Title of Invention

AN ENZYMATIC PROCESS FOR THE PREPARATION FATTY ACID HYDROXY ACID ESTER

Abstract

An enzymatic process for the preparation of fatty acid hydroxy acid ester such as palmitoyl lactic icid which comprises reacting respective long chain falty acid and hydroxy acid in equimolar amounts in presence of lipase in the range of 10-166 gLmole"1 and solvents such as ethylmethyl ketone, methylisobutyl ketone under agitation an recovering the corresponding eslcr by conventional methods such as herein described. Ref: USP: 3,141,030; 3,636,01 7; DE: 27;;.4059 CN: 85, 10898

Full Text

This invention relates to an enzymatic process for the preparation of fatty acid hydroxy acid ester. This invention particularly relates to an enzymatic process for the preparation of fatty acid hydroxy acid esters from fatty acids like lauric, myristic, palmitic and stearic acid and hydroxy acids like lactic, citric, and malic acid using immobilised lipaseffrom Mucor meihei and Porcine Pancreas. Fatty acid esters of hydroxy acid such as palmitoyl lactic acid are an interesting group of ionic, nontoxic, biocompatible surfactants with a wide range of applications in pharmaceuticals, cosmetics, petroleum and food industries ( Maag H., 1984. J. Am. Oil. Chem. Soc. 61: 259-267). Few procedures are available for the preparation of palmitoyl lactic acid by chemical methods. The following table lists these methods cited in the literature. (Table Removed) The major drawbacks of the chemical methods for the preparation of palmitoyl lactic acid are : 1. In all the procedures cited, products obtained are essentially mixtures of fatty acid esters of lactic acid like palmitoyl lactic acid and stearoyl lactic acid with residual unreacted fatty acids and lactic acid. 2. The conditions employed for the chemical esterification are drastic like high temperature and pressures. 3. Use of toxic solvents like pyridine and catalysts like H2SO4 and p-toluene sulphonic acid. 4. Yields are relatively low. 5. More unit operations resulting in cost ineffective procedures. 6. Protection of carboxyl group of lactic acid and activation of palmitic acid and final deprotection of lactic acid are required. 7. Removal of water from lactic acid is required. The present invention involves an enzymatic procedure for the preparation of fatty acid hydroxy acid ester which has many advantages over chemical synthesis. They are performed under milder reaction conditions of temperature and pH. No enzymatic method has been cited in the literature till date for the preparation ester such as palmitoyl lactic acid. The present invention provides an enzymatic process where the yields of the ester are enhanced when fatty acid is reacted with hydroxy acid in a nonpolar solvent in the presence of Upases from Mucor meihei and Porcine Pancreas. New principles underlying and advantages of the invention are : Esterification was achieved in a nonpolar solvent under low water environment and milder reaction conditions. The reaction although slow, yielded a clean product without any side reaction. Use of enzyme eliminated the need for the protection of carboxyl groups of hydroxy acid and its subsequent removal after esterification thereby reducing the cost of operation and tedious workout procedures. Activation of fatty acid is also not required. Recovery and reuse of the enzyme will give a cost effective process. The method does not involve removal of water from hydroxy acid. It does not involve employment of toxic solvents. The yields are almost comparable to that of chemical methods. Accordingly, the present invention provides an enzymatic process for the preparation of fatty acid hydroxy acid ester such as palmitoyl lactic acid which comprises reacting respect.ve long chain fatty acid and hydroxy acid in equimolar amounts in presence of lipase in the range of 10-166 gLmole"1 and solvents such as ethylmethyl ketone, methylisobutyl ketone under agitation and recovering the corresponding ester by conventional methods such as herein described. In an embodiment of the present invention, the hydroxy acid employed may be lactic acid, citric acid and malic acid. another embodiment of the present invention, the long chain fatty acid employed may be lauric acid, myristic acid, palmitic acid and stearic acid preferably palmitic acid. 