Purification and some properties of NAD-degrading purine nucleosidase from Aspergillus niger.

An enzyme which degrades NAD at the adenine-ribose linkage has been purified from the mycelial extract of Aspergillus niger. NADP, deamido-NAD, and purine nucleosides and nucleotides were also susceptible to the hydrolytic cleavage. Pyrimidine- and nicotinamide-ribose linkages were not attacked. The substrate specificity showed that the enzyme may be classified as a N-ribosyl-purine ribohydrolase (EC 3.2.2.1). The enzyme had a maximum activity in the pH range of 4.0-4.5 toward NAD. The Km values for NAD, 5'-AMP, and inosine were 3.0, 2.9 and 1.6mM respectively.     

Isolation,purification and properties of tannase from Aspergillus niger van Tieghem

Tannase (tannin acyl hydrolase E.C. 3.1.1.20) has been isolated from Aspergillus niger van Tieghem and purified 29-fold. The enzyme had a temperature optimum of 60^°C, pH optimum of 6.0 with a second peak at pH 4.5, K_m of 0.20mM and V_max of 5.0mol min^–1 mg^–1protein.     

Aspergillus niger alpha-N-acetylgalactosaminidase: isolation, purification and properties

alpha-N-acetylgalactosaminidase has been isolated from liquid culture of micromycete Aspergillus niger and purified 600 times by ammonium sulphate precipitation followed by ion exchange and gel-filtration chromatography on TSK-gels with specific activity 10.5 U/mg of protein. The preparation was homogenic: its molecular mass by the data of gel-filtration on Sepharose 6B was 430 kDa, on PAAGE in the system of DDSNa--70 kDa. That gives every reason to suppose oligomeric structure of the enzyme molecule. The carbohydrate component, including mannose, galactose, glucosamine and two nonidentified hexosamines was observed in alpha-N-acetylgalactosaminidase. Thermo- and pH- optima were 60 degrees C and pH 3.5, respectively. The enzyme was thermo- and pH-stable, resistant in storage. The enzyme was found to exhibit strict specificity in respect ofglycon. It was shown that enzyme was competitively inhibited by substrate and reaction product. Km and Vmax with respect to nitrophenyl substrate were 1.25 mM and 10.5 mkmole/min/mg of protein. The activity of glycosidase tested was independent of the presence of metal ions. The presence of carboxylic group of C-terminal aminoacid and imidazol group of hystidine in active centre of molecule was established. A number of natural and synthetic substrates were able to activate (50-200%) production of A. niger alpha-N-acetylgalactosaminidase.     

Partial purification and some properties of gamma-glutamyl transpeptidase from human bile.

1. Gamma-Glutamyl transpepetidase ((5-glutamyl)-peptide: amino acid 5-glutamyltransferase, EC 2.3.2.2) from human bile has been partially purified using protamine sulphate treatment, DEAE-cellulose chromatography and Sephadex G-200 filtration. The procedure resulted in 150-fold increase in specific acitivity with a 37% yield. 2. The partially purified enzyme showed a single zone of enzyme activity by polyacrylamide gel electrophoresis and eluted in the inner volume of Sephadex G-200. 3. The enzyme had a pH optimum of 8.1 and Km of 1.52 mM using gamma-glutamyl p-nitroanilide as substrate. 4. The effects of cations and different gamma-glutamyl acceptors on the activity of the enzyme are reported. 5. As bile gamma-glutamyl transpeptidase appears to be soluble in the absence of detergents, it is suggested that bile may prove to be a useful source for further studies of the kinetic properties and physiological role of human gamma-glutamyl transpeptidase.     

Partial purification and some properties of lipoxygenase from Saccharomyces cerevisiae

A partially purified lipoxygenase extract was obtained from the yeast Saccharomyces cerevisiae by precipitation with solid (NH₄)SO₄ at 20% to 80% saturation. The enzyme had two pH optima, at pH 8.0 and 10.0, with respective apparent K _m values of 13 and 9.5 m. At both pH optima, the lipoxygenase demonstrated highest substrate specificity towards linoleic acid, followed by linolenic acid; although the enzyme had less specificity towards mono-linolein than di-linolein at pH 8.0, the reverse was true at pH 10.0.     

Partial purification and some properties of shikimate dehydrogenase from tomatoes

The partial purification of shikimate dehydrogenase (SDH) from tomato fruit was achieved by precipitation with ammonium sulphate, and chromatography on DEAE-cellulose and hydroxyapatite. The enzyme has a MW of 73000, shows an optimum at pH 9.1 and K_m values of 3.8 × 10^?5 M and 1.0 × 10^?5 M with shikimic acid and NADP as substrates. NADP could not be replaced by NAD. The tomato enzyme is competitively inhibited by protocatechuic acid with a K_i value of 7.7 × 10^?5 M. On the other hand, cinnamic acid derivatives and 2-hydroxybenzoic acid were ineffective. At 50° for 5 min the SDH is inactivated by 85%. The activity was inhibited by pCMB and N-ethylmaleimide, suggesting a requirement for SH groups. The inactivation plot of oxidation by pCMB was biphasic, and NADP decreased the reactivity of sulphydryl groups to the reagent. The activation energy was found to be 14.2kcal/mol. The properties of the SDH are discussed in relation to the enzymes from other sources.     

