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In recent decades, enzyme-based biocatalytic systems have garnered increasing interest in industrial and applied research for catalysis and organic chemistry. Many enzymatic reactions have been applied to sustainable and environmentally friendly production processes, particularly in the pharmaceutical, fine chemicals, and flavor/fragrance industries. However, only a fraction of the enzymes available has been stepped up towards industrial-scale manufacturing due to low enzyme stability and challenging separation, recovery, and reusability. In this context, immobilization and co-immobilization in robust support materials have emerged as valuable strategies to overcome these inadequacies by facilitating repeated or continuous batch operations and downstream processes. To further reduce separations, it can be advantageous to use multiple enzymes at once in one pot. Enzyme co-immobilization enables biocatalytic synergism and reusability, boosting process efficiency and cost-effectiveness. Several studies on multi-enzyme immobilization and co-localization propose kinetic advantages of the enhanced turnover number for multiple enzymes. This review spotlights recent progress in developing versatile biocatalytic cascade systems by multi-enzyme co-immobilization on environmentally friendly biopolymers and nanostructured materials and their application scope in the chemical and biotechnological industries. After a succinct overview of carrier-based and carrier-free immobilization/co-immobilizations, co-immobilization of enzymes on a range of biopolymer and nanomaterials-based supports is thoroughly compiled with contemporary and state-of-the-art examples. This study provides a new horizon in developing effective and innovative multi-enzymatic systems with new possibilities to fully harness the adventure of biocatalytic systems. [ABSTRACT FROM AUTHOR]

Enzymes play an important role in diverse industries and are critical components of many industrial products, yet, their application is limited due to their sensitivity to environmental conditions, recovery challenges, and susceptibility to inhibition. Immobilizing enzymes onto a suitable support matrix imparts higher resistance and improves operational flexibility, recyclability, and reusability. Lignin, a renewable and abundant biopolymer derived from the paper and pulp industry, has emerged as one of the prominent materials to be incorporated in support matrices. The distinctive characteristics of lignin include high mechanical strength, ease of separation, chemical stability, robust matrix for securing enzyme binding, biocompatibility, and ease of surface functionalization, making it a promising alternative to traditional synthetic materials. Research studies suggest the effectiveness of various lignin-based materials for immobilizing enzymes and significantly improving their stability, reusability, and catalytic activity. This article critically examines the unique properties of lignin and highlights significant contributions made in the development of enzyme immobilization for biocatalysis and biosensing applications. Additionally, the roles of hybrid materials, multienzyme immobilization, and innovative strategies like interfacial activation and enzyme shielding are discussed for overcoming the current challenges and developing sustainable, efficient, and robust biocatalytic and biosensing processes for industrial applications. [Display omitted] • Lignin is a promising support material for immobilizing diverse enzymes. • Lignin offers an advantage of high mechanical strength and biocompatibility. • Lignin offers an ecofriendly alternative to traditional methods. • Lignin based immobilization holds great potential in biocatalysis and biosensing [ABSTRACT FROM AUTHOR]

Among different microbes, fungi are proficient candidates for the extracellular synthesis of iron nanoparticles. For biogenic synthesis of iron nanoparticles, a thermophilic mould Myceliophthora thermophila BJTLRMDU7 was used in this study. Mycogenic magnetic nanoparticles were used for phosphatase immobilization and therapeutic applications such as antimicrobial and antimalarial activity. Firstly, the phosphatase was immobilized on biogenic iron nanoparticles with an efficiency of >56 %. Immobilized enzyme was optimally active at 60 °C and pH 5. Immobilized phosphatase was recycled using external magnetic field up to 4th cycle retaining >50 % activity. The immobilized phosphatase efficiently released inorganic phosphate from different flours such as wheat, maize and gram at 37 °C and 60 °C. There was continuous increase in the release of inorganic phosphorus from all samples with incubation time at 37 °C and slight reduction at 60 °C. These nanoparticles showed the effective antimicrobial activity against Bacillus subtilis , Escherichia coli and Myceliophthora thermophila. Further, the synthesized iron nanoparticles showed antimalarial potential against Plasmodium falciparum. Biogenic nanoparticles did not exhibit hemolytic activity and cytotoxicity. Therefore, biogenic iron nanoparticles could be used as a suitable matrix for immobilization of enzymes and safe therapeutics. • Mycogenic magnetic nanoparticles showed potential as novel matrix for phosphatase immobilization and recycling. • Immobilized phosphatase showed efficient hydrolysis of food samples. • Biogenic FeNPs showed potent antibacterial and antifungal activity. • Biogenic iron nanoparticles are safe and biocompatible. [ABSTRACT FROM AUTHOR]

Enzyme immobilization is an effective strategy for achieving efficient and sustainable enzyme catalysis. As a kind of promising enzyme-loading materials, the systematic research on zirconium based metal organic frameworks (Zr-MOFs) about immobilization performance at molecular level is still in its initial stage. In this work, UiO-66 was functionalized with various groups (-H, -NH 2 , -COOH, -OH, -2OH) for the immobilization of cytochrome c (Cyt c) and antioxidant enzyme catalase (CAT). Then the effects of surface-functionalized UiO-66 derivatives on the loading efficiency, enzyme stability and catalysis kinetics were systematically investigated. In addition, the affinity constants of Cyt c and CAT towards UiO-66-series MOFs carriers were also compared. The results have shown that hydroxyl group functionalized UiO-66 represents the highest enzyme loading capacity, enhanced activity and improved stability for Cyt c and CAT possibly due to high surface area and suitable microenvironments as well as enhanced affinity towards the enzymes provided by the introduction of a single hydroxyl group. Our research would foresee immense potential of MOFs in engineering biocatalysts. • The binding affinity of UiO-66 series MOFs with enzymes was investigated. • Both Cyt C and CAT immobilized UiO-66-OH exhibit the most catalytic efficiency. • The mechanisms of loading efficiency, activity and stability were explored. [ABSTRACT FROM AUTHOR]

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 (Fe 3 O 4 @silica-NH 2) aiming for higher stability, recoverability, reusability, and applicability in proteolysis and as a microbial rennet-like milk-clotting enzyme. The nanobiocatalyst developed (Fe 3 O 4 @silica-TLN) displays hydrolytic activity on a synthetic TLN substrate and, apparently, was fully recovered from reaction media by magnetic decantation. More importantly, Fe 3 O 4 @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, Fe 3 O 4 @silica-TLN catalyzed the digestion of bovine serum albumin. In conclusion, Fe 3 O 4 @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). • A new nanobiocatalyst derived from the microbial metalloprotease thermolysin (TLN) was developed and studied. • In the presence of calcium ions, Fe 3 O 4 @silica-TLN is active after exposure to 60 °C for 48 h or storages at 4 °C for 80 days and at r.t. for 42 days. • Fe 3 O 4 @silica-TLN efficiently proteolyses milk-caseins and BSA with the advantages of stability, recoverability, and reusability. • The developed nanobiocatalyst also acted as a stable and recoverable milk-clotting microbial enzyme, being reused up to 11 cycles. [ABSTRACT FROM AUTHOR]

Utilizing carbonic anhydrase (CA) to catalyze CO 2 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 CO 2 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 CO 2 conversion and utilization technologies. [Display omitted] • Carbonic anhydrase (CA) was immobilized onto delignified bamboo cellulose. • Immobilization increased both the optimal temperature and pH of CA. • The CA activity post-immobilization demonstrated effective retention. • The immobilized CA exhibited sustained reusability and high stability. • CA coupled with cellulose enhanced microalgal growth and biomass accumulation. [ABSTRACT FROM AUTHOR]

