Novel Phospholipase C with High Catalytic Activity from a Bacillus stearothermophilus Strain: An Ideal Choice for the Oil Degumming Process

Abstract

A novel thermoactive phosphatidylcholine-specific phospholipase C (PC-PLC_Bs) was identified from Bacillus stearothermophilus isolated from a soil sample from an olive oil mill. Enhanced PLC_Bs production was observed after 10 h of incubation at 55 °C in a culture medium containing 1 mM of Zn²⁺ with an 8% inoculum size and 6 g/L glucose and 4/L yeast extract as the preferred carbon energy and nitrogen sources, respectively. PLC_Bs was purified to homogeneity by heat treatment, ammonium sulfate fractionation, and anion exchange chromatography, resulting in a purification factor of 17.6 with 39% recovery. Interestingly, this enzyme showed a high specific activity of 8450 U/mg at pH 8–9 and 60 °C, using phosphatidylcholine PC as the substrate, in the presence of 9 mM sodium deoxycholate and 0.4 mM Zn²⁺. Remarkable stability at acidic and alkali pH and up to 65 °C was also observed. PLC_Bs displayed a substrate specificity order of phosphatidylcholine > phosphatidylethanolamine > phosphatidylserine > sphingomyelin > phosphatidylinositol > cardiolipin and was classified as a PC-PLC. In contrast to phospholipases C previously isolated from Bacillus strains, this PLC_Bs substrate specificity was correlated to its hemolytic and anti-bacterial potential against erythrocytes and Gram-positive bacterial membranes, which are rich in glycerophospholipids and cardiolipin. An evaluation of PLC_Bs soybean degumming process efficiency showed that the purified enzyme reduced the phosphorus content to 35 mg/kg and increased the amount of diacylglycerols released, indicating its ability to hydrolyze phospholipids in the crude soybean oil. Collectively, PLC_Bs could be considered as a potential catalyst for efficient industrial oil degumming, advancing the edible oil industry by reducing the oil gum volume through transforming non-hydratable phospholipids into their hydratable forms, as well as through generating diacylglycerols, which are miscible with triacylglycerols, thereby reducing losses.

1. Introduction

Phospholipases C (PLCs) are classified as phosphodiesterase enzymes that hydrolyze the phosphodiester bond of glycerophospholipids, releasing diacylglycerols and water-soluble phosphorylated headgroups as products. Their preference toward phosphatidylcholine (PC) or phosphatidylinositol (PI) results in the identification of two classes: PC-specific PLC (PC-PLC) and PI-specific PLC (PI-PLC). PLCs are expressed and produced by plants and animal cells, as well as several yeasts and bacteria [1]. They have been isolated from a wide variety of Gram-positive and Gram-negative bacteria as virulence factors, especially Legionella pneumophila, Listeria monocytogenes, Mycobacterium tuberculosis, and Bacillus spp. (B. spp.) [2].

The most studied and characterized extracellular PLCs are those from B. cereus and B. subtilis [3]. Bacillus PLCs are 28.5 kDa monomeric enzymes containing three zinc ions in the active site and a choline binding site [4]. One of the zinc sites is specific for zinc and binds it tightly, while the other sites bind Zn²⁺ less tightly and can accept Mg²⁺, Mn²⁺, Ca²⁺, Ni²⁺, Cu²⁺, etc. [5].

The crystal structure of PLC from B. cereus (PLC_Bc) revealed that three residues, Glu4, Tyr56, and Phe66, form the choline-binding pocket and contribute to substrate recognition and specificity by interacting with the substrate’s positive charge. Moreover, like PC, headgroups of phosphatidylethanolamine (PE) or phosphatidylserine (PS) can bind to the choline-binding site of PLC_Bc, explaining the ability of this enzyme to hydrolyze other phospholipids [4].

Compared to those from mammals and plants, microbial PLCs, especially those from Bacillus strains, can be easily extracted and purified [6]. These enzymes exhibit special biochemical characteristics, such as their ability to hydrolyze various phospholipids, their high catalytic activities under extreme pH and temperature conditions, and their thermostability [2,6,7]. These characteristics promote the use of PLCs in several industrial processes that need a high operation temperature and pH and increased product solubility to raise the reaction rate and the process yield [8]. The production of emulsifiers and structured phospholipids with higher nutritional values and vegetable oil degumming are among the processes that favor PLCs compared to chemical processes [2,6].

Crude vegetable oil is a complex mix of triglycerides, phospholipids, sterols, colored pigment, tocopherols, and free fatty acids, which form a deposit gum during storage that causes problems for these oils and, especially, for food applications. Moreover, dark-colored oils and the generation of off-flavors in edible oils are mainly due to phospholipids [8]. For successful oil refinement, the removal of phospholipids is a crucial step, allowing one to obtain final high-quality oil that contains less than 10 mg/kg of phosphorus. During industrial refining, hydratable phospholipids are removed by water degumming, whereas the non-hydratable forms are eliminated thought acidic or alkalic chemical products characterized by a reduced yield and high costs [2]. Therefore, enzymatic oil degumming is a suitable process that offers safe biological- and eco-friendliness during oil refining, in which phospholipases are used to convert the non-hydratable phosphatides into a hydratable form [6,8]. The phospholipases used in industrial degumming are essentially PLA₁ and PLA₂: Lecitase^® (pancreatic PLA₂), Lecitase^® Novo (PLA₁ from Fusarium oxysporum), Lecitase^® Ultra (PLA₁ from Thermomyces lanuginosus/Fusarium oxysporum), LysoMax^® (PLA₂/LAT from Streptomyces violaceoruber), and Purifine^® (recombinant PLC from B. anthracis expressed in Pichia pastoris) [6]. Both PLA₁ and PLA₂ release free fatty acids that increase the oil acidity and, consequently, reduce the quality of refined oils. Thus, PLCs are preferred in such industrial processes. In addition, diacylglycerols (DAGs) released by PLCs from vegetable oils have nutritional value as they provide the essential linoleic acid involved in the biosynthesis pathways of ω-6 and ω-3 fatty acids [2].

