Carbonic anhydrase encapsulation using bamboo cellulose scaffolds for efficient CO2 capture and conversion.

Utilizing carbonic anhydrase (CA) to catalyze CO2 hydration offers a sustainable and potent approach for carbon capture and utilization. To enhance CA's reusability and stability for successful industrial applications, enzyme immobilization is essential. In this study, delignified bamboo cellulose served as a renewable porous scaffold for immobilizing CA through oxidation-induced cellulose aldehydation followed by Schiff base linkage. The catalytic performance of the resulting immobilized CA was evaluated using both p-NPA hydrolysis and CO2 hydration models. Compared to free CA, immobilization onto the bamboo scaffold increased CA's optimal temperature and pH to approximately 45 °C and 9.0, respectively. Post-immobilization, CA activity demonstrated effective retention (>60 %), with larger scaffold sizes (i.e., 8 mm diameter and 5 mm height) positively impacting this aspect, even surpassing the activity of free CA. Furthermore, immobilized CA exhibited sustained reusability and high stability under thermal treatment and pH fluctuation, retaining >80 % activity even after 5 catalytic cycles. When introduced to microalgae culture, the immobilized CA improved biomass production by ~16 %, accompanied by enhanced synthesis of essential biomolecules in microalgae. Collectively, the facile and green construction of immobilized CA onto bamboo cellulose block demonstrates great potential for the development of various CA-catalyzed CO2 conversion and utilization technologies.

Removal of benzo[a]pyrene from the soil by adsorption coupled with degradation on saponin-modified bentonite immobilized crude enzymes.

Bentonite is a non-metallic mineral with montmorillonite as the main component. It is an environmentally friendly mineral material with large reserves, wide distribution, and low price. Bentonite can be easily modified organically using the surfactant saponin to obtain saponin-modified bentonite (Sap-BT). This study investigates the immobilization of crude enzymes obtained from Trametes versicolor by physical adsorption with Sap-BT. Thus, saponin-modified bentonite immobilized crude enzymes (CE-Sap-BT) were developed to remove benzo[a]pyrene. Immobilization improves the stability of free enzymes. CE-Sap-BT can maintain more than 80% of activity at 45 °C and after storage for 15 d. Additionally, CE-Sap-BT exhibited a high removal rate of benzo[a]pyrene in soil, with 65.69% after 7 d in highly contaminated allotment soil and 52.90% after 6 d in actual soil contaminated with a low concentration of benzo[a]pyrene at a very low laccase dosage (0.1 U/3 g soil). The high catalytic and removal performance of CE-Sap-BT in contaminated sites showed more excellent practical application value.

Polydopamine-functionalized polyethersulfone membrane: A paradigm advancement in the field of α-amylase stability and immobilization.

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.

Covalent Lysozyme Immobilization on Enzymatic Cellulose Nanocrystals.

Nanostructured materials represent promising substrates for biocatalysts immobilization and activation. Cellulose nanocrystals (CNCs), accessible from waste and/or renewable sources, are sustainable and biodegradable, show high specific surface area for anchoring a high number of enzymatic units, and high thermal and mechanical stability. In this work, we present a holistic enzyme-based approach to functional antibacterial materials by bioconjugation between the lysozyme from chicken egg white and enzymatic cellulose nanocrystals. The neutral CNCs were prepared by endoglucanase hydrolysis from Avicel. We explore the covalent immobilization of lysozyme on the enzymatic CNCs and on their TEMPO oxidized derivatives (TO-CNCs), comparing immobilization yields, materials properties, and enzymatic activities. The materials were characterized by X-ray diffractometry (XRD), attenuated total reflectance Fourier Transform infrared spectroscopy (ATR-FTIR), bicinchoninic acid (BCA) assay, field-emission scanning electron microscopy (FE-SEM) and dynamic light scattering (DLS). We demonstrate the higher overall efficiency of the immobilization process carried out on TO-CNCs, based on the success of covalent bonding and on the stability of the isolated biocojugates.

