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An important enzyme for both the pharmaceutical and food industries is L-asparaginase (EC 3.5.1.1). This enzyme is produced by a wide variety of microorganisms. However, their potential use as sources of L-asparaginase at industrial scale is limited if glutaminase and urease are also produced. This is mainly due to the complexity and expenses of the purification process required to obtain l-asparaginase which make the production system inefficient. In order to select L-asparaginase highly producing strains, lacking detectable glutaminase and urease activity different isolation steps were established and rapid tests are the first recommended. However such studies need to be completed with quantitative analysis. Moreover, additional molecular studies can also confer useful information regarding the biotechnological potential of the selected strains. The aim of this paper is to correlate the microbiologic and biochemical tests with genetic approaches in order to improve the selection process of biotechnologically relevant Bacillus strains. [ABSTRACT FROM AUTHOR]

Type I L-asparaginase from Bacillus licheniformis Z-1 (BlAase) was efficiently produced and secreted in Bacillus subtilis RIK 1285, but its low yield made it unsuitable for industrial use. Thus, a combined method was used in this study to boost BlAase synthesis in B. subtilis. First, fifteen single strong promoters were chosen to replace the original promoter P43, with PyvyD achieving the greatest BlAase activity (436.28 U/mL). Second, dual-promoter systems were built using four promoters (PyvyD, P43, PaprE, and PspoVG) with relatively high BlAase expression levels to boost BlAase output, with the engine of promoter PaprE-PyvyD reaching 502.11 U/mL. The activity of BlAase was also increased (568.59 U/mL) by modifying key portions of the PaprE-PyvyD promoter. Third, when the ribosome binding site (RBS) sequence of promoter PyvyD was replaced, BlAase activity reached 790.1 U/mL, which was 2.27 times greater than the original promoter P43 strain. After 36 h of cultivation, the BlAase expression level in a 10 L fermenter reached 2163.09 U/mL, which was 6.2 times greater than the initial strain using promoter P43. Moreover, the application potential of BlAase on acrylamide migration in potato chips was evaluated. Results showed that 89.50% of acrylamide in fried potato chips could be removed when combined with blanching and BlAase treatment. These findings revealed that combining transcription and translation techniques are effective strategies to boost recombinant protein output, and BlAase can be a great candidate for controlling acrylamide in food processing. [ABSTRACT FROM AUTHOR]

This manuscript describes enhancement of soluble production, auto-cleavage analysis and assessment of acrylamide mitigation potential of Tk2246, a plant-type L-asparaginase from Thermococcus kodakarensis. The gene encoding Tk2246 was cloned and expressed in Escherichia coli. Recombinant Tk2246 was produced mainly in insoluble form. Various strategies were utilized to enhance the soluble production, which significantly increased the soluble yield. Interestingly, recombinant Tk2246 was produced even without addition of the inducer, though relatively in a lower amount. To our surprise, Tk2246 was produced in partially cleaved form when the inducer was not added in the culture. When applied for acrylamide mitigation, Tk2246 reduced the acrylamide formation more than 80% in French fries, chapati and yeast-leavened bread. In addition to acrylamide mitigation, Tk2246 exhibited antistaling activity without loss of sensory properties of the food. High activity, thermostability and efficient acrylamide reduction capability make Tk2246 a potential candidate for industrial applications. [ABSTRACT FROM AUTHOR]

L-asparaginase (ASNase) is an aminohydrolase currently used in the pharmaceutical and food industries. Enzyme immobilization is an exciting option for both applications, allowing for a more straightforward recovery and increased stability. High surface area and customizable porosity make carbon xerogels (CXs) promising materials for ASNase immobilization. This work describes the influence of contact time, pH, and ASNase concentration on the immobilization yield (IY) and relative recovered activity (RRA) using the Central Composite Design methodology. The most promising results were obtained using CX with an average pore size of 4 nm (CX-4), reaching IY and RRA of 100%. At the optimal conditions (contact time 49 min, pH 6.73, and [ASNase] 0.26 mg·mL−1), the ASNase-CXs biocomposite was characterized and evaluated in terms of kinetic properties and operational, thermal, and pH stabilities. The immobilized ASNase onto CX-4 retained 71% of its original activity after six continuous reaction cycles, showed good thermal stability at 37 °C (RRA of 91% after 90 min), and was able to adapt to both acidic and alkaline environments. Finally, the results indicated a 3.9-fold increase in the immobilized ASNase affinity for the substrate, confirming the potential of CXs as a support for ASNase and as a cost-effective tool for subsequent use in the therapeutic and food sectors. [ABSTRACT FROM AUTHOR]

L-asparaginase is an important enzyme in the pharmaceutical field used as treatment for acute lymphoblastic leukemia due to its ability to hydrolyze L-asparagine, an essential amino acid synthesized by normal cells, but not by neoplastic cells. 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 produced by eukaryotic microorganisms with low glutaminase activity. This work aimed to identify the L-asparaginase gene sequence from Penicillium sizovae, a filamentous fungus isolated from the Brazilian Savanna (Cerrado) soil with low glutaminase activity, and to biosynthesize higher yields of this enzyme in the yeast Komagataella phaffii. The L-asparaginase gene sequence of P. sizovae was identified by homology to L-asparaginases from species of Penicillium of the section Citrina: P. citrinum and P. steckii. Partial L-asparaginase from P. sizovae, lacking the periplasmic signaling sequence, was cloned, and expressed intracellularly with highest enzymatic activity achieved by a MUT+ clone cultured in BMM expression medium; a value 5-fold greater than that obtained by native L-asparaginase in P. sizovae cells. To the best of our knowledge, this is the first literature report of the heterologous production of an L-asparaginase from a filamentous fungus by a yeast. [ABSTRACT FROM AUTHOR]

The use of magnetic nanoparticles (MNPs) in biomedical applications has been wildly opted due to their unique properties. Here, we designed MNPs loaded with erlotinib (ERL/SPION-Val-PEG) and conjugated them with anti-mucin16 (MUC16) aptamer to introduce new image-guided nanoparticles (NPs) for targeted drug delivery as well as non-invasive magnetic resonance imaging (MRI) contrast agents. Also, the combination of our nanosystem (NS) along with L-Asparaginase (L-ASPN) led to synergistic effects in terms of reducing cell viability in ovarian cancer cells, which could suggest a novel combination therapy. The mean size of our NS was about 63.4 ± 3.4 nm evaluated by DLS analysis and its morphology was confirmed using TEM. Moreover, the functional groups, as well as magnetic properties of our NS, were examined by FT-IR and VSM tests, respectively. The loading efficacy of erlotinib on MNPs was about 80% and its release reached 70.85% over 7 days in the pH value of 5.4. The MR images and flow cytometry results revealed that the cellular uptake of ERL/SPION-Val-PEG-MUC16 NPs in cells with MUC16 overexpression was considerably higher than unarmed NPs. In addition, T2-weight MR images of ovarian cancer-bearing mice indicated significant signal intensity changes at the tumour site 4 h after intravenous injection compared to the non-target MNPs. Our data suggest ERL/SPION-Val-PEG NPs as an image-guided co-drug delivery system for ovarian cancer. [ABSTRACT FROM AUTHOR]

