Enzymatic synthesis of structured DHA phospholipids in a solvent- free medium with potential effects in the treatment of neurodegenerative diseases

doi:10.21203/rs.3.rs-4821561/v1

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Enzymatic synthesis of structured DHA phospholipids in a solvent- free medium with potential effects in the treatment of neurodegenerative diseases

https://doi.org/10.21203/rs.3.rs-4821561/v1

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Phospholipids containing docosahexaenoic acid (DHA) esterified at the sn-2 position of glycerol could contribute to the prevention of neurodegenerative diseases such as Alzheimer's. Optimizing the synthesis bioprocesses for these structured DHA lipids is essential to ensure their bioavailability, allowing them to cross the blood-brain barrier and be efficiently incorporated into neuronal membranes. Lipases and phospholipases are gaining attention due to their role as biocatalysts in selectively modifying and producing structured lipids. In particular, enzymatic processes in solvent-free media are highly valued for their ecological, economic, and technological benefits. Immobilization and post-immobilization techniques are indispensable for obtaining highly stable biocatalysts in solvent-free media. The immobilized derivative of phospholipase Quara® LowP (QlowP-C18) is the optimal catalyst for synthesizing di-substituted DHA phospholipids, achieving a yield of 58%. The post-immobilization technique increases the stability of QlowP-C18 threefold, allowing it to be reused for up to five reaction cycles at 40ºC.

Docosahexaenoic acid (DHA)

Glycerophosphocholine (GPC)

Esterification

2-didocosahexaenoyl-sn-glycero-3-phosphocholine (Di-DHA-PC)

Phospholipases

Interfacial adsorption

Docosahexaenoic acid (DHA), an essential omega-3 polyunsaturated fatty acid present in brain tissues, plays a crucial role in the development and proper functioning of the brain. It has been observed that adding omega-3, particularly DHA, to the diet has neuroprotective effects and neuromodulatory properties, which is especially relevant in the context of aging (Dangour & Uauy, 2008). The ability to develop DHA formulations that can cross the blood-brain barrier (BBB) and be efficiently incorporated into neuronal cell membranes is essential for these benefits (Ting et al., 2014).

In this regard, lysophospholipids containing DHA esterified at the sn-2 position of glycerol are of great interest, as they are postulated to enhance the bioavailability of this fatty acid (Burri et al., 2012; Parmentier et al., 2007). The absorption of DHA in the form of phospholipids could be particularly advantageous, allowing DHA to pass unaltered through the gastrointestinal tract, enter the bloodstream as an sn-2 lysophospholipid, and finally cross the BBB to be incorporated into neuronal membranas (Hachem & Nacir, 2022). It has been suggested that the lysophosphatidylcholine molecule could be the preferred carrier of DHA to cross the BBB and specifically deliver the fatty acid to the brain (Chouinard-Watkins et al., 2019; Semba, 2020; Sugasini et al., 2019). These DHA formulations could contribute to the prevention or reduction of neurodegenerative diseases such as Alzheimer's (Pencreac’h et al., 2011, 2013).

Currently, commercially valuable lipids, such as those enriched in polyunsaturated fatty acids (PUFAs), are often found in low concentrations and mixed with lipids of lesser commercial interest (Uprety et al., 2022). This is due to the difficulty in developing methods that allow for the production of structured lipids rich in these fatty acids with high purity and yield (Castejón & Señoráns, 2020; Q. Xu et al., 2023). For this reason, lipases and phospholipases have garnered significant interest in the food industry, as they can be used as biocatalysts for the modification and production of new fats and oils, facilitating the creation of functional and nutraceutical foods. Lipases have the ability to selectively modify the fatty acid composition of lipids, either by incorporating different fatty acids, restructuring their positions, or generating new TAGs and other derivatives of interest such as PLs or monoglycerides (MAGs), particularly 2-MAGs of omega-3 PUFAs (Moreno-Perez et al., 2016, 2018; Y. Zhang et al., 2018). To achieve this, lipases can carry out various types of synthesis reactions, including acidolysis, alcoholysis, esterification, and transesterification. Similarly, the hydrolysis and ethanolysis of natural oils can be very interesting alternatives for obtaining omega-3 derivatives both in their free fatty acid (FFA) form (Fernández-Lorente et al., 2011) and as ethyl esters (EE) (Moreno-Pérez et al., 2014). Both products are frequently used as intermediates or acyl donors for the synthesis of other more desirable omega-3 derivatives. All these enzymatic processes collectively enable the creation of structured lipids with a high concentration of DHA and improved nutritional characteristics (Lopes et al., 2023).

