Extract Of Aspergillus Niger Research Articles

Biotransformation of isoeugenol to vanilla flavour metabolites and capsaicin in suspended and immobilized cell cultures of Capsicum frutescens: study of the influence of β-cyclodextrin and fungal elicitor

Suspended and immobilized cell cultures of Capsicum frutescens accumulated vanilla flavour metabolites (vanillin, vanillic acid and ferulic acid) when fed with isoeugenol. The addition of isoeugenol also stimulated its biotransformation to capsaicin. Maximum concentrations of vanillin accumulated at 566 μg/40 ml culture on the sixth day in isoeugenol (2.5 mM) treated cultures. This was twice the increase recorded in suspended cultures on the same day. The formation of ferulic acid (58 μg) was observed on the fifth day in immobilized cultures treated with isoeugenol. The addition of β-cyclodextrin together with isoeugenol, each at 2.5 mM, resulted in increase in the accumulation of vanillin (919 μg/40 ml) on the fourth day. This was 1.62 times more than in cultures treated with isoeugenol alone. Isoeugenol treated immobilized cells, when challenged with aqueous mycelial extract of Aspergillus niger, yielded maximum vanillin concentrations (735 μg/40 ml culture) on the eighth day, whereas addition of medium filtrate of A. niger led to marginal increase in the vanillin (610 μg/40 ml). Accumulation of capsaicin reached a maximum, 121 and 92 μg, on the tenth day in isoeugenol and elicitor added cultures, respectively. Biotransformation of isoeugenol was more effective in immobilized cells, and this was further enhanced by the addition of β-cyclodextrin and fungal elicitor.

Elicitation of thiophene production by cultured hairy roots of Tagetes patula

Hairy roots of Tagetes patula were initiated by infecting the seedlings with Agrobacterium rhizogenes. The hairy roots were grown in liquid medium MS (Murashige and Skoog 1962) supplemented with sucrose. The roots were treated with three different elicitors obtained from mycelial culture of Aspergillus niger, Rhizopus oligosporus and Pencillium notatum. Accumulation of biomass and the production of thiophenes were studied over a period of six weeks in culture. The HPLC separation profile of the thiophenes indicated the presence of several structurally different thiophenes. α-terthienyl being predominent. Maximum production of thiophene was recorded at the end of the fourth week in culture with a content of 0.138 % (w/w on dry weight basis). Treatment of hairy roots with mycelial extract of Aspergillus niger (1.5 % v/v) elicited an increase in thiophene content by 1.6 folds over the control.

Oxidation of medium-chain acyl-CoA esters by extracts of Aspergillus niger: enzymology and characterization of intermediates by HPLC.

The activities of beta-oxidation enzymes were measured in extracts of glucose- and triolein-grown cells of Aspergillus niger. Growth on triolein stimulated increased enzyme activity, especially for acyl-CoA dehydrogenase. No acyl-CoA oxidase activity was detected. HPLC analysis after incubation of triolein-grown cell extracts with decanoyl-CoA showed that beta-oxidation was limited to one cycle. Octanoyl-CoA accumulated as the decanoyl-CoA was oxidized. Beta-oxidation enzymes in isolated mitochondrial fractions were also studied. The results are discussed in the context of methyl ketone production by fungi.

Identification, characterization, and partial purification of glucoamylase from Aspergillus niger (syn A. ficuum) NRRL 3135.

The crude extracellular extract of Aspergillus niger (syn A. ficuum) NRRL 3135 contains glucoamylase (exo-1,4-alpha-D-glucanohydrolase, EC 3.2.1.2). The enzyme, a glycoprotein, was purified 7-fold by ion-exchange chromatography, chromatofocusing, and conconavalin A affinity chromatography. The molecular weight of the enzyme was estimated to be 90 kDa by SDS-PAGE and gel permeation chromatography. The pI of the enzyme was 3.4. The temperature optimum of the enzyme was 60 degrees C and the pH optimum was 5.0. The Vmax values for soluble starch, maltose, maltotriose, maltotretraose, maltopentaose, and isomaltose were 55.2, 11.7, 32.3, 47.8, 59.2, 12.5 nKat glucose/sec, respectively and the Km values for the same substrates were 0.09%, 0.67 mM, 0.76 mM, 0.76 mM, 0.68 mM, and 122.01 mM, respectively.

