Magnetic cross-linked enzyme aggregate based on ionic liquid modification as a novel immobilized biocatalyst for phytosterol esterification

Abstract

A lipase from Candida rugosa (CRL) is an efficient enzyme for phytosterol esterification. However, its poor stability limits its application. A novel cross-linked enzyme aggregate of CRL (CRL-FIL-CLEAs@Fe₃O₄) was designed by lipase surface modification with a functional ionic liquid (FIL) and immobilization with magnetic Fe₃O₄ nanoparticles as carriers simultaneously. The loading of CRL was 0.16 mg protein per mg CRL-FIL-CLEAs@Fe₃O₄. The hydrolysis expressed activity of CRL-FIL-CLEAs@Fe₃O₄ was 1.36 ± 0.04 U per mg CRL-FIL-CLEAs@Fe₃O₄. The hydrolysis specific activity of CRL-FIL-CLEAs@Fe₃O₄ (8.48 ± 0.25 U per mg protein) was significantly higher than that of conventional CRL-CLEAs (2.86 ± 0.26 U per mg protein). After 15 days of storage at room temperature, CRL-FIL-CLEAs@Fe₃O₄ exhibited 2.6 times higher activity (73.31%) than free lipases (28.52%). After eleven times of reuse, CRL-FIL-CLEAs@Fe₃O₄ still retained a high hydrolysis activity (83.42%). CRL-FIL-CLEAs@Fe₃O₄ was further applied in the solvent-free esterification reaction of phytosterol with oleic acid, and the conversion rate of phytosterol was up to 93.24% after 22 hours (the reaction temperature was 48 °C, the substrate molar ratio of oleic acids to phytosterols was 10 : 1, and the amount of biocatalysts and substrate phytosterol was 5 mg and 0.1 mmol, respectively). It was expected that a good reference could be provided for the more efficient and green production of food-grade phytosterol esters.