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Carbon mineralization is an emerging field of research in carbon sequestration. In this process, dissolved inorganic carbon reacts with mineral cations such as Ca2+ and Mg2+ to form stable carbonate minerals, enabling permanent carbon sequestration and storage. However, current mineralization methods predominantly rely on physicochemical approaches to expedite the mineralization of carbon. While effective, these methods require substantial chemical and energy consumption and may cause significant environmental impacts. Biomineralization has recently emerged as a sustainable alternative, leveraging biochemical reactions to catalyze CO2 mineralization. This research focuses on investigating the specific roles of various biomolecules in natural carbon biomineralization and exploring state-of-the-art biomimetic carbon mineralization techniques, including whole-cell microbially induced carbonate precipitation (MICP) and cell-free systems, for carbon sequestration. In addition, we discuss various sources of mineral cations, ranging from natural minerals to industrial waste to seawater, along with their advantages and limitations. Our findings highlight the potential and feasibility of biological carbon mineralization processes to contribute towards sustainable carbon sequestration. However, we also identify challenges and propose future directions to guide further research and the application of these processes.

Bio‐catalysis represents a highly efficient and stereoselective method for the synthesis of valuable chiral compounds, however, the poor stability and limited reaction types of free enzymes restrict their wide application in industrial production. In this work, to overcome these problems, a multifunctional photoenzymatic nanoreactor CALB@COF‐Ir was developed through the encapsulation of Candida antarctica lipase B (CALB) in a photosensitive covalent organic framework COF‐Ir. This bio‐nanocluster serves as efficient catalysts in asymmetric dynamic kinetic resolution (DKR) of secondary amines to give a series of chiral amines in high yields (up to 99 %) and enantioselectivities (up to 99 % ee). The well‐designed COF‐Ir not only acts as safety cover to prevent CALB from deactivation but promotes racemization of secondary amines via photo‐induced hydrogen atom transfer (HAT) process. Photoelectric characterization and TDDFT calculation revealed that (ppy)2Ir units in COF‐Ir play crucial role in this photocatalytic system which enhance its photo‐redox properties through facilitating the separation between photoelectrons (e−) and holes (h+). Furthermore, the heterogeneous photoenzymatic nanoreactor could be recycled for five rounds with slight decline of catalytic reactivity. [ABSTRACT FROM AUTHOR]

Ethyl (S)-4-chloro-3-hydroxybutyrate ((S)-CHBE) is an important chiral intermediate in the synthesis of the cholesterol-lowering drug atorvastatin. Studying the use of SpyTag/SpyCatcher and SnoopTag/SnoopCatcher systems for the asymmetric reduction reaction and directed coupling coenzyme regeneration is practical for efficiently synthesizing (S)-CHBE. In this study, Spy and Snoop systems were used to construct a double-enzyme directed fixation system of carbonyl reductase (BsCR) and glucose dehydrogenase (BsGDH) for converting 4-chloroacetoacetate (COBE) to (S)-CHBE and achieving coenzyme regeneration. We discussed the enzymatic properties of the immobilized enzyme and the optimal catalytic conditions and reusability of the double-enzyme immobilization system. Compared to the free enzyme, the immobilized enzyme showed an improved optimal pH and temperature, maintaining higher relative activity across a wider range. The double-enzyme immobilization system was applied to catalyze the asymmetric reduction reaction of COBE, and the yield of (S)-CHBE reached 60.1% at 30 °C and pH 8.0. In addition, the double-enzyme immobilization system possessed better operational stability than the free enzyme, and maintained about 50% of the initial yield after six cycles. In summary, we show a simple and effective strategy for self-assembling SpyCatcher/SnoopCatcher and SpyTag/SnoopTag fusion proteins, which inspires building more cascade systems at the interface. It provides a new method for facilitating the rapid construction of in vitro immobilized multi-enzyme complexes from crude cell lysate. [ABSTRACT FROM AUTHOR]

The use of biocompatible ionic liquids (bio-ILs) as a "green" solvent with uniquely tunable cations and anions, specific selectivity, and many other desirable physicochemical properties in biocatalysis has shown great promises to address many challenges. A plethora of studies have demonstrated ILs possess a range of exceptional capabilities in coordinating biocatalysis such as stabilizing enzyme structure, enhancing enzyme-substrate interaction, increasing substrate solubility and product separation/removal, and improving enzyme immobilization/recycling. Therefore, bio-ILs have been explored for biocatalysis in a wide range of homogenous as well as multiphasic systems including biphasic, multiphasic, microemulsion, and whole-cell systems. This review provides an overview of IL-enabled biocatalysis including enzymatic and whole-cell reactions in homogeneous and multiphasic systems. First, the properties of IL concentration, specific ion effect, pH, viscosity, alkyl chain length, hydrophobicity, and polarity on the structures and activities of enzymes are discussed together with characterization techniques to elucidate the mechanisms of IL-enzyme interactions. Second, while homogenous biocatalysis systems containing ILs are briefly introduced and summarized, particular attention is given to multiphasic systems. Furthermore, recent progress in using bio-ILs to modify emerging new enzyme carrier materials including carbonaceous and magnetic nanomaterials, gel microspheres, and membranes as well as their effect on increasing enzyme stability and recycling are highlighted. Finally, we provide our expert opinion for more innovative research in the field and future outlook for the industrial application of ILs in biocatalysis. [ABSTRACT FROM AUTHOR]

Advancements in nanotechnology have ushered in nanosponges, biodegradable polyester structures with nanometersized cavities, designed for targeted drug delivery. Initially developed for topical applications, these nanosponges have evolved to be administered orally and intravenously. They exhibit a porous structure for controlled drug release, addressing issues like toxicity and poor bioavailability. Capable of carrying both hydrophilic and lipophilic drugs, nanosponges serve as versatile carriers for substances like enzymes, proteins, vaccines, and antibodies. Their characteristics include size and polarity control, crystalline forms, non-toxicity, stability in various conditions, suspension and regeneration capabilities, and a 3D structure for targeted capture and release. The composition involves a polymer, a cross-linking agent, and specific drug criteria. Advantages encompass targeted delivery, flavor masking, reduced side effects, water solubility, adjustable particle size, and easy commercial production. Disadvantages include limited encapsulation for larger molecules. The mechanism involves an open structure allowing prolonged release. Various types based on cyclodextrin offer unique properties. Factors affecting nanosponges include polymer nature, drug characteristics, complexation temperature, and degree of substitution. Preparation methods include solvent, ultrasound-assisted, melt, bubble electrospinning, and emulsion solvent diffusion methods. Comprehensive characterization includes drug entrapment efficiency, saturation state interaction, in vitro release studies, porosity measurement, and spectroscopic techniques. Applications span solubility enhancement, sustained delivery, oral and topical systems, protein delivery, protection from degradation, pollutant removal from water, cancer treatment, antiviral applications, enzyme immobilization, and modulation of drug release. [ABSTRACT FROM AUTHOR]

