Hirosuke Tatsumi

Electrochemical activity of an Fe(III)-reducing bacterium, Shewanella putrefaciens IR-1, in the presence of alternative electron acceptors

Cyclic voltammetry demonstrated that cells of Shewanella putrefaciens grown under anaerobic conditions without nitrate were electrochemically active. The electrochemical activity was inactivated reversibly by exposure to air, but not by nitrate. Lactate and an applied potential at +200 mV against an Ag/AgCl reference electrode restored the electrochemical activity. These findings can be used to improve the performance of a mediator-less microbial fuel cell using electrochemically active bacteria in the presence of nitrate.

Bioelectrocatalysis-based dihydrogen/dioxygen fuel cell operating at physiological pH

A biochemical fuel cell was constructed using H2 as fuel to produce H2O in the reaction with O2 at neutral pH and ambient temperature. The cell uses carbon felt as an electrode material for both the anode and the cathode and an anion exchange membrane as a separator. The anodic oxidation of H2 was accelerated by methyl viologen-mediated electrocatalysis with bacterial cells Desulfoibrio ulgaris (Hildenborough) as catalysts, and the cathodic reduction of O2 was accelerated by 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate)-mediated electrocatalysis with bilirubin oxidase as a catalyst. The bioelectrocatalytic systems allowed the cell to operate at 1.0 V with current 0.9 mA at an electrode of size 1.5 × 1.5 × 0.1 cm3. The cell voltage attained 1.17 V at open circuit, which is close to the standard electromotive force 1.23 V. The cell voltage–current behavior is interpretable by linear sweep voltammetry using the same electrode system. On this basis, the electrochemistry behind the performance of the biochemical fuel cell is discussed.

Pre-steady-state Kinetics for Hydrolysis of Insoluble Cellulose by Cellobiohydrolase Cel7A

Background: The molecular understanding of factors that limit enzymatic hydrolysis of cellulose remains incomplete. Results: Pre-steady-state analysis of cellulolytic activity provides rate constants for basic steps of the overall reaction. Conclusion: Slow dissociation of inactive enzyme-cellulose complexes governs the hydrolytic rate at pseudo-steady state. Significance: Kinetic constants elucidate molecular mechanisms and structure-function relationships for cellulases. The transient kinetic behavior of enzyme reactions prior to the establishment of steady state is a major source of mechanistic information, yet this approach has not been utilized for cellulases acting on their natural substrate, insoluble cellulose. Here, we elucidate the pre-steady-state regime for the exo-acting cellulase Cel7A using amperometric biosensors and an explicit model for processive hydrolysis of cellulose. This analysis allows the identification of a pseudo-steady-state period and quantification of a processivity number as well as rate constants for the formation of a threaded enzyme complex, processive hydrolysis, and dissociation, respectively. These kinetic parameters elucidate limiting factors in the cellulolytic process. We concluded, for example, that Cel7A cleaves about four glycosidic bonds/s during processive hydrolysis. However, the results suggest that stalling the processive movement and low off-rates result in a specific activity at pseudo-steady state that is 10–25-fold lower. It follows that the dissociation of the enzyme-substrate complex (half-time of ∼30 s) is rate-limiting for the investigated system. We suggest that this approach can be useful in attempts to unveil fundamental reasons for the distinctive variability in hydrolytic activity found in different cellulase-substrate systems.

Ion-transfer voltammetry at 1,6-dichlorohexane|water and 1,4-dichlorobutane|water interfaces.

The usefulness of 1,6-dichlorohexane (1,6-DCH) and 1,4-dichlorobutane (1,4-DCB) as organic solvent (O) for ion-transfer voltammetry at O|water (W) interface has been examined, and the results are compared with those with 1,2-dichloroethane (1,2-DCE). The width of potential window of the 0.1M tetraoctylammonium tetrakis(4-chlorophenyl)borate (O)|0.05M Li(2)SO(4) (W) interface increased in the sequence: O = 1,6-DCH > 1,4-DCB > 1,2-DCE. The voltammetric behavior of the transfer of various cations and anions at the 1,6-DCH|W and 1,4-DCB|W interfaces has been shown to be of reversible nature, and the midpoint potentials or the reversible half-wave potentials have been determined. The midpoint potentials of hydrophilic ions have also been determined by the analysis of anodic final rise or cathodic final decent of the voltammograms with the O|W interfaces, where the W contains a salt of the hydrophilic ion. Also, the effect of ion-pair formation in O on the midpoint potentials has also been discussed.

