Search

Despite the increasing demand for efficient and sustainable chemical processes, the development of scalable systems using biocatalysis for fine chemical production remains a significant challenge. We have developed a scalable flow system using immobilized enzymes to facilitate flavin-dependent biocatalysis, targeting as a proof-of-concept asymmetric alkene reduction. The system integrates a flavin-dependent Old Yellow Enzyme (OYE) and a soluble hydrogenase to enable H 2 -driven regeneration of the OYE cofactor FMNH 2 . Molecular hydrogen was produced by water electrolysis using a proton exchange membrane (PEM) electrolyzer and introduced into the flow system via a designed gas membrane addition module at a high diffusion rate. The flow system shows remarkable stability and reusability, consistently achieving >99% conversion of ketoisophorone to levodione. It also demonstrates versatility and selectivity in reducing various cyclic enones and can be extended to further flavin-based biocatalytic approaches and gas-dependent reactions. This electro-driven continuous flow system, therefore, has significant potential for advancing sustainable processes in fine chemical synthesis., (© 2024. The Author(s).)

Background and Objectives: Despite promising effects of individualized music listening (IML) for people with dementia, the individualized nature and heterogeneity of reactions to IML remain underexplored. We aimed to develop an empirically derived typology of directly observed reactions of people with dementia to IML and propose recommendations for tailoring the intervention to the respective types., Research Design and Methods: An ideal-type analysis was conducted on 108 video recordings of 45 people with mainly severe dementia (78% female, mean age of 83.02 years, all White participants) listening to recorded individualized music. Dimensions were identified for capturing similarities and differences between types., Results: The analysis yielded 10 types of reactions ("expressing and sharing joy," "self-disclosure stimulated by music," "concentrated, absorbed listening," "blissful enjoyment," "experience of the music as bittersweet," "sharing memories," "releasing tension," "tensing up and rejecting," "predominant search for social exchange," "no interpretable reaction") and 3 dimensions ("valence" from negative to positive, "arousal" from calm to activated, "communicative activity" from defensive/resistant to proactive), resulting in a three-dimensional coordinate system, providing a holistic representation and facilitating a systematic contrast of identified reaction types to IML., Discussion and Implications: Reactions to IML were influenced by the interaction with the project staff, who monitored the sessions. Based on these observations, we propose recommendations for tailoring both the behavior of the monitoring person (e.g., engaging in synchronous activities like clapping along) and the setup of the intervention (e.g., communal vs individual listening) to each type, which may improve the effects of IML., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

High cell density cultivation is an established method for the production of various industrially important products such as recombinant proteins. However, these protocols are not always suitable for biocatalytic processes as the focus often lies on biomass production rather than high specific activities of the enzyme inside the cells. In contrast, a range of shake flask protocols are well known with high specific activities but rather low cell densities. To overcome this gap, we established a tailor-made fed-batch protocol combining both aspects: high cell density and high specific activities of heterologously produced enzyme. Using the example of an industrially relevant amine transaminase from Bacillus megaterium, we describe a strategy to optimize the cultivation yield based on the feed rate, IPTG concentration, and post-induction temperature. By adjusting these key parameters, we were able to increase the specific activity by 2.6-fold and the wet cell weight by even 17-fold compared to shake flasks. Finally, we were able to verify our established protocol by transferring it to another experimenter. With that, our optimization strategy can serve as a template for the production of high titers of heterologously produced, active enzymes and might enable the availability of these catalysts for upscaling biocatalytic processes., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Introduction: COVID-19 causes a considerable degradation of pulmonary function to the point of an acute respiratory distress syndrome (ARDS). Over the course of the disease the gas exchange capability of the lung can get impaired to such an extent that extracorporeal membrane oxygenation (ECMO) is needed as a life-saving intervention. In patients COVID-19 as well as ECMO may cause severe coagulopathies which manifest themselves in micro and macro thrombosis. Previous studies established D-dimers as a marker for critical thrombosis of the ECMO system while on admission increased D-dimers are associated with a higher mortality in COIVD-19 patients. It is therefore crucial to determine if COVID-19 poses an increased risk of early thrombosis of the vital ECMO system., Methods: 40 patients who required ECMO support were enrolled in a retrospective analysis and assigned into 2 groups. The COVID group consist of 20 COVID-19 patients who required ECMO support (n = 20), whereas 20 ECMO patients without COVID-19 were assigned to the control group. D-dimers, fibrinogen, antithrombin III (AT III), lactate dehydrogenase (LDH) and platelet count were analysed using locally weighted scatterplot smoothing and MANOVAs., Results: The analysis of both groups shows highly significant differences in the dynamics of hemostasis. The increase in D-dimers that is associated with thrombosis of the ECMO systems occurs in COVID-19 patients around 2 days earlier (p = 2,8115 10-11) while fibrinogen is consumed steadily. In the control group fibrinogen levels increase rapidly after ten days with a plateau phase of around five days (p = 1,407 10-3) . Both groups experience a rapid increase in AT III after start of support by ECMO (p = 5,96 10-15). In the COVID group platelet count decreased from 210 giga/l to 130 giga/l within eight days, while in the same time span in the control group platelets decreased from 180 giga/l to 105 giga/l (p = 1,1 10-15). In both groups a marked increase in LDH beyond 5000 U/l occurs (p = 3,0865 10-15)., Conclusion: The early increase in D-dimers and decrease in fibrinogen suggests that COVID-19 patients bear an increased risk of early thrombosis of the ECMO system compared to other diseases treated with ECMO. Additionally, the control group shows signs of severe inflammation 10 days after the start of ECMO which were absent in COVID-19 patients.

To reduce carbon dioxide emissions, carbon-neutral fuels have recently gained renewed attention. Here we show the development and evaluation of process routes for the production of such a fuel, the cyclic acetal 4,5-dimethyl-1,3-dioxolane, from glucose via 2,3-butanediol. The selected process routes are based on the sequential use of microbes, enzymes and chemo-catalysts in order to exploit the full potential of the different catalyst systems through a tailor-made combination. The catalysts (microbes, enzymes, chemo-catalysts) and the reaction medium selected for each conversion step are key factors in the development of the respective production methods. The production of the intermediate 2,3-butanediol by combined microbial and enzyme catalysis is compared to the conventional microbial route from glucose in terms of specific energy demand and overall yield, with the conventional route remaining more efficient. In order to be competitive with current 2,3-butanediol production, the key performance indicator, enzyme stability to high aldehyde concentrations, needs to be increased. The target value for the enzyme stability is an acetaldehyde concentration of 600 mM, which is higher than the current maximum concentration (200 mM) by a factor of three., (© 2023. The Author(s).)

