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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. [ABSTRACT FROM AUTHOR]

Lowering the adverse effects of climate change requires advanced methods to decrease atmospheric levels of carbon dioxide. Here, we review the use of carbonic anhydrase nanobiocatalysts for carbon dioxide sequestration, with emphasis on inorganic, organic, and polymeric nanomaterials. Inorganic nanomaterials include metal and metal oxide, carbon-based, and nonmetallic nanomaterials. Hybrid nanomaterials comprise metal–organic frameworks and nanoflowers. Factors influencing the properties of nanobiocatalysts such as interactions between carbonic anhydrase and the nanoscale support are presented. The immobilization of carbonic anhydrase onto nanomaterials overcomes the limitations associated with its free form, such as short shelf life, challenging separation, and poor reusability. We discuss the potential for large-scale applications. [ABSTRACT FROM AUTHOR]

One of the commercial applications of the pectinase enzyme is pectinization and clarification of fruit juices and extraction of phenolic compounds from the fruits. Pectinase immobilization is one of the stabilization methods that makes it possible to separate the enzyme from the reaction and reuse it, thus reducing the cost of using the enzyme in the industry. In this study, the pectinase enzyme from Aspergillus niger was immobilized on hydrophilic silica aerogel by the adsorption method. Adsorption involves hydrogen, hydrophobic, and van der Waals interactions between solid carriers and adsorbed molecules, and due to their simplicity, high catalytic activity, and reusability of carriers after inactivation of immobilized enzymes, has higher economic potential, compared to other immobilization methods. Properties of silica aerogel containing pectinase were determined by Fourier Transform Infrared Spectroscopy (FTIR) and Field Emission Scanning Electron Microscope (FESEM) analysis. Effective parameters in enzyme immobilization (immobilization time, enzyme to support ratio, and pH) were studied using the Response Surface Methodology (RSM) and the application of immobilized enzyme in the extraction of phenolic compounds from the grape pomace was investigated. Results obtained from properties analysis and measurement of immobilization efficiency showed effective immobilization of the enzyme on the silica aerogel supports. Optimal conditions for immobilization were obtained at pH 5, enzyme to support ratio of 0.1, and time of 89.2 minutes. The immobilization efficiency in these conditions was 78.1%. Experimental results and modeling were not significantly different, which showed the adequacy of the model. Extraction of phenolic compounds from the grape pomace by pectinase immobilized on silica aerogel supports, increased by 78.7% and free radical scavenging by 55.7%, compared to the case without enzymatic treatment. [ABSTRACT FROM AUTHOR]

Effective enzyme stabilization through immobilization is essential for the functional usage of enzymatic reactions. We propose a new method for synthesizing elastic hydroxyapatite microgel (E-HAp-M) materials and immobilizing lipase using this mesoporous mineral via the ship-in-a-bottle-neck strategy. The physicochemical parameters of E-HAp-M were thoroughly studied, revealing that E-HAp-M provides efficient space for enzyme immobilization. As a model enzyme, lipase (LP) was entrapped and then cross-linked enzyme structure, preventing leaching from mesopores, resulting in highly active and stable LP/E-HAp-M composites. By comparing LP activity under different temperature and pH conditions, it was observed that the cross-linked LP exhibited improved thermal stability and pH resistance compared to the free enzyme. In addition, they demonstrated a 156% increase in catalytic activity compared with free LP in hydrolysis reactions at room temperature. The immobilized LP maintained 45% of its initial activity after 10 cycles of recycling and remained stable for over 160 days. This report presents the first demonstration of a stabilized cross-linked LP in E-HAp-M, suggesting its potential application in enzyme-catalyzed processes within biocatalysis technology. [ABSTRACT FROM AUTHOR]

The enzyme market is growing constantly, and lipases, in their free and immobilized forms, constitute an important part of this market. This study aimed to analyze and discuss current market scenarios using data from the literature, focusing on lipases and the feasibility of their use in the immobilized form. It also compares the cost of biodiesel production using two commercial lipases: Eversa Transform 2.0 (free lipase) and Novozyme 435 (immobilized lipase). The results show that the European and North American enzyme markets are the most prominent worldwide, with Denmark and the USA as the major exporters and importers, respectively. Lipases can be used in a wide variety of fields, and immobilization brings many advantages to this enzyme class. Finally, we show the use of immobilized enzymes and lipases in biorefinery and food industries and carry out a comparative cost analysis to produce biodiesel using a free and immobilized lipase. We concluded that the enzyme market, in which lipases play an important role, is extensive, and that immobilized lipases seem to be excellent biocatalysts with feasible costs for industrial applications. [ABSTRACT FROM AUTHOR]

Oxidative stress is implicated in the pathophysiology of several illnesses. Catalase (CAT), an antioxidant enzyme, remains a prime target for oxidative stress related therapies. Unfortunately, the labile nature, difficulty in recovering and reusing, and insufficient delivery systems limit its use in therapy. This study used a simple biomineralization process for the synthesis of vaterite porous calcium carbonate (CaCO 3) microspheres (MS) for the development of cross-linked immobilized (CAT-MS). Scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), particle size analysis, and zeta-potential measurements were used for CaCO 3 -MS characterization. CAT was immobilized efficiently with a high immobilization yield (99 %). The immobilized enzyme had a higher Km than free CAT with a 1.7 fold increase in Vmax and Kcat. CAT-MS displayed a longer half-life, efficient reusability, enhanced storage stability, and excellent stability against various denaturants compared to free CAT. Furthermore, CAT-MS demonstrated negligible cytotoxicity in the fibroblast cell line (BJ1), where CAT-MS recorded higher cell viability than free CAT, (p< 0.05) indicating exceptional biocompatibility. This study raises the potential of an eco-friendly approach that enforces wide and safe catalase application in intracellular therapy for treating oxidative stress-associated diseases. [Display omitted] • Catalase-loaded CaCO 3 microspheres (CAT-MS) were synthesized by a modified process. • High immobilization yield and prominent loading efficiency were achieved. • CAT-MS had a longer half-life and enhanced thermal and storage stability. • immobilization improved reusability and stability against various denaturants. • CAT-MS demonstrated high biocompatibility in vitro cell line. [ABSTRACT FROM AUTHOR]

