Purification Of L-asparaginase Research Articles

Use of Microbial L-asparaginase in Cancer Treatment -A Detail Review

Abstract The exceptional anticancer effects of l-asparaginases, which hydrolyze plasma l-asparagine and l-glutamine, have drew a lot of interest in recent years. This enzyme is effective against acute lymphoblastic leukemia (ALL) and lymphosarcoma, as well as ALL in children, neoplasia, and various other cancer .The l-asparaginase anticancer application technique has achieved amazing advances in modern oncology by depleting plasma l-asparagine to suppress cancer cell development, notably in youngsters. As bacterial alternative enzyme sources for anticancer therapy, high levels of l-asparaginase enzyme production by Escherichia coli, Erwinia species, Streptomyces, and Bacillus subtilis species are very desirable. Purification of L-asparaginase by Ammonium Sulphate precipitation and various chromatographic technique methods, and improvement of enzyme purity by dialysis confirmation of molecular weight with SDS page.

Neurotoxic Acrylamide Mitigation By L-Asparaginase Enzyme Extracted From Actinomycetes Species.

Acrylamide is a chemical that is generated in starchy foods, coffee, and bread, etc during high-temperature cooking processes, such as frying, roasting, and baking and is a major food safety concern. Acrylamide toxicity targets the nervous and reproductive systems, causing symptoms like muscle weakness and numbness therefore there is a need to solve this problem by biological method. Actinomycetes are an excellent source of L-asparaginase, an enzyme which specifically catalyses the breakdown of amino acid L-asparagine to aspartic acid and ammonia. If starchy food products are not treated with L- asparginase enzyme then at high temperature L-asparagine present in food converts to acrylamide. Hence, water and soil samples were obtained from Uran, Sai, and Panvel regions and Actinomycetes were isolated onto Starch Casein Agar plates. Biochemical analyses revealed negative results for TSI, nitrate reduction, and citrate but positive for catalase and starch hydrolysis. Noteworthy enzymatic activity at 2.66µg/ml/min was observed. Optimization was performed with various parameters. Production and purification of L-asparaginase by submerged fermentation, followed by ammonium precipitation, dialysis, and subsequent application to pretreat potatoes. After incubation and frying, high-performance liquid chromatography analysis exhibited a reduction in acrylamide levels. The study's meticulous approach, from isolation to application, addresses critical food safety issues, marking a significant contribution.

Cloning and Purification of L-Asparaginase from Enterobacter carcerogenus

In this study, the gene coding for EcL-ASP from Enterobacter carcerogenus was identified in full sequence and cloned into a mesophilic organism. The gene encoding L-asparaginase was transferred to the pET-28a (+) vector to ensure its expression in Escherichia coli BL21 (DE3) pLysS. The recombinant protein containing the N-terminal histidine tail (6xHis tag) was purified by Nickel affinity chromatography. As a result of SDS-PAGE, it was determined that the purified protein consisted of a single type of polypeptide. In the theoretical calculation, the subunit molecular weight of the recombinant protein containing the histidine tail was found to be 37 kDa. It was observed that the cloned enzyme had low L-glutaminase activity. The pH and temperature at which the recombinant enzyme showed the best activity were determined as 7.0 and 37 °C, respectively. From the drawn Lineweaver-Burk graph, it is estimated that the Km value is 0.06 mM and the Vmax value is 666.7 U mg-1 protein.

Integrated Lab-Scale Process Combining Purification and PEGylation of l-Asparaginase from Zymomonas mobilis

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 costs of the process. This parameter is critical, especially for biosimilar products that do not have patent protection. The integration of protein purification to its conjugation could potentially reduce total costs. Following this hypothesis, in this work, the purification of l-asparaginase from Zymomonas mobilis, produced recombinantly in Escherichia coli, linked to its conjugation with a PEG of 12 kDa, was studied. The effect of operating parameters on cell rupture by high-pressure homogenization was analyzed, and the optimal values for the conditions in this study were determined. The chromatographic capture and final purification steps were also studied. It was possible to elute the protein from ion-exchange chromatography, used as final purification, in the optimal buffer for the PEGylation step. This result allowed us to eliminate the buffer change step that is usually carried out at the beginning of the PEGylation process. The conjugate obtained by this continuous process showed higher thermal stability and slower protein degradation compared to the native protein. This continuous process not only has great potential to reduce costs but could also be useful for studying the effect of different parameters on the overall performance of the process. This knowledge is especially important to establish a quality by design approach at the industrial scale.

Development of a downstream process for purification and purity analysis of glutaminase free L-asparaginase using UPLC, DLS-ZP and DSC-TGA

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.

