Glucoamylase Biosynthesis by Cells of Aspergillus niger C58-III Immobilized in Sintered Glass and Pumice Stones

A simple method of A. niger C_58-III cell immobilization is described. This strain produces extracellular glucoamylase. According to the proposed method A. niger spores were first immobilized by adsorption in sintered glass Rasching rings (RR) or pumice stones (PS). Growing out from spores, A. niger cells produced extracellular glucoamylase. This technique facilitates the culture growth in a filamentous spongy structure of the supports with a continuous accumulation of biomass. After every 24 h it was possible to obtain culture liquid rich in glucoamylase. This procedure can be repeated 30 times using the same sample of immobilized A. niger culture without any loss of glucoamylase activity in the liquid medium. In a 96 h period immobilized A. niger cells produced 300 units × ml⁻¹ whereas a shake culture of this fungus produced only 186 units × ml⁻¹.     

Effect of the Reversion of Aspergillus niger Auxotrophic Mutants to Prototrophic Forms on their Glucoamylolytic Activity

Prototrophic revertants were obtained from 4 auxotrophic mutants A. niger, 2 of which originated from amylolytically highly active prototroph A. niger C, whereas 2 from weakly active prototrophs A. niger 23 and 71. A hundred of revertants randomly chosen from each group were initially evaluated with respect to total amylolytic activity. Ten revertants which in this respect reached the highest indices were closely studied in regard to the synthesis of glucoamylase. All of them showed a distinct increase in the activity of this enzyme in relation to their ancestor both auxotrophic and prototrophic. In the case of revertants, derivatives of A. niger C prototroph, maximal glucoamylase activity increased over 40%. Among the derivatives of A. niger 71 prototroph the increase in glucoamylase activity exceeded many times the level of glucoamylase activity of its prototrophic ancestor.     

Hydrolysis of Native Wheat and Corn Starch Granules by Glucoamylases from Aspergillus Niger and Rhizopus Niveus

The action of glucoamylase I and II (α-1,4-glucan glucohydrolase, E.C. 3.2.1.3) from Aspergillus niger and the glucoamylase from Rhizopus niveus on native wheat and corn starch granules was followed by using scanning electron microscopy (SEM) and by measuring the glucose released by enzymatic attack. Two distinct patterns of attack were observed. Glucoamylase I and the glucoamylase from R. niveus attacked the granule surface relatively uniformly, resulting in large disclike depressions. Glucoamylase II, while showing some disc-like depressions, produced small grooves (furrows) in the surface of the granule. Similar patterns were observed for both corn and wheat starch granules, except that attack by glucoamylase I and the glucoamylase from R. niveus on wheat starch granules also developed along the equatorial groove (not easily seen until the granules were exposed to enzyme solutions). Measuring glucose released indicated that hydrolysis by glucoamylase I and by the glucoamylase from R. niveus were nearly equal in extent and were about twice that by glucoamylase II.     

Improvement of Aspergillus niger Glucoamylase Thermostability by Directed Evolution

Directed evolution was used to improve the thermostability of Aspergillus niger glucoamylase (GA) expressed in Saccharomyces cerevisiae. A starch‐plate assay developed to screen GA mutants for thermostability gave results consistent with those of irreversible thermoinactivation kinetic analysis. Several thermostable multiply‐mutated GAs were isolated and characterized by DNA sequencing and kinetic analysis. Three new GA mutations, T62A, T290A and H391Y, have been identified that encode GAs that are more thermostable than wild‐type GA, and that improve thermostability cumulatively. These individual mutations were combined with the previously constructed thermostable site‐directed mutations D20C/A27C (forming a disulfide bond), S30P, and G137A to create a multiply‐mutated GA designated THS8. THS8 GA is substantially more thermostable than wild‐type GA at 80°C, with a 5.1 kJ/mol increase in the free energy of thermoinactivation, making it the most thermostable Aspergillus niger GA mutant characterized to date. THS8 GA and the singly‐mutated GAs have specific activities and catalytic efficiencies (k_cat/K_m) similar to those of wild‐type GA.     