Fe3O4@silica-thermolysin: A robust, advantageous, and reusable microbial nanobiocatalyst for proteolysis and milk-clotting.

Thermolysin (TLN) is a microbial highly-priced thermostable metallo-endoprotease with complementary substrate specificity to those of proteases widely used in science and industry for protein digestion and milk-clotting. This study is the first to immobilize TLN on aminated superparamagnetic nanoparticles (Fe3O4@silica-NH2) aiming for higher stability, recoverability, reusability, and applicability in proteolysis and as a microbial rennet-like milk-clotting enzyme. The nanobiocatalyst developed (Fe3O4@silica-TLN) displays hydrolytic activity on a synthetic TLN substrate and, apparently, was fully recovered from reaction media by magnetic decantation. More importantly, Fe3O4@silica-TLN retains TLN catalytic properties in the presence of calcium ions even after exposure to 60 °C for 48 h, storage at 4 °C for 80 days and room temperature for 42 days, use in proteolyses, and in milk-clotting for up to 11 cycles. Its proteolytic activity on bovine milk casein in 24 h furnished 84 peptides, of which 29 are potentially bioactive. Also, Fe3O4@silica-TLN catalyzed the digestion of bovine serum albumin. In conclusion, Fe3O4@silica-TLN showed to be a new, less autolytic, thermostable, non-toxic, magnetically-separable, and reusable nanobiocatalyst with highly attractive properties for both science (peptide/protein chemistry and structure, proteomic studies, and the search for new bioactive peptides) and food industry (cheese manufacture).

Unlocking of Hidden Mesopores for Enzyme Encapsulation by Dynamic Linkers in Stable Metal‒Organic Frameworks.

Mesoporous metal‒organic frameworks (MOFs) are promising supports for the immobilization of enzymes, yet their applications are often limited by small pore apertures that constrain the size of encapsulated enzymes to below 5 nm. In this study, we introduced labile linkers (4,4',4''-(2,4,6-boroxintriyl)-tribenzoate, TBTB) with dynamic boroxine bonds into mesoporous PCN-333, resulting in PCN-333-TBTB with enhanced enzyme loading and protection capabilities. The selective breaking of B-O bonds creates defects in PCN-333, which effectively expands both window and cavity sizes, thereby unlocking hidden mesopores for enzyme encapsulation. Consequently, this strategy not only increases the adsorption kinetics of small enzymes (<5 nm) such as cytochrome c (Cyt C) and horseradish peroxidase (HRP), but also enables the immobilization of various large-sized enzymes (>5 nm), such as glycoenzymes. The glycoenzymes@PCN-333-TBTB platform was successfully applied to synthesize thirteen complex oligosaccharides and polysaccharides, demonstrating high activity and enhanced enzyme stability. The dynamic linker-mediated enzyme encapsulation strategy enables the immobilization of enzymes exceeding the inherent pore size of MOFs, thus broadening the scope of enzymatic catalytic reactions achievable with MOF materials.

Aspergillus labruscus ITAL 22.223 xylanase - immobilization and application for the obtainment of corncob xylan targeting xylitol production.

Corncob is an agro-residue rich in lignocellulosic material that can be used for the xylitol production, through its enzymatic conversion obtaining fermentable sugars and their subsequent fermentation. In light of the above, this study targeted the immobilization of Aspergillus labruscus xylanase and the use of the derivative to hydrolyze the corncob xylan for the obtainment of xylose, and its subsequent use for the production of xylitol. The extracellular xylanase was immobilized using different supports (sodium alginate, DEAE-Cellulose, DEAE-Sephadex and CM-Sephadex). Among all supports used, the best results were obtained with the DEAE-Cellulose derivative showing an efficiency of immobilization of 97-99%, yield of 93-95% and recovered activity of 81-100%. The sodium alginate derivative showed 3 cycles of reuse, with drop in activity of about 65% in the 3rd cycle using both CaCl2 and MnCl2 as crosslinkers. The best enzymatic activity for the DEAE-Cellulose derivative was observed at 55ºC and pH 5.0. This derivative presented reuse of 10 cycles using commercial xylan as substrate, and 4 cycles using corncob xylan. This derivative was used in an enzymatic reactor to hydrolyze corncob xylan, obtaining 2.7 mg/mL of xylose after 48 h of operation under optimal condition of temperature and pH. The xylose obtained from the corncob was fermented by Candida tropicalis for 96 h with consumption of 60%. The HPLC analyses indicated a production of 1.02 mg/mL of xylitol with 48 h of fermentation. In conclusion, this is the first report on the immobilization of the A. labrucus xylanase as an alternative for the obtainment of xylose from corncob xylan, and the subsequent production of xylitol.