Most of the Bacillus PLCs used in the oil degumming operation are specific to PC and are also able to hydrolyze PE, which make up approximately 60–70% of the total phospholipids in vegetable oils. However, these PLCs have a low hydrolysis rate regarding PIs, which represent up to 24% of the total phospholipids in several vegetable oils [9]. A combination of PI-PLC with PC-PLC showed great efficiency in the degumming of crude oil; however, using a single enzyme with various substrate specificities is much more preferable [10].

PLCs purified from B. thuringiensis [2], B. licheniformis MTCC 7445 [6], and B. cereus [9] are PC-PLCs with interesting thermoactivity and stability with respect to pH and temperature, and they show important potential in the degumming of many crude oils. However, these PLCs are unable to hydrolyze PIs. Therefore, since Bacillus strains have advantageous rapid growth in various media and extracellular enzyme production, the research of new PLCs from these strains with the ability to hydrolyze PC, PE, and PI, as well as various phospholipid derivates, is a very interesting field that allows for the use of this enzyme in degumming applications. In line with this, the current research was focused on a B. stearothermophilus strain characterized by its ability to grow at extreme pH and temperature conditions and to produce thermophilic extracellular hydrolases (protease and lipase) [11,12]. We report the optimization of the production conditions of a new extracellular PLC, called PLC_Bs, from this strain. A biochemical characterization was carried out, and the correlation between substrate specificity and hemolytic and antimicrobial activities, as well as the degumming potential of the purified PLC_Bs, was also investigated.

2. Material and Methods

2.1. Materials and Reagents

Chromatography material (Mono Q-Sepharose, high-performance liquid chromatography coupled with evaporative light scattering detection), SDS-PAGE technique, pH-stat, and rotary shaker, were obtained from Bio-Rad (Hercules, CA, USA). The automated Edman degradation method was performed using a PROCISE instrument (Applied Biosystems, Foster City, CA, USA).

Chemicals were obtained from commercial sources. Yeast extract, tryptone powder, casein hydrolysate, Bacto-Peptone, glucose, fructose, maltose, mannose, ribose, starch, Rhodamine B, NaCl, NaHCO₃, Na₂HPO₄, KNO₃ and (NH4)₂SO₄, ZnCl₂, CaCl₂, sodium deoxycholate (NaDC), NaOH, and citric acid solution were purchased from Bio-Rad (Hercules, CA, USA). Sodium dodecyl sulfate (SDS), acrylamide, ammonium persulfate, N,N,N′,N′-tetramethyl ethylenediamine (TEMED), β-mercaptoethanol, coomassie brilliant blue R-250, and protein markers for molecular mass were also obtained from Bio-Rad (Hercules, CA, USA). All PLC substrates PC, PE, PI, PG, PS, and sphingomyelin (SM) were purchased from Sigma Aldrich (St. Quentin-Fallavier, France).

2.2. Phospholipase Production and Culture Condition Optimization

A thermophilic bacterial strain identified as B. stearothermophilus and previously isolated from soil samples from an olive oil mill (Jouf, Saudi Arabia) [11] was evaluated for its capability to hydrolyze PC. A plate assay in a solid medium that included PC and a colored indicator under UV, rhodamine B, was used for the initial screening of PLC activity according to a protocol from [13]. The bacterium was inoculated by injection on a solid medium consisting of agar (1.5 g), nutrient broth (1%, yeast extract and tryptone powder), NaCl (1%), PC (1‰), and rhodamine B (1‰). After incubating the culture plate for 24 h at 37 °C, the B. stearothermophilus strain presented orange fluorescence upon UV radiation, thus allowing the detection of phospholipase production.

Prior to fermentation, the studied strain was pre-cultured in nutrient broth for 8 h at 37 °C and 120 rpm. Then, the 8 h pre-culture was inoculated in a 300 mL culture flask containing 30 mL of the growth medium (10 g/L casein hydrolysate; 10 g/L Bacto Peptone; 5 g/L NaCl; 4 g/L glucose; 3 g/L NaHCO₃; 0.4 g/L Na₂HPO₄; 0.01 g/L ZnCl₂; initial OD of 0.2 at 600 nm, pH 7) [14]. The culture was incubated in a rotary shaker for 24 h at 37 °C and 120 rpm. The culture OD at 600 nm was determined to follow the bacterial growth. Every 2 h, samples were taken and centrifuged for 10 min at 9000× g. The obtained supernatants, regarded as crude enzymatic extracts, were used for enzyme testing.

Thereafter, optimum phospholipase production was studied via the pH-stat method using PC as the substrate. Using “one variable at a time”, several inoculated growth media were maintained at various temperatures (30–65 °C) and incubation times (0–24 h), with different carbon (fructose, glucose, maltose, mannose, ribose, and starch) or nitrogen (beef extract, casein hydrolysate, peptone, tryptone, yeast extract, KNO₃ and (NH₄)₂SO₄) sources and concentrations (2–10 g/L), and various metal ions (Zn²⁺, Mn²⁺, Mg²⁺, and Ca²⁺) at 1 mM concentration, and different inoculum volumes (2–10%).

2.3. Purification of PLCBs

The growth medium used to produce PLC_Bs contained 6 g/L yeast extract, 5 g/L NaCl, 4 g/L glucose, 3 g/L NaHCO₃, 0.4 g/L Na₂HPO₄, and 0.01 g/L ZnCl₂, with an initial OD of 0.2 at 600 nm, pH 7. Microbial cells were discarded by centrifugation (9000× g, 30 min) from 500 mL of a 10 h culture of B. stearothermophilus. The obtained crude phospholipase solution (480 mL) was initially treated for 10 min at 70 °C. After removal of denatured proteins by centrifugation (30 min, 10,000× g), the extracellular PLC was fractionated via (NH₄)₂SO₄ precipitation, and the active fraction precipitating between 40% and 70% saturation was resuspended in 15 mL of buffer A (25 mM Tris-HCl, pH 8.5, 2 mM benzamidine, and 25 mM NaCl), dialyzed overnight against the same buffer at 4 °C and, finally, poured into a column of Mono Q-Sepharose (2.5 cm × 20 cm) pre-equilibrated with buffer A. Once the eluent was free of proteins after washing with the same buffer, adsorbed proteins were eluted using a linear gradient of NaCl from 25 to 250 mM in buffer A at a flow rate of 0.5 mL/min, and 2 mL fractions were collected.

