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Background: L-asparaginase (L-ASNase, L-asparagine amidohydrolase, E.C.3.5.1.1) is an enzyme with wide therapeutic applicability. Currently, the commercialized L-ASNase comes from mesophilic organisms, presenting low specificity to the substrate and limitations regarding thermostability and active pH range. Such factors prevent the maximum performance of the enzyme in different applications. Therefore, extremophilic organisms may represent important candidates for obtaining amidohydrolases with particular characteristics desired by the biotechnological market. Objective: The present study aims to carry out a technological prospecting of patents related to the L-asparaginases derived from extremophilic organisms, contributing to pave the way for further rational investigation and application of such enzymes. Methods: This patent literature review used six patents databases: The LENS, WIPO, EPO, USPTO, Patent Inspiration, and INPI. Results: It was analyzed 2860 patents, and 14 were selected according to combinations of descriptors and study criteria. Approximately 57.14% of the patents refer to enzymes obtained from archaea, especially from the speciesPyrococcus yayanosii (35.71% of the totality). Conclusion: The present prospective study has singular relevance since there are no recent patent reviews for L-asparaginases, especially produced by extremophilic microorganisms. Although such enzymes have well-defined applications, corroborated by the patents compiled in this review, the most recent studies allude to new uses, such as the treatment of infections. The characterization of the catalytic profiles allows us to infer that there are potential sources still unexplored. Hence, the search for new L-ASNases with different characteristics will continue to grow in the coming years and, possibly, ramifications of the technological routes will be witnessed.

Cervical cancer is a leading cause of cancer death in women, occurring in more than a quarter of the developing countries of the world, with a slightly higher incidence in India. Many microbes producing L-asparaginase (ASNase) are used for the treatment of various cancers. However, previous studies have documented that long-term use of enzymes produced from these commercial strains triggers hypersensitivity in patients. Therefore, there is a need to find new microorganisms that produce L-asparaginase with anti-cancer properties, which can be used commercially in enzyme production. In the present study, of the 7 isolates, a single isolate of Lactobacillus plantarum had the highest enzyme production capacity. Purified enzyme can be obtained from fermented production medium by dialysis, thus showed a dose-depended cytotoxic effect on HeLa cells, as determined by MTT assay. The IC50 of the enzyme value was 0.75 IU µg/mL. This result indicates that L. plantarum producing L-asparaginase may be used for cancer treatment.

L-asparaginase (EC 3.5.11. L-asparagine amidohydrolase) is first enzyme, studied very intensively in human beings with regard to its anti-tumor potential against tumor of lymphoid precursor, acute lymphoblastic leukemia (ALL). The current drugs are suffering from many side effects like immune suppression, infertility, secondary neoplasm. The immunogenic complications associated with its present microbial sources Escherichia coli; Erwinia carotovora limits its medicinal frontier. So there exists a need of switching to novel natural sources to serve as non-immunogenic and better production sources of L-asparaginase. In the present study, four cultures of fungal endophytes viz. TSF-1, TSF-2, TSF-3 and TSF-4 selected on the basis of primary and secondary screening was carried on with L-asparagine as a sole carbon and nitrogen source and phenol red as pH indicator. The maximum protein content was observed to be present in TSF-2 i.e. 2.727 mg /mL and possessed maximum activity of 6.054 Units/ml. Sample was separated by SDS-PAGE, stained by silver staining, showed a single band with molecular weight of approximately ~45kDa.

