Enzymatic hydrolysis of p-nitroacetanilide: mechanistic studies of the aryl acylamidase from Pseudomonas fluorescens.

Aryl acylamidase (EC 3.1.5.13; AAA) catalyzes the hydrolysis of p-nitroacetanilide (PNAA) via the standard three-step mechanism of serine hydrolases: binding of substrate (K(s)), acylation of active-site serine (k(acyl)), and hydrolytic deacylation (k(deacyl)). Key mechanistic findings that emerged from this study include that (1) AAA requires a deprotonated base with a pK(a) of 8.3 for expression of full activity toward PNAA. Limiting values of kinetic parameters at high pH are k(c) = 7 s(-1), K(m) = 20 microM, and k(c)/K(m) = 340 000 M(-1) s(-1). (2) At pH 10, where all the isotope effects were conducted, k(c) is equally rate-limited by k(acyl) and k(deacyl). (3) The following isotope effects were determined: (D)()2(O)(k(c)/K(m)) = 1.7 +/- 0.2, (D)()2(O)k(c) = 3.5 +/- 0.3, and (beta)(D)(k(c)/K(m)) = 0.83 +/- 0.04, (beta)(D)k(c) = 0.96 +/- 0.01. These values, together with proton inventories for k(c)/K(m) and k(c), suggest the following mechanism: (i) The initial binding of substrate to enzyme to form the Michaelis complex is accompanied by solvation changes that generate solvent deuterium isotope effects originating from hydrogen ion fractionation at multiple sites on the enzyme surface. (ii) From within the Michaelis complex, the active site serine attacks the carbonyl carbon of PNAA with general-base catalysis to form a substantially tetrahedral transition state enroute to the acyl-enzyme. (iii) Finally, deacylation occurs through a process involving a rate-limiting solvent isotope effect, generating conformational change of the acyl-enzyme that positions the carbonyl bond in a polarizing environment that is optimal for attack by water.     

Purification and characterization of aryl acylamidase from Nocardia globerula

Aryl acylamidase was purified from an extract of N-acetyl-o-toluidine-induced cells of Nocardia globerula IFO 13510 in ten steps. The purified enzyme appeared to be homogeneous from analysis by polyacrylamide gel electrophoresis. The enzyme has a molecular mass of approximately 126 kDa and consists of two subunits which are identical in molecular mass. The purified enzyme catalyzed the hydrolysis of N-acetyl-o-toluidine to o-toluidine and acetic acid at a rate of 47.7 mumol.min-1.mg-1 at 35 degrees C. It also catalyzed the hydrolysis of various anilide derivatives and esters, as well as the transfer of an acetyl group to aniline as an acetyl acceptor. The purified enzyme was sensitive to thiol reagents such as HgCl2 and p-chloromercuribenzoate. The amino-terminal sequence (28 amino acid residues) of the enzyme was determined. Based on the substrate specificity of this enzyme, the pathway intermediates involved in the conversion of n-acetyl-o-toluidine to 4'-hydroxy-N-acetyl-o-toluidine are discussed.     

Purification and properties of pantohenase from Pseudomonas fluorescens.

Pantothenase (EC 3.5.1.22) from Pseudomonas fluorescens UK-1 was purified to homogeneity as judged by disc-gel electrophoresis and isoelectric focusing. The purification procedure consisted of four steps: DEAE-Sephadex chromatography, (NH4)2SO4 precipitation, hydroxyapatite chromatography and preparative polyacrylamide-gel electrophoresis. Gel filtration on Ultrogel AcA 34 was used to determine the molecular weight, and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to study the subunit molecular weight. The enzyme appeared to be composed of two subunits with mol.wts. of approx. 50000 each. The total mol.wt. of the enzyme was thus about 100000. The isoelectric point was 4.7 at 10 degrees C.     

Purification and some properties of intracellular esterase from Pseudomonas fluorescens

A novel esterase was found in Pseudomonas fluorescens cells and purified to homogeneity as determined by polyacrylamide gel electrophoresis. The esterase was extracted from the cells by freeze-thawing and hypotonic treatment. Purification was achieved by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-cellulose and benzylamine-agarose and then electrophoresis. The enzyme catalyzed the hydrolysis of methyl esters, such as methyl butyrate, but its hydrolyzing activity decreased with increase in the chain length of the alcohol moiety, and it did not catalyze the hydrolysis of triacylglycerols, such as triacetin. In contrast, the enzyme acted on various acyl residues in a series of methyl esters, such as dimethyl succinate, methyl methacrylate, and dimethyl malate. The optimum pH for activity of this enzyme with methyl butyrate was 7.0-8.5. The enzyme was inhibited by phenylmethylsulfonylfluoride. Its molecular weight was estimated as 48,000 by molecular sieve electrophoresis and gel filtration on Sephadex G-150.     

