Production and composition of an exocellular Nigeran-protein complex isolated from cultures of Aspergillus awamori.

Aspergillus awamori Nakazawa (QM873) hyphae maintained on a nitrogen-deficient medium produced an exocellular complex consisting of the wall alpha-glucan, nigeran (94%), water-soluble nigeran oligosacchrides (1 to 2%), protein (2 to 4%), and a small amount of beta-glycan (less than 1%). Nigeran was not covalently linked to protein. The complex appeared in the growth medium only under those temporal or nutritional conditions where the hyphal wall content to nigeran reached at least 30% of the cell dry weight. Rates of nigeran accumulation in the hypha cell wall, scanning electron microscopy of hyphae, and pulse-chase experiments with [14C]glucose suggested that the complex arises via displacement of a portion of the hyphal wall into the medium. The nigeran portion of the complex contained lamellar crystalline domains similar to those in the intact cell wall. Enzymic digestion of nigeran in the complex indicated that it has a degree of crystallinity lower than that observed with pure nigeran crystals grown in vitro. Therefore, polymerization in vivo resulted in somewhat less chain organization in the crystallite. Since this complex was isolated without any prior chemical or exogenous enzymatic treatment, it should be useful for studies of hyphal wall biogenesis and organization in this organism.     

泡盛曲霉植酸酶的酶学性质研究

泡盛曲霉植酸酶作为动物饲料添加剂具有广泛的应用前景。以半固体发酵方式培养泡盛曲霉AS3.324(Aspergillus awamori),并得到纯化的植酸酶。对其酶学性质研究表明:其反应最适温度为50～55℃,最适pH为5.5,在37℃下以植酸钠为底物的Km值为1.05nmol/L,Vmax为2.16μmol/(L.min)。EDTA基本不影响植酸酶活性;Ca2 、Mg2 、Mn2 对植酸酶活性有轻微的抑制作用;Fe2 、Zn2 对酶促反应有显著的抑制作用。对该酶的耐热性研究表明,在较高温度条件处理后,仍有较高残余酶活性,与当今商品化的植酸酶相比,有较强的耐热性。     

Lipase from marine Aspergillus awamori BTMFW032: production, partial purification and application in oil effluent treatment

Marine fungus BTMFW032, isolated from seawater and identified as Aspergillus awamori, was observed to produce an extracellular lipase, which could reduce 92% fat and oil content in the effluent laden with oil. In this study, medium for lipase production under submerged fermentation was optimized statistically employing response surface method toward maximal enzyme production. Medium with soyabean meal-0.77% (w/v); (NH(4))(2)SO(4)-0.1m; KH(2)PO(4)-0.05 m; rice bran oil-2% (v/v); CaCl(2)-0.05 m; PEG 6000-0.05% (w/v); NaCl-1% (w/v); inoculum-1% (v/v); pH 3.0; incubation temperature 35°C and incubation period-five days were identified as optimal conditions for maximal lipase production. The time course experiment under optimized condition, after statistical modeling, indicated that enzyme production commenced after 36 hours of incubation and reached a maximum after 96 hours (495.0 U/ml), whereas maximal specific activity of enzyme was recorded at 108 hours (1164.63 U/mg protein). After optimization an overall 4.6-fold increase in lipase production was achieved. Partial purification by (NH(4))(2)SO(4) precipitation and ion exchange chromatography resulted in 33.7% final yield. The lipase was noted to have a molecular mass of 90 kDa and optimal activity at pH 7 and 40°C. Results indicated the scope for potential application of this marine fungal lipase in bioremediation.     

Yellow Pigments of Aspergillus niger and Aspergillus awamori

Alkaline degradation of Aurasperone A, C₃₂H₂₆O₁₀, gave a binaphthyl (IIa), m.p. 255°C and acetone. (IIa) afforded a tetraacetate (IIb), C₃₂H₃₀O₁₂ m.p. 219°C and a tetramethyl ether (IId), C₂₈H₃₀O₈, m.p. 188°C. These facts along with the NMR spectra of aurasperone A and (IIb) confirm that aurasperone A is a dimeric 2-methyl-5-hydroxy-6,8-dimethoxy-4H-naphtho[2,3-b]pyran-4-one with asymmetric C-C linkage (7-10′ or 9-10′). The ether (IId) is not identical with 1,1′ ,3,3′ ?6,6′ ,8,8′-octamethoxy-4,4′-binaphthyl. Thus, it follows that (IId) is a 2,4′-binaphthyl and hence aurasperone A is 2,2′-dimethyl-5,5′- dihydroxy-6,6′,8,8′-tetrahydroxy-7,10′-bi[4H-naphtho[2,3-b]pyran-4-one] (I).     

Purification and Characterization of a Feruloylesterase from Aspergillus awamori

A feruloylesterase was purified from the extracellular broth of Aspergillus awamori grown on wheat bran culture. The purified enzyme gave a single band on SDS-polyacrylamide gel electrophoresis and isoelectric focusing, with an apparent M _r of 35,000 and a pI of 3.8, respectively. The substrate specificity of the purified enzyme differed obviously from that of acetylesterase of A. awamori. The enzyme bound to microcrystalline cellulose.     