Fungos endofiticos sao considerados um nicho em potencial na producao de enzimas de interesse biotecnologico. Entre elas destaca-se a asparaginase, utilizada no tratamento de leucemia aguda. Atualmente a enzima produzida em escala industrial e disponivel comercialmente e de origem bacteriana, entretanto, esta associada a ocorrencia de reacoes adversas, alem de sua producao ser intracelular, tornando o processo de extracao e purificacao dificil e oneroso. Muitos fatores fisico quimicos relacionados ao processo fermentativo, podem ser utilizados para otimizar a producao enzimatica por microrganismos, tais como diferentes fontes nutricionais, pH, temperatura, tempo de cultivo dentre outros. Neste contexto, o estudo teve como objetivo avaliar a influencia de diferentes tempos de cultivo e fontes de carbono na producao da enzima L-asparaginase por fungos endofiticos de Moringa oleifera. A influencia do tempo de incubacao foi avaliada cultivando os endofitos a 30 oC no meio especifico por 48, 72, 96 e 120 horas. Para avaliar a producao da enzima em diferentes fontes de carbono, os fungos foram cultivados a 30 °C por 120 horas em glicose, glicerol e etanol. A atividade enzimatica extracelular foi avaliada pela relacao entre o diâmetro medio do halo de degradacao e o diâmetro medio da colonia e expressa como indice enzimatico. A glicose foi a fonte de carbono preferencial para a maioria dos fungos, assim como o cultivo por 120 horas favoreceu maior producao da enzima. As variacoes no indice enzimatico nos diferentes parâmetros analisados (fonte de carbono e tempo de cultivo) mostram as individualidades fisiologicas de cada microrganismo, influenciando na producao de L-asparaginase pelos fungos, evidenciando que a producao enzimatica e variavel, estando relacionada com as diferentes condicoes de cultivo.

L-asparaginase (EC 3.5.1.1) is used for the treatment of acute lymphocytic leukemia and food processing of starch rich foods for reducing the acrylamide formation. In our current efforts, we have immobilized the purified glutaminase-free recombinant Lasparaginase II of Pectobacterium carotovorum MTCC 1428 from Escherichia coli BL21 (DE3) on glutaraldehyde activated chitosan beads. The purified recombinant L-asparaginase II has no partial glutaminase activity which is a pre-requisite to reduce the possibility of side effects during the course of anticancer therapy. In order to study the best conditions for the performance of free enzymes and immobilized enzymes, response surface methodology was used to optimize the pH and temperature of the process conditions. It was found that the most favorable pH and temperature for the free enzyme were pH 7.83 and 47.64°C while for the immobilized enzyme, the optimum pH and temperature levels were found to be 7.88 and 48.07 °C. Furthermore, the thermal and pH studies of free and immobilized enzymes were studied under various combinations of pH and temperature and finally the thermodynamic parameters of free and immobilized glutaminase-free recombinant asparaginase were evaluated.

L-asparaginase from Escherichia coli is an important therapeutic agent in the treatment of childhood acute lymphoblastic leukemia. In this study, the secretory production of a recombinant form of L...

L-asparaginase (ASNase) is an aminohydrolase enzyme widely used in the pharmaceutical and food industries. Although currently its main applications are focused on the treatment of lymphoproliferative disorders such as acute lymphoblastic leukemia (ALL) and acrylamide reduction in starch-rich foods cooked at temperatures above 100 °C, its use as a biosensor in the detection and monitoring of L-asparagine levels is of high relevance. ASNase-based biosensors are a promising and innovative technology, mostly based on colorimetric detection since the mechanism of action of ASNase is the catalysis of the L-asparagine hydrolysis, which releases L-aspartic acid and ammonium ions, promoting a medium pH value change followed by color variation. ASNase biosensing systems prove their potential for L-asparagine monitoring in ALL patients, along with L-asparagine concentration analysis in foods, due to their simplicity and fast response.

Objective: L-Asparaginase enzyme possesses a crucial role in the treatment of various hematologic malignancies. The current study focuses on homology modeling and interaction analysis of L-Asparaginase proteins belonging to Streptomyces albidoflavus (S. albidoflavus) with the essential ligand L-Asparagine and subsequent analysis with essential β-lactam antibiotic Cefotaxime. Methods: The process of understanding Asparaginase interactions primarily involved structure determination of WP_096097608, WP_095730301, which is achieved by GalaxyTBM, I-TASSER and SWISS-MODEL. Further, the S. albidoflavus Asparaginase proteins are subjected to GalaxySite and Autodock Vina of PyRx analysis. Results: The GalaxyTBM predicted structures of both the proteins are found promising on various validation studies. The two Asparaginase proteins exhibited high binding affinities of-6.8 and-6.5 kcal/mol with Cefotaxime and-5.1 and-4.9 kcal/mol towards Asparagine. The protein WP_096097608 residues forming hydrogen bonds with L-Asparagine are also analysed to involve in interaction with Cefotaxime on individual docking analysis. Conclusion: The current findings details the two S. albidoflavus Asparaginase proteins affinity towards L-Asparagine, hence can be assessed further for immunogenicity studies. In addition to the above findings, an attempt is made to find the L-Asparaginase binding possibilities with non-metals that identified an essential β-lactam antibiotic Cefotaxime to be an effective inhibitor. This study helps in understanding the interactions of L-Asparaginase with Cefotaxime, as intake of antibiotics between the phases of chemotherapy is observed to treat various infections and also as an antibiotic to microbes that utilize Asparaginase as a vital enzyme.

