Development of Quorum-Quenching Enzyme using Rational and Molecular Evolution Approaches = 합리적 설계 및 분자 진화 기술을 이용한 고기능 Quorum-Quenching 효소 개발

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Quorum sensing is a cell-cell communication mechanism that is involved in the regulation of biological functions like luminescence, virulence, and biofilm formation. Quorum quenching enzymes, which interrupt quorum sensing signaling through degradation of quorum sensing-molecules, have emerged as a new approach to control and prevent bacterial virulence and pathogenesis. In order to increase the potential of the quorum quenching enzymes for practical applications, several attempts have been made to enhance their catalytic properties using rational and molecular evolution approaches. A genetic circuit was constructed based on acylhomoserine lactone (AHL)-mediated quorum sensing signaling as a high-throughput screening system for molecular evolution of quorum-quenching enzymes. The genetic circuit was composed of lux-R, lux-I promoter, $\beta$-lactamase, and $\beta$-lactamase inhibitor, and designed to confer antibiotic resistance to host cells expressing an AHL-degrading enzyme, enabling a rapid screening of quorum quenching enzymes. Using the genetic circuit, the directed evolution of the AHL hydrolase from $\It{Bacillus sp}$. was attempted in an effort to develop quorum quenching enzymes with improved catalytic activity. As a result, improved enzymes with increased catalytic activity compared to the wild type enzyme were identified, and the genetic circuit was shown to be effective in screening of quorum quenching enzymes with high catalytic efficiency. A structure-based rational design of the enzyme was attempted for targeted quorum quenching against bacteria. The amino acid hot spots crucial to substrate specificity were predicted based on a computational modeling and sequence alignment at the active site of the AHL hydrolase. The involved amino acids in the hot spot positions were rationally substituted, and the substrate specificity about AHLs was effectively modified. Isolation of new quorum quenching reagents broaden a pool of candidates for control of b...
Advisors
Kim, Hak-Sungresearcher김학성researcher
Description
한국과학기술원 : 생명과학과,
Publisher
한국과학기술원
Issue Date
2010
Identifier
455356/325007  / 020037159
Language
eng
Description

학위논문(박사) - 한국과학기술원 : 생명과학과, 2010.08, [ vii, 83 p. ]

Keywords

Molecular Evolution; Protein Engineering; Quorum Quenching Enzyme; Quorum Sensing; Rational Design; 합리적 설계; 분자 진화; 단백질 공학; 쿼럼 퀀칭 효소; 쿼럼 센싱

URI
http://hdl.handle.net/10203/27705
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=455356&flag=dissertation
Appears in Collection
BS-Theses_Ph.D.(박사논문)
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