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.