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Article

Emulsion Systems Stabilized with Nonionic Emulsifier and Cross-Linked Polyacrylic Acid: A Promising Strategy to Enhance the Activity of Immobilized CALB

by
Joanna Siódmiak
1,*,†,
Jacek Dulęba
2,†,
Dominik Mieszkowski
2,
Piotr Bilski
3 and
Tomasz Siódmiak
2
1
Department of Laboratory Medicine, Faculty of Pharmacy, Ludwik Rydygier Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, 85-094 Bydgoszcz, Poland
2
Department of Pharmacy Practice, Faculty of Pharmacy, Ludwik Rydygier Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, 85-089 Bydgoszcz, Poland
3
Department of Pharmaceutical Technology, Faculty of Pharmacy, Ludwik Rydygier Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, 85-089 Bydgoszcz, Poland
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Catalysts 2025, 15(10), 916; https://doi.org/10.3390/catal15100916
Submission received: 8 August 2025 / Revised: 12 September 2025 / Accepted: 14 September 2025 / Published: 23 September 2025
(This article belongs to the Special Issue Enzyme and Biocatalysis Application)
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Abstract

The application of lipases in biphasic oil–water emulsions offers an efficient and sustainable alternative to conventional chemical synthesis. However, the natural immiscibility of these phases is a substantial limitation. To address this issue, we proposed a dual-stabilized emulsion system combining a nonionic emulsifier (Kolliphor® CS 20) and cross-linked polyacrylic acid (Carbopol® Ultrez 10), exceeding conventional single-stabilized systems. The activity of Candida antarctica lipase B (CALB), both in its free form and immobilized onto an IB-D152 support, was investigated in the prepared emulsion system. The olive oil emulsion stabilized with 10.0% Kolliphor® CS 20 and 0.1% Carbopol® Ultrez 10 significantly enhanced the lipolytic activity of immobilized CALB (156.27 ± 3.91 U/g of support), compared to the activity obtained in the emulsion stabilized only with 10.0% Kolliphor® CS 20 (71.11 ± 3.86 U/g of support). On the other hand, the activity of immobilized CALB in the emulsion containing 5.0% Kolliphor® CS 20 and 0.1% Carbopol® Ultrez 10 (62.22 ± 3.85 U/g of support) was lower than in the corresponding system without Carbopol® Ultrez 10 (72.03 ± 4.63 U/g of support), stabilized with only 5.0% Kolliphor® CS 20. Furthermore, immobilization onto IB-D152 led to lipase hyperactivation, with activity approximately eight-fold higher than that of free CALB. This dual emulsion stabilization strategy not only improves emulsion stability but also enhances lipase activity, offering new opportunities for scalable, high-performance biocatalysis using emulsions in industrial applications.

1. Introduction

The application of enzymes in biphasic water–oil systems represents an efficient and sustainable alternative to conventional chemical synthesis, particularly in the context of green chemistry. However, the natural immiscibility of these phases imposes significant limitations, affecting both the stability of the system and the catalytic efficiency. To overcome these limitations, amphiphilic molecules capable of stabilizing the water and oil phases, known as surfactants, are introduced into systems [1,2,3,4]. Among them, nonionic surfactants are of particular interest due to their lower toxicity compared to other surfactants, broad compatibility, and lack of ionization in aqueous solution [5,6]. When used to stabilize emulsions, they are also referred to as emulsifiers. The group of nonionic surfactants includes, among others, Ceteareth (polyethylene glycol (PEG) ethers of cetearyl alcohol) and Steareth (PEG ethers of stearyl alcohol), which are commonly used ingredients in cosmetic and pharmaceutical formulations [7]. Commercially available products of Ceteareth are, e.g., Kolliphor®, while Steareth is known as Brij®. Surfactants adsorb at the oil–water interface, forming a protective layer or barrier around the dispersed internal droplets in the emulsion [8]. These properties make them valuable additives in enzymatic catalysis, enabling increased enzyme activity. In this context, as some studies suggest, the concentration of the surfactant may play a significant role in modulating the lipase activity [9].
Besides emulsifiers, emulsion systems can also be stabilized with the addition of cross-linked polyacrylic acid (PAA) (commonly known as Carbopol®), which affects system viscosity. In these modified systems, Carbopol® polymer primarily contributes to the increase in the viscosity of the aqueous phases and slows down the motion of surfactant-coated droplets. This thereby improves the stability of the dispersed system. Furthermore, in the presence of surfactants, polymer/surfactant interactions may occur [10,11]. These interactions between polymer and surfactant can influence the formation of an optimal reaction medium and enzyme performance, thus enabling new capabilities in terms of biocatalysis. Among the polymer/surfactant systems, those containing nonionic surfactants seem to be very promising, especially in the context of lipase-catalyzed reactions where interfacial activation and enzyme stability are crucial for catalytic efficiency [12,13].
Candida antarctica lipase B (CALB) is one of the most widely used enzymes in biocatalysis due to its high catalytic activity and capability to catalyze reactions in both aqueous and non-aqueous media. This lipase operates at the water–oil interface [14,15,16]. The presence of an amphiphilic α-helix (referred to by some authors as a “lid”) in the structure allows, under hydrophobic conditions (e.g., in the presence of a hydrophobic surface), the enzyme’s conformation to change from a closed to an open form (interfacial activation phenomenon) [15,17]. The literature has shown that the addition of a surfactant to an emulsion system improves reaction efficiency [18]. Furthermore, it has been demonstrated that hydrophilic and hydrophobic groups in the surfactant affect the enzyme-support interaction as well as the enzyme itself, thereby influencing the biocatalyst’s activity and stability [19,20]. It should be noted that the above-mentioned effect depends on the type of enzyme, the support used for immobilization, and the properties of the surfactant. Enzyme immobilization is a preferred strategy for enhancing and maintaining the catalytic properties of the enzyme. Certain lipases, including CALB, exhibit high catalytic activity when immobilized on a commercial polyacrylic support [21,22].
Based on literature data [20], nonionic surfactants are able to enhance both enzyme stability and catalytic activity. Despite the extensive application of nonionic emulsifiers and cross-linked polyacrylic acid in various cosmetic and pharmaceutical formulations, their simultaneous use in emulsion systems applied in biocatalysis remains insufficiently studied. Therefore, in this study, we investigated the effect of a dual-stabilized emulsion system combining a nonionic emulsifier and cross-linked polyacrylic acid on the activity of CALB in its free form and immobilized onto an IB-D152 support. The dual emulsion stabilization strategy presented here contributes to the development of research on enzyme–emulsifier–polymer interactions in lipase-catalyzed reactions within a two-phase system. The proposed approach enables fine-tuning of enzyme activity. The combination of Kolliphor® CS 20 and Carbopol® Ultrez 10 offers an innovative stabilization method for lipase-based biocatalysis, compared to earlier stabilized systems that relied mainly on surfactants [9]. This dual mechanism not only improves the physical stability of the emulsion system but also enhances lipase catalytic activity. The proposed system demonstrated tunable effects depending on Kolliphor® CS 20 concentration in the presence of Carbopol® Ultrez 10, emphasizing the importance of emulsifier-polymer-enzyme interactions. This approach fills a clear gap in the literature, goes beyond the conventional single-stabilization concept, and offers new opportunities for scalable high-performance biocatalysis using emulsions in industrial applications, such as the hydrolysis of triglycerides and green chemistry. Moreover, the proposed approach makes a significant contribution to the methodology of optimizing enzyme performance in complex multiphase systems.

