Purification and characterization of methylamine oxidase induced in Aspergillus niger AKU 3302

Crude extract of Aspergillus niger AKU 3302 mycelia incubated with methylamine showed a single amine oxidase activity band in a developed polyacrylamide gel that weakly cross-reacted with the antibody against a copper/topa quinone-containing amine oxidase (AO-II) from the same strain induced by n-butylamine. Since the organism cannot grow on methylamine and the already known quinoprotein amine oxidases of the organism cannot catalyze oxidation of methylamine, the organism was forced to produce another enzyme that could oxidize methylamine when the mycelia were incubated with methylamine. The enzyme was separated and purified from the already known two quinoprotein amine oxidases formed in the same mycelia. The purified enzyme showed a sharp symmetric sedimentation peak in analytical ultracentrifugation showing S20,w0 of 6.5s. The molecular mass of 133 kDa estimated by gel chromatography and 66.6 kDa found by SDS-PAGE confirmed the dimeric structure of the enzyme. The purified enzyme was pink in color with an absorption maximum at 494 nm. The enzyme readily oxidized methylamine, n-hexylamine, and n-butylamine, but not benzylamine, histamine, or tyramine, favorite substrates for the already known two quinoprotein amine oxidases. Inactivation by carbonyl reagents and copper chelators suggested the presence of a copper/topa quinone cofactor. Spectrophotometric titration by p-nitrophenylhydrazine showed one reactive carbonyl group per subunit and redox-cyclic quinone staining confirmed the presence of a quinone cofactor. pH-dependent shift of the absorption spectrum of the enzyme-p-nitrophenylhydrazone (469 nm at neutral to 577 nm at alkaline pH) supported the identity of the cofactor with topaquinone. Nothern blot analysis indicated that the methylamine oxidase encoding gene is largely different from the already known amine oxidase in the organism.     

Amino acid sequence of cytochrome c fromAspergillus niger

Cytochrome c fromAspergillus niger consists of two forms, a major one (80%) with 111 amino acid residues and a minor one (20%) with 108 residues, missing the three N-terminal residues of the major one. The primary sequence ofA. niger cytochrome c was determined by standard spinning-cup Edman degradation of purified peptides and of pairs of peptides, from which the desired sequence was readily deduced by subtraction of common sequencies. Except for the extension and some variability at the N-terminal sequence, theA. niger protein conforms well with other cytochrome c structures.     

Regioselectivity enhancement by partial purification of lipase fromAspergillus niger

Lipase fromAspergillus niger was partially purified by a single step of ion-exchange chromatography and two major fractions I and II could be obtained. Two substrates, peracetylated 1-methyl and 1-thioethyl -D-glucopyranosides, were deacetylated by fraction II to give 3-OH derivatives as sole products with very high yield, respectively.     

Gene organization and molecular modeling of copper amine oxidase from Aspergillus niger: re-evaluation of the cofactor structure

Amine oxidase AO-I from Aspergillus niger AKU 3302 has been reported to contain topa quinone (TPQ) as a cofactor; however, analysis of the p-nitrophenylhydrazine-derivatized enzyme and purified active site peptides showed the presence of a carboxylate ester linkage of TPQ to a glutamate. The catalytic functionality of such a cross-linked cofactor has recently been shown unlikely by spectroscopic and voltammetric studies on synthesized model compounds. We have obtained resonance Raman spectra of native and substrate-reduced AO-I demonstrating that the catalytically active cofactor is unmodified TPQ. The primary structure of the enzyme (GenBank acc. no. U31869) has been reviewed and updated by repeated isolation and sequencing of AO-I cDNA. This allowed rectification of several errors that account for previously reported low homology to other amine oxidases in the regions around copper binding histididyl residues. The results were confirmed by cloning the ao-1 structural gene (GenBank acc. no. AF362473). Analysis of the gene 5'-upstream region of the gene revealed potential binding sites for an analog of NIT2, the nitrogen metabolism regulatory protein found in Neurospora crassa and other fungi. The molecular structure of AO-I was modeled by a comparative method using published crystal structures of amine oxidases as templates.     