Background: Biobased 5-(hydroxymethyl)furfural (5-HMF) is an important platform that offers numerous possibilities for upgrading to a range of chemical, material and fuel products. One reaction of special interest is the carboligation of 5-HMF into C 12 compounds, including 5,5'-bis(hydroxymethyl)furoin (DHMF) and its subsequent oxidation to 5,5'-bis(hydroxymethyl)furil (BHMF), due to their potential applications as building blocks for polymers and hydrocarbon fuels., Objectives: This study was aimed at evaluating the use of whole cells of Escherichia coli carrying recombinant Pseudomonas fluorescens benzaldehyde lyase as biocatalysts for 5-HMF carboligation, recovery of the C 12 derivatives DHMF and BHMF, and testing the reactivity of the carbonyl groups for hydrazone formation for potential use as cross-linking agents in surface coatings. The effects of different parameters on the reaction were investigated to find the conditions for achieving high product yield and productivity., Results: The reaction with 5 g/L 5-HMF using 2 g CDW /L recombinant cells in 10% dimethyl carbonate, pH 8.0 at 30 °C resulted in DHMF yield of 81.7% (0.41 mol/mol) at 1 h, and BHMF yield of 96.7% (0.49 mol/mol) at 72 h reaction time. Fed-batch biotransformation generated a maximum DHMF concentration of 53.0 g/L (or 26.5 g DHMF/g cell catalyst) with productivity of 10.6 g/L . h, after five feeds of 20 g/L 5-HMF. Both DHMF and BHMF reacted with adipic acid dihydrazide to form hydrazone that was confirmed by Fourier-transform infrared spectroscopy and 1 H NMR., Conclusion: The study demonstrates the potential application of recombinant E. coli cells for cost-effective production of commercially relevant products., (© 2023. The Author(s).)

In this work, two multi-enzyme catalysed processes to access a 1,3,4-substituted tetrahydroisoquinoline (THIQ), using either purified enzymes or lyophilised whole-cell catalysts, are presented. A key focus was the first step in which the reduction of 3-hydroxybenzoic acid (3-OH-BZ) into 3-hydroxybenzaldehyde (3-OH-BA) was catalysed by a carboxylate reductase (CAR) enzyme. Incorporation of the CAR-catalysed step enables substituted benzoic acids as the aromatic components, which can potentially be obtained from renewable resources by microbial cell factories. In this reduction, the implementation of an efficient cofactor regeneration system of both ATP and NADPH was crucial. Two different recycling approaches, either using purified enzymes or lyophilised whole-cells, were established and compared. Both of them showed high conversions of the acid into 3-OH-BA (>80%). However, the whole-cell system showed superior performance because it allowed the combination of the first and second steps into a one-pot cascade with excellent HPLC yields (>99%, enantiomeric excess (ee) ≥ 95%) producing the intermediate 3-hydroxyphenylacetylcarbinol. Moreover, enhanced substrate loads could be achieved compared to the system employing only purified enzymes. The third and fourth steps were performed in a sequential mode to avoid cross-reactivities and the formation of several side products. Thus, (1 R ,2 S )-metaraminol could be formed with high HPLC yields (>90%, isomeric content (ic) ≥ 95%) applying either purified or whole-cell transaminases from Bacillus megaterium ( Bm TA) or Chromobacterium violaceum ( Cv 2025). Finally, the cyclisation step was performed using either a purified or lyophilised whole-cell norcoclaurine synthase variant from Thalictrum flavum (Δ Tf NCS-A79I), leading to the formation of the target THIQ product with high HPLC yields (>90%, ic > 90%). As many of the educts applied are from renewable resources and a complex product with three chiral centers can be gained by only four highly selective steps, a very step- and atom efficient approach to stereoisomerically pure THIQ is shown., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The design of biocatalytic reaction systems is highly complex owing to the dependency of the estimated kinetic parameters on the enzyme, the reaction conditions, and the modeling method. Consequently, reproducibility of enzymatic experiments and reusability of enzymatic data are challenging. We developed the XML-based markup language EnzymeML to enable storage and exchange of enzymatic data such as reaction conditions, the time course of the substrate and the product, kinetic parameters and the kinetic model, thus making enzymatic data findable, accessible, interoperable and reusable (FAIR). The feasibility and usefulness of the EnzymeML toolbox is demonstrated in six scenarios, for which data and metadata of different enzymatic reactions are collected and analyzed. EnzymeML serves as a seamless communication channel between experimental platforms, electronic lab notebooks, tools for modeling of enzyme kinetics, publication platforms and enzymatic reaction databases. EnzymeML is open and transparent, and invites the community to contribute. All documents and codes are freely available at https://enzymeml.org ., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Most combinations of chemo- and biocatalysis take place in aqueous media or require a solvent change with complex intermediate processing. Using enzymes in the same organic solvent as the chemocatalyst eliminates this need. Here, it was shown that a complete chemoenzymatic cascade to form dioxolanes could be carried out in a purely organic environment. The result, including downstream processing, was compared with a classical mode, shifting solvent. First, a two-step enzyme cascade starting from aliphatic aldehydes to chiral diols (3,4-hexanediol and 4,5-octanediol) was run either in an aqueous buffer or in the potentially biobased solvent cyclopentyl methyl ether. Subsequently, a ruthenium molecular catalyst enabled the conversion to dioxolanes [e. g., (4S,5S)-dipropyl-1,3-dioxolane]. Importantly, the total synthesis of this product was not only highly stereoselective but also based on the combination of biomass, CO 2 , and hydrogen, thus providing an important example of a bio-hybrid chemical., (© 2022 The Authors. ChemSusChem published by Wiley-VCH GmbH.)

Background: Family caregivers are faced with a variety of challenges and burdens. Recent online studies showed that these burdens have increased even more during the corona pandemic. Here we conducted a low-threshold written survey to assess psychological distress in highly burdened family caregivers during the corona pandemic in Germany., Method: Both quantitative and qualitative analyses were conducted within the context of a tiered counselling intervention study for highly burdened family caregivers (N = 165) to assess their experience of burden, anxieties, worries and desires regarding care., Results: A quarter of all caregivers (26%) reported high fear of becoming infected with SARS-CoV‑2 and 50% feared their care recipient could get infected. Almost half of all caregivers reported (47%) that corona had a high impact on the care situation and increased the burden of caregiving (51%). A loss of professional support and less time for themselves were reported most often. The desire for additional support was frequently expressed. The level of care, the age of the caregiver and the previous use of care services were found to be predictors of the psychological distress experienced by care recipients., Conclusion: The results illustrate an increased burden related to caregiving during the corona pandemic. In order to cope with this complex additional burden, there is an even higher need for support to improve care and social participation for both care recipients and caregivers during the pandemic., (© 2022. The Author(s).)