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 24-72 h. In yet another embodiment of the present invention, the agitation may be effected at 100-250 rpm preferably at 150 rpm. In yet another embodiment of the present invention, the lipase employed may be in the range 10-160 gLmole"1 preferably 80 gLmole"1. Commercially available palmitic acid (99% pure from Sd. Fine Chemicals India Ltd.) and lactic acid (88% pure from Ranbaxy Chemicals) were used. All the solvents employed were obtained from SISCO Chemicals (I) Ltd. and were distilled once before use. Immobilised lipase preparation, Lipozyme IM-20 (Mucor meihei) was procured from M/s Novo Nordisk and Porcine Pancreas from Sigma Chemicals Co. (St. Louis Mo). The specific hydrolytic activity (Tris buffer, pH 7.0 and tributyrin substrate) units of the two lipases are 12.44 and 3.72 jimoles/min/mg protein respectively. The reactions were monitored by pippeting out at regular intervals aliquots (0.5 ml) of the reaction mixture into 10 ml of 0.01N NaOH and titrating against oxalic 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 palmitic acid 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. Since both lactic acid and palmitic acid titrated with NaOH, the theoretical litre value corresponding to the amount of palmitic acid employed was used as the initial value for the determination of percentage of esterification. The isolated product was characterized by recording a 'H NMR spectrum of the compound on the Bruker-DXP 500 NMR instrument operating at 20° C. The i sample was dissolved in CDCls-DMSO-de mixture and the signals were referenced to TMS. The chemical shift values are :- -CHa (palmitic acid) -0.85 ppm; -CH2 (palmitic acid) 1.23 ppm; -CHa (lactic acid) -1.42 ppm; -CHaCO (palmitic acid ) -2.1 ppm ;-CH2- (palmitic acid) -2.42 ppm; -CH- (lactic acid) -4.05 ppm; -CH-(lactylic acid) -4.15 ppm; -CH- (palmitoyl lactic acid ) -4.90 ppm; -CH (palmitoyl lactylic acid) -5.05 ppm. The *H NMR data confirmed the formation of the compound. The procedure employed for the reaction is as follows. A mixture of palmitic acid (0.02 M to 0.15 M) and lactic acid (0.02M to 0.15 M) was taken in a stoppered flask containing 5-20 ml of ethylmethyl ketone. To this enzyme was added at an enzyme/substrate ratio of 40-166 g L mole"1 in case of Mucor meihei and 1.7-75 % Lmole 'l in case of Porcine Pancreas and agitated at 30-50° C at 100-250 rpm for 12-100 h on a rotary shaker. The product work out was filtering the immobilised enzyme and evaporating the solvent to get a mixture of palmitoyl lactic acid, palmitic acid, lactic acid and some amount of palmitoyl lactylic acid. The process of the invention is described in detail in the example^given below which are illustrative only and should not be construed to limit the scope of the invention. EXAMPLE 1 To 10 ml of ethylmethyl ketone are added 0.06 M lactic acid, 0.06 M palmitic acid and immobilised lipase from Mucor meihei at an enzyme/substrate ratio of 41.67 g L mole'1 and the reaction mixture was agitated at 150 rpm at 37° C for 72 h. The product work out was filtering the immobilised enzyme and evaporating the solvent to get a mixture of palmitoyl lactic acid, palmitic acid, lactic acid and some amount of palmitoyl lactylic acid. Estimation by titration showed 24.48% of palmitoyl lactic acid formation (Table 1). EXAMPLE 2 To 10 ml of ethylmethyl ketone are added 0.06 M lactic acid, 0.06 M palmitic acid and immobilised lipase from Mucor meihei at an enzyme/substrate ratio of 166.7 g L mole"1 and the reaction mixture was agitated at 150 rpm at 37° C for 72 h. The product work out was filtering the immobilised enzyme and evaporating the solvent to get a mixture of palmitoyl lactic acid, palmitic acid, lactic acid and some amount of palmitoyl lactylic acid. Estimation by titration showed 50% of palmitoyl lactic acid formation (Table 1). EXAMPLE 3 To 10 ml of ethylmethyl ketone are added 0.09 M lactic acid, 0.09 M palmitic s acid and immobilised lipase from Mucor meihei at an enzyme/substrate ratio of 166.7 g L mole"1 and the reaction mixture was agitated at 150 rpm at 37° C for 72 h. The product work out was filtering the immobilised enzyme and evaporating the solvent to get a mixture of palmitoyl lactic acid, palmitic acid, lactic acid and palmitoyl lactylic acid. Estimation by titration showed 28.39% of palmitoyl lactic acid formation (Table 1). Table 1 Preparation of palmitoyl lactic acid using immobilized lipase from Mucor meihei (Table Removed) Unless otherwise stated substrate concentration refer to 0.06 M. a - 0.03 M b - 0.09 M c - Initial rate refer to slope of the initial part of the plot of the percentage esterification with time. EXAMPLE 4 To 10 ml of ethylmethyl ketone are added 0.06 M lactic acid, 0.06 M palmitic acid and immobilised lipase from Porcine Pancreas at an enzyme/substrate ratio of 41.75 g L mole"1 and the reaction mixture was agitated at 150 rpm at 37° C for 72 h. The product work out was filtering the immobilised enzyme and evaporating the solvent to get a mixture of palmitoyl lactic acid, palmitic acid, lactic acid and some amount of palmitoyl