Kinetic Study of Ethyl Hexanoate Synthesis Using Surface Coated Lipase from Candida Rugosa

Document Type : Research Article


1 Department of Chemical Engineering, MVGR College of Engineering, Vizianagaram, (Affiliated to JNTUK), INDIA

2 Department of Chemical Engineering, B V Raju Institute of Technology (Affiliated to JNTUH), Narsapur, Medak District, Telangana, INDIA


Kinetics of lipase-catalyzed esterification of hexanoic acid and ethyl alcohol using the solvent-free system, surface coated lipase from Candida rugosa, had been studied. The effect of various parameters such as reaction time, reaction temperature, reaction kinetics, water removal and feasibility of solvent-free system had been focused. Candida Rugosa lipase was more effective than other lipases when ethyl hexanoate was synthesized in n-hexane. The highest esterification yield after 72 h (93 %) was achieved at a pH of 5.2 and the esterification yield was reduced to 73% at pH 4.0. The values of the apparent kinetic parameters were computed as Vmax= 0.146 μmol/min/mg enzyme; KM, Acid = 0.296 M; KM, Alcohol = 0.1388 M; Ki, Acid = 0.40 M; and Ki, Alcohol = 0.309 M. The reaction rate could be described in terms of the Michaelis–Menten equation with a Ping-Pong Bi-Bi mechanism and competitive inhibition by both the substrates.


