Title: Development of Effective Lipase-Hybrid Nanoflowers Enriched with Carbon and Magnetic Nanomaterials for Biocatalytic Transformations
In the present study, hybrid nanoflowers (HNFs) based on copper (II) or manganese (II) ions were prepared by a simple method and used as nanosupports for the development of effective nanobiocatalysts through the immobilization of lipase B from Pseudozyma antarctica. The hybrid nanobiocatalysts were characterized by various techniques including scanning electron microscopy (SEM), energy dispersion spectroscopy (EDS), X-ray diffraction (XRD), Raman spectroscopy, and Fourier transform infrared spectroscopy (FTIR). The effect of the addition of carbon-based nanomaterials, namely graphene oxide and carbon nanotubes, as well as magnetic nanoparticles such as maghemite, on the structure, catalytic activity, and operational stability of the hybrid nanobiocatalysts was also investigated. In all cases, the addition of nanomaterials during the preparation of HNFs increased the catalytic activity and the operational stability of the immobilized biocatalyst. Lipase-based magnetic nanoflowers were effectively applied for the synthesis of tyrosol esters in non-aqueous media, such as organic solvents, ionic liquids, and environmental friendly deep eutectic solvents. In such media, the immobilized lipase preserved almost 100% of its initial activity after eight successive catalytic cycles, indicating that these hybrid magnetic nanoflowers can be applied for the development of efficient nanobiocatalytic systems. more »« less
Zhao, Hua; Campbell, Chante' D.(
, Journal of Chemical Technology & Biotechnology)
AbstractBACKGROUND
Knoevenagel condensation is an important tool for building carbon–carbon (CC) bonds, especially when catalyzed by enzymes to enable a potentially high chemo‐, regio‐ and/or stereoselectivity. Although many Knoevenagel condensation reactions are carried out in aqueous solutions, insoluble hydrophobic substrates often lead to poor catalytic efficiencies. The use of water‐miscible organic solvents improves the substrate solubilization, but usually induces activity suppression or inactivation of enzymes. There is a great need to develop alternative solvents for both substrate dissolution and enzyme compatibility in CC bond formation reactions.
RESULTS
Our group previously developed dual‐functionalized water‐mimicking ionic liquids (ILs) for the activation and stabilization of hydrolases (e.g. lipase and protease). In the present study, we evaluated the Knoevenagel condensation of 4‐chlorobenzaldehyde with acetylacetone, and found that porcine pancreas lipase in water‐mimicking ILs carrying ammonium, imidazolium and benzimidazolium cations enabled higher reaction rates (up to 3.22 μmol min−1 g−1lipase) and better yields thantert‐butanol, glymes and [BMIM][Tf2N]. Interestingly, tertiary amide solvents such asN‐methyl‐2‐pyrrolidone (NMP),N,N‐dimethylformamide (DMF) andN,N‐dimethylacetamide (DMAc) led to 8.2‐ to 11.1‐fold increases in the initial rate (up to 35.66 μmol min−1 g−1lipase) when compared with dual‐functionalized ILs, which is likely due to some synergistic effect of these tertiary amides with the lipase.
A glucose biofuel cell is presented using laser induced 3D graphene (LIG) substrate integrated with catalytic active nanomaterials for harnessing the biochemical energy of glucose. The LIG anode comprised glucose dehydrogenase immobilized on reduced graphene oxide and multiwalled carbon nanotubes (RGO/MWCNTs) nanocomposite for glucose oxidation. The LIG cathode is modified with RGO/MWCNTs and silver oxide (Ag 2 O) nanocomposites for the reduction of oxygen. The assembled biofuel cell exhibited a linear peak power response up to 18 mM glucose with sensitivity of 0.63 μW mM -1 cm −2 and exhibited good linearity (r 2 = 0.99). The glucose biofuel cell showed an open-circuit voltage of 0.365 V, a maximum power density of 11.3 μW cm −2 at a cell voltage of 0.25 V, and a short-circuit current density of 45.18 μA cm −2 when operating in 18 mM glucose. Cyclic voltammetry revealed the bioanode exhibited similar linearity for the detection of glucose. These results demonstrate that LIG based bioelectrodes offer great promise for diverse applications in the development of hybrid biofuel cell and biosensor technology.
