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Molecular Cloning of Extremely Thermostable Esterase Gene From Hyperthermophilic Archaeon Pyrococcus Furiosus in Escherichia Coli

Molecular Cloning of Extremely Thermostable Esterase Gene From Hyperthermophilic Archaeon Pyrococcus Furiosus in Escherichia Coli

M Ikeda, D S Clark

Biotechnol Bioeng. 1998 Mar 5;57(5):624-9.

PMID: 10099242

Abstract:

A genomic library of the hyperthermophilic archaeon Pyrococcus furiosus was constructed in Escherichia coli using pBluescript II SK(+) as a cloning vector. One positive clone exhibiting thermophilic ester-hydrolyzing activity was directly detected by an in situ plate assay using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-acetate. The plasmid isolated from the clone contained a 3.8 kb HindIII fragment from P. furiosus. Expression of active thermostable esterase in E. coli was independent of isopropyl-beta-D-thiogalactopyranoside, suggesting that the archaeal esterase gene was heterologously controlled by its own promoter sequence, not by the vector-located lac promoter. Assays of esterase activity in heat-treated extract of the recombinant E. coli showed the highest temperature optimum (100 degrees C) and thermostability (a half-life of 50 min at 126 degrees C) among esterases reported to date.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP3252366	5-Bromo-4-chloro-3-indolyl acetate		3252-36-6	Price

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