Azo dyes in textile industry effluents pose significant health and environmental risks. Laccase is an enzyme capable of degrading azo dyes, offering an environmentally friendly solution for treating textile wastewater. However, laccases need to be immobilized on specific carriers to enable effective reuse in batch reactors and continuous operation in flow-through reactors. This study employed silk fibroin nanofibers (SFNFs) obtained by mechanically grinding degummed silkworm silk as sustainable carriers to immobilize laccases through carbodiimide-mediated crosslinking. The immobilized laccases (SFNF-laccases) exhibited improved pH tolerance in the range of pH 3.0-8.0 with a smaller reduction in activity compared to free laccases (SFNF-laccases: 32.9 %, free laccases: 50.4 %). The thermal stability of immobilized laccases was also improved, showing 19, 13, and 9 % higher activities than those of free laccases at 40, 50, and 60 °C, respectively. After 8 days of storage, the activity of SFNF-laccases was 79 % of their activity immediately after immobilization, whereas free laccases retained only 29 % of their initial activity. In addition, SFNF-laccases maintained 73 % of their original operational activity in a flow-through reactor after 8 days. These results demonstrate the great potential of mechanically ground SFNFs as carriers of laccase and the resulting SFNF-laccases in industrial wastewater treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The combined technology of molecular imprinting and immobilization is a promising strategy for improving biocatalytic performance. In this work, a dual-functional cellulose-based hydrogel bioreactor with targeted recognition and transformation functions was innovatively designed by cellulose-based molecularly imprinted polymers (CM-MIPs) hydrogel coupled with layered double hydroxide immobilized enzyme (LDH-E). Firstly, CM-MIPs hydrogel was prepared by using phlorizin, 4-pinylpyridine, and cellulose microspheres as template molecule, functional monomer, and support material, respectively. Meanwhile, layered double hydroxide (LDH) taken as a carrier to immobilize β-glucosidase. The prepared LDH was layered sheet-like structure with ultra-thin thickness of approximately only 1.5 nm, and β-glucosidase was immobilized on both sides of the LDH. Further, the dual-functional bioreactor was constructed by the anion exchange, which possessed maximum targeted adsorption capacity to phlorizin of 15.25 mg/g, exhibited 1.2 folds higher transformation efficiency than LDH-E, and the phloretin content increased 26 folds to that in Lithocarpus litseifolius (Hance) Chu leaves extracts. Moreover, the transformation efficiency remained above 70 % even after five consecutive transformations. Overall, the dual-functional bioreactor has broad prospects for the application in targeted recognition and transformation of phytochemicals, and provides a new insight on multifunctional bio-based reactors in natural production field., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Novel hydrogel biocatalysts with immobilized lipase, stabilized by ionic liquids (ILs) of different hydrophobicity, were synthesized and evaluated. Variations of the time of immobilization and ratio of substrates during hydrogel synthesis were considered to obtain the most stable biocatalyst with the highest activity. Physicochemical characterization proved the success of the hydrogel synthesis and enzyme deposition on the surface of the support. Nevertheless, the key objective was to produce a biocatalyst for further application in ibuprofen methyl ester resolution, with the aim of obtaining an enantiomerically pure product. The hydrogel biocatalysts obtained in the presence of 5 wt% ILs after 8 h of immobilization achieved the highest activity recovery of 62 %. After 10 reaction cycles, enzymatic activity was still above 60 %, and the negative effect of pH and temperature on the activity of immobilized lipase was much lower than in the case of the free enzyme. After application of the catalyst in the resolution of ibuprofen methyl ester, the enantiomeric excess and conversion rate of the process were obtained for the dynamic kinetic resolution in isooctane. A conversion rate of 95 % was achieved due to the stabilization of the biocatalyst with IL and its resulting high catalytic activity. The study thus provides the pharmaceutical industry with a new potential approach with a strong scientific foundation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Integrating microfluidic devices and enzymatic processes in biocatalysis is a rapidly advancing field with promising applications. This review explores various facets, including applications, scalability, techno-commercial implications, and environmental consequences. Enzyme-embedded microfluidic devices offer advantages such as compact dimensions, rapid heat transfer, and minimal reagent consumption, especially in pharmaceutical optically pure compound synthesis. Addressing scalability challenges involves strategies for uniform flow distribution and consistent residence time. Incorporation with downstream processing and biocatalytic reactions makes the overall process environmentally friendly. The review navigates challenges related to reaction kinetics, cofactor recycling, and techno-commercial aspects, highlighting cost-effectiveness, safety enhancements, and reduced energy consumption. The potential for automation and commercial-grade infrastructure is discussed, considering initial investments and long-term savings. The incorporation of machine learning in enzyme-embedded microfluidic devices advocates a blend of experimental and in-silico methods for optimization. This comprehensive review examines the advancements and challenges associated with these devices, focusing on their integration with enzyme immobilization techniques, the optimization of process parameters, and the techno-commercial considerations crucial for their widespread implementation. Furthermore, this review offers novel insights into strategies for overcoming limitations such as design complexities, laminar flow challenges, enzyme loading optimization, catalyst fouling, and multi-enzyme immobilization, highlighting the potential for sustainable and efficient enzymatic processes in various industries., Competing Interests: Declaration of competing interest Authors report no conflict of interest on their parts., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Type of cross-linking agents influence the stability and active cross-linked enzyme aggregates (CLEA) immobilization. The information of molecular interaction between enzyme-cross linker is not well explored thus screening wide numbers of cross-linker is crucial in CLEA development. This study combined the molecular modeling and experimental optimization to investigate the influences of different cross-linking agents in developing CLEA of cyclodextrin glucanotranferase G1 (CGTase G1) for cyclodextrins (CDs) synthesis. Seven types of cross-linkers were tested and CGTase G1 cross-linked with chitosan (CS-CGTG1-CLEA) displayed the highest activity recovery (84.6 ± 0.26%), aligning with its highest binding affinity, radius of gyration and flexibility through in-silico analysis towards CGTase G1. CS-CGTG1-CLEA was characterized and showed a longer half-life (30.06 ± 1.51 min) and retained a greater thermal stability (52.73 ± 0.93%) after 30 min incubation at optimal conditions compared to free enzyme (10.30 ± 1.34 min and 5.51 ± 2.10% respectively). CS-CGTG1-CLEA improved CDs production by 33% and yielded cumulative of 52.62 g/L CDs after five cycles for 2 h of reaction. This study reveals that abundant of hydroxyl group on chitosan interacted with CGTase G1 surface amino acid residues to form strong and stable CLEA thus can be a promising biocatalyst in CDs production. • Different cross-linkers were screened using in-silico analysis for CGTase G1-CLEA. • Chitosan showed highest binding affinity with CGTase G1 as cross-linker. • Chitosan exhibited the highest activity recovery compared to other cross-linkers. • CGTase G1-CLEA demonstrated high thermal stability and longer half-life. • CGTase G1-CLEA yielded higher CDs production compared to free enzyme. [ABSTRACT FROM AUTHOR]

Magnetic nanoparticle coated with manganese‑aluminum layered double hydroxide (Fe3O4/Mg-Al-CO3-LDH) was prepared and used as porous support for ficin (EC 3.4.22.3) as a model enzyme. Structural characteristics were studied by XRD, FTIR, SEM and light scattering. The quantity of immobilized ficin on the mentioned LDH and non-magnetic LDH was measured and enzyme activity, stability and reusability were compared. Results revealed that the core and shell structure of Fe 3 O 4 /Mg-Al-CO 3 -LDH makes it better dispersion compared to the pristine Mg-Al-CO 3 -LDH. Ficin showed strong affinity to absorption of the surface of mentioned LDHs nanosheet especially magnetic LDH, confirmed that the existence of Fe 3 O 4 in the core structure of magnetic Fe 3 O 4 /Mg-Al-CO 3 -LDH caused better dispersion of LDH nanocrystal shell compared to pristine LDH moreover, enzyme which immobilized on the magnetic LDH supports, can be recovered by magnetic interaction. The storage stability of free ficin, immobilized ficin on the Mg-Al-CO 3 -LDH and Fe 3 O 4 /Mg-Al-CO 3 -LDH during a period of 120 days lost about 75%, 30%, and 20% of their initial activities, respectively. • Preparation of magnetic LDH as inorganic support to enzyme immobilization was discussed. • Immobilization efficiency of magnetic Mg/Al-CO3-LDH/ficin was inspected. • Merits of magnetizing of LDH as safe support to enzyme immobilization are highlighted. • Determination of optimum activity conditions of magnetic Mg/Al-CO3-LDH/ficin. [ABSTRACT FROM AUTHOR]

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. [Display omitted] [ABSTRACT FROM AUTHOR]

Aspergillus oryzae β-D-galactosidase (β-Gal) efficiently hydrolyzes sesaminol triglucoside into sesaminol, which has higher biological activity. However, β-Gal is difficult to be separate from the reaction mixture and limited by stability. To resolve these problems, β-Gal was immobilized on amino-functionalized magnetic nanoparticles mesoporous silica pre-activated with glutaraldehyde (Fe 3 O 4 @mSiO 2 -β-Gal), which was used for the first time to prepare sesaminol. Under the optimal conditions, the immobilization yield and recovered activity of β-Gal were 57.9 ± 0.3 % and 46.5 ± 0.9 %, and the enzymatic loading was 843 ± 21 U enzyme /g support. The construction of Fe 3 O 4 @mSiO 2 -β-Gal was confirmed by various characterization methods, and the results indicated it was suitable for heterogeneous enzyme-catalyzed reactions. Fe 3 O 4 @mSiO 2 -β-Gal was readily separable under magnetic action and displayed improved activity in extreme pH and temperature conditions. After 45 days of storage at 4 °C, the activity of Fe 3 O 4 @mSiO 2 -β-Gal remained at 92.3 ± 2.8 %, which was 1.29 times than that of free enzyme, and its activity remained above 85 % after 10 cycles. Fe 3 O 4 @mSiO 2 -β-Gal displayed higher affinity and catalytic efficiency. The half-life was 1.41 longer than free enzymes at 55.0 °C. Fe 3 O 4 @mSiO 2 -β-Gal was employed as a catalyst to prepare sesaminol, achieving a 96.7 % conversion yield of sesaminol. The excellent stability and catalytic efficiency provide broad benefits and potential for biocatalytic industry applications. [Display omitted] • The amino-functionalized magnetic mesoporous silica was pre-activated by glutaraldehyde. • Aspergillus oryzae β-D-galactosidase was successfully immobilized. • The immobilized enzyme displayed enhanced stability, substrate affinity, and recycling compared to the free enzyme. • The immobilized enzyme exhibited remarkable catalytic ability in the preparation of sesaminol. [ABSTRACT FROM AUTHOR]

