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.

中文翻译：

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SpyTag/SpyCatcher 环化和共价固定在增强头孢菌素 C 酰化酶稳定性方面的作用

摘要 通过将SpyTag 和SpyCatcher 分别与头孢菌素C 酰化酶(CCA) 的N-和C-末端融合，获得SpyRing 环化头孢菌素C 酰化酶(SRCCA)。结果表明，引入SpyRing（通过SpyTag和SpyCatcher进行头对尾环化）不影响SRCCA的活性中心（CCA和SRCCA的比活性分别为15.71 U/mg和13.11 U/mg） ）。此外，游离酶 SRCCA 的热稳定性、有机溶剂耐受性和变性剂耐受性也得到改善。由于乙醛琼脂糖载体有利于通过其具有最高赖氨酸残基密度的表面区域共价固定酶，SRCCA 允许其多点和定向固定，因为 SpyRing 含有非常丰富的赖氨酸残基，而 CCA 在赖氨酸残基方面非常差，固定是通过较少的酶支持键进行的。当酶载量为10 mg/g载体时，SRCCA的表达活性比CCA高22%。固定化的 SRCCA 的稳定性也显着提高；固定化 SRCCA 在 50 °C 下的半衰期为 125 min，约为固定化 CCA 半衰期的 5 倍。