WO2019175633A1 - Procédés de repliement de saccharose isomérase - Google Patents

Procédés de repliement de saccharose isomérase Download PDF

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WO2019175633A1
WO2019175633A1 PCT/IB2018/051734 IB2018051734W WO2019175633A1 WO 2019175633 A1 WO2019175633 A1 WO 2019175633A1 IB 2018051734 W IB2018051734 W IB 2018051734W WO 2019175633 A1 WO2019175633 A1 WO 2019175633A1
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refolding
sucrose isomerase
buffer
inclusion bodies
slase
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Saravanakumar IYAPPAN
Madhuri GAHI
Pavan Kumar Vadla
Banibrata Pandey
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Petiva Pvt Ltd
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Petiva Pvt Ltd
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    • 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
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y504/00—Intramolecular transferases (5.4)
    • C12Y504/99—Intramolecular transferases (5.4) transferring other groups (5.4.99)
    • C12Y504/99011—Isomaltulose synthase (5.4.99.11)

Definitions

  • the present disclosure relates to a method for refolding sucrose isomerase (Slase) of Pseudomonas mesoacidophila MX-45 from inclusion bodies produced during over expression of sucrose isomerase in a heterologous expression host.
  • Slase sucrose isomerase
  • Bacterial host expression systems such as Escherichia coli provide cost-effective manufacturing scale production of recombinant proteins.
  • IB Inclusion bodies
  • the aggregation of proteins is of significant concern in the biotechnology and pharmaceutical industries as proteins recovered as inclusion bodies are usually inactive. This represents a major problem as the incorrectly folded proteins recovered as inclusion bodies are practically useless for industrial applications. Hence, the proteins must be solubilized and refolded to recover their native structures having biological activities.
  • the inventors in the present instance have been able to invent a process for refolding of a recombinant sucrose isomerase, which gives extremely good yield of biologically active sucrose isomerase under the refolding conditions.
  • Sucrose isomerase used for production of trehalulose from sucrose.
  • Trehalulose is a naturally occurring isomer of sucrose that is valued as non-cariogenic and low glycemic sweetener with anti-oxidant property.
  • Trehalulose a-D-glucosylpyranosyl-l, l-D- fructofuranose
  • a structural isomer of sucrose a-d-glucosylpyranosyl- 1 ,2-b- ⁇ - fructofuranoside
  • sucrose a-d-glucosylpyranosyl- 1 ,2-b- ⁇ - fructofuranoside
  • it is absorbed more slowly and digested or metabolized completely and thus it attenuates insulin levels in blood stream. Due to its health benefits and no reported side effects, it can be an ideal sucrose substitute in diabetic foods and sport drinks.
  • the inventors have identified the above issues and addressed the same by inventing a process for refolding sucrose isomerase from Pseudomonas mesoacidophila MX-45.
  • the process provides a manner for recovery of bioactive sucrose isomerase (Slase) from inclusion bodies.
  • the present invention thus addressed the drawbacks of existing approaches to solve a long-standing problem of providing an efficient, cheap and industrially-scalable means for refolding sucrose isomerase, which in turn lowers the cost of production of trehalulose.
  • the technical problem to be solved in this invention is an improved method for refolding sucrose isomerase (Slase) of Pseudomonas mesoacidophila MX-45 from inclusion bodies produced during over-expression of sucrose isomerase in a heterologous expression host.
  • Slase sucrose isomerase
  • the invention provides an improved method for efficient, cheap and industrially- scalable means for refolding sucrose isomerase. Further, the method provides a manner for recovery of bioactive sucrose isomerase (Slase) from inclusion bodies. The invention lowers the cost of production of trehalulose.
  • the present invention relates to a method for obtaining bioactive recombinant trehalulose synthase or sucrose isomerase (Slase) from inclusion bodies.
  • the method comprising isolation of inclusion bodies from host cells overexpressing the recombinant sucrose isomerase, solubilization of inclusion bodies in a buffer with appropriate chaotrophs and solublizing agents, refolding (renaturation) of solublized sucrose isomerase into their native structures having sucrose isomerase activity, and recovery of the refolded recombinant sucrose isomerase.
  • Figure 1 depicts the vector map of pETl l-SI comprising modified nucleotide sequence encoding for sucrose isomerase derived from Pseudomonas mesoacidophila MX- 45.
  • Figure 2 depicts the vector map of pET23-SI comprising modified nucleotide sequence encoding for sucrose isomerase derived from Pseudomonas mesoacidophila MX- 45.
  • Figure 3A depicts the expression profile of control and recombinant Escherichia coli cells, which were induced for protein expression by addition of IPTG.
  • Figure 3B depicts identity analysis of recombinant protein by Western blot analysis.
  • Figure 4 depicts SDS-PAGE analysis of Slase refolding and purification from inclusion bodies.
