Cloning, nucleotide sequencing and characterization of a polyurethanase gene (pueB) from Pseudomonas chlororaphis

A second gene (pueB, polyurethane esterase B) encoding an extracellular pol yurethanase (PueB) was cloned from Pseudomonas chlororaphis into Escherichi a coli. The recombinant polyurethanase showed esterase activity when assaye d with various p-nitrophenyl substrates and lipase activity when assayed wi th triolein. Nucleotide sequencing of pueB showed an open reading frame of 1695 bp encoding a 60-kDa protein of 565 amino acid residues, with the seri ne hydrolase consensus sequence GXSXG and a C-terminal secretion signal (G- G-X-G-X-D-X-X-X). Unlike the PueA polyurethanase, PueB contains a putative N-terminal signal peptide. Comparison between the amino acid and nucleotide sequences of these two genes revealed that they share 42% and 59% identity respectfully. Parsimony analysis of the predicted amino acid sequences for the PueB. PueA, and other polyurethanase enzymes and similar lipase enzyme s was performed. Interestingly the polyurethanase enzymes do not form a sin gle cluster, but appear to be distributed among multiple lineages. These an alyses suggest that the polyurethanase enzymes thus far studied have evolve d from lipases, and are not derived from a single source. (C) 2001 Elsevier Science Ltd. All rights reserved.