Characterization of a thermostable carboxylesterase from the hyperthermophilic bacterium Thermotoga maritima.
The gene encoding carboxylesterase from the hyperthermophilic bacterium Thermotoga maritima (tm0053) was cloned. The recombinant protein (EST53) was overexpressed in Escherichia coli without its NH(2)-terminal hydrophobic region, and with a C-terminal hexahistidine sequence. The enzyme was purified to homogeneity by heat treatment, followed by Ni(2+) affinity chromatography, and then characterized. Among the p-nitrophenyl esters tested, the best substrate was p-nitrophenyl decanoate with K (m) and k (cat) values of 3.1 muM and 10.8 s(-1), respectively, at 60 degrees C and pH 7.5. The addition of O,O'-bis(2-aminoethyl)ethyleneglycol-N,N,N',N'-tetraacetic acid decreased the esterase activity, indicating that EST53 is dependent on the presence of Ca(2+) ion. In addition, its activity was inhibited by the addition of phenylmethylsulfonyl fluoride and diethyl pyrocarbonate, indicating that it contains serine and histidine residues, which play key roles in the catalytic mechanism. EST53 shows a relatively high degree of similarity to Burkholderia lipases that belong to family I.2 of the lipolytic enzymes, whereas the local sequence around the pentapeptide of EST53 is most similar to those of Bacillus lipases belonging to family I.4.
Kakugawa
Satoshi
S
Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
Fushinobu
Shinya
S
Wakagi
Takayoshi
T
Shoun
Hirofumi
H
D000595:Amino Acid Sequence; D001407:Bacillus / Q000235:genetics; D019117:Burkholderia / Q000235:genetics; D002118:Calcium / Q000494:pharmacology; D043182:Carboxylesterase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005591:Chemical Fractionation; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli; D006358:Hot Temperature; D008049:Lipase / Q000235:genetics; D008969:Molecular Sequence Data; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics
D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D010664:Phenylmethylsulfonyl Fluoride; D043182:Carboxylesterase; D008049:Lipase; D004047:Diethyl Pyrocarbonate; D002118:Calcium