Carbonic anhydrase (CA) has a promising application as a green and efficient biocatalyst for CO 2 capture, and many successful cases of immobilizing CA have been reported. However, CA antifouling coatings on metal for CO 2 sequestration have rarely been reported. Herein, dimeric CA from Sulfurihydrogenibium azorense (SazCA) with a ferritin tag, which was prepared by low-speed centrifugation with high yield, was adopted as a free enzyme and encapsulated in the sol-gel silica. The silica-immobilized CAs were dispersed into the commercialized metal-antifouling epoxy resin paint to obtain CA coated nickel foams, which had excellent stability, with 90 % and 67 % residual activity after 28 days of incubation at 30 °C and 60 °C, respectively. The CA coated nickel foams remained 60 % original activity after 6 cycles of use within 28 days. Then, a CA-microalgae carbon capture device was constructed using the CA coated nickel foams and Chlorella. The growth rate of Chlorella was significantly increased and the biomass of Chlorella increased by 29 % compared with control after 7 days of incubation. Due to the simple and cost-effective preparation process, sustainable and efficient CO 2 absorption, this easy-to-scale up CA coated nickel foam has great potential in CA assisted microalgae-based CO 2 capture and carbon neutrality. [Display omitted] • Highly expressed dimeric CA was simply purified by low-speed centrifugation. • A simple method for preparing immobilized CA antifouling coatings was proposed. • The CA coating has excellent stability and reusability for continuous CO 2 capture. • The CA coating can boost the growth of microalgae significantly. [ABSTRACT FROM AUTHOR]

In this study, casein@CoFe 2 O 4 was fabricated through a green synthesis methodology and applied to immobilize laccase. The constructed casein@CoFe 2 O 4 exhibited porous structures with distinct cavities and suitable magnetic properties. The abundance of aromatic functional groups on the surface renneted casein and possible π-type interaction between laccase and para-κ-casein resulted in a successful immobilization. The biocatalyst retained 50% of its initial activity after 24 reusability cycles, indicating stable immobilization of laccase onto the casein microstructures. The stability of laccase after immobilization was improved by 300% in comparison with the free enzyme, especially in basic pH values. The constructed laccase@casein@CoFe 2 O 4 was then incorporated to remove crystal violet (CV) as an environmentally harmful synthetic tri-phenylmethane dye. The prepared heterogeneous biocatalyst effectively diminished the antimicrobial activity of CV up to 81.3% in 40 min against some bacterial strains, resulting from the formation of more minor toxic metabolites identified by liquid chromatography coupled with mass spectroscopy after degradation procedure. The proposed green and feasible method for the preparation of magnetic casein aggregates has not been previously reported. The incorporation of casein, which acted as a molecular chaperon, resulted in a significant improvement in the enzymatic stability and exhibited appropriate reusability for the constructed biocatalytic system. • A novel method was established for synthesis of magnetic casein aggregates. • The immobilized laccase exhibited remarkable reusability. • Stability of laccase was enhanced after immobilization on casein@CoFe 2 O 4. • The immobilized laccase successfully removed crystal violet. • Biodegradation of crystal violet effectively diminished its micro-toxicity. [ABSTRACT FROM AUTHOR]

Here, we present highly porous, cellulose-based microspheres using (2,2,6,6-tetramethylpiperidine-1-oxyl) TEMPO-oxidized cellulose fibers (TOCFs) as starting materials. The TOCFs were first dissolved in a NaOH/urea solvent and transformed into microspheres via an emulsification method. The carboxyl groups on the surface of TOCFs were successfully carried on the cellulose-based microspheres, which provides them numerous reacting or binding sites, allowing them to be easily functionalized or immobilized with biomolecules for multi-functional applications. Furthermore, the introduction of magnetic nanoparticles awards these microspheres magnetic properties, allowing them to be attracted by a magnetic field. As a proof of concept, we demonstrate the application of using these carboxylate cellulose-based microspheres for enzyme immobilization. The cellulose-based microspheres can successfully create stable covalent bonds with enzymes after the activation of carboxyl groups. The enhanced pH tolerance, thermal stability, convenient recovery, and reusability position the emulsified microspheres as promising carriers for enzyme immobilization. [Display omitted] • TEMPO-oxidized cellulose was used as starting material for preparing microspheres. • The microspheres were prepared by emulsification of TOCF solution in paraffin oil. • The microspheres are highly porous with abundant carboxylate groups dispersed. • The microspheres can provide abundant binding sites for enzyme immobilization. • The immobilized enzyme showed improved stability with recyclability. [ABSTRACT FROM AUTHOR]

A review on the enzyme β-galactosidase from Kluyveromyces lactis is presented, from the perspective of its structure and mechanisms of action, the main catalyzed reactions, the key factors influencing its activity, and selectivity, as well as the main techniques used for improving the biocatalyst functionality. Particular attention was given to the discussion of hydrolysis, transglycosylation, and galactosylation reactions, which are commonly mediated by this enzyme. In addition, the products generated from these processes were highlighted. Finally, biocatalyst improvement techniques are also discussed, such as enzyme immobilization and protein engineering. On these topics, the most recent immobilization strategies are presented, emphasizing processes that not only allow the recovery of the biocatalyst but also deliver enzymes that show better resistance to high temperatures, chemicals, and inhibitors. In addition, genetic engineering techniques to improve the catalytic properties of the β-galactosidases were reported. This review gathers information to allow the development of biocatalysts based on the β-galactosidase enzyme from K. lactis , aiming to improve existing bioprocesses or develop new ones. • β-Galactosidase from Kluyveromyces lactis is a multimeric enzyme that depends on cations. • The enzyme suffers of diverse inhibitions. • The enzyme may be used in diverse reactions (hydrolysis, transglycosylation, glycosylation). • Immobilization and genetic tolls have been applied to improve the enzyme performance. [ABSTRACT FROM AUTHOR]

Papain is a cysteine protease from papaya, with many applications due to its broad specificity. This paper reviews for first time the immobilization of papain on different supports (organic, inorganic or hybrid supports) presenting some of the features of the utilized immobilization strategies (e.g., epoxide, glutaraldehyde, genipin, glyoxyl for covalent immobilization). Special focus is placed on the preparation of magnetic biocatalysts, which will permit the simple recovery of the biocatalyst even if the medium is a suspension. Problems specific to the immobilization of proteases (e.g., steric problems when hydrolyzing large proteins) are also defined. The benefits of a proper immobilization (enzyme stabilization, widening of the operation window) are discussed, together with some artifacts that may suggest an enzyme stabilization that may be unrelated to enzyme rigidification. • Advantages and problems of proteases immobilization are discussed. • Immobilization of papain in organic, inorganic and hydroid materials is discussed. • Most used groups to get covalent immobilization are presented. • Magnetic supports advantages are discussed. [ABSTRACT FROM AUTHOR]

Herein, our suggestion is to immobilize enzymes in-situ on absorbable shape-memory stents instead of injecting therapeutic enzymes into the blood. Chitosan (CHI)-based stents were tailored as novel support and the enzyme-immobilizing ability was elucidated using L-asparaginase (L-ASNase). For developing shape-memory stents, CHI-glycerol (GLY) solution was prepared and further blended with different ratios of polyethylene glycol (PEG), and polyvinyl alcohol (PVA). Afterward, the blends were modified by ionic crosslinking with sodium tripolyphosphate to obtain a shape-memory character. L-ASNase was included in the blends by using in-situ method before ionic crosslinking. The prepared stents, with or without L-ASNase, were comprehensively characterized by using several techniques. Collectively, immobilized L-ASNase exhibited much better performance in immobilization parameters than free one, thanks to its improved stability and reusability. For instance, CHI/GLY/PEG-3@L-ASNase retained about 70% of the initial activity after storage at 30 °C for 2 weeks, whereas the free form lost half of its initial activity. Besides, it retained 73.4% residual activity after 15 consecutive cycles. Most importantly, stent formulations exhibited ~60% activity in the bioreactor system after 4 weeks of incubation. Given the above results, shape-memory stents can be a promising candidate as a new platform for immobilization, especially in the blood circulation system. [Display omitted] • L-Asparaginase-containing shape-memory biodegradable stent formulations were prepared. • The stent formulations were characterized by FTIR, XRD, SEM, EDX, and thermal analysis. • The stent formulations showed porous morphology, high swelling ratio and mechanical behavior. • The L-Asparaginase-containing stent formulations showed good thermal and storage stability. • Most strikingly, the stent formulations exhibited 60% activity in mimicking blood circulation after 4 weeks incubation. [ABSTRACT FROM AUTHOR]