2.4. Protein Analysis

An estimation of the protein content, molecular weight, and purity of the studied PLC was performed via Bradford and SDS-PAGE techniques using 15% polyacrylamide gel under reducing conditions [15,16], whereas an automated Edman degradation method was performed using a PROCISE instrument (Applied Biosystems, Foster City, CA, USA) to determine the N-terminal sequence of the native PLC_Bs [17].

2.5. PLC_Bs Activity Measurement

The PLC activity was determined titrimetrically via a pH-stat method according to Zawaal et al. [18] in the presence of 10 mM sodium deoxycholate (NaDC) and 0.4 mM Ca²⁺ and at the designated temperature and pH (60 °C and pH 9). A 5 mM PC emulsion was used as the substrate. Under standard assay conditions, one unit of the PLC activity is defined as 1 μmole of phosphocholine produced per minute.

2.6. Catalytic Characteristics of PLC_Bs

PLC_Bs activity was also measured using cardiolipin, PE, PI, PG, PS, or sphingomyelin (SM) as a substrate to investigate the capability of the purified PLC to degrade diverse phospholipid substrates. The recorded data are expressed as percentages compared to the corresponding activity on the universal substrate, PC, under the optimal experimental conditions (60 °C and pH 9) in the presence of 10 mM NaDC and 0.4 mM CaCl₂. One unit of the PLC activity is defined as 1 μmole of phosphocholine produced per minute.

PLC_Bs’s requirement of metal ions was also investigated by measuring the hydrolysis rate of emulsified PC by PLC_Bs in the presence of various ions (Ca²⁺, Mg²⁺, Mn²⁺, and Zn²⁺) at different concentrations (0–1 mM) under optimal reaction conditions.

PLC_Bs’s apparent kinetic parameters (apparent K_m, apparent turnover k_cat and catalytic efficiency k_cat/K_m) were estimated from Lineweaver–Burk plots by measuring PLC_Bs-reaction rates as function of PC concentrations from 0.71 to 11.42 mM under standard assay conditions (in the presence of 10 mM NaDC and 0.4 mM Ca²⁺ at 60 °C and pH 9). The enzyme concentration used was 0.078 mg/mL.

2.7. Biotechnological Potential of PLC_Bs Oil Degumming

Soybean crude oil degumming was initially carried out using water following a slightly modified version of Yang et al.’s [19] method. In brief, crude soybean oil (SOYA, Yanbu–Kingdom of Saudi Arabia) (500 g) was heated to 70 °C under continuous mechanical stirring (500 rpm). Once the temperature (70 °C) was stabilized, water (15 mL) was added, and the reaction mixture was left to stir for 30 min. The stirring was stopped only after raising the oil temperature to 85 °C, and the sample was subsequently divided into Eppendorf tubes and centrifuged (for 10 min at 9000× g). The resulting supernatants were then stored at −20 °C for further experiments.

Likewise, as previously described by Jiang et al. [20], enzymatic degumming preceded by citric acid treatment was also performed with a slight adjustment. The crude soybean oil sample (500 g) was first heated to a temperature of approximately 60 °C under continuous agitation (500 rpm) and then mixed with NaOH 1 M (1.6 mL) and a 50% citric acid solution (0.4 mL). Afterward, distilled water (15 mL) and various enzyme amounts (25, 50, and 100 units) were added to the mixture, which was stirred continuously for 4 h at 500 rpm. Lastly, the enzyme was inactivated by heating the oil mixture for 10 min at 100 °C followed by centrifugation (for 10 min at 9000× g). The resulting supernatants were then stored at −20 °C for further use.

The phosphorus amount was measured in all samples according to a protocol by Jiang et al. [20], while high-performance liquid chromatography coupled with evaporative light scattering detection was performed to quantify DAGs in accordance with AOCS method Cd 11d-96 [20].

2.8. Antimicrobial Activity

PLC_Bs was tested against five bacterial species: Gram-positive Enterococcus faecalis ATCC 29122, Staphylococcus epidermidis ATCC 14990, Staphylococcus aureus ATCC 25923, Gram-negative Escherichia coli ATCC 25966, and Pseudomonas aeruginosa ATCC 27853 bacteria.

The antimicrobial activity of the purified PLC_Bs was evaluated by the well diffusion technique on nutrient agar medium according to Vanden Berghe et al. [21]. Petri dishes containing LB agar medium were uniformly inoculated with several bacterial cultures (10 µL of purified PLC_Bs per well). Then, the plates were incubated at 37 °C for 24 h. The zone of inhibition was measured in triplicate by comparing the control with the standard antibiotic. The inhibitory effect of PLC_Bs on bacterial growth was determined by measuring the inhibition zone in comparison with that of standard antibiotics (ampicillin) at 10 µg/well.

In the second stage, the antimicrobial activity was further characterized by calculating the colony-forming ability (in CFU) of bacteria incubated with various concentrations of the purified PLC_Bs from 0 to 120 µg/mL. PLC_Bs was mixed with 2 × 10⁷ CFU/mL bacteria in sterile brain heart infusion medium and incubated at 37 °C for 1 h under rotation. The bactericidal activity of PLC_Bs was calculated as the residual CFU value compared with that of the initial inocula. The IC₅₀ value corresponded to the PLC_Bs concentration that inhibited the growth of 50% of the initial inoculum.

