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 the 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) catalyzes hydrolysis of several beta-lactam antibiotic PNB esters to the corresponding free acid and PNB alcohol. This communication (i) describes cloning the pnbA gene, which encodes PNBCE, (ii) provides the nucleotide sequence of the pnbA open reading frame (ORF) and (iii) describes a method for efficiently expressing the ORF in Escherichia coli. The amino acid (aa) sequence, deduced from the nucleotide sequence of the pnbA ORF, matched an experimentally determined N-terminal aa sequence of B. subtilis PNBCE and also matched an active site sequence previously identified by biochemical analyses. Specific activity of PNBCE in crude 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.
Zock
J
J
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285.
Cantwell
C
C
Swartling
J
J
Hodges
R
R
Pohl
T
T
Sutton
K
K
Rosteck
P
P
McGilvray
D
D
Queener
S
S
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D004926:Escherichia coli; D015870:Gene Expression; D005798:Genes, Bacterial; D007700:Kinetics; D007769:Lactams; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015345:Oligonucleotide Probes; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013379:Substrate Specificity; D013816:Thermodynamics
D000900:Anti-Bacterial Agents; D007769:Lactams; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; C091617:4-nitrobenzyl esterase; D002265:Carboxylic Ester Hydrolases