L-asparaginase (a hydrolase converting L-asparagine to L-aspartic acid) was the first enzyme to be used in clinical practice as an anticancer agent after its approval in 1978 as a component of a treatment protocol for childhood acute lymphoblastic leukemia (ALL). Structural and biochemical properties of L-asparaginases have been extensively investigated during the last half century, providing an accurate structural description of the enzyme isolated from a variety of sources, as well as clarifying the mechanism of its activity. This review provides a critical assessment of the current state of knowledge of primarily structural, but also selected biochemical properties of "bacterial-type" L-asparaginases from different organisms. The most extensively studied members of this enzyme family are L-asparaginases highly homologous to one of the two enzymes from Escherichia coli (usually referred to as EcAI and EcAII). Members of this enzyme family, although often called bacterial-type L-asparaginases, have been also identified in such divergent organisms as archaea or eukarya. Over 100 structural models of L-asparaginases have been deposited in the Protein Data Bank during the last 30 years. One of the prime achievements of structure-centered approaches was the elucidation of the details of the mechanism of enzymatic action of this unique hydrolase that utilizes a side chain of threonine as the primary nucleophile. The molecular basis of other important properties of these enzymes, such as their substrate specificity, are still being evaluated. Results of structural and mechanistic studies of L-asparaginases are being utilized in efforts to improve the clinical properties of this important anticancer drug.

During the last quarter of a century, PEGylation has been used to enhance the clinical effectiveness of various therapeutic proteins. However, the conjugation process significantly increases the co...

Asparaginase, derived from microbial origin hydrolyses L-asparagine to L-aspartic acid. The enzyme finds principal use in the treatment of Acute Lymphoblastic Leukemia during childhood that primarily occurs between two to ten years of age. L-Asparaginase finds its use in management of haemopoietic disorders especially in pediatrics that is caused due to proliferation and enlargement of lymphoblast in bone marrow and in blood as well as other part of the body. L- Asparaginase from bacterial sources exhibit quaternary and tertiary structural forms. However for using it in therapeutic and clinical application it should not generate any fatal allergic reaction to the patient. Such effects can occur due to the enzyme associated L-Glutaminase activity and also due to the endotoxins from bacteria in enzyme preparations. Therefore, with the recent development in biotechnology with respect to production and purification techniques it is possible to get pure L- asparaginase from microbial origin. The present article provides an insight into the mechanism of action of L-Asparaginase as an anticancer agent and its industrial applications.

Several bacterial strains were isolated from soil and water in the current work and screened for glutaminase-free L-asparaginase activity. The selected strain was subjected to the production of glutaminase-free L-asparaginase and thereby purification. Before purification, pH scouting was performed, and 8.6 pH was the optimum working pH to carry out purification. The enzyme was purified using Sephadex G-200 gel filtration chromatography and up to 4.55-folds and its purity percentage was determined using UPLC. The purity of L-asparaginase was determined to be 99.2%. The enzyme was characterized using UV-Visible Spectrophotometer, DLS, ZP, DSC, TGA. The loss of the enzyme was 65% after sublimation, indicating its purity compared to the standard drug. The results report a developed process for the purification of L-asparaginase and its characterization, which can prove instrumental in the purification process of naturally occurring enzymes because today, most developed processes deal with recombinant enzymes.

It remains undeciphered how thermophilic enzymes display enhanced stability at elevated temperatures. Taking l-asparaginase from P. furiosus (PfA) as an example, we combined scattering shapes deduced from small-angle X-ray scattering (SAXS) data at increased temperatures with symmetry mates from crystallographic structures to find that heating caused end-to-end association. The small contact point of self-binding appeared to be enabled by a terminal short β-strand in N-terminal domain, Leu179-Val-Val-Asn182 (LVVN). Interestingly, deletion of this strand led to a defunct enzyme, whereas suplementation of the peptide LVVN to the defunct enzyme restored structural frameworkwith mesophile-type functionality. Crystal structure of the peptide-bound defunct enzyme showed that one peptide ispresent in the same coordinates as in original enzyme, explaining gain-of lost function. A second peptide was seen bound to the protein at a different location suggesting its possible role in substrate-free molecular-association. Overall, we show that the heating induced self-assembly of native shapes of PfA led to an apparent super-stable assembly.