In this study, oxidative stress marker (malondialdehyde, MDA) and antioxidant enzymes (glutathione (GSH), catalase (CAT)) levels in the liver and pancreas tissue and the histopathological effects of N-acetylcysteine (NAC) were investigated in l-asparaginase (l-ASP) induced liver and pancreatic damage in rats. Forty male albino rats were divided into four groups. The control group was intraperitoneally injected physiological saline (0.02 mL/g); NAC group was injected NAC (200 mg/kg, five days); l-ASP group was injected single-dose l-ASP (10,000 U/kg), and l-ASP + NAC group was injected NAC for five days following single-dose l-ASP (10,000 U/kg). The surgical operation was performed on all animals on the fifth day. There was no difference between the groups regarding tissue MDA, GSH, and CAT levels (p>.05, for all). In the group receiving NAC after l-ASP, there was a significant improvement in the liver and pancreas damage score than the l-ASP group. NAC was effective in reducing organ damage caused by l-ASP. [ABSTRACT FROM AUTHOR]

Bacterial L-asparaginases are amidohydrolases (EC 3.5.1.1) capable of deaminating L-asparagine and, with reduced efficiency, L-glutamine. Interest in the study of L-asparaginases is driven by their use as biodrugs for the treatment of acute lymphoblastic leukemia. Here, we report for the first time the description of the molecular structure of type II asparaginase from Escherichia coli in complex with its secondary product, L-glutamate. To obtain high-quality crystals, we took advantage of the N24S variant, which has structural and functional features similar to the wild-type enzyme, but improved stability, and which yields more ordered crystals. Analysis of the structure of the N24S-L–glutamate complex (N24S–GLU) and comparison with its apo and L-aspartate-bound form confirmed that the enzyme-reduced catalytic efficiency in the presence of L-glutamine is due to L-glutamine misfitting into the enzyme-binding pocket, which causes a local change in the catalytic center geometry. Moreover, a tight interaction between the two protomers that form the enzyme active site limits the capability of L-glutamine to fit into (and to exit from) the binding pocket of E. coli L-asparaginase, explaining why the enzyme has lower glutaminolytic activity compared to other enzymes of the same family, in particular the Erwinia chrysanthemi one. [ABSTRACT FROM AUTHOR]

L-asparaginase (ASNase) is an aminohydrolase enzyme widely used in the pharmaceutical and food industries. Although currently its main applications are focused on the treatment of lymphoproliferative disorders such as acute lymphoblastic leukemia (ALL) and acrylamide reduction in starch-rich foods cooked at temperatures above 100 °C, its use as a biosensor in the detection and monitoring of L-asparagine levels is of high relevance. ASNase-based biosensors are a promising and innovative technology, mostly based on colorimetric detection since the mechanism of action of ASNase is the catalysis of the L-asparagine hydrolysis, which releases L-aspartic acid and ammonium ions, promoting a medium pH value change followed by color variation. ASNase biosensing systems prove their potential for L-asparagine monitoring in ALL patients, along with L-asparagine concentration analysis in foods, due to their simplicity and fast response.

This study presents a novel and selective method for the determination of l-asparagine in diverse potato varieties under various storage conditions. L-asparagine levels serve as a crucial indicator for acrylamide formation, a hazardous substance in processed potato products. The fluorometric method utilized blue-emitting CDs (B-CDs), orange-emitting CDs (O-CDs), and the enzyme L-asparaginase for ratiometric detection of L-asparagine. Upon enzymatic hydrolysis of L-asparagine by L-asparaginase, liberated ammonia induced a pH increase in the reaction medium. This pH shift enhanced the fluorescence of B-CDs while simultaneously decreasing that of O-CDs, enabling sensitive and selective L-asparagine quantification. Comprehensive characterization of the CDs was performed using various spectroscopic techniques and transmission electron microscopy. The method demonstrated excellent sensitivity (LOD = 0.31 μM) and a wide linear range (1.0–50.0 μM). When the method was applied to potato samples, high recovery values (98.00–100.33 %) with low relative standard deviations (RSDs) were achieved, confirming the accuracy and precision of the method. The approach was employed to determine L-asparagine levels in three potato varieties (Lady Rosetta, Spunta, and Nicola) under different storage temperatures and durations. This method provides a valuable tool for monitoring L-asparagine content in potatoes, potentially aiding in the mitigation of acrylamide formation during processing. The robust performance and simplicity of the proposed technique make it suitable for routine analysis in both research and industrial applications within the potato industry. [Display omitted] • Ratiometric fluorescence detection for L-asparagine quantification. • Enzymatic L-asparaginase approach enables pH-based, selective detection. • Low detection limit (0.31 μM) and wide linear range (1.0–50.0 μM) achieved. • Successfully applied to three potato varieties under various storage conditions. • Potential for mitigating acrylamide formation in processed potatoes. [ABSTRACT FROM AUTHOR]

[Display omitted] • Novel dual carbon dot-based ratiometric fluorescence assay for L-asparaginase. • Wide linear range (20–2000 U L-1) with low detection limit (6.95 U L-1). • pH-dependent opposing fluorescence responses enhance sensitivity. • Excellent selectivity and recovery (96.15–99.75%) in human serum. • Promising method for clinical and food industry L-asparaginase detection. A novel fluorometric method for the determination of L-asparaginase, an enzyme crucial in cancer therapy and food industry applications, is presented. This sensitive and selective approach utilizes L-asparagine and two pH-sensitive carbon dots (blue-N-CDs and red-N-CDs) as probes. The interaction between L-asparagine and L-asparaginase liberates ammonia, causing an increase in pH. This pH change simultaneously decreases the fluorescence of blue-N-CDs while enhancing the emission of red-N-CDs, enabling ratiometric detection of L-asparaginase. Comprehensive characterization of both carbon dots and investigation of their response mechanism towards L-asparaginase were conducted using ultraviolet–visible spectrophotometry, fluorescence spectroscopy, and transmission electron microscopy (TEM) imaging techniques. The designed approach demonstrates outstanding linearity (20 to 2000 U L-1) and a low detection limit (6.95 U L-1) for L-asparaginase quantification. Moreover, when tested to human serum samples, the detection system exhibits outstanding selectivity and high recovery rates (96.15% to 99.75%) with low standard deviation, underscoring its suitability for practical implementation in clinical diagnostics. [ABSTRACT FROM AUTHOR]