Glucose oxidase is a crucial enzyme used in industries such as chemicals, pharmaceuticals, food, and biotechnology due to its ability to oxidize glucose into hydrogen peroxide and gluconic acid (C6H12O7). However, its limited and costly production necessitates strategies for improved efficiency. Immobilization, which involves attaching enzymes to surfaces, is a key method that enhances glucose oxidase's activity, stability, and reusability, making it more practical for repeated industrial use. This study comprehensively reviews glucose oxidase immobilization techniques, emphasizing their evolution, advanced methods, and diverse applications, particularly in biomedical fields. By immobilizing glucose oxidase, its stability, activity, and reusability are significantly enhanced, addressing the challenges of enzyme production and operational efficiency. The review discusses various immobilization methods, including physical adsorption, covalent binding, entrapment, and encapsulation, comparing their advantages and limitations. Incorporating nanomaterials and computational methods, such as molecular modeling and simulations, further optimizes the immobilization process, providing insights into enzyme-support interactions and improving performance in applications like biosensors, biofuel cells, and drug delivery systems. The study also explores future trends, including artificial intelligence and innovative support materials, to drive advancements in glucose oxidase immobilization. By integrating these techniques, the study aims to pave the way for more effective and versatile biomedical solutions, contributing to the ongoing evolution of enzyme immobilization technology. [ABSTRACT FROM AUTHOR]

Serine proteases have been receiving special attention from the industrial point of view, due to their thermo-stable properties and activity over wide ranges of pH. In the present investigation, a serine protease (49.3 kDa) from jackfruit latex has been purified using chromatographic techniques. Fold purification of the serine protease in the final purification step was 92.41 with 24% yield. The protease is completely inhibited by PMSF, a serine protease-specific inhibitor at a minimal concentration (100 µM). The purified serine protease was immobilized with sodium alginate (2.5%) and calcium chloride (0.3 M) solution. The kinetic studies of immobilized enzyme showed stable activity up to pH 7, and can withstand temperatures up to 45 °C. The immobilization process improves the catalytic efficiency of the enzyme over purified soluble enzyme (for Kcat 1.4 times and for Kcat/Km 1.21 times). Our results depicted that alginate mediated immobilization of serine protease greatly improves the pH and temperature optima which broadens the scope of usage of the enzyme further. [ABSTRACT FROM AUTHOR]

Immobilization is a key enabling technology in applied biocatalysis that facilitates the separation, recovery, and reuse of heterogeneous biocatalysts. However, finding a consensus immobilization protocol for several enzymes forming a multi‐enzyme system is extremely difficult and relies on a combinatorial trial‐and‐error approach. Herein, we describe a protocol in which 17 different carriers functionalized with different reactive groups are tested in a 96‐well microtiter plate to screen up to 21 immobilization protocols for up to 18 enzymes. This screening includes an activity and stability assay to select the optimal immobilization chemistry to achieve the most active and stable heterogeneous biocatalysts. The information retrieved from the screening can be rationalized using a Python‐based application CapiPy. Finally, through scoring the screening results, we find the consensus immobilization protocol to assemble an immobilized four‐enzyme system to transform vinyl acetate into (S)‐3‐hydroxybutyric acid. This methodology opens a path to speed up the prototyping of immobilized multi‐enzyme pathways for chemical manufacturing. [ABSTRACT FROM AUTHOR]

Biofuel cells (BFCs) offer an eco‐friendly route to convert biochemical energy into electricity. However, their performance is hindered by insufficient enzyme immobilization as well as limited electron transfer within the enzymatic electrode. While the incorporation of redox mediators (RMs) into enzyme layers has been shown to improve BFC performance through enhanced electron transfer, progress has plateaued in the last decade. Herein, a major breakthrough is presented realized by a novel strategy that exploits electron transfer‐intensified RM layers. Metal nanoparticles covalently bridged between neighboring RMs facilitate electron transfer ubiquitously. Electron transfer characteristics are enhanced not only within the RM layers themselves, but also at the glucose oxidase (GOx)/host electrode and GOx/GOx interfaces. This leads to a remarkable performance boost in the enzymatic anode. A hybrid BFC constructed with innovative anode and Pt‐based cathode exhibits a striking combination of high power output (2.3 and 8.5 mW cm−2 at 10 and 300 mmol L−1 glucose, respectively) and exceptional operational stability (≈80% and 47% power retention after 10 days and 1 month, respectively), outperforming all previously reported BFCs by a significant margin. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Spatial functionalization of hierarchically mesoporous metal–organic frameworks (HMMOFs) in a controllable manner is highly desirable yet challenging. Herein, microporous and mesoporous spaces as well as the external surface of nanoscale hierarchically mesoporous UiO-66-NH2 MOFs were site-separately modified to endow them with additional functionalities. Given the pore-filling effect of soft templates, the folic acid ligand was successfully grafted on the outer surface without reducing the pore size and available pore volumes, making the developed sensing nanoprobe competent to specifically identify cancer cells. The mesoporous support not only effectively protected the encapsulated enzymes but also facilitated the proximity between the substrates and enzymes by virtue of the spatial confinement effect. The Cu2+ ions introduced in microporous skeleton could synergistically activate the activity of horseradish peroxidase immobilized in mesopores. Thanks to such enhanced enzymatic activity and spatial functionalities, the designed nanoprobe could specifically and quantitatively check out FR-overexpressed cancer cells in a colorimetric and visible way. Through deliberately screening different functionalities in specific locations of HMMOFs, such a model is expected to set a guideline for the design of multiple-functional diagnostic platform for the detection of various cancers. [ABSTRACT FROM AUTHOR]

