Lipase B from Candida antarctica in Highly Saline AOT-Water-Isooctane Reverse Micelle Systems for Enhanced Esterification Reaction

Abstract

Butyl oleate synthesis by the lipase B from Candida antarctica (CalB) under extreme halophilic conditions was investigated in the present research through the AOT/Water/Isooctane reverse micellar system. The impact of aqueous content (${\mathrm{W}}_{\mathrm{o}}=\frac{\left[{\mathrm{H}}_2\mathrm{O}\right]}{\left[Surfactant\right]}$) and NaCl variation on the enzymatic activity of CalB in the butyl oleate reaction in reverse micelles was explored. The results indicated that, based on the increase of NaCl, it is remarkable to achieve higher enzymatic activity up to 444.85 $\frac{\mathsf{\mu }\mathrm{mol}}{\min }$ at 5 M NaCl and Wo = 10, as the best esterification conditions at pH 7.2 and 30 °C. However, it was clear that butyl oleate synthesis by lipase CalB increased based on the reduction in the average reverse micelle size, where reverse micelle sizes were determined by dynamic light scattering (DLS). This increase in butyl oleate synthesis demonstrated the potential of reverse micelles as systems that enhance mass transport phenomena in heterogeneous biocatalysis. Furthermore, reverse micelles are promising systems for extreme halophilic lipases research.

1. Introduction

Lipases (triacylglycerol ester hydrolases, E. C. 3.1.1. 3) are enzymes capable of physiologically catalyzing the hydrolysis of water-insoluble esters such as triglycerides [1]. There is great relevance for their use as biocatalysts in science, biotechnology, and industry due to their stability in organic solvents, diverse affinity and substrate recognition, selectivity, and catalytic capacity without the need to add costly cofactors due to their characteristic mechanism of action [2,3,4,5].

Lipases have been previously described in kinetic terms based on their catalytic mechanism called “interfacial activation” at the aqueous–organic interface [6,7]. It has been suggested that the so-called “interfacial activation” is due to the presence of a cap domain (lid domain) located near the active site, where conformational changes take place in the biocatalyst when a water-insoluble substrate interacts near the domain. However, experimental approaches have not demonstrated the association of this phenomenon when it occurs and the lid domain is present for all lipases and esterases [8]. Its catalytic activity is largely associated with a catalytic triad made up of serine (nucleophilic), histidine, and aspartate/glutamate (acidic residues) [9]. As an example, lipase B from Candida antarctica (CalB) has a relatively small “loop” that does not completely isolate the catalytic compartment. However, some lipases have more complex “lid” domains [10,11,12].

Thus, lipases play a remarkable role in industrial applications due to their high versatility [13,14,15]. It is expected that their global market size will exceed USD797.7 million by 2025 and grow at a compound annual growth rate (CAGR) of 6.2% from 2017 to 2025 [16]. Therefore, lipases represent the third most industrially used enzymes in the global market [17,18], as they offer a promising array of applications in food, oils, pharmaceuticals, cosmetics, paper, leather, detergents, and bioenergetics, among others [4,19,20,21,22].

It is considered that in lipase synthesis reactions, the medium should be mainly hydrophobic in order to favor substrate solubilization, containing low water proportion to shift the thermodynamic equilibrium in favor of synthesis over hydrolysis, and be heterogeneous in nature to support all the components of the medium and allow lipases an interface, which in most cases favors the catalytic performance of the enzymes [23]. By contrast, lipases from halophilic microorganisms have shown broad substrate specificity due to their type-, regio- and stereo-selectivity [24,25,26]. Halophilic enzyme complexes are capable of carrying out hydrolysis and synthesis reactions at high salt concentrations, exhibit thermostability, and adapt to a wide pH range [27,28].

