Nano-biocatalysts for food applications; immobilized enzymes within different nanostructures.

The rapid progress in modern technologies and paying more attention to food safety has prompted new green technologies superior than chemical methods in the food industry. In this regard, enzymes can decrease the usage of chemical reactions but they are sensitive to environmental effects (pH and temperature). In addition, enzymes are scarcely possible to be reused. Consequently, their application as natural catalysts is restricted. Using nanotechnology and the possibility of enzyme immobilization on nanomaterials has led to nanobiocatalysts, resulting from the integration of nanotechnology and biotechnology. Nanocarriers have individual features like nanoscale size, excellent surface/volume ratio, and diversity in construction to improve the activity, efficiency, stability, and storage stability of enzymes. Nanobiocatolysts have a wide range of applications in purification, extraction, clarification, production, and packaging of various products in the food industry. Furthermore, the application of nanobiocatalysts to identify specific components of food contaminants such as microorganisms or their metabolites, heavy metals, antibiotics, and residual pesticides has been successful due to the high accuracy of detection. This review investigates the integration of nanotechnology and food enzymes, the nanomaterials used to create nanobiocatalysts and their application, along with the possible risks and legal aspects of nanomaterials in food bioprocesses.

Molecule(s) of Interest: I. Ionic Liquids-Gateway to Newer Nanotechnology Applications: Advanced Nanobiotechnical Uses', Current Status, Emerging Trends, Challenges, and Prospects.

Ionic liquids are a potent class of organic compounds exhibiting unique physico-chemical properties and structural compositions that are different from the classical dipolar organic liquids. These molecules have found diverse applications in different chemical, biochemical, biophysical fields, and a number of industrial usages. The ionic liquids-based products and procedural applications are being developed for a number of newer industrial purposes, and academic uses in nanotechnology related procedures, processes, and products, especially in nanobiotechnology and nanomedicine. The current article overviews their uses in different fields, including applications, functions, and as parts of products and processes at primary and advanced levels. The application and product examples, and prospects in various fields of nanotechnology, domains of nanosystem syntheses, nano-scale product development, the process of membrane filtering, biofilm formation, and bio-separations are prominently discussed. The applications in carbon nanotubes; quantum dots; and drug, gene, and other payload delivery vehicle developments in the nanobiotechnology field are also covered. The broader scopes of applications of ionic liquids, future developmental possibilities in chemistry and different bio-aspects, promises in the newer genres of nanobiotechnology products, certain bioprocesses controls, and toxicity, together with emerging trends, challenges, and prospects are also elaborated.

Laccase-Functionalized Graphene Oxide Assemblies as Efficient Nanobiocatalysts for Oxidation Reactions.

Multi-layer graphene oxide-enzyme nanoassemblies were prepared through the multi-point covalent immobilization of laccase from Trametes versicolor (TvL) on functionalized graphene oxide (fGO). The catalytic properties of the fGO-TvL nanoassemblies were found to depend on the number of the graphene oxide-enzyme layers present in the nanostructure. The fGO-TvL nanoassemblies exhibit an enhanced thermal stability at 60 °C, as demonstrated by a 4.7-fold higher activity as compared to the free enzyme. The multi-layer graphene oxide-enzyme nanoassemblies can efficiently catalyze the oxidation of anthracene, as well as the decolorization of an industrial dye, pinacyanol chloride. These materials retained almost completely their decolorization activity after five reaction cycles, proving their potential as efficient nano- biocatalysts for various applications.

Immobilization of Bacillus amyloliquefaciens protease "Neutrase" as hybrid enzyme inorganic nanoflower particles: A new biocatalyst for aldol-type and multicomponent reactions.

Organic-inorganic hybrid nanoflowers (hNFs) with commercial protease "Neutrase" is proposed and characterized as efficient and green biocatalysts for promiscuous catalysis in aldol-type and multicomponent reactions. Neutrase hNFs [Neutrase-(Cu/Ca/Co/Mn)3(PO4)2] are straightforwardly prepared through mixing metal ion (Cu2+, Ca2+, Co2+ or Mn2+) aqueous solutions with Neutrase in phosphate buffer (pH 7.4, 10 mM) resulting in precipitation (3 days). The hNFs were characterized by various techniques including scanning electron microscopy (SEM), energy dispersive X-ray (EDX), element mapping, X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). In SEM images, the metal-Neutrase complexes revealed flower-like or granular structures after hybridization. The effect of metal ions and enzyme concentrations on the morphology and enzyme activity of the Neutrase-hNFs was examined. The synthesized Neutrase-Mn hNFs showed superior activity and stability compared to free Neutrase. Traditional organic CC coupling reactions such as aldol condensation, decarboxylative aldol, Knoevenagel, Hantzsch-type reactions and synthesis of 4H-pyran derivatives were used to test the generality and scope of Neutrase promiscuity, while optimizing conditions for the Neutrase-Mn hNF biocatalyst. Briefly, Neutrase-Mn3(PO4)2 hNFs showed excellent enzyme activity, stability and reusability, qualifying as effective reusable catalysts for coupling reactions under mild conditions.