A simple and nondestructive method for the separation of polysaccharides from β-glucosidase produced extracellularly by Aspergillus niger

An apparatus based on electrophoresis has been devised that removes noncovalently bound polysaccharides from extracellular proteins of Aspergillus niger with concomitant partial β-glucosidase purification and concentration. The apparatus consists of a series of three chambers separated by polyacrylamide gels. Dialyzed and concentrated crude extract of Aspergillus niger containing β-glucosidase was poured into the middle chamber, while smaller anodic and cathodic chambers contained buffer. When electric current was applied, negatively charged protein-polysaccharide complexes moved toward the anode. Most of the negatively charged proteins, including β-glucosidase, crossed the gel barrier into the anodic compartment, while neutral polysaccharides were either trapped in the gels or remained in the middle chamber. In this way, 125 ml of dialyzed and concentrated crude extract of Aspergillus niger was processed. Therefore, after 24 h of electrophoresis, 68% of the proteins and 90% of the β-glucosidase activity, but only negligible amounts of polysaccharide, were transferred to the anodic chamber. The removal of high-molecular-weight polysaccharide from β-glucosidase had a detrimental effect on the stability of the enzyme.

Effects of the addition of XAD-7 and of elicitor treatment on growth, thiophene production, and excretion by hairy roots of Tagetes patula

The effect of the addition of a solid adsorbent, XAD-7, on the growth of hairy roots of Tagetes patula and on their production and excretion of thiophenes is described in this article. Together with this, it was investigated whether or not the refreshment of the XAD and the medium makes any difference and finally, the combination of the above treatments with the addition of an elicitor (extract of Aspergillus niger) was tested for its effects. The growth of the hairy roots was slightly increased by the addition of XAD, but slightly decreased by the addition of the elicitor. The specific production of thiophenes remained unaffected in most cases; an increase in production was observed only when XAD and elicitor were added together. The excretion of the thiophenes, on the other hand, was strongly influenced by the addition of XAD-7 to the cultures. Without this resin, there was no measurable excretion at all, whereas the presence of XAD raised the excretion to 50% of the total thiophenes produced. When these experiments were extended to bubble columns, the same results came out, although there was a surprising difference between the reactor configuration with the XAD present in the medium in the reactor and the reactor configuration with an external column containing the XAD, through which the medium was constantly circulated. In the former, about 20% of the total production was excreted, while in the latter no thiophenes could be detected outside the hairy roots.

Purification and some properties of intracellular alpha-L-arabinofuranosidase from Aspergillus niger 5-16.

alpha-L-Arabinofuranosidase was purified from a cell-free extract of Aspergillus niger 5-16 by chromatographies on DEAE-Toyopearl, SP-Toyopearl, Ultro-gel AcA 44, Mono P, and TSK-Gel G3000SW. The final preparation thus obtained showed a single band on SDS-polyacrylamide gel electrophoresis. The molecular weight and isoelectric point were 67,000 by SDS-polyacrylamide gel electrophoresis and pH 3.5 by isoelectric focusing. The alpha-L-arabinofuranosidase contained amino acids in the order of Asx > Gly > Ala > Thr > Glx = Ser. The enzyme had maximum activity at pH 4.0 and 60 degrees C, and was stable from pH 4 to 7 and at temperatures up to 30 degrees C. The enzyme activity was not affected considerably by either metal ions or chemical reagents. The enzyme released arabinose from p-nitrophenyl-alpha-L-arabinofuranoside, O-alpha-L-arabinofuranosyl-(1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D- xylopyranose, and arabinan, but not from O-beta-D-xylopyranosyl-(1-->4)-O-[alpha-L-arabinofuranosyl- (1-->3)]-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose, O-beta-D-xylopyranosyl-(1-->2)-O-alpha-L-arabinofuranosyl-(1-->3)-O-beta -D- xylopyranosyl-(1-->4)-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose, gum arabic, or arabinoxylan. The limit of hydrolysis of arabinan was about 58% even when the enzyme was sufficiently in excess.

