Extremely thermostable esterases from the thermoacidophilic euryarchaeon Picrophilus torridus.
Two genes encoding esterases EstA and EstB of Picrophilus torridus were identified by the means of genome analysis and were subsequently cloned in Escherichia coli. PTO 0988, which is encoding EstA, consists of 579 bp, whereas PTO 1141, encoding EstB, is composed of 696 bp, corresponding to 192 aa and 231 aa, respectively. Sequence comparison revealed that both biocatalysts have low sequence identities (14 and 16%) compared to previously characterized enzymes. Detailed analysis suggests that EstA and EstB are the first esterases from thermoacidophiles not classified as members of the HSL family. Furthermore, the subunits with an apparent molecular mass of 22 and 27 kDa of the homotrimeric EstA and EstB, respectively, represent the smallest esterase subunits from thermophilic microorganisms reported to date. The recombinant esterases were purified by Ni2+ affinity chromatography, and the activity of the purified esterases was measured over a wide pH (pH 4.5-8.5) and temperature range (10-90 degrees C). Highest activity of the esterases was measured at 70 degrees C (EstA) and 55 degrees C (EstB) with short pNP-esters as preferred substrates. In addition, esters of the non-steroidal anti-inflammatory drugs naproxen, ketoprofen, and ibuprofen are hydrolyzed by both EstA and EstB. Extreme thermostability was measured for both enzymes at temperatures as high as 90 degrees C. The determined half-life (t1/2) at 90 degrees C was 21 and 10 h for EstA and EstB, respectively. Remarkable preservation of esterase activity in the presence of detergents, urea, and commonly used organic solvents complete the exceptional phenotype of EstA and EstB.
Hess
Matthias
M
Genomics Division, DOE Joint Genome Institute, Walnut Creek, CA, USA.
Katzer
Moritz
M
Antranikian
Garabed
G
D000595:Amino Acid Sequence; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000378:metabolism; D019843:Archaeal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D003902:Detergents / Q000494:pharmacology; D004795:Enzyme Stability; D004950:Esterases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004952:Esters / Q000378:metabolism; D019605:Euryarchaeota / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D019848:Gene Expression Regulation, Archaeal; D015971:Gene Expression Regulation, Enzymologic; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009596:Nitrophenols / Q000378:metabolism; D011489:Protein Denaturation; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000378:metabolism; D020539:Sequence Analysis, Protein; D012997:Solvents / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D014280:Triglycerides / Q000378:metabolism
D000894:Anti-Inflammatory Agents, Non-Steroidal; D019843:Archaeal Proteins; D003902:Detergents; D004952:Esters; D008670:Metals; D009596:Nitrophenols; D021122:Protein Subunits; D011994:Recombinant Proteins; D012997:Solvents; D014280:Triglycerides; C024719:4-nitrophenyl; D004950:Esterases