7-aminocephalosporanic acid (7-ACA) is an important intermediate compound for producing of most cephalosporin derivatives. It could be synthetized by conversion of cephalosporin C (CPC) in single step using cephalosporin acylase. The application enzymatic conversion is hampered by low activity of enzyme using CPC as the main substrate. Modification of AcyII genes from Pseudomonas SE83 produced S12 variant that could expressed CPC acylase with high-activityon CPC. Based on S12 variant sequence, a synthetic gene was constructed and subsequently transformed to E. coli. The expression of target proteins were performed using LB medium supplemented with ampicillin. Preliminary experiments showed that the target protein (CPC acylase) partially remained as the inclusion bodies. Furthermore to obtain high yield of the target proteins, we explored the factors influenced on protein expression, i.e. isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration, induction temperature, and induction time in a series of experiments. The result showed that the optimal condition of soluble cephalosporin acylase expression was induction with 0.1 mM IPTG, incubation temperature 20oC and incubated for 12 h