WO2019175632A1

WO2019175632A1 - Methods for refolding isomaltulose synthase

Info

Publication number: WO2019175632A1
Application number: PCT/IB2018/051733
Authority: WO
Inventors: Saravanakumar IYAPPAN; Humaira Parveen SHEIKH; Banibrata Pandey
Original assignee: Petiva Private Limited
Priority date: 2018-03-15
Filing date: 2018-03-15
Publication date: 2019-09-19

Abstract

The present disclosure provides methods for refolding isomaltulose synthase (ISase) of Pantoea dispersa UQ68J from inclusion bodies. The method comprises solubilizing inclusion bodies expressed in a host cell in a solubilization solution; diluting the solubilized sucrose isomerase to obtain a diluted sample; incubating the diluted sample in presence of a refolding buffer and purifying the refolded isomaltulose synthase. The invention represents an advancement over the prior art for efficient and cost-effective methods for obtaining biologically active isomaltulose synthase.

Description

METHODS FOR REFOLDING ISOMALTULOSE SYNTHASE

FIELD OF INVENTION

The present disclosure relates to a method for refolding isomaltulose synthase (ISase) of Pantoea dispersa UQ68J from inclusion bodies produced during over-expression of isomaltulose synthase in heterologous expression host.

BACKGROUND OF THE INVENTION

Industrial production of natural or modified proteins in genetically engineered cells has helped in expanding biotechnology industries. A number of proteins are currently being produced by recombinant DNA technology. Bacterial host expression systems, such as Escherichia coli provide cost-effective manufacturing scale production of recombinant proteins.

Though expression of recombinant proteins in bacterial hosts is cost effective, a major difficulty in the process is the precipitation of expressed proteins as insoluble intracellular precipitates which care also known as inclusion bodies. Inclusion bodies (IB) are non crystalline and amorphous structures. 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.

Various methods for correct refolding of proteins have been developed till date. The common approaches followed for commercial production of heterologous proteins include the following steps: -

• Expressing the protein which precipitates as bacterial inclusion bodies

• Lysing the cell and collecting the inclusion bodies

• Solubilizing the inclusion bodies in the solubilization buffer

• Refolding the protein back to its biologically active form using a refolding buffer

• Purifying the biologically active protein

The aforesaid generalized approach incorporates within itself a huge number of variables and hence, it has to be tailored to meet the needs of specific proteins. There is no universal protocol available for refolding proteins. It is an extremely difficult task to determine the correct refolding conditions of the aggregated proteins. Even after determination of correct refolding conditions, the refolding process is generally not effective due to low yield of biologically active proteins.

The inventors in the present instance have been able to invent a method for refolding of a recombinant isomaltulose synthase, which gives extremely good yield of biologically active isomaltulose synthase under the refolding conditions.

Isomaltulose synthase used for production of isomaltulose from sucrose. Isomaltulose is a naturally occurring isomer of sucrose that is valued as non-cariogenic and low glycemic sweetener. There is a huge demand of isomaltulose in the healthy lifestyle segment wherein the consumers demand a suitable alternative to sucrose for following a low glycemic diet and avoidance of significant blood sugar variation. Isomaltulose is also suitable to athletes who are interested in a slower glucose-fructose metabolic release.

The current methods used for refolding isomaltulose synthase from inclusion bodies after recombinant expression suffers provides low renaturation rates, which results in low activity. Subsequently, the yield of isomaltulose is low and the downstream processing cost is high.

The inventors have identified the above issues and addressed the same by inventing a method for refolding isomaltulose synthase from Pantoea dispersa UQ68J. The method provides a manner for recovery of bioactive isomaltulose synthase (ISase) from inclusion bodies.

Thus, 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 isomaltulose synthase, which in turn lowers the cost of production of isomaltulose.

SUMMARY OF THE INVENTION

Technical Problem

The technical problem to be solved in this invention is an improved method for refolding isomaltulose synthase from inclusion bodies during recombinant production.

Solution to the problem

The problem has been solved by inventing an improved method in which the inventors have devised 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 isomaltulose synthase.

Advantages of the invention The invention provides an improved method for efficient, cheap and industrially- scalable means for refolding isomaltulose synthase. Further, the method provides a manner for recovery of bioactive isomaltulose synthase (ISase) from inclusion bodies. The invention lowers the cost of production of isomaltulose.

