4. Conclusion
A low-cost and designable material, namely diatomite, was investigated in the immobilization of CRL and then used for the synthesis of various pine sterol esters. To further improve its efficiency, sulfonyl and octadecyl were employed as modifiers to regulate the hydrophobicity and the binding interaction between support and lipase. In addition, the Fe3O4 was also introduced to this system for quick and simple separation via the magnetic field. Interestingly, the enzyme loading and activity recovery of the single modification diatomite via octadecyl or sulfonyl group was lower than that of two functional groups modification diatomite, namely OSMD. It indicated that there may be a synergistic effect between these two modifiers, the octadecyl modification endowed a suitable hydrophobicity of diatomite, and the sulfonyl group changed the interaction between lipase and support which enhanced the enzyme loading of CRL. The optimization of the ratio of the modifiers suggested that the -SO3H/C18 (1:1.5) performed best with an enzyme loading and enzyme activity of 84.8 mg·g-1 and 54 U·g-1, respectively. Compared to free CRL, the thermal and storage stability of CRL@OSMD was significantly improved, which lays the foundation for the catalytic synthesis of phytosterol esters in a solvent-free system. The molar ratio of pine sterol and oleic acid, enzyme addition, reaction temperature, and time in the esterification reaction were optimized, and finally the reaction was carried out at a molar ratio of pine sterol to oleic acid of 1:6, an enzyme addition of 8.0 U·g-1 (concerning the mass of pine sterol), and at a temperature of 50°C for 48 h, which could ultimately achieve an esterification yield of 95.0%. After cycling the reaction 6 times, the esterification yield still reached more than 70.0%. This study shows that OSMD materials are good candidates for lipase immobilization and have great potential for application in further food biomanufacturing with low-cost and high efficiency.