In the literature, there are not many examples of phospholipid synthesis using lipases due to the high complexity of the reaction. Primarily, the synthesis of lysophosphatidylcholine (LPC) from glycerophosphatidylcholine (GPC) is described, either by direct esterification with free fatty acids (Hong et al., 2011; Kim & Kim, 2000) or by transesterification with fatty acid vinyl esters (Mnasri et al., 2017; Virto et al., 1999, p. 19; Virto & Adlercreutz, 2000). Additionally, the transesterification of soybean lecithin with ethyl esters of EPA and DHA using Rhizomucor miehei lipase in the presence or absence of hexane and additives has been reported. Under optimal conditions, high levels of EPA and DHA (45.7%) were incorporated into the phospholipids within 24 hours (Marsaoui et al., 2015). On the other hand, although fish oils are the main natural source of omega-3 PUFAs, the increasing demand for fish for the synthesis of these desirable derivatives has prompted the exploration of alternative sources, such as oilseed plant seeds, genetically modified plants, or primary marine sources like microalgae and cianobacteria (Castejón et al., 2019). In a recent study, the enzymatic transesterification of phospholipids using Schizochytrium sp. algal oil without pretreatment was carried out for the first time. After optimizing the reaction, a maximum incorporation of DHA into phosphatidylcholine of 39.1% was achieved at a temperature of 45°C (T. Zhang et al., 2023).

Biocatalytic processes generally gain greater interest when executed in the absence of solvents, as this is considered an ideal medium for developing sustainable processes (Malipeddi et al., 2011). In the case of phospholipid esterification, various factors affecting the esterification of GPC in solvent-free systems have been investigated (Kim & Kim, 2000). One of the main challenges in the modification of GPC is its low solubility in these media, as it is a highly polar molecule soluble in water and polar solvents, but poorly soluble in completely anhydrous organic media. Additionally, the lack of stability of immobilized enzymes in hydrophobic anhydrous media (e.g., oils) can be an additional drawback for the development of enzymatic processes in solvent-free systems (Abreu Silveira et al., 2019). Given these complications, it is necessary to develop immobilization and post-immobilization strategies that enable the production of highly active and stable biocatalysts capable of functioning efficiently under various reaction conditions (Guisan et al., 2001).

The solution to these problems lies in Enzyme Engineering. The development of enzyme stabilization strategies through immobilization and post-immobilization techniques has allowed the conversion of soluble and unstable biocatalysts into highly stable heterogeneous enzymatic derivatives in a single process (Guisan, 2006). Adsorption of lipases on hydrophobic supports is a very simple immobilization protocol that produces very interesting immobilized lipase derivatives, as the open and active form of the lipase molecules is stabilized by strong adsorption on the surface of the support. This results in lipase derivatives that are much more active (hyperactivation) and stable than other immobilized derivatives (Fernandez-Lorente, Cabrera, et al., 2008). For this type of immobilization, the use of commercial polyacrylic resins as immobilization supports for use in anhydrous reaction media has garnered considerable interest. In this publication, we will use the commercial resin Immobeads-C18 IB-ADS-3 (ChiralVision), with a particle size of 150–300 µm. These highly porous resins with octadecyl groups are ideal for the immobilization of lipases through interfacial adsorption, allowing for a high enzyme load due to their small pore size (58% pore volume), resulting in catalysts up to 10 times more active than those previously used (Facin et al., 2021).

On the other hand, several post-immobilization techniques can complement enzymatic immobilization to achieve better biocatalysts in terms of high activity, stability, specificity, and selectivity These techniques include coating enzymatic surfaces with polymers through covalent bonding, such as dextran aldehyde (DexCHO), or through ion exchange, for example with polyethyleneimine (PEI) (Bolivar et al., 2012). For lipases, it is highly advantageous to perform physicochemical modifications of immobilized derivatives by interfacial adsorption because in this type of immobilization, the open and active center is protected by the support from any surface modification of the immobilized lipase. This provides additional protection by creating a physical barrier between the enzyme and its environment, thereby insulating the active center without compromising it (Guisan, 2013; Martins et al., 2022; Palomo et al., 2013). In this work, dextran sulfate polymer will be used, known for its strongly anionic nature and its tendency to interact with cations present in the environment (Abian et al., 2002; Lin et al., 2005).