Several studies [29,30,31,32] have attempted to employ reverse micellar systems to integrate into nanoscale microemulsions an aqueous or polar material in continuous lipophilic organic media, where the microemulsion is structured with surfactants to meet the previously described criteria of the synthesis reactions [33]. Reverse micelles act as a biocompatible armor to host biocatalysts in an aqueous medium from the aggressive organic solvent, where this nanoreactor delimits an inner core of water and an outer organic solvent [34,35,36]. Therefore, reverse micelles are particularly favorable systems for lipases, due to the increased selective solubility of substrates due to the intimate liquid–liquid contact they maintain with the organic solvent and the polar core characteristics that provide a physiological environment for lipases [34]. Moreover, these systems might offer significant support to halophilic biocatalysts, since this class of enzymes has exhibited rapid and irreversible inactivation when exposed to conditions below 1 M NaCl/KCl [33].

It has been described in previous work [37] that lipase B from Candida antarctica (CalB) is suitable for ester synthesis reactions; however, its catalytic activity in the presence of high NaCl concentrations has been scarcely explored. In this sense, through this research, we hypothesize that reverse micelles are able to highlight for their versatile performance under low water conditions due to the presence of solvents in the bulk reaction medium or under extreme halophilic conditions such high salt concentration, with up to 5 M NaCl, emerging as an effective study model for the screening of robust biocatalysts such CalB and other halophilic lipases, due to the favorable characteristics that virtually simulate an intrinsic biological microenvironment at low water conditions in which some enzymes from halotolerant and halophilic microorganisms act.

2. Results and Discussions

2.1. Size Characterization of AOT/Water/Isooctane Reverse Micellar Systems

The injection method prepared the reverse micelles according to the ternary system (Section 3.1 in Materials and Methods). The reverse micellar system structured with the synthetic surfactant AOT (sodium salt of bis(2-Ethylhexyl) sulfosuccinate) at 100 mM concentration was prepared in isooctane with 50 mM phosphate buffer (pH 7.2), 100 mM oleic acid, 50 mM 1-butanol, and CalB lipase at 20 $\frac{\mathrm{mg}}{\mathrm{mL}}$, with diverse aqueous contents (${\mathrm{W}}_{\mathrm{o}}=\frac{\left[{\mathrm{H}}_2\mathrm{O}\right]}{\left[Surfactant\right]}$) from 5 to 20 and varying NaCl concentrations from 0 M to 5 M. The concentration of the AOT surfactant was used according to the concentration reported by several studies [38,39] and taking into consideration the structuring of the reverse micelles with a higher concentration of CMC reported in isooctane (1 mM) [40]; otherwise, the reverse micelles could be inhibited if the surfactant concentration is lower than CMC, and the system would be structured by free and dispersed AOT molecules [41].

Initially, the required operating range for Wo in reverse micellar systems was defined with Wo = 5 as the minimum limit and Wo = 20 as the maximum limit. The upper limit of aqueous content was established due to the favorable maintenance of several properties in nanoemulsions from a macroscopic scale, such as transparency, homogeneity, and isotropy [42]. During the exploration of higher aqueous contents, turbidity was noticeable, and the nanoemulsions tended to show instability in the isotropic property of the systems. This highlighted the aqueous content (Wo) as one of the most relevant factors of reverse micellar systems, where Wo promotes an aqueous microenvironment for protection against the denaturation of hydrolase enzymes in the organic phase where lipophilic substrates and products are found [38,43,44].

Likewise, the other critical components of these systems are surfactants and nonpolar organic solvents. The polar groups of the amphiphilic molecules generate their positioning towards the center, generating a polar core, and the aliphatic chains are directed towards the outside of the nonpolar organic phase [44,45,46]. In this sense, it is possible to assume as a model, that each reverse micelle in the system is a sphere [47].

