Conditions of the immobilization process: the reaction was carried out in 0.1 mol·L-1 phosphate buffer (pH 7.0) with enzyme addition of 100 mg·g-1 (mass of support) for 8 h at 30°C.
3.3Optimization ofCandida rugosa lipase immobilization conditions
Lipase immobilization efficiency on hydrophobic supports is generally influenced by enzyme concentration (enzyme addition), pH, temperature, and reaction time[47]. In investigating the factor of enzyme addition (Figure 5. A ), the results showed that the enzyme loading increased with the enzyme addition. However, considering the enzyme is relatively expensive, even though the enzyme loading and activity of the CRL@OSMD were still slightly increased at more than 100 mg·g-1, the optimum enzyme addition was set to 100 mg·g-1. As shown inFigure 5. B , the higher temperature would increase the collision between enzyme molecules and support, thus improving the immobilization rate, but too high temperature will also affect the stability of enzymes in the buffer solution. Taking the CRL loading and enzyme activity together as the evaluation index, 30°C was finally selected as the optimal immobilization temperature.
The pH of the buffer solution affects the surface charge of the CRL (Figure 5. C ), which in turn affects the electrostatic interaction of the enzyme protein with the sulfonic acid groups in the support. The results showed that the immobilization efficiency and enzyme activity were significantly increased at pH 6.5, indicating that the amino group of CRL is more likely to interact electrostatically with the sulfonic acid group after ion exchange under weakly acidic conditions.
Finally, the time was optimized and the results showed that the longer the immobilization process (Figure 5.D ), the higher the loading of CRL@OSMD, but by 10 h later, the support was saturated. Continued increase in time, on the contrary, led to a decrease in enzyme activity.
Finally, immobilized CRL with a loading of 84.8 mg·g-1 and enzyme activity of 54 U·g-1 was obtained after the reaction at 30°C for 10 h in phosphate buffer at pH 6.5 with an enzyme addition of 100 mg·g-1,which is significantly higher than that of some previous studies. For instance, Cabrera et al. studied the immobilization of CALB in a series of hydrophobic supports and found that only 30 mg·g-1loading was obtained with octadecyl resin as the support[48]. Similarly, Kurtovic et al. adsorbed CRL onto a highly hydrophobic octadecyl methacrylate resin by interfacial activation and only obtained an immobilized enzyme with a loading of 58.7 mg·g-1[49]. Thus, in contrast to many existing reports, OSMD exhibited its extreme advantages in enzyme loading.
Figure 5.Optimization of immobilization enzyme addition (A), temperature (B), pH (C), and time (D) of CRL@OSMD
3.4 Thestability study of immobilized CRL
To investigate the effect of temperature on CRL@OSMD, the residual enzyme activity of free CRL and CRL@OSMD at 50°C and 60°C were measured with the p-NPP assay. As illustrated in Figure S5 , as the temperature increased, the residual activity of both free CRL and CRL@OSMD exhibited a diminishing trend. However, it is noteworthy that the relative activity of CRL@OSMD remained conspicuously higher than that of free CRL. These findings suggested that under the experimental conditions, both CRL@OSMD and free CRL retained their activities at levels exceeding 90% when subjected to a 50°C treatment for 1 h.Figure S5 shows that the residual activity of free CRL and CRL@OSMD decreased with increasing temperature, but the relative activity of CRL@OSMD was significantly higher than that of free CRL. The results indicated that both the activity of CRL@OSMD and free CRL can be maintained above 90% at 50°C treatment for 1 h. However, when the temperature was increased to 60°C, a more pronounced decline in the activity of free CRL was observed, which may be due to the irreversible denaturation and inactivation of the secondary structure of CRL by the higher temperature. In stark contrast, CRL@OSMD exhibited remarkable resilience, maintaining its activity above 80% even after 30 minutes of exposure to the heightened temperature of 60°C. This observation serves as compelling evidence that CRL@OSMD boasts superior thermal stability compared to its free counterpart under the specified experimental conditions.
To investigate the storage stability of the immobilized enzyme, free CRL and immobilized CRL (CRL@OSMD) were stored at 4°C for one month and residual enzyme activity was tested every 5 days at the respective optimum pH and temperature and the initial enzyme activity was defined as 100%. As shown inFigure S5 , the enzyme activity of free CRL decreased rapidly to less than 50.0% after 10 days, and only 11.3% residual enzyme activity can be maintained after 30 days. In contrast, the storage stability of the OSMD immobilized enzyme was significantly higher than free CRL. After 5 days of storage, 93.2% of the initial enzyme activity was still preserved, and this level of activity endured above 50.0% even after the 30-day storage duration. These results unequivocally underscore the outstanding storage characteristics of the immobilized enzyme, highlighting its impressive reusability and ability to endure extended storage periods, making it well-suited to meet the demands of production requirements.
When p-NPP was used as a model substrate to test the repetitive hydrolysis activity of the immobilized enzyme, it was found that the hydrolysis effect of CRL@OSMD could still reach 86.2% after 10 times of reuse (Figure S6 ), which indicated that the immobilized enzyme had good operational stability and repetitive use performance.
3.5 Synthesis of pine sterol oleate with OSMD@CRL in a solvent-free system
Oleic acid, as a common monounsaturated ω-9 fatty acid, works synergistically with phytosterols to regulate blood lipid levels and effectively reduce hypercholesterolemia and cardiovascular disease[50]. In addition, our previous studies also found that oleic acid is one of the most efficient substrates for the esterification reaction due to its low melting point and its high solubility of phytosterols[50] (Figure 6 ). With excess oleic acid as a solvent, the additional costs and environmental concerns associated with the use of organic solvents can be avoided[51, 52]. Optimization of solvent-free esterification involves obtaining high conversions while avoiding excess reagents and catalysts and saving energy[53]. However, solvent-free reactions present specific challenges given the drastic changes that can occur in the reaction medium during the reaction processes[54]. Specific studies are needed to determine the optimal amounts of reagents and catalysts and the optimal temperatures under these conditions, where the thermodynamic and kinetic aspects converge toward high conversion[50].
