CN110643592A - Method for modifying whole cells by cholate-metal ion composite and application thereof - Google Patents
Method for modifying whole cells by cholate-metal ion composite and application thereof Download PDFInfo
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- CN110643592A CN110643592A CN201911036895.3A CN201911036895A CN110643592A CN 110643592 A CN110643592 A CN 110643592A CN 201911036895 A CN201911036895 A CN 201911036895A CN 110643592 A CN110643592 A CN 110643592A
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- 229910021645 metal ion Inorganic materials 0.000 title claims abstract description 55
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- 238000003756 stirring Methods 0.000 claims abstract description 9
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims abstract description 8
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- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 claims description 3
- 150000001491 aromatic compounds Chemical class 0.000 claims description 3
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- -1 iron ion Chemical class 0.000 claims description 3
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- VMSNAUAEKXEYGP-YEUHZSMFSA-M sodium glycodeoxycholate Chemical compound [Na+].C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 VMSNAUAEKXEYGP-YEUHZSMFSA-M 0.000 claims description 2
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- YXHRQQJFKOHLAP-FVCKGWAHSA-M sodium;2-[[(4r)-4-[(3r,5r,8r,9s,10s,12s,13r,14s,17r)-3,12-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]ethanesulfonate Chemical compound [Na+].C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 YXHRQQJFKOHLAP-FVCKGWAHSA-M 0.000 claims description 2
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- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
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Abstract
The invention discloses a method for modifying whole cells by cholate-metal ion composite modification and application thereof. The method comprises the following steps: preparing a 100-solution 600mmol/L metal ion solution, and stirring and mixing the solution with a Tris-HCl heavy suspension strain cell solution with the pH of 7.1-8.0 and the concentration of 0.1mol/L to obtain a mixed solution A; preparing 50-300 mmol/L surfactant aqueous solution, and dropwise adding the surfactant aqueous solution into the mixed solution A at the dropwise adding rate of 5-20 mu L/s while stirring at room temperature until the mixed solution A is turbid to obtain mixed solution B; and centrifuging the mixed solution B to obtain a precipitate, washing the precipitate for 1-3 times by using Tris-HCl with the pH of 7.1-8.0 and the concentration of 0.05mol/L, and washing away unreacted components to obtain the cholate-metal ion compound modified whole cell. The method adopts an aqueous phase solution mixing method for preparation, and has the advantages of simple operation, mild conditions, rapid synthesis, low cost, high cell stability, high intracellular enzyme catalysis efficiency, and both biological and physical catalytic activities.
Description
Technical Field
The invention relates to the technical field of microbial catalysis, in particular to a method for modifying whole cells by cholate-metal ion composite modification and application thereof.
Background
Biocatalysis is divided into microbial catalysis and enzyme catalysis. The biological catalysis of the microorganism has the characteristics of environmental protection and high efficiency. Compared with enzymes, the biological catalysis of the microorganisms does not need to add expensive cofactors, can realize the combined catalysis of a plurality of enzymes, and has incomparable advantages of enzyme catalysis. However, in a complex catalytic system, microorganisms face the disadvantages of large mass transfer resistance of cell membranes, easy cell death, low catalytic efficiency, difficult product recovery and the like. The traditional field of immobilized technical materials (such as calcium carbonate microspheres, activated carbon particles, polyurethane particles, etc.) is limited to the protection of cells by embedding immobilized materials, and the immobilized materials can cause further increase of mass transfer resistance, including the mass transfer resistance between solutions and materials and the mass transfer resistance in materials. In addition, the interaction mode between the nano-immobilization material field (such as metal organic framework MOFs, polydopamine coating) and the cell can be divided into: 1. adsorption of cell membranes to materials; 2 covalent binding of cell membranes to the material; 3. constructing a micro-matrix on the surface of the cell membrane. Due to the diversity of microorganism species, the components of cell membranes on cell surfaces vary widely, and the nano-immobilization materials under these action forms are often only suitable for one or a few microorganisms and applied to a single catalytic system. Therefore, an immobilization material capable of fusing with microbial cell membranes is developed, so that the immobilization material is embedded in the microbial cell membranes, the mass transfer resistance of substances can be reduced by utilizing the characteristics of the material, and the catalytic activity of cells is improved.
