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L-asparaginase (L-ASNase) is an enzyme that shows targeted activity against Acute Lymphoblastic Leukemia (ALL) and similar lymphoid neoplasms by facilitating the breakdown of asparagine into L-aspartic acid, thereby reducing L-asparagine levels in leukemic cells. However, its therapeutic potential is hindered by its associated toxicity, leading to complications, such as thrombosis, hemorrhage, thrombocytopenia, fibrinolysis, hypersensitivity reactions, and the development of Posterior Reversible Encephalopathy Syndrome (PRES). This review compiles documented cases of PRES linked to treating B and T cell acute lymphoblastic leukemia in children using L-ASNase. Although this pathology is rare, understanding its management is crucial within ASNase-based chemotherapy protocols. As PRES lacks a specific treatment, focusing on symptomatic management becomes pivotal. Therefore, comprehending the underlying causes during L-ASNase treatment for acute lymphoblastic leukemia is essential. Understanding the etiology and clinical symptoms of this illness is critical for early diagnosis and treatment. The cases of PRES described in this review include instances in which this syndrome has appeared after the administration of L-ASNase in children. In some cases, PRES developed during induction therapy, while in others, it occurred during the reinduction phase. These cases resolved days after discontinuation of L-ASNase. The findings suggest a close relationship between drug administration and the appearance of brain lesions, as evidenced by the disappearance or decrease of these lesions when the drug was eliminated from the bloodstream.
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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.
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Asparaginase , Simulação por Computador , Penicillium , Asparaginase/química , Asparaginase/imunologia , Asparaginase/metabolismo , Penicillium/imunologia , Penicillium/enzimologia , Sequência de Aminoácidos , Proteínas Fúngicas/química , Proteínas Fúngicas/imunologia , Proteínas Fúngicas/metabolismo , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/química , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/química , Humanos , Aspergillus/imunologia , Aspergillus/enzimologia , Escherichia coli/genética , Dickeya chrysanthemi/enzimologia , Dickeya chrysanthemi/imunologia , Modelos MolecularesRESUMO
Thermostability is an important and desired feature of therapeutic proteins and is critical for the success or failure of protein drugs development. It can be increased by PEGylation-binding of poly(ethylene glycol) moieties-or glycosylation-post-translational modification to add glycans. Here, the thermostability and thermodynamic parameters of native, PEGylated, and glycosylated versions of the antileukemic enzyme crisantaspase were investigated. First-order kinetics was found to describe the irreversible deactivation process. Activation energy of the enzyme-catalyzed reaction (E*) was estimated for native, PEGylated, and glycosylated enzyme (10.2, 14.8, and 18.8 kJ mol-1 respectively). Half-life decreased progressively with increasing temperature, and longer half-life was observed for PEG-crisantaspase (87.74 min) at 50 °C compared to the native form (9.79 min). The activation energy of denaturation of PEG-crisantaspase (307.1 kJ mol-1) was higher than for crisantaspase (218.1 kJ mol-1) and Glyco-crisantaspase (120.0 kJ mol-1), which means that more energy is required to overcome the energy barrier of the unfolding process. According to our results, PEG-crisantaspase is more thermostable than its native form, while Glyco-crisantaspase is more thermosensitive.
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Asparaginase , Polietilenoglicóis , Glicosilação , Termodinâmica , Temperatura , Cinética , Estabilidade EnzimáticaRESUMO
Aim: To review the available literature about heterologous expression of fungal L-asparaginase (L-ASNase). Materials & methods: A search was conducted across PubMed, Science Direct, Scopus and Web of Science databases; 4172 citations were identified and seven articles were selected. Results: The results showed that heterologous expression of fungal L-ASNase was performed mostly in bacterial expression systems, except for a study that expressed L-ASNase in a yeast system. Only three publications reported the purification and characterization of the enzyme. Conclusion: The information reported in this systematic review can contribute significantly to the recognition of the importance of biotechnological techniques for L-ASNase production.
