Enhancement of stability and catalytic activity of phospholipase A1 at high temperature or in organic solvents by evolutionary molecular engineering = 방향성 진화를 이용한 phospholipase A1의 열안정성과 유기용매 내성의 증가

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The thermal stability and catalytic activity of phospholipase $A_1$ from Serratia sp. MK1 were improved by an evolutionary molecular engineering. Two thermostable mutants were isolated after sequential rounds of error-prone PCR to introduce random mutations and filter-based screening of the resultant mutant library, and identified as having six (mutant TA3) and seven (mutant TA13) amino acid substitutions, respectively. Different types of the substitutions were found in two mutants, resulting in the increase of nonploar residues (mutant TA3) or changes between side chains within polar or charged residues (mutant TA13). The wild-type and mutant enzymes were purified, and the effect of temperature on their stability and catalytic activity was investigated. The $T_m$ values of TA3 and TA13 were increased by 7 and 11℃, respectively. Compared to the wild-type enzyme, the temperature optimum for activity was about 10℃ higher and the specific activity was significantly higher at the temperature range examined. The stability and activity of phospholipase $A_1$ were also enhanced by the same approach that used for thermostability evolution, except that the positive nine variants from the first round (30% DMSO, 6 h) were in vitro recombined by DNA shuffling in preparing the second library. Three mutants (SA8, SA17 and SA20) were isolated after exposure to 50% DMSO for 36 h and activity assay on the screening gel containing 30% DMSO. Mutants SA8, SA17 and SA20 were 3.0, 3.9, 2.6-fold more stable in 50% DMSO and 4.6, 5.5, 4.0-fold more active in 30% DMSO than the wild-type phospholipase $A_1$. Three mutants were also more stable in all organic solvents tested, compared with the wild-type enzyme. Thus, evolutionary molecular engineering was found to be an effective and efficient approach to increasing stability without compromising enzyme activity. Moreover, the more stable and more active enzymes could be obtained when the pressures on two properties (i.e. stability and ac...
Advisors
Rhee, Joon-Shick이준식
Description
한국과학기술원 : 생물과학과,
Publisher
한국과학기술원
Issue Date
2000
Identifier
157746/325007 / 000965210
Language
eng
Description

학위논문(박사) - 한국과학기술원 : 생물과학과, 2000.2, [ viii, 120 p. ]

Keywords

유기용매; 방향성 분자 진화; 효소활성; 포스포리파제 A1; Organic solvents; Directed evolution; Catalytic activity; Thermostability; Phospholipase A1; 열안정성

URI
http://hdl.handle.net/10203/27474
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=157746&flag=dissertation
Appears in Collection
BS-Theses_Ph.D.(박사논문)
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