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Journal of Microbiology and... May 2022L-asparaginase (E.C. 3.5.1.1) purified from bacterial cells is widely used in the food industry, as well as in the treatment of childhood acute lymphoblastic leukemia....
L-asparaginase (E.C. 3.5.1.1) purified from bacterial cells is widely used in the food industry, as well as in the treatment of childhood acute lymphoblastic leukemia. In the present study, the L-asparaginase gene was cloned into the pGEX-2T DNA plasmid, expressed in BL21 (DE3) pLysS, and purified to homogeneity using Glutathione Sepharose chromatography with 7.26 purification fold and 16.01% recovery. The purified enzyme exhibited a molecular weight of ~33.6 kDa with SDS-PAGE and showed maximal activity at 50°C and pH 8.0. It retained 95.1, 89.6%, and 70.2% initial activity after 60 min at 30°C, 40°C, and 50°C, respectively. The enzyme reserved its activity at 30°C and 37°C up to 24 h. The enzyme had optimum pH of 8 and reserved 50% activity up to 24 h. The recombinant enzyme showed the highest substrate specificity towards L-asparaginase substrate, while no detectable specificity was observed for L-glutamine, urea, and acrylamide at 10 mM concentration. THP-1, a human leukemia cell line, displayed significant morphological alterations after being treated with recombinant L-asparaginase and the IC of the purified enzyme was recorded as 0.8 IU. Furthermore, the purified recombinant L-asparaginase improved cytotoxicity in liver cancer HepG2 and breast cancer MCF-7 cell lines, with IC values of 1.53 and 18 IU, respectively.
Topics: Asparaginase; Burkholderia pseudomallei; Enzyme Stability; Escherichia coli; Humans; Recombinant Proteins; Substrate Specificity
PubMed: 35354764
DOI: 10.4014/jmb.2112.12050 -
PloS One 2019L-asparaginase (ASNase) from Escherichia coli is currently used in some countries in its PEGylated form (ONCASPAR, pegaspargase) to treat acute lymphoblastic leukemia...
L-asparaginase (ASNase) from Escherichia coli is currently used in some countries in its PEGylated form (ONCASPAR, pegaspargase) to treat acute lymphoblastic leukemia (ALL). PEGylation refers to the covalent attachment of poly(ethylene) glycol to the protein drug and it not only reduces the immune system activation but also decreases degradation by plasmatic proteases. However, pegaspargase is randomly PEGylated and, consequently, with a high degree of polydispersity in its final formulation. In this work we developed a site-specific N-terminus PEGylation protocol for ASNase. The monoPEG-ASNase was purified by anionic followed by size exclusion chromatography to a final purity of 99%. The highest yield of monoPEG-ASNase of 42% was obtained by the protein reaction with methoxy polyethylene glycol-carboxymethyl N-hydroxysuccinimidyl ester (10kDa) in 100 mM PBS at pH 7.5 and PEG:ASNase ratio of 25:1. The monoPEG-ASNase was found to maintain enzymatic stability for more days than ASNase, also was resistant to the plasma proteases like asparaginyl endopeptidase and cathepsin B. Additionally, monoPEG-ASNase was found to be potent against leukemic cell lines (MOLT-4 and REH) in vitro like polyPEG-ASNase. monoPEG-ASNase demonstrates its potential as a novel option for ALL treatment, being an inventive novelty that maintains the benefits of the current enzyme and solves challenges.
Topics: Asparaginase; Binding Sites; Cell Line, Tumor; Cell Survival; Chromatography, Gel; Enzyme Stability; Humans; Polyethylene Glycols; Precursor Cell Lymphoblastic Leukemia-Lymphoma
PubMed: 30753228
DOI: 10.1371/journal.pone.0211951 -
Haematologica Oct 2023For several decades, asparaginase has been considered world-wide as an essential component of combination chemotherapy for the treatment of childhood acute lymphoblastic...
For several decades, asparaginase has been considered world-wide as an essential component of combination chemotherapy for the treatment of childhood acute lymphoblastic leukemia (ALL). Discovered over 60 years ago, two main unmanipulated asparaginase products originated from primary bacteria sources, namely Escherichia coli and Erwinia chrysanthemi, have been available for clinical use. A pegylated product of the Escherichia coli asparaginase was subsequently developed and is now the main product used by several international co-operative groups. The various asparaginase products all display the same mechanism of action (hydrolysis of circulating asparagine) and are associated with similar efficacy and toxicity patterns. However, their different pharmacokinetics, pharmacodynamics and immunological properties require distinctive modalities of application and monitoring. Erwinia chrysanthemi asparaginase was initially used as a first-line product, but subsequently became a preferred second-line product for children who experienced immunological reactions to the Escherichia coli asparaginase products. An asparaginase product displaying the same characteristics of the Erwinia chrysanthemi asparaginase has recently been produced by use of recombinant technology, thus securing a preparation available for use as an alternative, or as a back-up in case of shortages, for the non-recombinant product. The long journey of the Erwinia chrysanthemi asparaginase product as it has developed throughout the last several decades has made it possible for almost every child and adult with ALL to complete the asparaginase-based protocol treatment when an immunological reaction has occurred to any Escherichia coli asparaginase product.
