The pectic enzymes of Aspergillus niger. A second exopolygalacturonase

1. A second exopolygalacturonase was separated from a mycelial extract of Aspergillus niger with a 265-fold purification and a recovery of 1%. 2. Unlike the first exopolygalacturonase this enzyme showed no requirement for metal activators, nor was it inhibited by chelating agents. 3. The two exopolygalacturonases were also distinguished by their pH optima and stability. 4. The enzyme progressively removed the terminal galacturonic residues from alpha-(1-->4)-linked galacturonide chains, converting digalacturonic acid, trigalacturonic acid and tetragalacturonic acid into galacturonic acid. Galacturonic acid was also released from pectic acid but complete digestion was not achieved.     

Biosynthesis of cellulolytic enzymes by Aspergillus niger A.n. 33 and its selectants

The aim of the investigations was to obtain — from the parent strain Aspergillus niger A.n. 33 — selectants with an increased ability of cellulolytic enzymes biosynthesis. Own selection methods allowed to receive two selectants A.n. 33/2 and A.n. 33/20 characterized by enhanced activities of saccharifying cellulase (respectively 0.11 and 0.14 FPU/cm³), endo-beta-1,4-glucanase (15.4 and 21.8 U/cm³) and cellobiase (0.6 and 1.4 IU/cm³) as compared with the parent strain (FPA — 0.09 IU, CMC — 8.2 U and CB — 0.1 IU/cm³). Moreover, the selectants differed in shape and size of conidial heads, in shape and colour of conidia, as well as in structure and shape of hyphae. Enzyme preparations obtained after ultrafiltration of liquid cultures were characterized by following activites: FPA-4–16 IU, CMC — 900–1800 U, CB — 60–120 IU and xylanase — 250–280 IU/cm³.     

Intracellular location of enzymes involved in citrate production by Aspergillus niger.

The intracellular distribution and maximal activities of nine enzymes involved in the biosynthesis and degradation of citric acid in Aspergillus niger were determined under conditions of growth and of citric acid production. Under these conditions the intracellular location of the enzymes in most cases resembled that described for other filamentous fungi. Pyruvate carboxylase was found predominantly or exclusively in the cytosol. A single isoenzyme of NADP-isocitrate dehydrogenase was present, which appeared to be localised in the mitochondrion. No significant differences in maximal enzyme activities were observed except for NADP-isocitrate dehydrogenase, which showed decreased activity in production-phase mycelia. The results obtained support the scheme proposed by C.P. Kubicek for the intracellular organisation of citric acid formation but provide little evidence that this process is controlled at the level of the biosynthesis of any of the enzymes examined here.     

Ochratoxin A production by strains of Aspergillus niger var. niger.

In a survey of the occurrence of ochratoxin A (OA)-positive strains isolated from feedstuffs, two of the 19 isolates of Aspergillus niger var. niger that were studied produced OA in 2% yeast extract-15% sucrose broth and in corn cultures. This is the first report of production of OA by this species.     

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.     

Catalytic degradation of amygdalin by extracellular enzymes from Aspergillus niger

Amygdalin is a controversial anti-tumor natural product that has been used as an alternative cancer drug for many years. The anti-tumor mechanism and metabolism of amygdalin have been the focus of many studies. However, previous studies by our group demonstrated that amygdalin itself has no anti-tumor activity, but rather the active ingredients were determined to be amygdalin degradation products. To screen novel drugs with anti-tumor activity, the extracellular enzymes from Aspergillus niger were used to degrade amygdalin. Within 4 h of the catalytic reaction at 37°, amygdalin was rapidly degraded into four products. The products were then extracted and purified by column chromatography. By comparing the HPLC chromatograms, ¹H NMR, ¹³C NMR and MS data, the products were identified as mandelonitrile, prunasin, benzaldehyde and phenyl-(3,4,5-trihydroxy-6-methyl-tetrahydro-pyran-2-yloxy)-acetonitrile (PTMT), a novel hydroxyl derivative of prunasin. Furthermore, pharmacology studies of these compounds demonstrated that 10 mg/kg of PTMT significantly suppressed the growth of S-18 tumor cells within 11 days in a concentration-dependent manner.     

