Abstract

Penicillin G acylase (PGA) is one of the most important enzymes in the pharmaceutical industry. It is utilized in the process for production of semi-synthetic penicillins. Several different penicillin acylases with various characteristic have been isolated from different bacteria and identification of bacterial isolates harboring PGA enzyme with higher industrial compatibilities is of high interest. The aim of this study is to screen for PGA producing Escherichia coli isolates as well as the cloning and recombinant expression of PGA for high level enzyme production. Bacteria isolated from environmental and clinical samples were identified by standard microbiological tests and then E. coli isolates were subjected to DNA extraction and PCR screening using primers designed on conserved region of PGA genes. The PCR product from a positive isolate were cloned and subjected to sequencing. The gene encoding for full length PGA was expressed in E. coli under the T7 promoter. PCR screening identified several PGA positive E. coli. One of the positive isolates was cloned in pGEM -T easy vector. Sequencing of the cloned gene revealed that the gene encoding Penicillin G acylase from this wild E. coli isolate contains an open reading frame of 2538 nucleotide encoding 846 amino acids. Analysis of the sequencing results showed that the PGA gene is highly conserved among E. colistrains. Recombinant expression of PGA from wild isolate in E. coli BL 21 resulted in a high level expression of recombinant PGA which appeared as a dense band in SDS-PAGE analysis of induced culture.

Key words: PGA, cloning, sequencing, recombinant expression.

Abbreviation

PGA, Penicillin G acylase; 6-APA, aminopenicillanic acid; PAA,phenyl acetic acid; EMB, eosin-methylene blue; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; PCR, polymerase chain reaction;IPTG,  isopropyl--D-thiogalactopyranoside; BLAST, basic local alignment search tool; PDAB, p-dimethyl-aminobenzaldehyde