WO2021256674A1 - Procédé permettant d'augmenter la stabilité d'une protéine cible immobilisée dans une nanoparticule de silice par traitement salin - Google Patents

Procédé permettant d'augmenter la stabilité d'une protéine cible immobilisée dans une nanoparticule de silice par traitement salin Download PDF

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WO2021256674A1
WO2021256674A1 PCT/KR2021/004311 KR2021004311W WO2021256674A1 WO 2021256674 A1 WO2021256674 A1 WO 2021256674A1 KR 2021004311 W KR2021004311 W KR 2021004311W WO 2021256674 A1 WO2021256674 A1 WO 2021256674A1
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silica
immobilized
target protein
protein
salt
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Korean (ko)
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조병훈
임균택
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경상국립대학교산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/14—Peptides being immobilised on, or in, an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B33/00—Silicon; Compounds thereof
    • C01B33/113—Silicon oxides; Hydrates thereof
    • C01B33/12—Silica; Hydrates thereof, e.g. lepidoic silicic acid
    • C01B33/18—Preparation of finely divided silica neither in sol nor in gel form; After-treatment thereof
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/01—Hydro-lyases (4.2.1)
    • C12Y402/01001—Carbonate dehydratase (4.2.1.1), i.e. carbonic anhydrase
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
    • C01P2004/00—Particle morphology
    • C01P2004/60—Particles characterised by their size
    • C01P2004/64—Nanometer sized, i.e. from 1-100 nanometer
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]

Definitions

  • the present invention relates to a method for increasing the stability of a target protein immobilized on silica nanoparticles through salt treatment.
  • Enzymes are biocatalysts and have advantages in environmental friendliness and substrate specificity compared to conventional chemical catalysts. However, it reacts very sensitively to the surrounding environment, so its activity changes, recycling is difficult, and it is difficult to separate and purify from the reaction product, which limits its industrial use. To solve this problem, various studies have been made, and the most actively studied among them is the method of immobilizing an enzyme.
  • the immobilized enzyme can maintain its activity for a long time, can be used repeatedly, and can be usefully used in industrial fields because the product and the enzyme are easy to separate, so it is possible to overcome the disadvantages of the existing enzyme.
  • the stability of the enzyme can be increased in reactions in organic solvents and high-temperature reactions, it is possible to develop various reaction systems.
  • the biological process using enzymes is performed under mild conditions to reduce energy consumption, and it is possible to improve the quality of products by suppressing by-products by the substrate specificity of the enzyme. In addition, it has the advantage of suppressing the generation of pollutants from an environmental point of view.
  • the enzyme immobilization method can be divided into a chemical method such as an ion exchange method, a covalent bonding method, and a crosslinking method, and a physical method such as an adsorption method, a blanket method, a microencapsulation method, and a membrane use method according to the binding method.
  • a chemical method such as an ion exchange method, a covalent bonding method, and a crosslinking method
  • a physical method such as an adsorption method, a blanket method, a microencapsulation method, and a membrane use method according to the binding method.
  • a sol-gel inorganic support linkage is formed around it, and since the enzyme is present in the pore, it can protect the enzyme from entanglement or modification, and the biggest problem in enzyme immobilization is ejection.
  • the silica structure formed by the sol-gel process is chemically stable and hydrophilic, has low synthesis cost, and has excellent mechanical strength and thermal stability compared to enzymes immobilized by organic polymers, etc., and has the advantage of being protected from bacteria. .
  • the shape of the enzyme is easily generated due to the harsh synthetic conditions and reagents applied when the sol-gel material is prepared. Recently, research on methods for producing and applying biomimetic silica by discovering biological-derived organic molecules (proteins, peptides, etc.) involved in the formation of babiosilica are being conducted.
  • Agglomerated silica powder can be formed at room temperature and neutral pH through biomimetic silica synthesis, and enzyme immobilization is possible at the same time.
  • an additional high-temperature process is required after the gel is formed, and it has the disadvantage that it takes a long time.