Elicitation of thiophene production by hairy roots of Tagetes patula

Various biotic and abiotic elicitors were assayed for their effects on thiophene production by hairy roots of Tagetes patula. The biotic elicitors tested were extracts of the fungi Aspergillus niger, Fusarium oxysporum, Penicillium expansum, and Phytophthora megasperma, of the bacteria Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, and yeast extract. The abiotic stress factors were the addition of calcium ions to the medium, Ethephon ( a precursor of ethene), and a high pH. In general, the biotic elicitors were effective, although the extent to which elicitation occurred differed from elicitor to elicitor. The highest production increase was attained with the extract of Aspergillus niger, an increase of 85% compared to the control. Only yeast extract had no positive effect on thiophene production. The abiotic elicitors tested had only small effects on thiophene production: the calcium ions induced a slight increase, the Ethephon only had a negative effect on production, and the high pH yielded a temporarily high concentration but the roots were no longer viable after this treatment.

Purification and Characterization of Intracellular α-Glucuronidase fromAspergillus niger5–16

Two kinds of α-glucuronidases, CM-I and CM-II, were purified from a cell-free extract of Aspergillus niger 5–16. Molecular weights of both CM-I and CM-II were 130,000 by SOS–PAGE, and 150,000 by gel filtration. The optimum temperature and pH for both enzyme reactions were 60°C and 4.8. Both en-zymes were stable up to 50°C, and between pHs 4.5 to 7.0 for 1 hr. Both enzymes were strongly inhibited by Ag+, Hg2+, Pb2+, Sn2+, Fe2+, Fe3+, Al3+, sodium dodecyl sulfate, monoiodoacetic acid, and N-bromosuccinimide. The Km of CM-I toward glucuronosyl-xylotriose and 4-O-methyl-glucuronosyl-xylotriose were 0.77 and 0.37, and those of CM-II were 0.82 and 0.47 mM, respectively. The Vmax of CM-I toward glucuronosyl-xylotriose and 4-0-methyl-glucuronosyl-xylotriose were 5.20 and 1.42, and those of CM-II were 17.1 and 4.68μmol of glucuronic acid formed/min/mg of protein, respectively.

Purification and properties of a 4-nitrophenylphosphatase from Aspergillus niger

A 4-nitrophenylphosphatase (EC 3.1.3.41) was identified in extracts of Aspergillus niger. The production of this activity was decreased by growth on a phosphatelimiting medium and was greatest in a medium supplemented with corn steep liquor. The phosphatase activity was purified by hydrophobic, ion-exchange, and molecular sieve chromatography. The purified enzyme has a native size of approximately 80,000, polypeptide subunits with sizes of 37,000 upon denaturation, and a p I of 4.6. The activity was optimal at pH 8.0 and was stimulated by Mg 2+ and to a lesser extent by Mn 2+ but was inhibited by Zn 2+ and Ca 2+. The enzyme was highly specific for 4-nitrophenyl phosphate as substrate, having a K m of 0.77 m m and a turnover number of 108 s −1. The purified enzyme did not hydrolyze any of 22 sugar phosphates, mononucleotides, or other phosphocompounds tested. A small, but reproducible, amount of activity was measured using 5′-DNA phosphate as a substrate. Although some similarities exist to three previously characterized 4-nitrophenylphosphatases from Saccharomyces cerevisiae, the enzyme from A. niger is distinctly different from at least two of these activities.