Purification and some properties of Pseudomonas fluorescens lipase

Lipase (triacylglycerol lipase, EC 3.1.1.3) has been purified from Pseudomonas fluorescens wild strain by chromatography on DEAE-cellulose and octyl-Sepharose CL-4B. The yield was 21% and the specific activity of the purified enzyme 4780 U/mg protein. It showed a Mr of about 45 x 10(4) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is active over a wide pH range and at 50-55 degrees C.     

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.     

Purification, crystallization and properties of triacylglycerol lipase from Pseudomonas fluorescens.

Triacylglycerol lipase of Pseudomonas fluorescens was purified from the crude enzyme by ammonium sulfate precipitation and chromatographies on Sephadex G-75 and DEAE-cellulose. The crystallization of the lipase was successfully carried out. The purified lipase was demonstrated to be homogenous on disc electrophoresis and its molecular weight was calculated to be 32 000 by gel filtration. The optimum pH for hydrolysis of sesame oil was 7.0. The enzyme was stable up to 40 degrees C under the condition of pH 7.0 for 30 min and had more than 80% of the remaining activity between pH 5.0--11.0 at 37 degrees C for 60 min. The lipase was strongly inhibited by iodine and partially inhibited by FeCl3 and N-bromosuccinimide, and showed the most activity on tricaproyglycerol, among the triacylglycerols used.     

Purification and characterization of adhesive exopolysaccharides from Pseudomonas putida and Pseudomonas fluorescens

In this study, the adhesive exopolysaccharides of strains of Pseudomonas putida and P. fluorescens, both isolated from freshwater epilithic communities, were examined with regard to their chemical composition, biosynthesis, and their role in adhesion. Electron microscopy showed that both strains were enrobed in fibrous glycocalyces and that these structures were involved in attachment of the cells to a solid surface and as structural matrices in the microcolony mode of growth. In batch culture experiments most of the extracellular polysaccharide of both strains was found to be soluble in the growth medium rather than being associated with bacterial cells. Exopolysaccharide was synthesized during all phases of growth, but when growth was limited by exhaustion of the carbon source, exopolysaccharide synthesis ceased whereas exopolysaccharide synthesis continued for some time after cessation of growth in nitrogen-limited cultures. Exopolysaccharide from both strains was isolated and purified. Pseudomonas putida synthesized an exopolysaccharide composed of glucose, galactose, and pyruvate in a ratio of 1:1:1; the P. fluorescens polymer contained glucose, galactose, and pyruvate in a ratio of 1:1:0.5, respectively. Polymers from both strains were acetylated to a variable degree.     

Purification and properties of a NAD-linked 1,2-propanediol dehydrogenase from propane-grown Pseudomonas fluorescens NRRL B-1244

