The media and related reagents used in the following examples are as follows:
10 XYNB (1L): dissolving 134gYNB (containing ammonium sulfate and no amino acid) in 1LddH2O, dissolved at 50 ℃ or lower.
BMGY (1L): dissolving yeast extract 10g and peptone 20g in 700ml ddH2O, sterilized at 121 ℃ for 20min, cooled to room temperature, and added with 100mL of 1mol/L potassium phosphate buffer (pH6.0), 100mL of 10 XYNB, 2mL of 500 XB, and 100mL of 10 GY.
BMMY (1L): dissolving yeast extract 10g and peptone 20g in 700ml ddH2O, sterilized at 121 ℃ for 20min, cooled to room temperature, and added with 100mL of 1mol/L potassium phosphate buffer (pH6.0), 100mL of 10 XYNB, 2mL of 500 XB, and 100mL of 10 XM.
25% polyvinyl alcohol olive oil emulsion (1L): 3g of polyvinyl alcohol was weighed, 150mL of distilled water was added thereto, and the mixture was dissolved by heating and stirring to obtain a 2% solution. 50mL of olive oil was added thereto, and the mixture was stirred with a high-speed tissue masher for 3 to 4 times for 10 seconds each time.
Olive oil-MMH-RB plate (1L): adding 15g of agar into 800mL of olive oil emulsion, sterilizing at 121 ℃ for 20min, cooling to 60 ℃, adding 100mL of 10 XYNB, 2mL of 500 XB, 100mL of 10 XM, 100mL of 10 XB and 10mL of 100 XH, mixing uniformly, and pouring into a plate.
Glyceryl tricaprylate polyvinyl alcohol emulsion with certain concentration: 3g of polyvinyl alcohol was weighed, 150mL of distilled water was added thereto, and the mixture was dissolved by heating and stirring to obtain a 2% solution. Stirring with a 2% polyvinyl alcohol solution and tricaprylin at a volume ratio of 3:1 for 4-5 times (20 s each time) by using a miniature high-speed homogenizer until white emulsion appears, and slightly heating or increasing stirring times and time if the emulsion is not uniform or layered.
Tricaprylin plate (1L): 10g of yeast extract and 20g of peptone in 900ml of ddH2Dissolving in O, adding tricaprylin polyvinyl alcohol emulsion with a certain concentration, diluting to a constant volume of 1L with deionized water (10 XD is required and no constant volume is required), mixing, adding 1.5% (w/v) agar powder, sterilizing at 121 deg.C for 20min, adding 10 XD after sterilization, and flattening to obtain the final product which can be stored at 4 deg.C.
Example 1
Extraction of saccharomyces cerevisiae eby100 genome, comprising the following steps:
(1) cerevisiae eby100 was purchased from Invitrogen. Firstly, activating strains on YPD plates, picking single colonies in YPD shake flasks, and culturing at 30 ℃ overnight.
(2) The thalli in logarithmic growth phase is taken and centrifuged at 5000rpm/min for 5min, the thalli is washed once by sterile distilled water, and the supernatant is removed.
(3) Adding 5ml of wall-breaking buffer solution (100mmol/LTris-HCl, pH8.0,10mmol/LEDTA, 1% SDS), mixing, and keeping the temperature at 65 ℃ for 1 h.
(4) Centrifuging at 10000rpm for 15min, and collecting supernatant.
(5) Adding phenol chloroform isoamyl alcohol (25:24:1) for 2 times, and centrifuging at 5000rpm for 7min each time.
(6) The upper layer extract was added with 6. mu.L NaseA (10mg/mL), and precipitated with 3-fold ethanol overnight (0.3 mol/L sodium acetate, pH 5.2). Centrifuge at 12000rpm for 30 min.
(7) Washing with 75% ethanol for 2 times, and air drying.
(8) Dissolving with 50 μ L sterile ultrapure water.
Example 2
The construction of a Rhizopus Oryzae Lipase (ROL) Pichia pastoris cell surface display system comprises the following steps:
A. construction of surface display recombinant plasmid vector pPICZ alpha A-ROL
The Rhizopus oryzae lipase gene ROL is cloned and fused with plasmid pPICZ alpha A. Primers were designed based on the plasmid pPIC9K-ROL containing the Rhizopus oryzae lipase gene sequence, and were synthesized by Shanghai Bioengineering Co., Ltd.
