Enzymatic Production of Phosphatidylserine Using a Phospholipase D Immobilized via a Composite Polysaccharide Strategy

Abstract

Phosphatidylserine (PS), a valuable phospholipid, is widely used in food, pharmaceutical and cosmetic industries. Its enzymatic synthesis, catalyzed by phospholipase D (PLD) via transphosphatidylation under mild conditions, has drawn considerable attention. However, the industrial use of free PLD is limited by poor stability, difficult recovery, and high cost. To address these challenges, a ternary composite carrier—integrating the flexibility of chitosan, the stability of cellulose, and the macroporosity of agarose—was constructed for immobilizing the PLD from Streptomyces antibioticus (saPLD). The resulting saPLD@chitosan–cellulose–agarose biocatalyst demonstrated enhanced immobilization efficiency, catalytic performance, and stability across varying pH and temperatures. After eight consecutive batches of usage, the PS yield of saPLD@chitosan–cellulose–agarose reached over 60% of that from the first batch. Thus, this study established a new method for preparing immobilized saPLD, and developed a robust and promising platform for the efficient and sustainable production of PS.

1. Introduction

Phosphatidylserine (PS) is a phospholipid with significant applications in the food, pharmaceutical, and cosmetic industries. It represents a key constituent in liposomal drug delivery systems, acts as a biocompatible surfactant in cosmetic formulations, and is widely employed as a dietary supplement for its functional health benefits. Furthermore, emerging evidence suggests that PS supplementation may ameliorate cognitive deficits and memory impairment in elderly patients affected by neurodegenerative and age-related conditions, such as Parkinson’s disease, Alzheimer’s disease, dementia, epilepsy, and geriatric depression [1,2,3]. PS has historically been sourced from animal tissues, such as bovine brain or cortical matter. However, the utilization of these animal-derived materials poses significant risks for transmissible infectious diseases, rendering them unsuitable for human therapeutic or nutraceutical applications [4,5]. PS could also be extracted from soybean, egg yolk, vegetable oils, and other biomass [6]. However, the extraction process was hindered by a lengthy preparation phase, challenging separation and purification steps, and ultimately, a low final yield [7]. In contrast to conventional extraction from natural sources, the utilization of enzymes in the production of PS has garnered significant attention due to its ability to operate under mild reaction conditions and its environmental sustainability [8].

The phospholipase D (PLD) (EC 3.1.4.4) catalyzes two distinct reactions: (i) the hydrolysis of PC to phosphatidic acid (PA) and choline by cleaving the phosphodiester bond, and (ii) a transphosphatidylation reaction, which transfers the phosphatidyl moiety to an acceptor. The transphosphatidylation of phosphatidylcholine (PC) with L-serine, catalyzed by PLDs, represents an efficient route for the PS production [9,10,11]. Up to now, PLDs have been found to be abundant in plants, animals, and microorganisms [12]. Among them, PLDs from Streptomyces strains—such as S. antibioticus [13], Streptomyce sp. PMF [14], and S. chromofuscus [15]—have attracted significant attention. Streptomyces PLDs are the preferred catalysts for the PS production, owing to their simpler preparation, broader substrate specificity, and superior transphosphatidylation activity, compared with PLDs from other sources [16]. However, the industrial application of free PLDs is currently limited by high costs, primarily resulting from low production yields and difficulty in reusability. In addition, the free PLDs render the system vulnerable to deactivation by environmental factors, including temperature and pH. These factors have been demonstrated to exert a substantial effect on the efficiency of PS production [17].

Enzyme immobilization on/in solid supports is a promising strategy to overcome these limitations, offering advantages such as enhanced stability, easier handling, and reusability. Over recent decades, a wide range of materials—including polymer resins, carbon nanotubes, hydrogels, ordered mesoporous silica, gold nanoparticles, and magnetic nanoparticles—have been employed to prepare immobilized PLDs for this purpose [18,19,20,21,22,23]. Polysaccharides, which function as carriers for immobilizing enzymes, have drawn much attention recently. Such polysaccharide-based carriers are easily available and facile to fabricate. They exhibit desirable properties for biocatalysis, including insolubility in aqueous environments, biocompatibility, non-toxicity, biodegradability, and physiological inertness. Thus, using polysaccharides as the carrier for enzyme immobilization has already been regarded as a highly promising, effective, and economical biotechnological process for applications in environmental monitoring, food, biotransformation, and pharmaceutical industries [24]. However, the use of single polysaccharides as the carrier for enzyme immobilization presents certain limitations, primarily concerning structural mechanical strength, mass transfer efficiency, and chemical stability [25].

