OVERPRODUCTION AND PURIFICATION OF GLUTARYL 7-AMINO CEPHALOSPORANIC ACID ACYLASE

The gene encoding glutaryl 7-amino cephalosporanic acid acylase (GL-7A CA acylase) from Pseudomonas sp. 130 has been cloned and expressed in Escherichia coli using a high-level expression system. The specific ac tivity of the acylase in the crude extract of cells in this system is approximately 10 times that in the previous one. The overproduced enzy me can be easily isolated within 3 days to a purity of over 90% by a s imple and inexpensive two-step preparative chromatographic method with an overall yield of nearly 50%. The deletion of the signal peptide an d mutation in the alpha-subunit of the acylase have little influence o n its posttranslational processing and its kinetic parameters. (C) 199 8 Academic Press.