Showing papers on "Aspergillus niger published in 1999"

1.8 and 1.9 A resolution structures of the Penicillium amagasakiense and Aspergillus niger glucose oxidases as a basis for modelling substrate complexes.

Abstract: Glucose oxidase is a flavin-dependent enzyme which catalyses the oxidation of β-d-glucose by molecular oxygen to δ-­gluconolactone and hydrogen peroxide. The structure of the enzyme from Aspergillus niger, previously refined at 2.3 A resolution, has been refined at 1.9 A resolution to an R value of 19.0%, and the structure of the enzyme from Penicillium amagasakiense, which has 65% sequence identity, has been determined by molecular replacement and refined at 1.8 A resolution to an R value of 16.4%. The structures of the partially deglycosylated enzymes have an r.m.s. deviation of 0.7 A for main-chain atoms and show four N-glycosylation sites, with an extended carbohydrate moiety at Asn89. Substrate complexes of the enzyme from A. niger were modelled by force-field methods. The resulting model is consistent with results from site-directed mutagenesis experiments and shows the β-d-glucose molecule in the active site of glucose oxidase, stabilized by 12 hydrogen bonds and by hydrophobic contacts to three neighbouring aromatic residues and to flavin adenine dinucleotide. Other hexoses, such as α-­d-­glucose, mannose and galactose, which are poor substrates for the enzyme, and 2-deoxy-d-glucose, form either fewer bonds or unfavourable contacts with neighbouring amino acids. Simulation of the complex between the reduced enzyme and the product, δ-gluconolactone, has provided an explanation for the lack of product inhibition by the lactone.

Penicillium amagasakiense and Aspergillus niger glucose oxidases as a basis for modelling substrate complexes

Abstract: # 1999 International Union of Crystallography Printed in Denmark ± all rights reserved Glucose oxidase is a avin-dependent enzyme which catalyses the oxidation of -d-glucose by molecular oxygen to -gluconolactone and hydrogen peroxide. The structure of the enzyme from Aspergillus niger, previously re®ned at 2.3 AE resolution, has been re®ned at 1.9 AE resolution to an R value of 19.0%, and the structure of the enzyme from Penicillium amagasakiense, which has 65% sequence identity, has been determined by molecular replacement and re®ned at 1.8 AE resolution to an R value of 16.4%. The structures of the partially deglycosylated enzymes have an r.m.s. deviation of 0.7 AE for main-chain atoms and show four N-glycosylation sites, with an extended carbohydrate moiety at Asn89. Substrate complexes of the enzyme from A. niger were modelled by force-®eld methods. The resulting model is consistent with results from site-directed mutagenesis experiments and shows the -d-glucose molecule in the active site of glucose oxidase, stabilized by 12 hydrogen bonds and by hydrophobic contacts to three neighbouring aromatic residues and to avin adenine dinucleotide. Other hexoses, such as -d-glucose, mannose and galactose, which are poor substrates for the enzyme, and 2-deoxy-d-glucose, form either fewer bonds or unfavourable contacts with neighbouring amino acids. Simulation of the complex between the reduced enzyme and the product, -gluconolactone, has provided an explanation for the lack of product inhibition by the lactone. Received 25 November 1998 Accepted 2 March 1999

Oxalic acid production by Aspergillus niger: an oxalate-non-producing mutant produces citric acid at pH 5 and in the presence of manganese.

Abstract: The external pH appeared to be the main factor governing oxalic acid production by Aspergillus niger. A glucose-oxidase-negative mutant produced substantial amounts of oxalic acid as long as the pH of the culture was 3 or higher. When pH was decreased below 2, no oxalic acid was formed. The activity of oxaloacetate acetylhydrolase (OAH), the enzyme believed to be responsible for oxalate formation in A. niger, correlated with oxalate production. OAH was purified from A. niger and characterized. OAH cleaves oxaloacetate to oxalate and acetate, but A. niger never accumulated any acetate in the culture broth. Since an A. niger acuA mutant, which lacks acetyl-CoA synthase, did produce some acetate, wild-type A. niger is apparently able to catabolize acetate sufficiently fast to prevent its production. An A. niger mutant, prtF28, previously isolated in a screen for strains deficient in extracellular protease expression, was shown here to be oxalate non-producing. The prtF28 mutant lacked OAH, implying that OAH is the only enzyme involved in oxalate production in A. niger. In a traditional citric acid fermentation low pH and absence of Mn2+ are prerequisites. Remarkably, a strain lacking both glucose oxidase (goxC) and OAH (prtF) produced citric acid from sugar substrates in a regular synthetic medium at pH 5 and under these conditions production was completely insensitive to Mn2+.