6 -Hydroxylation of Steroids by Extracts of Aspergillus niger

Growing cultures of Aspergillus niger hydroxylate progesterone at the 6α and 11αpositions. The properties of the 6β-hydroxylase activity were studied in extracts of this organism using 11α hydroxyprogesterone as substrate. Maximum 6β-hydroxylase activity was obtained at pH 6·5. EDTA and thiophenol inhibited hydroxylation, while NADPH, 2-oxoglutarate and pyruvate stimulated it. Cobalt and cadmium ions inhibited the 6β-hydroxylase activity. The feasibility of introducing a hydroxyl group into the 6β-position of various steroid compounds was tested: the 3-Δ4 configuration appeared to exert a directional effect for 6β-hydroxylation, while an 11β-hydroxyl group exerted some kind of steric hindrance against 6β-hydroxylation.

Adenosine nucleotides and ATP synthesizing enzymes in conidia of Aspergillus niger

In crude conidia extracts of Aspergillus niger, 650 pmol AMP/mg protein was found but no ADP or ATP could be detected. Cell‐free extracts from the conidia produce ATP in the presence of AMP and phosphoenolpyruvate. The system is Mg2+ ‐dependent and highly stimulated by increases in the viscosity of the incubation media. The examination of enzymes involved in the ATP synthesis, revealed the presence of adenylate kinase (EC 2.7.4.3) and pyruvate kinase (EC 2.7.1.40) activity. The pyruvate kinase is stimulated in viscous incubation media. A 30% polyethyleneglycol concentration was found to be an optimal viscous medium. Adenylate kinase was unaffected by viscosity changes.

Mutagenicities of 61 flavonoids and 11 related compounds.

The mutagenicities of 61 flavonoids (naturally occurring flavonoid aglycones and flavonal glycosides and synthetic flavonoids) and those of 11 compounds structurally related to flavonoids were tested with Salmonella typhimurium strains TA100 and TA98. Among the 22 flavone derivatives tested, only wogonin was strongly mutagenic, while five derivatives, apigenin triacetate, acacetin, chrysoeriol, pedalitin, and pedalitin tetraacetate, were only weakly mutagenic. Two bisflavonyl derivatives, neither of which has a 3-hydroxyl group, were not mutagenic. Of the 16 flavonol derivatives tested, all except 3-hydroxyflavone and the tetra- and penta-methyl ethers of quercetin were mutagenic. Of the five flavanone derivatives tested, only 7,4-dihydroxyflavanone was mutagenic, showing weak activity. Of the four flavanolol derivatives tested, hydrorobinetin and taxifolin were weakly mutagenic. Of the six isoflavone derivatives tested, tectorigenin was weakly mutagenic. Of the 11 compounds in the miscellaneous group structurally related to flavonoids, only isoliquiritigenin was mutagenic, showing weak activity. For the emergence of strong mutagenicity, the double bond between positions 2 and 3 and the hydroxyl group at position 3 are required, except in wogonin, which does not have a hydroxyl group at position 3 but is strongly mutagenic to TA100. The 3-O-acetyl ester of flavonol, quercetin, was mutagenic with S9 mix, but 3-O-methyl ethers were not. Six flavonol glycosides, three quercetin glycosides and three kaempferol glycosides were mutagenic after preincubation with "hesperidinase," a crude extract of Aspergillus niger. Of 66 flavonoid agylcones and compounds structurally related to flavonoids, quercetin was the strongest mutagen. The carcinogenicity of this compound should be clarified because it is ubiquitously found in vegetables.

Purification, properties, kinetics, and mechanism of beta-N-acetylglucosamidase from Aspergillus niger.

