Structural and functional analysis of pullulan hydrolyzing alpha-amylase and conversion of the substrate specificity
| Project/Area Number |
10660096
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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 |
応用微生物学・応用生物化学
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| Research Institution | University of Shizuoka |
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Principal Investigator |
SAKAI Hiroshi University of Shizuoka, Graduate School of Nutritional and Environmental Sciences, Professor, 大学院・生活健康科学研究科, 教授 (10092214)
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| Project Period (FY) |
1998 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2000: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1999: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1998: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | Structure and function of enzyme / Protein engineering / Site directed mutagenesis / Subsite / Substrate specificity / Crystallization / Inclusion body / Amylase / pETシステム / 大量発現系 / 基質結合部位 / 速度論的解析 / ホモロジーモデリング / 酵素の安定性 |
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| Research Abstract |
Thermoactinomyces vulgaris R-47 alpha-amylase II (TVA II) hydrolyzes not only starch but also pullulan. This enzyme hydrolyzes glucosidic linkage next to maltose unit of various oligosaccharides whether this is α-1,4 or α-1,6 glucosidic linkage. Previous studies suggested that HKYDT sequence from 202 to 206 is important to the substrate recognition. In this study, those amino acid residues are mutated to Trp and Ala, and the properties of those enzymes are examined kinetically. The results are summarized as follows. (1) All the Trp mutant enzymes lost almost all the activity, suggesting the importance of those residues. (2) The activities of H201A, D205A, and T206A did not so decrease compared to that of wild enzyme. Those residues may not be important to the activity. (3) The Km values of H202A mutant for low molecular weight substrates decreased greatly. (4) The Km values of K203A mutant for all the substrate examined increased remarkably. (5) The kcat values of Y204A mutant drastically decreased for all the substrates. Those evidences can be explained considering the 3D structure of TVA II as follows. The side chain of H202 and Y204 interact with the sugars that enter subsite-2 and subsite-1, respectively. The side chain of K203 stabilizes the substrate binding site by forming hydrogen bond network with the main chain. To obtain large amount of the protein for crystallization, we tried to construct an over-expression vector using pET system. Large amount of protein was produced in Escherichia coli cells harboring the plasmid, but in an inclusion body. The limited amount of the pure enzyme obtained by the previous methods prevented detailed examination of the crystallization condition. To compare the structure of TVA II with other pullulan hydrolyzing enzymes, we cloned a pullulanase gene from a thermophilic bacterium Thermus thermophilus HB8.
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Report
(4 results)
Research Products
(3 results)
