Novel oligomannose-type sugar chains derived from glucose oxidase of Aspergillus niger.

The primary structure of the N-linked sugar chains of glucose oxidase from Aspergillus niger was investigated. These sugar chains were released from the polypeptide backbone by hydrazinolysis, and the reducing ends of the sugar chains were pyridylaminated. HPLC of the pyridylamino sugar chains with an amide-silica column showed at least seven sugar chain peaks. Chemical and exoglycosidase digestion and 400 lMHz H-NMR studies of the sugar chains of lower molecular weight showed that these were novel oligomannose-type sugar chains, (Man)5-7 (GlcNAc)2, with the structure: +/- Man alpha 1----3Man alpha 1----3(Man alpha 1----6)Man alpha 1----6(+/- Man alpha 1----3Man alpha 1---3)Man )Man beta 1----4GlcNAc beta 1----4GlcNAc.     

Purification and characterization of a beta-glucuronidase from Aspergillus niger.

A beta-glucuronidase from Pectinex Ultra SP-L, a commercial pectolytic enzyme preparation from Aspergillus niger, was purified 170-fold by ion-exchange chromatography and gel filtration. Apparent M(r) of the purified enzyme, estimated by denaturing gel electrophoresis and size-exclusion chromatography, were 68,000 and 71,000, respectively, indicating that the enzyme is a monomeric protein. It released uronic acids not only from p-nitrophenyl beta-glucosiduronic acid (PNP-GlcA) but also from acidic galactooligosaccharides carrying either beta-D-glucosyluronic or 4-O-methyl-beta-D-glucosyluronic residues at the nonreducing termini through beta-(1-->6)-glycosidic linkages. The enzyme exhibited a maximal activity toward these substrates at pH 3.0. A regioisomer, 3-O-beta-glucosyluronic acid-galactose, was unsusceptible to the enzyme. The enzyme did act on a polymer substrate, releasing uronic acid from the carbohydrate portion of a radish arabinogalactan-protein modified by treatment with fungal alpha-L-arabinofuranosidase. The enzyme produced acidic oligosaccharides by transglycosylation, catalyzing the transfer of uronic acid residues of PNP-GlcA and 6-O-beta-glucosyluronic acid-galactose to certain exogenous acceptor sugars such as Gal, N-acetylgalactosamine, Glc, and xylose.     

The effect of beta-glucuronidase and chitinase on the cell wall of Aspergillus niger and Aspergillus fumigatus.

The effects of beta-glucuronidase and chitinase have been tested on the hydrolysis of the cell walls of the economically important fungi, Aspergillus niger and Aspergillus fumigatus. The extent of wall hydrolysis was measured by assaying for total reducing sugars, N-acetyl sugars and protoplast production. Maximum reducing sugar release was attained after 40 min incubation, both with beta-glucuronidase supplemented with chitinase and beta-glucuronidase alone, whereas N-acetyl sugar release reached a maximum at 80 min incubation. beta-Glucuronidase was effective in releasing protoplasts from both species of Aspergillus. This release was enhanced by adding chitinase to the incubation medium at 0 and 20 min, but with addition at 60, 80 and 100 min increase in protoplast yield was much reduced. The results of re-incubation experiments with chitinase suggest that this enzyme may in some way be inhibited during the later stages of incubation. Pronase used in combination with beta-glucuronidase slightly enhanced protoplast release.     

Glycosylation of nascent polypeptide chains in Aspergillus niger

Polysomes were isolated from Aspergillus niger and were characterized on sucrose gradients in several ways. First, they were found to be susceptible to degradation by treatment with RNase or EDTA. Second, they were labeled after treating mycelia with short pulses of [³H]uridine or [³H]leucine prior to polysome isolation. Third, they were capable of stimulating incorporation of [³H]leucine into trichloroacetic acid-precipitable material in a chick reticulocyte cell-free protein-synthesizing system. When isolated [³H]leucine pulse-labeled polysomes were treated with either EDTA-RNase or puromycin, 80–90% of the radioactivity was released, indicating that only the nascent polypeptide chains were labeled. After exposing mycelia for 1 min to [¹⁴C]mannose, the polysomes were exclusively labeled, indicating that initial glycosylation takes place on nascent polypeptide chains. Preincubation of mycelia with 2-deoxyglucose followed by pulse-labeling with [³H]leucine and [¹⁴C]mannose showed that 2-deoxy-d-glucose inhibits both protein synthesis and glycosylation. However, similar preincubation with tunicamycin caused an 80% drop in [¹⁴C]mannose label in the polysomes, but only a 10–20% drop of [³H]leucine label, suggesting that glycosylation of nascent chains in A. niger involves an oligosaccharide-lipid intermediate, since it has been shown that tunicamycin inhibits the synthesis of such an intermediate. When isolated polysomes were placed into an in vitro glycosylating mixture containing Mn²⁺, GDP-[¹⁴C]mannose, and smooth membranes from A. niger nascent chains were labeled. This reaction was shown to be dependent on addition of polysomes to the mixture and was not inhibited by 2-deoxy-d-glucose or tunicamycin. Both in vivo and in vitro glycosylated nascent chains were found to have about the same size range, and so it is suggested that in vitro no new oligosaccharide chains were synthesized, but preexisting chains were extended.     

