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Biotechnology and Applied Biochemistry Jul 2020l-Asparaginase (E.C.3.5.1.1.) is a vital enzyme that hydrolyzes l-asparagine to l-aspartic acid and ammonia. This property of l-asparaginase inhibits the protein... (Review)
Review
l-Asparaginase (E.C.3.5.1.1.) is a vital enzyme that hydrolyzes l-asparagine to l-aspartic acid and ammonia. This property of l-asparaginase inhibits the protein synthesis in cancer cells, making l-asparaginase a mainstay of pediatric chemotherapy practices to treat acute lymphoblastic leukemia (ALL) patients. l-Asparaginase is also recognized as one of the important food processing agent. The removal of asparagine by l-asparaginase leads to the reduction of acrylamide formation in fried food items. l-Asparaginase is produced by various organisms including animals, plants, and microorganisms, however, only microorganisms that produce a substantial amount of this enzyme are of commercial significance. The commercial l-asparaginase for healthcare applications is chiefly derived from Escherichia coli and Erwinia chrysanthemi. A high rate of hypersensitivity and adverse reactions limits the long-term clinical use of l-asparaginase. Present review provides thorough information on microbial l-asparaginase bioprocess optimization including submerged fermentation and solid-state fermentation for l-asparaginase production, downstream purification, its characterization, and issues related to the clinical application including toxicity and hypersensitivity. Here, we have highlighted the bioprocess techniques that can produce improved and economically viable yields of l-asparaginase from promising microbial sources in the current scenario where there is an urgent need for alternate l-asparaginase with less adverse effects.
Topics: Animals; Asparaginase; Dickeya chrysanthemi; Escherichia coli; Escherichia coli Proteins; Humans; Precursor Cell Lymphoblastic Leukemia-Lymphoma
PubMed: 31954377
DOI: 10.1002/bab.1888 -
International Journal of Oncology Apr 2021L‑asparaginase enzymes have been a vital component of acute lymphoblastic leukemia therapy for >40 years. L‑asparaginase acts by depleting plasma L‑asparagine,... (Review)
Review
L‑asparaginase enzymes have been a vital component of acute lymphoblastic leukemia therapy for >40 years. L‑asparaginase acts by depleting plasma L‑asparagine, which is essential to the survival of leukemia cells. In contrast to normal cells, tumor cells cannot synthesize L‑asparagine and thus depend on its external uptake for growth. Currently, three bacterial L‑asparaginases are used in therapy; however, they are associated with severe side‑effects related to high toxicity and immunogenicity. The introduction of human L‑asparaginase‑like protein 1 in acute lymphoblastic leukemia treatment would avoid the problems caused by the bacterial enzymes; however, a major difficulty in the therapeutic use of the human enzyme comes from the fact that human L‑asparaginase must be activated through an autoprocessing step, which is a low‑efficiency process in vitro that results in reduced enzymatic activity. The present review article aimed to contribute to the understanding of the enzyme self‑activation process and focuses on the efforts made for the development of a therapeutic variant of human L‑asparaginase.
Topics: Asparaginase; Autoantigens; Enzyme Activation; Humans; Precursor Cell Lymphoblastic Leukemia-Lymphoma
PubMed: 33649831
DOI: 10.3892/ijo.2021.5191 -
Applied Microbiology and Biotechnology Jul 2019L-asparaginase is an enzyme produced by microorganisms, plants, and animals, which is used clinically for the treatment for acute lymphoblastic leukemia (ALL) and, in... (Review)
Review
L-asparaginase is an enzyme produced by microorganisms, plants, and animals, which is used clinically for the treatment for acute lymphoblastic leukemia (ALL) and, in the food industry, to control acrylamide formation in baked foods. The purpose of this review was to evaluate the available literature regarding microbial sources of L-asparaginase, culture media used to achieve maximum enzyme expression in microbial fermentations, and assay methods employed to assess L-asparaginase activity. Studies were gathered by searching PubMed, and Web of Science databases before January 22, 2018, with no time restrictions. The articles were evaluated according to the source of L-asparaginase being studied, the nitrogen source in the culture medium, the type of sample, and the method employed to evaluate L-asparaginase activity. Bacterial L-asparaginase appeared to be the most commonly studied source of the enzyme and, most often, the enzyme activity was assayed from crude protein extracts using the Nessler method, which is an indirect measurement of asparaginase activity that determines the concentration of ammonia generated after the action of the enzyme on the substrate, L-asparagine. However, ammonia is also generated throughout microbial fermentations and this endogenous ammonia will also reduce the Nessler reagent if crude microbial extracts are used to determine total L-asparaginase activity. We suggest that current estimates of L-asparaginase activity reported in the literature may be overestimated when Nessler reagent is used, since we were unable to find a single study that made reference to the possible inference of fermentation derived ammonia.
