Development of Biocatalytic Functional Column Electrolytic Method for Application to High-Performance Biotransformation and Absolute and Sensitive Analysis.

开发生物催化功能柱电解方法，应用于高性能生物转化和绝对灵敏分析。

• 批准号: 10660110
• 负责人: KANO Kenji
• 金额: $ 2.3万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10660110/
• 关键词: Nicotinic Acid; 6-Hydroxynicotinic Acid; Column Electrolysis; Electrochemical Complete Hydroxylation; Nicotinic Dehydrogenase; Absolute and Sensitive Analysis; Biotransformation; Electron Transfer Mediator

Focusing our attention to a biotransformation system from nicotinic acid (NA) into 6-hydroxynicotinic acid (6HNA) by Pseudomonas fluorescens TN-5, the electron transfer from the cell to column electrodes has been realized using ferricyanide as an exogenous electron acceptor. The hydoxylation is remarkably accelerated using such exogenous electron transfer mediator, in place of the native final electron acceptor OィイD22ィエD2. In addition, the succeeding reaction of 6HNA can be completely suppressed by the use of the exogenous electron transfer mediator.Based on the results, an electrochemically accelerated and 100% biotransformation system has been realized on our column electrolytic system, where Pseudomonas fluorescens TN-5 cells were immobilized on a column electrode. Such complete biotransformation of NA into 6HNA is the first case. This method would be applied to the production of 6HNA as an important starting material of pesticides.In addition, this method can be successfully applied to the evaluation of the number of NA dehydrogenase in a single cell, where the enzyme immobilized on the column is reduced with NA and after then the reduced enzyme is electrochemically re-oxidized.Based on a similar principle, NA is bioelectrochemically oxidized at Pseudomonas fluorescens TN-5 cell-immobilized column electrode. The coulometric analysis has allowed highly sensitive and absolute determination of NA down to 0.2 pmol.

利用荧光假单胞菌（PseudomonasfluorescensTN-5）将烟酸（nicotinicacid，NA）转化为6-羟基烟酸（6-hydroxynicotinicacid，6 HNA），以铁氰化物为外源电子受体，实现了电子从细胞到柱电极的转移。使用这种外源电子转移介体代替天然最终电子受体O β D22 β D2显著加速了羟基化。在此基础上，将荧光假单胞菌TN-5固定在柱电极上，建立了一个电化学加速的100%生物转化体系。NA如此完全生物转化为6 HNA是第一例。该方法可应用于农药重要原料6-HNA的生产，并可成功地应用于单细胞中NA脱氢酶数量的测定，即用NA还原固定在柱上的酶，然后将还原后的酶电化学再氧化。NA在荧光假单胞菌TN-5细胞固定化柱电极上被生物电化学氧化。库仑分析允许高灵敏度和绝对测定NA下降到0.2 pmol。

项目成果

Kenji Kano: "Role of 2-Amino-3-Carboxy-1, 4-Naphthoquinone, a Strong Growth Stimulator for Bifidobacteria, as an Electron Transfer Mediator for NAD(P)^+Regeneration in Bifidobacterium longum"Biochim.Biophys.Acta. 1428(2). 241-250 (1999)

Kenji Kano：“2-氨基-3-羧基-1, 4-萘醌（双歧杆菌的强生长刺激剂）作为长双歧杆菌 NAD(P)^ 再生的电子转移介体的作用”Biochim.Biophys.Acta。

Kenji Kano et al: "Amperometric Determination of NAD (P) H with Peroxidase-Based HィイD22ィエD2OィイD22ィエD2-Sensing Electrodes and Its Application to Isocitrate Dehydrogenase Activity in Serum"J. Electroanal. Chem.. 478(1). 33-39 (1999)

Kenji Kano 等人：“用基于过氧化物酶的 D22 传感电极测定 NAD (P) H 及其在血清中异柠檬酸脱氢酶活性中的应用”J. Electroanal。

Kenji Kano: "Bioelectrochemical Transformation of Nicotinic Acid into 6-Hydroxynicotinic Acid on Pseudomonas fluorescence TN5-Immobilized Column Electrolytic Flow Systems"J.Mol.Catal.B : Enzymatic. 8(4/6). 265-273 (2000)

Kenji Kano：“在假单胞菌荧光 TN5 固定柱电解流系统上将烟酸生物电化学转化为 6-羟基烟酸”J.Mol.Catal.B：酶促。

Kenji Kano: "Amperometric Determination of NAD(P)H with Peroxidase-Based H_2O_2-S*ng Electrodes and Its Application to Isocitrate Dehydrogenase Activity in Serum"J.Electroanal.Chem.. 478(1). 33-39 (1999)

Kenji Kano：“基于过氧化物酶的 H_2O_2-S*ng 电极对 NAD(P)H 的电流测定及其在血清中异柠檬酸脱氢酶活性中的应用”J.Electroanal.Chem. 478(1)。

Kenji Kano: "Bioelectrochemical Transformation of Nicotinic Acid into 6-Hydroxynicotinid Acid on Pseudomonas fluorescence TN5-Immobilized column Electrolytic Flow Systems"J.Mol.Catal : Enzymatic. 8(4/6). 265-273 (2000)

Kenji Kano：“在假单胞菌荧光 TN5 固定柱电解流系统上将烟酸生物电化学转化为 6-羟基烟酸”J.Mol.Catal：酶促。