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.

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.

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).

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.

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.

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.

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.

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.

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.

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.