2.9. Direct Hemolytic Activity

In order to test the direct hemolytic activity of purified PLC_Bs, mixtures of various enzyme concentrations (0 to 3 nM) and 1 mL of washed erythrocytes in PBS were incubated for 30 min at 37 °C. Ice-cold PBS (9 mL) was added to stop the reaction, followed by centrifugation at 4 °C (20 min at 1500 g). The quantification of the hemolytic effect of PLC_Bs was carried out by measuring the hemoglobin liberated at 530 nm against 100% hemolysis produced by adding Triton X-100 (1%) [22].

3. Results and Discussion

3.1. Optimization of Medium Compounds for PLC_Bs Production

Incubation Time

The effect of the incubation time on PLC production was assessed from 2 h to 24 h, as presented in Figure 1. A significant increase in biomass dry weight and phospholipase activity was observed during the first few hours of bacterial culture (Figure 1). The highest biomass (8.61 g) observed after 10 h of incubation was proportional to the phospholipase enzyme production, with an optimum of 117.5 U/mL. Thereafter, gradual decreases in enzymatic activity (up to 2.5 U/mL) and biomass dry weight (up to 4 g) were recorded at 24 h. No dramatic change in the pH of the culture medium was noticed during the incubation period (24 h). The medium was slightly acidified to pH 6.75 in the first two hours, and then it was progressively alkalized to pH 7.97 within the 24 h cultivation period (Figure 1).

It is well-known that the incubation time greatly affects the production of microbial enzymes and varies from 24 h to a week depending on the type of micro-organism and other culture conditions. The production of PLC_Bs in a short period of time seems very advantageous as it avoids its degradation by protease enzymes produced by different strains of Bacillus, which generally takes place from 16 h to 72 h [23,24]. This short production time was previously described with PI-PLC purified from a culture broth of B. thuringiensis and B. cereus starting at 3 h of cultivation and B. thuringiensis serovar kurstaki starting at 4 h of cultivation [25,26]. Maximal PLC production was reached at 10 h with B. mycoides strain 970 [7].

3.2. Temperature

The effect of temperature on the enzyme production was assessed in a wide temperature range (from 30 to 65 °C), and the results show that the maximum PLC activity (178 U/mL) occurred at 55 °C (Figure 2). It is worth noting that, above 55 °C, enzymatic activity was not strongly affected. Indeed, even at 60 or 65 °C incubation, the PLC activity was slightly reduced to 155 and 108 U/mL, respectively. This attractive property encourages the use of this enzyme in several industrial applications that require thermophilic PLCs, such as degumming effectuated at a high temperature [2]. To our knowledge, this thermotolerance property is unique to the isolated strain of B. stearothermophilus among other PLC-producing Bacillus strains, which grow at 30–37 °C [2,3,25].

3.3. Effect of Carbon and Nitrogen Sources

Since carbon catabolism has great practical importance for fermentation, the classical ‘one variable at a time’ method was adopted in order to enhance PLC_Bs production. As the preferred carbon energy source of the majority of bacteria, especially the Bacillus genre [27], glucose (4 g/L) with a maximal PLC activity of 237.5 U/mL (Figure 3A) was chosen. By varying glucose concentrations, PLC activity reached 273 U/mL at 6 g/L and decreased slowly at 8 and 10 g/L glucose in the culture medium (Figure 3C). The use of other sugar as carbon and energy sources by B. stearothermophilus can be due to its ability, like other Bacillus, to degrade several polysaccharides via hydrolysis through extracellular carbohydrates. Indeed, the Bacillus genome exhibits multiple operons made by genes encoding enzymes degrading monosaccharides, disaccharides, and polysaccharides. The hydrolyzed polysaccharides are transported into Bacillus cells and further catabolized as single-carbon and energy sources via glycolysis or pentose phosphate pathways [28].

Both nature and the characteristics of nitrogen sources have a predominant role in the metabolism of bacteria. Nitrogen participates in the biomass synthesis of enzymes, nucleotides, and secondary metabolites for micro-organism growth and metabolism [29]. Generally, the preferred nitrogen source of almost all bacteria is ammonium ions or glutamine. In Bacillus bacteria, glutamine is the preferred nitrogen source and the precursor of all nitrogen-containing compounds. Bacillus can also utilize nitrite, nitrate, or urea as nitrogen sources and incorporate these inorganic nitrogen into biomolecules [29]. In line with this, the effect of several organic and inorganic nitrogen sources on PLC production by the B. stearothermophilus strain was assessed, and the obtained data revealed the highest enzymatic activity (200.5 U/mL) with the yeast-extract-supplemented medium (Figure 3B). The PLC production was affected by nitrogen sources in the following order: yeast extract > tryptone > peptone > casein hydrolysate > beef extract > KNO₃ > (NH₄)₂SO₄ (Figure 3B). The variation in yeast extract concentrations in the B. stearothermophilus culture medium showed maximal PLC activity (237 U/mL) at a final concentration of 6 g/L. At higher concentrations, the enzymatic activity decreased significantly (Figure 3D). According to Khusro et al. [30], yeast extract plays an important role in bacterial enzyme production due to the presence of elements such as abundant amino acids, vitamins, nucleosides, minerals, and polypeptides, which are essential growth factors. In addition, the supplementation of a rich organic nitrogen source provides additional reducing power by increasing the carbon uptake [31]. Furthermore, due to the nutrient-rich properties of yeast extract, as well as its low-cost production and safety, this nitrogen source is considered to have an ideal nutrient composition for use by the fermentation industry [32]. Similar results were obtained with PLC production by B. thuringiensis isolated from Tunisian industrial effluent. In addition to yeast extract, this production was dependent on the presence of a source of phosphorus (K₂HPO₄) at 2.5 g/L in the culture medium [2]. From the B. thuringiensis serovar kurstaki strain, peptone at 5.38 g/L was found to be the most appropriate nitrogen source for PI-PLC production [25]. In addition, PLC activity was slightly decreased when yeast extract was replaced by protein hydrolysates (peptone or tryptone) as the nitrogen source (Figure 3B). This could be related to the difference in composition of these hydrolysates compared to yeast extract. However, the reduced PLC activity in the presence of an inorganic nitrogen source (34.5 U/mL and 20 U/m L for KNO₃ and (NH₄)₂SO₄, respectively) could be explained by the fact that some amino acids cannot be synthesized from inorganic nitrogen sources, as described in several fungi strains [33].