R1(5 '-3', the same applies below): CCGGAATTCATGGTTCCTGTTTCTGGTAAATCTG, preceded by an EcoRI cleavage site.
R2：TCCCCGCGGATGATGATGATGATGATGCAAACAGCTTCCTTCGTTGATATCA, the SacII cleavage site and His-tag protein (underlined) were added to the above.
Performing PCR amplification by using the synthesized primers, wherein a PCR reaction system comprises the following steps: 1 μ L of plasmid pPIC9K-ROL, 1 μ LR1 primer, 1 μ LR2 primer, 25 μ LPremixExtaq, 22 μ LddH2And O. The amplification conditions were: denaturation at 94 deg.C for 2 min; denaturation at 94 ℃ for 30 s; annealing at 55 deg.C for 1 min; extending at 72 ℃ for 1 min; repeating the cycle for 30 times; extending at 72 ℃ for 10 min; the reaction was stopped and the temperature was maintained at 4 ℃. And purifying the PCR product by using a gel recovery kit to obtain the rhizopus oryzae lipase ROL gene with the His-tag protein gene.
The rhizopus oryzae lipase ROL gene and pPICZ alpha A are cut by EcoRI and SacII and purified and recovered. T4DNA ligase is used for connection to construct a surface display recombinant plasmid pPICZ alpha A-ROL.
B. Construction of Yeast expression vector pPICZ alpha A-ROL-Histag-Sed1p
(1) PCR amplification of mature Anchorin Gene Sed1p
Primers synthesized by Shanghai Czeri bioengineering, Inc. were designed according to the known gene sequence (GeneID:851649) of Saccharomyces cerevisiae Sed1p protein.
S1: TCCCCGCGGCAATTTTCCAACAGTACATCTGCTTCT, the SacII cleavage site was added previously.
S2: CTAGTCTAGATTATAAGAATAACATAGCAACACCAGC, Xba I cleavage sites were added previously.
And performing PCR amplification by using the synthesized primers by using the extracted saccharomyces cerevisiae EBY100 genome DNA as a template. And (3) PCR reaction system: 1 μ L Saccharomyces cerevisiae EBY100 genomic DNA, 1 μ LS1 primer, 1 μ LS2 primer, 25 μ LPremixExtaq, 22 μ LddH2And O. The amplification conditions were: denaturation at 94 deg.C for 5 min; denaturation at 94 ℃ for 30 s; annealing at 56 deg.C for 30 s; extending at 72 ℃ for 1 min; repeating the cycle for 30 times; extending at 72 ℃ for 10 min; the reaction was stopped and the temperature was maintained at 4 ℃. The PCR product was purified by a gel recovery kit to obtain the mature dockerin gene Sed1p.
(2) Sed1p and pPICZ alpha A-ROL are cut by SacII and Xba I and purified and recovered by a kit, and are connected by T4DNA ligase to obtain a recombinant Pichia pastoris surface display expression plasmid pPICZ alpha A-ROL-Histagg-Sed 1p. The resulting pPICZ alpha A-ROL-Histag-Sed1p plasmid was transformed into the E.coli host TOP 10F. Transformants were selected on plates containing 50. mu.g/ml Zeocin LLB, transformants positive for Zeocin resistance were selected, plasmids were extracted and sequenced, indicating that the muramyl sed1p gene sequence was correctly inserted. The construction method is shown in FIG. 1 (a).
C. Expression of recombinant plasmid pPICZ alpha A-ROL-Histag-Sed1p in Pichia pastoris
The plasmid pPICZ alpha A-ROL-Histag-Sed1p was linearized with the restriction enzyme SacI. Electrically transforming Pichia pastoris GS115, growing on YPD plate containing 100. mu.g/mLzeocin for 72h, picking up resistance positive transformant, i.e. transformant integrated with fusion gene sequence, dropping to high resistance YPD plate containing 400. mu.g/mLzeocin, and picking up larger strain, i.e. transformant containing more copies.
Example 3
The construction of a Candida Antarctica Lipase B (CALB) Pichia pastoris surface display system comprises the following steps:
A. construction of surface display recombinant plasmid vector pGAPZ alpha A-CALB
Cloning candida antarctica lipase B gene CALB, and fusing with pGAPZ alpha A. A primer is designed according to a gene sequence of candida antarctica lipase B, and is synthesized by Shanghai biological engineering Co.
