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.

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.

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.

Dissecting dual specificity: Identifying key residues in L-asparaginase for enhanced acute lymphoid leukemia therapy and reduced adverse effects.

L-asparaginase from Escherichia coli (EcA) has been used for the treatment of acute lymphoid leukemia (ALL) since the 1970s. Nevertheless, the enzyme has a second specificity that results in glutaminase breakdown, resulting in depletion from the patient's body, causing severe adverse effects. Despite the huge interest in the use of this enzyme, the exact process of glutamine depletion is still unknown and there is no consensus regarding L-asparagine hydrolysis. Here, we investigate the role of T12, Y25, and T89 in asparaginase and glutaminase activities. We obtained individual clones containing mutations in the T12, Y25 or T89 residues. After the recombinant production of wild-type and mutated EcA, The purified samples were subjected to structural analysis using Nano Differential Scanning Fluorimetry, which revealed that all samples contained thermostable molecules in their active structural conformation, the homotetramer conformation. The quaternary conformation was confirmed by DLS and SEC. The activity enzymatic assay combined with molecular dynamics simulation identified the contribution of T12, Y25, and T89 residues in EcA glutaminase and asparaginase activities. Our results mapped the enzymatic behavior paving the way for the designing of improved EcA enzymes, which is important in the treatment of ALL.

Synergistic effects of ternary mixture formulation and process parameters optimization in a sequential approach for enhanced L-asparaginase production using agro-industrial wastes.

A novel ternary mixture of inexpensive and nutrient-rich agro-substrates comprising groundnut de-oiled cake, corn gluten meal, and soybean meal has been explored to enhance the L-asparaginase production in solid-state fermentation. To achieve the aim, a hybrid strategy was implemented by utilizing a combination of a mixture design and artificial neural networks. The study initiated with the judicious selection of the agro-substrates based on their low C/N content in comparison to the control using the CHNS elemental analysis. The mixture composition of soybean meal (49.0%), groundnut de-oiled cake (31.5%), and corn gluten meal (19.5%) were found optimum using the simplex lattice mixture design. The agro-industrial substrates mix revealed synergistic effects on the L-asparaginase production than either of the substrates alone. The maximum L-asparaginase activity of 141.45 ± 5.24 IU/gds was observed under the physical process conditions of 70% moisture content, autoclaving period of 30 min and 6.0 pH by adopting the machine learning-derived artificial neural network (ANN) methodology. The ANN modeling showed excellent prediction ability with a low mean squared error of 0.7, a low root mean squared error of 0.84, and a high value of 0.99 for regression coefficient. Moisture content (%) was assessed to be the most sensitive process parameter in the global sensitivity analysis. The net outcome from the two sequential optimization designs is the selection of the ideal mixture composition followed by the optimum physical process parameters. The application of the enzyme demonstrated significant cytotoxicity against leukemia cell line and therefore exhibited an anti-cancer effect. The present study reports a novel mixture combination and methodology that can be used to lower the cost and enhance the production of L-asparaginase using an agro-industrial substrate mixture.

Biochemical characterization of glutaminase-free L-asparaginases from Himalayan Pseudomonas and Rahnella spp. for acrylamide mitigation.

L-asparaginase having low glutaminase activity is important in clinical and food applications. Herein, glutaminase-free L-asparaginase (type I) coding genes from Pseudomonas sp. PCH182 (Ps-ASNase I) and Rahnella sp. PCH162 (Rs-ASNase I) was amplified using gene-specific primers, cloned into a pET-47b(+) vector, and plasmids were transformed into Escherichia coli (E. coli). Further, affinity chromatography purified recombinant proteins to homogeneity with monomer sizes of ~37.0 kDa. Purified Ps-ASNase I and Rs-ASNase I were active at wide pHs and temperatures with optimum activity at 50 °C (492 ± 5 U/mg) and 37 °C (308 ± 4 U/mg), respectively. Kinetic constant Km and Vmax for L-asparagine (Asn) were 2.7 ± 0.06 mM and 526.31 ± 4.0 U/mg for Ps-ASNase I, and 4.43 ± 1.06 mM and 434.78 ± 4.0 U/mg for Rs-ASNase I. Circular dichroism study revealed 29.3 % and 24.12 % α-helix structures in Ps-ASNase I and Rs-ASNase I, respectively. Upon their evaluation to mitigate acrylamide formation, 43 % and 34 % acrylamide (AA) reduction were achieved after pre-treatment of raw potato slices, consistent with 65 % and 59 % Asn reduction for Ps-ASNase I and Rs-ASNase I, respectively. Current findings suggested the potential of less explored intracellular L-asparaginase in AA mitigation for food safety.