EFFECTS OF β-GLUCOSIDASE ACTIVITY ON THE CHARACTERISTICS OF AROMATIC RED RICE WINE

The pigment in the bran layer of aromatic red rice (Oryza sativa var. Indica, Tapol) is rather stable, has a characteristic red color just like that of grape wine and has a peak of absorbance at 530 nm at an acidic pH. A commercial saccharifying agent, glucoamylase AN-2, produced by Aspergillus niger was fractionated to separate glucoamylase and β-glucosidase activities by column chromatography on CM Sephadex C-50. The red rice wine made from uncooked, unpolished aromatic red rice using the fractionated, β-glucosidase-free preparation of glucoamylase had a characteristic red color. By contrast, red rice wine made with glucoamylase AN-2, which contained β-glucosidase activity, was inferior in color. The red pigment of aromatic red rice wine was decolorized and glucose originating from the red pigment was released by enzymatic digestion with the fractionated preparation of β-glucosidase. The partial decolorization of aromatic red rice wine was ascribed to the enzymatic action of β-glucosidase that was present in glucoamylase AN-2. Thus β-glucosidase activity has an undesirable effect on the brewing of aromatic red rice wine.     

Fumonisin B2 production by Aspergillus niger from grapes and natural occurrence in must

Aspergillus niger has been recently found to produce fumonisin B₂ (FB₂). Thirty-one strains belonging to four Aspergillus species isolated from grape were evaluated for FB₂ production on agar plates. Four out of eight strains of A. niger produced FB₂ (29–293 µg g^?1). None of the strains of A. uvarum (n = 7), A. tubingensis (8) and A. carbonarius (8) produced detectable amounts of toxin. The capability to produce FB₂ was also confirmed by some A. niger strains artificially inoculated on grape berries. Natural occurrence of FB₂, at levels of 0.01 and 0.4 µg ml^?1, was found in two samples of must collected in Apulian cellars in 2007. This is the first report of FB₂ contamination in must. These findings suggest that there is a potential risk of exposure to FB₂ in the grape–wine chain for consumers and that A. niger may represent the major fumonisin-producing species among black Aspergilli occurring on grapes.     

Genetic Engineering of white Shochu-Koji to achieve Higher Levels of Acid-Stable α-Amylase and Glucoamylase and other Properties when used for Shochu Making on a Laboratory Scale

The genes for acid-stable α-amylase and glucoamylase were cloned from white shochu-koji, Aspergillus kawachii. Both genes were used to transform the parent strain of white shochu-koji which carried a dominant selective marker gene, amdS, that originated from A. oryzae. Three lines of transformants were identified that secreted about 6-fold more acid-stable α-amylase activity and about 7-fold more glucoamylase activity than the parent strain in liquid culture. In solid culture, all three transformants had 2-fold higher acid-stable α-amylase activity and 2.4-fold higher glucoamylase activity than the parent strain. When koji was prepared on a laboratory scale, acid-stable α-amylase activity was 5.7-fold higher and glucoamylase activity was 3.8-fold higher than when the parent strain was used. Shochu was produced with a koji ratio of 33% or 10% using one line of transformants on a laboratory scale. Even with a koji ratio of 10%, the weight of the mash obtained with the transformant decreased to almost the same extent as with a koji ratio of 33% and the parent strain. Levels of flavour compounds in shochu produced with koji of the transformant were higher than in the shochu prepared with koji of the parent strain. In particular, levels of isoamyl acetate and β-phenethyl acetate were as high as 12.9 mg/litre and 3.8 mg/litre, respectively.     

Optimization of Aspergillus niger Fermentation for the Production of Glucose Oxidase

A number of nutritional factors influencing glucose oxidase (EC 1.1.3.4) production by Aspergillus niger NCIM 545 were studied. The synthesis of glucose oxidase by A. niger was investigated in two steps using submerged fermentation at 30 ± 2 °C and 180 rpm for 96 h. Primarily, nutritional components were selected by one-factor-at-a-time method, and the significance of each component with respect to glucose oxidase production was identified by Plackett–Burman design (seven variables including six nutritional viz. sucrose, sodium nitrate, peptone, calcium carbonate, magnesium sulfate, and potassium dihydrogen phosphate, and one dummy or unassigned variable were studied with eight experiments). In the second step, concentration of most significant factors and their interaction were studied with response surface methodology (central composite design). Each variable in the design was studied at five different levels, with all variables taken at a central coded value of zero. Considerable amount of glucose oxidase was produced from A. niger species with sucrose as the carbon source, sodium nitrate as the inorganic nitrogen source, and peptone as the organic nitrogen source. Glucose oxidase activity increased remarkably by 28.93 fold (from 0.00993 to 0.29 U ml⁻¹) with CaCO₃-supplemented media. The outcome of Plackett–Burman design showed CaCO₃, peptone, and MgSO₄ as significant parameters. Further optimization using a three-factor central composite design with 20 experiments increased yield of glucose oxidase from 0.29 to 2.05 U ml⁻¹ (sevenfold) with a decrease in cultivation time from 96 to 72 h.     

