Title of Invention

PROCESS OF PREPARING A REVERSIBLY SOLUBLE MODIFIED CONJUGATED LIPASE ENZYME

Abstract A process of preparation of reversibly soluble-insoluble conjugated lipase enzyme which can be effectively used for the enzyamatic hydrolysis of castor oil.The proposed method is for higher activity than the native enzyme.The enzyme lipase is conjugated to a copolymer using a functionalizing agent.The enzyme so obtained can be readily used to facilitate the enzymatic hydrolysis of castor oil at higher rates of reaction and in a cost effective manner.
Full Text F O R M - 2
THE PATENTS ACT, 1970
(39 OF 1970)

COMPLETE SPECIFICATION (See Section 10)



1. TITLE OF INVENTION
PROCESS OF PREPARING A REVERSIBLY SOLUBLE MODIFIED CONJUGATED LIPASE ENZYME
Registrar, Mumbai University Institute of Chemical Technology
Nathalal Parikh Marg, Matunga, Mumbai - 400 019,
State of Maharashtra, India, an Indian University
The following specification particularly describes the nature of the invention and the manner in which it is to be performed.
08-01-2003

PROCESS OF PREPARING A REVERSIBLY SOLUBLE MODIFIED CONJUGATED LIPASE ENZYME
Field of the invention
The present invention relates to the conjugation of enzymes. More particularly, the invention relates to conjugation of the enzyme lipase to a reversibly soluble-insoluble copolymer and to a process of hydrolyzing substrate such as fats or oils using the said conjugated enzyme.
Background of the invention
The demand for industrial enzymes, particularly of microbial origin, is ever increasing owing to their applications in a wide variety of processes. Enzyme mediated reactions are attractive alternatives to tedious and expensive chemical methods. Enzymes find great use in large number of fields such as food, dairy, pharmaceutical, detergent, textile, and cosmetic industries.
In the above scenario enzymes such as proteases and amylases have dominated the world market for a long time due to their hydrolytic reactions for proteins and carbohydrates. However, with the realization of biocatalytic potential of microbial lipases in both aqueous and non-aqueous media in the last decade, industrial fronts have largely shifted towards utilizing this enzyme for a variety of reactions of immense importance.
The enantio-selective and regio-selective nature of lipases has been utilized for the resolution of chiral drugs, fat modification, synthesis of cocoa butter substituents, biofuels, and for synthesis of personal care products and flavor enhancers.
Research has been carried out on plant lipases (Martin and Peers, 1953; Theimer and Rosnitschek, 1978; Mohankumar et al., 1990), animal lipases (Moreau et al., 1988; Winkler et al., 1990; Carriere et ai, 1991) and microbial lipases, particularly bacterial and fungal (Yadav et al., 1998; Yamaguchi et al., 1973; Yamaguchi etal., 1991; Suzuki et al., 1986). Although pancreatic lipases have been traditionally used for various purposes, it is now well established that microbial lipases are preferred for commercial applications due to their multi-fold properties, easy extraction procedures and unlimited supply.
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Lipases are spontaneously soluble in aqueous solutions (as a result of their globular protein nature), but their natural substrates (i.e., fats and oils) are insoluble in aqueous solutions. Although use of a proper organic solvent or an emulsifier helps overcoming the problem of intimate contact between the substrate and the enzyme, the practical use of lipases in such pseudo-homogeneous reaction systems poses technological difficulties (viz. contamination of the products with residual enzymatic activity) and economic difficulties (viz. use of the enzyme for a single reactor pass). The former leads to constraints on the product purity level, because the final characteristics of the product depend on such post-processing conditions as storage time and temperature. The latter leads to constraints on the process efficiency level, because the useful life of the enzyme is restricted to the space-time of the reactor (on the assumption that the space-time is small compared with the time scale associated with deactivation of the enzyme). In both the cases, part of the over-all potential enzymatic activity is lost.
The modification of lipase provides remedial measures from such constraints at the product as well as the process level.
Lipases have been modified by using various strategies to achieve specific behavior and functionalities in aqueous as well as non-aqueous environment. Modification studies on most of the other enzymes have been concentrated on enabling the recovery and reuse of the enzyme; however, unlike the other enzymes the aim of lipase-modifications have been equally divided into achieving enhanced efficiency and functionality in non-aqueous environments and to enable recovery and reuse of the enzyme.
Modification of lipases by use of functional polymers has been shown to endow them with new properties without destroying their native functions, thus providing useful materials for application in different fields. Some of the most significant strategies employed for modification of lipases have been; adsorption on a carrier, entrapment, cross-linked enzyme crystals (CLEC), covalent binding on inert support, conjugation to
reversibly soluble-insoluble polymeric carriers, surfactant coating and reverse
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micelles. The principal aim of the first five techniques has been to enable the enzyme to be recovered and reused in repeated reaction cycles, while that of the other two has been to utilize the enzyme in organic solvents. It should be however, noted that many studies utilized non-covalent and covalent binding of enzymes onto inert supports to answer both, the utilization in non-aqueous media as well as reuse.
The use of lipase on industrial scale is not yet very common, mainly because of the high cost normally associated with them.
Immobilizing lipases, to a certain extent enabled their consideration as economically feasible industrial catalysts.
United States Patent 6,162,623 (Grote, et al., 2000) describes processes for preparing and using immobilized lipases.
United States Patent 6,156,548 (Christensen, et al., 2000) describes immobilization of enzymes with a fluidized bed for use in an organic medium.
United States Patent 6,025,171 (Fabian, et al., 2000) talks about immobilizing enzymes and processing triglycerides with immobilized lipase.
However, the loss in activities and mass-transfer limitations commonly associated with immobilized lipases has still limited their utilization to few certain special applications. The mass-transfer limitations with immobilized lipases are especially pronounced in the case of bi-phasic systems where the substrate is usually in the form of micro-emulsion droplets.
In addition, there are several engineering problems associated with the use of insoluble enzymes for insoluble substrates and/or products. For example, of the two most
commonly used immobilized enzyme reactors: the continuous stirred tank reactor
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and the packed bed reactor, the utilization of the later is not feasible due to the high risks of plugging. Additionally, the desired, plug-flow characteristic of the fluid through the packed bed, in the case of high molecular weight soluble substrates and/or products, is difficult as a result of fluid dispersion and channeling, leading to irregular and unpredictable diffusion of reactants to the reaction site and temperature gradients in the reactor. Another disadvantage of using immobilized enzymes is that during the immobilization of lipase one has to critically monitor the hydrophilicity of the immobilized enzyme entity to ensure adequate hydrophilic-hydrophobic balance in the enzyme system, which not only governs activity, but also dictates the specificity of the biocatalyst.
The conjugation of enzymes with soluble-insoluble polymers using carbodimide (Tyagi R, Roy I, Agarwal R, Gupta MN. Biotechnol Appl Biochem, 28(3) 201-6 (998)) has been reported. However, it does not exemplify the conjugation of lipases.
Enzymes are primarily used to facilitate the enzymatic hydrolysis of oils especially castor. Traditionally, fat hydrolysis has been performed at extremely high temperatures (240 - 270°C) and pressures (700 - 750 psig). This process is unsuitable for the hydrolysis of castor oil since the castor fatty acid (Ricinoleic acid) is susceptible to degradation under these conditions. Therefore, even when castor is abundantly found in India, the production and utilization of castor fatty acids has been mired.
Alternatively prior art utilizes the saponification-acidulation process for the hydrolysis of castor oil. However even this process has the limitations of poor quality of product (development of heavy coloration) and large quantity of associated effluent.
Enzymatic hydrolysis provides a promising alternative for the hydrolysis of castor oil due to the following advantages:
Low energy consumption due to ambient operating conditions
Avoids degradation of thermo labile fatty acid products
Non-contamination of reaction mixture from colored compounds and hence ensures high quality product.
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However, the cost of the enzyme and the lower rates of reaction, have hindered the progress of this technology. The enzymatic hydrolysis of castor oil follows a logarithmic pattern against time, indicating a reduced enzyme activity in the later stages of the reaction. The fall in the rate of the reaction can be reduced by subjecting the substrate to large quantities of the enzyme and hence it is necessary to use it in an immobilized form for recovery and reuse.
Lipases are interfacially active enzymes and hence utilization of the enzyme in an insoluble form, where the oil-water interface is limited, is undesirable.
Use of immobilized lipase enzyme for efficient hydrolysis of oil or fats is well known in the prior art.
For example United States Patent Number 6,258,575 (Shimizu, et al., 2001) discloses a batch type process for hydrolysis of the oil or fat substrate using the insoluble immobilized lipase enzyme. This invention describes a process with insoluble immobilized enzyme in a column reactor. It proposes improved economics of the process by increasing the rate of the reaction due to continuous glycerol removal and insoluble enzyme immobilization to enable enzyme recovery and reuse.
United States Patent 5,932,458 (Piazza, Jr., et al., 1999) describes a method of fat and oil splitting using an immobilized lipase catalyst.
United States Patent 5,288,619 (Brown, et al., 1994) describes an enzymatic method for preparing transesterified oils using a 1-, 3-positionally specific lipase.
United States Patent 5,089,403 (Hammond, et al., 1992) describes a process for enzymatic hydrolysis of fatty acid triglycerides with oat caryopses having active oat lipase associated with the outer surfaces.