Studies in pectic enzymes of parasitic fungi VI. Factors affecting the secretion of pectic enzymes byAlternaria tenuis

The effects of various cultural factors on the secretion of three pectic enzymes (PP, PG and PE) byAlternaria tenuis on synthetic media were studied. The results can be summarised as follow:

1. 1.
   No definite correlation between the secretion of pectic enzymes, pH of the medium and the growth of the fungus was observed.     
   
   

Adenosine nucleotides and ATP synthesizing enzymes in conidia of Aspergillus niger

We have examined the effects of the enzyme inhibitors 2,4,6-trinitrobenzene sulfonic acid (TNBS) and 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) on ethylene and CO₂ production in apple and tomato fruit discs. In the past these inhibitors have been used to inhibit membrane bound enzyme systems in various animal tissues. The amino reactive inhibitor TNBS was shown to decrease ethylene production in tomato discs without affecting rates of respiration; similar results were obtained with apple. The effects of the sulfhydryl reactive inhibitor DTNB were not as clearcut as TNBS. There was little effect of DTNB on ethylene production in tomato discs, however, in apple discs ethylene production was significantly reduced. DTNB also reduced the respiration rate in apple discs, although not to the same extent as ethylene production. The inhibition of DTNB was reversed by a brief treatment with dithioerythritol. The results indicate that ethylene production takes place at the cell surface.     

A new method for immobilization of pectic enzymes

Summary Polygalacturonase and pectinesterase have been successfully immobilized on gamma alumina by activation of the support with glutaraldehyde at pH 3.0. The half life of the enzymes increased by four and two fold compared to the immobilization on gamma alumina without activation.     

Hydrolysis and transformation of cellulose with Aspergillus niger IBT-90 enzymes

Hydrolysis and transformation of Fibrenier cellulose (USA) with enzymes from Aspergillus niger IBT-90 was studied. The process was performed at 50°C and pH 4.8 for 24 h using an enzyme complex either as a properly diluted culture filtrate or as a mixture of isolated and purified enzymes from A.niger IBT-90. In the latter experiments, enzyme-substrate ratios expressed as units of activity per 1 g of cellulose were as follows: endoglucanase E₁ and E₂, 40; β-glucosidase, 40 and cellobio-hydrolase, 2. Cellulose concentration was 5%. It was proved that the crude celluloytic complex from A. niger IBT-90 exhibits higher efficiency in the decomposition of cellulose in comparison to the mixture of enzymes isolated from this complex, as was revealed in assays of reducing sugars and determinations of light transmission throughout cellulose fibres using a computer analysis of the microscopic image. Comparison of both the endoglucanases E₁ and E₂ showed that the first enzyme is more active against cellulose. It liberated more reducing sugars and caused more significant decomposition of fibres. The predominant effect of the endoglucanase E₂ was a smoothing of the fibre surface. The cellobiohydrolase split a cellulose fibre into many short fibres.     

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.     

Relationship between citric acid production and accumulation of phytate-degrading enzymes in Aspergillus niger mycelia.

The ability of eight strains of Aspergillus niger to produce citric acid by the solid surface method were found to correlate with their capabilities to synthesize intracellular enzymes which degrade phytates (phytase and acid phosphatase). Another high correlation was observed between phytase and acid phosphatase activities bound to the cell walls of mycelia.     

Recovery of pectolytic enzymes produced by solid state culture of Aspergillus niger

Pectinases are enzymes with a wide range of applications in the food and drink industries. In the present work, the extraction of pectinases produced by Aspergillus niger in a solid state fermentation system was investigated. The purpose was to reduce enzyme losses in the fermented solids and at the same time obtain a crude extract as concentrated as possible. Initially the performances of stirred tank and fixed bed extractors were compared. Polygalacturonase activity and viscosity reducing capacity obtained in the stirred tank system were 105% and 15% superior, respectively. Repeated extractions and multiple stage countercurrent extraction were studied, employing stirred tanks. It was possible to observe that three stages were enough for total recovery of the enzymes contained in the solids. The final enzyme extract obtained by counter-current extraction with three stages showed a polygalacturonase activity 81% higher than the one obtained by one-stage extraction.