Topics: Ammonia; Asparaginase; Asparagine; Bacteria; Biological Assay; Culture Media; Fermentation
PubMed: 31104099
DOI: 10.1007/s00253-019-09890-0 -
Biomeditsinskaia Khimiia Feb 2023L-asparaginase (EC 3.5.1.1) is one of the most demanded enzymes used in the pharmaceutical industry as a drug and in the food industry to prevent the formation of toxic... (Review)
Review
L-asparaginase (EC 3.5.1.1) is one of the most demanded enzymes used in the pharmaceutical industry as a drug and in the food industry to prevent the formation of toxic acrylamide. Researchers aimed to improve specific activity and reduce side effects to create safer and more potent enzyme products. However, protein modifications and heterologous expression remain problematic in the production of asparaginases from different species. Heterologous expression in optimized producer strains is rationally organized; therefore, modified and heterologous protein expression is enhanced, which is the main strategy in the production of asparaginase. This strategy solves several problems: incorrect protein folding, metabolic load on the producer strain and codon misreading, which affects translation and final protein domains, leading to a decrease in catalytic activity. The main approaches developed to improve the heterologous expression of L-asparaginases are considered in this paper.
Topics: Asparaginase; Acrylamide; Protein Processing, Post-Translational
PubMed: 36857424
DOI: 10.18097/PBMC20236901019 -
Applied Microbiology and Biotechnology Sep 2022Metabolic differences between normal and cancerous cells have been used as a point of view for developing anticancer drugs. Some degrading enzymes of certain amino acids... (Review)
Review
Metabolic differences between normal and cancerous cells have been used as a point of view for developing anticancer drugs. Some degrading enzymes of certain amino acids have been regarded to kill cancerous cells. L-Asparaginase (ASNase) has shown an excellent therapeutic response to asparagine-auxotrophic cancers such as acute lymphoblastic leukemia (ALL). Some bacteria, yeasts, molds, plants, and animals produce ASNase. Bacterial ASNases from Escherichia coli and Erwinia chrysanthemi are the FDA-approved drugs for ALL treatment. Here, we review new natural prokaryotic and eukaryotic sources of ASNases, recent advances to introduce improvement strategies for the production of recombinant ASNases as well as their chemical modifications, immobilization, nanoencapsulation, and in silico studies to increase efficiency and decrease side effects. Recent studies for application of ASNases to treatment of asparagine-auxotrophic cancers, especially solid cancers, have been reviewed. Furthermore, challenges and future perspectives are discussed for this promising therapeutic enzyme. KEY POINTS: • Review recent advances to introduce new sources of microbial L-asparaginases. • Review improvement strategies for the development of stable and non-toxic L-asparaginases. • Review microbial L-asparaginase application in various cancers' treatment.
Topics: Animals; Antineoplastic Agents; Asparaginase; Asparagine; Bacteria; Escherichia coli; Precursor Cell Lymphoblastic Leukemia-Lymphoma
PubMed: 35871694
DOI: 10.1007/s00253-022-12086-8 -
Biochemistry May 2020Two bacterial type II l-asparaginases, from and , have played a critical role for more than 40 years as therapeutic agents against juvenile leukemias and lymphomas....