United States Patent 5,032,515 (Tanigaki, et al., 1991) describe the hydrolysis process of fat or oil with lipase, characterized by maintaining the glycerol concentration in the aqueous phase of the reaction system constant within a range of 10 to 40% by weight to prevent inactivation of the lipase in the reaction system.
It is evident that the attempts in prior art were made to carry out the enzymatic hydrolysis of oils by using the lipase enzyme in an immobilized form, which has its own disadvantages as listed earlier, or to critically monitor the conditions of reaction to avoid rapid deactivation of the enzyme.
Objects of the invention
The object of the present invention is to provide a process of conjugating the lipase enzyme with a reversibly soluble-insoluble functionalized copolymer that is industrially feasible and economically attractive.
Another object of the present invention is to overcome the drawbacks of prior art by providing modification of the enzyme to enable its usage in a soluble form during the course of the reaction and enable its recovery after the reaction by precipitating it out in an insoluble form by changing the pH of the reaction mixture.
A further object of the present invention to economize the process by reuse of the enzyme in the hydrolysis of oils and fats and for the purpose a reversibly soluble lipase, which enables the availability of the enzyme in a soluble form during the reaction and an insoluble form after the reaction for convenient recovery of the enzyme.
A further object of the invention to further economize the process by increasing the rates of the reaction by improved mass transfer by enabling the availability of the enzyme catalyst in the soluble form.
Summary of the invention