Towards development of biobetter: L-asparaginase a case study.

BACKGROUND: L-asparaginase (ASNase) has played a key role in the management of acute lymphoblastic leukaemia (ALL). As an amidohydrolase, it catalyzes the hydrolysis of L-asparagine, a crucial step in the treatment of ALL. Various ASNase variants have evolved from diverse sources since it was first used in paediatric patients in the 1960s. This review describes the available ASNase and approaches being used to develop ASNase as a biobetter candidate. SCOPE OF REVIEW: The review discusses the Glycosylation and PEGylation techniques, which are frequently used to develop biobetter versions of the majority of the therapeutic proteins. Further, it explores current ASNase biobetters in therapeutic use and discusses the protein engineering and chemical modification approaches that were employed to reduce immunogenicity, extend protein half-life, and enhance protease stability of ASNase. Emerging strategies like immobilization and encapsulation are also highlighted as potential pathways for improving ASNase properties. MAJOR CONCLUSIONS: The purpose of the development of ASNase biobetter is to achieve a novel therapeutic candidate that could improve catalytic efficiency, in vivo stability with minimum glutaminase (GLNase) activity and toxicity. Modification of ASNase by immobilization and encapsulation or by fusion technologies like Albumin fusion, Fc fusion, ELP fusion, XTEN fusion, etc. can be exploited to develop a novel biobetter candidate suitable for therapeutic approaches. GENERAL SIGNIFICANCE: This review emphasizes the importance of biobetter development for therapeutic proteins like ASNase. Improved ASNase molecules have the potential to significantly advance the treatment of ALL and have broader implications in the pharmaceutical industry.

Polyoxazoline-Conjugated l-Asparaginase: An Antibody-Production-Free Therapeutic Agent for Acute Lymphoblastic Leukemia.

l-asparaginase (ASNase), an enzyme that catalyzes the hydrolysis of l-asparagine into l-aspartic acid, is frequently used as a medication for acute lymphoblastic leukemia (ALL). However, when derived from bacterial sources, this enzyme can elicit side effects, including allergic or hypersensitivity reactions, owing to immune responses. Here, we describe the synthesis of polyoxazoline-conjugated ASNase (POx-ASNase) and investigate its enzyme activity, anticancer efficacy, immunogenicity, and retention in the bloodstream. The water-soluble POx was coupled with surface lysine residues of ASNase using a bifunctional cross-linker. The average number of polymers bound to each enzyme was determined as 10. Although the enzymatic activity of POx-ASNase decreased to 56% of that of native ASNase, its temperature and pH dependencies remained unaltered. Remarkably, the lyophilized powder form of POx-ASNase retained its catalytic ability for 24 months. POx-ASNase demonstrated nearly identical anticancer efficacy compared to naked ASNase against leukemia and lymphoma cells (MOLT-4, CLBL-1, and K562) while displaying no cytotoxicity toward normal cells. Animal experiments conducted using rats revealed that the POx decoration suppressed the generation of anti-ASNase IgM and IgG antibodies with no detection of anti-POx antibodies. The half-life within the bloodstream extended to 34 h, representing a 17-fold increase compared to unmodified ASNase. These findings suggest that POx-ASNase serves as an anticancer therapeutic agent, characterized by the absence of antibody production and notably extended circulation persistence.

Characterization of the Type 2 L-Asparaginase Gene in Thermohalophilic Bacterial from Wawolesea Hot Springs, Southeast Sulawesi, Indonesia.