Asparaginase is a common treatment for the most common type of leukemia in children. These treatments generally use asparaginase sourced from bacteria. Some people can experience bad reactions to these treatments. One way that has been explored to avoid this is to use asparaginase sourced from fungi because they are more similar to humans. However, fungi produce less asparaginase than bacteria. This review looks into ways that the production of fungal asparaginases can be made more productive.
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Antineoplásicos , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Asparaginase/genética , Asparaginase/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Bactérias/metabolismo , Antineoplásicos/uso terapêuticoRESUMO
L-asparaginases from bacterial sources have been used in antineoplastic treatments and the food industry. A type II L-asparaginase encoded by the N-truncated gene ansZP21 of halotolerant Bacillus subtilis CH11 isolated from Chilca salterns in Peru was expressed using a heterologous system in Escherichia coli BL21 (DE3)pLysS. The recombinant protein was purified using one-step nickel affinity chromatography and exhibited an activity of 234.38 U mg-1 and a maximum catalytic activity at pH 9.0 and 60 °C. The enzyme showed a homotetrameric form with an estimated molecular weight of 155 kDa through gel filtration chromatography. The enzyme half-life at 60 °C was 3 h 48 min, and L-asparaginase retained 50% of its initial activity for 24 h at 37 °C. The activity was considerably enhanced by KCl, CaCl2, MgCl2, mercaptoethanol, and DL-dithiothreitol (p-value < 0.01). Moreover, the Vmax and Km were 145.2 µmol mL-1 min-1 and 4.75 mM, respectively. These findings evidence a promising novel type II L-asparaginase for future industrial applications.
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The search for new drug-producing microorganisms is one of the most promising situations in current world scientific scenarios. The use of molecular biology as well as the cloning of protein and compound genes is already well established as the gold standard method of increasing productivity. Aiming at this increase in productivity, this work aims at the cloning, purification and in silico analysis of l-asparaginase from Fusarium proliferatum in Komagataella phaffii (Pichia pastoris) protein expression systems. The l-asparaginase gene (NCBI OQ439985) has been cloned into Pichia pastoris strains. Enzyme production was analyzed via the quantification of aspartic B-hydroxamate, followed by purification on a DEAE FF ion exchange column. The in silico analysis was proposed based on the combined use of various technological tools. The enzymatic activity found intracellularly was 2.84 IU/g. A purification factor of 1.18 was observed. The in silico analysis revealed the position of five important amino acid residues for enzymatic activity, and likewise, it was possible to predict a monomeric structure with a C-score of 1.59. The production of the enzyme l-asparaginase from F. proliferatum in P. pastoris was demonstrated in this work, being of great importance for the analysis of new methodologies in search of the production of important drugs in therapy.
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Heterologous expression of L-asparaginase (L-ASNase) has become an important area of research due to its clinical and food industry applications. This review provides a comprehensive overview of the molecular and metabolic strategies that can be used to optimize the expression of L-ASNase in heterologous systems. This article describes various approaches that have been employed to increase enzyme production, including the use of molecular tools, strain engineering, and in silico optimization. The review article highlights the critical role that rational design plays in achieving successful heterologous expression and underscores the challenges of large-scale production of L-ASNase, such as inadequate protein folding and the metabolic burden on host cells. Improved gene expression is shown to be achievable through the optimization of codon usage, synthetic promoters, transcription and translation regulation, and host strain improvement, among others. Additionally, this review provides a deep understanding of the enzymatic properties of L-ASNase and how this knowledge has been employed to enhance its properties and production. Finally, future trends in L-ASNase production, including the integration of CRISPR and machine learning tools are discussed. This work serves as a valuable resource for researchers looking to design effective heterologous expression systems for L-ASNase production as well as for enzymes production in general.