Figure 1 shows the average diameters of the reverse micelles obtained using DLS measurements with variations of the aqueous content from 5 to 20 and of the NaCl concentration from 0 M to 5 M. In the systems with 0 M NaCl, the reverse micelles had a proportional and relatively linear increase, where the size distribution behaves as a function of the aqueous content in which these diameters correspond to 4.61 nm ± 0.22 (Wo = 5), 6.98 nm ± 0.21 (Wo = 10), 7.82 nm ± 0.09 (Wo = 15), and 9.40 nm ± 0.38 (Wo = 20). In parallel, the results showed that the reverse micelle sizes were not altered by the entry of the enzyme into the aqueous core [48,49]; however, changes in reverse micelle morphology were virtually complex to determine experimentally in the scope of this work. Therefore, characterization using DLS was carried out employing a biocatalyst in all assays in order to keep the composition constant with the esterification kinetics.

The sizes described above correspond to those reported in a study [50] that explored the stability in AOT/Water/Isooctane systems through molecular dynamics (MD) and DLS. The behavior of the size distributions revealed that at Wo = 5 and Wo = 20, the reverse micelles were able to reach sizes of 5 nm and 9–10 nm, respectively. On the other hand, other authors demonstrated [51] the use of AOT/Water/Isooctane reverse micelle systems as functional nanoreactors for the synthesis of protein nanoparticles, where the sizes of the reverse micelles at Wo = 20 are close to 9 nm. Although some studies have shown that water diffusion in the reverse micelles could be negatively compromised by the solvent and its conformational structure, making the size be determined by dynamic light scattering (DLS) larger than that estimated by molecular dynamics (MD) or other methods [50]; thus, DLS could detect average distribution structures corresponding to temporary or transient reverse micelles, which are neither detected nor measured in simulations (MD) due to the preset display time estimated to be in the order of one to a few nanoseconds. In this context, another result convergent with the literature was the average distributions obtained using DLS at Wo = 10 (6.98 nm ± 0.21) from our work in comparison to another study [52], where their AOT/Water/Isooctane reverse micelle system at Wo = 10 without NaCl showed structures of sizes close to 6.8 nm.

In the case of aqueous contents below 5, behaviors different from those projected based on the spherical model have been reported, i.e., several studies [48,50,53,54] have simulated variations in the morphology of the reverse micellar systems and the behavior of the components that structure these nanoreactors. In their reports, they demonstrated that during simulations (MD), at Wo < 5, the structures of the reverse micelles converge to ellipsoidal, cylindrical, and toroidal morphologies, among others. Although, as aqueous content (Wo > 5) increases, the reverse micelles tend to morphologically stabilize to spheres. They showed that the location of AOT surfactant molecules displayed changes as a function of decreasing aqueous content (Wo < 5), and the H₂O molecules acquired uncharacteristic bulk properties of water, where hydrophobic AOT tails partially entered the aqueous core of the reverse micelles and confined water molecules interacted in closer proximity to each other and solvated the polar groups of AOT [53,54].

In this context, studies determined alterations in the protonation of the intramicellar aqueous core, where they indicate increases in the internal electric potential of the reverse micelles due to the dissociated AOT surfactant cations (Na⁺) at the interface [55,56]; although, theoretically they demonstrated decay in the electric potential and increases in the dielectric constant of the medium based on the increase of the aqueous content. Consequently, the migration of protons (H⁺) as a function of the increase in Wo was preferentially directed towards the interface and Na⁺ towards the interior of the reverse micellar system [57], generating an important protonation gradient capable of altering the hydrogen potential (pH), which generates an aqueous nucleus of higher alkalinity.

Taking into account the above, it is possible to associate a relative pH alteration in the aqueous phase composed of the phosphate buffer added to the reverse micellar systems designed for butyl oleate synthesis reactions with CalB. Moreover, the average size distribution of reverse micelle in AOT/Water/Isooctane systems reported in the literature converges with those obtained experimentally in this work; however, the average reverse micelle distribution acquires a divergent behavior as a function of increasing NaCl concentration (Figure 1 and Figure 2).

