Application of repeated aspartate tags to improving extracellular production of Escherichia coli l-asparaginase isozyme II

Asparaginase isozyme II from Escherichia coli is a popular enzyme that has been used as a therapeutic agent against acute lymphoblastic leukemia. Here, fusion tag systems consisting of the pelB signal sequence and various lengths of repeated aspartate tags were devised to highly express and to release active asparaginase isozyme II extracellularly in E. coli. Among several constructs, recombinant asparaginase isozyme II fused with the pelB signal sequence and five aspartate tag was secreted efficiently into culture medium at 34.6 U/mg cell of specific activity. By batch fermentation, recombinant E. coli produced 40.8 U/ml asparaginase isozyme II in the medium. In addition, deletion of the gspDE gene reduced extracellular production of asparaginase isozyme II, indicating that secretion of recombinant asparaginase isozyme II was partially ascribed to the recognition by the general secretion machinery. This tag system composed of the pelB signal peptide, and repeated aspartates can be applied to extracellular production of other recombinant proteins.     

Studies of the kinetics of oxidation of cytochrome c by cytochrome c oxidase: comparison of the reactions of purified and membrane-bound oxidase

l-Asparaginase (EC 3.5.1.1.) activity has been detected in crude extracts of Lupinus arboreus young leaves, root tips, flower buds, and developing seeds. The enzyme was also present in Lupinus angustifolius root tips, developing nodules, and developing seeds. The asparaginase from each of these tissues had the same electrophoretic mobility on polyacrylamide gels and a K_m of 6–8 mm for asparagine. In extracts other than those of the developing seeds, asparaginase activity was dependent upon the inclusion of K⁺ ion and a sulfhydryl protectant in the extraction buffer. No asparaginase activity was detected in mature leaves, in the plant fraction of nodules that were fixing nitrogen, nor in root tissue further than 1.5 cm from the root tip. Asparaginase has been purified 326- and 230-fold from L. arboreus and L. angustifolius developing seeds, respectively. A molecular weight of 75,000 was obtained by gel filtration. An apparent K_m of 6.6 and 7.0 mm for asparagine was determined for the purified L. arboreus and L. angustifolius asparaginases, respectively. Of the amides, nitriles, and hydroxamates examined, the L. arboreus enzyme hydrolyzed only l-asparagine and dl-aspartyl hydroxamate. This same enzyme was inhibited by d-asparagine, 5-diazo-4-oxo-l-norvaline, dl-aspartyl hydroxamate, d-and l-aspartate, 3-cyano-l-alanine, glycine, and cysteine. Glutamine, glutamine analogs, and a number of other amino acids, amides and amines did not inhibit the L. arboreus asparaginase.     

Purification and Characterization of Glutaminase Free Asparaginase from Enterobacter cloacae: In-Vitro Evaluation of Cytotoxic Potential against Human Myeloid Leukemia HL-60 Cells

Asparaginase is an important antileukemic agent extensively used worldwide but the intrinsic glutaminase activity of this enzymatic drug is responsible for serious life threatening side effects. Hence, glutaminase free asparaginase is much needed for upgradation of therapeutic index of asparaginase therapy. In the present study, glutaminase free asparaginase produced from Enterobacter cloacae was purified to apparent homogeneity. The purified enzyme was found to be homodimer of approximately 106 kDa with monomeric size of approximately 52 kDa and pI 4.5. Purified enzyme showed optimum activity between pH 7–8 and temperature 35–40°C, which is close to the internal environment of human body. Monovalent cations such as Na⁺ and K⁺ enhanced asparaginase activity whereas divalent and trivalent cations, Ca²⁺, Mg²⁺, Zn²⁺, Mn²⁺, and Fe³⁺ inhibited the enzyme activity. Kinetic parameters K_m, V_max and K_cat of purified enzyme were found to be 1.58×10⁻³ M, 2.22 IU μg^-1 and 5.3 × 10⁴ S^-1, respectively. Purified enzyme showed prolonged in vitro serum (T_1/2 = ~ 39 h) and trypsin (T_1/2 = ~ 32 min) half life, which is therapeutically remarkable feature. The cytotoxic activity of enzyme was examined against a panel of human cancer cell lines, HL-60, MOLT-4, MDA-MB-231 and T47D, and highest cytotoxicity observed against HL-60 cells (IC₅₀ ~ 3.1 IU ml^-1), which was comparable to commercial asparaginase. Cell and nuclear morphological studies of HL-60 cells showed that on treatment with purified asparaginase symptoms of apoptosis were increased in dose dependent manner. Cell cycle progression analysis indicates that enzyme induces apoptosis by cell cycle arrest in G0/G1 phase. Mitochondrial membrane potential loss showed that enzyme also triggers the mitochondrial pathway of apoptosis. Furthermore, the enzyme was found to be nontoxic for human noncancerous cells FR-2 and nonhemolytic for human erythrocytes.     