Chitosan is one of the most abundant natural polymer worldwide, and due to its inherent characteristics, its use in industrial processes has been extensively explored. Because it is biodegradable, biocompatible, non-toxic, hydrophilic, cheap, and has good physical-chemical stability, it is seen as an excellent alternative for the replacement of synthetic materials in the search for more sustainable production methodologies. Thus being, a possible biotechnological application of Chitosan is as a direct support for enzyme immobilization. However, its applicability is quite specific, and to overcome this issue, alternative pretreatments are required, such as chemical and physical modifications to its structure, enabling its use in a wider array of applications. This review aims to present the topic in detail, by exploring and discussing methods of employment of Chitosan in enzymatic immobilization processes with various enzymes, presenting its advantages and disadvantages, as well as listing possible chemical modifications and combinations with other compounds for formulating an ideal support for this purpose. First, we will present Chitosan emphasizing its characteristics that allow its use as enzyme support. Furthermore, we will discuss possible physicochemical modifications that can be made to Chitosan, mentioning the improvements obtained in each process. These discussions will enable a comprehensive comparison between, and an informed choice of, the best technologies concerning enzyme immobilization and the application conditions of the biocatalyst. [Display omitted] • Different methods for using Chitosan as a support for enzymatic immobilization are discussed. • Chemical and physical modifications can improve Chitosan properties. • Key factors to consider are the magnitude of the enzyme-support interaction and the operating conditions. • The choice of modification route will mainly depend on the properties that need improving. • Biocatalysts based on Chitosan supports can be used for industrial applications. [ABSTRACT FROM AUTHOR]

In the present study, two different modified starches; microporous starch (MPS) and cationic microporous starch (CMPS) were synthesized. The granules of MPS that distributed regularly were destroyed after the etherification reaction. The data depicted that the immobilization of horseradish peroxidase (HRP) on CMPS revealed highest immobilization efficiency (86%) at 100 mg of CMPS at pH = 6.0 and 100 units of enzyme. After 10 reuses of the CMPS-HRP, it retained 66% of initial activity. The soluble HRP showed broad pH optimum of 6.0–7.0, which changed to sharp pH = 6.0 for CMPS-HRP. Soluble-HRP and CMPS-HRP showed temperature optima at 30 °C and 40 °C, respectively. The CMPS-HRP showed high thermal stability up to 50 °C compared to the soluble HRP (40 °C). The Km values of soluble HRP and CMPS-HRP were 6.6 and 10.8 mM for H 2 O 2 and 34 and 41.6 mM for guaiacol, respectively. CMPS-HRP showed higher affinity toward various substrates than the soluble-HRP. CMPS-HRP showed more resistance against heavy metals, urea, isopropanol, Triton X-100 and trypsin than soluble enzyme. The CMPS-HRP showed higher ability to remove phenol and p -chlorophenol compared to soluble-HRP. [ABSTRACT FROM AUTHOR]

Human tyrosine hydroxylase (hTH) has key role in the production of catecholamine neurotransmitters. The structure, function and regulation of hTH has been extensively researched area and the possibility of enzyme replacement therapy (ERT) involving hTH through nanocarriers has been raised as well. However, our understanding on how hTH may interact with nanocarriers is still lacking. In this work, we attempted to investigate the immobilization of hTH on magnetic nanoparticles (MNPs) with various surface linkers in quantitative and mechanistic detail. Our results showed that the activity of hTH was retained after immobilization via secondary and covalent interactions as well. The colloidal stability of hTH could be also enhanced proved by Dynamic light scattering and Zeta potential analysis and a homogenous enzyme layer could be achieved, which was investigated by Raman mapping. The covalent attachment of hTH on MNPs via aldehyde or epoxy linkers provide irreversible immobilization and 38.1 % and 16.5 % recovery (ER). The hTH-MNPs catalyst had 25 % ER in average in simulated nasal electrolyte solution (SNES). This outcome highlights the relevance of immobilization applying MNPs as a potential formulation tool of sensitive therapeutic enzymes offering new opportunities for ERT related to neurodegenerative disorders. • The binding of human tyrosine hydroxylase on magnetic nanoparticles was developed. • Physical and chemical binding of hTH was systematically investigated. • Detailed physico-chemical characterization of the binding linkers was performed. • Retained activity of hTH bounded on MNPs with amino-linker had 38 % of native form. • hTH-MNPs formulas could mean new alternative in enzyme replacement therapies. [ABSTRACT FROM AUTHOR]

Immobilization technology plays an important role in enhancing enzyme stability and environmental adaptability. Despite its rapid development, this technology still encounters many challenges such as enzyme leakage, difficulties in large-scale implementation, and limited reusability. Drawing inspiration from natural paired molecules, this study aimed to establish a method for immobilized α-glucosidase using artificial antibody-antigen interaction. The proposed method consists of three main parts: synthesis of artificial antibodies, synthesis of artificial antigens, and assembly of the artificial antibody-antigen complex. The critical step in this method involves selecting a pair of structurally similar compounds: catechol as a template for preparing artificial antibodies and protocatechualdehyde for modifying the enzyme to create the artificial antigens. By utilizing the same functional groups in these compounds, specific recognition of the antigen by the artificial antibody can be achieved, thereby immobilizing the enzymes. The results demonstrated that the immobilization amount, specific activity, and enzyme activity of the immobilized α-glucosidase were 25.09 ± 0.10 mg/g, 5.71 ± 0.17 U/mg protein and 143.25 ± 1.71 U/g carrier , respectively. The immobilized α-glucosidase not only exhibited excellent reusability but also demonstrated remarkable performance in catalyzing the hydrolysis of 4-methylumbelliferyl-α-D-glucopyranoside. • A biomimetic strategy for immobilizing enzymes was proposed. • Catechol was used as a template for synthesizing artificial antibodies. • Enzyme modified with protocatechualdehyde was used as artificial antigen. • Artificial antibody can affinity recognize catechol structural fragments of antigen. [ABSTRACT FROM AUTHOR]

As one of the most important industrial enzymes, α-amylase is widely used in food processing, such as starch sugar and fermentation, bringing high added value to industry of more than a trillion dollars. We developed a multi-enzyme system (Glu&Gox@Cu-MOF-74) prepared by embedding α-glucosidase (Glu) and glucose oxidase (Gox) into the biomimetic metal-organic framework Cu-MOF-74 using in situ encapsulation within 15 min at room temperature for efficient and sensitive detection of α-amylase activity. Benefitting from the remarkable peroxidase-mimicking property and rigid skeleton of Cu-MOF-74, the biocatalytic platform exhibited excellent cascade activity and tolerance in various extremely harsh environments compared to natural enzymes. On this basis, a cascade biocatalytic platform was constructed for the detection of α-amylase activity with wide linear range (5–100 U/L) and low limit of detection (1.45 U/L). The colorimetric cascade scheme is important for the sensitive and selective determination of α-amylase in complex fermentation samples, and the detection time is short (∼0.5 h). This work provides new ideas for the detection of α-amylase based on the cascade amplification method. • A multi-enzyme system, Glu&Gox@Cu-MOF-74, was synthesized by in situ encapsulation. • A rapid and sensitive fluorescent assay was constructed for α-amylase activity detection. • A low limit of detection (1.45 U/L) and high selectivity were achieved. • The α-amylase assay was used for detection in fermentation samples. [ABSTRACT FROM AUTHOR]

Polyethylene terephthalate (PET) is one of the most produced plastics globally and its accumulation in the environment causes harm to the ecosystem. Polyethylene terephthalate hydrolyse (PETase) is an enzyme that can degrade PET into its monomers. However, free PETase lacks operational stabilities and is not reusable. In this study, development of cross-linked enzyme aggregate (CLEA) of PETase using amylopectin (Amy) as cross-linker was introduced to solve the limitations of free PETase. PETase-Amy-CLEA exhibited activity recovery of 81.9 % at its best immobilization condition. Furthermore, PETase-Amy-CLEA exhibited 1.37-, 2.75-, 2.28- and 1.36-fold higher half-lives than free PETase at 50 °C, 45 °C, 40 °C and 35 °C respectively. Moreover, PETase-Amy-CLEA showed broader pH stability from pH 5 to 10 and could be reused up to 5 cycles. PETase-Amy-CLEA retained >70 % of initial activity after 40 days of storage at 4 °C. In addition, lower K m of PETase-Amy-CLEA indicated better substrate affinity than free enzyme. PETase-Amy-CLEA corroded PET better and products yielded was 66.7 % higher than free PETase after 32 h of treatment. Hence, the enhanced operational stabilities, storage stability, reusability and plastic degradation ability are believed to make PETase-Amy-CLEA a promising biocatalyst in plastic degradation. • PETase-Amy-CLEA gave high activity recovery. • PETase-Amy-CLEA showed better thermal, pH and storage stabilities than free PETase. • PETase-Amy-CLEA showed better PET degradation than free PETase. • Molecular docking validated the precipitation results in CLEAs' preparation. • Molecular dynamic validated the results of thermal stability. [ABSTRACT FROM AUTHOR]

