FORM 2
THE PATENT ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rulel3)
1. TITLE OF THE INVENTION:
Commercially viable process for enhancing enzyme activity
2. APPLICANT (S)
(a) NAME: MERCURY LABORATORIES LIMITED
(b)NATIONALITY: Indian Company incorporated under the Indian Companies ACT, 1956
(c) ADDRESS: 2/13 & 2/14, Gorwa Industrial Estate, Gorwa, Baroda - 390 016, Gujarat, India.
3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it is to be performed.

Technical Field of the Invention:
The present invention relates to a commercially viable process for enhancing the activity of an enzyme than their initial activity.
Background of the Invention:
Enzymes are constructed from chain of amino acids. As an unfolded chain, the enzyme has no activity. Only the folded structure forms the catalytic or active site. However this folded structure will generally be held together by non-covalent interactions such as ionic bridges, hydrogen bonds, hydrophobic and hydrophilic interactions and so on. The enzyme has an optimµM shape or flexibility and will hold this ideal shape at the optimµM temperature.
As temperature increases over the optimµM, increases the chances of breakdown of three-dimensional structure of the enzyme. As the heat in the system increases, the vibrational energy of enzyme also increases, putting strain on the weak interactions that hold the enzyme together. At higher temperature these bonds get apart and the three dimensional structure of enzymes destabilizes, this is called denaturation. Other forces that also disrupt these bonds will have same effect: extremes of pH, extreme concentrations of salt and so on.
The stabilization of enzyme activity is standing problem in all areas of technology where enzymes are likely to be used. Stability in this sense stands for resistance to decrease in activity prior to usage e.g. under storage conditions and contains active and inactive form.
Enzyme stability problems are most important when the enzyme containing composition or additive formulated with water or is used in aqueous solutions. The production of hµMan enzyme pharmaceuticals in bacteria by genetic engineering often results in the accµMulation of improperly folded enzymes called inclusion bodies, which have little or no biological activity. All intermediates as well as the unfolded enzyme can form misfolded intermediates that can reduce the yield of the native
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enzyme. There is considerable evidence that the intermediates have many hydrophobic amino acid residues exposed to the surface, which causes their aggregation. These enzymes after isolation and purification from the host cells have to be completely unfolded, or denatured, and subsequently refolded or renatured so that the enzymes regain their bioactivity.
The ability to overproduce natural or modified enzymes in genetically engineered cells has helped create an expanded biotechnology industry. While some enzymes have been expressed in an active state, others have been expressed in an inactive, misfolded configuration. A nµMber of attempts have been made to cause refolding of enzyme to an active state by adding materials such as sucrose, glycerin, Propylene glycol, Polyethylene glycol, synthetic polymers, detergents, cyclodextrins etc.
Cyclodextrins (CDs) have been reported to be useful for stabilization, solubilization, enhancement of bioavailability, purification of certain enzymes. It reduces the loss in many drugs during storage, heating, freeze drying, protects against oxidation, photo degradation, hydrolysis, dehydration etc. Szejtli, J., et al, "Cyclodextrins in Pharmacy'T63-164 (1994), has disclosed that addition of CD to an aqueous solution of protein prevents aggregation by interacting with aromatic amino acid of protein.
US Patent 5,728,804 discloses the use of cyclodextrin to renature the protein where alpha-CD, HP-beta-CD, gamma-CD claimed to renature the carbonic anhydrase B & alpha amylase.
US Patent 5,298,410 discloses the use of beta-CD as cryoprotectant to increase the shelf life of protein formulations.
US Patent 5,563,057 describes the method for refolding misfolded enzymes with detergents and cyclodextrins.
US Patent 5,997,856 discloses the method and compositions for solubilization and stabilization of polypeptide, proteins using cyclodextrins.
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Despite the foregoing, a clear and pressing need exists for folding aids which enhances the activity of inactive enzyme in addition to active enzyme to give more than 100% activity.
SµMmary of the Invention:
The present invention provides a method for enhancing the activity of the enzyme comprising said enzyme contacting in an aqueous mediµM with an amount of cyclodextrin (CD) effective to renature said inactive enzyme in addition to active enzyme to get the enhanced activity more than 100 %. The aqueous solution is buffered to about pH 5-9, with a Tris buffer or salt form, and it is free of other refolding aids, including sucrose, glycerin, Propylene glycol, Polyethylene glycol, synthetic polymers, and detergents. The interaction is carried out at temperature range of 15-37°C, although higher temperatures may be employed for thermally-resistant enzymes. After a sufficient period of time the initial activity of enzymes are enhanced upto l85%.