Directed enzyme prodrug therapy (DEPT) strategies show promise in mitigating chemotherapy side effects during cancer treatment. Among these, the use of immobilized enzymes on solid matrices as prodrug activating agents (IDEPT) presents a compelling delivery strategy, offering enhanced tumor targeting and reduced toxicity. Herein, we report a novel IDEPT strategy by employing a His-tagged Leishmania mexicana type I 2′-deoxyribosyltransferase (His-LmPDT) covalently attached to glutaraldehyde-activated magnetic iron oxide nanoparticles (MIONPs). Among the resulting derivatives, PDT-MIONP3 displayed the most favorable catalyst load/retained activity ratio, prompting its selection for further investigation. Substrate specificity studies demonstrated that PDT-MIONP3 effectively hydrolyzed a diverse array of 6-oxo and/or 6-amino purine 2′-deoxynucleosides, including 2-fluoro-2′-deoxyadenosine (dFAdo) and 6-methylpurine-2′-deoxyribose (d6MetPRib), both well-known prodrugs commonly used in DEPT. The biophysical characterization of both MIONPs and PDT-MIONPs was conducted by TEM, DLS, and single particle ICPMS techniques, showing an ideal nanosized range and a zeta potential value of −47.9 mV and −78.2 mV for MIONPs and PDT-MIONPs, respectively. The intracellular uptake of MIONPs and PDT-MIONPs was also determined by TEM and single particle ICPMS on HeLa cancer cell lines and NIH3T3 normal cell lines, showing a higher intracellular uptake in tumor cells. Finally, the selectivity of the PDT-MIONP/dFAdo IDEPT system was tested on HeLa cells (24 h, 10 µM dFAdo), resulting in a significant reduction in tumoral cell survival (11% of viability). Based on the experimental results, PDT-MIONP/dFAdo presents a novel and alternative IDEPT strategy, providing a promising avenue for cancer treatment. [ABSTRACT FROM AUTHOR]

The use of organic-inorganic hybrid nanoflowers as a support material for enzyme immobilization has gained significant attention in recent years due to their high stability, ease of preparation, and enhanced catalytic activity. However, a major challenge in utilizing these hybrid nanoflowers for enzyme immobilization is the difficulty in handling and separating them due to their low density and high dispersion. To address this issue, magnetic nanoflowers have emerged as a promising alternative enzyme immobilization platform due to their easy separation, structural stability, and ability to enhance catalytic efficiency. This review focuses on different methods for designing magnetic nanoflowers, as well as future research directions. Additionally, it provides examples of enzymes immobilized in the form of magnetic nanoflowers and their applications in environmental remediation, biosensors, and food industries. Finally, the review discusses possible ways to improve the material for enhanced catalytic activity, structural stability, and scalability. The magnetic hybrid nanoflowers embedded with enzymes exhibit superior catalytic activity, excellent stability, and ease of separation which can be used in a wide range of applications. Magnetic nanoflowers can be used as a novel platform for enzyme immobilization. There are three different approaches to the synthesis of efficient magnetic nanoflower. The magnetic nanoflowers provides excellent stability and good reusability of enzymes. The hybrid biocatalyst was applied in biotransformation, environmental, and food applications. The challenges and their remedies of hybrid biocatalyst have been discussed. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract Effective enzyme stabilization through immobilization is essential for the functional usage of enzymatic reactions. We propose a new method for synthesizing elastic hydroxyapatite microgel (E-HAp-M) materials and immobilizing lipase using this mesoporous mineral via the ship-in-a-bottle-neck strategy. The physicochemical parameters of E-HAp-M were thoroughly studied, revealing that E-HAp-M provides efficient space for enzyme immobilization. As a model enzyme, lipase (LP) was entrapped and then cross-linked enzyme structure, preventing leaching from mesopores, resulting in highly active and stable LP/E-HAp-M composites. By comparing LP activity under different temperature and pH conditions, it was observed that the cross-linked LP exhibited improved thermal stability and pH resistance compared to the free enzyme. In addition, they demonstrated a 156% increase in catalytic activity compared with free LP in hydrolysis reactions at room temperature. The immobilized LP maintained 45% of its initial activity after 10 cycles of recycling and remained stable for over 160 days. This report presents the first demonstration of a stabilized cross-linked LP in E-HAp-M, suggesting its potential application in enzyme-catalyzed processes within biocatalysis technology.

Encapsulating the enzyme in metal–organic frameworks (MOFs) is a convenient method to prepare MOF-enzyme biocomposite. In this study, Candida antarctica lipase B (CAL-B) was chosen to immobilize in Cu-BTC MOF under ultrasound irradiation. CAL-B was immobilized in Cu-BTC under ultrasound at 21 kHz and 11.4 W/cm2 and incubation. 98% of CAL-B was immobilized in Cu-BTC with 99 U/mg activity (threefold more active than the free CAL-B). The prepared biocomposite was characterized using FT-IR, XRD, TGA, SEM, EDX, and BET. The thermal and solvent stability of CAL-B@Cu-BTC was investigated. It was found that at a temperature of 55 ℃, CAL-B@Cu-BTC maintains its activity even after 2 h of incubation. Furthermore, in the presence of 20% and 50% concentrations of MeCN, THF, and DMF, CAL-B@Cu-BTC was found to have an activity of over 80%. A prepared biocatalyst was used to synthesize 1,3,4,5-tetrasubstituted pyrazole derivatives (50–75%) in a one-pot vessel, by adding phenyl hydrazine hydrochlorides, benzaldehydes, and dimethyl acetylenedicarboxylate. [ABSTRACT FROM AUTHOR]

Process intensification is crucial for industrial implementation of biocatalysis and can be achieved by continuous process operation in miniaturized reactors with efficiently immobilized biocatalysts, enabling their long-term use. Due to their extremely large surface-to-volume ratio, nanomaterials are promising supports for enzyme immobilization. In this work, different functionalized nanofibrous nonwoven membranes were embedded in a two-plate microreactor to enable immobilization of hexahistidine (His 6)-tagged amine transaminases (ATAs) in flow. A membrane coated with Cu2+ ions gave the best results regarding His 6 -tagged ATAs immobilization among the membranes tested yielding an immobilization yield of up to 95.3 % for the purified N -His 6 -ATA-wt enzyme. Moreover, an efficient one-step enzyme immobilization process from overproduced enzyme in Escherichia coli cell lysate was developed and yielded enzyme loads up to 1088 U mL−1. High enzyme loads resulted in up to 80 % yields of acetophenone produced from 40 mM (S)-α-methylbenzylamine in less than 4 min using a continuously operated microreactor. Up to 81 % of the initial activity was maintained in a 5-day continuous microreactor operation with immobilized His 6 -tagged ATA constructs. The highest turnover number within the indicated time was 7.23·106, which indicates that this immobilization approach using advanced material and reactor system is highly relevant for industrial implementation. [Display omitted] • A microreactor with nonwoven nanofiber membrane was developed. • Nanofibers with various functional groups were tested for enzyme immobilization. • His6-tagged amine transaminase was immobilized on membranes with metal ions. • One-step purification and immobilization with very high enzyme loads was reached. • Long-term stability of microreactor system and high turnover numbers were obtained. [ABSTRACT FROM AUTHOR]

