Purification and characterization of glucoamylase of Aspergillus terreus NA-170 mutant

A catabolite-derepressed mutant strain of Aspergillus terreus NA-170 was isolated by multi-step mutagenesis. The mutant produced an appreciable yield of glucoamylase in the culture medium. The enzyme was purified and was shown to be homogeneous. It hydrolysed successive glucose residues from the non-reducing end of starch molecules. The purified enzyme had an optimum pH of 5·0 and was stable over the pH range 3·0–7·0. It was highly active over a broad temperature range, 30–75°C, with optimal activity at 60°C. The molecular weight was 70 000 as determined by Sephadex G-200 filtration. The enzyme showed a decrease in K _m values with increasing chain length of the substrate molecule.     

Phytase from Aspergillus terreus

1) Aspergillus terreus No. 9A-1 was cultivated by a shaking method and the optimal cultural conditions for the phytase production were concluded as follows: Composition of medium; rice bran 30 g, ammonium sulfate 3 g, distilled water 1.0 liter; initial pH 5.5; shaking condition; 50 ml of medium/500 ml vol. flask; 120 oscil./min, 90 hr.

2) Phytase from Asp. terreus was purified by ammonium sulfate precipitation, acetone precipitation and chromatography on SE-Sephadex C-50 and Sephadex G-200 columns. The enzyme was purified about 520-folds with the yield of 20% from the broth. The purified enzyme was homogeneous by column chromatography, ultracentrifugation and electrophoresis.

3) This purified preparation of phytase showed following properties, a) Optimal pH for the reaction was 4.5; b) optimal temperature for the reaction was about 70°C; c) the enzyme was stable in the range of pH from 1.2 to 9.0     

Purification of glucoamylase fromAspergillus terreus

Glucoamylase from a rice bran culture ofAspergillus terreus was purified by chromatography on DEAE-cellulose and concanavatin A-Sepharose. A homogenous monomer resulted after SDS-PAGE electrophoresis. The enzyme was a glycoprotein, molecular weight, 86,000 with 7.5% (w/w) carbohydrate content.

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Résumé La glucoamylase d'Aspergillus terreus cultivee sur son de riz a été purifiée par chromatographie sur DEAE-cellulose et sur A-sepharose à concavaline. On a obtenu un monomère homogène après électrophorèse sur SDS-PAGE. L'enzyme est une glycoproténe d'un poids moléculaire de 86,000 avec un contenu en polysacchardie de 7.5% (p/p).

     

Emodin O-methyltransferase from Aspergillus terreus

Emodin O-methyltransferase, an enzyme catalyzing methylation of the 8-hydroxy group of emodin, was identified in the mould Aspergillus terreus IMI 16043, a (+)-geodin producing strain. The enzyme catalyzed the formation of questin from emodin and S-adenosyl-l-methionine. By chromatography on DEAE-cellulose, Phenyl Sepharose, Q-Sepharose, Hydroxyapatite, and CM-cellulose, emodin O-methyltransferase was purified to apparent homogeneity. The purified protein had a molecular weight of 322 kDa as estimated by gel filtration and 53.6 kDa as estimated by gel electrophoresis under denaturing conditions, suggesting that the active enzyme was a homohexamer. The enzyme showed pI 4.4 and optimum pH 7–8. Magnesium ion or manganese ion was not an absolute requirement, nor increased the enzyme activity. The enzyme had strict substrate specificity and very low Km values for both emodin (3.4×10^-7 M) and S-adenosyl-l-methionine (4.1×10^-6 M).Abbreviations EOMT emodin O-methyltransferase from A. terreus - SAM S-adenosyl-l-methionine - PAGE polyacrylamide gel electrophoresis     

New Sesquiterpenes from Aspergillus terreus Thom

Four neutral sesquiterpenes were isolated from the culture filtrate of of Aspergillus terreus THOM NO. 14 which produces two sesquiterpene antibiotics, terrecyclic acid A and terrecyclol. On the basis of spectroscopic data the structures of four neutral sesquiterpenes were elucidated. One of these is quadrone, a known antitumor substance, but isoquadrone, 8-hydroxyquadrone and 6-hydroxyisoquadrone are new sesquiterpenes. Although terrecyclic acid A exhibits antimicrobial and antitumor activities, these four scarcely show these activities.     

