| Project/Area Number |
17380065
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied biochemistry
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| Research Institution | Kyoto University |
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Principal Investigator |
INOUYE Kuniyo Kyoto University, Graduate School of Agriculture, Professor (10223249)
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| Co-Investigator(Kenkyū-buntansha) |
YASUKAWA Kiyoshi Kyoto University, Graduate School of Agriculture, Associate Professor (30397559)
TAKITA Teisuke Kyoto University, Graduate School of Agriculture, Assistant Professor (70263126)
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| Project Period (FY) |
2005 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥11,460,000 (Direct Cost: ¥10,800,000、Indirect Cost: ¥660,000)
Fiscal Year 2007: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000)
Fiscal Year 2006: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 2005: ¥6,100,000 (Direct Cost: ¥6,100,000)
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| Keywords | enzyme / Protein engineering / reaction control / Peptide / food / protease / thermolysin / site-directed mutagenesis |
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| Research Abstract |
Thermolysin (TLN) is a metalloproteinase with high activity and a thermophilic and halophilic enzyme. The activity increases exponentially with salt and does 13 times with 4 M NaCl. It is utilized for aspartame synthesis, and its Stabilization and activation are desired.
1. Ser residues located on the TLN surface were converted to Asp individually. The resulting variants were as active as the wild-type enzyme (WT), and more halophilic than WT. For the variants S53D and S65D, in which Ser53 and Ser65 were changed to Asp, respectively, the thermostability was much improved at 10 mM Ca^<2+>, but, the WT stability at 100 mM Ca^<2+> was similar to that of the variants, suggesting that the variants enhanced the stability at 10 mM Ca^<2+> with higher affinity of Ca^<2+> to Asp than Ser.
2. The variant G8C/N60C/S65P/L144S was activated and stabilized 5-10 times higher than WT. This stabilization is given by summation of the effects due to 4 single mutations.
3. The auto-cleavage site was identified as G1y154-Leu155. The variant L155A was 4 times stable than WT at 90℃.
4. The X-ray crystallographic study of TLN at 4 M NaCl showed that N-domain of TLN moved 1.5 Angstrom far from C-domain on adding 4 M NaCl and shifted to the open-form, which might be related to the higher activity observed at high salt concentrations.
5. A mutant strain producing TLN variant A73V was isolated. It showed higher activity to peptide substrate than WT, and also showed higher performance in aspartame synthesis than WT.
As shown above, we designed, expressed, and characterized TLN variants which could be potentially utilized for aspartame synthesis.
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