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.