Culture condition for production of glucoamylase from rice bran byAspergillus terreus

Summary Optimal conditions for the production of glucoamylase from rice bran usingAspergillus terreus in stationary culture were a medium containing 20 g rice bran/l, 0.3% (w/v) (NH₄)₂SO₄ and 0.2% (w/v) peptone at 30°C with an initial pH of 3.0. Enzymatic activity was maximal after 4 d. Glucose was the major reducing sugar produced by hydrolysis of starch. Carbohydrates favouring induction of glucoamylase were, in order: maltose, starch, cellobiose, lactose, glucose, fructose and galactose. Amino acids, in particular glycine, lysine, isoleucine and histidine, were vital for glucoamylase synthesis. Tween 80 and Triton X-100 enhanced the growth but suppressed glucoamylase synthesis.

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Conditions de culture pour la production de glucoamylase à partir de son de riz parAspergillus terreus

Résumé Les conditions optimales pour la production de glucoamylase à partir de son de riz en utilisantAspergillus terreus en culture en état stationnaire, consistent en un milieu contenant 20 g de son de riz par litre, 0.3 % (poids/vol.) de (NH₄)₂ SO₄ et 0.2 % (poids/vol.) de peptone, à 30 °C avec un pH initial de 3.0. L'activité enzymatique est maximum après 4 jours. Le glucose est le principal sucre réducteur produit par hydrolyse de l'amidon. Les hydrates de carbone qui favorisent l'induction de la glucoamylase, sont, dans l'ordre: le maltose, l'amidon, la cellobiose, le lactose, le glucose, le fructose et le galactose. Les acides aminés, en particulier la glycine, la lysine, l'isoleucine et l'histidine sont vitales pour la synthèse de glucoamylase. Le tween 80 et le triton X-100 augmentent la croissance mais suppriment la synthèse de glucoamylase.

     

Purification and Properties of beta-Glucosidase from Aspergillus terreus

A beta-glucosidase (EC 3.2.1.21) from the fungus Aspergillus terreus was purified to homogeneity as indicated by disc acrylamide gel electrophoresis. Optimal activity was observed at pH 4.8 and 50 degrees C. The beta-glucosidase had K(m) values of 0.78 and 0.40 mM for p-nitrophenyl-beta-d-glucopyranoside and cellobiose, respectively. Glucose was a competitive inhibitor, with a K(i) of 3.5 mM when p-nitrophenyl-beta-d-glucopyranoside was used as the substrate. The specific activity of the enzyme was found to be 210 IU and 215 U per mg of protein on p-nitrophenyl-beta-d-glucopyranoside and cellobiose substrates, respectively. Cations, proteases, and enzyme inhibitors had little or no effect on the enzyme activity. The beta-glucosidase was found to be a glycoprotein containing 65% carbohydrate by weight. It had a Stokes radius of 5.9 nm and an approximate molecular weight of 275,000. The affinity and specific activity that the isolated beta-glucosidase exhibited for cellobiose compared favorably with the values obtained for beta-glucosidases from other organisms being studied for use in industrial cellulose saccharification.     

