| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥300,000)
Fiscal Year 2007: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2006: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2005: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
Exo-β-glucosaminidase gene from Amycolatopsis orientalis was cloned, sequenced, and expressed in Streptomyces lividans TK24. The purified enzyme from the culture supernatant was tested for its reaction specificity. The enzyme was found to hydrolyze the β-1,4-glycosidic linkage of G1cN-G1cNAc in addition to G1cN-G1cN. Site-directed mutagenesis of Asp469 and Glu541 revealed that Asp469 is a proton donor and Glu541 a nuclephile. To more efficiently analyze the emzymatic reaction, we used continuous flow ESI-MS. The time-courses of the enzymatic hydrolysis of oligosaccharide substrates obtained by ESI-MS were found to be consistent with those obtained by HPLC. Furthermore, the amounts of the enzyme and substrate required for such a time-course determination were much lower than those required for determination by HPLC. The continuous flow ESI-MS was found to be efficient for characterizing the enzymatic reaction.
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