Enzymatic Synthesis Of Cephalexin In Recyclable Aqueous Two-phase Systems Composed By Two PH Responsive Polymers

Cephalexin, an widely used cephalosporin antibiotics, is synthesized by using immobilized penicillin acylase catalyzing reaction of7-amino-3-deacetoxy cephalosporanic acid (7-ADCA) and phenyl glycine methyl ester (PGME). However, product accumulation may inhibit the enzyme efficiency. The synthesis of Cephalexin in novel recyclable aqueous two-phase systems (ATPS) was investigated in this study.By adding different salts with dfferent concentrations to adjust the partition of Cephalexin in pH-pH response ATPS PADB/PADBA and thermo-pH response ATPS PNB/PADB· In PADB/PADBA ATPS, the partition coefficient of Cephalexin is1.31with50mM KC1. In PNB/PADB ATPS, the partition coefficient of Cephalexin is0.73with220mM KSCN.Two pH-response polymers PADBA and PMDB were used to construct the pH-response ATPS, then calculate the bimodal curve. In addition, the effect of different concentration of polymers, pH and temperature on the partition of Cephalexin were studied, and the partition coefficient of Cephalexin was above1.2.The partition behavior of7-ADCA, PGME and Cephalexin in PADBA/PMDB ATPS were studied. In the presence of20mM NaCl, the partition coefficient of Cephalexin,7-ADCA and PGME were2.57,0.21and0.94, respectively. In addition, the relationship between the electrostatic potential difference and the concentration of salts was studied, the electrostatic potential difference is positively associated with the partition coefficient of Cephalexin.Effect of pH, temperature, enzyme dosage and substrate concentration ratio on synthesis of Cephalexin in single aqueous phase were optimized by means of designing orthogonal experiments and some single factor experiments. The optimal conditions:pH5.8, temperature20℃, enzyme dosage3.6U/mL and PGME:7-ADCA3. The reaction reached equilibrium at3.5h with73.58%yield of Cephalexin with30mM7-ADCA and90mM PGME.Synthesis of Cephalexin in ATPS and single system was compared. Higher Cephalexin yield was obtained in ATPS. In addition, synthesis in ATPS was improved at higher substrates concentrations, obtaining yield of98.2%with50mM7-ADCA and150mM PGME. Even reducing acyl donor to nucleophile ratio to2, the yield can still above90%. PADBA/PMDB recycling aqueous two-phase systems are suitable for synthesis of Cephalexin.