Electrochemical impedance detection of DNA hybridization based on dendrimer modified electrode

Bioactive ultrathin films with the incorporation of amino-terminated G4 PAMAM dendrimers have been prepared via layer-by-layer self-assembly methods on a gold electrode and used for the DNA hybridization analysis. Surface plasmon resonance (SPR), X-ray photoelectron spectroscopy (XPS), and electrochemical impedance spectroscopy (EIS) are used to characterize the successful construction of the multicomponent film on the gold substrate. The dendrimer-modified surfaces improve the immobilization capacity of the probe DNA greatly, compared to the AET (2-aminoethanethiol) SAM sensor surfaces without dendrimer molecules. DNA hybridization analysis is monitored by EIS. The dendrimer-based electrochemical impedance DNA biosensor shows high sensitivity and selectivity for DNA hybridization assay. The multicomponent films also display a high stability during repeated regeneration and hybridization cycles.     

Continuous indirect electrochemical regeneration of galactose oxidase

The development of an indirect anaerobic electrochemical regeneration of galactose oxidase (GOase) allows the prevention of the undesired production of the enzyme inhibitor hydrogen peroxide, which is generated under aerobic regeneration conditions during synthetic applications of GOase. The pH optimum for the electrochemical regeneration of GOase with polyethyleneglycol-modified ferrocene mediators in carbonate buffer is 10.8. Total turnover numbers achieved by either electrochemical or aerobic regeneration of GOase are almost the same. The electrochemical regeneration is half as fast as the aerobic regeneration. It is not necessary to work under anaerobic conditions, because at pH 10.8 the aerobic regeneration of GOase is prevented. The enzyme can be stabilized most effectively by immobilization on an aminopropylated polysiloxane (DELOXAN) via the glutaric dialdehyde procedure with good activity yields up to 37%. Buffers containing amino groups proved to be fatal for long-term GOase stability.     

Bi‐Functional Iron‐Only Electrodes for Efficient Water Splitting with Enhanced Stability through In Situ Electrochemical Regeneration

Scalable and robust electrocatalysts are required for the implementation of water splitting technologies as a globally applicable means of producing affordable renewable hydrogen. It is demonstrated that iron‐only electrode materials prove to be active for catalyzing both proton reduction and water oxidation in alkaline electrolyte solution with superior activity to that of previously established bi‐functional catalysts containing less abundant elements. The reported bi‐functionality of the iron electrodes is reversible upon switching of the applied bias through electrochemical interconversion of catalytic species at the electrode surface. Cycling of the applied bias results in in‐situ electrochemical regeneration of the catalytic surfaces and thereby extends the catalyst stability and lifetime of the water electrolyzer. Full water splitting at a current density of I = 10 mA cm^?2 is achieved at a bias of ≈2 V, which is stable over at least 3 d (72 one hour switching cycles). Thus, potential‐switching is established as a possible strategy of stabilizing electrode materials against degradation in symmetrical water splitting systems.     

Improved strategies for electrochemical 1,4-NAD(P)H2 regeneration: A new era of bioreactors for industrial biocatalysis

Industrial enzymatic reactions requiring 1,4-NAD(P)H₂ to perform redox transformations often require convoluted coupled enzyme regeneration systems to regenerate 1,4-NAD(P)H₂ from NAD(P) and recycle the cofactor for as many turnovers as possible. Renewed interest in recycling the cofactor via electrochemical means is motivated by the low cost of performing electrochemical reactions, easy monitoring of the reaction progress, and straightforward product recovery. However, electrochemical cofactor regeneration methods invariably produce adventitious reduced cofactor side products which result in unproductive loss of input NAD(P). We review various literature strategies for mitigating adventitious product formation by electrochemical cofactor regeneration systems, and offer insight as to how a successful electrochemical bioreactor system could be constructed to engineer efficient 1,4-NAD(P)H₂-dependent enzyme reactions of interest to the industrial biocatalysis community.     

Cofactor Regeneration in Biocatalysis in Organic Media

Methods used for the regeneration of cofactors in organic media are reviewed. Substratedriven regeneration methods include the use of a second substrate of the same enzyme and the use of a second enzyme and its substrate. The use of mediators in oxidoreductions is described and examples of photochemical and electrochemical regeneration methods are presented. General problems and possibilities of cofactor regeneration in organic media are discussed.     