Overview of the invention

Accordingly, the present disclosure provides methods for obtaining bioactive recombinant isomaltulose synthase from inclusion bodies.

The method comprising isolation of inclusion bodies from E. coli cells overexpressing the recombinant isomaltulose synthase; solubilization of inclusion bodies in a buffer with appropriate chaotrophs and solublizing agents, refolding (renaturation) of solublized isomaltulose synthase into their native structures having isomaltulose synthase activity, and recovery of the refolded recombinant isomaltulose synthase.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts the vector map of pETl l-IS comprising modified nucleotide sequence encoding for isomaltulose synthase derived from Pantoea dispersa UQ68J.

Figure 2 depicts the vector map of pETl5-IS comprising modified nucleotide sequence encoding for isomaltulose synthase derived from Pantoea dispersa UQ68J.

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 samples from different stages of ISase refolding from inclusion bodies: Lane 1: Molecular weight standard; Lane 2: Soluble ISase (control); Lane 3: Crude cell lysate (2 pl); Lane 4: Soluble fraction of lysate (2 pl); Lane 5: Pellet fraction of lysate (2 pl); Lane 6: Washed IB (2 pg); Lane 7: Urea solubilized IB (1 pg); Lane 8: Refolded SIM (1 pg); Lane 9: Refolded and purified ISase (1 pg). The gel was stained with CBB R-250.

Figure 5 depicts Michaelis-Menten plot for kinetic analysis of refolded ISase.

Figure 6 illustrates the activity and bioconversion kinetics of purified soluble and refolded ISase.

Figure 7 depicts the purity of the refolded ISase.

Figure 8 depicts the pH optima for native and refolded ISase. The closed circles represent native enzyme and open circles represent refolded ISase. Figure 9 depicts the temperature optima for native and refolded ISase. The closed circles are for native ISase and open circles are for refolded ISase.

Figure 10 depicts the solubilization and refolding conditions for preparation of bioactive isomaltulose synthase form inclusion bodies.

BRIEF DESCRIPTION OF SEQUENCE LISTING

SEQ ID NO:l is the modified nucleotide sequence encoding isomaltulose synthase of Pantoea dispersa UQ68J.

SEQ ID NO:2 is the amino acid sequence of isomaltulose synthase of Pantoea dispersa UQ68J.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods belong. Although any methods and compositions similar or equivalent to those described herein can also be used in the practice or testing of the methods and compositions, representative illustrative methods and compositions are now described.

Where a range of values is provided, it is understood that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within by the methods and compositions. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within by the methods and compositions, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods and compositions.

It is appreciated that certain features of the methods, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the methods and compositions, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. It is noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements or use of a "negative" limitation. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

As used herein,“buffered solution” refers to a solution which resists changes in pH by the action of its acid-base conjugate components.

As used herein, 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 chaotropic 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. Examples of chaotropic agents include but are not limited to, guanidine hydrochloride, urea, alkaline hydroxide (e.g., sodium or potassium hydroxide) and combination thereof.

As used herein, the term "comprises" or "comprising" is generally used in the sense of include, that is to say permitting the presence of one or more features or components.

As used herein, the term“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.

As used herein, the term“properly folded” or“biologically active” ISase or other recombinant protein and the like refers to a molecule with a biologically active conformation.

As used herein, 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. Thus, 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.

As used herein, the term“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.

The term“recombinant strain” refers to a host cell which has been transfected or transformed with the expression constructs or vectors of this invention.

The term “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.

The term“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.

The term“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.

The term“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. The term“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.

The term "pH buffer" as used herein refers to any organic or inorganic compound or combination of compounds that will maintain the pH of a solution.

The term“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.

The term“translation” refers the process of translating the sequence of a messenger RNA (mRNA) molecule to a sequence of amino acids during protein synthesis. The genetic code describes the relationship between the sequence of base pairs in a gene and the corresponding amino acid sequence that it encodes. In the cell cytoplasm, the ribosome reads the sequence of the mRNA in groups of three bases to assemble the protein.

As used herein, the term“L-Arginine buffer” refers to a buffer solution comprising 50 mM Tris-HCl, 150 mM NaCl and 0.4 mM L-Arginine at pH 7.4.

