When nanozymes meet enzyme: Unlocking the dual-activity potential of integrated biocomposites.

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.

Microfluidic based continuous enzyme immobilization: A comprehensive review.

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.

Magnetic nanoflowers: a hybrid platform for enzyme immobilization.

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.

Magnetic nanoflowers can be used as a novel platform for enzyme immobilization.There are three different approaches to the synthesis of efficient magnetic nanoflower.The magnetic nanoflowers provides excellent stability and good reusability of enzymes.The hybrid biocatalyst was applied in biotransformation, environmental, and food applications.The challenges and their remedies of hybrid biocatalyst have been discussed.

Integrated strategies for enzyme assisted extraction of bioactive molecules: A review.

Conventional methods of extracting bioactive molecules are gradually losing pace due to their numerous disadvantages, such as product degradation, lower efficiency, and toxicity. Thus, in light of the rising demand for these bioactive, enzymes have garnered much attention for their efficiency in extraction. However, enzyme-assisted extraction is also plagued with a high capital cost that cannot justify the extraction yields obtained. In order to mitigate these problems, enzyme-assisted extraction can be consorted with non-conventional methods. This review includes current progress concerning the combined approaches while converging the recent advancements in the field that outperformed conventional extraction processes. It also highlights the design of biocatalyst and key parameters involved in the effective extraction of bioactive molecules. An integrated approach for efficiently extracting polyphenols, essential oils, pigments, and vitamins has been comprehensively reviewed. Furthermore, the different immobilization strategies have been discussed for large-scale implementation of enzymes for extraction. The integration of advanced non-conventional methods with enzyme-assisted extraction will open new avenues to enhance the overall extraction efficiency.

The untapped potential of magnetic nanoparticles for forensic investigations: A comprehensive review.

With a growing interest in precise and sensitive diagnosis for criminal investigations, nanoparticles (NPs) have intrigued scientific minds working in the field of forensic science due to their exceptional properties. Magnetic nanoparticles (MNPs) have emerged as a powerful tool for improving forensic analysis due to their super magnetic behavior combined with smaller dimensions. MNP-based applications can benefit criminologists to solve criminal mysteries with greater precision and pace. This review highlights the different types of MNP-based applications and their developmental and implicational aspects of forensic science. It also renders insight into the future prospects of a splendid blend of nanotechnology and forensic science, leading to a better scientific analysis.

Enzyme embedded microfluidic paper-based analytic device (µPAD): a comprehensive review.

Low-cost paper-based analytical devices are the latest generation of portable lab-on-chip designs that offers an innovative platform for the on/off-site analysis (biosensing) of target analytes, especially in rural and remote areas. Recently, microfluidic paper-based analytical devices (µPADs) have attained significant recognition owing to their exciting fundamental features such as: ease of fabrication, rapid operation, and precise interpretations. The incorporation of enzymes with paper-based analytical devices significantly improves analytical performance while exhibiting excellent chemical and storage stability. In addition to that, these devices are highly compact, portable, easy-to-use, and do not require any additional sophisticated equipment for the detection and quantification of target analytes. This review provides a holistic insight into design, fabrication, and enzyme immobilization strategies for the development of enzyme-µPADs, which enables them to be widely implemented for in-field analysis. It also highlights the recent application of enzyme-µPADs in the area of: biomedical, food safety, and environmental monitoring while exploring the mechanisms of detection involved. Further, in order to improve the accuracy of analysis, researchers have designed a smartphone-based scanning tool for multi-variant point-of-care devices, which is summarized in the latter part of the review. Finally, the future perspectives and outlook of major challenges associated with enzyme-µPADs are discussed with their possible solutions. The development of enzyme integrated µPADs will open a new avenue as an exceptional analytical tool to explore various applications.HIGHLIGHTSEnzyme embedded paper-based analytical devices are a revolution in the field of biosensing.The design, fabrication, and enzyme immobilization on µPADs have been comprehensively discussed.The application of enzyme-µPADs food safety, environmental monitoring, and clinical diagnostic have been reviewed.Smartphones can be used as an on-site, user-friendly, and compact next-gen scanning tool for biosensing.

Biological metal organic framework (bio-MOF) of glucoamylase with enhanced stability.

