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The use of organic-inorganic hybrid nanoflowers as a support material for enzyme immobilization has gained significant attention in recent years due to their high stability, ease of preparation, and enhanced catalytic activity. However, a major challenge in utilizing these hybrid nanoflowers for enzyme immobilization is the difficulty in handling and separating them due to their low density and high dispersion. To address this issue, magnetic nanoflowers have emerged as a promising alternative enzyme immobilization platform due to their easy separation, structural stability, and ability to enhance catalytic efficiency. This review focuses on different methods for designing magnetic nanoflowers, as well as future research directions. Additionally, it provides examples of enzymes immobilized in the form of magnetic nanoflowers and their applications in environmental remediation, biosensors, and food industries. Finally, the review discusses possible ways to improve the material for enhanced catalytic activity, structural stability, and scalability.

Integrating enzymes and nanozymes in various applications is a topic of significant interest. The researchers have explored the encapsulation of enzymes using diverse nanostructures to create nanomaterial-enzyme hybrids. These nanomaterials introduce unique properties that contribute to the additional activity along with the stabilization of enzymes in immobilized form, enabling a cascade of second-order reactions. This review centers on dual-activity nanozymes, providing insights into their applications in biosensors and biocatalysis. These applications leverage the enhanced catalytic activity and stability offered by dual-activity nanozymes. These nanozymes find promising applications in fields like bioremediation, offering eco-friendly solutions for mitigating environmental pollution while showing potential in medical diagnostics. The review delves into various techniques for creating enzyme-nanozyme hybrid catalysts, including adsorption, encapsulation, and incorporation methods. The review also addresses the challenges that must be overcome, such as overlapping catalytic surfaces and disparities in reaction rates in multi-enzyme cascade reactions. It concludes by presenting strategies to tackle these issues and offers insights into the field's promising future, suggesting that machine learning may drive further advancements in enzyme-nanozyme integration. This comprehensive exploration illuminates the present and charts a promising course for future innovations in the seamless integration of enzymes and nanozymes, heralding a new era of catalytic possibilities., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Shamraja Nadar reports administrative support was provided by the Institute of Chemical Technology. Shamraja Nadar reports a relationship with the Institute of Chemical Technology that includes: employment. Shamraja Nadar has patent pending to NA. Authors have no conflict of interest If there are other authors, they 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 Elsevier B.V. All rights reserved.)

Conventional techniques for enzyme immobilization suffer from suboptimal activity recovery due to insufficient enzyme loading and inadequate stability. Furthermore, these techniques are time-consuming and involve multiple steps which limit the applicability of immobilized enzymes. In contrast, the use of microfluidic devices for enzyme immobilization has garnered significant attention due to its ability to precisely control immobilization parameters, resulting in highly active immobilized enzymes. This approach offers several advantages, including reduced time and energy consumption, enhanced mass-heat transfer, and improved control over the mixing process. It maintains the superior structural configuration in immobilized form which ultimately affects the overall efficiency. The present review article comprehensively explains the design, construction, and various methods employed for enzyme immobilization using microfluidic devices. The immobilized enzymes prepared using these techniques demonstrated excellent catalytic activity, remarkable stability, and outstanding recyclability. Moreover, they have found applications in diverse areas such as biosensors, biotransformation, and bioremediation. The review article also discusses potential future developments and foresees significant challenges associated with enzyme immobilization using microfluidics, along with potential remedies. The development of this advanced technology not only paves the way for novel and innovative approaches to enzyme immobilization but also allows for the straightforward scalability of microfluidic-based techniques from an industrial standpoint., Competing Interests: Declaration of competing interest Authors report no conflict of interest on their parts., (Copyright © 2023 Elsevier B.V. All rights reserved.)