Effect of methionine sulfoximine on asparaginase activity and ammonium levels in pea leaves

In developing leaves of Pisum sativum the levels of ammonium did not change during the light-dark photoperiod even though asparaginase (EC 3.5.1.1) did; asparaginase activity in detached leaves doubled during the first 2.5 hours in the light. When these leaves were supplied with 1 millimolar methionine sulfoximine (MSX, an inhibitor of glutamine synthetase, GS, activity) at the beginning of the photoperiod, levels of ammonium increased 8-to 10-fold, GS activity was inhibited 95%, and the light-stimulated increase in asparaginase activity was completely prevented, and declined to less than initial levels. When high concentrations of ammonium were supplied to leaves, the light-stimulated increase of asparaginase was partially prevented. However, it was also possible to prevent asparaginase increase, in the absence of ammonium accumulation, by the addition of MSX together with aminooxyacetate (AOA, which inhibits transamination and some other reactions of photorespiratory nitrogen cycling). AOA alone did not prevent light-stimulated asparaginase increase; neither MSX, AOA, or elevated ammonium levels inhibited the activity of asparaginase in vitro. These results suggest that the effect of MSX on asparaginase increase is not due solely to interference with photorespiratory cycling (since AOA also prevents cycling, but has no effect alone), nor to the production of high ammonium concentration or its subsequent effect on photosynthetic mechanisms. MSX must have further inhibitory effects on metabolism. It is concluded that accumulation of ammonium in the presence of MSX may underestimate rates of ammonium turnover, since liberation of ammonium from systems such as asparaginase is reduced by the effects of MSX.     

Enzymes in cancer: Asparaginase from chicken liver

1. A procedure for partial purification of asparaginase from chicken liver is presented. 2. The bulk of the enzyme is located in the soluble fraction of chicken liver. 3. Molecular weights of chicken-liver asparaginase and of the guinea-pig serum enzyme, estimated by gel filtration, were 306000 and 210000 respectively. The Michaelis constants (K_m) at 37° and pH8·5 were 6·0×10⁻⁵m and 7·2×10⁻⁵m respectively. 4. At 50° the chicken-liver enzyme was moderately stable, some activity being lost by aggregation; in dilute electrolyte solutions the activity rapidly diminished. 5. The anti-lymphoma effect of guinea-pig serum in mice carrying the 6C3HED tumour was confirmed. Chicken-liver asparaginase also showed an effect but in this case the enzyme preparation had to be administered repeatedly. 6. Guinea-pig serum asparaginase was stable for several days in mouse blood, after intraperitoneal injection, whereas chicken-liver asparaginase rapidly disappeared. 7. Aspartic acid β-hydrazide was shown to be a competitive inhibitor of chicken-liver asparaginase with K_i approx. 5·6×10⁻⁴m. In mice it produced an anti-lymphoma effect, as reported previously.     

