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  • Conventional assays rely on a comparable transformation

    2019-07-16

    Conventional assays rely on a comparable transformation of the target analytes and the structurally similar (but not identical) proxy substrates and are commonly used to measure individual enzyme activities in (pre-treated) intact sludge flocs (Burgess and Pletschke, 2008a, Gessesse et al., 2003, Yu et al., 2007). These heterogeneous samples, consisting of viable and dead cells, EPS and other components (Arnosti et al., 2014, Flemming and Wingender, 2010) are however dynamic micro-environments, which challenges the analysis of enzymatic transformation processes. Cell-free systems produced via cell disruption, centrifugation and filtration steps therefore might be more appropriate to study enzymatic variety and activity as they simplify the complex environment and eliminate dynamic cellular processes. Studies with extracted native enzymes from activated sludge are rare and so far mainly focused on the extraction of specific enzymes from the sludge EPS, which were then monitored with selected reporter assays (Gessesse et al., 2003, Nabarlatz et al.,). This approach neglects the involvement of intracellular enzymes, which inarguably exhibit a high metabolic versatility for the degradation of micropollutants (Fischer and Majewsky, 2014). The critical step for the establishment of cell-free systems is the transfer of (native) enzymes from the activated sludge matrix into a buffered, particle-free solution where they are tangible for downstream protein analytical techniques like (native) fractionation and purification, activity testing, enzymatic digestion/modification and identification techniques (e.g. proteomics). The extraction of soluble proteins from disrupted microbial AR-A014418 is naturally leads to a loss of functionality, since extraction efficiencies vary for different proteins, such as e.g. integral membrane enzymes, which cannot be co-extracted without greater experimental efforts (Speers and Wu, 2007). Therefore, the aims of this study were to:
    Material and methods
    Results & discussion
    Conclusions
    Acknowledgements We thank Sandro Castronovo for providing support and the activated sludge samples and all ATHENE project partners for the valuable discussions. Financial support from the European Research Council (ERC) through the EU-Project ATHENE (Grant agreement 267897) is gratefully acknowledged.
    Introduction Cu2+ is a crucial element to human body as an important part of certain enzymes and plays a prominent role in the various physiological processes [1], [2], [3]. Galactose oxidase is a copper-activated enzyme and have a greatly vital role in the metabolism of galactose [4]. The most common disorder resulting from deficiency in the activity of galactose oxidase is classic galactosemia, which would disrupt normal metabolism of galactose [5]. Galactosemia patients must restrict their galactose ingestion from milk or dairy products. Some serious symptoms such as liver disfunction and kidney problems usually appear after drinking milk without properly treated for infants with galactosemia [6]. Therefore, galactosemia is regarded as one of the most common metabolic disorders of newborn babies. However, much of the work is focused on determining the amount of galactose in the blood rather than measuring the amount of galactose oxidase to urge the galactosemia patients to restrict milk intake. Therefore, it is greatly demanded to determinate galactose oxidase in biological matrix. Colorimetric detection and chromatography are the most commonly used method for galactosemia diagnose, however, it is time consuming and tedious [7], [8]. Fluorescence-based detection techniques have arouse the interest of researchers, because they exhibit excellent stability, high sensitivity and low cost. Many sensors using organic fluorophore have been developed with admirable sensitivity and selectivity; however, their preparation usually requires complicated molecular design and elaborate organic synthesis [9], [10], [11]. Many metal nanoclusters and carbon-based nanocrystals have been applied to Cu2+ sensors, however, the most of them are easily disturbed by some biomolecules, which are strongly binding with Cu2+, such as GSH, ATP and EDTA [12], [13], [14], [15]. Therefore, the accessible fluorescent materials for the rapid, sensitive and accurate detection of Cu2+ and galactose oxidase need to be developed.