<b>Background and Objective:</b> Type 2 L-asparaginase enzyme can be used as a cancer therapy agent and prevent acrylamide formation in food products. Enzymes produced by thermohalophilic bacteria can provide high activity at high temperatures so they are needed on an industrial scale. Hence, this study aims to determine the characteristics of the gene encoding type 2 L-asparaginase enzyme in the thermohalophilic bacterial isolate CAT3.4. <b>Materials and Methods:</b> This research is a type of exploratory research. The characteristics of the gene encoding type 2 L-asparaginase were determined using the PCR technique using the primer pairs AsnBac2-F2 (5'-CTCACGGGAATCTCCATAACTC-3') and AsnBac2-R2 (5'CAGCGATGTAACAGACAGCATC-3'). The characterization process was carried out in stages: Isolation of genomic DNA using a modified alkali-lysis method, nucleotide and protein similarity analysis using BLASTn analysis on the NCBI website, construction of a phylogenetic tree using the MEGAX program, restriction enzyme mapping and amino acid analysis using the Bioedit program. <b>Results:</b> The characterization results showed that the PCR product has a size of 1594 bp with a CDS of 1128 bp, has a similarity value of 100% with <i>Bacillus subtilis</i>, has seven restriction enzymes as molecular markers for the type 2 L-asparaginase gene at the species level: <i>Bsr</i>GI, <i>Dra</i>I, <i>Eco</i>RV, <i>Hind</i>III, <i>Hpy</i>CH4IV , <i>Ssp</i>I and <i>Tai</i>I, have dominant hydrophilic regions and are in the same subclass as <i>Bacillus subtilis</i> strain GOT9. <b>Conclusion:</b> The target gene was similar to the gene encoding type 2 L-asparaginase from <i>Bacillus subtilis</i> with a max identity of 98.85%, query coverage value of 100% and E-value of 0.

Assessment of structural behaviour of a new L-asparaginase and SAXS data-based evidence for catalytic activity in its monomeric form.

The present study reports the structural and functional characterization of a new glutaminase-free recombinant L-asparaginase (PrASNase) from Pseudomonas resinovorans IGS-131. PrASNase showed substrate specificity to L-asparagine, and its kinetic parameters, Km, Vmax, and kcat were 9.49 × 10-3 M, 25.13 IUmL-1 min-1, and 3.01 × 103 s-1, respectively. The CD spectra showed that PrASNase consisted of 18.5 % helix, 21.5 % antiparallel sheets, 4.2 % parallel sheets, 14 % turns, and rest other structures. FTIR was used for the functional characterization, and molecular docking predicted that the substrate interacts with serine, alanine, and glutamine in the binding pocket of PrASNase. Differing from known asparaginases, structural characterization by small-angle X-ray scattering (SAXS) and analytical ultracentrifugation (AUC) unambiguously revealed PrASNase to exist as a monomer in solution at low temperatures and oligomerized to a higher state with temperature rise. Through SAXS studies and enzyme assay, PrASNase was found to be mostly monomer and catalytically active at 37 °C. Furthermore, this glutaminase-free PrASNase showed killing effects against WIL2-S and TF-1.28 cells with IC50 of 7.4 µg.mL-1 and 5.6 µg.mL-1, respectively. This is probably the first report with significant findings of fully active L-asparaginase in monomeric form using SAXS and AUC and demonstrated the potential of PrASNase in inhibiting cancerous cells, making it a potential therapeutic candidate.

Rhizobium etli has two L-asparaginases with low sequence identity but similar structure and catalytic center.

The genome of Rhizobium etli, a nitrogen-fixing bacterial symbiont of legume plants, encodes two L-asparaginases, ReAIV and ReAV, that have no similarity to the well characterized enzymes of class 1 (bacterial type) and class 2 (plant type). It has been hypothesized that ReAIV and ReAV might belong to the same structural class 3 despite their low level of sequence identity. When the crystal structure of the inducible and thermolabile protein ReAV was solved, this hypothesis gained a stronger footing because the key residues of ReAV are also present in the sequence of the constitutive and thermostable ReAIV protein. High-resolution crystal structures of ReAIV now confirm that it is a class 3 L-asparaginase that is structurally similar to ReAV but with important differences. The most striking differences concern the peculiar hydration patterns of the two proteins, the presence of three internal cavities in ReAIV and the behavior of the zinc-binding site. ReAIV has a high pH optimum (9-11) and a substrate affinity of ∼1.3 mM at pH 9.0. These parameters are not suitable for the direct application of ReAIV as an antileukemic drug, although its thermal stability and lack of glutaminase activity would be of considerable advantage. The five crystal structures of ReAIV presented in this work allow a possible enzymatic scenario to be postulated in which the zinc ion coordinated in the active site is a dispensable element. The catalytic nucleophile seems to be Ser47, which is part of two Ser-Lys tandems in the active site. The structures of ReAIV presented here may provide a basis for future enzyme-engineering experiments to improve the kinetic parameters for medicinal applications.