Partial purification and some properties of beta-phosphoglucomutase from Lactobacillus brevis

A phosphoglucomutase (beta-phosphoglucomutase) specific for beta-glucose 1-phosphate, which catalyzes the beta-glucose 1-phosphate:glucose 6-phosphate interconversion, was 560-fold purified from Lactobacillus brevis strain L6. The isoelectric point of beta-phosphoglucomutase was 3.8 and it had an apparent molecular weight of 29,000 estimated by gel chromatography. The enzyme required a divalent cation (Mn2+ greater than Mg2+ greater than Ni2+ greater than Co2+) and beta-glucose 1,6-bisphosphate for activity. The equilibrium constant Ke for the reaction beta-D-glucose 1-phosphate in equilibrium D-glucose 6-phosphate at 30 degrees C and pH 6.7 is 18.5. beta-phosphoglucomutase had a pH optimum between 6.3 and 6.8 and appeared to be quite specific: alpha-glucose 1-phosphate, alpha- or beta-galactose 1-phosphate and alpha- or beta-N-acetylglucosamine 1-phosphate did not substitute for beta-glucose 1-phosphate. Double reciprocal plots of the data from initial velocity studies at five beta-glucose 1-phosphate concentrations (10 to 100 microM) and four beta-glucose 1,6-bisphosphate concentrations (0.125 to 1.0 microM) showed that the apparent Michaelis constants for beta-glucose 1-phosphate and beta-glucose 1,6-bisphosphate were related to the concentrations of beta-glucose 1,6-bisphosphate and beta-glucose 1-phosphate, respectively, in such a way as to suggest a ping-pong mechanism. The same conclusion was obtained when substrate-velocity relationships were investigated at fixed ratio of both substrates: the Lineweaver-Burk plots showed linear lines and no parabolic ones. The "true" Km for beta-glucose 1-phosphate and beta-glucose 1,6-bisphosphate were found to be about 12 and 0.8 microM, respectively.     

Glycoprotein enzymes secreted by Aspergillus niger: purification and properties of alpha-glaactosidase.

An alpha-galactosidase (alpha-D-galactoside galactohydrolase [EC 3.2.1.22]) was purified to homogeneity from the culture filtrate of Aspergillus niger. The enzyme had an apparent molecular weight of 45,000 and was a glycoprotein. Radioactive enzyme was prepared by growing cells in [14C]fructose and this enzyme was used to prepare 14C-labeled glycopeptides. The glycopeptides emerged from Sephadex G-50 between stachyose and the glycopeptide from ovalbumin. Based on calibration of the column with various-sized dextran oligosaccharides, the glycopeptides appeared to have a molecular weight of 1,200 to 1,400. Analysis of the glycopeptide(s) indicated that it contained mannose and N-acetylglucosamine (GlcNAc) in an approximate ratio of 3 or 4 to 1. Assuming that there are two GlcNAc residues in the oligosaccharide and based on the molecular weight of the glycopeptide, the oligosaccharide probably contains eight to nine sugar residues. Alks probably attached to the protein by a GlcNAc leads to asparagine linkage. The purified alpha-galactosidase was most active on raffinose (Km = 5 x 10--4 M, Vmax = 3 mumol/min per mg of protein), but also showed good activity on p-nitrophenyl-alpha-D-galactoside ans somewhat less activity on stachyose and melibitol. The enzyme also hydrolyzed guar flour and locust bean gum, but did not attack the p-nitrophenyl glycosides of beta-galactose, alpha- or beta-glucose, or alpha- or beta-mannose.     

Purification and properties of a cellulase from Aspergillus niger.

A cellulolytic enzyme was isolated from a commercial cellulase preparation form Aspergillus niger. A yield of about 50mg of enzyme was obtained per 100g of commerial cellulase. The isolated enzyme was homogeneous in the ultracentrifuge at pH 4.0 and 8.0, and in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis but showed one major and two minor bands in disc gel electrophoresis. No carbohydrate was associated with the protein. Amino acid analysis revealed that the enzyme was rich in acidic and aromatic amino acids. Data from the amino acid composition and dodecyl sulphate/polyacrylamide-gel electrophoresis indicated a molecular weight of 26000. The purified enzyme was active towards CM-cellulose, but no activity towards either cellobiose or p-nitrophenyl beta-D-glucoside was detected under the assay conditions used. The pH optimum for the enzyme was pH 3.8-4.0, and it was stable at 25 degrees C over the range pH 1-9; maximum activity (at pH 4.0) was obtained at 45 degrees C. The cellulase was more stable to heat treatment at pH 8.0 than at 4.0. Kinetic studies gave pK values between 4.2 and 5.3 for groups involved in the enzyme-substrate complex.     

An efficient and optimized purification procedure for the superoxide dismutase from Aspergillus niger. Partial characterization of the purified enzyme

Cu, Zn-superoxide dismutase was isolated from Aspergillus niger mycelia, harvested at the mid-logarithmic growth phase. The purification scheme aimed at the optimization of the ethanol/chloroform extraction (Tsuchihashi extraction) through response surface methodology. Upon optimum extraction conditions, it was possible to obtain electrophoretically pure enzyme preparations, by the application of one step anion exchange chromatography. The enzyme yield of this simple purification procedure was above 75% while the specific activity of the final preparation was among the highest reported for eucariotic microorganisms. The purified enzyme exhibited similar physicochemical characteristics with other Aspergillus sp. superoxide dismutases revealing an apparent tetrameric structure with a subunit molecular weight of 19 kDa, and a pl of 5.95.This revised version was published online in October 2005 with corrections to the Cover Date.