脱氢酶生物传感器研究关键技术与进展

自然界中依赖烟酰胺类辅酶(NAD+或NADP+)的脱氢酶是氧化还原酶中最重要的一类,基于此类酶的生物传感器应用前景广阔,近年来发展迅速。构建这类传感器需要两项关键技术,即氧化型辅酶在电极表面的再生和辅酶固定化。本文介绍了辅酶电化学再生的主要方法、辅酶固定化的常见手段,以及相关的研究进展。     

繁殖世代对水稻植株农艺性状及稻米品质的影响研究

以国家长期库躬存种质为对照,选择在生产上一直种植的同一品种为高繁殖世代材料,一起进行田间种植以了解两者之间在田间出苗率、生育期、植株农艺性状及稻米品质上是否存在差异。结果表明,在6个供试粳稻品种中,有5个品种的高繁殖世代种质,其稻米垩白品质出现了退化现象,而其他稻米品质特性没有出现明显的变化。来自库存的种质,其每穗粒数都比一直在生产上种植的种质少,这可能与母本长时间低温贮藏有关。此外,来自库存的种质,其田间出苗率也相对较低。     

Electrochemical Regeneration of Oxidoreductases for Cell-free Biocatalytic Redox Reactions

Oxidoreductases represent a highly interesting and versatile class of biocatalysts for specific reduction, oxidation, and oxyfunctionalization reactions. Since oxidoreductases depend on cofactors and coenzymes to supply or withdraw redox equivalents released during the catalytic process, their application in cell-free environments requires external supply with these redox equivalents. Next to enzymatic approaches, a variety of non-enzymatic regeneration strategies have been developed. This review focuses on electrochemical methods for the in situ regeneration of nicotinamide cofactors as well as flavin- and heme-coenzymes, developed for synthetic application. The fields of electrochemical biosensors as well as biofuel cells are not discussed in detail. Electrochemical approaches bear much promise and in some cases are more efficient and more versatile than enzymatic regeneration approaches.     

Electrochemical regeneration of Fe(III) to support growth on anaerobic iron respiration.

Here we describe artificial help for the respiratory electron flow supporting anaerobic growth of Thiobacillus ferrooxidans through exogenous electrolysis. Flux between H(2) and a anode through cells was accomplished with electrochemical regeneration of iron. The electrochemical help resulted in a 12-fold increase in yield compared with the yield observed in its absence.     

Establishing japonica rice suspensions retaining a high regeneration potential after 14 months of culture

We report here a method of ‘two cycles of selection’ for rapidly establishing rice embryogenic cell suspensions which had high regeneration potential even after long-term culture (up to 14 months). Embryogenic calli were induced from immature embryos of 17 genotypes of cultivated japonica rice (Oryza sativa L.) and tested for their regeneration potential. Five of the 17 genotypes showed relatively high regeneration frequencies (27%–45%) and were selected. This process was the first cycle selection, namely, the selection for responding genotypes. The second cycle of selection, or the selection for responding individual callus, was then conducted. Calli from different immature embryos of the same genotype were found to differ in regeneration potential, therefore calli with high regeneration potential were selected again in each of the five genotypes. Five finely-dispersed and rapidly-growing embryogenic cell suspensions, one for each of the genotypes, were established from the calli chosen by the two cycles of selection. The regeneration potential of these cell suspensions was tested every two months over a period of 14 months. All of the five genotypes retained high regeneration frequency (above 62%) over the whole period tested.     

Monascus kaoliang CBS 302.78 immobilized in polyurethane foam using iso-propanol as co-substrate: Optimized immobilization conditions of a fungus as biocatalyst for the reduction of ketones

Monascus kaoliang was selected after a microbial screening as a highly active and selective whole cell catalyst for the reduction of ketones. In the present paper we describe the optimum growing conditions and an interesting immobilization procedure by adsorption in polyurethane foams (PUFs). This methodology is easy to perform and the immobilized catalyst is active, stable and reusable. The use of different co-substrates for cofactor regeneration was also tested and iso-propanol (i-PrOH) was found as the best co-substrate, as it leads to a catalyst reusable for 17 cycles, displaying better NADH regeneration properties than others e.g., glucose (10 cycles) or saccharose (6 cycles). The reduction of different prochiral ketones showed that the ketone reductase activity of this mould follows the Prelog’s rule and kinetic experiments demonstrated that the process follows a pseudo-first kinetic order.     

