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Chemical and Biochemical Engineering Quarterly, Vol. 28 No. 3, 2014.

Original scientific paper

https://doi.org/10.15255/CABEQ.2013.1904

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

X. Ma ; State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, P. R. China
E. Su ; Enzyme and Fermentation Technology Laboratory, College of Light Industry Science and Engineering, Nanjing Forestry University, Nanjing 210037, P. R. China
S. Deng ; State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, P. R. China
Y. Xie ; State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, P. R. China
D. Wei ; State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, P. R. China

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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.

Keywords

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

Hrčak ID:

128036

URI

https://hrcak.srce.hr/128036

Publication date:

10.10.2014.

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