Topics: Child; Adult; Humans; Asparaginase; Dickeya chrysanthemi; Drug Hypersensitivity; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Escherichia coli; Antineoplastic Agents
PubMed: 37470157
DOI: 10.3324/haematol.2022.282324 -
Clinical Laboratory Feb 2022Asparaginase (ASP), a chemotherapy component in the acute lymphoblastic leukemia (ALL) treatment, could impair normal coagulation state. Usually, a decline in the levels...
BACKGROUND
Asparaginase (ASP), a chemotherapy component in the acute lymphoblastic leukemia (ALL) treatment, could impair normal coagulation state. Usually, a decline in the levels of several coagulation factors occurs which ultimately could lead to thrombotic events and abnormal coagulation tests. In this study, we aimed to compare the effects of two different subtypes of ASP, pegylated asparaginase (PEG-ASP) and L-asparaginase (L-ASP) on coagulation markers and test among 40 pediatric patients with ALL.
METHODS
In this cohort study a total of 40 pediatric patients with newly diagnosed ALL were enrolled and divided into two groups by simple randomization. In group A, 20 patients received PEG-ASP while in group B, 20 patients received L-ASP during the induction treatment. Coagulation markers included prothrombin time (PT), partial thrombin time (PTT), protein-C (Pr-C), protein-S (Pr-S), and antithrombin III (ATIII) and were assessed before start and after of induction chemotherapy.
RESULTS
Coagulation profile including PT, PTT, INR, Pr-C, Pr-S, and ATIII before start of treatment were not statistically significant between the two groups. Anticoagulant factors decreased significantly after consuming both drugs. Tests for PT and INR of those who took L-ASP decreased significantly. Overall, when comparing the changes of the six studied factors, ATIII and Pr-C were the significant factors which were different between groups.
CONCLUSIONS
ASP has a negative effect on anticoagulant factors including (ATIII, Pr-C, Pr-S). Additionally, the negative effect of L-ASP on anticoagulant factors was more prominent than PEG-ASP. Therefore, the risk of thrombosis probably was negligible in PEG-ASP in comparison with L-ASP.
Topics: Asparaginase; Child; Cohort Studies; Humans; Polyethylene Glycols; Precursor Cell Lymphoblastic Leukemia-Lymphoma
PubMed: 35142188
DOI: 10.7754/Clin.Lab.2021.210502 -
Current Microbiology Jul 2023L-asparaginase is a tetrameric enzyme from the amidohydrolases family, that catalyzes the breakdown of L-asparagine into L-aspartic acid and ammonia. Since its discovery... (Review)
Review
L-asparaginase is a tetrameric enzyme from the amidohydrolases family, that catalyzes the breakdown of L-asparagine into L-aspartic acid and ammonia. Since its discovery as an anticancer drug, it is used as one of the prime chemotherapeutic agents to treat acute lymphoblastic leukemia. Apart from its use in the biopharmaceutical industry, it is also used to reduce the formation of a carcinogenic substance called acrylamide in fried, baked, and roasted foods. L-asparaginase is derived from many organisms including plants, bacteria, fungi, and actinomycetes. Currently, L-asparaginase preparations from Escherichia coli and Erwinia chrysanthemi are used in the clinical treatment of acute lymphoblastic leukemia. However, they are associated with low yield and immunogenicity problems. At this juncture, endophytic fungi from medicinal plants have gained much attention as they have several advantages over the available bacterial preparations. Many medicinal plants have been screened for L-asparaginase producing endophytic fungi and several studies have reported potent L-asparaginase producing strains. This review provides insights into fungal endophytes from medicinal plants and their significance as probable alternatives for bacterial L-asparaginase.
Topics: Asparaginase; Antineoplastic Agents; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Bacteria; Fungi
PubMed: 37450223
DOI: 10.1007/s00284-023-03392-z -
International Journal of Biological... Feb 2021L-asparaginase is an enzyme capable of hydrolyzing the asparagine to aspartic acid and ammonia. L-asparaginase is widely used in the treatment of acute lymphoblastic...
