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EstA from Arthrobacter nitroguajacolicus Rü61a, a Thermo- and Solvent-Tolerant Carboxylesterase Related to Class C β-Lactamases

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Abstract

The estA gene encoding a novel cytoplasmic carboxylesterase from Arthrobacter nitroguajacolicus Rü61a was expressed in Escherichia coli. Sequence analysis and secondary structure predictions suggested that EstA belongs to the family VIII esterases, which are related to class C β-lactamases. The S-x-x-K motif that in β-lactamases contains the catalytic nucleophile, and a putative active-site tyrosine residue are conserved in EstA. The native molecular mass of hexahistidine-tagged (His6) EstA, purified by metal chelate affinity chromatography, was estimated to be 95 kDa by gel filtration, whereas the His6EstA peptide has a calculated molecular mass of 42.1 kDa. The enzyme catalyzes the hydrolysis of short-chain phenylacyl esters and triglycerides, and shows weak activity toward 2-hydroxy- and 2-nitroacetanilide. Its catalytic activity was inhibited by the serine-specific effector phenylmethylsulfonyl fluoride, and by Cd2+ and Hg2+ ions. Maximum activity of His6EstA was observed at a pH of 9.5 and a temperature of 50°C to 60°C. The enzyme was fairly thermostable. After 19 days at 50°C and after 24 hours at 60°C, its residual relative esterase activity toward phenylacetate was still 53% and 30%, respectively. Exposure of His6EstA to buffer-solvent mixtures showed that the enzyme was inactivated by several high log P (hydrophobic) solvents, whereas it showed remarkable stability and activity in up to 30% (by volume) of polar (low log P) organic solvents such as dimethylsulfoxide, methanol, acetonitrile, acetone, and propanol.

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Acknowledgment

The authors thank Dr. Katja Parschat, Münster, for critical reading of this article.

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  1. Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstraße 3, D-48149, Münster, Germany

    Marcus Schütte & Susanne Fetzner

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  1. Marcus Schütte
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  2. Susanne Fetzner
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Correspondence to Susanne Fetzner.

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Schütte, M., Fetzner, S. EstA from Arthrobacter nitroguajacolicus Rü61a, a Thermo- and Solvent-Tolerant Carboxylesterase Related to Class C β-Lactamases. Curr Microbiol 54, 230–236 (2007). https://doi.org/10.1007/s00284-006-0438-2

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  • DOI: https://doi.org/10.1007/s00284-006-0438-2

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