Efficient Immobilization of Streptomyces gobitricini Lipase for Sustainable Lipid Degradation and Wastewater Bioremediation

Abstract

Immobilized lipases are emerging as highly effective biocatalysts due to their enhanced operational stability, reusability, and promising environmental applications, particularly in the bioremediation of lipid-contaminated wastewater. In this study, the lipase from Streptomyces gobitricini (Lip_S.g) was immobilized on various supports, with calcium carbonate demonstrating the highest immobilization efficiency (82.66 ± 5.5%). Optimal conditions were achieved using a lipase concentration of 3500 U·g^–1 support, resulting in a retained activity of 92.67 ± 3.05%. The immobilized Lip_S.g showed significantly improved storage stability, maintaining 68.33% of its initial activity after 120 days at 4 °C, compared to only 29.7% for the free enzyme. It also exhibited greater tolerance to alkaline pH and high temperatures, with maximum activity at pH 9.0 and thermal stability up to 70 °C. Substrate specificity tests on oil-based substrates revealed improved catalytic performance in the immobilized form, likely due to enhanced substrate accessibility. In practical wastewater treatment trials, the immobilized enzyme achieved complete lipid removal by day 9, in contrast to the free enzyme, which achieved only 50% removal. Moreover, marked reductions in chemical oxygen demand and residual lipid levels further validated its bioremediation efficacy. These results position immobilized Lip_S.g as a robust, eco-friendly biocatalyst with strong potential for industrial applications in the treatment of oil-laden wastewater.

1. Introduction

Wastewater from various processes is often laden with fats, oils, grease, sulfates, and phosphates, along with elevated biological and chemical oxygen demand, making it a significant challenge for wastewater treatment systems. , These substances form a thin layer on the water’s surface, hindering oxygen exchange and reducing oxygen saturation, which slows down biological degradation. Additionally, oily wastes tend to solidify at lower temperatures, leading to blockages in pipes and drains, as well as causing unpleasant odors.

Various wastewater treatment processes are employed, typically involving physical, chemical, and biological methods. Conventional physicochemical techniques include gravity separation, dissolved air flotation, coagulation, and flocculation. While gravity separation effectively removes free oil, it is ineffective for smaller droplets and emulsified oils. Treating emulsified oil typically involves heating, acidification, polymer addition, and pH adjustment to promote flocculation, followed by sludge dewatering. The main disadvantage of conventional methods is their low efficiency. However, their performance can be enhanced by incorporating biological strategies, which provide a more sustainable and effective alternative. Cost-effective biological treatments using lipolytic enzymes, particularly lipases, are often applied after physicochemical treatments, offering a promising alternative to these pretreatment steps. ,

Lipases (EC 3.1.1.3), members of the serine hydrolase family, possess a core structure of eight parallel β-strands forming a twisted central β-sheet surrounded by several α-helices. Their catalytic triad comprises a nucleophilic serine, histidine, and either glutamate or aspartate residues, supported by an oxyanion hole that stabilizes the oxygen ion intermediate formed during catalysis. , These enzymes are notable for their exceptional catalytic activity, high substrate specificity, and versatility in catalyzing diverse reactions, which can vary depending on the reaction medium. Lipases participate in numerous reactions, including esterification, transesterification, interesterification, aminolysis, alcoholysis, and acidolysis, particularly in nonaqueous environments. Lipases are produced by animals, plants, and especially microorganisms. Microbial lipases offer significant advantages such as their abundance, the ease of genetic modification in producing microorganisms, and their distinct characteristics and specificities. Most microbial lipases are extracellular and are predominantly produced by bacterial and fungal species. For biotechnological applications, extracellular lipases from bacteria and fungi are preferred due to their ease of production and cost-effectiveness.

Lipases play a crucial role in the hydrolysis of waste oil and fat by catalyzing reactions at the interface between the oil and water phases. They efficiently convert complex, long-chain, water-insoluble triglycerides into simpler free fatty acids. The use of microbial lipases for degrading oil and fat in wastewater has emerged as a promising strategy. However, directly adding lipases to the reaction medium can result in their solubilization, making recovery challenging. Additionally, fluctuations in the reaction parameters, such as temperature and pH, may cause enzyme denaturation and activity loss. Immobilizing lipases on insoluble substrates addresses these issues, enhancing their stability, allowing for reuse across multiple cycles, and improving the overall efficiency and cost-effectiveness of the process.

Enzyme immobilization is a highly effective technique that enhances enzyme stability and reusability while preserving functionality under extreme conditions such as high or low pH, elevated temperatures, or the presence of organic solvents. This approach also significantly improves the economic feasibility of enzyme use in industrial applications. The enhanced stability of immobilized enzymes is attributed to multipoint covalent bonding between the enzyme and the support material, which creates numerous interactions that ensure strong attachment and durability. This prevents enzyme detachment from the surface, maintaining functionality over time. The nature of both the enzyme and the support material plays a critical role in determining the properties of the immobilized enzymes. An ideal support should exhibit chemical, physical, and biological stability during the immobilization process and under the reaction conditions. This allows the biocatalyst to achieve optimal mechanical, chemical, biochemical, and kinetic properties.

Enzyme immobilization can be achieved using various methods broadly classified into physical and chemical approaches. Physical methods include adsorption, entrapment, and encapsulation, while chemical methods involve cross-linking and covalent bonding. Recently, hybrid techniques that combine these approaches have gained popularity in enzyme immobilization processes.