Other experimental approaches suggest that [41,45,58] two different regions with divergent water structures may exist in the polar core of the reverse micelle, where one region, with reduced mobility, has a structured water layer around the polar heads of the surfactant, while the other region consists of free-spinning bulk water in the hydrophilic inner core of the reverse micelle. According to Figure 1A,B, it is evident that with increasing NaCl concentration in the reverse micellar systems, the size distribution decreases even at the maximum limit of the aqueous content (Wo = 20), where this behavior is remarkably different in Figure 1B across all NaCl variations at the maximum aqueous content (Wo = 20) and which has the highest statistically significant difference (p < 0.05). The screening of this behavior started by supplying low concentrations of NaCl (0.125, 0.25, and 0.5 M) in the phosphate buffer, where it is possible to notice that by slightly increasing the concentration of the electrolyte (NaCl) in the aqueous phase of the reverse micelles, a reduction in the distribution of the average sizes is observed (Figure 1A). The first evidence described above suggested that the presence of NaCl can have several effects on the hydrophilic core of the reverse micellar systems, such as the exchange between favoring or limiting their dissolution by reverse micelles [59].

Extrapolating the previous descriptions, in Figure 2 it is possible to notice that with the characterization of the range average size distribution of the reverse micelles in the concentrations with excess electrolyte (NaCl > 1 M), the behavior maintained a relative linearity between the increases of the aqueous content (Wo). In general, the average distribution of the reverse micelle sizes was observed to decrease as a function of increasing NaCl concentration (Figure 2). According to the increase of NaCl, significant changes (p < 0.05) in the sizes are observed and are interesting in parallel with the increase of aqueous content.

It can be suggested that the localization of NaCl is in the polar core region or dissolved in water, so increasing the NaCl concentration exerts a sequestration effect on water molecules, and the reverse micelles lose stability due to NaCl hydration, where the sequestration exerted by NaCl would pull both molecules out of the reverse micellar structure generating a decrease in the average size distribution. Furthermore, it has been reported [42] that the role of NaCl in the reverse micelle with an interactive attraction effect between water droplets generates the interface layer to reach higher rigidity due to the packing of the polar groups. In this sense, the structural rigidity of the reverse micelle is a function of the increase of solubilized NaCl in the polar core [60] (Figure 2). This is in agreement with related studies [61] of the Span-20/Water/Mineral Oil system that suggests increases in the interfacial elasticity of the reverse micelles with increasing NaCl content; however, above 0.04 M NaCl they observed a gradual decrease in interfacial elasticity due to the salting out process of the polar heads of the surfactant. This approach, addressed by several authors [62,63,64], reveals that the maximum solubility of NaCl in reverse micelles can be explained through the processes of salting in and salting out, where the progressive addition of NaCl has the ability to decrease the interaction of the aqueous core with the interfacial layer of the reverse micelle forming a higher rigidity. Consistent with these descriptions, studies [65] targeting AOT-based reverse micellar systems suggest that the optimal NaCl concentration may be the electrolyte concentration precisely needed for the electrostatic interaction range to be such that the polar heads of the adjacent AOT surfactant completely ignore each other. Within this context, based on the behaviors observed in Figure 2 with increasing NaCl concentration, the concept of Debye length (k⁻¹) can be introduced, where k⁻¹ may become smaller than the average internal charge distance between AOT molecules in the surfactant film at the optimum salinity.

Considering the above observations, the behaviors characterized in Figure 1 and Figure 2 are convergent with literature reports, where the increase of NaCl in the polar core, through DLS measurements, leads to demonstrate again that the progressive addition of NaCl possesses the potential to reduce the interaction of the polar core with the interfacial layer of the reverse micelle making it of higher rigidity and fixing the average size distribution. This is consistent with other related studies [66,67] that project the definition of rigidity through the phenomenon of electrostriction, i.e., in systems composed of electrolytes or dielectrics, it may be similar to the volumetric compression of mechanical systems, which generate rigidity or that the elastic spring constant increases as the material becomes denser and rigid. In addition, since electrostriction occurs when a salt dissolves in a solvent, the volume of the resulting solution is usually different from the sum of the volumes of the individual components [66].