Bioremediation of organic pollutants by laccase-metal-organic framework composites: A review of current knowledge and future perspective.

Immobilized laccases are widely used as green biocatalysts for bioremediation of phenolic pollutants and wastewater treatment. Metal-organic frameworks (MOFs) show potential application for immobilization of laccase. Their unique adsorption properties provide a synergic effect of adsorption and biodegradation. This review focuses on bioremediation of wastewater pollutants using laccase-MOF composites, and summarizes the current knowledge and future perspective of their biodegradation and the enhancement strategies of enzyme immobilization. Mechanistic strategies of preparation of laccase-MOF composites were mainly investigated via physical adsorption, chemical binding, and de novo/co-precipitation approaches. The influence of architecture of MOFs on the efficiency of immobilization and bioremediation were discussed. Moreover, as sustainable technology, the integration of laccases and MOFs into wastewater treatment processes represents a promising approach to address the challenges posed by industrial pollution. The MOF-laccase composites can be promising and reliable alternative to conventional techniques for the treatment of wastewaters containing pharmaceuticals, dyes, and phenolic compounds. The detailed exploration of various immobilization techniques and the influence of MOF architecture on performance provides valuable insights for optimizing these composites, paving the way for future advancements in environmental biotechnology. The findings of this research have the potential to influence industrial wastewater treatment and promoting cleaner treatment processes and contributing to sustainability efforts.

Nitrilotriacetic Acid Functionalized Microgels for Efficient Immobilization of Hyaluronan Synthase.

Enzymes play a vital role in synthesizing complex biological molecules like hyaluronic acid (HA). Immobilizing enzymes on support materials is essential for their efficient use and reuse in multiple cycles. Microgels, composed of cross-linked, highly swollen polymer networks, are ideal for enzyme uptake owing to their high porosity. This study demonstrates the immobilization of His6-tagged hyaluronan synthase from Pasteurella multocida (PmHAS) onto nitrilotriacetic acid functionalized microgels using different bivalent ions (Ni2+, Co2+, Mn2+, Mg2+, and Fe2+) via metal affinity binding. The results indicate that using Ni2+ yields the microgels with the highest enzyme uptake and HA formation. The immobilized PmHAS enables repetitive enzymatic production, producing high molecular weight HAs with decreasing dispersities in each step. Furthermore, the highest reported yield of HA with high molecular weight for immobilized PmHAS is achieved. This system establishes a foundation for continuous HA formation, with future works potentially enhancing PmHAS stability through protein engineering.

Enzymatic Routes for Chiral Amine Synthesis: Protein Engineering and Process Optimization.

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.

Immobilization of His6-tagged amine transaminases in microreactors using functionalized nonwoven nanofiber membranes.

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 (His6)-tagged amine transaminases (ATAs) in flow. A membrane coated with Cu2+ ions gave the best results regarding His6-tagged ATAs immobilization among the membranes tested yielding an immobilization yield of up to 95.3 % for the purified N-His6-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 His6-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.

Efficient and rapid digestion of proteins with a dual-enzyme microreactor featuring 3-D pores formed by dopamine/polyethyleneimine/acrylamide-coated KIT-6 molecular sieve.