Magnetic metal-organic frameworks immobilized enzyme-based nano-biocatalytic systems for sustainable biotechnology.

Nanobiocatalysts, in which enzyme molecules are integrated into/onto multifunctional materials, such as metal-organic frameworks (MOFs), have been fascinating and appeared as a new interface of nanobiocatalysis with multi-oriented applications. Among various nano-support matrices, functionalized MOFs with magnetic attributes have gained supreme interest as versatile nano-biocatalytic systems for organic bio-transformations. From the design (fabrication) to deployment (application), magnetic MOFs have manifested notable efficacy in manipulating the enzyme microenvironment for robust biocatalysis and thus assure requisite applications in several areas of enzyme engineering at large and nano-biocatalytic transformations, in particular. Magnetic MOFs-linked enzyme-based nano-biocatalytic systems offer chemo-regio- and stereo-selectivities, specificities, and resistivities under fine-tuned enzyme microenvironments. Considering the current sustainable bioprocesses demands and green chemistry needs, we reviewed synthesis chemistry and application prospects of magnetic MOFs-immobilized enzyme-based nano-biocatalytic systems for exploitability in different industrial and biotechnological sectors. More specifically, following a thorough introductory background, the first half of the review discusses various approaches to effectively developed magnetic MOFs. The second half mainly focuses on MOFs-assisted biocatalytic transformation applications, including biodegradation of phenolic compounds, removal of endocrine disrupting compounds, dye decolorization, green biosynthesis of sweeteners, biodiesel production, detection of herbicides and screening of ligands and inhibitors.

Magnetic nanomaterials assisted nanobiocatalysis to abate groundwater pollution.

Magnetic nanoparticles are of great interest for research as they have a wide range of applications in biotechnology, environmental science, and biomedicine. Magnetic nanoparticles are ideal for magnetic separation, improving catalysis's speed and reusability by immobilizing enzymes. Nanobiocatalysis allows the removal of persistent pollutants in a viable, cost-effective and eco-friendly manner, transforming several hazardous compounds in water into less toxic derivatives. Iron oxide and graphene oxide are the preferred materials used to confer nanomaterials their magnetic properties for this purpose as they pair well with enzymes due to their biocompatibility and functional properties. This review describes the most common synthesis methods for magnetic nanoparticles and their performance of nanobiocatalysis for the degradation of pollutants in water.•Magnetic nanomaterials have been synthesized for their application in nanobiocatalysis and treating groundwater.•The most used method for magnetic nanoparticle preparation is the co-precipitation technique.•Peroxidase and oxidase enzymes have great potential in the remotion of multiple contaminants from groundwater.

Progress of research on graphene and its derivatives in bone and cartilage repair.

In recent years, graphene and its derivatives have gained wide attention in the biomedical field due to their good physicochemical properties, biocompatibility, and bioactivity. Its good antibacterial, osteoinductive and drug-carrying properties make it a promising application in the field of orthopedic biomaterials. This paper introduces the research progress of graphene and its derivatives in bone tissue engineering and cartilage tissue engineering and presents an outlook on the future development of graphene-based materials in orthopedics.

Corrigendum: Progress of research on Graphene and its derivatives in bone and cartilage repair.

[This corrects the article DOI: 10.3389/fbioe.2023.1185520.].

Nanostructured supports for multienzyme co-immobilization for biotechnological applications: Achievements, challenges and prospects.

