2.1. Immobilization of RML and Aminated RML on Different Supports
RML and chemically aminated RML (NH
2-RML) were immobilized on different carriers previously activated with different functional groups (
Figure 1).
Immobilization of RML on aldehyde functionalized-agarose (Gx-RML) retrieved 47% of immobilization yield after 20 h of incubation at pH 10, 4 °C with 0.6 UI/mg specific activity. The long-time incubation of RML at relatively harsh conditions (pH 10) lowered the specific activity of the final derivative. This is probably related to the low amount of Lys groups in the enzyme surface that limits the enzyme-support multipoint attachment. As previously reported, chemical amination of lipases, after reversible immobilization on octyl-agarose, introduces new primary amino groups with pK
b value lower than enzyme original Lys residues, thus increasing the number of possible bonds between the enzyme and support at milder pH values (pH 8–9 approx.). It has been well documented that chemical amination leads to a four-fold increment in the number of amine groups on the surface of RML [
24,
25]. As a result, the immobilization yields, as well as the derivative stability, are expected to be increased. Effectively, the immobilization of NH
2-RML on aldehyde functionalized-agarose showed 80% of immobilization yield, which is 1.7-fold higher than immobilization of native RML on this support (
Table 1).
After the desorption of both modified and non-modified RML from octyl-agarose, the corresponding free enzymes were also immobilized on CNBr-activated agarose. Immobilization of RML on this support (88%) was performed shortly after 30 min, retrieving a specific activity of 1.9 UI/mg (
Table 1). This immobilization protocol is usually carried out to immobilize the free enzyme on a solid heterogeneous support without promoting a multipoint covalent stabilizing immobilization [
26]. The linking of the NH
2-RML enzyme on this support was also performed at the same condition, resulting in only 54% immobilization yield and the specific activity of 1.6 UI/mg (
Table 1). Probably, the lower specific activity of the aminated RML after immobilization (CNBr-NH
2-RML) can be attributed to the deleterious effect of chemical amination of RML on its activity.
Oriented immobilization of RML and NH
2-RML on epoxy functionalized support was also carried out (
Table 1). In this case, the epoxy groups of the support were partially modified by a ring-opening reaction of epoxide moieties with iminodiacetic acid (IDA) followed by chelating the newly introduced carboxylate groups with Ni
2+ ions (Ni-IDA) [
18,
27].
This heterofunctional support bearing IDA-chelated Ni2+ groups were then used for covalent immobilization of both RML and NH2-RML via a two-step mechanism of immobilization. Firstly, the enzyme is adsorbed on the surface of the support by ionic interaction of the imidazole ring of histidine moieties and Ni2+ of IDA groups. After that, the covalent attachment of the adsorbed enzyme is performed by promoting the reaction between nucleophilic groups (mainly amine groups) of RML in the neighboring of adsorption site and the epoxy groups of the support.
The structure of RML obtained from the Protein Data Bank (pdb code 3TGL), shows six residues of histidine at the positions of 42,108, 143, 207, 217, and 257. Initial adsorption of the enzyme via ionic interaction is assumed to be driven and oriented by the histidine residue number 42 (H42), which is the only accessible histidine group on the surface of RML. The immobilization yields were 73% and 43% for modified and native RML, respectively producing specific activities of 0.6 UI/mg for RML and 1.1 UI/mg and NH2-RML. The strength of RML-support interaction after oriented immobilization was also examined by incubation of the immobilized preparations in a solution containing 300 mM EDTA at 25 °C for 24 h. At this condition, Ni2+ ions are expected to be removed by chelation with EDTA molecules leading to desorption of RML molecules. The Bradford and activity assays showed a negligible amount of the enzyme in desorption solution, confirming very strong covalent attachment of RML and NH2-RML onto the support.
Immobilization of RML and aminated RML on glyoxyl agarose in the presence of DTT was also performed. It has been previously reported that enzyme linkage on glyoxyl agarose using DTT is directed via the most reactive amine group on the enzyme surface (e.g., the terminal amino) [
28].
This strategy allows the enzyme to be immobilized in a milder condition (pH 8, 25 °C), thus preserving the catalytic performances of the final derivatives. After the initial stability improvement, further incubation of the derivatives at higher pH (pH 9–10) would improve the reactivity of both amino groups of the Lys residues and chemically introduced amine groups that finally promote the multipoint covalent attachment of RML. As reported in
Table 1, immobilization of RML on this support resulted in a negligible yield (11%), while in the case of modified NH
2-RML, immobilization yield was 59%, producing specific activity almost four-fold higher than the activity of native RML.