Biocatalytic membranes have great potential in various industrial sectors, with the immobilization of enzymes being a crucial stage. Immobilizing enzymes through covalent bonds is a complex and time-consuming process for large-scale applications. Polydopamine (PDA) offers a more sustainable and eco-friendly alternative for enzyme immobilization. Therefore, surface modification with polydopamine as mussel-inspired antifouling coatings has increased resistance to fouling. In this study, α-amylase enzyme was covalently bound to a bioactive PDA-coated polyethersulfone (PES) membrane surface using cyanuric chloride as a linker. The optimal activity of α-amylase enzyme immobilized on PES/PDA membrane was obtained at temperature and pH of 55°C and 6.5, respectively. The immobilized enzyme can be reused up to five reaction cycles with 55 % retention of initial activity. Besides, it maintained 60 % of its activity after being stored for five weeks at 4°C. Additionally, the immobilized enzyme demonstrated increased Michaelis constant and maximum velocity values during starch hydrolysis. The results of the biofouling experiment of various membranes in a dead-end cell demonstrated that the PES membrane's water flux increased from 6722.7 Lmh to 7560.2 Lmh after PDA modification. Although α-amylase immobilization reduced the flux to 7458.5 Lmh due to enhanced hydrophilicity, compared to unmodified membrane. The findings of this study demonstrated that the membrane produced through co-deposition exhibited superior hydrophilicity, enhanced coating stability, and strong antifouling properties, positioning it as a promising candidate for industrial applications. [Display omitted] • α-Amylase was covalently immobilized onto polydopamine-functionalized polyethersulfone membrane. • Surface modification with polydopamine generated a mussel-inspired coating to inhibit fouling. • The immobilization technique effectively preserved the activity of α-amylase enzyme. • The biocatalyst membrane exhibited superior stability and strong antifouling properties. [ABSTRACT FROM AUTHOR]

Abstract Nucleotides, such as uridine 5′‐monophosphate (5′‐UMP) and cytidine 5′‐monophosphate (5′‐CMP), are heavily demanded as food additives, dietary supplements, and medicinal intermediates. Although enzymatic phosphorylation is an effective approach to producing these nucleosides, its practical application is challenging due to the need for the expensive enzymes and coenzyme guanosine‐5′‐triphosphate (GTP). Here we prepared biocompatible calcium‐based nanoflowers incorporated with cascade enzymes, uridine‐cytidine kinase (UCK) and acetate kinase (ACK), for the production of nucleotides. The spatial distribution of these enzymes was optimized to maximize their catalytic performances. The most active nanoflowers (Ca3(PO4)2& ACK)& UCK that display the rate‐limiting enzyme UCK on the outer layer were used in a packed‐bed reactor for continuous synthesis of 5′‐UMP and 5′‐CMP. The catalytic performance of the catalyst retained over 80% within 10 h, showing good operational stability.

Sayali Shantaram Vikhrankar,1 Seema Satbhai,2 Priyanka Kulkarni,2 Ranjit Ranbhor,3 Vibin Ramakrishnan,4 Prashant Kodgire1 1Biosciences and Biomedical Engineering, Indian Institute of Technology Indore, Indore, MP, India; 2Sevit Healthcare Private Limited, Pune, MH, India; 3Pergament & Cepeda LLP, Florham Park, NJ, USA; 4Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, IndiaCorrespondence: Ranjit Ranbhor, Pergament & Cepeda LLP, 25A Hanover Road, Florham Park, NJ, 07932, USA, Tel +1 973 967 0490, Email ranjitranbhor@gmail.comAbstract: Chiral amines are essential motifs in pharmaceuticals, agrochemicals, and specialty chemicals. While traditional chemical routes to chiral amines often lack stereoselectivity and require harsh conditions, biocatalytic methods using engineered enzymes can offer high efficiency and selectivity under sustainable conditions. This review discusses recent advances in protein engineering of transaminases, oxidases, and other enzymes to improve catalytic performance. Strategies such as directed evolution, immobilization, and computational redesign have expanded substrate scope and enhanced efficiency. Furthermore, process optimization guided by techno-economic assessments has been crucial for establishing viable biomanufacturing routes. Combining state-of-the-art enzyme engineering with multifaceted process development will enable scalable, economical enzymatic synthesis of diverse chiral amine targets.Keywords: chiral amines, biocatalysis, protein engineering, ω-transaminases, sitagliptin, enzyme immobilization, co-factor regeneration

Dendritic mesoporous silica nanoparticles (DMSNs) are a new class of solid porous materials used for enzyme immobilization support due to their intrinsic characteristics, including their unique open central–radial structures with large pore channels and their excellent biocompatibility. In this review, we review the recent progress in research on enzyme immobilization using DMSNs with different structures, namely, flower-like DMSNs and tree-branch-like DMSNs. Three DMSN synthesis methods are briefly compared, and the distinct characteristics of the two DMSN types and their effects on the catalytic performance of immobilized enzymes are comprehensively discussed. Possible directions for future research on enzyme immobilization using DMSNs are also proposed.

Hydrogen‐bonded organic frameworks (HOF) represent an emerging category of organic structures with high crystallinity and metal‐free, which are not commonly observed in alternative porous organic frameworks. These needle‐like porous structure can help in stabilizing enzymes and allow transfer of molecules between enzymes participating in cascade reactions for enhanced substrate channelling. Herein, we systematically synthesized and investigated the stability of HOF at extreme conditions followed by one‐pot encapsulation of single and bi‐enzyme systems. Firstly, we observed HOF to be stable at pH 1 to 14 and at high temperatures (up to 115 °C). Secondly, the encapsulated glucose oxidase enzyme (GOX) showed 80 % and 90 % of its original activity at 70 °C and pH 11, respectively. Thirdly, transient time close to 0 seconds was observed for HOF encapsulated bi‐enzyme cascade reaction system demonstrating a 4.25‐fold improvement in catalytic activity when compared to free enzymes with enhanced substrate channelling. Our findings showcase a facile system synthesized under ambient conditions to encapsulate and stabilize enzymes at extreme conditions. [ABSTRACT FROM AUTHOR]

