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

Koki Ejima, Jian Liu, Yasuhiro Oshima, Kazutake Hirooka, Shoko Shimanuki, Yukako Yokota, Hisashi Hemmi, Toru Nakayama, Tokuzo Nishino

Research output: Contribution to journalArticlepeer-review

23 Citations (Scopus)

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°C and pH 7.0 for 30 min. The recombinant enzyme catalyzed the hydrolysis of p-nitrophenyl (p-NP) esters with C 2-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 (∼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-nitropnenol release or a hysteretic behavior of SshEstI with these substrates.

Original languageEnglish
Pages (from-to)445-451
Number of pages7
JournalJournal of Bioscience and Bioengineering
Volume98
Issue number6
DOIs
Publication statusPublished - 2004 Dec

Keywords

  • Burst kinetics
  • Esterase
  • Hormone-sensitive lipase
  • Hysteresis
  • Sulfolobus shibatae
  • Thermostability
  • α/β hydrolase

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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