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L-asparaginase is used as one of the prime chemotherapeutic agents to treat acute lymphoblastic leukemia. The present work aimed to study the endophytic fungal diversity of Grewia hirsuta and their ability to produce L-asparaginase. A total of 1575 culturable fungal endophytes belonging to four classes, Agaricomycetes, Dothideomycetes, Eurotiomycetes, and Sordariomycetes, were isolated. The isolates were grouped into twenty-one morphotypes based on their morphological characteristics. Representative species from each group were identified based on their microscopic characteristics and evaluation of the ITS and LSU rDNA sequences. Most of the fungal endophytes were recovered from the leaves compared to other plant parts. Diaporthe sp. was the predominant genus with a colonization frequency of 8.62%. Shannon-Wiener index for diversity ranged from 2.74 to 2.88. All the plant parts showed similar Simpson's index values, indicating a uniform species diversity. Among the sixty-three fungal endophytes screened, thirty-two were identified as L-asparaginase-producing isolates. The enzyme activities of fungal endophytes estimated by the nesslerization method were found to be in the range of 4.65-0.27 IU/mL with Fusarium foetens showing maximum enzyme activity of 4.65 IU/mL. This study for the first time advocates the production of L-asparaginase from Fusarium foetens along with the endophytic fungal community composition of Grewia hirsuta. The results indicate that the fungal endophyte Fusarium foetens isolated in the present study could be a potent source of L-asparaginase.
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Grewia , Plantas Medicinais , Asparaginase/genética , Endófitos/genéticaRESUMO
Microbial L-asparaginase is well known for its application in food industries to reduce acrylamide content in fried starchy food. L-asparaginase produced by Arctic actinomycetes Streptomyces koyangensis SK4 was purified and studied for biochemical characterization. The L-asparaginase was purified with a yield of 15.49% and final specific activity of 179.77 IU/mg of protein. The enzyme exhibited a molecular weight of 43 kDa. The optimum pH and temperature for maximum activity of the purified enzyme were 8.5 °C and 40 °C, respectively. The enzyme expressed maximum activity at an incubation period of 30 min and a substrate concentration of 0.06 M. The enzyme has a low Km value of 0.041 M and excellent substrate specificity toward L-asparagine. The enzyme activity was inhibited by metal ions Ba2+ and Hg2+, while Mn2+ and Mg2+ enhanced the activity. The study evaluated the acrylamide reduction potential of L-asparaginase from Streptomyces koyangensis SK4 in potato chips. The blanching plus L-asparaginase treatment of potato slices resulted in a 50% reduction in acrylamide content. The study illustrated an effective acrylamide reduction strategy in potato chips using L-asparaginase from a psychrophilic actinomycete. Besides the acrylamide reduction potential, L-asparaginase from Streptomyces koyangensis SK4 also did not exhibit any glutaminase or urease activity which is an outstanding feature of L-asparaginase to be used as a chemotherapeutic agent.
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Asparaginase , Streptomyces , Asparaginase/genética , Asparaginase/metabolismo , Acrilamida/química , Acrilamida/metabolismo , Streptomyces/metabolismo , TemperaturaRESUMO
Acute lymphoblastic leukemia (ALL) is the most common cancer among children worldwide, characterized by an overproduction of undifferentiated lymphoblasts in the bone marrow. The treatment of choice for this disease is the enzyme L-asparaginase (ASNase) from bacterial sources. ASNase hydrolyzes circulating L-asparagine in plasma, leading to starvation of leukemic cells. The ASNase formulations of E. coli and E. chrysanthemi present notorious adverse effects, especially the immunogenicity they generate, which undermine both their effectiveness as drugs and patient safety. In this study, we developed a humanized chimeric enzyme from E. coli L-asparaginase which would reduce the immunological problems associated with current L-asparaginase therapy. For these, the immunogenic epitopes of E. coli L-asparaginase (PDB: 3ECA) were determined and replaced with those of the less immunogenic Homo sapiens asparaginase (PDB:4O0H). The structures were modeled using the Pymol software and the chimeric enzyme was modeled using the SWISS-MODEL service. A humanized chimeric enzyme with four subunits similar to the template structure was obtained, and the presence of asparaginase enzymatic activity was predicted by protein-ligand docking.
