Abstract: Cephalosporin C (CPC) acylase is the key enzyme for the production of 7-amino-cephalosporanic acid (7-ACA) in one-step enzyme process. The improved CPC acylase mutant gene from Pseudomonas sp. Strain SE83 was codon optimized according to Pichia pastoris codon bias and named as SECA. An endogenous signal sequence DSE4 was used as the signal peptide and mediated the secretion of SECA in recombinant P. pastoris strain G/DSECA for the first time. In comparion with the control strain G/MFCA harbouring the similar expression cassette except that the signal sequence DSE4 was replaced by α-factor, 65% and 44% higher CPC acylase activity in intracellular and extracellular were obtained in G/DSECA, respectively. Recombinant strains G/DSEL and G/MFL containing β-galactosidase (lacZ) as the report gene were constructed to further investigate the secretion efficiency of DSE4, and G/DSEL showed higher intracellular and extracellular β-galactosidase activity than G/MFL did.