If the appropriate immobilization method and carrier support are not selected, partial decreases in the activity of enzymes may occur after immobilization. Herein, to overcome this challenge, an excitation mechanism that enables energy transfer was proposed. Modified upconverting nanoparticles (UCNPs) were constructed and the important role of near-infrared (NIR) excitation in enhancing the catalytic activity of the enzyme was demonstrated. For this purpose, UCNPs were first synthesized via the hydrothermal method, functionalized with isocyanate groups, and then, PEG-L-ASNase was immobilized via covalent binding. UCNPs with and without PEG-L-ASNase were extensively characterized by different methods. These supports had immobilization yield and activity efficiency of >96 % and 78 %, respectively. Moreover, immobilized enzymes exhibited improved pH, thermal, and storage stability. In addition, they retained >65 % of their initial activity even after 20 catalytic cycles. Biochemical and histological findings did not indicate a trend of toxicity in rats due to UCNPs. Most importantly, PEG-L-ASNase activity was triggered approximately 5- and 2-fold under in vitro and in vivo conditions, respectively. Overall, it is anticipated that this pioneering work will shed new light on the realistic and promising usage of NIR-excited UCNPs for the immobilization of enzymes in expensive and extensive applications. [Display omitted] • Upconverting nanoparticles (UCNPs) were designed for PEG-L-ASNase immobilization. • UCNPs had rod-shaped particles and particle size from 80.8 ± 27.2 nm. • The immobilized enzyme showed high stability and reusability. • No biochemical or histological toxicity due to UCNPs was observed in rats. • PEG-L-ASNase activity was triggered 2-fold as in vivo under NIR irradiation. [ABSTRACT FROM AUTHOR]

Immobilized enzymes have received incredible interests in industry, pharmaceuticals, chemistry and biochemistry sectors due to their various advantages such as ease of separation, multiple reusability, non-toxicity, biocompatibility, high activity and resistant to environmental changes. This review in between various immobilized enzymes focuses on lipase as one of the most practical enzyme and chitosan as a preferred biosupport for lipase immobilization and provides a broad range of studies of recent decade. We highlight several aspects of lipase immobilization on the surface of chitosan support containing various types of lipase and immobilization techniques from physical adsorption to covalent bonding and cross-linking with their benefits and drawbacks. The recent advances and future perspectives that can improve the present problems with lipase and chitosan such as high-price of lipase and low mechanical resistance of chitosan are also discussed. According to the literature, optimization of immobilization methods, combination of these methods with other techniques, physical and chemical modifications of chitosan, co-immobilization and protein engineering can be useful as a solution to overcome the mentioned limitations. [ABSTRACT FROM AUTHOR]

Lipases belong to α/β hydrolases that cause hydrolytic catalysis of triacylglycerols to release monoacylglycerols, diacylglycerols, and glycerol with free fatty acids. Lipases have a common active site that contains three amino acid residues in a conserved Gly-X-Ser-X-Gly motif: a nucleophilic serine residue, an acidic aspartic or glutamic acid residue, and a basic histidine residue. Lipase plays a significant role in numerous industrial and biotechnological processes, including paper, food, oleochemical and pharmaceutical applications. However, its instability and aqueous solubility make application expensive and relatively challenging. Immobilization has been considered as a promising approach to improve enzyme stability, reusability, and survival under extreme temperature and pH environments. Innumerable supporting material in the form of natural polymers and nanostructured materials is a crucial aspect in the procedure of lipase immobilization used to afford biocompatibility, stability in physio-chemical belongings, and profuse binding positions for enzymes. This review outlines the unique structural and functional properties of a large number of polymers and nanomaterials as robust support matrices for lipase immobilization. Given these supporting materials, the applications of immobilized lipases in different industries, such as biodiesel production, polymer synthesis, additives, detergent, textile, and food industry are also discussed. [ABSTRACT FROM AUTHOR]

Enzyme immobilization using inorganic materials has been shown to preserve enzyme activity improving and improve their practical applications in biocatalytic process designs. Proper immobilization methods have been used to obtain high recycling and storage stability. In this study, we compared the activity and stability of in situ or crosslink-immobilized enzymes in a CaCO 3 biomineral carrier. More than 30% of the initial enzyme activity was preserved for both the systems after 180 days upon 15 activity measurements at room temperature, confirming the improved stability of these enzyme systems (100 mM phosphate buffer, pH 8.0); however, differences in enzyme loading, activity, and characteristics were observed for each of these methods. Each system exhibited efficacy of 80% and 20%, respectively. Based on the same amount of immobilized enzyme (0.2 mg), the specific activities of hydrolysis of p -nitrophenyl butyrate substrate at room temperature of in situ immobilized carboxyl esterase (CE) and crosslinked CE were 11.37 and 7.63 mM min−1 mg−1, respectively (100 mM phosphate buffer, pH 8.0). Moreover, based on the kinetic behavior, in situ immobilized CE exhibited improved catalytic efficiency (Vmax Km−1) of the enzyme, exhibiting 4-fold higher activity and efficiency values than those of the CE immobilized in CaCO 3. This is the first study to describe the stabilization of enzymes in CaCO 3 and compare the enzyme kinetics and efficiencies between in situ immobilization and crosslinking in CaCO 3 carriers. [ABSTRACT FROM AUTHOR]

In the present study, ultrasound irradiation was utilized to synthesize a novel zinc metal-organic framework (MOF). Scanning electron microscopic images, exhibited homogenous morphology with a nano-sized distribution of the Zn-MOF structure as also confirmed by X-ray diffraction patterns. Following, physical immobilization of Lepidium draba peroxidase (LDP) were optimized on the Zn-MOF in phosphate buffer (50 mM, pH 6.5), ratio amount of MOF/enzyme; 7/1 after shaking for 15 min at 25 °C, with high protein loading of 109.9 mg/g and immobilization yield of 93.3%. Immobilized enzyme (IE) exhibited more than 330% enhanced specific activity and also exhibited more than 150% specific affinity to its substrate (3,3′,5,5′-tetramethylbenzidine) with respect to the free enzyme (FE). Optimum temperature of the IE was obtained at 20 °C while its was 25 °C for the FE, and thermostability of the IE augmented at temperature of 30 °C and 40 °C by the factors of 104 and 108% respectively. pH stability under neutral and basic condition and storage stability of the IE improved with respect to the FE as well as its structural stability (T m ; 73 °C for IE vs. 63 °C for FE). Furthermore, immobilization is accompanied with alteration on the enzyme structure as revealed by the intrinsic and extrinsic fluorescence spectra. • New Zn-MOF nanostructure was synthesized using ultrasound irradiations. • Recombinant LDP was successfully immobilized on the nanostructure. • Specific activity, storage stability and structural stability of the enzyme were improved upon immobilization. [ABSTRACT FROM AUTHOR]

Enzyme immobilization can increase enzyme reusability to reduce cost of industrial production. Ginkgo biloba leaf extract is commonly used for medical purposes, but it contains ginkgolic acid, which has negative effects on human health. Here, we report a novel approach to solve the problem by degrading the ginkgolic acid with immobilized-laccase, where core/shell composite nanoparticles prepared by coaxial electrospraying might be first applied to enzyme immobilization. The core/shell Fe 3 O 4 /nylon 6,6 composite nanoparticles (FNCNs) were prepared using one-step coaxial electrospraying and can be simply recovered by magnetic force. The glutaraldehyde-treated FNCNs (FNGCNs) were used to immobilize laccase. As a result, thermal stability of the free laccase was significantly improved in the range of 60–90 °C after immobilization. The laccase-immobilized FNGCNs (L-FNGCNs) were applied to degrade the ginkgolic acids, and the rate constants (k) and times (τ 50) were ~0.02 min−1 and lower than 39 min, respectively, showing good catalytic performance. Furthermore, the L-FNGCNs exhibited a relative activity higher than 0.5 after being stored for 21 days or reused for 5 cycles, showing good storage stability and reusability. Therefore, the FNGCNs carrier was a promising enzyme immobilization system and its further development and applications were of interest. Unlabelled Image • Fe 3 O 4 /nylon composite nanoparticles (FNCNs) were prepared by coaxial electrospraying. • The core/shell FNCNs can be simply recovered by magnetic force. • Laccase immobilization was carried out by the glutaraldehyde-treated FNCNs (FNGCNs). • Degradation of ginkgolic acids was achieved by the laccase-immobilized FNGCNs. • The laccase-immobilized FNGCNs showed good storage ability and reusability. [ABSTRACT FROM AUTHOR]

