Enzymes as Catalysts in Industrial Biocatalysis: Advances in Engineering, Applications, and Sustainable Integration

Abstract

Enzymes are highly selective and efficient biological catalysts that play a critical role in modern industrial biocatalysis. Their ability to operate under mild conditions and reduce environmental impact makes them ideal alternatives to conventional chemical catalysts. This review provides a comprehensive overview of advances in enzyme-based catalysis, focusing on enzyme classification, engineering strategies, and industrial applications. The six major enzyme classes—hydrolases, oxidoreductases, transferases, lyases, isomerases, and ligases—are discussed in the context of their catalytic roles across sectors such as pharmaceuticals, food processing, textiles, biofuels, and environmental remediation. Recent developments in protein engineering, including directed evolution, rational design, and computational modeling, have significantly enhanced enzyme performance, stability, and substrate specificity. Emerging tools such as machine learning and synthetic biology are accelerating the discovery and optimization of novel enzymes. Progress in enzyme immobilization techniques and reactor design has further improved process scalability, reusability, and operational robustness. Enzyme sourcing has expanded from traditional microbial and plant origins to extremophiles, metagenomic libraries, and recombinant systems. These advances support the integration of enzymes into green chemistry and circular economy frameworks. Despite challenges such as enzyme deactivation and cost barriers, innovative solutions continue to emerge. Enzymes are increasingly enabling cleaner, safer, and more efficient production pathways across industries, supporting the global shift toward sustainable and circular manufacturing.

1. Introduction

The increasing demand for environmentally responsible manufacturing practices has catalyzed the widespread integration of biocatalysis into industrial operations, as illustrated in Figure 1. Biocatalysis, which involves the use of enzymes to facilitate chemical reactions, offers a cleaner, safer, and more efficient alternative to traditional chemical methods [1]. These biological catalysts excel in specificity and reaction speed, helping industries transition toward greener, more sustainable production systems. One of the defining features of enzymes is their ability to drive reactions with high selectivity [2]. This means that they can target specific substrates and convert them into desired products without forming unwanted by-products. Such precision helps minimize material waste, reduce purification steps, and lower overall energy consumption, making enzymatic processes attractive from both ecological and economic standpoints [3].

Industrial sectors have capitalized on these advantages by employing enzymes in diverse applications. In areas such as pharmaceuticals, food processing, textiles, and biofuel production, enzymes play a pivotal role in transforming raw materials under mild conditions. Many of these enzymes are naturally derived, while others are engineered to tolerate extreme operational environments, such as elevated temperatures or acidic and alkaline conditions [4]. Unlike synthetic chemical catalysts, enzymes function optimally in water-based systems and under ambient conditions [5]. This not only lowers operational hazards but also enables safer workspaces and reduces dependency on toxic solvents and high-energy inputs. The mild processing requirements contribute to the preservation of sensitive compounds and align with modern principles of sustainable manufacturing [6]. A key factor in the growing relevance of industrial enzymes is their structural diversity. Enzymes are categorized into families based on the reactions they catalyze, such as hydrolases, transferases, and oxidoreductases, each with specialized roles [7]. For instance, enzymes like proteases and cellulases are widely used in detergents and food applications, while oxidoreductases are critical in environmental and textile industries for oxidation-based processes [8].

Historically, the use of enzymes dates back to ancient practices like fermentation, long before their molecular roles were understood. The industrial-scale use of enzymes began with crude extracts from plants and animals, though production was limited by low efficiency and high cost [9]. A turning point came with the discovery that microorganisms could be cultivated to produce enzymes in bulk through fermentation. The advent of recombinant DNA technology in the late 20th century marked a revolutionary shift in enzyme production [10]. Scientists gained the ability to isolate genes coding for specific enzymes and express them in microbial hosts such as Escherichia coli and Saccharomyces cerevisiae. This advancement dramatically improved enzyme yield, purity, and adaptability, allowing for a broader range of applications across industries. Subsequent innovations in protein engineering, particularly techniques like directed evolution and rational design, have allowed researchers to fine-tune enzyme properties for specific tasks. Directed evolution mimics natural selection to identify beneficial mutations, while rational design uses structural data to modify enzymes for enhanced stability, substrate specificity, or catalytic performance. These approaches have expanded enzyme usability under challenging industrial conditions [11]. The sustainability of a process may be measured in a variety of ways. A life cycle assessment (LCA) considers the environmental effect of a complete process, from raw material manufacture to manufacturing, usage, and disposal. However, preparing an LCA can be exceedingly labor-intensive and requires specialized knowledge if fundamental data on the materials used are not available in conventional databases. As a result, an LCA is less commonly used in research groups to rapidly and efficiently examine the environmental effect of a process in development. Furthermore, typical LCA substance databases are still developed for the assessment of chemical processes, and many chemicals crucial to bioprocessing are not yet available [12].

Today, computational tools and artificial intelligence have further accelerated enzyme innovation. Machine learning algorithms and molecular modeling allow researchers to predict enzyme behavior, screen potential modifications, and simulate catalytic processes before actual experiments begin [13]. These technologies reduce development time and cost, enabling faster integration of customized enzymes into production systems. In addition to biological and computational advancements, the physical handling of enzymes has also evolved. Immobilization techniques, where enzymes are bound to solid supports or are encapsulated in materials, have significantly improved enzyme stability and reusability. This approach supports continuous processing in large-scale operations, lowers operational costs, and integrates well with modern reactor technologies [14]. Collectively, these advancements have cemented enzymes as indispensable components of sustainable, next-generation industrial practices.

2. Methodology

This review was conducted through a systematic and comprehensive search of peer-reviewed literature from 2003 to 2025 using databases including PubMed, Science Direct, Web of Science, and Google Scholar. The search employed Boolean combinations of the following keywords: “industrial enzymes,” “biocatalysis,” “enzyme immobilization,” “protein engineering,” “synthetic biology,” “directed evolution,” and “sustainable manufacturing.” Inclusion criteria focused on experimental studies, reviews, and case studies relevant to enzyme production, stabilization, and application in industrial settings. Preference was given to high-impact journals and research that emphasized technological innovations, enzyme optimization, or real-world industrial feasibility. Non-English articles and studies not directly related to industrial biocatalysis were excluded. The collected literature was categorized thematically into enzyme classification, source diversification, engineering strategies, industrial applications, and immobilization techniques to ensure a structured and balanced discussion of current advances and future prospects.

3. Enzyme Classes in Industrial Biocatalysis

In 1961, the IUB published the first edition of the Enzyme Classification (EC) and Nomenclature List. This nomenclature is based on assigning a four-digit code to enzymes with the following meaning: (i) the first number identifies the main enzyme class; (ii) the second digit indicates the subclass; (iii) the third number denotes the sub-subclass; and (iv) the fourth digit is the enzyme’s serial number in its sub-subclass. The following six enzyme types were found, classified according to the kind of reaction catalyzed: oxidoreductases (EC 1), transferases (EC 2), hydrolases (EC 3), lyases (EC 4), isomerases (EC 5), and ligases (EC 6). Despite multiple updates to the 1961 edition, the six designated classes have remained unchanged. The classification reflects the enzyme’s catalytic mechanism and helps guide its industrial application [15].

Oxidoreductases catalyze electron transfer reactions and play crucial roles in redox-based industrial processes [16]. Laccases are a prominent example, capable of oxidizing phenolic and non-phenolic substrates [17]. These multi-copper enzymes are employed in dye decolorization, lignin degradation, and waste detoxification. Their ability to act on a broad range of aromatic compounds makes them highly valuable in environmental biotechnology and green chemistry.