3.4. Effect of Metal Ions and Inoculum Concentration

Micro-organisms require specific metallic ions such as potassium, magnesium, manganese, zinc, and molybdenum for growth and function [34]. Metal ions in a bacterial medium are an important factor that affects extracellular enzyme production as they act as inducers, particularly for metalloenzymes and the structure, function, and metabolism of nucleic acid. It has also been reported that cations in the bacterial medium are required to reduce electrostatic repulsion between lipids and proteins, thus increasing the membrane stability [35].

The effect of different metal ions on PLC_Bs production was also investigated (Figure 4A). The maximum PLC enzymatic activity (272.5 U/mL) was obtained when the medium was supplemented with 1 mM of Zn²⁺ (Figure 4A). However, the PLC activity detected in the absence of metal ions is probably due to the presence of these ions in other medium compounds, especially complex nitrogen sources. Indeed, yeast extract is a complex mixture of amino acids, vitamins, several element minerals, and polypeptides [36]. The measured PLC activity in the presence of 1 mM of tested metal ions could be attributed to the fact that PLCs are a class of lipolytic enzymes that require cations for enzymatic hydrolysis potential. In fact, several PLCs produced by Bacillus strains require a millimolar concentration of Mg²⁺, Zn²⁺, or Ca²⁺. PLCs produced by B. thuringiensis (PLC_Bt) need 0.5 mM of Mg²⁺ in the medium to achieve a maximum level of 6000 U/mg [2]. PLC_Bc is defined as a PLC that contains three zinc ions in the active site [37]. PLCs produced by B. licheniformis are metal-ion-dependent enzymes that require Mn²⁺ and Mg²⁺ for catalysis [6]. The reduced enzymatic activity measured with 1 mM of Mn²⁺ (Figure 4A) could be attributed to the inhibition of the PLC [2].

On the other hand, as shown in Figure 4B, the rate of enzyme production significantly increased as the inoculum volumes increased until reaching the maximal activity (427 U/mL ± 3.5) at 8% inoculum size. At larger inoculum volumes, the enzyme production was clearly reduced (387.5 U/mL ± 3.5), which might be attributed to the more intense nutrient consumption in the culture medium. In fact, several inhibitory compounds could be produced by the bacterial culture, resulting in inhibition of its growth and development.

3.5. PLC_Bs Purification and Biochemical Characterization

Purification of PLC_Bs

In order to produce PLC_Bs, the identified thermophilic bacterial strain B. stearothermophilus was cultured in the optimized medium. PLC_Bs was purified according to the protocol described in the Materials and Methods section. After heat treatment of the supernatant culture at 70 °C for 10 min, the (NH₄)₂SO₄ precipitation fractionation was applied from 40% to 70% (w/v), followed by dialysis overnight, and it was finally injected into a Mono Q-Sepharose column. After washing the column, adsorbed proteins were eluted by a linear gradient of NaCl from 25 to 250 mM in buffer A. Eluted fractions with PLC activity tested under standard conditions (Figure 5A) were analyzed via SDS PAGE, showing a single band with an apparent molecular mass of approximately 28 kDa (Figure 5B).

Table 1. Purification steps of PLC_Bs.

Purification Step	Total Activity (U) a	Protein (mg) b	Specific Activity (U/mg)	Activity Recovery (%)	Purification Factor
Culture supernantant	204,000	425	480	100	1
Heat treatment (70 °C, 10 min)	163,200	115	1419	80	2.95
(NH4)2 SO4 fractionation (40–70%)	122,400	41.5	2950	60	6.14
Mono Q-Sepharose	79,560	9.41	8450	39	17.6

^a: One phospholipase activity is defined as 1 μmole of fatty acid liberated under assay conditions. ^b: Protein concentration was measured using the Bradford method [15].

summarizes the specific activity and recovery rates of PLC_Bs after each purification step. PLC_Bs was purified with a recovery rate of 39% and a purification factor of 17.6 (Table 1). Interestingly, the high specific activity of the pure PLC_Bs was measured (8450 U/mg) using PC as the substrate under standard conditions in the presence of 0.4 mM Zn²⁺. In the same line, PLC_Bt showed a high catalytic activity of approximately 6000 U/mL on PC as the substrate and 0.5 mM Mg²⁺ using the pH-stat method [2]. Specific activities with lower levels were reported with PLC from B. thuringiensis KT159186 (1042 U/mg) and Bacillus licheniformis MTCC7445 (398 U/mg) when the chromogenic substrate p-nitrophenylphosphorylcholine was used [3,6].

The first 39 amino acid residues from the PLCBs N-terminal sequence were determined. The obtained sequence “WSAEDKHKEGVNSHLWVVNRAIDIMSRNTTLVKQDRVAL” exhibited a high similarity of 97% [38] with the PLC of B. thuringiensis and B. cereus [39] and a similarity of 95% with the PLCBt from the B. thuringiensis MC28 strain [40], suggesting that PLCBs belongs to the PLC family (Figure 6).

3.6. Biochemical Characterization of PLC_Bs

3.6.1. Effect of Temperature and pH on PLC_Bs Activity and Stability

The pH activity profile of the PLC_Bs, shown in Figure 7A, indicates that the highest activity of PLC_Bs was recorded at pH 7–8. In addition, PLC_Bs remained highly stable at a pH range of 7 to 10 after pre-incubation at room temperature for 1 h, with a residual activity of more than 95% (Figure 7A). The same behavior was observed with PLC_Bt, which has maximal enzymatic activity at pH 8.5 and was stable at pH values 8 and 9 after incubation at room temperature for 1 h [2]. Wang et al. [7] reported that the PLC from B. mycoides was stable over a broad range of pH 5 to 9.5 and fully active at pH 7 and 7.5. The PLC activity of the B. licheniformis MTCC 7445 strain showed marked activation at alkaline pH values (8–10) and maintained 90% or 80% of its residual activity at pH 11 or 12, respectively [6]. According to Yuan et al. [40], the pH stability of enzymes is related to their tertiary structure, which becomes less ordered at acidic and alkaline pH but is maintained by rigid interactions between helices.

