Characterization of Prolyl Oligopeptidase from Hyperthermophilic Archaeon Thermococcus sp. NA1(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

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The prolyl oligopeptidase TNA1_POP was found to be encoded in the genome of the hyperthermophilic archaeon Thermococcus sp. NA1 and showed high similarities to its archaeal homologs(76-83%). The enzyme was found to be a single polypeptide composed of 616 amino acids with conserved signature domains. A recombinant TNA1_POP expressed in Escherichia coli was capable of hydrolyzing succinyl-Ala-Pro-p-nitroanilide(Suc-Ala-Pro-pNA) with temperature and pH optimums of 80℃ and 7, respectively. TNA1_POP activity appeared to be significantly activated by pre-incubation at 80℃ and 90℃ with the optimum temperature unchanged. The heat-activated enzyme exhibited a k_<cat> approximately twofold higher than that of the unheated enzyme, however, both enzymes showed the same K_m. TNA1_POP was thermostable at 80℃ retaining 80% of its heat-activated activity even after 23h, but it lost its enzymatic activity at 90℃ with a half-life of 3h. The loss of the enzymatic activity at 90℃ seemed to be caused by the autodegradation of the enzyme, not by thermal denaturation, as supported by circular dichroism spectropolarimetry. Autodegradation fragments ranging from 2 to 18 kDa were mapped by matrix-assisted laser desorption/ionization time-of-flight(MALDI-TOF) mass spectrometry.
社団法人日本生物工学会の論文
2007-03-25