2. Results and Discussion

2.1. The Influence of Kolliphor® CS 20 Concentration on Immobilized CALB Lipolytic Activity

To evaluate the impact of emulsifier concentration on the lipolytic activity of immobilized CALB, emulsions composed of olive oil and water (o/w emulsions) with varying amounts of emulsifier were used. A nonionic emulsifier, Kolliphor® CS 20, was applied in the study at concentrations of 2.5%, 5.0%, 7.5%, and 10.0%. The immobilized form of lipase was obtained by immobilizing free CALB onto polyacrylic matrix support containing carboxylic acid functional groups (IB-D152), forming an ionic (cationic) bond between the lipase and the support. Catalytic activity studies were carried out in phosphate buffer (100 mM, pH 7.4) at 37 °C for 30 min (see Section 3.2.3 for details). Lipolytic activity was determined based on the hydrolysis of triglycerides in olive oil, catalyzed by immobilized CALB. Based on the assessed lipase activity (U), the relative activity (Arel) was calculated. The results are presented in Figure 1.
Analysis of the results revealed that the highest lipase activity was observed in emulsions containing 5.0%, 7.5%, and 10.0% of the nonionic emulsifier Kolliphor® CS 20 with relative activity Arel = 100.0 ± 5.1%, Arel = 98.8 ± 3.9%, and Arel = 98.8 ± 7.0%, respectively. The lowest activity, within the tested concentration range, was noticed for the emulsion with 2.5% emulsifier (Arel = 81.8 ± 4.1%). At this stage of the research, no inhibitory effect of increasing Kolliphor® CS 20 concentration on the lipolytic activity of CALB was observed. Furthermore, lipase activity did not rise with increasing emulsifier concentrations in the range of 5.0% to 10.0%. The enzyme activity in the emulsion stabilized with 5.0% emulsifier was 72.03 ± 4.63 U/g of support, whereas in the emulsion containing 10.0% emulsifier, it was 71.11 ± 3.86 U/g of support. Regarding the amount of CALB immobilized onto the tested support, in accordance with our previous work [21], the lipase loading onto IB-D152 was 25.2 mg/g of support.
As part of the preliminary research, additional experiments were conducted to investigate the effect of emulsion homogenization (containing 5.0% of Kolliphor® CS 20) on the lipolytic activity of immobilized lipase. The results showed that lipase activity was comparable in both homogenized and unhomogenized systems. Relative activity in the homogenized emulsion was Arel = 97.0 ± 3.6%, whereas in the unhomogenized emulsion it was Arel = 100.0 ± 2.4%. The value of 100% relative activity corresponds to the highest enzymatic activity (U) registered in the particular study (the effect of homogenization of emulsions containing 5.0% of Kolliphor® CS 20 on immobilized CALB activity). We believe that the observed results may be influenced by factors such as the type of emulsifier, its concentration, and the immobilized form of the enzyme. It can be assumed that in both homogenized and unhomogenized emulsions, substrate availability at the interface, and thus enzymatic activity, was at a comparable level (p > 0.05). Based on these observations, the unhomogenized emulsion system was selected for further studies.
Literature reports indicate that nonionic surfactants improve the structural stability of lipase through hydrophobic interactions and hydrogen bonding [9]. It is also emphasized that the concentration of nonionic surfactants plays an important role in modulating lipase activity. Moreover, interactions between the nonionic surfactants and lipases are pH-dependent, as the pH of the reaction medium can influence both the strength and character of these interactions. Nonionic surfactants are particularly important in systems involving immobilized lipase, whether used during the immobilization procedure or as a component of the reaction medium. The complexity of interactions between lipases, substrates, and surfactants arises from several factors, such as micelle formation by surfactants, the associated concentration of free and micellar substrate, and their accessibility at the interface. The molecular structure of the emulsifier, especially its hydrophobic and hydrophilic parts, also plays a significant role. At low concentrations, nonionic surfactants typically stabilize lipases via hydrophobic interactions and enhance substrate solubilization. However, at higher concentrations, substrate solubilization within micelles may reduce substrate concentration at the interface, leading to decreased reaction rates. Furthermore, lipase removal from the interface at high surfactant concentrations has been noted [23]. Other studies have shown that nonionic surfactants such as polyoxyethylene alkyl ethers can form micelles with various shapes in aqueous solutions. Importantly, it has been shown that hydrophobic interactions and hydration (hydrophilic effect of hydrogen bonding) are the main driving forces responsible for the self-assembly of nonionic surfactants. Compared to ionic surfactants, nonionic surfactants show weak intermolecular interactions through dispersion forces and hydrogen bonds [24].
The results presented in our study demonstrate that a concentration of 5.0–10.0% Kolliphor® CS 20 ensures optimal activity of the immobilized CALB, without inducing any inhibitory effect.