Enzyme-based biosensors commonly utilize the drop-casting method for their surface modification. However, the drawbacks of this technique, such as low reproducibility, coffee ring effects, and challenges in mass production, hinder its application. To overcome these limitations, we propose a novel surface functionalization strategy of enzyme crosslinking via inkjet printing for reagentless enzyme-based biosensors. This method includes printing three functional layers onto a screen-printed electrode: the enzyme layer, crosslinking layer, and protective layer. Nanomaterials and substrates are preloaded together during our inkjet printing. Inkjet-printed electrodes feature a uniform enzyme deposition, ensuring high reproducibility and superior electrochemical performance compared to traditional drop-casted ones. The resultant biosensors display high sensitivity, as well as a broad linear response in the physiological range of the serum phosphate. This enzyme crosslinking method has the potential to extend into various enzyme-based biosensors through altering functional layer components. [ABSTRACT FROM AUTHOR]

Organic matter-induced mineralization is a green and versatile method for synthesizing hybrid nanostructured materials, where the material properties are mainly influenced by the species of natural biomolecules, linear synthetic polymer, or small molecules, limiting their diversity. Herein, we adopted dendrimer poly(amidoamine) (PAMAM) as the inducer to synthesize organosilica-PAMAM network (OSPN) capsules for mannose isomerase (MIase) encapsulation based on a hard-templating method. The structure of OSPN capsules can be precisely regulated by adjusting the molecular weight and concentration of PAMAM, thereby demonstrating a substantial impact on the kinetic behavior of the MIase@OSPN system. The MIase@OSPN system was used for catalytic production of mannose from D-fructose. A mannose yield of 22.24% was obtained, which is higher than that of MIase in organosilica network capsules and similar to that of the free enzyme. The overall catalytic efficiency (kcat/Km) of the MIase@OSPN system for the substrate D-fructose was up to 0.556 s−1·mmol−1·L. Meanwhile, the MIase@OSPN system showed excellent stability and recyclability, maintaining more than 50% of the yield even after 12 cycles. [ABSTRACT FROM AUTHOR]

A solution of porcine pancreas lipase was adsorbed using activated carbons derived from both raw cocoa husk and fermented cocoa husk (ACCPr). The resulting immobilized biocatalysts, referred to as ACCPrI and ACCI, were subjected to characterization for textural parameters, thermal and spectroscopic analyses, and enzymatic immobilization capacity. The surface area, pore volume, and hydrolytic activity yield were increased by 5, 15, and 9% respectively, in addition to the selectivity in the adsorption of the enzyme with 52 kDa, when using the fermented biomass in the production of the biocatalyst. Activated carbon with suitable characteristics for enzyme immobilization was able to be obtained using this simple and sustainable method. The integral use of biomass is facilitated by this technology, and the reduction of environmental impacts caused by inadequate disposal of fermented residues is achieved. [ABSTRACT FROM AUTHOR]

Horseradish peroxidase is an enzyme commonly used in wastewater treatment due to its ability to oxidize a wide range of organic compounds, including phenols. The use of peroxidases immobilized onto magnetite nanoparticles improves the enzyme's stability and catalytic activity, but also facilitates their simply separation and reuse. In the present study, a covalent immobilization of horseradish peroxidase onto polyethylene glycol-modified magnetite nanoparticles via glutaraldehyde was performed in order to prepare a promising bio-catalyst for phenol removal from wastewater. The efficiency of the immobilized enzyme in phenol removal in the presence of hydrogen peroxide and polyethylene glycol as stabilizer was measured. The general toxicity of bio-treated water (Allium cepa test) was also investigated. All analyses were performed in parallel for the free enzyme. The immobilized enzyme showed the highest activity at a temperature of 50 °C and pH 7.0 and retained 50% of its activity after four washing cycles. Additionally, its storage stability was higher compared to the free form, as well as its tolerance against inactivation to heavy metals and organics-induced inhibition. Since the tested enzymatic system was very efficient in phenol removal from water (75% for 2 h), along with the substantial reduction of the toxicity of the tested water (48%), it can be considered as an environmentally acceptable bio-catalyst for phenol-containing wastewater treatment. [ABSTRACT FROM AUTHOR]

Light-induced electron transfer can broaden the substrate range of metalloenzyme. However, the efficiency of photo-enzyme coupling is limited by the poor combination of photosensitizer or photocatalyst with enzyme. Herein, we prepared the nano-photocatalyst MIL-125-NH 2 @Ru(bpy) by in site embedding ruthenium pyridine-diimine complex [Ru(bpy) 3 ]2+ into metal organic frameworks MIL-125-NH 2 and associated it with multicopper oxidase (MCO) laccase. Compared to [Ru(bpy) 3 ]2+, the coupling efficiency of MIL-125-NH 2 @Ru(bpy) 3 for enzymatic oxygen reduction increased by 35.7%. A series of characterizations confirmed that the amino group of laccase formed chemical bonds with the surface defects or hydrophobic groups of MIL-125-NH 2 @Ru(bpy) 3. Consequently, the tight binding accelerated the quenching process and electron transfer between laccase and the immobilized ruthenium pyridine-diimine complex. This work would open an avenue for the synthesis of MOFs photocatalyst towards photo-enzyme coupling. [ABSTRACT FROM AUTHOR]

In this work, the diamine oxidase (DAO) produced from pea seedlings was partially purified using ammonium sulphate precipitation, dialysis, and gel filtration chromatography. Partially purified DAO was immobilized covalently with the help of a cross-linker glutaraldehyde onto the chitosan- modified gold electrode. The properties of immobilized enzyme were evaluated by optimum pH, optimum temperature, activity recovery, and recyclability using putrescine dihydrochloride as substrate. After immobilization of DAO, optimum pH ranges from 6.0 to 7.0 did not change and optimum temperature of enzyme changed from 34 to 38°C in comparison to free DAO. The immobilized DAO preserved 81% activity of free DAO and permitted increased stability and reusability of the enzyme than its native form. After 15 cycles of usage, the immobilized DAO maintained around 51% of its original activity, and this activity prolonged for 30 days at 4°C. [ABSTRACT FROM AUTHOR]

