Title of Invention

A PROCESS FOR PREPARATION OF ESTERS USING AN INTERFACIALLY ACTIVE ENZYME

Abstract

A process for the preparation of esters using an interracially active enzyme, which comprises a) preparation of a mixture of water and substrates such as herein described in an appropriate buffer; b) generation of colloidal dispersion of air in water by pumping air into the said matrix as obtained in step (a) above at a temperature ranging between 5 to 60°C ,to obtain a biphasic phase consisting of an aqueous phase and a non-aqueous phase; c) addition of an interracially active enzyme such as herein described to the said colloidal dispersion to catalyse the reaction; d) maintaining the reaction phase volume constant for an appropriate period of time; e) contacting the said interracially active enzyme with the said colloidal dispersion to obtain enriched factions of the products; recovering the esters from the said enriched fractions of the products by known methods.

Full Text

This invention relates to a process for the preparation of esters using interfacially active enzymes. More particularly, this invention relates to a process for the preparation of esters using interfacially active enzymes to obtain enhanced activity at the interface. Esterification is an important reaction in oleochemical industry with products of confectionary, cosmetic and dietary importance. In recent times lipases are utilized in oleochemical industry for their ability to function in organic solvent. By performing a lipase mediated esterification reaction in organic solvents besides retaining the advantages of enzyme catalyzed reactions, hydrolysis reactions and side reactions are kept at a minimum. Enzyme catalyses in organic solvents has the advantages of physiological reaction temperatures, ease of dissolving water-insoluble lipase substrates and excellent stereo- and regio-selectivity. Near absence of water in the organic solvent shifts the reaction equilibrium towards condensation but depresses the enzyme activity considerably. With the present day access on "green" processes, considerable emphasis is laid on processes which reduce the cost intensive and environment threatening organic solvents. Lipases by their make up a belong to the class of enzymes known as interfacially active enzymes. The activity of these enzymes increases several fold when the substrate is presented as a surface rather than a monomer. Solubiliza- tion of the substrate, as achieved in lipase reactions in organic solvents, has the disadvantages of losing this property of interfacial activation of lipases. Several media engineering strategies were adapted with an aim to bptimise the substrate-enzyme interactions for maximal mass action, while retaining the organic bulk phase. Following are some of the approaches made : (A) Water in oil emulsions or reverse micelles : In these colloidal suspensions the enzyme is present in the discontinuous aqueous phase and the substrates are dissolved in the bulk and continuous non-polar phase. Presence of excess (>95 volume %) organic solvent and emulsifiers to stabilise the reverse micelles effect the reaction efficiency considerably (K Martinek, AV Levashov, N Klyachko, Y Khmelnitski and IV Berezin (1986) Eur J.Biochem. 155, 453). (B) Biphasic mixtures of water and organic solvents : This medium is similar to reverse micellar system, except for the absence of emulsifiers. This system has the same limitations as A (G Carrea, (1984) TIBTECH 2, 102) (C) Monolayers of substrates : Since the physical properties, such as surface pressure, area etc. could be controlled, this system has become important to study the enzyme kinetics. Due to poor surface area to volume ratios interaction between enzyme and the substrate is poor, hence was never considered important for commercial application (E.Pinho Melo et.al. (1995) Biochemistry 34, 1615). (D) Heterogeneous eutectic mixtures: A eutectic can be described as a mixture of two or more compounds which, at a well defined composition, displays a minimum value for the melting temperature I the corresponding phase diagram. Presence of adjuvants is essential. Relatively new and unknown media and much less is known on the behaviour of enzymes in these heterogeneous eutectic mixtures. Some synthetic reactions involving amino acids showed promise (I Gill and E Vulfson (1994) TIBTECH 12, 118) (E) Supercritical fluids, e.g., carbon dioxide : A promising non-aqueous system, offers several advantages such as a, higher diffusivity of the substrates than in organic solvents;b, carbon dioxide could be easily removed from the reaction products; c,environmentally