Single-step purification and characterization of recombinant aspartase of Aeromonas media NFB-5.

Aspartase (L-aspartate ammonia-lyase; EC 4.3.1.1) catalyzes the reversible amination of fumaric acid to produce L-aspartic acid. Aspartase coding gene (aspA) of Aeromonas media NFB-5 was cloned, sequenced, and expressed with His tag using pET-21b⁺ expression vector in Escherichia coli BL21. Higher expression was obtained with IPTG (1.5 mM) induction for 5 h at 37 °C in LB medium supplemented with 0.3% K2HPO4 and 0.3% KH2PO4. Recombinant His tagged aspartase was purified using Ni-NTA affinity chromatography and characterized for various biochemical and kinetic parameters. The purified aspartase showed optimal activity at pH 8.5 and 8.0 in the presence and absence of magnesium ions, respectively. The optimum temperature was determined to be 35 °C. The enzyme showed apparent K(m) and V(max) values for L-aspartate as 2.01 mM and 114 U/mg, respectively. The enzyme was stable in pH range of 6.5-9.5 and temperature up to 45 °C. Divalent metal ion requirement of enzyme was efficiently fulfilled by Mg²âº, Mn²âº, and Ca²âº ions. The cloned gene (aspA) product showed molecular weight of approximately 51 kDa by SDS-PAGE, which is in agreement with the molecular weight calculated from putative amino acid sequence. This is the first report on expression and characterization of recombinant aspartase from A. media.

A missense mutation causes aspartase deficiency in Yersinia pestis.

It is established that cells of Yersinia pestis, the causative agent of bubonic plague, excrete l-aspartic acid at the expense of exogenous l-glutamic acid during expression of the low-calcium response. Results of enzymic analysis provided here suggest that a previously defined deficiency of aspartase (AspA) accounts for this phenomenon rather than an elevated oxaloacetate pool. The only known distinction between most sequenced isolates of aspA from Y. pestis and the active gene in Yersinia pseudotuberculosis (the immediate progenitor of Y. pestis) is a single base transversion (G.C-->T.A) causing replacement of leucine (encoded by UUG) for valine (encoded by GUG) at amino acid position 363. The gene from Y. pestis KIM possesses a unique second transversion (G.C-->T.A) at amino acid 146 causing substitution of aspartic acid (encoded by GAU) with tyrosine (encoded by UAU). We show in this study that Y. pestis expresses aspA as cross-reacting immunological material (CRIM). Functional and inactive aspA of Y. pseudotuberculosis PB1 and Y. pestis KIM, respectively, were then cloned and expressed in AspA-deficient Escherichia coli. After purification to near homogeneity, the products were subjected to biochemical analysis and found to exhibit similar secondary, tertiary and quaternary (tetrameric) structures as well as comparable Michaelis constants for l-aspartic acid. However, the k(cat) of the Y. pestis CRIM of strain KIM is only about 0.1 % of that determined for the active AspA of Y. pseudotuberculosis. Return of valine for leucine at position 363 of the Y. pestis enzyme restored normal turnover (k(cat) 86+/-2 s(-1)) provided that the amino acid substitution at position 146 was also reversed. These observations have important implications for understanding the nature of the stringent low-calcium response of Y. pestis and its role in promoting acute disease.

[Optimization of culture media and conditions for cultivating Erwinia sp., a producer of fumarase and aspartase]. / Optimizatsiia sredy i uslovii kul'tivirovaniia produtsenta fumarazy i aspartazy Erwinia sp.

Mathematical methods of experimental design were used to determine the optimal concentrations of nutrient medium components, aeration conditions, and pH providing for the maximum biomass yields, as well as fumarase and aspartase activities, during submerged cultivation of Erwinia sp. The data showed that different concentrations of carbon source (molasses) and pH of the nutrient medium were required to reach the maximum yields of fumarase and aspartase. Calculations suggested that the combination of these optimized factors would result in 3.2-, 3.4-, and 3.8-fold increases in the Erwinia sp. biomass, aspartase activity, and fumarase activity yields, respectively. The experimental data were consistent with these estimates to a 80% accuracy.

Purification and characterization of thermostable aspartase from Bacillus sp. YM55-1.