As used herein, the term“non-detergent sulfobetaines buffer” or“NDSB buffer” refers to a buffer solution comprising 50 mM HEPES, 240 mM NaCl, lmM KC1, 0.25 mM MnCl₂ at pH 7.5.

As used herein, the term“sorbitol buffer” refers to a solution comprising 50 mM TAPS, 1.5 M sorbitol, 240 mM NaCl and 1 mM KC1 at pH 8.5.

As used herein, the term“sucrose buffer” refers to a solution comprising 50 mM Tris- HCl, 0.2 mM sucrose, 150 mM NaCl, 20% Glycerol and 5 mM MnCh at pH 11.2.

As used herein, the term“arginine-glycerol buffer” refers to a solution comprising 50 mM Tris-HCl, 150 mM NaCl, 0.4 M L-Arginine-HCl and 10% glycerol at pH 7.4.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses an improved method for preparation of soluble and active recombinant isomaltulose synthase (ISase) from inclusion bodies produced during over-expression of isomaltulose synthase in heterologous expression host.

The inventors have conducted intensive experiments and have inventing an improved process 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 isomaltulose synthase. Effectiveness of invention

For the first time, the inventors have devised methods for obtaining biologically active isomaltulose synthase expressed as inclusion bodies in Escherichia coli. The properties of the refolded recombinant isomaltulose synthase are comparable to native isomaltulose synthase. The same can be evidenced from the following factors:

1. The kinetic profile of the refolded ISase is comparable to native ISase (Figure 5).

2. The conversion ability of the refolded ISase is comparable to native ISase (Figure 6).

3. The pH optima of the refolded ISase is comparable to native ISase at pH 6.0 (Figure

8).

4. The temperature optima of the refolded ISase is comparable to native ISase at 35°C (Figure 9).

The inventive approach used in the present invention has led to the development of methods which can enable cheap production of isomaltulose as the production cost for the enzyme would drastically become cheaper. Moreover, the enzyme exhibits comparable characteristics, which is an essential for industrial scale production.

Before the processes and methods of the present disclosure are described in greater detail, it is to be understood that the invention is not limited to particular embodiments and may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the methods and compositions will be limited only by the appended claims.

In one embodiment, the invention provides a recombinant host cell for expression of isomaltulose synthase. For preparing the recombinant host cell, the gene encoding for isomaltulose synthase (ISase) of Pantoea dispersa UQ68 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 pETlla and pETl5b vector and further transformed into Escherichia coli JM109 (DE3) host cell. In another embodiment, the present disclosure provides a method for producing biologically active recombinant isomaltulose synthase (ISase) from inclusion bodies comprising the steps of:

a. solubilizing inclusion bodies expressed in a host cell in a solubilization solution comprising a denaturant and a pH buffer, wherein the denaturant is selected from a group comprising urea and guanidine HC1;

b. diluting the solubilized isomaltulose synthase to obtain a diluted sample;

c. incubating the diluted sample in presence of a refolding buffer selected from a group comprising L-arginine buffer, NDSB buffer, sorbitol buffer, sucrose buffer and arginine-glycerol buffer; and

d. purifying the refolded isomaltulose synthase.

In another embodiment, the biologically active recombinant isomaltulose synthase (ISase) comprises the amino acid sequence as set forth in SEQ ID NO:2.

In another embodiment, the invention provides for isolation of isomaltulose synthase expressed as inclusion bodies in Escherichia coli. The recombinant isomaltulose synthase 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. Optionally the cell lysate is stored at -80°C or processed further for inclusion body preparation.

In one embodiment, the composition of lysis buffer is 50 mM Tris-HCl at pH8.0.

In another embodiment, the inclusion bodies collected are washed using wash buffer.

In one embodiment, inclusion bodies are washed by resuspending lysis buffer and recollecting by high speed centrifugation or TFF. Fysis buffer may contain detergents or salts or chaotrophs or a combination thereof. In yet another embodiment, detergent can be any detergent, typically, Triton X-100^® or Tween^®, and its concentration can be between 0.001-10% (w/v), preferably 1%. In yet another embodiment, salt can be any salt, preferably NaCl or KC1 between 0.01-2 M concentrations, preferably 1.0 M. In yet another embodiment, 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.

