Characterization and reaction mechanism of pig liver α-D-galactosidase = 돼지 간 α-D-galactosidase 의 특성 및 반응 메카니즘에 관한 연구

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$\alpha$-D-Galactosidase (E.C. 3.2.1.22) of pig liver was pruified by ammonium sulfate fractionation, DEAE-cellulose and Sephadex G-200 column chromatography. With these steps of purification the enzyme was purified about 70-fold over the crude enzyme showing specific activity of 106.6 units per mg of protein. The partially purified $\alpha$-galactosidase showed the maximum activity at pH 3.5, and the temperature at 50$^\circ$C. And the Na-citrate buffer at 50 mM concentration was demonstrated as the best buffer system. The enzyme exhibited maximum stability at pH 5.0-6.0 and deactivated completely within 2 hours at $60\,^\circ\!C$. The kinetic constants, i.e., Km and Vmax of this enzyme using PNPG as substrate was 1.37 mM and 14.71 n moles per minute, respectively. Both $\alpha$-D-galactose and melibiose exhibited competitive inhibition mode. Among the various metal ions, $Ag^+$ and $Hg^{2+}$ showed strong inhibition on enzyme activity. The observation of the effect of pH on Km and Vmax indicates that the carboxyl and histidine group may be involved in the active site of the enzyme. The inhibition of the enzyme by photooxidation again supports the participation of histidine group in the active site. Considering the above results, a two-step mechanism was postulated for the action of pig liver $\alpha$-D-galactosidase. Here the aglycone is cleaved by the concerted action of carboxyl and imidazolium groups. This is followed by reaction with an acceptor molecule (R` OH), which may be water or an aliphatic alcohol, resulting in hydrolysis or transfer products.
Advisors
Lee, Hyun-Jae이현재
Description
한국과학기술원 : 생물공학과,
Publisher
한국과학기술원
Issue Date
1979
Identifier
62419/325007 / 000771147
Language
eng
Description

학위논문 (석사) - 한국과학기술원 : 생물공학과, 1979.2, [ vii, 61 p. ]

Keywords

단백질 분리.

URI
http://hdl.handle.net/10203/27828
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=62419&flag=dissertation
Appears in Collection
BS-Theses_Master(석사논문)
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