The increasing use of sustainable manufacturing technologies in the industry presents a constant challenge for the development of suitable biocatalysts. Traditionally, improved biocatalysts are developed either using protein engineering (PE) or enzyme immobilization (EI). However, these approaches are usually not simultaneously applied. In this work, we designed and validated an enzyme improvement platform, Immobilized Biocatalyst Engineering (IBE), which simultaneously integrates PE and EI, with a unique combination of improvement through amino acid substitutions and attachment to a support material, allowing to select variants that would not be found through single or subsequent PE and EI improvement strategies. Our results show that there is a significant difference on the best performing variants identified through IBE, when compared to those that could be identified as soluble enzymes and then immobilized, especially when evaluating variants with low enzyme as soluble enzymes and high activity when immobilized. IBE allows evaluating thousands of variants in a short time through an integrated screening, and selection can be made with more information, resulting in the detection of highly stable and active heterogeneous biocatalysts. This novel approach can translate into a higher probability of finding suitable biocatalysts for highly demanding processes. • An enzyme improvement platform was generated: Immobilized Biocatalyst Engineering. • IBE simultaneously integrates protein engineering and enzyme immobilization. • Over 1800 mutants were immobilized using a high-throughput immobilization strategy. • IBE allows the immobilization to be included in the screening and selection steps. • IBE identified variants that would not be selected using traditional screening. [ABSTRACT FROM AUTHOR]

Alcohol dehydrogenases/reductases catalyze enantioselective syntheses of versatile chiral compounds relying on direct hydride transfer from cofactor to substrates, or to an intermediate and then to substrates. Since most of the substrates catalyzed by alcohol dehydrogenases/reductases are insoluble in aqueous solutions, increasing interest has been turning to organic-aqueous systems. However, alcohol dehydrogenases/reductases are normally instable in organic solvents, leading to the unsatisfied enantioselective synthesis efficiency. The behaviors of these enzymes in organic solvents at an atomic level are unclear, thus it is of great importance to understand its structure-based mechanisms in organic-aqueous systems to improve their relative stability. Here, we summarized the accessible structures of alcohol dehydrogenases/reductases in Protein Data Bank crystallized in organic-aqueous systems, and compared the structures of alcohol dehydrogenases/reductases which have different tolerance towards organic solvents. By understanding the catalytic behaviors and mechanisms of these enzymes in organic-aqueous systems, the efficient enantioselective syntheses mediated by alcohol dehydrogenases/reductases and further challenges are also discussed through solvent engineering and enzyme-immobilization in the last decade. Unlabelled Image • Alcohol dehydrogenases/reductases (SDRs) are powerful and valuable biocatalysts. • SDRs are unstable in organic solvents. • Structure-based mechanisms of SDRs in orgainc-aqueous systems are discussed. • Recent applications of SDRs in enantioselective syntheses in organic-aqueous reactions are summarized and discussed. [ABSTRACT FROM AUTHOR]

Despite the widespread use in various industries, enzyme's instability and non-reusability limit their applications which can be overcome by immobilization. The nature of the enzyme's support material and method of immobilization affect activity, stability, and kinetics properties of enzymes. Here, we report a comparative study of the effects of inorganic support materials on immobilized enzymes. Accordingly, immobilization of enzymes on nanoinorganic support materials significantly improved thermal and pH stability. Furthermore, immobilizations of enzymes on the materials mainly increased K m values while decreased the V max values of enzymes. Immobilized enzymes on nanoinorganic support materials showed the increase in ΔG value, and decrease in both ΔH and ΔS values. In contrast to weak physical adsorption immobilization, covalently-bound and multipoint-attached immobilized enzymes do not release from the support surface to contaminate the product and thus the cost is decreased while the product quality is increased. Nevertheless, nanomaterials can enter the environment and increase health and environmental risks and should be used cautiously. Altogether, it can be predicated that hybrid support materials, directed immobilization methods, site-directed mutagenesis, recombinant fusion protein technology, green nanomaterials and trailor-made supports will be used increasingly to produce more efficient immobilized industrial enzymes in near future. [ABSTRACT FROM AUTHOR]

In recent years, the synthesis and application of green and sustainable products have become global ecological and societal issues. Based on the principles of green chemistry, the application of different biocatalysts not only produce target products and decreases side effects but also enhances the selectivity and activity. Enzyme-based biocatalysts are very interesting due to their high catalytic performance, eco-friendly reaction systems, and selectivity. Immobilization is demonstrated as a favorable approach to improve the stability and recyclability of enzymes. Among different supports, porous and crystalline materials, covalent organic frameworks (COFs), represent an interesting class of support matrices for the immobilization of different enzymes. Owing to tunable physicochemical characteristics, a high degree of crystallinity, large specific surface area, superior adsorption capacity, pre-designable structure and marked stability, COFs might consider as perfect host materials for improving the desirable properties of enzymes, such as poor stability, low operational range, lack of repeatability, and products/by-products inhibition for large-scale applications. The enzyme-incorporated COFs have emerged as one of the hopeful ways to constitute tailor-made biocatalytic systems, which can be employed in an array of reactions. Highly porous nature of many COFs led to increased process output in contrast to other micro/nanoparticles. The enzymes can be integrated into COFs through different techniques, including physical adsorption and direct covalent attachment between the enzyme molecules and COFs or through a cross-linking agent. Herein, we discuss and highlight the synthesis methods, properties, and functionalization of COFs and the recent literature for the application of these materials in enzymes immobilization. Main approaches for immobilization of enzymes into COFs and the catalytic applications of these materials are also presented. This study offers new avenues to address the limitations of traditional enzyme immobilization supports as well as delivers new possibilities to construct smart biocatalytic systems for diverse biotechnological applications. [ABSTRACT FROM AUTHOR]

Micro/nanomotors (MNMs), both self-propelled actuators and external fields-promoted machines, have joined forces in the past decade to accomplish versatile tasks such as precise detection and targeted cargo delivery with adequate propulsion and desirable locomotion. Amongst, enzyme-driven MNMs have been able to differentiate themselves from others owing to their distinct characteristics, such as absence of chemical fuel, enhanced cellular uptake and the possibility to be easily conjugated with many therapeutics, including both small molecules and biologics, displaying superior efficacy, enhanced specificity and diminished side effects. In the present review, we aim to highlight and summarize recent advances in enzyme-driven MNMs, particularly to provide an in-depth discussion focusing on the enzyme linking approaches onto those MNMs and motion control strategies of such MNMs with advantages and limitations thereof. Conclusions and future perspectives are also provided in brief. Unlabelled Image [ABSTRACT FROM AUTHOR]

A recombinant β-xylosidase (rBxTW1) from the ascomycete Talaromyces amestolkiae and a mutant derived from it, with mostly synthetic activity, have been immobilized as magnetic cross-linked enzyme aggregates (mCLEAs). The mCLEAs of rBxTW1 kept the excellent hydrolytic and O -transxylosylating activities of the free enzyme and had improved thermal and pH stability. The mCLEAs of the mutant also maintained or improved the catalytic properties of the soluble enzyme, synthetizing the O -xylosides of vanillin and (−)-epigallocatechin gallate, and the N - and S -xyloside of 3,5-dibromo-1,2,4-triazole and thiophenol, respectively. The mCLEAs were recyclable across 4 cycles of synthesis of the O -xylosides through a green and highly selective process. The magnetic properties of the scaffold used for immobilization may allow the easy recovery and reuse of the biocatalyst even from reactions containing insoluble lignocellulosic biomass. • A β-xylosidase and its mutant multi-glycoligase were immobilized as mCLEAs. • mCLEAs showed improved catalytic activity and stability to pH and temperature. • mCLEAs of β-xylosidase were able to hydrolyze p NPX as well as natural substrates. • mCLEAs were successfully tested in O-, S- and N-glycosylation of acceptors. • mCLEAs keep their activity in at least 4 cycles of glycosylation. [ABSTRACT FROM AUTHOR]

Enzymes are the highly versatile bio-catalysts having the potential for being employed in biotechnological and industrial sectors to catalyze biosynthetic reactions over a commercial point of view. Immobilization of enzymes has improved catalytic properties, retention activities, thermal and storage stabilities as well as reusabilities of enzymes in synthetic environments that have enthralled significant attention over the past few years. Dreadful efforts have been emphasized on the renewable and synthetic supports/composite materials to reserve their inherent characteristics such as biocompatibility, non-toxicity, accessibility of numerous reactive sites for profitable immobilization of biological molecules that often serve diverse applications in the pharmaceutical, environmental, and energy sectors. Supports should be endowed with unique physicochemical properties including high specific surface area, hydrophobicity, hydrophilicity, enantioselectivities, multivalent functionalization which professed them as competent carriers for enzyme immobilization. Organic, inorganic, and nano-based platforms are more potent, stable, highly recovered even after used for continuous catalytic processes, broadly renders the enzymes to get efficiently immobilized to develop an inherent bio-catalytic system that displays higher activities as compared to free-counter parts. This review highlights the recent advances or developments on renewable and synthetic matrices that are utilized for the immobilization of enzymes to deliver emerging applications around the globe. Unlabelled Image • Organic matrices, inorganic matrices, and nanomaterials have proved to be versatile enzyme immobilization carriers. • Intensive efforts have been focused on recent advances based on the engineering of enzymes using renewable scaffolds.. • Multiple applications and advantages of immobilized enzymes in various emerging fields are being reviewed. • Immobilized enzymes have shown high thermal and storage stability, retention activity, as compared to their native forms. • Immobilized enzymes are, robust, stable, more recoverable, and resistant to environmental changes than free counterparts. [ABSTRACT FROM AUTHOR]