Chinese patent application No. 201710234386.6 discloses a method for preparing a surfactant-enzyme nanocomposite catalyst, which encapsulates enzyme by controlling the surface activity and the concentration of metal ions, and then catalyzes in a water-oil two-phase system. The method is simple to operate, the catalytic effect is obviously improved, but the method utilizes spherical materials with surface activity and metal ions to form about 500nm to encapsulate the enzyme, the enzyme is fixed in the internal environment of the spherical materials, the encapsulation technology in the sphere is utilized to provide stable catalytic environment for enzyme catalysis, and the immobilization of cells cannot be realized due to the small sphere size.
The chinese patent application No. 201810825757.2 discloses a method for preparing magnetic-loaded ionic liquid microsphere immobilized cells, which can realize rapid separation and protect cell activity. However, the method relies mainly on sodium alginate, chitosan and Ca2+The formed microspheres realize direct embedding of microorganisms, and the microspheres have large size (3.6 mm), so that the mass transfer efficiency of the microorganisms embedded in the material is reduced. No action occurs between the immobilized material and the microbial cell membrane, and the mass transfer efficiency of the substance into and out of the cell membrane is not changed.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for modifying whole cells by cholate-metal ion composite and application thereof. The method adopts an aqueous phase solution mixing method for preparation, and has the advantages of simple operation, mild conditions, rapid synthesis, low cost, high cell stability, high intracellular enzyme catalysis efficiency, and both biological and physical catalytic activities.
A method for modifying whole cells with cholate-metal ion complexes, comprising the steps of:
and 3, centrifuging the mixed solution B to obtain a precipitate, washing the precipitate for 1-3 times by using Tris-HCl with the pH of 7.1-8.0 and the concentration of 0.05mol/L, and washing away unreacted components to obtain the compound modified whole cell.
The improvement is that the surfactant is any one of sodium deoxycholate, sodium taurodeoxycholate, sodium glycodeoxycholate or sodium chenodeoxycholate.
The metal ions are any one of cobalt ions, calcium ions, zinc ions, manganese ions, barium ions, copper ions, nickel ions, tin ions, magnesium ions or iron ions.
The improvement is that the strain cell is any one of gram-negative bacteria, gram-positive bacteria or yeast or a combination of a plurality of strain cells.
As a modification, the stirring speed in step 1 and step 2 was 200 ~ 800 rpm.
As an improvement, the added OD of the cell resuspension solution of the strain described in step 1600The concentration was 1 ~ 10 OD.
The improvement is that in the step 3, the centrifugal separation is carried out for 5 ~ 10min, and the rotating speed during the centrifugal separation is 1000 ~ 5000 rpm.
The cholate-metal ion compound modified whole cell is applied to catalyzing aromatic compounds or aliphatic compounds in a pure water system, an organic/water miscible system or an organic/water immiscible system.
Has the advantages that:
compared with the prior art, the invention provides a cholate-metal ion composite modification method for whole cells and application thereof, which adopts two technical means of fusing cell membrane modification and immobilized cells by self-assembly of a normal-temperature water-phase surfactant and metal ions, and compared with the immobilized cells prepared by the traditional immobilized materials, because the cholate-metal ion composite contains the surfactant, the cholate-metal ion composite can generate mosaic action with cell membranes on the surfaces of the cell membranes, effectively reduces mass transfer resistance among the immobilized materials and transmembrane resistance of the cell membranes under the condition of not damaging the integrity of the cell membranes, improves the transmembrane mass transfer efficiency of the cell membranes, and enables enzymes to perform substance conversion more quickly;
meanwhile, compared with the damage of the traditional surfactant (such as Tween, sodium dodecyl benzene sulfonate, quaternary ammonium compound and the like) to cell membranes, the material does not cause the cell lysis and death, so that the enzyme can exert higher catalytic activity in a stable cell microenvironment. The surfactant-metal ion modified microorganism whole-cell catalyst has both biological catalytic activity and physical catalytic activity of the surfactant, and is simple and mild in synthesis process, low in cost and environment-friendly. Therefore, the immobilized whole-cell catalyst has wide significance and prospect by utilizing the lower mass transfer resistance and the surfactant characteristic of the immobilized material.