2.2. The Influence of Carbopol® Ultrez 10 Added to the Emulsion on the Lipolytic Activity of Immobilized CALB

To investigate the effect of Carbopol® Ultrez 10 (cross-linked PAA, Carbomer) as a second emulsion-stabilizing agent on the lipolytic activity of immobilized CALB, the mentioned Carbopol® was added to emulsions containing 5.0% Kolliphor® CS 20. Lipase activity was evaluated in emulsion systems with and without the addition of Carbopol® Ultrez 10. Enzymatic activity (U) was determined based on the hydrolysis of triglycerides in olive oil and expressed as relative activity—Arel, where the designated value of 100% relative activity corresponds to the highest enzymatic activity (U) recorded in the particular study. Experiments were carried out in phosphate buffer (100 mM, pH 7.4) at 37 °C for 30 min (a full description of the procedure is provided in Section 3.2.3). The results showed that the addition of 0.1% Carbopol® Ultrez 10 led to a decrease in the activity of immobilized lipase. In the presence of Carbopol® Ultrez 10, the activity was 62.22 ± 3.85 U/g of support (Arel = 86.4 ± 6.5%), compared to 72.03 ± 4.63 U/g of support (Arel = 100.0 ± 0.0%) in the control emulsion without it. This reduction in the activity may suggest an inhibitory effect of Carbopol® Ultrez 10 on the enzyme’s catalytic performance (p < 0.05). Several factors may have contributed to the observed decrease in lipase activity, including enhanced emulsion viscosity, lipase-Carbopol® interactions, and/or protein binding to the polymer, as well as changes in ionic strength or pH. It is important to note that free CALB was immobilized onto a polyacrylic matrix support (IB-D152) containing carboxylic acid functional groups, forming ionic (cationic) bonds between the lipase and the support. Potential electrostatic interactions between the support and Carbopol® Ultrez 10 should also be taken into consideration. However, further research needs to be performed to clarify the mechanisms underlying the reduction in lipase activity observed in this experiment.
In the literature, it has been pointed out that as the viscosity of the medium increases, enzyme mobility decreases (the enzyme’s access to the substrate is limited), which in turn leads to reduced catalytic activity [25]. Additionally, electrostatic interactions between enzymes and polyelectrolytes are considered crucial, as they affect both catalytic activity and conformational stability [26]. For example, interactions between the negatively charged groups of polyacrylic acid and the positively charged amino acid residues (such as arginine and lysine) in the lid region of lipase are believed to promote its opening [27]. Literature data also indicate that Carbopol® inhibits trypsin activity through a mechanism involving a reaction in which polyacrylic acid deprives trypsin of calcium ions. This action leads to the formation of a thermodynamically unstable enzyme and accelerates the autodegradation process. Furthermore, additional inhibitory mechanisms may also be involved [25,28]. Shortall et al. [29] demonstrated that increasing polyacrylic acid (PAA) concentration significantly decreased the activity of Candida antarctica lipase B. In contrast, Mohamed et al. [30] reported that the Carbopol 934-based gel caused rapid activation of Mucor racemosus LII lipase, with a substantial increase in enzymatic activity, reaching up to 800%. This effect (hyperactivation) was attributed to the immobilization of LII within the gel matrix. Thiele et al. [31] described a system composed of polyacrylic acid (PAA), sodium lauryl ether sulfate (SLES), and a protease enzyme in the presence of Ca2+/Mg2+ ions. They highlighted the role of interactions through electrostatic bridging between PAA and SLES, with Ca2+ ions. The resulting bridged SLES/PAA/Ca2+, due to its interfacial dynamic activity, exhibited strong solubilization capacity for poorly water-soluble immobilized proteins, leading to an enhanced cleaning effect. Consequently, the protease showed improved performance in a complex aqueous medium. Numerous studies have examined the effects of different surfactants, such as SDS (sodium dodecyl sulfate), Tween-80, Triton X-100, and arabic gum, on CALB activity, used, however, as sole stabilizers in emulsion systems [32].
In the next stage of the project, the activity of both immobilized and free lipases was tested in emulsion systems containing Carbopol® Ultrez 10, at concentrations of 0.1% and 0.05%, along with a 5.0% addition of nonionic emulsifier (Kolliphor® CS 20) (Figure 2). Analysis of the results showed that the activity of immobilized lipase, with the addition of Carbopol® Ultrez 10 at a concentration of 0.1% and 0.05%, remains almost unchanged (Arel = 100.0 ± 15.7% and Arel = 100.0 ± 9.4%, respectively). Regarding free lipase, a similar activity profile was observed between the emulsion containing 0.05% Carbopol® Ultrez 10 (Arel = 16.3 ± 5.3%), and the emulsion with 0.1% Carbopol® Ultrez 10 (Arel = 10.2 ± 3.3%). Negligibly higher activity was observed in the emulsion stabilized with 0.05% Carbopol® (p > 0.05). Based on the obtained results, which indicate no statistically significant differences in enzymatic activity between emulsions stabilized with 0.05% and 0.1% Carbopol®, the influence of increased viscosity does not appear to be substantial. However, this aspect requires more extensive analysis. Additionally, interactions between Carbopol® and lipase should also be regarded as a potential factor influencing enzyme activity. It should be emphasized that the presence of a nonionic emulsifier may affect the character of interactions between Carbopol® and lipase in the above-mentioned system. Further studies are planned to resolve the mechanism by which Carbopol® Ultrez 10 affects CALB activity in the presence of a nonionic emulsifier.
It should be emphasized that the activity of immobilized lipase (expressed as relative activity) in an emulsion containing 0.1% Carbopol® Ultrez 10 was approximately ten-fold higher than the activity of free lipase tested in an emulsion of the same composition. However, in the emulsion containing 0.05% Carbopol® Ultrez 10, immobilized lipase was characterized by circa six-fold higher activity compared to free lipase. These results may indicate a positive effect of immobilization of lipase onto the IB-D152 support, enhancing its lipolytic activity. The immobilized enzyme exhibited hyperactivation, as the obtained biocatalyst showed a higher catalytic activity than the free form of lipase. Similar findings (hyperactivation of lipolytic activity; activity retention of 178%) were described in our previous work [21] in which we also tested immobilized CALB onto the IB-D152 support, but the studied emulsion was created using a different emulsifier—gum arabic.
According to the literature, nonionic surfactants, due to their lack of electrostatic interaction capability, typically do not induce conformational changes in the protein. Moreover, they have been shown to enhance enzymatic activity, despite partial protein unfolding. It is assumed that the positive effect of zero-net-charge surfactants may result from the fact that amphiphiles tend to help open the lid over the active site, similar to phospholipids [33]. It has also been reported that nonionic surfactants can modulate electrostatic interactions between the support and the lipase, thereby enhancing the activity of immobilized enzymes. This effect has been observed when surfactants were used either during the immobilization process or as components of the reaction medium [23]. Recent literature highlights that hydrogen bonding is the main factor controlling the interaction between water-soluble polymer chains (including PAA) and nonionic surfactants. Additionally, substantial hydrophobic attraction was observed between polymer chains and hydrophobic parts of surfactant molecules. Importantly, the PAA chains have been shown to bind to C8E5 (pentaethylene glycol n-octyl ether) surfactant clusters due to beneficial intermolecular interactions [24].
The experimental data presented in this study showed that the addition of Carbopol® to an emulsion system stabilized with 5.0% Kolliphor® CS 20 affects the inhibition of the enzyme’s catalytic activity. However, increasing the Carbopol® concentration from 0.05% to 0.1% does not lead to a further reduction in CALB activity.

2.3. Influence of Kolliphor® CS 20 Concentration on Carbopol® Ultrez 10 Stabilized Emulsion

The lipolytic activity of CALB was evaluated in emulsion systems containing Kolliphor® CS 20 at concentrations of 2.5%, 5.0%, 7.5%, and 10.0%. Each emulsion also included 0.1% Carbopol® Ultrez 10. Experiments were carried out in phosphate buffer (100 mM, pH 7.4) at 37 °C for 30 min (a detailed description of the procedure is provided in Section 3.2.3). Enzymatic activity (U) results were expressed as relative activity (Arel). The activity of both free and lipase immobilized onto an IB-D152 support was tested. The results are presented in Figure 3.
Analysis of the data revealed that the highest catalytic activity of immobilized lipase was obtained in the emulsion containing 10.0% Kolliphor® CS 20 and 0.1% Carbopol® Ultrez 10, reaching an activity of 156.27 ± 3.91 U/g of support, compared to emulsions with lower emulsifier concentrations (2.5%, 5.0%, and 7.5%). The activity of immobilized lipase (expressed as relative activity—Arel) in the emulsion containing 10.0% Kolliphor® CS 20 and 0.1% Carbopol® Ultrez 10 was approximately two-fold as high as the activity values noted in the other tested emulsion systems (0.1% Carbopol® Ultrez 10 and an appropriate concentration of Kolliphor® CS 20). This significant increase in lipase activity may be attributed to the stabilizing effect of the higher emulsifier concentration on the emulsion structure. Similarly, in the case of free lipase, the highest activity was documented in the emulsion containing 10.0% of Kolliphor® CS 20 emulsifier and 0.1% Carbopol® Ultrez 10. It should be mentioned that the activity of immobilized CALB in the emulsion containing 10.0% of Kolliphor® CS 20 and 0.1% of Carbopol® Ultrez 10 (Arel = 100.0 ± 0.0%) was approximately eight-fold higher than that of free CALB (Arel = 11.8 ± 5.5%), suggesting a hyperactivation effect upon immobilization on the IB-D152 support.
The data indicate that the catalytic activity of immobilized CALB can be modulated by adjusting the concentration of the tested emulsifier (Kolliphor® CS 20) in emulsions containing 0.1% Carbopol® Ultrez 10. As previously mentioned, the use of olive oil emulsion (oil-in-water) stabilized with a 10.0% of nonionic emulsifier (Kolliphor® CS 20) and an additional gelling agent (0.1% Carbopol® Ultrez 10) enabled a significant enhancement of the lipolytic activity of immobilized CALB (156.27 ± 3.91 U/g of support), compared to the emulsion stabilized with 10.0% Kolliphor® CS 20 alone (71.11 ± 3.86 U/g of support). On the other hand, the activity of immobilized CALB in the emulsion containing 5.0% Kolliphor® CS 20 and 0.1% Carbopol® Ultrez 10 (62.22 ± 3.85 U/g of support) was lower than in the corresponding system without Carbopol® Ultrez 10 (72.03 ± 4.63 U/g of support), stabilized only with 5.0% Kolliphor® CS 20. These findings demonstrate a strong dependence of immobilized CALB activity on nonionic emulsifier (Kolliphor® CS 20) concentration in the presence of Carbopol® Ultrez 10. Therefore, among the tested emulsions, the system containing 10.0% Kolliphor® CS 20 and 0.1% Carbopol® Ultrez 10 showed the most promising and optimal properties.
Kunche and Natarajan [24] examined the interactions between PAA and the nonionic surfactant pentaethylene glycol n-octyl ether PEGOE (C8E5) in aqueous solution. They indicated that the intermolecular interactions of the PAA chain with the polar groups of the surfactant aggregate are more potent and advantageous than those with the nonpolar hydrocarbon groups. This suggests that the PAA chains adsorb on the surface of the micelle formed by the nonionic surfactant (C8E5), without penetrating the micelle core. At higher surfactant concentration, the PAA polymer chains can interact more favorably and bind with surfactant molecules by hydrogen bonds. This phenomenon can be observed because the PAA chains have an extended conformation, allowing them to form a significant number of hydrogen bonds with more surfactant molecules. Carboxyl groups of the PAA chains are located near the polar groups of the surfactant and are adsorbed on the micelle surface. Solely the polar groups of the surfactant molecules interact favorably with water molecules, as water does not penetrate the hydrocarbon core of the micelle, where the nonpolar groups are located. In another study [34], it was pointed out that the sorption of nonionic surfactant octaethylene glycol monooctyl ether (C8E8) within the polymer film (polyacrylic acid modified with hydrophobic sidechains mimicking carbomer polymer) was found to be significant. Additionally, it was observed that as the concentration of surfactant increased, water pockets filled with micelles of surfactant were formed. This contributes to the swelling of the polymer film and the loss of its stability.
Based on these findings, we assume that the emulsion system stabilized with cross-linked polyacrylic acid and a high concentration of nonionic emulsifier (10.0% Kolliphor® CS 20) contributes to greater substrate (olive oil) availability for the immobilized CALB acting at the water/oil interface, thereby improving the catalytic activity of the above-mentioned lipase B. Furthermore, this dual-stabilized formulation not only improves emulsion stability but also promotes lipase-favorable polymer–emulsifier interactions. The combination of 10.0% Kolliphor® CS 20 and 0.1% Carbopol® Ultrez 10 provided optimal conditions for immobilized CALB activity.