Transient Kinetics and Rate-Limiting Steps for the Processive Cellobiohydrolase Cel7A: Effects of Substrate Structure and Carbohydrate Binding Domain

Cellobiohydrolases are exoacting, processive enzymes that effectively hydrolyze crystalline cellulose. They have attracted considerable interest because of their role in both natural carbon cycling and industrial enzyme cocktails used for the deconstruction of cellulosic biomass, but many mechanistic and regulatory aspects of their heterogeneous catalysis remain poorly understood. Here, we address this by applying a deterministic model to real-time kinetic data with high temporal resolution. We used two variants of the cellobiohydrolase Cel7A from Hypocrea jecorina , and three types of cellulose as substrate. Analysis of the pre-steady-state regime allowed delineation rate constants for both fast and slow steps in the enzymatic cycle and assessment of how these constants influenced the rate of hydrolysis at quasi-steady state. Processive movement on the cellulose strand advanced with characteristic times of 0.15-0.7 s per step at 25 °C, and the rate was highest on amorphous substrate. The cellulose binding module was found to raise this rate on crystalline, but not on amorphous, substrate. The rapid processive movement signified high intrinsic reactivity, but this parameter had marginal influence on the steady-state rate. This was because dissociation and association were slower and, hence, rate limiting. Specifically, the dissociation from the strand was found to occur with characteristic times of 45-100 s. This meant that dissociation was the bottleneck, except at very low substrate loads (0.5-1 g/L), where association became slower.

Mechanistic study of the autoxidation of reduced flavin and quinone compounds

The mechanism of the autoxidation of reduced flavin and quinone compounds was investigated through semiquantitative analysis of voltammetric waves of the reduction of dioxygen (O2) catalyzed by flavin and quinone adsorbed on electrode surfaces, where the redox equilibrium among the oxidized, (flavo)semiquinone and fully reduced states is easily controlled by the electrochemical method. The analysis provided evidence that the semiquinone radical plays a predominant role in the autoxidation. The generation of a superoxide anion radical as a product of the one-electron transfer from the semiquinone to O2 was confirmed by the reduction of ferricytochrome c. The fact that the catalytic reduction wave of O2 increases with pH was ascribed to the increase in the semiquinone formation constant. The mechanism of the reoxidation of reduced flavoprotein glucose oxidase with O2 was also examined. The result supports the semiquinone-dependent one-electron transfer as the mechanism.

Origin of initial burst in activity for Trichoderma reesei endo-glucanases hydrolyzing insoluble cellulose.

The kinetics of cellulose hydrolysis have longbeen described by an initial fast hydrolysis rate, tapering rapidly off, leading to a process that takes days rather than hours to complete. This behavior has been mainly attributed to the action of cellobiohydrolases and often linked to the processive mechanism of this exo-acting group of enzymes. The initial kinetics of endo-glucanases (EGs) is far less investigated, partly due to a limited availability of quantitative assay technologies. We have used isothermal calorimetry to monitor the early time course of the hydrolysis of insoluble cellulose by the three main EGs from Trichoderma reesei (Tr): TrCel7B (formerly EG I), TrCel5A (EG II), and TrCel12A (EG III). These endo-glucanases show a distinctive initial burst with a maximal rate that is about 5-fold higher than the rate after 5 min of hydrolysis. The burst is particularly conspicuous for TrCel7B, which reaches a maximal turnover of about 20 s−1 at 30 °C and conducts about 1200 catalytic cycles per enzyme molecule in the initial fast phase. For TrCel5A and TrCel12A the extent of the burst is 2–300 cycles per enzyme molecule. The availability of continuous data on EG activity allows an analysis of the mechanisms underlying the initial kinetics, and it is suggested that the slowdown is linked to transient inactivation of enzyme on the cellulose surface. We propose, therefore, that the frequency of structures on the substrate surface that cause transient inactivation determine the extent of the burst phase.