  • Korean Patent No. 0837375 discloses cationic surfactants such as benzoalkonium chloride, myristalkonium chloride, cetylpyridinium chloride, and cetyltrimethyl ammonium bromide.
  • cationic surfactants such as benzoalkonium chloride, myristalkonium chloride, cetylpyridinium chloride, and cetyltrimethyl ammonium bromide.
  • a 'method for preparing an enzyme-immobilized silica' using cetyltrimethyl ammonium chloride is disclosed, but silica nano
  • silica nano There is no disclosure of a method for increasing the stability of a target protein immobilized on a particle.
  • the present invention has been derived from the above needs, and the present inventors have prepared a bovine carbonic anhydrase (bCA) coding gene or a fluorescent protein DsRed (red fluorescent protein) coding gene; and a biomimetic silica-forming peptide ( E. coli strain was transformed with a recombinant vector containing the coding sequence of the fusion protein in which the silica forming peptide) R5 coding sequence was sequentially linked to express the fusion protein bCA-R5 or DsRed-R5, and the fusion protein was immobilized on silica synthesis.
  • bCA bovine carbonic anhydrase
  • DsRed red fluorescent protein
  • a fusion protein for this purpose, by mixing a fusion protein, various salts (CsCl, LiCl, NaCl, KCl, RbCl, NaF, NaBr, NaI or NaNO 3 ) and a hydrolyzed silica precursor (TMOS), bCA or DsRed protein is immobilized on silica nano Particles were prepared.
  • the fusion protein bCA-R5 or DsRed-R5 was treated with high temperature (60° C.) on the immobilized silica nanoparticles.
  • high temperature 60° C.
  • the present invention provides the stability of the target protein immobilized on silica nanoparticles, characterized in that it comprises the step of mixing a fusion protein, a salt, and a silica precursor of the target protein and silica forming peptide provides a way to increase
  • the present invention comprises the steps of transforming a host cell with a recombinant vector comprising a coding sequence of a fusion protein in which a target protein coding gene and a silica forming peptide coding sequence are sequentially linked; culturing the transformed host cell to induce expression of a fusion protein of the target protein and the silica-forming peptide R5 and obtain the same; and mixing a salt and a silica precursor with the obtained fusion protein; provides a method for producing silica nanoparticles with increased stability of the immobilized target protein.
  • the present invention provides silica nanoparticles with increased stability of the immobilized target protein prepared by the above preparation method.
  • the present invention provides a composition for increasing the stability of a target protein immobilized on silica nanoparticles containing cesium chloride (CsCl) as an active ingredient.
  • CsCl cesium chloride
  • the method of the present invention is a bioprocess, food, pharmaceutical, bioprocess using an immobilized enzyme It may be usefully used in related industries.
  • bCA-R5 A is a fusion protein bCA-R5 (A) in which bovine carbonic anhydrase (bCA) and silica-forming peptide R5 are coupled, and DsRed-R5 (B), a fusion protein in which red fluorescent protein DsRed and silica-forming peptide R5 are coupled.
  • DsRed-R5 B
  • CsCl Cesium chloride, LiCl: lithium chloride (Lithium chloride), NaCl: sodium chloride (Sodium chloride), KCl: potassium chloride (Potassium chloride), RbCl: rubidium chloride (Rubidium chloride), NaF: sodium fluoride (Sodium) fluoride), NaBr: sodium bromide, NaI: sodium iodide (Sodium iodide), NaNO 3 : sodium nitrate.
  • Figure 3 shows bCA-R5@silica particles synthesized in conditions not treated with salt (CsCl) and bCA-R5@silica (0.1 M CsCl) particles synthesized in conditions in which salt (CsCl) was not treated during the immobilization of the fusion protein bCA-R5. This is a photograph observed with a runner electron microscope (SEM).