L-asparaginase is an enzyme capable of hydrolyzing the asparagine to aspartic acid and ammonia. L-asparaginase is widely used in the treatment of acute lymphoblastic leukemia (ALL) and other cancers. Here, for the first time, the effects of a novel yeast L-asparaginase from Yarrowia lipolytica were studied on human lung (A549) and breast cancer (MCF7) cell lines as the solid cancer cell lines in terms of cell growth and metastasis inhibition. Functional analysis showed the L-asparagine deprivation mediated anti-proliferation effects by apoptosis induction and changes in the expression of target genes involved in apoptosis and migration pathways. The qRT-PCR analysis showed the higher expression levels of pro-apoptosis genes, including Bax, P53, caspase 3, caspase 8, and down-regulation of Bcl-2 anti-apoptotic gene in treated cells. On the other hand, there was no increase in ROS production in the treated cells. However, L-asparaginase treatment was able to significantly induce autophagy activation in A549 cells. Besides, wound healing assay showed that L-asparaginase could considerably inhibit the migration of A549 and MCF7 cells. Taken together, our results suggested that Yarrowia lipolytica L-asparaginase might be considered for enzyme therapy against breast and lung cancers.
Topics: A549 Cells; Apoptosis; Asparaginase; Autophagy; Breast; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Lung; Lung Neoplasms; MCF-7 Cells; Reactive Oxygen Species; Yarrowia
PubMed: 33358949
DOI: 10.1016/j.ijbiomac.2020.12.141 -
Brazilian Journal of Microbiology :... Dec 2016l-asparaginase (EC 3.5.1.1) is an enzyme that catalysis mainly the asparagine hydrolysis in l-aspartic acid and ammonium. This enzyme is presented in different... (Review)
Review
l-asparaginase (EC 3.5.1.1) is an enzyme that catalysis mainly the asparagine hydrolysis in l-aspartic acid and ammonium. This enzyme is presented in different organisms, such as microorganisms, vegetal, and some animals, including certain rodent's serum, but not unveiled in humans. It can be used as important chemotherapeutic agent for the treatment of a variety of lymphoproliferative disorders and lymphomas (particularly acute lymphoblastic leukemia (ALL) and Hodgkin's lymphoma), and has been a pivotal agent in chemotherapy protocols from around 30 years. Also, other important application is in food industry, by using the properties of this enzyme to reduce acrylamide levels in commercial fried foods, maintaining their characteristics (color, flavor, texture, security, etc.) Actually, l-asparaginase catalyzes the hydrolysis of l-asparagine, not allowing the reaction of reducing sugars with this aminoacid for the generation of acrylamide. Currently, production of l-asparaginase is mainly based in biotechnological production by using some bacteria. However, industrial production also needs research work aiming to obtain better production yields, as well as novel process by applying different microorganisms to increase the range of applications of the produced enzyme. Within this context, this mini-review presents l-asparaginase applications, production by different microorganisms and some limitations, current investigations, as well as some challenges to be achieved for profitable industrial production.
Topics: Animals; Antineoplastic Agents; Asparaginase; Drug Industry; Fermentation; Food Industry; Humans; Industrial Microbiology
PubMed: 27866936
DOI: 10.1016/j.bjm.2016.10.004 -
Cell Death & Disease May 2023Extranodal NK/T-cell lymphoma (NKTL) is a rare and aggressive form of extranodal lymphoma with a poor prognosis. Currently, there are very limited treatment options for...
Extranodal NK/T-cell lymphoma (NKTL) is a rare and aggressive form of extranodal lymphoma with a poor prognosis. Currently, there are very limited treatment options for patients with advanced-stage disease or those with relapsed/recurrent disease. Here we show that Chiauranib, an orally small molecule inhibitor of select serine-threonine kinases (aurora B, VEGFRs, PDGFR, CSF1R, c-Kit), inhibited NKTL cell proliferation, induced cell cycle arrest, as well as suppressed the microvessel density in vitro and in vivo similar as in other types of cancer cells. Surprisingly, Chiauranib unfolded a new effect to induce apoptosis of NKTL cells by triggering AIF-dependent apoptosis other than the traditional cyt-c/caspase mitochondrial apoptosis pathway. The knockdown of AIF in vitro and in vivo dramatically blocked the efficacy of Chiauranib on NKTL. Mechanistically, the release of AIF from mitochondria is due to the upregulation of VDAC1 by the AKT-GSK3β pathway and activation of calcium-dependent m-calpain, which promotes the cleavage of VDAC1 and therefore permits the release of AIF. Notably, the low expression of Bax in both NKTL cells and patient tissues restrained the cyt-c release. It resulted in the inhibition of cyt-c/caspase mitochondrial pathway, suggesting that drugs targeting this traditional pathway may not be effective in NKTL. Furthermore, we found that L-asparaginase triggered CD95 (Fas/Apo-1)-caspase 8-caspase 3 apoptotic pathway in NKTL cells, and combination of Chiauranib and L-asparaginase exhibited a synergistic effect, suggesting a feasibility to combine these two drugs for effective treatment of NKTL. This study demonstrates Chiauranib's positive efficacy toward NKTL through the activation of the AIF-dependent apoptosis pathway for the first time. The novel and multi-targets of Chiauranib and the synergistic effect with L-asparaginase may provide a promising therapy for NKTL patients.
