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

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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 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 (∼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.

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Materials

5-Bromo-4-chloro-3-indolyl acetate (X-acetate), isopropyl β-D-thiogalactoside (IPTG), cetyltrimethylammonium bromide (CTAB), and p-NP esters of acetate, butyrate, valerate, caprylate, and palmitate were obtained from Sigma, St. Louis, MO, USA. The restriction enzyme BsaI was purchased from New England Biolabs, Beverly, MA, USA. Other restriction enzymes were purchased from Takara Shuzo, Shiga, and from Toyobo, Osaka. Q Sepharose Fast Flow, Mono Q HR10/10, and Superdex 200 HR10/30 columns were

Molecular cloning, sequencing, and sequence similarity

The genomic gene library of S. shibatae strain DSM5389 was screened for the expression of thermostable esterase. Among 12,000 ampicillin-resistant transformants, three colonies, denoted as XA1, XA2, and XA3, were found to produce esterases which were stable after overnight incubation at 60°C. Restriction enzyme analysis showed that these clones arose from different alleles of the archaeon. Preliminary specificity studies showed that these esterase activities were distinct from that of the

Acknowledgements

This work was supported, in part, by the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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