In this study, the free saPLD was successfully immobilized onto a composited polysaccharide carrier composed of chitosan, cellulose, and agarose. The ratios of the three types of polysaccharides were optimized, and the optimal conditions for the immobilization process were systematically determined. The resulting biocatalyst, designated as saPLD@chitosan–cellulose–agarose, was evaluated for its thermal and pH stability in comparison to the free saPLD. Furthermore, its batch catalysis efficiency was also characterized.

2. Materials and Methods

2.1. Materials and Reagents

Standard samples of PC (≥99%, from soybean) and PS (≥97%, from soybean) were provided by Sigma-Aldrich (St. Louis, MO, USA). Soybean lecithin (PC content ≥ 90%) was supplied by Yuanye Bio (Shanghai, China). L-serine was ordered from Solarbio Tech (Beijing, China). Chitosan (S52550, Mw = 150 kDa, DD ≥ 95%) was supplied by Yuanye Bio (Shanghai, China). All other chemicals and reagents mentioned in this study were of analytical grade.

2.2. Preparation of Immobilized Carriers

To prepare the chitosan carriers, 4.0 g of chitosan powder was dissolved in 100 mL of a 5% acetic acid solution under constant stirring at 60 °C for 1 h. The solution was subsequently subjected to sonication to remove air bubbles. Using an extrusion method, the chitosan–acetic acid solution was added dropwise into a coagulation bath consisting of 2 M NaOH and anhydrous ethanol with a ratio of 3:1 (v/v). The resulting carriers were immersed for 3 h to complete solidification.

The fabrication of the chitosan–cellulose composite carriers involved the dissolution of a mixture of chitosan and cellulose powders, with a total mass of 4 g, in 100 mL of a 5% acetic acid solution. The specific mass ratios employed in this process were 3:1, 4:1, and 5:1, respectively. The formation and coagulation of the polysaccharide carriers were performed as mentioned above.

Chitosan–cellulose–agarose composite carriers were prepared using three powder components with total mass fixed at 4 g. The mass ratios of chitosan, cellulose, and agarose were set to 4:1:0.25, 4:1:0.5, and 4:1:1 respectively. The polysaccharide carriers underwent formation and coagulation as mentioned above.

2.3. Characterization of the Carriers

The scanning electron microscopy Apreo (SEM, Thermo Fisher Scientific, Waltham, MA, USA) was utilized to detect the surface morphology of the carriers. The dry carriers were mounted on a stub using carbon tape and then sputter-coated with a thin layer of gold prior to SEM analysis to enhance conductivity. The surface morphologies of the carriers were finally examined by SEM. Fourier transform infrared spectroscopy (FT-IR) was employed to identify the functional groups of the carriers. The measurements were carried out via the KBr pressed-pellet method. FT-IR spectra of the samples were recorded on a Bruker TENSOR spectrophotometer (Karlsruhe, Germany) over a wavenumber range of 4000 to 400 cm⁻¹.

2.4. Optimum Synthesis Conditions of Immobilized saPLDs

The saPLD was produced following the protocol reported by Mao et al. [26]. Namely, a single colony of each recombinant B. subtilis WB600 strain was selected from a LB agar plate and precultured in 50 mL of LB medium at 37 °C with shaking at 200 rpm for 12 h. Subsequently, 2 mL of freshly prepared seed culture was inoculated into 100 mL of LB medium in 250 mL shake flasks and fermented at 37 °C with shaking at 200 rpm for 48 h. Then, the culture was subjected to centrifugation at 4000 g and 4 °C for 5 min, and the supernatant was transferred to the purification stage. The supernatant was applied to a Ni-NTA agarose resin column that had been equilibrated with a buffer composed of 20 mM Tris-HCl (pH 7.4), 20 mM imidazole and 500 mM NaCl. Following the removal of unwanted protein by washing buffer, the target protein was finally eluted with 20 mM Tris-HCl (pH 7.4) containing 500 mM imidazole and 500 mM NaCl. The purity of saPLD was determined by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The concentrated purified protein was utilized for the purpose of further immobilization.