; Thus according to the present invention, there is provided a process of preparing a reversibly soluble modified conjugated lipase enzyme comprising steps of:
(a) providing a buffer solution;
(b) providing a functionalized copolymer;
(c) adding a lipase enzyme to the buffer solution before or after step (b); and
(d) precipitating conjugated enzyme by altering the pH of the solution to predetermined levels.
The reversibly soluble-insoluble enzyme so obtained can be readily used to facilitate the enzymatic hydrolysis of oils especially castor oil at higher rates of reaction and in a cost effective manner.
According to another aspect of the present invention there is provided a process for the hydrolysis of oils or fats, using reversibly soluble modified conjugated lipase enzyme prepared by the process described in the foregoing paragraphs, comprising steps of
a) providing a first liquid oil or fat phase, above a water-containing soluble enzyme second phase such that there is distinct interface between the said first and second phases.
b) mixing the two phases and allowing the mass to settle to form two phases: upper phase comprising partially hydrolyzed oil and lower phase comprising water containing glycerol and soluble enzyme.
c) separately removing the phases from the column.
d) treating the said lower phase with an acid to precipitate out the enzyme for reuse.
Detailed description
(A) PROCESS DETAILS FOR THE PREPARATION OF THE CONJUGATED LIPASE ENZYME:
I) Primary steps in the process of conjugating the lipase enzyme:
The process of conjugation of lipase enzyme comprises steps of:
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1. Dissolving the enzyme and functionalized copolymer in buffer solution.
2. Providing adequate agitation at low temperature.
3. Precipitating the conjugated enzyme by change in pH.
The functionalized copolymer is provided by treating the copolymer with a cross-linking/functionalizing agent.
The solutions used in the preparation of the conjugated lipase enzyme are maintained at pH 7.0, so that the enzyme does not precipitate prior to conjugation.
The biological source of the lipase enzyme used for the present invention is a genetically engineered species of Aspergillus oryzae.
II) Sequence of addition of reactants:
There are two possible sequences of the addition of the reactants depending on whether the copolymer is prefunctionalized or it is functionalized in situ by addition of cross-linking/functionalizing agent.
When the copolymer is functionalized in situ, the sequence of addition of reactants/process steps are as follows:
(a) providing said buffer solution;
(b) adding said lipase enzyme and dissolving it in said buffer solution;
(c) adding said copolymer and said cross-linking/functionalizing agent;
(d) agitating the mass at low temperature to produce conjugated lipase enzyme; and
(e) precipitating said conjugated enzyme by altering the pH of the solution to predetermined levels.
Alternatively, prefunctionalized copolymer can also be used i.e. the copolymer is
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functionalized prior to the addition of the enzyme. In such a case the sequence of addition of reactants/process steps are:
(a) providing said buffer solution;
(b) adding said copolymer and said cross-linking/functionalizing agent thereby prefunctionalizing said copolymer;
(c) adding said lipase enzyme;
(d) agitating the mass at low temperature to produce conjugated lipase enzyme; and
(e) precipitating said conjugated enzyme by altering the pH of the solution to predetermined levels.
Here, the sequence is maintained in the order of the enzyme followed by the copolymer followed by the addition of the functionalizing agent. It is critical that the enzyme and the cross-linking agent should not be added simultaneously without the presence of copolymer to avoid intra-molecular cross-linking between the enzyme and the functionalizing agent.
The functionalizing agent conjugates a carboxylic function to an amine function generating a peptide linkage. The chances of intra-cross-linking amongst the copolymer molecules during the activation step are zero, due to the lack of the amine functions in copolymers used in the present invention.
Ill) Optimization of process of modifying the lipase enzyme:
The process of modifying the lipase enzyme is optimized with respect to parameters that include the type of copolymer, the functionalizing agent, the ratio of copolymer to the functionalizing agent, the ratio of the lipase enzyme to functionalized copolymer, the method of modifying the enzyme using the functionalized copolymer and the range of pH governing the solubility of the modified enzyme.
The optimization was carried out on the basis of the amount and the activity of the
bound enzyme. The bound enzyme quantity (C) was estimated indirectly by
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subtracting the enzyme quantity in the washings (A) from the total enzyme quantity added (B).
C =B -A
IV) Details of the parameters that require optimization in the process of modification of the lipase enzyme:
Type of copolymer
The reversibly soluble-insoluble polymers, which can be used to conjugate the enzyme, include, hydroxypropylmethyl cellulose acetate succinate, copolymers of methacrylic acid-methylmethacrylate and copolymers of acrylic-methacrylic acid esters.
An example of copolymer of acrylic-methacrylic acid esters is Eudragit RL-100.
The preferred copolymer used for modifying the enzyme is the anionic methacrylic acid-methyl methacrylate copolymer known as Eugradit L-100. The polymer has pH dependent solubility insoluble at 6.5. Its carboxyl content ranges from 46.0-50.6%. The carboxyl to ester ratio of the polymer is 1:1. The molecular weight is 135KD.
Functionalizing / Cross-Linking agent
The functionalizing agent used for modifying the copolymer for conjugation of enzyme
is selected from a group of carboxyl to amino cross linking carbodiimides such as 1 -
Ethyl-3-(3-Dimethylaminopropyl) Carbodi-imide (EDC), N-cyclohexyl-N"-[2-(4-
morpholinyl)-ethyl]-carbodiimide, N-cyclohexyl-N"-[2-(4-morpholinyl)-ethyl]-
carbodiimide-methyl-p-toluene-sulfonate and the like. Preferably a water-soluble carbodimide like EDC can be used.
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Ratio of copolymer to functionalizing agent
The amount of bound activity is significant in the range of 0.1 to 1.0 of the EDC to copolymer ratio, preferably in the ratio of 0.15 to 0.8 and most preferably 0.2.
Ratio of the enzyme to functionalized copolymer
The ratio of enzyme to functionalized copolymer affects the bound enzyme activity as well as the loss of absolute enzyme activity due to conjugation. The bound enzyme activity is significant and the loss of activity was minimum when the enzyme to functionalized copolymer ratio was from about 5 to 20, preferably 8 to 15 and most preferably 10.
Range of pH governing the solubility of the modified enzyme
The enzyme is gradually solubilized for use when the pH is increased from 4.5 to 7.0. At the end of the reaction, the enzyme is insolubilized by lowering the pH from 7.0 to 4.5.
Temperature of agitation
The agitation is preferably carried out at low temperatures of between 0 to 30°C, more preferably between 2 to 10°C and most preferably at 4°C.
Differentiation between the bound and the unbound enzyme
The conjugated bound enzyme can be differentiated from the unbound enzyme in that the bound enzyme is more susceptible to the effect of solvent than the unbound enzyme. In addition, the temperature optima of the conjugated bound enzyme as compared to the unbound enzyme indicate that the properties of the lipase have been altered due to conjugation.
The following data and discussion indicates that the end properties of the final product are significantly different from the individual ingredients and the process of modification
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of the enzyme is not a result of mere admixture of the ingredients such as enzyme, copolymer and functionalizing agent.
(B) Process details for hvdrolvsing the substrate using the conjugated lipase enzyme:
I) Primary steps in the process of hydrolysis
The process of hydrolysis of oils or fats using a conjugated lipase enzyme comprises steps of
1. Modifying the lipase using functionalized copolymer to give a conjugated lipase enzyme;
2. Reacting the oil or fat with the conjugated lipase enzyme;
3. Recovering and reusing the conjugated lipase enzyme by change in pH.
An oil or fat phase is provided in a column, which has been fed with a water-containing soluble enzyme in an adequate quantity from the top, at the bottom of the column in the form of droplets such that there are distinct interfaces between the phases at the top and bottom. The phases are separately removed from the column i.e. partially hydrolyzed oil containing mono, di- and tri-glycerides with fatty acids from the top and water containing glycerol and soluble enzyme from the bottom after the contact in column, in such a way that the water is treated so as to recover/reuse the enzyme from the water containing the soluble enzyme. The top oil phase is continuously fed to the vacuum distillation for the recovery of the fatty acid and un-hydrolyzed oil is fed to the next stage, which is identical in construction and the water containing the soluble enzyme is fed from the top. The number of contacting stages could be 2 or more to give the desired extent of hydrolysis and/or desired level of glycerol concentration in the water. The water is then treated with an acid to precipitate out the enzyme by lowering of the pH for reuse and the water can be processed further for glycerol recovery. The process provides enhanced rate of hydrolysis due to the elimination of mass transfer resistance.