Transferases catalyze the transfer of functional groups between molecules. They are important in pharmaceutical and food industries for creating complex molecules. Transaminases, for instance, are used in the stereoselective synthesis of chiral amines [18]. Glycosyltransferases are applied in the synthesis of glycosidic compounds such as oligosaccharides and glycoproteins. These reactions are typically highly specific and proceed under mild conditions, making them well-suited for sensitive bioprocesses.

Hydrolases, which catalyze the cleavage of chemical bonds via water addition, are among the most commercially important enzymes [19]. Key subclasses include proteases, lipases, and cellulases. Proteases catalyze the breakdown of proteins into peptides and amino acids and are widely used in detergents, food processing, and leather softening [20]. In detergents, proteases degrade protein stains, enhancing cleaning efficiency even at low temperatures. In food, they are used for improving texture and flavor, such as in cheese ripening and hydrolyzed protein production [21]. Lipases catalyze the hydrolysis of ester bonds in triglycerides, converting fats into glycerol and free fatty acids. These enzymes are extremely versatile, functioning in both aqueous and non-aqueous environments [22]. In biodiesel production, lipases facilitate transesterification reactions, converting fats into fatty acid methyl esters. In the food industry, they are used for modifying fats and enhancing flavors in dairy products. Their stereoselective properties are also exploited in pharmaceutical synthesis [23]. Cellulases hydrolyze β-1,4-glycosidic bonds in cellulose, breaking it down into glucose or cellobiose units. They are essential in industries dealing with lignocellulosic biomass, such as biofuel production, textile finishing, and paper recycling [24]. In the biofuel sector, cellulases convert agricultural waste into fermentable sugars for ethanol production. In textiles, they are used for fabric softening and de-pilling. Cellulases also contribute to improving the quality and smoothness of recycled paper.

Lyases facilitate the addition or removal of functional groups to or from double bonds without hydrolysis or oxidation [25]. These enzymes are valuable for constructing carbon–carbon or carbon–oxygen bonds. Aldolases and decarboxylases are notable lyases used in the synthesis of building blocks for pharmaceuticals and fine chemicals. Their utility in stereoselective reactions makes them ideal for use in asymmetric synthesis pathways [26].

Isomerases catalyze the structural rearrangement of molecules, converting one isomer into another. A prime industrial application is glucose isomerase, used to produce high-fructose corn syrup from glucose. Isomerases are also employed in carbohydrate metabolism and in the synthesis of intermediates for bioplastics. Their specificity and efficiency reduce the need for additional processing steps and contribute to cost-effective manufacturing [27].

Ligases join two molecules through covalent bond formation, often using ATP as a cofactor. Though less common in large-scale industries, ligases are essential in biotechnological applications such as molecular diagnostics, gene cloning, and synthetic biology. DNA ligases are used in nucleic acid assembly, while other emerging ligases are being explored for protein conjugation and peptide synthesis. Their role in constructing complex biopolymers is gaining traction in specialized applications [28].

The boundaries between enzyme classes are becoming increasingly fluid due to advances in protein engineering and synthetic biology. Researchers are designing hybrid enzymes that combine multiple catalytic activities within a single protein, enhancing efficiency and reducing processing steps. Computational tools and AI-driven platforms now guide the discovery of novel enzymes, enabling the rapid deployment of biocatalysts for emerging industrial needs. In August 2018, a new class was introduced. This new class includes translocases (EC 7) and was created to describe enzymes that catalyze the passage of ions or molecules across membranes or their separation within them. As a result, certain enzymes formerly categorized in other classes, such as EC 3.6.3, are now included in the EC 7 class [15].

4. Sources of Industrial Enzymes

Industrial enzymes are obtained from a wide range of biological sources, including microorganisms, plants, animals, extremophiles, and, more recently, through recombinant and synthetic biology platforms, as shown in

Table 1. Comparative overview of industrial enzyme sources.

Source	Advantages	Limitations
Microorganisms	High yield, scalable, easy genetic manipulation, and cost-effective	Some enzymes may lack post-translational modifications
Plants	Naturally occurring, and used in food and traditional medicine	Seasonal availability, low yield, and complex purification
Animals	High substrate specificity, and historically used	Ethical concerns, and lower sustainability
Extremophiles	Exceptional stability (high temp, pH, salinity), and ideal for extreme conditions	Harder to culture, and lower expression yields
Recombinant Systems	High yield, customizable, consistent quality, and scalable production	Requires infrastructure and expertise in genetic engineering
Metagenomics	Access to uncultured organisms, high biodiversity, and novel functions	Complex screening and expression optimization
Synthetic Biology	Precision design, multifunctionality, and creation of new-to-nature enzymes	Expensive, still emerging, requires computational and molecular expertise

. The choice of source significantly influences enzyme properties such as stability, specificity, and suitability for different industrial applications. Below is a discussion of the major sources of industrial enzymes.

4.1. Microbial Sources

Microorganisms serve as the most extensively utilized and reliable sources of industrial enzymes, with bacteria, fungi, and yeasts being particularly preferred. Their advantages include rapid growth rates, ease of genetic modification, low production costs, and the ability to secrete substantial amounts of extracellular enzymes directly into the culture medium [29]. Bacterial strains such as Bacillus subtilis, Bacillus amyloliquefaciens, and Streptomyces species are known for producing a diverse array of enzymes, including proteases, amylases, and cellulases [30]. In the fungal domain, species like Aspergillus niger and Trichoderma reesei are prominent producers of cellulases, pectinases, and xylanases, which find widespread applications in industries such as food processing, textiles, and paper manufacturing [31]. Meanwhile, yeasts such as Saccharomyces cerevisiae and Pichia pastoris are widely employed for the production of enzymes like invertases and alcohol dehydrogenases, and they are gaining prominence in recombinant enzyme production due to their eukaryotic expression systems, which allow for proper protein folding and post-translational modifications [32]. Notably, microbial enzymes exhibit enhanced stability and activity under extreme industrial conditions, such as high temperatures and variable pH levels, making them particularly well-suited for use in large-scale biocatalytic processes [33].

4.2. Plant-Derived Enzymes

Plants have long been recognized as valuable sources of enzymes, particularly in the realms of food processing and traditional medicine [34]. Notable plant-derived enzymes include papain from papaya (Carica papaya), which is widely used for meat tenderization and in medical applications such as wound debridement, and bromelain from pineapple (Ananas comosus), which is employed in pharmaceuticals for its anti-inflammatory properties [35]. Additionally, enzymes such as amylases and proteases extracted from germinating grains like barley play essential roles in brewing and baking industries [36]. However, despite their beneficial applications, plant enzymes are often limited by factors such as seasonal availability, labor-intensive and complex extraction processes, and generally low yields. Consequently, their use tends to be confined to niche or specialty applications where such limitations can be managed or tolerated [37].

4.3. Animal-Derived Enzymes

Enzymes derived from animal tissues, particularly from digestive organs, have traditionally played a significant role in various industries, notably in food processing and medicine [38]. Common examples include trypsin and chymotrypsin, both extracted from the pancreas, which are utilized in peptide synthesis and pharmaceutical manufacturing. Pepsin, obtained from the stomach lining, is commonly used in protein hydrolysis and cheese production [39]. Another important animal-derived enzyme is rennet, which contains chymosin and is essential for milk coagulation in traditional cheese-making processes [40]. Despite their longstanding industrial relevance, the use of animal enzymes has declined due to growing concerns over zoonotic disease transmission, potential immunogenic reactions in end users, and ethical considerations related to animal welfare. As a result, there has been a notable shift toward replacing animal-based enzymes with microbial or recombinant alternatives, which offer greater safety, scalability, and ethical acceptability [41].

