Search Results (461)

Biocatalysis is one of the oldest fields that has been used in industrial applications, with one of the earliest purposeful examples being the mass production of acetic acid from an immobilized Acinetobacter strain in the year 1815. Efficiency, specificity, reduced reaction times, lower overall costs, and environmental friendliness are some advantages biocatalysis has over conventional chemical synthesis, which has made biocatalysis increasingly used in industry. We highlight three necessary fields that are fundamental to advancing industrial biocatalysis, including biocatalyst engineering, solvent engineering, and mechanistic engineering. However, the fundamental mechanism of enzyme function is often overlooked or given less attention, which can limit the engineering process. In this review, we describe how mechanistic enzymology benefits industrial biocatalysis by elucidating key fundamental principles, including the k_cat and k_cat/K_m parameters. Mechanistic enzymology presents a unique field that provides in-depth insights into the molecular mechanisms of enzyme activity and includes areas such as reaction kinetics, catalytic mechanisms, structural analysis, substrate specificity, and protein dynamics. In line with the objective of protein engineering to optimize enzyme activity, we summarize a range of strategies reported in the literature aimed at improving the product release rate, the chemical step of catalysis, and the overall catalytic efficiency of enzymes. Further into this review, we delineate kinetic solvent viscosity effects (KSVEs) as a very efficient, cost-effective, and easy-to-perform method to probe different aspects of enzyme reaction mechanisms, including diffusion-dependent kinetic steps and rate-limiting steps. KSVEs are cost-effective because simple kinetic enzyme assays, such as the Michaelis–Menten kinetic approach, can be combined with them without the need for specialized and costly equipment. Other techniques in protein engineering and genetic engineering are also covered in this review. Additionally, we provide information on solvent systems in enzymatic reactions, details on immobilized biocatalysts, and common misconceptions that misguide enzyme design and optimization processes. Full article

: This study reports the development of a sustainable biocatalyst system for free fatty acid (FFA) production from cocoa bean shell (CBS) oil using Burkholderia cepacia lipase (BCL). CBS was explored as both a support material and a reaction substrate. Six immobilized systems were prepared using organic (CBS), inorganic (silica), and hybrid (CBS–silica) supports via physical adsorption or covalent binding. Among them, the covalently immobilized enzyme on CBS (ORG-CB) showed the most balanced performance, achieving a catalytic efficiency (Ke) of 0.063 mM⁻¹·min⁻¹ (18.6% of the free enzyme), broad pH–temperature tolerance, and over 50% activity retention after eight reuse cycles. Thermodynamic analysis confirmed enhanced thermal resistance for ORG-CB (Ed = 32.3 kJ mol⁻¹; ΔH‡ = 29.7 kJ mol⁻¹), while kinetic evaluation revealed that its thermal deactivation occurred faster than for the free enzyme under prolonged heating. In application trials, ORG-CB reached 60.1% FFA conversion from CBS oil, outperforming the free enzyme (49.9%). These findings validate CBS as a dual-function material for enzyme immobilization and valorization of agro-industrial waste. The results also reinforce the impact of immobilization chemistry and support composition on the operational and thermal performance of biocatalysts, contributing to the advancement of green chemistry strategies in enzyme-based processing. Full article

Unspecific peroxygenases (UPOs) are promising biocatalysts for oxyfunctionalizations in future sustainable economies and can be efficiently immobilized on the cell surface of their heterologous production yeast. This immobilization has versatile uses, ranging from the mL to m³ scale; but the production of the yeast surface displayed UPOs, and their handling has yet to be optimized to advance sustainable industrial processes in light of the UN’s sustainable development goals. Here, we present optimized production protocols for surface-displayed UPOs for shaken and stirred systems in different scales and describe suitable storage conditions and a sterilization method. We utilized one-factor-at-a-time and design of experiments approaches. We were able to streamline published protocols for shaken flask cultivations to achieve a 60% increase in volumetric activity, using reduced amounts of media. We also show at least a doubling of final activity for bioreactor cultivations by utilizing a different medium than the industry standard. Finally, we present a novel, robust protocol for parallelized methanol-induced enzyme production in Komagataella phaffii in a BioLector XT^® reactor. Enzyme activity did not decrease and even increased by our recommended sterilization method and during storage over 87 days. This study aims to advance the yeast surface display immobilization method by providing methods for efficient production, storage and utilization of this promising biocatalyst. Full article