C1: CCGGAATTCATGAAGCTACTCTCTCTGACCGGTG, preceded by an EcoRI cleavage site.
C2: AAGGAAAAAAGCGGCCGCTGGGGGTGACGATGCCGGAGC, preceded by a Not I cleavage site.
PCR amplification with synthetic primers using Candida antarctica genomic DNA as template). And (3) PCR reaction system: 1 uL Candida antarctica genomic DNA, 1 uLC 1 primer, 1 uLC 2 primer, 25 uLPremixExtaq, 22 uLddH2And O. The amplification conditions were: denaturation at 94 deg.C for 2 min; denaturation at 94 ℃ for 30 s; annealing at 55 deg.C for 1 min; extending at 72 ℃ for 1 min; repeating the cycle for 30 times; extending at 72 ℃ for 10 min; the reaction was stopped and the temperature was maintained at 4 ℃. And purifying the PCR product by using a gel recovery kit to obtain the Candida antarctica lipase B gene.
The Candida antarctica lipase B gene and pGAPZ alpha A are cut by EcoR I and Not I, purified and recovered. T4DNA ligase is used for connection to construct a surface display recombinant plasmid pGAPZ alpha A-CALB.
B. Construction of Yeast expression vector pGAPZ alpha A-CALB-Flag-Sed1p
(1) PCR amplification of mature Anchorin Gene Sed1p
Primers were designed according to the known gene sequence of Saccharomyces cerevisiae Sed1p protein, and were synthesized by Shanghai Jie-Bioengineering Co., Ltd.
SC1：AAGGAAAAAAGCGGCCGCGATTACAAGGATGACGACGATAAGATGAAATTATCAACTGTCCTATTATCT, preceded by a Not I cleavage site and a FLAG tag protein (underlined).
SC 2: CTAGTCTAGATTATAAGAATAACATAGCAACACCAG, Xba I cleavage sites were added previously.
And performing PCR amplification by using the synthesized primers by using the extracted saccharomyces cerevisiae EBY100 genome DNA as a template. And (3) PCR reaction system: 1 μ L Saccharomyces cerevisiae EBY100 genomic DNA, 1 μ LSC1 primer, 1 μ LSC2 primer, 25 μ LPremixExtaq, 22 μ LddH2And O. The amplification conditions were: denaturation at 94 deg.C for 5 min; denaturation at 94 ℃ for 30 s; annealing at 56 deg.C for 30 s; extending at 72 ℃ for 1 min; repeating the cycle for 30 times; extending at 72 ℃ for 10 min; the reaction was stopped and the temperature was maintained at 4 ℃. The PCR product was purified by a gel recovery kit to obtain the mature dockerin gene Sed1p.
(2) Sed1p and pGAPZ alpha A-CALB were digested with Not I and Xba I, purified and recovered with a kit, and ligated with T4DNA ligase to obtain recombinant Pichia pastoris surface display expression plasmid pGAPZ alpha A-CALB-Flag-Sed 1p. The obtained pGAPZ alpha A-CALB-Flag-Sed1p plasmid was transformed into the E.coli host TOP 10F. Transformants were selected on plates containing 50. mu.g/ml Zeocin LLB, transformants positive for Zeocin resistance were selected, plasmids were extracted and sequenced, and the result indicated that the muramyl Sed1p gene sequence was correctly inserted. The construction method is shown in FIG. 1 (b).
C. Expression of recombinant plasmid pGAPZ alpha A-CALB-Flag-Sed1p in Pichia pastoris
The plasmid pGAPZ. alpha.A-CALB-Flag-Sed 1p was linearized with the restriction enzyme SacI. Electrically transforming Pichia pastoris GS115, growing on YPD plate containing 100. mu.g/mLzeocin for 72h, picking up resistance positive transformant, i.e. transformant integrated with fusion gene sequence, dropping to high resistance YPD plate containing 400. mu.g/mLzeocin, and picking up larger strain, i.e. transformant containing more copies.