Therefore, to meet the growing interest in DHA-phospholipids with enhanced bioavailability, the use of immobilized lipases and phospholipases is proposed for the synthesis of highly pure structured DHA-phospholipids through direct esterification of GPC with DHA acid in anhydrous solvent-free media. Specifically, the synthesis of 1,2-di-DHA-phosphatidylcholine (Di-DHA-PC) is sought, as mentioned earlier, as the most effective form to deliver DHA to the brain (Thies et al., 1992, 1994).

Materials

Docosahexaenoic acid (DHA) was obtained from the enzymatic hydrolysis of Anchovy oil, which was purchased in capsules from NuaBiological at a Pharmacy (Madrid, Spain) and glycerophosphocholine (GPC) was purchased from Cayman Chemical Company (Michigan, USA). Soluble lipases such as Lipozyme® TL from Thermomyces lanuginosa (TLL), Candida antarctica B (CALB), Palatasa® 20000 L (PALA), Eversa® Transform 2.0 (NS40), and phospholipases Lecitase® Ultra (LECI) and Quara® LowP (QlowP) were kindly donated by Novozymes (Bagsvaerd, Denmark). Immobeads-C18 IB-ADS-3 (C18) immobilization support was provided by ChiralVision (Leiden, The Netherlands) and DEAE-Sepharose purification support from Sigma-Aldrich. Triton X-100, hexadecyltrimethylammonium bromide (CTAB), sodium chloride, p-nitrophenyl butyrate (pNPB) and 3Å pore size molecular sieve (2-3 mm bead) were provided by Sigma-Aldrich. Methanol, chloroform, ammonium hydroxid, and acetonitrile were purchased from VWR Chemicals (Pennsylvania, USA) and Sigma-Aldrich. 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine (Di-DHA-PC) standard was purchased from Avanti Polar Lipids (Alabama, USA). All other reagents and solvents used were of analytical grade.

Methods

Determination of the Activity of Different Soluble and Immobilized Lipases

The activity assay was carried out using a spectrophotometer with a thermostatted cell and continuous magnetic stirring (500 rpm) for 2 minutes. The increase in absorbance at 348 nm (ε = 5,150 M^-1cm^-1) produced by p-nitrophenol (pNP) released after the hydrolysis of the 0.4 mM p-nitrophenyl butyrate (pNPB) substrate in 25 mM sodium bicarbonate at pH 8.5 was measured at 25ºC. In the case of phospholipase QlowP, this increase was measured in 10 mM sodium phosphate buffer at pH 7. To initiate the reaction, 0.1 ml of soluble lipases/phospholipases (blank or supernatant) and their immobilized preparations (suspension) were added to 2.5 ml of the substrate solution. Enzyme activity was calculated as µmol of pNPB hydrolyzed per minute per mg of enzyme (IU) under the described conditions.

Immobilization of Lipases/Phospholipases on the Hydrophobic Support Immobeads-C18

Four different lipases (TLL, NS40, PALA, and CALB) and two phospholipases (LECI and QlowP) were immobilized on the hydrophobic support Immobeads-C18 by interfacial adsorption. These enzymes are adsorbed onto this type of support in their open and hyperactive form (Bastida et al., 1998). Using the Immobeads-C18 support, enzymatic preparations with maximum loading were achieved. The protocol established in a previous study (Garcia-Quinto et al., 2023) was followed to achieve this maximum loading. However, the QlowP-C18 derivative was not optimized previously and has been optimized in this study.

Firstly, the purification of QlowP-C18 was carried out using DEAE-Sepharose support through ion exchange. For this, 20 g of DEAE support was added to a buffered solution containing 1000 mg of enzyme in a 10 mM sodium phosphate buffer at pH 7 (determined to be 6 mg/ml according to the Bradford assay results). Purification proceeded for 24 hours at 25°C with continuous agitation.

To monitor the progress, the activity of both the suspension and supernatant was periodically measured using the p-NPB assay. Once all the enzyme was adsorbed onto the DEAE support, desorption was initiated. A 0.1 M NaCl solution in 158 ml of phosphate buffer was prepared, and the DEAE support containing the purified enzyme was introduced. The solution was stirred for 2.5 hours, resulting in complete desorption of the enzyme.

Following desorption, the supernatant containing the purified enzyme was collected to continue with the immobilization process. To the purified enzyme volume, 0.02% CTAB was added. Subsequently, 5 g of Immobeads-C18 immobilization support, pre-moistened, was introduced. The mixture was maintained at 25°C with continuous agitation for 96 hours. Throughout the immobilization process, the p-NPB assay was performed to monitor the enzyme's activity.

