Purification and preliminary characterization of L-asparaginase from Erwinia aroideae NRRL B-138.

L-Asparaginase (L-asparagine amidohydrolase EC 3.5.1.1) from Erwinia aroideae NRRL B-138 has been purified to apparent homogeneity by ammonium sulphate precipitation, chromatography on sulfopropyl-sephadex C-50 and sephadex G-200 with 22% recovery and 567-fold purification. The enzyme obtained from sulfopropyl-sephadex C-50 was unstable and lost activity within a few hours. Addition of glycerol helped in restoring the activity of the enzyme. The enzyme has an apparent molecular mass of approximately 155 kDa and has four subunits of identical molecular mass of approximately 38 kDa. The K(m) for L-asparagine is 2.8 x 10(-3) M. Enzyme shows optimal activity at 45 degrees C and pH 8.2. Energy of activation as determined from Arrhenius plot was 9.1 kcal/mol. Substrate L-asparagine and analogue L-glutamine, D-asparagine and 6 diazo-5-oxo-L-norleucine provide full protection to the enzyme against thermal denaturation.

Stabilization, purification, and characterization of glutamate synthase from Clostridium pasteurianum.

Clostridium pasteurianum possesses a high level of glutamate synthase (EC 1.4.1.14) activity and cell yield when grown on 4 mM ammonium chloride and molasses as the sole nitrogen and carbon sources, respectively. The enzyme activity is stabilized by addition of alpha-ketoglutarate, EDTA, and 2-mercaptoethanol. Ammonium sulfate precipitation and single-step combined gel and ion-exchange chromatography followed by fractional dialysis yield a homogeneous protein with 40% recovery of the glutamate synthase activity. The native enzyme (Mr congruent to 590,000) gives five different subunits (as dimers) upon SDS gel electrophoresis. The enzyme has been characterized for pH and temperature optimum, substrate specificity, Kmapp values, energy of activation, half-life, and thermal stabilization. Metal ions and citric acid cycle metabolites do not affect the enzyme activity. Glutamate synthase shows fluorescence maximum at 370 nm when excited at 280 nm. The fluorescence is quenched upon the addition of NADH. Spectroscopic examination of the enzyme gave absorption maximum at 280 and none at 380 and 440 nm, indicating the absence of iron and flavin. The absence of iron and flavin was also confirmed by atomic absorption, chemical analysis, and fluoroscopy, respectively. The C. pasteurianum enzyme differs from that of other aerobic bacterial sources.

Purification and characterization of glutamine synthetase from Clostridium pasteurianum.

Glutamine synthetase from Clostridium pasteurianum grown on molasses as the sole carbon source and ammonium chloride as the nitrogen source has been purified to homogeneity (45-fold) with 32% recovery. The procedure involves ammonium sulfate precipitation and chromatography on a combined Sepharose 4B/DEAE-Sephadex A-50 column. The purified enzyme being very unstable was stabilized by the addition of 25% (v/v) glycerol. The enzyme has an unusually high molecular weight of 1 X 10(6) and 20 subunits of Mr 50 000 each, as determined by gel filtration and sodium dodecyl sulfate gel electrophoresis, respectively. It has an absorption maximum at 280 nm and a fluorescence emission maximum at 380 nm when excited at 280 nm. Its substrate binding pattern as studied by fluorescence quenching studies is different from that of the Escherichia coli enzyme. Both the gamma-glutamyltransferase and synthetase activities reside in the same protein as the ratio of the two activities at each step of purification remains constant and the enzyme exhibits optimal transferase and synthetase activities at the same pH (7.2) and temperature (50 degrees C). The thermal stabilities of both activities were also similar, and decay of both the activities at 50 degrees C ran parallel. The enzyme shows stabilization by substrates, as L-glutamate, Mg2+, and ATP + Mg2+ protected both the synthetase and gamma-glutamyltransferase activities against thermal inactivation. Storage in 25% (v/v) glycerol enhanced the thermal stability of glutamine synthetase. Metal ion requirement and substrate specificity of the enzyme have been examined. Maximum synthetase activity occurs when [Mg2+]: [ATP] = 2. The Km app values are as follows (in parentheses): ATP (0.34 mM), NH2OH (0.4 mM in the synthetase reaction and 4.1 mM in the transferase reaction), glutamine (14.7 mM), ADP (3.8 X 10(-4) mM), arsenate (2.5 mM), and L-glutamate (3.4 mM, 22.2 mM). The enzyme exhibits negative cooperativity in the binding of glutamate. Amino acids such as L-serine, glycine, L-alanine, and L-aspartic acid inhibit the enzyme.

