A Novel Purification Process of Sardine Lipases Using Protein Ultrafiltration and Dye Ligand Affinity Chromatography

Abstract

Protein purification is often performed for various applications. However, enzyme purification processes typically involve multiple steps that reduce yield and increase production costs. To overcome these challenges, we developed a novel three-step process to purify a lipase from whole sardine viscera (WSV), leveraging protein properties and the structural affinity of lipases for dye ligands. A crude extract of the viscera (CEV) was obtained by grinding the whole viscera in 50 mM phosphate buffer (pH 7.0, Solution B) followed by centrifugation (6000× g; 30 min, 0 °C). Lipolytic activity (3.3 U/mg) was recorded only in the supernatant. The purification process began with ammonium sulfate fractionation (30–50% saturation), increasing lipolytic activity in the precipitate (PF30-50) to 32.9 U/mg. PF30-50 was then ultrafiltered using a 30 KDa MWCO membrane, where 5% of semi-purified lipases (SPLSV) was retained with an activity of 156.5 U/mg (UF30). Finally, the SPLSV was injected into a column packed with dye ligand affinity adsorbent, pre-equilibrated with 1.0 M ammonium sulfate in buffer A. The WSV lipase was eluted using a step gradient to progressively reduce salt concentration. SDS-PAGE analysis revealed a single band of purified lipase from sardine viscera (PLSV) corresponding to a molecular weight of 123.4 kDa, with a specific activity of 266.4 U/mg. The combination of ammonium sulfate precipitation, ultrafiltration, and dye-ligand affinity chromatography provides a scalable and reproducible approach with potential industrial relevance, particularly in biocatalysis and waste valorization contexts.

1. Introduction

Lipases (triacylglycerol acylhydrolase, EC 3.1.1.3) are enzymes that catalyze the hydrolysis of triglycerides (edible oil) have demonstrated to be one of the most outstanding biocatalysts, which play a prospective role in multi-dimensional industrial applications [1]. These enzymes are primarily responsible for the hydrolysis of acylglycerides. However, there are a large number of different substrates, such as esters of high and low molecular weight, amides, thioesters, esters of polyols or polyacids, which are useful for the same group of enzymes [2]. These enzymes are present in animals, plants and microorganisms. However, depending on the source of lipase they express different properties of specificity and activity [3]. At the present time, the main source of these lipolytic enzymes are microorganisms; however, only a few of them have been recognized as safe (GRAS) by regulatory agencies [4]. On the other hand, long-chain polyunsaturated fatty acids (PUFAs) do not appear as good substrates for most of the commercially available lipases [5]. Lipases from marine organisms display different specificities for PUFAs as substrates [6,7]. Lipases from cod (Gadus morrhua), rohu (Labeo rohita Hamilton), sea bream (Pragus major), atlantic salmon (Salmo salar), rainbow trout (Oncorhynus mykiss), Monterey sardine (Sardinops sagax), Chinook salmon (Oncorhynchus tshawytscha), and New Zealand hoki (Macruronus novaezelandiae) have shown high specificity for long-chain PUFA [8,9,10]. In addition to this, the protocols reported for purification of fish lipases exceed four or more steps [10,11,12,13].

Due to the high degree of importance of lipases, currently researchers are still focused on the characterization of their performance. This interest is mainly in the mechanism of catalysis, 3D structure, and lipase gene sequence cloning [14]. Purification costs of enzymes are the main limiting factor in the development of new processes and the economic performance of the industrial-scale production [15]. This concern is especially true for commodities like common oils and fats or products thereof, which are traded at a relatively low price.

