| Project/Area Number |
58850198
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| Research Category |
Grant-in-Aid for Developmental Scientific Research
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| Allocation Type | Single-year Grants |
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| Research Field |
発酵工学
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| Research Institution | Osaka University |
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Principal Investigator |
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| Project Period (FY) |
1983 – 1985
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| Project Status |
Completed (Fiscal Year 1985)
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| Budget Amount *help |
¥14,200,000 (Direct Cost: ¥14,200,000)
Fiscal Year 1985: ¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1984: ¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 1983: ¥7,200,000 (Direct Cost: ¥7,200,000)
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| Keywords | Enzyme reactor / Coupled continuous enzyme reaction / Macromolecular derivative of coenzyme / Poly(ethylene glycol)-bound NAD(P) / ポリエチレングリコール結合NADP / 耐熱性酵素 / デヒドロゲナーゼ |
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| Research Abstract |
Continuous utilization of dehydrogenase activity requires the retention and regeneration of coenzymes such as NAD and NADP. We have prepared poly(ethylene glycol)-bound NAD (PEG-NAD) as a macromolecular NAD derivative for enzyme reactors. PEG-NAD has good cofactor activity for various dehydrogenases. In particular, Thermus thermophilus malate dehydrogenase and Bacillus stearothermophilus lactate dehydrogenase show high activities for PEG-NAD. Therefore, we used these thermostable enzymes for our enzyme reactor.
The two enzymes and PEG-NAD were retained in the reactor with an ultrafiltration membrane, and the substrates (pyruvate and malate) were fed continuously. The continuous coupled two-enzyme reaction was operated for 7 - 21 d at different temperatures. Half-lives of the L-lactate-production rate of the reactor were 9.7, 6.2, 5.4 and 3.6 d at 30, 40, 45 and 50゜C, respectively. The decrease in the activity of the enzymes and PEG-NAD in the reactor during continuous operation was also measured. The enzymes were more stable than PEG-NAD, and the stability of PEG-NAD limit the stability of the reactor. The stability of the enzymes and PEG-NAD in the reactor was different from that in the buffer solution.
Poly(ethylene glycol)-bound NADP (PEG-NADP) was also prepared by selective alkylation of the 6-amino group of NADP. PEG-NADP thus obtained has good cofactor activity for various dehydrogenases and is applicable in a continuous enzyme reactor.
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