4.4. Extremophiles as Enzyme Sources

Extremophiles are microorganisms that thrive in harsh environmental conditions, including extreme temperatures, acidity, salinity, or pressure. The enzymes they produce, known as extremozymes, are remarkable for their ability to maintain stability and catalytic activity under conditions that would typically denature conventional enzymes [42]. For example, thermophilic bacteria such as Thermus aquaticus produce heat-stable enzymes like Taq polymerase, which is indispensable in polymerase chain reaction (PCR) techniques in molecular biology [43]. Similarly, halophiles and acidophiles yield enzymes that function efficiently in high-salt or acidic environments, making them particularly valuable in industrial processes such as leather processing and bioleaching. Owing to their resilience and adaptability, extremozymes are becoming increasingly important in industrial biotechnology and molecular biology, where robust enzyme performance under stress is often essential for process efficiency and reliability [44].

4.5. Recombinant Enzyme Production

Recombinant DNA technology has revolutionized enzyme production by enabling the expression of enzymes from diverse biological sources using genetically modified microbial hosts [45]. This method offers a highly controlled, scalable, and cost-effective alternative to traditional extraction techniques. Common host organisms used in recombinant systems include Escherichia coli, Pichia pastoris, and Bacillus subtilis, which are well-characterized and amenable to large-scale fermentation [46]. By inserting genes encoding enzymes from plants, animals, or extremophiles into these microbial hosts, it is possible to achieve high-yield enzyme expression in bioreactors. Furthermore, recombinant technology allows for the co-expression of multiple enzymes and the engineering of enzyme variants with improved characteristics such as greater stability, specificity, or activity [47]. As a result, recombinant enzyme production has become the standard approach in industrial biotechnology, effectively replacing conventional extraction methods for most commercial and research applications.

4.6. Metagenomic and Environmental Sources

Metagenomics is an innovative approach that involves the direct extraction and analysis of DNA from environmental samples such as soil, water, and compost, enabling the discovery of novel enzymes without the need to culture the source organisms [48]. This technique has significantly expanded the known diversity of enzymes by revealing previously inaccessible enzymes from uncultured microorganisms. Through high-throughput screening of metagenomic libraries, researchers can identify enzymes with highly specialized functions, including those that exhibit resistance to harsh conditions such as solvents, extreme temperatures, or oxidative stress [49]. As a result, metagenomics represent a powerful frontier in enzyme discovery and are increasingly being integrated into industrial research and development pipelines to uncover new biocatalysts with potential for a wide range of applications [50].

4.7. Synthetic Biology and Artificial Systems

Synthetic biology offers transformative possibilities in enzyme engineering by enabling the design of custom enzymes and synthetic metabolic pathways through advanced genome editing and modular gene assembly. Tools such as CRISPR/Cas9 allow for precise and targeted modifications within microbial genomes, enhancing the efficiency and yield of enzyme production [51]. Beyond natural enzymes, synthetic biology also facilitates the creation of artificial enzymes, or “synzymes,” which are engineered by combining catalytic domains from different sources to achieve multifunctional properties not found in nature [52]. Additionally, cell-free protein expression systems enable the synthesis of enzymes outside of living organisms, providing greater flexibility in controlling reaction conditions and avoiding issues related to cellular toxicity [53]. These synthetic approaches are revolutionizing the field of biocatalysis by pushing the boundaries of what enzymes can achieve, paving the way for novel industrial, pharmaceutical, and environmental applications.

5. Industrial Applications

Industrial enzymes have transformed manufacturing by enabling sustainable, energy-efficient, and highly specific processes. Used across industries such as food, textiles, pharmaceuticals, and environmental remediation, these biocatalysts boost productivity, reduce environmental impact, and enhance product quality, as illustrated in Figure 2.

5.1. Food and Beverage Industry

The food and beverage industry is one of the most mature and extensive users of enzymes. In the dairy sector, enzymes such as rennet (a protease) are used for milk coagulation in cheese production [54]. Lactase is employed to hydrolyze lactose into glucose and galactose, making dairy products suitable for lactose-intolerant individuals [55]. Lipases enhance flavor development in cheese through fat modification [56]. In baking, enzymes like amylases break down starch into simpler sugars, improving dough handling, bread volume, and shelf life. Xylanases weaken the flour’s hemicellulose structure, enhancing dough elasticity and reducing stickiness. Glucose oxidase strengthens gluten networks by oxidizing glucose into gluconic acid and hydrogen peroxide, improving dough stability [57]. The brewing industry relies on enzymes to convert starches and proteins in malted grains into fermentable sugars and amino acids. Amylases and proteases optimize the mashing process, while beta-glucanases reduce wort viscosity, improving filterability and fermentation rates [58]. These enzymatic interventions enhance product clarity and consistency. For fruit juice clarification, enzymes such as pectinases are essential, as pectinases degrade the pectin content of fruit cell walls, reducing juice viscosity and facilitating filtration [59]. Additional enzymes like cellulases and hemicellulases improve extraction efficiency by breaking down structural polysaccharides [60]. Yakult Honsha Co., Ltd. of Japan and Friesland Food Domo of the Netherlands, among others, have carried out the commercial-scale, enzyme-catalyzed manufacture of galacto-oligosaccharides (GOS), a profitable probiotic with digestive health advantages and usage as low-calorie sweeteners [61].

5.2. Textile and Leather Industry

In the textile industry, enzymes are now used instead of harsh chemicals for processes like bio-polishing. For example, cellulase enzymes remove tiny fibers from the surface of cotton, making the fabric smoother, brighter in color, and less likely to pill [62]. This process works under gentle conditions, helping to protect the fabric. Another step, called desizing, removes starch coatings added during weaving. Amylase enzymes break down the starch without harming the fabric. This method is faster, uses less water and energy, and avoids strong chemical treatments [63]. In leather production, enzymes have also replaced harmful chemicals. During dehairing, protease enzymes break down hair and root proteins to clean the hides [64]. In the bating step, proteases and lipases are used to soften the leather and improve its texture. These enzyme-based methods are safer for workers and better for the environment [65].

5.3. Detergent Industry

The detergent industry is one of the largest consumers of industrial enzymes. Surfactants are the major components of detergents that remove stains from clothing during the laundry process. Surfactants are most active at high temperatures, and heating laundry water requires a lot of energy, especially in cold nations. Furthermore, surfactants discharged into the environment after washing are hazardous to aquatic organisms unless they are removed in effective wastewater treatment plants. Enzymes may destroy stains at low washing temperatures and are less harmful than surfactants. They are used as additives in detergents [66]. Proteases, lipases, amylases, and cellulases are combined into enzyme cocktails for stain removal. Proteases target protein-based stains, lipases degrade fats and oils, amylases remove starchy residues, and cellulases restore fabric brightness by degrading microfibrils [67]. Modern detergents function effectively at lower temperatures and neutral pH values, thanks to advanced enzyme formulations. These eco-friendly detergents conserve energy, protect fabrics, and perform efficiently under mild washing conditions, making them popular in both household and industrial settings [68].

Bacillus licheniformis strain-MA1 developed an alkaline protease that was acceptable for industrial uses. The capacity of B. licheniformis strain-MA1 to make protease was optimized by utilizing multi-factorial designs (Plackett–Burman and Box–Behnken). The optimization approach increased enzyme production 9.6-fold over the initial medium. The highest alkaline protease production was achieved after 72 h at a pH of 7.0, 35 °C, and 150 rpm. The protease was most active at 50 °C and at a pH of 9.0, with excellent thermal and pH stability. The protease demonstrated significant catalytic efficiency and substrate affinity while maintaining a low activation energy (E_a). In addition, the thermodynamic properties of the protease enzyme (enthalpy, free energy, and entropy) were studied, revealing its remarkable thermal stability. At 70 °C, the thermal deactivation constant (kd) was 4.75 times greater than at 50 °C. The protease’s higher t0.5, D-values, and activation energy for thermal denaturation (Ed) suggested greater thermal stability and, hence, promise for use in industrial operations. The protease was more compatible with washing detergents at 40 °C than at 50 °C. In the presence of EDTA, the protease enzyme maintained 93.6% of its activity. Furthermore, the crude enzyme successfully dissolved the gelatin layer of X-ray film waste after 1 h, allowing for recycling and reuse [69], as illustrated in Figure 3.