In the present study, a new bacterial strain, Bacillus halotolerans OHEM18 was significantly found to produce extracellular L-asparaginase. L-asparaginase was purified using ammonium sulfate precip...

Polymersomes are self-assembled nanostructures with high loading capacity, possibility to deliver hydrophilic as well as hydrophobic drugs and stealth characteristic resulting in low immunogenicity...

Among the antitumor drugs, bacterial enzyme L-asparaginase has been employed as the most effective chemotherapeutic agent in pediatric oncotherapy especially for acute lymphoblastic leukemia. In previous study, the L-asparaginase from Erwinia chrysanthermy was expressed in Escherichia coli BL21(DE3). The recombinant L-asparaginase was produced from recombinant E.coli BL21(DE3) under different cultivation conditions (inducer concentration, inoculum concentration and KH2PO4 concentration). The optimized conditions by response surface methodology using face centered central composite design. The analysis of variance coupled with larger value of R2 (0.9) showed that the quadratic model used for the prediction was highly significant (p < 0.05). Under the optimized conditions, the model produced L-asparaginase activity of 123.74 U/ml at 1.03 mM IPTG, 3% (v/v) inoculum and 0.5% (w/v) KH2PO4. Recombinant protein was purified by two step using gel filtration and DEAE chromatography. The purified L-asparaginase had a molecular mass of 37 kDa with specific activity of 462 U/mg and identified by MALDI-TOF mass spectrometry. Results of MALDI-TOF analysis confirmed that recombinant protein was L-asparaginase II. Recombinant L-asparaginase has antiproliferative activity with K562 cell line. In conclusion, this study has innovatively developed cultivation conditions for better production of recombinant L-asparaginase in shake flask culture.

Background: L-asparaginase is one of the most widely used chemotherapeutic agents for the treatment of a variety of lymphoproliferative disorders and particularly acute lymphoblastic leukemia. Due to increased applications of L-asparaginase in several industrial fields including food processing and medical fields, its production needs to be increased to several folds. Objective: The aim was (i) to identify the significant factors which affect L-asparaginase production by Streptomyces fradiae NEAE-82 and (ii) to achieve higher production of L-asparaginase. Methods: Sixteen assigned factors and three dummy factors were screened using Plackett-Burman experimental design to determine the most important factors for the production of L-asparaginase by Streptomyces fradiae NEAE-82. Results: L-asparagine was determined to be the most significant positive independent factor (P-value 0.0092) affecting L-asparaginase production by Streptomyces fradiae NEAE-82 followed by pH and NaCl with significant P-values of 0.0133 and 0.0272; respectively. These factors were further optimized by Box-Behnken experimental design. The optimized fermentation conditions, which resulted in the maximum L-asparagine activity of 53.572 UmL-1 are g/L: dextrose 4, L-asparagine 15, KNO3 2, MgSO4.7H2O 0.5, K2HPO4 1, FeSO4.7H2O 0.02, NaCl 0.2, ZnSO4 0.01 and inoculum size 2 %, v/v for 7 days incubation at temperature 37°C, agitation speed 100 rpm, pH 8.5. Conclusion: A total of 3.41-fold increase in the production of L-asparaginase was achieved in the medium after statistical improvement (53.572 UmL-1) as compared to the unoptimized basal medium used prior to the application of Plackett-Burman (15.704 UmL-1).

Magnetic nanoparticles are well known as facile and effective support for enzyme immobilization since they have a high surface area, large surface-to-volume ratio, easy separation, a fast and high ...

pH and temperature are the most predominant parameters affecting protein macromolecular properties like activity and stability. Optimisation of these parameters is crucial for their industrial appl...

Two bacterial type II l-asparaginases, from Escherichia coli and Dickeya chrysanthemi, have played a critical role for more than 40 years as therapeutic agents against juvenile leukemias and lympho...