N-bromosuccinimide oxidation of a glucoamylase from Aspergillus saitoi

1. In order to elucidate the structure-function relation of a glucoamylase [EC 3.2.1.3, alpha-D-(1 leads to 4) glucan glucohydrolase] from Aspergillus saitoi (Gluc M1), the reaction of Gluc M1 with NBS was studied. 2. The tryptophan residues in Glu M1 were oxidized at various NBS/Gluc M1 ratios. The enzymatic activity decreased to about 80% of that of the native Gluc M1 with the oxidation of the first 2 tryptophan residues. The oxidation of these 2 tryptophan residues occurred within 0.2-0.5 s. On further oxidation of ca. 4-5 more tryptophan residues of Glu M1, the enzymatic activity of Gluc M1 decreased to almost zero (NBS/Gluc M1 = 20). Thus, the most essential tryptophan residue(s) is amongst these 4-5 tryptophan residues. 3. 7.5 tryptophan residues were found to be eventually oxidized with increasing concentrations of NBS up to NBS/Gluc M1 = 50. This value is comparable to the number of tryptophan residues which are located on the surface of the enzyme as judged from the solvent perturbation difference spectrum with ethylene glycol as perturbant. 4. In the presence of 10% soluble starch, about 5 tryptophan residues in Gluc M1 were oxidized at an NBS/Gluc M1 ratio of 20. The remaining activity of Glu M1 at this stage of oxidation was about 76%. On further oxidation, after removal of soluble starch, the enzymatic activity decreased to zero with the concomitant oxidation of 2 tryptophan residues. The results indicated that the essential tryptophan residue(s) is amongst these 2 tryptophans. 5. The UV difference spectrum induced by addition of maltose and maltitol to Gluc M1 showed 4 troughs at 281, 289, 297, and 303 nm. The latter 3 troughs were probably due to tryptophan residues of Gluc M1 and decreased with NBS oxidation.     

Purification and properties of dihydrogeodin oxidase from Aspergillus terreus

The last step of (+)-geodin biosynthesis is a phenol oxidative coupling, which is one of the most important reactions in biosynthesis of natural products. The enzyme named dihydrogeodin oxidase catalyzes the regio- and stereospecific phenol oxidative coupling reaction to form (+)-geodin from dihydrogeodin. The enzyme was purified from the cell-free extract of Aspergillus terreus, a (+)-geodin producer, by ammonium sulfate fractionation, acid treatment, and column chromatographies on DEAE-cellulose, Hydroxyapatite, chromatofocusing, and Toyopearl HW-55S. The purified enzyme was homogeneous as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 153,000 by gel filtration on a Toyopearl HW-55S column and 76,000 by SDS-polyacrylamide gel electrophoresis, indicating that the enzyme is a dimer. The purified enzyme showed an intense blue color and had absorption maxima at 280 and 600 nm, which suggested it to be a blue copper protein. The copper content was found to be 8 atoms per subunit by atomic absorption analysis and no significant amount of other metals was detected by ICP emission spectrometry. The electron paramagnetic resonance spectrum showed the presence of type 1 and type 2 copper atoms in the enzyme molecule. Sodium azide and ethylxanthate inhibited the enzyme activity, but potassium cyanide and diethyldithiocarbamate, both known as potent copper enzyme inhibitors, were not inhibitory.     

Crystallization and Characterization of Polyamine Oxidase from Aspergillus terreus

A thin-layer flow cell system for the determination of l-ascorbic acid by an ascorbate electrode was constructed and several components of this system were investigated. The most preferable conditions for optimum operation of the system were as follows: injection volume 150 μ1, delay coil length 60 cm, flow rate 1 ml/min, temperature 20°C, cell spacer thickness 0.2 mm. The linear response region was 0.2-3.0 mm and 0.02-0.5 mm (original l-ascorbic acid concentration) in the cases of pure oxygen and atmospheric oxygen bubbling, respectively. The relative standard variation at 1.5 mm l-ascorbic acid was 3.1 % for 20 successive assays. The measuring time was 2–3 min for each of these assays.     

Abnormal form of Aspergillus terreus isolated from mycotic abscesses

A remarkable outer cell-wall thickening (up to 1.5 m) was observed on septate hyphae obtained from pus collected from multiple abscesses of a 25-year-old female patient. Ultrastructural examination of the hyphae showed a thick electron dense layer of microfibrillar material surrounding the electron transparent cell wall. The organism was able to grow only on hypertonic media upon initial isolation but on later subculture it grew on normal isotonic media. The thick microfibrillar material diminished progressively upon subculture but could be demonstrated in 7 day secondary cultures in isotonic liquid medium. There, microfibrillar bridges appeared to bind hyphae together. The observations suggested that this microfibrillar material was a true extracellular component. The immunological status of the patient was not examined, but her 10 year history of multiple mycotic abscesses and dermatophytoses suggested some abnormalities.     