The handling of conventional enzyme- metal organic framework (MOF) composites is big challenge due to their nano-sized and lightweight structure with low density. Also, conventional MOFs are derived from non-renewable petroleum feedstock which makes them inherent toxic and non-biodegradable. To overcome these difficulties, recently, green, renewable framework material composite, biological metal-organic frameworks (bio-MOFs) have intrigued as a novel class of porous materials. Here, glucoamylase was encapsulated within ZIF-8 in presence of functionalized carboxymethylcellulose (CMC) at mild aqueous conditions. The successful formation of glucoamylase bio-MOF was confirmed by Fourier transform infrared (FT-IR), X-Ray Diffraction (XRD) and scanning electron microscopy (SEM). In thermal stability, glucoamylase bio-MOF exhibited 187 % enhanced thermal stability in the temperature range of 55-75 °C as compared to native form. Further, glucoamylase bio-MOF was recycled for 5 cycles and compared their activity with traditional glucoamylase MOF. Glucoamylase bio-MOF showed significantly improved recyclability which was attributed by adhesive nature of CMC. Finally, the conformational change occurred in enzyme after immobilization was determined by FT-IR data tools.

A rapid self-assembled hybrid bio-microflowers of alpha-amylase with enhanced activity.

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 (Vmax) 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.

Enzyme embedded metal organic framework (enzyme-MOF): De novo approaches for immobilization.

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.

Immobilization of proline activated lipase within metal organic framework (MOF).

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, Km values for lipase-proline MOF were found to be lower, whereas, it exhibited higher Vmax 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.

Entrapment of surfactant modified lipase within zeolitic imidazolate framework (ZIF)-8.

Mostly, enzyme activity is reduced after immobilization of enzyme within MOF due to unfavourable conformational changes occurred during the immobilization procedure. In this context, lipase was activated by surfactants (in order to get a highly active enzyme) followed by encapsulation within zeolitic imidazolate framework (ZIF)-8 via one-pot facile self-assembly method. The prepared lipase-SDS ZIF-8 exhibited 250% enhanced activity compared to native form. The prepared biocomposite was characterized and analysed by X-ray diffraction (XRD), Fourier transform infrared (FT-IR) and scanning electron microscopy (SEM). Thermo-stability was determined for prepared lipase-SDS ZIF-8 biocomposite in the range of 50-70 °C, which showed more than two-folds enhanced stability in terms of half-life. Further, immobilized lipase retained 76% of residual activity even after six repetitive cycles and, it showed 91% residual activity after twenty days of long term storage. Finally, lipase-SDS ZIF-8 was tested for chemical stability in polar denaturing solvents which showed excellent stability as compared to free lipase.

One pot synthesis of α-amylase metal organic framework (MOF)-sponge via dip-coating technique.

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.

Magnetic-metal organic framework (magnetic-MOF): A novel platform for enzyme immobilization and nanozyme applications.

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.

Enzyme assisted extraction of biomolecules as an approach to novel extraction technology: A review.

An interest in the development of extraction techniques of biomolecules from various natural sources has increased in recent years due to their potential applications particularly for food and nutraceutical purposes. The presence of polysaccharides such as hemicelluloses, starch, pectin inside the cell wall, reduces the extraction efficiency of conventional extraction techniques. Conventional techniques also suffer from low extraction yields, time inefficiency and inferior extract quality due to traces of organic solvents present in them. Hence, there is a need of the green and novel extraction methods to recover biomolecules. The present review provides a holistic insight to various aspects related to enzyme aided extraction. Applications of enzymes in the recovery of various biomolecules such as polyphenols, oils, polysaccharides, flavours and colorants have been highlighted. Additionally, the employment of hyphenated extraction technologies can overcome some of the major drawbacks of enzyme based extraction such as longer extraction time and immoderate use of solvents. This review also includes hyphenated intensification techniques by coupling conventional methods with ultrasound, microwave, high pressure and supercritical carbon dioxide. The last section gives an insight on application of enzyme immobilization as a strategy for large scale extraction. Immobilization of enzymes on magnetic nanoparticles can be employed to enhance the operational performance of the system by multiple use of expensive enzymes making them industrially and economically feasible.

Combi-metal organic framework (Combi-MOF) of α-amylase and glucoamylase for one pot starch hydrolysis.

The multi-enzyme biocatalyst allows to run in vitro multi-step cascade reactions in single pot. An efficient combi-metal organic frameworks (combi-MOF) of α-amylase and glucoamylase for one pot starch hydrolysis was constructed by mixing zinc acetate and 2­methylimmidazole with enzyme mixture in one pot under biocompatible conditions. The prepared combi-MOF was characterized and analyzed by powder X-ray diffraction (PXRD), Fourier transform infrared (FT-IR), thermogravimetric analysis (TGA) and scanning electron microscopy (SEM). Thermo-stability was evaluated for combi-MOF in the range of 55 to 75°C which showed three folds improved stability in terms of half-life. In kinetic parameter studies, rate of starch hydrolysis (Vmax) of combi-MOF was found to be enhanced after co-immobilization. Further, combi-MOF was recycled in batch mode which retained up to 52% residual activity after five successive cycles of reuse. In addition to that, combi-MOF exhibited extraordinary storage stability till 24days. At the end, starch hydrolytic activity of combi-MOF was tested for different sources of starch (corn, rice, wheat and potato) which exhibited higher rate of hydrolysis than mixture of free enzymes due to spatially co-localized multi-enzymatic systems.