Hydrogel coatings, with natural tissue‐like mechanical and biological compatibility, demonstrate promise for bulk material applications. However, existing coating methods lack efficiency and control, especially when dealing with complex geometries, diverse substrate materials, and varying sizes. In this study, an enzyme‐immobilized surface‐catalyzed cross‐link approach is proposed for the growth of hydrogel coatings on multiple substrates, accommodating various materials and shapes. The hydrogel coating is formed through the catalytic action of interface‐immobilized enzymes, which induce the crosslinking of hyaluronic acid, and its mechanical properties are further enhanced by introducing a secondary polyacrylamide network. This approach enables the controlled growth of conformal double network hydrogel coatings with a desired thickness on a wide range of substrate surfaces and devices. Notably, the hydrogel coating exhibits remarkable lubricity and anti‐thrombotic properties, especially desire for medical intervention devices. This advancement offers a universal route to impart biocompatible soft interfaces to bulk materials or devices. [ABSTRACT FROM AUTHOR]

Diabetes is an important public health problem all over the world that affects the quality of life, and its late diagnosis leads to serious complications such as stroke, blindness, limb amputations, and kidney failure. Fabrication of economical and user‐friendly biosensor systems allows the rapid and early detection of prediabetes and type 2 diabetes. Accordingly, a novel, amino‐functionalized, conjugated, alternating polymer was synthesized, characterized, and coated on a glassy carbon electrode (GCE) to be used as an immobilization platform for the glucose oxidase (GOx) enzyme. The decline in oxygen level due to the enzymatic reaction at a potential of −0.7 V vs. silver/silver chloride (Ag/AgCl) is screened as a biosensor response. The proposed biosensor displayed high selectivity and specificity for glucose with a broad linear range, a low detection limit, good reproducibility and repeatability, and a long shelf life. Surface modifications were characterized using cyclic voltammetry (CV) and field emission scanning electron microscopy (FE‐SEM) techniques. The real‐life sample application of the constructed biosensor to determine the glucose amount was performed in human serum. [ABSTRACT FROM AUTHOR]

The lactose intolerance—as a limiting factor for dairy milk consumption—has a high prevalence worldwide. Dairy milk and milk-derived products are major sources of multiple inorganic compounds and nutrients and thus are considered to be functional foods. β-galactosidases are able to hydrolyze lactose and are therefore widely applied for the production of lactose-free products. In addition, they are capable of the synthesis of galacto-oligosaccharides (GOSs); thus, the dairy industry has a special interest in applying them for the enrichment of dairy products with prebiotic GOSs. In this work, we studied two commercially available β-galactosidase products: Saphera 2600L and Nola Fit 5500. Both enzyme solutions contain a recombinant β-galactosidase of Bifidobacterium bifidum and have already been authorized for food industrial application, but the information about their hydrolytic and/or synthetic activities is only limited. After immobilization on chitosan beads, the enzymes were used for lactose hydrolysis and simultaneous synthesis of GOSs, by performing the reactions in pasteurized milk (skim milk). Both immobilized β-galactosidase exhibited elevated lactose hydrolysis (vmax increased from ~ 1 to ~ 4 mM/min) and GOS synthesis as compared to the free enzymes. The enzyme-coated beads were efficiently re-used at least 15 cycles; the residual lactose concentration was < 2 mg/ml after each cycle. After treatment, GOSs were present in ≤ 9% of the total sugar content, indicating that the prepared low-lactose milks were enriched in prebiotic GOSs. The application of immobilized Saphera 2600L and Nola Fit 5500 β-galactosidases may be implemented for the large-scale production of GOS-enriched low-lactose milk. [ABSTRACT FROM AUTHOR]

The immobilization of Aspergillus niger Lipase (E.C.3.1.1.3) via covalent binding on epoxy-activated chitosan-coated silver oxide nanoparticles (Ag2O/Chitosan) under optimized conditions was studied. Ultraviolet–visible spectroscopy, Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction spectral analysis (XRD), Transmission electron microscopy (TEM), Field-emission scanning electron microscopy (FE-SEM), and energy-dispersive X-Ray Microanalysis (EDAX) were employed to characterize the size, structural composition, crystallinity, surface morphologies, & percentage elemental composition of prepared Ag2O NPs & Ag2O/Chitosan nanocomposites. Optimal immobilization conditions (coupling time (16 h), pH (7.8), activating agent concentration (4% v/v), enzyme concentration (0.6 mg/ml)) were evaluated to acquire maximum expressed activity of bound lipases. The optimum catalytic activity of immobilized & solubilized lipases was exhibited at pH 8.5, 60 °C, and pH 7.0, 45 °C respectively. Thermal stability of immobilized lipases (Ag2O/Chitosan-EPI@Lip) preserved 76.47% of catalytic activity at 60 °C after 2 h of incubation while solubilized lipases retained only 40.51% of catalytic activity under similar conditions. Kinetic parameters (Km, Vmax) of immobilized (39.69 µM, 21.32 µM/min) & native lipase (34.12 µM, 21.83 µM/min) were determined, demonstrating the enhanced efficacy of lipase after immobilization. Moreover, bound lipases exhibited remarkable reusability and storage stability. Thus, the present methodology suggests that (Ag2O/Chitosan-EPI@Lip) bioconjugate would be a potential biocatalyst for promoting sustainable productions & advanced technologies. [ABSTRACT FROM AUTHOR]

N-glycosylation of recombinant proteins using bacterial glycosylation system has proven to be a valuable although developing tool ultimately applicable to various industries. When used for enzyme engineering, it offers the possibility of increased stability or immobilization route and thus increasing effectiveness of e.g. biotransformation or other biocatalysis procedures. One such promising enzyme is alcohol dehydrogenase (ADH) for use in redox biotransformation reactions. Given the current possibilities of recombinant enzyme production, including major advances in glycoengineering and glycoprotein production in bacterial organisms, the aim of this work was the production of thermotolerant ADH from Rhodococcus ruber (RrADH) in glycosylated form in Escherichia coli. We have successfully developed a dual plasmid expression system enabling glycosylation of target proteins utilizing a glyco-tag approach. We were able to produce RrADH in soluble form and at the same time we detected a bacterial glycan conjugated to RrADH as well as the activity of the enzyme. The glycan bound to recombinant enzyme can be used for oriented covalent immobilization of the enzyme, which would increase the potential for its practical application in biotransformation of various compounds. [ABSTRACT FROM AUTHOR]

