Title of Invention

"AN ENZYMATIC PROCESS FOR THE PREPARATION OF PHENOLIC ESTERS"

Abstract

This invention relates to an enzymatic process for the preparation of phenolic esters which comprises reacting organic acids having carbon chain length C2 to C20 with phenols or substituted phenols in presence of lipase enzyme 15 to 115 µ moles/min/mg at a temperature in the range 37°C to 60°C optionally with solvents or by maintaining water content less than 0.05 ml per 100 ml of reaction mixture by adding non polar solvent, recovering and purifying the corresponding phenolic ester by conventional solvent extraction methods such as herein described.

Full Text

This invention relates to an enzymatic process for the preparation of phenolic esters. This invention particularly relates to an enzymatic process for the preparation of phenolic esters of organic acids from phenol and substituted phenols like cresols and guaiacol particularly m- and p-cresols and organic acids of carbon chain length C2-C20 using lipases from Mucor melhei and Porcine Pancreas. Phenolic esters from low molecular weight and high molecular weight acids possess high flavour value as they exhibit strong fruity and flavour notes. Cresyl esters of low and high molecular weight acids possess unique flavour characteristics with a combination of sweety floral and fruity odours. Many known esters of cresols like 0-tolyllaurate, p-tolylacetate, 0-tolylisobutyrate, p-cresylisovalerate, p-cresylcaprylates and 0-tolylsalicylate are very much desired for their flavour notes as they are used extensively in bakery,confectionery and beverage products like baked goods, chewing gum, frozen dairy products , gelatin, pudding, cheese ,soft candy and non-alcoholic beverages. Presently only chemical procedures are available for the preparation of these compounds which involve activation of cresols and acids. In case of cresols sodium salts are invariably used. The acids used vary from their anhydrides to their acid chlorides. In some cases esterification catalysts like phosphorus oxychlorides are used. All these procedures result in cost-intensive processes with cumbersome work-up procedures. These are the major drawbacks of these chemical methods. The following table illustrates the chemical methods of preparation of these compounds and their main drawbacks: (Table Removed) The major drawbacks of the chemical methods for the preparation of substituted phenolic esters, especially cresylic esters are : 1. The conditions employed for the chemical estehfication are drastic like high temperatures. 2. Use of solvents like methanol and catalysts like phosphorus oxychlorides which are hazardous to deal with , lead to cumbersome work-up procedures and conthbute to pollution. 3. More unit operations resulting in cost intensive procedures. 4. Activation of carboxyl group of organic acids through derivatisation to acyl halides or employment of anhydrides and activation of cresols by employing sodium salts. Till date no enzymatic method for the preparation of these compounds are available. This patent proposal discusses for the first time an enzymatic method for the preparation of esters of phenols and substituted phenols, particularly the latter. The present invention involves an enzymatic procedure for the preparation of esters of phenols and substituted phenols like cresols and guaiacol with organic acids which has many advantages over chemical synthesis. The main advantages of the present invention are: 1. Milder reaction conditions. 2. Use of immobilised lipase facilitating the easy recovery of the catalyst. 3. Reuse of the enzyme. 4. Clean product without any side reaction. 5. Cost effective procedure. 6. An ecofriendly process which involves pollution free work out procedures without the use of toxic solvents. 7. Derivatization to activate phenols and organic acids are not required. 8. Fewer unit operations The present invention provides an enzymatic process where phenols or substituted phenols are reacted with organic acids with or without the presence of non-polar solvents in the presence of lipases from Mucor miehei and Porcine Pancreas. New principles underlying and advantages of the invention are: Esterification was achieved by using lipases from an animal and microbial source with or without the use of a nonpolar solvent under low water environment and milder reaction conditions. Use of enzyme eliminated the need for the protection of carboxyl groups of acids and activation of cresols resulting in a cost-effective process and elimination of tedious workout procedures. Recovery and reuse of the enzyme gives a cost effective process. The yields are comparable to that of chemical methods. Accordingly, the present invention provides an enzymatic process for the preparation of phenolic esters which comprises reacting respective organic acids having carbon chain length C2 to C20 with phenols or substituted phenols in presence of lipase enzyme 15 to 115 µ moles/min/mg at a temperature in the range 37°C to 60°C optionally with solvents or by maintaining water content less than 0.05 ml per 100 ml of reaction mixture by adding non polar solvent, recovering and purifying the corresponding phenolic ester by conventional solvent extraction methods such as herein described. In an embodiment of the present invention, the phenol employed may be m- cresol, p- cresol, phenol, guaiacol, catechol and resorcinol . In an another embodiment of the present invention, the organic acid of carbon chain length C2 to C20 like acetic, propionic, butyric, valeric, isovalenc, octanoic, decanoic, lauhc, myristic, palmitic and stearic acids may be employed. In yet another embodiment of the present invention, the lipase used may be immobilised lipase from Mucor meihei, Candida cylindrecea, Pseudomonos fluorescens, wheat germ. Porcine Pancreas and chicken liver esterase. In yet another embodiment of the present invention, the reaction may be effected at for a period of 24-100 h. In yet another embodiment of the present invention the phenols or the organic acids employed may be in the concentration range 0.02M-0.1M/0.002 mole to 0.05 mole depending on the presence or absence of the solvent. In still another embodiment of the present invention the non - polar solvent employed may be n-heptane, chloroform, n-hexane, petroleum ether (40-60° C fraction) In yet another embodiment of the present invention, the lipase employed may be in the range of activity units 15 to 115 fimoles/min/mg of immobilised enzyme. Commercially available organic acids (99% pure from Sd. Fine Chemicals India Ltd., Aldrich Chemical Company Ltd., Sigma Chemical Company Ltd) and substituted phenols (98% pure from Ranbaxy Chemicals, Loba chemie Ltd., SISCO Research Laboratories pvt Ltd., India) were used. All the solvents employed were obtained from SISCO Research Laboratories pvt Ltd., India and were distilled once before use. Immobilised lipase preparation, Lipozyme IM-20 {Mucor meihei -MML) was procured from M/s Novo Nordisk and Porcine Pancreas (PPL) from Sigma Chemicals Co. (St. Louis Mo). The specific hydrolytic activity (Tris buffer, pH 7.0 and tributyhn substrate) units of the two lipases are 12.44 and 3.72 famoles/min/mg protein or 0.77 and 1.13 fimoles/min/mg of immobilised enzyme respectively. In case of long chain fatty acids the reactions were monitored by pippeting out at regular intervals aliquots (0.5 vn\) of the reaction mixture into 10 ml of 0.01 N NaOH and titrating against O acid using phenolphthalein indicator. This gives the amount of NaOH reacted with the unreacted acid. A blank was also performed. The difference between the above two gave the amount of NaOH corresponding to unreacted fatty acids and hence the concentration of the ester formed. The percentage of esterification was determined from the difference between the initial value and value at specific reaction time. In case of low molecular weight acids the decrease in acid content showed the extent of esterification . The isolated product was characterized by recording a 1H NMR spectrum of the compound on the Bruker-DXP 500 NMR instrument operating at 20° C. The sample was dissolved in CDCIa-DMSO-de mixture and the signals were referenced to TMS. The 1H NMR data confirmed the formation of the compound. A typical procedure employed for the reaction is as follows. A mixture of substituted phenol (0.02 M to 0.1 M or 0.002 to 0.05 mole) and organic acid (0.02M to 0.1 M or 0.002 to 0.05 mole) was taken as such or in a stoppered flask containing 5-20 ml of n-heptane, when solvent was employed or in a two-necked round bottomed flask containing 100-200 ml of a non-polar solvent, fitted with a Soxhelet apparatus filled with a desiccant and with a condenser. To this enzyme was added possessing activity of 15-50 µmoles/min/mg of immobilised enzyme in case of Mucor meihei and 50-115 µmoles/min/mg of immobilised enzyme in case of Porcine Pancreas and agitated at 30-65° C at 100-250 rpm for 12-100 h on a rotary shaker or refluxed for a period of 24-72 hr. The product work out was by separation of the immobilised enzyme by filtration and treating the filtrate with saturated sodium bicarbonate to remove the unreacted acid and evaporating the solvent to get a mixture of the phenol and the phenyl ester. The process of the invention is described in detail in the examples given below which are illustrative only and should not be construed to limit the scope of the invention. EXAMPLE-1 To 10 ml of n-heptane are added 0.052 M p-cresol 0.0546M acetic acid and immobilised lipase from Porcine pancreas of activity units 68.3 µmoles/min/mg of immobilised lipase and the reaction mixture was agitated at 175 rpm at 50° C for 66 h. The product work out was by filtenng the immobilised enzyme and removing the unreacted acid by washing with saturated sodium bicarbonate solution and evaporating the solvent to get a mixture of unreacted p-cresol and p-cresyl acetate