A carboxylesterase from the thermoacidophilic archaeon Sulfolobus solfataricus P1; purification, characterization, and expression.
The carboxylesterase, a 34 kDa monomeric enzyme, was purified from the thermoacidophilic archaeon Sulfolobus solfataricus P1. The optimum temperature and pH were 85 degrees C and 8.0, respectively. The enzyme showed remarkable thermostability: 41% of its activity remained after 5 days of incubation at 80 degrees C. In addition, the purified enzyme exhibited stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme has broad substrate specificity towards various PNP esters and short acyl chain triacylglycerols such as tributyrin (C4:0). Among the PNP esters tested, the best substrate was PNP-caprylate (C8) with Km and kcat values of 71 microM and 14,700 s(-1), respectively. The carboxylesterase gene consisted of 915 bp corresponding to 305 amino acid residues. We demonstrated that active recombinant S. solfataricus carboxylesterase could be expressed in Escherichia coli. The enzyme was identified as a serine esterase belonging to mammalian hormone-sensitive lipases (HSL) family and contained a catalytic triad composed of serine, histidine, and aspartic acid in the active site.
Park
Young-Jun
YJ
Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon 200-701, Republic of Korea.
Choi
Soo Young
SY
Lee
Hee-Bong
HB
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D001483:Base Sequence; D043182:Carboxylesterase / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D002384:Catalysis; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006358:Hot Temperature; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000201:enzymology*
D019843:Archaeal Proteins; D011994:Recombinant Proteins; D043182:Carboxylesterase