benign and d, inexpensive raw material. Behaviour of enzymes in supercritical carbon dioxide is poorly understood and application in the context of enzyme mediated esterifications is in experimental stage (O Nakamura (1990) TIBTECH 8,288; 0 Aaltmen and R.Markuu (1991) CHEMTECH 21,240) (F) Membrane reactors : The nonpolar bulk solvent dis solving the substrates and the polar phase containing the enzyme are physically separated by a polymeric membrane that prevents mixing of the bulk phases but provides a interface for interaction. The system is developed as a continuous process. Very few studies were attempted using enzymes and require careful membrane selection and optimization. (G) Adsorption of substrates on solid matrices : This technique is adapted to improve the mass action between the enzyme and the substrates. Requires preloading of polar substrate on a insoluble matrix. The bulk solvent is still an organic solvent, hence has the same limitations as the other methods, which use organic solvent. ( M Berger, K Laumen and MP Schneider (1992) JAOCS, 69,955) (H) Neat organic solvents : This reaction phase uses the liquid nature of one of the substrates at reaction temperatures to dissolve the other substrates. Since the substrate becomes the bulk phase, no organic solvent is added to the reaction. Requires constant removal of catalytically produced water from the reaction. Limited to the reactions involving liquid substrates at the reaction temperatures and also has same limitations as the bulk organic solvent systems (SM Kim and JS Rhee (1991) JAOCS, 68,499). Except C and E all other methods employ organic solvents and lipase behaviour in E is largely unknown. All these reaction media contain two or more solvents, besides the substrates and the enzyme. In all these systems organic solvent / non-organic solvent interfaces exist and the studies emphasized on the reaction yields but the properties and extent of the interface was not described. Lipase reactions do occur on monomeric substrates albeit slowly. Optimal disposition and extensive interfacial area are very important for performing the lipase mediated reactions. Foam is a colloidal dispersion of air in water. In foam, water is the continuous phase and air is the discontinuous phase. In terms of volume occupancy, water occupies only a fractional volume compared to the bulk air phase. The major advantage of foam is enormous surface area it offers, similar to clay particles. Extensive information on physical properties of foams is available. Foams are formed due to decrease in surface tension at air-water interface by surface active compounds. Lipase substrates are amphiphilic in nature i.e., they have polar and non-polar portions separated in the same molecule and demonstrate surface excess property i.e., they preferentially partition to aqueous /non-aqueous interface and thereby decrease the surface tension. In the present invention this aspect of the lipase substrates is utilized to generate a biphasic phase made of air and water to conduct the lipase reactions. Water is essential for enzyme reactions, including lipase reactions. It is a constant debate on how much water is necessary in an esterification or a transesterification reaction. The role of the added water in organic solvents could either help retain the enzyme structure and/or help generate a biphasic mixture more conducive for lipase action. In literature there is no consensus of observation as to how much water is needed for a best product yield. Since lipase reaction are reversible and have preference for the interfaces, the quality and composition of the interface is crucial for reaction rates. The amount of water is important to that extent that it influences these parameters. Further, the physical properties of water immediately adjascent to the interface are significantly different compared to the bulk water properties. Lipase interaction with the interface occurs in this interfacial water, which may extend for tens of angstroms. Under such circumstances and in the presence of appropriate substrates, favorable reaction is esterification. Since the products of esterification are also water insoluble, they do not leave the interface. The products could be conveniently extracted at the end of the reaction. The main object of the invention is to provide a new method , to conduct reactions catalysed by interfacially active enzymes such as lipases, phospholipases etcwherein (a) no organic solvents are used; (b) the method takes advantage of intrinsic surface active properties of the substrates. The method involves dispersion of substrate in a colloidal