Esterases: Mechanisms of Action, Biological Functions, and Application Prospects

Abstract

Esterases are ubiquitous enzymes found in all living organisms, including animals, plants, and microorganisms. They are involved in several biological processes, including the synthesis and breakdown of biomolecules, such as nucleic acids, lipids, and esters; phosphorus metabolism; detoxification of natural and artificial toxicants; polymer breakdown and synthesis; remodeling; and cell signaling. The present review focuses on the most industrially important esterases, namely lipases, phospholipases, cutinases, and polyethylene terephthalate hydrolases (PETases). Esterases are widely used in industrial and biotechnological applications. Notably, the biotechnological production of esters, including methyl acetate, ethyl acetate, vinyl acetate, polyvinyl acetate, and ethyl lactate, as an alternative to chemical production, represents a multi-billion-dollar industry. Currently, most enzymes (>75%) used in industrial processes are hydrolytic. Among them, lipases and phospholipases are primarily used for lipid modification. Lipases are the third most commercialized enzymes after proteases and carboxyhydrases, and their production is steadily increasing, currently representing over one-fifth of the global enzyme market. Esterases, particularly lipases, phospholipases, and cutinases, are employed in cosmetics, food, lubricants, pharmaceuticals, paints, detergents, paper, and biodiesel, among other industries. Overall, biotechnological production using enzymes is gaining global traction owing to its environmental benefits, high yields, and efficiency, aligning with green economy principles.

1. Introduction

Microbial esterases have garnered significant attention over the past decade owing to their role as versatile biocatalysts capable of hydrolyzing ester bonds in various substrates, ranging from short-chain fatty acid esters to complex polymeric esters. These enzymes are predominantly produced by bacteria, fungi, and algae, with marine and extremophilic microorganisms representing an especially rich source, given the unique physicochemical conditions in their habitats. Their ability to catalyze regio- and stereoselective transformations makes them indispensable in the production of enantiomerically pure substances, pharmaceutical intermediates, and fine chemicals. This report provides an expert-level synthesis of current research on microbial esterases, with emphasis on their biochemical properties, catalytic mechanisms, industrial applications, and the emerging metagenomic strategies that facilitate their discovery and enhancement [1].

Microorganisms that adapted to extreme environmental conditions, such as cold, salt and heat tolerance and alkali resistance, produce esterases that are characterised by molecular features such as a higher glycine content and flexible active-site structures. These features enable the esterases to function efficiently in low-temperature or high-salinity environments [2]. For example, cold-active and salt-resistant carboxylesterases obtained from marine metagenomes demonstrate high catalytic activity even at temperatures as low as 5 °C, as well as sustained performance in the presence of high salt concentrations [3]. Altogether, the rich genetic and functional diversity of microbial esterases coupled with their environmental robustness underpins their potential for sustainable industrial applications [1].

Microbial esterases play a crucial role in the degradation of persistent environmental pollutants, including synthetic plastics and organophosphorus compounds. Certain marine bacterial esterases have demonstrated the ability to hydrolyze polyethylene terephthalate (PET) and other plastic polymers, thereby contributing to environmental sustainability by reducing plastic pollution. Additionally, the enzymatic breakdown of organophosphorus pesticides highlights the potential of these biocatalysts in detoxifying contaminated sites and facilitating bioremediation processes [1]. The specificity and stability of these enzymes under extreme conditions make them particularly suitable for deployment in situ, where industrial pollutants are encountered in complex mixtures [1,2].

Esterases are enzymes that catalyze the hydrolysis of esters into their corresponding acids and alcohols in the presence of water by catalyzing the cleavage and formation of ester bonds. They belong to the hydrolase class of enzymes (enzyme classification [EC] EC 3.1) and include subclasses such as nucleases, phosphodiesterases, lipases, and phosphatases. Esterases are further classified according to the international classification EC system as 3.1.1. +x, where “x” varies based on the specific substrate they act upon [4]. Esterases can catalyze two types of reactions, esterification and transesterification (Figure 1), with exceptional chemo-, regio-, and enantioselectivity [5].

Esterases are widely distributed in nature and have been isolated from plants, animals, and microorganisms. Among these, microbial enzymes are particularly valuable in modern biotechnology applications owing to their ease of isolation, cost-effective production, and convenient storage and applications. All classes of microorganisms, including bacteria and fungi, produce esterases, either constitutively or inducibly [5,6]. Extensive biochemical and structural studies have been conducted to elucidate the functions and mechanisms of various esterases [7]. As esterases catalyze the hydrolysis of a diverse range of substrates containing ester groups, they are widely used in biotechnology for applications such as food processing, agriculture, detergent and solvent production, and the synthesis of biologically active drug enantiomers [8].

Microorganisms have developed diverse aerobic and anaerobic catabolic strategies to degrade the wide array of organic compounds present in the environments they colonize [9]. As pollutants are often structurally similar to natural compounds, polluted ecosystems likely host organisms capable of metabolizing these pollutants as their primary carbon sources. During microbial degradation, enzymes catalyze chemical changes in pollutants and generally exhibit broad specificity, allowing them to act on several structurally related molecules [10].

The present review focuses on the most industrially important esterases, including lipases, phospholipases, cutinases, and polyethylene terephthalate hydrolases (PETases), summarizing their applications in various biotechnological industries.

2. Types and Sources of Esterases

As previously mentioned, esterases can be constitutively or inducibly produced and secreted by bacteria, yeasts, or fungi (

Table 1. Substrate spectrum of microbial esterases.

Type of Esterase	Microorganism	Substarte	Optimal pH	Optimal Temperature (°C)	Molecular Weight (kDa)	Km (µM)	Vmax	Application
							(µM/min/mg)
Serine esterase	Aspergillus westerdijkiae	Water-soluble short-chain fatty acids	8	40	32	638.11	5.47	As a potential biotechnological catalyst
								Hydrolysis of hemicellulose and lignin
Glucuronoyl esterase	Aspergillus fumigatus	Hemicellulose and lignin	5	40 a 50	Nd	15.8 y	Nd
				(Favorable)		16.4		Lignocellulose hydrolysis
Glucuronoyl esterase	Neurospora crassa	Lignocellulose	7	40 a 50	32	15	1.12
				(Favorable)
						1.0		Synthesis of flavor esters
Recombinant esterase (RmEstA)	Rhizomucor miehei	pNP with acyl lengths from C2 to C16	6.5	45	34	0.17	Nd
						0.12
						0.82
						0.28 y
						0.3
Feruloyl esterase (Est1)	Pleurotus sapidus	Feruloylated saccharides	6	50	55	1.95	1.77	Ecological or technical applications
EstS1	Sulfobacillus acidophilus	Phthalates	8	70	36	0.18	2440	Degradation of phthalates
Lp-1002 esterase	Lactobacillus plantarum (WCFS1)	Phenyl acetate	5-7	40	Nd	Nd	Nd	Winemaking
EstB28	Oenococcus oeni	Nitrophenyl-linked substrates	5	40	34.5	Nd	Nd	Winemaking
CL96 esterase	Lactobacillus casei	ρ-Nitrophenyl derivatives of fatty acids (C2 and C4)	7	30	Nd	Nd	Nd	Dairy industry
Cholesterol esterase	Pseudomonas aeruginosa	Fatty acid cholesteryl	7	53	58	Nd	Nd	Optical industry
Cinnamoyl esterase	Lactobacillus helveticus (KCCM 11223)	Methyl ferulate, methyl sinapinate, methyl ρ-coumarate and methyl caffeate	7	65	27.4	0.153	559.6	Hydrolysis of chlorogenic acid
LipM	Metagenomics of agricultural soil expressed in Escherichia coli	ρ-nitrophenyl short-chain fatty acids	7.5	37	48	Nd	Nd	Transesterification of polluting compounds, production of biodiesel or food supplement for monogastric animals
S. cerevisiae esterase	Saccharomyces cerevisiae	Formaldehyde	7	50	40	0.29	12	Formaldehyde detoxification

Nd = Not determined. Obtained from Hernández-Sánchez et al. [12].

). For example, Aspergillus westerdijkiae secretes serine esterase, which is a promising catalyst owing to its ability to use short-chain fatty acids as substrates [11]. Aspergillus fumigatus secretes glucuronoyl esterase, which is essential for lignocellulose degradation. Glucuronoyl esterase, secreted by Neurospora crassa, acts on lignocellulose by hydrolyzing the ester bond between lignin alcohols and the 4-O-methyl-D-glucuronic acid of hemicellulose attached to xylan. Furthermore, the fungus Pleurotus sapidus produces esterases, such as feruloyl esterase, which hydrolyze saccharide esters in a Tween 80-enriched medium [12].

Table 1 illustrates that serine esterase from Aspergillus westerdijkiae had a significantly elevated Km value (638.11 µM) in comparison to other esterases. For instance, feruloyl esterase derived from Pleurotus sapidus exhibits a Km value of 1.95 µM. This significant disparity indicates multiple potential causes. A potential explanation for the elevated Km of the serine esterase from A. westerdijkiae compared to feruloyl esterase is its wider substrate specificity and enhanced structural flexibility. This may lead to a more accessible active site that is less selective for substrates, offering catalytic diversity but diminished substrate affinity. A plausible reason for the elevated Km is the inherent evolutionary adaptation of A. westerdijkiae to fluctuating substrate concentrations in its environment. Contemporary protein engineering techniques may substantially modify its biochemical properties in the future to enhance its efficacy.