5.4. Paper and Pulp Industry

In the paper and pulp industry, enzymes are employed for bio-bleaching to reduce reliance on chlorine-based chemicals. Xylanases and laccases modify lignin and hemicellulose structures, aiding in chromophore removal. This enzymatic treatment yields brighter paper with reduced environmental impact [70]. The deinking of recycled paper uses lipases and esterases to break down ink binders, releasing ink particles from fibers. Enzymatic deinking enhances fiber quality, reduces surfactant use, and lowers wastewater production [71]. Pitch control involves removing sticky resinous substances from wood pulp using lipases. Pitch deposits disrupt machine operations and reduce paper quality. Enzymatic treatments prevent pitch agglomeration and deposition, supporting cleaner processing [72].

5.5. Pharmaceuticals and Fine Chemicals

In the pharmaceutical industry, enzymes are vital for chiral synthesis, producing enantiomerically pure drug compounds [73]. Transaminases, ketoreductases, and hydrolases convert prochiral substrates into active pharmaceutical ingredients (APIs) with desired stereochemistry, improving efficacy and safety. Enzymes also aid in drug intermediate production [74]. Nitrile hydratases and amidases, for example, are used in the synthesis of acrylamide and nicotinamide. These processes proceed under mild, aqueous conditions, offering a green alternative to traditional synthesis methods [75].

5.6. Biofuels and Biorefining

In biofuels and biorefining, enzymes enable the conversion of lignocellulosic biomass into fermentable sugars via enzymatic hydrolysis [76]. Cellulases, hemicellulases, and beta-glucosidases work together to degrade cellulose and hemicellulose into glucose and xylose, which are then fermented into ethanol or other fuels. The efficiency of hydrolysis depends on enzyme loading, substrate composition, and pretreatment [60]. Engineered enzymes now exhibit enhanced stability under high temperatures and resistance to inhibitors, making biofuel production more cost-effective. Biorefineries also use enzymes to produce organic acids, bioplastics, and bio-based chemicals. These biocatalysts enable specific, mild, and sustainable transformations compatible with integrated processing schemes, promoting circular bioeconomies [77].

Microbial lipases have sparked widespread attention due to their capacity to withstand high concentrations of organic solvents. Burkholderia cepacia lipase (BCL) is assessed for methanol tolerance in terms of conformational stability and catalytic activity in transesterification experiments. This lipase appears to be more tolerant than the homologous and better-known enzyme from Burkholderia glumae. In the presence of 50% methanol, the Tm of the BCL unfolding transition is higher than 60 °C, as measured by far-UV circular dichroism (CD) and intrinsic fluorescence. The protein unfolds at a low pH, and the organic solvent influences the denatured state under acidic circumstances. The protein performs well in transesterification experiments after prolonged incubation at high methanol concentrations. BCL is extremely resistant to methanol and has particularly good conformational stability under transesterification conditions. These characteristics highlight BCL as a promising enzyme for the biofuel sector [78], as illustrated in Figure 4 and Figure 5.

5.7. Waste Management and Bioremediation

In waste management and bioremediation, enzymes degrade pollutants and convert waste into less harmful or reusable forms. Laccases and peroxidases break down dyes, phenols, and hydrocarbons in contaminated water and soil, acting on a wide range of toxic compounds [79]. Enzymes such as lipases, proteases, and cellulases are used in organic waste treatment to accelerate the degradation of food, agricultural, and municipal waste. This improves composting efficiency, increases biogas yield in anaerobic digestion, and enhances the recyclability of waste materials [80]. Industrial enzymes are redefining modern manufacturing, offering precise and eco-friendly alternatives to conventional chemical processes. Continued innovation in enzyme technology will further expand their industrial applications and impact across sectors [6,81].

6. Advances in Enzyme Engineering

Enzyme engineering is a vital part of modern biotechnology, enabling the creation of more efficient and resilient biocatalysts for industrial use. Natural enzymes often struggle under harsh conditions like high temperatures, salinity, or organic solvents. To overcome these challenges, enzyme engineering modifies enzymes at the genetic and protein levels. Over the past two decades, a variety of strategies from experimental to computational have emerged to enhance or redesign enzyme functions.

6.1. Rational Design

Rational design is a structure-guided approach that involves the intentional alteration of amino acid residues within an enzyme to improve its properties. This method requires detailed knowledge of the enzyme’s three-dimensional structure, active site geometry, and reaction mechanism [82]. Tools such as X-ray crystallography, cryo-electron microscopy, and nuclear magnetic resonance (NMR) spectroscopy are used to resolve enzyme structures at an atomic resolution. Using computational modeling and molecular docking, researchers can predict how changes in amino acid composition will affect substrate binding, catalytic turnover, or structural stability [83]. For example, introducing disulfide bonds or stabilizing hydrogen-bond networks can enhance thermostability, while modifying charged residues in the active site can improve pH tolerance or catalytic specificity. Rational design has proven highly effective for improving enzymes such as lipases, proteases, and dehydrogenases used in food processing, pharmaceuticals, and biofuel production [84].

6.2. Directed Evolution

Directed evolution mimics the laboratory’s natural selection process to evolve enzymes with improved or novel properties. Unlike rational design, it does not require prior structural knowledge [85]. Instead, it generates an extensive library of enzyme variants through error-prone PCR, DNA shuffling, or saturation mutagenesis. These libraries are then subjected to high-throughput screening or selection processes to identify variants with desirable traits. Once a promising candidate is found, it can serve as the parent for subsequent rounds of mutation and screening, allowing for iterative improvement. Directed evolution has yielded significant success stories, including developing enzymes with dramatically increased thermal stability, substrate range, or activity in organic solvents [86]. The 2018 Nobel Prize in Chemistry was partly awarded for pioneering work in this field, underscoring its transformative impact on enzyme engineering [87].

6.3. Computational Modeling and Machine Learning

Integrating computational tools into enzyme engineering has vastly improved the speed and accuracy of protein design [88]. Molecular dynamics (MD) simulations, quantum mechanics/molecular mechanics (QM/MM) models, and energy minimization algorithms help predict specific mutations’ structural and functional consequences. These simulations can reveal the flexibility of enzyme active sites, identify unstable regions, and model enzyme–substrate interactions in detail [89]. Machine learning (ML) and artificial intelligence (AI) have further enhanced predictive capabilities. By training algorithms on extensive datasets of enzyme sequences, structures, and functions, ML models can suggest mutations likely to improve performance [90]. Deep learning approaches, including generative adversarial networks (GANs) and transformer-based models, are now being applied to design entirely new protein sequences or to predict optimal combinations of mutations. These tools have significantly reduced the trial-and-error burden in laboratory experiments [91].

6.4. Synthetic Biology and CRISPR-Based Engineering

Synthetic biology provides a framework for building novel biological systems using standardized genetic parts and modular design principles [92]. In enzyme engineering, it enables the assembly of synthetic operons, regulatory elements, and complete biosynthetic pathways within microbial hosts. These engineered systems allow the fine-tuned control of enzyme expression, activity, and cofactor availability [93]. CRISPR-Cas systems have refined synthetic biology by allowing precise genome editing in microbial production strains [94]. Genes encoding engineered enzymes can be inserted directly into high-expression loci, while unwanted metabolic pathways can be knocked out to reduce competition for cellular resources. This enables the development of tailor-made microbial factories that efficiently produce enzymes under specific industrial conditions. Moreover, synthetic biology enables the co-expression of multiple enzymes, facilitating the assembly of multistep biocatalytic processes within a single cell [95].