Example 4
The construction and identification of the rhizopus oryzae lipase and candida antarctica lipase B surface co-display recombinant gene engineering bacteria comprise the following steps:
A. construction of surface co-display recombinant gene engineering bacteria GS115/pGAPZ alpha A-RCS
A competent cell was prepared from the recombinant Pichia pastoris GS 115/pPICZ. alpha.A-ROL-Sed 1p, which exhibits Rhizopus Oryzae Lipase (ROL) on the surface.
The expression vector pGAPZ alpha A-CALB-Flag-Sed1p was linearized by restriction with the restriction enzyme SacI, and the complete linearization was checked by electrophoresis. After the expression vector is completely linearized, the expression vector is transformed into competent cells of recombinant Pichia pastoris GS115/pPICZ alpha A-ROL-Sed1p and is coated on a YPDS plate containing Zeocin antibiotic. And screening YPDS plates containing Zeocin antibiotics with different concentrations to obtain high-copy recombinant bacteria. Transformants were picked, spotted on olive oil-MMH-RB plates and tricaprylin plates of a certain concentration, and the larger hydrolytic circle bacteria were picked (FIGS. 2(a), (b)), and the surface co-display strain was designated GS 115/pGAPZ. alpha.A-RCS for subsequent culture.
B. Identification of surface co-display recombinant gene engineering bacteria GS115/pGAPZ alpha A-RCS
(1) Fluorescence analysis of surface co-display recombinant gene engineering bacteria
Inoculating positive transformant GS115/pGAPZ alpha A-RCS into 50mLBMGY culture medium, and performing shaking culture at 30 ℃ and 180rpm for 16-20h to OD600To 2-6. Collecting thallus by centrifugation, suspending in BMMY culture medium, and diluting to OD600And (5) continuing the shaking culture, and supplementing 0.5% methanol into the BMMY culture medium every 24h for induction expression. After fermentation for 72h, respectively carrying out immunoreaction by anti-Histag and anti-Flag antibodies, and then carrying out fluorescence microscopy on GS115/pGAPZ alpha A-RCS, GS115/pPICZ alpha A-ROL-Sed1p, GS115/pGAPZ alpha AA-CALB-Sed1p was analyzed and compared with the control strain GS 115. The detection results of the culture induction and the fluorescent antibody incubation under the same conditions at the ultraviolet 488nm and the ultraviolet 555nm are shown in figure 3. The lipase recombinant bacteria GS115/pPICZ alpha A-ROL-Sed1p and GS115/pGAPZ alpha A-RCS containing histidine tags show green fluorescence under 488nm blue light excitation, while the blank bacteria GS115 without histidine tags cannot be combined with a fluorescent antibody, so that no fluorescence is displayed. Similarly, the lipase recombinant bacteria GS115/pGAPZ alpha A-CALB-Sed1p containing the Flag tag and GS115/pGAPZ alpha A-RCS show red fluorescence under the excitation of green light at 555nm, while the blank bacteria GS115 without the Flag tag can not be combined with a fluorescent antibody, so that no fluorescence is shown. This result demonstrates that both lipases ROL and CALB are successfully displayed anchored on the Pichia cell surface by the anchor protein Sed1p.
(2) Analysis of surface co-display recombinant Pichia cell wall protein
Culturing a double-lipase co-display strain GS115/pGAPZ alpha A-RCS in a shake flask, and taking a blank strain Pichia pastoris GS115, a single-lipase surface display strain GS115/pPICZ alpha A-ROL-Sed1p and GS115/pGAPZ alpha A-CALB-Sed1p as negative controls. Cell wall proteins of the above strains were extracted by laminarinase and subjected to the WesternBlot analysis. As shown in FIG. 4, FIG. 4(a) shows the results of Western-blot immunoblotting using an anti-His tag antibody. Lanes 2 and 3 are cell wall proteins of recombinant bacteria GS 115/pGAPZ. alpha.A-RCS and GS 115/pPICZ. alpha.A-ROL-Sed 1p showing ROL, respectively, and have an obvious band with a size of about 75kDa, which is consistent with a theoretical value, and in addition, the cell wall protein of the recombinant bacteria GS115 in lane1 is used as a negative control, and no band is formed. Similarly, FIG. 4(b) is the result of Western-blot immunohybridization using an anti-Flag-tagged antibody. Lanes 1 and 2 show cell wall proteins of CALB-displaying recombinant bacteria GS115/pGAPZ alpha A-RCS and GS115/pGAPZ alpha A-CALB-Sed1p, and similarly, a band is evident around 75kDa and is consistent with a theoretical value, and no band is formed by using the cell wall protein of GS115 bacteria in lane3 as a negative control. This result again demonstrates the success of the construction of surface co-display recombinant pichia pastoris.