A novel class of protein from wheat which inhibits xylanases

Abstract: We have purified a novel class of protein that can inhibit the activity of endo-beta-1,4-xylanases. The inhibitor from wheat (Triticum aestivum, var. Soisson) is a glycosylated, monomeric, basic protein with a pI of 8.7-8.9, a molecular mass of 29 kDa and a unique N-terminal sequence of AGGKTGQVTVFWGRN. We have shown that the protein can inhibit the activity of two family-11 endo-beta-1, 4-xylanases, a recombinant enzyme from Aspergillus niger and an enzyme from Trichoderma viride. The inhibitory activity is heat and protease sensitive. The kinetics of the inhibition have been characterized with the A. niger enzyme using soluble wheat arabinoxylan as a substrate. The Km for soluble arabinoxylan in the absence of inhibitor is 20+/-2 mg/ml with a kcat of 103+/-6 s-1. The kinetics of the inhibition of this reaction are competitive, with a Ki value of 0.35 microM, showing that the inhibitor binds at or close to the active site of free xylanase. This report describes the first isolation of a xylanase inhibitor from any organism.

Production of Aspergillus xylanase by lignocellulosic waste fermentation and its application

Abstract: Strains of Aspergillus terreus and A. niger, known to produce xylanase with undetectable amounts of cellulase, were studied for xylanase (EC 3.2.1.8) production on various lignocellulosic substrates using solid state fermentation. Of the lignocellulosic substrates used, wheat bran was the best for xylanase production. The effects of various parameters, such as moistening agent, level of initial moisture content, temperature of incubation, inoculum size and incubation time, on xylanase production were studied. The best medium for A. terreus was wheat bran moistened with 1:5 Mandels and Strenberg mineral solution containing 0·1% tryptone, at 35 °C, and at inoculum concentration 2×107−2×108 spores 5 g−1 substrate; forA. niger, the best medium was wheat bran moistened with 1:5 Mandels and Strenberg mineral solution containing 0·1% yeast extract, at 35 °C, and at an inoculum concentration of 2×107−2×108 spores 5 g−1 substrate. Under these conditions, A. terreus produced 68·9 IU ml−1 of xylanase, and A. niger, 74·5 IU ml−1, after 4 d of incubation. A crude culture filtrate of the two Aspergillus strains was used for the hydrolysis of various lignocellulosic materials. Xylanase preparations from the two strains selectively removed the hemicellulose fraction from all lignocellulosic materials tested.

Expression of two major chitinase genes of Trichoderma atroviride (T. harzianum P1) is triggered by different regulatory signals.

Abstract: Regulation of the expression of the two major chitinase genes, ech42 (encoding the CHIT42 endochitinase) and nag1 (encoding the CHIT73 N-acetyl-β-d-glucosaminidase), of the chitinolytic system of the mycoparasitic biocontrol fungus Trichoderma atroviride (= Trichoderma harzianum P1) was investigated by using a reporter system based on the Aspergillus niger glucose oxidase. Strains harboring fusions of the ech42 or nag1 5′ upstream noncoding sequences with the A. niger goxA gene displayed a glucose oxidase activity pattern that was consistent under various conditions with expression of the native ech42 and nag1 genes, as assayed by Northern analysis. The expression product of goxA in the mutants was completely secreted into the medium, detectable on Western blots, and quantifiable by enzyme-linked immunosorbent assay. nag1 gene expression was triggered during growth on fungal (Botrytis cinerea) cell walls and on the chitin degradation product N-acetylglucosamine. N-Acetylglucosamine, di-N-acetylchitobiose, or tri-N-acetylchitotriose also induced nag1 gene expression when added to mycelia pregrown on different carbon sources. ech42 expression was also observed during growth on fungal cell walls but, in contrast, was not triggered by addition of chitooligomers to pregrown mycelia. Significant ech42 expression was observed after prolonged carbon starvation, independent of the use of glucose or glycerol as a carbon source, suggesting that relief of carbon catabolite repression was not involved in induction during starvation. In addition, ech42 gene transcription was triggered by physiological stress, such as low temperature, high osmotic pressure, or the addition of ethanol. Four copies of a putative stress response element (CCCCT) were found in the ech42 promoter.