Smart hydrogels hold much potential for biocatalysis, not only for the immobilization of enzymes, but also for the control of enzyme activity. We investigated upper critical solution temperature-type poly N-acryloyl glycinamide (pNAGA) hydrogels as a smart matrix for the amine transaminase from Bacillus megaterium (BmTA). Physical entrapment of BmTA in pNAGA hydrogels results in high immobilization efficiency (>89 %) and high activity (97 %). The temperature-sensitiveness of pNAGA is preserved upon immobilization of BmTA and shows a gradual deswelling upon temperature reduction. While enzyme activity is mainly controlled by temperature, deactivation tended to be higher for immobilized BmTA (≈62-68 %) than for free BmTA (≈44 %), suggesting a deactivating effect due to deswelling of the pNAGA gel. Although the deactivation in response to hydrogel deswelling is not yet suitable for controlling enzyme activity sufficiently, it is nevertheless a good starting point for further optimization., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Objectives: Diisocyanates are a chemical group that are widely used at workplaces in many sectors. They are also potent skin- and respiratory sensitizers. Exposure to diisocyanates is a main cause of occupational asthma in the European Union. To reduce occupational exposure to diisocyanates and consequently the cases of diisocyanate-induced asthma, a restriction on diisocyanates was recently adopted under the REACH Regulation in the European Union., Methods: A comprehensive evaluation of the data on occupational exposure to the most important diisocyanates at workplaces was made and is reported here. The diisocyanates considered are methylene diphenyl diisocyanate (MDI), toluene diisocyanate (TDI), and hexamethylene diisocyanate (HDI), accounting for more than 95% of the market volume in the EU. The exposure assessment is based on data from Chemical Safety Reports (CSRs) of REACH Registration Dossiers, workplace air monitoring data from Germany, from the UK Health and Safety Executive (HSE), and literature data relevant for the EU, and the USA., Results: Occupational exposure to diisocyanates is particularly relevant in: (i) C.A.S.E. applications (Coatings, Adhesives, Sealants, Elastomers), (ii) production of polyurethanes (PUs) (e.g. slab-stock foam), (iii) handling of partly uncured PU products (e.g. cutting, demoulding, spray application of foam), and (iv) when diisocyanates/PUs are heated (e.g. hot lamination, foundry applications/casting forms). Ranking of the reported data on inhalation to diisocyanate exposure at workplaces (maximum values) leads to following order: (i) HDI and its oligomers in coatings, (ii) MDI in spray foam applications, (iii) TDI in manufacture of foam, (iv) TDI in manufacture of PUs and PU composite materials, (v) TDI in adhesives, (vi) MDI in adhesives, (vii) MDI in manufacture of PUs and PU composite materials, (viii) TDI in coatings, (ix) MDI in manufacture of foam, and (x) HDI in adhesives., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Occupational Hygiene Society.)

Dermal exposure is an important exposure route for occupational exposure and risk assessment. A fluorescence method has been developed to quantify occupational dermal exposure based on a visualization technique, using Tinopal SWN as a fluorescent tracer. The method was developed within the framework of a large experimental study, the SysDEA project. In SysDEA, dermal exposure was measured with different methods for 10 simulated exposure situations by sampling powder and liquid formulations containing Tinopal SWN on coveralls and patches and subsequently chemically analysing them. For the fluorescence method, photographs of exposed volunteers who performed the experiments were taken inside a room which consisted of an optimized arrangement of several UV irradiating tube light brackets, reflective and non-reflective backgrounds for maximum light diffusion and a camera. Image processing analysis software processed these photographs to obtain corresponding light intensity in terms of summed pixel values. To be able to estimate the amount of Tinopal SWN, 25% of the measured data from the SysDEA experiments were used to calibrate by correlating the summed pixel values from the photographs to actual measured exposure values using a second order regression model. For spraying both high and low viscosity liquids, showing uniformly distributed exposure patterns, strong Pearson correlation coefficients (R > 0.77) were observed. In contrast, the correlations were either inconsistently poor (R = -0.17 to 0.28 for pouring, rolling high viscosity liquid, manually handling objects immersed in low viscosity liquid and handling objects contaminated with powder), moderate (R = 0.73 for dumping of powder), or strong (R = 0.83 and 0.77 for rolling low viscosity liquid and manually handling objects immersed in high viscosity liquid). A model for spraying was developed and calibrated using 25% of the available experimental data for spraying and validated using the remaining 75%. Under given experimental conditions, the fluorescence method shows promising results and can be used for the quantification of dermal exposure for different body parts (excluding hands) for spraying-like scenarios that have a more uniform exposure pattern, but more research is needed for exposure scenarios with less uniform exposure patterns. For the estimation of exposure levels, the surface loading limit should be lower than 1.5░µg/cm2 (a lower limit could not be quantified based on experiments conducted in this study) on a large surface, like a coverall, which should be ideally perpendicular to the camera., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Occupational Hygiene Society.)

Pyridoxal 5'-phosphate (PLP)-dependent enzymes are a versatile class of biocatalysts and feature a variety of industrial applications. However, PLP is light sensitive and can cause inactivation of enzymes in certain light conditions. As most of the PLP-dependent enzymes are usually not handled in dark conditions, we evaluated the effect of visible light on the activity of PLP-dependent enzymes during production as well as transformation. We tested four amine transaminases, from Chromobacterium violaceum , Bacillus megaterium , Vibrio fluvialis and a variant from Arthrobacter species as well as two lysine decarboxylases, from Selenomonas ruminantium and the LDCc from Escherichia coli . It appeared that five of these six enzymes suffered from a significant decrease in activity by up to 90 % when handled in laboratory light conditions. Surprisingly, only the amine transaminase variant from Arthrobacter species appeared to be unaffected by light exposure and even showed an activation to 150 % relative activity over the course of 6 h regardless of the light conditions., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. ChemCatChem published by Wiley-VCH GmbH.)

Accessing aldehydes from carboxylate moieties is often a challenging task. In this regard, carboxylate reductases (CARs) are promising catalysts provided by nature that are able to accomplish this task in just one step, avoiding over-reduction to the alcohol product. However, the heterologous expression of CARs can be quite difficult due to the excessive formation of insoluble protein, thus hindering further characterization and application of the enzyme. Here, the heterologous production of the carboxylate reductase from Nocardia otitidiscaviarum (NoCAR) was optimized by a combination of i) optimized cultivation conditions, ii) post-translational modification with a phosphopantetheinyl transferase and iii) selection of an appropriate expression strain. Especially, the selection of Escherichia coli tuner cells as host had a strong effect on the final 110-fold increase in the specific activity of NoCAR. This highly active NoCAR was used to reduce sodium benzoate to benzaldehyde, and it was successfully assembled with an in vitro regeneration of ATP and NADPH, being capable of reducing about 30 mM sodium benzoate with high selectivity in only 2 h of reaction., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