Example 5
The surface co-display recombinant gene engineering bacteria GS115/pGAPZ alpha A-RCS lipase activity determination process comprises the following steps:
the screened double lipase display strain GS115/pGAPZ alpha A-RCS is cultured in a shake flask, and the optimal induction time and the highest enzyme activity of the strain are examined by using an optimal reaction substrate pNPD (4-nitrophenyldodecanoate) of lipase. 1000. mu.L of enzyme activity assay system: 0.05mol/L of LTris-HCLbufferfercH is 7.5850 mu L, 100 mu L of bacterial liquid with a certain concentration is diluted, and 13.5mmol/L of pNPD50 mu L.
After the recombinant strain was induced to express for 144 hours, 6,000g of the cells were collected by centrifugation at room temperature, washed 3 times with 50mM pH7.5 PBS buffer, and finally resuspended in 50mM pH7.5 PBS buffer. Adjusting OD of different samples600And (3) preliminarily obtaining recombinant bacteria GS115/pGAPZ alpha A-RCS for co-displaying lipase, separately displaying recombinant bacteria GS115/pPICZ alpha A-ROL-Sed1p and GS115/pGAPZ alpha A-CALB-Sed1p of lipase and bacterial suspension of a control strain Pichia pastoris GS115, and reacting for 15min at 45 ℃, pH8.0 and proper reaction speed. And an absorbance method is selected for measuring the activity of the displayed lipase. 1 enzyme activity unit (IU) is defined as: the amount of enzyme required to hydrolyze one μmol of p-nitrophenol formed by the substrate p-nitrophenol ester pNPD per minute.
As shown in figure 5, the enzyme activity of the lipase displayed on the surface of Pichia pastoris increases with the increase of induction time, the activity is highest when the surface co-displayed lipase GS115/pGAPZA-RCS is cultured for 144h, the highest enzyme activity reaches 686U/g of stem cells, the lipase activities of ROL and CALB displayed on the surface alone are 217U/g of stem cells and 379U/g of stem cells respectively, and the surface co-displayed lipase activity is obviously higher than that of ROL and CALB displayed on the surface alone.
Example 6
The surface co-display type whole-cell catalyst is used for catalyzing grease transesterification to prepare biodiesel, and the steps are as follows:
A. preparation of surface co-display whole-cell catalyst: the surface co-display pichia pastoris engineering bacteria produce enzyme which is secreted outside cells, the target product is thallus, and the purpose of induction expression is to obtain cells with high enzyme activity as much as possible. The induction expression method of the recombinant pichia pastoris engineering bacteria comprises the following steps:
(1) the screened GS115/pGAPZ alpha A-RCS genetically engineered bacteria are cultured on a YPD plate for 2d at 30 ℃. Selecting single colony, inoculating into 500mL shake flask containing 150mLBMGY culture medium, culturing at 28 deg.C and 200r/min to OD600＝2～6。
(2) Centrifuging at room temperature at 3000 Xg for 5min to obtain thallus, removing supernatant, and re-suspending thallus with new BMMY culture medium to OD600Approximately equal to 1.0.
(3) Add 400mL of resuspended BMMY culture to a 1L shake flask, cover the mouth with 4 layers of sterile gauze, continue shaking table culture at 28 ℃ and 200 r/min.
(4) The induction was maintained by adding methanol to the flask every 24h, with the final concentration of 0.5% methanol.
(5) After 72 hours of induction of BMMY medium, the cells were collected by centrifugation at 6000 rpm.
(6) And washing the collected thalli for 3 times by using a buffer solution, and then using the thalli as a whole cell catalyst for subsequent use.
(7) The lipase activity of the surface co-display was determined as described in example 5.