Electrocatalytic properties of Acetobacter aceti cells immobilized on electrodes for the quinone-mediated oxidation of ethanol

Abstract Carbon paste electrodes on which intact cells of Acetobacter aceti (IFO3284) are immobilized behind a dialysis membrane produce a catalytic current for the oxidation of ethanol in the presence of 2-methyl-5,6-dimethoxy benzoquinone (Q 0 ). Analysis of the catalytic current reveals that the catalytic activity of the bacterial cells is due to alcohol dehydrogenase (ADH) existing in the cytoplasmic membranes. The membrane-bound ADH catalyzes the oxidation of ethanol by the use of Q 0 penetrating the membranes, in which Q 0 is reduced by accepting electrons from the enzyme. The reduced form of Q 0 , in turn, reaches the electrode and is oxidized there. Thus, Q 0 serves as an electron transfer mediator between the intact cells and the electrode. The mediated bioelectrocatalysis behavior can be described by an equation of the catalytic current derived for an enzyme-based electrocatalysis. Performance of the electrode as an ethanol sensor is compared with that of an electrode modified with purified ADH. An ADH deficient strain and the ADH mutant harboring ADH gene are also examined as biocatalysts in place of A. aceti .

An amperometric enzyme biosensor for real-time measurements of cellobiohydrolase activity on insoluble cellulose

An amperometric enzyme biosensor for continuous detection of cellobiose has been implemented as an enzyme assay for cellulases. We show that the initial kinetics for cellobiohydrolase I, Cel7A from Trichoderma reesei, acting on different types of cellulose substrates, semi-crystalline and amorphous, can be monitored directly and in real-time by an enzyme-modified electrode based on cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium (Pc). PcCDH was cross-linked and immobilized on the surface of a carbon paste electrode which contained a mediator, benzoquinone. An oxidation current of the reduced mediator, hydroquinone, produced by the CDH-catalyzed reaction with cellobiose, was recorded under constant-potential amperometry at +0.5 V (vs. Ag/AgCl). The CDH-biosensors showed high sensitivity (87.7 µA mM(-1) cm(-2)), low detection limit (25 nM), and fast response time (t(95%) ≈ 3 s) and this provided experimental access to the transient kinetics of cellobiohydrolases acting on insoluble cellulose. The response from the CDH-biosensor during enzymatic hydrolysis was corrected for the specificity of PcCDH for the β-anomer of cello-oligosaccharides and the approach were validated against HPLC. It is suggested that quantitative, real-time data on pure insoluble cellulose substrates will be useful in attempts to probe the molecular mechanism underlying enzymatic hydrolysis of cellulose.

Discoidin Domain of Chitosanase Is Required for Binding to the Fungal Cell Wall

Previously, we reported properties of a glycosylase belonging to GH-8 glycosyl hydrolase (GH) and having both chitosanase and glucanase activities. This enzyme (D2), whose molecular mass (86 kDa) was the largest among the GH-8 group, has its catalytic domain at the N-terminal region, and discoidin domain (DD) at the C-terminal region. Although various chitosanases, chitinases and glucanases have been known, DD is unique to the D2 enzyme. Glucanase and chitinase, but not chitosanase, are known to have functional domain such as carbohydrate-binding module, besides catalytic domain. Accordingly, function of the DD of D2 chitosanase was analyzed, using zygomycete cell wall containing chitosan, glucan and chitin as the basic constituents. The DD specifically and tightly bound to chitosan, but did not participate in affinity for glucan and chitin. Deletion of the DD caused marked reduction in absorbability to cell wall and in hydrolytic activity toward chitosan and glucan. These results suggest that the DD is concerned in binding of the enzyme to cell wall and in effective digestion of the insoluble substrate, through hydrolysis of not only chitosan but also coexisting glucan. Thus, this is the first example of chitosan-binding domain among various carbohydrate-binding modules reported thus far.