In nature, enzymes conventionally operate under aqueous conditions. Because of this, aqueous buffers are often the choice for reaction media when enzymes are applied in chemical synthesis. However, to meet the demands of an industrial application, due to the poor water solubility of many industrially relevant compounds, an aqueous reaction system will often not be able to provide sufficient substrate loadings. A switch to a non-aqueous solvent system can provide a solution, which is already common for lipases, but more challenging for biocatalysts from other enzyme classes. The choices in solvent types and systems, however, can be overwhelming. Furthermore, some engineering of the protein structure of biocatalyst formulation is required. In this review, a guide for those working with biocatalysts, who look for a way to increase their reaction productivity, is presented. Examples reported clearly show that bulk water is not necessarily required for biocatalytic reactions and that clever solvent systems design can support increased product concentrations thereby decreasing waste formation. Additionally, under these conditions, enzymes can also be combined in cascades with other, water-sensitive, chemical catalysts. Finally, we show that the application of non-aqueous solvents in biocatalysis can actually lead to more sustainable processes. At the hand of flowcharts, following simple questions, one can quickly find what solvent systems are viable., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Exposure information is a critical element in various regulatory and non-regulatory frameworks in Europe and elsewhere. Exposure science supports to ensure safe environments, reduce human health risks, and foster a sustainable future. However, increasing diversity in regulations and the lack of a professional identity as exposure scientists currently hamper developing the field and uptake into European policy. In response, we discuss trends, and identify three key needs for advancing and harmonizing exposure science and its application in Europe. We provide overarching building blocks and define six long-term activities to address the identified key needs, and to iteratively improve guidelines, tools, data, and education. More specifically, we propose creating European networks to maximize synergies with adjacent fields and identify funding opportunities, building common exposure assessment approaches across regulations, providing tiered education and training programmes, developing an aligned and integrated exposure assessment framework, offering best practices guidance, and launching an exposure information exchange platform. Dedicated working groups will further specify these activities in a consistent action plan. Together, these elements form the foundation for establishing goals and an action roadmap for successfully developing and implementing a 'European Exposure Science Strategy' 2020-2030, which is aligned with advances in science and technology.

There is a principal need for more precise methodology with regard to the determination of occupational dermal exposure. The goal of the Systematic analysis of Dermal Exposure to hazardous chemical Agents at the workplace project was therefore to generate scientific knowledge to improve and standardize measurement methods for dermal exposure to chemicals at the workplace. In addition, the comparability of different measurement methods was investigated. Different methods (body sampling by means of coveralls and patches, hand sampling by means of gloves and washing, and head sampling by means of headbands and wiping) were compared. Volunteers repeatedly performed a selection of tasks under standardized conditions in test chambers to increase the reproducibility and decrease variability. The selected tasks were pouring, rolling, spraying, and handling of objects immersed in liquid formulations, as well as dumping and handling objects contaminated with powder. For the chemical analysis, the surrogate test substance Tinopal SWN was analyzed by means of a high-performance liquid chromatographic method using a fluorescence detector. Tinopal SWN was either applied as a solid product in its pure form, or as a low and high viscosity liquid containing Tinopal SWN in dissolved form. To compare the sampling methods with patches and coveralls, the exposure values as measured on the patches were extrapolated to the surface areas of the respective parts of the coverall. Based on this extrapolation approach, using the patch method resulted in somewhat higher exposure values compared to using a coverall for all exposure situations, but the differences were only statistically significant in case of the liquid exposure situations. Using gloves resulted in significantly higher exposure values compared to hand wash for handling immersed objects, rolling, and handling contaminated objects, and slightly higher (not significant) exposure values during pouring and spraying. In the same context, applying wipe sampling resulted in higher exposure values than using a headband, which was at least partly due to extrapolation of the wipe results to the surface area of the headband. No 'golden standard' with regard to a preferred measurement method for dermal exposure could be identified from the methods as investigated in the current study., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Occupational Hygiene Society.)

Multi-step biocatalytic reactions have gained increasing importance in recent years because the combination of different enzymes enables the synthesis of a broad variety of industrially relevant products. However, the more enzymes combined, the more crucial it is to avoid cross-reactivity in these cascade reactions and thus achieve high product yields and high purities. The selective control of enzyme activity, i.e., remote on-/off-switching of enzymes, might be a suitable tool to avoid the formation of unwanted by-products in multi-enzyme reactions. This review compiles a range of methods that are known to modulate enzyme activity in a stimulus-responsive manner. It focuses predominantly on in vitro systems and is subdivided into reversible and irreversible enzyme activity control. Furthermore, a discussion section provides indications as to which factors should be considered when designing and choosing activity control systems for biocatalysis. Finally, an outlook is given regarding the future prospects of the field.

Alcohol dehydrogenases are of high interest for stereoselective syntheses of chiral building blocks such as 1,2-diols. As this class of enzymes requires nicotinamide cofactors, their application in biotechnological synthesis reactions is economically only feasible with appropriate cofactor regeneration. Therefore, a co-substrate is oxidized to the respective co-product that accumulates in equal concentration to the desired target product. Co-product removal during the course of the reaction shifts the reaction towards formation of the target product and minimizes undesired side effects. Here we describe an atom efficient enzymatic cofactor regeneration system where the co-product of the ADH is recycled as a substrate in another reaction set. A 2-step enzymatic cascade consisting of a thiamine diphosphate (ThDP)-dependent carboligase and an alcohol dehydrogenase is presented here as a model reaction. In the first step benzaldehyde and acetaldehyde react to a chiral 2-hydroxy ketone, which is subsequently reduced by to a 1,2-diol. By choice of an appropriate co-substrate (here: benzyl alcohol) for the cofactor regeneration in the alcohol dehydrogenases (ADH)-catalyzed step, the co-product (here: benzaldehyde) can be used as a substrate for the carboligation step. Even without any addition of benzaldehyde in the first reaction step, this cascade design yielded 1,2-diol concentrations of >100 mM with optical purities ( ee , de ) of up to 99%. Moreover, this approach overcomes the low benzaldehyde solubility in aqueous systems and optimizes the atom economy of the reaction by reduced waste production. The example presented here for the 2-step recycling cascade of (1 R ,2 R )-1-phenylpropane-1,2-diol can be applied for any set of enzymes, where the co-products of one process step serve as substrates for a coupled reaction.

The economically efficient utilization of NAD(P)H-dependent enzymes requires the regeneration of consumed reduction equivalents. Classically, this is done by substrate supplementation, and if necessary by addition of one or more enzymes. The simplest method thereof is whole cell NADPH regeneration. In this context we now present an easy-to-apply whole cell cofactor regeneration approach, which can especially be used in screening applications. Simply by applying citrate to a buffer or directly using citrate/-phosphate buffer NADPH can be regenerated by native enzymes of the TCA cycle, practically present in all aerobic living organisms. Apart from viable-culturable cells, this regeneration approach can also be applied with lyophilized cells and even crude cell extracts. This is exemplarily shown for the synthesis of 1-phenylethanol from acetophenone with several oxidoreductases. The mechanism of NADPH regeneration by TCA cycle enzymes was further investigated by a transient isotopic labeling experiment feeding [1,5- 13 C]citrate. This revealed that the regeneration mechanism can further be optimized by genetic modification of two competing internal citrate metabolism pathways, the glyoxylate shunt, and the glutamate dehydrogenase.

