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