An ultra-long-acting L-asparaginase synergizes with an immune checkpoint inhibitor in starvation-immunotherapy of metastatic solid tumors.

Metastasis stands as the primary contributor to mortality associated with tumors. Chemotherapy and immunotherapy are frequently utilized in the management of metastatic solid tumors. Nevertheless, these therapeutic modalities are linked to serious adverse effects and limited effectiveness in preventing metastasis. Here, we report a novel therapeutic strategy named starvation-immunotherapy, wherein an immune checkpoint inhibitor is combined with an ultra-long-acting L-asparaginase that is a fusion protein comprising L-asparaginase (ASNase) and an elastin-like polypeptide (ELP), termed ASNase-ELP. ASNase-ELP's thermosensitivity enables it to generate an in-situ depot following an intratumoral injection, yielding increased dose tolerance, improved pharmacokinetics, sustained release, optimized biodistribution, and augmented tumor retention compared to free ASNase. As a result, in murine models of oral cancer, melanoma, and cervical cancer, the antitumor efficacy of ASNase-ELP by selectively and sustainably depleting L-asparagine essential for tumor cell survival was substantially superior to that of ASNase or Cisplatin, a first-line anti-solid tumor medicine, without any observable adverse effects. Furthermore, the combination of ASNase-ELP and an immune checkpoint inhibitor was more effective than either therapy alone in impeding melanoma metastasis. Overall, the synergistic strategy of starvation-immunotherapy holds excellent promise in reshaping the therapeutic landscape of refractory metastatic tumors and offering a new alternative for next-generation oncology treatments.

Identification of a thermostable L-asparaginase from Pyrococcus yayanosii CH1 and its application in the reduction of acrylamide.

L-asparaginase (ASNase, E.C. 3.5.1.1) catalyzes the deamination of L-asparagine to L-aspartic acid and ammonia and is widely used in medicine to treat acute lymphocytic leukemia. It also has significant applications in the food industry by inhibiting acrylamide formation. In this study, we characterized a thermostable ASNase from the hyper thermophilic strain, Pyrococcus yayanosii CH1. The recombinant enzyme (PyASNase) exhibited maximal activity at pH 8.0 and 85 °C. Moreover, PyASNase demonstrated promising thermostability across temperatures ranging from 70 to 95 °C. The kinetic parameters of PyASNase for L-asparagine were a Km of 6.3 mM, a kcat of 1989s-1, and a kcat/Km of 315.7 mM-1 s-1. Treating potato samples with 10 U/mL of PyASNase at 85 °C for merely 10 min reduced the acrylamide content in the final product by 82.5%, demonstrating a high efficiency and significant advantage of PyASNase in acrylamide inhibition.

Expression, characterization and cytotoxicity of recombinant l-asparaginase II from Salmonella paratyphi cloned in Escherichia coli.

L-asparaginase is a remarkable antineoplastic enzyme used in medicine for the treatment of acute lymphoblastic leukemia (ALL) as well as in food industries. In this work, the L-asparaginase-II gene from Salmonella paratyphi was codon-optimized, cloned, and expressed in E. coli as a His-tag fusion protein. Then, using a two-step chromatographic procedure it was purified to homogeneity as confirmed by SDS-PAGE, which also showed its monomeric molecular weight to be 37 kDa. This recombinant L-asparaginase II from Salmonella paratyphi (recSalA) was optimally active at pH 7.0 and 40 °C temperature. It was highly specific for L-asparagine as a substrate, while its glutaminase activity was low. The specific activity was found to be 197 U/mg and the kinetics elements Km, Vmax, and kcat were determined to be 21 mM, 28 µM/min, and 39.6 S-1, respectively. Thermal stability was assessed using a spectrofluorometer and showed Tm value of 45 °C. The in-vitro effects of recombinant asparaginase on three different human cancerous cell lines (MCF7, A549 and Hep-2) by MTT assay showed remarkable anti-proliferative activity. Moreover, recSalA exhibited significant morphological changes in cancer cells and IC50 values ranged from 28 to 45.5 µg/ml for tested cell lines. To investigate the binding mechanism of SalA, both substrates L-asparagine and l-glutamine were docked with the protein and the binding energy was calculated to be -4.2 kcal mol-1 and - 4.4 kcal mol-1, respectively. In summary, recSalA has significant efficacy as an anticancer agent with potential implications in oncology while its in-vivo validation needs further investigation.