Flow biocatalysis has emerged as an empowering tool to boost the potential of enzymatic reactions towards more automatized, sustainable, and generally efficient synthetic processes. In the last fifteen years, the increasing number of biocatalytic transformations carried out in continuous flow exemplified the benefits that this technology can bring to incorporate biocatalysis into industrial operations. This perspective aims to capture in a nutshell the available methodologies for flow biocatalysis as well as to discuss the current limitations and the future directions in this field. Article highlights: Integration of enzymatic reactions into flow reactors is an exponentially growing technology. Flow biocatalysis can produce valuable molecules for food, cosmetics, and pharmaceuticals in a more eco-friendly manner. An interdisciplinary effort is needed to overcome the current challenges for industrial flow biocatalysis. [ABSTRACT FROM AUTHOR]

The merging of biocatalysis with continuous-flow chemistry opens up new opportunities for sustainable and efficient chemical synthesis. Cofactor-dependent enzymes are essential for various industrially attractive biocatalytic reactions. However, implementing these enzymes and biocatalytic reactions in industry remains challenging due to the inherent cost of cofactors and the requirement for their external supply in significant quantities. The development of efficient, low cost, simple and versatile methods for cofactor immobilization can address this important obstacle for biocatalysis in flow. This review explores recent progress in cofactor immobilization for biocatalysis by analyzing advantages and current limitations of the available methods that comprise covalent tethering, ionic adsorption, physical entrapment, and hybrid variations thereof. Moreover, this review analyzes all these immobilization techniques specifically for their utilization in continuous-flow chemistry and provides a perspective for future work in this area. This review will serve as a guide for steering the field towards more sustainable and economically viable continuous-flow biocatalysis. Highlights: Integration of cofactor immobilization along with enzyme immobilization can greatly enhance the sustainability of biocatalysis in flow. Various cofactor immobilization methodologies are available, each of them demonstrating a unique set of advantages and limitations. Hybrid co-immobilization methodologies for cofactors and enzymes seem to be promising for the generation of a more universal platform for biocatalysis in flow. [ABSTRACT FROM AUTHOR]

The number of biocatalyzed reactions at industrial level is growing rapidly together with our understanding on how we can maximize the enzyme efficiency, stability and productivity. While biocatalysis is nowadays recognized as a greener way to operate in chemistry, its combination with continuous processes has lately come up as a powerful tool to enhance process selectivity, productivity and sustainability. This perspective aims at describing the recent advances of this technology and future developments leading to smart, efficient and greener strategies for process optimization and large-scale production. [ABSTRACT FROM AUTHOR]

Low‐cost ceramic microfiltration membranes were explored for immobilizing lipases. Expensive commercial ceramics (e.g., alumina, zirconia) have advantages like chemical robustness, but high sintering temperatures limit their usability. To overcome this, researchers used Fuller's earth clay with rice husk ash to create cost‐effective membranes. However, fewer reactive sites on ceramic surfaces hinder enzyme immobilization. To address this, 3‐aminopropyltriethoxysilane and glutaraldehyde activated the membrane for covalent lipase binding. The immobilized membrane retained activity for over five 1‐h reaction cycles with a minimal performance decline of around 60 %. After five cycles, it retained over 40 % of the initial activity. These results show promise for using low‐cost ceramic membranes in various industries, such as in food, beverages, pharmaceuticals, and chemical production. [ABSTRACT FROM AUTHOR]

The practical application of enzymes as a biocatalyst still faces serious challenges, namely inactivation and loss. Immobilized enzyme technology can significantly improve the recoverability, stability and environmental adaptability of enzymes. Magnetic nanocomposites are widely used in enzyme immobilization because of their magnetic properties and the composite properties of various nanomaterials. In this work, the research progress of magnetic nanocomposites as enzyme carriers is reviewed and the future development direction is prospected. [ABSTRACT FROM AUTHOR]

Background: In recent years, the production of inclusion bodies that retain substantial catalytic activity was demonstrated. These catalytically active inclusion bodies (CatIBs) are formed by genetic fusion of an aggregation-inducing tag to a gene of interest via short linker polypeptides. The resulting CatIBs are known for their easy and cost-efficient production, recyclability as well as their improved stability. Recent studies have outlined the cooperative effects of linker and aggregation-inducing tag on CatIB activities. However, no a priori prediction is possible so far to indicate the best combination thereof. Consequently, extensive screening is required to find the best performing CatIB variant. Results: In this work, a semi-automated cloning workflow was implemented and used for fast generation of 63 CatIB variants with glucose dehydrogenase of Bacillus subtilis (BsGDH). Furthermore, the variant BsGDH-PT-CBDCell was used to develop, optimize and validate an automated CatIB screening workflow, enhancing the analysis of many CatIB candidates in parallel. Compared to previous studies with CatIBs, important optimization steps include the exclusion of plate position effects in the BioLector by changing the cultivation temperature. For the overall workflow including strain construction, the manual workload could be reduced from 59 to 7 h for 48 variants (88%). After demonstration of high reproducibility with 1.9% relative standard deviation across 42 biological replicates, the workflow was performed in combination with a Bayesian process model and Thompson sampling. While the process model is crucial to derive key performance indicators of CatIBs, Thompson sampling serves as a strategy to balance exploitation and exploration in screening procedures. Our methodology allowed analysis of 63 BsGDH-CatIB variants within only three batch experiments. Because of the high likelihood of TDoT-PT-BsGDH being the best CatIB performer, it was selected in 50 biological replicates during the three screening rounds, much more than other, low-performing variants. Conclusions: At the current state of knowledge, every new enzyme requires screening for different linker/aggregation-inducing tag combinations. For this purpose, the presented CatIB toolbox facilitates fast and simplified construction and screening procedures. The methodology thus assists in finding the best CatIB producer from large libraries in short time, rendering possible automated Design-Build-Test-Learn cycles to generate structure/function learnings. [ABSTRACT FROM AUTHOR]