  • Figure 4 is the result of confirming the thermal (60 °C, 48 hours) stability of the immobilized bCA enzyme according to the salt treatment during immobilization of the fusion protein bCA-R5
  • A is a chloride salt (LiCl, NaCl, KCl, RbCl, CsCl) treatment and a result of a then measuring the residual activity (residual activity) of immobilized bCA
  • B is the result of measuring a residual activity after treated with Sodium salt (NaF, NaCl, NaBr , NaI or NaNO 3) immobilized bCA
  • C is These are the results of measuring the residual activity of the immobilized bCA enzyme after treatment with various concentrations (0.01 M, 0.1 M, 0.5 M and 1 M) to confirm the optimal concentration of CsCl, which exhibited the best residual activity of the immobilized bCA enzyme.
  • Statistical analysis was performed using t- test, and the asterisk(*) just above the bar graph is
  • FIG. 6 is a result showing the fold change in half life of the bCA enzyme immobilized in bCA-R5@silica and bCA-R5@silica (salt) according to the pH conditions of the dialysis buffer of the fusion protein bCA-R5. .
  • the present invention provides a target protein immobilized on silica nanoparticles, characterized in that it comprises the step of mixing a fusion protein, a salt, and a silica precursor of the target protein and silica forming peptide
  • a method for increasing the stability of a target protein immobilized on silica nanoparticles characterized in that it comprises the step of mixing a fusion protein, a salt, and a silica precursor of the target protein and silica forming peptide
  • target protein refers to a protein to be immobilized on silica nanoparticles.
  • the target protein may be any one protein selected from the group consisting of medical, research and industrial proteins, for example, enzymes, fluorescent proteins, antigens, antibodies, cell receptors, structural proteins, serum and cellular proteins, preferably may be an enzyme protein or a fluorescent protein, more preferably carbonic anhydrase or DsRed (red fluorescent protein), but is not limited thereto.
  • the carbonic anhydrase may preferably be bovine carbonic anhydrase (bCA), but is not limited thereto.
  • the bovine carbonic anhydrase and DsRed may consist of the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 3, respectively, and encode the bovine carbonic anhydrase and DsRed
  • the gene may consist of the nucleotide sequence of SEQ ID NO: 2 and SEQ ID NO: 4, respectively, but is not limited thereto.
  • the silica-forming peptide may preferably be a silica-forming peptide R5 consisting of the amino acid sequence of SEQ ID NO: 5, but is not limited thereto.
  • the silica-forming peptide R5 is a peptide consisting of 19 amino acid residues including lysine, arginine and serine found in diatoms, and functions as a template and catalyst for silica formation together with polyamines.
  • the salt may be an alkali metallic salt, preferably cesium chloride (CsCl), lithium chloride (LiCl), sodium chloride (Sodium) chloride, NaCl), potassium chloride (KCl), rubidium chloride (RbCl), sodium fluoride (NaF), sodium bromide (NaBr), sodium iodide (Sodium iodide, NaI) or sodium nitrate (Sodium nitrate, NaNO 3 ) may be, and more preferably, cesium chloride, but is not limited thereto.
  • CsCl cesium chloride
  • LiCl lithium chloride
  • NaCl sodium chloride
  • KCl potassium chloride
  • RbCl rubidium chloride
  • NaF sodium fluoride
  • NaBr sodium bromide
  • sodium iodide Sodium iodide, NaI
  • sodium nitrate sodium nitrate, NaNO 3
  • the silica precursor may be tetramethyl orthosilicate (TMOS) or tetraethyl orthosilicate (TEOS), preferably TMOS, but this not limited
  • transforming the host cell with a recombinant vector comprising the coding sequence of the fusion protein in which the target protein coding gene and the silica forming peptide coding sequence are sequentially linked;
  • silica nanoparticles Mixing a salt and a silica precursor to the obtained fusion protein; a method for producing silica nanoparticles with increased stability of the immobilized target protein, and a method for increasing the stability of the immobilized target protein prepared by the method Silica nanoparticles are provided.
  • the silica-forming peptide may preferably be a silica-forming peptide R5 consisting of the amino acid sequence of SEQ ID NO: 5, but is not limited thereto.