  • Lane 1 Crude lysate
  • Lane 2 Cell lysate supernatant
  • Lane 3 Insoluble fraction of lysate
  • Lane 4 Soluble fraction during IB washing
  • Lane 5 Washed IB
  • Lane 6 Urea solubilized IB (2 ug)
  • Lane 7 Refolded SI(2 pg)
  • Lane 8 Refolded and purified SI (3 ug)
  • Lane 9 Internal control protein of Slase (2 pg)
  • Lane M Molecular weight standard. The proteins were separated on 12% SDS-PAGE and gel was stained with Coomassie brilliant blue (CBB R-250).
  • Ligure 5 depicts Michaelis-Menten plot for kinetic analysis of refolded Slase.
  • Ligure 6 depicts the purity of the refolded Slase.
  • Ligure 7 depicts the pH optima for native and refolded Slase.
  • the closed circles represent native enzyme and open circles represent refolded Slase.
  • Ligure 8 depicts the temperature optima for native and refolded Slase.
  • the closed circles are for native Slase and open circles are for refolded Slase.
  • Ligure 9 depicts the solubilization and refolding conditions for preparation of bioactive Slase form inclusion bodies.
  • SEQ ID NO: 1 is the modified nucleotide sequence encoding sucrose isomerase of Pseudomonas mesoacidophila MX-45.
  • SEQ ID NO:2 is the amino acid sequence of sucrose isomerase of Pseudomonas mesoacidophila MX-45.
  • “buffered solution” refers to a solution which resists changes in pH by the action of its acid-base conjugate components.
  • the term“denaturant” or“chaotropic agent” refers to a compound that, in a suitable concentration in aqueous solution, is capable of changing the spatial configuration or conformation of polypeptides through alterations at the surface thereof so as to render the polypeptide soluble in the aqueous medium.
  • the alterations may occur by changing, e.g., the state of hydration, the solvent environment, or the solvent-surface interaction.
  • the concentration of chaotropic agent will directly affect its strength and effectiveness.
  • a strongly denaturing chaotropic solution contains a chaotropic agent in large concentrations which, in solution, will effectively unfold a polypeptide present in the solution effectively eliminating the proteins secondary structure. The unfolding will be relatively extensive, but reversible.
  • a moderately denaturing chao tropic solution contains a chaotropic agent which, in sufficient concentrations in solution, permits partial folding of a polypeptide from whatever contorted conformation the polypeptide has assumed through intermediates soluble in the solution, into the spatial conformation in which it finds itself when operating in its active form under endogenous or homologous physiological conditions.
  • chaotropic agents include but are not limited to, guanidine hydrochloride, urea, alkaline hydroxide (e.g., sodium or potassium hydroxide) and combination thereof.
  • invention or“present invention” as used herein is a non limiting term and is not intended to refer to any single embodiment of the particular invention but encompasses all possible embodiments as described in the specification.
  • the term“properly folded” or“biologically active” Slase or other recombinant protein and the like refers to a molecule with a biologically active conformation.
  • the term“purified” or“pure recombinant protein” and the like refer to a material free from substances which normally accompany it as found in its recombinant production and especially in prokaryotic or bacterial cell culture.
  • the terms refer to a recombinant protein which is free of contaminating DNA, host cell proteins or other molecules associated with its in-situ environment.
  • the terms refer to a degree of purity that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 98% or more.
  • inclusion bodies refers to dense intracellular masses of aggregated polypeptide of interest, which constitute a significant portion of the total cell protein, including all cell components. In some cases, but not all cases, these aggregates of polypeptide may be recognized as bright spots visible within the enclosure of the cells under a phase-contrast microscope at magnifications down to 1 ,000-fold.
  • the term“host cell” includes an individual cell or cell culture which can be, or has been, a recipient for the subject of expression constructs. Host cells include progeny of a single host cell.
  • Host cell can be any expression host including prokaryotic cell such as but not limited to Escherichia coli, Bacillus subtilis, Pseudomonas putida, Corynebacterium glutamicum or eukaryotic system, such as, but not limited to Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha.
  • prokaryotic cell such as but not limited to Escherichia coli, Bacillus subtilis, Pseudomonas putida, Corynebacterium glutamicum or eukaryotic system, such as, but not limited to Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha.
  • recombinant strain refers to a host cell which has been transfected or transformed with the expression constructs or vectors of this invention.
  • expression cassette denotes a gene sequence used for cloning in expression vectors or in to integration vectors or integrated in to coding or noncoding regions of chromosome of the host cell in a single or multiple copy numbers, where the expression cassette directs the host cell's machinery to make RNA and protein encoded by the expression cassette.
  • expression construct is used here to refer to a functional unit that is built in a vector for the purpose of expressing recombinant proteins/peptides, when introduced into an appropriate host cell, can be transcribed and translated into a fusion protein which is displayed on the cell wall.
