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Structural and biochemical characterization of a novel thermophilic Coh01147 protease

Proteases play an essential role in living organisms and represent one of the largest groups of industrial enzymes. The aim of this work was recombinant production and characterization of a newly identified thermostable protease 1147 from thermophilum indigenous Cohnella sp. A01. Phylogenetic tree a...

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Autores principales: Tarrahimofrad, Hossein, Meimandipour, Amir, Arjmand, Sareh, Beigi Nassiri, Mohammadtaghi, Jahangirian, Ehsan, Tavana, Hossein, Zamani, Javad, Rahimnahal, Somayyeh, Aminzadeh, Saeed
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7310833/
https://www.ncbi.nlm.nih.gov/pubmed/32574185
http://dx.doi.org/10.1371/journal.pone.0234958
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author Tarrahimofrad, Hossein
Meimandipour, Amir
Arjmand, Sareh
Beigi Nassiri, Mohammadtaghi
Jahangirian, Ehsan
Tavana, Hossein
Zamani, Javad
Rahimnahal, Somayyeh
Aminzadeh, Saeed
author_facet Tarrahimofrad, Hossein
Meimandipour, Amir
Arjmand, Sareh
Beigi Nassiri, Mohammadtaghi
Jahangirian, Ehsan
Tavana, Hossein
Zamani, Javad
Rahimnahal, Somayyeh
Aminzadeh, Saeed
author_sort Tarrahimofrad, Hossein
collection PubMed
description Proteases play an essential role in living organisms and represent one of the largest groups of industrial enzymes. The aim of this work was recombinant production and characterization of a newly identified thermostable protease 1147 from thermophilum indigenous Cohnella sp. A01. Phylogenetic tree analysis showed that protease 1147 is closely related to the cysteine proteases from DJ-1/ThiJ/PfpI superfamily, with the conserved catalytic tetrad. Structural prediction using MODELLER 9v7 indicated that protease 1147 has an overall α/β sandwich tertiary structure. The gene of protease 1147 was cloned and expressed in Escherichia coli (E. coli) BL21. The recombinant protease 1147 appeared as a homogenous band of 18 kDa in SDS-PAGE, which was verified by western blot and zymography. The recombinant protein was purified with a yield of approximately 88% in a single step using Ni-NTA affinity chromatography. Furthermore, a rapid one-step thermal shock procedure was successfully implemented to purify the protein with a yield of 73%. Using casein as the substrate, K(m), and k(cat), k(ca)t/K(m) values of 13.72 mM, 3.143 × 10(−3) (s(-1)), and 0.381 (M(-1) S(-1)) were obtained, respectively. The maximum protease activity was detected at pH = 7 and 60°C with the inactivation rate constant (kin) of 2.10 × 10–3 (m(-1)), and half-life (t(1/2)) of 330.07 min. Protease 1147 exhibited excellent stability to organic solvent, metal ions, and 1% SDS. The protease activity was significantly enhanced by Tween 20 and Tween 80 and suppressed by cysteine protease specific inhibitors. Docking results and molecular dynamics (MD) simulation revealed that Tween 20 interacted with protease 1147 via hydrogen bonds and made the structure more stable. CD and fluorescence spectra indicated structural changes taking place at 100°C, very basic and acidic pH, and in the presence of Tween 20. These properties make this newly characterized protease a potential candidate for various biotechnological applications.
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spelling pubmed-73108332020-06-26 Structural and biochemical characterization of a novel thermophilic Coh01147 protease Tarrahimofrad, Hossein Meimandipour, Amir Arjmand, Sareh Beigi Nassiri, Mohammadtaghi Jahangirian, Ehsan Tavana, Hossein Zamani, Javad Rahimnahal, Somayyeh Aminzadeh, Saeed PLoS One Research Article Proteases play an essential role in living organisms and represent one of the largest groups of industrial enzymes. The aim of this work was recombinant production and characterization of a newly identified thermostable protease 1147 from thermophilum indigenous Cohnella sp. A01. Phylogenetic tree analysis showed that protease 1147 is closely related to the cysteine proteases from DJ-1/ThiJ/PfpI superfamily, with the conserved catalytic tetrad. Structural prediction using MODELLER 9v7 indicated that protease 1147 has an overall α/β sandwich tertiary structure. The gene of protease 1147 was cloned and expressed in Escherichia coli (E. coli) BL21. The recombinant protease 1147 appeared as a homogenous band of 18 kDa in SDS-PAGE, which was verified by western blot and zymography. The recombinant protein was purified with a yield of approximately 88% in a single step using Ni-NTA affinity chromatography. Furthermore, a rapid one-step thermal shock procedure was successfully implemented to purify the protein with a yield of 73%. Using casein as the substrate, K(m), and k(cat), k(ca)t/K(m) values of 13.72 mM, 3.143 × 10(−3) (s(-1)), and 0.381 (M(-1) S(-1)) were obtained, respectively. The maximum protease activity was detected at pH = 7 and 60°C with the inactivation rate constant (kin) of 2.10 × 10–3 (m(-1)), and half-life (t(1/2)) of 330.07 min. Protease 1147 exhibited excellent stability to organic solvent, metal ions, and 1% SDS. The protease activity was significantly enhanced by Tween 20 and Tween 80 and suppressed by cysteine protease specific inhibitors. Docking results and molecular dynamics (MD) simulation revealed that Tween 20 interacted with protease 1147 via hydrogen bonds and made the structure more stable. CD and fluorescence spectra indicated structural changes taking place at 100°C, very basic and acidic pH, and in the presence of Tween 20. These properties make this newly characterized protease a potential candidate for various biotechnological applications. Public Library of Science 2020-06-23 /pmc/articles/PMC7310833/ /pubmed/32574185 http://dx.doi.org/10.1371/journal.pone.0234958 Text en https://creativecommons.org/publicdomain/zero/1.0/ This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 (https://creativecommons.org/publicdomain/zero/1.0/) public domain dedication.
spellingShingle Research Article
Tarrahimofrad, Hossein
Meimandipour, Amir
Arjmand, Sareh
Beigi Nassiri, Mohammadtaghi
Jahangirian, Ehsan
Tavana, Hossein
Zamani, Javad
Rahimnahal, Somayyeh
Aminzadeh, Saeed
Structural and biochemical characterization of a novel thermophilic Coh01147 protease
title Structural and biochemical characterization of a novel thermophilic Coh01147 protease
title_full Structural and biochemical characterization of a novel thermophilic Coh01147 protease
title_fullStr Structural and biochemical characterization of a novel thermophilic Coh01147 protease
title_full_unstemmed Structural and biochemical characterization of a novel thermophilic Coh01147 protease
title_short Structural and biochemical characterization of a novel thermophilic Coh01147 protease
title_sort structural and biochemical characterization of a novel thermophilic coh01147 protease
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7310833/
https://www.ncbi.nlm.nih.gov/pubmed/32574185
http://dx.doi.org/10.1371/journal.pone.0234958
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