*2.8.6. Reaction temperature*

Temperature changes effect on different parameters including enzyme stability, affinity, and preponderance of the competing reactions [71]. Temperature normally affects lipase activity, and high temperatures usually increase the initial transesterification rate. However, high reaction temperatures deactivate the enzyme due to its protein nature [35]. The optimal temperature used in transesterification reactions is mainly based on considering properties of feedstock, such as melting behavior at different temperatures as well as the reaction system that is with or without solvent. In a solvent-free system, the temperature is maintained high enough to keep the substrates in liquid state [40].

The optimal temperature for the most immobilized lipases ranges from 30–60°C, while it tends to be lower for free lipases. Heat stability of lipase also depends on whether a substrate is present. This is because substrates remove excess water from the immediate vicinity of the enzyme, hence limiting its overall conformational mobility. Ishihara et al. [116] studied the effect of temperature on vanillyl alcohol acylation with nonanoic acid to give vanillyl nonanoate in *n*-hexane solvent medium. The authors found that the optimum temperature for enzymatic acylation was 70°C. Higher temperatures than 70°C lead to decrease the conversion yield due to the deactivation of enzyme at high temperature. The effect of temperature on the synthesis of capsiate analog by lipase-catalyzed esterification of vanillyl alcohol and conjugated linoleic acid (CLA) was presented [47]. The range of temperature tested was from 30 to 60°C. The results demonstrated that the yield increased when the temperature increased from 30 to 50°C. However, when temperature increased to 60°C, there is no increase effect on the yield.

### *2.8.7. Enzyme concentration*

Normally, as the enzyme concentration increases, the reaction equilibrium will be shifted quickly toward the synthesis [117]. However, for economic reasons, it is important to reduce the enzyme loading and the reaction time. In addition, the presence of high enzyme concentration in the reaction medium may increase the probability of its collision with the substrate subsequently enhancing the reaction rate [118]; however, after reaching certain enzyme concentration, the conversion yield was constant. Carrin et al. [117] reported that during the Lipozyme TL-IM-catalyzed acidolysis of sunflower oil with palmitic acid and stearic acid mixture, the extent of palmitic and stearic acids incorporation was enhanced by increasing the amount of enzyme in the reaction; however, when the enzyme concentration was greater than 8% by weight of substrates, there was no significant increase in the esterification yield. The effects of lipase concentration on the synthesis of capsiate analog were depicted in the work of [47].
