a Reaction conditions: 1 mmol pine sterol, 6 mmol fatty acid, 8 U/g (based on the mass of pine sterol) CRL@OSMD, 50°C, 48 h.
b The first number in parentheses represents the number of carbon atoms of the fatty acid, and the second number represents the number of carbon-carbon double bonds of the fatty acid.
c Mixing oleic, linoleic, and linolenic acids in equimolar ratios to simulate edible oils.
3.7 Process scale-up andreusability studies
To explore the scalability of the pine sterol ester synthesis process developed above, the amount of substrate was scaled up to 10 times, and a final esterification of 94.3% of pine sterol ester was obtained, demonstrating the scalable production potential of the new process for the synthesis of pine sterol esters catalyzed by the immobilized enzymes in the study. Moreover, the reusability of the catalyst was investigated under the optimized conditions, and the results are shown inFigure S6 . It can be seen that the esterification yield decreased with the increase of cycles, and could still be maintained above 70.9% after 6 cycles. The decrease in esterification yield is most likely due to the unavoidable loss of the immobilized enzyme during the separation from the reaction system, as well as denaturation and inactivation of the enzyme during the reaction. For instance, Zhang et al. suggested that only 82.0% activity of CRL immobilized with a hollow cubic carbon can be maintained in the second cycle[67]. However, in this study, our novel-designed OSMD immobilization can maintain the residual activity as high as 90.6% in two cycles, which suggests that the novel-designed OSMD in this study is a good candidate in lipase immobilization and has great potential for application in further food biomanufacturing.