The microreactor with two types of immobilized enzymes, exhibiting excellent orthogonal performance, represents an effective approach to counteract the reduced digestion efficiency resulting from the absence of a single enzyme cleavage site, thereby impacting protein identification. In this study, we developed a hydrophilic dual-enzyme microreactor characterized by rapid mass transfer and superior enzymatic activity. Initially, we selected KIT-6 molecular sieve as the carrier for the dual-IMER due to its three-dimensional network pore structure. Modification involved co-deposition of polyethyleneimine (PEI) and acrylamide (AM) as amine donors, along with dopamine to enhance material hydrophilicity. Remaining amino and double bond functional groups facilitated stepwise immobilization of trypsin and Glu-C. Digestion times for bovine serum albumin (BSA) and bovine hemoglobin (BHb) on the dual-IMER were significantly reduced compared to solution-based digestion (1 min vs. 36 h), resulting in improved sequence coverage (91.30% vs. 82.7% for BSA; 90.24% vs. 89.20% for BHb). Additionally, the dual-IMER demonstrated excellent durability, retaining 96.08% relative activity after 29 reuse cycles. Enhanced protein digestion efficiency can be attributed to several factors: (1) KIT-6's large specific surface area, enabling higher enzyme loading capacity; (2) Its three-dimensional network pore structure, facilitating faster mass transfer and substance diffusion; (3) Orthogonality of trypsin and Glu-C enzyme cleavage sites; (4) The spatial effect introduced by the chain structure of PEI and glutaraldehyde's spacing arm, reducing spatial hindrance and enhancing enzyme-substrate interactions; (5) Mild and stable enzyme immobilization. The KIT-6-based dual-IMER offers a promising technical tool for protein digestion, while the PDA/PEI/AM-KIT-6 platform holds potential for immobilizing other proteins or active substances.

Microtiter Plate Immobilization Screening for Prototyping Heterogeneous Enzyme Cascades.

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.

Nanopore-regulated in situ polymerization for synthesis of homogeneous heparan sulfate with low dispersity.

The biological activities of heparan sulfate (HS) are intimately related to their molecular weights, degree and pattern of sulfation and homogeneity. The existing methods for synthesizing homogeneous sugar chains of low dispersity involve multiple steps and require stepwise isolation and purification processes. Here, we designed a mesoporous metal-organic capsule for the encapsulation of glycosyltransferase and obtained a microreactor capable of enzymatically catalyzing polymerization reactions to prepare homogeneous heparosan of low dispersity, the precursor of HS and heparin. Since the sugar chain extension occurs in the pores of the microreactor, low molecular weight heparosan can be synthesized through space-restricted catalysis. Moreover, the glycosylation co-product, uridine diphosphate (UDP), can be chelated with the exposed metal sites of the metal-organic capsule, which inhibits trans-cleavage to reduce the molecular weight dispersity. This microreactor offers the advantages of efficiency, reusability, and obviates the need for stepwise isolation and purification processes. Using the synthesized heparosan, we further successfully prepared homogeneous 6-O-sulfated HS of low dispersity with a molecular weight of approximately 6 kDa and a polydispersity index (PDI) of 1.032. Notably, the HS generated exhibited minimal anticoagulant activity, and its binding affinity to fibroblast growth factor 1 was comparable to that of low molecular weight heparins.

Immobilized Multi-Enzyme/Nanozyme Biomimetic Cascade Catalysis for Biosensing Applications.

Multiple enzyme-induced cascade catalysis has an indispensable role in the process of complex life activities, and is widely used to construct robust biosensors for analyzing various targets. The immobilized multi-enzyme cascade catalysis system is a novel biomimetic catalysis strategy that immobilizes various enzymes with different functions in stable carriers to simulate the synergistic catalysis of multiple enzymes in biological systems, which enables high stability of enzymes and efficiency enzymatic cascade catalysis. Nanozymes, a type of nanomaterial with intrinsic enzyme-like characteristics and excellent stabilities, are also widely applied instead of enzymes to construct immobilized cascade systems, achieving better catalytic performance and reaction stability. Due to good stability, reusability, and remarkably high efficiency, the immobilized multi-enzyme/nanozyme biomimetic cascade catalysis systems show distinct advantages in promoting signal transduction and amplification, thereby attracting vast research interest in biosensing applications. This review focuses on the research progress of the immobilized multi-enzyme/nanozyme biomimetic cascade catalysis systems in recent years. The construction approaches, factors affecting the efficiency, and applications for sensitive biosensing are discussed in detail. Further, their challenges and outlooks for future study are also provided.

