Thermostable esterase from a thermoacidophilic archaeon: purification and characterization for enzymatic resolution of a chiral compound.
Homolog to lipolytic enzymes having the consensus sequence Gly-X-Ser-X-Gly, from the Sulfolobus solfataricus P2 genome, were identified by multiple sequence alignments. Among three potential candidate sequences, one (Est3), which displayed higher activity than the other enzymes on the indicate plates, was characterized. The gene (est 3) was expressed in Escherichia coli, and the recombinant protein (Est3) was purified by chromatographic separation. The enzyme is a trimeric protein and has a molecular weight of 32 kDa in monomer form in its native structure. The optimal pH and temperature of the esterase were 7.4 and 80 degrees C respectively. The enzyme showed broad substrate specificities toward various p-nitrophenyl esters ranging from C2 to C16. The catalytic activity of the Est3 esterase was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF) and diethyl p-nitrophenyl phosphate. Based on substrate specificity and the action of inhibitors, the Est3 enzyme was estimated to be a carboxylesterase (EC 3.1.1.1). The enzyme with methyl (+/-)-2-(3-benzoylphenyl)propionate-hydrolyzing activity to (-)-2-(3-benzoylphenyl)propionic acid displayed a moderate degree of enantioselectivity. The product, (-)-2-(3-benzoylphenyl)propionic acid, rather than its methyl ester, was obtained in 80% enantiomeric excess (e.e.(p)) at 20% conversion at 60 degrees C after a 32-h reaction. This result indicates that S. solfataricus esterase can be used for application in the synthesis of chiral compounds.
Kim
Seonghun
S
School of Environmental Science and Engineering, Pohang University of Science and Technology, Pohang, Korea. seonghun.kim@hotmail.com
Lee
Sun Bok
SB
D000595:Amino Acid Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D003902:Detergents; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D004950:Esterases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007660:Ketoprofen / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012997:Solvents; D013237:Stereoisomerism; D013379:Substrate Specificity; D016944:Sulfolobus / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
D004269:DNA, Bacterial; D003902:Detergents; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D012997:Solvents; D007660:Ketoprofen; D004950:Esterases