Differential Expression of Three α-Galactosidase Genes and a Single β-Galactosidase Gene from Aspergillus niger

Abstract: A gene encoding a third α-galactosidase (AglB) from Aspergillus niger has been cloned and sequenced. The gene consists of an open reading frame of 1,750 bp containing six introns. The gene encodes a protein of 443 amino acids which contains a eukaryotic signal sequence of 16 amino acids and seven putative N-glycosylation sites. The mature protein has a calculated molecular mass of 48,835 Da and a predicted pI of 4.6. An alignment of the AglB amino acid sequence with those of other α-galactosidases revealed that it belongs to a subfamily of α-galactosidases that also includes A. niger AglA. A. niger AglC belongs to a different subfamily that consists mainly of prokaryotic α-galactosidases. The expression of aglA, aglB, aglC, and lacA, the latter of which encodes an A. niger β-galactosidase, has been studied by using a number of monomeric, oligomeric, and polymeric compounds as growth substrates. Expression of aglA is only detected on galactose and galactose-containing oligomers and polymers. The aglB gene is expressed on all of the carbon sources tested, including glucose. Elevated expression was observed on xylan, which could be assigned to regulation via XlnR, the xylanolytic transcriptional activator. Expression of aglC was only observed on glucose, fructose, and combinations of glucose with xylose and galactose. High expression of lacA was detected on arabinose, xylose, xylan, and pectin. Similar to aglB, the expression on xylose and xylan can be assigned to regulation via XlnR. All four genes have distinct expression patterns which seem to mirror the natural substrates of the encoded proteins.

New PCR method to differentiate species in the Aspergillus niger aggregate

Abstract: The DNA that encodes the 5.8S gene of the ribosomal RNA and the two intergenic spacers ITS1 and ITS2 of the two proposed type strains of the Aspergillus niger aggregate (A. niger and Aspergillus tubingensis) have been sequenced. By comparison of sequences we have found that both species could be differentiated by RsaI digestion of the PCR products of the mentioned regions. This method could be a useful tool in the identification of strains of the A. niger aggregate, especially in studies that involve a large number of isolates.

Microbial production of citric acid

Abstract: Citric acid is the most important organic acid produced in tonnage and is extensively used in food and pharmaceutical industries. It is produced mainly by submerged fermentation using Aspergillus niger or Candida sp. from different sources of carbohydrates, such as molasses and starch based media. However, other fermentation techniques, e.g. solid state fermentation and surface fermentation, and alternative sources of carbon such as agro-industrial residues have been intensively studied showing great perspective to its production. This paper reviews recent developments on citric acid production by presenting a brief summary of the subject, describing micro-organisms, production techniques, and substrates, etc.

Regulation of the feruloyl esterase (faeA) gene from Aspergillus niger.

Abstract: Feruloyl esterases can remove aromatic residues (e.g., ferulic acid) from plant cell wall polysaccharides (xylan, pectin) and are essential for complete degradation of these polysaccharides. Expression of the feruloyl esterase-encoding gene (faeA) from Aspergillus niger depends on D-xylose (expression is mediated by XlnR, the xylanolytic transcriptional activator) and on a second system that responds to aromatic compounds with a defined ring structure, such as ferulic acid and vanillic acid. Several compounds were tested, and all of the inducing compounds contained a benzene ring which had a methoxy group at C-3 and a hydroxy group at C-4 but was not substituted at C-5. Various aliphatic groups occurred at C-1. faeA expression in the presence of xylose or ferulic acid was repressed by glucose. faeA expression in the presence of ferulic acid and xylose was greater than faeA expression in the presence of either compound alone. The various inducing systems allow A. niger to produce feruloyl esterase not only during growth on xylan but also during growth on other ferulic acid-containing cell wall polysaccharides, such as pectin.