Enhancing the Catalytic Activity of Thermo-Asparaginase from Thermococcus sibiricus by a Double Mesophilic-like Mutation in the Substrate-Binding Region.

L-asparaginases (L-ASNases) of microbial origin are the mainstay of blood cancer treatment. Numerous attempts have been performed for genetic improvement of the main properties of these enzymes. The substrate-binding Ser residue is highly conserved in L-ASNases regardless of their origin or type. However, the residues adjacent to the substrate-binding Ser differ between mesophilic and thermophilic L-ASNases. Based on our suggestion that the triad, including substrate-binding Ser, either GSQ for meso-ASNase or DST for thermo-ASNase, is tuned for efficient substrate binding, we constructed a double mutant of thermophilic L-ASNase from Thermococcus sibiricus (TsA) with a mesophilic-like GSQ combination. In this study, the conjoint substitution of two residues adjacent to the substrate-binding Ser55 resulted in a significant increase in the activity of the double mutant, reaching 240% of the wild-type enzyme activity at the optimum temperature of 90 °C. The mesophilic-like GSQ combination in the rigid structure of the thermophilic L-ASNase appears to be more efficient in balancing substrate binding and conformational flexibility of the enzyme. Along with increased activity, the TsA D54G/T56Q double mutant exhibited enhanced cytotoxic activity against cancer cell lines with IC90 values from 2.8- to 7.4-fold lower than that of the wild-type enzyme.

Quality comparison of a state-of-the-art preparation of a recombinant L-asparaginase derived from Escherichia coli with an alternative asparaginase product.

L-asparaginase (ASNase) is a protein that is essential for the treatment of acute lymphoblastic leukemia (ALL). The main types of ASNase that are clinically used involve native and pegylated Escherichia coli (E. coli)-derived ASNase as well as Erwinia chrysanthemi-derived ASNase. Additionally, a new recombinant E. coli-derived ASNase formulation has received EMA market approval in 2016. In recent years, pegylated ASNase has been preferentially used in high-income countries, which decreased the demand for non-pegylated ASNase. Nevertheless, due to the high cost of pegylated ASNase, non-pegylated ASNase is still widely used in ALL treatment in low- and middle-income countries. As a consequence, the production of ASNase products from low- and middle-income countries increased in order to satisfy the demand worldwide. However, concerns over the quality and efficacy of these products were raised due to less stringent regulatory requirements. In the present study, we compared a recombinant E. coli-derived ASNase marketed in Europe (Spectrila®) with an E. coli-derived ASNase preparation from India (Onconase) marketed in Eastern European countries. To assess the quality attributes of both ASNases, an in-depth characterization was conducted. Enzymatic activity testing revealed a nominal enzymatic activity of almost 100% for Spectrila®, whereas the enzymatic activity for Onconase was only 70%. Spectrila® also showed excellent purity as analyzed by reversed-phase high-pressure liquid chromatography, size exclusion chromatography and capillary zone electrophoresis. Furthermore, levels of process-related impurities were very low for Spectrila®. In comparison, the E. coli DNA content in the Onconase samples was almost 12-fold higher and the content of host cell protein was more than 300-fold higher in the Onconase samples. Our results reveal that Spectrila® met all of the testing parameters, stood out for its excellent quality and, thus, represents a safe treatment option in ALL. These findings are particularly important for low- and middle-income countries, where access to ASNase formulations is limited.

L-Asparaginase delivery systems targeted to minimize its side-effects.