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. The reversible thermosensitivity of L-asparaginase (ASNase) fused to an elastin-like polypeptide (ASNase-ELP) allows for forming a pharmaceutical depot that exhibits zero-order release kinetics following a single intra-tumoral administration. This depot effectively and selectively sustains L-asparagine (Asn) catabolism, thereby inducing tumor cells to enter a state of starvation. Simultaneously, anti-programmed cell death protein-1 antibody (aPD-1) inhibits the binding of PD-1 to its corresponding receptor, PD-L1, and enhances the recognition of major histocompatibility complex class I (MHC-I) molecules by the T-cell receptor (TCR). Consequently, aPD-1 elicits a robust immune response against the tumor cells, synergistically enhancing the anti-metastatic efficacy in conjunction with the starvation mechanism. [Display omitted] [ABSTRACT FROM AUTHOR]

Data on optimization of a laboratory method for determining the level of L-asparaginase in blood serum are presented. It is shown that the method allows quantification of L-asparaginase activity in patient blood serum in the range of enzyme activity 19.531 – 1250 U/L using the same calibration curve for high (>78.125 U/L) and low values of enzyme activity (19.531 – 78.125 U/L), which leads to significant acceleration of the analysis procedure at almost the same measurement accuracy. Validation of the conformity of measurements between the modified and original method using a comparative analysis of calibration curves built under the conditions of the corresponding protocols showed that the quantitative determinations had comparable accuracy. The results can be successfully used for drug monitoring in oncological and oncohematological practice. [ABSTRACT FROM AUTHOR]

Abstract L-Asparaginase (L-ASNase) is a biopharmaceutical used for acute lymphoblastic leukaemia (ALL) treatment, dramatically increasing the patients’ chance of cure. However, its production and distribution in developing countries were disrupted because of its low profitability, which caused great concern among patients. This study evaluates the feasibility of combining fractional precipitation and aqueous two-phase systems (ATPS) to purify L-ASNase from a low-grade product, commercially known as Acrylaway® L. The ATPS purification results were not particularly expressive compared to the two-step purification process composed of ethanol precipitation and gel filtration, which was able to recover the target molecule with a purification factor over 5 fold. Thus, we studied a purification process capable of manufacturing pharmaceutical grade L-ASNase from a commercially available low-grade raw material; however, improvements regarding its throughput must be achieved, and high purity is the first step to apply it as a new biopharmaceutical product. The proposed process could pose as a short-time solution to mitigate its shortage while a cost-effective production plant is being developed.

Endophytic fungi have been in the spotlight as a reservoir of novel agents with diverse bioactivities. Similarity in chemical diversity with the host plant makes them an amenable target for industrial interventions. A wide range of compounds as secondary metabolites and enzymes are manufactured inside the endophytic fungal factory. However, utilization of endophytic fungi as industrially imperative enzyme producers has been a scarce event. The present study was conducted to bio-prospect the fungal endophytes present in the high altitude medicinal plants of Uttarakhand, as industrially imperative enzyme producers. A total of 58 different endophytic isolates were obtained from Pinus sabiniana, Cinnamomum tamala, Cinnamomum verum, Ocimum tenuiflorum and Rhododendron arboreum. Endophytic fungal colonization was highest, 31%, in Pinus sabiniana. The pure isolates were further explored for the production of amylases, cellulases, proteases and L-asparaginase. Out of 58 isolates, 40 isolates exhibited potent enzyme productivity. #7PSSTB isolate was considered as a superlative contender on account of its relatively higher production of all the three enzymes viz. amylases, cellulases, proteases. Partial purification of #7PSSTB extract showed compelling enzymatic activity corroborating the existence of exogenous enzyme in the extract. Interestingly, #9 RASTB, #11 RASTB and #17 RASTB exhibited the production of therapeutically imperative L-asparaginase enzyme. The present study puts the spotlight on endophytic diversity in the high altitude medicinal plants as a source of enzymes of industrial interest. Production of L-asparaginase paves the way of pharmaceutical intervention to explore anti-oncogenic effects in the endophytic fungal repository of high altitude regions.

Cancer is among the leading causes of death worldwide, with no effective and safe treatment to date. This study is the first to co-conjugate the natural compound cinchonain Ia, which has promising anti-inflammatory activity, and L-asparaginase (ASNase), which has anticancer potential, to manufacture nanoliposomal particles (CALs). The CAL nanoliposomal complex had a mean size of approximately 118.7 nm, a zeta potential of −47.00 mV, and a polydispersity index (PDI) of 0.120. ASNase and cinchonain Ia were encapsulated into liposomes with approximately 93.75% and 98.53% efficiency, respectively. The CAL complex presented strong synergistic anticancer potency, with a combination index (CI) < 0.32 in two-dimensional culture and 0.44 in a three-dimensional model, as tested on NTERA-2 cancer stem cells. Importantly, the CAL nanoparticles demonstrated outstanding antiproliferative efficiency on cell growth in NTERA-2 cell spheroids, with greater than 30- and 2.5-fold increases in cytotoxic activity compared to either cinchonain Ia or ASNase liposomes, respectively. CALs also presented extremely enhanced antitumor effects, reaching approximately 62.49% tumor growth inhibition. Tumorized mice under CALs treatment showed a survival rate of 100%, compared to 31.2% in the untreated control group (p < 0.01), after 28 days of the experiment. Thus, CALs may represent an effective material for anticancer drug development.

