Excellent performance separation of trypsin by novel ternary magnetic composite adsorbent based on betaine-urea- glycerol natural deep eutectic solvent modified MnFe2O4-MWCNTs

The effective trypsin purification methods should be established since trypsin plays a crucial role in biosome. In this work, a novel ternary magnetic composite adsorbent (MnFe2O4-MWCNTs@B-U-G) with the features of strong specific selectivity, good adsorption effect, simple and efficient separation process, no secondary pollution brought in was prepared by integrating the superior physicochemical properties of ternary based natural deep eutectic solvent, multi-walled carbon nanotubes and MnFe2O4. The property, composition and microtopography structure of MnFe2O4-MWCNTs@B-U-G were characterized in detail. Combined with magnetic solid-phase extraction, MnFe2O4-MWCNTs@B-U-G was utilized to adsorb trypsin. Response surface methodology experiment was prepared under Box-Behnken design to optimize the adsorption conditions and the results showed that the practical maximum adsorption capacity for trypsin was 1020.1 mg g-1. Besides, the adsorption isotherms, adsorption kinetics, regeneration studies and method validation studies were investigated systematically to evaluate the established adsorption separation system. Mechanism exploration proved that electrostatic interaction, hydrogen bonding interaction and chelation interaction were the dominant forces for the high-performance adsorption of trypsin. The activity of trypsin after elution had been analyzed by UV-vis spectrophotometer and CD spectrometer with three methods, which illustrated that the enzyme activity, conformation and secondary structure of trypsin did not change significantly during the adsorption-desorption process. In addition, the proposed method was successful and practical applicability to isolation trypsin from crude bovine pancreas. As a result, due to the superiority of the MnFe2O4-MWCNTs@B-U-G, the proposed method not only exhibites high-performance adsorption of trypsin, but also provides a green and sustainable potential value in the adsorption of biomacromolecule.