  • refolding agent refers to compounds or a combination of compounds and/or conditions which assist during the process of correctly folding of a protein that is improperly folded, unfolded or denatured.
  • refolding buffer refers to compounds or a combination of compounds and/or conditions which assist during the process of correctly folding of a protein that is improperly folded, unfolded or denatured. Further, the buffer helps in maintaining the pH of the solution during the process of refolding.
  • promoter refers a DNA sequences that define where transcription of a gene begins. Promoter sequences are typically located directly upstream or at the 5' end of the transcription initiation site. RNA polymerase and the necessary transcription factors bind to the promoter sequence and initiate transcription.
  • pH buffer refers to any organic or inorganic compound or combination of compounds that will maintain the pH of a solution.
  • transcription refers the process of making an RNA copy of a gene sequence. This copy, called a messenger RNA (mRNA) molecule, leaves the cell nucleus and enters the cytoplasm, where it directs the synthesis of the protein, which it encodes.
  • mRNA messenger RNA
  • translation refers the process of translating the sequence of a messenger RNA (mRNA) molecule to a sequence of amino acids during protein synthesis.
  • mRNA messenger RNA
  • the genetic code describes the relationship between the sequence of base pairs in a gene and the corresponding amino acid sequence that it encodes.
  • the ribosome reads the sequence of the mRNA in groups of three bases to assemble the protein.
  • the present invention discloses an improved method for preparation of soluble and active recombinant sucrose isomerase (Slase) expresses as inclusion bodies during over expression of sucrose isomerase in heterologous expression host.
  • Slase sucrose isomerase
  • the inventors have conducted intensive experiments and have inventing an improved method by devising combinations of solubilizing solutions and refolding buffers. Further, the process parameters such as pH and temperature have been optimized to get an optimum yield of biologically active sucrose isomerase.
  • sucrose isomerase expressed as inclusion bodies in Escherichia coli.
  • the properties of the refolded recombinant sucrose isomerase are comparable to native sucrose isomerase. The same can be evidenced from the following factors:
  • the invention provides a recombinant host cell for expression of sucrose isomerase.
  • sucrose isomerase Slase
  • the gene encoding for sucrose isomerase (Slase) of Pseudomonas mesoacidophila MX-45 was modified for enhanced expression in Escherichia coli.
  • the gene was synthesized using gene synthesis approach.
  • the modified gene sequence is represented as SEQ ID NO: 1.
  • the modified gene was cloned into a pET vector, more specifically a pETl la and pET23a vector and further transformed into Escherichia coli JM109 (DE3) host cell.
  • the present disclosure provides a method for producing biologically active sucrose isomerase comprising the steps of:
  • a refolding buffer comprising a pH buffer, a salt moiety and a refolding agent, wherein the refolding agent is selected from a group comprising L-arginine, urea, calcium chloride, sucrose, t- octylphenoxypolyethoxyethanol (Triton X-100 TM ) and glycerol; and
  • the biologically active recombinant sucrose isomerase comprises the amino acid sequence as set forth in SEQ ID NO:2.
  • the invention provides for isolation of sucrose isomerase expressed as inclusion bodies in Escherichia coli.
  • the recombinant sucrose isomerase expressed as inclusion bodies are isolated by disrupting the host cells.
  • the cells are resuspended in a lysis buffer with pH 5-9, preferably pH 6-8.
  • the buffer strength may be between 0.01-2.0 M. Salts like NaCl or KC1 may also be included in the lysis buffer.
  • the cell lysis can be carried out by any known method in prior art. In brief the cell lysis can be carried out by mechanical methods such as high-pressure homogenizer, freeze-thaw cycling, french press, or sonication, or enzymatic or chemical methods such as lysozyme or detergents.
  • the cell lysis is carried out under reduced temperature conditions, generally less than 4 - l0°C.
  • the inclusion bodies are collected by centrifugation.
  • the cell lysate is stored at -80°C or processed further for inclusion body preparation.
  • the inclusion bodies collected are washed using wash buffer.
  • inclusion bodies are washed by resuspending lysis buffer and recollecting by high speed centrifugation or TFF.
  • Lysis buffer may contain detergents or salts or chaotrophs or a combination thereof.
  • detergent can be any detergent, typically, Triton X100 ® or Tween ® , and its concentration can be between 0.001-10% (w/v), preferably 1%.
  • salt can be any salt, preferably NaCl or KC1 between 0.01-2 M concentrations, preferably 1.0 M.
  • chaotroph can be any chaotroph, preferably urea or guanidium hydrochloride (Gdn-HCl) between 0.01-10 M, preferably 2 M (e.g. 1 M urea).