In the practice of motor skills in general, errors in the execution of movements may go unnoticed when a human instructor is not available. In this case, a computer system or robotic device able to detect movement errors and propose corrections would be of great help. This paper addresses the problem of how to detect such execution errors and how to provide feedback to the human to correct his/her motor skill using a general, principled methodology based on imitation learning. The core idea is to compare the observed skill with a probabilistic model learned from expert demonstrations. The intensity of the feedback is regulated by the likelihood of the model given the observed skill. Based on demonstrations, our system can, for example, detect errors in the writing of characters with multiple strokes. Moreover, by using a haptic device, the Haption Virtuose 6D, we demonstrate a method to generate haptic feedback based on a distribution over trajectories, which could be used as an auxiliary means of communication between an instructor and an apprentice. Additionally, given a performance measurement, the haptic device can help the human discover and perform better movements to solve a given task. In this case, the human first tries a few times to solve the task without assistance. Our framework, in turn, uses a reinforcement learning algorithm to compute haptic feedback, which guides the human toward better solutions.

Peroxygenases offer attractive means to address challenges in selective oxyfunctionalisation chemistry. Despite their attractiveness, the application of peroxygenases in synthetic chemistry remains challenging due to their facile inactivation by the stoichiometric oxidant (H 2 O 2 ). Often atom inefficient peroxide generation systems are required, which show little potential for large scale implementation. Here we show that visible light-driven, catalytic water oxidation can be used for in situ generation of H 2 O 2 from water, rendering the peroxygenase catalytically active. In this way the stereoselective oxyfunctionalisation of hydrocarbons can be achieved by simply using the catalytic system, water and visible light., Competing Interests: Competing Financial Interest The authors declare no competing financial interests.

Chemoenzymatic and enzymatic cascade reactions enable the synthesis of complex stereocomplementary 1,3,4-trisubstituted tetrahydroisoquinolines (THIQs) with three chiral centers in a step-efficient and selective manner without intermediate purification. The cascade employs inexpensive substrates (3-hydroxybenzaldehyde and pyruvate), and involves a carboligation step, a subsequent transamination, and finally a Pictet-Spengler reaction with a carbonyl cosubstrate. Appropriate selection of the carboligase and transaminase enzymes enabled the biocatalytic formation of (1R,2S)-metaraminol. Subsequent cyclization catalyzed either enzymatically by a norcoclaurine synthase or chemically by phosphate resulted in opposite stereoselectivities in the products at the C1 position, thus providing access to both orientations of the THIQ C1 substituent. This highlights the importance of selecting from both chemo- and biocatalysts for optimal results., (© 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The asymmetric reductive amination of ketones represents an elegant and convenient way to obtain chiral amines. Recently, several examples have been reported in which isolated imine reductases (IREDs) have been used for this type of reaction leading to promising results. In this work we focus on the applicability of whole cell biocatalysts (recombinant E. coli cells heterologously overexpressing an IRED) to simplify its preparation and to cut on catalyst production costs. Thirteen IREDs were screened towards six different ketones, using methylamine as amine donor. The targeted amines were formed with low to very high conversions and good to excellent stereoselectivity, depending on both, the ketone amine pair used for the reaction, as well as the applied IRED. It was further proven that a micro-aqueous reaction environment was applicable showing similar activity trends for the various reductive aminations but predominantly reduced conversions. A preparative scale experiment in a buffered environment was conducted leading to 93% conversion and 99% stereoselectivity of the product (1S,3R)-N,3-dimethylcyclohexylamine. As the whole cells intrinsic glucose dehydrogenase could be used for cofactor regeneration, no enzyme addition had to be applied, making this biocatalyst formulation particularly cost efficient., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Recent advances in biocatalysis have strongly boosted its recognition as a valuable addition to traditional chemical synthesis routes. As for any catalytic process, catalyst's costs and stabilities are of highest relevance for the economic application in chemical manufacturing. Employing biocatalysts as whole cells circumvents the need of cell lysis and enzyme purification and hence strongly cuts on cost. At the same time, residual cell wall components can shield the entrapped enzyme from potentially harmful surroundings and aid to enable applications far from natural enzymatic environments. Further advantages are the close proximity of reactants and catalysts as well as the inherent presence of expensive cofactors. Here, we review and comment on benefits and recent advances in whole cell biocatalysis., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The development of novel enzymes for biocatalytic processes requires knowledge on substrate profile and selectivity; this can be derived from databases and from publications. Often, these sources lack time-course data for the substrate or product, and an unambiguous link between experiment and enzyme sequence. The lack of integrated, original data hampers the comprehensive analysis of enzyme kinetics and the evaluation of sequence-function relationships. In order to accelerate enzyme engineering, BioCatNet integrates protein sequence, protein structure, and experimental data for a given enzyme family. BioCatNet explicitly assigns the enzyme sequence to the experimental data, which consists of information on reaction conditions and time-course data. BioCatNet facilitates the consistent documentation of reaction conditions, the archiving of time-course data, and the efficient exchange of experimental data among collaborators. Data integration is demonstrated for three case studies by using the TEED (Thiamine diphosphate-dependent Enzymes Engineering Database)., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The catalytic asymmetric synthesis of chiral 2-hydroxy ketones by using different thiamine diphosphate dependent enzymes, namely benzaldehyde lyase from Pseudomonas fluorescens (PfBAL), a variant of benzoylformate decarboxylase from Pseudomonas putida (PpBFD-L461A), branched-chain 2-keto acid decarboxylase from Lactococcus lactis (LlKdcA) and a variant of pyruvate decarboxylase from Acetobacter pasteurianus (ApPDC-E469G), was studied. Starting with the same set of substrates, substituted benzaldehydes in combination with different aliphatic aldehydes, PfBAL and PpBFD-L461A selectively deliver the (R)- and (S)-2-hydroxy-propiophenone derivatives, respectively. The (R)- and (S)-phenylacetylcarbinol (1-hydroxy-1-phenylacetone) derivatives are accessible in a similar way using LlKdcA and ApPDC-E469G, respectively. In many cases excellent stereochemical purities (>98 % enantiomeric excess) could be achieved. Hence, the regio- and stereochemistry of the product in the asymmetric aliphatic-aromatic cross-benzoin reaction can be controlled solely by choice of the appropriate enzyme or enzyme variant., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Context: Persistent low back pain (LBP) is a common complaint among women during and after pregnancy, and its effects on quality of life can be disabling., Objective: To evaluate the effectiveness of osteopathic manipulative therapy (OMTh; manipulative care provided by foreign-trained osteopaths) in women with persistent LBP and functional disability after childbirth., Methods: A pragmatic randomized controlled trial was conducted among a sample of women with a history of pregnancy-related LBP for at least 3 months after delivery. Participants were identified from the general population in Germany. By means of external randomization, women were allocated to an OMTh group and a waitlist control group. Osteopathic manipulative therapy was provided 4 times at intervals of 2 weeks, with a follow-up after 12 weeks. The OMTh was tailored to each participant and based on osteopathic principles. The participants allocated to the control group did not receive OMTh during the 8-week study; rather, they were put on a waiting list to receive OMTh on completion of the study. Further, they were not allowed to receive any additional treatment (ie, medication, physical therapy, or other sources of pain relief) during the study period. The main outcome measures were pain intensity as measured by a visual analog scale and the effect of LBP on daily activities as assessed by the Oswestry Disability Index (ODI)., Results: A total of 80 women aged between 23 and 42 years (mean [SD], 33.6 [4.5] years) were included in the study, with 40 in the OMTh group and 40 in the control group. Pain intensity decreased in the OMTh group from 7.3 to 2.0 (95% CI, 4.8-5.9; P<.001) and in the control group from 7.0 to 6.5 (95% CI, -0.2 to -0.9; P=.005). The between-group comparison of changes revealed a statistically significant improvement in pain intensity in the OMTh group (between-group difference of means, 4.8; 95% CI, 4.1-5.4; P<.001) and level of disability (between-group difference of means, 10.6; 95% CI, 9.9-13.2; P<.005). The follow-up assessment in the OMTh group (n=38) showed further improvement., Conclusion: During 8 weeks, OMTh applied 4 times led to clinically relevant positive changes in pain intensity and functional disability in women with postpartum LBP. Further studies that include prolonged follow-up periods are warranted. (German Clinical Trials Register: DRKS00006280.).

