estA, a gene coding for a cell-bound esterase from Paenibacillus sp BP-23,is a new member of the bacterial subclass of type B carboxylesterases

Screening of a gene library from Paenibacillus sp. BP-23 generated in Esche richia coli led to identification of a clone that directed the production o f lipolytic activity. From the sequencing data, we found an open reading fr ame encoding a protein of 485 amino acids with an estimated molecular mass of 53 kDa and a pI of 5.1. Absence of a signal peptide indicated that it wa s a cell-bound protein. Sequence analysis showed that the protein contained the signature G-X-1-S-X-2-G included in most serine-esterases and lipases. The cloned protein showed high homology with enzymes belonging to the bact erial subclass of type B carboxylesterases. The enzyme had a significant pr eference for esters of short-chain fatty acids and showed the kinetics beha viour of a true esterase. Maximum activity was found at pH 7.5 and 37 degre es C, although the enzyme was active in the pH range 6.0-9.0 and at tempera tures up to 45 degrees C. As expected for a serine-esterase, activity was i nhibited by phenylmethylsulphonyl fluoride. (C) 2000 Editions scientifiques et medicales Elsevier SAS.