L-asparaginase (L-ASP) is one of the key enzymes used in therapeutic applications, particularly to treat Acute Lymphocytic Leukemia (ALL). L-asparagine is a non-essential amino acid, which means that it can be synthesized by the body and is not required to be obtained through the diet. The synthesis of L-asparagine occurs primarily in the liver, but it also takes place in other tissues throughout the body. In contrast, leukemic cells cannot synthesize L-asparagine due the absence of L-asparagine synthetase and should obtain it from circulating sources for protein synthesis and cell division processes to ensure their vital functions. L-ASP catalyzes the deamination process of L-asparagine amino-acid into aspartic acid and ammonia, depriving leukemic cells of asparagine. This leads to decreased protein synthesis and cell division in tumor cells. However, using L-ASP has side effects, such as hypersensitivity or allergic reaction, antigenicity, short half-life, temporary blood clearance, and toxicity. L-ASP immobilization can minimize the side effects of L-ASP by stopping the immune system from attacking non-human enzymes and improving the enzyme's performance. The first strategy includes modification of enzyme structure, such as covalent binding (conjugation), adsorption to the support material and cross-linking of the enzyme. The chemical modification of residues, often nonspecific, changes the enzyme's hydrophobicity and surface charge, lowering the enzyme's activity. Also, the first strategy exposes the enzyme's surface to the environment. This eliminates its performance and does not allow targeted delivery of the enzyme. The second strategy is based on the entrapment of the enzyme inside the protecting structure or encapsulation. This strategy offers the same benefits as the first. Still, it also enables reducing toxicity, prolonging in vivo half-life, enhancing stability and activity, enables a targeted delivery and controlled release of the enzyme. Compared to the first strategy, encapsulation does not modify the chemical structure of the enzyme since L-ASP is only effective against leukemia in its native tetrameric form. This review aims to present state of the art in L-ASP formulations developed for reducing the side effects of L-ASP, focusing on describing improvements in their safety. The primary focus in the field remains to be improving the overall performance of the L-ASP formulations. Almost all encapsulation systems allow reducing immune response due to screening the enzyme from antibodies and prolonging its half-life. However, the enzyme's activity and stability depend on the encapsulation system type. Therefore, the selection of the right encapsulation system is crucial in therapy due to its effect on the performance parameters of the L-ASP. Biodegradable and biocompatible materials, such as chitosan, alginate and liposomes, mainly attract the researcher's interest in enzyme encapsulation. The research trends are also moving towards developing formulations with targeted delivery and increased selectivity.

Mutations in asparaginase II from E. coli and implications for inactivation and PEGylation.

All clinically-used asparaginases convert L-asparagine (L-Asn) to l-aspartate (L-Asp) and l-glutamine (L-Gln) to L-glutamate (L-Glu), which has been useful in reducing bioavailable asparagine and glutamine in patients under treatment for acute lymphoblastic leukemia. The E. coli type 2 L-asparaginase (EcA2) can present different sequences among varying bacterial strains, which we hypothesized that might affect their biological function, stability and interchangeability. Here we report the analysis of two EcA2 provided by the public health system of a middle-income country. These enzymes were reported to have similar specific activity in vitro, whereas they differ in vivo. Protein sequencing by LC-MS-MS and peptide mapping by MALDI-ToF-MS of their tryptic digests revealed that Aginasa™ share similar sequence to EcA2 from E. coli strain BL21(DE3), while Leuginase™ has sequence equivalent to EcA2 from E. coli strain AS1.357. The two amino acid differences between Aginasa™ (64D and 252 T) and Leuginase™ (64 N and 252S) resulted in structural divergences in solution as accessed by small-angle X-ray scattering and molecular dynamics simulation trajectories. The conformational variability further results in dissimilar surface accessibility with major consequences for PEGylation, as well as different susceptibility to degradation by limited proteolysis. The present results reveal that the sequence variations between these two EcA2 variants results in conformational changes associated with differential conformational plasticity, potentially affecting physico-chemical and biological properties, including proteolytic and immunogenic silent inactivation.

The effects of nature-inspired amino acid substitutions on structural and biochemical properties of the E. coli L-asparaginase EcAIII.

The Escherichia coli enzyme EcAIII catalyzes the hydrolysis of L-Asn to L-Asp and ammonia. Using a nature-inspired mutagenesis approach, we designed and produced five new EcAIII variants (M200I, M200L, M200K, M200T, M200W). The modified proteins were characterized by spectroscopic and crystallographic methods. All new variants were enzymatically active, confirming that the applied mutagenesis procedure has been successful. The determined crystal structures revealed new conformational states of the EcAIII molecule carrying the M200W mutation and allowed a high-resolution observation of an acyl-enzyme intermediate with the M200L mutant. In addition, we performed structure prediction, substrate docking, and molecular dynamics simulations for 25 selected bacterial orthologs of EcAIII, to gain insights into how mutations at the M200 residue affect the active site and substrate binding mode. This comprehensive strategy, including both experimental and computational methods, can be used to guide further enzyme engineering and can be applied to the study of other proteins of medicinal or biotechnological importance.