B. Surface co-display type whole-cell catalyst for catalyzing transesterification of grease
Adding 5g of grease and 0.18g of methanol into a 50mL triangular flask with a plug according to the molar ratio of 1:1, adding a certain amount of deionized water to control the water content of a reaction system to be 50-80%, weighing a surface co-display recombinant bacterium GS115/pGAPZA-RCS whole-cell catalyst with the total enzyme amount of 60-120U, and adding the whole-cell catalyst into the triangular flask. Placing the system in a constant temperature culture oscillator at 35-50 ℃, carrying out methyl esterification reaction at 200r/min, adding methanol with a molar ratio of 1:1 in batches at intervals of 12 hours, taking 100 mu L of supernatant at regular intervals, carrying out gas chromatography analysis on a sample after centrifugation, and calculating the methyl ester yield of the grease transesterification reaction.
1) Surface co-display type whole-cell catalyst for catalyzing methyl esterification of soybean oil
5g of commercially available soybean oil and 0.18g of methanol are added into a 50mL triangular flask with a plug according to the molar ratio of 1:1, a surface co-display recombinant bacterium GS115/pGAPZ alpha A-RCS whole-cell catalyst with the total enzyme amount of 80U is added, and water is added to adjust the water content of the reaction system to 70%. Reacting at the reaction temperature of 45 ℃ and the shaking rotation speed of 200r/min, and adding methanol in batches at the molar ratio of 1:1 for 12h, 24h and 48h respectively. 100 μ L of the supernatant was collected at regular intervals, and after centrifugation, the sample was subjected to gas chromatography, and the methyl ester yield of the transesterification reaction was shown in FIG. 6 as a function of time. When the reaction is carried out for 60 hours, the yield of the methyl ester can reach 96.0 percent at most.
2) Surface co-display type whole-cell catalyst for catalyzing methyl esterification of rapeseed oil
5g of commercially available rapeseed oil and 0.18g of methanol are added into a 50mL triangular flask with a plug according to the molar ratio of 1:1, a surface co-display recombinant bacterium GS115/pGAPZ alpha A-RCS whole-cell catalyst with the total enzyme amount of 80U is added, and water is added to adjust the water content of a reaction system to 70%. Reacting at the reaction temperature of 45 ℃ and the shaking rotation speed of 200r/min, and adding methanol in batches according to the molar ratio of 1:1 for 12h, 24h and 48h respectively. When the reaction is carried out for 60 hours under the condition, the yield of the methyl ester is 94.8 percent.
3) Surface co-display type whole-cell catalyst for catalyzing methylesterification of jatropha curcas seed oil (jatropha curcas oil)
5g of jatropha curcas seed oil subjected to solvent extraction and degumming treatment and 0.18g of methanol are added into a 50mL triangular flask with a plug, a surface co-display recombinant bacterium GS115/pGAPZ alpha A-RCS whole-cell catalyst with the total enzyme amount of 100U is added, and water is added to adjust the water content of a reaction system to 60%. Reacting at the reaction temperature of 40 ℃ and the shaking rotation speed of 200r/min, and adding methanol in batches according to the molar ratio of 1:1 for 12h, 24h and 48h respectively. When the reaction is carried out for 60 hours under the condition, the yield of the methyl ester is 93.2 percent.
4) Surface co-display type whole-cell catalyst for catalyzing methyl esterification of waste catering grease
5g of the pretreated restaurant waste grease and 0.18g of methanol are added into a 50mL triangular flask with a stopper, a surface co-display recombinant bacterium GS115/pGAPZ alpha A-RCS whole-cell catalyst with the total enzyme amount of 100U is added, and water is added to adjust the water content of the reaction system to 60%. Reacting at the reaction temperature of 40 ℃ and the shaking rotation speed of 200r/min, and adding methanol in batches according to the molar ratio of 1:1 for 12h, 24h and 48h respectively. When the reaction is carried out for 60 hours under the condition, the yield of the methyl ester is 94.0 percent.
5) Surface co-display type whole-cell catalyst for catalyzing methyl esterification of microalgae grease
Adding 5g of microalgae oil extracted from chlorella and 0.18g of methanol into a 50mL triangular flask with a plug, adding a surface co-display recombinant bacterium GS115/pGAPZ alpha A-RCS whole-cell catalyst with the total enzyme amount of 120U, and adding water to adjust the water content of a reaction system to 60%. Reacting at the reaction temperature of 45 ℃ and the shaking rotation speed of 200r/min, and adding methanol in batches according to the molar ratio of 1:1 for 12h, 24h and 48h respectively. When the reaction is carried out for 60 hours under the condition, the yield of the methyl ester is 95.5 percent.