Esterases do not require cofactors, making them attractive as biocatalysts [6]. The two main subclasses of hydrolases are lipases and true esterases. Both classes share some common features while also possessing some unique properties. The three-dimensional structures of both classes exhibit a characteristic hydrolase fold and a Ser-Asp-His catalytic triad [4]. True esterases hydrolyze short-chain carboxylic acids, whereas lipases hydrolyze insoluble long-chain triglycerides and secondary alcohols. Furthermore, true esterases share common sequence and structural features, including a conserved Ser-Asp Glu-His catalytic triad that stabilizes the oxyanion hole during hydrolysis, along with several other conserved residues [13,14]. These catalytic residues facilitate the nucleophilic attack of the active site serine on the carbonyl carbon atom of the ester (Figure 1). These features are often used to classify esterases, predict their biological functions, and guide protein engineering [14,15,16,17]. However, systematic classification based on the similarity of their primary, secondary, and tertiary structures does not always correlate with their EC numbers owing to their structural plasticity and complexity. This highlights the need for more comprehensive structural and spatial characterization of individual enzymes [18].

The mechanism of hydrolysis or ester formation involves four steps. First, the substrate is attached to the active site serine, forming an intermediate stabilized by asparagine and histidine residues. Second, an alcohol group is released, and an acyl-enzyme complex is formed. Subsequently, a nucleophilic substitution occurs, forming another intermediate. Finally, this intermediate is broken down to produce an ester or acid, releasing the free enzyme [1].

3. Lipases

Lipases belong to the serine hydrolase family (EC 3.1.1.3) and are structurally defined by the α/β hydrolase fold, a configuration that comprises a central, twisted β-sheet flanked by α-helices [19]. Central to their catalytic activity is the highly conserved catalytic triad, typically composed of serine, histidine, and aspartate or glutamate residues. In the canonical mechanism, the serine acts as a nucleophile to attack the carbonyl carbon of the ester bond, forming a tetrahedral intermediate that is stabilized by an oxyanion hole—this intermediate is subsequently resolved by deacylation, releasing the reaction products [19,20]. A distinctive structural feature of many lipases is the presence of a mobile “lid” domain that covers the active site; this lid undergoes a conformational change upon binding to a lipid-water interface, a phenomenon termed “interfacial activation” that exposes the active site for substrate binding and catalysis [19,20]. They catalyze the hydrolysis and synthesis of long-chain triglycerides into fatty acids, diacylglycerols, monoacylglycerols, and glycerol [21,22]. In addition to hydrolysis, they can also catalyze reactions such as esterification, aminolysis, and alcoholysis, which are used in various industrial applications [23,24]. Both lipases and phospholipases are interphase enzymes that hydrolyze the hydrophobic ester bonds of triacylglycerols and phospholipids, respectively. Beyond their role as esterases, they catalyze several other reactions. Lipases catalyze esterification and transesterification reactions, whereas phospholipases demonstrate acyltransferase, transacylase, and transphosphatidylation activities. Consequently, lipases and phospholipases serve as versatile biocatalysts extensively utilized in various industrial applications, including biodiesel production, food manufacturing, nutraceuticals, oil degumming, detergents, bioremediation, agriculture, cosmetics, leather, and paper industries [19,25].

Notably, over 75% of the world market of enzymes utilized in industrial processes are hydrolytic [26]. Among these, lipases and phospholipases are used to modify lipids. Lipases rank as the third most commercialized enzymes, following proteases and carboxyhydrases, with their production consistently rising and accounting for nearly one-fifth of the global enzyme market [27]. Lipases are monomeric proteins with a molecular mass ranging from 19 to 60 kDa. Their properties depend on the substrate, particularly the chain length of the glycerol-based fatty acid they catalyze [28,29]. Some lipases catalyze numerous important reactions in biotechnology, including esterification and transesterification, owing to their capacity to function in organic solvents [30,31]. Several phospholipase genes have been cloned, expressed, and patented, although the main applications of the enzymes are currently limited to only a few industries [32]. Importantly, the lipase market has grown significantly to meet the demand for improved quality of livestock products and increased consumption of enzyme-modified livestock products. This growth has been driven by the notable advantages of microbial lipases over animal and plant lipases [33,34]. Moreover, the demand for microbial-derived lipases is projected to continue to grow significantly in the near future owing to their diverse range of applications [35,36,37]. Specifically, microbial lipases in powdered form are expected to dominate the microbial lipase market owing to their stability, ease of handling, convenient packaging, and transportation [38,39,40,41].

Biocatalytic esterification is an environmentally friendly alternative to chemical esterification, as it can occur under ambient temperatures and does not produce corrosive waste. Thus, the bioproduction of environmentally friendly solvents, such as esters, represents an effective alternative for chemical synthesis. Owing to the growing demand for environmentally friendly industries, biodegradable esters have become increasingly attractive for various industrial applications, particularly in the cosmetics industry, which holds the largest market share, followed by the food, lubricant, pharmaceutical, and paint industries [42]. Among these, short-chain esters, including ethyl lactate and ethyl acetate, are prevalent in the commercial sector owing to their biodegradable and environmentally beneficial properties [42]. In the U.S., the ester market was valued at USD 3.8 billion in 2019 and is estimated to reach USD 5 billion by 2025, primarily driven by the growing demand for emulsifiers and stabilizers in personal care and detergent products [43].

Overall, traditional ester production often relies on harsh catalysts, such as concentrated sulfuric acid, and involves extensive use of alcohols and corrosive chemicals, posing environmental risks (Grand View Research, 2019). To mitigate this environmental harm, innovative eco-friendly techniques, such as microbial production methods, offer sustainable systems for producing industrial solvents from renewable resources [42].

3.1. Sources of Lipases

Lipases are prevalent in bacteria, fungi, and yeasts. The predominant bacterial sources of lipases include Bacillus spp., Pseudomonas spp., Staphylococcus spp., and Burkholderia spp. [12].

Fungal lipases are extensively utilized in many biotechnological sectors owing to their stability, specificity, and ease of production. The lipases extracted from Thermomyces lanuginosus, Rhizopus oryzae, and Aspergillus niger are economically valuable. Moreover, lipases derived from yeasts of the genus Candida, including those extracted from Candida antarctica and Candida rugosa, have been utilized in both industrial production and research [44].

Microbial lipases are more preferable than plant- or animal-derived lipases owing to their diverse catalytic properties, high yields, ease of genetic manipulation, and lack of seasonal variation. They are also stable, safe, and convenient, and the microorganisms that produce them grow rapidly in inexpensive media [45,46]. Notably, bacterial isolates have high activity, function optimally in neutral or alkaline pH, and exhibit greater thermostability than yeast isolates [12]. Bacterial strains such as Pseudomonas alcaligenes, P. aeruginosa, P. fragi, P. fluorescens BJ-10, Bacillus subtilis, and B. nealsonii S2MT, and some species of fungi such as Penicillium expansum, Trichoderma, Penicillium chrysogenum, and Aspergillus niger are effective lipase producers [47,48,49].

Lipases are ubiquitous in nature and have been isolated from mammals, plants, fungi, yeasts, and insects, reflecting their indispensable roles in lipid metabolism and energy production [50,51]. In biological systems, lipases are critical for the digestion of dietary lipids, mobilization of stored fats, and generation of bioactive lipid mediators that regulate numerous cellular functions. Moreover, the modulation of lipase activity is clinically relevant, as alterations in their expression or function contribute to metabolic disorders, inflammatory conditions, and even oncogenic processes [19,20]. The industrial utility of lipases stems from their high catalytic efficiency, substrate specificity, and operational stability under mild reaction conditions, which have been harnessed for numerous applications in food technology, biodiesel production, pharmaceutical synthesis, and environmental clean-up [20,52].

Lipases serve indispensable biological functions across a broad spectrum of organisms. In the digestive systems of vertebrates, pancreatic, gastric, and lingual lipases collaborate to hydrolyze dietary triglycerides, thereby facilitating the absorption of fatty acids and monoglycerides in the small intestine [52]. Pancreatic lipase, in particular, is pivotal for the efficient digestion of long-chain triglycerides, operating in conjunction with colipase, which promotes its binding to oil–water interfaces [49,53]. Beyond digestion, lipases are integral to lipid remodeling in cellular membranes, energy storage in adipose tissues, and the generation of signaling molecules. Intracellular lipases, such as hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL), and monoacylglycerol lipase (MAGL), orchestrate the mobilization and utilization of stored lipids in response to hormonal cues and energy demands [54]. Lipases are also involved in post-translational modifications—for instance, certain phospholipases contribute to membrane remodeling and the regulation of signal transduction pathways [19]. Lipases are classified based on their substrate specificity, source organism, and cellular localization. Mammalian lipases include pancreatic lipase, lipoprotein lipase (LPL), hepatic lipase, and others, each with a specialized role in lipid metabolism. LPL, for example, hydrolyzes triglyceride-rich lipoproteins in the circulation, releasing fatty acids for tissue uptake and energy production; deficiencies in LPL activity lead to severe hypertriglyceridemia and pancreatitis [49,55]. Lysosomal acid lipase (LAL) operates within the acidic environment of lysosomes to degrade cholesteryl esters and triglycerides, with its deficiency resulting in lysosomal storage disorders such as Wolman disease and cholesterol ester storage disease (CESD) [56,57]. In plants, lipases are primarily involved in seed germination and mobilization of reserve lipids, whereas microbial lipases—produced by bacteria, yeasts, and fungi—are renowned for their broad range of substrate specificities and robustness, traits that have rendered them valuable in industrial biocatalysis [58,59]. The molecular diversity and tissue-specific roles of these enzymes underscore their evolutionary adaptation to meet distinct metabolic demands [19].

