Characterization Of L-asparaginase Research Articles

The Characterization of L-Asparaginase with Low L-Glutaminase Activity Produced by the Marine Pseudomonas sp. Strain GH-W2b

L-asparaginase (ASNase) hydrolyzes L-asparagine to L-aspartic acid and ammonia and has been used as an antitumor agent for the treatment of acute lymphoblastic leukemia. ASNase has also been used to mitigate the suspected carcinogenic effects of acrylamide in foods. Commercial ASNases currently used in the pharmaceutical and food industries are produced by microorganisms, such as bacteria and fungi. However, their toxicity and poor thermal stability limit their application. Therefore, identifying novel sources of ASNase is critical. In the present study, we identified an asparaginase-producing marine bacterial strain, GH-W2b, as a Pseudomonas species. Based on the plate assay results, GH-W2b produced ASNase with marginal L-glutaminase (GLNase) activity, which has been reported to cause adverse effects in clinical ASNases. The ASNase activity of GH-W2b was maximized at 50–65 °C and pH 7.0–8.5. Notably, the activities were consistent at a wide range of NaCl concentrations (0–15%) at 37 °C. In addition, compared to the control (no pre-incubation), ASNase activities were retained (>87%) by 2 h pre-incubation at 4–37 °C. Overall, our results suggest that GH-W2b ASNase has the potential to serve as a candidate for the development of salt-tolerant and/or alternative ASNases in pharmaceutical and food products.

Response surface methodology based optimized production, purification, and characterization of L-asparaginase from Fusarium foetens.

L-asparaginase is used as one of the prime chemotherapeutic agents to treat acute lymphoblastic leukemia. L-asparaginase obtained from bacteria exhibits hypersensitive reactions including various side effects. The present work aimed to optimize growth parameters for maximum production of L-asparaginase by Fusarium foetens through response surface methodology, its purification, and characterization. The optimization of L-asparaginase production by Fusarium foetens was initially done through a one-factor-at-a-time method. L-asparaginase production was further optimized using a central composite design based response surface methodology. The maximum L-asparaginase activity of 12.83 IU/ml was obtained under the following growth conditions; temperature-27.5°C, pH-8, inoculum concentration-1.5 × 106 spores/ml, and incubation period-7 days. In comparison with the unoptimized growth conditions (4.58IU/ml), the optimization led to a 2.65-fold increase in the L-asparaginase activity. The L-asparaginase from Fusarium foetens was purified 15.60-fold, with a yield of 39.89% using DEAE-cellulose column chromatography. After purification, the L-asparaginase activity was determined to be 127.26 IU/ml and the specific activity was found to be 231.38 IU/mg. The molecular mass was estimated to be approximately 37kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme showed optimum activity at pH 5, and a temperature of 40°C. The enzyme showed 100% specificity towards L-asparagine and no activity towards L-glutamine. Its activity was enhanced by Mn2+, Fe2+, and Mg2, while it was inhibited by β-mercaptoethanol and EDTA. The Km and Vmax of the purified L-asparaginase were found to be 23.82 mM and 210.3 IU/ml respectively. The results suggest that Fusarium foetens could be a potent candidate for the bioprocessing of L-asparaginase at a large scale.

Isolation and characterization of l-asparaginase from seaweeds collected from Rameswaram coast, Southeast India

Isolation and characterization of l-asparaginase from seaweeds collected from Rameswaram coast, Southeast India

Characterization of L-asparaginase from Streptomyces koyangensis SK4 with acrylamide-minimizing potential in potato chips.

Microbial L-asparaginase is well known for its application in food industries to reduce acrylamide content in fried starchy food. L-asparaginase produced by Arctic actinomycetes Streptomyces koyangensis SK4 was purified and studied for biochemical characterization. The L-asparaginase was purified with a yield of 15.49% and final specific activity of 179.77 IU/mg of protein. The enzyme exhibited a molecular weight of 43 kDa. The optimum pH and temperature for maximum activity of the purified enzyme were 8.5 °C and 40 °C, respectively. The enzyme expressed maximum activity at an incubation period of 30 min and a substrate concentration of 0.06 M. The enzyme has a low Km value of 0.041 M and excellent substrate specificity toward L-asparagine. The enzyme activity was inhibited by metal ions Ba2+ and Hg2+, while Mn2+ and Mg2+ enhanced the activity. The study evaluated the acrylamide reduction potential of L-asparaginase from Streptomyces koyangensis SK4 in potato chips. The blanching plus L-asparaginase treatment of potato slices resulted in a 50% reduction in acrylamide content. The study illustrated an effective acrylamide reduction strategy in potato chips using L-asparaginase from a psychrophilic actinomycete. Besides the acrylamide reduction potential, L-asparaginase from Streptomyces koyangensis SK4 also did not exhibit any glutaminase or urease activity which is an outstanding feature of L-asparaginase to be used as a chemotherapeutic agent.

