Title of Invention

"AN IMPROVED PROCESS FOR THE PREPARATION OF AMYL ESTERS FROM AMYL ALCOHOL"

Abstract

An improved process for the preparation of amyl esters from amyl alcohol characterized in using amyl alcohol and carboxylic acid in the ratio ranging 1:1 to 8:1 , said process comprises reacting amyl alcohol with carboxylic acids of carbon chain length C2 - C5 as described herein, at a n alcohol to acid ratio as defined above, in presence of a hydrolyzing enzyme selected from lipase and esterase wherein the range of enzyme substrate ratio employed was 5-50g L/mol, at a neutral pH in a non polar solvent, and recovering the product by conventional methods as described herein.

Full Text

This invention relates to an improved process for the preparation of amyl esters from amyl alcohol. This invention particularly relates to an improved process for the preparation of amyl esters with carboxylic acids of carbon chain length C2 - C5, particularly amyl acetate, amyl propionate, amyl butyrate, amyl isobutyrate and amyl valerate. Isoamyl acetate has a strong banana flavour and hence is desired very much in food industry. Annual demand in USA alone amounts to about 74,000 Kg (Welsh FW, Murray WD & Williams RE, CRC Crit. Rev. Biotechnol, 9: 105-169, 1989). Of the different flavour esters, this compound is one among the largely produced and used. The chemical synthesis of amyl esters involves the esterification of amyl alcohol with carboxylic acids in the presence of sulphuric acid as a catalyst. Chemical synthesis has drawbacks like strenuous reaction conditions, tedious workout procedures, increased pollution, and cost-intensive procedures. Traditional natural sources (i.e., plant materials) suffer from the diminishing supplies of raw materials, expense of isolation and variability in the amount and quality of the final product from different geographical sources. These concerns have resulted in price premium for natural source chemicals and a search for alternative supplies of desired compounds. Chemical synthesis lacks stereoselectivity and in some cases the yields are low, leading to the formation of byproducts, which makes product recovery difficult. One method of alleviating this problem has been the development of biotechnological processes for the production of specific flavour and fragrance chemicals. There are two biotechnological approaches for the synthesis of flavour compounds: Microbiological and Enzymatic methods. Enzymatic syntheses are typically performed at more moderate temperature, pressure, and pH than similar chemical syntheses. Enzymatic procedures are desirable due to milder conditions, environmental friendly, with easy workout procedures resulting into less pollution. Use of non-aqueous solvents for the ester synthesis by enzyme catalysis offers several advantages: 1. Increased solubility of non-polar substrates and products 2. Shifting thermodynamic equilibria to ester synthesis over hydrolysis 3. Suppression of water dependent side-reactions 4. Alteration in substrate- and enantio-selectivity 5. Immobilization is often unnecessary because enzymes are insoluble in organic solvents 6. Enzymes can be recovered by simple filtration 7. Ease of product recovery from low boiling, high vapour pressure solvents 8. Enhanced thermostability of the enzymes 9. Elimination of microbial contamination 10. Potential for enzymes to be used directly within a new or existing chemical process Few procedures are available for the preparation of isoamyl acetate using enzymes (Table 1). However, these procedures have the following drawbacks: l.High enzyme concentrations were used, which will not be cost-effective, and 2.Various enzyme concentrations and molar ratios of alcohol and acids (isoamyl alcohol and acetic acid) were employed with yields not more than 80%. Table 1 (Table Removed) Commercial isoamyl alcohol is a mixture of 3 primary isomers namely n-amyl alcohol, 2-methyl-l-butanol, and 3-methyl-l-butanol (isoamyl alcohol). Amyl alcohol obtained from M/s. Sarabhai M. Chemicals exhibited the following proportions of the primary alcohols as determined by Gas chromatography: n-Amyl alcohol = 7.17% 2-methyl-l-butanol = 0.14% 3-methyl-l-butanol = 92.69 % The sample of isopentyl alcohol obtained from Fluka showed the following composition: n-Amyl alcohol = 0.61 % 2-methyl-l-butanol = 5.22 % 3-methyl-l-butanol = 94.06% Optically pure S (-) 2-methyl-l-butanol was obtained from M/s. Sigma Chemicals. Acetic acid, heptane, and other solvents employed were obtained from SISCO Chemicals (I) Ltd. All the solvents were used after distilling once. Lipozyme IM-20 (which is a Mucor miehei lipase immobilized on Duolite weak ion exchange resin) procured from M/s. Novo Nordisk was employed. It