L-asparaginase: a promising chemotherapeutic agent

This article comprises detailed information about L-asparaginase, encompassing topics such as microbial and plant sources of L-asparaginase, treatment with L-asparaginase, mechanism of action of L-asparaginase, production, purification, properties, expression and characteristics of l-asparaginase along with information about studies on the structure of L-asparaginase. Although L-asparaginase has been reviewed by Savitri and Azmi (2003), our effort has been to include recent and updated information about the enzyme covering new aspects such as structural modification and immobilization of L-asparaginase, recombinant L-asparaginase, resistance to L-asparaginase, methods of assay of L-asparagine and L-asparaginase activity using the biosensor approach, L-asparaginase activity in soil and the factors affecting it. Also, side-effects of L-asparaginase treatment in acute lymphoblastic leukemia (ALL) have been discussed in the current review. L-asparaginase has been and is still one of the most widely studied therapeutic enzymes by researchers and scientists worldwide.     

Antiproliferative activity of L-asparaginase of Tetrahymena pyriformis on human breast cancer cell lines

Purified L-asparaginase of Tetrahymena pyriformis is a multi-subunit enzyme exhibiting protein kinase activity as well. The enzyme's L-asparaginase activity is affected by its phosphorylation state. Both native and dephosphorylated L-asparaginase show antiproliferative activity on three breast cancer cell lines (T47D, BT20 and MCF-7) and on Walker 256 cells. These cells do not possess measurable L-asparaginase or L-asparagine synthetase activity. When T47D cells are treated for different times with L-asparaginase and then placed in fresh medium, the growth of cells treated for 1, 3, or 6 hours is initiated and parallels control curve, while the growth of cells treated for 24 or 48 hours with L-asparaginase stays at the same inhibitory level (24 h treatment) or continues to drop (48 h treatment). Addition of D-asparagine, a competitive inhibitor of T. pyriformis L-asparaginase, counteracts the antiproliferative activity of L-asparaginase, indicating that L-asparaginase and not the kinase activity is responsible for that effect.     

Indicator dye based screening of glutaminase free L-asparaginase producer and kinetic evaluation of enzyme production process

Abstract

Several soil isolates from 1 g of soil sample were isolated and screened for the production of L-asparaginase. Primary screening was performed using rapid plate assay; dye indicator studies were conducted, and phenol red with 0.005% concentration was found to be optimum. The secondary screening was carried out using the Nesslerization method. The bacteria screened for L-asparaginase production with no glutaminase activity was identified as Bacillus subtilis. Crude L-asparaginase enzyme was partially purified 1.57 folds of purity and 110 U/mg of specific activity. The glutaminase-free L-asparaginase activity was also confirmed using LC-MS analysis. The presence of mass peaks at 147.0 in the reaction mixture suggested an absence of glutaminase activity. An optimized medium obtained comprised of Dextrose 1.5 g/L, K₂HPO₄ 1.2 g/L, L-asparagine 15 g/L, and Tryptone 5 g/L. The highest L-asparaginase activity was observed at 6.0 pH and 30 °C. Kinetic parameters associated with biomass and L-asparaginase production were also studied. The computed values were µ_m 0.104 h^?1, X_m 6g/L P₀ 1.7U/mL P_m 8.2 U/mL Y_X/S 4 g-cell/g-glucose µP_m 0.35 h^?1 q_p 5.46 U/g/h Y_P/x 13.6667 U/g-cell. The novel bacterial isolates showed promise as a potential glutaminase-free L-asparaginase producer, which can prove to be of industrial applications.     

L-asparaginase of Tetrahymena pyriformis is associated with a kinase activity

Most of L-asparaginase activity of Tetrahymena pyriformis was found to be present in microsomal membranes from which it has been purified to homogeneity (Tsirka, S.A.E. and Kyriakidis, D.A. Mol. Cell. Biochem. 83: 147–155, 1988). The native enzyme has a relative molecular weight of approximately 200 kDa, while under denaturing conditions the enzyme exhibits. a subunit size of 39 kDa. Aminoacid analysis and an oligopeptide from N-terminal sequence have been determined. Dephosphorylation of L-asparaginase by alkaline phosphatase results in an activation of its catalytic activity. This enzyme also exhibits intrinsic phosphorylation activity with a Km value for ATP of 0.5 mM. Autophosphorylation with -^32P ATP of purified L-asparaginase results in the phosphorylation of tyrosine residues as well as in loss of its activity. Mg²⁺ and Ca²⁺ added together act synergistically to stimulate the kinase activity by more than 160%. The polyamines putrescine, spermidine and spermine activate the kinase approximately 100%, while neither cAMP or cGMP have any effect. These results indicate that this membrane protein with dual L-asparaginase/kinase activity must play an important role in regulating the intracellular levels of L-asparagine in Tetrahymena pyriformis.     