Cloning and molecular characterization of a soluble epoxide hydrolase from Aspergillus niger that is related to mammalian microsomal epoxide hydrolase.

Abstract: Aspergillus niger strain LCP521 harbours a highly processive epoxide hydrolase (EH) that is of particular interest for the enantioselective bio-organic synthesis of fine chemicals. In the present work, we report the isolation of the gene and cDNA for this EH by use of inverse PCR. The gene is composed of nine exons, the first of which is apparently non-coding. The deduced protein of the A. niger EH shares significant sequence similarity with the mammalian microsomal EHs (mEH). In contrast to these, however, the protein from A. niger lacks the common N-terminal membrane anchor, in line with the fact that this enzyme is, indeed, soluble in its native environment. Recombinant expression of the isolated cDNA in Escherichia coli yielded a fully active EH with similar characteristics to the fungal enzyme. Sequence comparison with mammalian EHs suggested that Asp(192), Asp(348) and His(374) constituted the catalytic triad of the fungal EH. This was subsequently substantiated by the analysis of respective mutants constructed by site-directed mutagenesis. The presence of an aspartic acid residue in the charge-relay system of the A. niger enzyme, in contrast to a glutamic acid residue in the respective position of all mEHs analysed to date, may be one important contributor to the exceptionally high turnover number of the fungal enzyme when compared with its mammalian relatives. Recombinant expression of the enzyme in E. coli offers a versatile tool for the bio-organic chemist for the chiral synthesis of a variety of fine chemicals.

In-vitro susceptibility of Aspergillus spp. isolates to amphotericin B and itraconazole

Abstract: The MICs of amphotericin B and itraconazole for 230 isolates of Aspergillus spp., comprising 156 Aspergillus fumigatus, 20 Aspergillus terreus, 22 Aspergillus flavus, 17 Aspergillus nidulans and 15 Aspergillus niger, were determined by a broth microdilution method with RPMI 1640 medium. No isolate was detected with an MIC of amphotericin B >2 mg/L. Itraconazole MICs >16 mg/L were detected for four Aspergillus fumigatus and one Aspergillus nidulans isolates.

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

Abstract: 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, Km of 0.20 mM and Vmax of 5.0 μmol min−1 mg−1protein.

Susceptibility of wheat and Aspergillus niger phytases to inactivation by gastrointestinal enzymes.

Abstract: The activity of wheat and Aspergillus niger phytases was determined following preincubation for 60 min at 37 degrees C alone or in the presence of pepsin or pancreatin to examine their ability to survive in the gastrointestinal tract. At pH 3.5 both phytases were stable, but at pH 2.5 wheat phytase rapidly lost activity. Following preincubation at pH 3.5 in the presence of 5 mg of pepsin/mL, A. niger phytase retained 95% of its original activity, whereas only 70% of the wheat phytase activity was recovered. The stability of A. niger phytase in the presence of pepsin was the same at pH 2.5 as at pH 3.5. Results similar to those with pepsin at pH 3.5 were obtained following preincubation of the phytases in the presence of pancreatin at pH 6.0.

Sequence Analysis, Overexpression, and Antisense Inhibition of a β-Xylosidase Gene, xylA, from Aspergillus oryzae KBN616

Abstract: β-Xylosidase secreted by the shoyu koji mold, Aspergillus oryzae, is the key enzyme responsible for browning of soy sauce. To investigate the role of β-xylosidase in the brown color formation, a major β-xylosidase, XylA, and its encoding gene were characterized. β-Xylosidase XylA was purified to homogeneity from culture filtrates of A. oryzae KBN616. The optimum pH and temperature of the enzyme were found to be 4.0 and 60°C, respectively, and the molecular mass was estimated to be 110 kDa based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The xylA gene comprises 2,397 bp with no introns and encodes a protein consisting of 798 amino acids (86,475 Da) with 14 potential N-glycosylation sites. The deduced amino acid sequence shows high similarity to Aspergillus nidulans XlnD (70%), Aspergillus niger XlnD (64%), and Trichoderma reesei BxII (63%). The xylA gene was overexpressed under control of the strong and constitutive A. oryzae TEF1 promoter. One of the A. oryzae transformants produced approximately 13 times more of the enzyme than did the host strain. The partial-length antisense xylA gene expressed under control of the A. oryzae TEF1 promoter decreased the β-xylosidase level in A. oryzae to about 20% of that of the host strain.

