Genetic manipulation of penicillin acylase in bacillus sp.

Abstract

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 2.8 - 3.1 Kb in length. Partial digestion of pMLV101 with restriction enzyme HpaII produce a DNA fragment (F1) of about 2.8 Kb in size. The F1 DNA was found to contain the PAC gene. Ligation product of F1 and the plasmid vector pACYC184 (pBA2, pBA3, pBA4 and pBA32) made the Escherichia coli (E. coli) transformants produced PAC enzyme. The recombinant plasmid of F1 and the shuttle vector pHV33-2, pHBA33, also conferred both E. coli and B. subtilis host cells to produce PAC. All E. coli subclones (pBA2, pBA3, pBA4, pBA32 and pHBA33) produced PAC at about the same level of enzyme activety as E. coli which carried the plasmid pMLV101. The recombinant B. subtilis which carried plasmid pHBA33 produced PAC extracellularly, but, its activity was slightly lower than that of B. megaterium UN-1.