6.5. De Novo Enzyme Design

De novo enzyme design represents one of the most ambitious and exciting frontiers in enzyme engineering. Rather than modifying existing enzymes, this approach involves designing entirely new protein scaffolds from scratch to catalyze reactions not found in nature [96]. Using advanced algorithms such as Rosetta Design, researchers can generate theoretical protein backbones, place catalytic residues in precise spatial arrangements, and simulate folding pathways to ensure stability and activity [96]. Although still in its early stages, de novo design has created artificial enzymes for Kemp elimination, Diels Alder reactions, and carbon–carbon bond formation. These synthetic enzymes have the potential to catalyze complex chemical transformations previously achievable only through traditional catalysis, opening new avenues in green chemistry and pharmaceutical synthesis [97].

6.6. Cell-Free Protein Synthesis

Cell-free protein synthesis (CFPS) systems are an emerging platform that enables enzyme production and testing without the constraints of living cells. CFPS involves cell extracts containing the necessary transcriptional and translational machinery to synthesize proteins in vitro [98]. This approach offers several advantages, including the rapid expression of enzyme variants, precise control over reaction conditions, and elimination of issues such as cellular toxicity or product inhibition. When combined with high-throughput screening and synthetic biology, CFPS allows the fast prototyping of enzyme designs, including those with toxic intermediates or non-natural amino acids [99]. Researchers can also fine-tune parameters such as pH, ionic strength, and cofactor concentration, enabling the detailed optimization of enzyme performance in controlled environments [100].

7. Limitations of Industrial Enzymes

Enzymes are not the best choice for catalysis in several large-scale applications since they are often unstable, have a limited shelf life, and can be readily deactivated by a variety of methods. Enzymes are also very sensitive to different severe process conditions, and significant technical issues develop when using enzymes in industrial applications, rendering them essentially unreliable. In contrast to traditional heterogeneous chemical catalysts, most enzymes perform better when dissolved in water in homogeneous catalysis systems. Furthermore, standard techniques of enzyme recovery and reuse are extremely difficult to implement. These many difficulties can be avoided or at least mitigated by employing various immobilization strategies [101].

8. Immobilized Enzymes and Reactor Engineering

Combining enzyme immobilization with advanced reactor designs enhances biocatalytic efficiency in the industry. Immobilizing enzymes on solid supports enables reuse, improves stability, and simplifies processing [102,103], as shown in

Table 2. Advantages and disadvantages of various methods for enzyme immobilization.

Immobilization Methods	Advantages	Disadvantages
Adsorption	Simple preparation and operation, cost-effective, and capable of regeneration	Weak bonding, limited activity and selectivity
Entrapment	High activity, robust bonds, and low costs	The preparation and operation are difficult and irreversible
Encapsulation	Simple and efficient preparation, cost-effective, highly active, and capable of regeneration	Lack of specificity and the presence of weak bonding
Covalent Binding	Strong binding with a high level of activity and specificity	Preparation is a challenging and expensive process that cannot be regenerated
Crosslinking	Intense activity, robust binding, and cost-effectiveness	Strong bonds and affordable; the process of preparation and operation is difficult and irreversible

. Physical or chemical methods, as illustrated in Figure 6, affect enzyme performance and longevity. Below is a brief overview of key immobilization techniques.

8.1. Physical Methods

8.1.1. Adsorption

In this method, enzymes are immobilized through weak, reversible interactions including hydrogen bonding, ionic attractions, hydrophobic forces, and Van der Waals interactions [104]. Common support materials used include ion exchange resins, silica-based compounds, activated carbon, alumina, ceramics, controlled-pore glass, and natural polymers such as cellulose and agarose, as well as some industrial-grade substrates [105]. This method is cost-effective and preserves enzyme activity; however, it may suffer from desorption under changing pH or ionic strength [106].

8.1.2. Entrapment

Entrapment is a physical immobilization technique where enzymes are confined within polymeric matrices or fibrous networks, formed by polymerizing a monomer–enzyme mixture [22]. This method stabilizes enzymes, protects them from denaturation, and allows for modification of the microenvironment (e.g., pH, polarity) to enhance activity. However, it may suffer from mass transfer limitations due to restricted substrate diffusion, reduced enzyme loading capacity, and possible weakening of the matrix during polymer formation [107]. To overcome these drawbacks, cross-linking agents are often added to reinforce structural integrity and improve mechanical stability [108].

8.1.3. Encapsulation

Encapsulation is a form of enzyme entrapment where the enzyme is enclosed within a spherical, semi-permeable membrane [109]. In this approach, the enzyme is surrounded by a polymeric shell that retains it within the internal cavity, while still allowing substrate molecules to enter and products to exit through the membrane. This method is particularly advantageous for biosensor applications, as it preserves the native structure of the enzyme and maintains its catalytic efficiency over time [110]. Additionally, encapsulation is a cost-effective technique suitable for producing compact enzymatic systems. Nevertheless, this method presents certain challenges, including the potential for enzyme leakage through inadequately sized pores and limited diffusion of substrates and products across the membrane. These diffusion constraints and stability issues highlight the need for ongoing refinement of encapsulation technologies [105].

8.2. Chemical Methods

8.2.1. Covalent Binding

This technique involves the immobilization of enzymes through the formation of strong covalent bonds. These bonds arise from chemical interactions between functional groups on the enzyme’s amino acid residues such as those found in lysine, cysteine, aspartic acid, and glutamic acid and reactive groups like carboxyl, hydroxylamine, phenol, and imidazole [111]. Covalent attachment offers enhanced structural rigidity, significantly improving enzyme stability and ensuring robust binding to the support material [104]. This strong linkage helps maintain the enzyme’s native conformation, protecting it from denaturation caused by harsh conditions including high temperatures, extreme pH levels, or organic solvents. For effective immobilization, selecting a support material rich in reactive functional groups is essential to ensure efficient enzyme attachment and activity retention [14].

8.2.2. Cross Linking

Crosslinking is a support-free method of enzyme immobilization that enhances enzyme stability by chemically linking enzyme molecules together. This process involves the use of crosslinking agents, which react with specific amino acid residues on the enzyme surface to form intermolecular and intramolecular bonds, resulting in the formation of cross-linked enzyme aggregates [112]. The primary purpose of crosslinkers is to shield the enzyme from environmental stressors, preserving its structural integrity and functional performance [113]. Immobilization occurs when the enzyme is exposed to a medium containing the crosslinking reagent, promoting the formation of a stable enzyme network.

8.3. Supporting Matrix

Immobilizing enzymes onto solid carriers or matrices is a widely used approach to improve their durability, facilitate repeated use, and simplify recovery from reaction systems [114]. However, the effectiveness of this method relies heavily on selecting an appropriate support material. The selection process must consider several properties, as illustrated in Figure 7. The following section outlines some of the most frequently utilized support materials in enzyme immobilization.

The following variety of materials are employed as supports for enzyme immobilization, each offering distinct advantages based on their structural and chemical properties:

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Polymeric materials: Synthetic polymers like polyacrylamide, polyethyleneimine, and polyvinyl alcohol are frequently used due to their structural flexibility, chemical adaptability, and ability to form porous networks that accommodate enzyme molecules while maintaining catalytic activity [115].

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Inorganic substrates: Materials such as silica, alumina, and zeolites serve as excellent immobilization platforms owing to their high surface area, durability, and resistance to harsh chemical environments, making them suitable for industrial biocatalysis [116].

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Membrane-based supports: Membranes made from compounds like cellulose acetate and polyamide act as selective barriers, enabling the compartmentalization of enzymes and substrates. These membranes facilitate controlled and continuous catalytic processes, especially in flow systems.