Effect of oxygen enrichment on morphology, growth, and heterologous protein production in chemostat cultures of Aspergillus niger B1-D.

Abstract: The response of steady state chemostat cultures of a recombinant Aspergillus niger (B1-D), secreting both a heterologous enzyme (Hen Egg White Lysozyme [HEWL]) and a native enzyme (Glucoamylase), to varying levels of O2 enrichment of the process gas was evaluated. Formation of both the native and the foreign enzyme increased with increasing O2 supply. Conversely, biomass levels and total extracellular protein levels were generally not increased under O2 enriched conditions. Two distinct micromorphologies were apparent in these cultures, one, typically seen under O2 limiting conditions (i. e. at 0 and 10% enrichment levels), tended to be represented by long, sparsely branched hyphal elements, with low percentages of "active" length (i. e. how much of the hypha is cytoplasm filled); whilst, a second micromorphology, typical of O2 enriched cultures at 30 and 50% O2 enrichment, was represented by shorter hyphal elements, with more branching and a higher % "active" length. At these higher O2 levels, formation of a yellow pigment occurred, and signs of culture autolysis were noted. At 50% enrichment, a "stranded" aggregate morphology was apparent, possibly as a response to a hyperoxidant state. Production of both the native enzyme and HEWL correlated well with a simple morphological measure (tip number) or, with % "active" length. It is proposed the morphological changes noted in the cultures were associated with the increased production of both HEWL and glucoamylase.

Dephosphorylation of Phytate by Using the Aspergillus niger Phytase with a High Affinity for Phytate