Thermal stability of the multiple charge isoforms of inulase fromAspergillus niger

Summary The three fractions of inulase differing in isoelectric pH were isolated from a strain ofAspergillus niger by isoelectric focussing and characterized in their temperature stability. These fractions had distinguishable thermal stability. Among them, one fraction (pI 5.2) having main activity showed a high thermal stability comparable to that of the Novozyme 230 (Novo A/S, Denmark) at 60°C.     

Purification and properties of two forms of glucoamylase fromAspergillus niger

A. niger produced α-glucosidase, α-amylase and two forms of glucoamylase when grown in a liquid medium containing raw tapioca starch as the carbon source. The glucoamylases, which formed the dominant components of amylolytic activity manifested by the organism, were purified to homogeneity by ammonium sulfate precipitation, ion-exchange and two cycles of gel filtration chromatography. The purified enzymes, designated GA1 and GA2, a raw starch digesting glucoamylase, were found to have molar masses of 74 and 96 kDa and isoelectric points of 3.8 and 3.95, respectively. The enzymes were found to have pH optimum of 4.2 and 4.5 for GA1 and GA2, respectively, and were both stable in a pH range of 3.5–9.0. Both enzymes were thermophilic in nature with temperature optimum of 60 and 65°C, respectively, and were stable for 1 h at temperatures of up to 60°C. The kinetic parametersK _m andV showed that with both enzymes the branched substrates, starch and amylopectin, were more efficiently hydrolyzed compared to amylose. GA2, the more active of the two glucoamylases produced, was approximately six to thirteen times more active towards raw starches compared to GA1.     

Stability and identification of active-site residues of carboxymethylcellulases fromAspergillus niger andCellulomonas biazotea

Determination of the apparent pK _a's of purified carboxymethylcellulases fromAspergillus niger andCellulomonas biazotea at different temperatures and in the presence of dioxane indicated two side chain carboxyl groups which controlled the limiting rate in both organisms. The thermostability of both enzymes slightly decreased with increasing pH from 5 to 7.5 but was unaffected in the presence of 0.5 mmol/L Mn²⁺. The CMCase fromC. biazotea had an activation energy of 35 kJ/mol and a half-life of 89 min in the presence of 8 mol/L urea at 40°C. The half-life of CMCase fromA. niger in 8 mol/L urea and at 37°C was 125 min as determined by a 0–9 mol/L transverse urea gradient PAGE. The CMCases fromA. niger andC. biazotea had the same thermostabilities in the absence of CMC although the enzyme from the former was more thermostable in the presence of the substrate. The CMCase fromA. niger was also more efficient in hydrolyzing CMC than the enzyme fromC. biazotea.     

Some characteristics of a raw starch digestion inhibitory factor fromAspergillus niger

Summary The effect of an inhibitory factor (IF) fromAspergillus niger 19 on raw starch digestion by pure glucoamylase I of blackAspergillus, pure glucoamylae ofRhizopus niveus, bacterial -amylase, fungal -amylase and various combination was investigated. The IF caused higher inhibition of raw starch hydrolysis by the combined action of glucoamylase and fungal -amylase than of hydrolysis by the individual enzymes. A protein moiety of IF might play an active part in this inhibition phenomenon. The IF was bound to starch granules, preventing hydrolysis by the enzymes, and caused decreased raw starch hydrolysis yields.     