  • the inclusion bodies can be washed in any order with washing buffers containing detergent(s) or salt(s) or chaotroph(s) or a combination thereof.
  • the composition of the wash buffer is 50 mM Tris-HCl, 1 M NaCl, 2 M urea and 1% Triton X-100 ® at pH 8.0.
  • the washed inclusion bodies are incubated in a solubilization solution containing a denaturant.
  • the incubation takes place under conditions of concentration, incubation time, and incubation temperature that will allow solubilization of desired amount or substantially all the recombinant Slase.
  • the solubilization can be done at a variety of temperatures.
  • the incubation temperature for the solubilization is room temperature. In another embodiment, the incubation is carried out at room temperature for 2-6 hrs. The incubation can also be carried out at lower temperature, for example, at 4-40°C for 2-24 hrs.
  • the solubilization solution is a urea solubilization solution.
  • the composition of the urea solubilization solution is 50 mM Tris- HC1, 8.0 M urea at pH 8.0.
  • the solubilization solution is a Guanidine-Hydrochloride solution.
  • the composition of the Guanidine-Hydrochloride solution is 50 mM Tris-HCl, 6 M Guanidine-Hydrochloride at pH 8.0.
  • the concentration of solubilized inclusion bodies is adjusted, the reaction mixture is diluted and then incubated in a refolding buffer.
  • the solubilized inclusion body mixture is clarified to remove insoluble debris.
  • the clarification can be carried out by any convenient means like filtration of centrifugation. Clarification is done at low temperature, e.g., 4-40°C.
  • the clarified mixture is then diluted to achieve the appropriate protein concentration for refolding. Protein concentration can be determined using any convenient technique, such as Bradford assay or light absorption at 280 nm (A 280 ).
  • the inclusion body solution is first diluted with refolding buffer to reduce the chaotroph and protein concentration.
  • the inclusion body solution is diluted to about 10-100 fold or about 10-50 fold or about 10-25 fold with a refolding buffer.
  • the final protein concentration after dilution may be about 0.01 -4 mg/mL.
  • the refolding buffer generally contains a pH buffer, a divalent cation, refolding enhancer or an agent that prevents aggregation or sub molar concentrations of denaturants and detergents.
  • the inclusion body solution is slowly added over a period of about 2-24 h or about 4-10 h to the refolding buffer. After completing the addition of inclusion body solution, the refolding may be continued for 4-48 h. In certain embodiments, it may be about 10-18 h.
  • the refolding is generally carried out at a temperature of about 4-37°C. In certain embodiments, the temperature is about l0-20°C. In one embodiment, the solubilized inclusion bodies are diluted to 20 volumes using Tris-HCl refolding buffer.
  • composition of the Tris-HCl refolding buffer is 50 mM Tris-HCl and 150 mM NaCl at pH range of 6-7.4.
  • solubilized inclusion bodies are incubated in Tris-HCl refolding buffer for 16 hours at a temperature between 4 -l0°C.
  • the solubilized inclusion bodies are diluted to 20 volumes using glycerol refolding buffer.
  • composition of the glycerol refolding buffer is 50 mM Tris- HCl, 10% Glycerol and 150 mM NaCl at pH range of 6-7.4.
  • solubilized inclusion bodies are incubated in glycerol refolding buffer for 16 hours at a temperature between 4 -l0°C.
  • the solubilized inclusion bodies are diluted to 20 volumes using sucrose refolding buffer.
  • the composition of the sucrose refolding buffer is 50 mM Tris- HCl, 200 mM sucrose and 150 mM NaCl at pH range of 6-7.4.
  • solubilized inclusion bodies are incubated in sucrose refolding buffer for 16 hours at a temperature between 4 -l0°C.
  • the solubilized inclusion bodies are diluted to 20 volumes using L- Arginine refolding buffer.
  • the composition of the L- Arginine refolding buffer is 50 mM Tris-HCl, 0.4 M L- Arginine and 150 mM NaCl at pH range of 6-7.4.
  • the solubilized inclusion bodies are incubated in L- Arginine refolding buffer for 16 hours at a temperature between 4 -l0°C.
  • the solubilized inclusion bodies are diluted to 40 volumes using urea refolding buffer.
  • the composition of the urea refolding buffer is 50 mM Tris- HC1, 150 mM NaCl and 1.0 M urea at pH range of 6-9.
  • solubilized inclusion bodies are incubated in urea refolding buffer for 16 hours at a temperature between 4 -l0°C.
  • solubilized inclusion bodies are diluted to 40 volumes using Triton X-100 refolding buffer.
  • composition of the Triton X-100 refolding buffer is 50 mM Tris-HCl, 150 mM NaCl and 0.5% Triton-X 100 at pH range of 6-9.
  • solubilized inclusion bodies are incubated in Triton X- 100 refolding buffer for 16 hours at a temperature between 4-lO°C.
