METHODS FOR REFOLDING SUCROSE ISOMERASE FIELD OF THE INVENTION
The present disclosure relates to a method for refolding sucrose isomerase (SIase) of Pseudomonas mesoacidophila MX-45 from inclusion bodies produced during over-expression of sucrose isomerase in a heterologous expression host.
BACKGROUND OF THE INVENTION
Industrial production of natural or modified proteins in genetically engineered cells has helped in expanding the 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 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 (α-D-glucosylpyranosyl-1,1-D-fructofuranose), a structural isomer of sucrose (α-d-glucosylpyranosyl-1,2-β-D-fructofuranoside) can be used as a sugar substitute because of its sweet taste, similar physical and organoleptic properties of sucrose. Moreover, 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 current methods used for refolding sucrose isomerase from inclusion bodies after recombinant expression suffers provides low renaturation rates, which results in low activity. Subsequently, the yield of trehalulose is low and the downstream processing cost is high.
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 (SIase) 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 sucrose isomerase, which in turn lowers the cost of production of trehalulose.

SUMMARY OF THE INVENTION
Technical Problem
The technical problem to be solved in this invention is an improved method for refolding sucrose isomerase (SIase) of Pseudomonas mesoacidophila MX-45 from inclusion bodies produced during over-expression of sucrose isomerase in a heterologous expression host. Solution to the problem
The problem has been solved by inventing improved methods in which the inventors have devised combinations of solubilizing solutions and refolding buffers. Further, the process parameters such as pH and temperature have been identified to get an optimum yield of biologically active sucrose isomerase. Advantages of the invention
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 (SIase) from inclusion bodies. The invention lowers the cost of production of trehalulose. Overview of the invention
Accordingly, the present invention relates to a method for obtaining bioactive recombinant trehalulose synthase or sucrose isomerase (SIase) 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.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts the vector map of pET11-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 SIase 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 μg); Lane 8: Refolded and purified SI (3 ug); Lane 9: Internal control protein of SIase (2 μg)); Lane M: Molecular weight standard. The proteins were separated on 12% SDS-PAGE and gel was stained with Coomassie brilliant blue (CBB R-250).
Figure 5 depicts Michaelis-Menten plot for kinetic analysis of refolded SIase.
Figure 6 depicts the purity of the refolded SIase.
Figure 7 depicts the pH optima for native and refolded SIase. The closed circles represent native enzyme and open circles represent refolded SIase.
Figure 8 depicts the temperature optima for native and refolded SIase. The closed circles are for native SIase and open circles are for refolded SIase.
Figure 9 depicts the solubilization and refolding conditions for preparation of bioactive SIase form inclusion bodies.
BRIEF DESCRIPTION OF SEQUENCE LISTING
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.
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” SIase 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.
DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses an improved method for preparation of soluble and active recombinant sucrose isomerase (SIase) expresses as inclusion bodies during over-expression of sucrose isomerase in heterologous expression host.
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.
Effectiveness of invention
For the first time, the inventors have devised a method for obtaining biologically active 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:
1. The kinetic profile of the refolded SIase is comparable to native SIase (Figure 5).
2. The pH optima of the refolded SIase is comparable to native SIase at pH 6.0 (Figure 7).
3. The temperature optima of the refolded SIase is comparable to native SIase at 35°C (Figure 8).
The inventive approach used in the present invention has led to the development of a method which can enable cheap production of trehalulose 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 sucrose isomerase. For preparing the recombinant host cell, the gene encoding for sucrose isomerase (SIase) 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 pET11a and pET23a vector and further transformed into Escherichia coli JM109 (DE3) host cell.
In another embodiment, the present disclosure provides a method for producing biologically active sucrose isomerase 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 sucrose isomerase to obtain a diluted sample;
c. incubating the diluted sample in presence 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™) and glycerol; and
d. purifying the refolded sucrose isomerase.
In another embodiment, the biologically active recombinant sucrose isomerase (SIase) comprises the amino acid sequence as set forth in SEQ ID NO:2.
In another embodiment, 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 KCl 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 - 10°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 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. Lysis buffer may contain detergents or salts or chaotrophs or a combination thereof. In yet another embodiment, detergent can be any detergent, typically, Triton X100® 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 KCl 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 SIase. 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 Tris-HCl, 6 M Guanidine-Hydrochloride at pH 8.0.
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., 4-40ºC. 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 (A280).
After adjusting the concentration, 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 10-20°C.

