1997 Fiscal Year Final Research Report Summary
Analyzes of the mechanisms of thermophilic and alkalophilic properties of aqualysin I,a protease from an extreme thermophile
| Project/Area Number |
08456046
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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 |
応用微生物学・応用生物化学
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| Research Institution | The University of Tokyo |
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Principal Investigator |
MATSUZAWA Hiroshi The University of Tokyo, Graduate School of Agricultural and Life Sciences, Professor, 大学院・農学生命科学研究科, 教授 (00011966)
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| Project Period (FY) |
1996 – 1997
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| Keywords | protein engineering / protease / heat-stable protease / aqualysin I / metal ion / site-directed mutagenesis / mutant enzyme |
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| Research Abstract |
Aqualysin I is a thermophilic and alkalophilic protease ; in the presence of calcium ion, the optimum temperature for the proteolytic activity is 80ﾟC,and the enzyme is stable at 80ﾟC for 3h. The optimum pH for the enzyme activity is about 10, and the enzyme is stable at pH 12 and 4ﾟC for long time. In order to investigate the structural properties which gives these characteristics to the enzyme, we have done this study, and obtained the following resuits.
(1) The effects of rare earth metals on the heat-stability of the enzyme were exzmined. Depending on the ionic radii (0.93 to 1.06 angstrom) and independent of the positive charges, some rare earth metals showed the effect like Ca^<2+> (0.98 angstrom). La^<3+> was more effective than Ca^<2+>. The binding constant of La^<3+> with the enzyme was about ten times larger than that of Ca^<2+>, suggesting that the binding constant of metals is important for the stabilization of the enzyme.
By random mutagenesis with PCR,mutations were introduced in the coding region of aqualysin I,and Escherichia coli cells were transformed with recombinant plasmids carrying the gene for the enzyme and its mutants. Mutant Clones, which seemed to produce mutant enzymes showing proteolytic activity at low temperatures or at low pHs, were selected. Such clones were obtained at the frequency of 10^<-3> or less, but purified enzymes from the clones did not show the expected properties.
Replacement of Asn219 with serine increased the catalytic defficiency (K_<cat/>K_m) about twice as much as that of the wild type at the temperature rage of 10 to 90ﾟC.Such a replacement resulted in a significant change of K_<cat/> rather K_<m'> indicating that the side chain in the vicinity of the catalytic residue Ser222 affects the catalytic rate constant.
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