| 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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| 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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