Physicochemical Modifications of Immobilized Enzymes

Amination

The chemical amination of the derivatives was carried out according to the literature described in the bibliography (Fernandez-Lorente, Godoy, et al., 2008; Rodrigues et al., 2009). One gram of enzyme immobilized on Immobeads-C18 was added to 10 mL of 1 M ethylenediamine (EDA) with a pH of 4.75 and continuous agitation. Then, solid 1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) was added to the suspension until reaching a final concentration of 10 mM. After 90 minutes of incubation at 25°C with gentle agitation, the immobilized preparations were filtered and vacuum-washed using a porous filter plate.

Adsorption of Anionic Polymer

The immobilized and aminated derivatives were coated with a layer of the anionic polymer sodium dextran sulfate of different sizes (8000 and 100000 Da) by adding 100 mg of dextran sulfate per gram of derivative. To do this, the solid polymer was dissolved in 10 mL of 10 mM sodium acetate buffer at pH 5, a gram of aminated enzymatic derivative was added, and the mixture was stirred at room temperature for 2 hours (Abian et al., 2002). Additionally, derivatives modified with two layers of dextran were prepared (DEX 100000+8000), first by coating the aminated derivative with the larger polymer (DEX 100000) and then repeating the procedure to coat this same already modified derivative with the smaller polymer (DEX 8000). All obtained derivatives were filtered and stored in the refrigerator for future use.

Drying of Lipases/Phospholipases Derivatives

The enzymatic derivatives were washed and dried before use, as they were employed in reactions in anhydrous media. Consequently, the derivatives were washed with increasing volumes of water and acetone using a sintered glass funnel until completely dry. For each gram of derivative, 50 mL of each of the following solutions were used: 100% water; different water solutions (70:30, 50:50, and 30:70, v/v); and 100% acetone.

Enzymatic Synthesis of Structured Phospholipids from Docosahexaenoic Acid

The esterification reaction with DHA was carried out in an anhydrous solvent-free medium. The reaction mixture consisted of 250 mg of dry lipase or phospholipase derivative, 30 mg of GPC, 0.25 g of molecular sieve with a pore size of 3 Å, and 2.5 mL of free DHA. The free DHA was previously obtained through the enzymatic hydrolysis of anchovy oil. Additionally, the effect of adding 30% butanone relative to the final volume was studied. The reactions were conducted in glass vials (final volume of 2.5 mL) at 60°C with constant stirring in an incubator at 150 rpm. Prior to adding the derivative, the vials were incubated and stirred for 15 minutes to increase the solubility of GPC in the reaction medium. For each experiment, a negative control without the lipase or phospholipase derivative was performed under the same conditions. The synthesis of DHA phospholipids was monitored by TLC (using the same mobile phase as described in (Garcia-Quinto et al., 2023)) and then quantified by HPLC by taking samples from the supernatant. For TLC, 60 μL of the reaction medium were taken and diluted in 900 μL of chloroform, and then, for HPLC, 100 μL of this solution were taken and diluted in 900 μL of methanol/acetonitrile (80:20 v/v; the mobile phase used in HPLC). The experiments were performed in triplicate, and the standard deviation was always less than 5%.

Stability of Physicochemically Modified Derivatives for the Synthesis of DHA Phospholipids

The stability of the derivatives modified with the post-immobilization strategy using dextran sulfate was evaluated in the esterification reaction of GPC with DHA following the methodology outlined below. Both the modified derivatives and the unmodified derivative were incubated for up to two days under specific reaction conditions: anhydrous solvent-free medium (DHA) and 40ºC. To initiate each pre-incubated reaction, the GPC substrate was first mixed with 1 mL of reaction medium (DHA) and stirred for 15 minutes. Subsequently, this mixture was added to the incubated reaction solution, which already contained an additional 1.5 mL of DHA. The activity of the derivatives was compared to that of a non-incubated reaction (considered as 100% activity). The stability of each derivative was determined by quantifying the Di-DHA-PC product in aliquots taken at 24 hours. The analysis of the products was carried out using HPLC.