was found to possess a specific hydrolytic activity of 20 p. moles / min / mg protein at 37°C (Tris buffer pH 7.0, Tributyrin as substrate). The esterification reaction was monitored by titrating aliquots of the reaction mixture against 0.01 N NaOH using phenolphthalein as an indicator at regular intervals of time. The analysis of reaction mixture was also carried out by subjecting the samples to gas chromatographic analysis (Fig. 1) using Shimadzu gas chromatograph (GC 15-A) equipped with a Carbowax 20M column (10 ft length, 0.125 inch dia) and a flame ionization detector. Nitrogen was used as carrier gas with a flow rate of 30 ml/min. Column oven, injection port and detector temperatures were maintained at 100°C (iso), 200°C and 250°C, respectively. The present methodology for the preparation of isoamyl acetate involves mild reaction conditions and easy workout procedures resulting in a desirable method of preparation of this compound with high yields. The novelty of this procedure, besides the employment of enzymes in non-polar solvents, is the difference in esterification of the three isomeric alcohols. Although the GC analysis showed the proportion of alcohols, as being 92.7%, 0.14% and 7.2% (isoamyl alcohol, 2-methyl-l-butanol and n-amyl alcohol, respectively), the proportion of esters formed as determined by H1 NMR analysis showed the proportion of isoamyl acetate, 2-methyl-l-butyl acetate and n-amy! acetate to be 67.23%, 3.83% and 28.94%, respectively (Fig. 2). Accordingly the present invention provides an improved process for the preparation of amyl esters from amyl alcohol characterized in using amyl alcohol and carboxylic acid in the ratio ranging 1:1 to 8:1 , said process comprises reacting amyl alcohol with carboxylic acids of carbon chain length C 2 - C 5 as described herein . at an alcohol to acid ratio as defined above , in presence of a hydrolyzing enzyme selected from lipase and esterase wherein the range of enzyme to substrate ratio employed was 5-50 g L/mol , at a neutral pH in a non polar solvent , and recovering the product by conventional methods as described herein. In an embodiment of the present invention the alcohols used are compounds such as n-amyl alcohol, 2-methyl-l-butanol and 3-methyl-1-butanol (isoamyl alcohol). In an another embodiment of the invention the enzyme used is a hydrolyzing enzyme such as lipase from sources such as Mucor miehei, Candida cylindracea, Pseudomonas fluorescence, Rice bran lipase, Wheat germ lipase, Chicken liver esterase and Porcine pancreatic lipase. In still another embodiment of the present invention wherein the molar ratio of alcohol to acid employed was in the range of ?fc: 1 to 1:1 In yet another embodiment of the invention the acid employed is a carboxylic acid with carbon chain length C2 - C5 such as acetic acid, propionic acid, butyric acid, valeric acid, isovaleric acid and their isomers and their anhydride derivatives and their mixtures thereof. In still another embodiment of the invention the non-polar solvents employed are such as heptane, hexane, isooctane, cyclohexane, dioxan, dichloromethane, chloroform, petroleum spirit (40-60°C, 60-80°C and 80-100°C) diisopropyl ether and amyl alcohol. In a typical reaction, amyl alcohol (0.02 -1.0 M) was treated with acetic acid (0.02 -1.0M) in 5 - 50 ml n-heptane solvent along with 5 mg - 2 g of lipase. The reaction mixture was agitated at 50 - 300 rpm and at 25 - 60°C for 2-120 hr. The reaction mixture was decanted and the solvent evaporated to get the compound. The process of 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 in a 50 ml stoppered conical flask are added 0.06 M amyl alcohol, 0.06 M acetic acid, and 35 mg of immobilized lipase from Mucor miehei and the reaction mixture was agitated at 150 rpm and at 37°C for 72 hr. After the reaction, the reaction mixture was decanted to separate the enzyme and the solvent was shaken with aqueous saturated bicarbonate to remove the residual acid and the solvent was evaporated to get the compound. Estimation by titration of acetic acid and GC analysis of the reaction mixture showed 96% and 95.82% of isoamyl acetate formation, respectively (Table 2). Example 2 To 10 ml of buffer (0.1 M phosphate buffer, pH 7.0) saturated heptane in a 50 ml stoppered conical flask are added 0.125 M amyl alcohol, 0.125 M acetic acid, and 35 mg of immobilized lipase from Mucor miehei and the reaction mixture was agitated at 150 rpm and at 37°C for 72 hr. After the reaction, the reaction mixture was decanted to separate the enzyme and the solvent was shaken with aqueous saturated bicarbonate to remove the residual acid and the solvent was evaporated to get the compound. Estimation by titration of acetic acid showed 59% of