In recent times, exploration of endophytes for L-asparaginase production is gradually gaining momentum. This work deals with studies on the production of L-asparaginase from Talaromyces pinophilus, an endophytic fungus isolated from the rhizomes of Curcuma amada. L-asparaginase production was carried out by Submerged Fermentation (SmF) followed by Solid-state Fermentation (SSF). A liquid medium was designed and optimized using Plackett-Burman Design and Response Surface Methodology (RSM), under SmF. Additionally, optimal concentrations of various metal salts were incorporated in the optimized liquid medium, by one-factor-at-a-time experiments. To further enhance L-asparaginase production, SSF was carried out using Polyurethane Foam (PUF) as inert support impregnated with the optimized liquid medium. Effects of PUF cube volume, mass of PUF, moisture content, initial medium pH, and incubation temperature on the enzyme production in SSF were optimized by one-factor-at-a-time approach.L-asparaginase production enhanced from 80.8 U/mL in the unoptimized medium to 94.4 U/mL in the optimized medium under SmF. Enzyme production further increased to 120.3 U/mL under SSF by using PUF soaked in the optimized liquid medium. This study highlights the benefits of carrying out SSF with PUF, using the same liquid medium optimized for SmF - a novel approach to enhance the enzyme yield (in our case an increase of about 27% was observed). To the best of our knowledge, this is the first report on the production of L-asparaginase by both SmF and SSF, from an endophyte Talaromyces pinophilus isolated from the rhizomes of Curcuma amada.

L-asparaginase is extensively used in the treatment of acute lymphoblastic leukemia and several other lymphoproliferative diseases. In addition to its biomedical application, L-asparaginase is also of prospective use in food industry to reduce the formation of acrylamide, which is classified as probably neurotoxic and carcinogenic to human, and in biosensors for determination of L-asparagine level in medicine and food chemistry. The importance of L-asparaginases in different fields, disadvantages of commercial ferments, and the fact that they are widespread in nature stimuli the search for biobetter L-asparaginases from new producing microorganisms. In this regard, extremofile microorganisms exhibit unique physiological properties such as thermal stability, adaptability to extreme cold conditions, salt and pH tolerance and so provide one of the most valuable sources for novel L-asparaginases. The present review summarizes the recent results on studying the structural, functional, physicochemical and kinetic properties, stability of extremophilic L-asparaginases in comparison with their mesophilic homologues.L-asparaginaza nashla shirokoe primenenie v lechenii ostrogo limfoblastnogo leĭkoza i riada drugikh limfoproliferativnykh zabolevaniĭ. Pomimo bioterapii ferment ispol'zuetsia v pishchevoĭ industrii dlia snizheniia urovnia neĭrotoksicheskogo i kantserogennogo soedineniia akrilamida, a takzhe dlia sozdaniia biosensorov s tsel'iu analiza urovnia L-asparagina pri leĭkozakh i v pishchevoĭ industrii. Ogromnaia nauchno-prakticheskaia znachimost', imeiushchiesia nedostatki, vyiavlennye pri ispol'zovanii kommercheskikh preparatov L-asparaginaz, i shirokaia ikh rasprostranennost' v prirode stimulirovali poisk novykh istochnikov L-asparaginaz. Odnimi iz naibolee perspektivnykh v étom plane predstavliaiutsia ékstremofil'nye mikroorganizmy, estestvennymi usloviiami obitaniia kotorykh iavliaiutsia povyshennye i ponizhennye temperatury, vysokaia solenost', kislye i shchelochnye znacheniia rN sredy. Rezul'taty issledovaniĭ strukturno-funktsional'nykh, fiziko-khimicheskikh, kineticheskikh kharakteristik, stabil'nosti L-asparaginaz ékstremofil'nykh mikroorganizmov svidetel'stvuiut o perspektivnosti primeneniia imenno étikh fermentov v biologii i meditsine.

