Molecular cloning of extremely thermostable esterase gene from hyperthermophilic archaeonPyrococcus furiosus inEscherichia coli

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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‐β‐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°C) and thermostability (a half‐life of 50 min at 126°C) among esterases reported to date. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 624‐629, 1998.