The chitosan, chitosan–cellulose, and chitosan–cellulose–agarose carriers were firstly crosslinked with varying concentrations of glutaraldehyde (0.01%, 0.05%, 0.1%, 0.5%, and 1.0%) for 1 h at 20 °C. Thereafter, the carriers were adsorbed with saPLDs for a period of 3 h at 20 °C to yield immobilized saPLDs. Subsequently, the recoveries of their enzyme activities were measured according to Section 2.5 to ascertain the optimal glutaraldehyde concentration.

The crosslinking of chitosan, chitosan–cellulose, and chitosan–cellulose–agarose carriers were conducted at the optimal glutaraldehyde concentrations for a duration of 1 h at a range of temperatures (20, 30, 40, 50, and 60 °C). Thereafter, the carriers were adsorbed with saPLDs for a period of 3 h at 20 °C to yield immobilized saPLDs. Subsequently, the recoveries of their enzyme activities were measured according to Section 2.5 to ascertain the optimal crosslinking temperature.

The chitosan, chitosan–cellulose, and chitosan–cellulose–agarose carriers were crosslinked at their optimal glutaraldehyde concentration and crosslinking temperatures for different time periods (1, 2, 3, and 4 h). Thereafter, the carriers were adsorbed with saPLDs for a period of 3 h at 20 °C to yield immobilized saPLDs. Finally, the recoveries of their enzyme activities were measured according to Section 2.5 to investigate the optimal crosslinking time.

The crosslinking of chitosan, chitosan–cellulose, and chitosan–cellulose–agarose carriers were conducted at the glutaraldehyde concentration, crosslinking temperature, and crosslinking time that were optimal for the process. Subsequently, the immobilized saPLDs were prepared by adsorption at different temperatures (10, 20, 30, 40, 50, and 60 °C) for 3 h. The recoveries of their enzyme activities were then measured according to Section 2.5 to ascertain the optimal adsorption temperature.

The carriers were first crosslinked under their respective optimal conditions, and the adsorption was conducted for different durations (1, 2, 3, and 4 h) at 20 °C. The recoveries of their enzyme activities were then measured according to Section 2.5 to ascertain the optimal adsorption time.

2.5. Measurements of PLD Activity

The yield of PS from L-serine and PC was utilized to determine the transphosphatidylation activity of saPLD@chitosan–cellulose–agarose. The reaction mixture was prepared by mixing 1.5 mL of PC solution (0.022 M) in ethyl acetate and 1 mL of 0.2 M sodium acetate buffer (pH 6.0) containing 12 mg of L-serine. The reaction was initiated by adding either 1 mL of free saPLD or 1 g of the immobilized saPLD@chitosan–cellulose–agarose. The reaction mixture was incubated at 50 °C for 20 min. The reaction was then terminated by adding 4 mL of chloroform/methanol (2:1, v/v) solution, followed by extraction of the phospholipids. The mixture was centrifuged at 12,000× g for 10 min, and the lower phase was analyzed by high-performance liquid chromatography (HPLC). HPLC analysis was performed on an Agilent Technologies system (Palo Alto, CA, USA) equipped with a ZORBAX Rx-SIL silica gel column (5 μm, 4.6 × 250 mm) and an ultraviolet detector, with a mobile phase of acetonitrile/methanol/phosphoric acid (95:5:0.8, v/v/v) at a flow rate of 0.3 mL/min. The column temperature was maintained at 25 °C. The PS was detected by monitoring the absorbance at 205 nm. One unit (U) of PLD activity was defined as the amount of enzyme required to produce 1 μmol of PS per minute under the specified assay conditions.

2.6. Enzymatic Properties of Free saPLD and saPLD@chitosan–cellulose–agarose

The effects of temperature on the activity of free saPLD and saPLD@chitosan–cellulose–agarose were investigated by conducting reactions at various temperatures (30, 40, 50, 60, and 70 °C). The highest PS yield was defined as 100% to calculate the relative enzyme activities under other conditions, thereby determining the optimal catalytic temperature. The thermostabilities of free saPLD and the saPLD@chitosan–cellulose–agarose were determined by incubating them without substrate at 40, 50, and 60 °C for 1 h. Subsequently, their residual activities were measured under the standard assay condition.

