Magnetic cross-linked lipase aggregates coupled with ultrasonic pretreatment for efficient synthesis of phytosterol oleate

In this study, coupling with substrate ultrasonic pretreatment technology, magnetic cross-linked enzyme ag-gregates (CRL-FIL-CLEAs@Fe3O4) was used as novel biocatalyst for efficient preparation of phytosterol oleate in solvent-free reaction system. The CRL loading was 0.16 mg protein/mg CRL-FIL-CLEAs@Fe3O4. The effects of ultrasonic parameters, catalyst dosage, substrate molar ratio, and reaction temperature on the synthesis of phytosterol esters were studied. The results showed that the catalytical properties of CRL-FIL-CLEAs@Fe3O4 (7.09 +/- 0.11 mu mol phytosterol/mg protein. hour) was significantly improved compared with natural lipase from Candida rugosa (CRL, 5.13 +/- 0.21 mu mol phytosterol/mg protein. hour). Simultaneously, ultrasonic pretreatment greatly enhanced substrate mass transfer, further reducing the esterification time. The max conversion rate 94.72% of phytosterol was obtained after 9 h reaction under optimum condition (the molar ratio of oleic acid to phytosterol was 6:1, the concentration of CRL-FIL-CLEAs@Fe3O4 was 10 wt% of the phytosterol mass). After five recycles, the activity of CRL-FIL-CLEAs@Fe3O4 (80.02%) was still 2.68 times higher than that of natural lipase system (29.91%). Meanwhile, the novel immobilized lipase (CRL-FIL-CLEAs@Fe3O4) before and after reaction was characterized by SEM, AFM, VSM, TEM, and TGA. The results showed that CRL-FIL-CLEAs@Fe3O4 was a robust and recyclable biocatalyst for phytosterol esterification.