Cloning and expression of aspartase and asparaginase from Pseudomonas PO7111

The aspartase and asparaginase genes of an industrial strain of Pseudomonas (PO7111) were cloned in the wide host range cosmid vector pJRD203 and identified by complementation of aspartase and asparaginase mutants. Subclones containing the asparaginase gene overproduced the enzyme 9-fold, but aspartase clones were highly unstable suggesting that aspartase overproduction conferred a severe growth disadvantage.     

Modification of recombinant asparaginase from Erwinia carotovora with polyethylene glycol 5000

A method for polyethylene conjugation with recombinant asparaginase has been developed to improve therapeutically important properties of enzyme. Methoxy-p-nitrophenyl carbamate of polyethylene glycol with molecular weight 5000 was employed as the modification reagent. Optimization of the pegylation procedure resulted in high level of enzyme modification. Under 4.5 molar excess of the modification reagent more than 10 molecules of methoxy-polyethylene bound per one asparaginase molecular. The modified asparaginase retained 57% of initial activity. A simple and efficient pegylation procedure described in this work can be used for production of asparaginase with improved therapeutic properties.     

Elucidation of the Specific Function of the Conserved Threonine Triad Responsible for Human l-Asparaginase Autocleavage and Substrate Hydrolysis

Our long-term goal is the design of a human l-asparaginase (hASNase3) variant, suitable for use in cancer therapy without the immunogenicity problems associated with the currently used bacterial enzymes. Asparaginases catalyze the hydrolysis of the amino acid asparagine to aspartate and ammonia. The key property allowing for the depletion of blood asparagine by bacterial asparaginases is their low micromolar K_M value. In contrast, human enzymes have a millimolar K_M for asparagine. Toward the goal of engineering an hASNase3 variant with micromolar K_M, we conducted a structure/function analysis of the conserved catalytic threonine triad of this human enzyme. As a member of the N-terminal nucleophile family, to become enzymatically active, hASNase3 must undergo autocleavage between residues Gly167 and Thr168. To determine the individual contribution of each of the three conserved active-site threonines (threonine triad Thr168, Thr186, Thr219) for the enzyme-activating autocleavage and asparaginase reactions, we prepared the T168S, T186V and T219A/V mutants. These mutants were tested for their ability to cleave and to catalyze asparagine hydrolysis, in addition to being examined structurally. We also elucidated the first N-terminal nucleophile plant-type asparaginase structure in the covalent intermediate state. Our studies indicate that, while not all triad threonines are required for the cleavage reaction, all are essential for the asparaginase activity. The increased understanding of hASNase3 function resulting from these studies reveals the key regions that govern cleavage and the asparaginase reaction, which may inform the design of variants that attain a low K_M for asparagine.     

In silico characterization of a cyanobacterial plant-type isoaspartyl aminopeptidase/asparaginase

Asparaginases are found in a range of organisms, although those found in cyanobacteria have been little studied, in spite of their great potential for biotechnological application. This study therefore sought to characterize the molecular structure of an L-asparaginase from the cyanobacterium Limnothrix sp. CACIAM 69d, which was isolated from a freshwater Amazonian environment. After homology modeling, model validation was performed using a Ramachandran plot, VERIFY3D, and the RMSD. We also performed molecular docking and dynamics simulations based on binding free-energy analysis. Structural alignment revealed homology with the isoaspartyl peptidase/asparaginase (EcAIII) from Escherichia coli. When compared to the template, our model showed full conservation of the catalytic site. In silico simulations confirmed the interaction of cyanobacterial isoaspartyl peptidase/asparaginase with its substrate, β-Asp-Leu dipeptide. We also observed that the residues Thr154, Thr187, Gly207, Asp218, and Gly237 were fundamental to protein–ligand complexation. Overall, our results suggest that L-asparaginase from Limnothrix sp. CACIAM 669d has similar properties to E. coli EcAIII asparaginase. Our study opens up new perspectives for the biotechnological exploitation of cyanobacterial asparaginases.     