Acidophilic tannase from marine Aspergillus awamori BTMFW032

Aspergillus awamori BTMFW032, isolated from sea water, produced tannase as extracellular enzyme under submerged culture conditions. Enzyme with a specific activity of 2761.89 IU/mg protein, a final yield of 0.51 %, and a purification fold of 6.32 was obtained after purification to homogeneity by ultrafiltration and gel filtration. SDS-PAGE analyses under non- reducing and reducing conditions yielded a single band of 230 kDa and 37.8 kDa, respectively, indicating presence of six identical monomers. pI of 4.4 and 8.02 % carbohydrate content in the enzyme were observed. Optimal temperature was 30oC, although the enzyme was active at 5-80 oC. Two pH optima, pH 2 and pH 8, were recorded and the enzyme was stable only at pH 2.0 for 24 h. Methylgallate recorded maximal affinity and K(m) and V(max) were recorded, respectively, as 1.9 X 10?3 M and 830 micronmol/min. Impact of several metal salts, solvents, surfactants, and typical enzyme inhibitors on tannase activity were determined to establish the novelty of the enzyme. Gene encoding tannase isolated from A. awamori is 1.232 kb and nucleic acid sequence analysis revealed an open reading frame consisting of 1122 bp (374 amino acids) of one stretch in -1 strand. In-silico analyses of gene sequences and comparison with reported sequences of other species of Aspergillus indicated that the acidophilic tannase from marine A. awamori is differs from that of other reported species.     

Enzyme productivity from the protoplast regenerants of Aspergillus awamori

Protoplasts from the hydroiase enzyme producer Aspergillus awamori were isolated using Bulgarian enzyme preparations—Trichocease-SU and Xylanase. Among the regenerated colonies from protoplasts two were selected with enhanced productivity of xylanase, endoglucanase and β-glucosidase. The endoglucanase produced by the parent and one of the regenerants differ in their absorptivity, sensitivity to product inhibition and heat stability.     

Conversion of dextrinized cassava starch into ethanol using cultures of Aspergillus awamori and Saccharomyces cerevisiae

Aspergillus awamori and Saccharomyces cerevisiae have been used to convert dextrinized cassava root flour into ethanol. A batch culture of the combined microorganisms produced 4.3% alcohol by weight from 15% cassava flour slurry in 39 h. Two-stage continuous fermentation was done using A. awamori in an airlift fermenter and yeast in a tower fermenter. A residence time of 12.5 h for the first stage resulted in 12.5% sugar concentration and a saccharification efficiency of 88%. A residence time of 5.6 h for the second stage gave an alcohol concentration of 5.3% alcohol and a starch-into-ethanol conversion efficiency of 72.5%.     

Optimization of immobilization conditions for acid proteinase from Aspergillus awamori

To optimize the immobilization conditions for acid proteinase from Aspergillus awamori by covalent binding through glutaraldehyde, experiments were carried out using the Box-Wilson method. The optimization process was assessed on the basis of absolute activity A, coefficient of activity retention gamma and their product A gamma. The following conditions can be recommended: glutaraldehyde concentration 50--60 mg/g, enzyme concentration not less than 40 mg/g, time of glutaraldehyde treatment 2--2.5 hrs, immobilization time 2 hrs, pH about 4.0, and temperature 35--40 degrees C. Under these conditions A=220--230 U/g, gamma = 23--24% Agamma = 5,000--6,000.     

Characteristics of transxylosylation by beta-xylosidase from Aspergillus awamori K4

The Aspergillus awamori K4 beta-xylosidase gene (Xaw1) sequence was deduced by sequencing RT-PCR and PCR products. The ORF was 2,412 bp and the predicted peptide was 804 amino acids long, corresponding to a molecular weight of 87,156 Da. The mature protein was 778 amino acids long with a molecular weight of 84,632 Da. A homology search of the amino acid sequence revealed that it was very similar to the Aspergillus niger beta-xylosidase gene with only five amino acid differences. K4 beta-xylosidase had the same catalytic mechanism as family 3 beta-glucosidases, involving Asp in region A. At an early stage in the reaction with xylobiose and xylotriose, the hydrolysis rate was much lower than the transxylosylation rate, decreasing gradually as the substrate concentration increased, whereas the transxylosylation rate increased greatly. Aspergillus awamori K4 beta-xylosidase had broad acceptor specificity toward alcohols, hydroxybenzenealcohols, sugar alcohols and disaccharides. A consensus portion involving the hydroxymethyl group of the acceptor was confirmed in the major transfer products 1(4)-O-beta-D-xylosyl erythritol, (2-hydroxyl)-phenyl-methyl-beta-D-xylopyranoside, 6S-O-beta-D-xylosyl maltitol (S: sorbitol residue) and 6G-O-beta-D-xylosyl palatinose (G: glucosyl residue). This might suggest that the methylene in the hydroxymethyl group facilitates base-catalyzed hydroxyl group attack of the anomeric center of the xylosyl-enzyme intermediate.     

Comparative study of functional characteristics of mitochondrial preparations from cell cultures

A comparative study was performed of coupled oxidative phosphorylation in mitochondria and the composition of mitochondria from some cell cultures. The mitochondria from VNK-21 and Vero cells had a high degree of coupled oxidative phosphorylation, which was estimated by the respiratory control coefficient. The cell cultures can be regarded as a promising source of mitochondria to be used for toxicological control of the products of the microbiological synthesis.     

Comparative study of glycosidase from cattle liver and exoglycanase from Aspergillus awamori]

Anomerities of products were estimated for glucosidases from cattle liver and Aspergillus awamori. It was demonstrated that the enzyme from cattle liver is alpha-glucosidase and that from Asp. awamori is exogluconase. It was demonstrated that alpha-glucosidase hydrolyzes the C1--O bond in the course of reaction. delta-Lactone of gluconic acid is a competitive inhibitor for both enzymes. The secondary kinetic isotope effects for both enzymes were measured. The isotope effect for alpha-glucosidase is equal to 1, for exogluconase 1,1 for glycogen and 1,18 for maltose. Some aspects of mechanisms of both enzymes are discussed in terms of the data obtained.