Citation: Yepes, C.; Estévez, J.; Arroyo, M.; Ladero, M. Immobilization of an Industrial β-Glucosidase from Aspergillus fumigatus and Its Use for Cellobiose Hydrolysis. Processes 2022, 10, 1225. https://doi.org/10.3390/pr10061225 Academic Editor: Francisco José Hernández Fernández Received: 11 May 2022 Accepted: 17 June 2022 Published: 20 June 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). processes Article Immobilization of an Industrial β-Glucosidase from Aspergillus fumigatus and Its Use for Cellobiose Hydrolysis Clara Yepes 1 , Juliana Estévez 2 , Miguel Arroyo 1 and Miguel Ladero 2, * 1 Department of Biochemistry and Molecular Biology, Faculty of Biology, Complutense University Madrid (Spain), 28040 Madrid, Spain; yepesrodriguezclara@gmail.com (C.Y.); arroyo@bio.ucm.es (M.A.) 2 Chemical Engineering and Materials Department, Chemical Sciences School, Complutense University Madrid (Spain), 28040 Madrid, Spain; juliaestg@gmail.com * Correspondence: mladero@quim.ucm.es; Tel.: +34-91-3944164 Abstract: In this study, several covalent methods of immobilization based on acrylic supports, Schiff bases and epoxides have been applied to a commercial cocktail with a high β-glucosidase activity secreted by Aspergillus fumigatus. This cocktail was preliminary compared to a commercial secretome of Aspergillus niger, which was also subjected to the aforementioned immobilization methods. Due to its higher activity, the cocktail from A. fumigatus immobilized on ReliZyme™ HA403 activated with glutaraldehyde was employed for pNPG and cellobiose hydrolysis in diverse operational conditions and at diverse enzyme loadings, showing a very high activity at high enzyme load. A kinetic model based on the Michaelis–Menten hypothesis, in which double inhibition occurs due to glucose, has been selected upon fitting it to all experimentally retrieved data with the lowest-activity immobilized enzyme. This model was compared to the one previously established for the free form of the enzyme, observing that cellobiose acompetitive inhibition does not exist with the immobilized enzyme acting as the biocatalyst. In addition, stability studies indicated that the immobilized enzyme intrinsically behaves as the free enzyme, as expected for a one-bond low-interaction protein- support immobilization. Keywords: β-glucosidase; covalent immobilization; acrylic support; cellobiose hydrolysis; kinetic model; double competitive inhibition 1. Introduction Biomass can be defined as the matter that has its origin in carbon compounds generated by photosynthesis and the matter derived from it [1]. Its use as a renewable energy source has been widely developed with the aim of substituting, or at least reducing, the use of other energy sources such as oil or coal, which are not renewable and, therefore, their resources are limited. In fact, biomass is envisaged as a sustainable source of energy, materials, chemicals, food and feed in the framework of the integrated biorefinery. Due to the increasing need for energy and material resources, biomass has positioned itself as an alternative source to fossil fuels and derived chemicals and materials. It can be considered that solar energy is transformed in the biosphere with an efficiency of 0.1%, so it is estimated that 3.1021 J/year [2] is the energy that can be used as an energy source. Thus, annual biomass production is only an order of magnitude smaller than known fossil fuel reserves, which implies that the energy provided by biomass could completely supply human global needs. However, it must also be considered that not all the biomass generated corresponds to terrestrial ecosystems, as algae also participate in its generation. However, most of the available biomass is lignocellulosic biomass, mainly consisting of cellulose (up to 60%), diverse types of hemicelluloses (up to 40%) and lignin (accounting for 7–25%) [3]. The recalcitrance of lignocellulosic biomass (LCB) involves the application of several physical (such as grinding), chemical (acid or basic hydrolysis) and physicochemical (steam Processes 2022, 10, 1225. https://doi.org/10.3390/pr10061225 https://www.mdpi.com/journal/processes