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l-asparaginase (ASNase, EC 3.5.1.1) is an aminohydrolase enzyme with important uses in the therapeutic/pharmaceutical and food industries. Its main applications are as an anticancer drug, mostly for acute lymphoblastic leukaemia (ALL) treatment, and in acrylamide reduction when starch-rich foods are cooked at temperatures above 100 °C. Its use as a biosensor for asparagine in both industries has also been reported. However, there are certain challenges associated with ASNase applications. Depending on the ASNase source, the major challenges of its pharmaceutical application are the hypersensitivity reactions that it causes in ALL patients and its short half-life and fast plasma clearance in the blood system by native proteases. In addition, ASNase is generally unstable and it is a thermolabile enzyme, which also hinders its application in the food sector. These drawbacks have been overcome by the ASNase confinement in different (nano)materials through distinct techniques, such as physical adsorption, covalent attachment and entrapment. Overall, this review describes the most recent strategies reported for ASNase confinement in numerous (nano)materials, highlighting its improved properties, especially specificity, half-life enhancement and thermal and operational stability improvement, allowing its reuse, increased proteolysis resistance and immunogenicity elimination. The most recent applications of confined ASNase in nanomaterials are reviewed for the first time, simultaneously providing prospects in the described fields of application.

The immobilization strategy can promote greater enzyme utilization in applications by improving the overall stability and reusability of the enzyme. In this work, the L-asparaginase (L-ASNase) obtained from Escherichia coli was chosen as a model enzyme and immobilized onto the Fe 3 O 4 @Au-carboxymethyl chitosan (CMC) magnetic nanoparticles (MNPs) through adsorption. TEM, SEM, FT-IR, XRD, EDS, and TGA analyses were performed to examine the structure with and without L-ASNase. The yield of immobilized L-ASNase on Fe 3 O 4 @Au-CMC was found to be 68%. The biochemical properties such as optimum pH, optimum temperature, reusability, and thermal stability of the Fe 3 O 4 @Au-CMC/L-ASNase were comprehensively investigated. For instance, Fe 3 O 4 @Au-CMC/L-ASNase reached maximum activity at pH 7.0 and the optimum temperature was found to be 50 °C. The noticeably lower Ea value of the Fe 3 O 4 @Au-CMC/L-ASNase revealed the enhanced catalytic activity of this enzyme after immobilization. The Km and Vmax values were 3.27 ± 0.48 mM, and 51.54 ± 0.51 μmol min −1 for Fe 3 O 4 @Au-CMC/L-ASNase, respectively, which means good substrate affinity. The Fe 3 O 4 @Au-CMC/L-ASNase retained 65% of its initial activity even after 90 min at 60 °C. Moreover, it maintained more than 75% of its original activity after 10 cycles, indicating its excellent reusability. The results obtained suggested that this investigation highlights the use of MNPs as a support for the development of more economical and sustainable immobilized enzyme systems.

In this study, nanoniosome‐loaded Myristica fragrans' (MF) phenolic compounds (NLMP) were synthesized and characterized for their physical properties, and hepatoprotective effects on mice with liver toxicity induced by L‐asparaginase (LA) injection. According to the results, NLMP has a spherical shape with a 263 nm diameter, a zeta potential of −26.55 mV and a polydispersity index (PDI) of 0.192. The weight and feed intake of mice induced with hepatotoxicity were significantly (p ≤ 0.05) increased after they were treated with NLMP (2.5 mg/kg body weight of mice). In addition, the blood levels of triglyceride (TG), cholesterol (Chol), liver enzymes (aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP)) and total bilirubin were significantly (p ≤ 0.05) decreased. A significant increase (p ≤ 0.05) in the blood levels of the antioxidant defence system (glutathione peroxidase (GPX), superoxide dismutase (SOD) and catalase (CAT)) were also reported after NLMP treatment. NLMP was also led to a significant decrease (p ≤ 0.05) in inflammatory‐related gene expression of inducible nitric oxide synthase (iNOS) and Interferon‐gamma (IFN‐γ) in the liver, as well as a meaningful (p ≤ 0.05) increase in the expression of SOD as an antioxidant status biomarker. Consequently, the NLMP is recommended as a potential dietary supplement to alleviate the symptoms of LA‐induced hepatotoxicity.

