title: Molecular cloning and characterization of a thermostable carboxylesterase from an archaeon, Sulfolobus shibatae DSM5389: non-linear kinetic behavior of a hormone-sensitive lipase family enzyme.
abstract: A gene coding for an esterase (SshEstI, 915 bp in length) of the thermoacidophilic archaeon Sulfolobus shibatae DSM5389 was cloned, sequenced, and overexpressed in Escherichia coli JM109 cells as a soluble, catalytically active protein. The deduced amino acid sequence of SshEstI was consistent with a protein containing 305 amino acid residues with a molecular mass of 33 kDa. Sequence comparison studies indicated that SshEstI could be a member of the hormone-sensitive lipase family, in that it had the highest sequence similarity to esterases from Sulfolobus solfataricus (90% identity) and Archaeoglobus fulgidus (42%) and a lipase from Pseudomonas sp. B11-1 (38%). The recombinant enzyme was highly thermostable and retained more than 70% of its initial activity after incubation at 90 degrees C and pH 7.0 for 30 min. The recombinant enzyme catalyzed the hydrolysis of p-nitrophenyl (p-NP) esters with C2-C16 acyl chains but not the hydrolysis of triacylglycerides such as tributyrin and triolein. The enzymatic hydrolysis of p-NP acetate proceeded in a linear manner with time, whereas that of p-NP esters with acyl chains of C5 or longer showed a biphasic profile, where a rapid release of p-nitrophenol ( approximately 3 min) was followed by a slow, sustained release. These non-linear kinetics may be explained in terms of a very slow, presteady-state burst phenomenon of p-nitrophenol release or a hysteretic behavior of SshEstI with these substrates.
authors.affiliation: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba 6-6-07, Aramaki, Aoba-ku, Sendai, Miyagi 980-8579, Japan.
authors.forename: Koki
authors.initials: K
authors.lastname: Ejima
authors.forename: Jian
authors.initials: J
authors.lastname: Liu
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Oshima
authors.forename: Kazutake
authors.initials: K
authors.lastname: Hirooka
authors.forename: Shoko
authors.initials: S
authors.lastname: Shimanuki
authors.forename: Yukako
authors.initials: Y
authors.lastname: Yokota
authors.forename: Hisashi
authors.initials: H
authors.lastname: Hemmi
authors.forename: Toru
authors.initials: T
authors.lastname: Nakayama
authors.forename: Tokuzo
authors.initials: T
authors.lastname: Nishino