Esterification is a key transformation in the production of lubricants, pharmaceuticals, and fine chemicals. Conventional processes employing homogeneous acid catalysts suffer from limitations such as corrosive byproducts, energy-intensive separation, and poor catalyst reusability. This review provides a comprehensive overview of heterogeneous catalytic systems, including ion exchange resins, zeolites, metal oxides, mesoporous materials, and others, for improved ester synthesis. Recent advances in membrane-integrated reactors, such as pervaporation and nanofiltration, which enable continuous water removal, shifting equilibrium and increasing conversion under milder conditions, are reviewed. Dual-functional membranes that combine catalytic activity with selective separation further enhance process efficiency and reduce energy consumption. Enzymatic systems using immobilized lipases present additional opportunities for mild and selective reactions. Future directions emphasize the integration of pervaporation membranes, hybrid catalyst systems combining biocatalysts and metals, and real-time optimization through artificial intelligence. Modular plug-and-play reactor designs are identified as a promising approach to flexible, scalable, and sustainable esterification. Overall, the interaction of catalyst development, membrane technology, and digital process control offers a transformative platform for next-generation ester synthesis aligned with green chemistry and industrial scalability. Full article

The growing need for developing safer and more effective methods for obtaining enantiomers of chiral compounds, particularly those with pharmacological activity, highlights the potential of biocatalysis as an appropriate pharmaceutical research direction. However, low catalytic activity and stability of free enzymes are often among the substantial limitations to the wide application of biocatalysis. Therefore, to overcome these obstacles, new technological procedures are being designed. In this study, we present optimized protocols for the immobilization of Candida antarctica lipase B (CALB) on an octyl- agarose support, ensuring high enantioselectivity in an organic reaction medium. The immobilization procedures (with drying step), including buffers with different pH values and concentrations, as well as the study of the influence of temperature and immobilization time, were presented. It was found that the optimal conditions were provided by citrate buffer with a pH of 4 and a concentration of 300 mM. The immobilized CALB on the octyl-agarose support exhibited high catalytic activity in the kinetic resolution of (R,S)-1-phenylethanol via enantioselective transesterification with isopropenyl acetate in 1,2-dichloropropane (DCP), as a model reaction for lipase activity monitoring on an analytical scale. HPLC analysis demonstrated that the (R)-1-phenylethyl acetate was obtained in an enantiomeric excess of ee_p > 99% at a conversion of approximately 40%, and the enantiomeric ratio was E > 200. Thermal and storage stability studies performed on the immobilized CALB octyl-agarose support confirmed its excellent stability. After 7 days of thermal stability testing at 65 °C in a climatic chamber, the (R)-1-phenylethyl acetate was characterized by enantiomeric excess of ee_p > 99% at a conversion of around 40% (similar values of catalytic parameters to those achieved using a non-stored lipase). The documented high catalytic activity and stability of the developed CALB-octyl-agarose support allow us to consider it as a useful tool for enantioselective transesterification in organic medium. Full article

This study discloses the noncovalent immobilization of a bienzyme cascade composed of glucose oxidase (GOx) and horseradish peroxidase (HRP) onto magnetically responsive polyamide microparticles (PA MPs). Porous PA6, PA4, and PA12 MPs containing iron fillers were synthesized via activated anionic ring-opening polymerization in suspension, alongside neat PA6 MPs used as a reference. Four hybrid catalytic systems (GOx/HRP@PA) were prepared through sequential adsorption of HRP and GOx onto the various PA MP supports. The initial morphologies of the supports and the hybrid biocatalysts were characterized by SEM, followed by evaluation of the catalytic performance using a two-step glucose oxidation cascade process. Among all systems, the GOx/HRP@PA4-Fe complex exhibited the highest activity, being approximately 1.5 times greater than the native enzyme dyad, followed by the PA6-supported system with slightly inferior performance. All systems obeyed Michaelis–Menten kinetics, with the immobilized cascades displaying higher Kₘ and Vₘₐₓ values than the non-immobilized enzyme pair while maintaining comparable catalytic efficiencies, CE (CE = k_cat/Kₘ). Subsequently, the immobilized and native enzyme systems were employed for the polymerization of aniline. According to UV–VIS, complete monomer conversion was achieved within 24 h for selected catalysts, and FTIR analysis confirmed the formation of polyaniline in the emeraldine base form without the use of template molecules. These findings highlight the potential of Fe-containing polyamide microparticles as efficient supports for the sustainable, enzyme-mediated synthesis of intrinsically conductive aromatic polymers. Full article