C. Reusability of surface co-display type whole-cell catalyst
5g of commercially available soybean oil and 0.18g of methanol are added into a 50mL triangular flask with a plug according to the molar ratio of 1:1, a surface co-display recombinant bacterium GS115/pGAPZ alpha A-RCS whole-cell catalyst with the total enzyme amount of 80U is added, and water is added to adjust the water content of the reaction system to 70%. Reacting at the reaction temperature of 45 ℃ and the shaking rotation speed of 200r/min, and adding methanol in batches according to the molar ratio of 1:1 for 12h, 24h and 48h respectively.
After 60 hours of reaction, the cells were collected by centrifugation at 6000rpm, washed with deionized water for 3 times and reused, and the results of catalyzing methyl esterification of soybean oil are shown in fig. 7. The yield of methyl ester of the whole-cell catalyst for catalyzing the transesterification reaction of the grease after being repeatedly used for 10 times is still kept above 80 percent, which shows that the catalyst has better repeated use performance.
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Claims (5)
1. A double-lipase cell surface co-display engineering bacterium is characterized in that: the saccharomyces cerevisiae cell wall protein Sed1p is used as an anchoring protein, rhizopus oryzae lipase and candida antarctica lipase B are used as double-lipase target proteins and are jointly displayed on the surface of a pichia pastoris cell, and the constructed double-lipase cell surface co-display recombinant yeast engineering bacteria are constructed.
2. The method for constructing the double-lipase cell surface co-display engineering bacteria as claimed in claim 1, wherein Sed1p is used as an anchoring protein, the anchoring protein is connected with the N end of rhizopus oryzae lipase, and rhizopus oryzae lipase ROL is fixed on the surface of a pichia pastoris cell by a covalent bond mode through an N end repetitive sequence of the anchoring protein to realize induced expression, so that recombinant yeast for independently displaying rhizopus oryzae lipase is obtained; and then preparing the recombinant yeast which independently displays rhizopus oryzae lipase into competent cells, electrically transforming the constructed candida antarctica lipase B expression plasmid pGAPZ alpha A-CALB-Flag-set 1p into the recombinant yeast competent cells which independently display ROL, and screening to obtain the recombinant yeast positive transformant which co-displays ROL and CALB on the surface.
3. The method for constructing the double-lipase cell surface co-display engineering bacterium according to claim 2, which comprises the following steps:
(1) cloning a gene sequence of a target protein rhizopus oryzae lipase ROL into an expression vector to obtain a surface display recombinant plasmid vector; anchoring protein geneSed1pCloning to a surface display recombinant plasmid vector to form a fusion gene between a saccharomyces cerevisiae cell wall protein gene and a target protein rhizopus oryzae lipase gene to obtain a rhizopus oryzae lipase cell surface display expression plasmid taking a saccharomyces cerevisiae cell wall protein Sed1p as an anchoring protein;
(2) electrically converting the rhizopus oryzae lipase cell surface display expression plasmid into pichia pastoris, and screening according to a screening marker on the expression vector to obtain a recombinant pichia pastoris positive transformant for independently displaying rhizopus oryzae lipase;
(3) preparing a recombinant yeast which independently displays rhizopus oryzae lipase ROL into a competent cell;
(4) and (2) constructing a cell surface display expression plasmid pGAPZ alpha A-CALB-Flag-Sed1p of the Candida antarctica lipase B by adopting the same method as the step (1), electrically converting the pGAPZ alpha A-CALB-Flag-Sed1p into a recombinant yeast competent cell which independently displays the Rhizopus oryzae lipase, and screening positive transformants to obtain the recombinant yeast cell which co-displays the Rhizopus oryzae lipase ROL and the Candida antarctica lipase B cell surface.
4. The use of the double-lipase cell surface co-display engineering bacterium of claim 1 as a double-lipase cell surface co-display whole-cell catalyst in the preparation of biodiesel by grease transesterification.
5. The use of claim 4, wherein the oil and fat used for preparing biodiesel by catalytic transesterification is soybean oil, rapeseed oil, jatropha curcas oil or jatropha curcas oil, rubber seed oil, microalgae oil, and waste catering oil and fat.
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| 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 |
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