Adsorption is a simple and convenient immobilization method in which enzymes attach to a support via weak interactions, such as hydrogen bonds, van der Waals forces, and hydrophobic interactions. However, these weak bonds can result in enzyme leakage, particularly under industrial conditions with high concentrations of reagents or products. This issue can be mitigated by using hydrophobic supports or enzymes, which enhance hydrophobic interactions and provide stronger binding

Noncovalent immobilization of lipases is particularly efficient in nonaqueous media, as desorption is minimal due to the low solubility of lipases in organic solvents. This method is widely employed in industrial catalysis in water-immiscible solvents because it is simple, cost-effective, and reversible. It offers advantages such as high enzyme activity, low carrier material costs, minimal use of chemical additives, and the potential for support regeneration.

A variety of organic and inorganic materials, such as nanofibers, nanoparticles, and alginates, can be used for enzyme immobilization via adsorption. Among inorganic materials, calcium carbonate (CaCO₃) is one of the most widely used due to its low synthesis cost and natural abundance in biological substrates such as seashells, coral, and bones. This eco-friendly biomineral offers several advantages, including high loading capacity, attributed to its large pore volume, thermal stability, mechanical strength, and porous microstructure.

Streptomyces are well-studied soil bacteria with critical ecological, medical, and biotechnological roles. Despite their genomes encoding 50 to 80 potential lipolytic enzymes, relatively few studies have focused on the purification and characterization of Streptomyces lipases. ,, In previous work, Lip_S.g, a lipase from Streptomyces gobitricini, was purified and characterized, demonstrating its potential for application in ester synthesis and detergent formulations (unpublished data). The current study investigated the immobilization of LipS.g and its application in wastewater treatment. The immobilization process aimed to enhance the enzyme’s stability and reusability, positioning Lip_S.g as a promising candidate for sustainable wastewater treatment solutions.

2. Results and Discussion

2.1. Immobilization of Lip_S.g

2.1.1. Adsorption on Various Support Immobilization

The attachment of an immobilized biocatalyst is governed by physical and chemical interactions between the support material and the enzyme. Adsorption, the simplest immobilization method, relies on these interactions, where the physical and chemical groups of the support matrix bind to the enzyme to retain it.

Adsorption of Lip_S.g on different supports resulted in different yields of immobilized lipase activities. The highest yield was achieved with CaCO₃ at 82.66 ± 5.5%, highlighting its efficiency as a support material for Lip_S.g (Table ). Celite 545 also demonstrated potential as an alternative, with a relatively high yield of 59 ± 4.5%. In contrast, cellulose acetate and chitosan showed moderate yields of 34.33 ± 3.05 and 54.33 ± 4.04%, respectively. Silica gel produced the lowest yield, at 31.66 ± 3.51%. The current findings underscore the importance of support selection in achieving maximum immobilization efficiency, with CaCO₃ emerging as the most promising option for Lip_S.g lipase. Based on these results, CaCO₃ was selected for further analysis. The high yield of immobilized lipase activity on CaCO₃ could be attributed to its favorable ionic interactions and surface characteristics, which promote effective enzyme binding. With its high thermal stability, robust mechanical properties, and porous structure, CaCO₃ serves as an ideal biocompatible support for enzyme or protein delivery and immobilization. It effectively controls enzyme diffusion and release rates, enhancing performance. In contrast, the lower yield observed with Celite 545 may be due to its inability to provide an optimal chemical environment for efficient enzyme binding without modification, despite its large surface area. Similarly, materials such as cellulose acetate, chitosan, and silica gel demonstrated lower yields, likely due to weaker or less effective enzyme interactions, the need for surface modifications, or reduced stability under the reaction conditions.

1. Yield of Lip_S.g Immobilization on Various Supports .

support	yield of immobilized lipase activity (%)
CaCO3	82.66 ± 5.5****
celite 545	59 ± 4.5
cellulose acetate	34.33 ± 3.05**
chitosan	54.33 ± 4.04
silica gel	31.66 ± 3.51***

^aComparisons were performed between each immobilization support and all others combined. Significance levels are indicated as p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).

Although numerous studies have described lipases from Streptomyces strains, few have focused on their immobilization. For instance, the immobilization of Streptomyces sp. A3301 lipase demonstrated that the adsorption method using a sponge resulted in the highest enzyme activity (57.31 U·g^–1), followed by a scrub pad (20 U·g^–1), whereas gelatin and starch produced significantly lower activities. In addition, several studies have reported the immobilization of microbial lipases on various supports. For example, lipase from Staphylococcus aureus was efficiently immobilized on CaCO₃ with a yield of 86% compared to Celite 545 and silica gel, which exhibited 16 and 23%, respectively. Similar yields were obtained with Bacillus stearothermophilus lipase onto the same supports, varying from 89.33% onto CaCO₃ to 38.33% on Celite 545 (Table ).