isoamyl acetate formation (Table 2). Table 2 Effect of Substrate concentration * (Table Removed) Enzyme used : 35 mg; Solvent used : 10 ml n-heptane or B.S. (0.1 M phosphate Buffer, pH 7.0 saturated) heptane. Activity of the enzyme : 0.55 p, moles / min / mg immobilized enzyme. Example 3 To 10 ml of n-heptane in a 50 ml stoppered conical flask are added 0.12 M amyl alcohol, 0.06 M acetic acid, and 35 mg of immobilized lipase from Mucor miehei and the reaction mixture was agitated at 150 rpm and at 37°C for 72 hr. After the reaction, the reaction mixture was decanted to separate the enzyme and the solvent was shaken with aqueous saturated bicarbonate to remove the residual acid and the solvent was evaporated to get the compound. Estimation by titration of acetic acid and GC analysis of the reaction mixture showed 97% and 92.19% of isoamyl acetate formation, respeetively (Table 3). Table 3 Effect of Substrate molar ratio * (Table Removed) * Acid used : 0.06 M; Enzyme used : 35 mg; Activity of the enzyme = 0.55 \x moles/min/mg of immobilized enzyme. Example 4 To 10 ml of n-hexane in a 50 ml stoppered conical flask are added 0.06 M amyl alcohol, 0.06 M acetic acid, and 35 mg of immobilized lipase from Mucor miehei and the reaction mixture was agitated at 150 rpm and at 37°C for 72 hr. After the reaction, the reaction mixture was decanted to separate the enzyme and the solvent was shaken with aqueous saturated bicarbonate to remove the residual acid and the solvent was evaporated to get the compound. Estimation by titration of acetic acid showed 95% of isoamyl acetate formation (Table 4). Table 4 Effect of Solvents * (Table Removed) * Alcohol used : 0.06 M; Acid used : 0.06 M, Enzyme used : 35 mg. Activity of the enzyme = 0.55 ja moles/min/mg of immobilized enzyme. Example 5 To 10 ml of n-hexane in a 50 ml stoppered conical flask are added 0.25 M amyl alcohol, 0.25 M butyiic acid, and 50 mg of immobilized lipase from Mucor miehei and the reaction mixture was agitated at 150 rpm and at 37°C for 72 hr. After the reaction, the reaction mixture was decanted to separate the enzyme and the solvent was shaken with aqueous saturated bicarbonate to remove the residual acid and the solvent was evaporated to get the compound. Estimation by titration of butyric acid showed 86% of isoamyl butyrate formation (Table 5). Table 5 Synthesis of other Amyl esters (Table Removed) Enzyme used : 50 mg; Yields were after 24 hr. The main advantages of the present invention are: 1. Ester yields more than 90 % and upto 97 % are achieved. 2. Ease of enzyme recovery and product separation 3. Elimination of microbial contamination 4. No by-product formation 5. Reactions are performed at mild conditions 6. Ecofriendly process. We Claim : 1. An improved process for the preparation of amyl esters from amyl alcohol characterized in using amyl alcohol and carboxylic acid in the ratio ranging 1:1 to 8:1 , said process comprises reacting, amyl alcohol with carboxylic acids of carbon chain length C 1 - C 5 as described herein, at an alcohol to acid ratio as defined above , in presence of a hydrolyzing enzyme selected from lipase and esterase wherein the range of enzyme to substrate ratio employed was 5-50 g L/mol , at a neutral pH in a non polar solvent, and recovering the product by conventional methods as described herein. 2. An improved process as claimed in claim 1 wherein the alcohol used are selected from n-amyi alcohol , 2-methyl-l-butanol and 3-methyl-l-butanol (isoamyl alcohol). 3. An improved process as claimed in claim 1&2 wherein the carboxylic acid employed is selected from acetic acid , propionic acid , butyric acid , valeric acid, isovaleric acid and their isomers, anhydride derivatives and mixtures thereof. 4. An improved process as claimed in claim 1-3 wherein the hydrolyzing enzymes used obtained from Mucor miehei , Candida cylindracea , Pseudomonas flurescence , rice bran lipase , wheat germ lipase , Chicken liver esterase and porcine pancreatic lipase. 5. An improved process as claimed in claim 1-4 wherein the non-polar solvents employed are selected from heptane, hexane, isooctane, cyclohexane , dioxan, dicloromethane , chloroform , petroleum spirit ( 40 -60 ° C , 60-80 ° C and 80 -100 ° C ) diisopropyl ether and amyl alcohol. 6. An improved process for the preparation of amyl esters from amyl alcohol substantially as herein described with reference to examples .

Documents:

2152-del-1998-abstract.pdf

2152-del-1998-claims.pdf

2152-del-1998-complete-spacification(granted).pdf

2152-del-1998-correspondence-others.pdf

2152-del-1998-correspondence-po.pdf

2152-del-1998-description (complete).pdf

2152-del-1998-form-1.pdf

2152-del-1998-form-2.pdf

2152-del-1998-form-3.pdf

2152-del-1998-form-4.pdf