Structural studies on the O-glycosidically linked carbohydrate chains of glucoamylase G1 from Aspergillus niger

Glucoamylase G1 from Aspergillus niger contains an unusual type of carbohydrate-protein linkage, involving mannose O-glycosidically linked to serine and threonine. The majority of the neutral oligosaccharides of glucoamylase G1 are located in a region of about 70 amino acid residues which carries about 35 oligosaccharide units [(1983) Carlsberg Res. Commun. 48, 517-527]. Structural analysis was performed on the O-linked carbohydrates of a tryptic fragment from glucoamylase G1 comprising the segment characterized by a high degree of glycosylation. The carbohydrate structures released by trifluoroacetolysis were elucidated using sugar analysis, methylation analysis, mass spectrometry, chromium trioxide oxidation, digestion with alpha-mannosidase and 1H-NMR spectroscopy. The following structures could be identified. (formula; see text)     

The chemical mechanism of action of glucose oxidase from Aspergillus niger

Glucose oxidase from Aspergillus niger (EC 1.1.3.4) is able to catalyze the oxidation of -D-glucose with p-benzoquinone, methyl-1,4-benzoquinone, 1,2-naphthoquinone, 1,2-naphthoquinone-4-sulfonic acid, potassium ferricyanide, phenazine methosulfate, and 2,6-dichloroindophenol. In this work, the steady-state kinetic parameters, V ₁/K _B , for reactions of these substrates were collected from pH 2.5–8. Further, the molecular models of the enzyme's active site were constructed for the free enzyme in the oxidized state, the complex of -D-glucose with the oxidized enzyme, the complex of reduced enzyme with methyl-1,4-benzoquinone, the reduced enzyme plus 1,2-naphthoquinone-4-sulfonic acid, oxidized enzyme plus reduced 1,2-naphthoquinone-4-sulfonic acid (hydroquinone anion), and oxidized enzyme plus fully reduced 1,2-naphthoquinone-4-sulfonic acid.Combining the steady-state kinetic and structural data, it was concluded that Glu412 bound to His559, in the active site of enzyme, modulates powerfully its catalytic activity by affecting all the rate constants in the reductive and the oxidative half-reaction of the catalytic cycle. His516 is the catalytic base in the oxidative and the reductive part of the catalytic cycle. It was estimated that the pK _a of Glu412 (bound to His559) in the free reduced enzyme is 3.4, and the pK _a of His516 in the free reduced enzyme is 6.9.     

The chemical mechanism of action of glucose oxidase from Aspergillus niger

Glucose oxidase from Aspergillus niger (EC 1.1.3.4) is able to catalyze the oxidation of beta-D-glucose with p-benzoquinone, methyl-1,4-benzoquinone, 1,2-naphthoquinone, 1,2-naphthoquinone-4-sulfonic acid, potassium ferricyanide, phenazine methosulfate, and 2,6-dichloroindophenol. In this work, the steady-state kinetic parameters, V1/K(B), for reactions of these substrates were collected from pH 2.5-8. Further, the molecular models of the enzyme's active site were constructed for the free enzyme in the oxidized state, the complex of beta-D-glucose with the oxidized enzyme, the complex of reduced enzyme with methyl-1,4-benzoquinone, the reduced enzyme plus 1,2-naphthoquinone-4-sulfonic acid, oxidized enzyme plus reduced 1,2-naphthoquinone-4-sulfonic acid (hydroquinone anion), and oxidized enzyme plus fully reduced 1,2-naphthoquinone-4-sulfonic acid. Combining the steady-state kinetic and structural data, it was concluded that Glu412 bound to His559, in the active site of enzyme, modulates powerfully its catalytic activity by affecting all the rate constants in the reductive and the oxidative half-reaction of the catalytic cycle. His516 is the catalytic base in the oxidative and the reductive part of the catalytic cycle. It was estimated that the pKa of Glu412 (bound to His559) in the free reduced enzyme is 3.4, and the pKa of His516 in the free reduced enzyme is 6.9.     