Comprehensive insight into exploring the potential of microbial enzymes in cancer therapy: Progress, challenges, and opportunities: A review.

Microbial enzymes are crucial catalysts in various industries due to their versatility and efficiency. The microbial enzymes market has recently expanded due to increased demand for many reasons. Among them are eco-friendly solutions, developing novel microbial strains with enhanced enzymes that perform under harsh conditions, providing sustainability, and raising awareness about the benefits of enzyme-based products. By 2030, the global enzyme market is expected to account for $525 billion, with a growth rate of 6.7 %. L-asparaginase and L-glutaminase are among the leading applied microbial enzymes in antitumor therapy, with a growing market share of 16.5 % and 9.5 %, respectively. The use of microbial enzymes has opened new opportunities to fight various tumors, including leukemia, lymphosarcoma, and breast cancer, which has increased their demand in the pharmaceutical and medicine sectors. Despite their promising applications, commercial use of microbial enzymes faces challenges such as short half-life, immunogenicity, toxicity, and other side effects. Therefore, this review explores the industrial production, purification, formulation, and commercial utilization of microbial enzymes, along with an overview of the global enzyme market. With ongoing discoveries of novel enzymes and their applications, enzyme technology offers promising avenues for cancer treatment and other therapeutic interventions.

Immobilization of l-asparaginase on genipin cross-linked chitosan beads shows better acrylamide diminution in cassava chips: Process optimization and characterization.

Glutaraldehyde is the conventionally used cross-linker for the activation and cross-linking of support matrices used in enzyme immobilization. However, the toxic nature of glutaraldehyde makes it unsafe for food applications, propelling the need for nontoxic cross-linkers. Genipin reacts with the primary and secondary amines generating a dark-blue colored pigment and is an attractive alternative to glutaraldehyde as a cross-linker for enzyme immobilization. Apart from its excellent cross-linking properties, genipin possesses added advantages over glutaraldehyde such as proven health benefits, biocompatibility, and biodegradability. The present study explores the application of chitosan beads cross-linked with the natural and nontoxic agent, genipin, for immobilizing l-asparaginase, aimed at its subsequent use in mitigating acrylamide formation in food products. The immobilized l-asparaginase exhibited improved functionalities such as stability, reusability, and reduction in acrylamide formation in deep-fried cassava chips. One of the limitations observed during application in the food process was the mechanical fragility of the chitosan beads during speedy stirring. This can be overcome by increasing the concentration and time of contact of the coagulant bath during the formation of chitosan beads. The drying of the enzyme-bound chitosan beads will also lead to shrinkage and prevent breakage during stirring. This study conclusively demonstrated the applicability of immobilizing l-asparaginase on genipin cross-linked chitosan beads in food-related processes.

Kinetics characterization of a low immunogenic recombinant l-asparaginase from Phaseolus vulgaris with cytotoxic activity against leukemia cells.

l-asparaginases play a crucial role in the treatment of acute lymphoblastic leukemia (ALL), a type of cancer that mostly affects children and teenagers. However, it is common for these molecules to cause adverse reactions during treatment. These downsides ignite the search for novel asparaginases to mitigate these problems. Thus, this work aimed to produce and characterize a recombinant asparaginase from Phaseolus vulgaris (Asp-P). In this study, Asp-P was expressed in Escherichia coli with high yields and optimum activity at 40 °C, pH 9.0. The enzyme Km and Vmax values were 7.05 mM and 1027 U/mg, respectively. Asp-P is specific for l-asparagine, showing no activity against l-glutamine and other amino acids. The enzyme showed a higher cytotoxic effect against Raji than K562 cell lines, but only at high concentrations. In silico analysis indicated that Asp-P has lower immunogenicity than a commercial enzyme. Asp-P induced biofilm formation by Candida sp. due to sublethal dose, showing an underexplored potential of asparaginases. The absence of glutaminase activity, lower immunogenicity and optimal activity similar to physiological temperature conditions are characteristics that indicate Asp-P as a potential new commercial enzyme in the treatment of ALL and its underexplored application in the treatment of other diseases.