Most enzymatic esterification studies consider the molar ratio of reagents, biocatalyst addition, reaction time, and temperature as the main variables that determine the reaction yield[50]. Moreover, there are two possible strategies to change the esterification equilibrium: (1) use an excess amount of one of the reagents or (2) remove one of the product mediators (water) from the reaction[55]. As the relatively low cost of oleic acid and it can act as the solvent of the solvent-free system, the ratio of oleic acid: pine sterol was selected from 1:4 to 1:8. As shown in the results of Figure 6. A , the yield can only reach 53.4% and 65.7% at ratios of 1:4 and 1:5 which can largely be attributed to the incomplete dissolution of pine sterol in the reaction medium. Nonetheless, it’s worth noting that excessive quantities of oleic acid can also exert a detrimental influence on esterification efficiency. This is likely attributable to a reduction in the relative concentration of pine sterol within the oleic acid medium, leading to a diminished likelihood of substrate access to the enzyme’s active center. The optimal esterification yield was achieved when the molar ratio of pine sterol to oleic acid was maintained at 1:6. This ratio takes into consideration the intricate dynamics introduced by the reaction medium, resulting in the highest efficiency.
The amount of biocatalyst in the reaction is limited by the dispersing capacity of the stirring system and the filtration capacity of the system[56-58]. The amount of enzyme addition in the system does not affect the final yield of the ester in equilibrium, but it does affect the reaction rate. Reaction product-induced lipase inhibition may occur under specific circumstances of the reaction[55, 59]. As shown in the results of Figure 6. B , the lower yield of the reaction at an enzyme addition of 5.0 U·g-1 (relative to the mass of pine sterol) is most likely due to a decrease in the rate of the reaction, as well as product inhibition. The efficiency of the esterification reaction remained essentially the same at enzyme additions above 8.0 U·g-1, which was finally chosen given the high cost of biocatalysts.
The temperature has a positive effect on the energy of the reagents, favoring the effective number of collisions leading to product formation[60-62]. However, collateral effects may occur - high temperatures may cause conformational changes in the enzyme, leading to an increase (or loss) of enzyme catalytic activity[61, 63]. Thus, incremental increases in reaction kinetics due to increased temperatures may be offset by reductions in the catalytic activity of lipases[64]. Temperature also affects the solubility of the reagents and viscosity reduction, which means that there are considerable changes in the reaction medium, and these changes affect the apparent equilibrium position because only dissolved reactants are involved in the thermodynamic process, and only dissolved substrates can be contacted by the enzyme[65]. The optimal temperature for enzymatic esterification should facilitate proper diffusion of the reagents in the medium, thus maintaining the performance of the biocatalyst. As shown inFigure 6. C , the low yield at 35°C was attributed to the poor solubilization of the pine sterols and the high mass transfer resistance of the medium due to the high viscosity of oleic acid at a low temperature, which resulted in the diffusion of substrates to the active site of CRL@OSMD became more difficult. In addition, the temperature above 55°C may also have a negative effect on the esterification yield due to the denature of CRL@OSMD. Taking into account the catalytic activity of the immobilized enzyme and the efficiency of the esterification reaction, 50°C was finally selected as the optimum reaction temperature. Reaction time affects the efficiency of product production and reduces the number of times the biocatalyst can be recycled, and reaching reaction equilibrium cannot always be pursued to maximize the benefits of immobilized enzyme-catalyzed esterification. The esterification reaction yield can reach 95.0% at 48 h (Figure 6.D ), and further increasing the reaction time did not have a significant effect on the esterification yield. Moreover, only a conversion of 91.1% of phytosterols can be obtained even though the dry air was introduced to regulate water activity at 72 h[66], which may increase the production cost. Fortunately, in this study, we found that our modified diatomite with long-chain alkyls, namely OSMD, can prevent the water molecules from adhering to the surface and thus beneficial to the reaction without dry air treatment.
Figure 6. Optimization of molar ratio (A), enzyme addition (B), temperature (C) and time (D) of the esterification reaction
3. 6Substrate scope investigation of immobilized CRL
To investigate the substrate scope of the immobilized lipase (CRL@OSMD), several medium or long-chain fatty acids, such as linoleic acid (C18:2), linolenic acid (C18:3), lauric acid (C12:0), and decanoic acid (C10:0) were selected as the acyl donors. As summarized in Table 3 , both saturated and unsaturated fatty acids can be used as the acyl donors of the esterification of pine sterol with phytosterol conversions at close to or above 90%. However, as the high melting point of the long-chain (above C12) saturated fatty acids are not beneficial to the solubility of pine sterol and the transfer mass effect of the reactions, only lauric acid and decanoic acid were selected as the model of saturated fatty acid with a yield of 92.33 ± 1.29% and 90.45 ± 1.70%. To further promote the industrial production processes, edible oil, which contains oleic acid, linoleic acid, and linolenic acid was also selected as the substrate in the synthesis of pine sterol esters, and the results showed that the yield can reach as high as 94.14 ± 1.37%. This indicates that our novel-designed immobilized enzyme (CRL@OSMD) can catalyze the esterification reaction of pine sterols with excellent substrate applicability, which can be adapted to the needs of processing and production.
Table 3 . Substrate scope investigation