The effect of the sugar source on citric acid production by Aspergillus niger

Summary Under otherwise identical fermentation conditions, the sugar source has been shown to have a marked effect on citric acid production by Aspergillus niger. Sucrose was the most favourable source, followed by glucose and fructose and then lactose. No citric acid was produced from galactose. Strong relationships were observed between citric acid production and the activities of certain enzymes in myccelial cell-free extracts prepared from fermentation samples. When sucrose, glucose, or fructose was the sugar source pyruvate carboxylase activity was high, but 2-oxoglutarate dehydrogenase activity was not detected. When galactose was the sugar source pyruvate carboxylase activity was low, but 2-oxoglutarate dehydrogenase activity was high. It is suggested that whereas glucose and fructose repress 2-oxoglutarate dehydrogenase, thereby causing accumulation of citric acid, galactose does not. The activity of aconitase showed a direct relationship to the citric acid production rate. Thus, the activity was highest when sucrose was the sugar source, and lowest when galactose was the source. It is suggested that when large amounts of citric acid are lost from the cell the activity of aconitase increases as a response to the diminished intracellular supply of its substrate.     

The action of different biocides on the phosphatase activity of Aspergillus niger

Metalorganic and quarternary ammonium compounds when added to culture medium inhibited growth of Aspergillus niger mycelium and activity of neutral and alkaline phosphatase. A quarternary ammonium compound, ethonium, and a tin-organic compound, tributyl oxide, exerted an inhibiting effect on activity of acid phosphatase which amounted to 54% of the total phosphatase activity in mycelium and 94% in the culture liquid. The rest of biocides induced lysis of intracellular membranes, phosphatase release from lysosomas, which made acid phosphatase activity higher. Being introduced into the mycelium homogenate the above compounds inhibited activity of the acid phosphatase. The same biocides inhibited extracellular acid phosphatase in the culture liquid. Recommendations are given on the use of a number of substances as means for protection of industrial materials from biolesions.     

Mode of action of pectin lyase A of Aspergillus niger on differently C(6)-substituted oligogalacturonides

A thorough investigation of the mode of action of Aspergillus niger (4M-147) pectin lyase A (PLA) on differently C(6)-substituted oligogalacturonides is described. PLA appeared to be very specific for fully methyl-esterified oligogalacturonides: removal of the methyl-ester or changing the type of ester (ethyl esterification) or transamidation resulted in (almost) complete loss of conversion. The PLA activity increased with increasing length of the substrate up to a degree of polymerization (DP) of 8 indicating the presence of at least eight subsites on the enzyme. Product analysis demonstrated the formation of several Delta 4,5 unsaturated products and their saturated counterparts. The Delta 4,5 unsaturated trimer was the main product up to DP 8. For DP 9 and 10 Delta 4,5 unsaturated tetramer was the major product. Based upon the bond cleavage frequencies, a provisional subsite map was calculated, which supports the presence of eight subsites. By limited alkaline de-esterification of fully methyl-esterified pentamer and hexamer two sets of partially methyl-esterified pentamers (x and y methyl groups) and hexamers (a and b methyl groups) were prepared. Matrix-assisted laser desorption/ionization time of flight mass spectroscopy (MALDI-TOF MS) analysis demonstrated that the methyl-ester distribution was fully random. Using these partially methyl-esterified oligogalacturonides as substrates for PLA a 10-fold decrease in reaction rate was recorded compared with the fully methyl-esterified counterparts. Analysis of the methyl-ester distribution of the products showed that PLA tolerates carboxyl groups in the substrate binding cleft. At either subsite +2, +4, or -1 to -4 a free carboxyl group could be tolerated, whereas methyl-esters were obligatory at subsite +1 and +3. So PLA is capable to cleave the bond between a methyl-esterified and a non-esterified galacturonic acid residue, where the newly formed Delta 4,5 unsaturated non-reducing end residue always contains a methyl-ester.     

