Study On Enzymatic Esterification For Ethyl Lactate Synthesis In Organic Media

Ethyl lactate has numerous attractive advantages including being 100% biodegradable, easy to recycle, non-corrosive, non-carcinogenic and non-ozone depleting. Ethyl lactate has replaced solvents including toluene, acetone and xylene, which have resulted in the workplace being made a great deal safer. Moreover, ethyl lactate is an important flavor ester in Distilled Spirits. Lipases have been employed for direct esterification and transesterification reactions in organic solvents. Lipase catalyzed biotransformations are gaining importance because of their substrate specificity, their milder reaction conditions and the relatively lower energy requirement.Lactic acid and ethanol are intersoluble completely and lactic acid is insoluble in most organic solvents. Moreover, lactic acid contains both hydroxyl and carboxylic acid functional groups, so that this substance can undergo self-esterification to form linear polyesters and lactones. The acidity constant of lactic acid(Kα ) is 83.2×10-5,which is greater than other fatty acid(For examples, the acidity constant of formic acid and acetic acid is 17.7×10-5 and 1.75× 10-5 , respectively ). On this basis, the goal of this work was to study the direct enzymatic esterification of lactic acid with ethanol. The thorough study was carried by mathematic method, and the dynamics model was constructed.Firstly, ethyl lactate was synthesized by lactic acid and ethanol, the best solvent is Tert-buty alcohol and the best lipase is Novozym 435. The optimization of the reaction was studied in detail, when the molar ratio(acid:alcohol) is 1:8 , the temperature is 60℃, the concentration of acid and lipase is 0.3mol/L and 55g/mol and the revolutions is 200 rpm, a yield of 77% was reached. Novozym 435 can be used continuously for six times and a yield of 60% can be reached.Secondly, Response surface methodology based on a three-variable central composite rotatable design was used to determine the effect of acid concentration (0.1-0.5mol/L), enzyme concentration (15⁵5g/mol), incubation period (24⁷2h) on the esterification reaction and enzymatic activity. Even with a low enzyme concentration of 15 g/mol, 60% yield was observed, which were in good agreement with the experimental yield.Thirdly, immobilization of lipase with diatomaceous earth was studied. Temperature, pH. ratio (enzyme to diatomaceous), time and revolutions were determined with cross-over method. The best result is T=30℃, pH=7.5, ratio=1:9,time=5h and revolutions= 200rpm.Finally, Kinetics of lipase-catalyzed esterification of lactic acid and ethanol have beeninvestigated. 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. The values of the apparent kinetic parameters were computed as: V_max = 14.5 μmol/(min*g); K_A = 0.0134 mol/L; K_B =0.0367 mol/L; K_iA= 0.0107 mol/L and K_iB = 0.275 mol/L. lactic acid, being a short-chain polar acid, causes enzyme inactivation more than ethanol.