Crosslinked enzyme aggregates of L-asparaginase (A-CLEAs) were prepared using genipin, a non-toxic crosslinker. The process parameters for the preparation of A-CLEAs were optimized using a 3-level factorial design, which predicted the optimized conditions to be 60 mg/mL BSA, 2 mg/mL genipin and a crosslinking time of 4 h. The model which was validated experimentally, resulted in a recovery of 90% activity of L-asparaginase. While the optimum pH shifted to a slightly acidic range, the optimum temperature remained unchanged. A-CLEAs displayed the best pH stability at pH 9 (48%) after 24 h of incubation. At 50 °C, the deactivation rate constant (K d) of A-CLEA decreased by 24%. A-CLEAs showed excellent reusability, retaining 79% of their initial activity after 10 cycles of use. The formation of a dark-blue coloured pigmented A-CLEA and the presence of the carboxymethyl group of genipin in FTIR spectrum confirmed the crosslinking reaction. The increase in the β-sheet content of A-CLEA, correlated with its improved stability. The improved thermostability with regards to structural integrity was well established by TGA-DSC analyses. Morphologically, A-CLEAs showed a highly clustered ball-like appearance. As a model example, the ability of genipin crosslinked A-CLEAs to reduce acrylamide in sweet potato chips was checked. The asparagine content of sweet potato was found to be 2906 ± 15.02 mg/kg as quantified by HPTLC. Analysis of acrylamide by GC-FID showed a 94% reduction in sweet potato chips on contact of sweet potato slices with A-CLEAs for 40 min. [Display omitted] • The non-toxic genipin was used as a crosslinker to form L-asparaginase CLEAs. • First report on preparation of L-asparaginase CLEAs. • CLEAs retained 79% of its initial activity after 10 cycles of use. • CLEAs were used to reduce acrylamide formation in sweet potato chips. [ABSTRACT FROM AUTHOR]

L-asparaginase Rhodospirillum rubrum (RrA) is an enzyme (amidohydrolases; EC 3.5.1.1) that catalyzes the L-asparagine hydrolysis reaction to form L-aspartic acid. Due to the shortcomings of existing L-asparaginases from Esherichia coli (EcA) and Erwinia chrysanthemi (ErA), RrA may turn out to be a new promising drug for the treatment of leukemia. RrA has a low homology with EcA and ErA, which makes the enzyme potentially less immunogenic. RrA has pronounced antitumor activity on a number of leukemia cells. However, there is a need to improve the biocatalytic properties of the enzyme. So, in this study, the RrA conjugates with polyamines with different molecular architectures were developed to regulate the catalytic properties of the enzyme. Linear polyethyleneimine (PEI), branched polyethyleneimine, modified with polyethylene glycol (PEI-PEG), and spermine (Spm) were used to obtain conjugates with RrA. It was discovered by gel permeation chromatography that Spm allows the most active tetrameric form of RrA to be obtained and stabilized. Molecular docking was used to study the binding of spermine to RrA subunits. The activity of the RrA conjugates with Spm and PEI-PEG was 23–30% higher than the native enzyme. The pH optimum of the conjugates shifted from 9.0 to 8.5. The conjugates had higher stability: Spm and PEI-PEG reduced the inactivation constant (kin) more than two-fold upon incubation at 53 °C. The conjugate RrA-PEI-PEG reduced the accessibility of trypsin to the protein surface and reduced kin by eight times. The modification of RrA with polyamines made it possible to obtain enzyme preparations with improved biocatalytic properties. These conjugates represent interest for further study as potential therapeutic agents.

L-asparaginase has proven itself as a potential anti-cancer drug and in the mitigation of acrylamide formation in the food industry. In the present investigation, a novel utilization of niger (Guizotia abyssinica) de-oiled cake as the sole source for the cost-effective production of L-asparaginase was evaluated and compared with different agro-substrates in solid-state fermentation. The substrate provided a favorable C/N content for the L-asparaginase production as evident from the chemical composition (CHNS analysis) of the substrate. The influential process parameters viz; autoclaving time, moisture content, temperature and pH were optimized and modeled using machine-learning based artificial neural network (ANN) and statistical-based response surface methodology (RSM). The maximum enzyme activity of 34.65 ± 2.18 IU/gds was observed at 30.3 min of autoclaving time, 62% moisture content, 30 °C temperature and 6.2 pH in 96 h. A 1.36 fold improvement in enzyme activity was observed on utilizing optimized parameters. In comparison with RSM, the ANN model showed superior prediction with a low mean squared error of 0.072, low root mean squared error of 0.268 and 0.99 value of regression coefficient. The present study demonstrates the novel utilization of inexpensive and readily available agro-industrial waste for the development of cost-effective L-asparaginase production process. [ABSTRACT FROM AUTHOR]

L-asparaginase catalyzes the hydrolysis of l-asparagine to l-aspartic acid and ammonia. It has application in the treatment of acute lymphoblastic leukemia in children, as well as in other malignancies, in addition to its role as a food processing aid for the mitigation of acrylamide formation in the baking industry. Its use in cancer chemotherapy is limited due to problems such as its intrinsic glutaminase activity and associated side effects, leading to an increased interest in the search for novel L-asparaginases without L-glutaminase activity. This study reports the cloning and expression of an L-asparaginase contig obtained from whole metagenome shotgun sequencing of Sardinella longiceps gut microbiota. Purified recombinant glutaminase-free L-asparaginase SlpA was a 74 kDa homodimer, with maximal activity at pH 8 and 30 °C. Km and Vmax of SlpA were determined to be 3.008 mM and 0.014 mM/min, respectively. SlpA displayed cytotoxic activity against K-562 (chronic myeloid leukemia) and MCF-7 (breast cancer) cell lines with IC50 values of 0.3443 and 2.692 U/mL, respectively. SlpA did not show any cytotoxic activity against normal lymphocytes and was proved to be hemocompatible. Pre-treatment of biscuit and bread dough with different concentrations of SlpA resulted in a clear, dose-dependent reduction of acrylamide formation during baking. Key points: • Cloned and expressed L-asparaginase (SlpA) from fish gut microbiota • Purified SlpA displayed good cytotoxicity against K-562 and MCF-7 cell lines • SlpA addition caused a significant reduction of acrylamide formation during baking [ABSTRACT FROM AUTHOR]

Acrylamide, a II A carcinogen, widely exists in fried and baked foods. L-asparaginase can inhibit acrylamide formation in foods, and enzymatic stability is the key to its application. In this study, the Escherichia coli L-asparaginase (ECA) stable variant, D60W/L211R/L310R, was obtained with molecular dynamics (MD) simulation, saturation mutation, and combinatorial mutation, the half-life of which increased to 110 min from 60 min at 50 °C. Furthermore, the working temperature (maintaining the activity above 80%) of mutation expanded from 31 °C–43 °C to 35 °C–55 °C, and the relative activity of mutation increased to 82% from 65% at a pH range of 6–10. On treating 60 U/mL and 100 U/g flour L-asparaginase stable mutant (D60W/L211R/L310R) under uncontrolled temperature and pH, the acrylamide content of potato chips and bread was reduced by 66.9% and 51.7%, which was 27% and 49.9% higher than that of the wild type, respectively. These results demonstrated that the mutation could be of great potential to reduce food acrylamide formation in practical applications. [ABSTRACT FROM AUTHOR]