preparation of air in water, which enhances the reaction rates, e.g., esterification, by facilitating maximal contact of enzyme with the substrate. The colloidal dispersion may be discontinuous air or gas phase in continuous water or liquid phase, similar to foams. Enzymes, as mentioned here, encompass all the enzymes which demonstrate enhanced activity at an interfaces, where one of the bulk phase is water. The classes of reactions catalysed by the enzymes involve hydrolysis, condensation and transesterifi-cation reactions. Conducting reactions of lipase in substrate foams or colloidal dispersion of air in water has the advantage of presenting the substrate for efficient mass action. Lipase substrates are insoluble in water and their surface active properties make them occupy the interface exclusively. Since substrate interface is an essential feature for lipase action, by considering air as the non-aqueous phase, use of organic solvents is avoided. This is a considerable advan- tage since presence of organic solvent not only depresses the activity of the enzymes but also poses problems in their disposal. Bulk of the enzyme is present in an aqueous environment hence its activity is not reduced. This advantage enhances the life of the enzyme and also lipases from other sources could be utilized, which were inefficient in organic solvents. Accordingly, the present invention provides a process for the preparation of esters using an interracially active enzyme, which comprises a) preparation of a mixture of water and substrates such as herein described in an appropriate buffer; b) generation of colloidal dispersion of air in water by pumping air into the said matrix as obtained in step (a) above at a temperature ranging between 5 to 60°C ,to obtain a biphasic phase consisting of an aqueous phase and a non-aqueous phase; c) addition of an interracially active enzyme such as herein described to the said colloidal dispersion to catalyse the reaction; the chemical reaction is carried out in a reactor with variable volume and have controlled air flow, humidity and temperature facility, d) maintaining the reaction phase volume constant for an appropriate period of time; e) contacting the said interracially active enzyme with the said colloidal dispersion to obtain enriched factions of the products; a) recovering the esters from the said enriched fractions of the products by known methods.. According the a feature of the invention substrates used for the preparation of esters are those suitable for use with interfacially active enzymes. Such substrates include the substrates of lipase such as alcohols and acids, and also all chemicals which have a esterifiable acid or alcohol. For example, the substrates might include all carbohydrates both monosaccharides and oligosaccharides, amino acids both natural and unnatural and nucleotide bases. According to another feature, all interfacially active enzymes used may be selected from lipases and proteases, phospholipases and enzymes involved in synthesis of compounds such as phosphoplipids, secondary metabolites derived from phospholipids. According to another feature, the enzymes used may be selected from those whose site of action is an interface formed by an aqueous and an non-aqueous, e.g. air or solid, phase. According to another feature of the process the operating temperature conditions could be varied from 5°C, to 60°C wherein the enzyme remains active and the substrate quality is not affected. According to another feature of the said process the humidity conditions of the air pumped into the reaction may range upto 100% relative humidity. According to yet another feature of the process wherein source of interfacially active enzyme may be selected from bacterial sources viz. Candida rugosa, C.cylindracea. Bacillus sp. etc. or from fungal source viz. Mucor sp. Or from higher organisms viz. porcine etc. According to yet another feature of the present process According to yet another feature of the present process the bulk non-aqueous phase is the gaseous phase. According to yet another feature of the present process the chemical reaction is carried out in a reactor with variable volume facility and a facility for controlled air flow, humidity and temperature. According to yet another feature of the present process use of organic solvent is avoided by providing air as a bulk non-aqueous phase. According to yet another feature of the present process substrates used are amphiphillic in nature providing polar and non-polar portion separated in the same molecule to facilitate decrease in the surface tension. According to yet another feature of