Purification and characterization of a new thermostable carboxylesterase from the thermoacidophilic archaeon S. solfataricus P1

Purification and characterization of a new thermostable carboxylesterase from the thermoacidophilic archaeon S. solfataricus P1

  We report that a new thermostable carboxylesterase from the thermoacidophilic archaean Sulfolobus solfataricus P1 (DSM1616) with a molecular mass of 28 kDa was purified to electrophoretic homogeneity and characterized. The purified enzyme showed a specific activity of 1,018 units/㎎ in the hydrolysis of p-nitrophenyl(PNP) caprylate at 60℃, pH 5.0. The maximum activity was observed at 74℃ and the optimum pH for the enzymatic activity was 5.0. The molecular mass of the enzyme was the smallest among those of archaeal esterases reported, and also the optimum pH for the enzymatic activity was the lowest. Among the PNP esters tested the best substrate was PNP-caprylate (C？) with K<SUB>m</SUB> and k<SUB>cat</SUB> values of 71.4 mM and 3779 s？¹,however no lipase activity was detected. The N-terminal amino acid sequence of the enzyme determined by automated Edman degradation. A gene encoding the carboxylesterase was cloned from the cDNA of S. solfataricus P1 (DSM1616). The determined DNA sequence carries 780 bp, and encodes the putative 260 amino acid sequence of carboxylesterase.