| Project/Area Number |
12650792
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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 |
生物・生体工学
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| Research Institution | Waseda University |
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Principal Investigator |
KINO Kuniki School of Science and Engineering, Professor, 理工学部, 教授 (60318764)
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| Co-Investigator(Kenkyū-buntansha) |
KIRIMURA Kohtaro School of Science and Engineering, Professor, 理工学部, 教授 (90195412)
宇佐美 昭次 早稲田大学, 理工学部, 教授 (50063508)
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2002: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2001: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2000: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Keywords | Glucosyl transfer enzyme / Glucosyl condensing enzyme / Glucosidase / Glycoside / Arbutin / Enzyme screening / Gene expression / Maltose phosphorylase |
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| Research Abstract |
In this study, the novel method of the glucoside production using microbial enzymes was investigated. Xanthomonas campestris WU-9701 produces a novel enzyme catalyzing α-anomer-selective glucosylation. Using this enzyme, α-anomer-selective glucosylation of alcoholic and phenolic -OH groups was performed. Moreover, it was identified that the enzyme has also an activity of α-anomer-selective glucosylation of -SH group. This enzyme was purified and characterized, and the properties of this enzyme were clarified. The gene (xgtA) encoding the enzyme catalyzing α-anomer-selective glucosylation was cloned. The nucleotide sequence of xgtA shows homologies to those of several glucosidases. In addition, the 3D structure of XgtA was predicted. The gene xgtA was highly expressed in Escherichia coli, and the recombinant enzyme produced in E. coli was utilized to the production of α-arbutin, a useful glucoside. The synthesis of a glucoside by condensing was investigated. In the case of commercial enzymes, α-arbutin was synthesized by α-glucosidase of Aspergillus niger from hydroquinone and glucose. It was cleared that maltose was synthesized using glucose, and α-arbutin was synthesized by glucosetranslation from this maltose. Arbutin was similarly synthesized by β-glucosidase of A. niger from hydroquinone and glucose. Through the screening, strain (YS003) was obtained. A glucose condensing activity was not confirmed, but YS003 produced an enzyme catalyzing β-anomer-selective glucosylation of hydroquinone. On the other hand, it was identified that a commercial maltose phosphorylase (EC 2.4.1.8, MPase) has an activity of the production of α-glucoside using maltose as a glucosyl donor. It was confirmed that the glucosylation of an alcoholic -OH group by MPase.
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