The cholate-metal ion complex contains metal ions, and can further coordinate and regulate the fluidity of cell membranes. According to the invention, a part of cholate-metal ion complex is embedded in a microbial cell membrane, and the whole microorganism is fixed by combining with a network structure of the cholate-metal ion complex, so that the cholate-metal ion complex can protect the microorganism, improve the tolerance of the microorganism, endow the special performance of a microbial material, and can be applied to various biological catalysis systems.
The results are several, as follows: 1. the invention reduces the transmembrane resistance of cell membranes and improves the yield of products of cell biocatalysis; 2. the invention overcomes the mass transfer resistance of the traditional immobilized material and improves the catalysis efficiency of the microorganism; 3. endows the microbial surfactant with the characteristics, overcomes the damage and death of cells caused by the pure use of the surfactant, and improves the catalytic efficiency and tolerance of the cells; 4. the invention overcomes the defect that the nano material can only modify a single cell, has strong universality, and can be combined with various microorganisms (gram-negative bacteria, gram-positive bacteria, eukaryotic cells and the like); 5. the invention can play a good catalytic effect aiming at various catalytic systems (pure water system, organic/water mutual soluble system and organic/water immiscible system); 6. the invention obviously reduces the usage amount of the organic cosolvent in an organic/water mutual soluble system; 7. the invention enhances the tolerance of cells, such as the tolerance of the cells to pH and the repeated utilization rate of the cells.
Drawings
FIG. 1 shows the cholate-metal ion complex modified Escherichia coli in example 2E.coliA morphological map after BL21(DE 3);
FIG. 2 is a scanning electron microscope image of single cells obtained by washing whole cells modified with cholate-metal ion complexes step by step with ethanol solutions of different concentrations in example 3, wherein (a) the strain is Escherichia coli, (b) the strain is Bacillus subtilis, and (c) the strain is Saccharomyces cerevisiae;
FIG. 3 is a comparison of the permeability of the cell membrane of the microorganism in example 4 under different treatments;
FIG. 4 is a comparison of the catalytic activity of the complex of example 5 (E.coli) with E.coli at different temperatures;
FIG. 5 is a comparison of the catalytic activity of the complex of example 5 (E.coli) with E.coli at different co-solvent usage levels;
FIG. 6 is a comparison of the catalytic activity of the complex of example 5 (E.coli) with E.coli at different pH;
FIG. 7 is a comparison of catalytic performance of Escherichia coli treated with different materials in an organic/water miscible system in comparative example 1;
FIG. 8 is a comparison of catalytic performances of Escherichia coli treated with different materials in an organic/water immiscible system in comparative example 2;
Detailed Description
The invention will be further described with reference to the accompanying drawings and specific embodiments
EXAMPLE 1 cultivation of the Strain and Gene expression
Escherichia coliE.coliBL21(DE3) belongs to the genus Bacillus and is available from Beijing Baiolai Boke technology, Inc.
Escherichia coliE.coliTrans1-T1 for transformation and replication of plasmid, E.coliE.coliBL21(DE3) was used for gene expression and strain fermentation, and plasmid pET28a was purchased from holo-type gold. The cytochrome P450 monooxygenase is given to professor Liu of Beijing university of chemical engineering, the whole gene sequence is synthesized by Nanjing Dulkraceae, PCR primer is designed, PCR amplification is carried out, agarose gel electrophoresis recovery is carried out, Nco I and Hind III cutting enzyme cutting vector pET28a and P450 gene sequence are connected by T4DNA ligase, recombinant plasmid pET28a-P450 is obtained, and the recombinant plasmid pET28a-P450 is transformed into escherichia coliE.coliTrans1-T1 for preservation of recombinant plasmids, transformation into E.coliE.coliObtaining recombinant strains from BL21(DE3) competenceE.coliP450 for the expression of cytochrome P450 monooxygenase.
The primers for PCR amplification are all designed by using Snapgene software.