Reuse of Phospholipase Derivatives in the Esterification Reaction

For catalyst reuse studies, the enzymatic esterification reaction was also carried out in glass vials (final volume of 2.5 mL) at 40ºC in an anhydrous solvent-free medium. The process involved reusing the derivative for the maximum number of possible cycles under optimized reaction conditions. After the optimal reaction time, the reaction volume was carefully filtered to recover the catalyst for reuse in the new reaction mixture. To effectively separate the derivative from the reaction medium, it had to be washed and dried several times with hexane and acetone to remove any remaining substrates or products. Subsequently, new substrates (DHA and GPC) were added along with the reused catalyst, ensuring that the weight of the derivative was controlled to avoid loss in successive cycles. This process was repeated as many times as possible until the enzyme completely lost its activity. The residual activity of the catalyst in each cycle was calculated based on the 100% activity of cycle 0, determined from the percentage of Di-DHA-PC formed in 24 hours. The analysis of the product peaks was performed using HPLC.

Product Analysis

Quantitative Analysis by High-Performance Liquid Chromatography (HPLC)

High-performance liquid chromatography analyses were conducted using HPLC system (Spectra Physic SP 100 pump coupled with a Spectra Physic SP 8450 UV detector) employing a normal phase column (LiquidPurple Sil, 250 x 4.6 mm, 5 μm) with a pre-filter column (0.5 μm disc and seal). Additionally, aliquots were pre-filtered using a 4 mm/0.45 μm PVDF syringe filter. Elution of products occurred at a flow rate of 1 ml/min utilizing a methanol/acetonitrile (80:20 v:v) mobile phase. UV detection was set at 205 nm due to the molecules' double bond content, which promotes significant absorbance at that wavelength. Synthesis yields were calculated based on the areas corresponding to different concentrations of DHA phosphatidylcholine and lysophosphatidylcholine, with retention times of 4.4 minutes (Di-DHA-PC) and 5.1 minutes (DHA-LPC), respectively. A calibration curve was performed using standard concentrations ranging from 0.05 mg/mL to 1 mg/mL. HPLC analyses were conducted in triplicate, and the standard deviation consistently remained below 5%.

Immobilization of Lipases/Phospholipases with Maximum Enzyme Loading

The four lipases and two phospholipases were immobilized through hydrophobic interfacial adsorption on Immobeads-C18 support with the maximum allowable enzyme loading, following the protocol described in the article by Garcia-Quinto et al., 2023 (Garcia-Quinto et al., 2023). The enzyme loading per gram of support varied depending on the enzyme and the achieved immobilization yield. TLL, PALA, and NS40 exhibited an enzyme loading of 180 mg of protein per gram of support, achieving an 90% immobilization yield after 72 hours. CALB and LECI reached a final enzyme loading of 170 mg of lipase/phospholipase per gram of support after 48 hours of continuous agitation. Phospholipase QlowP achieved a loading of 160 mg per gram of support after 72 hours.

Enzymatic Synthesis of Di-DHA-PC by Direct Condensation of DHA and GPC in Solvent-free System

The course of the esterification of GPC with DHA in a solvent-free system catalyzed by immobilized QlowP phospholipase with maximum enzymatic load via hydrophobic adsorption onto Immobeads-C18 support was studied. As seen in Figure 1, the reaction begins with the formation of a first product or intermediate product known as DHA lysophospholipid (DHA-LPC), which reaches maximum yield at 3 hours with 74%. Over time, this intermediate product gradually disappears, transforming into the final reaction product, which is the Di-DHA-phosphatidylcholine (Di-DHA-PC), reaching maximum yield at 48 hours with 58%. It was observed that the progression of the reaction closely resembled that described in the esterification of oleic acid and GPC in the study by Garcia-Quinto et al., 2023 (Garcia-Quinto et al., 2023).

In this research, the reaction was carried out in a solvent-free system with a substrate molar ratio of 1/62 (GPC/DHA) and a temperature of 60°C. Under these conditions, it was possible to incorporate DHA at the sn-2 position of GPC, resulting in the formation of Di-DHA-PC as the sole reaction product. Next, we will study whether the reaction products can vary depending on the catalyst and reaction conditions used.

Profile of DHA Phospholipid Synthesis Catalyzed by Different Lipases and Phospholipases Immobilized on C18 in Solvent-Free Media

Next, the esterification reaction of GPC with DHA catalyzed by the different immobilized lipases and phospholipases from section 3.1 was studied. The kinetics of formation of mono- and di-substituted DHA phospholipids were investigated independently. Figure 2a shows the formation of DHA-LPC for all immobilized biocatalysts, while Figure 2b shows the formation of Di-DHA-PC.

