Shin-ichiro Suye

First comparative reaction mechanisms of β-estradiol and selected environmental hormones in a redox environment

Abstract This work describes the first comparative electrochemical behavior of β-estradiol and selected important environmental hormones, specifically alkylphenols (APs). Most of the concerns about environmental hormones in humans center on their interference with estrogens, an ovarian steroid. APs are believed to have high affinity for biological receptors by generating conformational changes that can be detected by other macromolecules. However, no mechanistic or molecular evidence has been established for these studies. Here we show, an electrochemical mechanism of estradiol and selected APs including bisphenol-A (BPA), nonylphenol (NP), and diethylstilbestrol (DES) using cyclic voltammetry (CV), rotating disk electrode (RDE) and controlled potential coulometry (CPC). We observed a striking similarity in the electrochemistry of these synthetic xenobiotics and estradiol, a natural estrogen. The complete reaction mechanisms for the oxidation of NP, DES and estradiol were found to follow EC–EC scheme. Unlike most aromatic hydroxy phenols, our results showed that using bulk electrolysis with CPC, both estradiol and DES generated a ketone and quinone intermediate products, respectively. These products were isolated and confirmed using mass spectrometry. This study may provide insights into the origin of the in vivo molecular recognition since there is little steric or electronic hindrance expected at reactor sites between the APs and estradiol.

Surface display of organophosphorus hydrolase on Saccharomyces cerevisiae.

The gene encoding organophosphorus hydrolase (OPH) from Flavobacterium species was expressed on the cell surface of Saccharomyces cerevisiae MT8–1 using a glycosylphosphatidylinositol (GPI) anchor linked to the C‐terminal region of OPH. Immunofluorescence microscopy confirmed the localization of OPH on the cell surface, and fluorescence intensity measurement of cells revealed that 1.4 × 104 molecules of OPH per cell were displayed. Seventy percent of OPH whole‐cell activity was detected on the cell surface by protease accessibility assay. The activity of OPH was highly dependent on cell growth conditions. The maximum activity was obtained when cells were grown in a synthetic dextrose medium lacking tryptophan (SD‐W) buffered by 2‐[4‐(2‐hydroxyethyl)‐1‐piperazinyl]ethanesulfonic acid (HEPES, 200 mM, pH 7.0) at 20 °C, and cobalt chloride was added at 0.1 mM. S. cerevisiae MT8–1 displaying OPH which exhibited a higher activity than Escherichia coli displaying OPH using the ice nucleation protein (INP) anchor. The use of S. cerevisiaeMT8–1, which has a “generally regarded as safe (GRAS)” status, as a host for the easy expression of the OPH gene provides a new biocatalyst useful for simultaneous detoxification and detection of organophosphorus pesticides.

Layer-by-layer assembly of enzymes and polymerized mediator on electrode surface by electrostatic adsorption

Abstract Through layer-by-layer adsorption (LBL) technique, multilayer film was prepared from enzymes, diaphorase (DI) and glucose-6-phosphate dehydrogenase (G6PDH) and polyelectrolytes. The adsorption interface morphology was directly observed by atomic force microscopy, and the immobilization amount and layer thickness were characterized from quartz crystal microbalance which showed formation of nanoscale multilayer structure and linear mass increase which depended on alternate adsorption cycles. In order to construct a new mediated bi-enzyme biosensor system, polymerized mediator, DI and G6PDH were immobilized on carbon electrode surface by using LBL method. Electrochemical experiments indicated highly efficient electron transfer by the polymerized mediator. Two enzymes kept their activities after immobilization, and the electrode immobilized by mediator and enzymes showed sensitive response to glucose-6-phosphate in the presence of free NAD+, and high stability during long period of storage.

Electrochemical reduction of immobilized NADP+ on a polymer modified electrode with a co-polymerized mediator

An electrode chemically modified by the amino group (Polyaminoaniline, PAA) was prepared and the immobilization of the co-polymerized viologen (Alg-V) and of NADP+ on the electrode, was investigated. NADP+ can not be reduced electrocatalytically on the viologen immobilized PAA-modified electrode. On the other hand, a cathodic peak in the cyclic voltammogram of the NADP+ immobilized PAA-modified electrode appeared at −1.2 V vs. SCE. corresponding to the reduction wave of free NADP+ on the electrode. The anodic wave of NADP dimer was not observed in the presence of Alg-V and ferredoxin NADP+ reductase (FRD). A conjugated reaction, coupling the electrochemical regeneration of NADPH on the electrode and a glutathione reductase reaction was performed using Alg-V, FRD, and oxidized form of glutathione. The conjugated redox reaction was successful with the NADP+ immobilized PAA-modified electrode. Under given conditions, the conversion ratio of reduced glutathione (GSH) from oxidized glutathione (GSSG) reached 100% after 2.0 h of incubation at 37°C and the concentration of GSH accumulated in the reaction mixture was 1.0 mM.

Electron-transfer function of NAD+-immobilized alginic acid

Polymerized NAD+ (Alg-NAD+) was prepared and its electrochemical properties were investigated. NAD+ has been covalently immobilized at the carboxyl group of alginic acid using water soluble carbodiimide (EDC) and then Alg-NAD+s of various NAD+ density were obtainable depending on NAD+ concentration in the reaction mixture. Absorbance of 260 nm of Alg-NAD+s showed that 3.4 to 17.6% of carboxyl groups of alginic acid were coupled with NAD+. The coenzyme activity of immobilized NAD+ has reached 80 to 90% on each Alg-NAD+. A cathodic peak in the cyclic voltammogram of Alg-NAD+ appeared at -1.2 V (vs. SCE) corresponding to the reduction wave of free NAD+. The anodic wave of NAD dimer was not observed in the presence of 2.0 mM methyl viologen and 5 units of diaphorase and NAD+ immobilized on the composite electrode could be reduced to the normal NADH. The ratio of apparent diffusion coefficient (Dapp.) of Alg-NAD+ and free NAD+ was evaluated from the variation of ipc with the square root of sweep rate (v 1/2). Despite the high molecular weight of Alg-NAD+, Dapp. Alg-NAD+/Dapp. free NAD+ are larger than that expected. These results indicate that electron transfer occurred effectively between each NAD+ molecule immobilized onto the polymer chain. It is also confirmed by a conjugated redox enzyme reaction with Alg-NAD+.