Mitigation of acrylamide in fried food systems using a combination of zein-pectin hydrocolloid complex and a food-grade l-asparaginase.

Acrylamide, a Maillard reaction product, formed in fried food poses a serious concern to food safety due to its neurotoxic and carcinogenic nature. A "Green Approach" using L-Asparaginase enzyme from GRAS-status bacteria synergized with hydrocolloid protective coating could be effective in inhibiting acrylamide formation. To fill this void, the present study reports a new variant of type-II L-asparaginase (AsnLb) from Levilactobacillus brevis NKN55, a food-grade bacterium isolated using a unique metabolite profiling approach. The recombinant AsnLb enzyme was characterized to study acrylamide inhibition ability and showed excellent specificity towards L-asparagine (157.2 U/mg) with Km, Vmax of 0.833 mM, 4.12 mM/min respectively. Pretreatment of potato slices with AsnLb (60 IU/mL) followed by zein-pectin nanocomplex led to >70% reduction of acrylamide formation suggesting synergistic effect of this dual component system. The developed strategy can be employed as a sustainable treatment method by food industries for alleviating acrylamide formation and associated health hazard in fried foods.

Bacterial cellulose films for L-asparaginase delivery to melanoma cells.

L-asparaginase (L-ASNase) is an enzyme that catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia and is used to treat acute lymphoblastic leukemia. It is also toxic to the cells of some solid tumors, including melanoma cells. Immobilization of this enzyme can improve its activity against melanoma tumor cells. In this work, the properties of bacterial cellulose (BC) and feasibility of BC films as a new carrier for immobilized L-ASNase were investigated. Different values of growth time were used to obtain BC films with different thicknesses and porosities, which determine the water content and the ability to adsorb and release L-ASNase. Fourier transform infrared spectroscopy confirmed the adsorption of the enzyme on the BC films. The total activity of adsorbed L-ASNase and its release were investigated for films grown for 48, 72 or 96 h. BC films grown for 96 h showed the most pronounced release as described by zero-order and Korsmayer-Peppas models. The release was characterized by controlled diffusion where the drug was released at a constant rate. BC films with immobilized L-ASNase could induce cytotoxicity in A875 human melanoma cells. With further development, immobilization of L-ASNase on BC may become a potent strategy for anticancer drug delivery to superficial tumors.

Towards a dependable data set of structures for L-asparaginase research.

The Protein Data Bank (PDB) includes a carefully curated treasury of experimentally derived structural data on biological macromolecules and their various complexes. Such information is fundamental for a multitude of projects that involve large-scale data mining and/or detailed evaluation of individual structures of importance to chemistry, biology and, most of all, to medicine, where it provides the foundation for structure-based drug discovery. However, despite extensive validation mechanisms, it is almost inevitable that among the ∼215 000 entries there will occasionally be suboptimal or incorrect structure models. It is thus vital to apply careful verification procedures to those segments of the PDB that are of direct medicinal interest. Here, such an analysis was carried out for crystallographic models of L-asparaginases, enzymes that include approved drugs for the treatment of certain types of leukemia. The focus was on the adherence of the atomic coordinates to the rules of stereochemistry and their agreement with the experimental electron-density maps. Whereas the current clinical application of L-asparaginases is limited to two bacterial proteins and their chemical modifications, the field of investigations of such enzymes has expanded tremendously in recent years with the discovery of three entirely different structural classes and with numerous reports, not always quite reliable, of the anticancer properties of L-asparaginases of different origins.