The oxidation of 5‐hydroxymethylfurfural (HMF) to 2,5‐diformylfuran (DFF) is a key reaction in valorizing biomass. DFF is hardly soluble in water, while HMF is often obtained from biorefineries in crude wet organic fractions. Thus, the reaction is challenging for both biocatalysis performed in aqueous media, and for chemocatalysis where the presence of water often results in catalyst poisoning. Galactose oxidase (GalOx) can selectively oxidize HMF to DFF and displays promising activity in aqueous‐organic media. In this study, GalOx was immobilized on ten carriers, assessing the immobilization yield, activity, and stability. Covalently immobilized GalOx catalyzed the oxidation of HMF to DFF in neat and water‐saturated EtOAc, and in biphasic systems of various water contents. At 50 % v/v H2O, the reaction was conducted at a semi‐preparative scale (50 mL) with no adverse effect on DFF yield. Some limitations arise, such as enzyme deactivation, and adsorption of DFF to the support, particularly in the aqueous fraction. Future options to upgrade the route may include designed stable enzymes under the presence of HMF/DFF, and the setup of microaqueous systems where DFF adsorption is minimized. The use of wet EtOAc media would be a promising approach in future biorefineries employing inexpensive crude wet organic fractions. [ABSTRACT FROM AUTHOR]

Utilizing covalent organic framework (COF) as a hypotoxic and porous scaffold to encapsulate enzyme (enzyme@COF) has inspired numerous interests at the intersection of chemistry, materials, and biological science. In this study, we report a convenient scheme for one‐step, aqueous‐phase synthesis of highly crystalline enzyme@COF biocatalysts. This facile approach relies on an ionic liquid (2 μL of imidazolium ionic liquid)‐mediated dynamic polymerization mechanism, which can facilitate the in situ assembly of enzyme@COF under mild conditions. This green strategy is adaptive to synthesize different biocatalysts with highly crystalline COF "exoskeleton", as well evidenced by the low‐dose cryo‐EM and other characterizations. Attributing to the rigorous sieving effect of crystalline COF pore, the hosted lipase shows non‐native selectivity for aliphatic acid hydrolysis. In addition, the highly crystalline linkage affords COF "exoskeleton" with higher photocatalytic activity for in situ production of H2O2, enabling us to construct a self‐cascading photo‐enzyme coupled reactor for pollutants degradation, with a 2.63‐fold degradation rate as the poorly crystalline photo‐enzyme reactor. This work showcases the great potentials of employing green and trace amounts of ionic liquid for one‐step synthesis of crystalline enzyme@COF biocatalysts, and emphasizes the feasibility of diversifying enzyme functions by integrating the reticular chemistry of a COF. [ABSTRACT FROM AUTHOR]

The catalytic transformation (hydrolysis) of complex lignocellulosic polysaccharides into fermentable monosaccharides is crucial for producing bioethanol. Hydrolysis of various lignocellulosic biomasses can be achieved via traditional acid treatment (hydrochloric and sulfuric acid) and enzymatic catalysis. Considering the high specificity, selectivity, and prospective biocatalytic performance, enzyme biocatalysts are the preferred choice for an efficient and eco-friendlier deconstruction of lignocellulosic biomasses into simple fermentable sugars. Enzymes are a fascinating class of biodegradable catalysts that consume low energy and catalyze functioning under ambient environmental conditions. Recently, applying immobilized enzymes has emerged as a prodigious option in lignocellulosic biomass valorization. Immobilizing various enzymes, including cellulase, cellobiose, β-glucosidase, laccase, and xylanase, on nano-support materials to constitute nanobiocatalyst has shown increased biocatalytic efficiency and stability. Furthermore, enzyme immobilization on magnetic nanomaterials promotes facile retrieval, separation, and recycling of biocatalysts. Consequently, the development and implementation of nanobiocatalytic systems offer an eco-friendly and cost-effective approach for the catalytic deconstruction of lignocellulosic biomass. This review article provides a comprehensive insight into contemporary developments and the application of nanomaterial-immobilized biocatalysts to catalyze the degradation of lignocellulosic biomasses. After a succinct overview of characteristics of key lignocellulosic biorefinery enzymes and immobilization techniques, and novel material supports, the application of nanomaterial-immobilized cellulase-based biocatalysts to catalyze the degradation and conversion of lignocellulosic biomasses is thoroughly vetted by compiling with state-of-the-art examples. Moreover, reactors for immobilized cellulase systems, major challenges, research gaps, and future endeavors are also directed. In conclusion, this study provides a new horizon to bridging the main knowledge gaps and explicit barriers of these bioprocesses to harnessing the full adventure of enzyme immobilization to realize sustainable biorefinery. [ABSTRACT FROM AUTHOR]

The escalating demand for processed foods has led to the widespread industrial use of glucose isomerase (GI) for high-fructose corn syrup (HFCS) production. This reliance on GIs necessitates continual Co2+ supplementation to sustain high catalytic activity across multiple reaction cycles. In this study, Serratia marcescens GI (SmGI) was immobilized onto surfaces of the metal-organic framework (MOF) material MOF (Co)-525 to generate MOF (Co)-525-GI for use in catalyzing glucose isomerization to generate fructose. Examination of MOF (Co)-525-GI structural features using scanning electron microscopy-energy dispersive spectroscopy, Fourier-transform infrared spectroscopy, and ultraviolet spectroscopy revealed no structural changes after SmGI immobilization and the addition of Co2+. Notably, MOF (Co)-525-GI exhibited optimal catalytic activity at pH 7.5 and 70 °C, with a maximum reaction rate (Vmax) of 37.24 ± 1.91 μM/min and Km value of 46.25 ± 3.03 mM observed. Remarkably, immobilized SmGI exhibited sustained high catalytic activity over multiple cycles without continuous Co2+ infusion, retaining its molecular structure and 96.38% of its initial activity after six reaction cycles. These results underscore the potential of MOF (Co)-525-GI to serve as a safer and more efficient immobilized enzyme technology compared to traditional GI-based food-processing technologies. [ABSTRACT FROM AUTHOR]

Excessive CO2 and food shortage are two grand challenges of human society. Directly converting CO2 into food materials can simultaneously alleviate both, like what green crops do in nature. Nevertheless, natural photosynthesis has a limited energy efficiency due to low activity and specificity of key enzyme D‐ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO). To enhance the efficiency, many prior studies focused on engineering the enzymes, but this study chooses to learn from the nature to design more efficient reactors. This work is original in mimicking the stacked structure of thylakoids in chloroplasts to immobilize RuBisCO in a microreactor using the layer‐by‐layer strategy, obtaining the continuous conversion of CO2 into glucose precursor at 1.9 nmol min−1 with enhanced activity (1.5 times), stability (≈8 times), and reusability (96% after 10 reuses) relative to the free RuBisCO. The microreactors are further scaled out from one to six in parallel and achieve the production at 15.8 nmol min−1 with an energy conversion efficiency of 3.3 times of rice, showing better performance of this artificial synthesis than NPS in terms of energy conversion efficiency. The exploration of the potential of mass production would benefit both food supply and carbon neutralization. [ABSTRACT FROM AUTHOR]