In conventional preparation of enzyme embedded organic-inorganic hybrid flower-like nanostructures, usually it requires three days which is time-consuming and limits their widespread applications. In this context, alpha-amylase hybrid bio-microflowers were prepared by simple, efficient and rapid method in the presence of chitosan via inotropic gelation and biomineralization approach. The hybrid bio-microflowers was synthesized within 6 h with ∼140 % enhanced catalytic activity. The prepared hybrid bio-microflowers were characterized by FT-IR, SEM, and PXRD. The hybrid bio-microflowers exhibited higher rate of reaction (V max ) and outstanding thermo-stability (in the temperature range 55-75 °C). Further, hybrid bio-microflowers showed magnificent reusability (upto eight cycles) and long-term storage stability (for about 30 days). In the end, in starch hydrolytic study, immobilized alpha-amylase exhibited higher hydrolytic potential towards corn, wheat and potato starch as compared to free form. This designed hybrid bio-microflowers can be employed as an engineered biocatalyst in industrial applications., Competing Interests: Declaration of Competing Interest Authors have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The immobilization of enzyme with enhanced catalytic activity is the major challenge. In this work, we have activated the lipase in the presence of proline and successfully immobilized into zeolitic imidazolate framework (ZIF)-8 by biomineralization method. The prepared lipase-proline MOF exhibited 135% enhanced catalytic activity as compared to free counterpart. Further, it exhibited four-fold improved thermal stability with respect to native enzyme after immobilization. In Michaelis-Menten kinetic studies, K m values for lipase-proline MOF were found to be lower, whereas, it exhibited higher V max than lipase-MOF and free lipase. The lipase-MOF and lipase-proline MOF were showed 56% and 72% residual activity, respectively after six cycles of reuse., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The porous material has been considered as a potential candidate for immobilizing enzymes. Recently, metal organic framework (MOF) has been emerged as a hybrid organic inorganic material with unique intrinsic properties such as well-defined pore structure, excellent chemico-thermal stability, and extremely high surface areas which make them as a suitable scaffold for enzyme immobilization. The outstanding improvement in catalytic performance, high enzyme loading capacity, remarkable interaction between enzyme and MOF are the key features of the novel enzyme-MOF biocomposites. Amongst different immobilization approaches of enzyme-MOF composite development, de novo strategy received immense attention due to rapid, facile, mild immobilization procedure which exhibits potentially superior catalytic activity and extraordinary operational stability. This review presents a holistic insight of two different de novo strategies i.e. co-precipitation and biomineralization with state-of-art examples. Further, the recent developments in enzyme-MOF composites along with their potential features and characteristics are exploited in terms of catalytic activity, thermal/chemical stability, Michaelis-Menten kinetics, recyclability and storage stability. The advanced de novo strategies such as multi-enzyme catalytic system and magnetic enzyme-MOF are explored in the latter part of highlights., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Enzyme embedded metal-organic frameworks (MOFs) are blooming in the field of enzyme immobilization on account of their superior catalytic efficiency, thermal and chemical stability as compared to native enzyme. However, the separation and recovery of enzyme embedded MOF composite is quite challenging due to its nanometer size, high dispersity and low density which limits the reusability. In this work, α-amylase embedded zeolite imidazole framework (ZIF)-67 was fabricated onto melamine sponge using surfactant assisted dip coating technique. This was achieved in single pot making the method facile and rapid. α-Amylase MOF-sponge was analysed and characterized by FT-IR, XRD, SEM and TGA. We demonstrated that the encapsulation could maintain the active conformational structure of α-amylase after immobilization procedure with excellent bioactivity. Further, apart from merits of framework shielding, it offers mass transfer limitation for macromolecular substrate (like starch) which was determined and expressed as effectiveness factor (η) by using Michaelis-Menten enzyme kinetics. At the end, recycling studies were carried out for α-amylase MOF-sponge which showed 32% residual activity after six consecutive cycles. It is believed that this strategy has great potential in the field of continuous biotransformation (catalysis), biosensing and biomedicine application., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Enzymes are nature's catalysts which are highly selective and efficient. Recently, metal organic frameworks (MOFs) have captivated great attention as an attractive porous support matrix for immobilization