The Improved Antineoplastic Activity of Thermophilic L-Asparaginase Tli10209 via Site-Directed Mutagenesis.

Amino acid deprivation therapy (AADT) is a novel anticancer therapy, considered nontoxic and selective. Thermophilic L-asparaginase enzymes display high stability and activity at elevated temperatures. However, they are of limited use in clinical applications because of their low substrate affinity and reduced activity under physiological conditions, which may necessitate an improved dosage, leading to side effects and greater costs. Thus, in an attempt to improve the activity of L-Asn at 37 °C, with the use of a semi-rational design, eight active-site mutants of Thermococcus litoralis DSM 5473 L-asparaginase Tli10209 were developed. T70A exhibited a 5.11-fold increase compared with the wild enzyme in physiological conditions. Double-mutant enzymes were created by combining mutants with higher hydrolysis activity. T70A/F36Y, T70A/K48L, and T70A/D50G were enhanced by 5.59-, 6.38-, and 5.58-fold. The immobilized enzyme applied in MCF-7 breast cancer cells only required one-seventh of the dose of the free enzyme to achieve the same inhibition rate under near-infrared irradiation. This provides a proof of concept that it is possible to reduce the consumption of L-Asn by improving its activity, thus providing a method to manage side effects.

A novel type IIb L-asparaginase from Latilactobacillus sakei LK-145: characterization and application.

We succeeded in homogeneously expressing and purifying L-asparaginase from Latilactobacillus sakei LK-145 (Ls-Asn1) and its mutated enzymes C196S, C264S, C290S, C196S/C264S, C196S/C290S, C264S/C290S, and C196S/C264S/C290S-Ls-Asn1. Enzymological studies using purified enzymes revealed that all cysteine residues of Ls-Asn1 were found to affect the catalytic activity of Ls-Asn1 to varying degrees. The mutation of Cys196 did not affect the specific activity, but the mutation of Cys264, even a single mutation, significantly decreased the specific activity. Furthermore, C264S/C290S- and C196S/C264S/C290S-Ls-Asn1 almost completely lost their activity, suggesting that C290 cooperates with C264 to influence the catalytic activity of Ls-Asn1. The detailed enzymatic properties of three single-mutated enzymes (C196S, C264S, and C290S-Ls-Asn1) were investigated for comparison with Ls-Asn1. We found that only C196S-Ls-Asn1 has almost the same enzymatic properties as that of Ls-Asn1 except for its increased stability for thermal, pH, and the metals NaCl, KCl, CaCl2, and FeCl2. We measured the growth inhibitory effect of Ls-Asn1 and C196S-Ls-Asn1 on Jurkat cells, a human T-cell acute lymphoblastic leukemia cell line, using L-asparaginase from Escherichia coli K-12 as a reference. Only C196S-Ls-Asn1 effectively and selectively inhibited the growth of Jurkat T-cell leukemia, which suggested that it exhibited antileukemic activity. Furthermore, based on alignment, phylogenetic tree analysis, and structural modeling, we also proposed that Ls-Asn1 is a so-called "Type IIb" novel type of asparaginase that is distinct from previously reported type I or type II asparaginases. Based on the above results, Ls-Asn1 is expected to be useful as a new leukemia therapeutic agent.

A Structural In Silico Analysis of the Immunogenicity of L-Asparaginase from Penicillium cerradense.

L-asparaginase is an essential drug used to treat acute lymphoid leukemia (ALL), a cancer of high prevalence in children. Several adverse reactions associated with L-asparaginase have been observed, mainly caused by immunogenicity and allergenicity. Some strategies have been adopted, such as searching for new microorganisms that produce the enzyme and applying protein engineering. Therefore, this work aimed to elucidate the molecular structure and predict the immunogenic profile of L-asparaginase from Penicillium cerradense, recently revealed as a new fungus of the genus Penicillium and producer of the enzyme, as a motivation to search for alternatives to bacterial L-asparaginase. In the evolutionary relationship, L-asparaginase from P. cerradense closely matches Aspergillus species. Using in silico tools, we characterized the enzyme as a protein fragment of 378 amino acids (39 kDa), including a signal peptide containing 17 amino acids, and the isoelectric point at 5.13. The oligomeric state was predicted to be a homotetramer. Also, this L-asparaginase presented a similar immunogenicity response (T- and B-cell epitopes) compared to Escherichia coli and Dickeya chrysanthemi enzymes. These results suggest a potentially useful L-asparaginase, with insights that can drive strategies to improve enzyme production.