Cellulase Enzyme from Aspergillus niger

Cellulase enzyme was produced by a selected strain of Aspergillus niger isolated from deteriorated wood and grown on different carbon sources. Filter paper gave the highest yield, followed by carboxymethyl cellulose (CMC). Cellobiose as well as glucose gave a low yield, while the yield from lactose was negligible. The concentration of filter paper cellulose that induced the maximum yield of the enzyme was 1%. Both soluble cellulose (CMC) and cotton cellulose treated with phosphoric acid (swollen) were easily hydrolyzed by cellulase; an increase in cellulase concentration lead to more hydrolysis of CMC and gave linearity in the reaction velocity. At certain concentrations of the enzyme, increase in CMC concentration, (up to 1%) resulted in more reducing sugar. Beyond this point no more hydrolysis occur.     

Ultrasound effects on invertase from Aspergillus niger

Ultrasound effects on the release and activity of invertase from Aspergillus niger cultivated in a medium containing sucrose and peptone and in another with sugar-cane molasses and peptone were investigated. Irradiation was conducted for periods of 2–10 min. with waves of amplitude 20 and 40 using an ultrasound processor of 20 kHz. Product formation was determined as reducing equivalents formed by time units using 3,5-dinitrosalicylic acid. Total and specific activities of the culture supernatants were compared in the presence and absence of sonication. Both amplitudes promoted a significant increase of total invertase activity in the time periods investigated and the highest values were obtained with an amplitude of 20. Ultrasound irradiation caused cell disruption, thus releasing invertase and, after 4 min, activation of the enzyme also occurred. The best conditions for production, extraction and activation of invertase were in molasses medium containing peptone and irradiation with ultrasound waves at 20 for 8 min. This method showed high efficiency for the extraction and activation of invertase from A. niger as well as a great potential for use in industrial processes.     

Substrate specificity and mode of action of a cellulase from Aspergillus niger.

The mode of action and substrate specificity of a cellulase purified from Aspergillus niger were examined. The enzyme showed little capacity to hydrolyse highly ordered cellulose, but readily attacked soluble cellulose derivatives and amorphous alkali-swollen cellulose. Activity towards barley glucan and lichenin was greater than with CM-cellulose. Low activity was detected with CM-pachyman (a substituted beta-1,3-glucose polymer) and xylan. Activity towards yeast glucan, mannan, ethlene glycol chitin, glycol chitosan, laminarin, polygalacturonic acid and pectin could not be demonstrated. Cellobiose and p-nitrophenyl beta-D-glucoside were not hydrolysed, whereas the rate of hydrolysis of the higher members of the reduced cellulodextrins increased with chain length. The central bonds of cellotetraosylsorbitol and cellopentaosylsorbitol were the preferred points of clevage. Kinetic data indicated that the specificity region of the cellulase is five glucose units in length. The evidence indicates that the cellulase is an endoglucanase.     

Properties of cellobiase from Aspergillus niger

Summary Cellobiase enzyme was partially purified from the culture filtrate of Aspergillus niger AS-101 and the general and kinetic properties of the enzyme were examined. The enzyme was unstable on storage. However, it was protected by the addition of BSA, glycerol or sodium azide. Addition of glycerol also protected the enzyme from denaturation due to freezing and thawing. Effect of thiol group reagents revealed the presence of — SH groups at the active site of the enzyme. Different modulators such as metal ions and macroionic compounds illustrated varying effects on the purified cellobiase. Offprint requests to: A. Singh     

The effect of different carbon sources on the sugar composition of the mycelium of Aspergillus niger

The effect of different carbon sources on the sugar concentration and composition of theAspergillus niger mycelium has been studied at different days of incubation. No positive correlation could be drawn among dry weight, growth rate, and concentration of sugar and with substrate and sugar composition of mycelium. Glucose was the predominant sugar in all the cases.

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Zusammenfassung Die Wirkung von verschiedenen Kohlenhydratquellen an Zuckerkonzentration und Zusammensetzung des Myzels ofAspergillus niger war untersucht an verschiedenen Tagen der Inkubation. Keine positive Korrelation konnte festgestellt werden unter Trockengewicht, Wachstumsrate, Zuckerkonzentration, Nährboden und Zuckerbildung des Myzels. Glukose war der vorherrschende Zucker in allen Fällen.