One-Pot Production of l-threo-3-Hydroxyaspartic Acid Using Asparaginase-Deficient Escherichia coli Expressing Asparagine Hydroxylase of Streptomyces coelicolor A3(2)

We developed a novel process for efficient synthesis of l-threo-3-hydroxyaspartic acid (l-THA) using microbial hydroxylase and hydrolase. A well-characterized mutant of asparagine hydroxylase (AsnO-D241N) and its homologous enzyme (SCO2693-D246N) were adaptable to the direct hydroxylation of l-aspartic acid; however, the yields were strictly low. Therefore, the highly stable and efficient wild-type asparagine hydroxylases AsnO and SCO2693 were employed to synthesize l-THA. By using these recombinant enzymes, l-THA was obtained by l-asparagine hydroxylation by AsnO followed by amide hydrolysis by asparaginase via 3-hydroxyasparagine. Subsequently, the two-step reaction was adapted to one-pot bioconversion in a test tube. l-THA was obtained in a small amount with a molar yield of 0.076% by using intact Escherichia coli expressing the asnO gene, and thus, two asparaginase-deficient mutants of E. coli were investigated. A remarkably increased l-THA yield of 8.2% was obtained with the asparaginase I-deficient mutant. When the expression level of the asnO gene was enhanced by using the T7 promoter in E. coli instead of the lac promoter, the l-THA yield was significantly increased to 92%. By using a combination of the E. coli asparaginase I-deficient mutant and the T7 expression system, a whole-cell reaction in a jar fermentor was conducted, and consequently, l-THA was successfully obtained from l-asparagine with a maximum yield of 96% in less time than with test tube-scale production. These results indicate that asparagine hydroxylation followed by hydrolysis would be applicable to the efficient production of l-THA.     

Regulation of Escherichia colil-asparaginase II and l-aspartase by the fnr gene-product

l-asparaginase II and l-aspartase, which are known to be regulated by oxygen in Escherichia coli, are shown, by an examination of known fnr mutants, to be under the control of the fnr gene product. Mutants obtained from a procedure devised to select for asparaginase II-deficiency are fnr⁻.     

Diurnal variation of asparaginase in developing pea leaves

Levels of asparaginase activity from developing pea leaves (Pisum sativum) were found to change on a daily basis, increasing during the light period and decreasing in the dark. During extended periods of light, high levels of activity were maintained, while prolonged dark reduced activity to a low value. Half-expanded leaves exhibited the greatest change in activity over the photoperiod. Very young or mature leaves displayed little or no diurnal variation in asparaginase activity.     

Extracellular Asparaginase from Candida utilis,Its Properties as Glycoprotein and Antitumor Activities

Purified Candida asparaginase was proved to be homogeneous by gel filtration, ultra-centrifugation and disc electrophoresis. The enzyme was found to have properties as glycoprotein containing mannose. The ratio of mannose to protein was 1 to 2 in purified enzyme. Specific activity was 5500 units per nag of protein. Isoelectric point was pH 4 to 4.5 and sedimentation coefficient was found to be about 8.2 S. Antitumor activity of Candida asparaginase was inferior to E. coli enzyme. It was thought as the reason why the Candida asparaginase had less affinity to l-asparagine and it was cleared faster from the blood than E. coli asparaginase.     

Mycobacterium tuberculosis Exploits Asparagine to Assimilate Nitrogen and Resist Acid Stress during Infection

Mycobacterium tuberculosis is an intracellular pathogen. Within macrophages, M. tuberculosis thrives in a specialized membrane-bound vacuole, the phagosome, whose pH is slightly acidic, and where access to nutrients is limited. Understanding how the bacillus extracts and incorporates nutrients from its host may help develop novel strategies to combat tuberculosis. Here we show that M. tuberculosis employs the asparagine transporter AnsP2 and the secreted asparaginase AnsA to assimilate nitrogen and resist acid stress through asparagine hydrolysis and ammonia release. While the role of AnsP2 is partially spared by yet to be identified transporter(s), that of AnsA is crucial in both phagosome acidification arrest and intracellular replication, as an M. tuberculosis mutant lacking this asparaginase is ultimately attenuated in macrophages and in mice. Our study provides yet another example of the intimate link between physiology and virulence in the tubercle bacillus, and identifies a novel pathway to be targeted for therapeutic purposes.     