In yet another embodiment, 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.

In a further embodiment, 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 ISase. The solubilization can be done at a variety of temperatures.

In one embodiment, 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.

In one embodiment, the solubilization solution is a urea solubilization solution. In a further embodiment, the composition of the urea solubilization solution is 50 mM Tris- HCl, 8.0 M urea at pH 8.0.

In another embodiment, the solubilization solution is a Guanidine-Hydrochloride solution. In a further embodiment, the composition of the Guanidine-Hydrochloride solution is 50 mM sodium carbonate, 3.5 M Guanidine-Hydrochloride, 150 mM NaCl, 0.5 mM EDTA at pH 11.2.

In a further embodiment, 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 (A₂so).

After adjusting the concentration, the inclusion body solution is first diluted with refolding buffer to reduce the denaturant and protein concentration. The inclusion body solution is diluted to about 10- lOO-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 using urea solubilization solution (50 mM Tris-HCl, 8.0 M urea at pH 8.0).

In another embodiment, the solubilized inclusion bodies are diluted using Guanidine- HC1 solubilization solution (50 mM sodium carbonate, 3.5 M Guanidine-Hydrochloride, 150 mM NaCl, 0.5 mM EDTA at pH 11.2).

The diluted solutions are further incubated in a refolding buffer solution for allowing the solubilized protein to refold.

In one embodiment, the isomaltulose synthase is refolded in 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.

In another embodiment, the solubilized inclusion bodies are incubated in L-Arginine refolding buffer for 16 hours at a temperature between 4 -l0°C.

In another embodiment, the solubilized inclusion bodies are diluted to 20 volumes using non-detergent-sulfobetaines (NDSB) refolding buffer.

In another embodiment, the composition of the non-detergent-sulfobetaines refolding buffer is 50 mM HEPES, 240 mM NaCl, 1 mM KC1 and 0.25 mM MnCk at pH 7.5. In another embodiment, the solubilized inclusion bodies are incubated in non- detergent- sulfobetaines refolding buffer overnight at a temperature between 4°C.

In one embodiment, the solubilized inclusion bodies are diluted to 20 volumes using sorbitol refolding buffer.

In another embodiment, the composition of the sorbitol refolding buffer is 50 mM TAPS, 1.5 M Sorbitol 240 mM NaCl and 1 mM KC1 at pH 8.5.

In another embodiment, the solubilized inclusion bodies are incubated in sorbitol refolding buffer overnight at a temperature at 4 °C.

In one embodiment, the solubilized inclusion bodies are diluted to 20 volumes using sucrose refolding buffer.

In another embodiment, the composition of the sucrose refolding buffer is 50 mM Tris-HCl, 0.2 M Sucrose, 150 mM NaCl, 20% Glycerol and 5 mM MnCl₂ at pH 6.0.

In another embodiment, the solubilized inclusion bodies are incubated in sucrose refolding buffer overnight at a temperature at 4 °C.

In another embodiment, the composition of the sucrose refolding buffer is 50 mM Tris-HCl, 0.2 M Sucrose, 150 mM NaCl, 20% Glycerol and 5 mM MnCk at pH 6.0.

In one embodiment, the solubilized inclusion bodies are diluted to 40 volumes using arginine-glycerol refolding buffer.

In another embodiment, the composition of the arginine-glycerol refolding buffer is 50 mM Tris-HCl, 150 mM NaCl, 0.4 M L-Arginine.HCl and 10% glycerol at pH 7.4.

In another embodiment, the solubilized inclusion bodies are incubated in sucrose refolding buffer for 24 hours at a temperature between 4-6°C.

In another embodiment, after completion of the refolding reaction, properly folded ISase may be exchanged with suitable buffer, concentrated and further purified to produce biologically active ISase. 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 ISase 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).

In one embodiment, ion exchange chromatography (IEC) is used for recovery and purification of recombinant ISase.

In another embodiment, anion exchange chromatography is used. The chromatographic resin is derivatized with diethlyaminoethyl (DEAE) or quaternary ammonium (Q-) group. The exact conditions for IEC depends on the chromatography media selected. Generally, loading conditions will have low ionic strength. In an embodiment, the present dused Q-Sepharose anionic exchange media to further purify the protein.