Figure 2 shows that the increase in the structural rigidity of the reverse micelles depends on the increase in NaCl concentration or the increase in the ionic strength of the solution, where the electrostriction phenomenon is contrasted in the regions closer to 5 M NaCl of the reverse micellar systems. In this sense, NaCl exerts a constriction pressure of the order of hundreds of MPa on the solvent molecules surrounding the reverse micellar system [68], virtually reducing the volume occupied by the solvent in the system (Figure 2).

2.2. Enzymatic Esterification Reactions in Reverse Micellar AOT/Water/Isooctane

The compositions of the AOT/Water/Isooctane system were extrapolated to the experimental conditions that gave rise to the model reactions of butyl oleate synthesis using the lipase CalB, i.e., the compositions of the reactive species described in Section 3.1 of Materials and Methods were kept constant by operating the enzymatic reactions under isothermal and isobaric conditions of 30 °C, 101.325 kPa, 1000 rpm, and for 5 h. As a rule of thumb, 10% substrate conversion should not be exceeded during enzymatic reactions in order to determine the reaction rate $\left(\frac{\mathsf{\mu }\mathrm{mol}}{\min }\right)$ [69]. It is important to point out that the determination of the enzymatic activity of the reverse micellar system was estimated during the initial rate by determining the synthesis of butyl oleate during the first 60 min of reaction through the depletion of oleic acid, where there was a linear relationship between the reaction rate and time; however, another relevant criterion in these systems is the enzyme–substrate affinity (Km), where several reports [70,71] have pointed out that the increase of Km is commonly found in reverse micelles according to the electrostatic interaction of the biocatalyst with the surfactant.

The effect of NaCl (0 M–5 M) concentration and Wo (5–20) on the reaction rates in the reverse micelle system structured by the synthetic surfactant AOT was initially evaluated by TLC plates (data not shown). It was qualitatively proven that the reverse micellar systems were able to provide favorable reaction conditions by allowing CalB to maintain activity at the organic–aqueous interface, where a larger surface area due to the reduced size of the reverse micelle would increase the contact area [72].

The first treatments without NaCl in the AOT reverse micellar systems showed that, in the absence of NaCl and in the presence of reduced water (Wo = 5–10), CalB had the capacity to perform butyl oleate synthesis; however, a detriment in enzymatic activity was observed when Wo was increased to 15 and 20. This is consistent with other studies [73,74] indicating that aqueous content (Wo) regulates the structure of water molecules through the reverse micelle size, where at low Wo levels, higher enzyme stability is expected and all water is arranged around the polar head of the surfactant, and, therefore, reduced hydration levels may determine the catalytic parameters of the protected enzymes. Thus, the higher enzyme stability at reduced Wo levels may be associated with a more rigid conformation of the enzyme in the reverse micelles, i.e., water is present only with a molecular lubricant function to maintain the structure and flexibility of the enzyme and the reverse micelle [75,76]. On the other hand, at higher Wo levels, structural modifications of water in the reverse micelle can lead to higher enzymatic activity [77]. However, until particularly high Wo values are reached, bulk water can lead to a loss of stability and enzyme activity [45,78,79].

Based on the above, the advantage of employing isooctane as the organic solvent composing the bulk region is clear, due to the fact that nonpolar solvents are suitable for lipase stability, as they do not sequester the essential water molecules from the enzyme active site and do not generate the enzymatic misfolding that occurs through perturbations, which are capable of producing changes in conformational flexibility [80,81]. In this regard, enzymes essentially need water bound to their surface to show conformational flexibility and enzymatic activity. However, by gradually increasing the NaCl content, relatively enhanced butyl oleate esterification results were appreciated.

