(The) purification and catalytic properties of Acc Ⅰ restriction endonuclease = Acc Ⅰ 제한효소의 정제와 촉매적 성질에 관한 연구

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A large number of site-specific restriction endonucleases have been isolated from a wide variety of microorganisms because of their extensive usefulness in the structural analysis of DNA molecules, and for the construction of recombinant DNA in vitro. Through this thesis, I present a procedure of purification of restriction endonuclease AccI from Acinetobacter calcoaceticus ATCC 23055. I also describe the determination of physical and catalytic properties of this enzyme. For the purification of the enzyme, 300 g cells of wet weight were used and were broken by French press at 20,000 p.s.i.. After ammonium sulfate fractionation, the enzyme was further purified by heparin agarose column chromatography, DEAE-sephadex A-50 column chromatography, Affi - Gel Blue column chromatography, phosphocellulose column chromatography, and finally hydroxyl apatite column chromatography. The isolated enzyme has shown to have site-specific restriction endonuclease activity on the AccI recognition sequence which has a degeneracy i.e., : d (5``-G-T-A-T-A-C-3``) or d(5``-G-T-C-G-A-C-3``). AccI endonuclease has a single polypeptide species and its molecular weight of subunit was $45,000 \pm 1,000$ daltons, as judged by 10\% polyacrylamide gel electrophoresis in the presence of 0.1\% sodium dodecyl sulfate. In order to study the catalytic properties of AccI endonuclease, I was used (3H)-labeled DNA by HpaII methylase as a substrate. The enzyme showed maximum activity at pH values between 8.0 and 11.0, and in the presence of 5.0 to 20.0 mM magnesium chloride. But AccI endonuclease did not require salt (NaCl) and 2-mercaptoethanol for its activity. Furthermore the enzyme retained its maximum activity to the concentration of 100 mM sodium chloride and to the concentration of 300 mM 2-mercaptoethanol.
Advisors
Yoo, Ook-Joonresearcher유욱준researcher
Description
한국과학기술원 : 생물공학과,
Publisher
한국과학기술원
Issue Date
1985
Identifier
64479/325007 / 000831003
Language
eng
Description

학위논문(석사) - 한국과학기술원 : 생물공학과, 1985.2, [ 53 p. ]

Keywords

단백질 분리.

URI
http://hdl.handle.net/10203/28189
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=64479&flag=dissertation
Appears in Collection
BS-Theses_Master(석사논문)
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