Immobilization and Characterization of L-Asparaginase over Carbon Xerogels.

L-asparaginase (ASNase) is an aminohydrolase currently used in the pharmaceutical and food industries. Enzyme immobilization is an exciting option for both applications, allowing for a more straightforward recovery and increased stability. High surface area and customizable porosity make carbon xerogels (CXs) promising materials for ASNase immobilization. This work describes the influence of contact time, pH, and ASNase concentration on the immobilization yield (IY) and relative recovered activity (RRA) using the Central Composite Design methodology. The most promising results were obtained using CX with an average pore size of 4 nm (CX-4), reaching IY and RRA of 100%. At the optimal conditions (contact time 49 min, pH 6.73, and [ASNase] 0.26 mg·mL−1), the ASNase-CXs biocomposite was characterized and evaluated in terms of kinetic properties and operational, thermal, and pH stabilities. The immobilized ASNase onto CX-4 retained 71% of its original activity after six continuous reaction cycles, showed good thermal stability at 37 °C (RRA of 91% after 90 min), and was able to adapt to both acidic and alkaline environments. Finally, the results indicated a 3.9-fold increase in the immobilized ASNase affinity for the substrate, confirming the potential of CXs as a support for ASNase and as a cost-effective tool for subsequent use in the therapeutic and food sectors.

Screening for the Production and Characterization of L-asparaginase from Endophytic Fungi

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.

L-asparaginase from Soil actinomycetes: Optimization of process variables using Plackett Burman Method

Extensive exploration of soil microorganisms in search of novel metabolites have edified researchers on the available natural products for drug discovery. Actinomycetes are filamentous Gram positive bacteria capable of producing wide array of bioactive metabolites. High GC content in the DNA, makes the organism unique and peculiar from other microorganisms. The present article contains data on purification and characterization of L-asparaginase from soil actinomycetes. Soil samples were collected from different locations in Udupi fish market, Karnataka, India. A total of 106 isolates were screened through rapid plate assay method, out of which 17 were found active. Furthermore, a promising isolate FMCL-13A with maximum enzyme activity was chosen by submerged fermentation technique. Statistical designs viz., Placket-Burman (PB) was employed for optimization of various experimental parameters in order to enhance L-asparaginase production. The nutritional variables which affects the enzyme production by submerged fermentation from the active isolate was optimized by PB model.

ARTHROBACTER AS BIOFACTORY OF THERAPEUTIC ENZYMES

Therapeutic enzymes are proteins which can be used to treat rare and deadly diseases. They represent a small but profitable market. Therapeutic enzymes are superior to non-enzymatic drugs owing to their high specificity toward the target and also their ability to multiple substrate conversion. They are essential for speeding up all the metabolic processes and many a life-supporting chemical inter-conversions. Actinomycetes including Arthrobacter form an enormous reservoir of secondary metabolites and enzymes. The characterization of L-asparaginase, β-glucosidase, urate oxidase, methionine γ-lyase, acetyl cholinesterase, and arginase activities from actinomycetes Arthrobacter clearly demonstrate the potential of Arthrobacter as potent producer of therapeutic enzymes. These metabolic enzymes can be used either separately or in combination with other therapies for the treatment of several diseases such as leukemia, gout, asthma, and neurological disorders. The objective of this review is to compile the information on the application of therapeutic enzymes produced by Arthrobacter and their future prospects as drugs.

