GENETIC MANIPULATION OF PENICILLIN ACYLASE IN BACILLUS-SP

Localization of the Bacillus megaterium penicillin acylase (PAC) gene on plasmid pMLV101 was done by using the transposition inactivation technique. The PAC gene was found to be about 3 Kb in length. Partial digestion of pMLV101 with restriction enzyme HpaII produced a DNA fragment (F1) of 2.8 Kb. The F1 DNA and the plasmid vector pACYC184 (pBA3 and pBA32) allowed Escherichia coli transformants to produce PAC enzyme. A recombinant plasmid of F1 and the shuttle vector pHV33-2, pHBA33, allowed both E. coli and B. subtilis host cells to produce PAC. All E. coli subclones (pBA3, pBA32 and pHBA33) produced about the same level of PAC enzyme activity as did E. coli which carried the plasmid pMLV101. Recombinant B. subtilis which carried plasmid pHBA33 produced PAC extracellularly, but its activity was slightly lower than that produced by B. megaterium UN-1.