## 3 Results and Discussion
### Optimization of the GE Synthesis  
For GE synthesis, the olive oil-derived FFAs and FAMEs were used as model substrates to optimize the lipase-catalyzed reaction of esterification and transesterification (Figure 1). The reaction was carried out in a solvent system consisting of a tert-amyl alcohol and DMSO mixture as described elsewhere [11; 40; 41] for 50 h at 55 \({}^{\circ}\)C with shaking at 100 rpm, and the progress of the reaction was monitored by TLC analysis.  
The effect of various reaction conditions, such as the molar ratio of the reactants, the reaction temperature and the type and the concentration of immobilized enzyme, on the conversion rate was studied. Two immobilized lipases, lipase from _C. antarctica_ B (CA lipase) and lipase from _C. rugosa_ (CR lipase), were used as catalysts for both esterification and transesterification reactions for GE production (Table 1). The use of immobilized lipases as catalysts for GE synthesis has been proposed by several researchers [9; 21; 42; 43; 44].  
Moreover, different molar ratios of olive oil FFAs (or FAMEs) to glucose were tested (Table 1), showing that the conversion rate increased with an increasing concentration of glucose, attaining its maximum value with CA lipase as the catalyst and a FAMEs:glucose ratio of 1:3 (Entry 6, Table 1 and Figure 2). Glucose can be considered as a good acyl acceptor for SE synthesis in non-conventional media, ensuring a high conversion rate, due to its relatively higher solubility compared with other sugars [45, 46, 47]. On the contrary, the use of sugars with a higher degree of polymerization adversely affects the conversion rate as a result of their very low solubility in organic solvents.  
Although the conversion rate obtained by utilizing FFAs as acyl donor was high, this was, in all cases, below to that obtained with FAMEs as the substrate, probably due to the water that was produced, which may react with GE, causing hydrolysis (Figure 1). On the contrary, the transesterification reaction carried out using FAMEs is almost irreversible, as methanol (produced as a byproduct) at elevated temperatures evaporates to prevent the reaction from reversing [48, 49]. CR lipase showed a lower conversion rate (Entries 7-12, Table 1) than CA lipase. Furthermore, the reusability of CA lipase was checked  
Figure 1: Enzyme-catalyzed synthesis of glucose esters (GEs) using free fatty acids (FFAs) or fatty acid methyl esters (FAMEs) as substrates.  
Figure 2: Reaction course over time of olive oil FAME conversion to glucose esters in different olive oil FAME:glucose ratios.  
for several reaction cycles for the synthesis of GE using olive oil FAMEs as substrate under the optimized reaction conditions. It was found that the conversion rate using the regenerated enzyme remained essentially the same for three reaction cycles, while in the fourth cycle, the conversion rate was reduced to 88% of the original, which is important for the sustainability of the process. The reaction conditions were further optimized using the lipase CA in different quantities as a catalyst, and it was found that a 100% conversion was obtained using 0.25 g of the lipase CA (Table 2, entry 4), while a higher enzyme quantity was not necessary.  
The results recorded in this study emphasized the feasibility of enzymatic synthesis of GEs, the conversion yield of which reached 100% using CA lipase as a biocatalyst. These results are in agreement with those reported by [PERSON] et al. [50], who demonstrated that glucose FA monoesters were synthesized (with up to 93% yields) using lipase B from \(C\). _antarctica_. High yields for GE enzymatic synthesis were also reported by [PERSON] et al. [51] using lipase-FeSO4 nanoparticles as a catalyst. Furthermore, [PERSON] et al. [20] reported that the highest SE yield was achieved using CA lipase as a catalyst, and glucose and palmitic acid as substrates.