Cephalosporin C acylase (CCA), an important industrial enzyme for the production of 7-aminocephalosporanic acid, has very limited and scattered surface lysine residues. A mutant of cephalosporin C acylase (mCCA) has been designed to fuse a poly-lysine tag to the C-terminal of the beta-subunit, which is far away from the active site. The free mCCA showed a near equal specific activity with the wild-type CCA, while a much higher activity recovery was obtained for the mCCA than its wild-type counterpart after immobilization on glyoxyl agarose support (73.3 versus 53.3 %). The mCCA's oriented immobilization enables it to obtain a higher substrate affinity and even a higher thermal stability than the wild-type enzyme. The improvement of stability might be attributed to the multipoint covalent attachment by the oriented enzyme immobilization via the adhered poly-lysine tag, which prevents the dissociation of the beta-subunit of CCA from the support.