For the formation of DHA-LPC (Fig. 2a), all enzymes exhibit a similar behavior, mainly differing in the rate of formation of this product. However, for the formation of Di-DHA-PC (Fig. 2b), there are clear differences in behavior depending on the catalyst used. As observed, only three enzymes are capable of synthesizing the final reaction product, with LECI and QlowP, two phospholipases, and NS40, a lipase, achieving yields around 60% at 168 hours. However, reaction yields for lipases PALA, TLL, and CALB are considerably low for the formation of Di-DHA-PC. It is noteworthy that when the reaction was performed with oleic acid, only CALB was unable to achieve good yields for Di-oleoyl-PC (Garcia-Quinto et al., 2023). These differences could be attributed to greater difficulty in esterifying DHA at the sn-2 position of the lysophospholipid, given that the nature and structure of DHA differ significantly from oleic acid. Additionally, several factors could hinder the process, such as substrate solubility, high temperatures, or the use of highly hydrophobic and viscous anhydrous reaction media.

Due to the sn-1,3 selectivity described for lipases by other authors in this type of reaction, it would be logical for the esterification of GPC to only produce sn-1 DHA-LPC as the sole reaction product (Liu et al., 2017). However, thanks to the immobilization strategy used, the natural sn-1,3 regioselectivity would have been altered for NS40, LECI, and QlowP after their immobilization on Immobeads-C18. These enzymes would now exhibit additional selectivity for the sn-2 position, with LECI and QlowP, possessing characteristics of phospholipases and a natural substrate being a phospholipid, showing greater affinity to esterify this position. This latter aspect is related to the sn-2 selectivity previously described in the literature for phospholipases (Cabezas et al., 2012). For the rest of the lipases, this effect was not observed as Di-DHA-PC is barely formed, indicating that the selectivity of each lipase is a key factor in the formation of the final product. From here, we chose QlowP as the optimal biocatalyst for the synthesis of di-substituted DHA phospholipids. This is because QlowP exhibits a significantly higher initial velocity compared to LECI: 10.89 versus 3.65 (mg/mL.h)*g, indicating greater activity for the reaction.

Effect of the Solvent on the Esterification of GPC with DHA Catalyzed by Immobilized Phospholipase QlowP

In the previous study (Garcia-Quinto et al., 2023), it was observed that one of the issues in the reaction was the poor solubility between the substrates used in the esterification. Therefore, the effect of solvent on their solubility was investigated. It was found that the presence of acetone in the medium favored the solubility of GPC in oleic acid. Now, with DHA, it will be checked if this also affects the reaction kinetics. Specifically, we will study how the addition of 30% of acetone influences the formation of the di-substituted DHA phospholipid. To do this, we will compare the reaction catalyzed by QlowP-C18 under two conditions: a solvent-free medium (Fig. 1 in section 3.2) and another with 30% acetone (Fig. 3).

As seen in Figure 3, the presence of butanone alters the enzyme's behavior, as no production of di-DHA-phospholipid is observed. Furthermore, while in the solvent-free reaction a maximum yield of 74% DHA-LPC was achieved at 3 hours (Fig. 1), in the presence of butanone the formation of DHA-LPC is slower and with a lower yield (51% DHA-LPC at 24 hours). Additionally, this intermediate reaction product exhibits low stability under these conditions, disappearing over time.

In view of the results, it could be inferred that the presence of butanone might be causing a decrease in enzymatic activity due to possible conformational changes that hinder substrate entry into the active site (Pizarro et al., 2012). Therefore, the use of butanone for the synthesis of di-substituted DHA phospholipids is ruled out due to its negative effect on reaction yield.

Stabilization Strategies for QlowP-C18 Using Post-Immobilization Techniques

After confirming that the QlowP-C18 derivative was the best for the formation of Di-DHA-PC in a solvent-free medium, the next objective was to stabilize the catalyst to enable its reuse over multiple cycles. Given that the reaction conditions are very harsh for enzymes, such as the use of anhydrous, highly hydrophobic and viscous media, and high temperatures, it was proposed to design stabilization strategies using post-immobilization techniques. To this end, a post-immobilization strategy was carried out, which consisted of coating the surface of the catalyst with the polymer dextran sulfate and analyzing how the polymer size affects the enzyme activity in the esterification of GPC with DHA.

For this purpose, the esterification of GPC with DHA in a solvent-free medium at 40ºC was studied using both the unmodified QlowP-C18 derivative (C18) and three derivatives modified with dextran sulfate: one with a small polymer (DEX 8000 Da), another with a large polymer (DEX 100000 Da), and a third with a double layer of polymers (DEX 100000+8000 Da). Figure 4 shows the percentages of synthesis of the final product, Di-DHA-PC, as the reaction progresses.