2. Insights into the Characterization of Immobilized Lipases from the Literature.

adsorption on various support immobilization
immobilized lipase	support	enzymatic activity	references
Streptomyces sp. A3301 lipase	sponge	57.31 U·g–1
	scrub pad	20 U·g–1
S. aureus ALA1	CaCO3	86%
	celite 545	16%
	silica gel	23%
B. stearothermophilus	caCO3	89.33%
	celite 545	38.33%
Optimal Conditions for Immobilization
B. stearothermophilus lipase	4500 U·g–1
S. aureus ALA1 lipase	3500 U·g–1
Thermal and pH Stability
	temperature (°C)	pH
immobilized Streptomyces sp. A3301 lipase on sponge	30 and 60 °C	7.0
immobilized B.stearothermophilus lipase on CaCO3	65%	40 °C for 1 h
immobilized S. aureus ALA1 lipase	62% activity at 78 °C	58% activity at a pH range of 6.0 to 11.0
immobilized lipases CALA and CALB from Candida antarctica	40 and 50 °C	6.0–8.0
immobilized Acinetobacter hemolyticus lipase on eggshell membrane	50% activity for 150 min at 50 °C, 120 min at 60 °C, and 80 min at 70 °C	7.0

2.1.2. Optimal Conditions for Immobilization

The multiple noncovalent interactions such as hydrogen bonding, hydrophobic interactions, and van der Waals forces between the support and the enzyme enhance the enzyme’s attachment and stability, reducing desorption. This underscores the importance of optimizing enzyme amount and incubation time during the immobilization process.

To identify the ideal conditions for immobilizing Lip_S.g in aqueous solutions, various concentrations of Lip_S.g, ranging from 1000 to 7000 U·g^–1 of CaCO₃, were tested. The data presented in Figure demonstrate the correlation between the initial activity of soluble lipase (units per gram of support) and the immobilization yield (%). At a concentration of 1000 U·g^–1 of the CaCO₃ support, the immobilization yield was 37.67 ± 3.51%, which significantly increased to 80.67 ± 4.04% at 2000 U·g^–1 of the CaCO₃ support. The highest yield, 92.67 ± 3.05%, was observed at 3500 U·g^–1. However, as the lipase concentration increased further, the immobilization yield declined, with values of 77.67 ± 3.05% at 5000 U·g^–1, 64.33 ± 2.08% at 6000 U·g^–1, and 50.67 ± 2.51% at 7000 U·g^–1 of CaCO₃ support. An enzyme concentration of 3500 U·g^–1, determined to be optimal, was used in all subsequent experiments. This trend highlights the existence of an optimal lipase concentration for immobilization, beyond which the yield decreases.

1.

Yields of immobilized Lip_S.g evaluated using different initial lipase units/g of CaCO₃ support. Lipase activity was determined by the pH-stat method in the presence of 2 mM calcium and at pH 9.0 and 50 °C using, as substrate, TC18 (olive oil) emulsion. One unit of the lipase activity is defined as 1 μmol of fatty acid produced per minute. The results are expressed as the mean ± the standard deviation of three independent measurements.

According to Ben Bacha et al., enzyme concentrations above 4500 U·g^–1 were not retained by the support and were likely lost during the washing process. Similar findings have been reported for immobilized lipases from B. stearothermophilus and S. aureus ALA1, which exhibited maximum immobilization yields at 4500 U·g^–1 and 3500 U·g^–1, respectively , (Table ).

2.1.3. Kinetic Analysis of Adsorption and Retention Capacity of Immobilized Lip_S.g on CaCO₃

Reaction time is a critical factor in enzyme immobilization, as insufficient interaction time can hinder binding, while excessive time may lead to enzyme denaturation. The adsorption kinetics of immobilized Lip_S.g on CaCO₃ revealed a gradual decrease in both the enzyme concentration and residual catalytic activity in the supernatant over time (Figure ). After 10 min, the Lip_S.g enzyme concentration dropped to 2.28 mg mL^–1, with activity decreasing to 3066.67 U mL^–1. This decline continued, reaching 0.17 and 113.33 U mL^–1 at 60 min. By 2 h, the enzyme concentration stabilized at 0.01 mg mL^–1 with an activity of 13.67 U mL^–1. These results indicated that the adsorption of Lip_S.g on CaCO₃ is a rapid process, with a significant decrease in both enzyme concentration and activity within the first 60 min. The decline was likely due to saturation of the CaCO₃ surface, limiting its capacity for further adsorption. Considering the rapid adsorption kinetics, the immobilized Lip_S.g collected after 60 min of contact with the support CaCO₃, when a sufficient amount of enzyme was bound and retained significant catalytic activity, was used for all subsequent experiments. In a related study, the highest enzyme activity (277 U·g^–1 of immobilization material) for lipase from Streptomyces sp. A3301 was achieved after 48 h of soaking in the enzyme solution. However, activity decreased to 170 U·g^–1 after 84 h (Table ).

2.

Kinetics of Lip_S.g and protein adsorption on CaCO₃. The yield of immobilized lipase activity was calculated as the percentage of adsorbed lipase activity relative to the total soluble lipase activity initially added to 1 g of the support. The supernatant collected after centrifugation was used to determine the lipase activity via a pH-stat method in the presence of 2 mM calcium and at pH 9.0 and 50 °C, using as substrate, TC18 (olive oil) emulsion. One unit of the lipase activity is defined as 1 μmole of fatty acid produced per minute. Data are expressed as the mean ± standard deviation of three independent measurements.