Production of L-malic acid with fixation of HCO3(-) by malic enzyme-catalyzed reaction based on regeneration of coenzyme on electrode modified by layer-by-layer self-assembly method.

Malic enzyme prepared and purified from Brevundimonas diminuta IFO13182 catalyzed the decarboxylation reaction of malate to pyruvate and CO2 using NAD+ as the coenzyme, and the reverse reaction was used in the present study for L-malic acid production with fixation of HCO3(-) as a model compound for carbon source. The L-malic acid production was based on electrochemical regeneration of NADH on a carbon plate electrode modified by layer-by-layer adsorption of polymer-bound mediator (Alginic acid bound viologen derivative, Alg-V), polymer-bound coenzyme (Alginic acid bound NAD+, Alg-NAD+), and lipoamide dehydrogenase (LipDH). Electrochemical reduction of immobilized NAD+ catalyzed by LipDH in a multilayer film was achieved, and the L-malic acid production with HCO3(-) fixation system with layer-by-layer immobilization of Alg-V/LipDH/Alg-NAD+/malic enzyme multilayer film on the electrode gave an L-malic acid production of nearly 11.9 mmol and an HCO3(-) fixation rate of nearly 47.4% in a buffer containing only KHCO3 and pyruvic acid potassium salt, using a cation exchange membrane. The total turnover number of NADH within 48 h was about 19,000, which suggests that efficient NADH regeneration and fast electron transfer were achieved within the multilayer film, and that the modified electrode is a potential method for the fixation of HCO3(-) without addition of free coenzyme.

Enhancement of cellulase activity by clones selected from the combinatorial library of the cellulose-binding domain by cell surface engineering

To improve the cellulolytic activity of a yeast strain displaying endoglucanase II (EG II) from Trichoderma reesei, a combinatorial library of the cellulose‐binding domain (CBD) of EG II was constructed by using cell surface engineering. When EG II degrades celluloses, CBD binds to cellulose, and its catalytic domain cleaves the glycosidic bonds of cellulose. CBD had a flat face, composed of five amino acids for binding. It was supposed that the three hydrophobic amino acid residues of the five amino acid residues were essential for binding to cellulose. Therefore, by improving the two remaining amino acid residues, construction of mutants with a combinatorial library of the two amino acids in CBD was carried out and binding ability and hydrolysis activity were measured. In the first screening by halo assay using the Congo Red staining method, about 200 of the 2000 colonies formed clear halos, and then five colonies with the clearest halos were finally selected. In the second screening, the binding ability of the five mutants to phosphoric acid‐swollen Avicel was measured. In addition, the measurement of hydrolysis activity toward carboxymethylcellulose (CMC) using the screened mutants was carried out. As a result, the mutated EG II exhibiting higher binding ability (1.5‐fold) had higher hydrolysis activity (1.3‐fold) compared to the parent EG II‐displaying yeast cell, demonstrating that CBD has confirmatively some effect on the cellulase activity through its binding ability of the enzyme to cellulose.

Construction of a Cultivation System of a Yeast Single Cell in a Cell Chip Microchamber

A novel single cell screening system was constructed using a yeast cell chip in combination with the yeast cell surface engineering [NanoBiotechnology 2005, 1 , 105–111]. Enzymes or functional proteins displayed on a yeast cell surface can be used as a protein cluster. To achieve high‐throughput screening of protein libraries on the cell surface, a catalytic reaction by a single cell‐surface‐engineered yeast cell was successfully carried out in the microchamber on the yeast cell chip. After screening, to replicate a target cell for use in measuring of activity, DNA sequencing, and preservation, a novel single cell cultivation system in the yeast cell chip was constructed. To avoid damage of the rapid dry up of medium in the microchamber array, the yeast cell chip was modified with a protection sheet, so that the modified chip was like a micro‐culture tank constructed on the yeast cell chip microchamber. As a result, single yeast cell cultivation in the yeast cell chip microchamber was observed, and the modified yeast cell chip was evaluated to be good for a single cell selection. The improvement showed that the single cell screening system coupled with the single cell cultivation using the modified yeast cell chip may be superior to that by a cell sorter for the isolation of a target cell and its practical use.

Yeast Cell-Surface Expression of Chitosanase from Paenibacillus fukuinensis

To produce chitoorigosaccharides using chitosan, we attempted to construct Paenibacillus fukuinensis chitosanase-displaying yeast cells as a whole-cell biocatalyst through yeast cell-surface engineering. The localization of the chitosanase on the yeast cell surface was confirmed by immunofluorescence labeling of cells. The chitosanase activity of the constructed yeast was investigated by halo assay and the dinitrosalicylic acid method.

Cloning of a Novel Dehalogenase from Environmental DNA

Cloning of pceA, the gene of tetrachloroethene (PCE)-reductive dehalogenase, was undertaken from environmental DNA. Two genes were amplified using PCR primer deduced from pceA. Functional expression of these genes was unsuccessful in Escherichia coli, but PceA1 synthesized in vitro was enzymatically active. In recombinant E. coli, PceA1 formed a complex with host DnaK and caused filamentous growth.