The results indicate that the modification of the catalyst has little influence on enzymatic activity, as the yields are very similar in all cases. The fact that the activity is not affected suggests that the polymer coating does not hinder the substrate from reaching the enzyme's active site. On the other hand, although a higher reaction temperature can decrease the viscosity of the fatty acid (Ifeduba & Akoh, 2014), we ultimately decided to conduct the reaction at 40ºC. This temperature is less harsh on the biocatalyst compared to 60ºC and is more sustainable at an industrial level. Additionally, maintaining the reaction at 40ºC helps preserve the stability of DHA, as this compound is highly susceptible to lipid peroxidation due to the high degree of unsaturation in its long carbon chain (Montine & Morrow, 2005).

Stability of the Different QlowP-C18 Derivatives Under the Reaction Conditions

After confirming the activity of the dextran sulfate-modified derivatives, it was investigated whether the different polymer sizes used in post-immobilization offer greater stability to the unmodified QlowP-C18 derivative. The four derivatives were incubated for up to two days at 40ºC in a solvent-free medium, following the protocol described in Materials and Methods.

Figure 5 shows that in all cases, the polymer coating improves the stability of the QlowP-C18 catalyst to varying degrees. The unmodified derivative (C18) loses 65% of its activity at 24 hours and completely loses it at 48 hours. In contrast, the double-layer derivative (DEX 100000+8000) maintains 94% of its activity until the end of the incubation period, while the DEX 100000-modified derivative retains only 35% of its activity at 48 hours. Finally, although the derivative with DEX 8000 shows greater stability at 24 hours compared to C18, it also loses all its activity by the end of the incubation period.

These results indicate that DEX 8000 does not provide an effective protective layer due to its insufficient size. In contrast, the double-layer with dextran sulfates of different sizes significantly enhances the enzyme stability under severe reaction conditions (anhydrous hydrophobic media, highly viscous, and at 40ºC). It seems that the combination of highly hydrophilic and viscous polymers prevents the exposure of internal hydrophobic pockets of the enzyme and reduces conformational changes on its surface (Abian et al., 2002). herefore, the use of DEX 100000+8000 is very useful to prevent direct interaction of the immobilized enzyme with the reaction medium. This demonstrates that post-immobilization physicochemical modification improves the stability of QlowP-C18, which is a crucial property for its future industrial applications.

Operational Stability of the QlowP-C18 Catalyst

Finally, the reusability capacity of the double-layer dextran sulfate derivative (DEX 100000+8000) is studied over several consecutive cycles of 24 hours at 40ºC in a solvent-free medium and compared with the unmodified derivative (C18). Cycle 0 is considered as 100% catalytic activity, corresponding to 28.25% production of Di-DHA-PC for the modified derivative and 28% for the unmodified derivative (Fig. 4 in section 3.5).

Figure 6 shows that the modified derivative exhibits greater operational capacity compared to the unmodified one. QlowP-C18 coated with DEX 100000+8000 is reused for up to 5 cycles, maintaining 51% activity in the fourth cycle, while the unmodified derivative shows only 17% in the same cycle. By the end of the cycles, the modified derivative produces 120.4 mg of Di-DHA-PC, compared to 85.45 mg from the unmodified derivative. This indicates that the immobilization and post-immobilization strategy increases the lifetime of the QlowP biocatalyst, making it more effective for the synthesis of di-substituted DHA phospholipids.

The reuse of the derivative has been made possible by our hydrophobic adsorption immobilization strategy, which has imparted greater stability to the QlowP enzyme compared to its soluble form. It is noteworthy that the esterification reaction of DHA with GPC was impossible under the same conditions using the soluble enzyme, failing to synthesize the desired product, Di-DHA-PC. Furthermore, our post-immobilization strategy with double-layer dextran sulfate has provided the enzyme with greater stability compared to the uncoated derivative. In the future, further post-immobilization strategies using polymers with different chemical structures will be explored to further enhance the catalytic properties of the derivative and increase the production of the desired phospholipid.