A thermostable aspartase was purified from a thermophile Bacillus sp. YM55-1 and characterized in terms of activity and stability. The enzyme was isolated by a 5-min heat treatment at 75 degrees C in the presence of 11% (w/v) ammonium sulfate and 100 mM aspartate, followed by Q-Sepharose anion-exchange and AF-Red Toyopearl chromatographies. The native molecular weight of aspartase determined by gel filtration was about 200,000, and this enzyme was composed of four identical monomers with molecular weights of 51,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Unlike Escherichia coli aspartase, the enzyme was not activated by the presence of magnesium ion at alkaline pH. At the optimum pH, the Km and Vmax were 28.5 mM and 700 units/mg at 30 degrees C and 32.0 mM and 2200 units/mg at 55 degrees C, respectively. The specific activity was four and three times higher than those of E. coli and Pseudomonas fluorescens enzymes at 30 degrees C, respectively. Eighty percent of the activity was retained after a 60-min incubation at 55 degrees C, and the enzyme was also resistant to chemical denaturants; 80% of the initial specific activity was detected in assay mixtures containing 1.0 M guanidine hydrochloride. The purified enzyme shared a high sequence homology in the N-terminal region with aspartases from other organisms.

Mutagenic investigation of conserved functional amino acids in Escherichia coli L-aspartase.

The potential importance of several functional amino acids in the activity of L-aspartase from Escherichia coli has been examined by site-directed mutagenesis. Amino acids whose importance in enzyme activity was suggested by chemical modification and pH dependence studies were chosen as candidates for investigation. The selection of the particular amino acid targets was guided by homology comparisons among the other sequenced bacterial L-aspartases and by the broader comparison among the fumarase-aspartase enzyme family. Substitution of the most highly conserved cysteine with either serine or alanine, or the most highly conserved histidine with leucine, had no significant effect on the activity of L-aspartase or on the sensitivity of these mutated L-aspartases to cysteine and histidine specific modifying reagents. However, alteration of each of the two conserved lysines to arginine did cause dramatic changes in the catalytic properties of the enzyme. Modification of lysine 54 results in the complete loss of enzyme activity. However, this activity loss appears to be related to changes in the subunit association properties of the arginine 54 mutant. Lysine 326 appears to be involved in substrate binding. Modification of this residue causes a 5-fold increase in the Km for aspartic acid, a drastic decrease in kcat/Km, and a change in the divalent metal ion requirements of the enzyme.

Determination of the subunit contact region of aspartase.

The subunits of tetrameric enzyme aspartase from Escherichia coli and Pseudomonas fluorescens were incapable of forming hybrid tetramers, suggesting that the subunit contact regions of these two enzymes are not conserved in spite of significant homology between the total sequences of the enzymes. To locate the subunit contact region, we modified cysteine residues of the intermediate species formed during the assembly of the subunits of aspartase from E. coli. Subunits modified with N-ethylmaleimide were unable to assemble into tetramers. Further experiments showed that Cys-88 was the primary residue that was modified. The sequence flanking Cys-88 is quite different from that in the the enzyme from P. fluorescens, suggesting that this region participates in the mutual recognition of subunits.

Characterization of intermediate species during the molecular assembly of aspartase.

Molecular assembly of aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) from Escherichia coli was studied during the reversible denaturation. Although previous studies [Tokushige, M., Eguchi, G., and Hirata, F. (1977) Biochem. Biophys. Acta 480, 479-488] were unable to identify intermediate species during the course of reversible denaturation of aspartase, temperature-controlled HPLC and cross-linking with dimethyl suberimidate of the renaturation products showed that monomeric, dimeric and trimeric species occupied over 80% of the total oligomeric molecules below 13 degrees C; unlike the tetramer, these intermediates were without the activity. The degree of active tetramer formation was a linear function of the restoration of the activity below 18 degrees C, while above 23 degrees C, the activity regain was less than 70% restoration of tetrameric molecules. Upon examination by fluorescence spectroscopy, structural changes during reconstitution exhibited such complex kinetics that the rapid formation of structured oligomers proceeds first with a half-time of less than 10 sec, followed by slow subunit association. These results strongly suggest that the tetramer formation is an essential prerequisite, though not sufficient for the active enzyme.

Design of a new automatic chromatography system for efficient enzyme purification equipped with a time-shared multiple activity analyzer.