Amylase Synthesis by Forced Heterocaryons of Aspergillus niger in Submerged Culture Conditions

From 43 auxotrophic mutants A. niger found earlier 21 forced heterocaryons were obtained. All were examined with respect to total amylolytic activity by the method of test-tube microculture, whereas 12 of them (with the highest indices of this activity) were also tested with regard to glucoamylase synthesis. In most cases the amylase activity of hererocaryons was much higher than that of their component strains of lower and frequently of a higher activity. A distinct increase of this activity was also shown by heterocaryons formed from auxotrophs, originating from them in relation to prototrophic parent cultures with a weak amylase activity. However, none of the heterocaryons equalled the level of amylase synthesis, reached by a highly active prototroph A. niger C in this respect.     

The effect of cellulase preparations on the chemical changes during the ensilage of grass in laboratory silos

Three experiments are described. In the first, cellulase prepared from Aspergillus niger was added at the rate of 4 g/kg to herbage treated with a variety of silage additives, formic acid, caproic acid, formalin, sodium metabisulphite and zinc bacitracin. The lowest cellulose contents and highest residual water soluble carbohydrate contents were found in the silages treated with formic acid and cellulase. Formic acid was included as a treatment in subsequent experiments. In the second experiment, the enzyme was added to herbage at two levels, 1.0 g/kg fresh grass and 4.0 g/kg fresh grass. Cellulose contents of the silages were significantly lower after 61 days at the higher rate of application of the enzyme. In the third experiment, two enzymes produced from Aspergillus niger were compared with two enzymes derived from Trichoderma viride. Under the conditions of the experiment the cellulase enzymes produced from T. viride were more active.     

Glucoamylase of Amylomyces rouxii

The production of glucoamylase by Amylomyces rouxii, a mold used in rice and cassava fermentations in the Orient, was demonstrated under solid substrate culture conditions. The enzyme purified by ammonium sulfate fractionation, gel filtration, and Sephadex ion exchange column chromatography appears homogeneous. A. rouxii glucoamylase is a glycoprotein and has an optimum pH around 4.5, optimum temperature 60°C, a molecular weight of 55,600 daltons, and K_m values of 15.8, 27.6, 16.8 mg/mL for soluble starch, glycogen, and amylopectin, respectively. Unlike the other fungal glucoamylases, which were found (from culture filtrates) to exist in multiple forms, A. rouxii glucoamylase, isolated from solid substrate fermentation, displayed only one form.     

AFM Images of Complexes between Amylose and Aspergillus niger Glucoamylase Mutants,Native and Mutant Starch Binding Domains: A Model for the Action of Glucoamylase

Atomic force microscopy has been used to investigate the complexes formed between high molecular weight amylose chains and Aspergillus niger glucoamylase mutants (E400Q and W52F), wild‐type A. niger starch binding domains (SBDs), and mutant SBDs (W563K and W590K) lacking either of the two starch binding sites. The images are interpreted in terms of a favourable binding between the amylose chains and the wild‐type SBDs, leading to the formation of ring‐like structures, in which parallel strands of the amylose molecule bind to both binding sites on the SBDs. The SBDs are seen to form a template for the assembly of an expanded amylosic double helix. This model for amylose‐SBD binding has been used to propose a molecular mechanism for the role of the SBD in the hydrolytic action of glucoamylase on starch granules. The SBDs are considered to recognise the ends of amylosic double helices formed by the short amylosic chains present as branches on the amylopectin molecules, and displayed on the face of crystalline lamellae. This allows the binding and immobilisation of chain ends by the SBD, facilitating binding and cleavage by the exo‐acting catalytic domain.     