2.4. The Effect of Emulsifier Polyoxyethylene Chain Length and Emulsifier Mixture on the Lipolytic Activity of CALB

In this study, we investigated the effect of the polyoxyethylene chain length of the emulsifier on the activity of both immobilized and free lipase, using two nonionic emulsifiers that differ in the number of ethylene oxide (EO) units: Kolliphor® CS 12 (macrogol cetostearyl ether 12) and Kolliphor® CS 20 (macrogol cetostearyl ether 20). The tested emulsions contained either Kolliphor® CS 12 or Kolliphor® CS 20 at a concentration of 5.0%. The experiments were carried out in phosphate buffer (100 mM, pH 7.4) at 37 °C for 30 min (a detailed description of the procedure is provided in Section 3.2.3). The activity of the lipases (free and immobilized onto the IB-D152 support) was assessed, the activity (U) was determined, and the results were expressed as relative activity (Arel) and presented in Figure 4.
Analysis of the data for the tested nonionic emulsifiers revealed no significant effect of polyoxyethylene chain length on the activity of either immobilized or free lipase (p > 0.05) (Figure 4). It is worth noting that the values of catalytic activity (expressed as relative activity) of free lipases concerning the activity of the immobilized form indicate a significant increase in the lipolytic activity of immobilized CALB. The lipolytic activity of immobilized CALB in the emulsion containing either Kolliphor® CS 12 (Arel = 98.4 ± 2.9%), as well as Kolliphor® CS 20 (Arel = 100.0 ± 0.0%) was approximately six-fold higher, compared to the activity of lipase in the free form (Arel = 16.4 ± 3.1% (Kolliphor® CS 12) and Arel = 16.4 ± 2.3% (Kolliphor® CS 20). These results confirm the hyperactivation of CALB upon immobilization onto the IB-D152 support. Based on these findings, it can be concluded that both emulsifiers provided similar reaction conditions for the activity of the catalytic protein. Literature reports indicate that, regarding ethylene glycol-based surfactants, increasing the chain length of ethylene oxide units (the polar part) leads to an increase in solubility in aqueous solutions, accompanied by a proportionate decrease in surface activity. The key parameter determining the physical properties of ethylene glycol-based surfactants in solution is the hydrophilic-lipophilic balance (HLB), particularly their solubility, micelle formation, and emulsifying ability [24]. While Kolliphor® CS 12 has an HLB value of 13, Kolliphor® CS 20 has an HLB value of 15 (data achieved from BASF company). The tested nonionic emulsifiers differ notably in the number of ethylene oxide (EO) units—one contains 12 units, while the other 20, as well as in HLB values. Despite these differences, both exhibit hydrophilic properties and form stable oil/water (o/w) emulsions. Based on the lipase activity results, it can be assumed that the interfacial films formed by these emulsifiers create similar conditions for enzyme activity at the interface. It should also be considered that the emulsifier concentration in the emulsions may influence the activity results. However, substrate availability appears to be a key factor determining enzyme activity. As shown in the study, similar catalytic activity was achieved regardless of the emulsifier used, likely due to comparable substrate availability. Further research is needed to determine whether HLB greatly impacts CALB activity.
As part of the project, the effect of emulsifier mixtures on the activity of both free and immobilized lipase was also investigated. Two emulsion systems were tested: one containing 4.0% SP Brij® S2 MBAL (polyoxyethylene (2) stearyl ether, HLB value: 4.9) and 1.0% Kolliphor® CS 20, and the other one containing 4.0% SP Brij® S2 MBAL and 1.0% Kolliphor® CS 20, as well as 0.1% Carbopol® Ultrez 10 (Figure 5). The emulsifier ratio was precisely adjusted to ensure the stability of the emulsion. With the use of the tested emulsifiers, oil-in-water (o/w) emulsions were created. For immobilized lipase, similar activity values were observed in the emulsion containing 0.1% Carbopol® Ultrez 10 (Arel = 98.3 ± 5.5%), and in the emulsion without Carbopol® Ultrez 10 (Arel = 100.0 ± 2.7%; CALB activity was 66.67 ± 3.33 U/g of support). In the case of free lipase, no significant differences were observed in catalytic activity, both in the emulsion system containing 0.1% Carbopol® Ultrez 10 and in the emulsion without this additive. The relative activity of the emulsion without Carbopol® Ultrez 10 was Arel = 21.7 ± 2.4%, while in the emulsion with Carbopol® Ultrez 10, Arel = 23.3 ± 3.7%. This study also confirmed the hyperactivation of immobilized lipase—the lipolytic activity of the immobilized form was around four-fold higher than the activity of free lipase. The use of a mixture of emulsifiers (SP Brij® S2 MBAL and Kolliphor® CS 20), both with and without the addition of Carbopol®, although beneficial in terms of emulsion stabilization, did not result in higher lipase activity compared to the activity observed in the emulsion stabilized with 10.0% Kolliphor® CS 20 and 0.1% Carbopol® Ultrez 10. Furthermore, even though the studies have not revealed benefits from using a mixture of emulsifiers, it appears justified to explore other combinations of emulsifiers to enhance CALB activity.
To clearly and concisely present the best results obtained from the research, we compared the enzymatic activity of immobilized lipase in three formulations: 5.0% Kolliphor® CS 20 (1); 4.0% SP Brij® S2 MBAL with 1.0% Kolliphor® CS 20 (2); and 10.0% Kolliphor® CS 20 with 0.1% Carbopol® Ultrez 10 (3). The results are expressed as residual activity, where 100% corresponds to the highest enzymatic activity (U) observed among all tested emulsions in the study. Immobilized CALB in an emulsion stabilized with 5.0% Kolliphor® CS 20 (1) showed a residual activity of 46.1 ± 3.0%. In an emulsion stabilized with a mixture of 4.0% SP Brij® S2 MBAL and 1.0% Kolliphor® CS 20 (2), immobilized lipase exhibited a residual activity of 42.7 ± 2.1%. Whereas, in emulsion with two stabilizing agents—10.0% Kolliphor® CS 20 and 0.1% Carbopol® Ultrez 10 (3)—the immobilized CALB demonstrated a residual activity of 100.0 ± 0.0%. Additionally, Table 1 presents the values of the enzymatic activity of immobilized CALB in five selected emulsion systems.
Only a few studies in the literature address the use of the nonionic emulsifier described in our work (Kolliphor® CS 20) and the Carbopol® Ultrez 10 additive as a promising strategy to enhance the activity of immobilized CALB. This highlights the novelty of our investigations. Therefore, the results presented here constitute an important contribution to research involving the development of optimal reaction conditions for biocatalysis. The impact of the structure of nonionic emulsifiers, including the number of ethylene oxide (EO) units, and the combined use of several emulsifiers in an emulsion system (as a mixture of emulsifiers in various proportions) on enzyme activity requires further research. This line of research will be continued in future studies.

