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  • br Material and Methods br

    2019-09-11


    Material and Methods
    Results and Discussion
    Conclusions Two chemical coupling agents, BTDE and CDI, were used to activate the free hydroxyl groups of plant cellulose powder and OPH from Flavobacterium ATCC 27551 immobilized on modified carriers by covalent linkages. The highest immobilization yields obtained in optimum conditions of effective parameters on enzyme immobilization onto epoxy and CDI activated cellulose were found to be 68.32% and 73.51%, respectively. The kinetic parameters were determined, and it was showed that the apparent Km values of the immobilized enzymes onto epoxy and CDI activated cellulose increased about 1.81 t and 2.06 t in comparison with the free OPH, respectively. Also, the maximum reaction rates of the immobilized enzymes using epoxy method and CDI agent were about 2.68 t and 3.32 t lower than that of free OPH, respectively. According to the results, the immobilized OPH revealed more thermal and storage stability compared with the soluble enzyme. The experiments showed that the native enzyme which was kept at 25 °C lost all of its original activity after 6.5 d, whereas the immobilized OPH onto epoxy and CDI modified cellulose preserved around 25% and 6% of their initial activity within one month at the same conditions, respectively. Additionally, it was indicated that the enzyme acquired more denaturation resistance against pH variations after immobilization, and the OPH preparations have more pH stability in alkaline buy b12 kinase relative to the acidic conditions. Generally, it was concluded that the enzyme immobilization onto epoxy modified cellulose showed more improvement in stability parameters relative to CDI modified cellulose. Furthermore, the reusability of immobilized preparations were studied and confirmed that after ten consecutive batch reactions, the relative activity of immobilized OPH using the CDI mediated covalent coupling was about 9% more than epoxy method. So, regarding the achieved outcomes, it is possible to choose the appropriate spacer arms for production of immobilized OPH over the cellulose surface depending on whether stability or reusability of resulted bioconjugates is desired, and it open the feasibility of various large-scale applications for biodegradation of organophosphate compounds.
    Introduction It has become increasingly apparent that seemingly identical cells in a population can actually be a heterogenous mixture differing at genome, transcriptome, and/or proteome levels. Analysis of this heterogeneity among cells within a population is critical to provide deeper understanding of cell behavior in response to intrinsic differences and extrinsic inputs in both normal and diseased states (Keating et al., 2018; Mincarelli, Lister, Lipscombe, & Macaulay, 2018; Wang & Navin, 2015). For example, lineage tracing of somatic mutations in the genome can provide detailed information about mosaicism in normal tissue (Biesecker & Spinner, 2013; D\'Gama & Walsh, 2018; Mincarelli et al., 2018). Furthermore, the ramifications of clonal evolution in the development of diseased states can be realized as mutations are traced to differences in single cells; for example, in formation of both solid and blood cancers, immunological responses, and neurodegenerative diseases (D\'Gama & Walsh, 2018; Keating et al., 2018; Mincarelli et al., 2018; Ortega et al., 2017; Verheijen, Vermulst, & van Leeuwen, 2018). The clinical relevance of cellular heterogeneity is increasingly realized as patient-specific therapies are targeted toward distinct mutations in individualized therapy (Keating et al., 2018; Ortega et al., 2017). Although the importance of single-cell analysis is clear, significant challenges must be overcome to obtain relevant and reliable results. These challenges stem from the small size of the single cell, with most mammalian cells ranging from ≤10 to 50μm in diameter and 1 to 100pL in volume (Alberts et al., 2008; Wang & Navin, 2015). Packed into this miniature environment is a dense assortment of nucleic acids, lipids, carbohydrates, proteins, and metabolites which can confound sample collection and assay readout (Table 1; Alberts et al., 2008; Ortega et al., 2017; Wang & Navin, 2015; Zeng, Miao, & Sun, 2018). Many targeted analytes such as key signaling proteins are present within this complex milieu at vanishingly small copy numbers (10–1000 molecules) (Huang et al., 2007) Additionally, the small size of the cell necessitates low detection limits and often prevents multiple sampling from the same cell, making it challenging to parse out technical variability of the assay method from true biological variability between cells (Keating et al., 2018; Macaulay, Ponting, & Voet, 2017). The ability to reliably characterize single cells is of utmost importance buy b12 kinase in basic biological understanding, disease etiology, and development of personalized medicine.