The refolded and purified ISase can be stored at 4°C or -30°C in solution. In some embodiments, the ISase is stored in buffer containing about 50 mM Tris-HCl and about 10-50% glycerol at a pH of about 7.0.

The inventors have observed that the ISase produced in this method can be stored at - 50 mM Tris-HCl, 50% glycerol at -30°C for more than 6 months.

After completion of refolding reaction, properly folded ISase is exchanged with suitable buffer, concentrated and further purified to produce biologically active ISase. The refolding protein may be exchanged with suitable buffer or concentrated by any convenient method such as ultrafiltration, diafiltration, dialysis and chromatography. The buffer exchange can 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.

The folded/ buffer exchanged ISase may be mixed with a suitable buffer and further purified by ion exchange chromatography (IEC). ISase may be purified in flow-through mode on cationic or anionic chromatography. The exact conditions for IEC depends on type of chromatography media selected, whether buffer exchange is required or not and the requirements of any later purification steps. In one embodiment of the present disclosure, Q-Sepharose anionic exchange media is used to further purify the protein.

The refolded and purified ISase can be stored at 4°C or -30°C in solution. The ISase is stored in a buffer containing about 50 mM Tris-HCl and about 10-50% glycerol at a pH of about 7.0. The ISase obtained by the present process is stable for more than 6 months when stored in 50 mM Tris-HCl, 50% glycerol at -30°C.

In a further embodiment, the product formation kinetics of refolded isomaltulose was studied. The purified recombinant ISase shows specific activity of 430 IU/mg (±5%), K_m of 165 mM, K_cat of 461 S ¹ and K_ca/K_m of 8,300 (M^S ¹), which is similar to native protein.

In further embodiments, the refolded isomaltulose synthase was studied to determine the pH and temperature optima. The reaction mixture containing sucrose and refolded isomaltulose synthase were incubated at different pH and temperature.

It was found that the enzyme had high activity between pH of 5-7.5, highest at 6.0. It was also found that recombinant isomerase had the highest activity between the temperature 20-45°C, highest at around 35°C.

It was observed that the refolded isomaltulose synthase has characteristics which are comparable to native isomaltulose synthase.

EXAMPLES

The following examples particularly describe the manner in which the invention is to be performed. But the embodiments disclosed herein do not limit the scope of the invention in any manner.

Example 1: Gene construction for expression of isomaltulose synthase in E. coli

Gene encoding for isomaltulose synthase (ISase) of Pantoea dispersa UQ68 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 sequence was cloned in to pUC57 using EcoRV restriction enzyme site to generate pUC57-IS construct. Cloned gene sequence was confirmed by sequence analysis.

The DNA fragment encoding for isomaltulose synthase was PCR amplified using gene specific primers, and sub cloned into pETl la using Ndel and BamHI restriction enzyme sites to generate pETl l-IS. The vector map of pETl l-IS is represented in Figure 1. In addition, the coding region was PCR amplified without stop codon using gene specific primers and sub cloned into E. coli expression vector pETl5b using Ndel and Hind III restriction enzymes to generate pETl5-IS-HIS construct expressing isomaltulose synthase with C-terminal 6x Histidine tag. The recombinant plasmid carrying isomaltulose synthase gene (pETll- IS and pETl5-IS) was confirmed by restriction digestion analysis and followed by DNA sequencing. The vector map of pETl5-IS is represented in Figure 2.

The isomaltulose synthase of Pantoea dispersa UQ68J comprises the amino acid sequence as set forth in SEQ ID NO:2.

Example 2: Development of recombinant E. coli with gene constructs

Recombinant plasmid DNA (pETl l-IS) was transformed into Escherichia coli expression host JM 109 (DE3) 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 OD₆oo in LB liquid or defined media without ampicillin and grown up to 0.6 OD₆oo 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.

Figure 3 depicts expression analysis of recombinant isomaltulose synthase in Escherichia coli.

Figure 3A depicts that control and recombinant Escherichia coli cells [JM109 carrying pETl l-IS] 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. Lane 10 and 11 are four hrs induced supernatant and pellet. Abbreviations are: M: Protein molecular weight marker and kDa = Kilo Dalton.