Despite all efforts invested in lipase purification, it is very clear that the parameter information indicates poor adsorption. It is well known that lipases have a highly hydrophobic area surrounding the active site; for this reason, purification can be pursued through affinity or by hydrophobic interaction chromatography techniques [16]. Lipases have a highly hydrophilic character but also can be attached to water insoluble substrates, which provides a stable behavior in polar and nonpolar environments [17]. Cibacron F3GA and related dyes are polyaromatic sulfonates that bind with substantial specificity and affinity to enzymes related directly with nucleotides and a number of other proteins [18,19]. After attached to a suitable insoluble substrate, these compounds find wide applications in dye-ligand affinity chromatography for protein separation and purification. It has been recognized that these dyes are capable to effect hydrophobic and electrostatic interactions, depending on conditions of the surrounding environment [20]. The study of the interactions of the dyes, salt, solvent and others small molecules imply the nature of the interaction of the dye with different types of groups from the proteins [21]. When affinity resins are employed, the purification process is reduced to a single step, so that it can lower substantially the cost of their production [22]. The functional groups of the dyes are ligands containing both hydrophobic and charged groups. Dye affinity chromatography has been used extensively in the protein purification from laboratory to large scale because of its low cost, accessibility, ease of immobilization, resilience to chemical and biological changes, as well as acceptable selectivity and capacity [23]. However, no studies have been reported for the application of these adsorbents on lipase purification. The purpose of this work was not to develop new methodologies, but to demonstrate the applicability and efficiency of this protocol for obtaining active, purified lipases with potential industrial relevance.

2. Materials and Methods

2.1. Materials

All reagents used in this research were reagent grade or better (Sigma-Aldrich, St Louis, MO, USA). Olive oil was purchased from Sigma-Aldrich, and Menhaden fish oil was a gift from Omega Protein (Hammond, LA, USA). For the chromatography separation, the affinity resin Amicon Blue Dye matrix A was used.

Fresh Monterey sardine (Sardinops sagax) from the Gulf of California was collected within 6 h of capture from the fishing vessel’s storage vault and immediately transported to the laboratory in portable coolers using alternate layers of crushed ice and salt. At the laboratory, sardines were measured, weighted and incised ventrally to manually extract the viscera. This material was stored at −10 °C until used.

2.2. Preparation of Crude Extract of Viscera (CEV)

A crude extract from whole sardine viscera was prepared according to the method previously published [24]: one part of viscera (186 g) was homogenized with 1.5 parts of purified ice and 1.5 parts of 50 mM, pH 7.0 phosphate buffer for 10 min. The homogenate was centrifuged at 6000× g, 0 °C for 30 min. The fat layer on the top was removed and the supernatant, considered as the crude extract of viscera (CEV), was recovered.

2.3. Lipase Activity Assay

Lipase activity was determined by titration of fatty acid released from the substrates: olive oil or Menhaden oil [25,26]. The reaction mixture consisted of 2 mL of 0.1 M sodium phosphate buffer pH 7.0, 1 mL of the oil substrate and 1 mL of the enzyme preparation. The mixture was incubated in 50 mL flasks in a shaking bath at 40 °C, 120 rpm for 30 min. The reaction was quenched by addition of 5 mL of 95% ethanol, followed by titration with 0.01 M NaOH solution using phenolphthalein as an indicator. All assays were made in triplicate. Controls were run in a similar way, but the enzyme preparation was added after termination of the reaction time. One unit (U) of lipase activity was defined as the amount required to release 1 μmol of fatty acids per minute at 40 °C.

2.4. Preparation of Semi-Purified Lipases (SPLSV)

The CEV was fractionally precipitated with ammonium sulfate using a method described previously [24]. Chilled CEV was placed in a 500 mL jacketed Celstir^® reactor and solid ammonium sulfate was added to attain 30% saturation (1.2 M). After stirring for 1 h at 4 °C the suspensions were centrifuged at 6000× g, 0 °C for 30 min. The precipitate was discarded and the supernatant (SF30) was taken to 50% saturation (2.2 M) with solid ammonium sulfate. After stirring 1.0 h at 4 °C, the suspension was centrifuged at 6000× g, 0 °C for 30 min. The precipitated fraction (PF30-50) was recovered and resuspended in 50 mM sodium phosphate pH 7.0 solution (buffer B) to test for lipase activity.

For further purification, PF30-50 was ultra-filtered as follows: 15 mL of the PF30-50 solution was placed into the sample reservoir of a 30,000 MWCO centrifugal filter (Amicon Ultra, Millipore Corp., Bedford, MA, USA) and centrifuged at 4000× g, 0 °C for 15 min. Lipolytic activity was assayed in the retentate and filtrate for the detection of the semi-purified lipase from sardine viscera (SPLSV). The retentate showed lipolytic activity whereas the filtrate did not show any residual activity.