L-asparaginase (L-asp) is used in the treatment of acute lymphocytic leukemia (ALL). L-asp catalyzes the L-asp into ammonia and L-aspartic acid through deamination. The formed ammonia finds to be toxic to the organism. Therefore, the search for less cytotoxic anti-proliferative L-asp is of great importance. Because of its costeffectiveness, consistency, and easiness of process control, microbial L-asparaginase is chosen over plant or animal enzymes. L-asp enzyme which was studied in the article was isolated from a newly discovered bacteria Myroides gitamensis. The biochemical characteristics and antiproliferative, cytotoxic effect of purified L-asp were studied in order to develop safer biochemically stable anti-leukemia medication. To provide comprehensive information on the characteristics of the isolated L-asparaginase enzyme analytical tools were utilized, including cancer cell lines, brine shrimps, enzyme reactions etc. MOLT-4 and K-562 cell lines showed an IC50 value of 91.41 IU/mL and 77.42 IU/mL, respectively. Cytotoxicity activity of L-asp on Artemia salina showed that at 24 hours (LC50 value of 217.0 µg/mL) and toxic at 48 hours (LC50 value of 126.4µg/ml). [ABSTRACT FROM AUTHOR]

Asparaginases (ASNases) are a large and structurally diverse group of enzymes ubiquitous amongst archaea, bacteria and eukaryotes, that catalyze hydrolysis of asparagine to aspartate and ammonia. Bacterial ASNases are important biopharmaceuticals for the treatment of acute lymphoblastic leukemia, although some patients experience adverse allergic side effects during treatment with these protein therapeutics. ASNases are currently divided into three families: plant‐type ASNases, Rhizobium etli‐type ASNases and bacterial‐type ASNases. This system is outdated as both bacterial‐type and plant‐type families also include archaeal, bacterial and eukaryotic enzymes, each with their own distinct characteristics. Herein, phylogenetic studies allied to tertiary structural analyses are described with the aim of proposing a revised and more robust classification system that considers the biochemical diversity of ASNases. Accordingly, based on distinct peptide domains, phylogenetic data, structural analysis and functional characteristics, we recommend that ASNases now be divided into three new distinct classes containing subgroups according to structural and functional aspects. Using this new classification scheme, 25 ASNases were identified as candidates for future new lead discovery. [ABSTRACT FROM AUTHOR]

L-asparaginases (L-ASNases, EC 3.5.1.1) are a family of enzymes that are widely used for the treatment of lymphoblastic leukemias. L-ASNase from Rhodospirillum rubrum (RrA) has a low molecular weight, low glutaminase activity, and low immunogenicity, making it a promising enzyme for antitumor drug development. In our work, the complex formation and covalent conjugation of the enzyme with synthetic or natural polycationic polymers was studied. Among non-covalent polyelectrolyte complexes (PEC), polyethyleneimine (PEI) yielded the highest effect on RrA, increasing its activity by 30%. The RrA-PEI complex had increased stability to trypsinolysis, with an inactivation constant decrease up to 10-fold compared to that of the native enzyme. The covalent conjugation of RrA with chitosan-PEI, chitosan-polyethylene glycol (chitosan-PEG), and chitosan-glycol resulted in an increase in the specific activity of L-asparagine (up to 30%). RrA-chitosan-PEG demonstrated dramatically (by 60%) increased cytotoxic activity for human chronic myeloma leukemia K562 cells in comparison to the native enzyme. The antiproliferative activity of RrA and its conjugates was significantly higher (up to 50%) than for that of the commercially available EcA at the same concentration. The results of this study demonstrated that RrA conjugates with polycations can become a promising strategy for antitumor drug development. [ABSTRACT FROM AUTHOR]

L-Asparaginase (ASNase) is used in medicine for neoplasms treatment and in food industry for mitigation of acrylamide in high-temperature processed food. In medicine, commercial ASNases have exhibited side effects and l-glutaminase (GLNase) activity affecting the clinical treatment. The aim of this work was to study a novel ASNase from Enterobacter sp. M55 isolated from Maras Salters in Peru, which was purified and biochemically characterised. This ASNase exhibited a Km of 5.71 mM and a Vmax of 0.16 µmol mL--1 min--1, as well as an optimum temperature and pH of 37 °C and 6, respectively. Moreover, a good activity (80 %) was observed at physiological pH. Likewise, the enzyme increased its activity by around 50% in presence of urea, glutathione, and glucose. Whilst in presence of serum compounds, it kept more that 60 % of activity. In addition, this ASNase showed low GLNase activity. [ABSTRACT FROM AUTHOR]

L-asparaginase is used in chemotherapy for acute lymphoblastic leukemia and other cancers. L-asparaginase derived from bacterial source triggers immune responses. The current study investigates Solanum nigrum as a novel and latent source of L-asparaginase to minimize immunological reactions. The antitumor activity of SN methanol extract was determined using the potato disc assay. InterPro Chimera and InterPro were used to predict the amino acid sequence of L-asparaginase and its anticancer activity. Purification of the enzyme was carried out to homogeneity of 1.51-fold with a recovery of 61.99%. At optimal conditions of 36.5°C, pH 8.6, and 8.5 g/mL substrate, fruit (crude extract) revealed an L-asparaginase titer of 48.23 U/mL. The molecular weight of the enzyme was calculated to be 32 ± 5 kDa using SDS PAGE. The fruit's total flavonoids and phenolic contents are 0.42 ±.030 g/mL and 94 ± 1.9 mg CAE, respectively. Anti-tumorigenic efficacy was determined to be 66% against Agrobacterium tumefaciens. Additionally, the extract possesses potent antifungal and antibacterial properties. Molecular docking provided the structural motifs and underlying interactions between L-asparaginase, N-acetylglucosamine, murine, and chitin. SN contains high levels of the enzyme L-asparaginase and phytochemicals, making it a potential source of anticancer drugs. [ABSTRACT FROM AUTHOR]

Red blood cells (RBCs) can act as carriers for therapeutic agents and can substantially improve the safety, pharmacokinetics, and pharmacodynamics of many drugs. Maintaining RBCs integrity and lifespan is important for the efficacy of RBCs as drug carrier. We investigated the impact of drug encapsulation by hypotonic dialysis on RBCs physiology and integrity. Several parameters were compared between processed RBCs loaded with l -asparaginase ("eryaspase"), processed RBCs without drug and non-processed RBCs. Processed RBCs were less hydrated and displayed a reduction of intracellular content. We observed a change in the metabolomic but not in the proteomic profile of processed RBCs. Encapsulation process caused moderate morphological changes and was accompanied by an increase of RBCs-derived Extracellular Vesicles release. Despite a decrease in deformability, processed RBCs were not mechanically retained in a spleen-mimicking device and had increased surface-to-volume ratio and osmotic resistance. Processed RBCs half-life was not significantly affected in a mouse model and our previous phase 1 clinical study showed that encapsulation of asparaginase in RBCs prolonged its in vivo half-life compared to free forms. Our study demonstrated that encapsulation by hypotonic dialysis may affect certain characteristics of RBCs but does not significantly affect the in vivo longevity of RBCs or their drug carrier function. A wide red blood cell (RBC) characterization demonstrated that ERYCAPS® hypotonic dialysis encapsulation process induces some changes to RBC features without substantially affecting their survival or their capacity to carry therapeutics. [Display omitted] [ABSTRACT FROM AUTHOR]

