Optimization of L-asparaginase production from endophytic Fusarium proliferatum using OFAT and RSM and its cytotoxic evaluation.

L-asparaginase from endophytic Fusarium proliferatum (isolate CCH, GenBank accession no. MK685139) isolated from the medicinal plant Cymbopogon citratus (Lemon grass), was optimized for its L-asparaginase production and its subsequent cytotoxicity towards Jurkat E6 cell line. The following factors were optimized; carbon source and concentration, nitrogen source and concentration, incubation period, temperature, pH and agitation rate. Optimization of L-asparaginase production was performed using One-Factor-At-A-Time (OFAT) and Response surface methodology (RSM) model. The cytotoxicity of the crude enzyme from isolate CCH was tested on leukemic Jurkat E6 cell line. The optimization exercise revealed that glucose concentration, nitrogen source, L-asparagine concentration and temperature influenced the L-asparaginase production of CCH. The optimum condition suggested using OFAT and RSM results were consistent. As such, the recommended conditions were 0.20% of glucose, 0.99% of L-asparagine and 5.34 days incubation at 30.50 °C. The L-asparaginase production of CCH increased from 16.75 ± 0.76 IU/mL to 22.42 ± 0.20 IU/mL after optimization. The cytotoxicity of the crude enzyme on leukemic Jurkat cell line recorded IC50 value at 33.89 ± 2.63% v/v. To conclude, the enzyme extract produced from Fusarium proliferatum under optimized conditions is a potential alternative resource for L-asparaginase.

Fungal L-asparaginase: Strategies for production and food applications.

L-asparaginase (L-asparagine amidohydrolase EC 3.5.1.1) is of great importance in pharmaceutical and food applications. This review aims to describe the production and use of fungal L-asparaginase focusing on its potential as an effective reducer of acrylamide in different food applications. Fungal asparaginases have been used as food additives and have gained importance due to some technical advantages, for example, fungi can grow using low-cost culture mediums, and the enzyme is extracellular, which facilitates purification steps. Research aimed at the discovery of new L-asparaginases, mainly those produced by fungi, have great potential to obtain cheaper enzymes with desirable properties for application in food aiming at the reduction of acrylamide.

Gene cloning and expression of the l-asparaginase from Bacillus cereus BDRD-ST26 in Bacillus subtilis WB600.

l-Asparaginase (ASN; EC 3.5.1.1) shows great commercial value because of its ability to reduce toxic levels of acrylamide in foods. To achieve high-efficiency production of l-asparaginase, an open reading frame of 978 bp encoding asparaginase (BcA) was amplified from Bacillus cereus BDRD-ST26, followed by its expression in Bacillus subtilis WB600, with the highest yield of 374.9 U/ml obtained using an amyE-signal peptide. A four-step purification protocol was used to purify BcA, resulting in a 15.1-fold increase in purification yield, with a specific activity of purified BcA at 550.8 U/mg and accompanied by detection of minimal l-glutaminase activity. Maximum BcA activity was detected at 50°C and pH 9.0 in 20 mM Tris-HCl buffer, with a half-life at 50°C of 17.35 min and a Km and kcat of 9.38 mM and 63.6 s-1, respectively. Compared with untreated potato strips, 72% acrylamide (2.35 mg/kg) was removed from potato strips pretreated with BcA. These results indicated that this novel BcA variant represents a potential candidate for application in the food-processing industry.

A novel bacterial type II l-asparaginase and evaluation of its enzymatic acrylamide reduction in French fries.

This study reports the identification of a novel bacterial type II l-asparaginase, abASNase2, from Aquabacterium sp. A7-Y. The enzyme contains 319 amino acids and shared 35% identity with Escherichia coli type II l-asparaginase (EcAII), a commercial enzyme trademarked Elspar® that is widely used for medical applications. abASNase2 had high specific activity (458.9U/mg) toward l-asparagine, very low activity toward l-glutamine and d-glutamine and no activity toward d-asparagine. The optimal enzymatic activity conditions for abASNase2 were found to be 50mM Tris-HCl buffer (pH 9.0) at 60°C. It was very stable in the pH range of 7.0-11.0 and exhibited up to 80% relative activity after 2h below 40°C. The Km and kcat of abASNase2 were 1.8×10-3M and 241.9s-1, respectively. In addition, abASNase2's ability to remove acrylamide from fried potato strips was evaluated. Compared to untreated potato strips (acrylamide content: 0.823±0.0457mg/kg), 88.2% acrylamide was removed in the abASNase2-treated group (acrylamide content: 0.097±0.0157mg/kg). These results indicate that the novel l-asparaginase abASNase2 is a potential candidate for applications in the food processing industry.

Cloning, expression, and characterization of L-asparaginase from a newly isolated Bacillus subtilis B11-06.

This study focused on the cloning, overexpression, and characterization of the gene encoding L-asparaginase (ansZ) from a nonpathogenic strain of Bacillus subtilis B11-06. The recombinant enzyme showed high thermostability and low affinity to L-glutamine. The ansZ gene, encoding a putative L-asparaginase II, was amplified by PCR and expressed in B. subtilis 168 using the shuttle vector pMA5. The activity of the recombinant enzyme was 9.98 U/mL, which was significantly higher than that of B. subtilis B11-06. The recombinant enzyme was purified by a two-step procedure including ammonium sulfate fractionation and hydrophobic interaction chromatography. The optimum pH and temperature of the recombinant enzyme were 7.5 and 40 °C, respectively. The enzyme was quite stable at a pH range of 6.0-9.0 and exhibited about 14.7 and 9.0% retention of activity following 2 h incubation at 50 or 60 °C, respectively. The Km for L-asparagine was 0.43 mM, and the Vmax was 77.51 µM/min. Results of this study also revealed the potential industrial application of this enzyme in reducing acrylamide formation during the potato frying process.

