A facile method to oriented immobilization of His-tagged BirA on Co³⁺-NTA agarose beads

A facile and economical method was established for the oriented immobilization of biotin ligase (BirA) on Co³⁺-NTA sepharose through H₂O₂ oxidation of Co²⁺ and His-tag. His-tag of the BirA were designed at both N-terminal (His-BirA) and C-terminal (BirA-His), respectively. Immobilization of the His-BirA was performed, realized to 92.85% using by 10 mM H₂O₂ without compromising catalytic activity. Because amounts of ions on matrix were far more than that of the immobilized BirA, EDTA should be used to remove residual ions before catalyzing, while it should be limited to lower than 30 mM, and imidazole ranging from 50 to 250 mM could be added in the catalytic system. When 10 mM EDTA and 50 mM imidazole were used, over 90% of substrates were obtained from the matrix. Moreover, the His-BirA showed higher immobilization rate than the BirA-His, while both of them appeared high catalytic abilities at pH ranging from 6.5 to 9.0, indicating versatile options in the biotinylation of proteins with different pH stabilities. Under the best catalytic conditions, the both immobilized His-BirA and BirA-His exhibited the same activity as the free. When the enzyme was incubated at different pH (pH 3.0, 4.0, 5.0, 10.0 and 11.0) and temperature (40 °C, 50 °C and 60 °C), the immobilized His-BirA showed less pH-sensitive, overall preferable thermo-stability than the free, making it a more desirable option for storage and transportation. More importantly, the reusability of the immobilized His-BirA implied a promising value in industrialization.