The present study aims at bioengineering of medium components using data and process centric approaches for enhanced production of L-asparaginase, an important biological molecule, by halotolerant Bacillus licheniformis PPD37 strain. To achieve this, first significant medium components were screened followed by optimisation of a combination of media components and culture conditions such as L-asparagine, MgSO4, NaCl, pH, and temperature. Optimisation study was carried out using statistical models such as response surface methodology (RSM) – process centric and artificial neural network (ANN) – data centric approaches. The production improved from 2.86 U/mL to 17.089 U/mL, an increase of approximately 6-times of the unoptimised L-asparaginase production. On comparing RSM and ANN models for optimised L-asparaginase production based on R2 value, mean absolute percentage error (MAPE), root mean square error (RMSE), and mean absolute deviation (MAD) values, the ANN model emerged as the superior one. As this is the first report to the authors best knowledge on development of inference system using RSM and ANN models for enhanced L-asparaginase production using a halotolerant bacteria, this study could lead to more in-depth and large-scale L-asparaginase production. [ABSTRACT FROM AUTHOR]

Microbial enzymes have gained interest for their widespread use in various industries and medicine due to their stability, ease of production, and optimization. Endophytic fungi in plant tissues produce a wide range of secondary metabolites and enzymes, which exhibit a variety of biological activities. The present review illustrates promising applications of enzymes produced by endophytic fungi and discusses the characteristic features of the enzymes, application of the endophytic fungal enzymes in therapeutics, agriculture, food, and biofuel industries. Endophytic fungi producing ligninolytic enzymes have possible biotechnological applications in lignocellulosic biorefineries. The global market of industrially important enzymes, challenges, and future prospects are illustrated. However, the commercialization of endophytic fungal enzymes for industrial purposes is yet to be explored. The present review suggests that endophytic fungi can produce various enzymes and may become a novel source for upscaling the production of enzymes of industrial use. [ABSTRACT FROM AUTHOR]

L-asparaginases prevent the formation of acrylamide, a substance commonly found in foods subjected to heat and that also contains reducing sugars and L-asparagine. This work aimed to select a strain of Aspergillus spp. able to produce L-asparaginase and to optimize the fermentation parameters, the partial purification and biochemical characterization were also performed. The Aspergillus oryzae IOC 3999 was selected due to its greater enzymatic activity: 1443.57 U/mL of L-asparaginase after 48 h of fermentation. The optimized conditions allowed for an increase of 223% on the L-asparaginase production: 2.9% lactose, 2.9% L-asparagine and 0.7% hydrolyzed casein, 0.152% KH2PO4, 0.052% KCl and MgSO4, 0.001% of CuNO3.3H2O, ZnSO4.7H2O and FeSO4.7H2O adjusted to pH 7.0; added a concentration of 5.05x106 spores/mL at 30 °C for 100 rpm. A purification factor of 2.11 was found and the molecular mass was estimated at 20.8 kDa. The enzyme showed optimum activity at 60 °C and pH 5 and stability at 50 °C for 1 h. The enzyme presented desirable biochemical characteristics, mainly the acid pH stability, indicating that the enzyme would work well in food matrices due to the closeness of pH, meaning that it could be a potential option for use in the food industry. [ABSTRACT FROM AUTHOR]

In this study, the gene coding for EcL-ASP from Enterobacter carcerogenus was identified in full sequence and cloned into a mesophilic organism. The gene encoding L-asparaginase was transferred to the pET-28a (+) vector to ensure its expression in Escherichia coli BL21 (DE3) pLysS. The recombinant protein containing the N-terminal histidine tail (6xHis tag) was purified by Nickel affinity chromatography. As a result of SDS-PAGE, it was determined that the purified protein consisted of a single type of polypeptide. In the theoretical calculation, the subunit molecular weight of the recombinant protein containing the histidine tail was found to be 37 kDa. It was observed that the cloned enzyme had low L-glutaminase activity. The pH and temperature at which the recombinant enzyme showed the best activity were determined as 7.0 and 37 °C, respectively. From the drawn Lineweaver-Burk graph, it is estimated that the Km value is 0.06 mM and the Vmax value is 666.7 U mg-1 protein. [ABSTRACT FROM AUTHOR]

L-asparaginases (EC 3.5.1.1) are a family of enzymes that catalyze the hydrolysis of L-asparagine to L-aspartic acid and ammonia. These proteins with different biochemical, physicochemical and pharmacological properties are found in many organisms, including bacteria, fungi, algae, plants and mammals. To date, asparaginases from E. coli and Dickeya dadantii (formerly known as Erwinia chrysanthemi) are widely used in hematology for the treatment of lymphoblastic leukemias. However, their medical use is limited by side effects associated with the ability of these enzymes to hydrolyze L-glutamine, as well as the development of immune reactions. To solve these issues, gene-editing methods to introduce amino-acid substitutions of the enzyme are implemented. In this review, we focused on molecular analysis of the mechanism of enzyme action and to optimize the antitumor activity. [ABSTRACT FROM AUTHOR]

L-asparaginase is a therapeutic enzyme that converts L-asparagine into ammonia and L-aspartate. L-asparaginase is used to treat acute lymphoblastic leukaemia. In food manufacturing industries, it is used to inhibit the acrylamide formation. The current investigation has been performed to isolate L-asparaginase producing fungi from different medicinal plants and soil samples, through serial dilution. A total number of 15 fungal isolates were obtained from soil samples and 6 endophytic fungi isolated from medicinal plants. By performing screening of L-asparaginase 67% of positive isolates were obtained from endophytes and soil samples. Optimization of L-asparaginase production was performed for parameters such as pH, temperature, carbon and nitrogen source, and it was found that pH 6, 30°C, 2 g of glucose, and 1 g of L-arginine is suitable for maximum enzyme production. By performing Sodium dodecyl sulphate polyacrylamide gel electrophoresis the molecular weight of an enzyme was determined to be approximately 11.2 kDa. [ABSTRACT FROM AUTHOR]

Several bacterial strains were isolated from soil and water in the current work and screened for glutaminase-free L-asparaginase activity. The selected strain was subjected to the production of glutaminase-free L-asparaginase and thereby purification. Before purification, pH scouting was performed, and 8.6 pH was the optimum working pH to carry out purification. The enzyme was purified using Sephadex G-200 gel filtration chromatography and up to 4.55-folds and its purity percentage was determined using UPLC. The purity of L-asparaginase was determined to be 99.2%. The enzyme was characterized using UV-Visible Spectrophotometer, DLS, ZP, DSC, TGA. The loss of the enzyme was 65% after sublimation, indicating its purity compared to the standard drug. The results report a developed process for the purification of L-asparaginase and its characterization, which can prove instrumental in the purification process of naturally occurring enzymes because today, most developed processes deal with recombinant enzymes.