Production and purification of statins from Aspergillus terreus strains

Lovastatin, mevastatin, pravastatin and monacolin J were produced using Aspergillus terreus strains. Mevastatin (170 mg/l) was obtained at 14 days from the A1 strain, lovastatin (256 mg/l) at 21 days from the A2 strain and pravastatin (270-300 mg/l) at 14 days from both the A1 and A2 strains grown on defatted soybean flour. Similar yields of monacolin J (5-10 mg/l) were detected for both strains. Fermentation carried out by adding glycerol to A1 7-d old cultures gave 244 mg lovastatin/l at 14 days employing whole soybean flour. A new extraction procedure was applied to an A2 19-d old culture on the mycelium and the culture filtrate separately. Recovery yield showed that 83% lovastatin was associated with the mycelium and 17% was free in the culture filtrate. © Rapid Science Ltd. 1998     

Induction of glucoamylase production by non-starchy carbohydrates inAspergillus terreus

Glucoamylase production inAspergillus terreus was induced, in order, by glucose, cellobiose, sorbitol, sucrose, -methyl mannoside and -methyl glucoside. Optimal induction was at 38°C, pH 4.0 and with 8 mg glucose/ml. Cycloheximide at 10 g/ml completely inhibited induction indicatingde novo protein synthesis was involved in induction of glucoamylase.

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Résumé La production de gluco-amylase est induite chezAspergillus terreus, en ordre décroissant, par le glucose, le celloboise, le sorbitol, le sucrose, l'-methylmannoside et l'-methylglucoside. L'induction est optimum à 38°C, pH 4.0 et en présence de 8 mg de glucose parml. La cycloheximide à 10 g par ml inhibe complètement l'induction, ce qui implique use synthèse de protéinede novo dans l'induction de la gluco-amylase.

     

Producton of single-cell protein from cellulose by Aspergillus terreus

Cellulose fermentation studies were conducted with a thermotolerant strain of Aspergillus terreus. Batch cultivation of A. terreus using purified or complex cellulose showed that 80-88% of the available cellulose was utilized in 30-36 h with an average doubling time of 7.5-8.3 h. The protein content in the biomass ranged from 23 to 38%. Semicontinuous cultivation studies, in which 90% of the biomass was withdrawn at the end of growth cycle, indicated that 84% of added cellulose was utilized with the biomass containing 32% crude protein. No loss in cellulose consumption, growth rate, or protein production occurred through two growth cycles. Continuous cultivation of A. terreus showed that 78-84% cellulose consumption occurred over growth temperatures ranging from 35 to 45 degrees C. Maximum specific growth rates (0.14 h(-1)) occurred at 40 and 45 degrees C with a minimum doubling time of 4.9 h.     

Action of glucoamylase from Aspergillus niger on phosphorylated substrate

Glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) from Aspergillus niger was purified to be free from alpha-amylase and phosphatase (glucose 6-phosphate as substrate). The phosphatase was well separated from the glucoamylase by phosphocellulose ion-exchange chromatography. The glucoamylase action was prevented by the esterified phosphate groups of the substrate. Thus, the extensive action of the glucoamylase on potato starch exposed the 6-posphorylglucosyl residue of the starch at the non-reducing terminal and large molecular weight limit dextrins remained. The concomitant action of the phosphatase was necessary for the complete degradation of the starch.     

Chemical and Physicochemical Properties of Phytase from Aspergillus terreus

Some chemical and physicochemical properties of the purified phytase preparation produced by Asp. terreus were investigated. From the results of the examination of amino acid analysis, it was suggested that there existed some components other than amino acids in the purified enzyme. Examination of the neutral sugar analysis, therefore, was made by gaschromatography, and it was found that the purified enzyme preparation contained mannose, galactose and a small amount of inositol.

The molecular weight of the enzyme was found to be 214,000 by the Archibald method, and 2.2~2.3×10⁵ by gel-filtration on a Sephadex G–200 column. It was found that by guanidine hydrochloride or by urea, the purified enzyme preparation was dissociated into only one kind of subunit. The native enzyme was supposed to be a homohexamer of the subunits whose molecular weight is 37,000.