Beta-N-Acetylglucosaminidase has been purified from an acetone extract of Aspergillus niger. The protein has a Mr = 149,000. It contains neither Mn2+, Zn2+, nor cysteine and exhibits no cation requirement for activity. Isoelectric focusing separates two isozymes; the major isoenzyme has a pI = 4.4. Both isozymes exhibit beta-N-acetylgalactosaminidase and beta-glucosidase, as well as glucosaminidase activity. The mechanism of action of this enzyme has been studied in detail using a variety of substrate structure/activity and kinetic experiments. Rate data plotted versus pH depends on the following ionization constants, respectively: for pKm, 2.95; for log Kcat, 7.6; and for log kcat/Km, 2.95 and 8.25. The kcat value of H2O/D2O for p-nitrophenyl-beta-N-acetylglucosaminide hydrolysis is 1.27 at pH 4.6 and 1.00 at pH 7.0. The rho value for the hydrolysis of para-substituted phenylglucosaminides is +0.36; rho for the hydrolysis of fluoro-substituted N-acetyl derivatives is -1.41. Two sulfur-containing substrate analogues, the 1-thioglucosaminide, and the N-thioacetyl derivative, exhibit either no or little substrate activity. The hydrolysis of the 2,4-dinitrophenyl-glucosaminide is not biphasic as indicated by stopped flow kinetic studies. These several results are interpreted to show that: 1) enzymatic nucleophilic catalysis is not employed by beta-N-acetylglucosaminidase; 2) the glycosidic oxygen is protonated very early in the reaction, perhaps even in the Michaelis complex; 3) the acetamido oxygen provides anchimeric assistance to hydrolysis via charge stabilization of the oxocarbonium ion (or via oxazoline formation); 4) additional charge stabilization is provided by an enzymic anion, perhaps a side chain carboxylate group. The role of the acetamido group is discussed and comparisons are made between lysozyme, beta-galactosidase, and beta-N-acetylglucosaminidase.

Immunogenic Properties of PR8 Influenza Virus after Treatment with Acid Protease

Brief exposure of PR8 influenza virus at pH 4.5 to extracts of Aspergillus niger abolished the red cell agglutinating and infectious capacities of the virus without significantly decreasing its antigenicity or immunogenicity. The results of gel electrophoresis indicated that the HA1 portion of the viral hemagglutinin had been degraded by treatment of virus with Aspergillus extract. The immunogenicity in mice of virus that had been exposed to A. niger extract appeared to be very broad, since vaccination of mice with such material gave protection against challenge with strains of influenza A virus representing all major serotypes as well as one type B influenza virus.

A novel phosphodiesterase from Aspergillus niger and its application to the study of membrane-derived oligosaccharides and other glycerol-containing biopolymers

A novel phosphodiesterase has been found in commercially available extracts of Aspergillus niger and has been partially purified by fractionation with acetone and chromatography on carboxymethylcellulose. The enzyme attacks glycerophosphodiester bonds with the liberation of free glycerol only. The synthetic substrate glucose 6-phospho-sn-1'(3')-glycerol is hydrolyzed with production of equivalent amounts of free glycerol and glucose 6-phosphate. Similarly, the enzymic hydrolysis of sn-glycero-3-phosphocholine liberates glycerol and phosphocholine. The hydrophilic head groups of membrane phospholipids of Escherichia coli are continuously transferred to a closely related family of oligosaccharides ("membrane-derived oligosaccharides") containing glucose as the sole sugar (van Golde, L. M. G., Schulman, H., and Kennedy, E. P. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 1368--1372). Oligosaccharide A-2 contains sn-1-glycerophosphate residues (derived from phosphatidylglycerol) in phosphodiester linkage. Treatment of this oligosaccharide with the phosphodiesterase led to the liberation of nearly all of the glycerol as free glycerol. Subsequent partial acid hydrolysis of the enzyme-treated oligosaccharide led to the recovery of glucose 6-phosphate in almost quantitative yield. The sn-1-glycerophosphate residues are therefore linked to position 6 of glucose units of the oligosaccharide. The activity of the enzyme is not restricted to glycerophosphodiesterases since it will hydrolyze phosphodiesters containing other polyols such as the synthetically prepared glucose 6-phospho-DL-1'(2'-hydroxy-3'-ethoxy)propane.

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.

Formation of nicotinic acid ribonucleoside by an enzyme preparation and growing mycelium of Aspergillus niger.