Production,purification, characterization,antioxidant and antiproliferative activities of extracellular L-asparaginase produced by Fusarium equiseti AHMF4

L-Asparaginase is an antileukemic agent that depletes L-asparagine “an important nutrient for cancer cells” through the hydrolysis of L-asparagine into L-aspartic acid and ammonia leading to leukemia cell starvation and apoptosis in susceptible leukemic cell populations. Moreover currently, bacterial L-asparaginase has been limited by problems of lower productivity, stability, selectivity and a number of toxicities along with the resistance towards bacterial L-asparaginase. Then the current work aimed to provide pure L-asparaginase with in-vitro efficacy against various human carcinomas without adverse effects related to current L-asparaginase formulations. Submerged fermentation (SMF) bioprocess was applied and improved to maximize L-asparaginase production from Fusarium equiseti AHMF4 as alternative sources of bacteria. The enzyme production in SMF was maximized to reach 40.78 U mL⁻¹ at the 7th day of fermentation with initial pH 7.0, incubation temperature 30 °C, 1.0% glucose as carbon source, 0.2% asparagine as nitrogen source, 0.1% alanine as amino acid supplement and 0.1% KH₂PO₄. The purification of AHMF4 L-asparaginase yielded 2.67-fold purification and 48% recovery with final specific activity of 488.1 U mg⁻¹ of protein. Purified L-asparaginase was characterized as serine protease enzyme with molecular weight of 45.7 kDa beside stability at neutral pH and between 20 and 40 °C. Interestingly, purified L-asparaginase showed promising DPPH radical scavenging activity (IC₅₀ 69.12 μg mL⁻¹) and anti-proliferative activity against cervical epitheloid carcinoma (Hela), epidermoid larynx carcinoma (Hep-2), hepatocellular carcinoma (HepG-2), Colorectal carcinoma (HCT-116), and breast adenocarcinoma (MCF-7) with IC₅₀ equal to 2.0, 5.0, 12.40, 8.26 and 22.8 μg mL⁻¹, respectively. The enzyme showed higher activity, selectivity and anti-proliferative activity against cancerous cells along with tiny cytotoxicity toward normal cells (WI-38) which indicates that it has selective toxicity and it could be applied as a less toxic alternative to the current formulations.     

In vitro alterations of L-asparaginase activity of Tetrahymena pyriformis by lipids

A membrane-bound L-asparaginase (EC 3.5.1.1) of Tetrahymena pyriformis was purified to homogeneity. The purified enzyme is a lipoprotein, since it is inactivated by phospholipase C and its activity is restored by the addition of naturally occuring lipids, such as phosphatidylcholine, triolein and oleyl acetate. The relative effectiveness of a variety of phospholipids, free saturated and unsaturated fatty acids, or neutral lipids, such as esters of fatty, acids and glycerides, with respect to the activation of purified L-asparaginase is compared. Enzyme activity is reconstituted in the presence of lipids and evidence for the formation of an enzyme-phospholipid complex is presented. The data of this report suggest that L-asparaginase may have a requirement for lipids that reconstitute a physiological hydrophobic environment, similar to the one existing in vivo.Abbreviations DPPC Dipalmitoylphosphatidylcholine - DPPE Dipalmitoylphosphatidylethanolamine - DMPC Dimyristoylphosphatidylcholine - PS Phosphatidylserine - PI Phosphatidylinositol - IPC Lysophosphatidylcholine - PC Phosphatidylcholine - PE Phosphatidylethanolamine     