辅因子再生研究进展

许多有价值的酶催化反应都需要辅因子的参与。因为辅因子价格昂贵,所以,在酶催化工业应用中,需要实现辅因子原位再生。经过几十年研究,出现了酶法、化学法、电化学法、光化学法和基因工程法等手段实现烟酰胺类辅因子（NAD（P）H）、ATP、糖核苷酸等辅因子再生。对辅因子再生研究中取得的进展以及存在的问题进行讨论。     

Recent developments in pyridine nucleotide regeneration

NAD(P)-dependent oxidoreductases are valuable tools for the synthesis of chiral compounds. Due to the high cost of the pyridine cofactors, in situ cofactor regeneration is required for preparative applications. In recent years, existing regeneration methodologies have been improved and new approaches have been devised. These include the use of newly discovered dehydrogenases that are stable in high contents of organic solvent and novel enzymes that can regenerate either the reduced or oxidized forms of the cofactor. The use of electrochemical methods has allowed cofactor regeneration for monooxygenases and natural or engineered whole-cell systems provide alternatives to approaches relying on purified enzymes.     

Reusability of avidin-biotinylated immunoglobulin Y columns in immunoaffinity chromatography

Reusability of avidin-biotinylated IgY columns for immunoaffinity chromatography was examined by repeated use and regeneration. An enzyme-linked immunosorbent assay-elution assay using CovaLink NH microtiter plates was used to find the optimal conditions for regeneration of columns. Actigel avidin-biotinylated IgY column retained about 90% of its initial IgG binding capacity after 50 cycles, with 0.1 M glycine-HCl buffer, pH 2.8, as eluent, requiring no regeneration. However, IgG binding capacity of UltraLink avidin-biotinylated IgY column gradually decreased to 75 and 65% after 10 and 20 cycles, respectively, with the commercial eluent, Actisep. Results from the CovaLink NH system agreed with those from UltraLink avidin-biotinylated IgY columns. The UltraLink avidin-biotinylated IgY column was regenerated twice, by applying 8 M guanidine-HCl, pH 1.6, to dissociate biotinylated IgY antibodies from the column. About 40 and 25% of IgG binding capacities remained after the first and second regeneration. By applying new biotinylated IgY to the treated columns, about 95 and 90% of the initial IgG binding capacity before any treatment were recovered. These results demonstrated that avidin-biotinylated IgY columns are reusable with or without regeneration depending on the avidin-immobilized matrix.     

Electrochemical Regeneration of Fe(III) To Support Growth on Anaerobic Iron Respiration

Here we describe artificial help for the respiratory electron flow supporting anaerobic growth of Thiobacillus ferrooxidans through exogenous electrolysis. Flux between H₂ and a anode through cells was accomplished with electrochemical regeneration of iron. The electrochemical help resulted in a 12-fold increase in yield compared with the yield observed in its absence.     

The effect of electrochemical regeneration upon the enzymatic catalysis of a thermodynamically unfavorable reaction

The association between enzymatic and electrochemical reactions, enzymatic electrocatalysis, had proven to be a very powerful tooth in both analytical and synthetic fields. However, most of the combinations studied have involved enzymatic catalysis of irreversible or quasi-irreversible reaction. In the present work, we have investigated the possibility of applying enzymatic electrocatalysis to a case where the electrochemical reaction drives a thermodynamically unfavorable reversible reaction. Such thermodynamically unfavorable reactions include most of the oxidations catalyzed by dehydrogenases. Yeast alcohol dehydrogenase (E.C. 1.1.1.1) was chosen as a model enzyme because the oxidation of ethanol is thermodynamically very unfavorable and because its kinetics are well known. The electrochemical reaction was the oxidation of NADH which is particularly attractive as a method of cofactor regeneration. Both the electrochemical and enzymatic reactions occur in the same batch reactor in such a way that electrical energy is the only external driving force. Two cases were experimentally and theoretically developed with the enzyme either in solution or immobilized onto the electrode's surface. In both cases, the electrochemical reaction could drive the enzymatic reaction by NADH consumption in solution or directly in the enzyme's microenvironment. However even for a high efficiency of NADH consumption, the rate of enzymatic catalysis was limited by product (acetaldedehyde) inhibition. Extending this observation to the subject of organic synthesis catalyzed by dehydrogenases, we concluded that thermodynamically unfavorable reaction and can only be used in a process if efficient NAD regeneration and product elimination are simultaneously carried out within the reactor.     