The front primer P450-F: catgCATGGGCAGCAGCCATCATCATC
Rear primer P450-R: ccCAAGCTTTCAGAGTCGGAGGGTCAGTCG
A500 mL shake flask was charged with 100 mL LB medium (formulation: 10g/L peptone, 5g/L yeast powder, 5g/L sodium chloride) and 200. mu.L kanamycin (25 mg/L) at an inoculum size of 1%, 200 r/min, 37 ℃ at a rotation speed of 200 r/min, and cultured to OD600=0.6 ~ 0.8.8, 50. mu.L isopropyl-. beta. -D-thiogalactoside (IPTG, 50 mmol/L) was added thereto for induction, and after induction, the resultant was cultured at 18 ℃ at 200 r/min for 24 hoursE.coliThe P450 was centrifuged at 6000 g for 5 min in a tabletop centrifuge, and then the cells were washed twice with disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH 7.0), and finally the E.coli cells were collected.
Bacillus cereus
Bacillus cereus QCG4, preserved in China center for type culture Collection with preservation number (CCTCC NO: M2018503). Belongs to the public strains.
A500 mL shake flask is filled with 100 mL LB medium (the formula of the LB medium is 10g/L peptone, 5g/L yeast powder and 5g/L sodium chloride), the inoculation amount is 1%, the rotation speed is 200 r/min, and the temperature is 30 ℃ for 24 h. The bacillus cereus cultured in the shake flask is centrifuged at 6000 g for 5 min in a desktop centrifuge, and then the bacillus cereus is washed twice by a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (pH 7.0), and finally the bacillus cereus is collected.
Saccharomyces cerevisiaeBY4741
Saccharomyces cerevisiaeBY4741 it belongs to the conventional strain, and is purchased from Wuhan vast Ling Biotech limited.
A500 mL shake flask was filled with 100 mL YPD medium (formulation: 10g/L peptone, 5g/L yeast powder, 20g/L glucose) at an inoculum size of 1%, a rotation speed of 200 r/min, and a temperature of 30 ℃ for 24 h. Culturing the finished Saccharomyces cerevisiae in a shake flaskBY4741 centrifuging at 6000 g for 5 min in a desktop centrifuge, washing thallus with disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (pH 7.0) twice, and collecting Saccharomyces cerevisiaeBY4741 the bacterial cells.
EXAMPLE 2 preparation of cholate-Metal ion immobilized cell Complex
The collected cells were prepared to 1 ~ 10OD using Tris-HCl (pH 7.2, 0.05 mmol/L) buffer600Concentration of solution a.
Deoxycholate was prepared as solution B at a concentration of 100 ~ 300 mmol/L.
Magnesium chloride was prepared as a solution C at a concentration of 200 ~ 800 mmol/L.
And mixing the solution A and the solution C, adding the solution B, stirring at room temperature for 15 ~ 60min, and washing twice with Tris-HCl (pH 7.2, 0.05 mmol/L) buffer solution to obtain the cholate-metal ion complex modified whole cell.
The cell bodies herein were any of those in example 1.
Wherein, the Escherichia coli modified by cholate-metal ion complex is marked as complex (Escherichia coli); see FIG. 1;
marking the bacillus subtilis modified by the cholate-metal ion compound as a compound (bacillus subtilis);
saccharomyces cerevisiae modified with cholate-metal ion complexes was designated as complex (Saccharomyces cerevisiae).
Example 3 Universal binding mechanism of cholate-Metal ion complexes to different cells
With E.coli as in example 1E.coliBL21(DE3) was immobilized target bacterial cells, and Escherichia coli modified with cholate-metal ion complex was obtained by the preparation of example 2E.coliBL21(DE3), see FIG. 1.
Sequentially washing the bacterial strains modified by the cholate-metal ion complex step by using ethanol solutions with the volume fractions of 30%, 50%, 80%, 90% and 100% in sequence, and washing off redundant materials to obtain single cholate-metal ion complex modified whole cells (figure 2 (a), wherein the bacterial strains are escherichia coli, figure 2 (b) is bacillus subtilis, and figure 2 (c) is saccharomyces cerevisiae). The complex is still combined with the cell membrane after multiple times of washing, which indicates that the material is stable after being embedded into the cell membrane, and the complex is better combined with the cell membrane. The cell membranes of different cells are formed into a plurality of compositions, and the cholate-metal ion compound can have a mosaic effect on the cell membranes of gram-positive bacteria (bacillus subtilis), gram-negative bacteria (escherichia coli) and eukaryotic cells (saccharomyces cerevisiae), and the cholate-metal ion compound also indicates that the cholate-metal ion compound can modify various cells and has good universality. In addition, the form embedded in the cell membrane is different from the combination effect of other nanometer materials and the cell membrane.