  • the recombinant vector is a target protein, wherein the cow-derived carbonic anhydrase coding gene and the silica-forming peptide R5 coding sequence are sequentially linked, or the target protein DsRed coding gene and silica are formed
  • the peptide R5 coding sequence may be sequentially linked, but it is not particularly limited thereto, and it may be constructed by sequentially linking a protein coding gene to be mass-produced by those skilled in the art and a silica-forming peptide R5 coding sequence.
  • bovine carbonic anhydrase, DeRed protein, salt and silica precursor are the same as described above.
  • the salt may be cesium chloride (CsCl), and the salt may be mixed to a final concentration of 0.05 to 1M, preferably 0.1M, but is not limited thereto. does not
  • the silica-forming peptide R5 is used to form silica nanoparticles in powder form at room temperature and neutral pH, and forms silica nanoparticles in powder form in the conventional chemical silica synthesis method It is not necessary to carry out the process of heat treatment at high temperature (about 600° C.) for a long time, and it is possible to improve the fact that silica is formed only in the form of a gel at a very slow rate or that a gel is not formed near neutral pH.
  • Recombinant refers to a cell in which the cell replicates, expresses a heterologous nucleic acid, or expresses a peptide, heterologous peptide or protein encoded by the heterologous nucleic acid.
  • Recombinant cells can express genes or gene segments not found in the native form of the cell, either in sense or antisense form.
  • Recombinant cells can also express genes found in cells in a natural state, but the genes are modified and re-introduced into cells by artificial means.
  • vector is used to refer to a DNA fragment(s), a nucleic acid molecule, that is delivered into a cell.
  • the vector replicates DNA and can be reproduced independently in a host cell.
  • carrier is often used interchangeably with “vector.”
  • the expression vector preferably comprises one or more selectable markers.
  • the marker is a nucleic acid sequence having a characteristic that can be selected by a conventional chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples thereof include, but are not limited to, ampicillin, tetracycline, and the like.
  • any host cell known in the art may be used, for example, E. coli BL21, E. coli JM109, E. coli RR1 , E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus genus strains such as Bacillus thuringiensis, and Salmonella typhimurium, Serratia marcesens and various Pseudomonas There are enterobacteriaceae and strains such as species.
  • the host cell according to an embodiment of the present invention is preferably Escherichia coli BL21 (DE3), but is not limited thereto.
  • the method of delivering the recombinant vector of the present invention into a host cell may be carried out by the CaCl 2 method, the Hanhan method (Hanahan, D., 1983 J. Mol. Biol. 166, 557-580) and the electroporation method.
  • the transformed host cell can be cultured in a medium suitable for expression of the fusion protein of the target protein and the silica-forming peptide R5 using a known technique.
  • a suitable culture medium can be obtained commercially or can be prepared according to the ingredients and composition ratios described in publications such as, for example, catalogs of the American Type Culture Collection, but is not limited thereto.
  • the preparation method of the present invention may further include isolating and purifying the fusion protein from the host cell in which the fusion protein of the target protein and the silica-forming peptide R5 is expressed.
  • the separation method may be separated from the medium by a conventional method including, for example, centrifugation, filtration, extraction, spray drying, evaporation or precipitation, but is not limited thereto.
  • the isolated protein can further be purified by known methods including chromatography (eg ion exchange, affinity, hydrophobicity and size exclusion), dialysis, electrophoresis, fractional dissolution (eg ammonium sulfate precipitation), SDS-PAGE or extraction. It can be purified through various methods.
  • the present invention provides a composition for increasing the stability of a target protein immobilized on silica nanoparticles containing cesium chloride (CsCl) as an active ingredient.
  • the target protein is as described above.
  • Escherichia coli TOP10 strain was used for gene recombination vector production, and E. coli BL21(DE3) strain was used for protein expression.
  • E. coli was cultured in LB (Luria-Bertani) medium at 37° C. and 180 rpm, and 50 ⁇ g/ml ampicillin was added as needed.
