## 3 Discussion  
### Immobilized Lipases and Media Tailoring for Sustainable Biocatalyzed Esterifications  
To enhance the performance of the biocatalyst, the immobilization of enzymes and, more specifically, lipases are of high industrial interest, and it has become a requirement [29]. Indeed, it was shown in numerous reports that through, e.g., adsorption or covalent binding with a solid carrier, some acyltransferases, such as the lipase B from _Candida antarctica_ (CaLB), could display improved activity, stability and reusability [30; 31; 32]. This phenomenon can be understood _inter alia_ as a gain of rigidity for the enzyme's tertiary structure, which limits protein unfolding and thus denaturation of the biocatalyst [33]. This is in practice true in the case of the Novozym 435\({}^{\text{\textregistered}}\) formulation that was recently qualified as the \"perfect immobilized biocatalyst\" in a review from [PERSON] et al. [34]. In parallel, the native CaLB has been demonstrated to be among the most stable commercially available lipases [35; 36]. By binding it to a macroporous acrylic polymer resin (Lewatit VP OC 16,000), resulting in the now highly reported Novozym 435\({}^{\text{\textregistered}}\), it increased drastically the performances of the biocatalyst [37]. In the current work, it might appear shocking that we use 50 g/L of the enzyme to reach the same value in titer of product. At least on the tube scale, it is likely that, in the case of Novozym 435\({}^{\text{\textregistered}}\), the costs associated with the immobilization might be fully compensated [38; 39; 40; 41; 42; 43; 44; 45; 46; 47; 48]. Indeed, the formulation can be recycled, using filtration, for sometimes up to 10 cycles, as a report from [PERSON] et al. demonstrates [49]. It is also important to remember that in such a bead-adsorbed enzyme formulation, the non-catalytic ballast of the carrier represents a tremendous portion of the biocatalyst's mass (90 to >99 wt.%) [37; 50]. Thus, it is remarkable that in our work, the immobilized versions of CaLB, such as Lipozyme 435\({}^{\text{\textregistered}}\) or Novozym 435\({}^{\text{\textregistered}}\), rivalized with the buffered formulations as they should normally contain a higher enzyme concentration. Interestingly, CLEA CA was, in our case, comparable to the other CaLB formulations. The Cross-Linked Enzyme Aggregates (CLEAs) first introduced by [PERSON] et al. [51], but also the Cross-Linked Enzyme Crystals (CLECs) [52], represent very interesting and more elegant alternatives to the petrol-based polymer-carriers. CLEAs are a result of firsthand the precipitation and physical aggregation of the enzymes, then secondhand the cross-linking of these aggregates with a cross-linking agent, which can be typically glutaraldehyde. This gives several advantages to CLEAs over their non-covalently immobilized analogues, such as the quasi-noneistent leaching of enzyme even under reportedly harsh conditions [53]. Due to the covalent inter-molecular binding, CLEAs and CLECs afford complete removal of the carriers, thus resulting in carrier-free immobilized enzymes. However, they also present limitations and challenges for their industrial-scale production, notably for the control of the enzyme's aggregation that can result in less active enzyme dimers [54]; thus, they  
Figure 5: Time course of SL production (g/L): STR with 3-bladed spiral propeller versus tube in orbital shaking. are, to this day, rather rarely produced in bulk. On a side note, it is important to mention that standard acrylic-bead-immobilized enzymes were easier to handle throughout the tube-scale synthesis and downstream processing, than their CLEA analogues. Herein, we reached an optimal titer after 48 h of reaction, which is like other reports dealing with enzymatic production of sugar esters in DESs [11] but much shorter than microbial fermentation to produce glycolipids.