Separation of Cephalosporin-C Acylase from Fermentation Broth with Polyethylene Glycol-Salt Aqueous Two-Phase Systems Sasmito Wulyoadi1, Nabilla Amara Denaputri2, Ahmad Wibisana1
1Research Center for Pharmaceutical Ingredients and Traditional Medicine, National Research and Innovation Agency of Indonesia, Cibinong, Bogor, Indonesia
2Biotechnology Study Program, Faculty of Science and Mathematics, Diponegoro University, Semarang, Indonesia
Abstract
This study investigates the separation of cephalosporin-C acylase (CCA) using an Aqueous Two-Phase System (ATPS) composed of polyethylene glycols (PEGs) and salts. CCA is an enzyme essential for the one-step bioconversion of cephalosporin-C into 7-amino cephalosporanic acid (7-ACA), a key intermediate for synthesizing broad-spectrum, semi-synthetic cephalosporin antibiotics. ATPS, a separation technique based on immiscible liquid phases-typically formed by two polymers or a polymer and a salt-offers advantages over conventional extraction methods, including simplicity, environmental friendliness, rapid separation with minimal enzyme denaturation, high selectivity and scalability. While ATPS has been widely applied to other enzymes like penicillin acylase, amylase, protease and pectinase, no studies, to our knowledge, have reported on CCA separation using this method.
Our study utilizes PEGs (400, 2000, 4000, 6000 and 10000) and salts (sodium sulfate, ammonium sulfate, sodium citrate and dipotassium hydrogen phosphate) to explore optimal ATPS conditions for CCA separation. Two critical factors in evaluating ATPS efficiency for CCA were yield (recovery percentage) and purification factor (purity relative to crude extract). The separation process involved CCA fermentation with Escherichia coli, cell lysis, and phase formation using PEG and salt concentration variations in a phosphate buffer (0.1 M, pH 8.5). Results showed that the highest CCA recovery was achieved with 21% PEG 2000, 5% K2HPO4, and 74% phosphate buffer, yielding 83.0% in the top phase with a purification factor of 1.80 relative to the crude extract.