Cyclodextrins can be visualized as a toroidal, hollow, truncated cone with their exterior is hydrophilic and the inner cavity is hydrophobic, which gives cyclodextrins the ability to admit hydrophobic residues on the enzyme surface. Cyclodextrins have been suggested to act as 'chaperone mimics' by enhancing enzyme refolding from denatured or even aggregated states.
Cyclodextrins property to inhibit the aggregation and ability to refold the enzyme and makes them highly effective enzyme folding agents and are relatively inexpensive and easily commercially available.
Brief Description of the Figures:
FIG. 1 is a graph depicting the aggregation kinetics in the absence and presence of cyclodextrins. Native enzyme (9.8 µM) was denatured in mixture of 8M Urea and 0.1 M HC1 for 18 hours. In this case, enzyme was diluted to 4.9 u-M and 4M Urea, with renaturation buffer, containing 0.88 µM beta-CD. Curve A (absence of beta-CD), Curve B (with beta.-CD).
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FIG.2 is a graph depicting inclusion complex of fungal alpha amylase and beta-CD in different molecular ratio and respective % activity enhanced. In each case, fungal alpha amylase concentration during complexation was 9.8 µM.
FIG. 3 is a plot of the UV absorbance of fungal alpha amylase and beta-CD in different molecular ratio showing hypochromic shift at 3:1 molecular ratio.
FIG.4 is plot of the fluorescence spectra of Native Enzyme and inclusion complex at molecular ratio 3:1 showing identical spectra.
FIG.5 is graph depicting the effect of enzyme concentration on reactivation efficiency at 3:1 molecular ratio.
Detailed Description of the Invention:
The present method may be employed to enhance the activity of enzymes, particularly fungal alpha amylase from Aspergillus oryzae. The amount of cyclodextrins (CDs) and the amount of enzyme may be varied, as given in examples below. Molecular ratio of Host: Guest (CDs: Enzyme) from 1:1 to 10:1 have been found to be effective, while the enzyme at concentrations chosen to minimize the aggregation and enhancement of activity, preferably at less than 1 mg/ml,
The aqueous medium is preferably buffered to the desired pH. The buffered medium may contain small amount of co solvents to dissolve the enzyme. The medium does not contain any other folding aid. Commercially available sample of enzymes are always a mixture of active (correctly folded) and inactive (denatured or incorrectly folded) forms. The amount of inactive enzyme in a sample may vary depending on the purification scheme, storage and transport conditions. Thus, the present refolding method is useful to enhancing the activity of inactive form present in a sample of enzyme to get activity more than that of initial activity.
The invention will be further described by reference to the following detailed examples wherein fungal alpha amylase from Aspergillus oryzae was obtained from Biocon India, Beta-Cyclodextrin and Tris buffer were obtained from S.D.Fine Chemicals, India.
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Inclusion complex of fungal alpha amylase and beta-CD were carried out in buffer (50 µM Tris, pH 8.5) at different Host: Guest (betaCD : fungal alpha amylase) molecular ratio from 1:1 to 10:1 at different temperature 15°C - 37°C. The enzyme concentration varied from 9.8 µM to 196 µM. The amount of active fungal alpha amylase was determined after 2 hours of slow stirring by assaying for the enzyme by its amylolytic activity. Inclusion complex was confirmed by UV and fluorescence spectra and correlated with activity enhanced. The molecular ratio 3:1 gives maximum enhancing activity.
Example 1. Aggregation inhibition studies
Fungal alpha amylase (9.8 µM) was denatured in a urea/acid mixture (8M urea, 0.1 M HC1) for 18 hours. This denatured fungal alpha amylase in Urea was rapidly diluted with 50 mM Tris buffer at pH 8.5, to 4.9 µM enzyme and 4 M urea, aggregation was observed immediately and was monitored by light scattering at 400 nm (FIG. 1, curve A). Aggregation increased with time and then stabilized after approximately 5 minutes. When the denatured enzyme was renatured in the presence of beta-CD under the same conditions, light scattering due to aggregation was significantly reduced. (FIG. 1, curves B). Inhibition of fungal alpha amylase aggregation was enhanced with beta-CD in the renaturation buffer.
Example 2. Enhancing the Activity in Excess of Initial Activity
Given enzyme sample may contain both active and inactive enzyme. The inactive enzyme may be present due to denaturation during enzyme purification and/or storage. If such a sample were subjected to refolding by the present method, then enhancement of the activity of the inactive enzyme, in addition to the activity of the initially active enzyme, can yield greater than 100% recovery of activity.
Fungal alpha amylase from Aspergillus oryzae was obtained from Biocon India. This sample should contain functional (active) and some amount of non-functional (inactive) enzyme, as explained above.
Inclusion complex of fungal alpha amylase and beta-CD were carried out in buffer
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(50 mM Tris, pH 8.5) at different Host: Guest (betaCD: fungal alpha amylase) molecular ratio such 1:1,2:1,3:1,4:1,5:1,6:1, 7:1,8:1,9:1 & 10:1 at 37°C. The enzyme Concentration kept same for all ratios (9.8 µM). The amount of active fungal alpha amylase was determined after 2 hours of slow stirring by assaying for the enzyme by its amylolytic activity. Results are given in Table 1 and FIG 2.