Purification of Dickeya solanil-Asparaginase and Study of the Influence of TiO2 and ZnO Nanoparticles on Its Enzymatic Activity

l-asparaginase catalyzes the hydrolysis of l-asparagine to l-aspartate and ammonia. In the present study, we describe the production, purification, and preliminary characterization of Dickeya solanil-asparaginase. The purification of l-asparaginase was done using ion-exchange chromatography on HiTrap Q-Sepharose Fast Flow followed by FPLC-gel filtration chromatography on Superdex 75 pg. The presence of the enzyme was confirmed by enzymatic activity measurements along with SDS-PAGE analysis. The purified l-asparaginase was shown to exhibit specificity towards l-asparagine, while being inactive against l-glutamine. This data allows us to suggest that the l-asparaginase from Dickeya solani might be clinically more relevant than, e.g., the l-asparaginase isolated from E. coli which hydrolyzed also l-glutamine and produces l-glutamate, a neurotoxic agent. We also demonstrated that the enzymatic activity was enhanced in the presence of TiO2 and ZnO nanoparticles, making them good candidates to improve l-asparaginase activity. Indeed, the results obtained show that the TiO2 nanoparticles increased the activity of l-asparaginase by a factor of 6.0 while the ZnO nanoparticles increased it twice.

Expression and purification of L-asparaginase from Escherichia coli and the inhibitory effects of cyclic dipeptides

L-asparaginase, a key enzyme involved in nitrogen metabolism, is an effective anti-tumour agent. Cyclic dipeptides, a group of compounds, contain several important biological functions. In this paper, we proposed a novel method for L-asparaginase expression and purification from Echerichia coli and determined the effect of cyclic dipeptides on the enzymatic activity of recombinant L-asparaginase. The gene ansB encoding L-asparaginase was amplified from the genome of E. coli BL21 (DE3) by polymerase chain reaction and sub-cloned into pET-15b vector to construct expressing plasmid pET-15b-ansB. The expression of recombinant protein was purified by affinity chromatography using a nickel resin followed by anion exchange chromatography. The purity and quality of the recombinant L-asparaginase were optimised. The results indicated that km for the recombinant L-asparaginase was 3.02 × 10−4 mol/L. Both cyclo-(Pro-Tyr) and cyclo-(Pro-Phe) could inhibit the activity of recombinant L-asparaginase at the level of 10−5 mol/L.

Media Optimization, Isolation and Purification of L-Asparaginase from Marine Isolate

Media Optimization, Isolation and Purification of L-Asparaginase from Marine Isolate

Biochemical characterization of L-asparaginase in NaCl-tolerant Staphylococcus sp. OJ82 isolated from fermented seafood.

L-Asparaginase from gram-positive bacteria has been poorly explored. We conducted recombinant overexpression and purification of L-asparaginase from Staphylococcus sp. OJ82 (SoAsn) isolated from Korean fermented seafood to evaluate its biotechnological potential as an antileukemic agent. SoAsn was expressed in Escherichia coli BL21 (DE3) with an estimated molecular mass of 37.5 kDa, determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Consistent with asparaginases in gram-negative bacteria, size-exclusion chromatography determined SoAsn as a homodimer. Interestingly, the optimal temperature of SoAsn was 37°C and over 90% of activity was retained between 37°C and 50°C, and its thermal stability range was narrower than that of commercial E. coli L-asparaginase (EcAsn). Both SoAsn and EcAsn were active between pH 9 and 10, although their overall pH-dependent enzyme activities were slightly different. The Km value of SoAsn was 2.2 mM, which is higher than that of EcAsn. Among eight metals tested for enzyme activity, cobalt and magnesium greatly enhanced the SoAsn and EcAsn activity, respectively. Interestingly, SoAsn retained more than 60% of its activity under 2 M NaCl condition, but the activity of EcAsn was reduced to 48%. Overall, the biochemical characteristics of SoAsn were similar to those of EcAsn, but its kinetics, cofactor requirements, and NaCl tolerance differed from those of EcAsn.

Optimization and Purifi cation of L-Asparaginase Produced by Streptomyces tendae TK-VL_333

Cultural factors affecting the production of L-asparaginase by Streptomyces tendae isolated from laterite soil samples of Guntur region were investigated on glycerol-asparagine-salts (modified ISP-5) broth. Optimal yields of L-asparaginase were recorded in the culture medium with the initial pH 7.0 incubated at 30 degrees C for 72 h. The strain utilized sucrose (2%) and yeast (2%) extract as carbon and nitrogen sources for L-asparaginase production. The productivity of L-asparaginase was slightly enhanced when the strain was treated with cell-disrupting agents like EDTA. The crude enzyme was purified to homogeneity by ammonium sulfate precipitation, Sephadex G-100 and CM-Sephadex G-50 gel filtration. By employing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of the enzyme was recorded as 97.4 kDa. This is the first report on production and purification of L-asparaginase from S. tendae.

