Expression and structural analysis of pemicillin G acylade gene of bacillus megaterium ATCC14945 and characterization of the gene product

Abstract

A gene coding for penicillin G acylase of Bacillus megaterium ATCC14945 was cloned into Escherichia coli HB101 by inserting Pst I -generated DNA fragments into the PstI site of pBR322. Recombinant clones were screened for the clear halo forming activity on the lawn of Staphylococcus aureus ATCC6538P using the enzymatic acylating reaction of 7-aminodeacetoxycephalosporanic acid (7ADCA) and D-($\alpha$ )-phenylglycine methylester. From PstI-generated 17.6 kb DNA fragment, the gene coding for penicillin G acylase was localized in 2.8 kb insert DNA into pUC19 by a series of subcloning experiments. By analyzing the uncleotide sequence, the open reading frame, 1,812 bases long, was found. the ploypeptide encoded by the open reading frame would be composed of 604 amino acid residues and have a molecular weight of 61,623 as calculated from the predicted amino acid composition. Upstream from ATG of penicillin G acylase gene there was a putative ribosome binding site, Shine-Dalgarno sequence. The promoter-like structure, -10 and -35 regions, which may function as promoter sequence in E. coli and vegetative Bacillus subtillis were followed by the coding sequence of penicillin G acylase gene. Following the stop coden, TAG, a stucture reminiscent of the E. cili rho independent transcription terminator was present. The G+C content of the coding sequence was found to be 37.4\%. When the penicillin G acylase gene was transferred into B. subtills the enzyme was produced more than 10-fold higher level compared with B. megaterium and mostly secreted into the cultured broth. The penicillin T acylase protein produced by B. subtilis harboring pUBC73 was concentrated from the cultured broth using Celite and purified to homogeneity by CM-Sepharose and DEAE-Sepharose columns. The purified protein was shown a single protein band on a 10\% SDS-polyacrylamide gel and the relative molecular wight of the subunit was 59,000. The native molecular weight was estimated to be 120,000 by gel filt...