Structure and stability of glucoamylase II fromAspergillus niger: A circular dichroism study

Glucoamylase II (EC 3.2.1.3) fromAspergillus niger has 31 % α-helix, 36 %Β- structure and rest aperiodic structure at pH 4.8 as analysed by the method of Provencher and Glockner (1981,Biochemistry, 20,33). In the near ultra-violet circular dichroism spectrum the enzyme exhibits peaks at 304, 289, 282 and 257 nm and troughs at 285, 277 and 265 nm respectively. The enzyme activity and structure showed greater stability at pH 4.8 than at pH 7.0, were highly sensitive to alkaline pH but less sensitive to acid pH values. The enzyme retained most of its catalytic activity and structure even on partial removal of carbohydrate moieties by periodate treatment but was less stable at higher temperatures and storage at 30‡C. Reduction of the periodate treated enzyme did not reverse the loss of stability. Binding of the synthetic substrate,p-nitrophenyl-α-D-glucoside, perturbed the environment around aromatic amino acids and caused a decrease in the ordered structure.     

The primary structure of Aspergillus niger acid proteinase A

The complete amino acid sequence of the acid proteinase A, a non-pepsin type acid proteinase from the fungus Aspergillus niger var. macrosporus, was determined by protein sequencing. The enzyme was first dissociated at pH 8.5 into a light (L) chain and a heavy (H) chain, and the L chain was sequenced completely. Further sequencing was performed with the reduced and pyridylethylated or aminoethylated derivative of the whole protein, using peptides obtained by digestions with Staphylococcus aureus V8 protease, trypsin, chymotrypsin, and lysylendopeptidase. The location of the two disulfide bonds was determined by analysis of cystine-containing peptides obtained from a chymotryptic digest of the unmodified protein. These results established that the protein consists of a 39-residue L chain and a 173-residue H chain that associate noncovalently to form the native enzyme of 212 residues (Mr 22,265). This is, to our knowledge, the first time that such a protein with a rather short peptide chain associated noncovalently has been found. No sequence homology is found with other acid or aspartic proteinases, except for Scytalidium lignicolum acid proteinase B, an enzyme unrelated to pepsin by sequence, which has about 50% identity with the present enzyme. These two enzymes, however, are remarkably different from each other in some structural features.     

Purification and the effect of manganese ions on the activity of carboxymethylcellulases fromAspergillus niger andCellulomonas biazotea

Carboxymethylcellulases (CMCases) fromAspergillus niger andCellulomonas biazotea were purified by a combination of ammonium sulfate precipitation, anion-exchange and gel-filtration chromatography with a 12- and 9-fold increase in the purification factor. The native and subunit molar mass of CMCase fromA. niger were 40 and 25–57 kDa, respectively, while those fromC. biazotea were 23 and 20–30 kDa, respectively. Low concentrations of Mn²⁺ activated the enzymes from both organisms (mixed activation) with apparent activation constants of 0.80 and 0.45 mmol/L of CMCases fromA. niger andC. biazotea, respectively, while at higher CMC concentrations Mn²⁺ inhibited the enzymes (mixed and partial uncompetitive inhibition). The reason for this complex behavior is that more than one Mn²⁺ bind to the same enzyme form with the apparent average inhibition constants of 2.7 and 1.3 mmol/L for CMCases fromA. niger andC. biazotea, respectively.     

Mapping the polysaccharide degradation potential of Aspergillus niger

ABSTRACT: BACKGROUND: The degradation of plant materials by enzymes is an industry of increasing importance. For sustainable production of second generation biofuels and other products of industrial biotechnology, efficient degradation of non-edible plant polysaccharides such as hemicellulose is required. For each type of hemicellulose, a complex mixture of enzymes is required for complete conversion to fermentable monosaccharides. In plant-biomass degrading fungi, these enzymes are regulated and released by complex regulatory structures. In this study, we present a methodology for evaluating the potential of a given fungus for polysaccharide degradation. RESULTS: Through the compilation of information from 203 articles, we have systematized knowledge on the structure and degradation of 16 major types of plant polysaccharides to form a graphical overview. As a case example, we have combined this with a list of 188 genes coding for carbohydrate-active enzymes from Aspergillus niger, thus forming an analysis framework, which can be queried. Combination of this information network with gene expression analysis on mono- and polysaccharide substrates has allowed elucidation of concerted gene expression from this organism. One such example is the identification of a full set of extracellular polysaccharide-acting genes for the degradation of oat spelt xylan. CONCLUSIONS: The mapping of plant polysaccharide structures along with the corresponding enzymatic activities is a powerful framework for expression analysis of carbohydrate-active enzymes. Applying this network-based approach, we provide the first genome-scale characterization of all genes coding for carbohydrate-active enzymes identified in A. niger.     

