3.1 Immobilization of Eversa® Transform and Eversa® Transform 2.0 in different carriers
After evaluating the behavior of lipases Transform Eversa and Eversa Transform 2.0 immobilized in the production of esters with hexane in a previous study [15] the production without solvents was investigated.
The enzymatic transesterification of sunflower oil with ethanol in a solvent-free medium to produce FAEE using two commercial lipases, Eversa® Transform and Eversa® Transform 2.0, was evaluated in this work.
The lipases were immobilized by interfacial adsorption on four hydrophobic supports with different characteristics Sepabeads-C18, Lewatit-DVB, Purolite-C18, and Purolite-DVB. In all cases, the enzyme loading of the adsorbed preparations was 20 mg ml− 1 of support, with immobilization yields higher than 95 wt%, as compared to the initial enzyme activity. Table 1 shows the activity and production performance of the FAEE for each of the catalysts studied.
Evaluating the performance of the lipase Eversa Transform (Table 1), it appears that the reaction activity varied between 88.61 U g− 1 min− 1 and 144.7 U g− 1 min− 1 using the different supports. Also, the yield of the ester after 24 h of reaction was different for the supports, producing 41.7 wt% with the lipase immobilized in Lewatit-DVB, this being the lowest production, 61.3 wt% using the Purolite-DVB support, 66.5 wt% with Sepabeads-C18 support, and 68.5 wt% for immobilization on Purolite-C18.
The immobilized Eversa transform 2.0 lipase showed a higher reaction rate than the Eversa lipase, resulting in values between 117.9 U g− 1 min− 1 and 208.3 U g− 1 min− 1. Lipase Eversa 2 also showed lower values in FAEE yield and activity when immobilized in Purolite DV (65.1 wt%) and Lewatit (55.1 wt%) and better results when immobilized in Purolite C18 (86 wt%) and Sepabeds C18 (98 wt%). The lipase Eversa Transform 2.0 stood out producing higher final content of FAEE (98 wt%) and in general, it had a greater activity compared to lipase Eversa Transform.
Both lipases Eversas immobilized on hydrophobic supports showed high enzymatic activities. High activity is due to the immobilization of the lipase in the open form. According to Fernandez-Lafuente [21], the immobilization of lipases on hydrophobic supports has been attributed to the interfacial activation of the lipases versus the support surface.
Four commercial resins were used as hydrophobic supports in this study. Purolite C-18 and Sepabeads C-18 are both methacrylic resin functionalized with octadecyl groups. Purolite-DVB and Lewatit DVB are methacrylic resins, non-functionalized divinylbenzene groups.
How observed the Table 1, the results obtained using lipases immobilized the Purolite and Seapabeads supports with octadecyl groups showed a higher FAEE content. The difference in the performance of the lipases immobilized with the different supports may be linked to the characteristic of the support itself, such as structure and properties [20]. Thus, the morphology and functional hydrophobic moieties of the support where the lipase is immobilized are determinants in the reaction.
The amount of octadecyl functional groups available at the surface of the polymer create a highly hydrophobic environment that allows for better activation of the lipase during the immobilization process [26]. The increase of octadecyl groups lead also to a decrease both in the pore volume and pore diameter size of the resin leads to a high density of immobilized enzyme available leading to higher enzyme activity [26, 35, 36].
The excellent immobilization performance of Eversa lipases in Seapabeds C18 was also observed in previous work, in which the enzymatic transesterification of sunflower oil with ethanol in hexane to produce FAEE was evaluated [15]. In this previous work, both lipases immobilized in Seapabeds also were able to recognize the three positions of the oil, thus allowing to achieve high yields close to 100 wt%.
Lipases usually are sn-1,3-regioselective due to these achieve esters yields of up to 66 wt%, here the immobilized lipases in Lewatit- DVB had FAEE yield was only 55 wt%, in this case, the immobilization in this support might have altered the selectivity of lipase however, further investigation is needed.