To identify the optimum pH of free saPLD and saPLD@chitosan–cellulose–agarose, their activities were determined with the standard assay condition in a pH range of 5.0–10.0. The highest PS yield was defined as 100% to calculate the relative enzyme activities under other conditions, thereby determining the optimal catalytic pH. To determine the pH stability, free saPLD and saPLD@chitosan–cellulose–agarose were pre-incubated at different pH (6.0, 7.0, and 8.0) for 7 d without substrate, and the residual activity was estimated under the standard assay condition.

2.7. Catalytic Batches of saPLD@chitosan–cellulose–agarose

An amount of 1.0 g of saPLD@chitosan–cellulose–agarose was introduced into the catalytic system described in Section 2.5. After each batch reaction, the saPLD@chitosan–cellulose–agarose was separated from the reaction mixture. The supernatant was subjected to extraction with chloroform/methanol (2:1, v/v). The recovered saPLD@chitosan–cellulose–agarose was reused in subsequent batches under identical conditions. Samples from each batch were analyzed by HPLC according to Section 2.5.

2.8. Statistical Analysis

All experiments were performed in triplicate, and the data were presented as the mean values accompanied by their corresponding standard deviations (SD). One-way analysis of variance (ANOVA) was conducted using SPSS 16.0 software (IBM, Chicago, IL, USA). Statistical analyses were conducted using Origin 2025 (OriginLab, Northampton, MA, USA). Duncan’s multiple range test was applied to evaluate significant differences between groups, with a significance level defined as p < 0.05.

3. Results and Discussion

3.1. Preparation of Immobilized Carriers

Chitosan and cellulose with different ratios (3:1, 4:1 and 5:1) were made into spherical carriers, and their formation process in alkaline solution is shown in Figure S1a. It was observed that a ratio of 3:1 resulted in the tailing of the microspheres, while a ratio of 5:1 produced microspheres that were excessively brittle and susceptible to fracture. At the ratio of 4:1, the carrier exhibited well-proportioned characteristics. However, when the ratio of chitosan to cellulose was 4:1, polysaccharide carriers still had shortcomings. The moist chitosan spheres contained a significant amount of water, making them prone to structural collapse during the drying process. Thus, the addition of other components was necessary to optimize the polysaccharide carrier [27]. Agarose was widely recognized as a good carrier for enzyme immobilization, offering notable biocompatibility. Agarose allowed facile functionalization and diverse immobilization strategies, owing to the abundance of hydroxyl groups on its surface. These groups can be chemically modified with various functional moieties, enabling different enzyme fixation mechanisms [28]. Therefore, chitosan, cellulose, and agarose in different ratios (4:1:1, 4:1:0.5 and 4:1:0.25) were made into spherical carriers, and their moulding in an alkaline solution was demonstrated in Figure S1b. These spherical structures exhibited uniformity and durability, comparable to those of pure chitosan and chitosan–cellulose carriers, thus meeting the criteria for subsequent experimental procedures.

3.2. Characterization of the Carriers

Chitosan carriers and chitosan–cellulose carriers were analyzed using SEM, and the results were shown in Figure 1a–d. The surface of the chitosan carrier was smooth and the porosity was small. The outer surface of the chitosan carrier was wrinkled, and it did not form a regular spherical shape. When the ratio of chitosan–cellulose was 3:1, the porosity of the carrier surface was uneven. When the ratio of chitosan–cellulose was 4:1, the surface of the carrier was wavy in a regular shape, and the porosity was uniformly distributed, which could theoretically provide better immobilization effects with PLD. When the chitosan to cellulose was 5:1, the carrier surface was easily broken, and thus could not meet the requirements for immobilization. When the ratio of the chitosan–cellulose carrier was 4:1, the surface of the carrier contained tiny pores, which facilitated mass transfer during the reaction [29]. Moreover, the surface exhibited a three-dimensional network structure, and the pores were significantly more uniform than those obtained at chitosan–cellulose ratios of 3:1 and 5:1.

As demonstrated in Figure 1e, when the ratio of chitosan–cellulose–agarose was 4:1:1, the surface of the carrier was relatively flat and porous. As shown in Figure 1g, when the ratio of chitosan–cellulose–agarose was 4:1:0.25, the chitosan–cellulose–agarose carrier exhibited irregular surface pores. As shown in Figure 1f, the carrier prepared at a chitosan–cellulose–agarose ratio of 4:1:0.5 exhibited a more porous and rougher surface, along with a looser spherical structure, than those prepared at ratios of 4:1:1 and 4:1:0.25, which suggested that this configuration might be more amenable to immobilization with saPLDs. In addition, the carrier prepared at a chitosan–cellulose–agarose ratio of 4:1:0.5 possessed a rougher outer surface and showed a particle-accumulated structure, which might be more favorable for the adsorption of PLDs [30].