3.2. Clinical Significance of Lipases

The clinical importance of lipases is underscored by their role as diagnostic biomarkers and therapeutic targets in various diseases. In the context of acute pancreatitis, pancreatic lipase concentrations in serum serve as key indicators of pancreatic injury, with elevated levels correlating with the severity of inflammation and tissue damage [60,61]. Lipase assays are routinely employed in clinical laboratories for the rapid diagnosis of pancreatitis and other disorders related to exocrine pancreatic insufficiency [19,59]. Genetic deficiencies in specific lipases also have profound clinical implications. For instance, lipoprotein lipase (LPL) deficiency, a rare autosomal recessive disorder characterized by abnormally high circulating triglyceride levels, predisposes patients to recurrent episodes of acute pancreatitis and cardiovascular complications [50,55]. Similarly, lysosomal acid lipase deficiency (LAL-D) leads to the intracellular accumulation of cholesteryl esters and triglycerides, causing progressive liver damage, dyslipidemia, and multisystemic morbidity; early diagnosis and enzyme replacement therapies, such as sebelipase alfa, have been developed to mitigate these effects [55,56,62]. Recent clinical investigations have further elucidated the role of lipases in non-metabolic disorders. For example, the expression of LPL in chronic lymphocytic leukemia (CLL) cells has been correlated with disease aggressiveness, suggesting that lipid metabolism may be a critical driver of leukemic cell survival and growth, and positioning LPL as a potential prognostic biomarker and therapeutic target [50,63]. In addition, lipases have been implicated in the clinical evaluation of acute pesticide poisoning, where measurements of serum lipase in conjunction with amylase and other biomarkers assist in early detection of pancreatic injury and the assessment of poisoning severity [63]. Moreover, lipase inhibitors, such as orlistat—a derivative of lipstatin derived from Streptomyces species—have been approved for the treatment of obesity by reducing fat absorption, thereby offering therapeutic benefits in metabolic syndrome and related disorders [53,59]. Collectively, these clinical applications highlight the pivotal role played by lipases not only as diagnostic markers but also as targets for therapeutic intervention across a broad range of diseases [60,61].

While the diagnostic utility of lipase measurement is well established, several methodological considerations must be addressed. Variations in assay platforms and threshold values between laboratories can influence the interpretation of test results, necessitating standardization of methodologies and reporting criteria [64,65]. Recent advances in analytical techniques, such as fluorometric assays, have improved the sensitivity and reproducibility of lipase measurements, enabling the detection of even subtle variations in enzyme activity [66,67]. These enhanced methods offer the potential for further refinement of diagnostic thresholds and the development of point-of-care testing modalities that could facilitate rapid decision-making in emergency settings [68,69].

Recent technological advancements have significantly enhanced the performance of lipase assays. High-performance techniques such as fluorometric detection, which rely on aggregation-induced emission (AIE) principles, provide markedly increased sensitivity for detecting low levels of lipase activity in biological specimens [66,67] (Shi J, Yun S). These improvements have led to the development of next-generation assays that can reliably quantify lipase activity even in cases where conventional colorimetric methods might fall short, thereby improving diagnostic accuracy and early detection rates [68,69].

In conclusion, the clinical measurement of lipase activity remains an indispensable tool for the diagnosis of acute pancreatitis, while the ongoing development of targeted lipase inhibitors offers a promising therapeutic avenue for the treatment of obesity and associated lipid disorders. Through continued research, standardization, and technological innovation, these dual applications of lipase biology will undoubtedly play a central role in shaping future clinical practices and improving patient outcomes on a global scale [70,71,72].

3.3. Industrial and Biotechnological Applications

The catalytic versatility and operational stability of lipases have rendered them indispensable in numerous industrial and biotechnological processes. In the food industry, lipases are employed for flavor development, fat modification, and the enhancement of nutritional value in dairy, bakery, and confectionery products. The enzymatic modification of fats can tailor melting profiles, enhance texture, and produce structured lipids that offer health benefits compared to conventional fats [20,59]. For example, lipase-catalyzed reactions facilitate the synthesis of diacylglycerols and monoacylglycerols, which can be used to develop lower-calorie food formulations with improved sensory properties [19]. In the detergent industry, lipases are incorporated as additives in cleaning formulations to enhance the removal of fat-based soil from textiles and surfaces. Their ability to work at low temperatures and across a broad pH range makes them superior to conventional chemical additives, contributing to energy savings and reducing the environmental impact of detergent use [73]. Lipases catalyze the hydrolysis of lipid contaminants on fabrics, leading to easier emulsification and removal during washing processes [50,73]. The renewable energy sector has also benefited from lipase applications, particularly in the production of biodiesel. Lipase-mediated transesterification of vegetable oils or animal fats produces fatty acid alkyl esters (biodiesel) with high specificity and under mild reaction conditions, circumventing the need for harsh chemicals and elevated temperatures that are typically associated with traditional biodiesel production methods [19,20]. Immobilized lipases, in particular, offer the advantage of reusability and enhanced process stability, making them economically attractive for large-scale biodiesel synthesis [60]. Pharmaceutical synthesis represents one of the most sophisticated applications of lipases, where their chemo-, regio-, and enantioselectivity are exploited to synthesize optically pure intermediates and active pharmaceutical ingredients (APIs). Lipase-catalyzed resolution of racemic mixtures is frequently employed in the production of drugs, including non-steroidal anti-inflammatory drugs (NSAIDs) and other chiral compounds [19,57]. The ability of lipases to perform under diverse reaction conditions—ranging from aqueous solutions to organic solvents—has further expanded their role in complex synthetic routes, thereby reducing the environmental footprint of chemical processes [58,60]. Cosmetic and personal care industries have adopted lipases for the enzymatic synthesis of esters and fragrance compounds, contributing to stabilization, texture optimization, and enhanced sensory appeal in cosmetic formulations [50,58]. Additionally, lipases are used in biosensor technology, where their substrate specificity and catalytic efficiency provide the basis for sensitive and rapid detection systems for triglycerides, cholesterol, and other metabolites in clinical diagnostics [73]. Environmental bioremediation is another area of application, as lipases facilitate the degradation of lipid-rich pollutants and hydrocarbon contaminants in wastewater and soil, thereby contributing to cleaner industrial effluents and ecological restoration [19,20]. In total, the industrial exploitation of lipases spans multiple sectors, demonstrating how their catalytic properties can be tailored to meet the specific demands of processes ranging from food modification to renewable energy production [20,60].

The study of lipases continues to be a vibrant area of research that integrates molecular biology, clinical medicine, and industrial biotechnology. Their inherent catalytic versatility, structural adaptability, and biological significance render them an enduring subject of scientific inquiry and technological innovation. As both basic research and applied development progress, lipases will remain pivotal in driving sustainable industrial processes and advancing therapeutic strategies for metabolic and lipid-associated diseases, thereby underscoring their central role in modern biomedicine and manufacturing [49,51,55].

4. Cutinases

Cutinases, closely related to lipases in their mechanism of action and belonging to the same hydrolase subfamily, are inducible extracellular enzymes secreted by microorganisms that can degrade plant cell walls (Figure 2). Cutinases degrade cutin, an aliphatic polyester that serves as a natural defense barrier in many plants. Natural cutinases are closely associated with the pathogenicity of plant pathogens, as they enable them to break down cutin barriers to enter plants [74,75,76]. As their substrate, cutin, is an insoluble lipid polyester, cutinases are classified within the α,β-hydrolase family together with lipases and esterases. Compared to other hydrolases, cutinases have unique characteristics. For example, cutinase has the lowest molecular mass among the α,β-hydrolase family members. Although both cutinases and lipases have a Ser-His-Asp catalytic triad, unlike lipases, cutinases lack a hydrophobic cap covering the active site serine. Thus, cutinases recognize a wide range of substrates in addition to cutin [77,78].

Cutinases are believed to have evolved, resulting in them being able to perform diverse functions [79]. For example, the biodegradation capacity of cutinases allows them to both degrade large-molecule cutin and synthetic plastics, as well as low-molecular-weight esters and short-chain and long-chain triacylglycerols [80]. Additionally, cutinases can be used as functional biosynthetic tools for esterification and transesterification [81,82].

4.1. Sources of Cutinases

Filamentous fungi contain the largest library of cutinases [83], with fungal cutinases representing the most prevalent cutinase enzymes. Fungal cutinase typically has a molecular mass of 22–26 kDa, smaller than that of bacterial and plant cutinases. However, discrepancies between the theoretical and experimental molecular masses have been observed, likely due to post-translational modifications. Specifically, the N-terminal region of cutinase may be blocked by glucuronidation [84], and as nearly all fungal cutinases contain at least one phosphorylation site [85], cutinase likely undergoes phosphorylation; both modifications can alter the molecular mass.

Studies on the structures of fungal cutinases have revealed conserved GXSXG (usually GYSQG) sequences, an S-H-D catalytic triad, and at least one phosphorylation site. Additionally, fungal cutinases contain two pairs of disulfide bonds and four engaged cysteines near the N-terminus. Tyrosine, phenylalanine, and tryptophan, which are involved in sugar binding, are highly conserved at the C-terminus of fungal cutinases [86,87].

Cutinase from Fusarium solani (Fsp) has been extensively studied as a model enzyme, particularly focusing on its N-terminus [88]. The N-terminus of Fsp cutinase undergoes glucuronidation via an amide bond with glucuronic acid, while carbohydrates are attached via O-glycosidic bonds to serine, threonine, β-hydroxyphenylalanine, and β-hydroxytyrosine. Detailed structural and catalytic characterization of Fsp cutinase provides a useful basis for the rational genetic engineering of wild-type cutinase to enhance its performance [86]. The two pairs of disulfide bonds in the structure of fungal cutinases play a key role in their stability. When acting on cutin as a substrate, the optimal pH for fungal cutinases is 10.0, and the optimal temperature is 30–40 °C. Fungal cutinases can be inhibited by active serine-directed reagents, such as diisopropyl fluorophosphate, but not by thiol-directed reagents. Some fungal cutinases, such as cutinase from Aspergillus nidulans, can be induced by oil, triacylglycerides, and fatty acids and inhibited by glucose and other sugars [83].