CHEMICAL MODIFICATION OF L-ASPARAGINASE FROM Cladosporium sp. FOR IMPROVED ACTIVITY AND THERMAL STABILITY

L-Asparaginase (ASNase), an antileukemia enzyme, is facing problems with antigenicity in the blood. Modification of L-asparaginase from Cladosporium sp. was tried to obtain improved stability and improved functionality. In our experiment, modification of the enzyme was tried with bovine serum albumin, ovalbumin by crosslinking using glutaraldehyde, N-bromosuccinimide, and mono-methoxy polyethylene glycol. Modified enzymes were studied for activity, temperature stability, rate constants (k_d), and protection to proteolytic digestion. Modification with ovalbumin resulted in improved enzyme activity that was 10-fold higher compared to native enzyme, while modification with bovine serum albumin through glutaraldehyde cross-linking resulted in high stability of L-asparaginase that was 8.5- and 7.62-fold more compared to native enzyme at 28°C and 37°C by the end of 24 hr. These effects were dependent on the quantity of conjugate formed. Modification also markedly prolonged L-asparaginase half-life and serum stability. N-Bromosuccinimide-modified ASNase presented greater stability with prolonged in vitro half-life of 144 hr to proteolytic digestion relative to unmodified enzyme (93 h). The present work could be seen as producing a modified L-asparaginase with improved activity and stability and can be a potential source for developing therapeutic agents for cancer treatment.     

Recombinant L-Asparaginase from Zymomonas mobilis: A Potential New Antileukemic Agent Produced in Escherichia coli

L-asparaginase is an enzyme used as a chemotherapeutic agent, mainly for treating acute lymphoblastic leukemia. In this study, the gene of L-asparaginase from Zymomonas mobilis was cloned in pET vectors, fused to a histidine tag, and had its codons optimized. The L-asparaginase was expressed extracellularly and intracellularly (cytoplasmically) in Escherichia coli in far larger quantities than obtained from the microorganism of origin, and sufficient for initial cytotoxicity tests on leukemic cells. The in silico analysis of the protein from Z. mobilis indicated the presence of a signal peptide in the sequence, as well as high identity to other sequences of L-asparaginases with antileukemic activity. The protein was expressed in a bioreactor with a complex culture medium, yielding 0.13 IU/mL extracellular L-asparaginase and 3.6 IU/mL intracellular L-asparaginase after 4 h of induction with IPTG. The cytotoxicity results suggest that recombinant L-asparaginase from Z. mobilis expressed extracellularly in E.coli has a cytotoxic and cytostatic effect on leukemic cells.     

Extracellular L-asparaginase production in solid-state fermentation by using sugarcane bagasse as support material

Abstract

L-asparaginase is an important enzyme used in the pharmaceutical and food industry, which can be produced by different microorganisms using low cost feedstocks. In this work, sugarcane bagasse (SCB) was used as support for enzyme production in solid-state fermentation (SSF) by A. terreus. Initially, the influence of the variables carbon and nitrogen sources on the enzyme production was studied following an experimental design carried out in Erlenmeyer flasks. Statistical analysis indicated the use of 0.54% of starch, 0% of maltose, 0.44% of asparagine, and 1.14% of glutamine in the medium, resulting in enzyme activity per volume of produced extract of 120.723?U/L. Then, these conditions were applied in a horizontal column reactor filled with SCB, producing 105.3?U/L of enzyme activity. Therefore, the potential of extracellular L-asparaginase enzyme production in the column reactor using sugarcane bagasse as support was demonstrated and it represents a system that can favor large scale production.     

The Role of Solvent Interactions in Protein Conformatio

Abstract

For many years, the ease with which the pH of enzyme reactions could be varied has led to innumerable reports of the pH-dependence of the kinetic parameters of such reactions. These studies have been provided with a veneer of respectability by suggestions that they allow deductions to be made about the nature of the ionizing groups upon which the catalytic activity of the enzyme depends. It is the purpose of this article to summarize some of the reasons why a large proportion of pH-dependence studies yields little information about the real pK_a-values of ionizing groups in enzymes, and still less about the identity of these groups.     

Physicochemical properties and antiproliferative activity of recombinant Yersinia pseudotuberculosis L-asparaginase

The physicochemical, catalytic, and antiproliferative activity of a recombinant L-asparaginase from Yersinia pseudotuberculosis (YpA) have been studied. The following results were obtained: the K _M value for L-asparagine is 17 ± 0.9 ??M, the optimal temperature is 60°C, pH is 8.0, pI is 5.4 ± 0.3, the L-glutaminase activity is no more than 5?C6% of the L-asparaginase activity, and the antiproliferative activity on the Fisher L5178y lymphadenosis cell line comprised T/C = 136% (p < 0.001) at a 15% recovery rate. The described characteristic allows one to regard YpA as an antitumor enzyme with biological features similar to the L-asparaginase of E. coli.     