L-asparaginase is an essential enzyme used in cancer treatment, but its production faces challenges like low yield, high cost, and immunogenicity. Recombinant production is a promising method to overcome these limitations. In this study, response surface methodology (RSM) was used to optimize the production of L-asparaginase 1 from Saccharomyces cerevisiae in Escherichia coli K-12 BW25113. The Box-Behnken design (BBD) was utilized for the RSM modeling, and a total of 29 experiments were conducted. These experiments aimed to examine the impact of different factors, including the concentration of isopropyl-b-LD-thiogalactopyranoside (IPTG), the cell density prior to induction, the duration of induction, and the temperature, on the expression level of L-asparaginase 1. The results revealed that while the post-induction temperature, cell density at induction time, and post-induction time all had a significant influence on the response, the post-induction time exhibited the greatest effect. The optimized conditions (induction at cell density 0.8 with 0.7 mM IPTG for 4 h at 30 °C) resulted in a significant amount of L-asparaginase with a titer of 93.52 μg/mL, which was consistent with the model-based prediction. The study concluded that RSM optimization effectively increased the production of L-asparaginase 1 in E. coli, which could have the potential for large-scale fermentation. Further research can explore using other host cells, optimizing the fermentation process, and examining the effect of other variables to increase production., (© 2024. Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i.)

A confirmed case of 41‑year‑old male with T‑cell precursor acute lymphoblastic leukemia was inducted to BFM 2002 protocol, induction phase 1 (this protocol includes injection daunorubicin, vincristine, L-asparaginase with triple intrathecal injection of methotrexate, cytosine, hydrocortisone, and oral prednisolone). The patient complained of epigastric pain with hepatosplenomegaly and laboratory investigation revealed deranged lipid profile with hypertriglyceridemia (HTG) as a major feature and hyperbilirubinemia and raised transaminase. He was diagnosed with a case of HTG with acute pancreatitis, a complication, due to L-asparaginase. Initially, his condition was treated with medical management, but reduction in triglyceride (TG) level was not as expected. It was decided then to manage the case with concomitant therapeutic plasma exchange therapy. One exchange not only reduced the TG level drastically but also the bilirubin level and the patient recovered and was able to start the next phase of the protocol. [ABSTRACT FROM AUTHOR]

The aim of the present study was to compare the efficacy of an L-asparaginase-based regimen and a CHOP regimen followed by radiotherapy as first-line treatments for newly diagnosed nasal type extranodal natural killer cell/T-cell lymphoma (ENKTL). A total of 69 patients received the CHOP regimen as the first-line treatment and 112 patients received the L-asparaginase-based regimen. All patients received radical radiotherapy following two cycles of chemotherapy. The overall response rates of the L-asparaginase-based and CHOP treatment groups were 90.18 and 72.46%, respectively (P=0.002). The one, two, and five-year overall survival (OS) rates and progression-free survival (PFS) rates of the L-asparaginase group were 96.0, 88.3, 65.1, 94.2, 79.8 and 50.0%, respectively. The one, two, and five-year OS and PFS rates of the CHOP group were 82.6, 61.9, 25.8, 63.8, 44.0 and 21.0%, respectively (P

Background: This study targets the enhanced production of l-asparaginase, an antitumor enzyme by Acinetobacter baumannii ZAS1. This organism is an endophyte isolated from the medicinal plant Annona muricata. Plackett–Burman design (PBD) and central composite design (CCD) were used for statistical optimization of media components. Results: The organism exhibited 18.85 ± 0.2 U/mL enzyme activities in unoptimized media. Eight variables: l-asparagine, peptone, glucose, lactose, yeast extract, NaCl, MgSO4, and Na2HPO4 were screened by PBD. Among them, only four factors—l-asparagine, peptone, glucose, and Na2HPO4—were found to affect enzyme production significantly (p < 0.05). Furthermore, the best possible concentrations and interactive effects of the components that enhance this enzyme's output were chosen by using CCD on these selected variables. The results revealed that an optimized medium produces a higher concentration of enzymes than the unoptimized medium. After optimizing media components, the maximum l-asparaginase activity was 45.59 ± 0.36 U/mL, around the anticipated value of 45.04 ± 0.42 U/mL. After optimization of process parameters, it showed a 2.41-fold increase in the production of l-asparaginase by the endophyte Acinetobacter baumannii ZAS1. Conclusion: The findings of this study indicated that an endophyte, Acinetobacter baumannii ZAS1 that produces l-asparaginase could be used to increase enzyme output. However, using the statistical methods Plackett-Burman design and central composite design of response surface methodology is a handy tool for optimizing media components for increased l-asparaginase synthesis. [ABSTRACT FROM AUTHOR]

Summary: Introduction. Erwinase® (native Erwinia chrysanthemi L-Asparaginase (nErA)) is an approved second-line treatment for acute lymphoblastic leukaemia (ALL) in children and adolescents, who develop hypersensitivity or neutralising antibodies to E.coli derived L-Asparaginases (ASNases). However, nErA has a short in vivo half-life requiring frequent dosing schedules in patients. In this study, nErA was covalently conjugated to PEG molecules with the aim of extending its half-life in vivo. Methods. Firstly, efficacy of this novel product PEG-nErA was investigated on human ALL cell lines (Jurkat, CCRF-CEM and CCRF-HSB2), in vitro. Secondly, its pharmacokinetic (PK) and pharmacodynamic (PD) characteristics were determined, in vivo (12 rats in each group). Results. It was found that the specific activity (U/mg of enzyme) and the kinetic constant (KM) of nErA remained unaltered post PEGylation. PEG-nErA was shown to have similar cytotoxicity to nErA (IC50: 0.06–0.17 U/mL) on human ALL cell lines, in vitro. Further, when compared to nErA, PEG-nErA showed a significantly improved half-life in vivo, which meant that L-Asparagine (Asn) levels in plasma remained depleted for up to 25 days with a four-fold lower dose (100 U/kg) compared with 72 h for nErA at 400 U/kg dose. Conclusion. Overall, this next generation product PEG-nErA (with improved PK and PD characteristics compared to nErA) would bring a significant advantage to the therapeutic needs of ALL patients and should be further explored in clinical trials. [ABSTRACT FROM AUTHOR]

