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  • br Conclusions Two chemical coupling agents BTDE and


    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 MK-571 sodium salt hydrate 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 Chemotherapy is at the forefront of treatment against cancer because of its high efficiency and convenience [1], [2]. Unfortunately, conventional chemotherapy has shown several side effects including poor bioavailability, high toxicity and non-specificity [3], [4], [5]. To address these problems, the developments of prodrug strategies for sidestepping side effects have been used to directly enhance therapeutic efficiency; these strategies usually involve drug delivery systems, which can be activated by endogenous or exogenous substances and release the anticancer drug in tumor tissues [6], [7], [8], [9]. However, in situ monitoring of the drug's release and localization in tumor is yet to be fully exploited. Prodrug might become a promising approach to integrate chemotherapy and diagnostics, and this approach has become attractive for activating and monitoring drug release at the unique tumor microenvironment, thereby the therapeutic effect of anticancer drugs could be significantly improved [10], [11], [12], [13], [14]. The special environments of tumor cells include intracellular reduction [15], [16], [17], lower pH [18], [19] hypoxia [20], [21], active oxygen species [22], [23] or over-expressed enzymes [24], [25]. Among them, over-expressed enzymes in cancer cells draw particular interest as an anticancer drug-releasing trigger (mechanism) because of the differences of the enzymes’ levels between tumor and normal cells. Cleavable prodrugs have shown excellent capability to mediate the release of drug molecules and fluorophore simultaneously via the cleavage of a single linker [26]. A series of cleavable linkers for prodrugs have been developed in recent years, which indicated that cleavable prodrugs are conductive to monitoring drug release and therapeutic effect as well as the trigger (usually biomarker) level [27], [28]. Recently, coumarin has been adopted as a suitable fluorophore in biochemistry, which show low cytotoxicity, high quantum yield, easy modification and some optical beneficial characteristics (ICT effect, photo-responsive feature, two-photon excitation and so on). Many coumarin derivatives have been widely reported as fluorescent probes for detection of different biomarkers or assessment of drug release [29], [30], [31].