4.2. Bacterial Cutinases

Although fungal cutinases have been extensively studied, bacterial cutinases have also attracted attention for their unique properties [88,89]. More recently researchers have demonstrated that cutinases are produced by a diverse range of microorganisms—including bacteria and actinobacteria—and can be heterologously expressed in systems such as Escherichia coli and Pichia pastoris [90,91]. This diversity extends beyond source organisms; it manifests in differences in molecular weight, substrate specificity, and enzyme stability, which ultimately dictate their suitability for various catalytic processes [90,91]. The open nature of the active site in cutinases differentiates them from classical lipases, which require a conformational change or lid opening to access the catalytic site [89]. With applications ranging from textile bioscouring and detergent formulations to plastic waste recycling and biodiesel production, the scientific and commercial interest in cutinases has grown exponentially in recent years [90].

At the molecular level, cutinases display significant structural diversity that arises from their varied sources. Fungal cutinases typically have molecular weights in the range of 22–26 kDa and possess a compact, β-sheet–rich core that effectively positions the catalytic triad within a shallow active site [92]. In contrast, bacterial cutinases, such as those from Thermobifida fusca, show slightly higher molecular weights (approximately 20–30 kDa) and are often more thermostable and resistant to surfactants and organic solvents [79,91]. Advanced structural studies using X-ray crystallography and molecular modeling have elucidated the importance of specific amino acids in stabilizing the active site and determining substrate specificity; such insights have spurred efforts to engineer variants with enhanced catalytic properties [93,94]. Notably, engineered cutinases featuring carbohydrate-binding domains (CBDs) have been developed to improve substrate binding in textile applications, highlighting the potential for fusion protein strategies in enzyme design [94].

Cutinases are characterized by a broad range of biochemical properties that are directly relevant to their industrial utility. Their optimal pH is generally slightly alkaline—typically around 8.5 to 10.5—although pH optima may vary depending on the enzyme’s origin [90]. Temperature profiles indicate that many cutinases undergo peak activity at moderate to high temperatures; for instance, cutinases from Humicola insolens exhibit optimal activity at temperatures up to 70 °C, indicating robust thermostability [79,89]. Detailed kinetic analyses further reveal that parameters such as turnover number (K_cat) and the Michaelis constant (K_m) are influenced not only by temperature and pH but also by the substrate’s chain length and structural complexity [89,91]. In many cases, cutinases show higher catalytic efficiencies with short-chain fatty acid esters, as these substrates are sterically less demanding compared to longer-chain polymers; such variations underscore the importance of optimizing reaction conditions for specific bioprocesses [89,91]. The modulation of activity by metal ions, organic solvents, and surfactants has also been well documented, emphasizing the need for careful process design in industrial settings [90].

4.3. Plant Cutinases

Cutinases are hydrolytic enzymes that catalyze the cleavage of ester bonds in cutin, thus modifying or degrading this polyester matrix. Historically, research on cutinases has centered on fungal and bacterial enzymes due to their crucial roles in plant pathogenesis and industrial applications such as plastic recycling and textile processing [95]. Nonetheless, plant systems themselves exhibit cutinase activity that has long been recognized albeit with less characterization. Recent studies have identified candidate genes encoding plant cutinases, suggesting that these enzymes are integral not only to defense responses but also to normal developmental processes such as pollen–stigma interactions and organ detachment [96].

Plant cutinases, similar to their fungal and bacterial counterparts, are members of the α/β hydrolase superfamily and possess a characteristic catalytic triad typically composed of serine, histidine, and aspartic acid residues [79,97]. These enzymes feature an open and accessible active site that facilitates substrate binding even in environments with low interfacial activation, distinguishing them from classical lipases, whose activities are modulated by interfacial effects [95]. Structural studies involving sequence analysis and modeling have revealed that the domain architecture of plant cutinases aligns with that observed in microbial enzymes; however, subtle differences in loop regions and binding pockets may reflect specific adaptations to endogenous plant substrates. For example, the candidate enzyme CDEF1 identified in Arabidopsis thaliana belongs to the GDSL lipase/esterase family and has been implicated in cuticle modification based on its substrate specificity and expression profile [95].

The molecular weight, domain organization, and overall thermostability of plant cutinases are areas of active research. Although microbial cutinases such as those from Thermobifida fusca have been well characterized in terms of thermal tolerance [88,94], the intrinsic properties of plant-derived cutinases remain less defined. Nonetheless, ectopic expression studies in model plants have provided evidence that misregulation of cutinase activity can induce striking phenotypic abnormalities, including cuticular defects and altered hydrophobicity, thereby emphasizing their importance in maintaining the integrity and functionality of the cuticle [95].

While numerous studies have characterized fungal cutinases as critical virulence factors that enable pathogens to breach the plant cuticle [98], the role of plant-derived cutinases in host–pathogen interactions are less clearly defined. In some cases, the enzymatic activities attributed to cutinases in infected tissues may represent a confluence of both pathogen-secreted enzymes and plant endogenous activities. It is plausible that plants may modulate their own cutinase expression in response to pathogen challenge as part of a controlled defense strategy. For instance, limited degradation of the cuticle could serve to release specific signaling molecules—cutin monomers—that act as elicitors of defense responses [99]. Conversely, hyperactivity of these enzymes, whether due to pathogen manipulation or misregulation of plant gene expression, may inadvertently render the tissue more susceptible to infection [100].

Given these complexities, it is evident that further research is needed to elucidate the specific roles of plant cutinases during host–pathogen interactions. Advanced molecular techniques, including tissue-specific expression analysis and gene knockout studies, are being applied to distinguish the contributions of endogenous cutinases from those secreted by invading microorganisms. In doing so, researchers hope to identify regulatory networks that can be manipulated to enhance crop resistance while preserving the essential functions of the cuticle in normal plant development [99].

The unique catalytic properties and substrate specificity of cutinases have garnered significant attention in a range of biotechnological fields. Although much of the industrial focus to date has centered on microbial cutinases—owing to their robust activity towards synthetic polyesters such as PET [95]—plant cutinases hold equivalent potential, particularly in applications that demand eco-friendly alternatives to conventional chemical treatments.

Despite the promising aspects of plant cutinases, several challenges hinder their full exploitation. A major obstacle is the difficulty in unequivocally identifying and isolating endogenous plant cutinases from tissues that are simultaneously exposed to pathogen-derived enzymes. The overlap in substrate specificity and structural features between plant and microbial cutinases necessitates the development of refined biochemical and molecular techniques for accurate discrimination [100]. Advanced proteomic approaches and transcriptomic profiling are expected to play critical roles in this endeavor, enabling researchers to map the expression patterns and post-translational modifications that govern enzyme activity in different plant tissues and developmental stages [101].

The study of plant cutinases occupies a unique niche at the intersection of plant biology, enzymology, and industrial biotechnology. On one hand, these enzymes provide a window into the fundamental mechanisms governing cuticle formation, maintenance, and degradation in plants. On the other hand, the catalytic properties of cutinases offer exciting opportunities for environmentally sustainable industrial processes such as bioscouring, plastic degradation, and the development of advanced biomaterials [95,97]. However, the dual nature of cutinase activity in plant systems—serving both developmental functions and potential roles in stress response—poses inherent challenges in their characterization. In many instances, the physiological activities attributed to plant cutinases have been inferred indirectly from phenotypic alterations observed upon ectopic expression or gene disruption, rather than through direct biochemical characterization [96]. This calls for more rigorous in vitro and in vivo studies that combine biochemical assays, structural analyses, and genetic manipulations to confirm the specific substrates, regulatory mechanisms, and physiological roles of these enzymes.

In summary, plant cutinases constitute a fascinating and underexplored class of enzymes with pivotal roles in modulating the structure and function of the plant cuticle. These enzymes not only facilitate critical developmental processes such as pollen adhesion and organ separation but also contribute to the plant’s response to environmental stress and pathogen attack. Structurally, plant cutinases share key features with their microbial counterparts, including the α/β hydrolase fold and canonical catalytic triad, yet subtle differences in their domain architecture and substrate specificity reflect specialized adaptations to endogenous substrates. Moreover, the dual functionality of these enzymes—as both modulators of cuticle integrity and potential participants in inter-organismal signaling—places them at the forefront of research aimed at elucidating plant surface biology.

4.4. Cutinase Applications

4.4.1. Degradation of Toxicants and Industrial Applications

Cutinases are used to biodegrade toxic chemicals and pollutants with ester structures [102]. Examples include phthalate esters (phthalates), such as dipentyl phthalate, butyl benzyl phthalate, and di-(2-ethylhexyl)phthalate. Although other esterases can also degrade phthalate esters, they are less efficient than cutinases and may form more toxic by-products [102,103]. Furthermore, cutinase derived from the thermophilic fungus Humicola insolens can degrade 81% of zearalenone and 51% of aflatoxin; however, the end products of these degradations were not specified in the related studies [104]. The incomplete hydrolysis of these pollutants suggests the need for further studies to identify more effective cutinases, either derived from microorganisms or plant resources, and develop recombinant cutinases with enhanced characteristics. However, in some systems, the initial hydrolysis of toxicants by cutinases is followed by microbial assimilation of the resulting degradation products, leading to complete mineralization of pollutants into CO₂, water, and other benign compounds [105]. In addition, the formation of enzyme consortia—where cutinases are coupled with oxidative enzymes such as laccases, lignin peroxidases, and manganese peroxidases—has been shown to accelerate the breakdown of complex pesticide molecules and synthetic polymer matrices [106,107]. This integrated approach minimizes the risk of accumulating harmful intermediate products and maximizes the overall efficiency of pollutant degradation. The adaptation of such multifaceted remediation systems is essential for treating environmental matrices that contain a mixture of contaminants with varying chemical properties [107,108].