3. Materials and Methods

3.1. Materials and Equipment

Olive oil, 2-propanol (IPA), SP Brij® S2 MBAL (Steareth-2), and triethanolamine were purchased from Sigma-Aldrich (Steinheim, Germany). Lipase B from Candida antarctica and polymeric support Immobead D152 (IB-D152) were bought from ChiralVision (Den Hoorn, The Netherlands). Carbopol® Ultrez 10 (Carbomer, cross-linked PAA) was taken from Lubrizol Advanced Materials Europe BVBA (Brussels, Belgium). Kolliphor® CS 12 (Ceteareth-12) and Kolliphor® CS 20 (Ceteareth-20) were gained from BASF SE (Ludwigshafen, Germany). Methanol, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium hydroxide solution (0.1 mol/L), acetone, orthophosphoric acid, and phenolphthalein were procured from POCH S.A. (Gliwice, Poland). The water used in the study was prepared with a Milli-Q Water Purification System (Millipore, Bedford, MA, USA). Formulations were prepared with a laboratory stirrer Eurostar Digital (IKA Werke, Staufen im Breisgau, Germany), whereas in the case of a homogenized emulsion with a 5.0% Kolliphor® CS 20, a homogenizer Ultra Turrax T 25 was additionally used (IKA Werke, Staufen im Breisgau, Germany). In the enzymatic studies, an incubator Unimax 1010 (Heidolph, Germany) and a pH-meter SevenMulti (Mettler-Toledo, Schwerzenbach, Switzerland) were used.

3.2. Methods

3.2.1. Immobilization of Lipase B from Candida antarctica onto IB-D152 Support

The procedure was performed according to the methodology described by the literature data [21] and data shared by ChiralVision (Den Hoorn, The Netherlands), with a few modifications. Briefly, 50.0 mg of IB-D152 support was placed into 2.0 mL centrifuge tubes (Eppendorf SE, Hamburg, Germany). To remove air from the pores in the beads, the supports were rinsed with 0.3 mL of 2-propanol and allowed to stand for 15 min. Next, the supports were rinsed on the filter with 1.5 mL of distilled water and added to tubes containing 10.0 mg of CALB suspended in 1.0 mL of 0.1 M phosphate buffer (pH 7.0). The suspension was mixed with a spoon in an ice bath for 5 min and then incubated overnight at 4 °C. Each immobilization experiment was conducted in triplicate. Control tests to assess the support effect were carried out without lipase.

3.2.2. Preparation of Formulations with Emulsifiers

The qualitative and quantitative compositions of formulations were presented in Table 2, Table 3, Table 4 and Table 5:
Preparation of Formulation with Carbopol® Ultrez 10
The calculated amount of water was weighed in a beaker. The Carbopol® Ultrez 10, in the concentration of 0.05–0.1% was carefully added to the water (on the surface) while stirring constantly with the Eurostar Digital laboratory stirrer equipped with a propeller stirrer, 4-bladed. In another beaker, the olive oil and emulsifier (Kolliphor® CS 20) or emulsifiers (Kolliphor® CS 20 with SP Brij® S2 MBAL) have been joined, followed by the placement of the beaker in the water bath at about 60 °C and stirring with a glass rod until the emulsifier (or emulsifiers) dissolve in the oil. Then, oil with emulsifier (or emulsifiers) was poured into the water with dispersed Carbopol® Ultrez 10 under continuous stirring. The emulsification process was performed for about 2 min at about 500 rpm. Finally, triethanolamine was added dropwise, stirring vigorously until a homogeneous consistency was achieved. All emulsion formulations were prepared in three independent batches. The qualitative and quantitative compositions of formulations were presented in Table 3, Table 4 and Table 5.
Preparation of Formulation Without Carbopol® Ultrez 10
The appropriate amounts of olive oil and emulsifier (Kolliphor® CS 20 or Kolliphor® CS 12) or emulsifiers (Kolliphor® CS 20 with SP Brij® S2 MBAL) were joined in a beaker, followed by the placement of the beaker in the water bath at about 60 °C and stirred with a glass rod until the emulsifier (or emulsifiers) fully dissolved in the oil phase. The calculated amount of water was weighed in another beaker and stirred with the Eurostar Digital laboratory stirrer equipped with a propeller stirrer, 4-bladed. Next, the solution of emulsifier (or emulsifiers) in the oil was poured into the water under continuous stirring. The emulsification process was performed for about 2 min at about 500 rpm. In the case of a homogenized formulation, the homogenizer Ultra Turrax T 25 was used. Homogenization process performed at the lowest speed (6500 rpm) for 5 min. All emulsion formulations were prepared in three independent batches. The qualitative and quantitative compositions of formulations were presented in Table 2 and Table 5.

3.2.3. Evaluation of the Lipolytic Activity of Immobilized Lipase B from Candida antarctica

The protocol of evaluation of lipolytic activity of CALB was excerpted from literature data [21,35,36] with some changes. The mixture was composed of: each of emulsion described in Section 3.2.2 (5.0 mL), phosphate buffer (2.0 mL, pH 7.4, 100 mM), and free lipase (10.0 mg suspended in 1.0 mL phosphate buffer, pH 7.4, 100 mM) or immobilized CALB (50.0 mg support in 1.0 mL phosphate buffer at pH 7.4, 100 mM). The hydrolysis of olive oil was performed in a shaking water bath (37 °C, 30 min, and 150 rpm). To inactivate the enzyme, the reaction was stopped by the addition of a methanol-acetone solution (1:1, total volume 10.0 mL). The liberated fatty acids were calculated based on the results of titration with NaOH solution (50 mM) with the application of phenolphthalein indicator. The analysis was carried out using a blind sample (without enzyme). One unit of CALB activity (U) was determined as the amount of lipase that hydrolyzed olive oil, liberating 1 μmol fatty acid per minute under the assay conditions. Relative activity (expressed in percentage) was calculated according to the following equation:
R e l a t i v e   a c t i v i t y = U s a m p l e U m a x i m u m × 100 %
where
  • Relative activity (Arel)—the ratio between the activity of the sample and the maximum activity of the sample under the test conditions (within the scope of a given experiment);
  • Usample—the activity of the sample under the test conditions;
  • Umaximum—the maximum activity of the sample under the test conditions.
In turn, residual activity (expressed in percentage) was calculated as the ratio between the immobilized CALB activity in the emulsion system and the maximum activity of immobilized lipase in the emulsion (among all emulsions tested). All analyses were performed in triplicate. The titration procedure was verified to confirm its accuracy and reproducibility.
Statistical analysis of the results was performed using Student’s t-test. The results of the p-value (p) are presented in the text above. Statistical significance was set at p < 0.05. Data are presented as means ± standard deviations.