2.2. Characterization of Immobilized Lip_S.g

2.2.1. Storage Stability

Enzyme immobilization technology is a powerful tool for modifying and enhancing catalytic properties, including enzyme activity, selectivity, stability under varying pH and temperature conditions, and reusability across multiple catalytic cycles. This capability is particularly evident in practical applications, as demonstrated by the storage stability of free and immobilized Lip_S.g. The storage stability of free and immobilized Lip_S.g at 4 and 25 °C (pH 8.0) over time highlighted a clear decline in enzymatic activity, with significant differences between the free and immobilized forms (Figure ). At 4 °C, both free and immobilized Lip_S.g showed minimal loss in activity during the first 40 days, with free lipase retaining 93.3 ± 3.51% and immobilized lipase maintaining 100 ± 0% activity by day 20. However, by day 120, free Lip_S.g activity dropped to 29.7 ± 2.51%, while immobilized lipase retained 68.33 ± 2.51% of its activity. In contrast, at 25 °C, the free Lip_S.g exhibited a more pronounced decline, with activity dropping from 100% to 4.66 ± 0.57% by day 90. Meanwhile, the immobilized Lip_S.g showed a slower decline, retaining 40.67 ± 2.08% of activity by day 120. These results indicate that immobilized Lip_S.g demonstrates greater stability than its free form, especially at higher temperatures. Storage at 4 °C provided the best preservation for both enzyme forms. As reported by several studies, immobilization is an effective strategy for preserving enzymatic activity and significantly prolonging enzyme shelf life, ensuring more consistent and extended performance in catalytic applications. Similar trends were observed in immobilized lipases from B. stearothermophilus and S. aureus.

3.

Storage stability of free and immobilized Lip_S.g: activity retention (%) at 4 and 25 °C, pH 8.0. The initial activity measured at J ₀ of storage represents 100%, and the residual activity at each subsequent incubation time point was determined by the pH-stat technique. The percentage activity was then calculated based on this initial activity using the devised formula ((residual activity/initial activity) × 100). Data are expressed as the mean ± standard deviation of three independent measurements. Comparisons were made between free and immobilized Lip_S.g at each incubation time point for 4 and 25 °C separately. Significance levels indicated as p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

2.2.2. Effect of Temperature and pH on Free and Immobilized Lip_S.g Stability and Activity

Immobilization enhances enzyme stability, making it well-suited for industrial applications, such as biotransformation and food processing. This process simplifies enzyme handling, prevents contamination of reaction products, and improves stability by creating a unique microenvironment around the enzyme. To assess these benefits, the stability and activity of immobilized Lip_S.g were tested against key physicochemical parameters and compared to those of the free form. The data in Figure .A illustrate the effect of pH on the activity of both forms. The lipolytic activity of free and immobilized Lip_S.g increased with pH, peaking at pH 9.0 with activities of 100 ± 0% for the free enzyme and 101 ± 1.73% for the immobilized enzyme. At pH 9.5, the immobilized form retained its maximum activity (101 ± 1.73%), while the free form exhibited a slight reduction (83.67 ± 3.51%). Beyond this pH, a decrease in activity was observed. At pH 10.0 and 11.0, the immobilized enzyme retained 85.33 ± 4.5% and 75 ± 2% activity, respectively, compared to a significant decrease in the free enzyme, which dropped to 28.33 ± 2.51% at pH 11.0. These results highlighted that immobilization preserved the optimal pH for enzymatic activity and conferred superior stability under alkaline conditions, showcasing the potential of immobilized Lip_S.g for applications requiring robust performance in challenging environments.

4.

Effect of pH on the activity (A) and the stability (B) of free and immobilized Lip_S.g. Stability was assessed by preincubating the enzyme in various buffer solutions at different pH values ranging from 2.0 to 13.0 for 48 h. The impact of temperature on the activity (C) and stability (D) of free and immobilized Lip_S.g. For temperature stability, the enzymes were preincubated at various temperatures for 1 h, followed by the activity measurement using the pH-stat method under standard conditions (using as substrate TC18 (olive oil) emulsion in the presence of 2 mM calcium and at pH 9.0 and 50 °C). The lipase activity was expressed as a percentage, where the maximal activity was considered to be 100%. The activity at each time point was calculated using the following formula: activity (%) = (activity at time point/initial activity) × 100. Data are expressed as the mean ± the standard deviation of three independent measurements. Comparisons were performed between free and immobilized LipS.g at each pH and temperature. Significance levels indicated as p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).

Figure B illustrates the pH stability of free and immobilized Lip_S.g, highlighting the enhanced stability of the immobilized enzyme across various pH values. The residual activity of immobilized Lip_S.g consistently exceeded that of its free form at all of the pH levels tested. Both forms exhibited increasing activity with increasing pH, peaking at 100 ± 0% for free Lip_S.g and 101.67 ± 2.88% for the immobilized enzyme at pH 8.0. At higher pH values, the free enzyme’s activity declined to 85 ± 2% at pH 11.0, while the immobilized enzyme maintained greater stability, with activity slightly decreasing to 91 ± 3.6% at pH 12.0. This demonstrated the superior robustness of immobilized Lip_S.g over a broader pH range, particularly under alkaline conditions, making it more suitable for a variety of biotechnological applications. These findings aligned with reported studies emphasizing the stabilizing effects of immobilization on pH stability, which enhances the enzymatic performance by maintaining activity and stability. The immobilization process on insoluble supports is known to significantly affect the ionization and dissociation states of the enzyme. It also alters the characteristics of the surrounding microenvironment, creating more favorable conditions for enzyme activity and stability. These combined effects contribute to the enhanced efficiency and stability observed for the immobilized Lip_S.g. Similar results have been observed in the immobilization of A. hemolyticus lipase onto eggshell membranes, which demonstrated significantly higher activity compared to the free enzyme, especially in acidic conditions.

