Characterization of ampicillin acylase : purification and study of active center = 앰피실린 아실리제의 효소 특성:분리정제 및 활성부위 구조 purification and study of active center

Ampicillin acylase, which is known to have a novel substrate spectrum, was purified to homogeneity from Pseudomonas melanogenum by the crude extract preparation and chromatography with S-Sepharose, hydroxyapatite, CMcellulose C-52, and CM-Sepharose. The enyme was purified 8847-fold from the crude extract with a final recovery of 12\%. The molecular weight of the native enyme was calculated to be 146,000 by Protein PAK-300 sw HPLC chromatography. SDS-polyacrylamide gel electrophoresis revealed that the enzyme consisted of two identical subunits with a molecular weight of 72.000. The enzyme was a glycoprotein containing 13\% total carbohydrate, and its isoelectric point was 7.2. The optimal temperature for enzymatic activity was found to be 37$^\circ$C with a pH optimum of 6.0. The enyme catalyzed both synthesis and hydrolsis of ampicillin and hydrolysis of the ester bond of phenylglycinemethylester hydrochloride substrate. The substrate specificity showed that the enyme required a free amino group on the $\alpha$ -carbon of the acyl group. The chemical modification of purified ampicillin acylase by N-bromosuccinimide and diethlpyrocarbonate resulted in time-dependent inactivation of the enzyme. Both substrates ampicillin and 6-aminopenicillanic acid protected the enzyme against inactivation, suggesting that the modification occurred near or at the active site. Amino acid analyses and other data indicated that two histidyl residues per subunit molecule were essential for cataltic activity.
Advisors
Byun, Si-Myungresearcher변시명researcher
Publisher
한국과학기술원
Issue Date
1989
Identifier
61467/325007 / 000835045
Language
eng
Description

학위논문(박사) - 한국과학기술원 : 생물공학과, 1989, [ ix, 108 p. ]

Keywords

효소 활성; 단백질 분리

URI
http://hdl.handle.net/10203/27282
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=61467&flag=t
Appears in Collection
BS-Theses_Ph.D.(박사논문)
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