Water quality is essential for safeguarding human health and ensuring the stability of ecosystems. Nonetheless, the rising prevalence of emerging contaminants, particularly pharmaceutical compounds, has raised serious environmental concerns due to their bioactivity, widespread use, persistence, and potential toxicity. Among these, acetaminophen (paracetamol) is one of the most frequently detected pharmaceutical pollutants in aquatic environments. Among the various degradation strategies explored, biological methods, especially those involving white-rot fungi, have shown substantial promise owing to their production of ligninolytic enzymes capable of degrading complex pollutants. This study investigates the use of laccases from Ganoderma parvulum, covalently immobilized on chitosan microspheres, for acetaminophen degradation. The immobilization involved a 10% crosslinking agent, 60-min crosslinking time, and 10,000 U/L enzyme concentration, resulting in an immobilization efficiency of 123%, 203%, and 218%, respectively. The immobilized enzymes displayed enhanced stability across pH 3–8 and temperatures between 20 and 60 °C. Biodegradation assays achieved 97% acetaminophen removal within four hours. Nuclear Magnetic Resonance (¹H NMR and COSY) confirmed structural transformation. The enzymes also retained over 95% catalytic activity after multiple reuse cycles. These findings highlight the novel application of laccases as efficient and reusable biocatalysts for pharmaceutical pollutant removal, providing valuable insights into the mechanisms of enzymatic environmental remediation. Full article

In the present investigation, an eco-friendly biocatalyst was developed using Pleurotus eryngii laccase (PeLac) through a copper (Cu)-based protein–inorganic hybrid system for the degradation of bisphenol A, a representative xenobiotic. After partial purification, the specific activity of crude PeLac was 92.6 U/mg of total protein. Immobilization of PeLac as Cu₃(PO₄)₂–Lac (Cu–PeLac) nanoflowers (NFs) at 4 °C resulted in a relative activity 333% higher than that of the free enzyme. The Cu–PeLac NFs exhibited greater pH and temperature stability and enhanced catalytic activity compared to free laccase. This enhanced activity was validated through improved electrochemical properties. After immobilization, Cu–PeLac NFs retained up to 8.7-fold higher residual activity after storage at 4 °C for 30 days. Free and immobilized laccase degraded bisphenol A by 41.6% and 99.8%, respectively, after 2 h of incubation at 30 °C. After ten cycles, Cu–PeLac NFs retained 91.2% degradation efficiency. In the presence of potent laccase inhibitors, Cu–PeLac NFs exhibited a 47.3-fold improvement in bisphenol A degradation compared to free PeLac. Additionally, the synthesized Cu–PeLac NFs demonstrated lower acute toxicity against Vibrio fischeri than Cu nanoparticles. This study presents the first report of PeLac immobilization through an eco-friendly protein–inorganic hybrid system, with promising potential for degrading bisphenol A in the presence of inhibitors to support sustainable development. Full article

Enzymes, as nature’s precision biocatalysts, hold transformative potential across industrial, environmental, and biomedical sectors. However, their instability, solvent sensitivity, and limited reusability in their free form necessitate advanced immobilization strategies to enhance their robustness and scalability. This review critically examines cutting-edge advancements in enzyme immobilization, focusing on the integration of artificial intelligence (AI), novel nanomaterials, and dynamic carrier systems to overcome the traditional limitations of mass transfer, enzyme leakage, and cost inefficiency. Key innovations such as metal–organic frameworks (MOFs), magnetic nanoparticles, self-healing hydrogels, and 3D-printed scaffolds are highlighted for their ability to optimize enzyme orientation, stability, and catalytic efficiency under extreme conditions. Moreover, AI-driven predictive modeling and machine learning emerge as pivotal tools for rationalizing nanomaterial synthesis, multi-enzyme cascade design, and toxicity assessment, while microfluidic systems enable precise biocatalyst fabrication. This review also explores emerging carrier-free strategies, including cross-linked enzyme aggregates (CLEAs) and DNA-directed immobilization, which minimize diffusion barriers and enhance substrate affinity. Despite progress, challenges persist in regards to eco-friendly nanomaterial production, industrial scalability, and real-world application viability. Future directions emphasize sustainable hybrid material design, AI-aided lifecycle assessments, and interdisciplinary synergies between synthetic biology, nanotechnology, and data analytics. By connecting laboratory innovation with industrial needs, this work provides a forward-thinking framework to harness immobilized enzymes for achieving global sustainability goals, particularly in bioremediation, bioenergy, and precision medicine. Full article