The effect of temperature on the activity and stability of free and immobilized Lip_S.g was investigated (Figure C,D). Figure C demonstrates that the lipolytic activity of both forms increased with temperature, peaking at 50 °C with values of 100 ± 0% for free Lip_S.g and 93.33 ± 2.31% for the immobilized enzyme. However, at temperatures above 50 °C, the activity of free lipase declined sharply, dropping to 55.67 ± 3.05% at 60 °C and further to 29.33 ± 2.08% at 70 °C. In contrast, the immobilized lipase exhibited greater thermal stability, retaining 85.66 ± 3.51% and 60.33 ± 3.05% activity at 60 and 70 °C, respectively. These results underscore the superior thermal stability of immobilized Lip_S.g, making it more suitable for industrial applications that require elevated temperatures.

Figure D highlights the thermal stability of both free and immobilized Lip_S.g, revealing that both forms maintained high residual activity at temperatures between 40 and 60 °C, with 100 ± 0% residual activity at 40 and 50 °C. However, at higher temperatures, the free enzyme’s stability decreased more rapidly. At 65 °C, the free enzyme retained only 73.33 ± 2.08% residual activity, whereas the immobilized enzyme remained stable with 99 ± 2.64% activity. At 80 °C, free Lip_S.g activity declined dramatically to 21 ± 2%, while the immobilized form maintained significant activity (61.67 ± 3.51%).

These results confirmed that immobilization enhanced the thermal stability of Lip_S.g, enabling it to retain activity at higher temperatures and making it more appropriate for industrial processes that require heat tolerance. This property makes the immobilized enzyme particularly suitable for industrial processes that require high heat tolerance. The improved thermal stability can be attributed to several factors, including the increased molecular rigidity induced by multipoint covalent attachment between the enzyme and the solid support. This rigidification helps to prevent thermal unfolding and maintains the active conformation of the enzyme under stress conditions. Moreover, the immobilization process creates a protected microenvironment around the enzyme, shielding it from external denaturing agents, such as heat or solvents. An additional contributing factor may be the increase in the hydrophobic surface area resulting from immobilization, which is known to enhance conformational flexibility while simultaneously stabilizing the enzyme structure. Similar effects have been observed and reported for various immobilized enzymes, such as lipases, esterases, and oxidases, supporting the relevance of these findings to Lip_S.g. However, the reduction in lipase activity at different pH and temperature conditions is likely due to enzyme denaturation, a process where the enzyme’s structure is disrupted, leading to a loss of catalytic function. Enzymes are sensitive to changes in pH and temperature, which can alter the active site and overall structure, thereby reducing their ability to bind substrates and catalyze reactions. High temperatures can cause unfolding of the enzyme, while deviations from the optimal pH can also distort its structure, both resulting in a decrease in activity. ,

Comparable findings have been previously described for immobilized lipases from different sources. For instance, the immobilized lipase of Streptomyces sp. A3301 on sponge exhibited thermal tolerance between 30 and 60 °C, with a slight reduction in activity at 70–80 °C, whereas the free form was active only between 30 and 50 °C, losing 50% activity at 60 °C. Likewise, the adsorption of B. stearothermophilus on CaCO₃ significantly enhanced its stability, maintaining 58% activity across a pH range of 6.0 to 11.0 after 48 h of incubation. The immobilized S. aureus lipase also demonstrated greater thermal stability, retaining approximately 62% activity at 78 °C, compared to the free form, which retained only 10% activity, further confirming the role of CaCO₃ immobilization in enhancing heat resistance. Similarly, immobilized lipases CALA and CALB from C. antarctica on supports such as octyl agarose, octyl-vinyl sulfone agarose, and amino-glutaraldehyde were fully active at 40 and 50 °C but became inactivated at 70 and 75 °C, respectively. Additionally, immobilized A. hemolyticus lipase on eggshell membrane retained 50% activity for 150 min at 50 °C, 120 min at 60 °C, and 80 min at 70 °C, demonstrating enhanced thermal stability due to immobilization (Table ).

2.2.3. Substrate Specificity

The substrate specificity of lipase, which refers to the enzyme’s preference toward different substrates, is largely influenced by its molecular structure, particularly the configuration of its active site and the nature of the substrate. A comparative analysis of free and immobilized Lip_S.g activities across various substrates was performed. For TC4, TC8, and TC18, both free and immobilized Lip_S.g displayed nearly identical activity, with values ranging from 91.66 ± 3.21 to 100 ± 0% (Table ). However, when tested with oils, immobilized Lip_S.g demonstrated slightly higher activity compared with the free form. For instance, on soybean oil, immobilized Lip_S.g achieved 87.66 ± 2.51%, compared to 84.66 ± 3.5% for the free form. Similarly, on sunflower oil, the activity of immobilized Lip_S.g (79.33 ± 4.71%) surpassed that of free Lip_S.g (77.33 ± 2.08%). This substrate preference and enhanced activity for oil-based substrates may be attributed to complementary and specific interactions between the substrate and the enzyme residues. This substrate preference and enhanced activity for oil-based substrates might be attributed to complementary and specific hydrophobic interactions between the enzyme and substrate molecules. Furthermore, enzyme immobilization can stabilize the active conformation of lipases, protect them against denaturation, and improve accessibility to large, hydrophobic substrates, which explains the observed better performance with oils. These findings indicated that while immobilization had a minimal effect on lipase activity for simple esters, it provided an improvement for oil-based substrates.