PEGylation is a reductive alkylation of a protein N-terminal/α-amine of protein with mPEG chain by reducing agent. To obtain quantitative and site-specific PEGylation, sodium cyanoborohydride is commonly used as a reducing agent. The reduction process of sodium cyanoborohydride produces highly poisonous hydrogen cyanide, which may render the final product toxic. Herein, we have studied various reducing agents such as dimethylamine borane, triethylamine borane, trimethylamine borane, pyridine borane, morpholine borane, 2-picoline borane, and 5-ethyl-2-methyl-pyridine borane were tested as alternatives to sodium cyanoborohydride for the PEGylation of L-asparaginase. The characterization of reacted pegaspargase was carried out by SDS-PAGE, Western blotting, SEC-HPLC, RP-HPLC, SEC-MALS, CD, enzyme activity, and cell proliferation assays using with lymphoblast cells and MTS/PMS as substrate. Pyridine borane was determined to be the best acceptable reducing agent for PEGylation in terms of purity and activity. As a result, instead of sodium cyanoborohydride, pyridine borane can be employed. [ABSTRACT FROM AUTHOR]

Background: Asparaginase (ASP), a chemotherapy component in the acute lymphoblastic leukemia (ALL) treatment, could impair normal coagulation state. Usually, a decline in the levels of several coagulation factors occurs which ultimately could lead to thrombotic events and abnormal coagulation tests. In this study, we aimed to compare the effects of two different subtypes of ASP, pegylated asparaginase (PEG-ASP) and L-asparaginase (L-ASP) on coagulation markers and test among 40 pediatric patients with ALL. Methods: In this cohort study a total of 40 pediatric patients with newly diagnosed ALL were enrolled and divided into two groups by simple randomization. In group A, 20 patients received PEG-ASP while in group B, 20 patients received L-ASP during the induction treatment. Coagulation markers included prothrombin time (PT), partial thrombin time (PTT), protein-C (Pr-C), protein-S (Pr-S), and antithrombin III (ATIII) and were assessed before start and after of induction chemotherapy. Results: Coagulation profile including PT, PTT, INR, Pr-C, Pr-S, and ATIII before start of treatment were not statistically significant between the two groups. Anticoagulant factors decreased significantly after consuming both drugs. Tests for PT and INR of those who took L-ASP decreased significantly. Overall, when comparing the changes of the six studied factors, ATIII and Pr-C were the significant factors which were different between groups. Conclusions: ASP has a negative effect on anticoagulant factors including (ATIII, Pr-C, Pr-S). Additionally, the negative effect of L-ASP on anticoagulant factors was more prominent than PEG-ASP. Therefore, the risk of thrombosis probably was negligible in PEG-ASP in comparison with L-ASP. [ABSTRACT FROM AUTHOR]

The disintegration of the amide link, causing L-Asparaginase catalyzes the hydrolysis process of L-Asparagine into ammonia and aspartic acid. The purposes of this study were to determine the optimal conditions for enzyme production and enzymatic activity, including their potential as a new anti-dengue of L-asparaginase from Klebsiella sp. The Nessler method used a UV-Visible spectrophotometer to determine the L-asparaginase activity by measuring ammonia levels produced by L-asparaginase catalysis. The ideal fermentation period was at the 48th hour, and the optimal substrate concentration was 10 g/L with an activity value of 49.572 U/mL, and the optimal FeCl2 concentration was 0.5 g/L with an activity value of 60.1183 U/mL, and protein content of 0.1878 mg/mL. The optimum conditions for L-asparaginase, which has an activity value of 61.1182 U/mL, were observed at pH 8 and 37 °C. The optimum cofactor concentration of FeCl2 was 0.5 mM, which resulted in an activity value of 106.5046 U/mL. Both the inhibition percentage and CC50 value were 80% and 167.15 µg/mL respectively, showing that the L-asparaginase has a high antidengue potential. [ABSTRACT FROM AUTHOR]

Enzymes play a vital role in the biological system as a catalyst for biochemical reactions. They have a wide range of commercial applications in many areas like the pharmaceutical industry, food industry, etc. l-asparaginase is one of the commercial enzymes with prime importance in anticancer therapy. It is mainly used in chemotherapy; however, it has the potential to cure autoimmune disorders and infectious diseases also. Previous studies reported the antimicrobial potential of l-asparaginase. Therefore, we have discussed the possibility and challenges of the antimicrobial application of l-asparaginase in the treatment of infectious diseases. This is followed by a discussion on the effective delivery of this enzyme using biopolymeric nanocarriers that ensure safe and on target action. The present article gives a perspective on the l-asparaginase molecule that could be developed/established as an approved antimicrobial drug in the future. [ABSTRACT FROM AUTHOR]

BACKGROUND: The treatment of acute lymphoblastic leukemia (ALL) uses the biopharmaceutical l‐asparaginase (ASNase) as the main medication. This drug, from bacterial origin (Escherichia coli or Erwinia chrysanthemi), depletes l‐asparagine (Asn) and secondarily l‐glutamine (Gln – GLNase activity) from the bloodstream, leading leukemic cells to die by deprivation of Asn. The use of ASNase is limited by the high incidence of adverse effects, which collectively can specifically impair quality of life of patients. Its high toxicity caused by the product of the hydrolysis of amino acids and the formation of anti‐ASNase antibodies often required treatment interruption, thus reducing the chances of cure and increasing the rates of disease relapse. RESULTS: In order to improve enzymatic activity, while reducing toxicity, we developed through directed evolution a double‐mutant ASNase from Erwinia chrysanthemi (Erw_DM), which has specific activity for Asn 46% higher than the wild‐type enzyme (Erw_WT). This makes it possible to reduce the amount of protein necessary for depletion of this amino acid and, consequently, the reduction of GLNase activity, considered toxic. In silico analysis showed that a lower number of epitopes was exposed, resulting in reduced recognition of the recombinant protein by antibody anti‐ASNase observed in vitro assay. Furthermore, we observed the same cytotoxic profile for the MOLT‐4 and REH ALL cell lines using 40% lower protein mass of Erw_DM to achieve the minimum enzyme activity required in the bloodstream during treatment. CONCLUSION: Altogether, our findings describe a potent and less immunogenic ASNase, an improvement that may alleviate treatment adverse effects developed in anti‐ALL therapy. © 2021 Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]