Rational immobilization of fragile enzymes on a scaffold is an efficient protocol to achieve robust biocatalyst, yet the construction remains challenging due to the trade‐off between enzyme activity and framework environment. Herein, an enzyme‐engineered assembly strategy enabling the in situ fusion of acetylcholinesterase (AChE) into Zn‐based metal‐organic frameworks (Zn‐MOF‐74) during the rapid nucleation and crystal defect growth process, is demonstrated. The Zn‐MOF‐74 composites can offer a hydrophilic framework microenvironment for maintaining the biological state of AChE, with flower‐like porous structure that allows for boosting the mass transfer process of substrates and products. Impressively, the apparent activity of AChE can be well maintained 90% after encapsulation, and the AChE@Zn‐MOF‐74 composites produced excellent stability in inhospitable environments. Given the structural integration, a robust AChE@Zn‐MOF‐74‐based biosensor is constructed for the sensitive detection of chlorpyrifos (organophosphorus pesticides) with a detection limit of 1.0 ng mL−1, enabling the monitoring of pesticide degradation extent in the water body. This study is anticipated to offer valuable insight into understanding the structure‐activity relationship of enzyme@MOF to encourage applications in the construction of robust biosensors for precision agriculture. [ABSTRACT FROM AUTHOR]

The objective of this study was to develop a bioprocess for lactose hydrolysis in diverse dairy matrices, specifically skim milk and cheese whey, utilizing column reactors employing a core–shell enzymatic system featuring β-galactosidase fused to a Cellulose Binding Domain (CBD) tag (β-galactosidase-CBD). The effectiveness of reactor configurations, including ball columns and toothed columns operating in packed and fluidized-bed modes, was evaluated for catalyzing lactose hydrolysis in both skim milk and cheese whey. In a closed system, these reactors achieved lactose hydrolysis rates of approximately 50% within 5 h under all evaluated conditions. Considering the scale of the bioprocess, the developed enzymatic system was capable of continuously hydrolyzing 9.6 L of skim milk while maintaining relative hydrolysis levels of approximately 50%. The biocatalyst, created by immobilizing β-galactosidase-CBD on magnetic core-shell capsules, exhibited exceptional operational stability, and the proposed bioprocess employing these column reactors showcases the potential for scalability. [ABSTRACT FROM AUTHOR]

Microfluidic devices have attracted much attention in the current day owing to the unique advantages they provide. However, their application for industrial use is limited due to manufacturing limitations and high cost. Moreover, the scaling-up process of the microreactor has proven to be difficult. Three-dimensional (3D) printing technology is a promising solution for the above obstacles due to its ability to fabricate complex structures quickly and at a relatively low cost. Hence, combining the advantages of the microscale with 3D printing technology could enhance the applicability of microfluidic devices in the industrial sector. In the present work, a 3D-printed single-channel immobilized enzyme microreactor with a volume capacity of 30 μL was designed and created in one step via the fused deposition modeling (FDM) printing technique, using polylactic acid (PLA) as the printing material. The microreactor underwent surface modification with chitosan, and β-glucosidase from Thermotoga maritima was covalently immobilized. The immobilized biocatalyst retained almost 100% of its initial activity after incubation at different temperatures, while it could be effectively reused for up to 10 successful reaction cycles. Moreover, a multi-channel parallel microreactor incorporating 36 channels was developed, resulting in a significant increase in enzymatic productivity. [ABSTRACT FROM AUTHOR]

Abstract A novel and versatile approach called “physical imprinting” is introduced to modulate enzyme conformation using mesoporous materials, addressing challenges in achieving improved enzyme activity and stability. Metal–organic frameworks with tailored mesopores, precisely matching enzyme size and shape, are synthesized. Remarkably, enzymes encapsulated within these customized mesopores exhibit over 1670% relative activity compared to free enzymes, maintaining outstanding efficiency even under harsh conditions such as heat, exposure to organic solvents, wide‐ranging pH extremes from acidic to alkaline, and exposure to a digestion cocktail. After 18 consecutive cycles of use, the immobilized enzymes retain 80% of their initial activity. Additionally, the encapsulated enzymes exhibit a substantial increase in catalytic efficiency, with a 14.1‐fold enhancement in kcat/KM compared to native enzymes. This enhancement is among the highest reported for immobilized enzymes. The improved enzyme activity and stability are corroborated by solid‐state UV–vis, electron paramagnetic resonance, Fourier‐transform infrared spectroscopy, and solid‐state NMR spectroscopy. The findings not only offer valuable insights into the crucial role of size and shape complementarity within confined microenvironments but also establish a new pathway for developing solid carriers capable of enhancing enzyme activity and stability.

Lipase-catalyzed organic reactions have been widely practiced in the past three decades. Especially interesting are insoluble/immobilized forms due to providing a possibility of facile use and recyclability, thus reducing process costs, and making the procedure more environmentally friendly. Carbon-based supports have been extensively exploited for this purpose, because of neutral and biodegradable nature and thermal and chemical stability. Their high specific surface area, characteristic surface morphology and lower mass transfer resistances play a vital role in the performance of the attached enzyme. This review paper presents an overview of the main aspects of lipase immobilized on multi-walled carbon nanotubes (MWCNTs). Moreover, different immobilization strategies to achieve a biocatalyst with improved performances are discussed. Furthermore, as lipases are considered to have high commercial worth for synthesis of valuable organic molecules, the second part of the paper is dedicated to the overview of the most important industrial sectors in which these nanobiocatalysts have been used. In specific, applications in biodiesel production, flavour ester synthesis and racemization are summarize

This editorial published in the journal Frontiers in Microbiology discusses extremozymes, which are enzymes obtained from organisms that thrive in extreme environmental conditions. The editorial highlights the unique stability and function of extremozymes in harsh conditions, making them valuable in various applications and industrial processes. It emphasizes the importance of exploring microbial diversity in extreme environments to uncover novel enzymes with unique properties. The document also provides a summary of research articles published on the topic, covering various aspects of extremozymes and their applications. Additionally, the article titled "doi: 10.3389/fbioe.2015.00148" explores the potential of using microorganisms for various applications in biotechnology, including biofuel production, waste management, and drug discovery. The authors emphasize the importance of understanding the microbial world and its potential in addressing global challenges such as food security, environmental sustainability, and healthcare. This article provides a comprehensive overview of current research and future prospects in the field of biotechnology. [Extracted from the article]