Due to the excellent yields obtained with both enzymes, Eversa® Transform and Eversa® Transform 2.0, immobilized in Seapabeads-C18, we chose to follow the investigation of the production of esters and the behavior of immobilized lipases only on this support.
3.2 Effect of Alcohol in the Transesterification of Sunflower Oil Catalyzed by Sepabeads–Eversa Lipases
To evaluate and expand the application of lipases in solvent-free transesterification reactions, we studied the use of two different types of alcohols as substrates to produce esters. Thus, the study of the effects of alcohol (ethanol and methanol) on the transesterification of sunflower oil with the Eversa® Transform and Eversa® Transform 2.0 lipases can be observed in Fig. 1.
Looking the Fig. 1 the reaction that had the fastest product formation, that is higher initial velocities and catalytic activities, was lipase Eversa® Transform 2.0 using methanol as substrate. With the same immobilized lipase, but using ethyl alcohol as a substrate, an initial velocity, and the catalytic activity in the formation of esters were lower. In 3 h, using the lipase Eversa Transform 2.0 is possible to get about 98 wt% FAME, but with ethanol 98 wt% FAEE is only achieved with 24 h of reaction, this synthesis is 8 times slower.
The Eversa® Transform enzyme (Fig. 1) showed lower yields in both FAME (0.5 wt%) and FAEE (66.5 wt%) transesterification of sunflower oil in solvent-free reaction. Because of the minimal formation of FAME using the immobilized lipase Eversa® Transform, the denaturation of this enzyme may have occurred due to the excess of methanol offered in this assay.
The performance of the Eversa® Transform enzyme showed lower yields in both FAEE and especially in FAME compared to the Eversa® Transform 2.0 enzyme. The ideal molar ratio of alcohol to oil in a reaction is highly dependent on specific characteristics of lipase and the type of alcohol used for synthesis. A study conducted by Mibielli [9] using a Lipase Eversa Transform 2.0 in its liquid form for the production of biodiesel, indicated in synthesis the preference of this lipase for a higher molar ratio methanol, they using a 7.2: 1 molar ratio of methanol to residual soybean oil as ideal for production in laboratory and pilot plant biodiesel production.
3.3 Effect of Molar Ratio Alcohol in the Transesterification of Sunflower Oil Catalyzed by Sepabeads–Eversa Lipases
To better investigate as alcohol concentration affects lipases and the formation of esters we tried to maximize the reaction using a different molar ratio of alcohol. The alternative molar ratios between oil and alcohol (methanol or ethanol) were based on previous studies [15].
In Fig. 2, it can be seen that using different concentrations of ethanol it is possible to increase the reaction speed and FAEE yield for the Eversa® Transform lipase. Using the lowest molar ratio 3: 1 (E: O) it is possible to achieve the total conversion of the oil to FAEE in 3 h of reaction.
However, with methanol, the behavior of Eversa Transform is completely different, even when using the lowest molar ratios of 3: 1 (M:O). In a previous study, Eversa Transform immobilized on Seapabeads-C18 was using for the transesterification of sunflower oil in an anhydrous medium using hexane as a solvent, the performance of the immobilized enzyme was higher, producing 100 wt% FAEE in 6 hours and 60 wt% FAME in 24 hours of reaction. In an investigation by Remonatto [8] using a Transform Eversa in its free form for the production of FAME from waste oils and methanol, esters content greater than 97 wt% were reached, however for the stability of the enzyme, the presence of water in the reaction medium was essential.
Is possible that the stability of the Eversa Transform immobilized was affected by the absence of organic solvents or water in the reactions studied in this work. Thus, in solvent-free transesterification reactions, anhydrous medium, and the presence of methanol, the immobilized Eversa® Transform on Seapabeads-C18 was unable to develop its catalytic power, probably due to its denaturation.