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Antineoplásicos , Leucemia-Linfoma Linfoblástico de Células Precursoras , Criança , Humanos , Asparaginase/genética , Asparaginase/uso terapêutico , Escherichia coli/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Asparagina , Proteínas Recombinantes de Fusão/uso terapêutico , Antineoplásicos/uso terapêuticoRESUMO
BACKGROUND: L-asparaginase (L-ASNase) is an essential component of chemotherapy strategies due to its differential action between normal and leukemic cells. Recently, concerns about the efficiency of commercial formulations administered in developing countries have been reported, and available methods have limitations for directly determining the quality of the formulation of the medications. PROCEDURE: We developed a cell-based protocol to analyze the activity of different L-ASNase formulations used in Colombia to induce apoptosis of the NALM-6 cell line after 24, 48, and 72 hours, using flow cytometry. Then we compared results and determined the statistically significant differences. RESULTS: Three statistically different groups, ranging from full to no activity against leukemic cells, using 0.05, 0.5, and 5.0 IU/ml concentrations, were identified. Group 1 (asparaginase codified [ASA]2-4) exhibited very low to no activity against B-cell acute lymphoblastic leukemia (B-ALL) cells. Group 2 (ASA6) exhibited intermediate-level activity, and group 3 (ASA1 and ASA5) exhibited high activity. CONCLUSIONS: Differences found between the therapeutic formulations of L-ASNase distributed in Colombia raise concerns about the quality of the treatment administered to patients in low- and middle-income countries. Therefore, we recommend a preclinical evaluation of formulations of L-ASNase in order to prevent therapeutical impacts on the outcome of ALL patients.
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Antineoplásicos , Asparaginase , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Antineoplásicos/uso terapêutico , Asparaginase/uso terapêutico , Linhagem Celular , Colômbia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológicoRESUMO
El objetivo del estudio fue realizar la caracterización bioinformática, así como optimizar la producción de L-asparaginasa extracelular de Bacillus sp. M62 aislada de las salinas de Maras (Cusco). Para ello, se verificó la producción de L-asparaginasa mediante el viraje del medio M9 modificado con azul de bromofenol 0.0075%, pH 7.4 a 37 °C por 72 h. A la vez, se extrajo el ADN genómico para amplificar los genes ribosómicos 16S y el gen ansA3. La secuencia aminoacídica codificada por el gen ansA3 se predijo mediante análisis bioinformático. La producción de L-asparaginasa intracelular y extracelular se evaluó a diferentes niveles de glucosa, L-asparagina, NaCl y pH en el medio M9 modificado. Adicionalmente, las actividades enzimáticas de L-asparaginasa y L-glutaminasa se determinaron mediante cuantificación del amonio liberado por el método de Nessler. Así, Bacillus sp. M62 produjo el viraje del medio M9 modificado, obtuvo alta similitud y cercanía evolutiva con Bacillus licheniformis, se encontró que el gen ansA3 amplificado codificaba para 319 aa, dentro de la cual se predijo una secuencia patrón del sitio activo (GFVITHGTDTM ) y 15 sitios inmunogénicos. La producción de L-asparaginasa extracelular fue superior a la intracelular, la que se optimizó de 0.37 U/mL (0.24 U/mg) a 2.15 ± 0.39 U/mL (0.63 U/mg). Finalmente, se encontró que Bacillus sp. M62 presenta L-asparaginasa extracelular con mínima actividad de L-glutaminasa.