Abstract: Phytic acid (myo-inositol hexakis dihydrogen phosphate) is the major storage form of phosphate in cereals, pollen, legumes, and oilseed. Phytic acid is considered to be an antinutritional factor since it chelates minerals such as magnesium, zinc, and calcium and may also react with proteins, therefore decreasing the bioavailability of protein and nutritionally important minerals. The use of phytase as a feed additive has been examined several times over the last 20 years, resulting in improved phosphorus availability from poultry and swine feed. However, the high cost of the enzyme, compared to the cost of inorganic phosphate, has prevented its universal use. Recently, there has been renewed interest in phytase due to the low-cost production of this enzyme by recombinant DNA technology and an increased concern for the environment. Phytase has the potential to reduce the amount of phosphate in poultry and swine wastes by enhancing phosphorus retention by the animal. Phytase, myo-inositol hexakisphosphate phosphohydrolase (EC. 3.1.3.8), catalyzes the hydrolysis of phytic acid to inositol polyphosphates and free orthophosphoric acid. Phytase-producing microorganisms comprise bacteria such as Bacillus subtilis (8), Pseudomonas sp. (3), and Escherichia coli (1); yeasts such as Schwanniomyces castellii (9) and Saccharomyces cerevisiae (6); and fungi such as Aspergillus ficuum (2) and Aspergillus terreus (14). The phytase produced by A. ficuum NRRL 3135 has been isolated and well characterized by Ullah and Gibson (10, 11). In addition, the cloning and expression of the phyA gene have been reported for A. ficuum (13), Aspergillus awamori (7), and A. terreus (5). Recently, Kostrewa et al. reported the crystal structure of phytase from A. ficuum (4). In this paper, we describe the purification and characterization of a phytase with a high affinity for phytate. The amount of orthophosphate released by this enzyme is compared with that released by the A. ficuum phytase. Aspergillus niger SK-57 was inoculated on solid media with wheat bran and cultivated at 30°C for 5 days. Proteins were extracted from solid-state fermentation (koji mold grown on sterilized wheat bran) by using cones with warm water. After filtration with filter paper (no. 2; ADVANTEC, Tokyo, Japan), the crude extract was desalted by using a Sartcon mini system (Sartorius) equipped with an ultrafilter (molecular weight cutoff, 10,000) and used for enzyme purification. Purification of phytase from A. niger SK-57 was done at 4°C. In step 1, the crude enzyme was applied to an anion-exchange DIAION HPA-75 column (5.6 by 30 cm; Mitsubishi Chemical, Tokyo, Japan) that had previously been equilibrated with 50 mM acetate buffer (pH 5.5). The column was washed with equilibration buffer, and the proteins were eluted with 0.3 M NaCl in 50 mM acetate buffer (pH 4.8). The peak fractions of phytase activity were pooled and concentrated by ultrafiltration through a UK-10 membrane having a molecular weight cutoff of 10,000 (ADVANTEC) and were dialyzed overnight against 50 mM acetate buffer (pH 4.9) at 4°C. In step 2, the concentrate obtained from step 1 was applied to an S Sepharose Fast Flow column (2.5 by 30 cm; Pharmacia Biotech, Uppsala, Sweden) that had previously been equilibrated with 50 mM acetate buffer (pH 4.9). The column was washed with equilibration buffer, and the proteins were eluted with 50 mM acetate buffer (pH 5.2). Fractions containing phytase activity were pooled and concentrated by ultrafiltration, as described for step 1. In step 3, the concentrate obtained from step 2 was applied to a TOYO-PEARL HW-55F column (2.0 by 60 cm; Tosoh, Tokyo, Japan) and equilibrated with 50 mM acetate buffer (pH 4.5). Fractions with high phytase activity were pooled and dialyzed overnight against 50 mM acetate buffer (pH 6.0) at 4°C. In step 4, the enzyme solution was applied to a Mono-P HR 5/20 column (Pharmacia) that had previously been equilibrated with 25 mM histidine-HCl buffer (pH 5.8), and the phytase was eluted with 10% polybuffer 74-HCl (pH 4.2). A purification profile of the phytase from A. niger SK-57 with solid-state fermentation is shown in Table ​Table1.1. The enzyme was purified 11-fold with a 2.5% yield from the crude extract. The specific activity of the purified enzyme was 158 U/mg of protein. The purified enzyme was shown as a single protein band on a sodium dodecyl sulfate (SDS)-polyacrylamide gel. The molecular masses of the native protein and the protein deglycosylated by endoglycosidase H were estimated to be approximately 60 and 55 kDa, respectively (Fig. ​(Fig.1),1), suggesting that the protein contains a small amount of carbohydrate. The isoelectric point determined by chromatofocusing was 4.7. TABLE 1 Summary of purification of A. niger SK57 phytase FIG. 1 SDS-polyacrylamide gel of the purified enzyme. Electrophoresis was performed on a slab of 12% polyacrylamide gel in 25 mM Tris-HCl–0.192 M glycine containing 0.1% SDS. The gel was stained for proteins with Coomassie brilliant blue ... To study the enzyme substrate