The synergistic combination of current biotechnological and nanotechnological research has turned to multienzyme co-immobilization as a promising concept to design biocatalysis engineering. It has also intensified the development and deployment of multipurpose biocatalysts, for instance, multienzyme co-immobilized constructs, via biocatalysis/protein engineering to scale-up and fulfil the ever-increasing industrial demands. Considering the characteristic features of both the loaded multienzymes and nanostructure carriers, i.e., selectivity, specificity, stability, resistivity, induce activity, reaction efficacy, multi-usability, high catalytic turnover, optimal yield, ease in recovery, and cost-effectiveness, multienzyme-based green biocatalysts have become a powerful norm in biocatalysis/protein engineering sectors. In this context, the current state-of-the-art in enzyme engineering with a synergistic combination of nanotechnology, at large, and nanomaterials, in particular, are significantly contributing and providing robust tools to engineer and/or tailor enzymes to fulfil the growing catalytic and contemporary industrial needs. Considering the above critics and unique structural, physicochemical, and functional attributes, herein, we spotlight important aspects spanning across prospective nano-carriers for multienzyme co-immobilization. Further, this work comprehensively discuss the current advances in deploying multienzyme-based cascade reactions in numerous sectors, including environmental remediation and protection, drug delivery systems (DDS), biofuel cells development and energy production, bio-electroanalytical devices (biosensors), therapeutical, nutraceutical, cosmeceutical, and pharmaceutical oriented applications. In conclusion, the continuous developments in nano-assembling the multienzyme loaded co-immobilized nanostructure carriers would be a unique way that could act as a core of modern biotechnological research.

Synthesis and Characterization of ZIF-67 Mixed Matrix Nanobiocatalysis for CO2 Adsorption Performance.

In this study, ZIF-67-based mixed matrix membrane was synthesized with a solution casting method using tetrahydrofuran as the solvent. The as-synthesized ZIF-67 was characterized using PXRD, TGA, ATR-FTIR, and BET analysis for the surface area measurements. The minimum 3 wt% loading of ZIF-67 was incorporated within a hydrophobic polymer to evaluate the CO2 adsorption performance of ZIF-67. The stability of ZIF-67 in pure water and inorganic solvents was investigated. The maximum CO2 adsorption of the ZIF-67 mixed-matrix membrane (MMM) was 0.5 mmol/g at 273 K, which is higher than that of the pure polymer. The fabricated ZIF-67-based mixed-matrix membrane showed higher CO2 capture even at lower MOF loading using THF. The current study highly recommends the combination of hydrophobic polysulfone and a water-stable ZIF-67 for CO2 capture from wet flue gases.

Armoring bio-catalysis via structural and functional coordination between nanostructured materials and lipases for tailored applications.

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.

Magnetic bio-nanocomposite catalysts of CoFe2O4/hydroxyapatite-lipase for enantioselective synthesis provide a framework for enzyme recovery and reuse.

Enzymatic catalysis is a sustainable alternative for cost-prohibitive catalysts based on noble metals and rare earths. Enzymes can catalyze selective reactions under mild conditions. Enzyme recovery after a reaction for its reuse is still a challenge for industrial application. Herein, a biocompatible magnetic nanocomposite is presented as alternative for enzyme stabilization and easy recovery. The magnetic core of CoFe2O4 provides capabilities for magnetic recovery. Two different functionalization methods based on adsorption of enzyme onto biocompatible hydroxyapatite (HAP) and through covalent bonding using a molecular spacer based on 3-Aminopropyl)triethoxysilane (APTES) have been evaluated. Both enzymatic bio-nanocomposites presented high selectivity for the transesterification reaction of racemic mixtures of (R,S)-1-phenylethanol, with complete conversion of (R)-1-phenylethanol enantiomer. Studies with different solvent and temperature had demonstrated high range of operation conditions due to enzyme stabilization provided by surface attachment. Meanwhile, magnetic properties allowed easy recovery through application of an external magnetic field for enzyme reuse. Results showed high stability of lipase covalently bond to CoFe2O4/HAP over several reaction cycles.

Tailoring a stable and recyclable nanobiocatalyst by immobilization of surfactant treated Burkholderia cepacia lipase on polyaniline nanofibers for biocatalytic application.

