SpyTag/SpyCatcher cyclization and covalent immobilization in enhancing cephalosporin C acylase stability

Abstract

A SpyRing cyclized cephalosporin C acylase (SRCCA) was obtained by fusing SpyTag and SpyCatcher to the N- and C- termini of cephalosporin C acylase (CCA), respectively. The results suggested that the introduction of the SpyRing (head-to-tail cyclization via SpyTag and SpyCatcher) did not affect the active center of the SRCCA (the specific activities of CCA and SRCCA are 15.71 U/mg and 13.11 U/mg, respectively). Also, the thermostability, organic solvents tolerance, and denaturant tolerance of the free enzyme SRCCA were improved. Since glyoxyl agarose carrier favors the covalent immobilization of enzymes through its surface regions having the highest lysine residues density, SRCCA permitted its multipoint and oriented immobilization because SpyRing is very rich in Lys residues, while CCA is quite poor in Lys residues and immobilization is via less enzyme support-bonds. When the enzyme loading amount was 10 mg/g carrier, the expressed activity of SRCCA was 22 % higher than that of CCA. The stability of the immobilized SRCCA was also significantly improved; the half-life of the immobilized SRCCA at 50 °C was 125 min, which was about 5 times the half-life of the immobilized CCA.