The stereoselective production of (R)- or (S)-2-butanol is highly challenging. A potent synthesis strategy is the biocatalytic asymmetric reduction of 2-butanone applying alcohol dehydrogenases. However, due to a time-dependent racemisation process, high stereoselectivity is only obtained at incomplete conversion after short reaction times. Here, we present a solution to this problem: by using a continuous process, high biocatalytic selectivity can be achieved while racemisation is suppressed successfully. Furthermore, high conversion was achieved by applying recombinant, lyophilised E. coli cells hosting Lactobacillus brevis alcohol dehydrogenase in a micro-aqueous solvent-free continuous reaction system. The optimisation of residence time (τ) and 2-butanone concentration boosted both conversion (>99%) and enantiomeric excess (ee) of (R)-2-butanol (>96%). When a residence time of only τ=3.1 min was applied, productivity was extraordinary with a space-time yield of 2278±29g/(L×d), thus exceeding the highest values reported to date by a factor of more than eight. The use of E. coli cells overexpressing an ADH of complementary stereoselectivity yielded a synthesis strategy for (S)-2-butanol with an excellent ee (>98%). Although conversion was only moderate (up to 46%), excellent space-time yields of up to 461g/(L×d) were achieved. The investigated concept represents a synthesis strategy that can also be applied to other biocatalytic processes where racemisation poses a challenge., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Thiamine diphosphate dependent enzymes are well known for catalyzing the asymmetric synthesis of chiral α-hydroxy ketones from simple prochiral substrates. The steric and chemical properties of the enzyme active site define the product spectrum. Enzymes catalyzing the carboligation of aromatic aldehydes to (S)-benzoins have not so far been identified. We were able to close this gap by constructing a chimeric enzyme, which catalyzes the synthesis of various (S)-benzoins with excellent enantiomeric excess (>99%) and very good conversion., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Thiamine diphosphate-dependent enzymes are broadly distributed in all organisms, and they catalyse a broad range of C-C bond forming and breaking reactions. Enzymes belonging to the structural families of decarboxylases and transketolases have been particularly well investigated concerning their substrate range, mechanism of stereoselective carboligation and carbolyase reaction. Both structurally different enzyme families differ also in stereoselectivity: enzymes from the decarboxylase family are predominantly R-selective, whereas those from the transketolase family are S-selective. In recent years a key focus of our studies has been on stereoselective benzoin condensation-like 1,2-additions. Meanwhile, several S-selective variants of pyruvate decarboxylase, benzoylformate decarboxylase and 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC) synthase as well as R-selective transketolase variants were created that allow access to a broad range of enantiocomplementary α-hydroxyketones and α,α'-dihydroxyketones. This review covers recent studies and the mechanistic understanding of stereoselective C-C bond forming thiamine diphosphate-dependent enzymes, which has been guided by structure-function analyses based on mutagenesis studies and from influences of different substrates and organic co-solvents on stereoselectivity., (© 2013 FEBS.)

Stereoselective reduction towards pharmaceutically potent products with multi-chiral centers is an ongoing hot topic, but up to now catalysts for reductions of bulky aromatic substrates are rare. The NADPH-dependent alcohol dehydrogenase from Ralstonia sp. (RADH) is an exception as it prefers sterically demanding substrates. Recent studies with this enzyme indicated outstanding potential for the reduction of various alpha-hydroxy ketones, but were performed with crude cell extract, which hampered its detailed characterization. We have established a procedure for the purification and storage of RADH and found a significantly stabilizing effect by addition of CaCl(2). Detailed analysis of the pH-dependent activity and stability yielded a broad pH-optimum (pH 6-9.5) for the reduction reaction and a sharp optimum of pH 10-11.5 for the oxidation reaction. The enzyme exhibits highest stability at pH 5.5-8 and 8-15°C; nevertheless, biotransformations can also be carried out at 25°C (half-life 80 h). Under optimized reaction parameters a thorough study of the substrate range of RADH including the reduction of different aldehydes and ketones and the oxidation of a broad range of alcohols was conducted. In contrast to most other known alcohol dehydrogenases, RADH clearly prefers aromatic and cyclic aliphatic compounds, which makes this enzyme unique for conversion of space demanding substrates. Further, reductions are catalyzed with extremely high stereoselectivity (>99% enantio- and diastereomeric excess). In order to identify appropriate substrate and cofactor concentrations for biotransformations, kinetic parameters were determined for NADP(H) and selected substrates. Among these, we studied the reduction of both enantiomers of 2-hydroxypropiophenone in more detail., (Copyright © 2013 Wiley Periodicals, Inc.)

We report the first rationally designed (S)-selective MenD from E. coli for the synthesis of functionalized α-hydroxy ketones. By mutation of two amino acids in the active site stereoselectivity of the (R)-selective EcMenD (ee > 93%) was inverted giving access to (S)-5-hydroxy-4-oxo-5-phenylpentanoate derivatives with stereoselectivities up to 97% ee.

