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1. Modifying Surface Charges of a Thermophilic Laccase Toward Improving Activity and Stability in Ionic Liquid

   https://doi.org/10.3389/fbioe.2022.880795  

   Stevens, Joseph C.; Shi, Jian (June 2022, Frontiers in Bioengineering and Biotechnology)

   The multicopper oxidase enzyme laccase holds great potential to be used for biological lignin valorization alongside a biocompatible ionic liquid (IL). However, the IL concentrations required for biomass pretreatment severely inhibit laccase activity. Due to their ability to function in extreme conditions, many thermophilic enzymes have found use in industrial applications. The thermophilic fungal laccase from Myceliophthora thermophila was found to retain high levels of activity in the IL [C 2 C 1 Im][EtSO 4 ], making it a desirable biocatalyst to be used for lignin valorization. In contrast to [C 2 C 1 Im][EtSO 4 ], the biocompatibility of [C 2 C 1 Im][OAC] with the laccase was markedly lower. Severe inhibition of laccase activity was observed in 15% [C 2 C 1 Im][OAc]. In this study, the enzyme surface charges were modified via acetylation, succinylation, cationization, or neutralization. However, these modifications did not show significant improvement in laccase activity or stability in [C 2 C 1 Im][OAc]. Docking simulations show that the IL docks close to the T1 catalytic copper, likely interfering with substrate binding. Although additional docking locations for [OAc] - are observed after making enzyme modifications, it does not appear that these locations play a role in the inhibition of enzyme activity. The results of this study could guide future enzyme engineering efforts by showing that the inhibition mechanism of [C 2 C 1 Im][OAc] toward M. thermophila laccase is likely not dependent upon the IL interacting with the enzyme surface. 

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2. Metastable states assisted homogeneous nucleation in supercooled liquid aluminum alloys: Insights from a phenomenologically coupled atomistic, phase-field, and machine learning investigation

   https://doi.org/10.1063/5.0249473  

   Hasan, Md Mahmudul; Choudhuri, Deep (February 2025, The Journal of Chemical Physics)

   Crystallization due to liquid → solid transformation is observed in many natural and engineering processes. Extant literature indicates that crystallization in supercooled liquids is initiated by precursory metastable phases or states, also called non-classical nucleation. For face-centered cubic (FCC) materials, latest experimental and computational studies suggest that metastable hexagonal-closed packed (HCP) structures facilitate equilibrium FCC formation. However, the underlying nucleation mechanism remains unclear. Here, we examine structural changes and energetic barriers associated with such a non-classical mechanism, by performing molecular dynamics (MD) simulations using pure Al, Al-0.5 at. %Cu, and Al-0.5 at. %Ni (all FCC-formers) and phenomenologically coupling MD results with phase-field (PF) modeling. Such a coupling involved initializing PF simulation domains and constructing Landau polynomials—consistent with MD observations. Unsupervised machine learning was utilized to capture nuclei structures from MD simulations, while neural networks helped in extracting equilibrium interfacial energies from PF modeling. Atomistic simulations showed that precursory nuclei are comprised of collection of metastable-HCP states with medium ranged ordering. The pockets of HCP states later transform to critical nuclei—containing an FCC core and an outer layer of HCP. PF modeling qualitatively replicated the precursory-to-critical nuclei transformation and showed that the energetic barriers between the precursory and critical nuclei are substantially smaller than predictions obtained from classical nucleation theory. Together, these observations permitted us to propose a holistic non-classical mechanism that links triangular motifs within Al-based supercooled liquids to the critical nuclei via in-liquid structural transformations. 

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3. Biocatalytic properties of cell surface display laccase for degradation of emerging contaminant acetaminophen in water reclamation

   https://doi.org/10.1002/bit.27214  

   Wu, Ying; Chen, Yingying; Wei, Na (December 2019, Biotechnology and Bioengineering)