Enzyme immobilization is a useful tool to produce biocatalysts with improved performance, such as high activity, high stability at operational conditions, and easy recovery and reuse. In this context, the production of second-generation ethanol using immobilized enzymes was reviewed. Emphasis was placed on the pre-treatment and/or hydrolysis steps to increase efficiency and reduce the cost of the process. In addition, the process design of bioethanol using immobilized enzymes was critically reviewed. In the enzymatic pre-treatment, laccases, manganese peroxidases, lignin peroxidases, and lytic polysaccharide monooxygenases are the main enzymes involved. When considering processes with heterogeneous biocatalysts, laccases are the most explored enzyme. In the hydrolysis step, cellulases and hemicellulases or a mixture of them are the main enzymes explored in the literature frequently using magnetic nanoparticles as support for enzyme immobilization. Although enzyme immobilization is a mature technology, the use of these biocatalysts is frequently limited to only one step of the bioethanol production process, such as pre-treatment or hydrolysis performed using Benchmark Technology. However, some recent papers have explored innovative design processes using a mixture of immobilized enzymes or co-immobilized enzymes to join some process steps, thereby decreasing the cost of enzymes and equipment.

The enhanced and direct immobilization of the enzyme horseradish peroxidase on poly(methyl methacrylate) (PMMA) microchannel surfaces to create a miniaturized enzymatic reactor for the biocatalytic oxidation of phenols is demonstrated. Enzyme immobilization occurs by physical adsorption after oxygen plasma treatment, which micro-nanotextures the PMMA surfaces. A five-fold enhancement in immobilized enzyme activity was observed, attributed to the increased surface area and, therefore, to a higher quantity of immobilized enzymes compared to an untreated PMMA surface. The enzymatic reaction yield reached 75% using a flow rate of 2.0 μL/min for the reaction mixture. Additionally, the developed microreactor was reused more than 16 times without affecting the enzymatic conversion yield. These results demonstrate the potential of microchannels with plasma micro/nanotextured surfaces for the rapid and facile fabrication of microfluidic enzymatic microreactors with enhanced catalytic activity and stability.

It has been observed that the immobilization of a phosphotriesterase enzyme (PTE) onto polyvinylidene fluoride (PVDF) membranes significantly decreased the enzyme activity, and this negative effect was attributed to the hydrophobic character of the membrane. The indirect indication of this reason was that the same enzyme immobilized on other membrane materials bearing hydrophilic character showed better performance. In this work, we provide direct evidence of the mechanism by immobilizing a PTE on a PVDF membrane hydrophilized by blending it with alkali lignin (AL). The PTE was immobilized on PVDF membrane by a covalent bond with the same procedure used in earlier studies to attribute changes in enzyme activity solely to the wettability properties (and not to the material chemistry). The activity of the PTE immobilized on the PVDF membrane hydrophilized with AL was 50% higher than that of the enzyme immobilized on the PVDF hydrophobic membrane. Further improvements of the membrane structure tailored for the development of a biocatalytic membrane reactor (BMR) were also promoted. In particular, the performance of the BMR was studied as a function of the thickness of the membrane, which allowed us to modulate the residence time into the enzyme-loaded membrane pores while maintaining the flow rate through the pores at a constant.

We revealed that the encapsulation of enzyme-immobilized silica particles in hollow-type spherical bacterial cellulose (HSBC) gels enables the use of the inside of HSBC gels as a reaction field. The encapsulation of horseradish peroxidase (HRP)-immobilized silica particles (Si-HRPs, particle size: 40–50 μm) within HSBC gels was performed by using a BC gelatinous membrane produced at the interface between Komagataeibacter xylinus suspension attached onto an alginate gel containing Si-HRPs and silicone oil. After the biosynthesis of the BC gelatinous membrane, formed from cellulose nanofiber networks, the alginate gel was removed via immersion in a phosphate-buffered solution. Si-HRP encapsulated HSBC gels were reproducibly produced using our method with a yield of over 90%. The pore size of the network structure of the BC gelatinous membrane was less than 1 μm, which is significantly smaller than the encapsulated Si-HRPs. Consequently, the encapsulated Si-HRPs could neither pass through the BC gelatinous membrane nor leak from the interior cavity of the HSBC gel. The activity of the encapsulated HRPs was detected using the 3,3′,5,5′-tetramethylbenzidine (TMB)-H2O2 system, demonstrating that this method can encapsulate the enzyme without inactivation. Since HSBC gels are composed of a network structure of biocompatible cellulose nanofibers, immune cells cannot enter the hollow interior, thus, the enzyme-immobilized particles encapsulated inside the HSBC gel are protected from immune-cell attacks. The encapsulation technique demonstrated in this study is expected to facilitate the delivery of enzymes and catalysts that are not originally present in the in vivo environment.

The ever-increasing presence of micropollutants necessitates the development of environmentally friendly bioremediation strategies. Inspired by the remarkable versatility and potent catalytic activities of microbial enzymes, researchers are exploring their application as biocatalysts for innovative environmental cleanup solutions. Microbial enzymes offer remarkable substrate specificity, biodegradability, and the capacity to degrade a wide array of pollutants, positioning them as powerful tools for bioremediation. However, practical applications are often hindered by limitations in enzyme stability and reusability. Enzyme immobilization techniques have emerged as transformative strategies, enhancing enzyme stability and reusability by anchoring them onto inert or activated supports. These improvements lead to more efficient pollutant degradation and cost-effective bioremediation processes. This review delves into the diverse immobilization methods, showcasing their success in degrading various environmental pollutants, including pharmaceuticals, dyes, pesticides, microplastics, and industrial chemicals. By highlighting the transformative potential of microbial immobilized enzyme biocatalysts, this review underscores their significance in achieving a cleaner and more sustainable future through the mitigation of micropollutant contamination. Additionally, future research directions in areas such as enzyme engineering and machine learning hold immense promise for further broadening the capabilities and optimizing the applications of immobilized enzymes in environmental cleanup.