The asymmetric mixed carboligation of aldehydes with thiamine diphosphate (ThDP)-dependent enzymes is an excellent example where activity as well as changes in chemo- and stereoselectivity can be followed sensitively. To elucidate the influence of organic additives in enzymatic carboligation reactions of mixed 2-hydroxy ketones, we present a comparative study of six ThDP-dependent enzymes in 13 water-miscible organic solvents under equivalent reaction conditions. The influence of the additives on the stereoselectivity is most pronounced and follows a general trend. If the enzyme stereoselectivity in aqueous buffer is already >99.9% ee, none of the solvents reduces this high selectivity. In contrast, both stereoselectivity and chemoselectivity are strongly influenced if the enzyme is rather unselective in aqueous buffer. For the S-selective enzyme with the largest active site, we were able to prove a general correlation of the solvent-excluded volume of the additives with the effect on selectivity changes: the smaller the organic solvent molecule, the higher the impact of this additive. Further, a correlation to log P of the additives on selectivity was detected if two additives have almost the same solvent-excluded volume. The observed results are discussed in terms of structural, biochemical and energetic effects. This work demonstrates the potential of medium engineering as a powerful additional tool for varying enzyme selectivity and thus engineering the product range of biotransformations. It further demonstrates that the use of cosolvents should be carefully planned, as the solvents may compete with the substrate(s) for binding sites in the enzyme active site.

A rapid TTC-based screening assay for ω-transaminases was developed to determine the conversion of substrates with a 2-hydroxy ketone motif. Oxidation of the compounds in the presence of 2,3,5-triphenyltetrazolium chloride (TTC) results in a reduction of the colourless TTC to a red-coloured 1,3,5-triphenylformazan. The enzymatic reductive amination of a wide range of various aliphatic, aliphatic-aromatic and aromatic-aromatic 2-hydroxy ketones can be determined by the decrease of the red colouration due to substrate consumption. The conversion can be quantified spectrophotometrically at 510 nm based on reactions, e.g. with crude cell extracts in 96-well plates. Since the assay is independent of the choice of diverse amine donors a panel of ω-transaminases was screened to detect conversion of 2-hydroxy ketones with three different amine donors: l-alanine, (S)-α-methylbenzylamine and benzylamine. The results could be validated using HPLC and GC analyses, showing a deviation of only 5-10%. Using this approach enzymes were identified demonstrating high conversions of acetoin and phenylacetylcarbinol to the corresponding amines. Among these enzymes three novel wild-type ω-transaminases have been identified., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Silica and silicates are widely used in nanomedicine with applications as diverse as medical device coatings to replacement materials in tissue engineering. Although much is known about silica and its synthesis, relatively few biomedical scientists fully appreciate the link that exists between its formulation and its resultant structure and function. This article attempts to provide insight into relevant issues in that context, as well as highlighting their importance in the material's eventual surface patterning/activation with alkoxy- and organo-silanes. The use of aminosilanes in that context is discussed at some length to permit an understanding of the specific variables that are important in the reproducible and robust aminoactivation of surfaces using such molecules. Recent investigative work is cited to underline the fact that although aminosilanization is a historically accepted mechanism for surface activation, there is still much to be explained about how and why the process works in the way it does. In the last section of this article, there is a detailed discussion of two classical approaches for the use of aminosilanized materials in the covalent immobilization of bioligands, amino-aldehyde and amino-carboxyl coupling. In the former case, the use of the homobifunctional coupler glutaraldehyde is explored, and in the latter, carbodiimides. Although these chemistries have long been employed in bioconjugations, it is apparent that there are still variables to be explored in the processes (as witnessed by continuing investigations into the chemistries concerned). Aspects regarding optimization, standardization and reproducibility of the fabrication of amino functionalized surfaces are discussed in detail and illustrated with practical examples to aid the reader in their own studies, in terms of considerations to be taken into account when producing such materials. Finally, the article attempts to remind readers that although the chemistry and materials involved are 'old hat', there is still much to be learnt about the methods involved. The article also reminds readers that although many highly specific and costly conjugation chemistries now exist for bioligands, there still remains a place for these relatively simple and cost-effective approaches in bioligand conjugate fabrication.

The bacterial sulfane dehydrogenase SoxCD is a distantly related member of the sulfite oxidase (SO) enzyme family that is proposed to oxidize protein-bound sulfide (sulfane) of SoxY as part of a multienzyme mechanism of thiosulfate metabolism. This study characterized the molybdenum cofactor of SoxCD1, comprising the catalytic molybdopterin subunit SoxC and the truncated c-type cytochrome subunit SoxD1. Electron paramagnetic resonance spectroscopy of the Mo(V) intermediate generated by dithionite reduction revealed low- and high-pH species with g and A((95,97)Mo) matrices nearly identical to those of SO, indicating a similar pentacoordinate active site in SoxCD1. However, no sulfite-induced reduction to Mo(V) was detected, nor could a strongly coupled (1)H signal or a phosphate-inhibited species be generated. This indicates that the outer coordination sphere controls substrate binding in SoxCD, permitting access only to protein-bound sulfur via the C-terminal tail of SoxY.

A new way to study the electrochemical properties of proteins by coupling front-face fluorescence spectroscopy with an optically transparent thin-layer electrochemical cell is presented. First, the approach was examined on the basis of the redox-dependent conformational changes in tryptophans in cytochrome c, and its redox potential was successfully determined. Second, an electrochemically induced fluorescence analysis of periplasmic thiol-disulfide oxidoreductases SoxS and SoxW was performed. SoxS is essential for maintaining chemotrophic sulfur oxidation of Paracoccus pantotrophus active in vivo, while SoxW is not essential. According to the potentiometric redox titration of tryptophan fluorescence, the midpoint potential of SoxS was -342 ± 8 mV versus the standard hydrogen electrode (SHE') and that of SoxW was -256 ± 10 mV versus the SHE'. The fluorescence properties of the thioredoxins are presented and discussed together with the intrinsic fluorescence contribution of the tyrosines.

It has been shown that excessive white light exposure during retinal microsurgery can induce retinal damage. To address this problem, one can illuminate the retina with a device that alternates between white, and less damaging limited-spectrum light. The surgeon is then presented with a fully colored video by recoloring the limited-spectrum light frames, using information from the white-light frames. To obtain accurately colored images, while reducing phototoxicity, we have developed a novel algorithm that monitors the quality of the recolored images and determines when white light may be substituted by limited-spectrum light. We show qualitatively and quantitatively that our system can provide reliable images using a significantly smaller light dose as compared to other state-of-the-art coloring schemes.

The periplasmic thiol-disulfide oxidoreductase SoxS is beneficial for the sulfur-oxidizing (Sox) phenotype of the facultative chemotrophic bacterium Paracoccus pantotrophus and is not part of the Sox enzyme system. SoxS combines features of thioredoxins, glutaredoxins and the thiol-disulfide oxidoreductases of the Dsb family in structure, target specificity and reaction. The structure of SoxS was solved in oxidized and reduced forms at 2.1 and 1.9 A resolution, respectively. SoxS revealed high structural homology to typical cytoplasmic bacterial thioredoxins. In contrast, SoxS contained the active-site motif Pro-Gly-Cys-Leu-Tyr-Cys that is not present in other thioredoxins. Interestingly, the sequence of this motif is closely related to the Pro-Gly-Cys-Pro-Tyr-Cys sequence of some glutaredoxins and to the Pro-Xaa-Cys-Xaa-Tyr-Cys sequences of some members of the DsbC and DsbG subfamilies of thiol-disulfide oxidoreductases. Furthermore, the proposed substrate of SoxS, the interprotein disulfide of SoxY, Cys110(Y)-Cys110(Y), is structurally similar to oxidized glutathione. However, SoxS is proposed to specifically reduce the interprotein disulfide between two SoxY subunits, releasing a heterodimeric SoxYZ as an active part of the sulfur-oxidation cycle.

