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  • The prospects of the use of this


    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 arginase inhibitor 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 (
    ACAT enzymes Acyl-CoA:cholesterol acyltransferases (ACATs), also known as sterol O-acyltransferases (SOATs), play important roles in cellular cholesterol homeostasis and are drug targets for therapeutic intervention of several diseases including atherosclerosis (reviewed in [1]), Alzheimer’s disease [2], [3], [4], [5] and cancer [6]. In mammals, two genes that encode two different proteins exist: Acat1 and Acat2 [7]. Along with acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1), ACAT1 and ACAT2 are founding members of the membrane-bound O-acyltransferase (MBOAT) enzyme family [8]. MBOATs are multi-span membrane enzymes that use long-chain or medium-chain fatty acyl-CoA as the first substrate, and catalyze the transfer of the fatty acyl group to the 3β-hydroxyl moiety of a certain hydrophobic substance as the second substrate. An MBOAT contains two active sites: a histidine within a long hydrophobic peptide region, and an asparagine located within a long hydrophilic peptide region. In humans, there are 11 MBOAT members, with similar catalytic mechanisms but with diverse biological functions. At present, there are no complete crystal structures for any members of the MBOAT family. For ACAT1 and ACAT2, the major sterol substrate is cholesterol; both enzymes use long chain fatty acyl-coenzyme A as the fatty acyl donor to convert cholesterol to cholesteryl esters. For ACAT1, the preferred fatty acyl-CoA is oleoyl coenzyme A [9]. Cholesterol is a lipid molecule; it partitions well within the phospholipid bilayer of various cell membranes. Unlike free (unesterified) cholesterol, the cholesteryl esters do not partition well within the lipid bilayer; instead, cholesteryl esters coalesce in aqueous medium and form cytoplasmic lipid droplets. The over accumulation of free cholesterol in the membranes can be cytotoxic to cells. Thus, a major function of ACATs is to protect against the unnecessary built up of free cholesterol within the cell membranes. ACAT1 is ubiquitously expressed in essentially all tissues examined; it is a resident enzyme at the endoplasmic reticulum (ER). ACAT2 is mainly expressed in the intestines and hepatocytes. It is also expressed in various other tissues, but at much lower levels than ACAT1. In intestines, ACAT2 provides cholesteryl esters for lipoprotein assemblies. In humans, both ACAT1 and ACAT2 are expressed in hepatocytes; the relative roles of ACAT1 and ACAT2 in human hepatic lipoprotein assembly are yet to be clarified. (reviewed in [1]). Homologs of ACAT1 and ACAT2 have been identified in yeast saccharomyces cerevisiae [10], [11] and other species.