Example 4 cell Membrane Permeability of cholate-Metal ion complexes after modification of Whole cells
And (4) utilizing an AO/PI kit to test the cell membrane permeability. AO (acridine orange) can penetrate living cell membrane to be combined with double-stranded DNA in cells, and shows fluorescence. PI is impermeable to the cell membrane of living cells.
Comparing fluorescence intensity through a compound stained by an AO/PI kit in unit time, and judging the permeability of a cell membrane, namely, the higher the AO fluorescence intensity is, the better the cell membrane permeability of a living cell is; higher PI fluorescence intensity indicates cell death of damaged cells.
And (3) comparing the cell membrane permeability of the compound of the immobilized cells, the cells treated by the single factor and the free cells of the comparison sample by using an AO/PI double-staining kit.
Each 500. mu.l of the test sample was taken, 20. mu.l of AO/PI staining solution was added, and after 5 minutes, the fluorescence intensity of the two stains was recorded by a fluorescence spectrometer to obtain FIG. 3.
From FIG. 3, it can be seen that the whole cells modified with cholate-metal ion complex all obtained the highest AO fluorescence intensity (i.e., the highest transmembrane mass transfer efficiency) and maintained the same low PI fluorescence intensity (low mortality) as the untreated whole cells, indicating that the modification was better than the single-factor treatment (using cholate alone or magnesium alone) of the microbial cells, i.e., increased cell membrane permeability without causing cell lysis.
Example 5 catalytic Performance of cholate-Metal ion complexes after modification of Whole cells
With E.coli as in example 1E.coliBL21(DE3) was immobilized target bacterial cells, and Escherichia coli modified with cholate-metal ion complex was obtained by the preparation of example 2E.coliBL21(DE3) as complex (E.coli).
Catalyzing 7-ethoxycoumarin by using the compound (escherichia coli) to produce 7-hydroxycoumarin;
the substrate concentration was controlled to 0.15g/L in total volume.
And dissolving the substrate by using a cosolvent dimethyl sulfoxide or N, N-dimethylformamide, and performing catalytic reaction to obtain an organic/water miscible system.
The catalytic reaction is carried out by dissolving the substrate by utilizing dioctyl phthalate, and the system is marked as an organic/water insoluble system.
The substrate is directly put into the reaction system to carry out catalytic reaction, and the system is marked as a pure water system.
The above substrates and their corresponding products were detected by High Performance Liquid Chromatography (HPLC) using an Agilent 1920 Infinity System, an Agilent fluorescence detector (FLD G1321B), an Agilent 5 HC-C18 column (250 mm. times.4.6 mm), mobile phase components A: water, B: and (3) acetonitrile. The sample detection time is 30min, and the gradient elution procedure is as follows: 0min, 60% of phase B; 5 min, 60% of phase B; 20 min, 100% phase B; 25 min, 60% of phase B; 30min, 100% B phase. The flow rate is 1 mL/min, the sample injection amount is 20 mu L, the column temperature is 25 ℃, and the ultraviolet light is 272 nm.
The catalytic activities of the compound (escherichia coli) in example 2 and escherichia coli for producing 7-hydroxycoumarin from 7-ethoxycoumarin are compared in an organic/water miscible system and a pure water system, wherein an organic cosolvent dimethyl sulfoxide (DMSO) accounts for 5% of the total reaction volume, and the reaction speed is 100 rpm.
As a result, as shown in FIG. 4, the yield of the complex (E.coli) was increased to 268.1% as compared with that of E.coli.
The complex (escherichia coli) and escherichia coli are catalyzed in an organic/water mutual soluble system and a pure water system to produce 7-hydroxycoumarin from 7-ethoxycoumarin, the dosage of an organic cosolvent dimethyl sulfoxide (DMSO) is adjusted, and a catalytic activity comparison is obtained, as shown in figure 5. Illustrating that the use of cholate-metal ion complexes reduces the amount of organic reagent used, as a co-solvent: the proportion of water is 1: 10 as reference, complex (E.coli) in cosolvent: the proportion of water is reduced to 1: at 50, the catalytic activity can be improved by 7 percent. Meanwhile, the catalytic activity of 80 percent of the composite standard can be maintained under the condition of completely not using an organic cosolvent. Wherein the addition amount of the 7-hydroxycoumarin is 0.15g/L under the total reaction volume, the reaction speed is 100 rpm, and the reaction temperature is 35 ℃.