The potential use of asparaginases has gained tremendous significance in the treatment of acute lymphoblastic leukemia (ALL). Earlier studies suggest L-asparaginases (L-ASP) extracted from Escherichia coli and Erwinia aroideae regulates L-asparagine (L-Asn) from the circulating blood. Prolonged exposure to these enzymes may lead to hypersensitivity reactions. So, it is important to find novel asparaginases with anti-cancer properties. The three-dimensional structure of L-ASP I from Vibrio campbellii was determined by homology modeling using EasyModeller v.4.0. The structure was validated with quality indexing tools and was deposited in Protein Model DataBase. Molecular docking was performed between L-ASP I and ligand substrate L-Asn to study enzyme-substrate interactions. Qualitative and quantitative analysis of L-ASP I enzyme was found to be reliable and stable with a significant protein quality factor (LG score: 7.129). The enzyme is a dimer, belongs to α/β class of proteins. The active sites comprises of N-glycosylation site and a catalytic triad (T14-S117-D92). The binding energy of the docked complex was calculated to be -7.45 kcal/mol. The amino acid T14 identified as a primary nucleophile essential for catalytic reaction. The enzyme L-ASP I of V. campbellii provides a detailed view of structure and functional aspects with ligand substrate L-Asn. This in silico investigation has explicitly demonstrated for the first time that cytosolic L-ASP Type I of V. campbellii to have a catalytic triad which was attributed only to periplasmic L-ASP Type II. Thus, L-ASP I can serve as anti-leukemic agent in the treatment, management and control of ALL.

OBJECTIVE: Long-term treatment with tyrosine kinase inhibitors (TKI) represents an effective cure for chronic myeloid leukemia (CML) patients and discontinuation of TKI therapy is now proposed to patient with deep molecular responses. However, evidence demonstrating that TKI are unable to fully eradicate dormant leukemic stem cells (LSC) indicate that new therapeutic strategies are needed to control LSC and to prevent relapse. In this study we investigated the metabolic pathways responsible for CML surviving to imatinib exposure and its potential therapeutic utility to improve the efficacy of TKI against stem-like CML cells. METHODS: Using complementary cell-based techniques, metabolism was characterized in a large panel of BCR-ABL+ cell lines as well as primary CD34+ stem-like cells from CML patients exposed to TKI and L-Asparaginases. Colony forming cell (CFC) assay and flow cytometry were used to identify CML progenitor and stem like-cells. Preclinical models of leukemia dormancy were used to test the effect of treatments. RESULTS: Although TKI suppressed glycolysis, compensatory glutamine-dependent mitochondrial oxidation supported ATP synthesis and CML cell survival. Glutamine metabolism was inhibited by L-asparaginases such as Kidrolase or Erwinase without inducing predominant CML cell death. However, clinically relevant concentrations of TKI render CML cells susceptible to Kidrolase. The combination of TKI with Lasparaginase reactivates the intinsic apoptotic pathway leading to efficient CML cell death. CONCLUSION: Targeting glutamine metabolism with the FDA-approved drug, Kidrolase in combination with TKI that suppress glycolysis represents an effective and widely applicable therapeutic strategy for eradicating stem-like CML cells. ispartof: MOLECULAR METABOLISM vol:55 ispartof: location:Germany status: published

L-asparaginase produced by various microorganisms which it used for the treatment of leukemia and starchy food industry. Screening of L-asparaginase producing bacteria from soil samples from Damietta different locations was done. The primary screening by qualitative methods using rapid plate assay determined that out of 25 isolates, 20 strains were producing L-asparaginase. The greatest pink color zones were observed for four bacterial isolates(A6, A11, C11 and C19) after 48 hrs. These four isolates that preliminary screened were grown in liquid media for static and submerged fermentation and then used for secondary screening by Nesslerization method. The best four isolates producing L-asparaginase were identified based on morphological, cultural and biochemical tests, as A6, C11 and C19 were identified as Bacillus species and A11 was identified as Staphylococcus species.

Enzymes play a vital role in the biological system as a catalyst for biochemical reactions. They have a wide range of commercial applications in many areas like the pharmaceutical industry, food industry, etc. l-asparaginase is one of the commercial enzymes with prime importance in anticancer therapy. It is mainly used in chemotherapy; however, it has the potential to cure autoimmune disorders and infectious diseases also. Previous studies reported the antimicrobial potential of l-asparaginase. Therefore, we have discussed the possibility and challenges of the antimicrobial application of l-asparaginase in the treatment of infectious diseases. This is followed by a discussion on the effective delivery of this enzyme using biopolymeric nanocarriers that ensure safe and on target action. The present article gives a perspective on the l-asparaginase molecule that could be developed/established as an approved antimicrobial drug in the future.