Main Subjects

[1] Berger R.G., Biotechnology of Flavours-the Next Generation, Biotechnol. Lett., 31: 1651–1659 (2009).
[2] Schrader J., Etschmann M.M.W., Sell D., Hilmer J.M., Rabenhorst J., Applied Biocatalysis for the Synthesis of Natural Flavour Compounds - Current Industrial Processes and Future Prospects, Biotechnol. Lett., 26: 463–472 (2004).
[3] Serra S., Fuganti C., Brenna E., Biocatalytic Preparation of Natural Flavours and Fragrances, Trends Biotechnol., 23:193–198 (2005).
[4] Armstrong D.W., Yamazaki H., Natural Flavours Production: A Biotechnological Approach, Trends Biotechnol., 4: 264–268 (1986).
[5] Gilles B., Yamazaki H., Armstrong D.W., Production of Flavour Esters: Immobilized Lipase, Biotechnol. Lett., 9: 709–714 (1987).
[6] Langrand G., Triantaphylides C., Baratti J., Lipase Catalyzed Formation of Flavor Esters, Biotechnol. Lett. 10: 549–554 (1988).
[7] Welsh F.W., Muray W.D., Williams R.E., Microbiological and Enzymatic Production of Flavour and Fragrance Chemicals, Critical Reviews in Biotechnology., 9: 105–169 (1989).
[8] Ahmed E.H., Raghavendra T., Madamwar D., A Thermostable Alkaline Lipase From A Local Isolate Bacillus Subtilis Eh 37: Characterization, Partial Purification, and Application in Organic Synthesis, Appl. Biochem. Biotechnol., 160(7): 2102-2113 (2010).
[9] Nogales J. M. R., Contreras E. R. E., Biosynthesis of Ethyl Butyrate Using Immobilized Lipase: A Statistical Approach, Process Biochem., 40(1): 63-68 (2005).
[10] Goto M., Kamiya N., Nakashio F., Enzymatic Interesterification of Triglyceride with Surfactant-Coated Lipase in Organic Media, Biotechnol. Bioeng., 45: 27–32 (1995).
[11] Goto M., Noda S., Kamiya N., Nakashio F., Enzymatic Resolution of Racemic Ibuprofen by Surfactant-Coated Lipases in Organic Media., Biotechnol Lett., 18: 839–844 (1996).
[12] Klibanov A.M., Dabulis K., Dramatic Enhancement of Enzymatic Activity in Organic Solvents by Lyoprotectants, Biotechnol. Bioeng., 41: 566-571 (1993).
[13] Wu J.C., He Z.M., Yao C.Y., Yu K.T., Increased Activity and Stability of Candida Rugosa Lipase in Reverse Micelles Formed by Chemically Modified Aot in Iso-Octane, J. Chem. Technol. Biotechnol., 76: 949-953 (2001).
[14] Tietz N.W., Fiereck E.A., A Specific Method for Serum Lipase Determination, Clinica Chimica Acta., 13: 352-358 (1996).
[15] Lavayre J., Verrier J., Baratti J., Stereospecific Hydrolysis by Soluble and Immobilized Lipases, Biotechnology and Bioengineering, 24: 2175 – 2187 (1982).
[16] Kamiya N., Goto M., Nakashio F., Surfactant Coated Lipase Suitable for the Enzymatic Resolution of Menthol as a Biocatalyst in Organic MediaBiotechnology Progress.,11: 270 –275 (1995).
[17] Basri M., Ampon K., Yunus W.M.Z.W., Razak C.N.A., Salleh A.B., Enzymic Synthesis of Fatty Esters by Hydrophobic Lipase Derivatives Immobilized on Organic Polymer Beads, Journal of the American Chemists Society., 72: 407-411(1995).
[18] Basri M., Ampon K., Yunus W.M.Z.W., Razak C.N.A., Salleh A.B., Synthesis of Fatty Esters by Poly Ethylene Glycol Modified LipaseJournal of Chemical Technology and Biotechnology. 64: 10-16 (1995).
[19] Lowry R.R., Tinsley I.J., Rapid Colorimetric Determination of Free Fatty Acids, J. Am. Oil Chem. Soc.,53: 470-474 (1976).
[20] Prapulla S.G., Divakar S., Karanth N.G., An Enzymatic Process for the Preparation of Low Molecular Weight Esters of Aromatic Alcohols, Indian Patent No. 3696/DEL/98 (1998).
[21] Suzana Ferreira-Dias., Georgina Sandoval., Francisco Plou., Francisco Valero., The Potential Use of Lipases in the Production of Fatty Acid Derivatives for the Food and Nutraceutical Industries, E. J. of Biotech., 16(3): 1-24 (2013).
[22] Kamiya N., Goto M., Nakashio F., Surfactant Coated Lipase Suitable for the Enzymatic Resolution of Menthol as a Biocatalyst in Organic Media, Biotechnology Progress., 11: 270-275(1995).
[23] Lokotch W., Fritsch K., Syldatk C., Resolution of D,L Menthol by Interesterification with Triacetin Using the Free and Immobilized Candia Cylindracea Lipase, Appl. Microbiol. Biotechnol., 31: 467-472 (1989).