In this study, we developed an innovative method for one-step enzyme purification and immobilization utilizing polysaccharide-based microspheres through a chitosan-binding module that mediated affinity adsorption. The chitosan-binding domain derived from Paenibacillus sp. IK-5 was genetically fused with multiple target enzymes (lysine decarboxylase, glutamate oxidase, and formate dehydrogenase), all of which were successfully expressed in soluble forms. Three distinct polysaccharide microspheres with optimized surface characteristics were engineered to facilitate the concurrent purification and immobilization of these fusion enzymes. Comprehensive characterization using organic elemental analysis, fluorescence microscopy, and thermogravimetric analysis confirmed the efficient immobilization of fusion enzymes. Remarkably, the immobilized enzymes demonstrated exceptional operational stability, maintaining over 80% of their initial catalytic activity after ten consecutive reuse cycles. This study establishes a robust and versatile platform for enzyme immobilization, providing significant advantages in biocatalyst engineering applications. Full article

Background: This study explored the biotransformation of (–)-isopulegol using immobilized cells of Rhodococcus rhodochrous IEGM 1362 to optimize the production of new bioactive compounds. Methods: An efficient biocatalyst based on R. rhodochrous IEGM 1362 cells immobilized in a macroporous polyvinyl alcohol (PVA) cryogel matrix was developed for the production of bioactive derivatives of (–)-isopulegol. The biological characteristics of the immobilized cells were investigated using scanning and transmission electron microscopy and energy-dispersive X-ray spectroscopy methods. Results: The use of the biocatalyst increased the overall yield of target products from 54% with free cells to 87% with immobilized cells in a single cycle. Major derivatives identified included (1R,2S,5R)-5-(hydroxymethyl)-2-(prop-1-en-2-yl)cyclohexanol and (1R,3R,4S)-3-hydroxy-4-(prop-1-en-2-yl)cyclohexanecarboxylic acid, both exhibiting potential pharmacological activity. The biocatalyst retained functional activity toward monoterpenoid over 13 exploitation cycles, meeting industrial biotechnology requirements. Immobilized cells were characterized by the absence of endogenous reserve inclusions (in particular lipids) and a high intracellular iron content. Conclusions: The developed immobilized biocatalyst is promising for scaling up the production of biologically active compounds. Full article

Surface display technology has revolutionized whole-cell biocatalysis by enabling efficient enzyme immobilization on microbial cell surfaces. Compared with traditional enzyme immobilization, this technology has the advantages of high enzyme activity, mild process, simple operation and low cost, which thus has been widely studied and applied in various fields. This review explores the principles, optimization strategies, applications in the food industry, and future prospects. We summarize the membrane and anchor protein structures of common host cells (Escherichia coli, Bacillus subtilis, and yeast) and discuss cutting-edge optimization approaches, including host strain genetic engineering, rational design of anchor proteins, innovative linker peptide engineering, and precise regulation of signal peptides and promoters, to maximize surface display efficiency. Additionally, we also explore its diverse applications in food processing and manufacturing, additive synthesis, food safety, and other food-related industries (such as animal feed and PET packaging degradation), demonstrating their potential to address key challenges in the food industry. This work bridges fundamental research and industrial applications, offering valuable insights for advancing agricultural and food chemistry. Full article