lactylic acid. Estimation by titration showed 51.85% of palmitoyl lactic acid formation (Table 2). EXAMPLE 5 To 10 ml of ethylmethyl ketone are added 0.06 M lactic acid, 0.06 M palmitic acid and immobilised lipase from Porcine Pancreas at an enzyme/substrate ratio of 16.7 g L mole"1 and the reaction mixture was agitated at 150 rpm at 37° C for 72 h. The product work out was filtering the immobilised enzyme and evaporating the solvent to get a mixture of palmitoyl lactic acid, palmitic acid, lactic acid and palmitoyl lactylic acid. Estimation by titration showed 49.18 % of palmitoyl lactic acid formation (Table 2). EXAMPLE 6 To 10 ml of ethylmethyl ketone are added 0.06 M lactic acid, 0.06 M palmitic acid and immobilised lipase from Porcine Pancreas at an enzyme/substrate ratio of 51.10 g L mole"1 and the reaction mixture was agitated at 150 rpm at 37° C for 72 h. The product work out was by filtering the immobilised enzyme, and evaporating the solvent to get a mixture of palmitoyl lactic acid, palmitic acid, lactic acid and palmitoyl lactylic acid. Estimation by titration showed 33.33% of palmitoyl lactic acid formation (Table 2). EXAMPLE 7 To 10 ml of ethylmethyl ketone are added 0.03 M lactic acid, 0.03 M stearic acid and immobilised lipase immobilised lipase from Mucor meihei at an enzyme/substrate ratio of 125.0 g L mole"1 and the reaction mixture was agitated at 150 rpm at 37° C for 72 h. The product work out was by filtering the immobilised enzyme, and evaporating the solvent to get a mixture of stearoyl lactic acid, stearic acid, lactic acid and stearoyl lactylic acid. Estimation by titration showed 40.0% of stearoyl lactic acid formation. EXAMPLE 8 To 10 ml of ethylmethyl ketone are added 0.06 M lactic acid, 0.06 M Stearic acid and immobilised lipase from Porcine Pancreas at an enzyme/substrate ratio of 40.0 g L mole"1 and the reaction mixture was agitated at 150 rpm at 37° C for 72 h. The product work out was by filtering the immobilised enzyme, and evaporating the solvent to get a mixture of stearoyl lactic acid, stearic acid, lactic acid and stearoyl lactylic acid. Estimation by titration showed 30.0% of stearoyl lactic acid formation. Table 2 Preparation of palmitoyl lactic acid using immobilized lipase from Porcine Pancreas (Table Removed) Unless otherwise stated substrate concentration refer to 0.06 M. a - 0.03 M b - 0.09 M c - Initial rate refer to slope of the initial part of the plot of the percentage esterification with time. 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. Reasonable cost effective procedure. 6. Does not involve the removal of water from lactic acid. 7. An ecofriendly process which involves pollution free work out procedures without the use of toxic solvents. 8. Reasonable yield of about 50%. 9. Deri vat ization to protect lactic acid, to activate palmitic acid and deactivation of lactic acid are not required. 10. Fewer unit operations. We claim : 1. An enzymatic process for the preparation of fatty acid hydroxy acid ester such as palmitoyl lactic acid which comprises reacting respective long chain fatty acid and hydroxy acid in equimolar amounts in presence of lipase in the range of 10-166 gLmole"1 and solvents such as ethylmethyl ketone, methylisobutyl ketone under agitation and recovering the corresponding ester by conventional methods such as herein described. 2. An enzymatic process as claimed in claim 1 wherein the hydroxy acid employed is such as lactic acid, citric acid, malic acid. 3. An enzymatic process as claimed in claims 1 to 2 wherein the long chain fatty acid employed is such as lauric acid, myristic acid, palmitic acid, stearic acid, preferably palmitic acid. 4. An enzymatic process as claimed in claims 1 to 3 wherein the lipase employed is such as immobilized Upases from Mucor meithei, Candida cylindreacea, Pseudomonos flourescens, wheat germ, Porcine Pancreas and Chicken liver esterase. 5. An enzymatic process as claimed in claims 1 to 4 wherein the agitation is effected at 100-250 rpm preferably at 150 rpm. 6. An enzymatic process as claimed in claims 1 to 5 wherein the lipase employed is in the range 10-160 gLmole"1 preferably 80 gLmole"1. 7. An enzymatic process as claimed in claims 1 to 6 wherein the reaction is effected for a period of 24-72 h. 8. An enzymatic process for the preparation of fatty acid hydroxy acid ester substantially as herein described with reference to the examples.

Documents:

1978-del-1998-abstract.pdf

1978-del-1998-claims.pdf

1978-del-1998-complete specificaation (granted).pdf

1978-del-1998-correspondence-others.pdf

1978-del-1998-correspondence-po.pdf

1978-del-1998-description (complete).pdf

1978-del-1998-form-1.pdf

1978-del-1998-form-2.pdf

1978-del-1998-form-4.pdf