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)₄-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_LC on amine-modified magnetic nanoparticles (NH₂-MNPs) via microbial transglutaminase-catalyzed bioconjugation for the development of the oriented-immobilized enzyme, namely, EK_LC@NH₂-MNP biocatalyst. Upon the site-specific immobilization, approximately 90% EK_LC 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_LC@NH₂-MNP biocatalyst was further applied to remove the His tag-(Asp)₄-Lys fusion partner from the His tag-(Asp)₄-Lys-(GLP-1)₃ substrate fusion protein, result suggested the EK_LC@NH₂-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_LC@NH₂-MNP biocatalyst is expected to promote the economical utilization of enterokinase in fusion protein cleavage.