  • solubilized inclusion bodies are diluted to 40 volumes using calcium chloride refolding buffer.
  • composition of the calcium chloride refolding buffer is 50 mM Tris-HCl, 150 mM NaCl and 2 mM CaCh at pH range of 6-9.
  • solubilized inclusion bodies are incubated in calcium chloride refolding buffer for 16 hours at a temperature between 4-lO°C.
  • solubilized inclusion bodies are diluted to 20 volumes using glycerol refolding buffer.
  • composition of the glycerol refolding buffer is 50 mM Tris-HCl and 10% Glycerol at pH 6.0.
  • solubilized inclusion bodies are incubated in glycerol refolding buffer for 20 hours at a temperature between 4-lO°C.
  • properly folded Slase may be exchanged with suitable buffer, concentrated and further purified to produce biologically active Slase.
  • the buffer exchange may be performed by size exclusion chromatography (SEC). Any size exclusion chromatography media, for example, Sephadex G-25 can be used. If desired the SEC step may be utilized for purification of folded protein as well as buffer exchange.
  • Recovery and purification of the recombinant Slase can employ various methods and known procedures such as, for example, salt and solvent fractionation, adsorption with colloidal materials, gel filtration, ion exchange chromatography, affinity chromatography, immunoaffinity chromatography, electrophoresis and high- performance liquid chromatography (HPLC).
  • IEC ion exchange chromatography
  • anion exchange chromatography is used.
  • the chromatographic resin is derivatized with diethlyaminoethyl (DEAE) or quaternary ammonium (Q-) group.
  • DEAE diethlyaminoethyl
  • Q- quaternary ammonium
  • the exact conditions for IEC depends on the chromatography media selected. Generally, loading conditions will have low ionic strength.
  • the refolded and purified Slase can be stored at 4°C or -30°C in solution.
  • the Slase is stored in buffer containing about 50 mM sodium acetate and about 10-50% glycerol at about pH 6.5.
  • the inventors have observed that the Slase produced in this method can be stored at -30°C in 50 mM sodium acetate, 50% glycerol for more than 6 months.
  • the product formation kinetics of refolded sucrose isomerase was studied.
  • the purified recombinant Slase shows specific activity of 981 U/mg ( ⁇ 5%), K m of 31 mM, K cat of 1055 S 1 and K ca /K m of 34,000 (M ⁇ 1 ), which is similar to native protein.
  • the refolded sucrose isomerase was studied to determine the pH and temperature optima.
  • the reaction mixture containing sucrose and refolded sucrose isomerase were incubated at different pH and temperature.
  • the enzyme had high activity between pH of 4-7.5, highest at 6.5. It was also found that recombinant isomerase had the highest activity between the temperature l0-50°C, highest at around 30°C. It was observed that the refolded sucrose isomerase has characteristics which are comparable to native sucrose isomerase. In some aspect like specific activity, the refolded isomerase exhibits better characteristics than native soluble sucrose isomerase.
  • Example 1 Gene construction for expression of sucrose isomerase in E. coli
  • sucrose isomerase Slase
  • the gene was synthesized using gene synthesis approach.
  • the modified gene sequence is represented as SEQ ID NO: 1.
  • the sequence was cloned in to pUC57 using EcoRV restriction enzyme site to generate pUC57-SI constructs. Cloned gene sequence was confirmed by sequence analysis.
  • the DNA fragment encoding for sucrose isomerase was PCR amplified using gene specific primers, and sub cloned into pETl la using Ndel and BamHI restriction enzyme sites to generate pETl l-SL
  • the vector map of pETl l-SI is represented in Figure 1.
  • the coding region was PCR amplified without stop codon using gene specific primers and sub cloned into E. coli expression vector pET23a using BamHI and Hind III restriction enzymes to generate pET23-SI-HIS construct expressing sucrose isomerase with C -terminal 6x Histidine tag.
  • the recombinant plasmid carrying sucrose isomerase gene (pETll-SI and pET23-SI) was confirmed by restriction digestion analysis and followed by DNA sequencing.
  • the vector map of pET23-SI is represented in Figure
  • the sucrose isomerase of Pseudomonas mesoacidophila MX-45 comprises the amino add sequence as set forth in SEQ ID NO:2.
  • Recombinant plasmid DNA (pETl 1 -SI) was transformed into Escherichia coli JM 109 expression host by electro-transformation method and grown on Luria-Bertani (LB) agar plates containing ampicillin (50 g/mL). Individual colonies were picked and grown on LB liquid or defined media containing ampicillin (75 g/mL) for overnight at 37°C.