Chemical modification of liquefying alpha-amylase: role of tyrosine residues at its active center.

Liquefying alpha-amylase from Bacillus amyloliquefaciens was inactivated by treatment with tetranitromethane and N-acetylimidazole. The loss of activity occurred with modification of five tyrosine residues. Preincubation of the enzyme with either the substrate or the competitive inhibitor at saturating levels provided complete protection against inactivation. However, the presence of substrate/inhibitor in the reaction mixture protected only two of the five modifiable tyrosine residues, suggesting the involvement of only two tyrosine residues at the active center. This was confirmed when hydroxylamine treatment of the acetylated enzyme fully restored the enzymatic activity. Both nitration and acetylation increased the apparent Km of the enzyme for soluble starch, which indicated that the tyrosine residues are involved in substrate binding. Reduction of nitrotyrosine residues to aminotyrosine residues failed to restore the enzymatic activity. So, the loss of activity on modification of tyrosine residues was ascribed to conformational perturbances and not simply to the changes in the ionic character of tyrosine residues.

Clostridium pasteurianum glutamine synthetase mechanism. Evidence for active site tyrosine residues.

Preliminary chemical modification studies indicated the presence of tyrosine, carboxyl, arginine, histidine and the absence of serine and sulfhydryl residues at or near the active site of Clostridium pasteurianum glutamine synthetase. The conditions for tyrosine modification with tetranitromethane were optimized. The inactivation kinetics follow pseudo-first-order kinetics with respect to enzyme and second order with respect to modifier per active site. There was no inactivation at pH 6.5 suggesting the absence of thiol oxidation. The synthetase and transferase reactions followed the same pattern of inactivation on enzyme modification and both were equally protected by glutamate plus ATP. Thus tyrosine residues are present at the active site of the enzyme and are essential for both transferase and synthetase activities.

Carboxypeptidase a immobilization on activated styrene-maleic anhydride systems.

The copolymer styrene-maleic anhydride (SMA) was activated to various forms to create enzyme coupling groups. Carboxypeptidase A (CPA) was Immobilized on these supports to enhance their thermal and chemical stability. Immobilized enzyme retained 60-70% of the original activity. When kept at 60 degrees C, while free enzyme was deactivated within 30 min, the immobilized enzyme retained 40% of initial activity at the end of 3 h. The half-life of free enzyme was only 21 min, while for immobilized enzyme it was enhanced up to 3 h. Also, the immobilized enzyme could be repeatedly used over 50 times retaining almost 50% of original activity.

Mechanistic studies on carboxypeptidase A from goat pancreas: role of arginine residue at the active site.

Chemical modification of carboxypeptidase Ag1 from goat pancreas with phenylglyoxal or ninhydrin led to a loss of enzymatic activity. The inactivation by phenylglyoxal in 200 mM N-ethylmorpholine, 200 mM sodium chloride buffer, pH 8.0, or in 300 mM borate buffer, pH 8.0, followed pseudo-first-order kinetics at all concentrations of the modifier. The reaction order with respect to phenylglyoxal was 1.68 and 0.81 in 200 mM N-ethylmorpholine, 200 mM NaCl buffer and 300 mM borate buffer, pH 8.0, respectively, indicating modification of single arginine residue per mole of enzyme. The kinetic data were supported by amino acid analysis of modified enzyme, which also showed the modification of single arginine residue per mole of the enzyme. The modified enzyme had an absorption maximum at 250 nm, and quantification of the increase in absorbance showed modification of single arginine residue. Modification of arginine residue was protected by beta-phenylpropionic acid, thus suggesting involvement of an arginine residue at or near the active site of the enzyme.

Active site studies on Bacillus amyloliquefaciens alpha-amylase (I).