Figure 3B depicts identity analysis of recombinant protein by Western blot analysis. Lane 1 and 2 depicts host cell lysate un-induced and induced. Lane 3 and 4 depicts recombinant strain uninduced and induced stage. Immuno -detection was carried our using protein specific antibodies. Example 3: Large Scale production of recombinant isomaltulose synthase

High cell density fermentation was used for large scale production of recombinant isomaltulose synthase 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 pre-culture 2 (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 medium 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%. When the ODeoo of the culture reached 10 - 15, connect the feed at 37.5 ml/hr (25ml/L/hr) flow rate. When OD₆oo of the culture reached 50 - 80, 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to the reactor to induce isomaltulose synthase expression, and the fermentation was continued for another 8 -10 hrs.

Example 4: Isolation and solubilization of inclusion bodies

The culture was harvested by centrifugation and then resuspended in 400 mL of lysis buffer (50 mM Tris-HCl, pH 8.0). 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 IB wash buffer (50 mM Tris-HCl, 1 M NaCl, 2 M urea, 1% Triton X-100^®, pH 8.0).

The washed inclusion bodies (4 g) were dissolved in 135 ml of Urea solubilization solution (50 mM Tris-HCl, 8.0 M urea, pH 8.0). Approximately 1.0 g of protein was recovered after the solubilization_·

Alternatively, the inclusion bodies were dissolved in Guanidine-Hydrochloride solution (50 mM Sodium carbonate, pH 11.2, 3.5 M Guanidine-Hydrochloride, 150 mM NaCl, 0.5 mM EDTA). The solution was clarified by centrifugation at 25,000 X g.

Example 5: Refolding of recombinant isomaltulose synthase in presence of L- Arginine

The inclusion bodies solubilized in the urea solubilization solution, as provided in Example 4, was adjusted for protein concentration of 2 mg/mL with urea solubilization solution (50 mM Tris-HCl, 8.0 M urea, pH 8.0). Refolding was performed by directly adding the clarified solution drop-wise at a rate of 0.5 mL/min to 20 volumes of refolding buffer (50 mM Tris-HCl, 150 mM NaCl, 0.4 M L-Arginine, pH 7.4) at 4-lO°C. The refolding was continued overnight, and temperature was maintained between 4 -lO°C.

Example 6: Refolding of recombinant isomaltulose synthase in presence of Non- detergent-sulfobetaines (NDSB)

The inclusion bodies solubilized in the urea solubilization solution, as provided in Example 4, was adjusted for protein concentration of 5 mg/ml with urea solubilization solution (50 mM Tris-HCl, 8.0 M urea, pH 8.0). Recombinant isomaltulose synthase was refolded by rapidly diluting the solubilized inclusion bodies in 25 volumes of refolding buffer containing NDSB 201 (50 mM HEPES, 240 mM NaCl, 1 mM KC1, 0.25 mM MnCk, pH 7.5) at 4°C. The refolding was carried out overnight and temperature was maintained at 4°C.

Example 7: Refolding of recombinant isomaltulose synthase in presence of Sorbitol

The inclusion bodies solubilized in the urea solubilization solution, as provided in Example 4, was adjusted for protein concentration of 5 mg/ml with urea solubilization solution (50 mM Tris-HCl, 8.0 M urea, pH 8.0). Recombinant isomaltulose synthase was refolded by rapidly diluting the solubilized inclusion bodies in 25 volumes of refolding buffer containing sorbitol (50 mM TAPS, 1.5 M Sorbitol 240 mM NaCl, 1 mM KC1, pH 8.5). The refolding was carried overnight, and temperature was maintained at 4°C.

Example 8: Refolding in presence of sucrose

The inclusion bodies solubilized in the Guanidine-Hydrochloride solubilization solution, as provided in Example 4, was adjusted for protein concentration of 2 mg/ml with the Guanidine-Hydrochloride solubilization buffer (50 mM Sodium carbonate, pH 11.2, 3.5 M Guanidine-Hydrochloride, 150 mM NaCl, 0.5 mM EDTA).