the present process substrates used are presented as a surface rather than a monomer for mass action. According to yet another feature of the present process the life of the enzyme is increased in the presence of an aqueous phase. According to yet another feature of the present process the rates of formation of esters are faster than the reaction occurring in the presence or absence of organic solvent . The present invention is further illustrated by the following examples which should not be construed to limit the scope of the invention in any manner. Example 1 : Synthesis of esters between fatty alcohols and fatty esters using Mucor meihie enzyme in a foam system : The foam reactions are performed in batches in a glass columns (2.5" X 10") fitted at one end with a sintered joint (G3) as an air inlet and the other end was left open . In a typical reaction volume of 5ml of Tris buffer (lOmM, pH 7.4), oleic acid and n-octanol were added at an equimolar concentration of 100 mM, while passing air (partially humidified) through the column. After the formation of foam, 0.05 ml of lipase ( Mucor miehei, 10,000L, soluble form, 6.4 mg /ml, a gift from Novo Nordisk) was added to the foam column. The height of the foam column is maintained constant during the reaction time. The reaction was quenched and extracted using dichlormethane. The dichloromethane was dried under a stream of nitorgen. The esters and unreacted oleic acid were separated and quantitated by GC (HP2 column on a HP5890 Series IIPlus gas chromatograph). The rate of formation of octyloleate was 5.4 mole/min/mg of protein. For comparison data on identical reaction was performed in heptane was presented. In foam reactions the esterification rates are several folds faster compared to heptane reactions. Initial rates of ester formation in foam were compared with few published studies using immobilized lipase preparations. Rate of formation of ester with decanoic acid and dodecanol in various solvents was in the range of 0.003-0.08 mol/min/mg of protein (RH Valivety, GA Johnston, CJ Suckling and PJ Hailing (1991) Biotech and Bioengg. 38, 1137). Formation of oleic acid esters with various alcohols in the absence of any organic solvents was in the range of 0.84-1.8 mol/min/mg of protein of lipase (M Habulin, V Krmelj and Z Knez (1996) 44, 338). In foams, the rate of formation of simple esters of oleic acid is faster than the reactions occurring in pure alcohols in the absence of any organic solvents or water (Fig.l). Similar reactions were also done with lipases from other sources such as Candida rugosa, C.cylindracea and porcine pancreas, with equal efficiency..lsl -1 -1 Substrates Solvent Rate, mol.min mg 1 Decanoic acid and Several 0.003-0.08 Dodecanol 2 Oleic acid and No solvent 0.84-1.8 various alcohols 3 Oleic acid and Foam 0.6-5.4 various alcohols Example 2. Synthesis of glycerides using glycerol and oleic acid with Mucor meihie enzyme: The foam reactions are performed in batches in a glass columns (2.5" X 10") fitted at one end with a sintered joint (G3) as an air inlet and the other end was left open. In an identical set up, esterification of glycerol with oleic acid was performed using Mucor meihei enzyme. In a batch process, oleic acid, doped with radioactive oleic acid obtained from Amersham Co., was used at 0.1M concentration and glycerol was used at 10% v/v in a 10ml reaction volume. Air was passed through the mixture keeping the height of the column constant. Air flow determines the stability of the foam. Too much flow will break the foam and does not allow the foam to form. Too less air flow causes the collapse of the foam column since the foam drainage would be in excess. The reaction was conducted at room temperature and the constant humidity of 80% was maintained in the air to prevent drying of the foam. The reaction was quenched using dichloromethane and the products were extracted in to di-chloromethane. The solvent is dried under a stream of nitrogen and the products were separated on HPTLC plates using appropriate solvent system. The separated product and the substrates were visualized using iodine vapour and were scraped for counting. The reaction comersion efficiency is quantitated as the amount of radioactivity present in the glycerides spots compared to the total counts added initially. Within 8h of reaction time 70% of the oleic acid is converted in to glycerides (Fig.2). Initially diglyceride formation is highest and after 3h triglyceride formation is more. This is justifiable since diglycerides are intermediate to the formation of triglycerides. Example 3: Synthesis of cholestryloleate by the enzyme