Putative esterase genes were isolated from environmental DNA by using pre-amplified inverse PCR. The sequence analysis of the isolated genes showed 32-80% amino acid sequence identities to known esterases/lipases in public databases. The isolated genes were subsequently expressed in recombinant Escherichia coli. Insoluble proteins were noted in the expression of the majority of the isolated genes. The findings suggest that it is difficult to isolate these genes by using activity-based screening with construction of metagenome library. For the enzymes characterized, we examined substrate specificity, optimal temperature, optimal pH, and thermal stability. The substrate specificity of all the enzymes was high for p-nitrophenyl acetate, but almost undetectable for p-nitrophenyl decanoate. The results indicate that the obtained enzymes are defined as esterases. The enzymes were active in a broad range of temperature. The optimum activity was observed at 25-70 degrees C and at pH 8.0-9.0. Some enzymes have moderate thermostability and would be useful for industrial enzymes. This study illustrates that pre-amplified inverse PCR, which is one of the sequence-based approach, is potentially applicable to the isolation of diverse genes from environmental DNA. (C) 2010 Elsevier Inc. All rights reserved.