4. Conclusions

Although Candida antarctica lipase B (CALB) is one of the most often used lipases in biocatalysis, new approaches are continuously sought to enhance its catalytic activity in two-phase systems. Studies of CALB lipolytic activity are largely conducted in emulsion systems stabilized using only a single emulsifier, which may not provide optimal stabilization of the two-phase systems. Therefore, we applied technological solutions utilized in pharmaceutical formulation and proposed the introduction of dual-stabilized emulsion systems to biocatalysis. Described in our work, the use of olive oil emulsion (oil-in-water) stabilized with both a nonionic emulsifier (Kolliphor® CS 20) at a concentration of 10.0% and an additional gelling agent (0.1% Carbopol® Ultrez 10) enabled a significant enhancement of the lipolytic activity of immobilized CALB (156.27 ± 3.91 U/g of support), compared to the activity obtained in the emulsion stabilized only with 10.0% Kolliphor® CS 20 (71.11 ± 3.86 U/g of support). On the other hand, the activity of immobilized CALB in the emulsion containing 5.0% Kolliphor® CS 20 and 0.1% Carbopol® Ultrez 10 (62.22 ± 3.85 U/g of support) was lower than in the corresponding system without Carbopol® Ultrez 10 (72.03 ± 4.63 U/g of support), stabilized with only 5.0% Kolliphor® CS 20. It should be mentioned that the activity of immobilized CALB in the emulsion containing 10.0% of Kolliphor® CS 20 and 0.1% of Carbopol® Ultrez 10 was approximately eight-fold higher than that of free CALB, indicating the hyperactivation of the lipase upon immobilization onto the IB-D152 support. These findings demonstrate that the proposed dual-stabilized emulsion system can significantly enhance the activity of immobilized CALB in a biphasic system. Further studies are necessary to clarify the mechanisms of the interactions between the lipase, the support, nonionic emulsifiers (and their mixtures), and cross-linked polyacrylic acid.

Author Contributions

Conceptualization, J.S. and T.S.; methodology, J.S., J.D. and P.B.; validation, J.S., J.D. and D.M.; formal analysis, J.S. and J.D.; investigation, J.D. and P.B.; resources, J.S. and T.S.; data curation, J.S. and D.M.; writing—original draft preparation, J.S. and J.D.; writing—review and editing, J.S., J.D., D.M. and P.B.; visualization, J.S. and D.M.; supervision, T.S.; project administration, J.S.; funding acquisition, T.S. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Excellence Initiative—Debuts, under the “Excellence Initiative—Research University” program, NCU in Toruń—6/2022/Debiuty3.

Data Availability Statement

The data are contained within this article.

Conflicts of Interest

The authors have no conflicts of interest to declare.