This study presents a design for green synthesis of silver nanoparticles and nanobioconjugates to enhance enzymatic activity and biosynthesis of biodiesel using free and immobilized enzymes. Enzymes were immobilized by covalent attachment with Ag-PDA complex, PDA bound MSN complex, PDA coated MSN functionalized aluminum nanoparticles, and PDA coated MSN functionalized Fe nanoparticles. 2.47 U/mL increase in the activity of Ag-PDA bound trypsin was observed. Conjugated trypsin retained 80–95% of its activity after 5 days of storage at 4 °C while unconjugated enzyme retained 45% activity. Trypsin immobilized on Ag-PDA complex, PDA coated MSN-Al nanoparticles, PDA bound MSN complex, and PDA coated MSN functionalized Fe nanoparticles retained a residual activity of 80.3%, 78.5%, 68.6% and 66.4% correspondingly after 5, 4, 4, and 3 consecutive operations, respectively. Excellent coupling of biocompatible silver nanoparticles with remarkable adhesive attributes of PDA deposition on silver nanoparticles made Ag-PDA complex very efficient for the immobilization of trypsin by providing a large surface area for the active binding to the trypsin molecules. Biodiesel was synthesized by using lipase immobilized cerium oxide, MSN supported iron, and silica nanosystems. Maximum of 80% biodiesel yield was produced by lipase immobilized cerium oxide nanoparticles. Immobilized nanoparticles were recovered and reused for biodiesel production with a low percentage biodiesel yield. [ABSTRACT FROM AUTHOR]

The entrapment of deep eutectic solvents (DES) and eutectic systems into porous scaffolds renders a new class of soft and nonvolatile materials called eutectogels that have recently stepped into the spotlight in different areas ranging from electronics to drug delivery. Recent progress in the use of DES in biocatalysis, where they have been demonstrated to improve substrate supply, conversion, and enzyme stability, has opened an unparalleled opportunity to exploit the merits of eutectogels for immobilizing biological catalysts. The resulting functional materials could outperform traditional hydrogels and ionic liquid gels, offering fresh perspectives to broaden the application scope of many enzymes. In this perspective, we go into the potential of eutectogels as innovative scaffolds that support biocatalytic reactions and discuss different applications where these systems could show plain benefits compared to traditional materials. Future directions for this newly developed technology are highlighted. [ABSTRACT FROM AUTHOR]

l‐Pipecolic Acid (l‐PA) is a valuable building block for the synthesis of pharmaceuticals such as anesthetics and immunosuppressants. Thus, more efficient and greener strategies are desired for its production. Herein, we have applied a previously engineered variant of the Lysine Cyclodeaminase from Streptomyces pristinaespiralis (e‐SpLCD) for the bioconversion of l‐Lysine into l‐PA. The reaction can be performed by the free e‐SpLCD reaching full conversion to 50 mM l‐PA. From a biotechnological perspective, the process scale‐up has been trialed in a SpinChem® reactor, albeit with lower conversion yields. To further enhance the biocatalyst stability, we present a detailed study of the e‐SpLCD immobilization on microparticles. This enabled the integration of the immobilized biocatalyst into a packed‐bed reactor for the continuous flow synthesis of l‐PA. The full conversion was achieved in 90 min, maintaining also high operational stability. Remarkably, the addition of exogenous cofactor was not needed for the flow reaction, although the long‐term operational stability was improved by the addition of NAD+. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Although trypsin is a protease naturally applicable in many processes, it is still possible to increase its efficiency and forms of use. Processes that increase their resilience to different conditions in the reaction medium can expand and/or refine their range of applications. Its performance in the presence of organic solvents, such as acetonitrile (ACN), has been indicated as a promising way to increase the efficiency of digestion processes, such as in sample treatment for MALDI-MS peptide mapping. The maintenance of the activity of trypsin immobilized on glyoxyl-agarose was herein demonstrated in different temperature and ACN ranges. Compared to the soluble, the immobilized enzyme was able to remain active above 50% ACN, where the soluble trypsin showed no activity. Although low, it was still possible to detect about 7% activity at 70% ACN when immobilized form was used. With 7.5% of ACN, the soluble enzyme has already shown loss of activity. The same only occurred with immobilized trypsin at 20% or more. Although the immobilized enzyme may have lower specific activity compared to the soluble one, as confirmed by the determined kinetic parameters, its possibility of reuse was confirmed, with no loss of activity for 5 cycles at 0% and 5% ACN. Added to this is the simplicity that the reaction can be interrupted, through the removal of the enzyme from the medium. What could prevent its own protein chain from contaminating the final product. With these characteristics added, trypsin in its immobilized form presents itself as a promising biocatalyst. [ABSTRACT FROM AUTHOR]

Hybrid nanomaterials have recently emerged as a new interface of nanobiocatalysis, serving as a host platform for enzyme immobilization. Enzyme immobilization in inorganic crystal nanoflowers and metal-organic frameworks (MOFs) has sparked the bulk of scientific interest due to their superior performances. Many breakthroughs have been achieved recently in the preparation of various types of enzyme@MOF and enzyme-hybrid nanoflower composites. However, it is unfortunate that there are few reviews in the literature related to enzyme@MOF and enzyme-hybrid nanoflower composites and their improved synthesis strategies and their applications in biotechnology. In this review, innovative synthetic strategies for enzyme@MOF composites and enzyme-hybrid nanoflower composites are discussed. Enzyme@MOF composites and enzyme-hybrid nanoflower composites are reviewed in terms of biotechnological applications and potential research directions. We are convinced that a fundamental study and application of enzyme@MOF composites and enzyme-hybrid nanoflower composites will be understood by the reader as a result of this work. The summary of different synthetic strategies for enzyme@MOF composites and enzyme-hybrid nanoflower composites and the improvement of their synthetic strategies will also benefit the readers and provide ideas and thoughts in the future research process. [ABSTRACT FROM AUTHOR]

