An Efficient Method for Recovering Recombinant Cephalosporin C Deacetylase from the Cytoplasm of E. coli Cells

In this study, several chemical treatment techniques were verified for releasing cytoplasmic cephalosporin C deacetylase (CAH) from recombinant Escherichia colt (E. colt) cells. Among all the chemical treatment methods, using cationic detergent, organic solvents and chelating agents as permeabilizing reagents exhibited limited capacities for extraction of cytoplasmic CAR from cells. Combined use of 5.0 % (v/v) Triton X-100 and 0.3 mol L-1 NaCl achieved better effect on CAH extraction, and the release yield of CAR reached approximately 88 %. The extraction parameters such as temperature, ionic strength, Triton X-100 concentration and time were separately optimized to enhance the process of CAR release. The optimal permeabilization conditions (Triton X-100: 2.0 %, v/v; extraction temperature and time: 30 degrees C and 14 h) resulted in 103 % of relative CAH yield when compared with an optimized sonication process. Release yield of more than 100 % indicated that sonication could inactivate a few CAR molecules in the extraction process. This chemical treatment method could avoid the inactivation of CAH caused by sonication, and thus improve the CAR extraction process.