Enhancement of the catalytic activity of an autodisplayed esterase = Autotransporter에 의하여 표면발현되는 에스테르가수분해효소의 촉매 활성의 증가

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For confirming esterase from Pseudomonas putida 3SK was an autotransporter protein, esterase activity of the whole cell and the supernatant versus culture time, effects of trypsin treatment and cellular localization in Escherichia coil were determined. The esterase activity profile of the whole cell corresponded well with the cell growth curve and majority of esterase activity was detected in the whole cell, revealing that enzyme was not secreted. Trypsin treatment of whole cells released a significant amount of esterase, indicating that the enzyme was located in the outer membrane with the catalytic domain exposed to the surface. We revealed that the enzyme was associated with the cellular membranes by cellular localization. The catalytic activity of this bacterial surface displayed esterase was enhanced by evolutionary molecular engineering. Five mutant esterases were isolated after error-prone PCR performed to introduce random mutations and screening on a tributyrin agar plate. We determined that these mutant esterases had about 4 amino acid substitutions and these substitutions were concentrated on two specific parts of their whole amino acid sequences. The catalytic activity of wild-type and mutant enzymes was determined; Ml, M2, M3, M4 and M5 had 5.7-, 3.8-, 4.3-, 1.8- and 4.1-fold increased catalytic activity compared to their wild-type enzyme, respectively. Thus, we found the autotransporter protein is effective expression system that could improve properties of enzymes by applying evolutionary molecular engineering. This evolved autotransporter enzyme gives more opportunities for using as whole cell catalyst.
Advisors
Rhee, Joon-Shick이준식
Description
한국과학기술원 : 생물과학과,
Publisher
한국과학기술원
Issue Date
2002
Identifier
173572/325007 / 020003579
Language
eng
Description

학위논문(석사) - 한국과학기술원 : 생물과학과, 2002.5, [ v, 37 p ]

Keywords

catalytic activity; esterase; surface display; autotransporter; directed evolution; 인위적 분자진화; 촉매활성; 에스테르가수분해효소; 표면발현; 오토트랜스포터

URI
http://hdl.handle.net/10203/27970
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=173572&flag=dissertation
Appears in Collection
BS-Theses_Master(석사논문)
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