Immobilization of Aspergillus niger Mycelium on Seeds for Glucoamylase Production

Mycelium of the glucoamylolytic mutant Aspergillus niger C-58-III was immobilized on wheat, rye, barley, pea, buckwheat and mustard seeds in repeated-batch flasks. After every 24th it was possible to obtain culture broth rich in glucoamylase. The highest yield of enzyme (66.4 U × ml⁻¹) was obtained on the mustard carrier. Immobilized cells were successfully reused with high level of enzyme formation being mantained for longer period (192h). Some of the variables influencing the enzymatic activity have been standardized. Enzyme productivity reached in immobilized cells of A. niger was 1.6-times higher in comparison with free cells.     

EXPRESSION OF THE STA2 GLUCOAMYLASE GENE OF SACCHAROMYCES CEREVISIAE IN BREWERS' YEAST

Two fragments of DNA containing the Saccharomyces cerevisiae STA2 glucoamylase gene, with differing lengths of 5î non-coding DNA, were separately subcloned into a yeast centromeric plasmid. Of these two subclones, only the shorter one (containing 127 base-pairs of 5î non-coding DNA) was able to confer glucoamylase production on a standard laboratory strain of S. cerevisiae. The longer subclone (containing 465 bp of 5î non-coding DNA) did, however, confer glucoamylase production on a strain of S. cerevisiae lacking a functional STA10 gene (which encodes a repressor of STA2 gene expression). All-yeast plasmids lacking bacterial DNA were constructed from the two STA2 subclones for the transformation of a lager brewing yeast. Only the shorter STA2 subclone conferred glucoamylase activity on this yeast. The level of enzyme activity was comparable to that produced by the same yeast strain containing STA2 expressed from the PGK1 (that is, PGK1) promoter.     

Synthesis of Glucoamylase by Somatic Diploids of Aspergillus niger in Submerged Culture Conditions

Out of twelve forced heterocaryons of A. niger, in nine cases somatic diploids were obtained comprising a total of 107 strains. All were initially examined with respect to general amylase activity in test-tube microcultures. Over 21% of all diploid recombinants reached a higher coefficient of amylase activity than the parent haploid highly active in this respect. Four most active diploids selected from this group were examined with regard to glucoamylase synthesis. Also in this case diploids were characterized by a certain predominance in their activity over the initial prototrophic haploid strain (maximally about 16%).     

Some Pectinolytic and Cellulolytic Enzyme Activities of Fungi Causing Rots of Cocoyams

Representative isolates of Aspergillus niger, Botryodiplodia theobromae, Corticium rolfsii, Geotrichum candidum, Fusarium oxysporum and F solani recovered from rotten cocoyams were studied for the production of pectinolytic and cellulolytic enzymes. All the isolates elaborated high levels of hydrolase, lyase and pectinesterase in cocoyam tissue medium and lyase and pectinesterase in pectin medium. There were no significant differences in the overall levels of lyase and pectinesterase activities produced by all the isolates in both media. The level of hydrolase, lyase and pectinesterase activities individually produced by A niger, B theobromae and C rolfsii in both media was significantly higher than that of any other isolate. The highest hydrolase activity was produced by C rolfsii in cocoyam medium while A niger produced the highest lyase activity in pectin medium. Maximum pectinesterase activity was obtained from B theobromae and C rolfsii in pectin medium. All the test isolates produced cellulase in a medium containing carboxymethyl cellulose with C rolfsii showing significantly high activity followed by F oxysporum and A niger. © 1997 SCI.     

Degradation of aflatoxin B1 by fungal laccase enzymes

The enzymatic degradation of aflatoxin B₁ (AFB₁) by white rot fungi through laccase production was investigated in different liquid media. A significant (P < 0.0001) correlation was observed between laccase activity and AFB₁ degradation exhibited by representatives of Peniophora and Pleurotus ostreatus cultivated in minimal salts (MSM) (r = 0.93) and mineral salts — malt extract (MSB–MEB) (r = 0.77) liquid media. Peniophora sp. SCC0152 cultured in MSB–MEB liquid medium supplemented with veratryl alcohol and sugarcane bagasse showed high laccase activity (496 U/L), as well as 40.45% AFB₁ degradation as monitored using high performance liquid chromatography. P. ostreatus St2-3 cultivated in MSM liquid medium supplemented with veratryl alcohol resulted in laccase activity of 416.39 U/L and 35.90% degradation of AFB₁. Aflatoxin B₁ was significantly (P < 0.0001) degraded when treated with pure laccase enzyme from Trametes versicolor (1 U/ml, 87.34%) and recombinant laccase produced by Aspergillus niger D15-Lcc2#3 (118 U/L, 55%). Aflatoxin B₁ degradation by laccase enzyme from T. versicolor and recombinant laccase enzyme produced by A. niger D15-Lcc2#3 coincided with significant (P < 0.001) loss of mutagenicity of AFB₁, as evaluated in the Salmonella typhimurium mutagenicity assay. The degradation of AFB₁ by white rot fungi could be an important bio-control measure to reduce the level of this mycotoxin in food commodities.     