In one embodiment, the solubilized inclusion bodies are diluted to 20 volumes using Tris-HCl refolding buffer.
In another embodiment, the composition of the Tris-HCl refolding buffer is 50 mM Tris-HCl and 150 mM NaCl at pH range of 6-7.4.
In another embodiment, the solubilized inclusion bodies are incubated in Tris-HCl refolding buffer for 16 hours at a temperature between 4 -10°C.
In one embodiment, the solubilized inclusion bodies are diluted to 20 volumes using glycerol refolding buffer.
In another embodiment, the composition of the glycerol refolding buffer is 50 mM Tris-HCl, 10% Glycerol and 150 mM NaCl at pH range of 6-7.4.
In another embodiment, the solubilized inclusion bodies are incubated in glycerol refolding buffer for 16 hours at a temperature between 4 -10°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, 200 mM sucrose and 150 mM NaCl at pH range of 6-7.4.
In another embodiment, the solubilized inclusion bodies are incubated in sucrose refolding buffer for 16 hours at a temperature between 4 -10°C.
In one embodiment, the solubilized inclusion bodies are diluted to 20 volumes using L-Arginine refolding buffer.
In another embodiment, 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 -10°C.
In one embodiment, the solubilized inclusion bodies are diluted to 40 volumes using urea refolding buffer.

In another embodiment, the composition of the urea refolding buffer is 50 mM Tris-HCl, 150 mM NaCl and 1.0 M urea at pH range of 6-9.
In another embodiment, the solubilized inclusion bodies are incubated in urea refolding buffer for 16 hours at a temperature between 4 -10°C.
In another embodiment, the solubilized inclusion bodies are diluted to 40 volumes using Triton X-100 refolding buffer.
In another embodiment, the 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.
In another embodiment, the solubilized inclusion bodies are incubated in Triton X-100 refolding buffer for 16 hours at a temperature between 4-10°C.
In another embodiment, the solubilized inclusion bodies are diluted to 40 volumes using calcium chloride refolding buffer.
In another embodiment, the composition of the calcium chloride refolding buffer is 50 mM Tris-HCl, 150 mM NaCl and 2 mM CaCl2 at pH range of 6-9.
In another embodiment, the solubilized inclusion bodies are incubated in calcium chloride refolding buffer for 16 hours at a temperature between 4-10°C.
In another embodiment, the solubilized inclusion bodies are diluted to 20 volumes using glycerol refolding buffer.
In another embodiment, the composition of the glycerol refolding buffer is 50 mM Tris-HCl and 10% Glycerol at pH 6.0.
In another embodiment, the solubilized inclusion bodies are incubated in glycerol refolding buffer for 20 hours at a temperature between 4-10°C.
In another embodiment, after completion of the refolding reaction, properly folded SIase may be exchanged with suitable buffer, concentrated and further purified to produce biologically active SIase. 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 SIase 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 SIase.
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 SIase can be stored at 4°C or -30°C in solution. In some embodiments, the SIase 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 SIase produced in this method can be stored at -30°C in 50 mM sodium acetate, 50% glycerol for more than 6 months.
In a further embodiment, the product formation kinetics of refolded sucrose isomerase was studied. The purified recombinant SIase shows specific activity of 981 U/mg (±5%), Km of 31 mM, Kcat of 1055 S-1 and Kcat/Km of 34,000 (M-1S-1), which is similar to native protein.
In further embodiments, 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.
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.

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.
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 sucrose isomerase in E. coli
Gene encoding for sucrose isomerase (SIase) 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 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 pET11a using Ndel and BamHI restriction enzyme sites to generate pET11-SI. The vector map of pET11-SI 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 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 2.
The sucrose isomerase of Pseudomonas mesoacidophila MX-45 comprises the amino acid sequence as set forth in SEQ ID NO:2.
Example 2: Development of recombinant Escherichia coli with gene constructs
Recombinant plasmid DNA (pET11-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 OD600 in LB liquid or defined media without ampicillin and grown up to 0.6 OD600 and the cells were induced for protein expression by addition of 0.5mM of IPTG (Isopropyl β-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 sucrose isomerase in E. coli. Figure 3A depicts that control and recombinant E. coli cells [JM109 carrying pET11-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. Lane 10 and 11 are four hrs induced supernatant and pellet of cell fractions of recombinant strains. Abbreviations used are: M : Protein molecular weight marker and kDa = Kilo Dalton.
Figure 3B depicts identity analysis of recombinant protein by Western blot analysis. Lane l 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%. When the OD600 of the culture