  • Overnight culture was re-inoculated into 0.1 OD 6 oo in LB liquid or defined media without ampicillin and grown up to 0.6 OD 6 oo and the cells were induced for protein expression by addition of 0.5mM of IPTG (Isopropyl b-D-l-thiogalactopyranoside) and incubated at 37°C. An aliquot of E. coli culture was collected at different time points. The cell lysate was subjected to SDS-PAGE and Western blot analysis to verify the protein expression.
  • IPTG Isopropyl b-D-l-thiogalactopyranoside
  • Figure 3 depicts expression analysis of recombinant sucrose isomerase in E. coli.
  • Figure 3 A depicts that control and recombinant E. coli cells [JM109 carrying pETl l-SI] were induced for protein expression by addition of 0.5 mM IPTG into media. Cells were lysed and supernatant and pellet fractions were subjected to 10 % SDS-PAGE. Lane 1 and 2 are uninduced and induced total cell lysate of control strain. Lane 3 and 4 are uninduced and induced total cell lysate of recombinant strain. Lane 6 and 7 are uninduced cell supernatant and pellet of cell fractions of recombinant strains.
  • Lane 8 and 9 are two hrs induced supernatant and pellet of cell fractions of recombinant strains.
  • Figure 3B depicts identity analysis of recombinant protein by Western blot analysis.
  • Lane 1 and 2 depicts host cell lysate in un-induced and induced stage.
  • Lane 3 and 4 depicts recombinant strain in uninduced and induced stage. Immuno-detection was carried our using protein specific antibodies.
  • Example 3 Large scale production of recombinant sucrose isomerase
  • High cell density fermentation was used for large scale production of recombinant sucrose isomerase as inclusion bodies in E. coli.
  • Seed culture for fermentation was prepared in 3 stages. First, 10 mL of LB broth was inoculated with glycerol stock and incubated at 37°C in shake flask to prepare pre-culture 1 (PC1). Then, 1 mL of PC1 was used to inoculate 25 mL of LB broth and incubated at 37°C for 5 h to prepare PC2. For seed culture, 100 mL of defined media or terrific broth was inoculated with 25 mL of PC2 and incubated overnight at 37°C.
  • the 100 mL of overnight seed culture was added to 900 mL of defined media or terrific broth in a fermenter with a working volume of 5 L.
  • the fermenter was maintained at 37°C with agitation rate being increased progressively from 250 to 1200 rpm; an aeration rate being increased progressively from 0.6 to 2.4 scfm and maintaining dissolved oxygen (DO) at a concentration greater than 20%.
  • DO dissolved oxygen
  • IPTG isopropyl-beta-D-thiogalactopyranoside
  • Example 4 Inclusion body preparation and solubilization
  • Cells were harvested by centrifugation at 10,000 X g for 10 min at 4°C in a high speed centrifuge (HITACHI-CR21GIII using R12A6 rotor). The cell pellet was washed with cold buffer (20 mM Tris-HCl, 5 mM EDTA, pH 8.0) and stored at -80°C.
  • the culture pellets were further resuspended in 400 mL of lysis buffer (50 mM Tris- HCl, pH 8.0) for use.
  • the cells were disrupted by passing the suspension through high pressure homogenizer (Constant systems) at 25 kpsi.
  • Inclusion bodies were collected by centrifugation. Inclusion bodies were further washed by 120 mL of inclusion body wash buffer (50 mM Tris-HCl, 1 M NaCl, 2 M urea, 1% Triton X-100 ® , pH 8.0).
  • the washed inclusion bodies (2 g) were dissolved in 65 mL of urea solubilization solution (50 mM Tris-HCl, 8.0 M urea, pH 8.0). Approximately l.Og of protein was recoverd after the solubilization.
  • the inclusion bodies were dissolved in Gdn-HCl solution (50 mM Tris-HCl, pH 8, 6 M Gdn-HCl). The solution was clarified by centrifugation at 25,000 X g for 30 min at 4°C.
  • Example 2 An initial screening experiment was performed to find the best refolding conditions for Slase.
  • the inclusion bodies, as described in Example 2 were solubilized in urea or Gdn.HCl solutions.
  • the urea/Gdn.HCl solubilized inclusion body (IB) was rapidly diluted into a 20-fold excess refolding buffer containing different refolding agents (given in table 1) at pH 6 or 7.4.
  • the final protein concentration was maintained at 100 pg/mL.
  • the refolding was carried out at 4-lO°C and continued overnight (16 h) at 4°C. After 16 h of incubation, the samples were centrifuged at 25,000 X g for 30 min at 4°C. The supernatant was collected and the activity of the Slase remained in the solution was measured.
  • the urea solubilized inclusion bodies from Example 2 was diluted to 4 mg/mL with urea solubilization solution.
  • Refolding was performed by directly adding the urea denatured Slase solution, drop-wise, to 40 volumes of refolding buffer (50 rriM Tris-HCl, 150 rriM NaCl, 1.0 M urea) between pH 6-9 at 4-lO°C.
