To achieve improved Lipase activity for the hydrolysis of various monoesters, a large number of mutant lipases from Acinetobacter species were constructed and analyzed in a high throughput manner by direct evolution. Enzyme libraries were created in Escherichia coli, transfected into Bacillus subtilis 168 by an electroporation method and expressed. The transfected library was screened using a Rhodamine B plate containing olive-oil and a phenol-red pH indicator plate containing glyceryl tricaprylate as substrates. One lipase variant, M58, which has five amino acid (S21F, 102G, S103F, D299E and N300H) mutations, was selected and characterized. The acyl chain-length selectivity of the in vitro expressed lipases against various p-nitrophenyl (p-NP) monoesters were compared by their relative hydrolysis rates. The result showed that lipase mutant M58 increased its selectivity for the short-C2 and C4 and the long-chain esters, C10, C12, C14 and C16. but decreased its selectivity for the middle-chain esters, C6 and C8. Wild-type and M58 lipases had been expressed in the presence and absence of its lipase-specific foldase (lif) in B. subtilis 168 and relative activity was measured. As a result, the activities of the wild-type and M58 lipases in the presence of lif increased by 1.8-fold, with enhanced stability. (C) 2004 Elsevier Inc. All rights reserved.