Molecular design of an enzyme reactor involving amplification for L-glutamate using biotin-avidin bioaffinity binding.

YAO Toshio, NANJYO Youko

doi:10.2116/bunsekikagaku.50.613

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ビオチン‐アビジン生物親和結合を用いる多孔性ガラス細孔内での酵素の連続固定化　第２報　　ビオチン‐アビジン生物親和結合を用いるＬ‐グルタミン酸に対する増幅型リアクターの設計
ビオチンーアビジン生物親和結合を用いるL-グルタミン酸に対する増幅型リアクターの設計
ビオチンアビジンセイブツシンワケツゴウオモチイル L グルタミンサンニタイスルゾウフクガタリアクターノセッケイ

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Description

To prepare an enzyme minireactor involving amplification for L-glutamate, multilayer co-immobilization of L-glutamate oxidase (GlOD) and glutamate dehydrogenase (GlDH) onto aminopropyl controlled-pore glass (NH₂-CPG) was carried out stepwise by the alternating binding of avidin and biotin-labeled enzymes. Amplification for L-glutamate is based on substrate recycling between coupled enzymes. The layer-by-layer structure of enzyme molecules immobilized into the pore-space of CPG was geometrically estimated from the immobilized amounts of avidin and biotin-labeled enzymes. The enzyme reactor with a greatest amplification was obtained by the alternating binding of avidin and biotin-labeled enzymes on a monolayer of biotin-labeled GlOD and GlDH crosslinked randomly with glutaraldehyde onto the NH₂-CPG; the amplification factor for L-glutamate was 606 at a flow rate of 20 μl min^-1, when a 0.1 M ammonium phosphate buffer (pH 7.5) containing 0.75 mM NADPH and 1 mM sodium azide was used as the carrier buffer in a FIA system. The calibration graph for the amplified response was linear over the range 5 × 10^-9∼5 × 10^-7 M L-glutamate for 10 μl injections. The detection limit was 3 × 10^-9 M (30 fmol) for L-glutamate.

Journal

BUNSEKI KAGAKU

BUNSEKI KAGAKU 50 (9), 613-618, 2001

The Japan Society for Analytical Chemistry

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