Electro-enzymatic reduction of dioxygen to water in the cathode compartment of a biofuel cell

Palmore, G. Tayhas R.; Kim, Hyug-Han

doi:10.1016/s0022-0728(99)00008-x

Cited by 297 publications

(135 citation statements)

References 39 publications

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“…Direct electron transfer from the electrode is less efficient than transfer via a mediator (58). For the application of laccase in the cathodic compartment of a biofuel cell, the rate of direct electron transfer is insufficient (31,47). Mediators, however, are mostly toxic, instable, or expensive.…”

Section: Discussionmentioning

confidence: 99%

Functional Expression of a Fungal Laccase in Saccharomyces cerevisiae by Directed Evolution

Bülter

Alcalde

Sieber

et al. 2003

Appl Environ Microbiol

256

170

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Laccase from Myceliophthora thermophila (MtL) was expressed in functional form in Saccharomyces cerevisiae. Directed evolution improved expression eightfold to the highest yet reported for a laccase in yeast (18 mg/liter). Together with a 22-fold increase in k cat , the total activity was enhanced 170-fold. Specific activities of MtL mutants toward 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine indicate that substrate specificity was not changed by the introduced mutations. The most effective mutation (10-fold increase in total activity) introduced a Kex2 protease recognition site at the C-terminal processing site of the protein, adjusting the protein sequence to the different protease specificities of the heterologous host. The C terminus is shown to be important for laccase activity, since removing it by a truncation of the gene reduces activity sixfold. Mutations accumulated during nine generations of evolution for higher activity decreased enzyme stability. Screening for improved stability in one generation produced a mutant more stable than the heterologous wild type and retaining the improved activity. The molecular mass of MtL expressed in S. cerevisiae is 30% higher than that of the same enzyme expressed in M. thermophila (110 kDa versus 85 kDa). Hyperglycosylation, corresponding to a 120-monomer glycan on one N-glycosylation site, is responsible for this increase. This S. cerevisiae expression system makes MtL available for functional tailoring by directed evolution.Directed evolution by random mutagenesis and recombination followed by screening or selection is a valuable tool for the engineering of enzymes (2,3,16,61). Functional gene expression in a suitable host is a prerequisite for directed evolution. Considering transformation efficiency, stability of plasmid DNA, and growth rate, Escherichia coli and Saccharomyces cerevisiae are best suited for these experiments. Heterologous expression in these hosts, however, is often limited by differences in the expression systems from the native organism (50). Different codon usage, missing chaperones, and posttranslational modifications such as disulfide bridges or glycosylation can all cause low expression levels and misfolded proteins that are degraded or driven into inclusion bodies (20). Finding the bottlenecks of a specific expression system requires consideration of many possibilities whose impact is hard to predict. Some incompatibilities between the expressed gene and heterologous host, such as codon usage or the recognition of signal sequences, can be overcome by changing the gene sequence. Thus, achieving functional expression is a good target for directed evolution (13,42,43).Laccases, like other ligninolytic enzymes, are notoriously difficult to express in nonfungal systems. The laccase from Myceliophthora thermophila (MtL) used in this work was previously expressed only in Aspergillus oryzae (6). Expression in S. cerevisiae has been reported for other laccase genes (11,33,34,60). Kojima et al. demonstrated expression of...

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Section: Discussionmentioning

confidence: 99%

Functional Expression of a Fungal Laccase in Saccharomyces cerevisiae by Directed Evolution

Bülter

Alcalde

Sieber

et al. 2003

Appl Environ Microbiol

256

170

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“…No stability information for these modified electrodes is provided in the paper. Palmore and Kim (1999) investigated the cathodic reduction of oxygen to water by the enzyme laccase in free solution, mediated by 2,2 -azinobis(3-ethylbenzothiazoline-6-sulphonate) (ABTS) at platinum and glassy carbon cathodes. A platinised platinum gauze hydrogen anode was used in the fuel cell design.…”

Section: Diffusive Met Enzymatic Electrodesmentioning

confidence: 99%

“…(13)- (15) 8. Power density of a prototype enzymatic biofuel cell plotted against cell potential to illustrate the cell voltage at which the power density reaches a maximum for different cathodes, and dramatically illustrating the enhancement possible through the use of enzymatic catalysis (after Palmore and Kim, 1999).…”

Section: Diffusive Met Enzymatic Electrodesmentioning

confidence: 99%

“…It was noted that the stability and activity of a hydrogen-oxidising anode provide a high performance; hence it is difficult for biochemical fuels to compete with the relatively high power density of gaseous hydrogen (Tsujimura et al, 2001a) and liquid fuels such as methanol. Biofuel cell research has shown success in investigating systems based on more complex fuel molecules (Palmore and Kim, 1999). Recent experiments have demonstrated the possibility of exceeding the hydrogen oxidation activity of platinum (Morozov et al, 2002), at least in the short-term.…”

Section: Non-diffusive Enzyme Electrodesmentioning

confidence: 99%

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Biofuel cells and their development

Bullen

Arnot

Lakeman

et al. 2006

Biosensors and Bioelectronics

937

545

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This review considers the literature published since 1994 on microbial and enzymatic biofuel cells. Types of biofuel cell are classified according to the nature of the electrode reaction and the nature of the biochemical reactions. The performance of fuel cells is critically reviewed and a variety of possible applications is considered. The current direction of development of biofuel cells is carefully analysed. While considerable chemical development of enzyme electrodes has occurred, relatively little progress has been made towards the engineering development biofuel cells. The limit of performance of biofuel cells is highlighted and suggestions for future research directions are provided.

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“…The microorganism from which it is extracted greatly determines the redox potential of the T 1 site which can vary from 430 mV vs. NHE up to 780 mV vs. NHE (Palmore & Kim, 1999). Laccase from Trametes versicolor is the most attractive one since redox potential of its T 1 site is ca.…”

Section: Reduction Of Oxygen Catalyzed By Laccasementioning

confidence: 99%