Recombinant isomaltulose synthase was refolded by rapidly diluting the solubilized inclusion bodies in 20 volumes of refolding buffer containing 50 mM Tris-HCl, 0.2 M Sucrose, 150 mM NaCl, 20% Glycerol and 5 mM MnCk, pH 6.0. The refolding was carried overnight, and temperature was maintained at 4°C.

Example 9: Large scale refolding in presence of arginine-glycerol refolding buffer

The cell pellet (50 g) from 2 L fermentation broth was lysed and the inclusion bodies were isolated and solubilized in urea solubilization buffer as described in example 4.

Isomaltulose synthase was refolded by rapidly diluting the urea solubilized inclusion bodies into a 40-fold excess buffer containing 0.4 M L- Arginine. HC1. The final concentration of solubilized IB was adjusted to 2 mg/ml with urea solubilization buffer, and 500 ml this solubilized solution was slowly added drop-wise at rate of 0.5 ml/min to a 20 L of Refold Buffer (50 mM Tris-HCl, 150 mM NaCl, 0.4 M L-Arginine.HCl, 10% glycerol, pH 7.4). The refolding was performed in cold room at 4 - 6°C with rapid mixing on a magnetic stirrer. The final protein concentration of the refolded sample was 50 pg/ml. The refolding was allowed to continue for 24 h at 4-6°C with gentle stirring. There was some aggregation in the sample during refolding and it was slightly hazy. However, the Aeon nm value for light scattering was insignificant. After 24 h of incubation at 4°C, the refolded sample (20 L) was filtered through a 0.45 pm cellulose acetate capsule filter (Sartoclean^® manufactured by Sartorius) with a flow rate of 60 ml/min to remove particulate matter. After the filtration, the clear solution was concentrated to 5L by Tangential flow filtration (Sartorius) equipped with 0.1 m² Hydrosart^® membrane (30,000 MWCO). The permeate flow rate was maintained at 40 ml/min during the process. After the volume reduction, diafiltration was carried out with 15 L of Exchange buffer (50 mM Tris-HCl, 10% glycerol, pH 7.4) to remove urea and L-arginine from refolded sample. During the diafiltration, protein aggregation was observed, and further buffer exchange had resulted in more aggregation. After completion of buffer exchange, the sample was further concentrated to 500 ml. At this stage, the refolded sample showed a specific activity of 109 IU/mg, only 30% of the native ISase. Hence, the concentrated sample was again dialyzed against 50 mM Tris-HCl, pH 7.0. The final enzyme had a specific activity of -300 IU/mg which was -30% less than recombinant soluble enzyme (Internal reference standard).

Example 10: Buffer exchange and purification

After 24 hrs of incubation at 4°C, the refolded sample (20 L) the refolded ISase from example 9 was subjected to a pre-filtration step. The sample was filtered through 0.45 pm cellulose acetate capsule filter (Sartoclean^® manufactured by Sartorius) and concentrated to 5 L by Tangential flow filtrations (Sartorius Hydrosart^®, 0.1 m², 30, 000 MWCO membrane). Polysorbitol, Tween-20 or Tween-80 may be added to a final concentration of 0.005% to the filtered solution. The concentrated sample was diafiltered against three volumes of Exchange buffer (50 mM Tris-HCl, 10% Glycerol, pH 7.4) to remove L-Arginine-HCl and Urea. The exchange buffer may include Tween-20 or Tween-80 at about 0.005%.

The refolded isomaltulose synthase was purified by Q-Sepharose FF^® ion exchange chromatography. First, the pH of the refolded sample was adjusted to 8.0 and then applied to Q-Sepharose FF^® column that had been equilibrated with 50 mM Tris-HCl, 10% Glycerol, pH 8.0. ISase at these conditions do not show binding to Q-Sepharose FF^® hence collected in flow through and wash. Polysorbital detergent and Tween-80^® was added to 0.001% to the purified recombinant ISase and the sample was further concentrated by TFF.

The recombinant ISase was concentrated to at least 3.0 mg/ml and stored at -30°C in 50 mM Tris-HCl, 50% glycerol, 0.001% Tween-80, pH 7.4.