Photocontrol of affinity chromatography: Purification of asparaginase by photosensitive AHA-gel

An agarose gel modified with N-(ω-aminohexytl)-L -aspartic acid (AHA) and spiropyran compound (AHA–spiropyran gel) was prepared and the photocontrolled binding and releasing of asparaginase were investigated with the AHA–spiropyran gel. Asparaginase was bound on the AHA–spiropyran gel under visible light and was released in the dark. The optimum conditions for photocontrolled binding and releasing of asparaginase were a 0.05M phosphate buffer concentration and pH 7.0. Seventy-five percent of the bound asparaginase was released from the AHA–spiropyran gel column in the dark. Ninetyfold purification of asparaginase was performed with the AHA–spiropyran gel Column.     

Effect of asparaginase on cell membranes of sensitive and resistants mouse lymphoma cells

Summary High concentrations ofEscherichia coli asparaginase (80 U/ml) altered the binding of concanavalin A (Con A) to L 5178Y murine lymphoma cells that are sensitive to the cytotoxic action of this enzyme. Incubation of the asparaginase sensitive line in asparagine-free media or media containingAcinetobacter glutaminase-asparaginase did not alter the Con A binding of these cells.Escherichia coli asparaginase had no effect on Con A binding of two asparaginase resistant L5178Y cell lines that were isolate and maintained in asparagine depleted or asparaginase containing medium. TheE. coli asparaginase preparation inhibited protein and glycoprotein biosythesis to comparable degrees. It did not have proteolytic or glycolytic activity.Escherichia coli asparaginases did not alter the binding of wheat germ, soybean or ricin agglutinins to any of these cell lines. These data suggest that high concentration ofE. coli asparaginase have a specific effect on the Con A receptor in the sensitive line. Results of the lecting binding studies were presented at the Federation meeting in Atlanta, GA, 1981. This work was supported by U.S. Public Health Service Grant CA20061, the Midwest Athletes Against Childhood Cancer Fund, and the Burroughs Wellcome Fund.     

Two routes for asparagine metabolism in Pisum sativum L.

Asparagine, a major transport compound, is metabolized in Pisum sativum by two enzymes, asparaginase (EC 3.5.1.1) and asparagine-pyruvate aminotransferase. The relative amount of the two enzymes varies between tissues. In developing seeds, there are very high levels of asparaginase but only trace amounts of the aminotransferase. Asparaginase is high in young leaves but falls rapidly during leaf growth; the aminotransferase remains high throughout development. Inhibitor studies with aminooxyacetate and methionine sulfoximine confirm that the aminotransferase is the main enzyme involved in asparagine utilisation in the leaf. Root tissue has low levels of asparaginase and only trace amounts of the aminotransferase. The asparaginase is potassium dependent, but is also partially activated by ammonium ions. The leaf aminotransferase has a lower K _m for asparagine (4.5 mM) than the leaf asparaginase (8 mM). The seed asparaginase has a lower K _m for asparagine (3 mM) than the leaf asparaginase.     