The aim of this study was to perform bioinformatics characterization and optimize the production of extracellular L-asparaginase from Bacillus sp. M62, isolated from the Maras salt ponds (Cusco). To achieve this, the production of L-asparaginase was verified by the change in color of modified M9 medium, containing 0.0075% bromophenol blue, at pH 7.4 and 37°C for 72 hours. Genomic DNA was extracted to amplify the 16S ribosomal genes and the ansA3 gene. The amino acid sequence encoded by the ansA3 gene was predicted using bioinformatic analysis. The production of intracellular and extracellular L-asparaginase was evaluated at different levels of glucose, L-asparagine, NaCl, and pH in modified M9 medium. Additionally, the enzymatic activities of L-asparaginase and L-glutaminase were determined by quantifying the released ammonium using the Nessler method. Bacillus sp. M62 showed the change in color of the modified M9 medium, high similarity, and evolutionary closeness to Bacillus licheniformis. The amplified ansA3 gene was found to encode for 319 amino acids, with a predicted active site pattern (GFVITHGTDTM) and 15 immunogenic sites. The production of extracellular L-asparaginase was found to be higher than intracellular L-asparaginase and was optimized from 0.37 U/mL (0.24 U/mg) to 2.15 ± 0.39 U/mL (0.63 U/mg). Finally, it was found that Bacillus sp. M62 presents extracellular L-asparaginase with minimal L-glutaminase activity.
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Helicobacter pylori has become the causal agent of multiple forms of gastric disease worldwide, including gastric cancer. The enzyme l-asparaginase (ASNase) has been studied as a virulence factor. In this work, we performed an in silico investigation to characterize the immunological profile of H. pylori ASNase (HpASNase) to ascertain the possible implication of HpASNase immunogenicity in the H. pylori virulence mechanism. We applied a workflow based on bioinformatics tools, which, by calculating the relative frequency of immunogenic T-cell and B-cell epitopes, allowed us to predict the immunogenicity and allergenicity of HpASNase in silico. We also visualized the epitopes by mapping them into the native structure of the enzyme. We report for the first time the T-cell and B-cell epitope composition that contributes to the immunogenicity of this HpASNase, as well as the regions that could generate a hypersensitivity response in humans. ASNase from H. pylori resulted in highly immunogenic and allergenic. The high immunogenicity of HpASNase could imply the pathogenic mechanisms of H. pylori. This knowledge could be important for the development of new drugs against H. pylori infections. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03359-0.
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L-asparaginase (ASNase) is an important biological drug used to treat Acute Lymphoblastic Leukemia (ALL). It catalyzes the hydrolysis of L-asparagine (Asn) in the bloodstream and, since ALL cells cannot synthesize Asn, protein synthesis is impaired leading to apoptosis. Despite its therapeutic importance, ASNase treatment is associated to side effects, mainly hypersensitivity and immunogenicity. Furthermore, degradation by plasma proteases and immunogenicity shortens the enzyme half-life. Encapsulation of ASNase in liposomes, nanostructures formed by the self-aggregation of phospholipids, is an attractive alternative to protect the enzyme from plasma proteases and enhance pharmacokinetics profile. In addition, PEGylation might prolong the in vivo circulation of liposomes owing to the spherical shielding conferred by the polyethylene (PEG) corona around the nanostructures. In this paper, ASNase was encapsulated in liposomal formulations composed by 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) containing or not different concentrations of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N [methoxy (polyethylene glycol)-2000] (DSPE-PEG). Nanostructures of approximately 142-202 nm of diameter and polydispersity index (PDI) of 0.069 to 0.190 were obtained and the vesicular shape confirmed by Transmission Electron Microscopy (TEM and cryo-TEM). The encapsulation efficiency (%EE) varied from 10% to 16%. All formulations presented activity in contact with ASNase substrate, indicating the liposomes permeability to Asn and/or enzyme adsorption at the nanostructures' surface; the highest activity was observed for DMPC/DSPE-PEG 10%. Finally, we investigated the activity against the Molt 4 leukemic cell line and found a lower IC50 for the DMPC/DSPE-PEG 10% formulation in comparison to the free enzyme, indicating our system could provide in vivo activity while protecting the enzyme from immune system recognition and proteases degradation.