Submerged fermentation, purification, and characterization of L-asparaginase from Streptomyces sp. isolated from soil

Purification and characterization of extracellular alkaline protease from Streptomyces sp. LCJ12A isolated from Pichavaram mangrovesM. Parthasarathy, J. Joel Gnanadoss

Characterization of L-asparaginase from marine-derived Aspergillus niger AKV-MKBU, its antiproliferative activity and bench scale production using industrial waste

L-asparaginase (LA), an enzyme with anticancer activities, produced by marine-derived Aspergillus niger was subjected to purification and characterization. The purified enzyme was observed to have molecular weight ∼90KDa. The enzyme retained activity over a wide range of pH, i.e. pH 4–10. The enzyme was quite stable in temperature range 20–40°C. Tween 80 and Triton X-100 were observed to enhance LA activity while inhibition of LA activity was observed in presence of heavy metals. The values for Km was found to be 0.8141 mM and Vmax was 6.228μM/mg/min. The enzyme exhibited noteworthy antiproliferative activity against various cancer cell lines tested. Successful bench scale production (in 5L bioreacator) of LA using groundnut oil cake as low cost substrate has also been carried out.

Production and characterization of l-asparaginase from native Bacillus sp.

Production and characterization of l-asparaginase from native Bacillus sp.

Partial purification and characterization of L-asparaginase from an endophytic Talaromyces pinophilus isolated from the rhizomes of Curcuma amada

l-Asparaginase is a commercially significant enzyme. There exists a demand for a broad variety of microbial l-asparaginases with characteristics compatible with its different applications. Endophytic microorganisms, in particular are emerging as potential sources of l-asparaginases.The current work involves partial purification and characterization of l-asparaginase from Talaromyces pinophilus, an endophytic fungus isolated from the rhizomes of Curcuma amada. Maximum enzyme activity could be achieved at pH 8.0 and with temperature 28°C. The enzyme Exhibits 95 % and 98% of its total activity at physiological pH and temperature, respectively. The enzyme activity is largely unhindered in the presence of metal ions such as Ca2+, Cu2+, Fe2+, Mg2+, Mn2+, Zn2+. Increase in the enzyme activity in the presence of thiol groups and reduction in the same upon addition of thiol blockers indicates the involvement of cysteine in the enzyme’s catalytic activity. The enzyme is a heterodimer of 62kDa and 39kDa. The enzyme has a Km of 6.4mM, its turnover number towards l-asparagine is 286.3s−1. The enzyme has 16% glutaminase activity; its Km towards glutamine is 13.3mM and turnover number is 54.6s−1.Our results highlight that l-asparaginase from endophytic Talaromyces pinophilus could be considered as potential candidate for clinical and industrial trials, owing to its efficiency and biochemical properties. To the best of our knowledge, this is the first report on partial purification and characterization of L-asparaginase from an endophyte.

Purification and characterization of l-asparaginase from Pseudomonas aeruginosa strain SN004: Production optimization by statistical methods

Cancer cells have an abnormally high requirement for the l-asparagine and cannot make enough l-asparagine due to lack of l-asparagine synthetase. Consequently, administration of l-asparaginase withdraws dependent cancer cells of their extracellular source of l-asparagine and prime to apoptosis. In this study, SN004 strain was screened from different places in Kerman sweage as the best l-asparaginase producer and identified based on 16S rRNA sequence as Pseudomonas aeruginosa. Statistical methods were used to evaluate and optimize the effect of diverse parameters including pH, yeast extract, l-Asparagine, glucose, MgSO4, K2HPO4 and NaCl on l-asparaginase production. The maximum amount of l-asparaginase produced (785 U/ml) from the optimized medium containing l-asparagine (0.5%), glucose (0.2%), NaCl (0.045%) and K2HPO4 (0.045%). Purified enzyme showed a single band around 34 kDa on SDS-Page. SN004 l-asparaginase was active between pH values of 4.5–7.0 with an optimum at pH 5.0. In the presence of Mg2+, Mn2+, and Na+, it was detected that there was an increase in the l-asparaginase activity, whereas complete loss in activity was perceived in the presence of HgCl2. These results indicated that l-asparaginase from P. aeruginosa SN004 can be examined for medical applications.