Through the butyl oleate synthesis reactions in the presence of NaCl as described above, it was evident that halotolerant enzymes catalyze by effectively competing with the electrolyte (NaCl) for hydration, and this property provides insight into their functioning in environments with low water activity, including organic solvents [82]. In this context, it has been shown that halophilic and halotolerant enzymes exhibit a marked excess of negatively charged amino acids on their surface, while about 68% correspond to aliphatic residues distributed over the surface [73,83].

High cation concentrations in halotolerant and halophilic organisms may, in part, be necessary to protect the negative charges on the protein surface, since otherwise the denaturation phenomenon at NaCl/KCl concentrations below 1 M may be irreversible [77,79]. Thus, the high density of negative charges on the surface of these enzymes increases the electrostatic repulsion between them and prevents the formation of aggregates and precipitates allowing them to remain soluble at concentrations at which most enzymes would aggregate [84,85,86,87]. Given the above descriptions, it is believed that gradual changes in the transition states of CalB might be occurring in Figure 2 due to the increase of solubilized NaCl in the polar core of the reverse micelles; however, at 5 M NaCl it is noticeable that, being close to the solubility limit of NaCl, CalB reaches the highest enzymatic activity (444.85 $\frac{\mathsf{\mu }\mathrm{mol}}{\min }\pm$37.40) at Wo = 10 for this particular reverse micellar system. However, as Wo increases, a significant decrease in enzyme activity is observed (Figure 3).

Based on the wide variation of NaCl in the reverse micellar systems, it is clear that CalB exhibits increases in catalytic activity even near NaCl saturation. However, although the concept of the loop covering the active site is a matter of debate, crystallographic research suggests that the α5 helix significantly influences the catalytic properties of CalB [88]. In this context, molecular dynamics simulation studies have demonstrated the influence of α-helix 4, α-helix 5, and α-helix 10 as highly flexible regions responsible for open and closed states of CalB, which clearly promote enzymatic changes that favor the access of substrates to the active site [12,89]. Therefore, it could be associated that depending on the increase of NaCl, significant increases in the catalytic activity of CalB was observed, which can be associated with an alteration in its secondary structure and which favor the access of substrates to the active site by α5 helix [86].

Several studies on micellar systems with dissolved salts have been addressed, such as research on Halobacterium salinarum [90], which has elucidated the molecular basis for the potential of glutamate dehydrogenase (Hs GluDH) to catalyze reactions under conditions of 4 M KCl, where rapid and irreversible inactivation by exposure to solutions below 1 M KCl has been reported. In particular, enzymes from Halobacterium salinarum have received additional interest through reverse micelle studies. The CTAB/Cyclohexane/1-butanol system has been characterized such that the activity of malate dehydrogenase (hMDH) towards oxaloacetic acid (OAA) [59] was higher at low salt concentration (0.05 M NaCl). Furthermore, the study [91] on the parameters of the reverse micellar system with effects on the enzymatic activity of halophilic p-nitrophenylphosphatase (pNPPase), through the organic medium CTAB/Cyclohexane/1-butanol, revealed that the enzyme tended to a higher stability in reverse micelles than in aqueous medium, requiring 1.2 M NaCl for maximum activity. In this regard, several research groups also directed their explorations toward hydrolysis reactions through pNPPase in reverse micellar systems [71,92]. Moreover, recombinant glucose dehydrogenase (GDH) from the halophilic archaeon Haloferax mediterranei overexpressed in Escherichia coli was solubilized in the reverse micellar system CTAB/Cyclohexane/1-butanol [73]. The results showed an adequate yield for the enzymatic activity displayed by the enzyme at 1 M NaCl as the optimum condition; however, even at a concentration of 0.125 M NaCl, the reverse micellar system functioned as a stabilizer of the enzyme. Nevertheless, the evidence described above through hMDH, pNPPase, and GDH opens a window of potential applications for halophilic biocatalysts in reverse micelles.

According to the previously described results of the AOT/Water/Isooctane system, in the present research work a complementary hypothesis was formulated, where it was envisioned that several phenomena associated to the mass transfer (enzymatic activity) are favored by the increase of the interfacial area of the reverse micelles, i.e., that the enzymatic activity is inversely proportional to the size of the reverse micelles.