of enzymes. An outstanding improvement in catalytic efficiency, enhanced chemical/thermal stability, easy accessibility to active sites, promising recyclability and high enzyme loading are some of the fascinating features of enzyme-MOF composites. Due to low density and high dispersion, the handling and separation of enzyme-MOF composites is challenging. Hence, metal organic framework with magnetic properties (magnetic-MOF) can be a potential candidate as it possesses sophisticated structural design integrated with magnetic properties. Specifically designed magnetic-MOF offers novel properties such as devisable composition, large surface area, easy loading and rapid collection which make it potential amenable for enzyme immobilization (magnetic-enzyme-MOF). This article highlights the different strategies for enzyme immobilization such as physical adsorption, covalent binding, co-ordination bonding and de novo encapsulation method. It further discusses about the artificial enzyme properties of magnetic-MOF coupled with enzyme to extend its application in biosensor., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The enzyme under lower-intensity ultrasonic irradiation leads to favorable conformational changes, thereby enhancing its activity. In this study, lipase activity was augmented upto 1.6-folds after ultrasonic treatment at 22kHz and 11.38Wcm -2 for 25min. This highly activated lipase was encapsulated within zeolite imidazolate framework-8 (ZIF-8) as a metal-organic framework (MOF) material via facile one-step biomineralization method by simply mixing aqueous solution of 2-methylimidazole (13.3mmol) and zinc acetate (1.33mmol) along with sonicated lipase within 10min at room temperature (28±2°C). The prepared lipase-MOF was characterized by using FT-IR, FT-Raman, XRD, BET, confocal scanning laser microscopy, TGA and SEM. Further, the thermal stability of lipase embedded MOF was evaluated in the range of 55-75°C on the basis of half-life which showed 3.2 folds increment as against free lipase. In Michaelis-Menten kinetics studies, sonicated lipase entrapped MOF showed nearly same K m and V max values as that of sonicated free lipase. Moreover, the immobilized lipase exhibited up to 54% of residual activity after seven successive cycles of reuse, whereas it retained 90% of residual activity till twenty-five days of storage. Finally, the conformational changes occurred in lipase after sonication treatment and encapsulation within MOF were analyzed by using FT-IR data analysis tools and fluorescent spectroscopy., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Pectinase was immobilized onto chitosan magnetic nanoparticles (CMNPs) by dextran polyaldehyde as a macromolecular cross-linking agent. The parameters like cross-linking concentration, time and CMNPs to enzyme ratio were optimized. Further, prepared magnetic pectinase nanobiocatalyst was characterized by FT-IR and XRD. The thermal kinetic studies for immobilized pectinase showed two folds improved thermal stability in the range of 55-75°C as compared to free form. The V max and K m values of immobilized pectinase were found to be nearly equal to native form which indicated that conformational flexibility of pectinase was retained even after immobilization. The residual activity of immobilized pectinase was 85% after seven successive cycles of reuse, while it retained upto 89% residual activity on storage of fifteen days which exhibited excellent stability and durability. The conformational changes in pectinase after immobilization were evaluated by FT-IR spectroscopy data analysis tools. Finally, magnetic pectinase nanobiocatalyst was employed for apple juice clarification which showed turbidity reduction upto 74% after 150min treatment., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Macromolecular cross-linked enzyme aggregates (M-CLEAs) of α-amylase were prepared by precipitation and subsequent cross-linking. The non-toxic, biodegradable, biocompatible, renewable polysaccharide based macromolecular cross-linkers viz. agar, chitosan, dextran, and gum arabic were used as a substitute for traditional glutaraldehyde to augment activity recovery toward macromolecular substrate. Macromolecular cross-linkers were prepared by periodate mediated controlled oxidation of polysaccharides. The effects of precipitating agent, concentration and different cross-linkers on activity recovery of α-amylase CLEAs were investigated. α-Amylase aggregated with ammonium sulphate and cross-linked by dextran showed 91% activity recovery, whereas glutaraldehyde CLEAs (G-CLEAs) exhibited 42% activity recovery. M-CLEAs exhibited higher thermal stability in correlation with α-amylase and G-CLEAs. Moreover, dextran and chitosan M-CLEAs showed same affinity for starch hydrolysis as of free α-amylase. The changes in secondary structures revealed the enhancements in structural and conformational rigidity attributed by cross-linkers. Finally, after five consecutive cycles dextran M-CLEAs retained 1.25 times higher initial activity than G-CLEAs., (Copyright © 2015 Elsevier B.V. All rights reserved.)