l -Asparaginase is a chemotherapeutic drug used in the treatment of acute lymphoblastic leukaemia (ALL), a malignant disorder in children. l -Asparaginase helps in removing acrylamide found in fried and baked foods that is carcinogenic in nature. l -Asparaginase is present in plants, animals and microbes. Various microorganisms such as bacteria, yeast and fungi are generally used for the production of l -asparaginase as it is difficult to obtain the same from plants and animals. l -Asparaginase from bacteria causes anaphylaxis and other abnormal sensitive reactions due to low specificity to asparagine. Toxicity and repression caused by bacterial l -asparaginase shifted focus to eukaryotic microorganisms such as fungi to improve the efficacy of l -asparaginase. Clinically available l -asparaginase has glutaminase and urease that may lead to side effects during treatment of ALL. Current work tested 45 fungal strains isolated from soil and agricultural residues. Isolated fungi were tested using conventional plate assay method with two indicator dyes, phenol red and bromothymol blue (BTB), and results were compared. l -Asparaginase activity was measured by cultivating in modified Czapek–Dox medium. Four strains have shown positive result for l -asparaginase production with no urease or glutaminase activity, among these C 7 has high enzyme index of 1.57 and l -asparaginase activity of 33.59 U/mL. l -Asparaginase production by C 7 was higher with glucose as carbon source and asparagine as nitrogen source. This is the first report focussing on fungi that can synthesize l -asparaginase of the desired specificity. Since the clinical toxicity of l -asparaginase is attributed to glutaminase and urease activity, available evidence indicates variants negative for glutaminase and urease would provide higher therapeutic index than variants positive for glutaminase and urease.

L-asparaginase (L-ASNase) is a key industrial enzyme significant for cancer therapy and the food industry for reducing dietary acrylamide. The hyperthermophilic L-ASNase from Thermococcus sibiricus (TsAI) was previously shown to exhibit high activity and thermostability and is promising for biotechnology. To gain insights into structure-functional relationships of TsAI, determination of the substrate specificity, kinetic parameters, structural characterization, and molecular docking were performed. TsAI characteristics were compared with the TsAID54G/T56Q mutant, which exhibited increased activity after a double mutation in the substrate-binding region. TsAI and TsAID54G/T56Q were found to display high activity towards D-asparagine—62% and 21% of L-asparaginase activity, respectively—and low L-glutaminase coactivity of ~5%. Restoring the mesophilic-like triad GSQ in the mutant resulted in a two-fold increase in activity towards L-asparagine compared with TsAI. Crystal structures of TsAI forms solved at 1.9 Å resolution revealed that double mesophilic-like mutation increased the flexibility of the loop M51-L57, located in close proximity to the active site. Structural superposition and mutational analysis indicate that mobility of this loop is essential for the activity of thermo-ASNases. Molecular docking, without taking into account the temperature factor, showed that, in contrast to L-asparagine interaction, D-asparagine orientation in the TsAI and TsAID54G/T56Q active sites is similar and not optimal for catalysis. Under real conditions, high temperatures can induce structural changes that reduce L-ASNase discrimination towards D-asparagine. Overall, the obtained structural and biochemical data provide a basis for a more detailed understanding of thermo-ASNase functioning and possibilities to engineer improved variants for future biotechnological application.

The bacterial L-asparaginase is a highly effective chemotherapeutic drug and a cornerstone of treatment protocols used for treatment the acute lymphoblastic leukemia in pediatric oncology. A potential actinomycete isolate, Streptomyces sp. strain NEAE-99, produces glutaminase-free L-asparaginase was isolated from a soil sample. This potential strain was identified as S. violaceoruber strain NEAE-99. The central composite design (CCD) approach was utilized for finding the optimal values for four variables including the mixture of soybean and wheat bran in a 1:1 ratio (w/w), the concentrations of dextrose, L-asparagine, and potassium nitrate under solid state fermentation conditions. Through the use of an artificial neural network (ANN), the production of L-asparaginase by S. violaceoruber has been investigated, validated, and predicted in comparison to CCD. It was found that the optimal predicted conditions for maximum L-asparaginase production (216.19 U/gds) were 8.46 g/250 mL Erlenmeyer flask of soybean and wheat bran mixture in a 1:1 ratio (w/w), 2.2 g/L of dextrose, 18.97 g/L of L-asparagine, and 1.34 g/L of KNO 3 . The experimental results (207.55 U/gds) closely approximated the theoretical values (216.19 U/gds), as evidenced by the validation. This suggests that the ANN exhibited a high degree of precision and predictive capability.

l-asparaginase is an enzyme used as treatment for acute lymphoblastic leukemia (ALL) due to its ability to hydrolyze l-asparagine, an essential amino acid synthesized by normal cells unlike neoplastic cells. The adverse effects of l-asparaginase formulations are associated with its glutaminase activity and bacterial origin; therefore, it is important to find new sources of l-asparaginase-producing eukaryotic microorganisms with low glutaminase activity. This work evaluated the biotechnological potential of filamentous fungi isolated from Brazilian Savanna soil and plants for l-asparaginase production. Thirty-nine isolates were screened for enzyme production using the plate assay, followed by measuring enzymatic activity in cells after submerged fermentation. The variables influencing l-asparaginase production were evaluated using Plackett–Burman design. Cell disruption methods were evaluated for l-asparaginase release. Penicillium sizovae 2DSST1 and Fusarium proliferatum DCFS10 showed the highest l-asparaginase activity levels and the lowest glutaminase activity levels. Penicillium sizovae l-asparaginase was repressed by carbon sources, whereas higher carbon concentrations enhanced l-asparaginase by F. proliferatum. Maximum enzyme productivity, specific enzyme yield and the biomass conversion factor in the enzyme increased after Plackett–Burman design. Freeze-grinding released 5-fold more l-asparaginase from cells than sonication. This study shows two species, which have not yet been reported, as sources of l-asparaginase with possible reduced immunogenicity for ALL therapy.