Substrate Affinity Is Not Crucial for Therapeutic L-Asparaginases: Antileukemic Activity of Novel Bacterial Enzymes.

L-asparaginases are used in the treatment of acute lymphoblastic leukemia. The aim of this work was to compare the antiproliferative potential and proapoptotic properties of novel L-asparaginases from different structural classes, viz. EcAIII and KpAIII (class 2), as well as ReAIV and ReAV (class 3). The EcAII (class 1) enzyme served as a reference. The proapoptotic and antiproliferative effects were tested using four human leukemia cell models: MOLT-4, RAJI, THP-1, and HL-60. The antiproliferative assay with the MOLT-4 cell line indicated the inhibitory properties of all tested L-asparaginases. The results from the THP-1 cell models showed a similar antiproliferative effect in the presence of EcAII, EcAIII, and KpAIII. In the case of HL-60 cells, the inhibition of proliferation was observed in the presence of EcAII and KpAIII, whereas the proliferation of RAJI cells was inhibited only by EcAII. The results of the proapoptotic assays showed individual effects of the enzymes toward specific cell lines, suggesting a selective (time-dependent and dose-dependent) action of the tested L-asparaginases. We have, thus, demonstrated that novel L-asparaginases, with a lower substrate affinity than EcAII, also exhibit significant antileukemic properties in vitro, which makes them interesting new drug candidates for the treatment of hematological malignancies. For all enzymes, the kinetic parameters (Km and kcat) and thermal stability (Tm) were determined. Structural and catalytic properties of L-asparaginases from different classes are also summarized.

L-Asparaginase Conjugates from the Hyperthermophilic Archaea Thermococcus sibiricus with Improved Biocatalytic Properties.

This study investigated the effect of polycationic and uncharged polymers (and oligomers) on the catalytic parameters and thermostability of L-asparaginase from Thermococcus sibiricus (TsA). This enzyme has potential applications in the food industry to decrease the formation of carcinogenic acrylamide during the processing of carbohydrate-containing products. Conjugation with the polyamines polyethylenimine and spermine (PEI and Spm) or polyethylene glycol (PEG) did not significantly affect the secondary structure of the enzyme. PEG contributes to the stabilization of the dimeric form of TsA, as shown by HPLC. Furthermore, neither polyamines nor PEG significantly affected the binding of the L-Asn substrate to TsA. The conjugates showed greater maximum activity at pH 7.5 and 85 °C, 10-50% more than for native TsA. The pH optima for both TsA-PEI and TsA-Spm conjugates were shifted to lower pH ranges from pH 10 (for the native enzyme) to pH 8.0. Additionally, the TsA-Spm conjugate exhibited the highest activity at pH 6.5-9.0 among all the samples. Furthermore, the temperature optimum for activity at pH 7.5 shifted from 90-95 °C to 80-85 °C for the conjugates. The thermal inactivation mechanism of TsA-PEG appeared to change, and no aggregation was observed in contrast to that of the native enzyme. This was visually confirmed and supported by the analysis of the CD spectra, which remained almost unchanged after heating the conjugate solution. These results suggest that TsA-PEG may be a more stable form of TsA, making it a potentially more suitable option for industrial use.

Improved L-Asparaginase Properties and Reusability by Immobilization onto Functionalized Carbon Xerogels.