PLC_Bs activity was measured under standard conditions at temperatures ranging from 30 to 70 °C. The results presented in Figure 7B show that PLC_Bs was active in this temperature interval, with a maximal activity recorded at 60 °C. In addition, PLC_Bs retained its full activity after heat treatment up to 50 °C for 1 h, while only 70% of its initial activity was lost at 70 °C. However, PLC_Bs activity was drastically affected after 1 h of exposure to 80 °C (Figure 7B). This remarkable thermostability was also observed with PLC_Bt, which presented an optimal activity at 55 °C and was stable at high temperature from 37 °C to 65 °C [2]. The maximal PLC activity from the B. licheniformis MTCC 7445 strain was found at 70 °C and was reduced to 80% at 80 °C. The thermostability profile indicates that this PLC exhibited stability at temperatures ranging from 50 °C to 70 °C and retained 70% of its activity at 80 °C [6]. Wang et al. [7] reported that the maximum activity of PLC from the B. mycoides strain 970 was 60 °C and that stability was exhibited at a temperature below 50 °C. PLC purified from the B. thuringiensis KT159186 strain was thermostable as it retained more than 80% of its initial activity after 60 min of heat treatment. At 60 °C and 70 °C, PLC activity was drastically affected and dropped to less than 15% [3]. This difference in thermostability could be explained by the fact that enzymes stable at high temperatures have a more rigid 3D structure, with a reduced entropy of unfolding and α-helixes maintained by hydrogen bonds, electrostatic and hydrophobic interactions, and disulfide bonds [41].

3.6.2. Bile Salt Dependence and Metal Ion Requirement

In order to investigate the bile salt dependence of PLC_Bs, enzymatic activity was determined by varying NaDC concentrations from 2 to 12 mM. Figure 8A shows that PLC_Bs displayed only 23% of its maximal activity without adding any bile salt to the reaction medium. A gradually increasing PLC activity was observed as the NaDC concentration increased to reach its maximal level at 9 mM (Figure 8A). Moreover, a larger concentration of bile salt (up to 12 mM) had no inhibitory effect on the PLC_Bs activity. This bile salt requirement was indicated with PLC_Bt, which displayed maximal specific activity in the presence of 7 mM NaDC [2]. PLC from B. cereus reached its maximal specific activity (1040 U/mg) with 0.15–0.3 mM NaDC. However, at 10 mM, NaDC competitively inhibited PLC_Bc by 50% [42]. An increase in PLC activity with an increasing bile salt concentration could occur as a result of an interaction between bile salts and the enzyme. This detergent can bind to the catalytic site and cause a conformational change that results in more active enzyme conformation. Bile salt can also interact with the phospholipid, generating a substrate with better binding properties, and/or bind with the released product, facilitating the hydrolysis rate [42].

Furthermore, as shown in Figure 8B, PLC_Bs exhibited a specific activity of around 2000 U/mg in the absence of any trace of ions in the reaction medium. Notably, bivalent cations at 0.4 mM seemed to be the optimal concentration that gave the maximal specific activity; however, the specific activity decreased when this concentration was exceeded. Zn²⁺ was found to be the best metal ion for reaching the maximal specific activity of PLC_Bs (8450 U/mL). At 0.4 mM Ca²⁺, PLC_Bs exhibited only 50% of its maximal specific activity while, at the same concentration of Mg²⁺ or Mn²⁺, the enzyme displayed up to 89% or 63% of its maximal activity, respectively (Figure 8B). Similarly, PLC_Bt activity was enhanced with 0.5 mM Mg²⁺, showing a maximum specific activity of 6000 U/mg [2], whereas B. licheniformis PLC required Mg²⁺ or Mn²⁺ to express its maximal activity [6]. These findings are supported by the crystal structure resolution of PLC_Bc, which displays three zinc ions in its active site that are closely associated with residues near to the catalytic site [43]. PLC_Bc thus belongs to the metalloenzymes family, possessing a tri-metal zinc center that plays a crucial role in catalytic hydrolysis [44]. Maximum activity occurs when the enzyme contains two or three Zn²⁺ per mole of protein. In addition, it has been reported that Mg²⁺ or Ca²⁺ can occupy the metal zinc center during phospholipid hydrolysis [5].

3.6.3. PLC_Bs Apparent Kinetic Parameters

In order to further characterize PLC_Bs, apparent kinetic parameters (apparent K_m, k_cat and k_cat/K_m) were estimated using Lineweaver–Burk plots after measuring reaction rates at various PC concentrations ranging from 0.71 to 11.42 mM under standard assay conditions (Supplementary file). PLC_Bs showed an apparent K_m value of 2.94 ± 0.02 mM, turnover k_cat of 66.46 ± 2.5 s⁻¹ and catalytic efficiency k_cat/K_m of 22.60 ± 4 s⁻¹ mM⁻¹. Using the same substrate, PC, the Michaelis constant K_m of PLC_Bs was less than PLCs from B. licheniformis MTCC 7445 which was 16 mM and similar to K_m from B. cereus (2.4 mM) [6,45]. The apparent turnover k_cat of PLC_Bc was only 1000 s⁻¹, while PLC_Bs presented k_cat was 66.46 s⁻¹. These differences can be related to the difference in the rate of PC hydrolysis resulting in various specific activities of only 2100 µmol/min/mg for PLC_Bc [45].