A new system equipped with a computer-controlled multiple activity analyzer has been developed for the efficient purification of multiple enzymes. The system consists of the following units: conventional enzyme fractionation system with a peristaltic pump, liquid chromatographic column, fraction collector, and uv monitor; computer-operated uv-vis spectrophotometer equipped with a thermo-regulated metal block and a flow-through type silica cuvette; personal computer; dot matrix printer; cooling facility; and automatic sampling-mixing system. The whole system is operated by a newly designed time-sharing computer program for periodic and repetitive sampling of the column eluants containing multiple kinds of enzymes and of designated assay mixtures for each enzyme and for measurement of the initial velocity of spectrophotometric signals. For example, a mixture of aspartase (EC 4.3.1.1) and malate dehydrogenase (EC 1.1.1.39) and also a mixture of these two enzymes and glutamate dehydrogenase (EC 1.4.1.3 or EC 1.4.1.4) were analyzed by the above system using gel permeation chromatography, and the two or three enzyme activities were repeatedly monitored within 4 min. Based on the above results further possibilities for the application of the system for a variety of purposes are discussed.

Kinetic studies of L-aspartase from Escherichia coli: substrate activation.

The enzyme L-aspartase from Escherichia coli was observed to have a time lag during the production of aspartic acid from fumarate and ammonia. This time lag is pH dependent, with little lag observed below pH 7.0 and a very extensive lag observed above pH 8.0. This time lag was also found to be dependent on both substrate and divalent metal ion concentrations and on the degree of proteolysis of L-aspartase. The observed lag, in the reaction examined in the amination direction, has been found to be correlated with the nonlinear kinetics seen at higher pH in the deamination direction. Both phenomena are consistent with a model in which there is a separate activator site for the substrate, L-aspartic acid, that is distinct from the enzyme active site. Occupation of this site by the substrate, or by various substrate analogues, eliminates both the nonlinearity and the time lag. The D isomer of aspartic acid, which does not bind at the active site, can bind at this newly identified activator site.

Purification of aspartase and aspartokinase-homoserine dehydrogenase I from Escherichia coli by dye-ligand chromatography.

Improved purification schemes are reported for the enzymes L-aspartase and aspartokinase-homoserine dehydrogenase I from Escherichia coli. Dye-ligand chromatography on commercially available dye matrices are incorporated as key steps in these purifications. Red A-agarose has a high affinity for L-aspartase, which is then eluted as a homogeneous protein fraction with 1 mM L-aspartic acid. Green A-agarose shows a high binding affinity for the bifunctional enzyme aspartokinase-homoserine dehydrogenase I. Purification is accomplished by elution with NADP+, followed by formation of a ternary complex with NADP and cysteine, a good competitive inhibitor of the homoserine dehydrogenase activity, and rechromatography on Green A-agarose. The final specific activity of each purified enzyme equaled or exceeded previously reported values, the overall yield of enzymes obtained was significantly higher, and these improved purification schemes were found to be more amenable to being scaled up for the production of large quantities of purified enzyme.

Purification, crystallization, and molecular properties of aspartase from Pseudomonas fluorescens.

Aspartase [L-aspartate ammonia-lyase, EC 4.3.1.1] of Pseudomonas fluorescens was highly purified to homogeneity and crystallized. The purified enzyme sedimented as a monodisperse entity upon ultracentrifugation with a s0(20),w value of 8.6S. Upon polyacrylamide gel electrophoresis (PAGE), the enzyme migrated as a single band. The molecular weight of the native enzyme was 173,000 +/- 3,000, as determined by sedimentation equilibrium analysis, and that of the enzyme subunit was determined to be 50,000 +/- 1,500 by sodium dodecyl sulfate (SDS)-PAGE. Cross-linking experiments using dimethyl suberimidate followed by SDS-PAGE indicated that the native enzyme was composed of four subunits with identical molecular weight. The amino acid composition of the enzyme was determined.

Sodium effect of growth on aspartate and genetic analysis of a Bacillus subtilis mutant with high aspartase activity.

Most strains of Bacillus subtilis, dervied from the 168 (Marburg) strain, grow slowly on aspartate as sole carbon source. We isolated a mutant (aspH) that grows rapidly on aspartate because it produces aspartase constitutively. Thus, aspartase is needed for rapid growth on aspartate, whereas aspartate-alpha-ketoglutarate aminotransferase is not needed, as was demonstrated by a mutant lacking that enzyme activity. By two--and three-factor crosses using PBSl transduction, the aspH mutation was located between the aroD and the lys markers of the genetic map. Although sodium ions do not affect growth on glucose or L-malate, they specifically stimulate growth on aspartate in both the parent and the aspH mutant strains. Enzyme activities of crude aspartase and fumarase and of purified aspartase do not increase in the presence of sodium. These results show that stimulation by sodium involves some reaction other than the enzymes catabolizing aspartate. The ease of purification from the aspH strain and the stability of aspartase suggest that the B. subtilis enzyme is particularly useful for aspartate determinations.