In this paper is described the optimization of the esterification reaction of butyl acetate synthesis catalyzed by Candida antarctica lipase B (Novozym 435). The reaction parameters temperature, substrate molar ratio, enzyme content, and added water, and their responses measured as conversion yields, were evaluated using central composite design and response surface methodology. The best acid concentration for the reaction without enzyme inactivation was determined to be 0.3 M. The optimal conditions for butyl acetate synthesis were found to be temperature of 40 degrees C; substrate molar ratio of 3:1 butanol:acetic acid; enzyme content of 7.5% of substrate wt.; added water 0.25% of substrate wt. Under these conditions, over 90% of conversion was obtained in 2.5 h. Enzyme reuse was tested performing three different treatments before each batch: washing the enzyme system with either n-hexane or water, or suspending the immobilized enzyme in water for 24 h. Direct enzyme reuse or washing with water produced a rapid decrease on enzyme activity, while washing with n-hexane allowed enzyme to be reused for 6 reactions cycles keeping around 70% of its activity. This fast and high yield of conversion represents a large improvement to previously reported results. (C) 2011 Elsevier Ltd. All rights reserved.