Mitochondrial therapy promotes regeneration of injured hippocampal neurons

Due to the inhibitory microenvironment and reduced intrinsic growth capacity of neurons, neuronal regeneration of central nervous system remains challenging. Neurons are highly energy demanding and require sufficient mitochondria to support cellular activities. In response to stimuli, mitochondria undergo fusion/fission cycles to adapt to environment. It is thus logical to hypothesize that the plasticity of mitochondrial dynamics is required for neuronal regeneration. In this study, we examined the role of mitochondrial dynamics during regeneration of rat hippocampal neurons. Quantitative analysis showed that injury induced mitochondrial fission. As mitochondrial dysfunction has been implicated in neurodegenerative diseases, we tested the possibility that the mitochondrial therapy may promote neuronal regeneration. Supplying freshly isolated mitochondria to the injured hippocampal neurons not only significantly increased neurite re-growth but also restored membrane potential of injured hippocampal neurons. Together, our findings support the importance of mitochondrial dynamics during regeneration of injured hippocampal neurons and highlight the therapeutic prospect of mitochondria to the injured central nervous system.     

Studies on genetic changes in rye samples (Secale cereale L.) maintained in a seed bank

The aim of this study was to identify genetic changes in rye seeds induced by natural ageing during long-term storage and consecutive regeneration cycles under gene bank conditions. Genomic DNA from four rye samples varying in their initial viability after one and three cycles of reproduction was analyzed by AFLP (amplified fragment length polymorphism) fingerprinting. Seven EcoRI/MseI primer combinations defined 663 fragments, and seven PstI/MseI primer combinations defined 551 fragments. The variation in the frequency of the seventy-four EcoRI/MseI bands was statistically significant between samples. These changes could be attributed to genetic changes occurring during storage and regeneration. However, the PstI/MseI fragments appeared to be uninfluenced by seed ageing, regeneration and propagation. A combined Principle Coordinate Analysis revealed differences between samples with different initial viability. We showed that materials with low initial viability differ in their response from highly viable ones, and that the changes exhibited in the former case are preserved through regeneration cycles.     

巴西蕉高效间接器官发生再生体系研究

香蕉间接器官发生再生体系是一种较好的突变育种和基因转化受体再生系统。本研究以巴西蕉低代试管苗茎段薄层切片、中代试管苗茎段薄层切片、吸芽薄层切片、雄花薄层切片为外植体,对巴西蕉间接器官发生再生体系进行了系统研究。研究结果表明,以MS为基本培养基,巴西蕉低代试管苗茎段薄层切片在0.5～1.0mg/L2,4-D+4～6mg/L6-BA激素条件下;雄花薄层切片在0.5～1.0mg/L2,4-D+0.2～0.5mg/LZT激素条件下能通过花菜体间接器官发生途径实现高效再生;同时,花菜体薄层切片在1.0mg/L2,4-D+5mg/L6-BA激素条件下,再次通过花菜体途径实现循环高效再生。本研究为建立香蕉高效间接器官发生再生体系和相关育种技术奠定了基础。     

Simultaneous production of 1,3-dihydroxyacetone and xylitol from glycerol and xylose using a nanoparticle-supported multi-enzyme system with in situ cofactor regeneration

Cofactor-dependent biotransformations often require consumption of a secondary substrate for cofactor regeneration. Alternatively, two synthetic reactions may be coupled together through cofactor regeneration cycles. Simultaneous production of value-added products from glycerol and xylose was realized in this work through an enzymatic NAD(H) regeneration cycle involving two enzymes. Glycerol dehydrogenase (GDH) catalyzed the production of 1,3-dihydroxyacetone (DHA) from glycerol, while xylose reductase (XR) enabled the reduction of xylose to xylitol using the protons released from glycerol. Both enzymes were immobilized with P(MMA-EDMA-MAA) nanoparticles. Interestingly, the immobilized multi-enzyme system showed much improved productivity and stability as compared to native enzymes, such that the total turnover number (TTN) reached 82 for cofactor regeneration while the yield reached 160g/g-immobilized GDH for DHA production.