Production,partial purification and properties of β‐mannanases obtained by solid substrate fermentation of spent soluble coffee wastes and copra paste using Aspergillus oryzae and Aspergillus niger

In order to achieve a higher added value of two galactomannan‐containing wastes, copra paste and spent coffee from the soluble coffee industry (SCW), solid substrate fermentation (SSF) was used. Filamentous fungi Aspergillus oryzae and A niger were used to evaluate the feasibility of producing β‐mannanase by SSF. A 2³ factorial design was used to select the best interaction among the two fungi, the two substrates and two fermentation times. The treatment ‘A niger–copra–2.5 days’ produced a significantly higher (p < 0.05) β‐mannanase activity, having five different isoforms of the enzyme, one of which was partially purified to a specific activity of 764 U mg⁻¹ (U = nmol of mannose released per second from a galactomannan substrate). Copra paste had a higher mannose/galactose ratio (14:1) than SCW (6:1), and low oil content, which led to higher β‐mannanase production from SSF. A β‐mannanase from SSF of copra produced by A oryzae was highly purified using acetone precipitation and cation exchange and size exclusion chromatographies. This enzyme had an MW of 110 kDa, a pI between 3.5 and 4.5 and a specific activity of 1760 U mg⁻¹; purification achieved was 90.7 times. The temperature and pH for optimal activity were 40 °C and 6.0 respectively. The optimal temperature was lower and the optimal pH higher than others previously reported (produced by submerged fermentation), which could be important for viscosity reduction of concentrated coffee extract in instant coffee manufacture. Copra is an interesting alternative for β‐mannanase production, since it is readily available in Mexico; moreover, the residue after SSF has a reduced galactomannan content and may be used for monogastric animal feed. © 2000 Society of Chemical Industry     

Multiplicity of Glucoamylase of Aspergillus oryzae Part 1. Separation and Purification of Three Forms of Glucoamylase

The glucoamylase system of Aspergillus oryzae cultured on wheat bran was separated into 3 active fractions by (NH₄)₂SO₄, rivanol and ethanol precipitation followed by ion exchange chromatography on DEAE-Sephadex A-25. One of these fractions, referred to as glucoamylase I was purified by further chromatography on hydroxyapatite gel and Sephadex G-200. The other two fractions referred to as glucoamylase II and III were purified by further gel filtration on Sephadex G-200. The purified glucoamylases were found to be homogeneous on 7.5% polyacrylamide gel electrophoresis and isoelectric focusing by carrier ampholites.     

Investigation of the use of ram horn hydrolysate for fungal protein production

Production of the fungus Aspergillus niger NRRL 330 was studied in submerged fermentation with ram horn hydrolysate (RHH) as substrate. The characteristics of RHH have been reported previously. The RHH was enriched by addition of glucose and KH₂PO₄. The effects of kinetic parameters on the biomass yield of the fungus were investigated. The optimal conditions for growth of A niger on RHH were initial pH 6.5, temperature 30 °C, fermentation time 96 h and agitation speed 150 rpm. Under these optimal conditions the initial carbohydrate content of RHH was reduced from 1.52 to 0.2% and the biomass yield was 8.9 g l^?1. The biomass contained about 48.1% protein, 5.2% fat and 9.4% ash (on a dry weight basis). The amino acid content of the biomass was compared with Food and Agricultural Organisation (FAO) and animal feed standards. The protein produced contained all the essential amino acids for animal feed, but the amounts of these amino acids were somewhat lower than those of FAO and soybean reference protein. However, the amino acid composition of the biomass was better than that of animal feed. The results with RHH were also compared with previously reported data on fungal mycelium grown on waste liquor substrate. In conclusion, it was found that RHH could be used as a substrate in the production of fungal protein for use as animal feed. Copyright © 2003 Society of Chemical Industry