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  • br Experimental br Results and discussion br


    Results and discussion
    Conclusions The fungus Mucor circinelloides MUT44, previously shown to have ene-reductase activity [11], possesses ten genes coding for putative ene-reductases belonging to the Old Yellow Enzymes family. Since the reduction of CC double bonds is one of the most important strategies for the production of compounds with up to two chiral centers [1], these Eribulin are very important for biocatalytic purposes. To this end, in silico studies can be used to gain information about the structural and functional properties of these new putative enzymes. For example, analysis of the primary sequence of the McOYEs, highlight that one isoform (McOYE10) is homolog to thermophilic-like bacterial OYEs whereas McOYE8 has a theoretical pI of 7.18 due to the presence of extra Arg/Lys residues distributed on the protein surface. Thus, these two isoforms are likely to have a higher stability at high temperatures (McOYE10) and at more basic pH (McOYE8) compared to the other McOYEs. From a structural point of view, the McOYEs share the TIM barrel scaffold typical of the OYEs family where structural variability is associated with the capping subdomain that is a flexible region involved in active site shaping and Eribulin also in NAD(P)H and substrate binding. Interestingly, the crystal structure of an OYE from Shewanella oneidensis in complex with p-hydroxybenzaldehyde shows that a second molecule of the substrate is present in a hydrophobic cleft next to the entry of the active site tunnel in the capping subdomain [50], indicating an important role of this region in substrate binding. According to our models, the volume can vary from 126.8 to 1587.4Å3 indicating that the fungal enzymes can accommodate substrates of different size and therefore possess different substrate selectivity. This hypothesis found a confirmation in the high versatility shown by M. circinelloides MUT44 in the reduction of compounds with different EWGs and different steric hindrance such as ketone, aldehyde, nitro and carboxylic group [11]. In conclusion, our data show that it is possible to develop and validate in silico methods to predict structural differences among isoenzymes from the same organism in order to select the most suitable catalyst for the desired application.
    Acknowledgments This work was partially funded by Compagnia San Paolo and Fondazione CRT (Italy).
    Main Text The singular solution to these limitations—stability, recovery, and recyclability—is immobilization, although chemical modification and protein engineering can also contribute. Immobilization is the attachment of an enzyme to a support or its inclusion in a matrix. The use of various immobilization strategies has shown that enzymes are better stabilized and can retain their activity when working in organic solvents, at extreme pH, and under mechanical stress. Immobilization of enzymes onto micrometer-sized particles also gives the possibility of easy separation of the catalyst from the products and thus provides access to recovery and recyclability. The ideal support matrix used for immobilization needs to be cheap, be controllable in structure, and provide high enzyme loading with minimal compromise to the activity. Substrate selectivity by the matrix could be an additional benefit. In practice, immobilization has been performed with a wide range of materials such as natural and synthetic polymers, as well as inorganic materials, such as zeolites, ceramics, celites, silica, glass, and activated carbon.2, 3 The choice of a particular material is predominantly dictated by the process conditions under which the supported enzyme has to work. Drawbacks of immobilization are an increase in the production costs and the incompatibility of materials with certain enzymes or applications. In addition, the reaction rate is often diminished as a result of mass-transfer limitations occurring in the system, in particular for the substrate reaching the enzyme and for the product diffusing out from the matrix. This behavior is due to enzyme-support interactions, which can block the accessibility of the active site.