Characterization of an alkali- and halide-resistant laccase expressed in E. coli: CotA from Bacillus clausii.

The limitations of fungal laccases at higher pH and salt concentrations have intensified the search for new extremophilic bacterial laccases. We report the cloning, expression, and characterization of the bacterial cotA from Bacillus clausii, a supposed alkalophilic ortholog of cotA from B. subtilis...

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Autores principales:	Søren Brander, Jørn D Mikkelsen, Kasper P Kepp
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/bcfcccf2331a4090927a6502cd4e96ea
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spelling	oai:doaj.org-article:bcfcccf2331a4090927a6502cd4e96ea2021-11-18T08:16:19ZCharacterization of an alkali- and halide-resistant laccase expressed in E. coli: CotA from Bacillus clausii.1932-620310.1371/journal.pone.0099402https://doaj.org/article/bcfcccf2331a4090927a6502cd4e96ea2014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24915287/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203The limitations of fungal laccases at higher pH and salt concentrations have intensified the search for new extremophilic bacterial laccases. We report the cloning, expression, and characterization of the bacterial cotA from Bacillus clausii, a supposed alkalophilic ortholog of cotA from B. subtilis. Both laccases were expressed in E. coli strain BL21(DE3) and characterized fully in parallel for strict benchmarking. We report activity on ABTS, SGZ, DMP, caffeic acid, promazine, phenyl hydrazine, tannic acid, and bilirubin at variable pH. Whereas ABTS, promazine, and phenyl hydrazine activities vs. pH were similar, the activity of B. clausii cotA was shifted upwards by ~0.5-2 pH units for the simple phenolic substrates DMP, SGZ, and caffeic acid. This shift is not due to substrate affinity (K(M)) but to pH dependence of catalytic turnover: The k(cat) of B. clausii cotA was 1 s⁻¹ at pH 6 and 5 s⁻¹ at pH 8 in contrast to 6 s⁻¹ at pH 6 and 2 s⁻¹ at pH 8 for of B. subtilis cotA. Overall, k(cat)/K(M) was 10-fold higher for B. subtilis cotA at pH(opt). While both proteins were heat activated, activation increased with pH and was larger in cotA from B. clausii. NaCl inhibited activity at acidic pH, but not up to 500-700 mM NaCl in alkaline pH, a further advantage of the alkali regime in laccase applications. The B. clausii cotA had ~20 minutes half-life at 80°C, less than the ~50 minutes at 80°C for cotA from B. subtilis. While cotA from B. subtilis had optimal stability at pH~8, the cotA from B. clausii displayed higher combined salt- and alkali-resistance. This resistance is possibly caused by two substitutions (S427Q and V110E) that could repel anions to reduce anion-copper interactions at the expense of catalytic proficiency, a trade-off of potential relevance to laccase optimization.Søren BranderJørn D MikkelsenKasper P KeppPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 6, p e99402 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Søren Brander Jørn D Mikkelsen Kasper P Kepp Characterization of an alkali- and halide-resistant laccase expressed in E. coli: CotA from Bacillus clausii.
description	The limitations of fungal laccases at higher pH and salt concentrations have intensified the search for new extremophilic bacterial laccases. We report the cloning, expression, and characterization of the bacterial cotA from Bacillus clausii, a supposed alkalophilic ortholog of cotA from B. subtilis. Both laccases were expressed in E. coli strain BL21(DE3) and characterized fully in parallel for strict benchmarking. We report activity on ABTS, SGZ, DMP, caffeic acid, promazine, phenyl hydrazine, tannic acid, and bilirubin at variable pH. Whereas ABTS, promazine, and phenyl hydrazine activities vs. pH were similar, the activity of B. clausii cotA was shifted upwards by ~0.5-2 pH units for the simple phenolic substrates DMP, SGZ, and caffeic acid. This shift is not due to substrate affinity (K(M)) but to pH dependence of catalytic turnover: The k(cat) of B. clausii cotA was 1 s⁻¹ at pH 6 and 5 s⁻¹ at pH 8 in contrast to 6 s⁻¹ at pH 6 and 2 s⁻¹ at pH 8 for of B. subtilis cotA. Overall, k(cat)/K(M) was 10-fold higher for B. subtilis cotA at pH(opt). While both proteins were heat activated, activation increased with pH and was larger in cotA from B. clausii. NaCl inhibited activity at acidic pH, but not up to 500-700 mM NaCl in alkaline pH, a further advantage of the alkali regime in laccase applications. The B. clausii cotA had ~20 minutes half-life at 80°C, less than the ~50 minutes at 80°C for cotA from B. subtilis. While cotA from B. subtilis had optimal stability at pH~8, the cotA from B. clausii displayed higher combined salt- and alkali-resistance. This resistance is possibly caused by two substitutions (S427Q and V110E) that could repel anions to reduce anion-copper interactions at the expense of catalytic proficiency, a trade-off of potential relevance to laccase optimization.
format	article
author	Søren Brander Jørn D Mikkelsen Kasper P Kepp
author_facet	Søren Brander Jørn D Mikkelsen Kasper P Kepp
author_sort	Søren Brander
title	Characterization of an alkali- and halide-resistant laccase expressed in E. coli: CotA from Bacillus clausii.
title_short	Characterization of an alkali- and halide-resistant laccase expressed in E. coli: CotA from Bacillus clausii.
title_full	Characterization of an alkali- and halide-resistant laccase expressed in E. coli: CotA from Bacillus clausii.
title_fullStr	Characterization of an alkali- and halide-resistant laccase expressed in E. coli: CotA from Bacillus clausii.
title_full_unstemmed	Characterization of an alkali- and halide-resistant laccase expressed in E. coli: CotA from Bacillus clausii.
title_sort	characterization of an alkali- and halide-resistant laccase expressed in e. coli: cota from bacillus clausii.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/bcfcccf2331a4090927a6502cd4e96ea
work_keys_str_mv	AT sørenbrander characterizationofanalkaliandhalideresistantlaccaseexpressedinecolicotafrombacillusclausii AT jørndmikkelsen characterizationofanalkaliandhalideresistantlaccaseexpressedinecolicotafrombacillusclausii AT kasperpkepp characterizationofanalkaliandhalideresistantlaccaseexpressedinecolicotafrombacillusclausii
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Characterization of an alkali- and halide-resistant laccase expressed in E. coli: CotA from Bacillus clausii.

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