Based on previous bioinformational analytical results [Shu ZY, et al. Biotechnol Prog 2009;25:409-16], four A. niger lipase (ANL) mutants, ANL-Ser84Gly, ANL-Asp99Pro, ANL-Lys108Glu and ANL-E alpha H (obtained by replacing the lid domain of ANL with the corresponding domain from A. niger feruloyl esterase), were constructed to screen out ANL mutants with oil-water interface independence. ANL-S84G displayed a pronounced interfacial activation, while ANL-D99P and ANL-K108E displayed no interfacial activation. The specific activity of ANL-S84G towards p-nitrophenyl esters decreased from 29.8% to 76.5% compared with that of ANL, while the specific activity of ANL-D99P towards p-nitrophenyl palmitate increased 2.2-fold. The thermostability of ANL-K108E was almost unchanged, while the thermostability of ANL-S84G and ANL-D99P significantly decreased compared with that of ANL. The construction of oil-water interface-independent ANL mutants would help to further understand the mechanism of lipase interfacial activation. (C) 2010 Elsevier Inc. All rights reserved.