II) Optimization of process of hvdrolvsinq the substrate using the conjugated lipase " enzyme:
The process of hydrolysing the substrate is optimized with respect to parameters that include the conjugated enzyme to substrate (oil or fat) ratio, oil (non-aqueous) to aqueous solution containing enzyme ratio, oil to buffer ratio, the mode of agitation, agitation speed and duration of reaction to achieve optimum % hydrolysis.
Conjugated enzyme to substrate (oil or fat) ratio
The conjugated enzyme to substrate (oil or fat) at the ratio of 0.25 for enzyme concentration: 2 mg/ml (7 Lipase Units/ ml), yields most optimum use of the enzyme in the hydrolysis process.
Oil (non-aqueous) to aqueous solution containing enzyme ratio
The extent of hydrolysis of oil, using the conjugated lipase is greater than 90 % when the non-aqueous to aqueous phase ratio is 3 to 0.25, preferably 0.25. The recoverable and reusable nature of the modified enzyme enabled usage of such a ratio.
Oil to buffer ratio
The optimum oil to buffer ratio for maximum yield of the fatty acids is 0.03.
Solvent Effect
In the case of the reaction in presence of isooctane, the rate of the reaction obtained with the conjugated enzyme, is higher in the initial stages of the reaction as compared to the free enzyme. However, as the reaction proceeds for a prolonged period, the rate of the reaction is found to be same with both the forms of the enzyme.
This enhancement in the enzyme activity is of immense significance in view of the reuse potential of the enzyme, and the potential for better mass transfer efficiencies of the conjugated reversibly soluble (pH-dependent) enzyme as compared to the enzyme
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immobilized on an insoluble carrier.
The enhancement in the initial rate of the reaction may be the result of certain morphological modifications of the copolymer-enzyme conjugate from that of native enzyme that effect alterations in the physico-chemical properties of the conjugate, such as imparting an interface-accumulating tendency to the conjugated moiety.
The conjugated enzyme is more prone to solvent induced deactivation as compared to the free enzyme and therefore even when the rate of hydrolysis is higher in the initial phase of the reaction, it eventually slowed down due to the deactivating effect of the solvent in the later stages.
Temperature effect
The temperature range optimum of the conjugated enzyme is 20 to 50°C, preferably 35°C as against the higher temperature optimum for the free enzyme (55°C). The conjugated enzyme loses all its activity at the temperature optimum of the free enzyme, indicating lower temperature stability. However, the activity and stability profiles with respect to time are similar for the bound and the free enzyme at their respective temperature optima. Hence, the entire hydrolysis process with the conjugated enzyme could be carried out at a lower temperature (35°C as compared to 55°C for the free enzyme) and with higher or similar rates of reaction.
The lower temperature optimum of the conjugated enzyme is highly beneficial in regards to the energy economy offered.
Selection and optimization of the mode of agitation
As the enzyme Lipase is known to be an Interfacially residing enzyme, generation of large interfacial area (Oil-Water interface) is likely to promote the availability of this enzyme at the interface and substantially improve the rate of hydrolysis.
Thus, two modes of agitation to generate large interfacial area were tried out. The 20
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mm diameter stator-rotor Homogeniser, working on the principle of fluid shear to generate large interfacial area was used in one set of experiments, whereas standard 6 bladed paddle impeller of 20mm diameter, working on the principle of fluid velocity to generate large interfacial area was used in another set of experiments.
When a Homogenizer is used for agitation the efficiency of power utilization for optimum rate of hydrolysis is significantly better as compared to a six-paddle impeller.
Although the rate of hydrolysis is optimum at the power to volume (PA/) ratio of 235 Watts/m3, the speed of homogenizer is optimized to a power to volume ratio of 70 Watts/m3 as the enzyme lost 15 % of its original activity in 1 hour at 235 Watts/m3.
Ill) Enzyme recovery and reusability
The reusability of the conjugated enzyme is assessed by performing successive hydrolysis reactions of a fresh substrate using the same enzyme and even after 10 cycles, approximately 92% of the original activity is preserved.
Description of figures
Figure 1 shows a flow diagram of the conjugation process when the copolymer is functionalized in situ.
Figure 2 shows a flow diagram of the conjugation process when the copolymer is prefunctionalized.
Figure 3 shows a graph of EDC to polymer ratio vs. Bounded activity when duration of immobilization is 1 hour. The graph illustrates the optimization of the amount EDC.
Figure 4 shows a graph of Enzyme to polymer ratio vs. Bounded activity and % Loss in original activity when duration of immobilization is 1 hour. The graph illustrates the optimization of the enzyme to copolymer ratio as function of the % loss in enzyme activity on binding and the amount of bound activity.
Figure 5 shows schematic representation of batch process of hydrolysis to validate the