In Fig. 3, the decrease of the molar ratio from 4: 1 (E:O) to 3: 1 (E:O) brought benefits by increasing the reaction speed, since Eversa® Transform 2.0 achieved higher esters content (> 98 wt%) in less reaction time, only 3 hours (MR 3: 1) compared if at 24 hours in the reaction using 4: 1 (E:O) molar ratio. However, with the lowest 2: 1 (E:O) molar ratio researched, the conversion of FAEE at the end of the reaction was the lowest, 81 wt% FAEE in 6 h.
The high conversion in FAEE or ethyl oleate (> 98 wt%) with both lipases Eversas immobilized was obtained in a short time, in 3 h of reaction. Chiaradia [37] obtained 85 wt% ethyl oleate using the ethanol/oil molar ratio of 3: 1, 7 wt% Candida Antarctica B lipase immobilized on magnetic poly (urea-urethane) nanoparticles at 50° C and stirring at 150 rpm for 4 h Ferrero [38] evaluated the production of ethyl esters from used frying oil and ethanol, using Pseudomonas fluorescen lipase immobilized on supports modified SBA-15 mesoporous systems, in a batch system, using the conditions, 4 wt% water, 1: 4 oil:ethanol molar ratio, 0.4 g enzyme immobilized, at 37 ° C and stirring at 180 rpm, where, after 24 hours of reaction, obtained a 90 wt% FAEE yield.
In the study of the effect of methanol on the transesterification reactions with the Eversa® Transform 2.0 (Fig. 3b) was investigated novels ratios molar. The lipase had already performed well in the assay using the 4: 1 (M:O) molar ratio, with this, established two new molar ratios: a highest 11: 1 (M:O) and a lowest 3: 1 (M:O), providing for the immobilized lipase a greater range to evaluate its performance.
As observed the Fig. 3b with the highest molar ratio 11: 1 (M:O), the Eversa Transform 2.0 did not better perform, not producing relevant FAME values (< 2 wt%). With the molar ratios 4: 1 (M:O) and 3: 1 (M:O) and in the reaction time of 3 hours, it reached high ester content (> 98 wt%), however, the initial velocity in the reaction was lower with the molar ratio 3: 1 (M:O). Molar ratio 4: 1 (M:O) produce good results in a short reaction time (3h), with a higher initial velocity and more importantly in a solvent-free and anhydrous medium. Facin [14] immobilized the lipase Eversa Transform 2.0 in flexible polyurethane (PU), where its necessary 24 hours to convert 91 wt% FAME in the operational conditions were 2 wt% of water, 2.0 eqv. of methanol, 300 ppm of NaOH, and 500 ppm of the enzymatic cofactor.
In the research of the Eversa Transform 2.0 immobilized on Seapabeads-C18 was using for the transesterification of sunflower oil in an anhydrous medium using hexane as a solvent, the performance of the immobilized enzyme in a higher molar ratio of methanol 12:1 (M:O) was better resulting in yield 80 wt% FAME, the solvent absence affects the excellent performance this immobilized lipase. Chang [11] investigated the methanol tolerance of liquid lipase Eversa Transform 2. According to with study the enhanced methanol tolerance of the enzyme may be attributed to the gentler environment created by the water. Because this the Eversa Transform 2 has preferred for esterification reaction that can water generated from esterification of FFA.
The analysis of the stability of Eversa® Transform 2.0 immobilized on Seapabeads-C18 in the presence of ethanol and methanol was previously studied [15]. For the Eversa Transform 2.0 immobilized on Sepabeads, the stability in the presence of ethanol was greater than methanol, in ethanol, in 7 h the lipase activity remained stable at 95 wt% but after 7 h of incubation in methanol, the derivative loses more than 50 wt% of its initial activity [15].
