Molecular cloning of extremely thermostable esterase gene from hyperthermophilic archaeon Pyrococcus furiosus in Escherichia coli
✍ Scribed by Masato Ikeda; Douglas S. Clark
- Publisher
- John Wiley and Sons
- Year
- 1998
- Tongue
- English
- Weight
- 86 KB
- Volume
- 57
- Category
- Article
- ISSN
- 0006-3592
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✦ Synopsis
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-4chloro-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--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.