Modification of liquefying alpha-amylase by diethylpyrocarbonate or its photo-oxidation in the presence of rose bengal caused rapid loss of enzyme activity. The photo-oxidation followed pseudo-first-order kinetics giving maximal value at pH 8.0. The photo-oxidized enzyme showed a characteristic increase in absorbance at 250 nm which was directly proportional to the extent of inactivation. Diethylpyrocarbonate at low concentration at pH 6.0 and 30 degrees C completely inactivated alpha-amylase. Inactivation followed pseudo-first-order kinetics. The reaction order with respect to inactivation by diethylpyrocarbonate-modified enzyme showed increased absorbance at 240 nm which was reversed completely upon treatment with NH2OH at 30 degrees C for 16 hr. Calculating the histidine residues being modified from the increase in absorbance at 240 nm showed that three residues were ethoxyformylated on treatment with diethylpyrocarbonate, of which only one was found at the active site. Substrate and competitive inhibitor protects the enzyme against both, photo-oxidation, and modification by diethylpyrocarbonate, confirming that histidine plays an essential role at the alpha-amylase active site.

An active center tryptophan residue in liquefying alpha-amylase from Bacillus amyloliquefaciens.

Liquefying alpha-amylase from Bacillus amyloliquefaciens was inactivated on treatment with N-bromosuccinamide. Preincubation of the enzyme with either of the substrate, or competitive inhibitor provided significant protection against inactivation. The relationship between activity loss and the number of tryptophan residues modified, as well as presence of substrate/inhibitor in the reaction mixture, demonstrated that only one of three modifiable tryptophan residues is at or near the active center. The apparent Km of the modified enzyme for soluble starch increased manifold, thus implicating the sensitive tryptophan residue in the substrate binding region of the enzyme.

Role of metal ions in goat carboxypeptidase A-catalysed hydrolysis of acyl peptides.

The carboxypeptidase A purified from goat pancreas has been found to have a molecular weight of 34,600 +/- 300. The enzyme is a zinc-protein and the molar ratio of zinc to enzyme protein is 1:1. Removal of zinc yields an inactive apocarboxypeptidase A. The loss of activity of the native enzyme and restoration of the activity of the apoenzyme run parallel with the zinc content of the protein, thus showing the essentiality of zinc for the enzymatic activity. The exact role of zinc in the enzyme catalysed hydrolysis of the acylpeptides has been investigated after preparing metallo proteins by substituting the zinc of carboxypeptidase A with Co2+, Mn2+, Ni2+, Fe2+, Cd2+, Hg2+, and Cu2+ and determining the kinetic parameters of such metalloproteins. These studies indicate that the metal ion is involved in both binding the substrate and polarising the peptide bond.

An active-site carboxyl group in liquefying alpha-amylase: specific chemical modification.

Bacillus amyloliquefaciens alpha-amylase activity is pH-dependent and the plot log (Vmax/Km) versus pH implicated a carboxyl group of aspartic acid/glutamic acid at the active site. Chemical modification of alpha-amylase with EDC confirmed this view. Further, analysis of inactivation kinetics showed that modification of a single carboxyl group led to complete loss of the enzymic activity.

Purification and characterisation of naphthalene oxygenase from Corynebacterium renale.

Naphthalene oxygenase has been purified 420-fold from naphthalene-adapted Corynebacterium renale. The purified enzyme was obtained in 32% yield and gave an apparent single band on polyacrylamide gel electrophoresis. It has a molecular weight of approximately 99,000 and contains two non-identical polypeptide chains of molecular weights 43,000 and 56,000. The enzyme requires molecular oxygen for its activity and gave a single products, cis-1,2-dihydroxy-1,2-dihydronaphthalene. The absorption spectrum of the enzyme protein shows a maximum at 278 nm and no absorption in the Soret region, indicating that it is non-heme protein. Absence of cytochrome P-450 was also confirmed when the protein, treated with CO, showed no absorption at 450 nm. The enzyme followed Michaelis-Menten kinetics with Km values of 2.9 mM and 1.42 mM for naphthalene and NADH respectively. It exhibited maximal activity at 30 degrees C and pH 6.4. Studies on the stoichiometry of the reaction showed that one molecule of oxygen is consumed for each molecule of NADH oxidised or product formed.