2.5. Preparation of Purified Lipases from Sardine Viscera (PLSV)

The concentrated SPLSV (100 μL) was loaded into a glass column (1.0 × 15 cm) packed with 4 mL of Dye Matrex Blue A (Amicon Millipore Corp., Bedford, MA, USA) pre-equilibrated with 1.0 M ammonium sulfate in 50 mM potassium phosphate pH 7.0 solution (buffer A). After the sample was applied, the non-adsorbed proteins were washed with buffer A at a flow rate of 1.0 mL/min by 15 min. The adsorbed proteins were eluted at a flow rate of 1.0 mL/min using two linear gradients with buffer B (50 mM potassium phosphate pH 7.0), programmed as follows: from 0% to 50% of buffer B in 5 min; held for 15 min at 50% of buffer B; from 50% to 100% of buffer B in 5 min; held for 20 min at 100%B. The protein concentration at the effluent of the column was determined by direct measurement of absorbance at 280 nm using a standard curve of BSA (Sigma-Aldrich). The fraction showing high lipase activity (PLSV) was dialyzed and concentrated with a centrifugal filter 10,000 MWCO (Amicon Ultra, Millipore Corp., Bedford, MA, USA) at 4000× g, 0 °C for 15 min.

2.6. Purity and Molecular Weight Determination

Gels of 14% polyacrylamide with 0.1% SDS were used to assess lipase purity and molecular weight [27]. High-range molecular weight markers (MWMs) (BioRad Laboratories Inc., Hercules, CA, USA) containing Myosin (200,000 Da), β-galactosidase (116,300 Da), Phosphorylase b (97,400 Da), bovine serum albumin (66,200 Da), and ovalbumin (45,000 Da) were ran as standard for the molecular weight determination of lipase. Protein concentration was determined by the method of Bradford [28] using bovine serum albumin (BSA) as standard (Sigma, St. Louis, MO, USA).

3. Results and Discussion

3.1. Crude Extract Activity

After grinding and centrifugation the whole viscera of sardine, lipase activity was assayed to the precipitate and supernatant. In the preparation of the enzyme extract, three phases were obtained: the fat in the upper layer (9.1% w/w), a precipitate at bottom (37.7% w/w), and the supernatant (53.2% w/w). The supernatant showed lipolytic activity (59.94 U/mL) with a protein concentration of 18.0 mg/mL and was considered as the CEV.

3.2. Semi-Purified Lipases (SPLSV)

When the CEV was fractionated with ammonium sulfate, when the supernatant was brought to 30% followed by precipitation to 50% saturation, the PF30-50 obtained showed a high lipolytic activity (16.80 U/mL). After PF30-50 was ultra-filtered using a 30 KDa MWCO membrane, about 95% of the solution was filtered and the activity was only found in the retentate with a value of 26.62 U/mL.

3.3. Purified Lipases from Sardine Viscera (PLSV)

Figure 1 and Figure 2 show both the signal from the UV detector (280 nm), which monitors the protein concentration, and the feed gradient of buffer B (50 mM phosphate buffer pH 7.0). The separation of four peaks was achieved, which were collected in 2 mL fractions and the lipolytic activity was determined after being diafiltered to eliminate ammonium sulfate. Peaks 1 and 2 were eluted in the wash with the loading buffer A solution (1.0 M ammonium sulfate), so it is clear that they do not show affinity for the adsorbent under this run condition. Peak 3 was eluted at 50% of buffer B, which corresponds to 0.5 M ammonium sulfate concentration, while peak number four was eluted at 100% of buffer B, which corresponds to 0.0 M ammonium sulfate. This fraction does not need dialysis because it was eluted at phosphate buffer pH 7.0 (buffer B). Figure 1 shows the separation of proteins of the supernatant fraction with ammonium sulfate 30% of saturation (SF30). As described above, four peaks were eluted with the program gradient established, but only peak 4 presented lipolytic activity. When the precipitated fraction of PF30-50 was injected in the column, we observed in Figure 2 a reduction in the concentration of peaks 1 and 2, and the lipolytic activity was detected on peak 4. The fractions collected in peak 4 presented lipolytic activity, so it was considered that this fraction is where the purified lipase of the sardine viscera was eluted (PLSV). With this series of experiments, it was possible to establish that the recovery of lipase activity was possible using the dye affinity adsorbent. Additionally, the supernatant from the precipitation with ammonium sulfate at 30% saturation (SF30) can be used, thus eliminating a step in the purification process.