NAD-dependent 1,2-propanediol dehydrogenase (EC 1.1.1.4) activity was detected in cell-free crude extracts of various propane-grown bacteria. The enzyme activity was much lower in 1-propanol-grown cells than in propane-grown cells of Pseudomonas fluorescens NRRL B-1244, indicating that the enzyme may be inducible by metabolites of propane subterminal oxidation. 1,2-Propanediol dehydrogenase was purified from propane-grown Ps. fluorescens NRRL B-1244. The purified enzyme fraction shows a single-protein band upon acrylamide gel electrophoresis and has a molecular weight of 760,000. It consists of 10 subunits of identical molecular weight (77,600). It oxidizes diols that possess either two adjacent hydroxy groups, or a hydroxy group with an adjacent carbonyl group. Primary and secondary alcohols are not oxidized. The pH and temperature optima for 1,2-propanediol dehydrogenase are 8.5 and 20-25 degrees C, respectively. The activation energy calculated is 5.76 kcal/mol. 1,2-Propanediol dehydrogenase does not catalyze the reduction of acetol or acetoin in the presence of NADH (reverse reaction). The Km values at 25 degrees C, pH 7.0, buffer solution for 1,2-propan1,2-propanediol dehydrogenase are 8.5 and 20-25 degrees C, respectively. The activation energy calculated is 5.76 kcal/mol. 1,2-Propanediol dehydrogenase does not catalyze the reduction of acetol or acetoin in the presence of NADH (reverse reaction). The Km values at 25 degrees C, pH 7.0, buffer solution for 1,2-propan1,2-propanediol dehydrogenase are 8.5 and 20-25 degrees C, respectively. The activation energy calculated is 5.76 kcal/mol. 1,2-Propanediol dehydrogenase does not catalyze the reduction of acetol or acetoin in the presence of NADH (reverse reaction). The Km values at 25 degrees C, pH 7.0, buffer solution for 1,2-propanediol and NAD are 2 X 10(-2) and 9 X 10(-5) M, respectively. The 1,2-propanediol dehydrogenase activity was inhibited by strong thiol reagents, but not by metal-chelating agents. The amino acid composition of the purified enzyme was determined. Antisera prepared against purified 1,2-propanediol dehydrogenase from propane-grown Ps. fluorescens NRRL B-1244 formed homologous precipitin bands with isofunctional enzymes derived from propane-grown Arthrobacter sp. NRRL B-11315, Nocardia paraffinica ATCC 21198, and Mycobacterium sp. P2y, but not from propane-grown Pseudomonas multivorans ATCC 17616 and Brevibacterium sp. ATCC 14649, or 1-propanol-grown Ps. fluorescens NRRL B-1244. Isofunctional enzymes derived from methane-grown methylotrophs also showed different immunological and catalytic properties.     

Some properties of mandelate racemase from Pseudomonas fluorescens

1. l-Mandelate dehydrogenase and mandelate racemase were partially purified from extracts of Pseudomonas fluorescens A-312 grown in media containing d-mandelate. 2. The activity of mandelate racemase, but not that of l-mandelate dehydrogenase, is greatly stimulated by Mg(2+), Mn(2+), Co(2+) and, though less effectively, by Ni(2+). Other metal ions are inactive or inhibitory. 3. Racemase activity is inhibited by phosphate, fluoride, pyrophosphate and EDTA. The inhibitions by pyrophosphate and EDTA are competitive with respect to the metal ion activator; those by phosphate and fluoride are competitive with respect to the substrate. 4. The addition of Mg(2+) diminishes the Michaelis constant of racemase. 5. The pH optimum of the racemase is at 7.8. The pH-activity curve of the dehydrogenase complex of enzymes has two peaks, at 7.0 and 8.2. 6. The enzymic racemization of d-mandelate is initially faster than that of l-mandelate. 7. The rates of oxidation of related substrates, catalysed by l-mandelate dehydrogenase, are in the decreasing order: l-p-hydroxymandelate; l-3,4-dihydroxymandelate; l-4-hydroxy-3-methoxymandelate. The racemase is active towards d-p-hydroxymandelate but inactive towards d-3,4-dihydroxymandelate and d-4-hydroxy-3-methoxymandelate. Since 4-hydroxy-3-methoxymandelate, and presumably also 3,4-dihydroxymandelate, arising from the metabolism of catechol-amines, have the d-configuration, the enzymes studied cannot be utilized for estimation of the last two acids in urine.     

A novel aryl acylamidase from Nocardia farcinica hydrolyses polyamide

An alkali stable polyamidase was isolated from a new strain of Nocardia farcinica. The enzyme consists of four subunits with a total molecular weight of 190 kDa. The polyamidase cleaved amide and ester bonds of water insoluble model substrates like adipic acid bishexylamide and bis(benzoyloxyethyl)terephthalate and hydrolyzed different soluble amides to the corresponding acid. Treatment of polyamide 6 with this amidase led to an increased hydrophilicity based on rising height and tensiometry measurements and evidence of surface hydrolysis of polyamide 6 is shown. In addition to amidase activity, the enzyme showed activity on p-nitrophenylbutyrate. On hexanoamide the amidase exhibited a K(m) value of 5.5 mM compared to 0.07 mM for p-nitroacetanilide. The polyamidase belongs to the amidase signature family and is closely related to aryl acylamidases from different strains/species of Nocardia and to the 6-aminohexanoate-cyclic dimer hydrolase (EI) from Arthrobacter sp. KI72.