PRODUCTION AND PURIFICATION OF β-GLUCOSIDASE FROM Aspergillus caespitosus USING ORA-PRO-NOBIS FIBER AS AN ALTERNATIVE SUBSTRATE

β-Glucosidases (E.C 3.2.1.21) catalyzes the hydrolysis of β-glycosidic bonds and have been applied in the bioconversion of lignocellulosic residues for ethanol production, the clarification of fruit juices, and the cleavage of β-1,3 and β-1,4 bonds in glucans present in barley, thereby improving the filtration process. They are also used in the wine industry to enhance aroma. The use of agro-industrial residues as substrates for large-scale β-glucosidase (β-GL) production has a positive impact on reducing process costs compared with the use of conventional culture media. However, the prospecting of new enzyme-producing microorganisms and the development of efficient purification strategies remain major challenges. This study aimed to optimize β-glucosidase production by Aspergillus caespitosus CCDCA 11593 under submerged fermentation using ora-pro-nobis (Pereskia aculeata Miller) leaf fiber as an alternative substrate and to evaluate enzyme purification by a sulfanilamide-functionalized ion-exchange cryogel column. The fungus was reactivated on malt extract agar for 10 days and used to inoculate submerged fermentations containing 10⁷ spores mL⁻¹. Different cultivation conditions were evaluated with respect to temperature (25 °C and 40 °C), pH (5.0 and 6.0), and incubation time (72 and 120 h), under agitation at 100 rpm. β-Glucosidase activity was determined using p-nitrophenyl-β-D-glucopyranoside as the substrate, while total protein content was quantified by the Bradford method. The crude extract obtained under the optimal production conditions was purified by ion-exchange cryogel column chromatography, operated at a flow rate of 1.0 mL min⁻¹, and five consecutive adsorption and elution cycles were evaluated. The highest specific β-glucosidase activity was obtained at 25 °C, pH 5.0, and 72 h, reaching 4.090 ± 0.081 U mg⁻¹, whereas cultures grown at 40 °C exhibited activities lower than 0.11 U mg⁻¹, highlighting the strong influence of temperature on enzyme production. During the purification step, the fraction eluted with four column volumes (16 mL) yielded the best results, with specific activity ranging from 0.475 U mg⁻¹ to 0.798 U mg⁻¹, a purification factor between 11.03 and 19.14, and a purification yield ranging from 2.01% to 3.92% over five consecutive cycles, demonstrating the high selectivity and good operational stability of the cryogel column. The successful use of ora-pro-nobis leaf fiber demonstrates the feasibility of valorizing an underutilized lignocellulosic biomass as a low-cost substrate for enzyme production, while the reproducibility of the chromatographic process highlights the potential reuse of the cryogel matrix in downstream processing. The results demonstrate, for the first time, the potential of A. caespitosus as a β-glucosidase producer using ora-pro-nobis leaf fiber as an alternative substrate. Furthermore, the sulfanilamide-functionalized cryogel column exhibited reproducible performance and high purification efficiency, representing a promising strategy for obtaining this enzyme for biotechnological and industrial applications.