Reversible immobilization of cellulase on gelatin for efficient insoluble cellulose hydrolysis.

Immobilized enzymes are one of the most common tools used in enzyme engineering, as they can substantially reduce the cost of enzyme isolation and use. However, efficient catalysis of solid substrates using immobilized enzymes is challenging, hydrolysis of insoluble cellulose by immobilized cellulases is a typical example of this problem. In this study, inspired by bees and honeycombs, we prepared gelatin-modified cellulase (BEE) and gelatin hydrogels (HONEYCOMB) to achieve reversible recycling versus release of cellulase through temperature-responsive changes in the triple-stranded helix-like interactions between BEE and HONEYCOMB. At elevated temperatures, BEE was released from HONEYCOMB and participated in hydrolytic saccharification. After 24 h, the glucose yields of both the free enzyme and BEE reached the same level. When the temperature was decreased, BEE recombined with HONEYCOMB to facilitate the effective separation and recycling of BEE from the system. The enzymatic system retained >70 % activity after four reuse cycles. In addition, this system showed good biocompatibility and environmental safety. This method increases the mass transfer capacity and enables easy recovery of immobilized cellulase, thereby serving as a valuable strategy for the immobilization of other enzymes.

Enzyme-Immobilized Porous Crystals for Environmental Applications.

Developing efficient technologies to eliminate or degrade contaminants is paramount for environmental protection. Biocatalytic decontamination offers distinct advantages in terms of selectivity and efficiency; however, it still remains challenging when applied in complex environmental matrices. The main challenge originates from the instability and difficult-to-separate attributes of fragile enzymes, which also results in issues of compromised activity, poor reusability, low cost-effectiveness, etc. One viable solution to harness biocatalysis in complex environments is known as enzyme immobilization, where a flexible enzyme is tightly fixed in a solid carrier. In the case where a reticular crystal is utilized as the support, it is feasible to engineer next-generation biohybrid catalysts functional in complicated environmental media. This can be interpreted by three aspects: (1) the highly crystalline skeleton can shield the immobilized enzyme against external stressors. (2) The porous network ensures the high accessibility of the interior enzyme for catalytic decontamination. And (3) the adjustable and unambiguous structure of the reticular framework favors in-depth understanding of the interfacial interaction between the framework and enzyme, which can in turn guide us in designing highly active biocomposites. This Review aims to introduce this emerging biocatalysis technology for environmental decontamination involving pollutant degradation and greenhouse gas (carbon dioxide) conversion, with emphasis on the enzyme immobilization protocols and diverse catalysis principles including single enzyme catalysis, catalysis involving enzyme cascades, and photoenzyme-coupled catalysis. Additionally, the remaining challenges and forward-looking directions in this field are discussed. We believe that this Review may offer a useful biocatalytic technology to contribute to environmental decontamination in a green and sustainable manner and will inspire more researchers at the intersection of the environment science, biochemistry, and materials science communities to co-solve environmental problems.

Surface Engineering of Superparamagnetic Graphene Oxide Nanosheet as a Chemically Tunable Platform for Facial Biofuel Production by Lipase.

