2012 Fiscal Year Final Research Report
Isomer-specific synthesis of phosphatidylinositol by engineered phospholipase D
| Project/Area Number |
22560770
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biofunction/Bioprocess
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| Research Institution | Nagoya University |
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Principal Investigator |
IWASAKI Yugo 名古屋大学, 生命農学研究科, 准教授 (50273214)
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| Project Period (FY) |
2010 – 2012
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| Keywords | 生体触媒工学 / ホスホリパーゼD / ホスファチジルイノシトール / 位置特異性 |
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| Research Abstract |
Attempts were made to brush up phosphatidylinositol-synthesizing phospholipase D using protein engineering technique. First, the positional specificity of the enzyme was improved by mutation at the residues that form the “inner wall” of the inositol binding pocket. It was fond that the substitutions of G186 and D189 were effective to improve the specificity, resulting in 93% 1-PI. Second attempt was to improve the thermostability. Random mutation was introduced to the residues with high B-factor. The resultant libraries were screened for enhanced thermostabiliy, and we have successfully isolated mutants with the desired property. Finally, another mutant with deletion at an unstable 9-residue loop was constructed. The constructed deletion mutant showed further improved stability.
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