reuse of the conjugate enzyme.
Figure 6 shows the proposed hydrolysis scheme.
Figure 7 shows a graph of Oil to buffer ratio vs. % Hydrolysis when reaction duration is 1 hour.
Figure 8 shows the profile for hydrolysis of castor oil in the presence and absence of isooctane with free lipase and Eudragit L100 conjugated lipase.
Figure 9 shows the effect of isooctane treatment on the activity of the free and Eudragit L100 conjgated lipase.
Figure 10 shows effect of temperature on free (native) and conjugated enzyme Protein to Substrate Ratio is 3 x 10-4 and duration of reaction is 1 hour.
Figure 11 shows influence of the amount of power dissipated per unit volume (PA/ in W/m3) on the hydrolysis of castor oil on using a laboratory stator - rotor type homogenizer when reaction duration is 1 hour.
Figure 12 shows influence of the amount of power dissipated per unit volume (PA/ in W/m3) on the hydrolysis of castor oil on using a paddle type impeller when raction duration is 1 hour.
Figure 13 shows effect of the speed of the rotor of the homogenizer on the activity of the enzyme.
Figure 14 shows robustness of the enzyme with respect to reaction duration.
EXAMPLES
The following illustrative but non-limiting examples exemplify one of the embodiments of the present invention.
EXAMPLE 1: Conjugation of Lipase Enzyme bv in situ functionalization of copolymer
The conjugation was carried out by cross-linking the enzyme to the copolymer using the bifunctional cross-linking agent EDC shown in Figure 1 of the accompanying drawings which shows a flow diagram representing the Enzyme - Eudragit conjugation process.
One gram of enzyme powder (crude enzyme as provided by the manufacturer,
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consisting of added stabilizers) was suspended in 200 ml of 0.2 M sodium phosphate buffer, pH-7.0. The suspension was centrifuged to remove the insoluble additives with the enzyme activity contained in the supernatant (total protein content of the supernatant was found to be 185 mg). To this, 100 ml of copolymer solution (1 mg/ml in 0.2 M sodium phosphate buffer, pH-7.0) was added. Finally 100 ml of the EDC solution (0.2 mg/ml in 0.2 M sodium phosphate buffer, pH-7.0) was added and the reaction mixture was stirred at 600 rpm using a propeller at 4°C for 1 hour. The reaction mixture was then acidified to pH-4.0 using 1 N HCI to precipitate the Eudragit L100 - Enzyme (EL-En) conjugate. The insoluble EL-En conjugate was washed twice with 250 ml of citro-phosphate buffer, pH-4.0. The conjugate was then redissolved in 100 ml of 0.2 M sodium phosphate buffer, pH-7.0 and was used as the bound enzyme for the hydrolysis studies. Lipase activity was determined by performing controlled hydrolysis of tributyrin substrate and analyzing the free fatty acid formed. One Lipase Unit of the enzyme activity was expressed as the amount of enzyme required to liberate one ^M of fatty acid per minute at the given conditions.
Protein content of different samples were determined by modified Folin Cio-Calteau method using Bovine Serum Albumin as standard curve.
The bound activity was estimated indirectly by subtracting the enzyme activity in washings from the total enzyme activity added for conjugation, by simple material balance.
Example 2: Conjugation of Lipase Enzyme by prefunctionalized copolymer
The conjugation was carried out by cross-linking the enzyme to the copolymer using the bifunctional cross-linking agent EDC shown in Figure 2 of the accompanying drawings which shows a flow diagram representing the Enzyme - Eudragit conjugation process
100 ml of copolymer solution (1 mg/ml in 0.2 M sodium phosphate buffer, pH-7.0) was added to 100 ml of the EDC solution (0.2 mg/ml in 0.2 M sodium phosphate buffer, pH-7.0) and the reaction mixture was stirred at 600 rpm using a propeller at 4°C for 10
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minutes. One gram of enzyme powder (crude enzyme as provided by the manufacturer, consisting of added stabilizers) was suspended in 200 ml of 0.2 M sodium phosphate buffer, pH-7.0. The suspension was centrifuged to remove the insoluble additives with the enzyme activity contained in the supernatant (total protein content of the supernatant was found to be 185 mg). This centrifuged suspension was added to copymer and EDC solution. The reaction mixture was stirred at 600 rpm using a propeller at 4°C for 1 hour. The reaction mixture was then acidified to pH-4.0 using 1 N HCI to precipitate the Eudragit L100 - Enzyme (EL-En) conjugate. The insoluble EL-En conjugate was washed twice with 250 ml of citro-phosphate buffer, pH-4.0. The conjugate was then redissolved in 100 ml of 0.2 M sodium phosphate buffer, pH-7.0 and was used as the bound enzyme for the hydrolysis studies.
Example 3: Experiments to demonstrate optimum EDC to copolymer ratio
Multiple experiments as described in example 1, were carried out, where the concentration of the 100 ml EDC solution was varied with all other quantities as described in example 1 in such a range that the EDC to copolymer ratio could be varied over a range of 0.1 to 1.0. Figure 3 shows that the bound enzyme activity increases from 230 Lipase units to 250 Lipase units, where the EDC to copolymer ratio increases from 0.1 to 0.2. Beyond this ratio of 0.2 up to a ratio of 0.8, the bound enzyme activity remains almost unchanged. When the EDC to copolymer ratio is increased beyond 0.8 the bound enzyme activity was found to reduce. Thus, keeping the aim of getting maximum bound activity with least quantity of the expensive functionalizing agent an optimum EDC to Co-polymer ratio was decided as 0.2. (Figure 3). The bound enzyme activity was estimated by following the procedure, as described in example 1.
Example 4: Experiments to demonstrate optimum enzyme to functionalized copolymer ratio
Multiple experiments as described in example 1, were carried out, where, keeping the enzyme quantity at 1 gm level, the Co-polymer quantity was varied in such a way that the enzyme to Copolymer weight ratio was varied over a range of 1 to 20. As shown in figure 4, the amount of enzyme binding to the polymer and the bound enzyme activity showed a linear increase with an increase in the enzyme to Copolymer ratio up to 10,
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beyond which the increase in the bound activity was in a much lesser proportion indicating lower binding efficiency. Thus from economical point of view, the optimum binding efficiency i.e. highest bound activity to % loss of original activity, ratio was obtained at enzyme to Co-polymer ratio of 10. The bound enzyme activity was estimated by the same procedure as described in example 1.
Example 5: Difference in enzyme activity of the conjugated enzyme prepared as per example 1 and example 2.
Two experiments have been conducted where 15.3 gm of castor oil was added to 5.1 ml of enzyme solution containing 2.53mg/ml of conjugated enzyme. Experiment A had conjugated enzyme prepared as per example 1 and experiment B had conjugated enzyme prepared as per example 2. Both the reaction mixtures were incubated for different durations with constants stirring. The progress of hydrolysis monitored by titrating the mixture after specified time intervals with 0.1 N, alcoholic NaOH solution to estimate the fatty acid formed. Table 1 gives the results of these experiments. Thus from Tablel, it can be concluded that the quality of the conjugated enzyme prepared as per example 2 is about 10 to 12% less as compared to the conjugated enzyme prepared as per example 1, for the hydrolysis of the castor oil.
Table 1

Properties Experiment A
Conjugated enzyme as per
Example 1 Experiment B
Conjugated enzyme as per
Example 2
Protein content in enzyme sample (mg/ml) 0.53 0.49