Novel antifungal agents,targets or therapeutic strategies for the treatment of invasive fungal diseases: a review of the literature (2005-2009)

BackgroundThe incidence and prevalence of serious mycoses continues to be a public health problem. Despite aggressive treatment with new or more established licensed antifungal agents, these infections are an important cause of morbidity and mortality, especially in immunocompromised patients.AimsTo critically review the literature regarding important new developments in the field of antifungal therapy both in the English and Spanish versions.MethodsThe search of the literature focused on different antifungal targets or mechanisms of action as well as new agents or strategies that could improve antifungal therapy.ResultsThe review produced a huge amount of information on the use of virulent factors such as growth, filamentation, pathogen tissue clearance, among others, as putative targets of antifungal activity. More recently, the chemical-genetic relationships for licensed agents as well as for other compounds have been provided by the identification of the genes related to the mechanism of action.ConclusionsAlthough the antifungal activity of numerous compounds has been examined, most of them are at the in vitro or animal models of efficacy stages. Therefore, further investigation should be carried out to realize the true clinical utility of these compounds.     

Isolation and screening of endophytes from the rhizomes of some Zingiberaceae plants for L-asparaginase production

Endophytes are described as microorganisms that colonize the internal tissues of healthy plants without causing any disease. Endophytes isolated from medicinal plants have been attracting considerable attention due to their high biodiversity and their predicted potential to produce a plethora of novel compounds. In this study, an attempt was made to isolate endophytes from rhizomes of five medicinal plants of Zingiberaceae family, and to screen the endophytes for L-asparaginase activity. In total, 50 endophytes (14 bacteria, 22 actinomycetes, and 14 fungi) were isolated from Alpinia galanga, Curcuma amada, Curcuma longa, Hedychium coronarium, and Zingiber officinale; of these, 31 endophytes evidenced positive for L-asparaginase production. All the L-asparaginase-positive isolates showed L-asparaginase activity in the range of 54.17–155.93 U/mL in unoptimized medium. An endophytic fungus isolated from Curcuma amada, identified as Talaromyces pinophilus, was used for further experiments involving studies on the effect of certain nutritional and nonnutritional factors on L-asparaginase production in submerged fermentation. Talaromyces pinophilus initially gave an enzyme activity of 108.95 U/mL, but gradually reduced to 80 U/mL due to strain degeneration. Perhaps this is the first report ever on the production of L-asparaginase from endophytes isolated from medicinal plants of Zingiberaceae family.     

L-asparaginase produced from soil isolates of Pseudomonas aeruginosa shows potent anti-cancer activity on HeLa cells

Among cancers, acute lymphoblastic leukemia (ALL) occurs in the children <15 years of age. L-asparaginase is an important therapeutic enzyme used for treating ALL. Owing to its therapeutic use and demand, microorganisms have been in use for many years to produce L-asparaginase on an industrial scale. Gram-negative bacteria (Serratia, Erwinia and Escherichia coli) species were used in L-asparaginase. However, earlier studies have documented that the long-term use of enzymes produced from these commercial strains induces hypersensitivity in patients. Therefore, there is a need to discover novel microbial strains producing L-asparaginase with anti-cancer properties, which can be employed for the commercial production of the enzyme. In this study, three strains of Pseudomonas aeruginosa (accession numbers LC425424 (P31), LC425425 (P32), and LC425426 (P34)) isolated from garden soil were screened for the invention of L-asparaginase. Fermented production media was dialyzed to attain the purified enzyme, thus showed a dose-depended cytotoxic effect on HeLa cells, as determined by MTT assay. The IC₅₀s of the different isolates were 86.73, 57.65, and 40.34 µg/mL. These results indicate that pseudomonal L-asparaginase may be used for cancer treatment.     

L-asparaginase-induced apoptosis in ALL cells involves IP3 receptor signaling

L-asparaginase treatment is used in the clinic to treat acute lymphoblastic leukemia (ALL) patients. Lee et al. (2019, Blood 133:2222-2232) demonstrated that L-asparaginase induces apoptosis by activating inositol 1,4,5-trisphosphate (IP₃)-induced Ca²⁺ signaling in a Huntingin-associated protein 1 (HAP1)-dependent manner. Moreover, HAP1 levels inversely correlate with the sensitivity of the ALL cells to L-asparaginase. HAP1 can therefore be used as biomarker for evaluating L-asparaginase resistance.     