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Magnetic nanoparticles: Iron oxide-based nanoparticles (e.g., Fe₃O₄) offer the dual benefit of enzyme immobilization and ease of recovery via magnetic separation. This feature enhances process efficiency and reduces operational costs by allowing enzyme reuse. Nanoparticles (NPs) with magnetic functionality form a unique subclass and will be explored in more detail in the following section [117].

8.4. Nano- and Micromaterials as Enzyme Immobilization Supports

Nano- and micro-immobilization have several advantages, including lower mass transfer resistance, effective enzyme loading, increased surface area, and reduced diffusional difficulties. However, it is crucial to examine the drawbacks, such as the cost of the manufacturing process, the high costs associated with large-scale applications, and the difficulties in separating the reaction media. The choice of nano supports must be carefully considered, since some may be challenging to handle while preventing diffusion difficulties. Furthermore, if the support lacks porosity, then the enzyme is exposed to the surrounding media. Several research studies have looked at nanomaterials as platforms for immobilizing enzymes and biomolecules, and the merits and downsides have been thoroughly reviewed [118].

Nanobiocatalysts (NBC) represent an innovative technological breakthrough that merges cutting-edge nanotechnology with biotechnology, leading to a powerful and synergistic integration of the two fields [102]. Nanoparticles, defined as materials with at least one dimension below 100 nm, can exist in various physical forms such as fused, aggregated, or agglomerated and may exhibit diverse morphologies including spherical, tubular, or irregular shapes [119,120,121]. Their unique nanoscale characteristics make them exceptionally suited for enzyme immobilization. Key advantages include a high surface-to-volume ratio, minimal diffusion resistance, and the capacity to achieve efficient enzyme loading. These properties are especially beneficial when working with large substrates, where mass transfer becomes a limiting factor [121]. Nanoparticles help alleviate such challenges by offering closer proximity and more accessible interaction sites for the enzymes. Their small size allows enzymes to retain functional mobility and efficient substrate access, even in complex reaction media, while also enhancing thermal and chemical stability [122]. When enzymes are immobilized onto nanoparticles, they often exhibit superior catalytic performance compared to their free counterparts. This improvement is partly attributed to their ability to maintain Brownian motion in aqueous environments, allowing them to behave similarly to free enzymes while benefiting from the enhanced stability and reusability provided by immobilization [123], as shown in Table 3.

8.4.1. Magnetic Nanoparticles

With the rapid progress in nanotechnology and its integration into fields like biotechnology and medicine, magnetic nanoparticles (MNPs) have emerged as highly promising materials for enzyme immobilization [103]. These nanoscale particles possess unique features such as superparamagnetism, high surface-area-to-volume ratios, ease of manipulation using external magnetic fields, and chemical modifiability, making them ideal platforms for biocatalyst attachment [124]. One of the key advantages of MNPs is the simplicity of recovering immobilized enzymes from reaction mixtures using a basic magnet, eliminating the need for complex separation techniques [123]. Their large surface area enables a high enzyme loading capacity while reducing mass transfer limitations. Moreover, surface functionalization allows for tailored chemical interactions, enhancing the binding efficiency and orientation of enzymes on the support. Studies have consistently shown that enzymes immobilized on magnetic nanoparticles maintain improved structural integrity, reduced denaturation, and heightened catalytic performance [117,125]. Despite their numerous benefits, the synthesis of uniformly dispersed, chemically functionalized magnetic nanoparticles, especially those bearing amine groups, remains a technical challenge, often requiring precise control over particle size and surface chemistry to ensure reproducibility and effectiveness in immobilization applications.

8.4.2. Non-Magnetic Nanoparticles

A variety of non-magnetic nanoparticles such as those made from gold, silver, silica, chitosan, and zirconia have been extensively utilized as carriers for enzyme immobilization. These nanoparticles provide a favorable environment for enzyme attachment and dispersion within the reaction medium, ensuring uniform catalytic activity throughout the system [125,126]. However, a key limitation of using non-magnetic supports is the difficulty associated with recovering the immobilized enzymes after the reaction. Unlike magnetic particles, these systems lack a built-in separation mechanism, making enzyme retrieval dependent on time-consuming methods like high-speed centrifugation, which may hinder efficiency in large-scale or repetitive applications [117].

8.4.3. Metal-Organic Frameworks

Both metallic and non-metallic hybrid materials have been developed for advanced enzyme immobilization applications. Among these, metal organic frameworks (MOFs) represent a unique class of crystalline structures formed through the coordination of metal ions with organic ligands [127]. These metal centers act as nodes, linking the organic molecules into repeating, cage-like networks that exhibit high levels of structural regularity and porosity. MOFs are particularly valued for their exceptionally large internal surface areas and tunable pore sizes, making them ideal candidates for accommodating enzymes within their frameworks [128]. Their well-ordered architecture allows for efficient enzyme loading and stabilization, while also enabling precise control over molecular interactions. In addition, mesoporous materials and organic carriers further enhance the potential for enzyme entrapment and confinement, supporting catalytic function through improved accessibility and minimal structural disruption.

8.4.4. Carbon Nanotubes

Carbon nanotubes (CNTs) are cylindrical nanostructures derived from rolled layers of graphene. As a relatively recent advancement in the field of nanomaterials, CNTs have attracted significant scientific interest owing to their unique structural configuration, exceptional conductivity, and outstanding thermal and mechanical resilience [129]. Their compatibility with biological systems further enhances their appeal for biotechnological applications. These characteristics make CNTs particularly advantageous for enzyme immobilization. Their high surface area, combined with excellent dispersibility in liquid media and a versatile range of chemical modification options, enables efficient enzyme attachment while preserving catalytic activity. As a result, CNTs have emerged as a highly effective platform for developing robust and high-performance biocatalysts [130].

Table 3. Enzyme immobilization for industrial and biomedical applications.

Enzyme	Nanomaterial	Application	Ref.
Urokinase-type plasminogen activator	Magnetic polyelectrolyte-based composites	Thrombolytic and anticoagulant properties	[131]
Urokinase-type plasminogen activator	Magnetic NPs	Enhanced thrombolysis rate in a microfluidic channel	[132]
Tissue plasminogen activator, streptokinase	Chitosan NPs	Treatment of thrombolytic disorder	[133]
Tissue plasminogen activator, streptokinase	Cu NPs	Restores blood flow in arterial thrombosis	[134]
Lipase	Zinc Ferrite NPs	Antibacterial activity against E. coli and S. aureas	[135]
Streptokinase	Alumina NPs	Thrombolytic colloid with prolonged action	[136]
Catalase, SOD, and glutathione peroxidase	Cu5.4O NPs	Cytoprotective effects against ROS-mediated damage	[133]
Tissue plasminogen activator	Magnetic iron oxide micro-rods	Enhanced thrombolysis after ischemic stroke	[137]
Urokinase	Chitosan NPs	Enhanced thrombolytic activity	[138]
Catalase, peroxidase xanthine oxidase	Polyethylene glycol and poly-lactic/polyglycolic acid NPs	Protection and vascular oxidative stress	[137]
Catalase, SOD	Polymeric NPs	Protection against inflammation	[132]
Catalase	Poly (lactic co-glycolic acid) NPs	Protection of neurons from oxidative damage	[139]
Candida antarctica lipase B	Carbon-based magnetic NPs	Biodiesel preparation	[140]
Purine nucleoside 2′-deoxyribosyltransferase from Trypanosoma brucei	Glutaraldehyde-activated magnetic microspheres	Pharmaceutical industry	[141]
Horseradish peroxidase	Fe3O4–NH2/hNFs	Decolorization of textile dyes	[142]
Lipase	ZnFe2O4@Mesoporous silica nano	Food industry	[143]

Table 3. Enzyme immobilization for industrial and biomedical applications.