reached 10 – 15, the feed was connected at 37.5 ml/hr (25ml/L/hr) flow rate. When OD600 of the culture reached 50 – 80, 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to the reactpr tp induce sucrose isomerase expression, and the fermentation was continued for another 8 -10 hr.
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 1.0g of protein was recoverd after the solubilization. In another experiment, 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 3: Refolding of sucrose isomerase inclusion bodies
An initial screening experiment was performed to find the best refolding conditions for SIase. 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 μg/mL. The refolding was carried out at 4-10°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 SIase remained in the solution was measured.
S. Refolding buffer pH Activity recovery (%)
No. Solubilizing buffer

50 mM Tris-HCl, 50 mM Tris-
8.0 M urea, pH HCl, pH 8, 6
8.0 M Gdn-HCl
6.0 54 1.4
1 50 mM Tris-HCl, 150 mM NaCl
7.4 1.5 1.5
50 mM Tris-HCl, 150 mM NaCl, 6.0 80 1.1
2
10% Glycerol 7.4 3.3 3
50 mM Tris-HCl, 150 mM NaCl, 6.0 12 16
3
200 mM Sucrose 7.4 50 7.32
50 mM Tris-HCl, 150 mM NaCl, 6.0 1.3 36
4
0.4 M L-Arginine 7.4 5 5.4
Table 1: Refolding screen for Urea or Gdn.HCl solubilized SIase
Maximum activity recovery was observed in refolding buffer containing 10% glycerol at pH 6.0. The screening results were used to develop a refolding method for SIase using glycerol as refolding agent. The refolding method was further optimized, and large-scale refolding procedure was developed which was described in Example 7. Apart from glycerol, SIase was refolded in presence of various refolding agents that are described in detail as further examples.
Example 4: Refolding in presence of 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 SIase solution, drop-wise, to 40 volumes of refolding buffer (50 mM Tris-HCl,
150 mM NaCl, 1.0 M urea) between pH 6-9 at 4-10°C. The refolding was continued for 16
h at 4-10°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 SIase
shows a specific activity of 659 IU/mg for sucrose when refolded at pH 6.0 in presence of
1 M urea.
Specific activity pH
(IU/mg)
6.0 659

7.0 402
8.0 193
9.0 4.93
Table 2: Influence of pH on SIase refolding in presence of 1 M urea
Example 5: Refolding in presence of Triton X-100:
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 SIase solution, drop-wise, to 40 volumes of refolding buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% Triton-X100) between pH 6-9 at 4-10°C. The refolding was continued for 16 h at 4-10°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 SIase shows a specific activity of 440 IU/mg for sucrose when refolded at pH 6.0 in presence of 0.5% Triton-X100.
Specific activity pH
(IU/mg)
6.0 440
7.0 347
8.0 5.90
9.0 0.02
Table 3: Influence of pH on Triton-X100 assisted refolding of SIase Example 6: Refolding in presence of Cacl2
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 SIase solution, drop-wise, to 40 volumes of refolding buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM Cacl2) between pH 6-9 at 4-10°C. The refolding was continued for 16 h at 4-10°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 SIase

shows a specific activity of 594 IU/mg for sucrose when refolded at pH 6.0 in presence of
2 mM Cacl2.
Specific activity pH
(IU/mg)
6.0 594
7.0 332
8.0 103
9.0 9.28
Table 4: Influence of pH on Cacl2 assisted refolding of SIase Example 7: Large scale refolding of SIase in presence of glycerol
Large scale optimized refolding process was developed for SIase using glycerol as a refolding agent. The refolded SIase was further purified by ion exchange chromatography near homogeneity. For large scale refolding of SIase, first, the cell pellet (42 g) from 2 L fermentation broth was lysed, and inclusion bodies were isolated from cell lysate as described in Examples 1 and 2 respectively.
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. The amount of solubilized IB was calculated by measuring the protein concentration spectrophotometrically at A280 nm using the molar extinction coefficient of SIase (119340 M-1cm-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. First, the final concentration of solubilized IB was adjusted to 1.75 mg/mL with urea solubilization buffer. Then, 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 4-10°C with rapid mixing on a magnetic stirrer. The final protein concentration of the refolded sample was maintained at 85 μg/mL. The refolding was allowed to continue for 20 h at 4-10°C with gentle stirring. The refolded sample (10