Host: Guest Ratio Initial % activity % Activity Enhanced
1:1 100% 166.68%
2:1 100% 173.90%
3:1 100% 185.00%
4:1 100% 117.70%
5:1 100% 131.95%
6:1 100% 105.27%
7:1 100% 108.52%
8:1 100% 118.88%
9:1 100% 112.60%
10:1 100% 115.29%
Table 1
The results above indicate that enzyme present in the commercially available sample was a mixture of active (correctly folded) and inactive (denatured or incorrectly folded) forms. The amount of inactive enzyme in a sample may vary depending on the purification scheme, storage and transport conditions. Thus, the present refolding method is useful to recover the inactive form present in a sample of enzyme.
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Example 3. Stoichiometry of CD complex:
Data of Inclusion complex of fungal alpha amylase and beta-CD at different Host: Guest molecular ratio shown in Table 1 suggests that molecular ratio 3:1 gives maximum activity and it is the most reactive ratio.
UV spectroscopy of inclusion complex of different molecular ratio also shows that molecular ratio 3:1 gives the hypochromic shift in UV absorbance which confirms the formation of complex. Graph of absorbance shown in FIG.3.
Fluorescence spectroscopy of inclusion complex of different molecular ratio shows identical fluorescence spectra as that of the native enzyme. This suggests that betaCD forms complex with denatured enzyme present in the original sample and the enzyme regains its native structure. Graph of fluorescence spectra shown in FIG.4.
Example 4. Effect of Enzyme Concentration
Effect of enzyme concentration on reactivation efficiency at molecular ratio 3:1 is shown in FIG. 5. In each case, refolding was allowed to occur for 2 hours. Enhancement of active enzyme decreased at higher enzyme concentrations due to increased enzyme aggregation.
Example 5. Effect of pH and Temperature on the inclusion complex
The effect of pH and temperature on betaCD- fungal alpha amylase complex is summarized in Table 2. In each case, the enzyme was refolded at molecular ratio 3:1 and at 9.8 µM concentration for 2 hours. Optimal yield of active enzyme was obtained
between 25°C- 37°C and under alkaline conditions.

pH Temp.(°C) Initial % Activity % Enhanced
8.5 15 100 107
8.5 25 100 170
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8.5 37 100 185
8.5 50 100 71
5.0 37 100 80
6.0 37 100 115
7.0 37 100 155
8.0 37 100 165
9.0 37 100 160
Table 2
Example 6. Effect of Ionic Strength
Fungal alpha amylase was refolded at 5 mg/mL in the presence of buffered cyclodextrin in 3:1 molecular ratio in 50 µM Tris buffer, pH 8.5 containing various amounts of NaCI. The results obtained after 2 hour of refolding at 37°C is summarized on Table 3

NaCI mM % Enhanced
5 115
10 119
25 124
50 120
250 118
500 108
Table 3
Fungal alpha amylase was refolded at 0.5 mg/mL in the presence of buffered cyclodextrin in 3:1 molecular ratio in 50 mM Tris buffer, pH 8.5 containing various amounts of CaCl2. The results obtained after 2 hour of refolding at 37°C is summarized on Table 4.
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CaCI2 mM % Enhanced
0.6 103
0.9 109
1.1 124
1.8 96
2.7 100
4.5 106
Table 4
This data demonstrates that ionic strength decreases the enhancement of fungal alpha amylase. In order to achieve higher yield, other conditions have to be optimized. However, even under non-optimal conditions, refolding in the presence of beta-CD significantly increases the amount of folded enzyme.
While the present invention is described above in connection with preferred or illustrated embodiments, there embodiment are not intended to be exhaustive or limiting of the invention. Rather, the invention and the claims are intended to cover all alternatives, modifications and equivalence included within its scope.
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We claim,
1. A commercially viable process for enhancing the activity of an enzyme comprising addition of cyclodextrin to an enzyme in an aqueous mediµM at a temperature range of 15 to 37°C wherein the said aqueous mediµM has an enzyme concentration in the range of 9.8-196µM and wherein the molar ratio of said cyclodextrin and said enzyme is in the range of 1:1 -10:1.
2. The process of claim 1, wherein the preferred molar ratio is 3:1.
3. The process of claim 1, wherein the said enzyme is fungal alpha amylase from Aspergillus oryzae.
4. The process of claim 1, wherein the cyclodextrin is beta-cyclodextrin.
5. The process of claim 1, wherein the said aqueous medium is buffered at about pH 5-9.
6. The process of claim 1, wherein the activity of the said enzyme is enhanced from 100% of the initial activity to enhanced activity of upto 185%.
Dated this 9,h day of December 2005

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Abstract
Invention relates to a commercially viable process for enhancing the activity of enzyme, particularly, fungal alpha amylase from Aspergillus oryzae in an aqueous medium more than 100% of their initial activity by adding beta-cyclodextrin in the said aqueous medium.
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