The FT-IR analysis of chitosan carrier and chitosan–cellulose carrier with varying ratios are presented in Figure S2. The chitosan carrier exhibited well-defined absorption peaks: a peak at 3301 cm⁻¹ corresponds to O–H and N–H stretching vibrations from side chains [31]. The peak at 2872 cm⁻¹ was assigned to C–H stretching vibrations [31]. The peak at 1649 cm⁻¹ was primarily attributed to C=O stretching vibrations [31,32,33]; the overlapping peak at 1577 cm⁻¹ was mainly due to N–H bending vibrations [32,33]; the peaks at 1422 cm⁻¹ and 1375 cm⁻¹ were associated with bending and rocking vibrations of methylene groups, respectively [31,32,33]. The overlapping peaks near 1025 cm⁻¹ were located within the region characteristic of the C–O stretching vibrations in chitosan [31,32,33]. The peak at 897 cm⁻¹ was related to bending vibrations of C–OH in the chitosan heterocyclic side chain. Upon blending chitosan with cellulose, the FT-IR of the chitosan–cellulose carrier remained largely similar to that of pure chitosan, which could be attributed to the high proportion of chitosan and spectral overlap between the chitosan and cellulose. However, with the addition of cellulose, the introduction of abundant O–H structures from cellulose molecules resulted in varying degrees of shift in the O–H absorption peak near 3272 cm⁻¹. These interacted with the abundant O–H and N–H groups in chitosan via intermolecular hydrogen bonding, leading to observable peak shifts. This hydrogen bonding represented a major form of interaction between chitosan and cellulose. The C–H absorption peak near 2870 cm⁻¹ also underwent a slight shift. This was because both chitosan and cellulose were polymer chains; during physical mixing, they formed a physical crosslinking network, which restricted the movement of chain segments and thus led to changes in the alkane-related absorption bands. A notable change was the peak around 1726 cm⁻¹, characteristic of the ester carbonyl (C=O) stretching vibration. Its intensity increased with higher cellulose content, possibly due to the formation of ester linkages between hydroxyl groups of cellulose and carbonyl or other reactive groups in chitosan during processing. The increased esterification degree with rising cellulose proportion suggested potential chemical crosslinking alongside physical interactions. Similarly, the peak near 1641 cm⁻¹ gained intensity with increasing cellulose content, which might be associated with the incorporation of non-conjugated carbonyl groups from cellulose. In summary, the interactions between chitosan and cellulose in blends with different crosslinking ratios were dominated by intermolecular hydrogen bonding and physical entanglement of chitosan–cellulose carrier chains. However, chemical interactions between hydroxyl and carbonyl groups might also occur to a certain extent. The functional group at 1629 cm⁻¹–1645 cm⁻¹ was -COOH, which was a characteristic functional group of polysaccharide carrier and could be bonded with -NH of the enzyme molecule [34,35]. In the enzyme immobilization system, -COOH could interact with -NH on the surface of enzyme molecules (such as ionic bond binding or amide bond covalent binding) to achieve enzyme immobilization. From the characterization results, the chitosan–cellulose carrier with a ratio of 4:1 exhibited a wider peak shape and a larger peak area at the characteristic peak corresponding to -COOH, indicating that the carrier with this ratio contained a more abundant content of -COOH that could bind to enzyme molecules. Therefore, it was considered that the chitosan–cellulose carrier with a ratio of 4:1 had a better binding effect with saPLDs [34]. With the further blending of agarose in different proportions, the infrared characteristics of the chitosan–cellulose–agarose carriers exhibited slight variations. For instance, the peak near 3281 cm⁻¹ also shifted noticeably, indicating that the introduction of agarose provided additional O–H structures to the system, thereby forming new intermolecular hydrogen bonds and altering the original hydrogen-bonding network and its strength. The peak of the polysaccharide C–O backbone near 1021 cm⁻¹ also shifted to varying degrees, which might be related to the incorporation of agarose [34].