Effects of carbohydrate depletion on the structure, stability and activity of glucose oxidase from Aspergillus niger

Glucose oxidase from Aspergillus niger was purified to homogeneity by hydrophobic interaction and ion-exchange chromatography. Approx. 95% of the carbohydrate moiety was cleaved from the protein by incubation of glucose oxidase with endoglycosidase H and alpha-mannosidase. Cleavage of the carbohydrate moiety effected a 24-30% decrease in the molecular weight and a reduction in the number of isoforms of glucose oxidase. No significant changes were observed in the circular dichroism spectra of the deglycosylated enzyme. Other properties, such as thermal stability, pH and temperature optima of glucose oxidase activity and substrate specificity were not affected. However, removal of the carbohydrate moiety marginally affected the kinetics of glucose oxidation and stability at low pH. From these results it appears that the carbohydrate chain of glucose oxidase does not contribute significantly to the structure, stability and activity of glucose oxidase.     

Reductive trapping of substrate to methylamine oxidase from Arthrobacter P1

Methylamine oxidase (EC 1.4.3.6) from Arthrobacter P1 was inactivated by NaCNBH3 in the presence of [14C]benzylamine, leading to the incorporation of 1 mol of radiolabeled substrate/mol of enzyme subunit at complete inactivation. By contrast, no labeling of enzyme was observed using [3H]NaCNBH3 as reductant. These results are analogous to those previously reported for the eukaryotic enzyme, bovine serum plasma amine oxidase [(1987) J. Biol. Chem. 262, 962-965]. The observed pattern of labeling is consistent with the presence of dicarbonyl cofactor at the active site of methylamine oxidase. Further, these studies suggest that our reductive trapping technique, in which the pattern of radiolabeling of an enzyme is compared using C-14 substrate vs tritiated reductant, may serve as a general assay for covalently bound dicarbonyl structures.     

The primary structure of D-amino acid oxidase from Rhodotorula gracilis

Summary The amino acid sequence of D-amino acid oxidase from Rhodotorula gracilis was determined by automated Edman degradation of peptides generated by enzymatic and chemical cleavage. The enzyme monomer contains 368 amino acid residues and its sequence is homologous to that of other known D-amino acid oxidases. Six highly conserved regions appear to have a specific role in binding of coenzyme FAD, in active site topology and in peroxisomal targeting. Moreover, Rhodotorula gracilis D-amino acid oxidase contains a region with a cluster of basic amino acids, probably exposed to solvent, which is absent in other D-amino acid oxidases.     

The primary structure of the flavoprotein D-aspartate oxidase from beef kidney.

The complete primary structure of the peroxisomal flavoenzyme D-aspartate oxidase from beef kidney has been determined by analyses of the peptides obtained through fragmentation of the carboxymethylated protein with trypsin, CNBr, heptafluorobutyric acid/CNBr and Staphylococcus aureus V8 protease. The protein consists of a single polypeptide of 338 residues, accounting for a M(r) of 37,305 for the apoprotein. A form of the enzyme lacking Lys-338 and therefore ending with Pro-337 has been detected. The N-terminal residue is blocked. Seven cysteines and no disulfide bridges are present. Residue 228 can be either Ile or Val. Thus, D-aspartate oxidase presents two types of heterogeneity in the polypeptide chain in addition to the one already described concerning the possible content of FAD or 6-hydroxyflavin adenine dinucleotide. Comparison of the primary structure of D-aspartate oxidase with other known sequences reveals that D-aspartate oxidase is homologous with D-amino acid oxidase (another flavo-oxidase) and does not present significant sequence similarities with any other protein, including flavoenzymes.