Cloning, expression, purification, and characterization of a thermostable esterase from the archaeon Sulfolobus solfataricus P1

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Title
Cloning, expression, purification, and characterization of a thermostable esterase from the archaeon Sulfolobus solfataricus P1
Author(s)
J K Nam; Young-Jun Park; H B Lee
Bibliographic Citation
Journal of Molecular Catalysis B: Enzymatic, vol. 94, pp. 95-103
Publication Year
2013
Abstract
A genomic library of the thermoacidophilic archaeon Sulfolobus solfataricus P1 was constructed using 3.5 kb BamHI-fragments into pUC118 vector and the recombinant plasmids were hosted in Escherichia coli. One positive clone showing thermostable esterase activity was directly selected on tributyrinemulsified agar plates. The open reading frame of the esterase gene isolated from this clone was composed of 942 nucleotides encoding 314 amino acids. The recombinant enzyme stably expressed in E. coli was purified to apparent homogeneity by two column chromatographies using butyl Sepharose followed by Q-Sepharose. The molecular mass of the enzyme, estimated to be approximately 35 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration, agreed with that deduced by sequence analysis (34,260 Da). Maximal activity was observed at 80 °C and pH 8.0. The enzyme was extremely stable without significant change in its activity up to 120 h at 50 °C, and even at 80 °C almost 30% of its activity remained after 120 h. Among the p-nitrophenyl esters (C4-C16) tested, the best substrate was p-nitrophenyl caprate (C10) with Km and kcat values of 24.0 mM and 2337 s-1, respectively. At 30 °C, the enzyme displayed remarkable stability against up to 90% methanol, ethanol, and 2-propanol, and also withstood, to a certain extent, 5% SDS and 8 M urea. Site-directed mutagenesis revealed that the enzyme contains a catalytic triad composed of Ser144, Asp266, and His295 in the active site. The S. solfataricus P1 esterase is an archaeal esterase grouped into family V as well as B-type esterases.
Keyword
Archaeal esteraseCatalytic triadFamily VSulfolobus solfataricus P1Thermostability
ISSN
1381-1177
Publisher
Elsevier
DOI
http://dx.doi.org/10.1016/j.molcatb.2013.05.019
Type
Article
Appears in Collections:
Division of Research on National Challenges > Environmental diseases research center > 1. Journal Articles
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