| Budget Amount *help |
¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000)
Fiscal Year 2012: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2011: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2010: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
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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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