As shown in Figure 2, the peak at 3167 cm⁻¹ was attributed to the -OH functional group. The -OH group could form interactions with the -NH groups present on PLD molecules. The peak area at this wavenumber was larger for the chitosan–cellulose–agarose carrier with a ratio of 4:1:0.5, indicating a greater abundance of accessible -OH groups available for binding with the PLDs. The peak area of -OH groups on the carrier surface is positively correlated with the efficiency of enzyme immobilization, indicating that a larger peak area of -OH groups is more conducive to the immobilization of enzyme molecules on the carrier [36]. For the chitosan–cellulose–agarose carrier, a large peak area at the characteristic wavenumber of -OH groups indicated a high abundance of accessible -OH groups on the carrier surface, which provided active binding sites for the crosslinking reaction with glutaraldehyde and further promoted the binding between the carrier and enzyme molecules. Therefore, it was concluded that the carrier with a 4:1:0.5 ratio of chitosan, cellulose, and agarose theoretically exhibited a good binding capacity for the free saPLD.

3.3. Optimization of saPLD Immobilization Conditions

After fermentation at 37 °C for 48 h, the enzyme activity of saPLD was determined to be 110.22 U/mL. After purification, the enzyme displayed a single band on SDS-PAGE, exhibiting a specific activity of 45.12 U/mg. As exhibited in Figure 3a, the immobilized saPLD with chitosan as the carrier showed the highest enzyme activity recovery of 65.68% at 0.05% glutaraldehyde, and then decreased with the increasing glutaraldehyde concentration to 45.08%; the immobilized saPLD with chitosan–cellulose (4:1) showed the highest enzyme activity recovery of 68.96% at 0.05% glutaraldehyde, and then decreased with the increasing glutaraldehyde concentration to 46.64%; the immobilized saPLD with chitosan–cellulose–agarose (4:1:0.5) as the carrier showed the highest enzyme activity recovery of 83.40% at the glutaraldehyde concentration of 0.10%, and then decreased with the increasing glutaraldehyde concentration. In this process, glutaraldehyde served as a crosslinking agent in enzyme immobilization. Specifically, this reagent reacted with amino groups on the enzyme surface to form Schiff bases. The strength of enzyme binding to the carrier was influenced by the concentration of glutaraldehyde. However, excessive concentrations of glutaraldehyde might lead to enzyme/protein deactivation, significantly reducing immobilization efficiency. The findings aligned with previous investigations examining the influences of glutaraldehyde concentration on the activity of immobilized enzymes [34]. In this study, at a glutaraldehyde concentration of 0.1%, the saPLD@chitosan–cellulose–agarose achieved an enzyme activity recovery exceeding 80%. The adsorbed saPLDs were intramolecularly and intermolecularly crosslinked via glutaraldehyde with appropriate concentration. This crosslinked network not only prevented the adsorbed enzymes from leaching from the chitosan–cellulose–agarose carriers, but also enhanced the stability of the saPLD@chitosan–cellulose–agarose [37]. As the glutaraldehyde concentration increased further, the enzyme activity recoveries progressively decreased. Due to the excessive glutaraldehyde, the amino groups of polysaccharide molecules were crosslinked with each other, which might decrease the porosity of the carrier surface, thus affecting the immobilization effect of the enzyme. In previous studies, it was demonstrated that when the volume fraction of glutaraldehyde was excessively high, the excess glutaraldehyde would undergo aldol condensation and attach to the surface of the carrier, thereby interfering with the binding between the enzyme and the immobilization carrier [38,39]. As demonstrated in the preceding data, the immobilized saPLD with a composite carrier composed of a 4:1:0.5 ratio of chitosan, cellulose, and agarose exhibited the maximum level of enzyme activity.

As illustrated in Figure 3b, the immobilized saPLD with chitosan as the carrier exhibited the highest enzyme activity recovery of 63.27% at the crosslinking temperature of 30 °C. Subsequently, the enzyme activity recovery exhibited a gradual decrease with an increase in crosslinking temperature. The immobilized saPLD with chitosan–cellulose (4:1) demonstrated the highest enzyme activity recovery of 73.80% at the crosslinking temperature of 30 °C. The immobilized saPLD, with chitosan–cellulose–agarose (4:1:0.5) serving as the composite carrier, exhibited the highest enzyme activity recovery of 80.42% at the crosslinking temperature of 20 °C. Thereafter, the enzyme activity recovery exhibited a gradual decrease in response to elevated temperatures, reaching 66.32% at 60 °C. The enzyme activity recovery during the immobilization process was found to be most significantly influenced by crosslinking temperature. Among these immobilized enzymes, those comprising chitosan–cellulose–agarose (4:1:0.5) as the composite carrier demonstrated consistent superiority in immobilization performance.