   Abstract The surface display laccase (SDL) biocatalyst, where the enzyme laccase is displayed on the surface of biological cells through synthetic biology, provides a new opportunity to develop sustainable technologies for removal of emerging contaminants from wastewater. This study vigorously characterized biocatalytic properties of the SDL in comparison to free laccase in removing emerging contaminant acetaminophen (APAP), with the aim to understand the effect of surface display on enzyme functionality and identify the strategy to overcome the potential limitation. The SDL could effectively remove APAP. Adding redox mediators substantially improved the removal efficiency. The Michaelis–Menten kinetic analysis showed that the redox mediator 2,2‐azinobis‐3‐ethylbenzothiazoline‐6‐sulfonate could overcome the limitation of APAP accessing the active site of laccase in the SDL biocatalyst. The APAP removal rate catalyzed by the SDL in real secondary wastewater effluent was higher than that in acetate buffer; comprehensive enzyme kinetic analysis provided clear evidence that there were redox mediating compounds in the wastewater. Analysis of transformation products revealed that surface display did not change laccase functionality in terms of APAP transformation mechanism. In addition, the SDL retained 88% of the initial activity after six repeated APAP biotransformation reactions. Results from this study provide a scientific basis for developing and implementing SDL as an innovative biocatalytic material for contaminant treatment applications. 

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4. Structural snapshots of base excision by the cancer-associated variant MutY N146S reveal a retaining mechanism

   https://doi.org/10.1093/nar/gkac1246  

   Demir, Merve; Russelburg, L. Peyton; Lin, Wen-Jen; Trasviña-Arenas, Carlos H.; Huang, Beili; Yuen, Philip K.; Horvath, Martin P.; David, Sheila S. (January 2023, Nucleic Acids Research)

   Abstract DNA glycosylase MutY plays a critical role in suppression of mutations resulted from oxidative damage, as highlighted by cancer-association of the human enzyme. MutY requires a highly conserved catalytic Asp residue for excision of adenines misinserted opposite 8-oxo-7,8-dihydroguanine (OG). A nearby Asn residue hydrogen bonds to the catalytic Asp in structures of MutY and its mutation to Ser is an inherited variant in human MUTYH associated with colorectal cancer. We captured structural snapshots of N146S Geobacillus stearothermophilus MutY bound to DNA containing a substrate, a transition state analog and enzyme-catalyzed abasic site products to provide insight into the base excision mechanism of MutY and the role of Asn. Surprisingly, despite the ability of N146S to excise adenine and purine (P) in vitro, albeit at slow rates, N146S-OG:P complex showed a calcium coordinated to the purine base altering its conformation to inhibit hydrolysis. We obtained crystal structures of N146S Gs MutY bound to its abasic site product by removing the calcium from crystals of N146S-OG:P complex to initiate catalysis in crystallo or by crystallization in the absence of calcium. The product structures of N146S feature enzyme-generated β-anomer abasic sites that support a retaining mechanism for MutY-catalyzed base excision. 

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5. Dynamics of ionic liquids in the presence of polymer-grafted nanoparticles

   https://doi.org/10.1039/c9nr04204k  

   Liu, Siqi; Liedel, Clemens; Tarakina, Nadezda V.; Osti, Naresh C.; Akcora, Pinar (October 2019, Nanoscale)

   null (Ed.)

   We incorporated polymer-grafted nanoparticles into ionic and zwitterionic liquids to explore the solvation and confinement effects on their heterogeneous dynamics using quasi-elastic neutron scattering (QENS). 1-Hexyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (HMIM-TFSI) mixed with deuterated poly(methyl methacrylate) (d-PMMA)-grafted nanoparticles is studied to unravel how dynamic coupling between PMMA and HMIM-TFSI influence the fast and slow diffusion characteristics of the HMIM + cations. The zwitterionic liquid, 1-butyl-3-methyl imidazole-2-ylidene borane (BMIM-BH 3 ) is critically selected and mixed with PMMA-grafted nanoparticles for comparison in this work as its ions do not self-dissociate and it does not couple with PMMA through ion-dipole interactions as HMIM-TFSI does. We find that long-range unrestricted diffusion of HMIM + cations is higher in well-dispersed particles than in aggregated particle systems, whereas the localized diffusion of HMIM + is measured to be higher in close-packed particles. Translational diffusion dynamics of BMIM-BH 3 is not influenced by any particle structures suggesting that zwitterions do not interact with PMMA. This difference between two ionic liquid types enables us to decouple polymer effects from the diffusion of ionic liquids, which is integral to understand the ionic transport mechanism in ionic liquids confined in polymer-grafted nanoparticle electrolytes. 

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