The complex (E.coli) was compared with E.coli for catalytic activity under different pH conditions, and the pH tolerance of the test sample was investigated. From FIG. 6, it was found that the complex (E.coli) has a good pH tolerance and can maintain 63.6% of the catalytic activity at the optimum pH 7 at pH 4 and 90.2% of the catalytic activity at the optimum pH 7 at pH 9. The reason is that the microenvironment constructed by the cholate-metal ion composite material has a better protective effect on modified cells. Wherein the addition amount of the 7-hydroxycoumarin is 0.15g/L under the total reaction volume, the organic cosolvent dimethyl sulfoxide DMSO is 5 percent of the total reaction volume, the reaction speed is 100 rpm, and the reaction temperature is 35 ℃.
Comparative example 1 comparison of catalytic Activity in organic/Water-miscible System and pure Water System
The compound (Escherichia coli), free Escherichia coli and Escherichia coli wrapped by calcium carbonate microspheres (preparation method reference document: research on production of apricot peel residue vinegar by immobilized microorganism cell fermentation) in example 2 were subjected to catalytic activity comparison of catalytic aromatic compounds in an organic/water miscible system. Wherein the adding amount of the 7-hydroxycoumarin is 0.15g/L under the total reaction volume, the organic cosolvent dimethyl sulfoxide DMSO is 5 percent of the total reaction volume, the reaction speed is 100 rpm, the reaction temperature is 35 ℃, and the reaction time is 2 hours each time. The experimental catalytic product detection method is shown in example 5. From FIG. 7, it was found that the complex (E.coli) had the best catalytic activity and recycling rate among the three. The catalytic activity of the escherichia coli wrapped by the calcium carbonate microspheres is reduced to a certain extent (only 77.6% of the escherichia coli), which is caused by excessive embedding of part of cells, and the mass transfer resistance of the substance is increased by the calcium carbonate material immobilized material.
Comparative example 2 comparison of catalytic Activity in organic/Water-insoluble System
The complex (Escherichia coli), free Escherichia coli and Escherichia coli wrapped by calcium carbonate microspheres (preparation method reference: study on immobilized microorganism cell fermentation for producing apricot peel residue vinegar) in example 2 were subjected to catalytic activity comparison in an organic/water-insoluble system. In an organic/water-insoluble system, the complex (Escherichia coli) in example 2 catalyzes 7-ethoxycoumarin to produce 7-hydroxycoumarin, and the yield of the complex is increased to 404.8% of that of free cells. Wherein the addition amount of the 7-hydroxycoumarin is 0.15g/L under the total reaction volume, the reaction speed is 100 rpm, the reaction temperature is 35 ℃, the dioctyl phthalate is selected as an organic phase, and the volume ratio of the organic phase to a water phase is 1: 4, the product and the substrate are present in the organic phase, each reaction time being 2 hours. The experimental catalytic product detection method is shown in example 5.
In the system, the surfactant property of the compound is retained, so that the compound has emulsification effect in two phases, an organic phase can exist in the system in a droplet form, the contact area of the two phases is increased, the mass transfer efficiency is improved, and the catalytic activity is improved, and particularly, see fig. 8.
Coli has low catalytic activity due to the immiscibility of the organic phase and the aqueous phase. The microporous structure of the calcium carbonate immobilizing material cannot transport fat-soluble compounds in the organic phase.
The above results show that the cholate-metal ion composite material has better mass transfer efficiency and better recycling rate than the common traditional immobilized material.
Compared with the patent of application No. 201710234386.6, the invention uses surface activity and metal ions to form a dandelion flower-shaped main network structure with micron scale (about 20 μm) for immobilizing cells, wherein part of the surface activity and metal ions are embedded and fused in cell membranes by nano unit 10 ~ 30nm particles and are part of the main network structure, and the network structure maintains the hydrophobic pore structure in the material, is very suitable for mass transfer of fat-soluble compounds, and does not cause imbalance of microenvironment in the cell membranes.