3. Substrate Specificity of Free and Immobilized Lip_S.g .

substrate	free LipS.g	immobilized LipS.g
TC4	92 ± 2	96 ± 2*
TC8	91.66 ± 3.21	92.33 ± 2.08
TC18	100 ± 0	100 ± 0
coconut oil	88.33 ± 3.05	89.33 ± 3.05
sesame oil	87.33 ± 3.05	85.33 ± 2.51
soybean oil	84.66 ± 3.5	87.66 ± 2.51
sunflower oil	77.33 ± 2.08	79.33 ± 4.71

^aThe lipase activity was expressed as a percentage, where the maximal activity was considered 100%. The activity at each time point was calculated using the following formula: activity (%) = (activity at time point/initial activity) × 100. Comparisons were performed between free and immobilized LipS.g with each substrate. Significance levels are indicated as p < 0.05 (*).

Similar findings have been reported for lipases immobilized on various supports. The S. aureus lipase immobilized on CaCO₃ exhibited the highest activity with corn seed oil, olive oil, coconut oil, sunflower oil, and palm oil. A preference for sunflower and palm oils (92%), corn seed oil (111%), and coconut oil (128% activity) was also recorded in the case of CaCO₃-immobilized lipase from B. stearothermophilus. Likewise, immobilized lipases CALA and CALB from C. antarctica, on supports such as octyl agarose, octyl-vinyl sulfone agarose, and amino-glutaraldehyde, displayed higher activity on p-NPB compared to the triacetin substrate. A similar trend was observed with the immobilized lipase CLR from Candida rugosa, showing variations in specific activities depending on the substrate used.

2.3. Comparative Bioremediation of Wadi Hanifa Water Using Free and Immobilized Lip_S.g

Wastewater containing high levels of lipids and oils poses significant environmental risks when untreated, including soil degradation, water pollution, and ecosystem disruptions. Therefore, effective wastewater treatment is crucial for mitigating these impacts and fostering a more sustainable environment. Common methods for treating edible oil wastewater include mechanical, physicochemical, and biological approaches, with biological treatments gaining favor due to their cost-effectiveness and low solid waste generation. Recently, enzyme-based bioremediation has emerged as a promising alternative, offering a highly targeted and efficient means of pollutant removal. Immobilized lipases, in particular, have proven to be effective tools for wastewater treatment due to their high stability and reusability. In this context, immobilized Lip_S.g was employed for the bioremediation of Wadi Hanifa water, demonstrating its capacity to hydrolyze all of the selected commercial oils. The results of these experiments are summarized in Table .

4. Wadi Hanifah Water Treatment with Immobilized and Free Lip_S.g .

	lipid content (%)		TOC (%)		COD (%)
incubation duration (days)	free LipS.g	immobilized LipS.g	free LipS.g	immobilized LipS.g	free LipS.g	immobilized LipS.g
0	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0
1	88 ± 3.05	62.66 ± 4.16****	111.66 ± 3.51	186.33 ± 5.13****	90 ± 4.58	54 ± 4.58****
2	81 ± 4.72	36.66 ± 3.51****	155.33 ± 5.86	259.66 ± 8.02****	75 ± 4	44.66 ± 3.51****
3	71 ± 4.16	21.66 ± 2.51****	191.33 ± 6.08	315.66 ± 11.01****	69 ± 3	27.16 ± 2.75****
4	67 ± 2.51	9.66 ± 1.15****	208.66 ± 4.50	347.33 ± 10.69****	58.336.11 ±	9.66 ± 1.15****
5	62 ± 3.51	3.13 ± 0.35****	226 ± 5.56	406.66 ± 8.50****	49 ± 4	3.83 ± 0.35****
6	60 ± 4.16	1.43 ± 0.30****	255.33 ± 8.02	450 ± 13.74****	44.66 ± 5.03	2.93 ±. 025****
7	55 ± 3	0.81 ± 0.06****	264.33 ± 8.32	499 ± 14.52****	42.33 ± 4.04	0.98 ± 0.12****
8	52 ± 2.51	0.43 ± 0.06****	275.66 ± 7.09	561 ± 15.09****	37.33 ± 4.5	0.37 ± 0.03****
9	51 ± 3	0 ± 0****	264 ± 11	597.33 ± 11.06****	34.33 ± 4.04	0.16 ± 0.12****
10	50 ± 4.5	0 ± 0****	309 ± 5.29	614 ± 9.53****	31 ± 2.64	0.19 ± 0.01****

^aComparisons were performed between immobilized and free Lip_S.g each day. Significance levels are indicated as p < 0.0001 (****). COD: Chemical oxygen demand; TOC: Total organic chloride.

The lipid degradation data reveal that immobilized Lip_S.g was significantly more efficient than the free form, achieving complete lipid removal (0% lipid content) by day 9, compared to 50 ± 4.50% lipid content with the free enzyme by day 10 (Table ). Immobilized Lip_S.g displayed a rapid degradation rate, reducing lipids to 9.66 ± 1.15% by day 4 and further to 0.43 ± 0.06% by day 8. In contrast, the free Lip_S.g demonstrated a slower, more gradual reduction, with lipid content decreasing to 67 ± 2.51% and 52 ± 2.51% on the same days. This enhanced efficiency of immobilized LipS.g can be attributed to improved enzyme stability, increased substrate accessibility, and greater resistance to denaturation resulting from immobilization. These properties underscored the suitability of immobilized Lip_S.g for industrial applications, particularly in wastewater treatment, where rapid and thorough lipid hydrolysis is essential. Similar findings have been reported for free and immobilized lipases from Bacillus aryabhattai, which achieved reductions in lipid content of 48 and 64%, respectively, after 120 h of dairy wastewater treatment. Likewise, the immobilized B. stearothermophilus and S. aureus lipases were also shown to efficiently reduce lipid content in wastewater.