A summary of the data obtained during the concentration and purification of PLSV is presented in

Table 1. Summary of the lipase purification procedure from the whole viscera of the sardine (Sardinops sagax).

	Weight (mg)	Protein (mg mL−1)	Activity (U mL−1)	Specific Activity (U mg−1)	Purification (Fold)	Recovery Yield (%)
CEV	28.9	18.0	59.94	3.33	1.0	100
PF30-50	3.28	0.64	16.80	32.95	7.9	89.5
SPLSV (UF30)	0.16	0.17	26.62	156.58	47.0	26.7
PLSV	0.05	0.02	5.32	266.40	80.0	13.8

CEV, crude extract of viscera; PF30-50, precipitates fraction with 30–50% of ammonium sulfate saturation; SPLSV, semi purified lipase; UF30, ultrafiltered fraction with 30,000 Da MWCO; PLSV, purified lipase of sardine viscera.

. Throughout the purification process, the specific activity of PLSV increased by 80.0-fold.

Very few studies have investigated fish lipases, reporting various purification methods and yields [29,30,31]. Most of the reports include dialysis for desalting NH₄ sulfate [11,29,30,31], as well as further purification through chromatographic resins and ion exchange: DEAE-Sephadex, Sephadex G-100 [11] cholate-EAH-Sepharose 4B [29,30], Q-Sepharose, Toyopearl-HW65F, Super Q-Toyopearl, Asahipak GF510HQ, and TSK gel DEAE-5PW [31]. In these studies, the yield after extraction and fractionation was calculated based on the total weight of viscera, with an average yield of 5.86%. The yield of the PLSV obtained after extraction and purification operation was 13.8%.

The lipolytic activity of PLSV was significantly increased (p < 0.05) when Menhaden oil was used as substrate. Such an increase was proportional (up to 3-fold) with the PUFA composition of the substrates. It is well known that fish lipases have greater specificity for PUFA than microbial lipases [8,32].

3.4. Purity and Molecular Weight of PLSV

According to experimental data of the relative movement of proteins in the polyacrylamide gel, the molecular weight and the relative distance were correlated. It was determined that the PLSV corresponds to a molecular weight of 123.4 kDa (Figure 3).

There are a few reports of purified lipases from marine organisms where the molecular weight of the lipases has been determined. The molecular weight of a lipase purified from Red Sea bream hepatopancreas was 64 kDa [31], and pancreatic bile salt lipase purified from cod showed a molecular weight of 60 kDa [30]. However, a lipase from the pyloric cecum extract of cod was reported with a molecular weight of 100 kDa [30].

4. Conclusions

Lipases extracted from sardine viscera exhibit high solubility in aqueous media and remain soluble up to 30% ammonium sulfate saturation (1.2 M). A 47-fold increase in lipolytic activity was achieved through fractional precipitation (30–50% ammonium sulfate saturation) followed by ultrafiltration using a 30 kDa molecular weight cut-off (MWCO) membrane. This process yielded a semi-purified lipase preparation (PLSV), as evidenced by the appearance of four distinct bands on SDS-PAGE. Further purification was accomplished in a single chromatographic step using a matrix with immobilized Cibacron Blue, exploiting the affinity of lipases for the dye in the presence of ammonium salts, which facilitated the separation from non-adsorbed proteins. The final purification process, comprising fractional precipitation, ultrafiltration, and affinity chromatography, resulted in an 80-fold increase in lipolytic activity with a recovery yield of 13.8%. The purified lipase from sardine viscera (PLSV) has a relatively large molecular mass (~100 kDa) compared to microbial lipases and exhibits higher specificity toward polyunsaturated fatty acids (PUFAs).