Sequence listing
<110> Nanjing university of industry
<120> cholate-metal ion composite modification method for whole cells and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
catgcatggg cagcagccat catcatc 27
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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cccaagcttt cagagtcgga gggtcagtcg 30
Claims (8)
1. A method for modifying whole cells by cholate-metal ion complexes, which is characterized by comprising the following steps:
step 1, preparing a 100-600mmol/L metal ion solution, and stirring and mixing the solution with a Tris-HCl resuspended strain cell solution with the pH of 7.1-8.0 and the concentration of 0.1mol/L to obtain a mixed solution A;
step 2, preparing 50-300 mmol/L surfactant aqueous solution, and dropwise adding the surfactant aqueous solution to the mixed solution A at a dropwise rate of 5-20 mu L/s while stirring at room temperature until the mixed solution A is turbid to obtain mixed solution B;
and 3, centrifuging the mixed solution B to obtain a precipitate, washing the precipitate for 1-3 times by using Tris-HCl with the pH of 7.1-8.0 and the concentration of 0.05mol/L, and washing away unreacted components to obtain the compound modified whole cell.
2. The method for modifying whole cells by using cholate-metal ion complexes, according to claim 1, wherein the surfactant is any one of sodium deoxycholate, sodium taurodeoxycholate, sodium glycodeoxycholate and sodium chenodeoxycholate.
3. The method of claim 1, wherein the metal ion is any one of cobalt ion, calcium ion, zinc ion, manganese ion, barium ion, copper ion, nickel ion, tin ion, magnesium ion, or iron ion.
4. The method for modifying whole cells by cholate-metal ion complexes as claimed in claim 1, wherein the strain cells are any one or a combination of gram-negative bacteria, gram-positive bacteria or yeast.
5. The method for modifying whole cells by cholate-metal ion complexes according to claim 1, wherein the stirring speed in step 1 and step 2 is 200 ~ 800 rpm.
6. The method of claim 1, wherein the added OD of the resuspension solution of the bacterial cells in step 1 is600The concentration was 1 ~ 10 OD.
7. The method for modifying whole cells by cholate-metal ion complexes of claim 1, wherein the centrifugation in step 3 is 5 ~ 10min, and the rotation speed is 1000 ~ 5000 rpm.
8. The use of the cholate-metal ion complex-modified whole cell obtained in claim 1 for catalyzing aromatic compounds or aliphatic compounds in a pure water system, an organic/water miscible system, or an organic/water immiscible system.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106939305A (en) * | 2017-04-12 | 2017-07-11 | 南京工业大学 | Preparation method and application of surfactant-enzyme nano composite catalyst |
| CN107099562A (en) * | 2017-06-27 | 2017-08-29 | 南京工业大学 | Method for producing phosphatidylserine by using immobilized phospholipase D |
| CN107299096A (en) * | 2017-06-21 | 2017-10-27 | 南京工业大学 | Preparation method and application of imidazole and derivative thereof modified surfactant-enzyme nano composite catalyst |
| CN108949658A (en) * | 2018-06-15 | 2018-12-07 | 江苏科技大学 | A kind of whole-cell catalyst and its preparation method and application increasing permeability |
-
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106939305A (en) * | 2017-04-12 | 2017-07-11 | 南京工业大学 | Preparation method and application of surfactant-enzyme nano composite catalyst |
| CN107299096A (en) * | 2017-06-21 | 2017-10-27 | 南京工业大学 | Preparation method and application of imidazole and derivative thereof modified surfactant-enzyme nano composite catalyst |
| CN107099562A (en) * | 2017-06-27 | 2017-08-29 | 南京工业大学 | Method for producing phosphatidylserine by using immobilized phospholipase D |
| CN108949658A (en) * | 2018-06-15 | 2018-12-07 | 江苏科技大学 | A kind of whole-cell catalyst and its preparation method and application increasing permeability |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024056853A1 (en) * | 2022-09-16 | 2024-03-21 | Université D'aix Marseille | Use of coumarin derivatives as tools for in vitro or ex vivo diagnosis of bacterial efflux |
| FR3139830A1 (en) * | 2022-09-16 | 2024-03-22 | Université D'aix Marseille | use of coumarin derivatives as in vitro or ex vivo diagnostic tools for bacterial efflux |
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