Paper Report for: Ejima_2004_J.Biosci.Bioeng_98_445
Reference
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 Ejima K, Liu J, Oshima Y, Hirooka K, Shimanuki S, Yokota Y, Hemmi H, Nakayama T, Nishino T Ref: J Biosci Bioeng, 98:445, 2004 : PubMed
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.
Ejima K, Liu J, Oshima Y, Hirooka K, Shimanuki S, Yokota Y, Hemmi H, Nakayama T, Nishino T (2004) 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 J Biosci Bioeng98: 445-51
Ejima K, Liu J, Oshima Y, Hirooka K, Shimanuki S, Yokota Y, Hemmi H, Nakayama T, Nishino T (2004) J Biosci Bioeng98: 445-51