Replacement of oxidizable residues predicted by QM-MM simulation of a fungal laccase generates variants with higher operational stability

Avelar, Mayra; Pastor, Nina; Ramirez-Ramirez, Joaquin; Ayala, Marcela

doi:10.1016/j.jinorgbio.2017.10.007

Cited by 9 publications

(12 citation statements)

References 60 publications

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“…In the case of free-radical generating reactions such as phenol oxidation, laccases may inactivate due to oxidation of relevant residues. Using a fungal laccase from C. gallica as a model, quantum mechanics/molecular mechanics (QM/MM) calculations were performed to identify which residues are susceptible to oxidation within the active site cavity of the T1 copper; where free radicals are generated after substrate-electron subtraction [197]. According to theoretical predictions, phenylalanine residues, particularly those exposed to the solvent, are prone to oxidation.…”

Section: Engineering Laccases: the Quest For A Better Biocatalystmentioning

confidence: 99%

Laccases: structure, function, and potential application in water bioremediation

et al. 2019

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The global rise in urbanization and industrial activity has led to the production and incorporation of foreign contaminant molecules into ecosystems, distorting them and impacting human and animal health. Physical, chemical, and biological strategies have been adopted to eliminate these contaminants from water bodies under anthropogenic stress. Biotechnological processes involving microorganisms and enzymes have been used for this purpose; specifically, laccases, which are broad spectrum biocatalysts, have been used to degrade several compounds, such as those that can be found in the effluents from industries and hospitals. Laccases have shown high potential in the biotransformation of diverse pollutants using crude enzyme extracts or free enzymes. However, their application in bioremediation and water treatment at a large scale is limited by the complex composition and high salt concentration and pH values of contaminated media that affect protein stability, recovery and recycling. These issues are also associated with operational problems and the necessity of large-scale production of laccase. Hence, more knowledge on the molecular characteristics of water bodies is required to identify and develop new laccases that can be used under complex conditions and to develop novel strategies and processes to achieve their efficient application in treating contaminated water. Recently, stability, efficiency, separation and reuse issues have been overcome by the immobilization of enzymes and development of novel biocatalytic materials. This review provides recent information on laccases from different sources, their structures and biochemical properties, mechanisms of action, and application in the bioremediation and biotransformation of contaminant molecules in water. Moreover, we discuss a series of improvements that have been attempted for better organic solvent tolerance, thermo-tolerance, and operational stability of laccases, as per process requirements.

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Section: Engineering Laccases: the Quest For A Better Biocatalystmentioning

confidence: 99%

Laccases: structure, function, and potential application in water bioremediation

et al. 2019

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“…However, this task is out of the scope of this work. Importantly, inactivation of laccases during detoxification of phenols has been widely reported and may be caused by enzyme entrapment within the synthetized polyphenols (Ba et al, 2013 ), attack of free radicals (products of the laccase catalytic reaction) to aromatic residues on the active site (Avelar et al, 2018 ), and the medium composition (Jurado et al, 2009 ).…”

Section: Resultsmentioning

confidence: 99%

Ethanol production by Escherichia coli from detoxified lignocellulosic teak wood hydrolysates with high concentration of phenolic compounds

Sierra-Ibarra

Alcaraz‐Cienfuegos

Vargas‐Tah

et al. 2021

Journal of Industrial Microbiology and Biotechnology

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Teak wood residues were subjected to thermochemical pretreatment, enzymatic saccharification, and detoxification to obtain syrups with a high concentration of fermentable sugars for ethanol production with the ethanologenic Escherichia coli strain MS04. Teak is a hardwood, and thus a robust deconstructive pretreatment was applied followed by enzymatic saccharification. The resulting syrup contained 60 g L−1 glucose, 18 g L−1 xylose, 6 g L−1 acetate, less than 0.1 g L−1 of total furans, and 12 g L−1 of soluble phenolic compounds (SPC). This concentration of SPC is toxic to E. coli, and thus two detoxification strategies were assayed: 1) treatment with Coriolopsis gallica laccase followed by addition of activated carbon and 2) overliming with Ca(OH)2. These reduced the phenolic compounds by 40 and 76%, respectively. The detoxified syrups were centrifuged and fermented with E. coli MS04. Cultivation with the over-limed hydrolysate showed a 60% higher volumetric productivity (0.45 gETOH L−1 h−1). The bioethanol/sugars yield was over 90% in both strategies.

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“…Moreover, during MD simulations it is usual to observe an increase in flexibility in the hydrophobic regions. Furthermore, ASP 266 and ASN 297 residues are located in regions without a defined secondary structure, such as loops or coils; corresponding to regions of the highest fluctuations in any protein [ 100 , 101 ]; as was observed during the analyses.…”

Section: Discussionmentioning

confidence: 99%

Recombinant laccase rPOXA 1B real-time, accelerated and molecular dynamics stability study

et al. 2021

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Background Laccases (EC 1.10.3.2) are multi-copper oxidoreductases with great biotechnological importance due to their high oxidative potential and utility for removing synthetic dyes, oxidizing phenolic compounds, and degrading pesticides, among others. Methods A real-time stability study (RTS) was conducted for a year, by using enzyme concentrates from 3 batches (L1, L3, and L4). For which, five temperatures 243.15, 277.15, 298.15, 303.15, 308.15, and 313.15 K were assayed. Using RTS data and the Arrhenius equation, we calculated the rPOXA 1B accelerated stability (AS). Molecular dynamics (MD) computational study results were very close to those obtained experimentally at four different temperatures 241, 278, 298, and 314 K. Results In the RTS, 101.16, 115.81, 75.23, 46.09, 5.81, and 4.83% of the relative enzyme activity were recovered, at respective assayed temperatures. AS study, showed that rPOXA 1B is stable at 240.98 ± 5.38, 277.40 ± 1.32 or 297.53 ± 3.88 K; with t1/2 values of 230.8, 46.2, and 12.6 months, respectively. Kinetic and thermodynamic parameters supported the high stability of rPOXA 1B, with an Ed value of 41.40 KJ mol− 1, a low variation of KM and Vmax, at 240.98 ± 5.38, and 297.53 ± 3.88 K, and ∆G values showing deactivation reaction does not occur. The MD indicates that fluctuations in loop, coils or loops with hydrophilic or intermediate polarity amino acids as well as in some residues of POXA 1B 3D structure, increases with temperature; changing from three fluctuating residues at 278 K to six residues at 298 K, and nine residues at 314 K. Conclusions Laccase rPOXA 1B demonstrated experimentally and computationally to be a stable enzyme, with t1/2 of 230.8, 46.2 or 12.6 months, if it is preserved impure without preservatives at temperatures of 240.98 ± 5.38, 277.40 ± 1.32 or 297.53 ± 3.88 K respectively; this study could be of great utility for large scale producers.

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Replacement of oxidizable residues predicted by QM-MM simulation of a fungal laccase generates variants with higher operational stability

Cited by 9 publications

References 60 publications

Laccases: structure, function, and potential application in water bioremediation

Laccases: structure, function, and potential application in water bioremediation

Ethanol production by Escherichia coli from detoxified lignocellulosic teak wood hydrolysates with high concentration of phenolic compounds

Recombinant laccase rPOXA 1B real-time, accelerated and molecular dynamics stability study

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