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

Authors
  • Ardila-Leal, Leidy D.1
  • Monterey-Gutiérrez, Pedro A.2
  • Poutou-Piñales, Raúl A.1
  • Quevedo-Hidalgo, Balkys E.3
  • Galindo, Johan F.4
  • Pedroza-Rodríguez, Aura M.5
  • 1 Laboratorio de Biotecnología Molecular, Grupo de Biotecnología Ambiental e Industrial (GBAI), Bogotá, D.C, Colombia , Bogotá (Colombia)
  • 2 Programa de Maestría y Doctorado en Educación Matemática, Bogotá, D.C, Colombia , Bogotá (Colombia)
  • 3 Laboratorio de Biotecnología Aplicada, Grupo de Biotecnología Ambiental e Industrial (GBAI), Bogotá, D.C, Colombia , Bogotá (Colombia)
  • 4 Universidad Nacional de Colombia, Bogotá, D.C, Colombia , Bogotá (Colombia)
  • 5 Laboratorio de Microbiología Ambiental y de Suelos, Grupo de Biotecnología Ambiental e Industrial (GBAI), Bogotá, D.C, Colombia , Bogotá (Colombia)
Type
Published Article
Journal
BMC Biotechnology
Publisher
Springer (Biomed Central Ltd.)
Publication Date
Jun 04, 2021
Volume
21
Issue
1
Identifiers
DOI: 10.1186/s12896-021-00698-3
Source
Springer Nature
Keywords
License
Green

Abstract

BackgroundLaccases (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.MethodsA 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.ResultsIn 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.ConclusionsLaccase 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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