THE BACILLUS-SUBTILIS PNBA GENE ENCODING P-NITROBENZYL ESTERASE - CLONING, SEQUENCE AND HIGH-LEVEL EXPRESSION IN ESCHERICHIA-COLI

p-Nitrobenzyl esters serve as protecting groups on intermediates in th e manufacture of clinically important oral beta-lactam antibiotics; de -esterification of the intermediates is required for synthesis of the final product. A Bacillus subtilis PNB carboxy-esterase (PNBCE) cataly zes hydrolysis of several beta-lactam antibiotic PNB esters to the cor responding free acid and PNB alcohol. This communication (i) describes cloning the pnbA gene, which encodes PNBCE, (ii) provides the nucleot ide sequence of the pnbA open reading frame (ORF) and (iii) describes a method for efficiently expressing the ORF in Escherichia coli. The a mino acid (aa) sequence, deduced from the nucleotide sequence of the p nbA ORF, matched an experimentally determined N-terminal aa sequence o f B. subtilis PNBCE and also matched an active site sequence previousl y identified by biochemical analyses. Specific activity of PNBCE in cr ude extracts was more than 90-fold greater in recombinant E. coli, as compared to B. subtilis. This increase in expression led to more than a 500-fold improvement in the efficiency of purification of PNBCE.