  • bovine carbonic anhydrase bCA
  • monomeric DsRed a red fluorescent protein
  • the bCA coding gene was chemically synthesized and DsRed was obtained from pDsRed-Monomer-N1, and the genes were amplified using the primers in Table 1.
  • the PCR product of each gene was first cloned into the pGEM-T Easy vector, and the sequence was confirmed through sequencing. These genes were cloned into pET-22b(+) vector using Nde I and Hind III restriction enzymes, respectively, to prepare pET-bCA and pET-DsRed.
  • pET-bCA and pET-DsRed were cut with Hind III and Xho I, respectively, and the gene fragment bound with the R5 primer of Table 1 was inserted between them.
  • pET-bCA-R5 and pET-DsRed-R5 were prepared.
  • a hexahistidine tag provided from the pET-22b(+) vector is fused to the C-terminus of the sequence and expressed.
  • Primer information for fusion protein cloning of target protein and silica-forming peptide R5 primer designation Primer sequence (5' ⁇ 3') (SEQ ID NO:) bCA F: CATATG AGCCACCACTG (6) R: AAGCTT CTTCGGGAAGCC (7) DsRed F: CATATG GACAACACCGAGGACG (8) R: AAGCTT CTGGGAGCCGGAGT (9) R5 F: AGCTTAGCAGCAAAAAAAATCTGGCTCCTATTCAGGCTCGAAAGGTTCTAAACGTCGCATTCTGC (10) R: TCGAGCAGAATGCGACGTTTAGAACCTTTCGAGCCTGAATAGGAGCCAGATTTTTTGCTGCTA (11)
  • the prepared recombinant vector pET-bCA-R5 or pET-DsRed-R5 was introduced into the E. coli BL21 (DE3) strain and cultured at 37° C. and 180 rpm. 25 °C (for DeRed) or 37 °C after addition of 0.1 mM (for DeRed) or 1 mM (for bCA) IPTG (isopropyl-bD-thiogalactopyranoside) when the cell concentration reached 0.6-0.8 at OD 600 (in the case of bCA) for 20 hours or 10 hours, respectively.
  • the cells were centrifuged for 10 minutes at 4°C and 4,000 x g to recover the cells, and the cells were washed with a lysis buffer (50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, pH 8.0). resuspended.
  • the suspended cells were disrupted by sonication in a cold state, and the lysate was centrifuged for 10 minutes at 4°C and 10,000 x g conditions. Thereafter, the supernatant was named as a soluble fraction (S), and the pellet was resuspended in the same volume of dissolution buffer and named as an insoluble fraction (IS).
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • nickel-nitrilotriacetic acid agarose beads (Ni 2+ -nitrilotriacetic acid) were added to the aqueous fraction of the lysate. agarose beads) were added to bind the protein, and then the non-specifically bound protein was removed using a wash buffer (50 mM sodium phosphate, 300 mM NaCl, 30 mM imidazole, pH 8.0).
  • a purified protein was obtained using an elution buffer (50 mM sodium phosphate, 300 mM NaCl, 250 mM imidazole, pH 8.0), and the purified fusion proteins bCA-R5 and DsRed-R5 were pH 8.0, respectively.
  • the buffer was exchanged by dialysis with 20 mM sodium phosphate buffer of 7.5 and 20 mM sodium phosphate buffer of pH 5.5.
  • the fusion proteins bCA-R5 and DsRed-R5 were mixed with a denaturing buffer (6 M guanidine hydrochloride GuHCl/20 mM sodium phosphate buffer, pH 7.5) and denatured by heating at 100° C. for 10 minutes. Absorbance at 280 nm was measured. The concentration of the protein was confirmed through the measured absorbance and the extinction coefficient at 280 nm calculated from the protein amino acid sequence. The extinction coefficient calculation was performed using ProtParam (http://web.expasy.org/protparam/).