In situ extraction of intracellular l-asparaginase using thermoseparating aqueous two-phase systems

The feasibility and generic applicability of directly integrating conventional discrete operations of cell disruption by high pressure homogenizer and the product capture by aqueous two-phase extraction (ATPE) system have been demonstrated for the extraction of intracellular l-asparaginase from E. coli. In a side-by-side comparison with the conventional ATPE process, including cell disruption, centrifugal clarification and following ATPE, purification of l-asparaginase via this novel in situ ATPE process yielded a product of l-asparaginase with a higher specific activity of 94.8 U/(mg protein) and a higher yield of 73.3%, both of which in the conventional ATPE process were 78.6 U/(mg protein) and 52.1%, respectively. In the purification of l-asparaginase (p I = 4.9), product–debris interactions commonly diminish its recovery. It was demonstrated that immediate extraction of l-asparaginase in ATPE systems when it is released at pH 5.0 during cell disruption effectively increased its recovery in the top phase due to the reduced interaction between l-asparaginase and cell debris and the reduced degradation by contaminated protease. In addition, no clarification step and/or disruptate storage are required in this in situ ATPE, which reduced the number of unit operations and thus shortened the overall process time. This novel process has a good potential for the separation of other intracellular biological products.

Direct process integration of cell disruption and fluidised bed adsorption in the recovery of labile microbial enzymes.

The practical feasibility and generic applicability of the direct integration of cell disruption by bead milling with the capture of intracellular products by fluidised bed adsorption has been demonstrated. Pilot-scale purification of the enzyme L-asparaginase from unclarified Erwinia chrysanthemi disruptates exploiting this novel approach yielded an interim product which rivalled or bettered that produced by the current commercial process employing discrete operations of alkaline lysis, centrifugal clarification and batch adsorption. In addition to improved yield and quality of product, the process time during primary stages of purification was greatly diminished. Two cation exchange adsorbents, CM HyperD LS (Biosepra/Life Technologies) and SP UpFront (custom made SP form of a prototype stainless steel/agarose matrix, UpFront Chromatography) were physically and biochemically evaluated for such direct product sequestration. Differences in performance with regard to product capacity and adsorption/desorption kinetics were demonstrated and are discussed with respect to the design of adsorbents for specific applications. In any purification of L-asparaginase (pI = 8.6), product-debris interactions commonly diminish the recovery of available product. It was demonstrated herein, that immediate disruptate exposure to a fluidised bed adsorbent promoted concomitant reduction of product in the liquid phase, which clearly counter-acted the product-debris interactions to the benefit of product yield.

A simple method for the isolation and purification of L-asparaginase from Enterobacter aerogenes.

L-Asparaginase from Enterobacter aerogenes was purified by a simple method involving sonication of the crude cell mass, gel filtration with Sephacryl S-100 as the separating material, followed by ultrafiltration. Recent methods involve complex purification procedures of 5-6 steps. The isolation process resulted in 10-fold purification of the enzyme with a specific activity of 55 IU/mg protein and recovery of 54%. The purity was tested by capillary electrophoresis, used for the first time for documenting the purification of L-asparaginase. The choice of the column material was critical in the purification process.

A rapid purification procedure for L-asparaginase from Vibrio succinogenes.

A simple procedure has been developed for the purification of L-asparaginase from Vibrio succinogenes. Only two steps of ion-exchange chromatography are required. A higher yield and higher specific activity are obtained than previously reported.

Affinity chromatography of E. coli L-asparaginase on silicate supports

1. The synthesis is proposed of an affinity sorbent for the purification of L-asparaginase using silicate materials as supports and the inhibitor D-asparagine as ligand. 2. By the method of affinity chromatography on a column and in bulk, from a crude preparation of L-asparaginase with a specific activity of 40 IU/mg a highly purified enzyme has been obtained with a specific activity of 200–230 IU/mg with a yield of about 90% in one stage.

Isolation and purification of L-asparaginase from Escherichia coli 671

A modified method for obtaining purified L-asparaginase fromE. coli 671 is proposed. The purification includes extraction with water from an acetone powder, acid-thermal treatment, fractionation with acetone and with polyethyleneglycol, and volume chromatography on DEAE-cellulose.

Crystalline l-Asparaginase from Escherichia coli B: I. PURIFICATION AND CHEMICAL CHARACTERIZATION

Abstract Purification of l-asparaginase from Escherichia coli B is described. The enzyme is crystallized from a partially purified preparation in the presence of cations and aqueous alcohol. Effective crystallization requires a minimum of 4 g atoms of cation to each mole of protein. The crystalline l-asparaginase is homogeneous by disc gel electrophoresis and ultracentrifugation and has a sedimentation coefficient (s020,w) of 7.6 S and a molecular weight (by sedimentation equilibrium) of 133,000 ± 5,000. On gel electrophoresis, the crystalline enzyme migrates similarly to the enzyme present in cell-free extracts. The affinity constants for l-asparagine and l-glutamine are 1.15 x 10-5 and 6.25 x 10-3 m, respectively. The amino acid composition indicates a minimum molecular weight of 33,300. The crystalline enzyme has an E1%278 of 7.1 and a specific activity of 300 ± 15 I.U. per mg, corrected for moisture. The isoelectric point, determined by cellulose acetate electrophoresis, is 5.2. No carbohydrate or phospholipid was detected in the enzyme. The purification procedure provides an over-all yield of about 25% from cell cake to crystals, and is adaptable to large scale isolation of the enzyme.

Purification of L-asparaginase from E. coli by specific adsorption and desorption

Purification of L-asparaginase from E. coli by specific adsorption and desorption