L-Asparaginase Production by Fungal Coculture Under Solid-State Fermentation: Biochemical Characterization and Application in French Fries for Acrylamide Mitigation

Abstract L-Asparaginase is widely used as an antineoplastic agent and as a biocatalyst to reduce acrylamide formation in thermally processed foods. Acrylamide is mainly generated through the Maillard reaction in carbohydrate-rich matrices, such as fried potatoes, and is considered a potential carcinogen. Although enzymatic pretreatment with L-asparaginase is the most effective mitigation strategy, its large-scale application remains limited by the high production costs associated with conventional enzymatic sources. This study assessed the production of L-asparaginase by the coculture of Rhizopus microsporus var. oligosporus and Penicillium camemberti in solid-state fermentation (SSF). The simplex-centroid design identified the optimal substrate formulation, equal proportions of cocoa bean shell, coffee parchment, and ora-pro-nobis leaves which increased enzyme production by 66%, yielding 8.69 U/g. Subsequent optimization using a Box–Behnken design improved the fermentation conditions (135 h at 26 °C and 68% initial moisture), resulting in an activity of 11.0 U/g. The crude enzymatic extract (CEE) exhibited optimal activity at pH 8.0, retained stability up to pH 9.0, and showed maximal thermal stability at 40 °C, maintaining 64% activity even at 70 °C. Enzyme activity was enhanced by Na⁺, Cu 2 ⁺, and Mn 2 ⁺ with increases of up to 182%, while Mg 2 ⁺ and Ca 2 ⁺ demonstrated inhibitory effects. The enzyme also displayed halotolerance, with optimal performance at 2 M NaCl, and was stimulated by ethanol, reaching 135% activity at 30%. Pretreatment of French fries with the CEE reduced acrylamide formation by up to 75%. These results demonstrate that L-asparaginase obtained via solid-state fermentation represents a cost-effective and practical biotechnological strategy for acrylamide mitigation, eliminating the need for purification steps. Graphical Abstract