Cephalosporin C acylase characterization and purification using chromatography techniques
Cephalosporin C Acylase is an enzyme that plays an important role in the one step conversion of Cephalosporin C into 7-aminocephalosporanic acid (7-ACA), a key intermediate for the synthesis of semisynthetic cephalosporin antibiotics. Purification is a method or process of removing unwanted protein molecules or impurities from protein of interest with the aim of increasing enzyme specific activity. The purification process begins by expressing Cephalosporin C Acylase using E. coli BL21 (DE3) followed by the cell lysis process. Characterization of Cephalosporin CrnAcylase was being examined in this study to get a profile of the enzyme before entering purification process. This study applied chromatography techniques for enzyme purification, but mainly focusing on a specific purification method, Immobilized Metal Affinity Chromatography. It is a widely used purification method to purify histidine tagged protein. Ion-exchange Chromatography was also conducted as one of the chromatography techniques for additional purification step. Purification process in this research study is capable to increase enzyme specific activity up torn4.56-fold and recover 63.9% yield. Result of protein bands analysis using SDS-PAGE techniques shown that recombinant CCA enzyme is consist of two subunits, ? (25kDA) and ? (58kDa) subunit. This analysis also shown that purification process is capable in removing impurities from enzyme of interest.
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