Mucor miehei in a foam system: In a batch process, 500 micromoles of cholesterol and oleic acid, doped with tritiated oleic acid ( from Amersham co.) each were mixed and suspended in 5ml of 10 mM Tris buffer (pH 7.4). The foam reactions are performed in batches in a glass columns (2.5" X 10") fitted at one end with a sintered joint (G3) as an air inlet and the other end was left open. The mixture was alldwed to foam by passing air through the system and to this foam column 0.05 ml of crude fungal lipase (Mucor meihie) was added ( Obtained from Novo Nordisk, India). The reaction was allowed to proceed at room temperature with a constant supply of humidified air (85%). At the end of the reaction the reaction was quenched and extracted using dichloromethane. The solvent is dried under a stream of nitrogen and the reaction products were separated from the substrates on a HPTLC using appropriate solvent system (dichlromethane and ethyl acetate at 1:1 v/v ratio). The substrates and the products were visualized with iodine vapour and then scarped for counting in a liquid scintillation counter. The percent conversion is expressed as the percent of counts present in the cholesteryloleate spot relative to the total counts present in the reaction. Authentic cholesteryloleate was synthesized by chemical means to confirm the Rf values. In 4 h of foaming 40 % of the oleic acid is converted into the cholesterol ester. We Claim: 1. A process for the preparation of esters using an inte Facially active enzyme, which comprises a) preparation of a mixture of water and substrates such as herein described in an appropriate buffer; b) generation of colloidal dispersion of air in water by pumping air into the said matrix as obtained in step (a) at a temperature ranging between 5 to 60°C ,to obtain a biphasic phase consisting of an aqueous phase and a non-aqueous phase; c) addition of an interracially active enzyme such as herein described to the said colloidal dispersion to catalyse the reaction; d) maintaining the reaction phase volume constant for an appropriate period of time; e) contacting the said interracially active enzyme with the said colloidal dispersion to obtain enriched factions of the products; f) recovering the esters from the said enriched fractions of the products by known methods. 2. A process as claimed in claim 1 wherein substrates used for the preparation of esters are suitable for use with interracially active enzymes. 3. A process as claimed in claim 1 to 2 wherein interracially active enzymes used may be selected from lipases and proteases, phospholipids and enzymes involved in synthesis of compounds such as phospholipids, secondary metabolites derived from phospholipids. 4. A process as claimed in claim 1 to 3 wherein source of inte Facially active enzyme may be selected from bacterial sources viz. Camdoda rugosa, C. cylindracea, Bacillus sp. etc. or from fungal source viz. Mucor sp. Or form higher organisms viz. porcine etc. 5. A process as claimed in claim 1 to 4 wherein the bulk non-aqueous phase is the gaseous phase 6. A process as claimed in claim 1 to 5 wherein the air is pumped in to the reaction mixture under controlled humidity and temperature. 7. A process as claimed in claim 1 to 6 wherein air is pumped into the reaction mixture at humidity level ranging between 50 to 100%. 8. A process as claimed in claim 1 to 7 wherein the appropriate buffer is used to control the pH of the reaction phase for optimum product yield. 9. A process as claimed in claim 1 to 8 wherein substrates used are amphiphillic in nature providing polar and non-polar portion separated in the some molecule to facilitate decrease in the surface tension. 10. A process as claimed in claim 1 to 9 wherein substrates used are presented as a surface rather than a monomer for mass action. 11.A process as claimed in claim 1 to 10 wherein the life of the enzyme used is increased in the presence of an aqueous phase. 12. A process as claimed in claim 1 to 11 wherein the rates of formation of esters are faster than the reaction occurring in the presence of absence of organic solvent. 13. A process for the preparation of esters using interracially active enzymes substantially as herein described with reference to the examples.

Documents:

610-del-1998-abstract.pdf

610-del-1998-claims.pdf

610-del-1998-complete specification (granted).pdf

610-del-1998-correspondence-others.pdf

610-del-1998-correspondence-po.pdf

610-del-1998-description (complete).pdf

610-del-1998-drawings.pdf

610-del-1998-form-1.pdf

610-del-1998-form-19.pdf

610-del-1998-form-2.pdf

610-del-1998-form-3.pdf

610-del-1998-petition-138.pdf