The cell-free extract of Aspergillus niger (AKU 3302) catalyzed the degradation of nicotinamide mononucleotide to an unidentified compound. This compound was isolated from the reaction mixture and identified as nicotinic acid ribonucleoside (NAR). When the mold was cultured on media containing nicotinic acid- and nicotinamide-14C, the ratio of radioactivity incorporated into NAR was 16 and 10% of the total activity in the mycelial extracts, respectively.

Gluconsäure bildende Enzyme bei Aspergillus niger

At least three gluconic acid forming enzymes were identified in cell-free extracts of Aspergillus niger: glucose oxidase (EC 1.1.3.4), a glucose dehydrogenase (EC 1.1.99.10), and an enzyme or a mixture of enzymes catalyzing the cleavage of 6-phosphogluconate into gluconate and inorganic phosphate. 2,6-dichlorphenolindophenol was one of the hydrogen acceptors in vitro of the glucose dehydrogenase. Some properties of this enzyme (Km values, pH-dependence, substrate and hydrogen acceptor specificity), as determined in cell-free extracts, were found to be in good agreement with properties described in literature for a glucose dehydrogenase which has been purified from Aspergillus oryzae. The formation of Pi from 6-phosphogluconate and other phosphate esters was found to have an optimum between pH 7 and 8 , and another below pH 4. This suggests that it is catalyzed by an alkaline and an acid phosphomonoesterase (EC 3.1.3.1, 3.1.3.2), both enzymes exhibiting only low substrate specificity. The influence of extraction and assay buffers on the activity of gluconate forming enzymes was investigated. Loss of activity during preparation of cell-free extracts, as calculated from loss of activity storage of cell-free extracts at 4 degrees C, was found to be lower than 4%. Purified glucose oxidase added before homogenization was found in the extract almost quantitatively.

Isolation, Properties, and Crystallization of an Iron-Chlorin Protein from Aspergillus niger

A green iron-chlorin protein was purified 160-fold from a lyophilized extract of Aspergillus niger by ion-exchange chromatography and gel filtration with a yield of 25%. The purified preparation appeared nearly homogeneous on sedimentation analysis and the sedimentation coefficient of the protein at infinite dilution was 13.4S. Its molecular weight was calculated to be 2.8--3.2 X 10(5) from sedimentation and gel filtration data. The ferric form of the protein had absorption maxima at 587.5 and 708 nm in the visible region and a Soret band at 404 nm. High-spin ESR signals of a rhombically distorted ferric iron-chlorin complex were observed at g = 6.5 and 5.3 together with unidentified, weaker signals at g = 4.3 and 2.0. The ferric form reacted readily with cyanide to give a complex showing absorption peaks at 422 and 632 nm and a shoulder at about 595 nm. When the protein combined with cyanide its high-spin ESR signals disappeared and low-spin signals at g = 1.88, 2.29, anous form having absorption maxima at 622 and about 410 nm. The rate of reduction by dithionite was slightly reduced by the presence of either nitrite or sulfite, and greatly accelerated by the presence of hydroxylamine. The reduced spectrum obtained in the presence of hydroxylamine had maxima at 620 and about 420 nm. The ferric cyanide complex did not show any spectral change on addition of dithionite. The green prosthetic group could be extracted with acidified acetone and the absorption maxima of the pyridine ferrihemochrome were at 413 and 599 nm. On removal of metal from the prosthetic group the characteristic spectrum of a chlorin was obtained. The absorption maxima of a solution of the chlorin in benzene were at 403, 501, 537, 576, 595, and 655 nm, the 655 nm band being strongest of those in the visible region. No significant amount of flavin was detected in the purified preparation. The iron-chlorin protein catalyzed methyl viologen-linked reduction of hydroxylamine and also that of nitrite at a slower rate under the same conditions, but not evidence that it reduced sulfite was obtained in the present study. The purified preparation also had high catalase [ec 1.11.1.6] activity. Crystalline material was obtained by gradual concentration of the purified preparation at about pH 6.