Cloning, expression and characterization of L-asparaginase from Pseudomonas fluorescens for large scale production in E. coli BL21.

l-Asparaginase (E.C. 3.5.1.1) is used as an anti-neoplastic drug in the treatment of acute lymphoblastic leukemia. l-Asparaginase from Pseudomonas fluorescens was cloned and overexpressed in E. coli BL21. The Enzyme was found to be a Fusion protein-asparaginase complex which was given a lysozyme treatment and sonication, and then was purified in a Sepharose 6B column. The enzymatic properties of the recombinant enzyme were studied and the kinetic parameters were determined with kilometre of 109.99 mM and Vmax of 2.88 µM/min. Recombinant enzyme showed pH optima at 6.3 and temperature optima at 34 °C. Asp gene was successfully cloned into E. coli BL21 which produced high level of asparaginase intracellularly with 85.25 % recovery of enzyme with a specific activity of 0.94 IU/mg protein. The enzyme was a tetramer with molecular weight of approximately 141 kDa.

Isolation, Partial Purification and Characterization of L-Asparaginase from Hedgehog Serum

The need to further explore other sources of therapy for leukemias prompted this current work. It’s already known that L-asparaginase possess a lot of potentials in the treatment of cancers of the white blood cell but the sources of these enzyme and its properties determines the efficacy of this drug. Generally, for an asparaginase to be ideally suited for use in anti-tumour therapy, it had to satisfy a variety of criteria. The organism that is selected should produce the asparaginase in high quantity or yield, and it should be capable of being isolated from simple mammalian sources. The procedures developed for purification of the enzyme should be as rapid and simplified as possible. The purified enzyme should have long term stability on storage, maximal activity at a physiological pH, and a Km for substrate below the concentration of the substrate in the blood. Therefore the aim of this present research was to partially purify and characterize this enzyme from Hedgehog serum in order to compare its anti-leukemic potentials with those from documented literature for possible future medical application. L-asparaginase was isolated and partially purified from Hedgehog serum which is also known to be a reservoir of many other useful proteins including anti venoms using a four step profile of ammonium sulphate fractionation, dialysis, and ion exchange and gel filtration chromatography. The enzyme gave an overall yield of 77.58%, optimum pH and temperature of 7.8 and 39oC respectively, and a Km of 0.0125 mM. Gel filtration gave an approximate molecular weight of 139,000 Da, while SDS PAGE indicated a subunit molecular weight of 36,000 and 34,600 Da respectively. The enzyme also catalyses the hydrolysis of glutamine though slightly. Mg2+ and Zn2+ serve as activators for Hedgehog serum L-asparaginase, while inhibition from heavy ions like Hg2+ was also observed. This work shows that mammalian sources of this enzyme could be more preferable for treatment of asparagine dependent tumours in time to come.

Production, Purification and Characterization of L-Asparaginase from Marine Endophytic Aspergillus sp. ALAA-2000 under Submerged and Solid State Fermentation

Among all endophytic fungi recovered from the marine soft sponge Aplysina fistularis, 72.2% were able to produce L-asparaginase. Among all obtained isolates, Aspergillus sp. ALAA-2000, the hyperactive producer for the anticancer agent, L-asparaginase, under submerged fermentation (SMF) and solid state fermentation (SSF) of different agriculture wastes was selected for the optimization of extraction process; optimization of physicochemical parameters, which affecting the production of L-asparaginases in SSF and optimization of the purified L-asparaginases parameters. Maximum L-asparaginase activity 23.34 U/ml was recovered from soybean with hot water at 40 °C and 150 rpm for 30 min under SSF and 30.64 U/ml under submerged fermentation using glucose as carbon source and asparagine as nitrogen source. Two types of L-asparaginase (AYA-1 and AYA-2) were purified from the culture supernatant of Aspergillus sp. ALAA-2000 through ammonium sulfate precipitation and gel filtration chromatography (sephadex G-200). Molecular weights of the enzymes were 25 kDa (AYA-1) and 31 kDa (AYA-2). The parameters of purified L-asparaginase were optimized for AYA-1 (pH 6.0, stable at 30°C to 50°C for 60 min, reaction time 15 min, and substrate concentration 1.275 mg/ml) and AYA-2 enzyme (pH 10, stable at 30°C to 70°C for 60 min, reaction time 15 min, and substrate concentration 1.275 mg/ml). Whereas inhibitors of metaloproteases, chelating agents EDTA, had no effect on L-asparaginase. These finding suggest that L-asparaginase was not metaloproteases.

