Amidst the diverse lifestyles of fungal species, endophytes emerge as intriguing organisms. These entities reside within plant tissues, causing no harm to their host. The spotlight on endophytic fungi intensified following the discovery of Taxomyces andreanae, isolated from Taxus brevifolia (Taxaceae). This fungus produces the diterpene paclitaxel ("Taxol®"), a well-known and extensively used anticancer agent (Stierle et al., 1993). Numerous studies have demonstrated the capacity of endophytic fungi to produce various enzymes, including L-Asparaginase (Chow and Ting, 2015, Gummadi, 2014, Pádua et al., 2018). Consequently, researchers explore endophytic fungi associated with plants inhabiting underexplored or extreme environments, believing that these environments harbor fungal communities with significant biotechnological potential (Hokama et al., 2016, Knight et al., 2003).

The Antarctica environment remains largely uncharted and exhibits extreme characteristics. These traits result from an array of life-limiting factors, including low temperatures, limited water availability, infrequent sunlight, recurring freeze-thaw cycles, meager annual precipitation, strong winds, high rates of sublimation and evaporation, and particularly intense solar radiation, notably ultraviolet light (Ugolini and Bockheim, 2007, Campbell and Claridge, 1988). Life in Antarctica comprises organisms that demonstrate remarkable levels of adaptation, exposed daily to diverse abiotic factors across various habitats (Ruisi et al., 2007). Among the Antarctic flora, species of mosses, such as Sanionia uncinata (Hedw.) Loeske and Polytrichastrum alpinum (Hedw.) G.L. Sm., stand out as some of the most abundant on King George Island in the South Shetland Islands, forming expansive carpets across significant portions of the region (Ochyra, 1998, Nakatsubo, 2002, Eights, 1833). Consequently, these organisms serve as excellent hosts for endophytic fungi within this extreme environment.

Fungi in Antarctica, alongside other microorganisms, play a dominant role in the food chains. Filamentous fungi and yeast-like forms in this environment exhibit a high degree of genetic plasticity, underscoring their adaptation to the extreme conditions prevalent in Antarctica (Rosa, 2019). Reports indicate the presence of species such as Aspergillus, Cladosporium, Penicillium, Pseudogymnoascus, Phaeosphaeria, Microdochium, Mortierella, and Purpureocillium, which hold potential as producers of bioactive secondary metabolites with diverse activities (Rosa, 2019). The fact that fungi in Antarctica endure constant extreme conditions suggests the possibility of novel biochemical pathways that enable them to synthesize compounds with as-yet-unknown properties. This opens avenues for potential applications in new treatments, such as those for Acute Lymphoblastic Leukemia (ALL). Notably, fungi from the genera Aspergillus, Fusarium, and Penicillium have been identified as producers of L-Asparaginase (L-ASNase) for this purpose (Rosa, 2019, Santiago et al., 2012, Cachumba, 2012, Costa-Silva, 2018, Da Rocha, 2019, Kumar and Sobha, 2012, Vala, 2018).

Acute Lymphoblastic Leukemia (ALL) is a malignant neoplasm of lymphocytic origin, recognized as the most prevalent form of cancer in children, accounting for approximately 25% of diagnoses among patients under 15 years of age (Steliarova-Foucher, 2017). L-Asparaginase, when used alongside corticosteroids and chemotherapy, serves as a vital medicinal intervention. This enzyme facilitates the hydrolysis of L-Asparagine, an essential amino acid critical for protein synthesis and not produced by leukemic cells. Depriving leukemic cells of free asparagine curbs cellular supplementation, inducing cell cycle arrest and ultimately triggering apoptosis in leukemic cells (Steliarova-Foucher, 2017, Kebriaei et al., 2002, Onciu, 2009). Following numerous advancements within the pharmaceutical industry, L-ASNase can now be synthesized within prokaryotic organisms, emerging as the most widely employed enzyme due to substantial bacterial-mediated production. However, L-ASNase originating from prokaryotic organisms has been directly linked to various side effects reactions (Panosyan et al., 2004, Saeed, 2017).

Numerous studies aim to comprehend the principal factors influencing the side effects induced by the use of L-Asparaginase produced by bacteria. One of the topics under scrutiny is the joint activity of L-Asparaginase with L-Glutaminase and Urease. This interaction culminates in diminished L-Asparaginase efficacy and holds the potential to trigger severe side effects, including hypersensitivity, thrombosis, coagulation disorders, pancreatitis, hyperglycemia, hepatotoxicity, central nervous system dysfunction, and immunological reactions involving antibody production. Furthermore, this interaction can lead to hyperammonemia, associated with extensive cerebral lesions due to the substantial release of ammonia facilitated by Urease (Greenberg et al., 1964, Bano and Sivaramakrishnan, 1980, Sarquis et al., 2004, Doriya and Kumar, 2016).

L-Asparaginase is present in various organisms; however, there is a demand for its production in eukaryotic systems that are both straightforward and rapid-growing, such as filamentous fungi (Sindhu and ManonmanI, 2018, Rahimzadeh et al., 2016, Muneer et al., 2020, Eisele et al., 2011, Benchamin et al., 2019). Fungi offer the advantage of efficient cultivation on a large scale within a short time frame. Furthermore, being eukaryotic, they possess several post-translational properties that make them a significant resource for L-Asparaginase production (Sarquis et al., 2004). Numerous studies have already documented the production of L-Asparaginases that are free from glutaminase and urease, achieved through filamentous fungi and yeasts. This achievement holds notable importance in the quest for enzymes with fewer side effects for patients (Hatamzadeh et al., 2020, Ashok et al., 2019, Freire et al., 2020).

Only a limited number of studies utilizing Antarctic organisms have concentrated on the quest for L-Asparaginase, and these efforts still fail to fully capture the significance of seeking this enzyme within these extreme organisms. This study's objective is to isolate and identify endophytic fungi from S. uncinata and P. alpinum, utilizing both morphological characterization and phylogenetic DNA analysis. Furthermore, the study aims to assess the potential of these fungi to produce the antileukemic enzyme L-Asparaginase, while ensuring its freedom from glutaminase and urease. The research also strives to identify the optimal conditions for maximizing enzyme production.