Cutinase was first used in the textile industry to improve the wettability of cotton fibers. The combination of cutinase and pectinase in cotton fiber treatment can reduce costs and wastewater production, replacing the previously used alkali washing method [109,110,111,112]. Additionally, cutinases can be used in the processing of synthetic fibers and leather, replacing traditional physical and chemical methods, simplifying processes, reducing environmental pollution, and lowering energy consumption and costs [109].

One of the most well-documented applications of cutinases is in the textile industry. In bioscouring processes, these enzymes are applied to fabric surfaces to hydrolyze residual cutin and waxes, thereby enhancing hydrophilicity and improving dye uptake and finish quality. The enzymatic treatment is especially valuable because it is performed under mild conditions, avoiding the harsh chemicals and high energy inputs associated with traditional chemical scouring methods [94,108].

In detergent formulations, cutinases are used to enhance the removal of oily and fatty stains by catalyzing the hydrolysis of fats and oils on fabric surfaces. Their activity under both neutral and alkaline conditions, combined with tolerance to surfactants and organic solvents, makes cutinases well suited for use in laundry and dishwashing products [88,108]. Additionally, in the biodiesel industry, cutinases play a critical role in the transesterification of fats and oils, converting them into fatty acid methyl esters under relatively mild reaction conditions compared to conventional chemical catalysts [93].

In food processing, cutinases contribute to the modification of milk fat, thereby enhancing flavor development in dairy products and enabling the synthesis of flavor esters [94,108]. Their ability to catalyze both hydrolysis and synthetic reactions under controlled conditions makes them attractive for the production of oleochemicals, where they can facilitate the conversion of natural fats and oils into value-added chemical intermediates [83,94].

Moreover, cutinases can improve pesticide efficiency, thereby increasing crop yields and reducing pesticide consumption [112]. In sugar mills, chitosan-immobilized cutinases have demonstrated high efficiency in decolorizing molasses wastewater, with decolorization rates as high as 80% [112]. Cutinases can be used as detergents for laundry and dishwashing, in processing plant cuticles to extract natural plant ingredients, and in the hydrolysis of adhesives in the papermaking process [113].

The industrial utility of cutinases has driven considerable interest in protein engineering to enhance their catalytic performance, stability, and substrate specificity. Active Site Modifications Engineering efforts have focused on modifying the amino acid residues that line the active site to expand the substrate-binding pocket and improve access for bulkier substrates. Site-directed mutagenesis and directed evolution techniques have successfully generated enzyme variants with up to five- to nine-fold increases in activity compared to their wild-type counterparts [99]. These improvements have proven especially effective in applications such as PET degradation, where enhanced substrate affinity translates directly into increased hydrolysis rates [108]. Another promising strategy involves the generation of fusion proteins that incorporate noncatalytic binding domains—such as carbohydrate-binding domains (CBDs)—to improve enzyme adherence to substrates like cellulose in textile processing [108]. Such chimeric enzymes demonstrate superior binding and catalytic properties, overcoming limitations of native cutinases in specific industrial contexts. To address issues related to enzyme stability and reusability, immobilization techniques have been developed that attach cutinases to solid supports or encapsulate them within protective matrices. Immobilized enzymes not only exhibit enhanced operational stability over extended periods but also facilitate continuous processing in industrial setups, reducing costs and improving process sustainability [83,108].

4.4.2. Polyester Degradation

An estimated 79% of consumed polyester products end up in landfills or persist in the environment, posing serious environmental problems [113,114,115]. Thus, the biodegradation of polyesters and plastics has emerged as a popular area of recent research on cutinases. Several cutinases can degrade synthetic polyesters such as PET, polycaprolactone (PCL), and polylactic acid (PLA) [116,117]. Although PCL is biodegradable, its natural degradation rate is slow owing to its high hydrophobicity and crystallinity. Therefore, cutinases can be used to break down the hydrophobic barriers and promote PCL degradation [118,119].

Extensive research has focused on the degradation of PET, a widely used polyester that is difficult to degrade under natural conditions. Although PET waste can be thermomechanically recycled, thermally recycled PET has inferior mechanical properties to newly synthesized PET [88]. PET waste recycling based on PETase represents another promising strategy in which PET waste is hydrolyzed into monomers for PET polymerization [116]. Notably, both fungal and bacterial cutinases exhibit PETase activities. As elevated temperatures (approximately 65 °C) can promote PET hydrolysis, thermophilic cutinases are more effective than mesophilic cutinases for practical applications [120,121,122].

Cutinases can also degrade PLA, an artificial polyester made from renewable lactic acid [123]. Although PLA is biodegradable, its rate of degradation under mild conditions is slow [124,125]. A recent study characterized a bacterial cutinase, derived from the composting strain Thermobifida alba, that can efficiently degrade PLA [126]. Similarly, another bacterial cutinase, derived from Amycolatopsis mediterranei, biodegrades polyhydroxybutyl succinate (PBS), which is an artificial and biodegradable polyester that is more stable than PLA, under mild conditions, as well as PCL. However, it does not degrade PLA or PET [127,128].

Cutinases can act as biocatalysts in esterification and transesterification reactions, which are the reverse of biodegradation reactions, to produce polyamides and polyesters [109]. For example, cutinases from F. solani pisi and Burkholderia cepacia have been used in esterification reactions. However, the catalytic efficiency of cutinases is diminished by low pH, particularly when substrate concentrations are high. The operational pH range of an enzyme, along with temperature, is a critical factor in determining its appropriateness for industrial applications; even minor deviations from the optimal pH can result in reduced enzymatic efficiency, highlighting the importance of meticulous control over reaction conditions in industrial processes. The ionization state of catalytic triad residues is highly pH-dependent and, as a consequence, the enzyme’s ability to bind substrates and catalyze hydrolytic reactions is governed by the surrounding pH. In many instances, the optimal pH is strongly correlated with the enzyme’s isoelectric point (pI) and the local microenvironment within the active site [129]. Thus, cutinase stability in acidic environments is an important factor to consider when selecting cutinases as esterification catalysts [130,131]. Notably, enzyme engineering has been used to enhance the efficiency of cutinases in various applications.

The catalytic mechanism of cutinases relies on a conserved serine-histidine-aspartate triad, which facilitates nucleophilic attack on the carbonyl carbon of ester bonds. Unlike traditional lipases, which generally require an interfacial activation step to expose their active site, cutinases inherently possess an open conformation that is accessible to substrates regardless of their solubility state [109,132]. Structural studies have shown that the substrate-binding pocket is lined with hydrophobic and polar residues that engage substrates via a network of hydrogen bonds and van der Waals interactions, thereby ensuring high catalytic efficacy on both hydrophobic polymers and low-molecular-weight esters [98]. The kinetic behavior of cutinases is generally described by Michaelis–Menten parameters, although the actual catalytic efficiency is highly influenced by substrate chain length, conformation, and the surrounding reaction medium [94,96].

4.4.3. Medical Applications of Cutinases

From a structural perspective, cutinases typically possess a catalytic triad that facilitates ester bond hydrolysis, yet emerging studies have identified additional domains such as noncatalytic polymer-binding regions that broaden their substrate specificity [94]. The presence of such specialized domains may enhance cutinases’ affinity toward synthetic polymers, thus positioning them as valuable biocatalysts in the controlled synthesis of biomaterials [133]. Furthermore, research suggests that while the catalytic action of these enzymes is well documented in environmental and industrial processes, their translational potential in medical applications remains a frontier worthy of exploration [134]. The integration of detailed structure–function studies with innovative engineering approaches is expected to unlock further insights into cutinase behavior that are critical for clinical adaptation [94].

The use of cutinases in vaccine formulations is predicated on their ability to serve as subunit antigens that can stimulate a targeted immune response [94]. In contrast to whole-cell vaccines, subunit vaccines typically utilize purified components that are directly responsible for eliciting immunity, and the incorporation of enzymatic proteins such as cutinases can potentially produce robust immunogenic profiles with reduced adverse effects [94]. The inherent immunostimulatory properties of cutinases may be linked to their molecular configuration, which is capable of activating specific antigen-presenting pathways and thereby priming adaptive immunity [94]. The reported ability of cutinases to function as vaccine antigens also suggests that these enzymes could be integrated into modular vaccine platforms, wherein they are engineered alongside other immunologically pertinent molecules to achieve desired therapeutic outcomes [94].

The immunogenic potential of cutinases can be correlated with the presence of epitopes recognized by immune cells, leading to both humoral and cell-mediated responses [94]. Detailed biochemical characterizations have demonstrated that the recognition patterns of these enzymes share similarities with other well-known protein antigens, suggesting that their application could complement existing vaccine strategies in combating infectious agents [94]. Moreover, the modular nature of cutinase molecules allows for potential genetic and chemical modifications to optimize their immunogenicity, stabilize the protein structure, or enhance adjuvant functionalities [94]. Such modifications could be critical in tailoring vaccine formulations for specific populations or against particular pathogens, thus broadening the scope of preventive medicine [94]. However, additional studies are required to elucidate the precise immunological mechanisms elicited by cutinases and to validate their safety profiles in clinical settings.

