Biochemical characterisation of a new thermotolerant L-asparaginase produced by Cunninghamella echinulata PA3S12MM isolated from the Atlantic Forest.

L-asparaginase is an amidohydrolase enzyme that catalyses the hydrolysis of L-asparagine into L-aspartic acid and ammonia. This enzyme has significant biotechnological applications, functioning as an antineoplastic agent in the pharmaceutical industry and as a tool for reducing acrylamide formation in the food industry. This study aimed to identify a filamentous fungus strain capable of synthesizing a new L-asparaginase and to perform its biochemical characterisation. Among the fungal isolates screened using plate assays and submerged fermentation, Cunninghamella echinulata strain PA3S12MM was identified as a promising L-asparaginase producer. The highest enzymatic induction was achieved using 5% (w/v) glucose as the carbon source and 0.4% (w/v) chicken feather as an agro-industrial nitrogen source, under incubation for 120 h. The enzyme exhibited an apparent Km app of 0.011 mmol L⁻¹ and a Vmax app of 126.5 mmol L⁻¹ min⁻¹ for the hydrolysis of L-asparagine. Partial purification revealed that the enzyme displayed optimal activity at pH 7.0 and a temperature of 85 °C. In terms of substrate specificity, the enzyme demonstrated 100% relative activity toward L-asparagine and 32.9% toward L-glutamine. Enzymatic activity was enhanced by β-mercaptoethanol but inhibited by Mn²⁺, L-cysteine, and dithiothreitol (DTT). These findings suggest that C. echinulata PA3S12MM is a promising candidate for large-scale biotechnological production of a new fungal L-asparaginase, with biochemical indicators of potential for applications in both pharmaceutical and food industries.