초고온성 고세균 써모코커스 NA1으로부터 아미노펩티다제의 클로닝, 특성, 응용

Abstract

Thermococcus sp. NA1 is one of the hyperthermophilic archaea collected from a deep-sea hydrothermal vent area at the PACMANUS field in the EAST Manus Basin. As a part of genomic research of the strain, a gene showing a high sequence similarity to deblocking aminopeptidases from Pyrococcus furiosus (86% identity) and P. horikoshii (83% identity) was revealed. The deblocking aminopeptidase gene comprises open reading frame of 1,044 bp, corresponding to polypeptides of 348 amino acids. To characterize the enzyme, the gene was cloned and overexpressed in Escherichia coli. The purified enzyme displayed aminopeptidase activity including deblocking activity. The optimum activity occurred at pH 7.5 and in the range of 90 to 100 °C. The enzyme activity and thermostability were extremely dependent on Co2+, which showed drastic inactivation (t1/2 <10 min) at 96 °C in the absence of Co2+. The specific activities of the enzyme toward the substrates, Leu-pNA and Met-pNA, were 18- and 50-fold higher than those of P. horikoshii deblocking aminopeptidase. When the enzyme was applied to sequence analysis of an N-terminal blocked peptide, alpha-MSH, sequential N-terminal hydrolysis of the peptide followed by MALDI-TOF mass analysis enabled the ladder sequencing of the peptide.