Purification and properties of a membrane-bound L-asparaginase of Tetrahymena pyriformis

L-Asparaginase activity reaches maximal values at the stationary phase of growth of Tetrahymena pyriformis and fluctuates upon the growth conditions and the composition of the medium. Most of the L-asparaginase activity (80%) is associated with the endoplasmic reticulum, and the remaining with the pellicles. Detergents either alone or in combination with NaCl up to 0.5 M concentration failed to solubilize L-asparaginase. Solubilization can be accomplished by means of either the chaotropic agents KSCN and NaClO₄, or 0.1 M sodium phosphate buffer pH 8.0, following pretreatment of the particulates with 2% w/v Triton X₁₀₀. L-Asparaginase has been purified to near homogeneity by hydrophobic and gel filtration chromatography. The native enzyme has a relative molecular weight of 230000. It is a multiple subunit enzyme, with subunit size of 39000. Its isoelectric point is at pH 6.8. It acts optimally at pH 8.6 with a Km of 2.2 mM. It does not hydrolyse L-glutamine and its reaction is inhibited competitively by D-aspartic acid and D-asparagine as well as by Ir asparagine analogues with substituents at the 0 position.     

Antitumor activity of L-asparaginase from Yersinia pseudotuberculosis

The cytotoxic activity of L-asparaginases from Yersinia pseudotuberculosis and from Erwinia carotovora were investigated in vitro using human T-lymphoblastic leukemia (Jurkat and Molt-4) and also solid tumor cell lines MCF-7 (human breast adenocarcinoma), LnCap (human prostate carcinoma), NGUK1 (rat Gasser node neurinoma). E.coli L-asparaginase produced by Medak (Germany) was used as a reference preparation. The data obtained indicate that Y. pseudotuberculosis L-asparaginase significantly inhibits growth of leukemic and solid tumor cells. Its antitumor activity is comparable to that of the reference preparation of L-asparaginase (Medak). These results suggest that the recombinant L-asparaginase can be used for the development of new preparations for the therapy of different types of tumors.     

Monitoring proteolytic processing events by quantitative mass spectrometry

Introduction: Protease activity plays a key role in a wide variety of biological processes including gene expression, protein turnover and development. misregulation of these proteins has been associated with many cancer types such as prostate, breast, and skin cancer. thus, the identification of protease substrates will provide key information to understand proteolysis-related pathologies.

Areas covered: Proteomics-based methods to investigate proteolysis activity, focusing on substrate identification, protease specificity and their applications in systems biology are reviewed. Their quantification strategies, challenges and pitfalls are underlined and the biological implications of protease malfunction are highlighted.

Expert commentary: Dysregulated protease activity is a hallmark for some disease pathologies such as cancer. Current biochemical approaches are low throughput and some are limited by the amount of sample required to obtain reliable results. Mass spectrometry based proteomics provides a suitable platform to investigate protease activity, providing information about substrate specificity and mapping cleavage sites.     

Design of experiments and artificial neural network linked genetic algorithm for modeling and optimization of L-asparaginase production by <Emphasis Type="Italic">Aspergillus terreus</Emphasis> MTCC 1782

The sequential optimization strategy for design of an experimental and artificial neural network (ANN) linked genetic algorithm (GA) were applied to evaluate and optimize media component for L-asparaginase production by Aspergillus terreus MTCC 1782 in submerged fermentation. The significant media components identified by Plackett-Burman design (PBD) were fitted into a second order polynomial model (R² = 0.910) and optimized for maximum L-asparaginase production using a five-level central composite design (CCD). A nonlinear model describing the effect of variables on L-asparaginase production was developed (R² = 0.995) and optimized by a back propagation NN linked GA. Ground nut oil cake (GNOC) flour 3.99% (w/v), sodium nitrate (NaNO₃) 1.04%, L-asparagine 1.84%, and sucrose 0.64% were found to be the optimum concentration with a maximum predicted L-asparaginase activity of 36.64 IU/mL using a back propagation NN linked GA. The experimental activity of 36.97 IU/mL obtained using the optimum concentration of media components is close to the predicted L-asparaginase activity of the ANN linked GA.