Overproduction and purification of glutaryl 7-amino cephalosporanic acid acylase.
The gene encoding glutaryl 7-amino cephalosporanic acid acylase (GL-7ACA acylase) from Pseudomonas sp. 130 has been cloned and expressed in Escherichia coli using a high-level expression system. The specific activity of the acylase in the crude extract of cells in this system is approximately 10 times that in the previous one. The overproduced enzyme can be easily isolated within 3 days to a purity of over 90% by a simple and inexpensive two-step preparative chromatographic method with an overall yield of nearly 50%. The deletion of the signal peptide and mutation in the alpha-subunit of the acylase have little influence on its posttranslational processing and its kinetic parameters.
Li
Y
Y
State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, Academia Sinica, China.
Jiang
W
W
Yang
Y
Y
Zhao
G
G
Wang
E
E
D000581:Amidohydrolases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D010399:Penicillin Amidase; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D000581:Amidohydrolases; D010399:Penicillin Amidase; C046586:glutarylamidocephalosporanic acid acylase