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

X. Ma, E. Su, S. Deng, Y. Xie and D. Z. Wei

Abstract
In this study, several chemical treatment techniques were verified for releasing cytoplasmic cephalosporin C deacetylase (CAH) from recombinant Escherichia coli (E. coli) 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 CAH 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 CAH 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 CAH release. The optimal permeabilization conditions (Triton X-100: 2.0 %, v/v; extraction temperature and time: 30 °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 CAH molecules in the extraction process. This chemical treatment method could avoid the inactivation of CAH caused by sonication, and thus improve the CAH extraction process.

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Keywords
chemical treatment method, Triton X-l00, extraction, cytoplasmic protein, Cephalosporin C deacetylase (CAH)