Nanostructured materials represent an interesting and novel class of support matrices for the immobilization of different enzymes. Owing to the high surface area, robust mechanical stability, outstanding optical, thermal, and electrical properties, nanomaterials have been rightly perceived as desired immobilization matrices for lipases immobilization with a wide array of biotechnological applications such as dairy, food technology, fine chemical, pharmaceutical, detergent, and oleochemical industries. Lipases immobilized on nanomaterials have demonstrated superior attributes than free counterparts, such as aggrandized pH and thermal stability, robustness, long-term stability, and the possibility of reuse and recycling in several times. Here we review current and state-of-the-art literature on the use of nanomaterials as novel platforms for the immobilization of lipase enzymes. The physicochemical properties and exploitation of a large number of new nanostructured materials such as carbon nanotubes, nano-silica, graphene/graphene oxide, metal nanoparticles, magnetic nanostructures, metal-organic frameworks, and hybrid nanoflowers as a host matrix to constitute robust lipases-based nanobiocatalytic systems are discussed. Conclusive remarks, trends, and future recommendations for nanomaterial immobilized enzymes are also given. [ABSTRACT FROM AUTHOR]

The Phenylketonuria (PKU) is an inborn defect of phenylalanine (Phe) metabolism, in which Phe accumulated in the blood causing alterations at the central nervous system. Therefore, the detection of PKU is very important for the early diagnosis of PKU patients. However, existing tests for PKU are time-consuming and require high-resource laboratories. In this study, a novel paper-based biosensor based on phenylalnine ammonia lyase (PAL) hybrid nanoflowers was constructed that provides a semi-quantitative output of the concentration of Phe from urine samples. PAL@Ca 3 (PO 4) 2 hybrid nanoflowers (PAL@NF) were first prepared using PAL and Ca2+. Synthesis conditions of the PAL@NF on the formation of the PAL@NF were optimized. The PAL@NF exhibited 90% activity recovery under optimal condition. Compared with free PAL, the PAL@NF displayed good storage stability and increased tolerance to proteolysis. After five consecutive operating cycles, the PAL@NF still retained 73% of its initial activity, indicating excellent reusability. Furthermore, the paper-based biosensor was able to detect Phe concentration in urine samples, and exhibited good linearity to the Phe concentrations in the range from 60 to 2400 μM and the response time was only about 10 min. Therefore, the paper-based biosensor can be a promising candidate as a biosensor for the detection of PKU. Unlabelled Image • PAL@Ca 3 (PO 4) 2 hybrid nanoflowers were successfully synthesized. • The PAL@Ca 3 (PO 4) 2 hybrid nanoflowers exhibited high activity and stability. • A paper-based biosensor based on the PAL hybrid nanoflowers for urinary Phe measurement was constructed for the first time. • The paper-based biosensor is expected to become a home-test monitoring for PKU. [ABSTRACT FROM AUTHOR]

In this study, serine alkaline protease from halotolerant alkaliphilic Salipaludibacillus agaradhaerens strain AK-R was purified and immobilized onto double mesoporous core-shell silica (DMCSS) nanospheres. Covalent immobilization of AK-R protease onto activated DMCSS-NH 2 nanospheres was more efficient than physical adsorption and was applied in further studies. DMCSS-NH 2 nanospheres showed high loading capacity of 103.8 μg protein/mg nanospheres. Relative to free AK-R protease, the immobilized enzyme exhibited shifts in the optimal temperature and pH from 60 to 65 °C and pH 10.0 to 10.5, respectively. While the soluble enzyme retained 47.2% and 9.1% of its activity after treatment for 1 h at 50 and 60 °C, the immobilized protease maintained 87.7% and 48.3%, respectively. After treatment for 2 h at pH 5 and 13, the immobilized protease maintained 73.6% and 53.4% of its activity, whereas the soluble enzyme retained 32.9% and 1.4%, respectively. Furthermore, the immobilized AK-R protease showed significant improvement of enzyme stability in high concentration of NaCl, organic solvents, surfactants, and commercial detergents. In addition, the immobilized protease exhibited a very good operational stability, retaining 79.8% of its activity after ten cycles. The results clearly suggest that the developed immobilized protease system is a promising nanobiocatalyst for various protease applications. • Alkaline protease was purified from halotolerant alkaliphilic Salipaludibacillus agaradhaerens strain AK-R. • AK-R protease was immobilized onto double mesoporous core-shell silica nanospheres by physical and covalent immobilization • Covalently immobilized AK-R protease exhibited enhancements of stability against temperature, pH, salt and organic solvents • Immobilized AK-R protease showed significant improvement of stability in a range of surfactants and commercial detergents • The developed immobilized alkaline protease is a promising nanobiocatalyst for various protease applications [ABSTRACT FROM AUTHOR]

In the recent past, numerous new types of nanostructured carriers, as support matrices, have been engineered to advance the traditional enzyme immobilization strategies. The current research aimed to develop a robust enzyme-based biocatalytic platform and its effective deployment in the industrial biotechnology sectors at large and catalysis area, in particular, as low-cost biocatalytic systems. Suitable coordination between the target enzyme molecules and surface pendent multifunctional entities of nanostructured carriers has led an effective and significant contribution in myriad novel industrial, biotechnological, and biomedical applications. As compared to the immobilization on planar two-dimensional (2-D) surface, the unique physicochemical, structural and functional attributes of nano-engineered matrices, such as high surface-to-volume ratio, surface area, robust chemical and mechanical stability, surface pendant functional groups, outstanding optical, thermal, and electrical characteristics, resulted in the concentration of the immobilized entity being substantially higher, which is highly requisite from applied bio-catalysis perspective. Besides inherited features, nanostructured materials-based enzyme immobilization aided additional features, such as (1) ease in the preparation or green synthesis route, (2) no or minimal use of surfactants and harsh reagents, (3) homogeneous and well-defined core-shell nanostructures with thick enzyme shell, and (4) nano-size can be conveniently tailored within utility limits, as compared to the conventional enzyme immobilization. Moreover, the growing catalytic needs can be fulfilled by multi-enzymes co-immobilization on these nanostructured materials-based support matrices. This review spotlights the unique structural and functional attributes of several nanostructured materials, including carbon nanotubes, graphene, and its derivate constructs, nanoparticles, nanoflowers, and metal-organic frameworks as robust matrices for laccase immobilization. The later half of the review focuses on the applied perspective of immobilized laccases for the degradation of emergent contaminants, biosensing cues, and lignin deconstruction and high-value products. [ABSTRACT FROM AUTHOR]

The ever-increasing applications of enzymes are limited by the relatively poor performance in harsh processing conditions. As a result, there are constant innovations in immobilization protocols for improving biocatalyst activity and stability. Bacterial spores are cheap to generate and highly resistant to environmental stress. The spore core is sheathed by an inner membrane, the germ cell wall, the cortex, outer membrane, spore coat and in some species the exosporium. The spore surface is anion-rich, hydrophobic and contains several reactive groups capable of interacting and stabilizing enzyme molecules through electrostatic forces, hydrophobic interactions and covalent bonding. The probiotic nature of spores obtained from non-toxic bacterial species makes them suitable carriers for the enzyme immobilization, especially food-grade enzymes or those intended for therapeutic use. Immobilization on spores is by direct adsorption, covalent attachment or surface display during the sporulation phase. Hindrances to the immobilization on spore matrix include the production rates, operational instability, and reduced catalytic properties due to conformational changes in enzyme. This paper reviews bacterial spore as a heterofunctional support matrix gives reasons why probiotic bacillus spores are better options and the diverse technologies adopted for spore-enzyme immobilization. It further suggests directions for future use and discusses the commercialization prospects. • Bacterial spores are anionic, hydrophobic and contain several reactive groups. • Enzyme immobilization on spores is by adsorption, covalent binding or in-situ display. • Probiotic Bacillus spores possess unique potentials to serve as enzyme support matrix. • Probiotic spores are cheap to generate and very resistant to environmental stress. • Immobilization on spores has therapeutic benefits and commercialization prospects. [ABSTRACT FROM AUTHOR]