Nucleotides, such as uridine 5′‐monophosphate (5′‐UMP) and cytidine 5′‐monophosphate (5′‐CMP), are heavily demanded as food additives, dietary supplements, and medicinal intermediates. Although enzymatic phosphorylation is an effective approach to producing these nucleosides, its practical application is challenging due to the need for the expensive enzymes and coenzyme guanosine‐5′‐triphosphate (GTP). Here we prepared biocompatible calcium‐based nanoflowers incorporated with cascade enzymes, uridine‐cytidine kinase (UCK) and acetate kinase (ACK), for the production of nucleotides. The spatial distribution of these enzymes was optimized to maximize their catalytic performances. The most active nanoflowers (Ca3(PO4)2& ACK)& UCK that display the rate‐limiting enzyme UCK on the outer layer were used in a packed‐bed reactor for continuous synthesis of 5′‐UMP and 5′‐CMP. The catalytic performance of the catalyst retained over 80% within 10 h, showing good operational stability. [ABSTRACT FROM AUTHOR]

Heterogeneous biocatalysis is highly relevant in biotechnology as it offers several benefits and practical uses. To leverage the full potential of heterogeneous biocatalysts, the establishment of well‐crafted protocols, and a deeper comprehension of enzyme immobilization on solid substrates are essential. These endeavors seek to optimize immobilized biocatalysts, ensuring maximal enzyme performance within confined spaces. For this aim, multidimensional characterization of heterogeneous biocatalysts is required. In this context, spectroscopic and microscopic methodologies conducted at different space and temporal scales can inform about the intraparticle enzyme kinetics, the enzyme spatial distribution, and the mass transport issues. In this Minireview, we identify enzyme immobilization, enzyme catalysis, and enzyme inactivation as the three main processes for which advanced characterization tools unveil fundamental information. Recent advances in operando characterization of immobilized enzymes at the single‐particle (SP) and single‐molecule (SM) levels inform about their functional properties, unlocking the full potential of heterogeneous biocatalysis toward biotechnological applications. [ABSTRACT FROM AUTHOR]

Only a few halophilic archaea producing carboxylesterases have been reported. The limited research on biocatalytic characteristics of archaeal esterases is primarily due to their very low production in native organisms. A gene encoding carboxylesterase from Halobacterium salinarum NRC-1 was cloned and successfully expressed in Haloferax volcanii. The recombinant carboxylesterase (rHsEst) was purified by affinity chromatography with a yield of 81%, and its molecular weight was estimated by SDS-PAGE (33 kDa). The best kinetic parameters of rHsEst were achieved using p-nitrophenyl valerate as substrate (KM = 78 µM, kcat = 0.67 s−1). rHsEst exhibited great stability to most metal ions tested and some solvents (diethyl ether, n-hexane, n-heptane). Purified rHsEst was effectively immobilized using Celite 545. Esterase activities of rHsEst were confirmed by substrate specificity studies. The presence of a serine residue in rHsEst active site was revealed through inhibition with PMSF. The pH for optimal activity of free rHsEst was 8, while for immobilized rHsEst, maximal activity was at a pH range between 8 to 10. Immobilization of rHsEst increased its thermostability, halophilicity and protection against inhibitors such as EDTA, BME and PMSF. Remarkably, immobilized rHsEst was stable and active in NaCl concentrations as high as 5M. These biochemical characteristics of immobilized rHsEst reveal its potential as a biocatalyst for industrial applications. [ABSTRACT FROM AUTHOR]

Several procedures were tested for the immobilization of the artificial d -selective amino acid dehydrogenase from Ureibacillus thermosphaericus (Ut DAADH) and its co-immobilization with the NADPH–regenerating glucose dehydrogenase (GDH). Based on the conversions of the reductive amination of phenylpyruvate, recyclability, batch-to-batch reproducibility, and immobilization costs, DAADH covalently attached onto Purolite® ECR8415F or co-immobilized with GDH on polyethylenimine-coated agarose were selected for optimizations. The non-desired substrate/product adsorption occurring in case of the Purolite® support, was avoided by increasing the volume of linkers employed for the covalent fixation of the enzyme. The more convenient to prepare immobilization variant, DAADH adsorbed onto the Purolite® resin also proved to be applicable. This preparation, despite presenting significant drop of 58.8% of its specific activity over 10 reaction cycles, still provided similar conversions with the covalently immobilized variant. As third effective preparation, the DAADH–GDH co-immobilized system, showed no activity loss over 10 reaction cycles. In this case the additional co-immobilization of the NADP+ cofactor provided self-sufficient biocatalysts only for limited cycles, after >3 consecutive reactions the conversion dropped with ∼60%, due to cofactor leakage. The synthetic utility of the immobilized DAADH was demonstrated by the 100 mg-scale reductive amination of phenylpyruvate, obtaining d -phenylalanine with 86% yield. [Display omitted] • Covalently immobilized DAADHs tested in reductive amination of phenylpyruvate. • Purolite® ECR8415F as optimal support for the covalent or adsorption–based immobilization. • DAADH and glucose dehydrogenase co-immobilized on agarose beads as effective biocatalyst. • Synthetic utility, for the synthesis of d -phenylalanine, tested at 100-mg scale. [ABSTRACT FROM AUTHOR]

It has been discovered that metal–organic frameworks (MOFs) have desirable qualities for the immobilization of enzymes, including a high surface area, significant interior pore volumes, and easily changeable pore size. Herein, the xylanase (Xyl) enzyme was immobilized for the first time to two different carrier supports, zeolitic imidazolate framework-67 (ZIF-67) and manganese-doped ZIF-67 (Mn/ZIF-67) by in situ method. The physicochemical characterizations of MOFs with and without Xyl were performed by FT-IR, XRD, SEM, and EDAX techniques. Xyl@ZIF-67 and Xyl@Mn/ZIF-67 were evaluated in terms of optimum temperature, optimum pH, kinetic parameters, thermal stability, reusability as well as juice clarification and compared with free Xyl. Optimum temperature values were 50 °C for Xyl@ZIF-67 and 70 °C for free Xyl and Xyl@Mn/ZIF-67. Optimum pH values for free Xyl, Xyl@ZIF-67, and Xyl@Mn/ZIF-67 were recorded as 6.0, 8.0, and 7.0, respectively. Km values for free Xyl, Xyl@ZIF-67, and Xyl@Mn/ZIF-67 were calculated as 3.139, 5.430, and 0.799 mg/mL, respectively, while Vmax values were calculated as 0.167, 0.226, and 0.062 µmol/min/mL, respectively. The results revealed that in comparison to the free Xyl, Xyl@ZIF-67, and Xyl@Mn/ZIF-67 exhibited more thermal resistance. After incubation at 70 °C for 120 min, the free Xyl remained at 28.7% of the activity, while the Xyl@ZIF-67 and Xyl@Mn/ZIF-67 remained at 85.7% and 40.0%, respectively. Moreover, after eight cycles, the Xyl@ZIF-67 and Xyl@Mn/ZIF-67 retained more than 70% of their initial activity. Further, the transmittance of apple juice was increased from 65.61 to 94.73% and from 77.80 to 84.13%, respectively, when Xyl@ZIF-67 and Xyl@Mn/ZIF-67 were used as biocatalysts. Overall, these findings indicated that the suggested Xyl@ZIF-67 and Xyl@Mn/ZIF-67 have a high potential for juice clarification as an efficient heterogeneous biocatalyst. [ABSTRACT FROM AUTHOR]