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L-Asparaginase (L-ASNase) is an enzyme applied in the treatment of lymphoid malignancies. However, an innovative L-ASNase with high yield and lower side effects than the commercially available preparations are still a market requirement. Here, a new-engineered Bacillus subtilis strain was evaluated for Aliivibrio fischeri L-ASNase II production, being the bioprocess development and the enzyme characterization studied. The pBS0E plasmid replicative in Bacillus sp and containing PxylA promoter inducible by xylose and its repressive molecule sequence (XylR) was used for the genetic modification. Initially, cultivations were carried out in orbital shaker, and then the process was scaled up to stirred tank bioreactor (STB). After the bioprocess, the cells were recovered and submitted to ultrasound sonication for cells disruption and intracellular enzyme recovery. The enzymatic extract was characterized to assess its biochemical, kinetic and thermal properties using L-Asparagine and L-Glutamine as substrates. The results indicated the potential enzyme production in STB achieving L-ASNase activity up to 1.539 U mL-1. The enzymatic extract showed an optimum pH of 7.5, high L-Asparagine affinity (Km = 1.2275 mmol L-1) and low L-Glutaminase activity (0.568-0.738 U mL-1). In addition, thermal inactivation was analyzed by two different Kinect models to elucidate inactivation mechanisms, low kinetic thermal inactivation constants for 25 ºC and 37 ºC (0.128 and 0.148 h-1, respectively) indicate an elevated stability. The findings herein show that the produced recombinant L-ASNase has potential to be applied for pharmaceutical purposes.
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Antineoplásicos , Produtos Biológicos , Aliivibrio fischeri , Antineoplásicos/química , Asparaginase/química , Asparaginase/genética , Asparaginase/uso terapêutico , Asparagina , Bacillus subtilis/genética , Glutaminase , Glutamina , Preparações Farmacêuticas , XiloseRESUMO
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.
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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.
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Asparaginase/isolamento & purificação , Aspergillus oryzae/metabolismo , Meios de Cultura , Estabilidade Enzimática , Fermentação , Temperatura Alta , Concentração de Íons de Hidrogênio , HidróliseRESUMO
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.
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Asparaginase , Leucemia-Linfoma Linfoblástico de Células Precursoras , Asparaginase/química , Bactérias/metabolismo , Humanos , Hidrólise , FilogeniaRESUMO
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.
Assuntos
Asparaginase/isolamento & purificação , Precipitação Fracionada/métodos , Antineoplásicos/isolamento & purificação , Estudos de Viabilidade , Cromatografia em Gel , Análise Custo-BenefícioRESUMO
A Leucemia Linfoide Aguda (LLA) é um câncer de maior incidência em crianças, e tem a Lasparaginase (ASNase) como fármaco amplamente utilizado no tratamento dos afetados. A ASNase catalisa a hidrólise do aminoácido L-asparagina (Asn), presente na corrente sanguínea, a ausência do aminoácido no meio extracelular leva à morte células leucêmicas, que necessitam deste aminoácido para as funções celulares. Fatores envolvendo a eficiência do tratamento com ASNase como reações adversas e curta meia-vida, principalmente devido ao reconhecimento pelo sistema imune e degradação por proteases, limitam a sua eficácia. A encapsulação da enzima em lipossomas pode conferir proteção à degradação, melhorar seu perfil farmacocinético e diminuir os efeitos adversos, de forma a melhorar o tratamento da LLA sendo este o objetivo desse trabalho. Lipossomas de DOPC (1,2-dioleoil-sn-glicero-3-fosfocolina) e DMPC (1,2-dimiristoil-snglicero-3-fosfocolina) foram desenvolvidos empregando-se o método de hidratação do filme lipídico e diferentes protocolos de preparo contendo ou não diferentes concentrações de 18:0 