3.6.4. PLC_Bs Substrate Specificity

The substrate specificity of the purified PLC_Bs was investigated by measuring its enzymatic activity toward various phospholipids with different headgroups. Figure 9 displays the relative PLC activity expressed as a percentage compared to the corresponding activity on the universal substrate, PC. PE, PI, PG, PS, SM, and cardiolipin were assessed as substrates. The recorded data show that the zwitterionic PC was the most effective substrate for PLC_Bs (Figure 9). Therefore, PLC_Bs can be classified as a PC-PLC. PE, PS and SM were hydrolyzed by PLC_Bs with relative activities of 44.5%, 29.5%, and 52%, respectively. A much lower hydrolysis level was observed with PI and cardiolipin (10.25% and 7.25%, respectively). The specificity order for PLC_Bs, PC > PE > PS > PG, is similar to that obtained with PLC_Bt, which preferred PC as the substrate and showed relative activities of 40%, 22%, and 18% for 1-palmitoyl-2-oleoyl-PE, 1-palmitoyl-2-oleoyl-PS, and 1-palmitoyl-2-oleoyl-PG, respectively [2]. Substrate specificity studies on PLCs produced by various Bacillus strains confirmed that PC is usually their preferred substrate. In contrast, there is a divergence regarding their ability to hydrolyze other types of phospholipids. Indeed, PLC from B. licheniformis MTCC 7445 demonstrated comparable hydrolysis rates of PI, PS, and PC (0.2–0.26 mM/min) but a highest affinity for SM [6]. The substrate specificity order PC >> PI > PE > PG was reported with PLC from B. thuringiensis KT159186 [3], while extracellular PLC from B. cereus D101 had an average affinity for PE and SM and low hydrolysis rate of PI, PS, PG, and cardiolipin [46]. PLC_Bc preferentially processed substrate PC over PE and PS, with catalytic efficiencies of 417, 300, and 47 mM⁻¹·s⁻¹, respectively [45]. The fact that PLC not only preferentially catalyzes the hydrolysis of PC but also processes PE and PS with correspondingly decreased catalytic efficiencies was explained by mutagenesis studies and correlated with available crystal structure analyses of PLC from B. cereus [4]. This study established that PLC_Bc affects catalysis by activating a water molecule during the attack on the phosphorus atom of a phosphodiester moiety. Residues Glu4, Asp55, and Glu146 around the active site ensure this attack by stabilizing the positive charge on the headgroups of PE and PS [4].

3.6.5. Correlation between Hemolytic and Antibacterial Activities and Substrate Specificity

Biological membranes are organized in the continuous bilayer of phospholipids, mainly SM, cardiolipin, and glycerophospholipids. In erythrocytes, PS, PE, and PI form the inner monolayer, while PC and SM are essentially located in the outer monolayer [47]. The major phospholipids in the Gram-negative bacterial membrane are PE, PG, and cardiolipin, while Gram-positive bacteria contain glycosylated DAGs and PG in their membranes [48]. Several phospholipases exhibit cytotoxicity toward cells or bacteria through an interaction and/or hydrolysis of their membrane phospholipids. In line with this, the behavior of PLC_Bs toward phospholipids in the biological membrane was investigated, since it is able to hydrolyze various phospholipids, such as PC, PE, and SM, with high preference toward PC.

The study of the direct hemolytic activity of PLC_Bs was carried out using human erythrocytes. Figure 10 shows that PLC_Bs exhibited a dose–response effect toward erythrocytes from 0.5 to 3 nM. At 0.75 nM, the hemolytic effect was 82.25%, and it reached 93.75% at 1.25 nM. This effect could be correlated to the erythrocyte’s membrane composition. Indeed, PC and SM in the outer membrane of human erythrocytes are the preferred phospholipids of PLC_Bs, exhibiting a relative activity of 100% and 60%, respectively. In contrast, PLC_Bc does not show any direct hemolytic activity alone while it acts in synergy with a sphingomyelinase to hydrolyze human membrane phospholipids. The inability of PLC_Bc to hydrolyze PC in the erythrocytes membrane is attributed to the lower accessibility of ester bonds, which link the fatty acyl chains, than that of the head group of PC [49]. PLC purified from B. mycoides strain 970 is described as non-hemolytic due to its inability to hydrolyze lecithin as a substrate [7].

The study of the antibacterial activity of PLC_Bs was carried out using the agar diffusion method.

Table 2. Antimicrobial activities of purified PLC_Bs via agar disc diffusion assay (IC₅₀, 50% inhibition concentration). The results represent the means of three independent experiments and are expressed as the mean ± SD (n = 3).

Strain	Gram	Inhibition Zone (mm)		IC50 (µg/mL)
		PLCBs	Ampicillin
Escherichia coli (ATCC 25922)	−	10.6 ± 0.57	22.6 ± 1.5	−
Pseudomonas aeruginosa (ATCC 27853)	−	5.6 ± 0.58	20 ± 0.7	−
Enterococcus faecalis (ATCC 29122)	+	21.6 ± 0.57	24.2 ± 0.7	15 ± 1.51
Staphylococcus epidermidis (ATCC 14990)	+	19.3 ± 1.52	26 ± 0.5	17.5 ± 1.61
Staphylococcus aureus (ATCC 25923)	+	20 ± 1	21.5 ± 1.4	11 ± 1.65

shows that PLC_Bs was effective against all the tested bacterial strains, especially Gram-positive ones, producing inhibition zone diameters ranging from 19 mm to 21 mm; these are comparable to those of the positive control, ampicillin (21–26 mm). PLC_Bs was, however, less effective against Gram-negative Escherichia coli and Pseudomonas aeruginosa strains, with inhibition zone diameters of 10.6 and 5.6, respectively. The antibacterial potential of PLC_Bs was further investigated by determining IC₅₀ values, which ranged between 11 and 17.5 µg/mL for Gram-positive bacterial strains, corresponding to 0.39 and 0.62 µM, respectively (Table 2). However, up to 120 µg/mL of PLC_Bs, the percentage of the CFU values of treated Escherichia coli and Pseudomonas aeruginosa was only 70% and 83%, respectively (data not shown), although their membranes contain mainly PE, PG, and cardiolipin, which are all hydrolysable by PLC_Bs. This finding can be explained by the presence of an additional protective barrier in the outer membrane of the tested Gram-negative bacteria that probably prevents PLC_Bs from reaching the cytoplasmic membrane [48,50].