The need for sustainable and efficient water decontamination methods has led to the increasing use of redox enzymes such as glucose oxidase (GOx). GOx immobilization on textile supports provides a promising alternative for catalyzing pollutant degradation in bio-Fenton (BF) and bio-electro-Fenton (BEF) systems. However, challenges related to enzyme stability, reusability, and environmental impact remain a concern. This communication paper outlines innovative strategies developed to address these challenges, notably the use of ecotechnologies to achieve efficient GOx immobilization while maintaining biocatalytic activity. Plasma ecoprocesses, amino-bearing biopolymer-chitosan, as well as a bio-crosslinker genipin have been used efficiently on conductive carbon and non-conductive polyester-PET nonwovens. In certain cases, immobilized GOx can retain high catalytic activity after multiple cycles, making them an effective biocatalyst for organic dye degradation (Crystal Violet and Remazol Blue) via bio-Fenton reactions, including total heterogeneous bio-Fention system. Moreover, the conductive carbon felt-based bioelectrodes successfully supported simultaneous pollutant degradation and energy generation in a BEF system. This work highlights the potential of textile-based enzyme immobilization for sustainable wastewater treatment, bio-electrochemical energy conversion, and also for bacterial deactivation. Future research will focus on optimizing enzyme stability and enhancing BEF efficiency for large-scale applications. Full article

Efficient hydrolysis of cellulose in agricultural waste (e.g., coconut fiber) is critical for biorefining processes such as second-generation bioethanol (2G ethanol) production. However, free cellulases suffer from low thermal stability and challenges in recovery. To address this, we developed cross-linked enzyme aggregates (CLEAs) combined with magnetic nanoparticles (magnetic CLEAs, m-CLEAs) to enhance enzyme stability and reusability. In this context, solutions of ethanol, acetone, and ammonium sulfate were used to prepare enzymatic aggregates, with subsequent use of glutaraldehyde and magnetic nanoparticles to obtain the biocatalysts. The addition of bovine serum albumin (BSA) protein was also tested to improve immobilization. Biocatalysts with ethanol and acetone performed better. Acetone (AC) and BSA yielded the highest enzymatic activities (287.27 ± 42.59 U/g for carboxymethyl cellulase (CMCase) with Celluclast; 425.37 ± 48.11 U/g for CMCase with Cellic CTec2). Magnetic nanoparticles were incorporated to expand the industrial applicability, producing m-CLEAs with excellent thermal stability and high catalytic activities. The m-CLEA–Celluclast–AC–BSA–GA 5% maintained 58% of its activity after 72 h at 70 °C. The m-CLEA–Celluclast-AC–BSA–GA 2.5% proved effective in hydrolyzing coconut fiber and isolated cellulose, producing up to 0.91 ± 0.01 g/L of glucose and 2.7 ± 0.15 g/L of glucose, respectively, after 72 h. Therefore, this approach supports sustainability by using coconut fiber, which is often discarded into the environment. Full article

The sustainable production of healthy structured lipids (SLs) using oils extracted from agro-industry by-products or non-conventional lipid sources is of utmost importance in the framework of a circular bioeconomy, toward a zero-waste goal. In this study, low-calorie triacylglycerols (TAGs) containing a long-chain (L) fatty acid (FA) at position sn-2 and medium-chain (M) FAs at positions sn-1,3 (MLM type SL) were obtained from virgin cold-pressed milk thistle (51.55% linoleic acid; C18:2), grapeseed (66.62% C18:2), and apricot kernel (68.61% oleic acid; C18:1) oils. Lipase-catalyzed acidolysis with capric acid (C10:0) or interesterification with ethyl caprate (C10 Ethyl) in solvent-free media were performed. In batch reactions, immobilized Rhizomucor miehei lipase (Lipozyme RM) was used as a biocatalyst. For all tested oils, new TAG (SL) yields, varying from 61 to 63%, were obtained after 6 h of interesterification. Maximum new TAG yields were reached after 6, 24, and 30 h of acidolysis with grapeseed (64.7%), milk thistle (56.1%), or apricot kernel (69.7%) oils, respectively. Continuous acidolysis and interesterification of grapeseed oil were implemented in a packed-bed bioreactor, catalyzed by immobilized Thermomyces lanuginosus lipase (Lipozyme TL IM). Throughout 150 h of continuous operation, no lipase deactivation was observed, with average SL yields of 79.2% ± 4.1 by interesterification and 61.5% ± 5.91 by acidolysis. Full article