  • TMOS acid-hydrolyzed 1 M tetramethyl orthosilicate
  • Salts added during silica synthesis are cesium chloride (CsCl), lithium chloride (LiCl), sodium chloride (NaCl), potassium chloride (KCl), rubidium chloride (RbCl), sodium fluoride (NaF), sodium bromide (NaBr), Sodium iodide (NaI) or sodium nitrate (NaNO 3 ) were all dissolved in distilled water, and the final concentration of the salt was 0.1 M when silica was formed.
  • distilled water was added in the same ratio, and 1 M TMOS was pre-hydrolyzed with 1 mM HCl for 20 minutes before silica synthesis.
  • the silica on which the fusion protein was immobilized was named bCA-R5@silica and DsRed-R5@silica, respectively, and the silica treated with salt during immobilization was named bCA-R5@silica (salt) and DsRed-R5@silica (salt), respectively. named.
  • the synthesized silica was washed twice with distilled water and then resuspended in 20 mM sodium phosphate buffer (pH 7.5).
  • the amount of silica formed upon immobilization of the fusion protein bCA-R5 was measured by ⁇ -silicomolybdate assay.
  • bCA-R5@silica and bCA-R5@silica (salt) were washed twice with distilled water and then resuspended in 1 ml of distilled water, and a sample of 100 ml was taken and mixed with 900 ml of 0.5 M NaOH for 1 hour. melted 40 ml of the dissolved sample, 160 ml of distilled water, and 800 ml of molybdate solution were mixed and dispensed in a 96-well plate, and absorbance was measured at 370 nm in a plate reader.
  • the preparation method of the molybdate solution is as follows: 1.35 ml of HCl (37%) is diluted in distilled water to make 40.3 ml, and 774.2 mg of ammonium heptamolybdate tetrahydrate (AHT) is dissolved in distilled water to make 9.7 ml of the solution. were mixed and the pH was adjusted to 1.12 with NaOH. For quantitative analysis, the calibration curve was performed with a silicon standard solution dissolved in 0.5 M NaOH.
  • the activity of bCA immobilized on silica was measured using a CO 2 hydration assay. After mixing 600 ⁇ l of 20 mM Tris buffer (100 ⁇ M phenol red, pH 8.3) kept cold and 10 ⁇ l of bCA sample, put it in a disposable cuvette, and put it in a spectrometer set at 4°C. 400 ⁇ l of a saturated solution of CO 2 kept cold was rapidly added and mixed, and the change in absorbance at 570 nm was measured. The time (t) for the absorbance to decrease from the absorbance 1.1 corresponding to pH 7.5 to 0.2, the absorbance corresponding to pH 6.5, was calculated.
  • the time taken by the natural CO 2 hydration reaction (t0; blank) was obtained using a dialysis buffer instead of the bCA sample, and the enzyme activity was determined using (t0-t)/t Calculated.
  • the activity of DsRed immobilized on silica was confirmed by measuring the fluorescence intensity.
  • each sample was heated at 60° C. and then the activity was measured, and the relative activity was compared with the activity of the non-heat-treated sample.
  • the half-life was calculated using the activity reduction data over time.
  • the fusion protein bCA-R5 was dialyzed against pH 5.5, 6.5, 7.5 or 8.0 buffer (20 mM sodium phosphate) to compare the effect of salt treatment according to pH conditions during protein immobilization.
  • Cesium chloride (CsCl) was used as the salt, and the final concentration was 0.1M.
  • Silica synthesized at each pH condition was washed twice with distilled water and then resuspended in the same pH 7.5 buffer (20 mM sodium phosphate), and stability was measured and half-life was calculated in the same manner as above.
  • bCA-R5@silica and bCA-R5@silica were washed twice with distilled water and dried at 60° C. for 24 hours. The dried silica sample was observed through a scanning electron microscope (SEM).
  • PDB ID: 1V9E for bCA and PDB ID: 2VAD for DsRed were used and visualized using the UCSF chimera program.