Enzyme immobilization can offer a range of significant advantages, including reusability, and increased selectivity, stability, and activity. In this work, a central composite design (CCD) of experiments and response surface methodology (RSM) were used to study, for the first time, the L-asparaginase (ASNase) immobilization onto functionalized carbon xerogels (CXs). The best results were achieved using CXs obtained by hydrothermal oxidation with nitric acid and subsequent heat treatment in a nitrogen flow at 600 °C (CX-OX-600). Under the optimal conditions (81 min of contact time, pH 6.2 and 0.36 g/L of ASNase), an immobilization yield (IY) of 100 % and relative recovered activity (RRA) of 103 % were achieved. The kinetic parameters obtained also indicate a 1.25-fold increase in the affinity of ASNase towards the substrate after immobilization. Moreover, the immobilized enzyme retained 97 % of its initial activity after 6 consecutive reaction cycles. All these outcomes confirm the promising properties of functionalized CXs as support for ASNase, bringing new insights into the development of an efficient and stable immobilization platform for use in the pharmaceutical industry, food industry, and biosensors.

RrA, an enzyme from Rhodospirillum rubrum, is a prototype of a new family of short-chain L-asparaginases.

L-Asparaginases (ASNases) catalyze the hydrolysis of L-Asn to L-Asp and ammonia. Members of the ASNase family are used as drugs in the treatment of leukemia, as well as in the food industry. The protomers of bacterial ASNases typically contain 300-400 amino acids (typical class 1 ASNases). In contrast, the chain of ASNase from Rhodospirillum rubrum, reported here and referred to as RrA, consists of only 172 amino acid residues. RrA is homologous to the N-terminal domain of typical bacterial class 1 ASNases and exhibits millimolar affinity for L-Asn. In this study, we demonstrate that RrA belongs to a unique family of cytoplasmic, short-chain ASNases (scASNases). These proteins occupy a distinct region in the sequence space, separate from the regions typically assigned to class 1 ASNases. The scASNases are present in approximately 7% of eubacterial species, spanning diverse bacterial lineages. They seem to be significantly enriched in species that encode for more than one class 1 ASNase. Here, we report biochemical, biophysical, and structural properties of RrA, a member of scASNases family. Crystal structures of the wild-type RrA, both with and without bound L-Asp, as well as structures of several RrA mutants, reveal topologically unique tetramers. Moreover, the active site of one protomer is complemented by two residues (Tyr21 and Asn26) from another protomer. Upon closer inspection, these findings clearly outline scASNases as a stand-alone subfamily of ASNases that can catalyze the hydrolysis of L-Asn to L-Asp despite the lack of the C-terminal domain that is present in all ASNases described structurally to date.

Homology modeling and molecular docking studies to decrease glutamine affinity of Yarrowia lipolytica L-asparaginase.

L-Asparaginase is a key component in the treatment of leukemias and lymphomas. However, the glutamine affinity of this therapeutic enzyme is an off-target activity that causes several side effects. The modeling and molecular docking study of Yarrowia lipolytica L-asparaginase (YL-ASNase) to reduce its l-glutamine affinity and increase its stability was the aim of this study. Protein-ligand interactions of wild-type and different mutants of YL-ASNase against L-asparagine compared to l-glutamine were assessed using AutoDock Vina tools because the crystal structure of YL-ASNase does not exist in the protein data banks. The results showed that three mutants, T171S, T171S-N60A, and T171A-T223A, caused a considerable increase in L-asparagine affinity and a decrease in l-glutamine affinity as compared to the wild-type and other mutants. Then, molecular dynamics simulation and MM/GBSA free energy were applied to assess the stability of protein structure and its interaction with ligands. The three mutated proteins, especially T171S-N60A, had higher stability and interactions with L-asparagine than l-glutamine in comparison with the wild-type. The YL-ASNase mutants could be introduced as appropriate therapeutic candidates that might cause lower side effects. However, the functional properties of these mutated enzymes need to be confirmed by genetic manipulation and in vitro and in vivo studies.

Structural and functional analyses of an L-asparaginase from Geobacillus thermopakistaniensis.