Transport and metabolic effects of α-aminoisobutyric acid in saccharomyces cerevisiae

α-Aminoisobutyric acid is actively transported into yeast cells by the general amino acid transport system. The system exhibits a K_m for α-aminoisobutyric acid of 270 μM, a V_max of 24 nmol/min per mg cells (dry weight), and a pH optimum of 4.1–4.3. α-Aminoisobutyric acid is also transported by a minor system(s) with a V_max of 1.7 nmol/min per mg cells. Transport occurs against a concentration gradient with the concentration ratio reaching over 1000:1 (in/out). The α-aminoisobutyric acid is not significantly metabolized or incorporated into protein after an 18 h incubation. α-Aminoisobutyric acid inhibits cell growth when a poor nitrogen source such as proline is provided but not with good nitrogen sources such as NH₄⁺. During nitrogen starvation α-aminoisobutric acid strongly inhibits the synthesis of the nitrogen catabolite repression sensitive enzyme, asparaginase II. Studies with a mutant yeast strain (GDH-CR) suggest that α-aminoisobutyric acid inhibition of asparaginase II synthesis occurs because α-aminoisobutyric acid is an effective inhibitor of protein synthesis in nitrogen starved cells.     

Role of γ‐glutamyltranspeptidase in the pathogenesis of Helicobacter pylori infection

γ‐Glutamyltranspeptidase and asparaginase have been shown to play important roles in Helicobacter pylori colonization and cell death induced by H. pylori infection. In this study, the association of γ‐glutamyltranspeptidase and asparaginase was elucidated by comparing activities of both deamidases in H. pylori strains from patients with chronic gastritis, gastric and duodenal ulcers, and gastric cancer. γ‐Glutamyltranspeptidase activities in H. pylori strains from patients with gastric cancer were significantly higher than in those from patients with chronic gastritis or gastric ulcers. There was a wide range of asparaginase activities in H. pylori strains from patients with gastric cancer and these were not significantly than those from patients with other diseases. To identify the contributions of γ‐glutamyltranspeptidase and asparaginase to gastric cell inflammation, human gastric epithelial cells (AGS line) were infected with H. pylori wild‐type and knockout strains and inflammatory responses evaluated by induction of interleukin‐8 (IL‐8). IL‐8 response was significantly decreased by knockout of the γ‐glutamyltranspeptidase‐encoding gene but not by knockout of the asparaginase‐encoding gene. Additionally, IL‐8 induction by infection with the H. pylori wild‐type strain was significantly decreased by adding glutamine during infection. These findings indicate that IL‐8 induction caused by γ‐glutamyltranspeptidase activity in H. pylori is mainly attributable to depletion of glutamine. These data suggest that γ‐glutamyltranspeptidase plays a significant role in the chronic inflammation caused by H. pylori infection.     

Effect of chemical and physical parameters on the production of L-asparaginase from a newly isolated Serratia marcescens SK-07

Aims: The objective of this study is to optimize the levels of carbon and nitrogen sources of the medium in shake flask experiments and evaluate the effect of pH and dissolved oxygen (DO) on the production of l ‐asparaginase from a newly isolated Serratia marcescens SK‐07 in a batch bioreactor. Methods and Results: Central composite rotatable design (CCRD) was applied to optimize the levels of carbon and nitrogen sources of the medium in shake flask experiments. The optimal levels of l ‐asparagine, glucose, yeast extract and peptone were found to be 4·93, 3·81, 3·65 and 1·47 g l^?1, respectively, and maximal l ‐asparaginase production of 25·02 U mg^?1 was obtained under these conditions. Among the carbon sources tested, l ‐asparagine was identified to be the most favourable carbon source for enhanced production of l ‐asparaginase. The maximum l ‐asparaginase production of 29·89 U mg^?1 was achieved in a batch bioreactor at initial pH of 6·5 (uncontrolled) and DO level of 40% in the culture. Conclusions: We have isolated, screened and identified the potential micro‐organism, S. marcescens, for the production of l ‐asparaginase. An overall 5·55‐fold increase in the production was achieved under optimal levels of carbon and nitrogen sources, DO level and at initial pH of 6·5 (uncontrolled). Significance and Impact of the Study: The experiments illustrate the importance of statistical method for optimization of carbon and nitrogen sources and study the effect of physical process parameters on the production of l ‐asparaginase in shake flask and bioreactor, respectively. This study would be helpful for bioprocess development of bacterial l ‐asparaginase production.