A novel esterase from Burkholderia gladioli which shows high deacetylationactivity on cephalosporins is related to beta-lactamases and DD-peptidases

The gene (estB) encoding for a novel esterase (EstB) from Burkholderia glad ioli (formerly Pseudomonas marginata) NCPPB 1891 was cloned in Escherichia coli. Sequence analysis showed an open reading frame encoding a polypeptide of 392 amino acid residues, with a molecular mass of about 42 kDa. Compari son of the amino acid sequence with those of other homologous enzymes indic ated homologies to beta -lactamases, penicillin binding proteins and DD-pep tidases. The serine residue (Ser(75)) which is located within a present cla ss A beta -lactamase motif ([F,Y]-X-[L,I,V,M,F,Y]-X-S-[T,V]-X-K-X-X-X-X-[A, G,L]-X-X-[L,C]) was identified by site-directed mutagenesis to represent th e active nucleophile. A second serine residue (Ser(149)) which is located w ithin a G-x-S-x-G motif which is typically found in esterases and lipases w as demonstrated not to play a significant role in enzyme function. The estB gene was overexpressed in E. coli using a tac promoter-based expression sy stem. Investigation of EstB protein with respect to the ability to hydrolys e beta -lactam substrates clearly demonstrated that this protein has no B-l actamase activity. The recombinant enzyme is active on triglycerides and on nitrophenyl esters with acyl chain lengths up to C6. The preference for sh ort chain length substrates indicated that EstB is a typical carboxylestera se. As a special feature EstB esterase was found to have high deacetylation activity on cephalosporin derivatives. (C) 2001 Elsevier Science B.V. All rights reserved.