Molecular cloning of extremely thermostable esterase gene from hyperthermophilic archaeon Pyrococcus furiosus in Escherichia coli.
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.
Ikeda
M
M
Department of Chemical Engineering, University of California, Berkeley, California 94720, USA.
Clark
D S
DS
D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D004950:Esterases / Q000235:genetics* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D011994:Recombinant Proteins; D004950:Esterases