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Catalysts 15 00916 g001
Figure 1. Effect of varying concentrations of Kolliphor® CS 20 in the emulsion on the catalytic activity of immobilized CALB. Reaction conditions: immobilized CALB onto IB-D152 (50.0 mg of support); phosphate buffer (100 mM, pH 7.4); the emulsion (unhomogenized) composed of Kolliphor® CS 20 (appropriate concentration), olive oil, and water; temperature 37 °C; incubation 30 min. Data are presented as means ± standard deviations of three independent analyses (n = 3). Error bars represent the standard deviations. The value of 100% relative activity corresponds to the highest value of enzymatic activity (U) documented in the particular study (the effect of Kolliphor® CS 20 concentration on immobilized CALB activity). The complete definition of relative activity can be found in Section 3.2.3.
Figure 1. Effect of varying concentrations of Kolliphor® CS 20 in the emulsion on the catalytic activity of immobilized CALB. Reaction conditions: immobilized CALB onto IB-D152 (50.0 mg of support); phosphate buffer (100 mM, pH 7.4); the emulsion (unhomogenized) composed of Kolliphor® CS 20 (appropriate concentration), olive oil, and water; temperature 37 °C; incubation 30 min. Data are presented as means ± standard deviations of three independent analyses (n = 3). Error bars represent the standard deviations. The value of 100% relative activity corresponds to the highest value of enzymatic activity (U) documented in the particular study (the effect of Kolliphor® CS 20 concentration on immobilized CALB activity). The complete definition of relative activity can be found in Section 3.2.3.
Catalysts 15 00916 g001
Catalysts 15 00916 g002
Figure 2. Effect of the concentration of Carbopol® Ultrez 10 added to the emulsion on the activity of CALB in both free and immobilized forms. Reaction conditions: immobilized CALB onto IB-D152 (50.0 mg of support) or free lipase (10.0 mg); phosphate buffer (100 mM, pH 7.4); the emulsion (unhomogenized) composed of 5.0% Kolliphor® CS 20 and Carbopol® Ultrez 10 (appropriate concentration), olive oil, and water; temperature 37 °C; incubation 30 min. Data are presented as means ± standard deviations of three independent experiments (n = 3). Error bars represent the standard deviations. 5.0% KCS 20 + 0.1% CL − 5.0% Kolliphor® CS 20 + 0.1% Carbopol® Ultrez 10; 5.0% KCS 20 + 0.05% CL − 5.0% Kolliphor® CS 20 + 0.05% Carbopol® Ultrez 10. The value of 100% relative activity corresponds to the highest enzymatic activity (U) documented in the particular study (the effect of Carbopol® Ultrez 10 concentration (0.05% and 0.1%) in emulsion on CALB activity). The complete definition of relative activity can be found in Section 3.2.3.
Figure 2. Effect of the concentration of Carbopol® Ultrez 10 added to the emulsion on the activity of CALB in both free and immobilized forms. Reaction conditions: immobilized CALB onto IB-D152 (50.0 mg of support) or free lipase (10.0 mg); phosphate buffer (100 mM, pH 7.4); the emulsion (unhomogenized) composed of 5.0% Kolliphor® CS 20 and Carbopol® Ultrez 10 (appropriate concentration), olive oil, and water; temperature 37 °C; incubation 30 min. Data are presented as means ± standard deviations of three independent experiments (n = 3). Error bars represent the standard deviations. 5.0% KCS 20 + 0.1% CL − 5.0% Kolliphor® CS 20 + 0.1% Carbopol® Ultrez 10; 5.0% KCS 20 + 0.05% CL − 5.0% Kolliphor® CS 20 + 0.05% Carbopol® Ultrez 10. The value of 100% relative activity corresponds to the highest enzymatic activity (U) documented in the particular study (the effect of Carbopol® Ultrez 10 concentration (0.05% and 0.1%) in emulsion on CALB activity). The complete definition of relative activity can be found in Section 3.2.3.
Catalysts 15 00916 g002
Catalysts 15 00916 g003
Figure 3. Effect of Kolliphor® CS 20 concentration on the catalytic activity of free and immobilized lipase in an emulsion system containing 0.1% Carbopol® Ultrez 10. Reaction conditions: immobilized CALB onto IB-D152 (50.0 mg of support) or free lipase (10.0 mg); phosphate buffer (100 mM, pH 7.4); the emulsion (unhomogenized) composed of Kolliphor® CS 20 (appropriate concentration) and 0.1% Carbopol® Ultrez 10, olive oil, and water; temperature 37 °C; incubation 30 min. Data are presented as means ± standard deviations of three independent experiments (n = 3). Error bars represent the standard deviations. 2.5% KCS 20 + 0.1% CL − 2.5% Kolliphor® CS 20 + 0.1% Carbopol® Ultrez 10; 5.0% KCS 20 + 0.1% CL − 5.0% Kolliphor® CS 20 + 0.1% Carbopol® Ultrez 10; 7.5% KCS 20 + 0.1% CL − 7.5% Kolliphor® CS 20 + 0.1% Carbopol® Ultrez 10; 10.0% KCS 20 + 0.1% CL − 10.0% Kolliphor® CS 20 + 0.1% Carbopol® Ultrez 10. The value of 100% relative activity corresponds to the highest enzymatic activity (U) documented in the particular study (the effect of Kolliphor® CS 20 concentration in emulsion stabilized by 0.1% Carbopol® Ultrez 10 on CALB activity). The complete definition of relative activity can be found in Section 3.2.3.
Figure 3. Effect of Kolliphor® CS 20 concentration on the catalytic activity of free and immobilized lipase in an emulsion system containing 0.1% Carbopol® Ultrez 10. Reaction conditions: immobilized CALB onto IB-D152 (50.0 mg of support) or free lipase (10.0 mg); phosphate buffer (100 mM, pH 7.4); the emulsion (unhomogenized) composed of Kolliphor® CS 20 (appropriate concentration) and 0.1% Carbopol® Ultrez 10, olive oil, and water; temperature 37 °C; incubation 30 min. Data are presented as means ± standard deviations of three independent experiments (n = 3). Error bars represent the standard deviations. 2.5% KCS 20 + 0.1% CL − 2.5% Kolliphor® CS 20 + 0.1% Carbopol® Ultrez 10; 5.0% KCS 20 + 0.1% CL − 5.0% Kolliphor® CS 20 + 0.1% Carbopol® Ultrez 10; 7.5% KCS 20 + 0.1% CL − 7.5% Kolliphor® CS 20 + 0.1% Carbopol® Ultrez 10; 10.0% KCS 20 + 0.1% CL − 10.0% Kolliphor® CS 20 + 0.1% Carbopol® Ultrez 10. The value of 100% relative activity corresponds to the highest enzymatic activity (U) documented in the particular study (the effect of Kolliphor® CS 20 concentration in emulsion stabilized by 0.1% Carbopol® Ultrez 10 on CALB activity). The complete definition of relative activity can be found in Section 3.2.3.
Catalysts 15 00916 g003
Catalysts 15 00916 g004
Figure 4. The effect of polyoxyethylene chain length of Kolliphor® CS 12 and Kolliphor® CS 20 used in emulsion on the lipolytic activity of CALB in immobilized and free form. Reaction conditions: immobilized CALB onto IB-D152 (50.0 mg of support) or free lipase (10.0 mg); phosphate buffer (100 mM, pH 7.4); the emulsion (unhomogenized) composed of 5.0% Kolliphor® CS 12 or Kolliphor® CS 20, olive oil, and water; temperature 37 °C; incubation 30 min. Data are presented as means ± standard deviations of three independent experiments (n = 3). Error bars represent the standard deviations. 5.0% KCS 12 − 5.0% Kolliphor® CS 12; 5.0% KCS 20 − 5.0% Kolliphor® CS 20. The value of 100% relative activity corresponds to the highest enzymatic activity (U) documented in the particular study (the effect of Kolliphor® CS 12 and Kolliphor® CS 20 on CALB activity). The complete definition of relative activity can be found in Section 3.2.3.
Figure 4. The effect of polyoxyethylene chain length of Kolliphor® CS 12 and Kolliphor® CS 20 used in emulsion on the lipolytic activity of CALB in immobilized and free form. Reaction conditions: immobilized CALB onto IB-D152 (50.0 mg of support) or free lipase (10.0 mg); phosphate buffer (100 mM, pH 7.4); the emulsion (unhomogenized) composed of 5.0% Kolliphor® CS 12 or Kolliphor® CS 20, olive oil, and water; temperature 37 °C; incubation 30 min. Data are presented as means ± standard deviations of three independent experiments (n = 3). Error bars represent the standard deviations. 5.0% KCS 12 − 5.0% Kolliphor® CS 12; 5.0% KCS 20 − 5.0% Kolliphor® CS 20. The value of 100% relative activity corresponds to the highest enzymatic activity (U) documented in the particular study (the effect of Kolliphor® CS 12 and Kolliphor® CS 20 on CALB activity). The complete definition of relative activity can be found in Section 3.2.3.
Catalysts 15 00916 g004
Catalysts 15 00916 g005
Figure 5. Effect of emulsion systems containing emulsifier mixtures, without and with the addition of Carbopol® Ultrez 10, on CALB activity. Reaction conditions: immobilized CALB onto IB-D152 (50.0 mg of support) or free lipase (10.0 mg); phosphate buffer (100 mM, pH 7.4); the emulsion (unhomogenized) composed of 4.0% SP Brij® S2 MBAL and 1.0% Kolliphor® CS 20 and olive oil and water; or 4.0% SP Brij® S2 MBAL and 1.0% Kolliphor® CS 20 and 0.1% Carbopol® Ultrez 10, olive oil and water; temperature 37 °C; incubation 30 min. Data are presented as means ± standard deviations of three independent experiments (n = 3). Error bars represent the standard deviations. 4.0% BJ + 1.0% KCS 20 − 4.0% SP Brij® S2 MBAL + 1.0% Kolliphor® CS 20; 4.0% BJ + 1.0% KCS 20 + 0.1% CL − 4.0% SP Brij® S2 MBAL + 1.0% Kolliphor® CS 20 + 0.1% Carbopol® Ultrez 10. The value of 100% relative activity corresponds to the highest enzymatic activity (U) documented in the particular study (the effect of emulsifier mixtures, without and with the addition of Carbopol® Ultrez 10 on CALB activity). The complete definition of relative activity can be found in Section 3.2.3.
Figure 5. Effect of emulsion systems containing emulsifier mixtures, without and with the addition of Carbopol® Ultrez 10, on CALB activity. Reaction conditions: immobilized CALB onto IB-D152 (50.0 mg of support) or free lipase (10.0 mg); phosphate buffer (100 mM, pH 7.4); the emulsion (unhomogenized) composed of 4.0% SP Brij® S2 MBAL and 1.0% Kolliphor® CS 20 and olive oil and water; or 4.0% SP Brij® S2 MBAL and 1.0% Kolliphor® CS 20 and 0.1% Carbopol® Ultrez 10, olive oil and water; temperature 37 °C; incubation 30 min. Data are presented as means ± standard deviations of three independent experiments (n = 3). Error bars represent the standard deviations. 4.0% BJ + 1.0% KCS 20 − 4.0% SP Brij® S2 MBAL + 1.0% Kolliphor® CS 20; 4.0% BJ + 1.0% KCS 20 + 0.1% CL − 4.0% SP Brij® S2 MBAL + 1.0% Kolliphor® CS 20 + 0.1% Carbopol® Ultrez 10. The value of 100% relative activity corresponds to the highest enzymatic activity (U) documented in the particular study (the effect of emulsifier mixtures, without and with the addition of Carbopol® Ultrez 10 on CALB activity). The complete definition of relative activity can be found in Section 3.2.3.
Catalysts 15 00916 g005
Table 1. The summary results of immobilized CALB activity depending on the used emulsion system.
Table 1. The summary results of immobilized CALB activity depending on the used emulsion system.
Emulsion SystemActivity
[U/ml] 		Specific Activity
[U/mg Lipase]		Activity
[U/g Support]		Residual
Activity [%] *
5.0% Kolliphor® CS 203.60 ± 0.232.86 ± 0.1872.03 ± 4.6346.1 ± 3.0
5.0% Kolliphor® CS 20 + 0.1% Carbopol® Ultrez 103.11 ± 0.192.47 ± 0.1562.22 ± 3.8539.8 ± 2.5
10.0% Kolliphor® CS 203.56 ± 0.192.82 ± 0.1571.11 ± 3.8645.5 ± 2.5
10.0% Kolliphor® CS 20 + 0.1% Carbopol® Ultrez 107.81 ± 0.206.20 ± 0.16156.27 ± 3.91100.0 ± 0.0
1.0% Kolliphor® CS 20 + 4.0% SP Brij® S2 MBAL3.33 ± 0.172.65 ± 0.1366.67 ± 3.3342.7 ± 2.1
Reaction conditions: immobilized CALB onto IB-D152 (50.0 mg of support); phosphate buffer (100 mM, pH 7.4); the appropriate unhomogenized emulsion composed of olive oil and water, as well as Kolliphor® CS 20 (5.0% or 10.0%) with or without 0.1% Carbopol® Ultrez 10 or 4.0% SP Brij® S2 MBAL with 1.0% Kolliphor® CS 20; temperature 37 °C; incubation 30 min. Data are presented as means ± standard deviations of three independent experiments (n = 3). * The value of 100% residual activity corresponds to the highest enzymatic activity (U) documented among all the performed experiments described in this paper. The complete definition of residual activity can be found in Section 3.2.3.
Table 2. Formulations containing Kolliphor® CS 20 at concentrations of 2.5–10.0%.
Table 2. Formulations containing Kolliphor® CS 20 at concentrations of 2.5–10.0%.
Emulsions with Kolliphor® CS 20
(1)
2.5% Kolliphor® CS 20
The quantitative composition [g]Type of stirrer
Olive oil25.0Eurostar Digital
Kolliphor® CS 201.25
Distilled waterad 50.0
(2)
5.0% Kolliphor® CS 20
The quantitative composition [g]Type of stirrer
Olive oil25.0Eurostar Digital
Kolliphor® CS 202.5
Distilled waterad 50.0
(3)
7.5% Kolliphor® CS 20
The quantitative composition [g]Type of stirrer
Olive oil25.0Eurostar Digital
Kolliphor® CS 203.75
Distilled waterad 50.0
(4)
10.0% Kolliphor® CS 20
The quantitative composition [g]Type of stirrer
Olive oil25.0Eurostar Digital
Kolliphor® CS 205.0
Distilled waterad 50.0
(5)
5.0% Kolliphor® CS 20 (homogenized)
The quantitative composition [g]Type of stirrer
Olive oil25.0Eurostar Digital
+
Ultra Turrax T 25
Kolliphor® CS 202.5
Distilled waterad 50.0
Latin abbreviation used: ad—ad (up to).
Table 3. Formulations containing 5.0% Kolliphor® CS 20 with Carbopol® Ultrez 10.
Table 3. Formulations containing 5.0% Kolliphor® CS 20 with Carbopol® Ultrez 10.
Emulsions Containing 5.0% Kolliphor® CS 20 and Carbopol® Ultrez 10
(1)
5.0% Kolliphor® CS 20 + 0.1% Carbopol® Ultrez 10
The quantitative
composition [g]		Type of stirrer
Olive oil25.0Eurostar Digital
Kolliphor® CS 202.5
Carbopol® Ultrez 100.05
Triethanolamineq.s.
Distilled water ad 50.0
(2)
5.0% Kolliphor® CS 20 + 0.05% Carbopol® Ultrez 10
The quantitative
composition [g]		Type of stirrer
Olive oil25.0Eurostar Digital
Kolliphor® CS 202.5
Carbopol® Ultrez 100.025
Triethanolamineq.s.
Distilled waterad 50.0
Latin abbreviation used: q.s.—quantum satis (a sufficient quantity), ad—ad (up to).
Table 4. Formulations containing Kolliphor® CS 20 at concentrations of 2.5–10.0% with 0.1% Carbopol® Ultrez 10.
Table 4. Formulations containing Kolliphor® CS 20 at concentrations of 2.5–10.0% with 0.1% Carbopol® Ultrez 10.
Emulsions Containing Kolliphor® CS 20 with 0.1% Carbopol® Ultrez 10
(1)
2.5% Kolliphor® CS 20 + 0.1% Carbopol® Ultrez 10
The quantitative
composition [g]		Type of stirrer
Olive oil25.0Eurostar Digital
Kolliphor® CS 201.25
Carbopol® Ultrez 100.05
Triethanolamine q.s.
Distilled waterad 50.0
(2)
5.0% Kolliphor® CS 20 + 0.1% Carbopol® Ultrez 10
The quantitative
composition [g]		Type of stirrer
Olive oil25.0Eurostar Digital
Kolliphor® CS 202.5
Carbopol® Ultrez 100.05
Triethanolamineq.s.
Distilled waterad 50.0
(3)
7.5% Kolliphor® CS 20 + 0.1% Carbopol® Ultrez 10
The quantitative
composition [g]		Type of stirrer
Olive oil25.0Eurostar Digital
Kolliphor® CS 203.75
Carbopol® Ultrez 100.05
Triethanolamineq.s.
Distilled waterad 50.0
(4)
10.0% Kolliphor® CS 20 + 0.1% Carbopol® Ultrez 10
The quantitative
composition [g]		Type of stirrer
Olive oil25.0Eurostar Digital
Kolliphor® CS 205.0
Carbopol® Ultrez 100.05
Triethanolamineq.s.
Distilled waterad 50.0
Latin abbreviation used: q.s.—quantum satis (a sufficient quantity), ad—ad (up to).
Table 5. Formulations containing single emulsifier—5.0% Kolliphor® CS 12 (1) or 5.0% Kolliphor® CS 20 (2); combination of 1.0% Kolliphor® CS 20, 4.0% SP Brij® S2 MBAL and 0.1% Carbopol® Ultrez 10 (3); and a formulation with 1.0% Kolliphor® CS 20 and 4.0% SP Brij® S2 MBAL, without Carbopol® Ultrez 10 (4) additive.
Table 5. Formulations containing single emulsifier—5.0% Kolliphor® CS 12 (1) or 5.0% Kolliphor® CS 20 (2); combination of 1.0% Kolliphor® CS 20, 4.0% SP Brij® S2 MBAL and 0.1% Carbopol® Ultrez 10 (3); and a formulation with 1.0% Kolliphor® CS 20 and 4.0% SP Brij® S2 MBAL, without Carbopol® Ultrez 10 (4) additive.
Emulsions Containing: Kolliphor® CS 12 or Kolliphor® CS 20; Mixtures of 1.0% Kolliphor® CS 20, 4.0% SP Brij® S2 MBAL and 0.1% Carbopol® Ultrez 10; Mixtures of 1.0% Kolliphor® CS 20 and 4.0% SP Brij® S2 MBAL
(1)
5.0% Kolliphor® CS 12
The quantitative composition [g]Type of stirrer
Olive oil25.0Eurostar Digital
Kolliphor® CS 122.5
Distilled waterad 50.0
(2)
5.0% Kolliphor® CS 20
The quantitative composition [g]Type of stirrer
Olive oil25.0Eurostar Digital
Kolliphor® CS 202.5
Distilled waterad 50.0
(3)
5.0% (Kolliphor® CS 20 + SP Brij® S2 MBAL) + 0.1% Carbopol® Ultrez 10
The quantitative composition [g]Type of stirrer
Olive oil37.5Eurostar Digital
SP Brij® S2 MBAL3.0
Kolliphor® CS 200.75
Carbopol® Ultrez 100.075
Triethanolamineq.s.
Distilled waterad 75.0
(4)
5.0% (Kolliphor® CS 20 + SP Brij® S2 MBAL)
The quantitative composition [g]Type of stirrer
Olive oil37.5Eurostar Digital
SP Brij® S2 MBAL3.0
Kolliphor® CS 200.75
Distilled waterad 75.0
Latin abbreviation used: q.s.—quantum satis (a sufficient quantity), ad—ad (up to).
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MDPI and ACS Style