and separation of the ester from unreacted phenol by distillation. Estimation by titration showed 66.9%of p-cresyl acetate formation in the reaction mixture. EXAMPLE-2 To 0.00225 mole p-cresol are added 0.0023 mole of acetic acid and immobilised lipase from Porcine pancreas of activity units 29.04 µmoles/min/mg of immobilised lipase and the reaction mixture was agitated at 175 rpm at 60° C for 24.5 h. The product work out was by filtering the immobilised enzyme and removing the unreacted acid by washing with saturated sodium bicarbonate solution and evaporating the solvent to get a mixture of unreacted p-cresol and p-cresyl acetate and separation of the ester from unreacted phenol by distillation. Estimation by titration showed 51.06%of p-cresyl acetate formation in the reaction mixture. EXAMPLE- 3 To 0.0022 mole p-cresol are added 0.0023 mole of lauhc acid and immobilised lipase from Porcine pancreas of activity units 28.5 µmoles/min/mg of immobilised lipase and the reaction mixture was agitated at 175 rpm at 63° C for 40.2 h. The product work out was by filtehng the immobilised enzyme and removing the unreacted acid by washing with saturated sodium bicarbonate solution and evaporating the solvent to get a mixture of unreacted p-cresol and p-cresyl laurate and separation of the ester from unreacted phenol by distillation. Estimation by titration showed 20.8%of p-cresyl laurate formation in the reaction mixture. EXAMPLE-4 To 0.00225 mole p-cresol are added 0.0023 mole acetic acid and immobilised lipase from Porcine pancreas of activity units 29.04 µmoles/min/mg of immobilised lipase and the reaction mixture was agitated at 175 rpm at 63° C for 63.2 h. The product work out was by filtering the immobilised enzyme and removing the unreacted acid by washing with saturated sodium bicarbonate solution and evaporating the solvent to get a mixture of unreacted p-cresol and p-cresyl acetate and separation of the ester from unreacted phenol by distillation. Estimation by titration showed 65.91 %of p-cresyl acetate formation in the reaction mixture. EXAMPLE- 5 To 0.0046 mole p-cresol are added 0.023 mole propionic acid and immobilised lipase from Porcine pancreas of activity units 56.5 µmoles/min/mg of immobilised lipase and 0.2 ml of 0.1 M phosphate buffer pH 7.0 and the reaction mixture was agitated at 175 rpm at 37° C for 56 h. The product work out was by filtering the immobilised enzyme and removing the unreacted acid by washing with saturated sodium bicarbonate solution and evaporating the solvent to get a mixture of unreacted p-cresol and p-cresyl propionate and separation of the ester from unreacted phenol by distillation. Estimation by titration showed 20.8%of p-cresyl propionate formation in the reaction mixture. EXAMPLE - 6 To 10 ml of n-heptane are added 0.00224 mole phenol and 0.0022 mole acetic acid and immobilised lipase from Porcine pancreas of activity units 56.5 fimoles/min/mg of immobilised lipase the reaction mixture was agitated at 175 rpm at 63° C for 48.5 h. The product work out was by filtering the immobilised enzyme and removing the unreacted acid by washing with saturated sodium bicarbonate solution and evaporating the solvent to get a mixture of unreacted phenol and phenyl acetate and separation of the ester from unreacted phenol by distillation. Estimation by titration showed 43.3%of phenyl acetate formation in the reaction mixture. EXAMPLE - 7 To 10 ml of n-heptane are added 0.052 guaiacol 0.0546M acetic acid and immobilised lipase from Mucor meihei of activity units 15.4µmoles/min/mg of immobilised lipase and the reaction mixture was agitated at 175 rpm at 50° C for 120 h. The product work out was by filtering the immobilised enzyme and removing the unreacted acid by washing with saturated sodium bicarbonate solution and evaporating the solvent to get a mixture of unreacted guaiacol and guaiacyl acetate and separation of the ester from unreacted phenol by distillation. Estimation by titration showed 65.7%of guaiacyl acetate formation in the reaction mixture. EXAMPLE - 8 To 150 ml of chloroform are at. ,k; 0.02 mole p-cresol, 0.02 mole acetic acid, 0.1 mL of 0.1M phosphate buffer pH 7.0 and immobilised lipase from Porcine pancreas of activity units 113 µmoles/min/mg of immobilised lipase and the reaction mixture was refluxed using a Soxhelet apparatus fitted to a condenser containing the molecular sieve ( 50 g) as the desiccant for 54 h. The product work out was by filtering the immobilised enzyme and removing the unreacted acid by washing with saturated sodium bicarbonate solution evaporating the solvent to get a mixture of unreacted p-cresol and p-cresyl acetate and separation of the ester from unreacted phenol by distillation. Estimation by titration showed 31.9 %of p-cresyl acetate formation in the reaction mixture. EXAMPLE - 9 To 150 ml of chloroform are added 0.01 mole p-cresol, 0.01 mole launc acid. 0.15 mL of 0.1 M phosphate buffer pH 7.0 and immobilised lipase from