[24] BasheerS., Mogi K., Nakajima M., Surfactant – Modified Lipase for the Catalysis of The Interesterification of Triglycerides and Fatty AcidsBiotechnology and Bioengineering., 45: 187 – 195 (1995).
[25] Huang S.Y., Chang H.L., Goto M., Preparation of Surfactant Lipase for the Esterification of Geraniol and Acetic Acid in Organic Solvents, Enzyme and Microbial Technology.,22: 552 – 557 (1998).
[26] Dordick J.S.,Enzyme Catalysis in Monophasic Organic Solvents, Enzyme and Microbiol Technology.,11: 194 – 211(1989).
[27] Laane C., Boeren S., Ves K., Veeger C., Rules for Optimization of Biocatalysis in Organic Solvents, Biotechnology and Bioengineering., 30: 81-87 (1987).
[28] Chulallaksnanukul W., Conderet J.S., Combes D., Kinetics of Geranyl Acetate Synthesis By Lipase-Catalyzed Transesterification, Enzyme Microbial Technology., 14: 293-298 (1992).
[29] Chulallaksnanukul W., Conderet J.S., Combes D., Geranyl Acetate Synthesis by Lipase Catalyzed Transesterification In Super Critical Carbondioxide, Enzyme and Microbial Technology., 15: 691-698 (1993).
[30] Gandhi N.N., Sawant S.B., Joshi J.B., Studies on the Lipozyme Catalyzed Synthesis of Butyl Laurate, Biotechnology and Bioengineering., 46: 1-12 (1995).
[31] Dordick J.S., Enzyme Catalysis in Monophasic Organic Solvents, Enzyme and Microbial Technology., 11: 194-211 (1989).
[32] Knez Z., Leitgeb M., Zavrsni D., Lavric B., Synthesis of Oleic Acid Esters with an Immobilized Lipase, Fat Science and Technology., 92: 169 – 172 (1990).
[33] Goderis H.L., Ampe G., Feyten M.P., Fouwé B.L., Guffens W.M., Van-Couwenbergh S.M., Tobback P.P., Lipase Catalyzed Ester Exchange Reactions in Organic Media with Controlled HumidityBiotechnology and Bioengineering., 30: 258-266 (1987).
[34] Ergan F., Trani M., Andre G., Production of Glycerides from Glycerol and Fatty Acid by Immobilized Lipase in Non-Aqueous Media, Biotechnology and Bioengineering., 35: 195-200 (1991).
[35] Gillies B., Yamazaki H., Armstrong D.W., Production of Flavour Esters by Immobilized Lipase, Biotechnology Letters., 9: 709-714(1987).
[36] Carta G., Gainer J.L., Gibson M.E., Synthesis of Esters Using a Nylon-Immobilized Lipase in Batch and Continuous ReactorsEnzyme and Microbiol Technology., 14: 904 – 910 (1992).
[37] Marty A., Chulalaksananukul W., Willemot R.M., Condoret J.S., Kinetics of Lipase-Catalyzed Esterification in Super Critical Co2Biotechnology and Bioengineering., 39: 273-280(1992).
[38] Rizzi M., Stylos P., Riek A., Reuss M., A Kinetic Study of Immobilized Lipase Catalyzing the Synthesis of Isoamyl Acetate by Transesterification in N-Hexane, Enzyme and Microbiol Technology., 14: 709-714(1992).
[39] Malcata F. X.., Reyes H.R., Garcia H.S., Hill C.G.,  Amundson C.,H.,  Kinetics and Mechanism of Reactions Catalyzed by Immobilized Lipases, Enzyme and Microbial Technology., 14: 426-446 (1992).
[40] Garcia T., Coteron A., Martinez M., Aracil J., Kinetic Model for the Esterification of Oleic Acid and Cetyl Alcohol Using Immobilized Lipase as Catalyst, Chemical Engineering Science., 55: 1411-1423 (2000).
[41] Martinelle M., Hult K., Kinetics of Acyl Transfer Reaction in Organic Media Catalyzed by Candida Antarctica Lipase BBiochimica et Biophysica Acta., 1251: 191-197 (1995).
[42] Segel I.H., “Enzyme Kinetics”, John Wiley & Sons Inc., New York (1975).
[43] Chulallaksnanukul W., Conderet J.,S., Delorme P., William R.,E.,  Kinetic Study of Esterification by Immobilized in Hexane, F.E.B.S. lett., 276: 181-184 (1990).
[44] Su G.D., Huang D.F.,  Han S.Y.,  Zheng S.P., Lin Y.,  Display of Candida Antarcticalipase B on Pichia Pastoris and its Application to Flavor Ester Synthesis, App. Microbiology and Biotech., 86(5): 1493-1501 (2010).
[45] Jin Z.,  Ntwali J., Han  S.Y., Zheng  S.P., Lin Y.,  Production of Flavor Esters Catalyzed by CALB-Displaying Pichia pastoris Whole-Cells in a Batch Reactor, J. of Biotech.,, 159(1-2): 108-114 (2012).
[46] Duan G., Ching C.B., Lim E., Ang C.H.,  Kinetic Study of Enantioselective Esterification of Letoprofen with n-propanol Catalyzed by an Lipase in an Organic Medium, Biotechnology Letters., 19: 1051-1055 (1997).