Example 9: Product formation kinetics of isomaltulose synthase

Sucrose isomerization activity of recombinant ISase was tested by an enzyme assay using sucrose as substrate. The pH and temperature optimum of the refolded isomaltulose is comparable to native soluble isomaltulose synthase enzyme. The reaction velocity was measured by incubating appropriately diluted ISase with various concentrations of sucrose in a 50 mM Citrate phosphate buffer pH 6.0 at 35°C for 15 min and measuring the production of isomaltulose 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 (Figure 5). The purified recombinant ISase had activity of 430 IU/mg (±5%), K_m of 165 mM, K_cat of 461 S ¹ and K_ca/K_m of 8300 (M^S ¹), which is comparable to native isomaltulose synthase.

The recombinant refolded ISase was also compared with native ISase for its ability to convert sucrose into isomaltulose. The results are depicted in Figure 6. The conversion ability of the enzyme is comparable to native enzyme.

Example 10: Characterization of refolded isomaltulose synthase

The refolded isomaltulose synthase was studied to determine the purity profile. It was found that after purification, the refolded ISase was extremely pure. The HPLC analysis is depicted in Figure 7.

The refolded isomaltulose synthase was studied to determine the pH and temperature optima for the same. The reaction mixture containing sucrose and refolded isomaltulose synthase were incubated at different pH (Figure 8) and temperature (Figure 9). It was found that the enzyme had high activity between pH of 5-7.5, highest at 6.0. It was also found that recombinant isomerase had the highest activity between the temperature 20-45 °C, highest at around 35°C.

Table 1: Comparison between the IRS and Refolded Isase

Table 1 depicts that the refolded isomaltulose synthase has characteristics which are comparable to native isomaltulose synthase.

Figure 10 depicts the solubilization and refolding conditions for preparation of bioactive isomaltulose synthase from inclusion bodies.

Claims

The claims:

1. A method for refolding isomaltulose synthase of Pantoea dispersa UQ68J from inclusion bodies, said method comprising the steps of:

a. solubilizing inclusion bodies expressed in a host cell in a solubilization solution comprising a denaturant and a pH buffer, wherein the denaturant is selected from a group comprising urea and guanidine-HCl;

b. diluting the solubilized isomaltulose synthase to obtain a diluted sample;

d. purifying the refolded isomaltulose synthase.

2. The method as claimed in claim 1, wherein the host cell is Escherichia coli.

3. The method as claimed in claim 1, wherein isomaltulose synthase of Pantoea dispersa UQ68J comprises the amino acid sequence of SEQ ID NO:2.

4. The process as claimed in claim 1, wherein the pH buffer is selected from a group comprising Tris-HCl buffer and sodium carbonate buffer.

5. The process as claimed in claim 1, wherein the solubilized isomaltulose synthase is diluted with refolding buffer in a ratio of 1 to 20.

6. The process as claimed in claim 1, wherein incubating is carried out for at least 16 hours at a temperature in the range of 4-lO°C.

7. The process as claimed in claim 1, wherein the pH of the refolding buffer ranges between 5.5 - 11.5.

8. The process as claimed in claim 1, wherein the solubilization solution comprises 50 mM sodium carbonate, 3.5 M guanidine-HCl, 150 mM NaCl and 0.5 mm EDTA.

9. The process as claimed in claim 1, wherein the L-arginine buffer comprises 50 mM Tris-HCl, 150 mM NaCl and 0.4 mM L-Arginine.

10. The process as claimed in claim 1, wherein the NDSB buffer comprises 50 mM HEPES, 240 mM NaCl, lmM KC1 and 0.25 mM MnCl₂.

11. The process as claimed in claim 1, wherein the sorbitol buffer comprises 50 mM TAPS, 1.5 M sorbitol, 240 mM NaCl and 1 mM KC1.

12. The process as claimed in claim 1, wherein the sucrose buffer comprises 50 mM Tris- HCl, 0.2 mM sucrose, 150 mM NaCl, 20% Glycerol and 5 mM MnCl₂.

13. The process as claimed in claim 1, wherein the arginine-glycerol buffer comprises 50 mM Tris-HCl, 150 mM NaCl, 0.4 M L-Arginine-HCl and 10% glycerol.

14. The process as claimed in claim 1, wherein the refolded isomaltulose synthase was purified by a process comprising the steps:

a. a prefiltration step of the sample through a cellulose acetate filter;

b. a diafiltration step of the sample to remove the denaturant; and

c. a purification step by ion-exchange chromatography.