Murthy, N. Sanjeeva; Upadhya, Rahul; Kosuri, Shashank; Tamasi, Matthew; Gormley, Adam J.(
, Transactions of the Society for Biomaterials)
Statement of Purpose
Hybrid nanoparticles in which a polymer is used to stabilize the secondary structure of enzyme provide a means to preserve its activity in non-native environments. This approach is illustrated here with horseradish peroxidase (HRP), an important heme enzyme used in medical diagnostic, biosensing, and biotechnological applications. Polymer chaperones in these polymer-enzyme complex (PEC) nanoparticles can enhance the utility of enzymes in unfavorable environments. Structural analysis of the PECs is a crucial link in the machine-learning driven iterative optimization cycle of polymer synthesis and testing. Here, we discuss the utility of small-angle X-ray scattering (SAXS) and quartz crystal microbalance with dissipation (QCMD) for evaluating PECs.
Materials and Methods
Six polymers were synthesized by automated photoinduced electron/energy transfer-reversible addition-fragmentation chain-transfer (PET-RAFT) polymerization directly in 96-well plates.1 Multiple molar ratios of enzyme:polymer (1:1, 1:5, 1:10, and 1:50) were characterized. HRP was mixed with the polymer and heated to 65 °C for 1 hr to form PECs. Enzyme assay and circular dichroism measurements were performed along with SAXS and QCMD to understand polymer-protein interactions. SAXS data were obtained at NSLS-II beamline 16-ID.
Results and Discussion
SAXS data were analyzed to determine the radius of gyration (Rg), Porod exponent and pair distance distribution functions (P(r)) (Figure 1). Rg, which corresponds to the size of the PEC nanoparticles, is sensitive to the polydispersity of the solution and does not change significantly in the presence of the polymer GEP1. Notably, the maximal dimension does not change as significantly upon heating to denaturation in the case of the PEC as it does with HRP alone. The effect of denaturation induced by heating seems to depend on the molar ratio of the polymer to enzyme. The Porod exponent, which is related to roughness, decreased from about 4 to 3 upon complexation indicating polymer binding to the enzyme’s surface. These were confirmed by modeling the structures of the HRP, the polymer and the PEC were modeled using DAMMIF/DAMMIN and MONSA (ATSAS software). The changes observed in the structure could be correlated to the measured enzymatic activity.
Figure 2 shows the evolution of the PEC when the polymer is deposited onto the enzyme immobilized on
Figure 1. P(r) plots for PEC vs. HRP before and after heating, illustrating the increased enzymatic stability due to polymer additives.
gold-coated QCM sensors. The plots show the changes in frequency (f) and dissipation (D) with time as HRP is first deposited and is followed by the adsorption of the polymer. Large f and D show that the polymer forms a complex with HRP. Such changes were not observed with negative controls, Pluronics and poly(ethylene glycol). Comparison of the data from free particles in solution with QCM data from immobilized enzymes, shows that the conformation of the complexes in solution and surface-bound HRP could be different. This way, we were able to explore the various states of complex formation under different conditions with different polymers.
Figure 2. QCMD data showing the interaction between the immobilized HRP and the polymer. 3rd and 5th harmonics are plotted (blue -f; red-D).
Conclusion
SAXS and QCMD data show that stabilization of the enzyme activity by inhibiting the unraveling of the secondary structure as seen in size, surface roughness, pair distribution function and percent helicity.
Acknowledgment
This work was supported by NSF grant 2009942.
References
[1] Tamasi, M, et al. Adv Intell Syst 2020, 2(2): 1900126.