Enzyme	Nanomaterial	Application	Ref.
Urokinase-type plasminogen activator	Magnetic polyelectrolyte-based composites	Thrombolytic and anticoagulant properties	[131]
Urokinase-type plasminogen activator	Magnetic NPs	Enhanced thrombolysis rate in a microfluidic channel	[132]
Tissue plasminogen activator, streptokinase	Chitosan NPs	Treatment of thrombolytic disorder	[133]
Tissue plasminogen activator, streptokinase	Cu NPs	Restores blood flow in arterial thrombosis	[134]
Lipase	Zinc Ferrite NPs	Antibacterial activity against E. coli and S. aureas	[135]
Streptokinase	Alumina NPs	Thrombolytic colloid with prolonged action	[136]
Catalase, SOD, and glutathione peroxidase	Cu5.4O NPs	Cytoprotective effects against ROS-mediated damage	[133]
Tissue plasminogen activator	Magnetic iron oxide micro-rods	Enhanced thrombolysis after ischemic stroke	[137]
Urokinase	Chitosan NPs	Enhanced thrombolytic activity	[138]
Catalase, peroxidase xanthine oxidase	Polyethylene glycol and poly-lactic/polyglycolic acid NPs	Protection and vascular oxidative stress	[137]
Catalase, SOD	Polymeric NPs	Protection against inflammation	[132]
Catalase	Poly (lactic co-glycolic acid) NPs	Protection of neurons from oxidative damage	[139]
Candida antarctica lipase B	Carbon-based magnetic NPs	Biodiesel preparation	[140]
Purine nucleoside 2′-deoxyribosyltransferase from Trypanosoma brucei	Glutaraldehyde-activated magnetic microspheres	Pharmaceutical industry	[141]
Horseradish peroxidase	Fe3O4–NH2/hNFs	Decolorization of textile dyes	[142]
Lipase	ZnFe2O4@Mesoporous silica nano	Food industry	[143]

8.5. Factors Influencing the Enzyme Immobilization

There are various factors that can influence the performance of enzyme immobilization. The selection of an appropriate support is a fundamental aspect of enzyme immobilization, as it provides the structural platform to which the enzyme is bound. Commonly utilized materials include agarose, silica gel, and various synthetic resins. Properties such as surface area, porosity, and mechanical and chemical stability of the support significantly influence the efficiency and durability of the immobilization process [14]. Prior to enzyme attachment, the support material typically undergoes activation to introduce reactive groups that can interact with the enzyme. This step often involves chemical treatments such as cross-linkers or physical modifications aimed at enhancing the affinity and strength of the enzyme–support interaction [104]. Enzymes can be immobilized using a range of approaches, including physical adsorption, covalent bonding, entrapment, and encapsulation. The choice of method depends on multiple factors, including the desired stability, operational conditions, and enzyme loading requirements. Each method presents specific trade-offs in terms of reusability, activity retention, and ease of preparation [144]. Maintaining optimal pH and temperature during immobilization is critical to preserving enzyme activity. Deviations from the enzyme’s ideal working conditions may lead to reduced catalytic function or denaturation, making it essential to tailor the process environment accordingly [114]. The amount of enzyme used and its loading density on the support directly impact the final catalytic output. Achieving an effective balance between enzyme concentration and surface availability is crucial for maximizing both efficiency and stability [145]. Care must be taken to avoid exposure to substances that may inhibit or irreversibly deactivate the enzyme during immobilization. These can include solvents, reactive chemicals, or impurities that interfere with the enzyme’s active site or structural integrity [146]. The performance and shelf life of immobilized enzymes are influenced by storage and working conditions. Parameters such as temperature, pH, moisture levels, and exposure to specific solvents must be carefully controlled to maintain enzyme functionality over time [147]. Nanoparticle size plays a critical role in the success of enzyme immobilization. Downsizing supports from the microscale to the nanoscale increases surface area and reduces mass transfer and diffusion limitations [148]. Smaller nanoparticles typically promote higher enzyme activity and better retention of catalytic properties due to enhanced surface interactions and more efficient substrate access.

9. Multi-Enzymatic Nano Biocatalyst

The development of multi-enzymatic nano biocatalysts is a fast evolving and profitable technology for the synthesis of fine chemicals and other high-value products. There are several ways and approaches for developing multi-enzyme NBCs, but the most successful is to immobilize these enzymes on various types of support materials. When designing multi-enzyme NBCs, several issues must be considered, including enzyme stability and reusability, as well as activity. It is well understood that natural processes within cells are caused by a cascade of enzymes [1]. A multi-enzyme cascade is a technique in which multiple enzymes are immobilized on a suitable carrier or support. However, enzymes can also be joined via a linker, which eliminates the need for a carrier or support [149]. To develop successful biocatalytic processes, Xu et al. investigated the use of various support materials for immobilizing numerous enzymes [112]. These include inert inorganic materials like silica and TiO₂, graphene, carbon nanotubes, metals and organic ligand complexes, DNA nanostructures, and various polymers. Zhang et al. described the categorization, synthesis conditions, functional properties, and commercial applications of enzyme-inorganic hybrid nanoflowers (HNF), a novel material for immobilizing numerous enzymes. They proposed that this is a significant new arena for the nano immobilization of several enzymes [150].

10. Limitations of Nanomaterials During Enzyme Immobilization

Direct exposure to nanomaterials with significant harmful potential can cause serious environmental and health concerns. For instance, handling pure powdered carbon nanotubes poses notable risks. Therefore, assessing the toxicity of specific nanomaterials is essential before employing nano-supports as a safety measure. Transition metal-based catalysts are generally costly due to their restricted applications, and they often require hazardous solvents as reaction media, which may cause additional secondary toxicity. Consequently, it is critical to monitor nanomaterials’ environmental impact and determine exposure levels during production. In a previous study, Artemia salina was used to assess the toxicity of untreated diclofenac and naproxen solutions, along with formulations modified using laccase either encapsulated or adsorbed onto NBCs, at room temperature [151]. The EC30 values for untreated naproxen and diclofenac were approximately 20% and 25%, respectively, reflecting the high toxicity of these pharmaceutical solutions. After immobilization with NBCs, the toxicity decreased significantly, with EC30 values ranging from 60% to 85%. Encapsulated laccase yielded higher EC30 values than adsorbed laccase, indicating reduced effluent toxicity and making encapsulation the more effective immobilization approach. To ensure the safe large-scale implementation of nanomaterials, industry, academia, and regulatory authorities must balance their benefits against potential hazards, safeguarding both environmental and human health [152].

11. Some Examples Where Immobilization Can Hardly Improve Enzyme Stability

Even though the potential of immobilization to solve many enzyme limitations is high, there are some instances where enzyme inactivation cannot be prevented by immobilization, because using an enzyme with greater rigidity or partitioning undesired compounds will not improve the situation. In these cases, the advantages of enzyme immobilization are limited, and it should be considered if its other advantages can justify the enzyme immobilization. One instance of this scenario could occur when an enzyme is deactivated through suicide inhibition [153,154]. If there is an inherent probability of enzyme inactivation during catalysis, immobilization is unlikely to significantly reduce this risk (although, in rare cases, better structural arrangements might arise by chance, no evidence has been reported to confirm this). In other cases, enzyme inactivation may primarily result from the loss of an ion, cofactor, or prosthetic group or from the oxidation of a specific residue. Immobilization could even accelerate this process if it alters the enzyme’s conformation, exposing more reactive groups to the surrounding medium or reducing its affinity for essential components, thereby facilitating their dissociation [155,156]. Even if the protein polymer becomes more stiff, enzyme stability will remain same. There have been some reports that increasing the number of enzyme-support links affects enzyme stability [157,158,159]. Similarly, in certain cases, excessive multi-point covalent attachment of multimeric enzymes might impair the enzyme assembly, necessitating the use of moderate degrees of multi-point covalent attachment to provide maximum immobilized enzyme stability [160]. As a result, understanding the main causes of enzyme inactivation can be a key point in determining whether immobilization can solve an enzyme’s stability problems and help make decisions about the convenience of using an immobilized enzyme, taking into account the other benefits discussed in the introduction.