The total organic chloride (TOC) content increased over time for both free and immobilized Lip_S.g, with the immobilized form exhibiting a significantly higher accumulation rate (Table ). By day 10, the TOC content reached 614 ± 9.53% following treatment with immobilized Lip_S.g, compared to 309 ± 5.29% for the free enzyme (Table ). This marked increase in organic chloride levels with immobilized Lip_S.g highlighted its enhanced catalytic activity. The data suggested that immobilized Lip_S.g efficiently hydrolyzed lipid substrates in wastewater, leading to the release of chlorinated byproducts.

The chemical oxygen demand (COD) decreased steadily for both free and immobilized Lip_S.g, with the immobilized enzyme showing a significant reduction (Table ). By day 10, immobilized LipS.g reduced COD efficiently up to 0.19 ± 0.01% compared to 31 ± 2.64% for the free form. The rapid decrease in COD observed with immobilized Lip_S.g, particularly during the first 5 days (from 100 to 3.83 ± 0.35%), highlighted its high efficiency in hydrolyzing organic matter in wastewater. In contrast, free Lip_S.g showed a slower COD decline, likely due to its reduced stability over time compared to the immobilized form. The exceptional performance of immobilized Lip_S.g underscored its suitability for industrial wastewater treatment applications, where rapid and thorough organic pollutant removal is critical. Similarly, COD reductions to 0.56% and 0.22% were reported for B. stearothermophilus and S. aureus lipases, respectively. These findings emphasize the potential of immobilized enzymes as a promising tool for bioremediation and highlight the need for further research to facilitate their large-scale application.

The residual activity of immobilized Lip_S.g was assessed over 15 cycles of use (Figure ). During the first five cycles, Lip_S.g demonstrated stable performance, retaining 100% activity (Figure ). However, a gradual decline in activity was observed from cycle 6 onward, with activity dropping to 95 ± 3.60, 83.67 ± 4.04, and 74 ± 2.64% in cycles 6, 7, and 8, respectively. The activity was further reduced to 63 ± 3.0, 45.33 ± 3.51, and 12% by cycles 10, 12, and 15, respectively (Figure ). Several factors, including the breaking of chemical bonds, enzyme desorption, or erosion of the support material, could explain this decline in residual activity. Similarly, immobilized A. hemolyticus lipase on eggshell membrane retained approximately 50% of its activity after 19 reuses.

5.

Enzyme activity retention after multiple uses of immobilized Lip_S.g. Data are expressed as the mean ± standard deviation of three independent measurements.

Effective wastewater treatment with immobilized lipases not only ensures the thorough removal of organic pollutants but also promotes sustainable industrial practices by enabling enzyme reuse and reducing waste generation. , Consequently, further research and development in this field hold the potential to revolutionize wastewater management and significantly mitigate environmental impacts. Although free and immobilized LipS.g exhibited nearly identical activities on simple model substrates (Table ), their performances diverged in Wadi Hanifa wastewater due to the complex effluent matrix: emulsified fats and micelles can obstruct substrate access for the free enzyme but are more readily hydrolyzed by immobilized lipase via interfacial activation on the support surface; inhibitory compounds such as heavy metals, surfactants, and phenolics present in real wastewater may bind to or denature the free enzyme, whereas immobilization helps preserving the enzyme’s active conformation and reduce nonspecific adsorption; and the high organic loadincluding proteins and polysaccharidescan compete with target lipids for binding to free enzyme, while the porous support of immobilized LipS.g creates a protective microenvironment that enhances substrate specificity and shields the active site from competitive interference.

3. Materials and Methods

3.1. Production and Immobilization of Lip_S.g

S. gobitricini strain was isolated from polluted mangrove soil, identified, and stored in the Botany and Microbiology Department-College of Science at King Saud University (Riyadh, Saudi Arabia), as previously described.

S. gobitricini culture conditions were 2% glucose and 2% yeast extract as carbon and nitrogen sources, respectively, as well as calcium, olive oil, and Tween, at 1%. After 84 h of S. gobitricini incubation in the appropriate medium at 45 °C on a rotary shaker at 270 g, the crude enzyme solution obtained after centrifugation (30 min at 12,000 rpm) was incubated for 15 min at 70 °C followed by a rapid cooling and subsequent centrifugation for 30 min at 12,000 rpm. Following ammonium sulfate fractionation (20–65%) of the obtained supernatant, the precipitate was resuspended in 25 mM Tris–HCl buffer containing 50 mM NaCl and 2 mM benzamidine, pH 8.0. The collected lipase solution after centrifugation at 12,000 rpm for 30 min was kept at 4 °C.

Various support materials (silica gel, chitosan cellulose acetate, Celite 545, and CaCO₃) were tested for immobilization assays according to Rosu et al. Briefly, 1 g of each powdered support was added to a lipase solution containing 4500 U mL^–1. After stirring for 2 h at 4 °C, 20 mL of chilled acetone was added, and the suspension was filtered through a Büchner funnel. The immobilized lipase was washed on the filter paper with an additional 20 mL of chilled acetone and dried in a vacuum desiccator for 8 h. The yield of immobilized lipase activity was calculated as the percentage of adsorbed lipase activity relative to the total soluble lipase activity initially added to 1 g of the support according to the following formula:

$$\mathrm{residual}\mathrm{activity}(\%)=(\mathrm{initial}\mathrm{free}\mathrm{enzyme}\mathrm{activity}/\mathrm{activity}\mathrm{after}\mathrm{immobilization})\times 100$$

where, activity after immobilization: Lipase activity measured after immobilization, initial free enzyme activity: Lipase activity measured before immobilization (considered as 100%).

