A 1.020-bp esterase gene, estQ encoding for a protein of 339 amino acids, was cloned from Aspergillus fumigatus and expressed in E. coli. EstQ exhibited the optimal activity around 40 degrees C and pH 9.0. In order to obtain more thermostable esterases, three mutants (A134T, V160T, A134T-V160T) were constructed by site-directed mutagenesis and also characterized for further research. Compared to A134T and V160T displaying their optimum activity at 40 degrees C, A134T-V160T exhibited a 5 degrees C higher optimal temperature and a longer half-life more than 24 times than that of WT at 50 degrees C. All the mutants displayed favorable effects on thermostability and retained 53-76% activity after pre-incubation for 30 min at 45 degrees C, about 20-40% higher than that of the WT. With an increase in K-m of the three mutants, a decrease in catalytic efficiency in k(cat)/K-m was observed in mutant V160T and A134T-V160T against p-nitrophenyl butyrate. Homology models of WT and A134T-V160T were built to understand the structure-function relationship. The analysis results showed that the improved thermostability may be due to the favorable interaction and additional hydrogen bonds formed in the mutants by substitution of hydrophobic residues with hydrophilic residues. This study provide useful theoretical reference for enzyme evolution in vitro. (C) 2014 Elsevier Inc. All rights reserved.