  • the refolding was continued for 16 h at 4-lO°C.
  • the sucrose isomerase activity of the refolded samples was analyzed by enzyme assay as described in Example 7.
  • the properly folded and biologically active Slase shows a specific activity of 659 IU/mg for sucrose when refolded at pH 6.0 in presence of 1 M urea.
  • the urea solubilized inclusion bodies from Example 2 was diluted to 4 mg/mL with urea solubilization solution. Refolding was performed by directly adding the urea denatured Slase solution, drop-wise, to 40 volumes of refolding buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% Triton-XlOO) between pH 6-9 at 4-lO°C. The refolding was continued for 16 h at 4-lO°C.
  • the sucrose isomerase activity of the refolded samples was analyzed by enzyme assay as described in Example 7.
  • the properly folded and biologically active Slase shows a specific activity of 440 IU/mg for sucrose when refolded at pH 6.0 in presence of 0.5% Triton-XlOO.
  • the urea solubilized inclusion bodies from example 2 was diluted to 4 mg/mL with urea solubilization solution. Refolding was performed by directly adding the urea denatured Slase solution, drop-wise, to 40 volumes of refolding buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM Cacl 2 ) between pH 6-9 at 4-lO°C. The refolding was continued for 16 h at 4-lO°C.
  • the sucrose isomerase activity of the refolded samples was analyzed by enzyme assay as described in Example 9.
  • the properly folded and biologically active Slase shows a specific activity of 594 IU/mg for sucrose when refolded at pH 6.0 in presence of 2 mM Cacl 2 .
  • the isolated inclusion body preparation weighing -8.74 g (wet weight), was solubilized in 8.0 M Urea at pH 8.0.
  • the pellet was resuspended in 100 mL of urea solubilization buffer (50 mM Tris-HCl, 8.0 M Urea, pH 8.0) and incubated at room temperature for 3 hours with gentle stirring. After solubilization, the extract was centrifuged at 25,000 X g at 20°C for 30 min to remove remaining insoluble cell debris.
  • urea solubilization buffer 50 mM Tris-HCl, 8.0 M Urea, pH 8.0
  • the amount of solubilized IB was calculated by measuring the protein concentration spectrophotometrically at A 2 80 nm using the molar extinction coefficient of Slase (119340 IVT'cnr 1 ), and a total of 850 mg of protein was recovered.
  • Sucrose Isomerase was refolded by rapidly diluting the urea solubilized IB into a 20-fold excess buffer containing 10% glycerol.
  • the final concentration of solubilized IB was adjusted to 1.75 mg/mL with urea solubilization buffer.
  • 500 mL of this solubilized solution was slowly added drop- wise at a rate of 1.0 mL/min with peristaltic pump to a 9.5 L of refolding buffer (50 mM Tris-HCl, 10% Glycerol, pH 6.0).
  • the refolding was performed in cold room at 4-lO°C with rapid mixing on a magnetic stirrer.
  • the final protein concentration of the refolded sample was maintained at 85 pg/mL.
  • the refolding was allowed to continue for 20 h at 4-lO°C with gentle stirring.
  • the refolded sample (10 L) was subjected to a pre-filtration step.
  • the sample was filtered through a 0.45 pm Sartoclean cellulose acetate capsule filter (Sartorius) with a flow rate of 60 mL/min to remove particulate matter.
  • the filtered solution was concentrated to 4.7 L by Tangential Flow Filtration (TFF) equipped with 0.1 m 2 Hydrosart (Sartorius) membrane (30,000 MWCO).
  • the permeate flow rate, 50 mL/min was maintained during the process.
  • 750 mg of protein was obtained with a protein concentration of 150 pg/mL.
  • the pH of the refolded sample was adjusted to 8.0 using diluted NaOH solution.
  • the refolded Slase was further purified by Q-Sepharose ® Fast Flow (GE Healthcare) ion exchange chromatography.
  • the concentrated solution (4.7 L), containing -750 mg of the refolded Slase, was applied to 150 ml, Q Sepharose® Fast Flow column (XK 26/40, GE Healthcare) with a flow rate of 3.5 mL/min at 4°C.
  • the column was pre equilibrated with ten column volumes of 50 mM Tris-HCl, pH 8.0.
  • the column was washed with four column volumes of equilibration buffer until the UV absorbance (OD 2 80 nm) returned to a stable baseline.
  • the bound proteins were eluted from column by step gradient with 100 mM, 150 mM and 400 mM of NaCl at pH 8.0. All peaks were collected and analyzed for Slase activity. Slase was found in the fraction eluted with 100 mM NaCl at nearly 12-15 mS/cm. The specific activity of these highly pure Slase was 752 IU/mg.
