Immobilization of enzymes of agroindustrial interest on hydroxyapatite nanoparticles: β-glucosidase, xylase and phytase

Abstract

Industrial processes are increasingly requiring environmentally sustainable technologies and the use of enzymes is relevant due to the selectivity and specificity of these biocatalysts. However, the application of soluble enzymes in catalytic processes is often impracticable due to the high cost of these biocatalysts and the catalytic instability under severe physical-chemical process conditions. In this sense, the immobilization of enzymes guarantees the reuse of these biomolecules and often improves their stability. Among the supports used as immobilizing agents, nanoparticles have been increasingly studied due to the possibility of creating structures with high surface area to adsorb proteins and also to demonstrate low resistance to mass transfer, thus ensuring good accessibility of the catalyst to the substrate. Hydroxyapatite (HA) is a solid inorganic potential for immobilizing enzymes, since it is non-toxic, has good chemical and physical resistance, good ability to interact with proteins and can be synthesized in the form of nanoparticles; however, it has been little explored as a support for enzymatic immobilization. Given this context, the objective of this research was to evaluate the immobilization of the enzymes β-glucosidase, xylanase and phytase on hydroxyapatite nanoparticles (HA), as a strategy to improve the catalytic efficiency and stability of these enzymes. These enzymes have wide application in different sectors of the agribusiness, such as biofuels, food, animal feed and drugs. For this, HA nanoparticles were first characterized in order to understand their composition, size, morphology and surface area. Then, a systematic study of the immobilization of these enzymes in HA was carried out. The biochemical aspects of enzymatic immobilization were investigated, such as the physicochemical conditions of adsorption and desorption, that helps to understand the type of chemical interaction between support and enzyme. Changes in enzymatic activity profile, thermo-stability, conversion capacity during enzymatic hydrolysis and reuse of these biomolecules were also evaluated. The results obtained showed that the β-glucosidase, phytase and xylanase enzymes were efficiently immobilized on the HA nanoparticles using a simple and fast adsorption protocol, which occur mainly through coordination interactions between the Ca2+ sites of the HA with the carboxylic acids (COO-) of the enzyme amino acids. The biochemical behavior of the enzymes in the presence of HA was evaluated under different physicochemical conditions of adsorption and desorption (pH and ionic strength), indicating a strong and highly stable interaction between enzymes and support, with immobilization yields close to 100% and recovered activities in the range of 70-100%. For β-glucosidase it was possible to recycle the immobilized enzyme and retain 70% of the initial activity during at least 10 hydrolysis cycles. For phytase, the immobilized enzyme showed broader activity profile as to pH and temperature, and higher stability at high temperatures than free enzyme, whose improvement in its properties suggested the potential of applying the immobilized form of phytase in animal feed. For xylanase, the immobilized enzyme demonstrated greater affinity for the HA support that was modified with Cu2+ ions, promoting chelation interaction with enzyme amino acids, generating derivatives with maintenance of their catalytic activity and with activity profile similar to that of the free enzyme. Finally, the three enzymes were immobilized on HA magnetic nanoparticles (synthesized with cobalt ferrite, CoFe2O4), demonstrating excellent recovery capacity from the reaction medium through the application of a magnetic field. The results obtained showed the potential of HA to act as a support for enzyme immobilization, creating derivatives with promising industrial applications, which can improve processes and make them more sustainable.