Phospholipids of Gram-positive bacterial membrane are mainly formed of glycosylated DAGs and PG, lysylPG, cardiolipin, and lysyl-cardiolipin, although some Gram-positive strains have a high content of PE [50,51]. This fact could explain the significant antimicrobial activity of PLC_Bs against Enterococcus faecalis, Staphylococcus epidermidis, and Staphylococcus aureus (Table 2). PLC_Bs seemed to interact with the anionic phospholipids PG and cardiolipin in the Gram-positive bacteria membrane. This interaction, together with the hydrolysis of phospholipids by PLC_Bs, could be a potential mechanism of membrane damage by disrupting bacterial membrane bilayer organization, consequently leading to toxicity [51].

3.6.6. Biotechnological Application of PLC_Bs: Oil Degumming

The presence of phospholipids in crude vegetable oils can lead to dark-colored oils and the generation of off-flavors during storage. Therefore, the removal of these phospholipids is an essential step toward producing high-quality finished oil that contains less than 10 mg/kg of phosphorus [8]. During the industrial oil refining process, water degumming removes hydratable phospholipids, while non-hydratable forms are eliminated by chemical or enzymatic treatment, which is usually carried out with phospholipases. Enzymatic degumming has several advantages, such as improving the yield; reducing the amount of acid, alkali, and wastewater; decreasing the negative effect on the environment; and reducing industrial operating costs [6]. Among phospholipases, PLCs are the most effective degumming candidates since they release oil-soluble sn-1,2- DAGs that have high nutritional value and water-soluble organophosphates [3].

Soybean oil, the most widely consumed vegetable oil, is rich in lysophospholipids, PC, PI, DAG, TAG, and glycolipids [52]. Since PLC_Bs was able to hydrolyze various phospholipids, soybean oil was used to evaluate its performance in the degumming process by measuring the phosphorus content and DAG content (Figure 11).

Water degumming allowed for a reduction in the phosphorus content to 111 mg/kg after 4 h of treatment compared to 820 mg/kg before any treatment (Figure 11A). However, enzymatic degumming using PLC_Bs seemed to be more efficient, with a residual phosphorus content of 35 mg/kg after 4 h of oil treatment with 100 U at 60 °C and a high shaking speed (500 rpm) (Figure 11A). Figure 11B reveals a small increase in DAG content after water treatment only, while this content increased significantly as the enzyme amount increased, reaching 3.5% when 100 U of PLC_Bs was used (Figure 11B). This finding clearly demonstrates the ability of PLC_Bs to hydrolyze phospholipids in the crude soybean oil with a subsequent release of DAGs as reaction products, since the relative concentrations of different phospholipid species in crude soybean oil are approximately 35–47%, 20–30%, 20–24%, and 9–15% for PC, PE, PI, and phosphatidic acid, respectively [9]. In addition, the highest degumming efficiency was achieved by using 100 U of PLC_Bs in combination with the water content (Figure 11C). Indeed, 19 mg/kg of residual phosphorus was reached after 4 h with 100 U of PLC_Bs while 34.5 and 24 mg/kg were obtained with 25 and 50 U of PLC_Bs, respectively (Figure 11C). This finding proves that water enhanced the affinity of the enzyme toward its substrate.

A few PLCs from Bacillus strains have been reported as being efficient in the degumming process using different oils. For instance, PLC from the B. licheniformis MTCC 7445 strain decreased the phosphorus level to 8, 9, and 10 mg/kg for sesame and castor oils, palm oil, and corn and sunflower oils, respectively, during a 4 h time period, while with citric acid treatment, the content of phosphorus in these crude oils decreased from approximately 200 to 36.5 mg/kg [6].

The recombinant PI-PLC enzyme from the B. thuringiensis strain is described as having a high potential for oil degumming. Indeed, PI-PLC_Bt decreased the phosphorus content of soybean oil down to 30.87 mg/kg after 2 h of treatment. A higher degumming efficiency of 89.42% with a final residual phosphorus content of 13.7 mg/kg was achieved by a combination of PI-PLC_Bt and water treatments. In addition, a marked increase in DAGs up to 94% was also observed in the case of PI-PLC_Bt [52]. In line with this, native PLC_Bc was shown to be more efficient than its recombinant form produced in Pichia pastoris for soybean oil degumming by hydrolyzing PC and PE, which make up 70% of the total soybean oil phospholipids. However, PLC_Bc mutants F66Y (PLCBcY) and F66W (PLCBcW) hydrolyzed PC in crude oil at a higher reaction rate compared to the wild-type PLC_Bc. PLCBcY also showed a higher efficiency, hydrolyzing 91% of PE compared to only 49% for the PLCBcW mutant [9].

4. Conclusions

A novel phospholipase C, “PLC_Bs”, produced by the B. stearothermophilus strain was purified and characterized. In addition to its high specific activity (8450 U/mg on PC as the substrate and in the presence of 0.4 mM Zn²⁺), PLC_Bs exhibited high thermoactivity and remarkably high stability in both acidic and alkaline conditions. Interestingly, in contrast to previously studied PLCs from Bacillus strains, this novel enzyme was able to hydrolyze cardiolipin and sphingomyelin. Collectively, the stability characteristics and substrate specificity of PLC_Bs greatly enhance its potential for use in crude soybean oil degumming. Compared to classical water degumming, degumming using PLC_Bs displayed a high efficiency and minimum residual phosphorus content, as well as an increased amount of DAGs. This degumming potential encourages the use of PLCBs in various industrial processes. However, since phospholipases, as sustainable biocatalysts for soybean oil degumming, are more costly than chemical catalysts, their immobilization on appropriate carriers should be assessed in order to decrease the enzyme cost by reusing PLCBs.