Polyaniline nanofibers were synthesized by the oxidative polymerization of aniline. Surfactant treated lipase from Burkholdaria cepacia was immobilized on these polyaniline nanofibers by adsorption. The activity of immobilized preparation was six times higher than that of free lipase with an enhanced dispersion in organic solvents. Five-level-four-factor central composite design was applied for the optimization of immobilization parameters (viz. reaction time, pH, stirring rate and enzyme-support ratio) which were evaluated on the basis of lipase loading and activity. The thermal stability of the lipase in the nanobioconjugate, demonstrated in terms of the half-life at 80 °C was almost sixteen-fold higher than in the free form. The reusability data revealed the utility of the nanoconjugate for seven consecutive cycles with a slow and gradual decline in the activity. However, the nanoconjugate retained almost 30% of their initial activity after seven cycles of reuse revealing its utility of in industrial applications. The nanoconjugate was used in the kinetic resolution of (RS)-1-(7-(3-chloro-2-hydroxypropoxy)benzofuran-2-yl) ethanone, racemic intermediate of an important ß-blocker (Befunolol), with a high conversion rate of 48.2%, 98% ee-value and enantioselectivity (E) of 188, which signify its importance as a nanobiocatalyst.

Nanostructured materials as a host matrix to develop robust peroxidases-based nanobiocatalytic systems.

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.

Engineering enzyme-coupled hybrid nanoflowers: The quest for optimum performance to meet biocatalytic challenges and opportunities.

The current industrial revolution signifies the high-value of biocatalysis engineering. Over the past decade, multiple micro- and nanostructured materials have been attempted for immobilization of enzymes to improve their catalytic properties. Conventional immobilization strategies result in improved stability, while insolubilized enzymes generally lost their activity compared to free counterparts. Recently, a new generation organic-inorganic hybrid nanoflowers with unique properties have received great attention as a novel and incentive immobilization approach owing to their simple fabrication, high biocatalytic efficiency, and enzyme stabilizing capability. The hybrid nanoflowers biocatalytic system implicates metal ions and biomolecules (enzymes). In contrast to free or conventionally immobilized enzymes, single enzyme or multi enzyme-incorporated flowers-like hybrid nanoconstructs demonstrated elevated catalytic activities and stabilities over a very broader range of experimental conditions, i.e., pHs, temperatures and salt concentration. This review discusses the recent developments in the fabrication strategies to diversifying nanoflowers, types, characteristics, and applications of organic-inorganic hybrid nanoflowers as a host platform to engineer different kinds of enzymes with requisite functionalities for biocatalysis applications in different sectors of the modern world. Based on experimental and theoretical literature data, the review is wrapped up with concluding remarks and an outlook in terms of upcoming challenges and prospects for their scale-up applications.

Stabilization of Laccase Through Immobilization on Functionalized GO-Derivatives.

This chapter deals with the use of functionalized derivatives of graphene oxide as nanoscaffolds for the immobilization and stabilization of laccase from Trametes versicolor. Covalent and noncovalent immobilization approaches are described, while a novel method for the development of laccase-based multilayer nanoassemblies is also presented. Various biochemical, spectroscopic, and microscopic techniques were applied to characterize the nanobiocatalytic systems in respect to their microstructure and catalytic performance. Laccase-GO nanosystems were characterized with FTIR spectroscopy in order to confirm the functionalization of the nanomaterials, as well as to interpret the nanomaterial-enzyme interactions, while the multilayer structure of laccase-based multilayer nanoassemblies was confirmed by atomic force microscopy. The nanobiocatalytic systems presented here demonstrated exceptional stability and reusability compared with the free enzyme form, leading to robust biocatalytic systems appropriate for various applications of industrial interest.

Chitosan-functionalized supermagnetic halloysite nanotubes for covalent laccase immobilization.

Halloysite nanotubes (HNTs) were modified with supermagnetic Fe3O4 (M-HNTs) and functionalized with chitosan (CTA) (termed as M-HNTs-CTA). Furthermore, M-HNTs-CTA were cross-linked using glutaraldehyde and applied for covalent laccase immobilization (M-HNTs-CTA-Lac). Facile-synthesized modified HNTs were structurally characterized by scanning electron microscopy, high resolution transmission electron microscopy, X-ray photoelectron spectroscopy, and thermogravimetric analyses. M-HNTs-CTA-Lac exhibited 92.74 mg/g of laccase immobilization capacity and 92% of activity recovery. Biochemical properties of M-HNTs-CTA-Lac exhibited higher pH and temperature stabilities, with exceptional reusability capabilities until the 11th cycle. Moreover, M-HNTs-CTA-Lac exhibited 87% of 2,2'-azinobis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS)-mediated Direct Red 80 (DR80) decolorization. By the 11th cycle, M-HNTs-CTA-Lac exhibited 33% DR80 decolorization. Therefore, M-HNTs-CTA can function as CTA-modified supermagnetic nonreactors for immobilization of biomacromolecules. The investigated M-HNTs-CTA-Lac are thus biocompatible and environment-friendly biocatalysts for degradation of textile waste, such as DR80, and can be rapidly retrieved from aqueous solution by a magnet after decontamination of environmental pollutants.