Activity of conjugated enzyme (Lipase unit/ml) 69 40
Specific Activity (Lipase unit/mg) 131 82.2
Time (hrs) % Hydrolysis % Hydrolysis
1
10 18 24 38.79 50.26 55.32 60.79 33.87 47.23 51.53 55.47
Example 6: Batch process to validate the reuse of the conjugate enzyme:
One gram of castor oil was suspended in 1 ml of the isooctane and was mixed with 0.2M sodium phosphate buffer, pH-7.0. To this the enzyme solution (free or conjugated enzyme), to maintain the enzyme to substrate ratio of 1.04 x 10"2 was added. The reaction mixture was then incubated for different durations with constant stirring to obtain a hydrolysis pattern with respect to time (Figure 8). In the studies without solvent, the isooctane was replaced with 1 ml phosphate buffer, pH-7.0. At the end of the incubation period the reaction was terminated with 20 ml methanol. Figure 5 is a schematic representation of batch process of hydrolysis to validate the reuse of the conjugate enzyme.
The acid value was determined by titrating the reaction mixture against 0.1 N sodium hydroxide.
Example 7: Experiment for hydrolysis of oil
Figure 6 shows flow sheet representing the proposed hydrolysis scheme for industrial use. An oil or fat phase is provided in a column, which has been fed with a water-

containing soluble enzyme in an adequate quantity from the top, at the bottom of the column in the form of droplets such that there are distinct interfaces between the phases at the top and bottom. The phases are separately removed from the column i.e. partially hydrolyzed oil containing mono, di- and tri-glycerides with fatty acids from the top and water containing glycerol and soluble enzyme from the bottom after the contact in column, in such a way that the water is treated so as to recover/reuse the enzyme from the water containing the soluble enzyme. The top oil phase is continuously fed to the vacuum distillation for the recovery of the fatty acid and un-hydrolyzed oil is fed to the next stage, which is identical in construction and the water containing the soluble enzyme is fed from the top. The number of contacting stages could be 2 or more to give the desired extent of hydrolysis and/or desired level of glycerol concentration in the water. The water is then treated with an acid to precipitate out the enzyme by lowering of the pH for reuse and the water can be processed further for glycerol recovery. Table 2 shows the results of vacuum distillation. The amount of oil taken is 50 gm.
Table 2

Process Weight of oil
(9) Amount of
enzyme
(ml) % Hydrolysis Acid value Ricinoleic acid
(9) Time (h)
1st Hydrolysis 50.005 16.66 38.75 50.14 - 1
1st Vacuum distillation 48.8 - - 8.1 5.71 3

nna
hydrolysis 36.868 11.66 40.38 52.258 - 1
Vacuum distillation 35.211 - - 8.12 5.669 3
3ra Hydrolysis 27.144 9.048 33.17 43.18 - 1
3rd Vacuum distillation 26.28 - - 10 3 3
4th Hydrolysis 16.801 5.46 26.26 34.38 - 1
4tn Vacuum distillation 18.506 - - 10.94 2.5 3
5th Hydrolysis 13.642 4.547 18 23.19 - 1
5tn Vacuum distillation 12.373 - - 3 3
Total % hydrolysis = 83.06%.
Number of vacuum distillation cycles— 5
Total time for hydrolysis = 5 hours
Total time for 83.06% hydrolysis is 5 hours.
Example 8: Experiment to demonstrate optimum oil to buffer ratio
Multiple experiments as described in example 5, were carried out, where the Oil (non¬aqueous) to buffer (aqueous) ratio was varied from 0.01 to 0.06. Figure 7 showed the maximum % hydrolysis was obtained when oil to buffer ratio was 0.03. Hence the optimum oil to buffer ratio for maximum yield of the fatty acid was found to be 0.03.
Example 9: Experiment to demonstrate Solvent effect
Multiple experiments were carried out as described in example 5 using free enzyme and conjugated enzyme in the presence of solvent (iso-octane) and in the absence of solvent (iso-octane).

Jn the case of the reaction in presence of isooctane, the rate of the reaction obtained with the conjugated enzyme, is higher in the initial stages of the reaction as compared to the free enzyme, (% hydrolysis increases from 40 % with the native enzyme to 55% with the conjugated enzyme in 6 hours) (for the same protein to substrate ratio i.e. 1.04 x 10"2). As the reaction proceeds for a prolonged period, the rate of the reaction is found to be same with both the forms of the enzyme (Figure 8). In the case of the reaction without isooctane, for the same enzyme to substrate ratio, the conjugated enzyme exhibits almost a three-fold improvement in the conversion values in the first hour itself (% hydrolysis increases from 13 % with the native enzyme, to 42 % with the conjugated enzyme), indicating a hyper activation in the activity of the enzyme due to the conjugation with the copolymer used.
This enhancement in the enzyme activity is of immense significance in view of the reuse potential of the enzyme, and the potential for better mass transfer efficiencies of the conjugated reversibly soluble (pH-dependent) enzyme as compared to the enzyme immobilized on an insoluble carrier.
The enhancement in the initial rate of the reaction may be the result of certain morphological modifications of the copolymer-enzyme conjugate from that of native enzyme that effect alterations in the physico-chemical properties of the conjugate, such as imparting an interface-accumulating tendency to the conjugated moiety.
The results indicated that the conjugated enzyme was more prone to solvent induced deactivation as compared to the free enzyme and therefore even when the rate of hydrolysis was higher in the initial phase of the reaction, it eventually slowed down due to the deactivating effect of the solvent in the later stages.
This explanation was supported by an experiment in which the free and conjugated enzymes were treated with isooctane over a period of 24 hours. The percentage relative activity of the free enzyme in the presence of solvent for the initial period (solvent induced activation) was found to be much higher than the conjugated enzyme (Figure - 9).