Porcine pancreas of activity uniis 113µmoles/min/mg of immobilised lipase and the reaction mixture was refluxed using a Soxhelet apparatus fitted to a condenser containing the molecular sieve ( 50 g) as the desiccant for 50 h. The product work out was by filtering the immobilised enzyme and removing the unreacted acid by washing with saturated sodium bicarbonate solution and evaporating the solvent to get a mixture of unreacted p-cresol and p-cresyl laurate and separation of the ester from unreacted phenol by distillation. Estimation by titration showed 18.29 %of p-cresyl laurate formation in the reaction mixture. EXAMPLE-10 To 150 ml of chloroform are added 0.02 mole p-cresol, 0.02 mole propionic acid acid, 0.1 mL of 0.1M phosphate buffer pH 7.0 and immobilised lipase from Porcine pancreas of activity units 113 µmoles/min/mg of immobilised lipase and the reaction mixture was refluxed using a Soxhelet apparatus fitted to a condenser containing the molecular sieve ( 50 g) as the desiccant for 42 h. The product work out was by filtering the immobilised enzyme and removing the unreacted acid by washing with saturated sodium bicarbonate solution and evaporating the solvent to get a mixture of unreacted p-cresol and p-cresyl propionate and separation of the ester from unreacted phenol by distillation. Estimation by titration showed 20.7 %of p-cresyl propionate formation in the reaction mixture. EXAMPLE-11 To 0.0046 mole m-cresol are added 0.023 mole acetic, 0.1 mL of 0.1 M phosphate buffer pH 7.0 and immobilised lipase from Porcine pancreas of activity units 84.75 µmoles/min/mg of immobilised lipase and the reaction mixture was agitated at 175 rpm for 62h at 37 °C. The product work out was by filtering the immobilised enzyme and removing the unreacted acid by washing with saturated sodium bicarbonate solution and evaporating the solvent to get a mixture of unreacted m-cresol and m-cresyl acetate and separation of the ester from unreacted phenol by distillation. Estimation by titration showed 12.3 %of m-cresyl acetate formation in the reaction mixture. The vahous reaction conditions employed and the estehfication achieved in the above examples are given in a summahzed form in Table 1. Table -1 Preparation of esters of phenols and substituted phenols using immobilized lipases from Porcine Pancreas and Mucor miehei (Table Removed) The main advantages of the present invention are: 1. Milder reaction conditions. 2. Use of immobilised lipase facilitating the easy recovery of the catalyst. 3. Reuse of the enzyme. 4. Clean product without any side reaction. 5. Cost effective procedure. 6. An ecofriendly process which involves pollution free work out procedures without the use of toxic solvents. 7. Dehvatization to activate phenols and organic acids are not required. 8. Fewer unit operations. We claim: 1. An enzymatic process for the preparation of phenolic esters which comphses reacting organic acids having carbon chain length C2 to C20 with phenols or substituted phenols in presence of lipase enzyme 15 to 115 µ moies/min/mg at a temperature in the range 37°C to 60°C optionally with solvents or by maintaining water content less than 0.05 ml per 100 ml of reaction mixture by adding non polar solvent, recovehng and purifying the corresponding phenolic ester by conventional solvent extraction methods such as herein described. 2. An enzymatic process as claimed in claim 1 wherein the phenol or substituted phenol employed is m-cresol, p-cresol, phenol, guaiacol, catechol and resorcinol. 3. An enzymatic process as claimed in claims 1 and 2 wherein the organic and employed is of carbon chain length C2 to C20 like acetic , propionic , butyric, valeric, isovalehc, octanoic, decanoic, lauric, myristic, palmitic and steahc acids. 4. An enzymatic process as claimed in claims 1-3 wherein the lipase enzyme used is of Mucor meihei, Candida cylindrecea, Pseudomonos fluorescens, wheat germ. Porcine Pancreas and chicken liver esterase origin and optionally in the immobilized form. 5. An enzymatic process as claimed in claims 1-4 wherein the reaction is effected for a pehod of 24-100 h. 6. An enzymatic process as claimed in claims 1-5 wherein the phenols or the organic acids employed may be in the concentration range 0.02M-0.1M/ 0.002 mole to 0.05 mole depending on the presence or absence of the solvent. 7. An enzymatic process as claimed in claims 1-6 wherein the non - polar solvent employed is n-heptane, chloroform, n-hexane, petroleum ether (40-60°C fraction). 8. An enzymatic process for the preparation of phenolic esters substantially as herein described with reference to the examples.

Documents:

1243-del-1999-abstract.pdf

1243-del-1999-claims.pdf

1243-del-1999-complete specification(granted).pdf

1243-del-1999-correspondence-others.pdf

1243-del-1999-correspondence-po.pdf

1243-del-1999-description (complete).pdf

1243-del-1999-form-1.pdf

1243-del-1999-form-2.pdf

1243-del-1999-form-4.pdf

1243-del-1999-form-9.pdf