Anticancer properties of highly purified L-asparaginase from Withania somnifera L. against acute lymphoblastic leukemia.

Withania somnifera L. has been traditionally used as a sedative and hypnotic. The present study was carried out for the purification, characterization, and in vitro cytotoxicity of L-asparaginase from W. somnifera L. L-Asparaginase was purified from the fruits of W. somnifera L. up to 95% through chromatography. The purified L-asparaginase was characterized by size exclusion chromatography, polyacrylamide gel electrophoresis (PAGE), and 2D PAGE. The antitumor and growth inhibition effect of the L-asparaginase was assessed using [3-(4, 5-dimethyl-thiazol-2yl)-2, 5-diphenyl-tetrazolium bromide] (MTT) colorimetric dye reduction method. The purified enzyme is a homodimer, with a molecular mass of 72 +/- 0.5 kDa, and the pI value of the enzyme was around 5.1. This is the first report of the plant containing L-asparaginase with antitumor activity. Data obtained from the MTT assay showed a LD(50) value of 1.45 +/- 0.05 IU/ml. W. somnifera L. proved to be an effective and a novel source of L: -asparaginase. Furthermore, it shows a lot of similarity with bacterial L-asparaginases EC-2.

Withania somnifera (Ashwagandha): a novel source of L-asparaginase.

Different parts of plant species belonging to Solanaceae and Fabaceae families were screened for L-asparaginase enzyme (E.C.3.5.1.1.). Among 34 plant species screened for L-asparaginase enzyme, Withania somnifera L. was identified as a potential source of the enzyme on the basis of high specific activity of the enzyme. The enzyme was purified and characterized from W. somnifera, a popular medicinal plant in South East Asia and Southern Europe. Purification was carried out by a combination of protein precipitation with ammonium sulfate as well as Sephadex-gel filtration. The purified enzyme is a homodimer, with a molecular mass of 72 +/- 0.5 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion chromatography. The enzyme has a pH optimum of 8.5 and an optimum temperature of 37 degrees C. The Km value for the enzyme is 6.1 x 10(-2) mmol/L. This is the first report for L-asparaginase from W. somnifera, a traditionally used Indian medicinal plant.

[Biological properties of L-asparaginase preparations from E. coli in cell cultures]. / Izuchenie biologicheskikh svoistv preparatov L-asparaginazy iz E. coli v kletochnykh kul'turakh.

Non-specific cytotoxicity and specific antitumor activity of 5 preparations of L-asparaginase from E. coli were studied. Two cell line, i.e. the asparagine-dependent (Berkitt lymphoma cells) and asparagin-independent (human ovary cancer cells) were used as the test-system. Incorporation of 3H-thimidine into DNA was the criterion of the preparation effect on the cells. Preparation I with the specific activity of 60-90 IU per 1 mg of protein obtained at the first stages of purification had high non-specific cytotoxicity. Preparation II obtained after further purification of preparation I, as well as preparation II without any stabilizer with the specific activity of 200 IU/mg were not inferior to the "Bayer" preparation by their biological properties. Addition of L-asparaginase to the preparation as a stabilizer of excessive glycine (preparation IV) increased its non-specific cytotoxicity and interfered with the study of its properties in the cell systems. Mannitol (preparation V) had no effect on the biological activity of L-asparaginase preparation.

L-Asparaginase of Klebsiella aerogenes. Activation of its synthesis by glutamine synthetase.

An L-asparaginase has been purified some 250-fold from extracts of Klebsiella aerogenes to near homogeneity. The enzyme has a molecular weight of 141,000 as measured by gel filtration and appears to consist of four subunits of molecular weight 37,000. The enzyme has high affinity for L-asparagine, with a Km below 10(-5) M, and hydrolyzes glutamine at a 20-fold lower rate, with a Km of 10(-3) M. Interestingly, the enzyme exhibits marked gamma-glutamyltransferase activity but comparatively little beta-aspartyl-transferase activity. A mutant strain lacking this asparaginase has been isolated and grows at 1/2 to 1/3 the rate of the parent strain when asparagine is provided in the medium as the sole source of nitrogen. This strain grows as well as the wild type when the medium is supplemented with histidine or ammonia. Glutamine synthetase activates the formation of L-asparaginase. Mutants lacking glutamine synthetase fail to produce the asparaginase, and mutants with a high constitutive level of glutamine synthetase also contain the asparaginase at a high level. Thus, the formation of asparaginase is regulated in parallel with that of other enzymes capable of supplying the cell with ammonia or glutamate, such as histidase and proline oxidase. Formation of the asparaginase does not require induction by asparaginase and is not subject to catabolite repression.

The properties and large-scale production of L-asparaginase from citrobacter.

An intracellular L-asparaginase with antitumour activity was purified from a strain of Citrobacter. The optimum conditions for enzyme production by fermentation on scales up to 2700 l were investigated. Highest enzyme yield was obtained in corn-steep liquor medium (9-2%, W/V) at 37 degrees C. Oxygen limitation was not necessary for high enzyme yield. A total recovery of 4-3% from nucleic-acid-free extract and a 180-fold increase in specific activity were obtained after purificaiton. The specific activity of the purified preparation was 45 i.u./mg protein. The enzyme hydrolysed D-asparagine and L-glutamine at 7 and 5%, respectively, of its activity toward L-asparagine, but L-glutaminase activity could be demonstrated only at substrate concentrations above 5 mM. The Km values for L-asparagine and D-asparagine were 2-6 X 10(-5) and 1-4 X 10(-4) respectively. The anti-lymphoma activity of the enzyme was demonstrated with Gardner lymphosarcoma and was found only slightly less potent that Crasnitin, the most active asparaginase so far tested in this system.