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Determination of concentration and activity of immobilized enzymesPriyanka Singh, Holly Morris, Alexei V. Tivanski, Amnon Kohen

Department of Chemistry, University of Iowa, Iowa City, IA 52242, USAInstructor: Prof.Liu

Student:黃德偉

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Outline• Immobilized DHFR

•DHFR catalyzes

•Microscopy image of immobilized DHFR on mica chip

•Radiogram of reverse-phase HPLC

•Fraction conversion versus time plot

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Immobilized DHFR

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DHFR catalyzesDHFR catalyzes the stereospecific transfer of hydride from C4 of NADPH to C6 of DHF, producing the product THF and oxidized cofactor NADP+

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Microscopy image of immobilized DHFR on mica chip

The distribution of immobilized enzyme on mica chip is random and might consist of regions with bundles and aNonuniform distribution

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Radiogram of reverse-phase HPLC

Illustrating the separation of product ([Ad-14C]NADP+) and reactant ([Ad-14C]NADPH).

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Fraction conversion versus time plot

The value found in this case was 6.7±0.6 s-1

(in good agreement with the reportedvalue of 8 to 12 s-1 for the same enzyme in solution

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The End