1,1'-Difunctionalised ferrocene derivatives have been studied, which contain groups suitable for chemisorption on gold substrates, namely -NC, -PR(2) as well as a range of sulfur-containing units like -NCS, -SR, and thienyl. Thin films on gold have been fabricated from solution with most of these adsorbate species. Film thickness, composition and structure were investigated primarily by X-ray photoelectron and near-edge X-ray absorption fine-structure spectroscopy. The quality of self-assembled monolayers fabricated from 1,1'-diisocyanoferrocene (1) and 1,1'-diisothiocyanatoferrocene (2) turned out to be superior to that of films based on the other adsorbate species investigated. In addition to the surface coordination behaviour of 1 towards gold substrates, relevant aspects of the molecular coordination chemistry of 1 have also been addressed, including the synthesis and characterisation of [(mu-1){Cr(CO)(5)}(2)], [Ag(2)(mu-1)(2)](NO(3))(2) x H(2)O and [(mu-1)(AuCl)(2)]. The crystal structure of the gold complex is governed by aurophilic interactions and can be taken as a model for the arrangement of 1 in self-assembled monolayers on gold.

The two chemically equivalent functional groups of 1,1'-diisocyanoferrocene each undergo a different specific reaction with the gold(I) acetylide [Au(C=C-Fc)], viz. ordinary coordination and extraordinary 1,1-insertion.

The heterodimeric hemoprotein SoxXA, essential for lithotrophic sulfur oxidation of the aerobic bacterium Paracoccus pantotrophus, was examined by a combination of spectroelectrochemistry and EPR spectroscopy. The EPR spectra for SoxXA showed contributions from three paramagnetic heme iron centers. One highly anisotropic low-spin (HALS) species (gmax = 3.45) and two "standard" cytochrome-like low-spin heme species with closely spaced g-tensor values were identified, LS1 (gz = 2.54, gy = 2.30, and gx = 1.87) and LS2 (gz = 2.43, gy = 2.26, and gx = 1.90). The crystal structure of SoxXA from P. pantotrophus confirmed the presence of three heme groups, one of which (heme 3) has a His/Met axial coordination and is located on the SoxX subunit [Dambe et al. (2005) J. Struct. Biol. 152, 229-234]. This heme was assigned to the HALS species in the EPR spectra of the isolated SoxX subunit. The LS1 and LS2 species were associated with heme 1 and heme 2 located on the SoxA subunit, both of which have EPR parameters characteristic for an axial His/thiolate coordination. Using thin-layer spectroelectrochemistry the midpoint potentials of heme 3 and heme 2 were determined: Em3 = +189 +/- 15 mV and Em2 = -432 +/- 15 mV (vs NHE, pH 7.0). Heme 1 was not reducible even with 20 mM titanium(III) citrate. The Em2 midpoint potential turned out to be pH dependent. It is proposed that heme 2 participates in the catalysis and that the cysteine persulfide ligation leads to the unusually low redox potential (-436 mV). The pH dependence of its redox potential may be due to (de)protonation of the Arg247 residue located in the active site.

The sulfur-oxidizing enzyme system (Sox) of the chemotroph Paracoccus pantotrophus is composed of several proteins, which together oxidize hydrogen sulfide, sulfur, thiosulfate or sulfite and transfers the gained electrons to the respiratory chain. The hetero-dimeric cytochrome c complex SoxXA functions as heme enzyme and links covalently the sulfur substrate to the thiol of the cysteine-138 residue of the SoxY protein of the SoxYZ complex. Here, we report the crystal structure of the c-type cytochrome complex SoxXA. The structure could be solved by molecular replacement and refined to a resolution of 1.9A identifying the axial heme-iron coordination involving an unusual Cys-251 thiolate of heme2. Distance measurements between the three heme groups provide deeper insight into the electron transport inside SoxXA and merge in a better understanding of the initial step of the aerobic sulfur oxidation process in chemotrophic bacteria.

Recent biochemical and genomic data differentiate the sulfur oxidation pathway of Archaea from those of Bacteria. From these data it is evident that members of the Alphaproteobacteria harbor the complete sulfur-oxidizing Sox enzyme system, whereas members of the beta and gamma subclass and the Chlorobiaceae contain sox gene clusters that lack the genes encoding sulfur dehydrogenase. This indicates a different pathway for oxidation of sulfur to sulfate. Acidophilic bacteria oxidize sulfur by a system different from the Sox enzyme system, as do chemotrophic endosymbiotic bacteria.

Sulfur dehydrogenase, Sox(CD)(2), is an essential part of the sulfur-oxidizing enzyme system of the chemotrophic bacterium Paracoccus pantotrophus. Sox(CD)(2) is a alpha(2)beta(2) complex composed of the molybdoprotein SoxC (43 442 Da) and the hybrid diheme c-type cytochrome SoxD (37 637 Da). Sox(CD)(2) catalyzes the oxidation of protein-bound sulfur to sulfate with a unique six-electron transfer. Amino acid sequence analysis identified the heme-1 domain of SoxD proteins to be specific for sulfur dehydrogenases and to contain a novel ProCysMetXaaAspCys motif, while the heme-2 domain is related to various cytochromes c(2). Purification of sulfur dehydrogenase without protease inhibitor yielded a dimeric SoxCD(1) complex consisting of SoxC and SoxD(1) of 30 kDa, which contained only the heme-1 domain. The heme-2 domain was isolated as a new cytochrome SoxD(2) of about 13 kDa. Both hemes of SoxD in Sox(CD)(2) are redox-active with midpoint potentials at E(m)1 = 218 +/- 10 mV and E(m)2 = 268 +/- 10 mV, while SoxCD(1) and SoxD(2) both exhibit a midpoint potential of E(m) = 278 +/- 10 mV. Electrochemically induced FTIR difference spectra of Sox(CD)(2), SoxCD(1), and SoxD(2) were distinct. A carboxy group is protonated upon reduction of the SoxD(1) heme but not for SoxD(2). The specific activity of SoxCD(1) and Sox(CD)(2) was identical as was the yield of electrons with thiosulfate in the reconstituted Sox enzyme system. To examine the physiological significance of the heme-2 domain, a mutant was constructed that was deleted for the heme-2 domain, which produced SoxCD(1) and transferred electrons from thiosulfate to oxygen. These data demonstrated the crucial role of the heme-1 domain of SoxD for catalytic activity, electron yield, and transfer of the electrons to the cytoplasmic membrane, while the heme-2 domain mediated the alpha(2)beta(2) tetrameric structure of sulfur dehydrogenase.