The polyester polyurethanase gene (pueA) from Pseudomonas chlororaphis encodes a lipase

A gene (pueA, polyurethane esterase A) encoding an extracellular polyuretha nase (PueA) was cloned from Pseudomonas chlororaphis into Escherichia coli. The enzyme secreted from E. coli showed esterase activity when assayed wit h p-nitrophenyl acetate. Subcloning of a 3.2-kb SalI-EcoRI fragment into a T7 RNA polymerase expression vector (pT7-6) produced a S-35-labeled protein of 65 kDa. Nucleotide sequencing of pueA showed an open reading frame enco ding a 65-kDa protein of 617 amino acid residues, with the serine hydrolase consensus sequence GXSXG. PueA was over-expressed using the pT7-6 vector t ransformed into E. coli BL21(DE3) and was purified in one step using Sephad ex G-75. (C) 2000 Federation of European Microbiological Societies. Publish ed by Elsevier Science B.V. All rights reserved.