Topics: Humans; Asparaginase; Naphthalenes; Quinolines; Lymphoma, T-Cell
PubMed: 37160920
DOI: 10.1038/s41419-023-05833-w -
Journal of Biomolecular Structure &... Jun 2022In the present study, a new bacterial strain, OHEM18 was significantly found to produce extracellular L-asparaginase. L-asparaginase was purified using ammonium sulfate...
In the present study, a new bacterial strain, OHEM18 was significantly found to produce extracellular L-asparaginase. L-asparaginase was purified using ammonium sulfate precipitation and QFF column to 3.84-fold with specific activity of 215.33 U/mg and its molecular mass was assessed to be 41.5 kDa. Maximum enzyme activity was determined at pH 8.2 and 40 °C and with retaining 70% of its activity after incubation for 1 h at 50 °C. Km and Vmax values were determined to be 0.0047 M and 92.74, respectively. Cytotoxicity test indicated a significant safety of L-asparaginase on Vero cells with selectivity against leukemia, breast cancer and hepatoma cells. NFS-60 cells was the most sensitive tumor cells to L-asparaginase with IC of 10.29 µg/ml and selectivity index of 30.61. This selectivity was recognized to be an apoptosis-dependent mechanism proven cell cycle arrest in sub-G1 phase and fragmentation of genomic DNA. L-asparaginase showed antioxidant activity against both DPPH and ABTS radicals with IC values of 64.07 and 177.1 mg/ml, respectively. These competitive advantage of bacterial L-asparaginase over than other sources is that it might be produced in large amounts through production in large-scale biofermenters, which decreases costs, besides having a sustainable bacterial source. Our findings established that the potent cytotoxic effect of L-asparaginase isolated from OHEM18 may be a promise candidate for further medicinal applications as an antioxidant and antitumor drug.Communicated by Ramaswamy H. Sarma.
Topics: Animals; Antineoplastic Agents; Antioxidants; Asparaginase; Bacillus; Chlorocebus aethiops; Enzyme Stability; Vero Cells
PubMed: 33228468
DOI: 10.1080/07391102.2020.1851300 -
Applied Biochemistry and Biotechnology Aug 2020In this presented work, magnetic poly(HEMA-GMA) nanoparticles were synthesized, characterized, and used for immobilization of an anti-leukemic enzyme L-asparaginase. The...
In this presented work, magnetic poly(HEMA-GMA) nanoparticles were synthesized, characterized, and used for immobilization of an anti-leukemic enzyme L-asparaginase. The average particle size of the synthesized magnetic nanoparticles was found to be as 117.5 nm. L-asparaginase was successfully immobilized onto the synthesized magnetic nanoparticles, and attached amount of L-asparaginase was found to be as 66.43 mg/g nanoparticle. The effects of the medium pH, temperature, and substrate concentration on the L-asparaginase activity were also tested. Optimum pH of the free and immobilized L-asparaginase was found to be as 7.5 and 6.5, respectively. Optimum temperature of the free L-asparaginase was 45 °C, while optimum temperature was shifted to 55 °C after immobilization onto the magnetic nanoparticles. Also, k value of free L-asparaginase (47,356 min) was calculated to be higher than that of immobilized L-asparaginase (497 min). Thermal stability of both asparaginase preparation was followed for 10 h, and at the end of the incubation time, free asparaginase almost lost its activity, while immobilized asparaginase protected 50% of its initial activity. Storage stabilities of free and immobilized asparaginase were also tested, and at the end of the 40 days storage, free asparaginase lost all of its activity, while immobilized asparaginase still showed 30% activity. Operational stability of immobilized asparaginase was tested for 8 successive usage, and immobilized asparaginase lost only 15% of its initial activity. In present study, activities of free and immobilized L-asparaginase were tested in artificial human serum medium, to foresee the in vivo efficiency, and it was demonstrated here that immobilized L-asparaginase protected its 74.74% of its initial activity in artificial serum medium.
Topics: Asparaginase; Catalysis; Enzyme Stability; Enzymes, Immobilized; Humans; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Magnetite Nanoparticles; Neoplasms; Particle Size; Temperature
PubMed: 32103470
DOI: 10.1007/s12010-020-03276-z