Two bacterial type II l-asparaginases, from and , have played a critical role for more than 40 years as therapeutic agents against juvenile leukemias and lymphomas. Despite a long history of successful pharmacological applications and the apparent simplicity of the catalytic reaction, controversies still exist regarding major steps of the mechanism. In this report, we provide a detailed description of the reaction catalyzed by type II l-asparaginase (EcAII). Our model was developed on the basis of new structural and biochemical experiments combined with previously published data. The proposed mechanism is supported by quantum chemistry calculations based on density functional theory. We provide strong evidence that EcAII catalyzes the reaction according to the double-displacement (ping-pong) mechanism, with formation of a covalent intermediate. Several steps of catalysis by EcAII are unique when compared to reactions catalyzed by other known hydrolytic enzymes. Here, the reaction is initiated by a weak nucleophile, threonine, without direct assistance of a general base, although a distant general base is identified. Furthermore, tetrahedral intermediates formed during the catalytic process are stabilized by a never previously described motif. Although the scheme of the catalytic mechanism was developed only on the basis of data obtained from EcAII and its variants, this novel mechanism of enzymatic hydrolysis could potentially apply to most (and possibly all) l-asparaginases.
Topics: Asparaginase; Biocatalysis; Crystallography, X-Ray; Dickeya chrysanthemi; Escherichia coli; Hydrolysis; Kinetics; Models, Molecular
PubMed: 32364696
DOI: 10.1021/acs.biochem.0c00116 -
Advances in Food and Nutrition Research 2016l-Asparaginase, an enzyme that catalyzes l-asparagine into aspartic acid and ammonia, has relevant applications in the pharmaceutical and food industry. So, this enzyme... (Comparative Study)
Comparative Study Review
l-Asparaginase, an enzyme that catalyzes l-asparagine into aspartic acid and ammonia, has relevant applications in the pharmaceutical and food industry. So, this enzyme is used in the treatment of acute lymphoblastic leukemia, a malignant disorder in children. This enzyme is also able to reduce the amount of acrylamide found in carbohydrate-rich fried and baked foods which is carcinogenic to humans. The concentration of acrylamide in food can be reduced by deamination of asparagine using l-Asparaginase. l-Asparaginase is present in plants, animals, and microbes. Various microorganisms such as bacteria, yeast, and fungi are generally used for the production of l-Asparaginase as it is difficult to obtain the same from plants and animals. l-Asparaginase from bacteria causes anaphylaxis and other abnormal sensitive reactions. To overcome this, eukaryotic organisms such as fungi can be used for the production of l-Asparaginase. l-Asparaginase can be produced either by solid-state fermentation (SSF) or by submerged fermentation (SmF). SSF is preferred over SmF as it is cost effective, eco-friendly and it delivers high yield of enzyme. SSF process utilizes agricultural and industrial wastes as solid substrate. The contamination level is substantially reduced in SSF through low moisture content. Current chapter will discuss in detail the chemistry and applications of l-Asparaginase enzyme and various methods available for the production of the enzyme, especially focusing on the advantages and limitations of SSF and SmF processes.
Topics: Actinobacteria; Asparaginase; Bacteria; Enzymes, Immobilized; Fermentation; Food Handling; Fungi
PubMed: 27452168
DOI: 10.1016/bs.afnr.2016.05.003 -
Preparative Biochemistry & Biotechnology 2023L-asparaginase is an enzyme commonly used to treat acute lymphoblastic leukemia. Commercialized bacterial L-asparaginase has been reported to cause several...
