Restriction mapping and localization of GL-7-ACA acylase gene.

This paper presents the results about the restriction mapping of recombinant plasmids pMR5 and pMR6 containing GL-7-ACA acylase gene from Pseudomonas sp. 130, gene localization and its expression under the control of different promoters, tet, tac or lac/tac, in Escherichia coli. The analysis of gel electrophoresis of pMR5 cleaved with several kinds of restriction enzymes indicated that there is no sites of EcoRI, HindIII and ClaI but the presence of following sites: one HpaI, two XhoI, three EamHI and four PstI on the cloned gene fragment. The restriction maps of pMR5 and pMR6 were determined by comparative digestion of various endonucleases. The gene of GL-7-ACA acylase was localized on a 3.0kb fragment of B2-B3-HpaI from the studies on a serial subcloning. Expression of subclones pMR9, pMR10 and pMR11 in E. coli was compared. Higher yield of acylase was obtained when the gene fragment was placed downstream of the tac promoter. The expression of Pseudomonas gene in E. coli was also discussed.