Cloning of GL-7-ACA acylase gene from Pseudomonas sp. 130 and its expression in Escherichia coli.

Using BamHI digested and dephosphorylated pBR322 as vector, a GL-7-ACA acylase gene from Pseudomonas sp. 130 chromosomal DNA was cloned and expressed in Escherichia coli C600. Seven positive clones were detected from 3205 recombinant plasmids with 32P-labeled oligonucleotide probes in situ hybridization. Out of them, three clones which produced active GL-7-ACA acylase were identified by radio-immunological assay, chemical test and chromatographic analysis of reaction mixture. The plasmid DNAs of recombinant pMR5, pMR6 and pMR7 were extracted. Analysis of gel electrophoresis indicated that pMR5 and pMR7 contained the same 6.8kb fragment and the size of the insert in pNR6 was 5.7 kb. The effects of various E. coli hosts on the expression of cloned GL-7-ACA acylase gene is also presented.