[Display omitted] A critical challenge in the enzymatic conversion of acylglycerols is the limited exposure of the enzyme dissolved in the aqueous solution to the hydrophobic substrate in the oil phase. Positioning the enzyme in a microenvironment with balanced hydrophobicity and hydrophilicity in Pickering emulsion will facilitate the acylglycerol-catalyzing reactions at the interface between the oil and liquid phases. In this work, to overcome the challenge of biphasic catalysis, we report a method to immobilize enzymes in polyethylene glycol (PEG)-based hydrogel microparticles (HMPs) at the interface between the oil and water phases in Pickering emulsion to promote the enzymatic conversion of acylglycerols. 3 wt% of HMPs can stabilize the oil-in-water Pickering emulsion for at least 14 days and increase the viscosity of emulsions. Lipase-HMP conjugates showed significantly higher hydrolytic activity in Pickering emulsion; HMP-immobilized lipase SMG1 showed an activity about three times that of free lipase SMG1. Co-immobilization of a lipase and a fatty acid photodecarboxylase from Chlorella variabilis (CvFAP) in Pickering emulsion enables light-driven cascade conversion of triacylglycerols to hydrocarbons, transforming waste oil to renewable biofuels in a green and sustainable approach. HMPs stabilize the Pickering emulsion and promote interfacial biocatalysis in converting acylglycerols to renewable biofuels. [ABSTRACT FROM AUTHOR]

In light of their outstanding properties, metal–organic frameworks appear to be the latest and most assured sorbent in CCUS technology. In this study, adsorption isotherms of pure CO2, CH4, and N2 at three different temperatures were obtained for HKUST-1, and it showed a maximum CO2 adsorption uptake of 8.25 mmol g−1 at 298 K and 30 bar. HKUST-1 was also employed as support to immobilize carbonic anhydrase (CA) derived from the bovine erythrocytes through cross-linked enzyme aggregates. CO2 hydration as well as its transformation into CaCO3 was studied using the CA immobilized HKUST-1 (CA/HKUST-1). The biocatalytic activity, thermal stability, reusability, and storage stability of CA/HKUST-1 were evaluated through para-nitrophenyl acetate hydrolysis. CA immobilized on HKUST-1 exhibits a fairly low Kcat/Km value than the free CA; regardless of the above, the reusability of CA/HKUST-1 for about 20 cycles makes it a suitable biocatalyst of CO2 hydration under industrial operating conditions. The results demonstrated HKUST-1 as potential support for CA immobilization for CO2 sequestration and high CO2 uptake adsorbent. [ABSTRACT FROM AUTHOR]

The main reasons that make mankind look for new sources of energy are decreasing dependence on fossil fuels and reducing pollution. Wastewater treatment with the help of microbial fuel cells is an area where these two goals can be combined. Microbial fuel cells, in which microorganisms catalyze the oxidation of organic substances, represent a new and promising alternative for electricity generation. The creation of an efficient cathode in such systems is the important problem. It is evident that in these applications an oxygen (air) electrode is a promising cathode. In this study, the oxygen biocathode based on the laccase enzyme Pleurotus ostreatus HK-35 was developed and its electrochemical properties were studied depending on the immobilization method of the enzyme on the surface of a carbon-graphite electrode and the type of the electrolyte. It was experimentally established that laccase injection using a sol-gel matrix was the effective method for immobilizing laccase on the surface of a carbon-graphite electrode. It was shown that the more efficient operation of the laccase-based biocathode was observed in a phosphate-citrate (pH 4.0) buffer solution, i.e., in an acidic environment.

Abstract 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.

The enzyme carbonic anhydrase (CA) has gainned considerable attention from the literature and the industry in the context of CO2 capture. CA immobilization in gas-liquid membrane contactors, and more specifically, on poly(ionic liquid) (PIL) composite membranes has been demonstrated to be a potential strategy to facilitate its industrial implementation. These membranes were comprised of a PIL layer coating on a porous hydrophobic polymeric support. In this work, the composition of the PIL layer was tuned by anion exchange to yield a variety of enzyme carriers. The following anions were compared: bromide [Br], acetate [Ac], tetrafluoroborate [BF4], and bis(trifluoromethylsulfonyl)imide [NTf2]. The surface morphology, chemistry, and properties of these composite membranes were characterized by SEM, EDX, ATR-FTIR, and water contact angle. The activity of the different biocatalytic composite membranes was determined by the p-nitrophenyl acetate hydrolysis model reaction. It was found that the anion exchange salts had a detrimental effect on the immobilized enzyme activity. In light of these results, the enzyme immobilization step was conducted after anion exchange. The resulting biocatalytic membranes displayed slight differences in immobilized enzyme activities and thermal stabilities following the order [Br]>[BF4]>[Ac]>[NTf2] and [BF4]>[Br]≈[Ac]>[NTf2], respectively. The differences were more pronounced and detrimental for the most hydrophobic anion, [NTf2]. Parallel trends were noted when the membranes were tested for CO2 absorption in a gas-liquid membrane contactor set-up suggesting that the CO2 mass transfer is strongly influenced by the activity of the immobilized enzymes. In addition, the effect of the absorption conditions, i.e., solvent flow rate, solvent saturation, and solvent concentration were evaluated. Under the best conditions, the novel biocatalytic membranes outperformed the commercial PVDF support by about a factor of 4 in terms of overall mass transfer coefficient. Such improvement would result in significant reductions in the required membrane area to capture CO2 by a gas-liquid membrane contactor.