Gamma-aminobutyric acid (GABA) is an vital neurotransmitter, and the reaction to obtain GABA through biocatalysis requires coenzymes, which are therefore limited in the production of GABA. In this study, polyacrylamide hydrogels doped with chitosan and waste toner were synthesized for glutamate decarboxylase (GAD) and coenzyme co-immobilization to realize the production of GABA and the recovery of coenzymes. Enzymatic properties of immobilized GAD were discussed. The immobilized enzymes have significantly improved pH and temperature tolerance compared to free enzymes. In terms of reusability, after 10 repeated reuses of the immobilized GAD, the residual enzyme activity of immobilized GAD still retains 100% of the initial enzyme activity, and the immobilized coenzyme can also be kept at about 32%, with better stability and reusability. And under the control of no exogenous pH, immobilized GAD showed good performance in producing GABA. Therefore, in many ways, the new composite hydrogel provides another way for the utilization of waste toner and promises the possibility of industrial production of GABA. [ABSTRACT FROM AUTHOR]

As an efficient and safe biocatalyst, enzymes have been widely used in many fields such as food, medicine and environmental governance, but it is difficult to realize further industrial application due to the poor environmental stability of free enzymes. Enzyme immobilization technology helps to improve the tolerance of free enzymes to sensitive environments and the stability during operation, and greatly reduces the application cost. This paper reviews the development and current situation of immobilization technology in the past five years, and summarizes the research progress of different immobilization methods (including traditional immobilization methods such as adsorption method and binding method and new immobilization methods such as coimmobilization enzyme method) and immobilization carriers (including natural material carriers, composite carriers and nanocarriers) in various fields. In general, compared with free enzymes, the immobilized enzyme system has been significantly improved in terms of stability and reusability. However, there are some shortcomings, such as lower recovery rate after immobilization, cumbersome and costly carrier synthesis pathway, and imperfect mechanism of immobilization enzyme.Finally, the development direction of the technology in the future was put forward based on these shortcomings. [ABSTRACT FROM AUTHOR]

In this study, artificial red blood cells (RBCs) for immobilization of l‐asparaginase are developed. The artificial red blood cells are prepared via a self‐assembly method and characterized by various methods. The prepared material shows a small hydrodynamic size of nearly 120 nm and a zeta potential of ‐7.2 ± 0.2 mV. After l‐asparaginase encapsulation, the particle size is nearly 295 nm and the zeta potential value is ‐9.8 ± 0.1. As expected, the immobilized enzyme shows better pH stability and thermal stability than the free enzyme. For instance, at 60 °C, the immobilized enzyme maintains 63.5% of the original activity, while the free enzyme retains only 27.6% of the original activity. Furthermore, the immobilized enzyme exhibits excellent reusability and storage stability, retaining 46.1% of its initial activity after 15 reuses. Overall, the developed artificial RBCs can potentially be applied as a novel support for immobilization of l‐asparaginase. [ABSTRACT FROM AUTHOR]

Peroxidase-based treatment that uses H 2 O 2 as the clean oxidant appears promising for the removal of aqueous phenolic endocrine disrupting chemicals (EDCs). However, oxidative degradation of active heme species at high H 2 O 2 concentrations leads to irreversible enzyme inactivation. The method of combining photocatalysis and enzyme biocatalysis could alleviate the issue by photocatalytic in-situ H 2 O 2 production. In this study, we constructed a photocatalyst-enzyme hybrid system that integrated the horseradish peroxidase with carbon vacancy-modified carbon nitride nanosheets (HRP@ACNNs). Attributed to the modified carbon nitride, the HRP@ACNNs exhibited higher substrate affinity and comparable catalytic efficiency compared to free HRP. This photocatalyst-enzyme hybrid system could offer H 2 O 2 continuously to sustain enzymatic catalysis without enzyme inactivation as well as stabilize the enzyme. The bisphenol A (BPA) removal experiments further confirmed the effectiveness in treating phenolic EDCs of the prepared HRP@ACNNs. [ABSTRACT FROM AUTHOR]

[Display omitted] Microgel–enzyme coatings were fabricated onto graphite substrates (highly oriented pyrolytic graphite or graphite- based screen-printed electrodes) via two-stage sequential procedure, which consists in adsorption of a pH- and temperature-sensitive copolymer microgel, followed by electrostatic immobilization of glucose oxidase into the microgel film. The microgel–enzyme coatings were examined by means of atomic force microscopy showing remarkable coverage of graphite surface by the microgels and visualizing the immobilized enzyme. Finally, amperometry was applied to reveal the effect of the polymeric (microgel) matrix on temperature behavior of glucose oxidase in the microgel– enzyme coatings. [ABSTRACT FROM AUTHOR]

Metal–organic framework materials (MOFs) and their derivatives are considered ideal immobilization carrier materials because of their large specific surface area, high porosity and excellent structural designability. Among them, ZIF-8 has great potential for immobilization of enzymes due to mild synthesis conditions, and good biocompatibility. However, conventional ZIF-8 crystals have poor separation and recovery efficiency due to their small pore size and poor acid stability, greatly limiting their application in enzyme immobilization and further application. Although the carbonization of ZIF-8 by pyrolysis has been shown to be one of the approaches that can enhance its chemical stability, this still does not effectively solve the problem of the difficulty of recycling. Herein, we developed a strategy of pre-carbonization immersion (immersion in aqueous FeSO4 solution before carbonization) to synthesize ordered macroporous ZIF-8-derived carbon materials with stable ferromagnetism (denoted as CZ-x-M-y, where x denotes the carbonization temperature and y denotes the concentration of the impregnated FeSO4 solution) and used them to immobilize lipases for biodiesel production. XRD analysis showed that the magnetic properties in the materials came from Fe3C species. We found that the magnetic carbon materials obtained by carbonization at 600 °C showed the best immobilization effect, where CZ-600-M-0.3 (using 0.3 mol·L−1 FeSO4 aqueous solution to soak ZIF-8 and carbonized at 600 °C) had the highest enzyme loading of 183.04 mg·g−1, which was 49.7% higher than that of the non-magnetic CZ-600. In addition, CZ-600-M-0.5 maintained the highest enzyme activity, which was 81.9% of the initial activity, after five batches of reuse. The stable magnetic support materials reported in this study have promising potential for the industrial application of immobilized lipase. [ABSTRACT FROM AUTHOR]

