Site-specific, covalent immobilization of an engineered enterokinase onto magnetic nanoparticles through transglutaminase-catalyzed bioconjugation.

Enterokinase (EK) is one of the most popular enzymes for the in vitro cleavage of fusion proteins due to its high degree of specificity for the amino-acid sequence (Asp)-Lys. Enzyme reusability is desirable for reducing operating costs and facilitating the industrial application of EK. In this work, we report the controlled, site-specific and covalent cross-linking of an engineered EK on amine-modified magnetic nanoparticles (NH-MNPs) via microbial transglutaminase-catalyzed bioconjugation for the development of the oriented-immobilized enzyme, namely, EK@NH-MNP biocatalyst. Upon the site-specific immobilization, approximately 90% EK enzymatic activity was retained, and the biocatalyst exhibited more than 85% of initial enzymatic activity regardless of storage or reusable stability over a month. The EK@NH-MNP biocatalyst was further applied to remove the His tag-(Asp)-Lys fusion partner from the His tag-(Asp)-Lys-(GLP-1) substrate fusion protein, result suggested the EK@NH-MNP possessed remarkable reusability, without a significant decrease of enzymatic activity over 10 cycles (P >  0.05). Supported by the unique properties of MNPs, the proposed EK@NH-MNP biocatalyst is expected to promote the economical utilization of enterokinase in fusion protein cleavage.