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

• Published in: Journal of molecular catalysis. B, Enzymatic Vol. 94; pp. 95 - 103
• Main Authors: Nam, Jae-Kyung, Park, Young-Jun, Lee, Hee-Bong
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.10.2013; Elsevier
• Subjects: active sites; agar; agarose; amino acids; Archaeal esterase; Bioconversions. Hemisynthesis; Biological and medical sciences; Biotechnology; catalytic activity; Catalytic triad; Escherichia coli; esterases; esters; ethanol; Family V; Fundamental and applied biological sciences. Psychology; gel chromatography; genes; genomic libraries; isopropyl alcohol; methanol; Methods. Procedures. Technologies; molecular weight; nucleotides; open reading frames; plasmids; polyacrylamide gel electrophoresis; sequence analysis; site-directed mutagenesis; sodium dodecyl sulfate; Sulfolobus solfataricus; Sulfolobus solfataricus P1; thermal stability; Thermostability; urea; Thermostability; Archaeal esterase; Family V; Catalytic triad; Sulfolobus solfataricus P1; Purification; Enzyme; Archaeobacteria; Esterases; Sulfolobus solfataricus; Thermal stability; Characterization; Sulfolobaceae; Bacteria; Hydrolases; Sulfolobales
• ISSN: 1381-1177; 1873-3158
• DOI: 10.1016/j.molcatb.2013.05.019

•An esterase gene was isolated from a genomic library of S. solfataricus P1.•The esterase gene was composed of 942 nucleotides encoding 314 amino acids.•The enzyme shows remarkable thermal and chemical stability.•Site-directed mutagenesis revealed a catalytic triad, Ser144-Asp266-His295.•The enzyme is an archaeal esterase grouped into family V as well as B-type. A genomic library of the thermoacidophilic archaeon Sulfolobus solfataricus P1 was constructed using 3–5kb 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 tributyrin-emulsified 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 35kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration, agreed with that deduced by sequence analysis (34,260Da). Maximal activity was observed at 80°C and pH 8.0. The enzyme was extremely stable without significant change in its activity up to 120h at 50°C, and even at 80°C almost 30% of its activity remained after 120h. Among the p-nitrophenyl esters (C4–C16) tested, the best substrate was p-nitrophenyl caprate (C10) with Km and kcat values of 24.0mM and 2337s−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 8M 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.