Enzymatic synthesis of fruit flavor esters by immobilized lipase from Rhizopus oligosporus optimized with response surface methodology

Short chain aliphatic esters have immense applications as flavors in fruit juices, cheeses, baked goods, candies, beverages and ice creams. Two such flavor esters (n-butyl acetate and n-propyl acetate) were synthesized enzymatically by transesterification of vinyl acetate with alcohols namely n-butanol and n-propanol, respectively, in solvent-free systems. These two synthesized esters occur naturally in various fruits like apple, strawberry, pear, etc. Lipase from Rhizopus oligosporus NRRL 5905 immobilized onto cross-linked silica gel was used for synthesis of the esters. The process parameters (reaction time, enzyme amount, additional water and shaking speed) were studied to achieve the highest yield of the esters. Maximum conversion of n-butyl acetate (50%) and n-propyl acetate (56%) were achieved after 24 h of reaction at 30 °C, at an enzyme concentration of 25% (w/v) of reaction mixture. This process was further optimized using response surface methodology (RSM) based on a three-level, four-variable central composite design (CCD). The optimum molar conversion of 54.6% using 27.5% enzyme concentration at 215 rpm and 26.5 °C for 28 h and 56.5% molar conversion using 29.8% enzyme concentration at 101 rpm and 28.2 °C for 28 h were achieved for n-butyl and n-propyl acetates, respectively. Immobilized lipase could be used for three cycles for synthesis of n-butyl acetate and n-propyl acetate, respectively, with almost 100% retention of molar conversion. The Km and Vmax values were determined to be 227 mM and 322 μmol/(g-h), respectively, for n-butyl acetate while for n-propyl acetate the respective values were 222 mM and 385 μmol/(g-h).