The analysis was performed in triplicate.

The optimal immobilization conditions were assessed by determining the adsorption kinetics and the yields of immobilized materials onto the best support. Lip_S.g adsorption kinetics were assessed on CaCO₃ as support, over 120 min period, every 10 min intervals. The supernatant collected after centrifugation at 8000 rpm for 5 min was used to determine the lipase activity of each sample.

Different initial activities (1000 to 7000 Units/g) of soluble lipase were used to calculate the yields of immobilized Lip_S.g on CaCO₃.

3.2. Characterization of Immobilized Lip_S.g

3.2.1. Storage Stability

The storage stability of the immobilized Lip_S.g was evaluated by measuring the residual activity of the enzyme stored at 4 and 25 °C at 10-day intervals over a 120-day period and compared to the starting activity. The pH-stat method was followed to determine titrimetrically the lipolytic activity at pH 9.0 and 50 °C using, as substrate, TC18 (olive oil: triolein) emulsion.

3.2.2. Lipase Activity Measurement

The lipase activity was determined titrimetrically via the pH-stat method according to Rathelot et al. in the presence of 2 mM calcium and at pH 9.0 and 50 °C, using as substrate, TC18 (olive oil) emulsion. Under these assay conditions, one unit of the lipase activity is defined as 1 μmol of fatty acid produced per minute.

Substrate specificity assays were performed using emulsions of TC4 (tributyrin) and TC8 (trioctanoin) as well as commercial oils (sunflower oil, soybean oil, sesame oil, and coconut oil) as substrates at 1%.

3.2.3. Protein Analysis

Protein quantitation was estimated by the Bradford method by measuring optical density at 595 nm and using BSA as the standard.

3.2.4. Effects of pH and Temperature on Lip_S.g Activity and Stability

Lipolytic activity was initially measured at 50 °C or at pH 9.0 across a pH range of 5.0 to 11.0 or a temperature range of 30 to 70 °C, respectively. The following buffers at 50 Mm were used: sodium acetate buffer (pH 4.0–6.0), potassium phosphate buffer (pH 7.0–8.0), Tris–HCl buffer (pH 8.5–9.0), and glycine–NaOH buffer (pH 10.0–12.0). Thermostability was investigated by incubating the enzyme preparations for 1 h at different temperatures ranging from 40 and 80 °C. Likewise, both free and immobilized Lip_S.g were incubated at room temperature for 48 h in different buffers (pH range of 3.0 to 12.0). The residual activity was measured after centrifugation using the standard assay method, as mentioned above. All measurements were performed in triplicate.

3.3. Bioremediation of Wadi Hanifa Water Using Free and Immobilized LipS.g

TOC, COD, and lipid content values were determined before and after treatment of Wadi Hanifah water (Al-Riyadh City, Saudi Arabia) with immobilized or free Lip_S.g. One-liter water samples were treated with 4500 U of either lipase and stirred at 200 rpm at room temperature. Following the American Public Health Association protocols, samples were collected at 1 day-intervals for 10 days. Distilled water was taken as a negative control for the comparison study. All experiments were conducted in triplicate.

3.4. Repeated Use of Immobilized Lipase

For each reuse cycle, immobilized LipS.g was recovered by centrifugation at 5,000g for 5 min, washed three times with 50 mM phosphate buffer (pH 8.0), and gently resuspended. Reusability was tested by treatment of one L of Wadi Hanifah water with recuperated immobilized LipS.g and stirred at 200 rpm at room temperature. Samples were then collected and analyzed, as previously reported in Section .

3.5. Statistical Analysis

All statistical analyses were performed using GraphPad Prism. Data are presented as the mean ± standard deviation (SD) from at least three independent measurements (n = 3). To assess differences in lipase immobilization efficiency among the tested supports (CaCO₃, Celite 545, Cellulose acetate, Chitosan, and Silica gel), comparisons were performed between each support and all others combined using an unpaired t test with Welch’s correction. Statistical comparisons between free and immobilized LipS.g were conducted using two-way ANOVA, followed by Sidak’s posthoc test for multiple comparisons. Statistical significance was indicated as p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).

4. Conclusions

The immobilization of Lip_S.g onto the CaCO₃ support demonstrated significant potential for enhancing stability and catalytic efficiency. Among the tested supports, CaCO₃ proved to be the most effective, achieving a high immobilization yield of 82.66 ± 5.5% and excellent stability. The immobilized Lip_S.g outperformed its free form in substrate hydrolysis, lipid degradation in wastewater, and COD reduction. Furthermore, it exhibited enhanced stability across broader pH and temperature ranges, as well as prolonged storage life. These findings underscored the potential of immobilized Lip_S.g for sustainable industrial wastewater treatment and a wide range of biotechnological applications.

The data underlying this study are not publicly available due to institutional restrictions and confidentiality agreements related to wastewater treatment sources. The data are available from the corresponding author upon reasonable request.

All authors contributed to the study’s conception and design. All authors have read and agreed to the submitted version of the manuscript.

The authors declare no competing financial interest.