The course of the DHA and GPC esterification reaction depends greatly on the lipase or phospholipase used, the immobilization strategy, and the experimental conditions, as all of these factors modulate the activity and selectivity of the synthesis reaction. Thanks to the immobilization strategy using hydrophobic adsorption on Immobeads-C18 support, derivatives with maximum enzymatic load are obtained, highly active and enabling the synthesis of structured DHA phospholipids in a solvent-free medium at 60ºC. The QlowP-C18 phospholipase derivative stands out as the most promising candidate, achieving a maximum synthesis yield of Di-DHA-PC of 58% in just 48 hours, with a maximum formation rate of 10.89 (mg/mL.h)*g. Despite the potential benefits of butanone to enhance GPC solubility, its use is ruled out due to potential adverse effects on catalytic activity. In the future, a new study on solvents will be considered, investigating the addition of ionic liquids that possess stabilizing properties for enzymes.

The post-immobilization technique with a double layer of dextran sulfate has tripled the stability of QlowP-C18. Additionally, it has been demonstrated that this derivative can be reused for up to five reaction cycles at 40ºC, achieving a total production of 120.4 mg of DHA-disubstituted phospholipid. Therefore, the immobilization and post-immobilization strategies used ensure proper enzyme orientation on the support, providing high stability and enabling operation under adverse reaction conditions such as solvent-free anhydrous media, highly hydrophobic and viscous environments, with low substrate solubility, and high temperatures.

In the future, these highly pure di-substituted DHA phospholipids could be employed as novel nutraceuticals aimed at preventing and/or treating neurodegenerative diseases associated with DHA deficiency in the brain (Karaki et al., 2022). Due to their high bioavailability, they would represent a very interesting alternative for incorporating DHA into neuronal membranes.

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Gloria Fernandez-Lorente reports financial support was provided by Spanish Scientific Research Council. Gloria Fernandez-Lorente reports a relationship with Spanish Scientific Research Council that includes: employment.

Acknowledgments

The authors gratefully acknowledge the financial support from the Ministry of Science and Innovation, Spain (Number project No. RTI2018-093583-B-I00). The authors are also grateful to Novozymes for generously donating the commercial enzymes. Ernestina García gratefully acknowledges the Spanish National Research Council for his predoctoral contract.

Author Contribution

Ernestina García-Quinto contributed to the methodology and investigation of the study. Ernestina García-Quinto wrote the original draft of the manuscript and also performed the review and editing. José M. Guisán and Gloria Fernández-Lorente contributed to the conceptualization and supervision of the research. Ernestina García-Quinto, José M. Guisán and Gloria Fernández-Lorente were involved in writing the original draft, as well as reviewing and editing the manuscript. Gloria Fernández-Lorente also carried out project administration and funding acquisition. All authors have read and approved the final submitted version.

Funding

Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This research was funded by the Spanish Ministry of Science and Innovation, Spain (Number project No. RTI2018-093583-B-I00).

Data availability

The authors are unable or have chosen not to specify which data has been used.

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Enzymatic synthesis of structured DHA phospholipids in a solvent- free medium with potential effects in the treatment of neurodegenerative diseases

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Abstract

Figures

Introduction

Materials and Methods

Materials

Methods

Determination of the Activity of Different Soluble and Immobilized Lipases

Immobilization of Lipases/Phospholipases on the Hydrophobic Support Immobeads-C18

Physicochemical Modifications of Immobilized Enzymes

Amination

Adsorption of Anionic Polymer

Drying of Lipases/Phospholipases Derivatives

Enzymatic Synthesis of Structured Phospholipids from Docosahexaenoic Acid

Stability of Physicochemically Modified Derivatives for the Synthesis of DHA Phospholipids

Reuse of Phospholipase Derivatives in the Esterification Reaction

Product Analysis

Quantitative Analysis by High-Performance Liquid Chromatography (HPLC)

Results and Discussion

Immobilization of Lipases/Phospholipases with Maximum Enzyme Loading

Enzymatic Synthesis of Di-DHA-PC by Direct Condensation of DHA and GPC in Solvent-free System

Profile of DHA Phospholipid Synthesis Catalyzed by Different Lipases and Phospholipases Immobilized on C18 in Solvent-Free Media

Effect of the Solvent on the Esterification of GPC with DHA Catalyzed by Immobilized Phospholipase QlowP

Stabilization Strategies for QlowP-C18 Using Post-Immobilization Techniques

Stability of the Different QlowP-C18 Derivatives Under the Reaction Conditions

Operational Stability of the QlowP-C18 Catalyst

Conclusions

Declarations

Acknowledgments

Author Contribution

Funding

Data availability

References

Additional Declarations

Supplementary Files

Status:

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