Genome sequence of Geobacillus thermopakistaniensis contains an open reading frame annotated as a type II L-asparaginase (ASNaseGt). Critical structural analysis disclosed that ASNaseGt might be a type I L-asparaginase. In order to determine whether it is a type I or type II L-asparaginase, we have performed the structural-functional characterization of the recombinant protein as well as analyzed the localization of ASNaseGt in G. thermopakistaniensis. ASNaseGt exhibited optimal activity at 52 °C and pH 9.5. There was a > 3-fold increase in activity in the presence of ß-mercaptoethanol. Apparent Vmax and Km values were 2735 U/mg and 0.35 mM, respectively. ASNaseGt displayed high thermostability with >80 % residual activity even after 6 h of incubation at 55 °C. Recombinant ASNaseGt existed in oligomeric form. Addition of ß-mercaptoethanol lowered the degree of oligomerization and displayed that tetrameric form was the most active, with a specific activity of 4300 U/mg. Under physiological conditions, ASNaseGt displayed >50 % of the optimal activity. Localization studies in G. thermopakistaniensis revealed that ASNaseGt is a cytosolic protein. Structural and functional characterization, and localization in G. thermopakistaniensis displayed that ASNaseGt is not a type II but a type I L-asparaginase.

Evaluation of the efficiency of thermostable L-asparaginase from B. licheniformis UDS-5 for acrylamide mitigation during preparation of French fries.

A thermostable L-asparaginase was produced from Bacillus licheniformis UDS-5 (GenBank accession number, OP117154). The production conditions were optimized by the Plackett Burman method, followed by the Box Behnken method, where the enzyme production was enhanced up to fourfold. It secreted L-asparaginase optimally in the medium, pH 7, containing 0.5% (w/v) peptone, 1% (w/v) sodium chloride, 0.15% (w/v) beef extract, 0.15% (w/v) yeast extract, 3% (w/v) L-asparagine at 50 °C for 96 h. The enzyme, with a molecular weight of 85 kDa, was purified by ion exchange chromatography and size exclusion chromatography with better purification fold and percent yield. It displayed optimal catalysis at 70 °C in 20 mM Tris-Cl buffer, pH 8. The purified enzyme also exhibited significant salt tolerance too, making it a suitable candidate for the food application. The L-asparaginase was employed at different doses to evaluate its ability to mitigate acrylamide, while preparing French fries without any prior treatment. The salient attributes of B. licheniformis UDS-5 L-asparaginase, such as greater thermal stability, salt stability and acrylamide reduction in starchy foods, highlights its possible application in the food industry.

Immobilization of l-asparaginase on chitosan nanoparticles for the purpose of long-term application.

Asparaginase holds significant commercial value as an enzyme in the food and pharmaceutical industries. This study examined the optimum and practical use of the l-asparaginase derived from Pseudomonas aeruginosa HR03. Specifically, the study focused on the effectiveness of the stabilized enzyme when applied to chitosan nanoparticles. The structure, size, and morphology of chitosan nanoparticles were evaluated in relation to the immobilization procedure. This assessment involved the use of several analytical techniques, including FT-IR, DLS, SEM, TEM, and EDS analysis. Subsequently, the durability of the enzyme that has been stabilized was assessed by evaluating its effectiveness under extreme temperatures of 60 and 70 °C, as well as at pH values of 3 and 12. The findings indicate that incorporating chitosan nanoparticles led to enhanced immobilization of the l-asparaginase enzyme. This improvement was observed in terms of long-term stability, stability under crucial temperature and pH conditions, as well as thermal stability. In addition, the optimum temperature increased from 40 to 50 °C, and the optimum pH increased from 8 to 9. Enzyme immobilization led to an increase in Km and a decrease in kcat compared to its free counterpart. Because of its enhanced long-term stability, l-asparaginase immobilization on chitosan nanoparticles may be a potential choice for use in industries that rely on l-asparaginase enzymes, particularly the pharmaceutical and food industries.