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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 -
BioDrugs : Clinical Immunotherapeutics,... Nov 2023Over the past few years, there has been a surge in the industrial production of recombinant enzymes from microorganisms due to their catalytic characteristics being... (Review)
Review
Over the past few years, there has been a surge in the industrial production of recombinant enzymes from microorganisms due to their catalytic characteristics being highly efficient, selective, and biocompatible. L-asparaginase (L-ASNase) is an enzyme belonging to the class of amidohydrolases that catalyzes the hydrolysis of L-asparagine into L-aspartic acid and ammonia. It has been widely investigated as a biologic agent for its antineoplastic properties in treating acute lymphoblastic leukemia. The demand for L-ASNase is mainly met by the production of recombinant type II L-ASNase from Escherichia coli and Erwinia chrysanthemi. However, the presence of immunogenic proteins in L-ASNase sourced from prokaryotes has been known to result in adverse reactions in patients undergoing treatment. As a result, efforts are being made to explore strategies that can help mitigate the immunogenicity of the drug. This review gives an overview of recent biotechnological breakthroughs in enzyme engineering techniques and technologies used to improve anti-leukemic L-ASNase, taking into account the pharmacological importance of L-ASNase.
Topics: Humans; Antineoplastic Agents; Asparaginase; Biological Factors; Biological Products; Escherichia coli; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Protein Engineering
PubMed: 37698749
DOI: 10.1007/s40259-023-00622-5 -
Paediatric Drugs Sep 2021Asparaginase therapy is a vital agent in the treatment of acute lymphoblastic leukemia (ALL), with increasing evidence of its high importance in high-risk ALL... (Review)
Review
Asparaginase therapy is a vital agent in the treatment of acute lymphoblastic leukemia (ALL), with increasing evidence of its high importance in high-risk ALL populations. However, despite the clear clinical and biological benefits of asparaginase therapy, many patients experience toxicities. A well-known treatment-limiting toxicity is asparaginase-associated pancreatitis (AAP). If severe, it necessitates discontinuation of asparaginase therapy, which can lead to a higher risk of relapse in patients with ALL. New protocols for ALL therapy have increased overall total doses of asparaginase therapy in select high-risk populations and have incorporated longer half-life formulations of pegylated asparaginase. Treatment drug monitoring has also allowed assurance of adequate levels of asparagine depletion throughout treatment. It is currently unknown if these changes will increase rates of AAP. Interestingly, important pharmacogenomics data, such as single nucleotide polymorphisms, can identify patients at the highest risk for severe AAP. The incidence of AAP in recent trials, current pharmacogenomic data that could further our understanding of the disease, and the importance of cautiously re-exposing patients to further asparaginase treatment after an initial episode of AAP are discussed.
Topics: Acute Disease; Antineoplastic Agents; Asparaginase; Child; Humans; Pancreatitis; Precursor Cell Lymphoblastic Leukemia-Lymphoma
PubMed: 34351604
DOI: 10.1007/s40272-021-00463-1 -
Blood Advances Jan 2022Asparaginase treatment is a mainstay in contemporary treatment of acute lymphoblastic leukemia (ALL), but substantial asparaginase-related toxicity may lead to...
Asparaginase treatment is a mainstay in contemporary treatment of acute lymphoblastic leukemia (ALL), but substantial asparaginase-related toxicity may lead to jeopardized protocol compliance and compromises survival. We investigated the association between risk of asparaginase-associated toxicities (AspTox) and asparaginase enzyme activity (AEA) levels in 1155 children aged 1.0 to 17.9 years, diagnosed with ALL between July 2008 and March 2016, and treated according to the Nordic Society of Pediatric Hematology and Oncology (NOPHO) ALL2008 protocol. Patients with ≥2 blood samples for AEA measurement drawn 14 ± 2 days after asparaginase administration were included (6944 trough values). AEA was measurable (or >0 IU/L) in 955 patients, whereas 200 patients (17.3%) had asparaginase inactivation and few AspTox recorded. A time-dependent multiple Cox model of time to any first asparaginase-associated toxicity adjusted for sex and age was used. For patients with measurable AEA, we found a hazard ratio (HR) of 1.17 per 100 IU/L increase in median AEA (95% confidence interval [CI], 0.98-1.41; P = .09). For pancreatitis, thromboembolism, and osteonecrosis, the HRs were 1.40 (95% CI, 1.12-1.75; P = .002), 0.99 (95% CI, 0.70-1.40; P = .96), and 1.36 (95% CI, 1.04-1.77; P = .02) per 100 IU/L increase in median AEA, respectively. No significant decrease in the risk of leukemic relapse was found: HR 0.88 per 100 IU/L increase in AEA (95% CI, 0.66-1.16; P = .35). In conclusion, these results emphasize that overall AspTox and relapse are not associated with AEA levels, yet the risk of pancreatitis and osteonecrosis increases with increasing AEA levels.
Topics: Adolescent; Antineoplastic Agents; Asparaginase; Child; Child, Preschool; Humans; Infant; Polyethylene Glycols; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Thromboembolism
PubMed: 34625787
DOI: 10.1182/bloodadvances.2021005631 -
Orthopadie (Heidelberg, Germany) Oct 2022Osteonecrosis occurs as an acute and long-term serious side effect in children, adolescents, and adults with acute lymphoblastic leukemia. It is associated with severe... (Review)
Review
Osteonecrosis occurs as an acute and long-term serious side effect in children, adolescents, and adults with acute lymphoblastic leukemia. It is associated with severe pain and reduced mobility, ultimately leading to joint destruction and significant long-term morbidity. The cumulative incidence ranges from 11 to 20% in adolescents and young adults. In symptomatic patients, multiple joints are frequently affected, which in turns poses a risk factor for the development of severe osteonecrosis. The genesis of leukemia-associated osteonecrosis is multifactorial. Risk factors include the use of corticosteroids and asparaginase. These exert their effects on the blood supply to the bone through hypercholesterolemia, hypertriglyceridemia, and hypertension. Bacteriemia, genetic susceptibility, and stem cell transplantation pose additional risk factors. The treatment of osteonecrosis is challenging and not evidence based. Preventive measurements have as yet mainly been tested in preclinical models.
Topics: Acute Disease; Adolescent; Asparaginase; Child; Hematopoietic Stem Cell Transplantation; Humans; Incidence; Osteonecrosis; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Young Adult
PubMed: 36069910
DOI: 10.1007/s00132-022-04301-1 -
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 -
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