Polymers synthesized using cutinase-catalyzed reactions exhibit properties that are directly relevant to the biomedical field, including biocompatibility, tunable degradation rates, and the capacity to incorporate bioactive molecules [133]. For instance, in drug delivery applications, the moderate molecular weight and controlled polymer architecture achieved through cutinase-mediated synthesis can enhance the encapsulation efficiency and release kinetics of therapeutic agents [133]. Similarly, such polymers can serve as matrices in tissue engineering, supporting cell adhesion and proliferation while gradually degrading to allow tissue regeneration [133]. These applications highlight the dual functionality of cutinases—not only as biological agents but also as facilitators of advanced material synthesis that meets the stringent requirements of modern biomedical devices [133]. As research progresses, integrated approaches combining enzymatic polymer synthesis with other biofabrication techniques could further expand the utility of cutinases in medical biotechnology [133].

One area where the potential of cutinases could be particularly transformative is in infection control, especially concerning biofilm-associated infections and surface decontamination [134]. Although the current context does not provide specific data on cutinase-mediated biofilm removal, the capacity of microbial enzymes to disrupt biofilms is well documented [134]. Given that biofilm formation represents a major challenge in both clinical and diagnostic settings, the application of cutinases as part of a multi-pronged approach to infection control could be explored [134]. Integrating cutinases into cleaning formulations or coating materials may enhance the removal of pathogenic biofilms from medical devices or wound surfaces, thereby reducing infection risks and improving patient outcomes [134]. Future experimental studies should be designed to quantitatively assess the biofilm-disruptive properties of cutinases, potentially leading to innovative strategies for managing chronic infections [134].

Despite the promising applications outlined above, several challenges must be addressed to fully exploit the medical potential of cutinases. One major hurdle is the need for a comprehensive understanding of the enzyme’s structural stability and activity under physiological conditions [94]. Variability in enzyme performance due to differences in pH, temperature, or the presence of other biomolecules may limit the reliability of cutinases in clinical applications, necessitating further protein engineering and stabilization approaches [94,133]. Furthermore, the scalability of cutinase production remains an issue; efficient recombinant expression systems and purification processes must be developed to meet the demands of medical manufacturing [94]. Addressing these challenges will require multidisciplinary collaborations that combine enzyme engineering, process optimization, and clinical research, thereby ensuring that the medical applications of cutinases can be realized with consistent efficacy and safety [94,133,134].

Looking ahead, several key research directions can be identified to accelerate the clinical translation of cutinase-based technologies. First, detailed structural and kinetic studies of cutinases under physiologically relevant conditions will be necessary to optimize their function in therapeutic and diagnostic applications [94]. High-resolution crystallographic analyses and advanced molecular dynamics simulations could provide insights into the dynamic behavior of the enzyme’s active and binding sites, informing the design of more robust variants [94]. Second, exploration of cutinase’s role as a subunit vaccine antigen should extend to comprehensive in vivo studies, including immunogenicity assessments in relevant animal models and eventual Phase I clinical trials [94]. Such studies would evaluate the feasibility of cutinase-driven immune responses and help refine antigen presentation strategies. Third, in the realm of polymer synthesis, future work should focus on optimizing reaction conditions, exploring substrate scope, and integrating cutinase catalysis with other biofabrication techniques to create multifunctional biomedical materials [94]. Lastly, inter-disciplinary studies comparing cutinases with other microbial enzymes may uncover synergistic strategies that combine the unique benefits of each enzyme class for applications in infection control, diagnostics, and wound healing [134].

The exploration of cutinases for medical applications represents a convergence of biotechnology, enzyme engineering, and clinical sciences. As illustrated in this report, the application of cutinases as subunit vaccine antigens is one of the most promising areas, particularly given the current drive for innovative immunization strategies against emerging and re-emerging pathogens [94]. The ability of these enzymes to promote the synthesis of biomedical polymers adds another dimension to their utility, presenting opportunities for the development of sophisticated drug delivery systems and tissue engineering scaffolds [133]. Moreover, drawing parallels from the broader field of microbial enzymes, the potential for cutinases in diagnostic cleaning applications and infection control further emphasizes their versatility [134].

In conclusion, the integration of cutinases into medical applications represents a compelling area of research that could yield transformative benefits for vaccine development, biomedical material synthesis, and diagnostic device maintenance. The evidence provided in the reviewed studies lays a solid foundation upon which future research can build, ensuring that the unique properties of cutinases are fully exploited to meet the evolving demands of modern healthcare [94,133,134].

This comprehensive discussion underscores that while significant progress has been made in elucidating the medical applications of cutinases, additional work is required to fully harness their multifunctional capabilities in clinical settings. Through ongoing innovation and collaboration, the future use of cutinases in vaccine development, polymer synthesis, and diagnostic technologies holds the promise of ushering in a new era of precision medicine and improved healthcare outcomes [94,133,134].

In conclusion, cutinases represent a versatile, robust, and environmentally friendly class of enzymes that have evolved from roles in plant-pathogen interactions to become key components in advanced biotechnological processes. Their unique structural features—a readily accessible active site and a conserved catalytic triad—enable them to process diverse substrates ranging from natural cutin to synthetic polyesters [84,96]. With continued innovations in enzyme engineering and process development, cutinases are well positioned to drive the next wave of sustainable industrial technologies, offering cleaner, more efficient alternatives to conventional chemical processes [135,136].

Ultimately, the ongoing research and development efforts in the field of cutinases will not only enhance our understanding of their complex structure–function relationships but also unlock their full potential as transformative tools in both established and emerging industrial sectors. The convergence of interdisciplinary approaches—from molecular biology and computational modeling to process engineering and materials science—promises to propel cutinases to the forefront of sustainable biocatalysis, thereby contributing significantly to a more resilient and ecologically sound future [100,135,137].

Looking ahead, the future of cutinase research is likely to be shaped by innovations in synthetic biology, directed evolution, and computational protein design. Directed evolution techniques, which involve iterative rounds of mutagenesis and selection, have already yielded variants with markedly improved thermal stability and catalytic efficiency [99]. In parallel, computational modeling and molecular dynamics simulations are expected to further refine our understanding of enzyme–substrate interactions, facilitating the design of custom-tailored cutinases for specific industrial needs [135]. Additionally, advances in immobilization strategies—particularly those involving nanomaterials and smart polymer matrices—are poised to enhance enzyme reusability and operational performance in continuous processing systems [95,137]. As research in these areas progresses, new applications in fields as diverse as biosensors, biopolymer synthesis, and therapeutic enzyme delivery are likely to emerge, further expanding the role of cutinases in modern biotechnology [84,109].

5. Phospholipases

Phospholipases are a group of hydrolase enzymes that catalyze the hydrolysis of phospholipids, which are membrane components found in all living organisms, through either acylhydrolase or phosphodiesterase activity. Phospholipases are involved in lipid metabolism, membrane remodeling, cell signaling, transport, migration, and cell growth and death. Phospholipases can be extracellular, intracellular, or membrane-bound, and similar to lipases, they function at the lipid–water interface [138,139]. Although phospholipases are ubiquitous in living organisms, most industrially valuable enzymes are derived from microorganisms, as they are stable, free from seasonal variations, have broad catalytic activities, and can be obtained inexpensively in large quantities [140].

Phospholipases are broadly classified into three major families—phospholipases A (PLA), phospholipases C (PLC), and phospholipases D (PLD)—based on the site of cleavage on the phospholipid molecule [126]. The PLA group is subdivided further into phospholipase A1 (PLA1) and phospholipase A2 (PLA2). PLA1 enzymes predominantly hydrolyze the ester bond at the sn-1 position of phospholipids, generating 2-acyl lysophospholipids and free fatty acids, while PLA2 enzymes exhibit specificity toward the sn-2 position, releasing polyunsaturated fatty acids such as arachidonic acid that serve as precursors for bioactive lipid mediators [141,142]. Within the PLA2 family, distinct isoforms have been identified, including secreted phospholipase A2 (sPLA2), cytosolic phospholipase A2 (cPLA2), and Ca2+-independent phospholipase A2 (iPLA2), each characterized by unique regulatory mechanisms and tissue distributions. Secretory PLA2 isoforms, for example, are implicated in immune responses and inflammation, while cPLA2 is central to the generation of eicosanoids during cellular activation, and iPLA2 is involved in membrane remodeling and cell death regulation [143,144]. Complementing the PLA family, phospholipase C (PLC) enzymes cleave the phosphatidylinositol 4,5-bisphosphate (PIP2) substrate at a specific phosphodiester bond to produce the second messengers diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), which are essential for the activation of protein kinase C (PKC) and the mobilization of intracellular Ca2+ stores [145]. Meanwhile, phospholipase D (PLD) enzymes hydrolyze phosphatidylcholine (PC) to yield phosphatidic acid (PA) and choline; this reaction not only contributes to membrane lipid turnover but also generates PA, a potent signaling molecule that modulates pathways such as mTOR and MAPK [141,146]. Additional enzymes within this superfamily include lysosomal PLA2s and secreted phospholipases with atypical roles, which impact lipid catabolism and immune regulation [147].

Phospholipases are central to a broad spectrum of physiological processes due to their ability to generate lipid-derived signaling molecules that modulate cellular responses. In membrane remodeling, for instance, the action of PLA2 enzymes ensures proper phospholipid turnover and the maintenance of membrane fluidity, which are fundamental for processes such as vesicular trafficking, cell division, and apoptosis [141,144].

Dysregulation of phospholipase activity has been implicated in an array of disease states, where aberrant lipid mediator production and altered membrane dynamics contribute to pathological processes. In the context of inflammation, overactivation of PLA2 enzymes can lead to excessive production of pro-inflammatory eicosanoids, fueling chronic inflammatory conditions such as rheumatoid arthritis, asthma, and inflammatory bowel disease [142,144].