This work evaluates different dendrimer-silica supports for the immobilization of enzymes by multipoint covalent binding. Thermolysin was immobilized on two dendrimers (PAMAM and carbosilane) with two different generations (zero (G0) and first (G1)). Results were compared with a control, a silica support functionalized with a monofunctional molecule. Dendrimers increased the number of available sites to bind the enzyme. Despite the enzyme was immobilized on all supports, G0 dendrimers immobilized a 30% more enzyme than G1. Thermolysin immobilized on G0 dendrimer supports showed the highest activity and could be employed in three consecutive hydrolysis cycles. Optimal immobilization time was 1 h while optimal protein loading was 25 mg enzyme/100 mg support. Enzyme activity was promoted when using 5 mg of immobilized enzyme at 750 rpm, 60 °C, and 2 h of hydrolysis. Under these conditions, the activity of thermolysin increased up to the 78% of the free enzyme activity. Kinetics of the hydrolysis reaction using the immobilized thermolysin was also studied and compared with the obtained using the free thermolysin. The addition of ZnCl 2 and NaCl during the immobilization procedure increased thermolysin activity in the second (22% more) and in the third (14% more) hydrolysis clycles. • Thermolysin has been successfully immobilized on dendrimer-silica supports. • Zero generation PAMAM and carbosilane dendrimers showed the highest immobilization. • Immobilized thermolysin was reutilized for different digestion cycles. • Enzyme immobilization in presence of salts improved thermolysin activity. • Supports activated by dendrimers promoted the immobilization and activity of enzyme. [ABSTRACT FROM AUTHOR]

Composite polycaprolactone–chitosan material was produced by an electrospinning method and used as a support for immobilization of tyrosinase by mixed ionic interactions and hydrogen bonds formation. The morphology of the fibers and enzyme deposition were confirmed by SEM images. Further, multivariate polynomial regression was used to model the experimental data and to determine optimal conditions for immobilization process, which were found to be pH 7, temperature 25 °C and 16 h process duration. Under these conditions, novel type of biocatalytic system was produced with immobilization yield of 93% and expressed activity of 95%. Furthermore, as prepared system was applied in batch experiments related to biodegradation of bisphenol A under various remediation conditions. It was found that over 80% of the pollutant was removed after 120 min of the process, in the temperature range 15–45 °C and pH 6–9, using solutions at concentration up to 3 mg/L. Experimental data collected proved that the stability and reusability of the tyrosinase were significantly improved upon immobilization: the immobilized biomolecule retained around 90% of its initial activity after 30 days of storage, and was still capable to remove over 80% of bisphenol A even after 10 repeated uses. By contrast, free enzyme was able to remove over 80% of bisphenol A at pH 7–8 and temperature range 15–35 °C, and retained less than 60% of its initial activity after 30 days of storage. Unlabelled Image • Effective immobilization of tyrosinase onto PCL-chitosan electrospun material • Optimization of immobilization conditions using response surface methodology • Application of biocatalytic systems produced for removal of bisphenol A • Total removal of BPA after 90 min of the process at temperature 25 °C and pH 7 • Improvement of enzyme stability and reusability upon immobilization [ABSTRACT FROM AUTHOR]

This paper has focused on making suitable carriers for the immobilization of α-amylase from Bacillus subtilis and lipase from Candida rugosa via adsorption on modified Na-sepiolite (SEP). Two modified carriers were prepared by changing the nature of SEP with cetrimonium bromide surfactant at concentrations below the CMC of the surfactant, to produce SEP with monolayer surfactant (MSEP) with hydrophobic properties and above the CMC of the surfactant to produce SEP with bilayer surfactant (BSEP) with hydrophilic properties. The enzymatic activity of immobilized lipase on MSEP (MSEPL) and immobilized α-amylase on BSEP (BSEPA) was successfully tested in the hydrolysis of olive oil and starch. The support modification and immobilization process were characterized by BET, XRD, and SEM techniques. The results demonstrated that the specific activity of MSEPL and BSEPA was 1.74 and 2.28 U/mg, respectively. The remained activity of MSEPL (56.7%) and BSEPA (40.4%) after their incubation at 60 °C for 4 h was much higher than that of free enzymes. The residual activity of MSEPL and BSEPA was 77.4% and 66.7%, after 30 days of storage at 4 °C. The MSEPL and BSEPA also showed good reusability, and their relative activities were 54.0% and 44.2% after ten cycles. Unlabelled Image • Make proper surfaces for immobilization of lipase and α-amylase by surfactant modification of SEP • The MSEPL and BSEPA showed the most activities among the other biocatalysts • The specific activity for the MSEPL and BSEPA was 1.74 and 2.28 U/mg, respectively • Immobilization improves the pH stability, thermal resistance, storage capacity and reusability of MSEPL and BSEPA [ABSTRACT FROM AUTHOR]

Nanostructured materials constitute an interesting and novel class of support matrices for the immobilization of peroxidase enzymes. Owing to the high surface area, robust mechanical stability, outstanding optical, thermal, and electrical properties, nanomaterials have been rightly perceived as immobilization matrices for enzyme immobilization with applications in diverse areas such as nano-biocatalysis, biosensing, drug delivery, antimicrobial activities, solar cells, and environmental protection. Many nano-scale materials have been employed as support matrices for the immobilization of different classes of enzymes. Nanobiocatalysts, enzymes immobilized on nano-size materials, are more stable, catalytically robust, and could be reused and recycled in multiple reaction cycles. In this review, we illustrate the unique structural/functional features and potentialities of nanomaterials-immobilized peroxidase enzymes in different biotechnological applications. After a comprehensive introduction to the immobilized enzymes and nanocarriers, the first section reviewed carbonaceous nanomaterials (carbon nanotube, graphene, and its derivatives) as a host matrix to constitute robust peroxidases-based nanobiocatalytic systems. The second half covers metallic nanomaterials (metals, and metal oxides) and some other novel materials as host carriers for peroxidases immobilization. The next section vetted the potential biotechnological applications of the resulted nanomaterials-immobilized robust peroxidases-based nanobiocatalytic systems. Concluding remarks, trends, and future recommendations for nanomaterial immobilized enzymes are also given. [ABSTRACT FROM AUTHOR]

Polypeptide-mediated silica mineralization is an attractive approach to prepare polypeptide/silica nanocomposites for enzyme immobilization. Herein, a facile approach for in situ immobilization of catalase (CAT) in polypeptide/silica nanocomposites is developed via the preparation of cross-linked polypeptide/enzyme microgels using an emulsion process followed by silica mineralization. The efficient protein immobilization under benign condition (25–28 °C, pH 7.0, 0.05 N) was evidenced by high immobilization yield (> 99%) and no protein leakage. Our data showed that the immobilized CAT exhibited prolonged reusability and storage stability compared to free one, suggesting that the composite networks not only provide suitable microenvironments to facilitate enzymatic reactions but also confine the enzyme macromolecules to prevent subunit dissociation. Star-shaped topology exhibited better coverage onto the enzyme than linear counterpart, leading to the superior reusability (relative activity >95% for 30 cycling number) and storage stability (relative activity >95% for 60 days) of the immobilized CAT (~ 14 mg/g of support). The substrate affinity and enzymatic reaction rate for the immobilized CAT were also influenced by silica content and polypeptide topology. This strategy may provide a feasible and inexpensive approach to fabricate polypeptide/silica nanocomposites, which would be promising materials in biotechnological fields such as enzyme immobilization. Unlabelled Image • A facile approach for in situ immobilization of enzyme in the polypeptide/silica nanocomposites • Preparation of polypeptide/enzyme microgels using an emulsion process followed by silica biomineralization. • Immobilized enzyme exhibiting prolonged reusability and storage stability • Substrate affinity and enzymatic reaction rate influenced by silica content and polypeptide topology. [ABSTRACT FROM AUTHOR]

Bacterial cellulose (BC) has emerged as an attractive adsorptive material for antimicrobial agents due to its fine network structure, its large surface area, and its high porosity. In the present study, BC paper was first produced and then lysozyme was immobilized onto it by physical adsorption, obtaining a composite of lysozyme-BC paper. The morphology and the crystalline structure of the composite were similar to that of BC paper as examined by scanning electron microscopy and X-ray diffraction, respectively. Regarding operational properties, specific activities of immobilized and free lysozyme were similar. Moreover, immobilized enzyme showed a broader working temperature and higher thermal stability. The composites maintained its activity for at least 80 days without any special storage. Lysozyme-BC paper displayed antimicrobial activity against Gram-positive and Gram-negative bacteria, inhibiting their growth by 82% and 68%, respectively. Additionally, the presence of lysozyme increased the antioxidant activity of BC paper by 30%. The results indicated that BC is a suitable material to produce bioactive paper as it provides a biocompatible environment without compromising the activity of the immobilized protein. BC paper with antimicrobial and antioxidant properties may have application in the field of active packaging. [Display omitted] • Bacterial cellulose paper is suitable for physical adsorption of lysozyme. • Lysozyme retained activity after incorporation in BC paper matrix. • Lys-BC biopaper presented antimicrobial and antioxidant activity. • Enzymatically active paper showed high stability and durability. • Lys-BC biopaper could be suitable for active packaging. [ABSTRACT FROM AUTHOR]