In a mouse model of breast cancer, asparagine bioavailability strongly influences metastasis and this is correlated with the production of proteins that regulate the epithelial-to-mesenchymal transition, which provides at least one potential mechanism for how a single amino acid could regulate metastatic progression. Tumour spread assisted by asparagine Not all cells that derive from a primary tumour contribute to metastasis—the progression of cancer into other parts of the body. Gregory Hannon and colleagues find determinants of metastatic potential in a mouse model of breast cancer. They find that expression of the metabolic enzyme asparagine synthetase is associated with metastasis formation. Decreasing asparagine availability by treatment with ʟ-asparaginase or by dietary restriction reduced the development of metastasis. The authors show that asparagine availability promotes an epithelial-to-mesenchymal transition, a process that has been linked to metastasis. Using a functional model of breast cancer heterogeneity, we previously showed that clonal sub-populations proficient at generating circulating tumour cells were not all equally capable of forming metastases at secondary sites 1 . A combination of differential expression and focused in vitro and in vivo RNA interference screens revealed candidate drivers of metastasis that discriminated metastatic clones. Among these, asparagine synthetase expression in a patient’s primary tumour was most strongly correlated with later metastatic relapse. Here we show that asparagine bioavailability strongly influences metastatic potential. Limiting asparagine by knockdown of asparagine synthetase, treatment with l -asparaginase, or dietary asparagine restriction reduces metastasis without affecting growth of the primary tumour, whereas increased dietary asparagine or enforced asparagine synthetase expression promotes metastatic progression. Altering asparagine availability in vitro strongly influences invasive potential, which is correlated with an effect on proteins that promote the epithelial-to-mesenchymal transition. This provides at least one potential mechanism for how the bioavailability of a single amino acid could regulate metastatic progression.

Pegaspargase (Oncaspar ® ), a pegylated form of native Escherichia coli -derived l -asparaginase (hereafter referred as E. coli l -asparaginase), is indicated in the USA and EU for the treatment of acute lymphoblastic leukaemia (ALL) as a component of multi-agent chemotherapy in paediatric and adult patients. Relative to E. coli l -asparaginase, pegaspargase has a prolonged circulation time, thereby offering less frequent administration. Moreover, pegylation of E. coli l -asparaginase may diminish the immunogenicity of the enzyme. Based on extensive evidence, intramuscular (IM) or intravenous (IV) administration of pegaspargase as a component of a multi-agent chemotherapy is an effective first-line treatment for paediatric and adult patients with ALL, as well as for the treatment of paediatric and adult patients with ALL and hypersensitivity to E. coli l -asparaginase. Pegaspargase had a manageable tolerability profile in paediatric and adult patients with newly diagnosed ALL, with the most commonly occurring adverse events being generally consistent to that seen with E. coli l -asparaginase. Pegaspargase treatment in patients with relapsed ALL and hypersensitivity to E. coli l -asparaginase had a similar tolerability profile to that observed in patients with newly diagnosed ALL. Given the potentially reduced immunogenicity and more convenient dosage regimen over E. coli l -asparaginase, pegaspargase remains an important and effective treatment option for paediatric and adult patients with ALL, including those with hypersensitivity to E. coli l -asparaginase.

Silent inactivation of L-asparaginase (L-Asp) represents rapid clearance of L-Asp by anti-L-Asp IgG antibodies without clinical symptoms. Measurement of L-Asp activity is the gold standard for diagnosis of silent inactivation, but this test is not commercially available in Japan as of 2023. We evaluated ex vivo and in vivo ammonia production in relation to L-Asp activity. Blood samples from ten adult patients treated with L-Asp were collected to measure ammonia levels and L-Asp activity before the first dose and 24 h after the last dose of L-Asp, during each cycle of treatment. Plasma ammonia levels were analyzed immediately and 1 h after incubation at room temperature, and ex vivo ammonia production was defined as the increase in ammonia concentration. Ex vivo ammonia production correlated with L-Asp activity ( R 2  = 0.741), and ammonia levels measured immediately after blood collection were moderately correlated with L-Asp activity ( R 2  = 0.709). One patient with extranodal NK/T-cell lymphoma showed an increase in ammonia levels during the first cycle, but no increase in ammonia levels or L-Asp activity after L-Asp administration during the second cycle. Both ex vivo and in vivo ammonia production and surrogate markers are used for L-Asp biological activity.