In this research, we utilized an efficient approach to synthesize superparamagnetic graphene oxide (SPGO) rapidly in a one-pot method using microwave irradiation of graphene oxide (GO), urea, and Fe(III) ion. Tannic acid (TA) was introduced to the surface of SPGO through a straightforward and eco-friendly process. Methods were devised to furnish GO nanosheets and modify their surfaces with TA in an environmentally friendly manner. Two series of nanosheets, namely, SPGO/TA-COOH and SPGO/TA-IM, were engineered on the surface and used for immobilizing lipase enzyme. Through various analytical tools, the unique biocatalysts SPGO/TA-COOH/L and SPGO/TA-IM/L were confirmed. These biocatalysts exhibited enhanced stability at high temperatures and pH levels compared with free lipase. They also demonstrated prolonged storage stability and reusability over four months and seven cycles, respectively. Furthermore, the catalytic activity of immobilized lipase showed minimal impairment based on kinetic behavior analysis. The kinetic constants of SPGO/TA-IM/L were determined as Vmax = 0.24 mM min-1, Km = 0.224 mM, and kcat = 0.8 s-1. Additionally, the efficiency of biocatalysts for biodiesel production from palmitic acid was studied, focusing on various reaction parameters, such as temperature, alcohol to palmitic acid molar ratio, water content, and lipase quantity. The esterification reaction of palmitic acid with methanol, ethanol, and isopropanol was tested in the presence of SPGO/TA-COOH/L and SPGO/TA-IM/L, and the corresponding esters were obtained with a yield of 30.6-91.6%.

Promotion of lactose isomerization to fructose and lactulose in one batch by immobilized enzymes on bacterial cellulose membranes.

The production of the sugars fructose and lactulose from lactose using the enzymes ß-galactosidase and glucose isomerase immobilized on bacterial cellulose (BC) membranes has been investigated. Lactose is hydrolyzed by ß-galactosidase at 30 °C to glucose and galactose at a high conversion rate, while at the same temperature, glucose isomerase is not effective in converting the produced glucose to fructose. The rate of the isomerization reaction of glucose to fructose at 70 °C has been studied. Two types of enzyme immobilization were investigated: immobilization in one stage and immobilization in two stages. The results showed that BC membrane increased three-fold the yield and the reaction rate of fructose and lactulose production from lactose. The noteworthy enhancement of BC membranes' impact on the isomerization reaction by immobilized enzymes grants permission for a novel research avenue within the context of white biotechnology development. Additionally, this effect amplifies the role of BC in sustainability and the circular economy.

A Hydrophilic Metal Azolate Coordination Polymer for In Situ Encapsulation of Haloalkane Dehalogenase with Enhanced Enzymatic Performance.

Encapsulating enzymes within metal-organic frameworks such as zeolitic imidazolate framework-8 (ZIF-8) has been demonstrated to enhance enzymatic performance under harsh conditions. However, by computer-aided analysis, we revealed that highly hydrophobic organic ligands and unfavorable metal ions could greatly impair the activity of haloalkane dehalogenase DhaA by directly interacting with the catalytic sites, causing an extremely low activity of DhaA after encapsulating within ZIF-8. We also found that the presence of a protecting polymer could protect DhaA from the damage of organic ligands and metal ions and that a positively charged amino acid could increase the DhaA activity. Based on the simulations and experimental observations, we have designed to coencapsulate DhaA with poly(vinylpyrrolidone) (PVP) and lysine (Lys) within the amorphous Co-based metal azolate coordination polymer (CoCP). The as-prepared immobilized enzyme (DhaA/PVP/Lys@CoCP) exhibited significantly increased activity (91.5 times higher than that of DhaA@ZIF-8), dramatically enhanced thermostability at 50-70 °C, greatly improved catalytic performance in several organic solvent solutions, and good recyclability (over 75% of the initial activity after 10 cycles). The superiority of the immobilized enzyme was also demonstrated with a substrate frequently detected in the real world. In addition to the protective effect of PVP and positive effect of Lys, experimental and computational investigations unveiled other two favorable aspects that contributed to the enhanced enzymatic performance: (1) high hydrophilicity of the immobilization material and (2) the use of Co2+ with a minimal negative effect on DhaA. The research has thus provided a promising immobilized DhaA with favorable catalytic performance and great potential in industrial applications.