Here the stability of Eversa® Transform immobilized on Seapabeads-C18 was study. The stability of Eversa Transform (Fig. 4) in the ethanol was better, in 7 days the lipase activity remained stable at 95 wt%, but with methanol, the activity reduced for 62 wt% of the original in 7 days. The answer found for stability to ethanol were similar for both Eversa lipases, but in the stability, of methanol, the performance of the Eversa® Transform 2.0 lipase was worse. So the Eversa Transform lipase had greater is stability than the Eversa Transform 2.0 lipase immobilized on Seapabeads-C18.
Transesterification reactions were carried out in the total absence of water and organic solvent, even so, both lipases showed good results (> 98 wt%) in the production of FAEE and FAME. The solvent-free reaction requires a much higher stabilization of the immobilized biocatalyst because anhydrous oils are a very deleterious medium [30]. Therefore, strategies are needed to increase the stability of the enzyme in the Sepabeads-C18 support. With greater stability of the enzyme in the Seapabeads-C18 support, it will be possible to protect the active center of the lipase from denaturation/inactivation.
Abreu [30] investigate the Thermomyces lanuginosus lipase (TLL) immobilization by interfacial adsorption on octadecyl (C-18) supports. The TLL immobilized on Purolite C-18 was 20 times less stable in anhydrous oil than in anhydrous hexane. As a strategy for improving the stability was made a mild PEGylation of immobilized TLL, greatly increased its stability in hexane anhydrous fully preserving the activity after 20 days, and in anhydrous oil, the PEGylated TLL-Purolite C-18 retained 65 wt% of its initial activity after six days compared to 10 wt% of the activity retained by the unmodified biocatalyst.
As the immobilized lipases, Eversa® Transform and Eversa® Transform 2.0 are more stable to ethanol when compared to methanol. Allied to the good performance of both immobilized lipases in the reactions of ethyl oleate production, these were selected for the study of the use cycles in transesterification reactions using sunflower oil and ethyl alcohol, in an organic solvent-free system.
3. Cycles of Use of the Eversa® Transform (A) Eversa® Transform 2.0 (B) lipases in the Synthesis of FAEE.
Was defined as the best reaction conditions for studying the use cycles in solvent-free sunflower oil transesterification reactions for the production of ethyl oleate, in the ethanol/sunflower oil molar ratio of 3:1 in 3 hours reaction for both Eversa lipases immobilized on Sepabeads-C18.
In Fig. 5 can observe that the immobilized lipase Eversa® Transform kept the FAEE content high (> 98 wt%) for 2 cycles, after that, it presented about 75 wt% for another 3 cycles (cycles 3, 4, and 5) and 70 wt% FAEE in cycle 6. The lipase Eversa® Transform 2.0 was able to maintain a high ester content in the first (98 wt%) and second (95 wt%) cycles, however, in the third it presented less than 20 wt% ethyl esters.
In a study previous [15] the reuse of Sepabeads–Eversa® Transform 2.0 in the ethyl oleate production an anhydrous medium using hexane as a solvent, has better results, allowing the use of immobilized lipase for 4 reaction cycles. The performance of immobilized lipase in a solvent-free medium is completely different than in an organic solvent medium. As already described, the stability of the derivatives in anhydrous oil is generally less. The modifications of enzymatic derivatives as the PEGylation study by Abreu [30] can be studied in future works in the Eversa Transform 2.0 lipase immobilized on Sepabeads-C18 as a strategy to improve lipase stabilization and achieve better results in its reuse in solvent-free reactions.
As a result of these results, it is evident that despite the high catalytic speed presented by the lipase Eversa® Transform 2.0 throughout the study in solvent-free reactions, an Eversa® Transform lipase is the one that presents the best performance in the transesterification reactions of sunflower oil with ethyl alcohol in a solvent-free medium. The Eversa® Transform lipase immobilized in Seapabeads-C18 to presenting high production of ethyl esters (> 98 wt%) and excellent reaction time for the production of the compounds of interest (3 hours), it also presents a possibility of reusing lipases for up to 5 cycles with relevant values of FAEE.