  • the pooled fractions (375 mL) were concentrated by Ultrafiltration using Macrosep (30,000 MWCO) centrifugal devices (Pall Corporation, USA) to 45 mL of final volume. The concentrated sample was then dialyzed against 20 mM sodium acetate, pH 6.5 at 4°C. The dialyzed sample was centrifuged at 20,000 X g at 4°C for 30 min to remove aggregated and particulate matter. The protein concentration was estimated at A 2 so nm using molar extinction coefficient, 119340 M 1 .
  • the protein was concentrated to 5.6 mg/mL in 20 mM sodium acetate buffer, pH 6.5 without any aggregation.
  • the specific activity of the final purified and dialyzed sample was 694 IU/mg.
  • the concentrated protein solution was stored in 50% (w/v) glycerol at - 30°C. Slase refolding and purification from inclusion bodies was monitored by SDS-PAGE analysis. The results are depicted in Figure 4.
  • Example 9 Product formation kinetics of refolded sucrose isomerase Sucrose isomerization activity of recombinant Slase was tested by an enzyme assay using sucrose as substrate. The reaction velocity was measured by incubating appropriately diluted Slase with various concentrations of sucrose in a 50-mM sodium acetate buffer pH 6.5 at l5°C for 15 min and measuring the production of Trehalulose by HPLC (Shimadzu, LC-20) on 4.6 X 150 mm Zorbax Carbohydrate column (Agilent) using acetonitrile water mix (80:20, v/v) as mobile phase. Results were plotted using Michaelis-Menten plot by PRISM statistical analysis program (see Figure 5).
  • the data are means for three enzyme reaction replicates with SEM.
  • the purified recombinant Slase shows specific activity of 981 U/mg ( ⁇ 5%), K m of 31 mM, Kcat of 1055 S 1 and K cat /K m of 34,000 (M _1 S _1 ).
  • the product formation kinetics are similar to native protein.
  • the refolded sucrose isomerase was studied to determine the pH and temperature optima for the same.
  • the reaction mixture containing sucrose and refolded sucrose isomerase were incubated at different pH ( Figure 7) and temperature ( Figure 8). It was found that the enzyme had high activity between pH of 4-7.5, highest at 6.5. It was also found that recombinant isomerase had the highest activity between the temperature 10- 50°C, highest at around 30°C.
  • Table 6 depicts that the refolded sucrose isomerase has characteristics which are comparable to native sucrose isomerase. In some aspect like specific activity, the refolded isomerase exhibits better characteristics than native soluble sucrose isomerase.
  • Figure 9 depicts the solubilization and refolding conditions for preparation of bioactive Slase form inclusion bodies.

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Abstract

La présente invention concerne des procédés de repliement de saccharose isomérase (SIase) de Pseudomonas mesoacidophila MX-45 issue de corps d'inclusion. Le procédé comprend la solubilisation des corps d'inclusion exprimés dans une cellule hôte dans une solution de solubilisation ; la dilution de la saccharose isomérase solubilisée pour obtenir un échantillon dilué ; l'incubation de l'échantillon dilué en présence d'un tampon de repliement comprenant un tampon de pH, une fraction de sel et un agent de repliement ; la purification de la saccharose isomérase repliée. L'invention représente une avancée par rapport à l'état de la technique pour des procédés efficaces et rentables en vue d'obtenir une saccharose isomérase biologiquement active.
PCT/IB2018/051734 2018-03-15 2018-03-15 Procédés de repliement de saccharose isomérase Ceased WO2019175633A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113957065A (zh) * 2021-11-30 2022-01-21 南京诺云生物科技有限公司 一种高转化率的蔗糖异构酶及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1630173A2 (fr) * 2004-08-27 2006-03-01 Bioceuticals Arzneimittel AG Procédé de récupération du G-CSF humain sous une forme biologiquement active à partir de corps d'inclusion
US9885030B2 (en) * 2012-04-17 2018-02-06 Petiva Private Limited Polynucleotide for recombinant expression of sucrose isomerase

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1630173A2 (fr) * 2004-08-27 2006-03-01 Bioceuticals Arzneimittel AG Procédé de récupération du G-CSF humain sous une forme biologiquement active à partir de corps d'inclusion
US9885030B2 (en) * 2012-04-17 2018-02-06 Petiva Private Limited Polynucleotide for recombinant expression of sucrose isomerase

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
RICHARD R .: "Burgess Refolding Solubilized Inclusion Body Proteins", METHODS IN ENZYMOLOGY, vol. 463, 2009, pages 259 - 282, XP009149876 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113957065A (zh) * 2021-11-30 2022-01-21 南京诺云生物科技有限公司 一种高转化率的蔗糖异构酶及其应用
CN113957065B (zh) * 2021-11-30 2023-09-19 南京诺云生物科技有限公司 一种高转化率的蔗糖异构酶及其应用

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