Encapsulation of lipase within metal-organic framework (MOF) with enhanced activity intensified under ultrasound.

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 Km and Vmax 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.

Ultrasound assisted intensification of enzyme activity and its properties: a mini-review.

Over the last decade, ultrasound technique has emerged as the potential technology which shows large applications in food and biotechnology processes. Earlier, ultrasound has been employed as a method of enzyme inactivation but recently, it has been found that ultrasound does not inactivate all enzymes, particularly, under mild conditions. It has been shown that the use of ultrasonic treatment at appropriate frequencies and intensity levels can lead to enhanced enzyme activity due to favourable conformational changes in protein molecules without altering its structural integrity. The present review article gives an overview of influence of ultrasound irradiation parameters (intensity, duty cycle and frequency) and enzyme related factors (enzyme concentration, temperature and pH) on the catalytic activity of enzyme during ultrasound treatment. Also, it includes the effect of ultrasound on thermal kinetic parameters and Michaelis-Menten kinetic parameters (km and Vmax) of enzymes. Further, in this review, the physical and chemical effects of ultrasound on enzyme have been correlated with thermodynamic parameters (enthalpy and entropy). Various techniques used for investigating the conformation changes in enzyme after sonication have been highlighted. At the end, different techniques of immobilization for ultrasound treated enzyme have been summarized.

Recent advances in enzyme extraction strategies: A comprehensive review.

The increasing interest of industrial enzymes demands for development of new downstream strategies for maximizing enzyme recovery. The significant efforts have been focused on the development of newly adapted technologies to purify enzymes in catalytically active form. Recently, an aqueous two phase system (ATPS) is emerged as powerful tools for efficient extraction and purification of enzymes due to their versatility, lower cost, process integration capability and easy scale-up. The present review gives an overview of effect of parameters such as tie line length, pH, neutral salts, properties of polymer and salt involved in traditional polymer/polymer and polymer/salt ATPS for enzyme recovery. Further, advanced ATPS have been developed based on alcohols, surfactants, micellar compounds to avoid tedious recovery steps for getting desired enzyme. In order to improve the selectivity and efficiency of ATPS, recent approaches of conventional ATPS combined with different techniques like affinity ligands, ionic liquids, thermoseparating polymers and microfluidic device based ATPS have been reviewed. Moreover, three phase partitioning is also highlighted for enzymes enrichment as a blooming technology for efficiently integrated bioseparation techniques. At the end, it includes an overview of CLEAs technology and organic-inorganic nanoflowers preparation as novel strategies for simultaneous extraction, purification and immobilization of enzymes.

Immobilization of pectinase onto chitosan magnetic nanoparticles by macromolecular cross-linker.

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 Vmax and Km 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.

Sonochemical Effect on Activity and Conformation of Commercial Lipases.

The enzyme under lower-intensity ultrasonic irradiation leads to favourable conformational changes, thereby enhancing its activity. The augmentation of activity of ultrasound-treated enzyme is strongly dependent on ultrasound intensity, duty cycle and exposure time, which was investigated for commercial lipases. Thermomyces lanuginosus (TL) lipase showed a 1.3-fold enhanced activity after irradiating at 22 kHz and 11.38 W cm-2 with 50 % duty cycle for 25-min ultrasonic treatment and 1.5-fold enhanced activity was observed for lipozyme (candida antarctica lipase B (CALB)) lipase, at 22 kHz and 15.48 W cm-2 with 66.67 % duty cycle for 20-min ultrasonic treatment. After sonication, thermodynamic parameters viz. E a, ΔH, ΔS and ΔG were evaluated and values were found to be significantly lower for both lipases. In addition, the changes in secondary structure due to sonication were investigated by using Fourier transform infrared (FT-IR), which revealed increase in a certain number of random coiled structure, loss of ß-sheets, ß-turns and α-helix content in TL lipase and CALB lipase. Also, fluorescence spectroscopy exhibited the increased number of tryptophan on surface of both lipases. Moreover, particle size distribution after sonication also helped to improve surface area and enhanced mass transfer, which contributed to improvement in lipase activity.