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  • AZD3264 br Results and discussion br Conclusions In this wor

    2019-09-11


    Results and discussion
    Conclusions In this work, we have conjugated two thermophilic enzymes (i.e., AMY and LASPO) to iron oxide NPs through different conjugation strategies obtaining efficient biocatalysts. We have demonstrated that these NP-enzyme systems can be successfully activated by an AMF in a “wireless” fashion. We have also shown that, notwithstanding the AMF activation, the temperature of the medium increases only slightly (Fig. 10). Indeed, non-thermophilic enzymes are able to work in the same pot with the NP-AMY systems. These results made us think that there is plenty of space among the suspended NP-enzyme systems, where a non-thermophilic enzyme can work at its optimal temperature. The prospects of the use of this novel approach for the selective local thermo-activation of enzymes include biomedical and biotechnological applications. As NPs could be engineered to gain access to AZD3264 through the endosomal compartment [47] or through non-endocytotic pathways [48], [49], [50] or by cell internalization techniques [51], [52], the use of NP-enzyme systems to remotely control cell metabolism or for the implementation of new alternatives for enzyme/pro-drug therapy could be envisioned. Our results also allow foreseeing a future implementation of AMF-enzyme activation for biocatalytic processes of industrial interest, as we showed it is possible to achieve a fine-tuning of the enzyme-NP interface to maximize the enzyme activation effect and its re-use. In particular, the two enzymes used are attractive for biotechnological applications. AMY is widely used in the starch industry for the conversion of starch to medium‐sized oligosaccharides. LASPO can be used for the production of d-aspartate from a racemic mixture of d,l-aspartate, a molecule employed in the pharmaceutical industry, for parenteral nutrition, as a food additive and in sweetener manufacture [53]. However, the industrial application of both enzymes is hampered by the high cost per enzymatic unit, which encourage exploring the use of thermophilic enzyme to improve their reusability due to their increased stability. However, their use introduces the need to heat the reaction media to higher temperatures in order to maximize the efficiency of the reaction. As we have shown that the magnetic NPs can generate enough local amount of thermal energy for the activation of both enzymes, a future implementation of AMF-enzyme activation should allow saving energy costs. Besides, as each enzyme was activated without raising the temperature of the reaction solution as a whole, the implementation of AMF-activation of multi-enzymatic processes of biotechnological interest will be also feasible.
    Materials and methods
    Acknowledgements Ilaria Armenia is a PhD student of the “Biotechnology, Biosciences and Surgical Technology” course at Università degli Studi dell\'Insubria. The Authors are in debt with Prof. Loredano Pollegioni for the gift of the enzymes LASPO and DAAO. Authors would like to also acknowledge the public funding from Fondo Social de la DGA, Spain (grupos DGA), and from Ministerio de la Economía y Competitividad del Gobierno de España for the public funding of Proyectos I D+i – Programa Estatal de Investigación, Desarrollo e Innovación Orientada a los Retos de la Sociedad, Spain (project n. BIO2017-84246-C2-1-R). The Authors thank Dr Gianluca Tomasello for the use of 3DPROTEINIMAGING (https://3dproteinimaging.com).
    Introduction The industrial use of enzymes has seen a significant rise in the last decade which is owed in a large extent to the ease with which enzymes can be genetically tailored [1,2]. Properties such as substrate scope, enantioselectivity, solvent tolerance and thermostability can be adapted via enzyme engineering leading to catalysts that have been improved on a multifactorial level [3]. In the chemical and pharmaceutical industry, biocatalysts are most often used for the installation of chirality and the late stage functionalization of scaffolds while in the production of aroma compounds enzymatic catalyst confer the added benefit of the ‘natural’ label as defined by the European flavor and food legislation. Biocatalytic strategies for the chiral resolution of esters and the synthesis of enantiopure alcohols and amines are particularly well established and are successfully applied in the biocatalytic production of pharmaceuticals such as sitagliptin [4], montelukast [5] and cipargamin [6]. Despite the increasing industrial and academic interest in harnessing enzymes for synthesis, our understanding of the structure–function relationship in proteins is still limited [7] and enzyme engineering is considered to be mostly a collection of case-studies [2]. However, recent protein optimization campaigns shed more light on selected enzyme families and highlight that more generalized engineering principles can be elucidated [8,9].