Optimization, purification, and characterization of L-asparaginase from Actinomycetales bacterium BkSoiiA

ABSTRACTActinobacteria are promising source of a wide range of important enzymes, some of which are produced in industrial scale, with others yet to be harnessed. L-Asparaginase is used as an antineoplastic agent. The present work deals with the production and optimization of L-asparaginase from Actinomycetales bacterium BkSoiiA using submerged fermentation in M9 medium. Production optimization resulted in a modified M9 medium with yeast extract and fructose as carbon and nitrogen sources, respectively, at pH 8.0, incubated for 120 hr at 30 ± 2°C. The crude enzyme was purified to near homogeneity by ammonium sulfate precipitation following dialysis, ion-exchange column chromatography, and finally gel filtration. The sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) revealed an apparent molecular weight of 57 kD. The enzyme was purified 95.06-fold and showed a final specific activity of 204.37 U/mg with 3.49% yield. The purified enzyme showed maximum activity at a pH 10.0 and was stable at pH 7.0 to 9.0. The enzyme was activated by Mn2+ and strongly inhibited by Ba2+. All these preliminary characterization suggests that the L-asparaginase from the source may be a tool useful to pharmaceutical industries after further research.

Extraction, purification and characterization of L-asparaginase from Penicillium sp. by submerged fermentation.

A fungal strain identified as Penicillium sp. was evaluated for its L–asparaginase enzyme production. The L-asparaginase enzyme was purified to homogeneity from Penicillium sp. that was grown on submerged fermentation. Different purification steps including salt precipitation, followed by separation on sephadex G-100-120 gel filtration and DEAE were applied to obtain pure enzyme preparation. The purified enzyme showed 13.97 IU/mg specific activity and 36.204% yield. The polyacrylmide gel electrophoresis of the pure enzyme exhibited one protein of 66 kDa. The enzyme showed maximum activity at 7.0 pH and 37°C and Km value 4.00 × 10-3 M. Key words: L – Asparaginase, fungi, Penicillium.

Biochemical Characterization of Recombinant L-Asparaginase (AnsA) from Rhizobium etli, a Member of an Increasing Rhizobial-Type Family of L-Asparaginases

We report the expression, purification, and characterization of L-asparaginase (AnsA) from Rhizobium etli. The enzyme was purified to homogeneity in a single-step procedure involving affinity chromatography, and the kinetic parameters K(m), V(max), and k(cat) for L-asparagine were determined. The enzymatic activity in the presence of a number of substrates and metal ions was investigated. The molecular mass of the enzyme was 47 kDa by SDS-PAGE. The enzyme showed a maximal activity at 50 degrees C, but the optimal temperature of activity was 37 degrees C. It also showed maximal and optimal activities at pH 9.0. The values of K(m), V(max), k(cat), and k(cat)/K(m) were 8.9 +/- 0.967 × 10⁻³ M, 128 +/- 2.8 U/mg protein, 106 +/- 2 s⁻¹, and 1.2 +/- 0.105 × 10⁴ M⁻¹s⁻¹, respectively. The L-asparaginase activity was reduced in the presence of Mn²⁺, Zn²⁺, Ca²⁺, and Mg²⁺ metal ions for about 52% to 31%. In addition, we found that NH₄⁺, L-Asp, D-Asn, and beta-aspartyl-hydroxamate in the reaction buffer reduced the activity of the enzyme, whereas L-Gln did not modify its enzymatic activity. This is the first report on the expression and characterization of the L-asparaginase (AnsA) from R. etli. Phylogenetic analysis of asparaginases reveals an increasing group of known sequences of the Rhizobialtype asparaginase II family.

Cloning, expression and characterization of l-asparaginase from Withania somnifera L. for large scale production

l-Asparaginase (E.C. 3.5.1.1) is used as a therapeutic agent in the treatment of acute childhood lymphoblastic leukemia. It is found in a variety of organisms such as microbes, plants and mammals. In plants, l-asparaginase enzymes are required to catalyze the release of ammonia from asparagine, which is the main nitrogen-relocation molecule in these organisms. An Indian medicinal plant, Withania somnifera was reported as a novel source of l-asparaginase. l-Asparaginase from W. somnifera was cloned and overexpressed in E. coli. The enzymatic properties of the recombinant enzyme were investigated and the kinetic parameters (Km, kcat) for a number of substrates were determined. The kinetic parameters of selected substrates were determined at various pH and the pH- and temperature-dependence profiles were analyzed. WA gene successfully cloned into E. coli BL21 (DE3) showed high asparaginase activity with a specific activity of 17.3 IU/mg protein.