Based on the above-mentioned concentrations of glutaraldehyde and temperatures, the crosslinking time was investigated. As demonstrated in Figure 3c, the immobilized saPLD on the chitosan carrier reached the maximum enzyme activity recovery of 74.36% at a crosslinking time of 2 h. For the chitosan–cellulose (4:1) and chitosan–cellulose–agarose (4:1:0.5) composite carriers, the highest enzyme activity recoveries were achieved at a crosslinking time of 3 h, reaching 81.74% and 89.01%, respectively. Beyond this point, the activity gradually decreased with extended crosslinking time, declining to 75.70% and 85.05% after 4 h of crosslinking, respectively. During the crosslinking process, glutaraldehyde reacted with the amino groups on the polysaccharide carriers, resulting in the formation of free aldehyde groups. The number of these active aldehyde groups was directly associated with the efficiency of enzyme immobilization. As the crosslinking time increased, the number of aldehyde groups on the carrier increased, thereby enhancing the amount of immobilized enzyme. However, beyond a certain point, excessive aldehyde groups might undergo side reactions with the enzyme, leading to conformational changes and a reduction in enzyme activity recovery. In a previous study, the recovered enzyme activity showed the lowest value at 2 h of crosslinking, leading to inhomogeneous aggregation and enzyme leakage. Prolonging the crosslinking duration improved the recovered enzyme activity, which peaked at 6 h, indicating enhanced stability. Subsequently, the recovered enzyme activity decreased, probably because prolonged exposure of the enzyme to glutaraldehyde resulted in its penetration into the interior of enzyme molecules. Glutaraldehyde reacted non-specifically with essential amino acid residues in the enzyme active center, thereby altering or destroying the precise conformation of the active site. Excessive crosslinking impaired the flexibility of the enzyme, and an optimal balance needed to be achieved between sufficient immobilization and activity preservation [40]. Based on the above data, when comparing the immobilized saPLD prepared with the three different carriers at their respective optimal crosslinking times, the chitosan–cellulose–agarose (4:1:0.5) composite carrier demonstrated the best performance in terms of enzyme activity recovery.

The chitosan, chitosan–cellulose (4:1), and chitosan–cellulose–agarose (4:1:0.5) composite carriers were crosslinked under their respective optimal conditions of glutaraldehyde concentration, crosslinking temperature, and crosslinking time. Subsequently, the immobilized saPLD was prepared by adsorption at different temperatures (10, 20, 30, 40, 50, and 60 °C) for 3 h to determine the optimal adsorption temperature. The results were presented in Figure S3a, and it was observed that the highest enzyme activity recovery of the immobilized saPLD prepared with all three carriers were achieved at an adsorption temperature of 20 °C, reaching 76.87%, 84.56%, and 86.40%, respectively. As the temperature increased, the enzyme activity recovery of immobilized saPLDs using chitosan, chitosan–cellulose (4:1), and chitosan–cellulose–agarose (4:1:0.5) as carriers all decreased; therefore, adsorption was conducted at 20 °C. In a previous study, it was confirmed that if the temperature was excessively high at the initial adsorption stage, the free enzyme might undergo thermal inactivation before binding to the carrier. When the temperature rose to a certain level, the high temperature endowed the adsorbed enzyme molecules with greater kinetic energy, making them easier to detach from the carrier surface and thus reducing the immobilization efficiency [38].

Thereafter, the immobilized saPLD was prepared by means of adsorption at 20 °C for varying time periods (1, 2, 3, and 4 h) with the objective of investigating their optimal adsorption time. As illustrated in Figure S3b, the immobilized saPLD, when administered with a carrier composed of chitosan–cellulose (4:1), exhibited the highest enzyme activity recovery of 84.78% at an adsorption time of 3 h. Both the saPLDs immobilized on chitosan and chitosan–cellulose–agarose (4:1:0.5) reached their maximum enzyme activity recovery at an adsorption time of 2 h, with values of 78.73% and 87.77%, respectively. Subsequently, their enzyme activity recoveries gradually decreased with prolonged adsorption time, dropping to 76.08% and 81.52% at 4 h, respectively, consistent with the trends reported in the literature [41,42].