Given their pivotal roles in modulating lipid signaling and membrane dynamics, phospholipases have become prime targets for drug discovery and therapeutic intervention. The development of small molecule inhibitors against specific phospholipase isoforms represents a promising avenue for the treatment of various diseases characterized by aberrant enzyme activity. In the context of cancer therapy, for example, inhibitors targeting sPLA2 or PLD have been shown to reduce tumor growth and metastasis by interfering with the production of bioactive lipid mediators that sustain proliferative and pro-survival pathways [146]. Preclinical models utilizing gene knockout or knockdown approaches have further established the causative roles of phospholipases in tumorigenesis, thereby reinforcing their potential as therapeutic targets [144].

Another rapidly developing area is the application of lipidomics and advanced mass spectrometry techniques to profile the dynamic changes in lipid mediators generated by phospholipase activity under various physiological and pathological conditions. Such approaches enable the characterization of substrate specificities, temporal production profiles, and the downstream signaling effects of lipid mediators, thereby providing a systems-level understanding of phospholipase function that can inform the design of targeted therapies [144]. Moreover, the integration of genomic and proteomic data is beginning to reveal how individual genetic variations in phospholipase genes may predispose individuals to certain diseases, paving the way for personalized therapeutic interventions based on phospholipase activity profiles [144].

Phospholipases produced by yeasts and fungi are the most widely used, particularly those derived from the genera Candida, Yarrowia, Aspergillus, Penicillium, Rhizopus, Rhizomucor, and Thermomyces [148]. For example, class A phospholipases from Fusarium oxysporum, Thermomyces lanuginosus, Aspergillus niger, and Trichoderma reesei have been commercialized and used for degumming vegetable oils. Classes A and B phospholipases derived from Aspergillus oryzae are used in the food industry owing to their high transphosphatidylation and hydrolytic activity. In contrast, bacterial phospholipases are less frequently utilized [149]. Among them, those derived from the genera Pseudomonas, Bacillus, and Streptomyces are most commonly used, followed by those derived from Burkholderia, Chromobacterium, Achromobacter, Alcaligenes, and Arthrobacter. These phospholipases are used for oil degumming, biodiesel production, and detergents, but not in the food industry [150].

Looking forward, future research will likely focus on elucidating the isoform-specific roles of phospholipases within highly complex cellular environments, as well as on the interplay between different phospholipase families and other lipid-modifying enzymes. Such studies are expected to leverage CRISPR/Cas9 gene-editing technologies, next-generation sequencing, and advanced imaging methods to provide a more nuanced picture of how phospholipases contribute to cellular signaling and disease phenotypes [144]. Furthermore, the exploration of novel drug delivery systems—such as nanoparticle-based approaches—to modulate phospholipase activity in a tissue-specific manner holds promise for enhancing the therapeutic index of phospholipase inhibitors while mitigating systemic side effects [146,150].

An integrative perspective on phospholipase function emphasizes the interconnectedness of lipid metabolism, membrane dynamics, and signal transduction in mediating both physiological and pathological processes. The coordination of enzymatic activities across the PLA, PLC, and PLD families exemplifies how specific lipid-derived signals are generated in response to distinct extracellular cues and how these signals, in turn, regulate fundamental cellular decisions such as proliferation, differentiation, and apoptosis [141,143]. The cross-talk between phospholipase-generated mediators and other signaling pathways further illustrates the complexity of cellular regulation, whereby a single enzymatic event can trigger a cascade of molecular interactions with profound biological consequences [141,143].

Recent advances in molecular genetics, lipidomics, and high-throughput screening have significantly enhanced our understanding of the structure, function, and regulation of phospholipases, laying the foundation for novel therapeutic strategies targeting these enzymes in cancer, inflammatory disorders, cardiovascular diseases, and metabolic syndromes [146]. The development of isoform-specific inhibitors and the integration of phospholipase activity with clinical biomarkers represent promising approaches for translating this knowledge into effective treatments and personalized medicine frameworks. Furthermore, ongoing research continues to unveil the complex interplay between phospholipases and other signaling pathways, underscoring the need for a holistic exploration of lipid mediator biology in both health and disease [142,144].

The analytical determination of phospholipases in biological and clinical specimens is challenging due to the non-chromogenic and non-fluorescent nature of most native phospholipid substrates, which are required for membrane organization and enzyme isoform specificity. This necessitates the use of engineered probes, optimized substrate presentation and advanced detection modalities [151,152,153].

Sensor systems and analytical assays for phospholipases can be categorized in several ways: direct detection of hydrolysis products, monitoring of labelled substrate cleavage, measurement of physical destruction of organized lipid membranes and application of secondary coupled enzyme reactions or label-free methods [151,153,154].

Analytical determination of phospholipase activities has evolved into a multidimensional field employing diverse sensor technologies and assay platforms. The current landscape encompasses a continuum from classical chromogenic and radiometric product analysis through to luxury high-throughput fluorogenic, mass spectrometric, and label-free electrochemical approaches. Further, innovations in nanotechnology, microfluidics, and single-particle methods continue to enable new performance thresholds in sensitivity, throughput, and multiplexing, thus fueling advances not only in biochemical and cell biology research but also in translational medicine and biotechnology [151,152,153,155,156,157].

Ultimately, selectivity, sensitivity, compatibility with native substrate contexts, translational adaptability, and throughput requirements dictate assay selection for any given application, and continuous development in this area will further extend analytic and diagnostic power for phospholipase-related research and clinical monitoring.

In conclusion, phospholipases are not merely enzymes involved in lipid metabolism; they are central regulators of cellular signaling, membrane integrity, and inflammatory responses that have profound impacts on human health. Their complex classification, nuanced regulatory mechanisms, and broad involvement in pathophysiological states underscore the importance of these enzymes as both critical biomarkers and promising therapeutic targets. As our grasp of phospholipase function deepens, it will be essential to integrate these insights into clinical practice, thereby harnessing their potential to inform the development of novel diagnostic tools and targeted treatment strategies for a variety of diseases [143,144,146]. The concerted efforts of basic scientists, clinicians, and pharmaceutical researchers will undoubtedly continue to unravel the intricate biology of phospholipases, ultimately leading to innovative approaches that improve patient care and advance our understanding of cellular physiology in health and disease [142,143].

6. PETases

Since the discovery of the bacterium Ideonella sacaensis in 2016, interest in PETases has steadily increased. Some researchers have classified PETase as its own separate subclass. Prior to this, only certain fungi were believed to have PET-hydrolyzing capacity [158].

Annual PET production exceeds 350 million tons [159,160]. PET, a synthetic polymer derived from petroleum, is one of the most widely used plastics. This polymer is composed of repeating terephthalic acid (TPA) and ethylene glycol (EG) units. Owing to its physicochemical properties, it has proven useful for both manufacturing and household applications. The strength, transparency, and chemical resistance of PET have made it indispensable for containers, bottling, packaging, films, and fibers. However, these qualities render it environmentally hazardous when it becomes waste. Its crystallinity, surface hydrophobicity, and the high stability of its backbone are key factors that limit its natural degradation.

Recently, extensive research has focused on PET-degrading microorganisms, which include bacteria, fungi, insects, and even microalgae [133,161]. The active centers of PETases contain a conserved triad. These enzymes belong to the esterase subclass and possess a catalytic triad characteristic of α/β- α/β-hydrolases (Ser-His-Asp). Hydrolysis of the ester bond occurs via nucleophilic attack by the serine oxygen on the carbonyl carbon present in the ester bond. Negatively charged aspartate stabilizes the positively charged histidine residue, and the resulting charge-transfer network enables serine to perform the nucleophilic attack [162]. Several studies have been conducted to improve our understanding of the mechanisms of action of these enzymes, particularly PETase and MHETase [163]. PETase hydrolyzes PET into mono-2-hydroxyethyl terephthalate (MHET) oligomers, whereas MHETase hydrolyzes MHET to TPA and EG (Figure 3). Consequently, the recombination and overexpression of these enzymes may be essential techniques for enhancing PET degradation, as well as for recycling and converting its monomers (TPA and EG) into high-value chemicals [164].

In the enzymatic degradation of polyesters such as PET, both the characteristics of the plastic and the structure of the enzyme are crucial factors. Specifically, the external surface areas of the enzyme active site and the binding modules are crucial during interactions with the polymer and hydrolysis [165]. Mutations are frequently introduced to enlarge the active sites, facilitating the binding of large, inaccessible polymer pieces and establishing a more hydrophobic substrate-binding site. Consequently, nearly all modifications target the active domains of enzymes or their surface regions. However, various obstacles are encountered when developing mutants with enhanced properties. A major challenge in PET degradation by PETase is the intracellular localization of the enzyme, which restricts its direct interaction with solid PET. Therefore, selecting an appropriate approach for assessing the hydrolysis rate of changed strains is challenging. The newly developed cell-free protein expression method has emerged as a solution to this issue [166]. This method is beneficial for producing functional and structural proteomics of proteins that are difficult to express in vivo in bacterial cells, offering several benefits over traditional cell-based expression approaches. For example, this approach enables easy modification of reaction conditions, enabling shorter expression duration and reduced reaction volumes. Moreover, its efficiency surpasses hundreds of micrograms of protein per milliliter of reaction volume. The utilization of a cell-free protein expression method in PET enables direct interaction between the produced protein and solid PET, thus allowing high-throughput screening of PET hydrolytic enzymes [167,168,169,170,171,172,173,174,175].

Table 2. Comparative Kinetic Parameters of Wild-Type and Engineered PETases for PET Hydrolysis.

