Optimization of cephalosporin C acylase immobilization using crosslinked enzyme aggregates technique

Abstract

Cephalosporin C acylase (CCA) is an essential enzyme for the one-step conversion of cephalosporin C into 7-aminocephalosporanic acid (7-ACA), an intermediate compound used to synthesize various semi-synthetic cephalosporin antibiotics. The industrial process prefers to use enzymes in immobilized form rather than soluble. A crosslinked enzyme aggregate (CLEAs) is a potential matrix-less enzyme immobilization technique to produce stable immobilized enzymes with high activity and low production costs. This study aimed to optimize the CCA immobilization using the CLEAs technique with Chitosan as a co-aggregate. The CCA lysate was obtained from harvesting CCA fermentation broth using a mutant strain of Escherichia coli through cell separation and lysis steps. Partially purified CCA by ammonium sulfate addition was conducted to obtain an active fraction of 20-60% saturation, followed by co-aggregation with Chitosan to form physical CCA aggregates. The aggregates were then immobilized by a crosslinking technique using glutaraldehyde to form CLEAs-CCA. Optimization of the immobilization process was carried out by Response Surface Methodology in three steps, (i) screening of the influencing factors, (ii) determining the level of the significant factors, and (iii) optimizing the immobilization condition. The CLEAs-CCA activity was used as a response parameter. Under optimum conditions, CLEAs-CCA activity obtained was 85.91 Ug-1.