This article provides a method to upgrade epoxy-functionalized carriers for covalent enzyme immobilization to selective carriers suitable for covalent immobilization of metal affinity-tagged enzymes without the need of preliminary enzyme purification. Affinity function doping of the epoxy-functionalized surface introduces an advanced possibility to avoid the costly and time-consuming downstream processes required for efficient immobilization on non-selective epoxy carriers. Our approach is based on the partial functionalization of surface epoxides via a proper diamine-derived linker and an ethylenediaminetetraacetic dianhydride-based chelator charged with cobalt ions. The solid macroporous carriers, doped with metal affinity functions, have both coordinative binding ability (rapid anchoring the metal affinity-tagged enzymes to the surface) and subsequent covalent bond-forming ability (preferred binding of the tagged enzyme to the surface after proper washing by the residual epoxide functions), enabling a single operation for the enrichment and immobilization of a recombinant phenylalanine ammonia-lyase from parsley fused to a polyhistidine affinity tag. The immobilized PcPAL was applied in the ammonia elimination of racemic phenylalanine, 4-chlorophenylalanine, and 4-bromophenylalanine to produce the corresponding d-phenylalanines, in addition to the formation of (E)-cinnamates, as well as in ammonia addition reactions to (E)-cinnamates, yielding the corresponding enantiopure l-phenylalanines. [ABSTRACT FROM AUTHOR]

Metal organic frameworks (MOFs) are multi-dimensional network of crystalline material held together by bonding of metal atoms and organic ligands. Owing to unique structural, chemical, and physical properties, MOFs has been used for enzyme immobilization to be employed in different catalytic process, including catalytic degradation of antibiotics. Immobilization process other than providing large surface provides enzyme with enhanced stability, catalytic activity, reusability, and selectivity. There are various approaches of enzyme immobilization over MOFs including physical adsorption, chemical bonding, diffusion and in situ encapsulation. In situ encapsulation is one the best approach that provides extra stability from unfolding and denaturation in harsh industrial conditions. Presence of antibiotic in environment is highly damaging for human in particular and ecosystem in general. Different methods such as ozonation, oxidation, chlorination and catalysis are available for degradation or removal of antibiotics from environment, however these are associated with several issues. Contrary to these, enzyme immobilized MOFs are novel system to be used in catalytic degradation of antibiotics. Enzyme@MOFs are more stable, reusable and more efficient owing to additional support of MOFs to natural enzymes in well-established process of photocatalysis for degradation of antibiotics aimed at environmental remediation. Prime focus of this review is to present catalytic degradation of antibiotics by enzyme@MOFs while outlining their synthetics approaches, characterization, and mechanism of degradation. Furthermore, this review highlights the significance of enzyme@MOFs system for antibiotics degradation in particular and environmental remediation in general. Current challenges and future perspective of research in this field are also outlined along with concluding comments. [ABSTRACT FROM AUTHOR]

Electrophoretic deposition is a powerful tool for depositing materials onto a substrate by using an electric field; its application in biotechnological areas, namely, electrophoretic protein deposition (EPD), is the most promising for, e.g., fabricating novel amperometric biosensors. Unfortunately, EPD suffers from several drawbacks due to coupled parasite electrochemical processes damaging the deposit; moreover, the nature of the deposition process, the deposit, and its stability are still controversial and unknown. The present research presents a deep investigation of the EPD processes conducted by using several electroanalytical techniques and an electrochemical quartz crystal microbalance (EQCM); notably, EPD was used here as a novel tool for performing an electrophoretically assisted, classical enzyme immobilization technique like co-crosslinking, thus permitting the immobilization of the desired protein in situ, i.e., exclusively onto the deposition electrode. An electrochemical study permitted the acquisition of useful insights about electrophoresis processes as well as solvent discharge and gas evolution at the deposition electrode; further, the use of appropriate current or potential pulse sequences, as investigated and improved in this study, together with fine-tuned chemical conditions, allowed the optimization of this novel EPD approach. Moreover, an EQCM study gave useful insights into the kinetics of the process, permitting a quantitative estimate of the deposit. [ABSTRACT FROM AUTHOR]

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Ionic additives affect the structure, activity and stability of lipases, which allow for solving common application challenges, such as preventing the formation of protein aggregates or strengthening enzyme–support binding, preventing their desorption in organic media. This work aimed to design a biocatalyst, based on lipase improved by the addition of ionic additives, applicable in the production of ethyl esters of fatty acids (EE). Industrial enzymes from Thermomyces lanuginosus (TLL), Rhizomucor miehei (RML), Candida antárctica B (CALB) and Lecitase®, immobilized in commercial supports like Lewatit®, Purolite® and Q-Sepharose®, were tested. The best combination was achieved by immobilizing lipase TLL onto Q-Sepharose® as it surpassed, in terms of %EE (70.1%), the commercial biocatalyst Novozyme® 435 (52.7%) and was similar to that of Lipozyme TL IM (71.3%). Hence, the impact of ionic additives like polymers and surfactants on both free and immobilized TLL on Q-Sepharose® was assessed. It was observed that, when immobilized, in the presence of sodium dodecyl sulfate (SDS), the TLL derivative exhibited a significantly higher activity, with a 93-fold increase (1.02 IU), compared to the free enzyme under identical conditions (0.011 IU). In fatty acids ethyl esters synthesis, Q-SDS-TLL novel derivatives achieved results similar to commercial biocatalysts using up to ~82 times less enzyme (1 mg/g). This creates an opportunity to develop biocatalysts with reduced enzyme consumption, a factor often associated with higher production costs. Such advancements would ease their integration into the biodiesel industry, fostering a greener production approach compared to conventional methods. [ABSTRACT FROM AUTHOR]

Two approximate analytical expressions based on third degree polynomial and Akbari–Ganji's method (AGM) were derived for the reaction/diffusion controlled kinetics of an immobilized enzyme ( IE ) systems. The approximation methods predict substrate concentration profile and effectiveness factor (η ) in a porous spherical particle. The reaction is assumed to follow reverse Michaelis–Menten ( rMM ) kinetics. The approximate methods predictions were comparable to that of numerical solution (using the Matlab finite difference function, b v p 4 c ) at wide range of ϕ 2 and y o especially at low ϕ 2 and high y o (polynomial equation) and low ϕ 2 and low y o (AGM equation). Although the approximate solution was derived for rMM kinetics, the results can be used to describe other important enzymatic reaction kinetics such as simple Michaelis–Menten ( M M) kinetics and MM with competitive product inhibition kinetics. A necessary design equation should be satisfied when using polynomial or AGM approximation for different enzyme kinetic equations. In this work, two examples of enzymatic reactions of industrial interest were studied, namely glucose-fructose isomerization follows rMM kinetics and hydrolysis of lactose follows Michaelis–Menten ( MM ) equation with competitive product (galactose) inhibition. Predictions of the developed third degree polynomial and AGM approximation equations agree with that of numerical solution, the percentage relative error for the effectiveness factor was less than 11 in comparison with the numerical solution. Good agreement between approximate and numerical estimations demonstrates the validity of these approximation methods. [ABSTRACT FROM AUTHOR]