12. Logistical Considerations for Industrial-Scale Immobilized Enzyme Applications

Some logistical issues may not be as important in an academic laboratory. However, they may be quite important to a factory. The first question is whether the user company wants to purchase a commercial biocatalyst or make their own biocatalyst. Purchasing biocatalysts from a specialist company may be simpler, but it limits the possibilities for tuning and developing the biocatalyst, and the user company must rely on the supplier’s good quality control. Making their own biocatalyst gives more opportunities to improve the enzyme features in the direction required by the company. However, this necessitates independently buying supports and enzymes, and the user company must be able to control the reproducible quality of these materials. In both circumstances, there is a danger of removing a specific support from the market, as was the case with Eupergit by Rohm and Hass. This necessitates the search for a comparable product on the market, as well as the re-optimization of the biocatalyst preparation. This potential is difficult to regulate, and many businesses may consider it too risky and prefer to utilize soluble enzymes. However, it is extremely unlikely that an immobilization support with a large customer base will be removed from the market unless it is due to pressure from a competitor, usually with some advantages over the former.

13. Enzyme Immobilization Cost

Enzyme immobilization studies should be evaluated based on the economic balance between product added value and enzyme costs. The cost of the support and immobilization process is often stated without considering important factors like loading capacity. Proper immobilization systems can increase enzyme activity and reduce solids in reactors. If enzyme immobilization is essential or reusing immobilized enzymes, the balance between product added value and enzyme costs must be evaluated. If the price of the product or substrate is high, soluble enzymes may be best, but if the immobilized enzyme alone is capable of carrying out the desired reaction at the desired yields and reactor productivity, immobilization becomes a necessity. In low-priced products, all costs are important, and enzyme immobilization may improve the competitiveness of the process. However, if only a very sophisticated immobilization protocol permits the preparation of a biocatalyst with the desired performance, the use of immobilized enzymes may be limited. Further investigation into enzyme immobilization is required to ensure reasonable costs [161].

14. Industrial and Economic Perspectives

A techno-economic study on β-glucosidase production via recombinant E. coli for second-generation ethanol plants in Brazil reported costs of 316 USD/kg, higher than fungal enzyme averages due to facility, raw material, and consumable costs. Sensitivity analysis suggested that optimizing scale, inoculation, and productivity could significantly lower costs, making E. coli a viable platform for tailored enzymatic cocktails in lignocellulosic biomass hydrolysis [162].

Some research has focused on the economic implications of the production of enzymes with possible uses in biodiesel synthesis [163]. To produce a reportable comparative validity result in these experiments, an ester synthesis productivity/enzyme cost normalization is required. The production of enzymes for ester syntheses via submerged or solid-state fermentation, followed by immobilization, has been extensively studied; optimizing these processes is critical for obtaining enzymes with high activity and stability at reasonable costs [164,165,166]. Other research has focused on the economic analysis of the enzymatic biodiesel synthesis process [167], often using commercial enzymes such as Novozym^® 435 [161]. In these circumstances, the enzymes are included in an economic analysis as an additional material cost (with a considerable influence on expenses). In reality, enzyme-catalyzed biodiesel synthesis procedures differ significantly from conventional chemical processes in that the downstream process is typically easier. To avoid enzyme deactivation, more sophisticated upstream processes may be required, such as the progressive addition of alcohol [168].

The introduction of a biocatalytic phase in the large-scale pharmaceutical manufacturing of the antidiabetic medication sitagliptin (R)-20, which replaced the previously employed transition-metal catalyzed process, was a watershed moment in the area of biocatalysis. An engineered transaminase enzyme was used to successfully install a chiral amine in the final step of drug synthesis, replacing the original rhodium-catalyzed asymmetric enamine hydrogenation and eliminating the need for high pressure (250 psi) hydrogenation, ligand optimization and synthesis for the chiral Rh-catalyst, and additional purification steps to remove the precious and toxic rhodium catalyst. The biocatalytic step improved yields (by 10–13%), productivities (53% increase, kg L⁻¹ per day), and enantioselectivity. The addition of the biocatalytic phase was an important turning point in enzyme engineering capabilities, necessitating an outstanding engineering achievement that included a ‘substrate-walking’ method as well as computer modeling, docking investigations, and guided evolution [169].

15. Future Trends and Perspectives

The future of industrial biocatalysis will be driven by innovations that improve reaction efficiency, process integration, and environmental sustainability. A key advancement is multi-enzyme cascade systems, where enzymes work sequentially to mimic natural metabolism, enabling the streamlined synthesis of complex, high-value compounds. Optimizing enzyme ratios, timing, and interactions will be essential for consistent industrial performance. Nanotechnology is expanding enzyme applications through engineered nanomaterials like magnetic nanocarriers and metal–organic frameworks. These improve enzyme immobilization, reduce mass transfer limitations, and enhance thermal stability and reaction rates. Nano-biocatalysts also facilitate controlled reactions in confined environments, benefiting biosensors, microfluidics, and smart reactors.

Aligning biocatalysis with circular economy principles is another priority. Enzymes will increasingly convert renewable biomass and industrial residues into biofuels, biopolymers, and bio-based chemicals, supporting low-emission, zero-waste production. Growing environmental regulations and consumer demand for sustainable products will further boost enzyme-driven processes. Hybrid catalytic platforms combining biological and chemical catalysts are gaining traction, merging enzyme specificity with synthetic catalyst robustness to enable novel, efficient reactions. Advances in synthetic biology, AI-guided protein engineering, and automated screening will accelerate the development of these integrated systems across pharmaceuticals, renewable energy, and other sectors. Industrial biocatalysis will increasingly intersect with machine learning, metabolic engineering, and systems biology. These fields will provide predictive models and automation tools to enhance enzyme discovery, design, and deployment. Modular, adaptable enzyme technologies will help industries meet demands for cleaner, safer, and more efficient production. Looking ahead, progress will depend on interdisciplinary collaboration to translate these laboratory innovations into scalable, sustainable solutions. By integrating enzymes into smart manufacturing platforms, biocatalysis will be central to the transition toward a circular, bio-based economy.

16. Conclusions

Biocatalysis has rapidly evolved into a key technology in modern industrial biotechnology, supported by major advancements in enzyme discovery, molecular engineering, immobilization techniques, and reactor design. These developments have enabled enzymes to perform efficiently in diverse industrial applications, offering a sustainable alternative to traditional chemical processes. However, several challenges persist, including enzyme instability under harsh conditions, high production costs, and difficulties in scaling up from laboratory to industrial operations. The broad applicability of enzymes spans critical sectors such as pharmaceuticals, food and beverage processing, biofuels, textiles, and environmental remediation. Their high selectivity, mild operating conditions, and reduced environmental impact make them ideal for supporting green chemistry initiatives and circular economy models. Enzyme-driven processes help minimize energy use, eliminate hazardous reagents, and convert renewable resources or waste into valuable products, contributing directly to sustainability goals. Looking forward, the integration of digital and biological technologies will be pivotal in overcoming existing limitations and unlocking new opportunities. Artificial intelligence, machine learning, and computational enzyme modeling are poised to revolutionize enzyme design and optimization. Future trends also point toward the development of hybrid catalytic platforms, enzyme cascades, and nanobiocatalysts that combine biological precision with enhanced functionality. These innovations are expected to drive the next wave of growth in biocatalysis, making it a central enabler of cleaner, smarter, and more resilient industrial systems.