Supermagnetically Tuned Halloysite Nanotubes Functionalized with Aminosilane for Covalent Laccase Immobilization.

Halloysite nanotubes (HNTs) were tuned with supermagnetic Fe3O4 (M-HNTs) and functionalized with γ-aminopropyltriethoxysilane (APTES) (A-M-HNTs). Gluteraldehyde (GTA) was linked to A-M-HNTs (A-M-HNTs-GTA) and explored for covalent laccase immobilization. The structural characterization of M-HNTs, A-M-HNTs, and A-M-HNTs-GTA-immobilized laccase (A-M-HNTs-GTA-Lac) was determined by X-ray photoelectron spectroscopy, field-emission high-resolution transmission electron microscopy, a magnetic property measurement system, and thermogavimetric analyses. A-M-HNTs-GTA-Lac gave 90.20% activity recovery and a loading capability of 84.26 mg/g, with highly improved temperature and storage stabilities. Repeated usage of A-M-HNTs-GTA-Lac revealed a remarkably consistent relative activity of 80.49% until the ninth cycle. The A-M-HNTs-GTA-Lac gave consistent redox-mediated sulfamethoxazole (SMX) degradation up to the eighth cycle. In the presence of guaiacol, A-M-HNTs-GTA-Lac gave elevated SMX degradation compared with 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) and syrinialdehyde. Therefore, the A-M-HNTs can serve as supermagnetic amino-functionalized nanoreactors for biomacromolecule immobilization. The obtained A-M-HNTs-GTA-Lac is an environmentally friendly biocatalyst for effective degradation of micropollutants, such as SMX, and can be easily retrieved from an aqueous solution by a magnet after decontamination of pollutants in water and wastewater.

Mesoporous Metal-Organic Framework with Well-Defined Cruciate Flower-Like Morphology for Enzyme Immobilization.

Metal-organic frameworks (MOFs) have recently emerged as a promising candidates for the immobilization of enzymes due to their diversified structures and porosity. However, a lack of good size and morphological control over the as-prepared MOFs has limited their practical applications in some cases. Herein, instead of zeolitic imidazolate framework-8 (ZIF-8) with the standard rhombic dodecahedral morphology, we successfully synthesize a novel mesoporous catalase@ZIF composite with cruciate flower-like morphology by embedding catalase molecules into uniformly sized ZIF crystals. With extraordinarily large mesopore size and high protein loading capacity, the catalase@ZIF composites with cruciate flower-like morphology exhibit 400% higher activity than that of catalase@ZIF composites with conventional rhombic dodecahedral morphology, and show higher reusability than conventional rhombic dodecahedral morphology. More importantly, we demonstrate for the first time that the biomacromolecules (proteins) can not directly regulate the crystal size, morphology, and crystallinity of ZIF-8. Moreover, the crystal morphology of ZIF has primary dependence on concentrations of 2-methylimidazole and Zn2+ ions, and can be directly controlled by adjusting concentrations of Zn2+ ions while keeping the high concentration of 2-methylimidazole.

Nanonets Derived from Turnip Mosaic Virus as Scaffolds for Increased Enzymatic Activity of Immobilized Candida antarctica Lipase B.

Elongated flexuous plant viral nanoparticles (VNPs) represent an interesting platform for developing different applications in nanobiotechnology. In the case of potyviruses, the virion external surface is made up of helically arrayed domains of the viral structural coat protein (CP), repeated over 2000 times, in which the N- and C-terminal domains of each CP are projected toward the exterior of the external virion surface. These characteristics provide a chemical environment rich in functional groups susceptible to chemical conjugations. We have conjugated Candida antarctica lipase B (CALB) onto amino groups of the external surface of the potyvirus turnip mosaic virus (TuMV) using glutaraldehyde as a conjugating agent. Using this approach, TuMV virions were transformed into scaffolds for CALB nanoimmobilization. Analysis of the resulting structures revealed the formation of TuMV nanonets onto which large CALB aggregates were deposited. The functional enzymatic characterization of the CALB-bearing TuMV nanonets showed that CALB continued to be active in the nanoimmobilized form, even gaining an increased relative specific activity, as compared to the non-immobilized form. These novel virus-based nanostructures may provide a useful new approach to enzyme nanoimmobilization susceptible to be industrially exploited.