Figure 4 shows the convergence of the results obtained by DLS and enzymatic activity in the AOT/Water/Isooctane reverse micellar system, where the variation of NaCl unfolds an effect on the reverse micelle sizes. In this sense, it is evident that reverse micelles provide stability in the encapsulation of halotolerant biocatalysts due to their high surface area, which can be controlled as a function of water content (Wo) and electrolyte content (NaCl), and tailor specific size dimensions according to the operational requirements of the enzyme. The premise of this hypothesis relies on the use of these nanoreactors in order to reduce the limitations of mass transport phenomena and increase the operational surface area and, thus, increase the synthesis titers in halotolerant and halophilic enzymes. Therefore, the reverse micelles described in this research work showed relative increase of diffusion characteristics with effect on the enzymatic activity (Figure 4) of CalB protected in these nanoscale reactors [93,94], where these systems showed the functionality of increasing the operative surface area.

3. Materials and Methods

3.1. Preparation of AOT/Water/Isooctane Reverse Micellar Systems with the Injection Method

The reverse micellar systems were prepared as nanoreactors by preparing the samples in 2 mL graduated glass screw vials (Agilent, Palo Alto, CA, USA) through the injection method (Figure 5). The preparation of AOT/Water/Isooctane reverse micellar systems was carried out in the vials with a final volume of 1 mL, which had in composition AOT 100 mM (sodium salt of bis(2-Ethylhexyl) sulfosuccinate) (99%, Sigma-Aldrich, St. Louis, MO, USA), oleic acid 100 mM (99%, Sigma-Aldrich, St. Louis, MO, USA), 1-butanol 50 mM (99%, Sigma-Aldrich, St. Louis, MO, USA), isooctane (2,2,4-trimethylpentane) (99%, Sigma-Aldrich, St. Louis, MO, USA), and CalB in an aqueous phase. CalB lipase (CAS 9001-62-1, Sigma-Aldrich, St. Louis, MO, USA) from Candida antarctica and expressed in Aspergillus niger was employed in liquid formulation with phosphate buffer and a constant concentration of 20 $\frac{\mathrm{mg}}{\mathrm{mL}}$ in all assays. The aqueous phase in the reverse micellar systems was made up of 50 mM phosphate buffers at pH 7.2 each with NaCl variation from 0 M to 5 M. The injection method (Figure 5) [41,95,96,97] is one of the most commonly used to prepare reverse micellar systems for biocatalysts. The method consists of injecting a relatively small portion of H₂O in solution into the organic phase of the previously prepared system.

Likewise, the organic phase is structured using various components such as surfactants, substrates, nonpolar solvents, and cosurfactants as stabilizers (if required). The method proposes a simple structural model, where it is assumed that the protein is solubilized in the “aqueous phase” (polar region) within the reverse micelle, and the other components are solubilized in the bulk region of the organic solvent (nonpolar region) due to the low aqueous solubility.

The formation of the nanoemulsions was carried out in a sonicator (Scientz, Ningbo China) for 20 min until transparent systems were obtained indicating homogeneous and isotropic distribution of the aqueous phase in the organic phase through the reverse micelles. All AOT/Water/Isooctane reaction systems were carried out in an Eppendorf ThermoMixer C (Eppendorf, Hamburg, Germany) under isothermal and isobaric conditions at 30 °C, 101,325 kPa, and 1000 rpm.

3.2. Size Determination of AOT/Water/Isooctane Reverse Micellar Systems using Dynamic Light Scattering (DLS)

The size distribution was estimated employing the Malvern ZetaSizer Nano ZS90 (Malvern Instruments, Ltd., Malvern, UK), with 12.5 mm plastic cells (Sigma-Aldrich, St. Louis, MO, USA) resistant to solvents (Figure 6A). Dynamic light scattering (DLS) allows measurement of Brownian motion in relation to particle size. Through this spectroscopic correlation, the particles are illuminated with lasers, and the intensity fluctuations of the scattered light are analyzed [99,100].