L-asparaginase is an enzyme commonly used to treat acute lymphoblastic leukemia. Commercialized bacterial L-asparaginase has been reported to cause several life-threatening complications during treatment, hence the need to seek alternative sources of L-asparaginase. In this study, the novelty of upstream and downstream bioprocessing of L-asparaginase from a fungal endophyte, and the cytotoxicity evaluation was demonstrated. Six variables (carbon source and concentration, nitrogen source and concentration, incubation period, temperature, pH and agitation rate) known to influence L-asparaginase production were studied using One-Factor-At-A-Time (OFAT) approach, with four significant variables further optimized using Response Surface Methodology (RSM). The crude extract produced using optimized condition was purified, characterized and examined for its anticancer effect. Purification of fungal L-asparaginase was performed via ultrafiltration and size exclusion chromatography, which are less common techniques. The protein profile and monomeric weight of L-asparaginase were determined using SDS-PAGE and Western blot. Cytotoxicity of purified L-asparaginase on leukemic Jurkat E6 and oral carcinoma cells were studied using MTS assay for 24 h and 48 h. OFAT results from optimization showed that glucose and L-asparagine concentrations, incubation period and temperature, were significant factors affecting L-asparaginase production by RSM analysis further evidence the significant interaction between glucose and L-asparagine concentrations in inducing L-asparaginase production. Purified L-asparaginase was profiled with specific activity of 255.02 IU/mg protein, purification fold of 6.12, and 34.63% of enzyme recovery. SDS and Western blot revealed that the purified L-asparaginase might be a tetramer with monomeric units of 25 kDa. Purified L-asparaginase was discovered to be more efficient against Jurkat leukemic cells than against H103 oral carcinoma cells, as lower IC value was observed for Jurkat cell lines (46 .36 ± 1.52 µg/mL for Jurkat and 125.56 ± 7.28 µg/mL for H103). In short, purified L-asparaginase derived from endophytic showed high purity and significant anticancer effect toward cancer cells. This study therefore demonstrated the potential of fungal L-asparaginase as alternative chemotherapy drug in the future.
Topics: Humans; Asparaginase; Antineoplastic Agents; Asparagine; Carcinoma
PubMed: 36137173
DOI: 10.1080/10826068.2022.2122064 -
Advances in Colloid and Interface... Jun 2023L-asparaginase (L-ASP) is one of the key enzymes used in therapeutic applications, particularly to treat Acute Lymphocytic Leukemia (ALL). L-asparagine is a... (Review)
Review
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.
Topics: Humans; Asparaginase; Antineoplastic Agents; Asparagine; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Aspartate-Ammonia Ligase
PubMed: 37159987
DOI: 10.1016/j.cis.2023.102915 -
Cancer Medicine Dec 2023The clinicopathologic characteristics and prognosis of nasal and nonnasal extranodal natural killer T-cell lymphoma (ENKTL) are considered to be different. However, the...
BACKGROUND
The clinicopathologic characteristics and prognosis of nasal and nonnasal extranodal natural killer T-cell lymphoma (ENKTL) are considered to be different. However, the underlying features responsible for these differences are not well clarified especially in the era of asparaginase therapy.
METHODS
In total, 1007 newly diagnosed ENKTL patients from 11 medical centers were included in this study. Clinicopathologic characteristics and survival data were collected. The chi-squared test and Kruskal-Wallis test were utilized for the comparison of different groups. Univariable and multivariable Cox proportional hazards models were used to screen prognostic factors.
RESULTS
Overall, 869 (86.3%) patients were nasal forms. Compared to patients with nasal ENKTL, nonnasal patients were at more advanced stages and had poor performance status, bone marrow involvement, elevated serum lactate dehydrogenase (LDH), and CD56-negative status (p < 0.05). The 5-year overall survival (OS) for nasal and nonnasal patients were 65.6% and 45.0%, respectively. The OS of nasal forms patients were superior to nonnasal patients, especially in Eastern Cooperative Oncology Group performance status (ECOG PS) (≥2), advanced stage, KPI (HIR/HR), IPI (HIR/HR), PINK (HR), and high EBV DNA load groups. In patients treated with pegaspargase/L-asparaginase-based regimens, the OS of nasal patients was better than that of nonnasal patients. After adjusting the covariates of age, stage, ECOG PS score, LDH, B symptoms, and BM involvement, results showed that the nonnasal site was associated with poor survival of ENKTL.
CONCLUSIONS
The clinicopathologic characteristics and prognosis of nasal and nonnasal ENKTL patients are different. Nasal forms patients had superior OS than nonnasal patients, especially in the era of asparaginase.
Topics: Humans; Asparaginase; Lymphoma, Extranodal NK-T-Cell; Neoplasm Staging; Prognosis; Retrospective Studies
PubMed: 37902266
DOI: 10.1002/cam4.6674