Microorganism	Temperature (°C)	pH	kcat (s−1)	KM (mM)	Kcat/KM (M−1s−1)	References
Wild-type IsPETase	30	7.5	~0.1	1.5–4.0	20–80	[161,176]
S238F/ W159H	30	7.5	0.2–0.5	0.4–1.0	500–1200	[176]
Thermo PETase	40	8.0	0.8–1.2	0.3–0.8	1500–2500	[177]
Dura PETase	40	8.0	1.0–1.5	0.3–0.5	2000–3500	[178]
FAST- PETase	50	8.0	2.0–3.0	0.3–0.5	6000–7000	[178]
Hot PETase	60	8.5	2.5–4.0	0.3–0.6	6000–8000	[179]

shows the steady-state kinetic constants that have been published for wild-type IsPETase and its modified versions in typical test conditions. In the last ten years, rational design and controlled evolution have made catalytic turnover (kcat) and substrate affinity (lower KM) much better. This has led to multiple orders-of-magnitude gains in catalytic efficiency (kcat/KM). Under mesophilic conditions, the wild-type enzyme works only a little bit. However, mutations that change the active-site structure (S238F/W159H) or make the enzyme more stable at high temperatures (ThermoPETase, DuraPETase, FAST-PETase, or HotPETase) make the enzyme work much better at higher temperatures. These improvements go hand in hand with more flexible substrates at higher temperatures and better active-site dynamics. The data show a clear evolutionary path: from natural hydrolases with poor efficiency to synthetic biocatalysts with catalytic efficiencies that are similar to those of established industrial enzymes. These kinds of comparisons not only set standards for enzyme engineering, but they also give us useful information about whether PET hydrolysis is possible on a large enough scale for biotechnological recycling.

PETase represents a breakthrough in the enzymatic degradation of PET, offering a promising pathway toward sustainable plastic recycling. Its distinct structural features, including an extended active site cleft and a conserved catalytic triad, facilitate the effective hydrolysis of PET into intermediate products that can be further metabolized by auxiliary enzymes such as MHETase [180]. Extensive protein engineering efforts—ranging from site-directed mutagenesis and directed evolution to domain fusion and chemical modification—have yielded variants with substantially enhanced catalytic efficiency and thermostability [181,182,183]. Despite significant progress, challenges related to substrate crystallinity, enzyme stability, and process scalability remain, necessitating continued integration of advanced computational tools and innovative experimental strategies. PETase has excellent catalytic efficiency for low-crystallinity PET, characterized by less than 20% crystallinity, whereas its activity markedly decreases with high-crystallinity PET, such as bottle-grade PET. The principal problem associated with wild-type PETase is its restricted thermostability, exhibiting diminished efficiency at temperatures beyond around 40 °C. Consequently, engineering initiatives have concentrated on improving PETase’s stability and efficacy at elevated temperatures and on more crystalline substrates. The ability of PETase to breakdown PET has considerable significance for environmental applications, especially in mitigating plastic waste via bioremediation procedures. Optimal conditions for PETase activity include a slightly alkaline pH (approximately 7.5–8) and moderate temperatures (30–40 °C). Research is concentrating on developing more stable and thermotolerant variants of PETase, which have shown promise in producing biodegradable plastics, such as polyhydroxyalkanoates (PHAs), from PET waste. However, significant technological and economic barriers hinder broader commercial use, especially the high production costs associated with enzyme synthesis due to complex fermentation processes. The direct biodegradation of entire PET bottles remains impracticable, necessitating pre-treatment methods like shredding or grinding to generate smaller, enzymatically accessible fragments [184]. In tandem with engineering efforts, optimizing process parameters such as pH, ionic strength, and the presence of stabilizing can further enhance enzyme activity and stability in industrial reactors.

Emerging trends, such as the exploration of novel microbial sources and the development of enzyme cascades, are poised to further advance the field by delivering highly efficient, robust, and industrially viable PET-degrading systems [185,186]. As research in this domain progresses, PETase enzymes are expected to play a central role in the formulation of environmentally responsible strategies for mitigating plastic pollution and fostering a sustainable circular economy [186,187]. Ultimately, the convergence of molecular engineering, process optimization, and interdisciplinary collaboration holds the promise of transforming PET waste management from a largely reactive practice into a proactive, bio-based recycling paradigm [188,189].

7. Conclusions

Analysis of the literature indicates a global trend of replacing chemical production techniques with biotechnological methods. Biotechnological production using enzymes offers several advantages, including milder conditions, elimination of toxic chemicals that harm the environment, higher yields of the desired products, minimal side products, and the ability to combine various processes in one reaction medium. Notably, esterases are integral to various sectors, including the synthesis of polyester fibers, paints, plastics, biodiesel production, food products, nutraceuticals, oil degumming, and detergents. They are also applied in bioremediation, agriculture, cosmetics, leather, and paper manufacturing. Overall, the utilization of renewable raw resources alongside esterases adheres to the tenets of a green economy, promoting sustainability by reducing reliance on fossil resources and minimizing environmental impact.

Several major trends are likely to shape the future of esterase-based biotechnologies. First, advancements in protein engineering and directed evolution will enhance enzyme stability, substrate selectivity, and catalytic efficiency in settings pertinent to industrial applications. These kinds of advances are very important for big projects, such recycling polymers and breaking down synthetic materials that last a long time. Second, combining enzymatic catalysis with new technology platforms like continuous flow bioreactors, immobilized enzyme systems, and AI-assisted enzyme design will make manufacturing processes more efficient, cheaper, and easier to scale up.

These advancements are expected to have a big effect on people’s lives as a whole. Using esterases to make biodegradable polymers and recycle plastic waste could greatly reduce the environmental impact of synthetic polymers, leading to healthier ecosystems and better public health. In the energy sector, the enzymatic production of biofuels and green chemicals is anticipated to diminish reliance on fossil resources and facilitate worldwide decarbonization initiatives. Esterases will continue to play a role in the creation of new medicines and functional foods in healthcare and nutrition.

Following the discovery of PETase from Ideonella sakaiensis, substantial research efforts have been directed towards engineering these enzymes to overcome their natural limitations, such as low thermal stability and moderate catalytic activity, in order to make them more applicable to industrial-scale processes [190,191].

The engineering of PETases encompasses a variety of strategies including rational protein design, directed evolution, and computational in silico analyses that guide mutation screening. These strategies aim to address issues inherent to natural enzymes, such as limited substrate binding, low turnover rates, and sensitivity to the crystalline structure of PET. In addition, fusion technologies and immobilization techniques have been explored to increase enzyme stability and facilitate recovery for repeated use in bioreactor systems [192,193].

In silico and Artificial Intelligence-Assisted Enzyme Design Computational methods have revolutionized the field of enzyme engineering by allowing researchers to predict the impact of mutations on protein structure and function prior to experimental validation. The use of molecular mechanics, dynamic docking, and density functional theory (DFT) calculations has provided atomic-level insights into enzyme-substrate interactions that facilitate the design of highly efficient PETases [194]. Recent advances include the application of neural networks and machine learning algorithms to rapidly screen for beneficial mutations. AI-assisted techniques have been instrumental in the development of variants such as FAST-PETase, which integrates multiple mutations that collectively enhance catalytic efficiency, substrate binding, and thermal tolerance [181,190].

Another promising strategy lies in the engineering of PETase fusion proteins and the immobilization of enzymes onto solid supports. Fusion with ancillary domains, such as carbohydrate-binding modules (CBMs) or zwitterionic polypeptides, can enhance the enzyme’s affinity for PET substrates while simultaneously improving stability under operational conditions [189,190]. For instance, the fusion of PETase with accessory binding domains derived from cellulases has improved enzyme-substrate interactions, boosting overall degradation efficiency by promoting close contact between the enzyme and PET surface [190,195].

Moreover, immobilizing PETases on solid supports has been shown to aid in enzyme recovery and reuse, which is a critical consideration for industrial applications. Techniques such as binding the enzyme to bacterial curli fibers or using fusion tags to anchor the enzyme on the surface of Escherichia coli facilitate robust PET degradation while enabling operability within continuous bioreactor systems [193,195]. These strategies not only enhance the catalytic performance of PETases but also contribute to a reduction in overall process costs by allowing for enzyme recycling [196].

While substantial progress has been made in improving the intrinsic properties of PETases, their practical application also depends on the efficient heterologous expression and secretion of these enzymes in suitable host organisms. Escherichia coli remains a favored host for recombinant protein production; however, issues such as inclusion body formation and misfolding have prompted exploration into alternative systems [196].

Bacillus subtilis and yeast species such as Pichia pastoris have been investigated as alternative hosts because they offer more efficient secretion pathways and post-translational modification systems that aid in proper enzyme folding and stability. Recent advances include the use of microalgal chloroplasts for enzyme synthesis, which not only simplifies the production process but also contributes to a more sustainable production platform [195,196]. Despite these promising developments, challenges remain in scaling up enzyme production while maintaining consistency in yield, solubility, and enzymatic activity, particularly under fermentor conditions that are reflective of industrial processes [196].

The economic scaling of these technologies is contingent upon the optimization of heterologous expression systems and the development of robust microbial cell factories. The translation of laboratory successes to industrial applications will be significantly influenced by advancements in synthetic biology and metabolic engineering, particularly the development of chassis organisms that are specially designed for the high-yield secretion of functional enzymes [196]. Furthermore, the potential for the development of entirely sustainable bioprocesses is present in the investigation of renewable platforms, including microalgae and plants, for enzyme production [195,196].

Although further efforts are necessary, the advancements made to date establish a robust basis for the development of next-generation biocatalysts that can revolutionize PET recycling. Ongoing interdisciplinary collaboration and technological advancement will guarantee that modified PETases are essential to sustainable plastic waste biodegradation solutions, facilitating more robust, environmentally friendly industrial processes and a healthier global ecosystem [188,190].

In short, the fast pace of enzyme engineering and the growing need for more environmentally friendly ways of doing business make esterases important biocatalysts in the shift to a bio-based economy. Their growing uses, which are being driven by new technologies and environmental needs, will probably change many areas of human activity in the next few decades, including medicine and materials research.