L-asparaginase is used as an antileukemic drug and as a food additive to reduce the risk of acrylamide formation. New L-asparaginases are required to reduce costs and avoid clinical side effects. Citrobacter freundii EGY-NE1represents a potential source of new L-asparaginase. We purified extracellular L-asparaginase from Citrobacter freundii EGY-NE1 (through ammonium sulfate precipitation, dialysis, Sephadex-G50 and DEAE-cellulose columns) to 5.83 fold and 25.76 % recovery. The purified L-asparaginase was a low molecular weight enzyme of 19 kDa. It was optimally active at 37 ˚C, pH 8 and 40 mM asparagine. The Km and Vmax of the enzyme were 0.0179 M and 2.66 U/ml, respectively. Ca2+, Mg2+, K+ and Ba+ 2 activated the enzyme, while Na+, Zn+ 2, EDTA, azide, tartrate and HgCl2 inhibited it. The crude and purified enzymes were immobilized by encapsulation in Ca-alginate; this improved their stability and reusability. The entrapped L-asparaginases on Ca-alginate were examined by scanning electron microscope; their protein diameters ranged from 42.17 to 47.37 nm and from 46.78 to 71.97 nm for the immobilized crude and purified enzymes, respectively. Both the immobilized enzymes kept their maximal activity for 10 minutes at 40 ˚C. After the 5th cycle of repeated use, the immobilized purified and crude enzymes kept 91% and 89 % of their activities, respectively.

Covalent conjugates of asparaginase with copolymers of chitosan-PEG, chitosan-glycol and chitosan-PEI were obtained and their antitumor activity on human chronic myeloma leukemia K-562 cells was determined.

L-asparaginase (E.C.3.5.1.1) hydrolyzes L-asparagine to L-aspartic acid and ammonia, which has been widely applied in the pharmaceutical and food industries. Microbes have advantages for L-asparaginase production, and there are several commercially available forms of L-asparaginase, all of which are derived from microbes. Generally, L-asparaginase has an optimum pH range of 5.0–9.0 and an optimum temperature of between 30 and 60 °C. However, the optimum temperature of L-asparaginase from hyperthermophilic archaea is considerable higher (between 85 and 100 °C). The native properties of the enzymes can be enhanced by using immobilization techniques. The stability and recyclability of immobilized enzymes makes them more suitable for food applications. This current work describes the classification, catalytic mechanism, production, purification, and immobilization of microbial L-asparaginase, focusing on its application as an effective reducer of acrylamide in fried potato products, bakery products, and coffee. This highlights the prospects of cost-effective L-asparaginase, thermostable L-asparaginase, and immobilized L-asparaginase as good candidates for food application in the future.

L-asparaginase is an enzyme that catalyzes the degradation of asparagine and successfully used in the treatment of acute lymphoblastic leukemia. L-asparaginase toxicity is either related to hypersensitivity to the foreign protein or to a secondary L-glutaminase activity that causes inhibition of protein synthesis. PEGylated versions have been incorporated into the treatment protocols to reduce immunogenicity and an alternative L-asparaginase derived from Dickeya chrysanthemi is used in patients with anaphylactic reactions to the E. coli L-asparaginase. Alternative approaches commonly explore new sources of the enzyme as well as the use of protein engineering techniques to create less immunogenic, more stable variants with lower L-glutaminase activity. This article reviews the main strategies used to overcome L-asparaginase shortcomings and introduces recent tools that can be used to create therapeutic enzymes with improved features.

Acute Lymphoblastic Leukemia (ALL) is the commonest malignancy in childhood with a second incidence peak in adulthood. Improvements in pediatric therapy including the addition of L-asparaginase (L-ASP) have enabled cure rates in excess of 90% to be achieved in children. More recently L-ASP-containing pediatric protocols are being used to treat younger adults with ALL and have improved survival by approximately 2-fold. However, a toxicity associated with L-ASP-containing therapy in ALL is venous thromboembolism (VTE) which is associated with significant morbidity in this patient population and results in interruptions in L-ASP therapy that can impact on survival outcomes. The incidence of VTE among adult patients with ALL receiving L-ASP containing therapy has been reported to be as high as 43%. Despite this, there is a lack of evidence-based recommendations for VTE prophylaxis in this clinical context; low-molecular weight heparin (LMWH) and/or AT replacement have mostly been used. The low-quality data and inconveniences associated with these VTE prophylaxis regimens highlight the need to evaluate alternatives such as direct oral anticoagulants for the prevention of L-ASP-associated VTE in ALL. This narrative will review the body of evidence on primary thromboprophylaxis in adult patients with ALL receiving L-ASP containing therapy.