L) was subjected to a pre-filtration step. The sample was filtered through a 0.45 μm 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 m2 Hydrosart (Sartorius) membrane (30,000 MWCO). The permeate flow rate, 50 mL/min was maintained during the process. After the filtration and concentration, 750 mg of protein was obtained with a protein concentration of 150 μg/mL. The pH of the refolded sample was adjusted to 8.0 using diluted NaOH solution.
Example 8: Purification of refolded SIase
The refolded SIase 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 SIase, 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 (OD280 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 SIase activity. SIase was found in the fraction eluted with 100 mM NaCl at nearly 12-15 mS/cm. The specific activity of these highly pure SIase 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 A280 nm using molar extinction coefficient, 119340 M-1cm-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. SIase 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 SIase was tested by an enzyme assay using sucrose as substrate. The reaction velocity was measured by incubating appropriately diluted SIase with various concentrations of sucrose in a 50-mM sodium acetate buffer pH 6.5 at 15°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).
Protein Km (mM) Vm ax Kcat Kcat/Km
(IU mg-1) (S-1) (M-1S-1)
Refolded 31 ± 8 981 ± 68 1055 3.4 X 104
Soluble (Native) 47± 13 1023 ± 84 1100 2.3 X 104
§. The data are means for three enzyme reaction replicates with SEM.
Table 5: Kinetic parameters for SIase
The purified recombinant SIase shows specific activity of 981 U/mg (±5%), Km of 31 mM, Kcat of 1055 S-1 and Kcat/Km of 34,000 (M-1S-1). The product formation kinetics are similar to native protein.
Example 10: Characterization of refolded sucrose isomerase
The refolded sucrose isomerase was studied to determine the purity profile. It was found that after purification, the refolded SIase was extremely pure. The HPLC analysis is depicted in Figure 6.
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.
S.No. Property Soluble Refolded
(Native)
1 Specific activity (IU/mg) 668 694
2 Optimum pH 6.5 6.5

3 Optimum temperature (°C) 30 30
4 Km (mM) 47± 13 31± 8
5 Vmax (IU/mg) 1023± 84 981±68
6 Enzymatic conversion of ~92 ~92
sucrose (%)
Table 6: Comparison between soluble and refolded SIase
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 SIase form inclusion bodies.

We claim:
1. A method for refolding sucrose isomerase of Pseudomonas mesoacidophila MX-
45 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 sucrose isomerase to obtain a diluted sample;
c. incubating the diluted sample in presence 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™) and glycerol;
and
d. purifying the refolded sucrose isomerase.
2. The method as claimed in claim 1, wherein the host cell is Escherichia coli.
3. The method as claimed in claim 1, wherein sucrose isomerase of Pseudomonas mesoacidophila MX-45 comprises the amino acid sequence of SEQ ID NO:2.
4. The method as claimed in claim 1, wherein the pH buffer is Tris-HCl.
5. The method as claimed in claim 1, wherein the salt moiety is NaCl.
6. The method as claimed in claim 1, wherein the solubilized sucrose isomerase is diluted with refolding buffer in a ratio of 1 to 20.
7. The method as claimed in claim 1, wherein incubating is carried out for at least 16 hours at a temperature in the range of 4-10°C.
8. The method as claimed in claim 1, wherein the pH of the refolding buffer ranges between 6.0 – 7.4.
9. The method as claimed in claim 1, wherein the solubilization solution comprises 50 mM Tris-HCl and 8.0 M urea.

10. The method as claimed in claim 1, wherein the refolding buffer comprises 50 mM Tris-HCl, 150 mM NaCl and 10% glycerol.
11. The method as claimed in claim 1, wherein the refolded sucrose isomerase 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.