affinity, the kinetic parameter of phytic acid was determined at pH 5.5 and 37°C. The apparent Michaelis constant (Km) of the phytase for phytic acid calculated from the Lineweaver-Burk plot was 18.7 ± 4.6 μM. This low Km shows a remarkably high affinity of the protein for phytic acid, higher than the Km values of 40 μM and 250 μM reported for A. ficuum by Ullah (11) and van Gorcom et al. (12), respectively. van Gorcom et al. demonstrated that the phytase from A. ficuum NRRL 3135 purified by Ullah contained two proteins, a phytase having a molecular mass of 85 kDa and an acid phosphatase having a molecular mass of 100 kDa (12). When we purified the phytase from A. ficuum NRRL 3135, we found the molecular mass and the Km of the phytase to be 85 kDa and 184.2 ± 12.5 μM, respectively (data not shown). The action of the purified enzyme in 0.1 M sodium acetate, pH 5.5, on several phosphate compounds was tested. The tested substrates were as follows: phytic acid, p-nitrophenylphosphate, d-glucose 6-phosphate, fructose 6-phosphate, d-myo-inositol 1,4,5-triskisphosphate, glycerophosphate, and ATP. Phytic acid was hydrolyzed at the fastest rate; the other phosphorylated compounds reached a maximum of about only 2% of the phytic acid hydrolysis rate. To investigate the pH optimum and pH stability, the phytase assay was performed at a pH range of 2 to 9 with a variety of buffers by standard assay. The phytase had a double pH optimum of pH 5.5 and pH 2.5 and was virtually inactive above pH 7.0. The activity at pH 2.5 was 60% less than that at pH 5.5. When the enzyme was incubated at various pH values at 37°C for 60 min in the absence of substrate and the residual activity was measured, the phytase was found to be stable at the pH range of 5 to 7. The temperature profile of purified phytase was determined from 4 to 60°C by standard assay at the given temperature. The optimum temperature was found to be 50°C. To investigate thermal stability, the phytase was incubated at 0 to 60°C for 60 min in 0.1 M acetate buffer, pH 5.5, and its activity was determined by standard assay. No less activity was observed from 0 to 30°C, while at 50°C only 30% of the activity remained. The N-terminal amino acid sequence analysis of the enzyme was determined to be Ser-Arg-Asn-Gln-Ser-Thr-Cys-Asp-Thr-Val-Asp-Gln-Gly-Tyr-Gln with a gas-phase sequencer (Applied Biosystems). To investigate the hydrolysis of Na phytate by the A. niger SK-57 and A. ficuum phytases, an enzymatic reaction was started by the addition of enzyme (0.01 U) to the assay mixture. The final concentration of phytate was 0.2 mM in 0.1 M acetate buffer, pH 5.5. From the incubation mixture, samples (0.4 ml) were removed periodically, and the reaction was stopped by adding 0.8 ml of freshly prepared acetone–5 N H2SO4–10 mM ammonium molybdate (2:1:1 [vol/vol/vol]). After mixing, 40 μl of 1.0 M citric acid was added to each tube. The orthophosphates released from phytate were determined (Fig. ​(Fig.2).2). The A. niger SK-57 phytase with a low Km released more orthophosphate, even at a lower substrate concentration, than the A. ficuum phytase with a high Km. The difference was about 8%. To demonstrate the performance of the enzyme, the concentration of a substrate necessarily has to be higher than its Km, and if an enzyme with a low Km and an enzyme with a high Km have the same maximum reaction rate (Vmax), the enzyme with the low Km, unlike the enzyme with the high Km, does not decrease the reaction rate, even at a lower substrate concentration. That is, when compared with the enzyme with the high Km, the enzyme with the low Km has an advantage in that it can maintain a sufficient degradation rate, even at a lower substrate concentration, thereby minimizing the amount of the remaining substrate. Accordingly, there is a demand for an inexpensive A. niger phytase with a low Km for phytic acid because phytase degrades phytic acid, an antitrophic factor contained in feed, thereby improving the nutritive value of the feed and simultaneously achieving an efficient utilization of phosphoric acid released by the degradation. FIG. 2 Time course of the release of phosphate from Na phytate by phytases. The enzyme activity was fixed at 0.01 U. Symbols: ○, A. ficuum phytase; ●, A. niger SK-57 phytase.

Purification and characterization of a highly enantioselective epoxide hydrolase from Aspergillus niger.

Abstract: The epoxide hydrolase from Aspergillus niger was purified to homogeneity using a four-step procedure and p-nitrostyrene oxide (pNSO) as substrate. The enzyme was purified 246-fold with 4% activity yield. The protein is a tetramer composed of four identical subunits of molecular mass 45 kDa. Maximum activity was observed at 40 degrees C, pH 7.0, and with dimethylformamide as cosolvent to dissolve pNSO. Hydrolysis of pNSO was highly enantioselective, with an E value (i.e. enantiomeric ratio) of 40 and a high regioselectivity (97%) for the less hindered carbon atom of the epoxide. This enzyme may be a good biocatalyst for the preparation of enantiopure epoxides or diols.