The DLS system, where the samples were analyzed, consists of a laser as a light source to illuminate the particles inside the sample cell and a 90° array detector to scan submicron particle sizes. The analyzer also displays the particle size distribution in both graphical (Figure 6B) and chart representations, which provide average sizes (nm) and standard deviations (SD), among other parameters.

3.3. Characterization of Synthesis Reactions in Reverse Micellar AOT/Water/Isooctane Systems

The quantitative determination of fatty acid consumption was carried out using the colorimetric method of Kwon–Rhee [101], where oleic acid was employed as substrate. The preparation of the cupric acetate-pyridine reagent was carried out by adjusting a 5% $\frac{w}{v}$ solution of copper (II) acetate-1-hydrate (99%, Merck, Darmstadt, Germany) at pH 6.1 with pyridine (99%, Sigma-Aldrich, St. Louis, MO, USA); the reagent was stored in an amber bottle in order to avoid photooxidation of the reagent. During the analysis of the samples from the kinetics, 50 $\mathsf{\mu }$L were extracted from the reaction system, and each was mixed appropriately with 450 $\mathsf{\mu }$L of isooctane and 250 $\mathsf{\mu }$L of the cupric acetate-pyridine reagent. Subsequently, the samples were vortexed vigorously (Daigger Scientific, Buffalo Grove, IL, USA) for 30 s each; centrifuged (Hermle, Baden-Württemberg, Germany) under conditions of 13,000 rpm, 5 min, 4 °C; and analyzed at a wavelength of 690 nm using an xMark^TM UV-Vis spectrophotometer (Biorad, Hercules, CA, USA) by placing 200 $\mathsf{\mu }$L in each well of the microplate (Corning Costar, New York, NY, USA). Lipase activity was determined based on the amount of fatty acid consumed compared to the standard curve, whereby one unit of enzyme activity (U) was defined as the amount of enzyme required to consume 1 $\mathsf{\mu }$mol of free fatty acid per minute under the conditions of the previously described reverse micellar systems [38,102]. All tests were run by triplicate.

4. Conclusions

Through the experiments performed, the effects on the variation of NaCl and aqueous content (Wo) in the AOT/Water/Isooctane reverse micellar system of the model reaction of butyl oleate synthesis by lipase CalB were demonstrated. However, it is clear that the reverse micellar system structured by the synthetic surfactant AOT provides a favorable environment for CalB to catalyze synthesis reactions under different salinity conditions in the presence of isooctane. Notably, it is highlighted that reverse micelles under 5 M NaCl operating conditions emerge as effective study models for the research of halotolerant and halophilic enzymes. To date, there are no reports on synthesis reactions of lipases in the presence of high amounts of NaCl for reverse micelle systems. Therefore, reverse micelles present an advantage for halophilic lipases, and, for this reason, it is also relevant to understand how mass transport phenomena condition certain types of reverse micelles to favor diffusion as a function of surface area.

To overcome various obstacles, biocatalysts require several modifications of their natural function through media and protein engineering. To address some of the catalytic modifications, the exploration of different solvents and unnatural substrates in the reaction systems should be examined, as each biocatalyst exhibits different catalytic properties for certain conditions. According to the evidence presented, lipases seem to be optimal and suitable candidates for synthesis reactions in reverse micellar systems due to their stability and activity in conditions as aggressive as those of the bulk reaction medium. Obviously, bioprocess engineering is an important complement to achieve biocatalyst modifications as well. For this reason, it is necessary to investigate the technical-economic feasibility of the use of reverse micelles in real industrial conditions, due to the lack of information. In view of the above results, we believe that reverse micellar systems are a breakthrough for biotechnological innovation on halophilic enzyme research. In addition, current efforts are focused on improving the engineering of the media to achieve higher titers in biocatalysis.