2.2. Thermal Stability of the Immobilized Derivatives
The effects of incubation at different temperatures (50–80 °C) on the activity of immobilized derivatives of RML and NH
2-RML in phosphate buffer (25 mM) at pH 7.0 were then studied (
Figure 2). The preparation obtained by the immobilization of RML on CNBr-activated support was used as reference biocatalyst because of its quite similar properties to the soluble enzyme [
29]. At 50 °C most of the derivatives remain completely active during 24 h incubation. Only epoxy-IDA-RML showed lower activity compared to CNBr-RML, showing negative the effect of immobilization of RML via histidine residue.
This observation was in agreement with our previous report on the immobilization of RML on the silica matrix via the same protocol [
19]. By increasing the temperature to 60 °C, a further decrease in the activity of the immobilized derivatives was observed. As reported in
Figure 2, CNBr-RML loses 62% of its activity. Gx-NH
2-RML and Gx-DTT-NH
2-RML were revealed to be the most thermostable preparations showing unaltered specific activities in the same condition. In general, the derivatives obtained from the immobilization of native RML showed lower thermal stabilities compared to the immobilized preparations of NH
2-RML. These results further confirm the significant effect of multipoint covalent attachment of enzymes on their thermal stabilities. By further increasing the temperature to 70 °C, Gx-NH
2-RML, Gx-DTT-RML, and Gx-DTT-NH
2-RML were the only derivatives still active after 24 h of incubation while CNBr-RML lost its whole activity at the same condition. The highest stability at this temperature was shown by Gx-NH
2-RML with retaining 47% of its initial activity. By incubating these active derivatives at 80 °C, it was observed that Gx-NH
2-RML and Gx-DTT-NH
2-RML still retained 13% and 9% of their initial activities, respectively. This great increment in enzyme stability can be attributed to the suitable rigidification of NH
2-RML on glyoxyl support in these immobilized preparations by multipoint covalent attachment. Glyoxyl agarose has been described as a very suitable support for enzyme-matrix multipoint covalent attachment [
30]. In fact, the differences in increased stability can be explained by an increase in the number of support-enzyme bonds that resulted in an intense multipoint attachment.
2.3. Stability of RML Derivatives in the Presence of Organic Solvents
Organic solvents, particularly those having log P values below 2, can strongly distort the required water-enzyme interaction, thus denaturing the enzyme structure and decreasing its catalytic activity [
31]. In order to investigate the effect of immobilization protocol on co-solvent stability, the immobilized derivatives of RML and NH
2-RML were incubated in the presence of three water-miscible solvents (20% of n-propanol, iso-propanol, and dioxane) for 24 h (
Figure 3). The immobilized derivatives showed improved stability compared to the reference derivative (CNBr-RML). In the presence of n-propanol and iso-propanol, CNBr-RML lost its whole activity while it still retained only 22% of its initial activity in the presence of dioxane. Incubation of the immobilized preparations in the presence of dioxane produced different results (
Figure 3). Epoxy-IDA-RML, Gx-NH
2-RML, and Gx-DTT-RML retained 72%, 76%, and 100% of their activities after 24 h of incubation in the presence of dioxane, respectively.
Conversely, Gx-RML, CNBr-NH
2-RML, and epoxy-IDA-NH
2-RML lost the major part of their activities in the same condition. Furthermore, a comparison of the obtained results from the co-solvent stability of aminated vs. native RML shows that the strength of enzyme-support linkage is not the only effective factor in the solvent stability of the derivatives. In fact, propanol seems to have the highest deleterious effect on the stability of the immobilized derivatives in which most of the preparations lost their complete activities after 24 h. The only stable derivative was Gx-NH
2-RML that showed very interesting results while maintaining 100% of its activity (
Figure 3). In 2-propanol, the reference derivative was the most unstable preparation, losing entirely its initial activity. On the other hand, the derivatives obtained from the immobilization of RML and NH
2-RML on glyoxyl by using DTT showed higher stabilities with 100% and 71% residual activities for Gx-DTT-RML and Gx-DTT-NH
2-RML, respectively.