Background/aim L-Asparaginase (L-ASNase) is used as a tumor-inhibitory drug on paediatric acute lymphoblastic leukemia (ALL). ERW-ASNase is commercialised as a lyophilized powder stable only for 8 hours once reconstituted and, consequently, the leftover is usually discarded. The aim of this study will be to analyse the stability of the reconstituted lyophilised ERW-ASNase. Materials and methods In the present study, we analysed the enzymatic stability of reconstituted ERW-ASNase after conservation in three different temperature conditions for 2 and 5 days. Results Our results show that ERW-ASNase is stable at 4°C, -20°C and -80°C for up to 5 days, retaining 95% of the initial enzymatic activity in all three storage temperatures tested. Conclusion It is feasible to reuse the remaining content of ERW-ASNase vial after reconstitution, which allows the optimization of the content of ERW-ASNase vials use and reduces the cost of this formulation usage, making it more accessible.

Amidases (L-asparaginase and L-glutaminase) catalyze the deamination process of L-asparagine and L-glutamine to their corresponding acidic form with ammonia releasing. Both enzymes are considered one of the most biomedical and biotechnologically important groups of enzymes, besides their international contributing as an important commercial products. L-asparaginase and L-glutaminase have been receiving more attention as antileukemic agent for treatment of acute lymphoblastic leukemia (ALL) and other types of cancer. On the other hand, these enzymes also used in food manufacture for their hydrolysis effect and is a possible way to decrease the amount of free L-asparagine in the preliminary ingredients of food making, thus minimize the imminent risk of causing neurotoxic and carcinogenic acrylamide compound which formed when food heated above 120 °C. Glutamic and aspartic acid are important amino acids in food processing achieve a delicious, fine, sour and umami taste beside their nutritional important to food. A recent review discusses the mode of action of L-asparaginase and L-glutaminase. Also, this review lists the sources of L-asparaginase and L-glutaminase, production optimization of enzymes, and uses of the two enzymes in cancer therapy and other industrial purposes.

Background and Objectives: L-asparaginase has become the backbone of acute lymphoblastic leukemia induction. In Berlin–Frankfurt–Munster (BFM) 95/2000 protocols, L-asparaginase was given twice weekly for initial 4 weeks. While sufficient L-asparaginase levels are important, there is no apparent correlation between high L-asparaginase levels and minimal residual disease (MRD). In view of toxicities of L-asparaginase, we planned to study the effect of dose and schedule of Escherichia coli-derived L-asparaginase on early MRD by phasing the same total dose, once a week over 8 weeks. Methods: This prospective, observational study enrolled 45 children and young adults ≤40 years. Modified BFM 95 protocol was followed. Weekly 5000 IU/m2 L-asparaginase was given intravenously, and MRD was analyzed at the end of 4 weeks (MRD1) and at 8 weeks (MRD2), using multicolor flow cytometry. MRD positive was defined as residual blasts ≥0.01%. Results: Thirty-one patients were eligible for final analysis. Nine could receive scheduled eight doses of L-asparaginase and 22 patients received less than eight doses. We analyzed age, gender, diagnosis, prednisone response, cytogenetics, central nervous system status, BFM risk group, MRD2, and relapse. L-asparaginase dose association was not statistically significant with respect to MRD2 (P = 0.237). There were no cases of pancreatitis, hypersensitivity, bleeding, or thrombosis. Reasons for patients receiving less than the scheduled eight doses were low serum fibrinogen levels and liver dysfunction. This study revealed 8 MRD1-negative and 13 MRD2-negative patients. Conclusion: L-asparaginase dose intensity does not affect early MRD. Phasing L-asparaginase over 8 weeks could lead to the achievement of more MRD2-negative status and thereby improve long-term outcome. This strategy may also reduce the incidence of adverse drug events.

Introduction: Natural killer (NK)/T-cell lymphomas are aggressive malignancies that present predominantly in nasal and adjacent sites (nasal subtype), occasionally in skin, gastrointestinal tract a...