Siódmiak, J.; Dulęba, J.; Mieszkowski, D.; Bilski, P.; Siódmiak, T. Emulsion Systems Stabilized with Nonionic Emulsifier and Cross-Linked Polyacrylic Acid: A Promising Strategy to Enhance the Activity of Immobilized CALB. Catalysts 2025, 15, 916. https://doi.org/10.3390/catal15100916

AMA Style

Siódmiak J, Dulęba J, Mieszkowski D, Bilski P, Siódmiak T. Emulsion Systems Stabilized with Nonionic Emulsifier and Cross-Linked Polyacrylic Acid: A Promising Strategy to Enhance the Activity of Immobilized CALB. Catalysts. 2025; 15(10):916. https://doi.org/10.3390/catal15100916

Chicago/Turabian Style

Siódmiak, Joanna, Jacek Dulęba, Dominik Mieszkowski, Piotr Bilski, and Tomasz Siódmiak. 2025. "Emulsion Systems Stabilized with Nonionic Emulsifier and Cross-Linked Polyacrylic Acid: A Promising Strategy to Enhance the Activity of Immobilized CALB" Catalysts 15, no. 10: 916. https://doi.org/10.3390/catal15100916

APA Style

Siódmiak, J., Dulęba, J., Mieszkowski, D., Bilski, P., & Siódmiak, T. (2025). Emulsion Systems Stabilized with Nonionic Emulsifier and Cross-Linked Polyacrylic Acid: A Promising Strategy to Enhance the Activity of Immobilized CALB. Catalysts, 15(10), 916. https://doi.org/10.3390/catal15100916

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