In summary, the chitosan–cellulose–agarose (4:1:0.5) carrier was cross-linked with 0.1% glutaraldehyde at 20 °C for 3 h, followed by adsorption of the free saPLD at 20 °C for 2 h, yielding saPLD@chitosan–cellulose–agarose with the highest enzyme activity recovery (87.77%).

3.4. Enzymatic Properties of saPLD@chitosan–cellulose–agarose

The optimal temperature for both saPLD@chitosan–cellulose–agarose and free saPLD was 60 °C, suggesting that the immobilization of the enzyme did not alter its optimal catalytic temperature. As demonstrated in Figure 4a, the enzyme thermostability was significantly enhanced upon saPLD@chitosan–cellulose–agarose. This marked protective effect of the carrier toward saPLD was evidenced by the stark contrast in residual activity at 60 °C: approximately 90% for the immobilized saPLD versus only approximately 20% for the free saPLD. The improved thermostability of saPLD@chitosan–cellulose–agarose was likely attributable to the chitosan–cellulose–agarose (4:1:0.5) carrier, which restricted unfavorable conformational changes and thereby shielded the enzyme’s active structure at elevated temperatures.

The experimental findings demonstrated that the optimal pH of both saPLD@chitosan–cellulose–agarose and free saPLD was 6.0, suggesting that the immobilization of the enzyme had no effect on the optimal pHAs exhibited in Figure 4b, the residual enzyme activities of the saPLD@chitosan–cellulose–agarose were 103.45%, 101.64%, and 98.95% after 7 d of incubation at pH 6.0, 7.0, and 8.0, respectively, while the residual enzyme activities of the free saPLD were 100%, 97.23% and 95.05%, respectively. The enhanced pH stability observed for saPLD@chitosan–cellulose–agarose was likely attributable to the confinement of saPLD molecules within the chitosan–cellulose–agarose (4:1:0.5) carrier, which reinforced the structural rigidity of the enzyme. A comparable observation was previously reported by Lambrecht et al., in which the immobilization of PLD onto octyl-Sepharose led to a notable enhancement in the pH tolerance of the enzyme. This phenomenon was hypothesized to originate from the protective role exerted by the support surface on both the PLD molecule and its catalytic sites, which alleviated the detrimental effects of pH variations within the reaction system on enzymatic performance [43].

3.5. Catalytic Batches of saPLD@chitosan–cellulose–agarose

As exhibited in Figure 5, after eight consecutive batches, the PS yield of saPLD@chitosan–cellulose–agarose retained over 60% of the initial batch’s yield. The decline in yield could be attributed to the impact of the reaction system’s solvent on the saPLD@chitosan–cellulose–agarose during the PS catalytic production process, resulting in a reduction in the PS yield. The observed decline in production yield over successive cycles was also attributed to the gradual deactivation of saPLD@chitosan–cellulose–agarose following repeated catalytic use. Additionally, partial loss of the immobilized enzyme during recovery steps—such as washing and centrifugation—represented another contributing factor. This phenomenon was consistent with previous reports on other immobilized enzyme systems, including the laccase-based magnetic nanoflowers [44]. When compared to previously reported nanoflower-based carriers for saPLD immobilization, the chitosan–cellulose–agarose (4:1:0.5) composite carrier developed in this work demonstrated a higher level of operational stability across multiple batch cycles [45]. In other investigations on immobilized PLD, the reusability exhibited a declining trend following seven consecutive reaction cycles, in which the hydrolytic activity remained above 40%, while the transphosphatidylation activity remained approximately 20% [46]. In summary, the saPLD@chitosan–cellulose–agarose represented a robust and promising biocatalytic platform for the efficient production of functional phospholipids, offering a cost-effective and sustainable strategy.

4. Conclusions

In this study, we successfully synthesized a novel biocatalyst, saPLD@chitosan–cellulose–agarose, by immobilizing saPLD onto a chitosan–cellulose–agarose (4:1:0.5) composite carrier. The saPLD@chitosan–cellulose–agarose demonstrated good thermostability and pH stability compared to its free counterpart. Furthermore, the saPLD@chitosan–cellulose–agarose catalyst retained over 60% of its initial PS yield after eight cycles of reuse, highlighting its exceptional reusability and potential for practical application. Collectively, this work established saPLD@chitosan–cellulose–agarose as a promising and efficient biocatalytic platform for the PS production.