초고온성 고세균 써모코커스 NA1으로부터 아미노펩티다제의 클로닝, 특성, 응용

Title
초고온성 고세균 써모코커스 NA1으로부터 아미노펩티다제의 클로닝, 특성, 응용
Alternative Title
A Deblocking Aminopeptidase from a Hyperthermophilic Archaeon Thermococcus sp. NA1: Cloning and Expression in Escherichia coli of the Gene, and Its Application to Peptide Sequence Analysis
Author(s)
이현숙; 조요나; 김윤재; 강성균; 김상진; 이정현
KIOST Author(s)
Lee, Hyun Sook(이현숙)Kang, Sung Gyun(강성균)Lee, Jung Hyun(이정현)
Publication Year
2006-06-24
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.
URI
https://sciwatch.kiost.ac.kr/handle/2020.kiost/30954
Bibliographic Citation
10-th International Symposium on the Genetics of Industrial Microorganisms, pp.1, 2006
Publisher
GIM
Type
Conference
Language
English
Publisher
GIM
Related Researcher
Research Interests

marine biotechnology,molecular microbiology,해양생명공학,분자미생물학

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