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
Section snippets
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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