  • the ratio of charged amino acids to the total surface area of the protein was calculated by applying the dssp algorithm (https://www3.cmbi.umcn.nl/xssp/).
  • the isoelectric point of the protein was calculated from the amino acid sequence using Compute pI/Mw (https://web.expas y.org/compute_pi/).
  • Silica formed upon immobilization of the fusion protein bCA-R5 was analyzed by ⁇ -silicomolybdate assay. As a result, silica of 2.75 ( ⁇ 0.1) g/L was formed when immobilization was carried out in the untreated condition, and 3.06 to 3.4 g/L of silica was formed in the condition in which the salt was treated, especially among various salts. It was confirmed that silica was formed at the highest concentration of 3.4 g/L when CsCl was treated (FIG. 2). That is, since the concentration of silica formed in the salt-treated condition increased by about 10% or more compared to the non-salt-treated condition, it was found that the amount of silica synthesis could be increased when the salt was treated during the protein immobilization process.
  • bCA-R5@silica and bCA-R5@silica were heated at 60° C. for 48 hours, and then the residual activity of the immobilized bCA enzyme was measured.
  • Example 5 Effect of salt treatment according to pH conditions upon immobilization of bCA enzyme
  • the property of the enzyme surface that can interact with silica was expected to be important, so the pH of the dialysis buffer of the fusion protein bCA-R5 was adjusted to have different surface charges. And since silica was not synthesized at a pH condition of 8.5 or higher, the pH adjustment range was set to 5.5 to 8.0, and bCA-R5@silica and bCA-R5@silica (0.1 M CsCl) synthesized at each pH condition were reacted at 60°C. The thermal stability of the immobilized bCA enzyme was analyzed by measuring the half-life.
  • the half-life of bCA-R5@silica (0.1 M CsCl) immobilized at pH 6.5, pH 7.5, and pH 8.0 was generally higher than that of pH 5.5.
  • the half-life increased by about 1.3 times after the addition of CsCl, showing the lowest stability increase, whereas at the pH 8.0 condition, the half-life increased by about 32 times, showing the highest stability increase (Fig. 6). Therefore, in order to increase the high stability of bCA-R5@silica (salt) compared to bCA-R5@silica, it was considered that the condition in which the surface charge of the enzyme was relatively negative during immobilization was considered optimal.
  • DsRed-R5@silica and DsRed-R5@silica were heated at 60° C. for 24 hours, and then the fluorescence intensity of the immobilized DsRed protein was measured. .
  • the residual fluorescence of the DsRed protein immobilized on DsRed-R5@silica was higher than that of DsRed-R5@silica, and through this, the salt treatment resulted in a higher stability of the immobilized DsRed-R5 as well as bCA-R5. It was also confirmed to be effective for improvement. Unlike the bCA-R5 result (FIG. 5C), it was confirmed that the residual fluorescence intensity of the immobilized DsRed protein increased in proportion to the concentration of CsCl treated during the protein immobilization process (FIG. 7).

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  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Inorganic Chemistry (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

Abstract

La présente invention concerne un procédé permettant d'augmenter la stabilité d'une protéine cible immobilisée dans une nanoparticule de silice par un traitement salin. Le procédé selon la présente invention peut être utilisé de manière utile dans, par exemple, les industries biologiques, les produits pharmaceutiques, les produits alimentaires et les processus biologiques qui utilisent des enzymes immobilisées.
PCT/KR2021/004311 2020-06-15 2021-04-07 Procédé permettant d'augmenter la stabilité d'une protéine cible immobilisée dans une nanoparticule de silice par traitement salin WO2021256674A1 (fr)

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KR1020200072553A KR102420704B1 (ko) 2020-06-15 2020-06-15 염 처리를 통해 실리카 나노입자에 고정화된 목적 단백질의 안정성을 증가시키는 방법

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CN114702832B (zh) * 2022-06-07 2022-08-23 江西中医药大学 玉米蛋白-二氧化硅复合物乳剂及其制备方法和应用

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