2.4. Fish Oil Hydrolysis
Many researchers have reported enrichment of PUFAs in fish oil by using lipases in free and immobilized forms [
32]. It has also been reported that small changes during immobilization strategy can alter the selectivity and activity of the immobilized enzyme. For example, the modulation of lipases selectivity in fish oil hydrolysis has been reported by oriented immobilization [
16]. Therefore, the selectivity of the immobilized derivatives of RML was examined in selective hydrolysis of fish oil in order to evaluate the effect of different immobilization strategies on the selectivity of RML. The reaction temperature and pH were the variable parameters assessed in this experiment by applying two temperatures (25 °C and 4 °C) and two pH value (pH 5 and 7). For the quantitation of activity and selectivity, an HPLC–UV analysis was performed. Both parameters were determined by measuring the releasing rate of EPA and DHA during the reaction. All derivatives displayed a significant preference for EPA in comparison to DHA. The observed selectivity has been previously reported for different lipases by several researchers.
Table 2 and
Table 3 show the results for the hydrolysis reactions in different conditions. The reported activities were calculated by the following equation:
The immobilized derivatives of RML and NH
2-RML presented different results in the reaction based on the type of immobilization procedure (
Table 2). For example, a broad range of activities (0.01–1.7) and selectivities (2.7–32.9) were observed by using different procedures and reaction conditions.
As can be seen from
Table 2, the highest catalytic efficiency of the derivatives was achieved with the reactions performed at pH 7.0 and 25 °C, presenting the most suitable condition for the application of the immobilized preparations. At this condition, the most active enzyme was NH
2-RML immobilized on epoxy-IDA with the catalytic efficiency of 1.7 followed by CNBr-RML with a catalytic efficiency of 0.5. The highest EPA/DHA selectivity (18.1) of biocatalysts at pH 7.0 and 25 °C was also obtained for epoxy-IDA-RML. For the other biocatalysts, low to moderate selectivities (ranging between 2.7 and 8.8) were observed. The reduction of pH value from 7 to 5 showed a negative effect on the catalytic efficiency of all the derivatives, while the EPA/DHA selectivities increased if compared to the selectivity values obtained at pH 7, 25 °C. In fact, the highest selectivity (22.1) at this condition was obtained for epoxy-IDA-NH
2-RML, while its catalytic efficiency was 1.1, which is 65% of catalytic efficiency of this derivative at pH 7, 25 °C. Further investigation was performed on the selected biocatalysts with the highest catalytic efficiency and selectivity to evaluate the effect of temperature on their functional properties (
Table 3). The results showed that lowering the temperature caused a remarkable improvement in the selectivity of the selected biocatalysts (CNBr-RM, epoxy-IDA-RML, and epoxy-IDA-NH
2-RML).
As a general trend, the reduction of the hydrolysis rate of the reaction due to the low temperature allows achieving higher selectivities. As shown in
Table 3, oriented immobilization of chemically aminated RML on epoxy-IDA greatly improves its selectivity at pH 5.0 and 4 °C. This improved selectivity accessed the production of almost 97% of pure EPA at the first stages of the reaction.
2.5. Recyclability of the Selected Biocatalysts in Fish Oil Hydrolysis
Recyclability of an immobilized enzyme is crucial to lowering the process economy in large-scale application of enzymes. The ability of the three selected derivatives for the repeated use in the hydrolysis of fish oil was examined for five cycles. The immobilized lipases were removed by filtration after each run (8 h), washed with cyclohexane, and reused for a new hydrolysis process under the same condition. The enzyme catalytic efficiency in the first cycle of the reaction was set as 100%, and then catalytic efficiency in the subsequent reactions was calculated accordingly. Experiments investigating recyclability indicated good capability of the immobilized biocatalysts to be used repeatedly, retaining 49–91% of their catalytic efficiencies after five cycles of the reaction (
Figure 4). For epoxy-IDA-NH
2-RML in the first three cycles, no loss of catalytic efficiency was appreciated, and after the fifth cycle, the enzyme lost only 9% of its catalytic efficiency.
For epoxy-IDA-RML, after the first two reuses, no relevant loss of catalytic efficiency was observed (−10% approx.), and it decreased up to 70% of retained catalytic efficiency after the fifth use. For CNBr-RML, the initial catalytic efficiency dropped up to the half after five cycles at the same condition. Altogether, the results clearly demonstrated the positive impact of oriented-multipoint covalent immobilization of aminated RML in order to improve the recyclability of biocatalyst in comparison with the results obtained from the of not-aminated RML derivatives.