It must be noted that although the conjugated enzyme demonstrated just 20 % hyper activation in the presence of the solvent, the rate of the reaction exhibited by it in the absence of the solvent and that exhibited by the free enzyme in the presence of solvent were almost the same. This indicated that the enzyme was getting hyper activated due to the changes occurring at its molecular level during the conjugation.
Example 10: Experiment to demonstrate Temperature effect
Multiple experiments were carried out as described in example 5 with free and conjugated enzyme where temperature of reaction mass was varied from 30°C to 70°C keeping oil(substrate) to enzyme ratio at optimum level as described in example 8.
The temperature optimum of the conjugated enzyme was 35°C as against the higher temperature optimum for the free enzyme (55°C). The conjugated enzyme was found to lose all its activity at the temperature optimum of the free enzyme, indicating lower temperature stability (Figure - 10). However, the activity and stability profiles with respect to time were similar for the bound and the free enzyme at their respective temperature optima. Hence, the entire hydrolysis process with the conjugated enzyme could be carried out at a lower temperature (35°C as compared to 55°C for the free enzyme) and with higher or similar rates of reaction.
The lower temperature optimum of the conjugated enzyme is highly beneficial in regards to the energy economy offered.
Example 11: Experiment to demonstrate mode of agitation
Multiple experiments were carried out as described in example 5 with conjugated enzyme using 20 mm Stator-Rotor type Homogenizer having a stator-rotar assembly with a clearance of 0.5 mm between the stator and the rotor and Six-Paddle type impeller also of 20 mm diameter, keeping oil(substrate) to enzyme ratio at optimum
25

Ievel(0.03) as in example 8.
When a homogenizer was used for agitation the efficiency of power utilization for optimum rate of hydrolysis was found to be significantly better (Figure 11) as compared to a six-paddle impeller (Figure 12) as seen from the higher % hydrolysis obtained at equivalent power dissipation level.
Although the rate of hydrolysis was optimum at the power to volume (PA/) ratio of 235 Watts/m3, the speed of homogenizer was optimized to a power to volume ratio of 70 Watts/m3 as the enzyme lost 15 % of its original activity in 1 hour at 235 Watts/m3 (Figure 13) of power dissipation level.
Example 12: Experiment to demonstrate enzyme recovery and reusability
The recovery of the enzyme at the end of the reaction was carried out by centrifugation of the reaction mixture at 10,000 RPM for 20 (min) to separate the two immiscible layers (oil and buffer containing enzyme)). The buffer layer was isolated and its pH was adjusted to 4.0 to precipitate the enzyme-copolymer conjugate. The precipitate was then washed with citro-phosphate buffer (pH 4.5) and redissolved in 0.2M sodium phosphate buffer, pH - 7.0 and reused as the enzyme source for the next reaction.
The repeated use of the conjugated enzyme can be expected to generate some deactivating effects over the enzyme. The robustness of the enzyme towards its recovery and reuse was examined with respect to time by conducting two sets of experiments -
In the first set, experiments were carried out using fresh conjugated enzyme as described in example 5 except that the oil(substrate) to enzyme ratio used, was 1.04 x 10"2 and the duration of the reaction was kept constant (24 hours). The enzyme was recovered and subjected to a second reaction (with the same amount of substrate as the first reaction), the duration of which was varied from 1 to 24 hours.

In the second set, experiments were carried out using fresh conjugated enzyme as described in example 5 except that the oil(substrate) to enzyme ratio used, was 1.04 x 10"2 The duration of the first batch of hydrolysis (using the fresh enzyme) was varied from 1 to 24 hours while that of the second batch using the recovered enzyme was kept constant (24 hours). The % hydrolysis obtained after the second reactions (using the reused enzyme) was determined to examine the stability and the catalytic ability of the recovered enzyme.
The enzyme reusability was examined by using the enzyme repeatedly for the hydrolysis of fresh substrate in a one-hour reaction under the optimized conditions of pH (7.0) and temperature (35°C). The oil(substrate) to enzyme ratio was maintained at 1.04 x10"2.
The described method for the recovery of the enzyme enabled approximately 98% recovery of the dry weight of the enzyme - copolymer conjugate. The recovered enzyme was found to be active over the variations of the time of the reaction. Both the sets of experiments conducted to establish the robustness of the conjugated enzyme indicated that the enzyme was highly robust and a maximum of 10 % gradual loss in the enzyme activity was observed (Figure 14). The reusability of the conjugated enzyme was assessed by performing successive hydrolysis reactions of a fresh substrate using the same enzyme and it was found that after 10 cycles, approximately 92% of the original activity was preserved.
Advantages of the process of the present invention:
To conjugate the lipase enzyme covalently to entities or carriers, capable of undergoing reversible phase transition in response to an external stimulus, such enzyme systems with reversible solubility being termed as Soluble - Insoluble (S-IS) enzyme systems.

To enable the enzyme to be in the solution form during the course of the reaction and in the insoluble form after the reaction for easy recovery.
To eliminate the mass transfer limitations by maintaining the enzyme in a soluble state during the reaction and then by applying an external stimulus, convert it into an insoluble state for easy recovery and reuse.
To obtain a Soluble - Insoluble (S-IS) enzyme systems that are industrially feasible and economically attractive.
To use the conjugated enzyme for fat hydrolysis instead of the traditional method of carrying it out at extremely high temperatures (240-270°C) and pressures (700-750 psig).
To facilitate the enzymatic hydrolysis of castor oil wherein the castor fatty acid is susceptible to degradation using the traditional method.
To facilitate the enzymatic hydrolysis of castor oil at higher rates of reaction and in a cost effective manner.
Use of the conjugated lipase enzyme for the catalysis of several processes such as hydrolysis, esterification, transesterification etc. yielding products of high commercial value such as fatty acids, glycerol, surfactants, perfumes etc.

We claim:
1. A process of preparing a reversibly soluble modified conjugated lipase enzyme
comprising steps of:
(a) providing a buffer solution;
(b) providing a functionalized copolymer;
(c) adding a lipase enzyme to the buffer solution before or after step (b); and
(d) precipitating conjugated enzyme by altering the pH of the solution to predetermined levels.

2. A process according to claim 1, wherein the said functionalized copolymer is provided by treating the copolymer with a cross-linking or functionizing agent.
3. A process according to any of claims 1 or 2, comprising steps of:

(a) providing said buffer solution;
(b) adding said lipase enzyme and dissolving it in said buffer solution;
(c) adding said copolymer and said cross-linking or functionalizing agent;
(d) agitating the mass at low temperature to produce conjugated lipase enzyme; and
(e) precipitating said conjugated enzyme by altering the pH of the solution to predetermined levels.
4. A process according to any of claims 1 or 2, comprising steps of:
(a) providing said buffer solution;
(b) adding said copolymer and said cross-linking and functionalizing agent thereby prefunctionalizing said copolymer;
(c) adding said lipase enzyme;

(d) agitating the mass at low temperature to produce conjugated lipase enzyme; and
(e) precipitating said conjugated enzyme by altering the pH of the solution to predetermined levels.