Ayare, Pooja J.; Gregory, Shawn A.; Key, Ryan J.; Short, Andrew E.; Tillou, Jake G.; Sitter, James D.; Yom, Typher; Goodlett, Dustin W.; Lee, Dong-Chan; Alamgir, Faisal M.; et al(
, Green Chemistry)
Homogeneous molecular catalysts are valued for their reaction specificity but face challenges in manufacturing scale-up due to complexities in final product separation, catalyst recovery, and instability in the presence of water. Heterogenizing these molecular catalysts, by attachment to a solid support, could transform the practical utility of molecular catalysts, simplify catalyst separation and recovery, and prevent catalyst decomposition by impeding bimolecular catalyst interactions. Previous strategies to heterogenize molecular catalysts via ligand-first binding to supports have suffered from reduced catalytic activity and leaching (loss) of catalyst, especially in environmentally friendly solvents like water. Herein, we describe an approach in which molecular catalysts are first attached to a metal oxide support through acidic ligands and then “encapsulated” with a metal oxide layer via atomic layer deposition (ALD) to prevent molecular detachment from the surface. For this initial report, which is based upon the well-studied Suzuki carbon–carbon cross-coupling reaction, we demonstrate the ability to achieve catalytic performance using a non-noble metal molecular catalyst in high aqueous content solvents. The catalyst chosen exhibits limited catalytic reactivity under homogeneous conditions due to extremely short catalyst lifetimes, but when heterogenized and immobilized with an optimal ALD layer thickness product yields >90% can be obtained in primarily aqueous solutions. Catalyst characterization before and after ALD application and catalytic reaction is achieved with infrared, electron paramagnetic resonance, and X-ray spectroscopies.
Aramfard, Mohammad; Kaynan, Ozge; Hosseini, Ehsan; Zakertabrizi, Mohammad; Pérez, Lisa M.; Asadi, Amir(
, Small)
Abstract
Dispersing carbon nanomaterials in solvents is effective in transferring their significant mechanical and functional properties to polymers and nanocomposites. However, poor dispersion of carbon nanomaterials impedes exploiting their full potential in nanocomposites. Cellulose nanocrystals (CNCs) are promising for dispersing and stabilizing pristine carbon nanotubes (pCNTs) and graphene nanoplatelets (pGnP) in protic media without functionalization. Here, the underlying mechanisms at the molecular level are investigated between CNC and pCNT/pGnP that stabilize their dispersion in polar solvents. Based on the spectroscopy and microscopy characterization of CNCpCNT/pGnP and density functional theory (DFT) calculations, an additional intermolecular mechanism is proposed between CNC and pCNT/pGnP that forms carbonoxygen covalent bonds between hydroxyl end groups of CNCs and the defected sites of pCNTs/pGnPs preventing re‐agglomeration in polar solvents. This work's findings indicate that the CNC‐assisted process enables new capabilities in harnessing nanostructures at the molecular level and tailoring the performance of nanocomposites at higher length scales.
@article{osti_10325565,
place = {Country unknown/Code not available},
title = {Development of Effective Lipase-Hybrid Nanoflowers Enriched with Carbon and Magnetic Nanomaterials for Biocatalytic Transformations},
url = {https://par.nsf.gov/biblio/10325565},
DOI = {10.3390/nano9060808},
abstractNote = {In the present study, hybrid nanoflowers (HNFs) based on copper (II) or manganese (II) ions were prepared by a simple method and used as nanosupports for the development of effective nanobiocatalysts through the immobilization of lipase B from Pseudozyma antarctica. The hybrid nanobiocatalysts were characterized by various techniques including scanning electron microscopy (SEM), energy dispersion spectroscopy (EDS), X-ray diffraction (XRD), Raman spectroscopy, and Fourier transform infrared spectroscopy (FTIR). The effect of the addition of carbon-based nanomaterials, namely graphene oxide and carbon nanotubes, as well as magnetic nanoparticles such as maghemite, on the structure, catalytic activity, and operational stability of the hybrid nanobiocatalysts was also investigated. In all cases, the addition of nanomaterials during the preparation of HNFs increased the catalytic activity and the operational stability of the immobilized biocatalyst. Lipase-based magnetic nanoflowers were effectively applied for the synthesis of tyrosol esters in non-aqueous media, such as organic solvents, ionic liquids, and environmental friendly deep eutectic solvents. In such media, the immobilized lipase preserved almost 100% of its initial activity after eight successive catalytic cycles, indicating that these hybrid magnetic nanoflowers can be applied for the development of efficient nanobiocatalytic systems.},
journal = {Nanomaterials},
volume = {9},
number = {6},
author = {Fotiadou, Renia and Patila, Michaela and Hammami, Mohamed Amen and Enotiadis, Apostolos and Moschovas, Dimitrios and Tsirka, Kyriaki and Spyrou, Konstantinos and Giannelis, Emmanuel P. and Avgeropoulos, Apostolos and Paipetis, Alkiviadis and Gournis, Dimitrios and Stamatis, Haralambos},
}
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