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polietilenogicol)-2000] (DSPE-PEG). Os lipossomas produzidos foram utilizados para encapsular a ASNase e os sistemas contendo ou não ASNase encapsulada foram caracterizados por espalhamento de luz dinâmico (DLS), potencial zeta, microscopia eletrônica de transmissão (MET) e criomicroscopia de transmissão. Adicionalmente, foram avaliados a taxa de encapsulação e o perfil de permeabilidade das vesículas à L-asparagina. As análises de DLS mostraram que as nanoestruturas formadas empregando-se agitação magnética a partir de sistemas contendo 10% e 20% de DSPE-PEG possuem diâmetro hidrodinâmico menor (~ 25 nm a 60 nm) que os mesmos sistemas sem o fosfolipídio peguilado (~190 nm a 222 nm), demonstrando a relação entre a diminuição do tamanho e o aumento da quantidade de fosfolipídio peguilado e possível formação de estruturas micelares ou bicelares. O emprego de agitação em vórtex para hidratação do filme lipídico, adição do antioxidante -tocoferol e redução da concentração de DSPE-PEG (5% e 10%) levou à formação de sistemas com diâmetro hidrodinâmico maior, sendo esse protocolo e concentrações de PEG definidos como padrão. As análises de MET comprovaram a formação de lipossomas com diâmetro hidrodinâmico semelhante ao observado por DLS; com a utilização da criomicroscopia foi possível observar os lipossomas sem deformações. Os lipossomas de DMPC/DSPE-PEG 10% apresentaram maior permeabilidade à L-asparagina ao longo do tempo e, portanto, poderiam funcionar como nanoreatores, depletando o aminoácido da circulação. Estudos in vitro com células tumorais devem ser realizados e em seguida estudos in vivo, para confirmar este potencial
L-asparaginase (ASNase) is a first-choice drug, combined with other drugs, in therapeutic schemes to treat Acute Lymphoblastic Leukemia (ALL) in children and adolescents. ASNase catalyzes the hydrolysis of L-asparagine (Asn) in the bloodstream; since ALL cells cannot synthesize this amino acid, protein synthesis is impaired leading to leukemic cells death by apoptosis. In spite of its therapeutic importance, treatment with ASNase is associated to side effects, mainly hypersensitivity and immunogenicity. Another drawback refers to degradation by plasma proteases that altogether with immunogenicity shortens the enzyme half-life. Encapsulation of ASNase in liposomes, vesicular nanostructures formed by the self-aggregation of phospholipids, is an attractive alternative that possibly will protect the enzyme from plasma proteases, resulting on better pharmacokinetics profile. In this work, we prepared by thin film hydration liposomal formulations of the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC) containing or not different concentrations of 18:0 1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG), and encapsulated ASNase by electroporation. The systems containing or not ASNase were analyzed by Dynamic Light Scattering, zeta potential and Electron Microscopy. The encapsulation efficiency and vesicles permeability were also evaluated. According to the DLS analysis, the nanostructures formed by film hydration under magnetic stirring employing 10% or 20% DSPE-PEG presented smaller hydrodynamic diameter (~ 25 nm to 60 nm) than the same systems without the pegylated phospholipid (~ 190 nm to 222 nm), demonstrating the relation between size and the amount of pegylated phospholipid that results in formation of micellar or bicellar structures. The protocol was stabilize by hydration of the lipid film under vortex agitation, addition of the antioxidant - tocopherol and reduction of the concentration of DSPE-PEG (5% and 10%), what altogether led to the formation of nanostructures of higher hydrodynamic diameter and monodisperse systems. TEM analyzes confirmed the formation of liposomes with hydrodynamic diameter similar to that observed by DLS; with the use of cryomicroscopy it was possible to observe the liposomes without deformations. Liposomes of DMPC/DSPE-PEG 10% showed permeability to L-asparagine over time and, therefore, could function as nanoreactors, depleting the circulating amino acid