5. A process according to any Of claims 1 to 4 wherein the cross linking agent is selected from a group of carboxyl to amino cross linking carbodiimides such as 1-Ethyl-3-(3-Dimethylaminopropyl) Carbodi-imide (EDC), N-cyclohexyl-N"-[2-(4-morpholinyl)-ethyl]-carbodiimide, N-cyclohexyl-N"-[2-(4-morpholinyl)-ethyl]-carbodiimide-methyl-p-toluene-sulfonate and the like.
6. A process according to claim 5 wherein the cross-linking agent is 1-Ethyl-3-(3-Dimethylaminopropyl) Carbodi-imide (EDC).
7. A process according to any of claims 1 to 4 wherein the copolymer is selected from hydroxypropylmethyl cellulose acetate succinate, copolymers of methacrylic acid-methylmethacrylate and copolymers of acrylic-methacrylic acid esters.
8. A process according to claim 7 wherein the copolymer is an anionic methacrylic acid- methyl methacrylate copolymer.
9. A process according to any of claims 1 to 4 wherein the weight ratio of lipase enzyme to functionalized copolymer is from 5 to 20.
10. A process according to claim 9 wherein said weight ratio is preferably from 8 to 15 and more preferably 10.
11. A process according to any of claims 1 to 4 wherein the enzyme is precipitated by changing the pH from 7.0 to 4.6.

12. A process according to any of claims 1 to 4 wherein the ratio of copolymer to the cross-linking/functionalizing agent is from 0.1 to 1.0.
13. A process according to claim 12 wherein the said ratio is 0.15 to 0.8 and preferably 0.2.
14.A process according to any of claims 1 to 4 wherein the said buffer solution is maintained at pH 7.0.
15. A process according to any of claims 3 and 4 wherein the said agitation is preferably carried out at low temperatures of between 0 to 30°C.
16. A process according to any of claims 3 and 4 wherein the said agitation is preferably carried out at low temperatures of between 2 to 10°C.
17. A process according to any of claims 3 and 4 wherein the said agitation is preferably carried out at low temperature of 4°C.
18. A process for the hydrolysis of oils or fats, using reversibly soluble modified conjugated lipase enzyme prepared by the process according to any one of claims 1 to 15, comprising steps of

(a) providing a first liquid oil or fat phase, above a water-containing soluble enzyme second phase such that there is distinct interface between the said first and second phases.
(b) mixing the two phases and allowing the mass to settle to form two phases: upper phase comprising partially hydrolyzed oil and lower phase comprising water containing glycerol and soluble enzyme.
(c) separately removing the phases from the column.
(d) treating the said lower phase with an acid to precipitate out the enzyme for reuse.

19. A process according to claim 18, wherein the said upper phase comprises partially hydrolyzed oil containing mono, di- and tri-glycerides with fatty acids.
20. A process according to any one of claims 18 and 19, wherein the said upper phase is continuously fed to vacuum distillation for separation of fatty acid and un-hydrolyzed oil.
21. A process according to anyone of claims 18 to 20, wherein the said un-hydrolyzed oil is repeatedly processed with lipase enzyme until desired extent of hydrolysis and/or desired level of glycerol concentration in the water is obtained.
22. A process according to claim 18, comprising further processing the said lower phase for glycerol recovery.
23. A process according to claim 18, wherein the said water-containing soluble enzyme phase is maintained at pH 7.0 to enable use of the enzyme in soluble form.
24. A process according to claim 18, wherein the enzyme is precipitated for reuse by changing the pH from 7.0 to 4.5.
25. A process according to claim 18, wherein the percentage of hydrolysis is more than 90 when the ratio of fat-or oil to enzyme is 3 to 0.25.
26. A process according to claim 18 wherein the said process is preferably carried out at temperature between 20 and 50°C.
27. A process according to claim 26, wherein the said temperature is preferably 35°C.

28. A process according to claim 18, wherein the hydrolysis of oil is carried out using homogenizer.
29. A process according to claim 28, wherein the said homogenizer is a 20mm diameter stator rotor type homogenizer.
30. A process of preparing a reversibly soluble modified conjugated lipase enzyme and a process for the hydrolysis of oils or fats, using the reversibly soluble modified conjugated lipase enzyme us substantially herein described in the text and examples 1 to 12.
Dated this 6th day of January 2003.
S. MAJUMDAR OfS. MAJUMDAR & CO. Applicants" Agent

Abstract
A process of preparation of reversibly soluble-insoluble conjugated lipase enzyme which can be effectively used for the enzymatic hydrolysis of castor oil The proposed method is for the modification of lipase into a form, which is reusable, and at the same time has higher activity than the native enzyme. The enzyme lipase is conjugated to a copolymer using a functionalizing agent. The enzyme so obtained can be readily used to facilitate the enzymatic hydrolysis of castor oil at higher rates of reaction and in a cost effective manner.
To
The Controller of Patents
The Patent Office Branch
34
Mumbai

Documents:

29-mum-2003-abstract(08-01-2003).pdf

29-mum-2003-cancelled pages(08-01-2003).pdf

29-mum-2003-claims(granted)-(08-01-2003).pdf

29-mum-2003-correspondence(01-08-2007).pdf

29-mum-2003-correspondence(ipo)-(28-08-2007).pdf

29-mum-2003-drawing(08-01-2003).pdf

29-mum-2003-form 1(08-01-2003).pdf

29-mum-2003-form 1(28-02-2003).pdf

29-mum-2003-form 18(03-11-2006).pdf

29-mum-2003-form 2(granted)-(08-01-2003).pdf

29-mum-2003-form 3(08-01-2003).pdf

abstract 1.jpg


Patent Number 214161
Indian Patent Application Number 29/MUM/2003
PG Journal Number 13/2008
Publication Date 28-Mar-2008
Grant Date 05-Feb-2008
Date of Filing 08-Jan-2003
Name of Patentee DIRECTOR, MUMBAI UNIVERSITY INSTITUTE OF CHEMICAL TECHNOLOGY
Applicant Address NATHALAL PARIKH MARG, MATUNGA, MUMBAI 400 019
Inventors:
# Inventor's Name Inventor's Address
1 PANDIT ANIRUDDHA B CHEMICAL ENGINEERING DIVISION, INSTITUTE OF CHEMICAL TECHNOLOGY, MUMBAI UNIVERSITY, NATHALAL PARIKH MARG, MATUNGA, MUMBAI 400 019
PCT International Classification Number C12N9/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA