Isolation and characterisation of Aspergillus awamori BS05, a root-knot-nematode-trapping fungus

Nematode-trapping fungi are important biocontrol agents against parasitic nematodes through adhesive or mechanical hyphal traps. Aspergillus awamori, a root-knot-nematode-trapping fungus from tomato rhizosphere soil, was identified based on morphology and molecular characteristics of internal transcribed spacer DNA sequence. Conidial heads were white to black brown, loosely globose, and 72–127 μm in diameter. Conidiophores usually arose from the foot cell of basal mycelium, straight, and 960–1730 × 10.2–13.4 μm, hyaline to pale brown, not constricted below the vesicles; vesicles hemispherical to elongate, 43–56 μm in diameter, black brown, fertile over the upper half to two-thirds. Aspergilla were biseriate, and metulae were variable, 12–26 × 3.8–4.7 μm; phialides were 8.2–9.4 × 2.5–3 μm. Conidia were globose or subglobose, 3.6–4.8 μm in diameter, rough, grey brown and parallel in chains. A. awamori BS05 showed 44.9% control efficacy against Meloidogyne incogtina in pot experiments which suggests it as a potential biocontrol agent against Meloidogyne. This is the first report on A. awamori as nematode-trapping fungus.     

SSF production,purification and characterization of chitin deacetylase from Aspergillus flavus

The production of an extracellular chitin deacetylase (CDA) produced by Aspergillus flavus under solid-substrate fermentation (SSF) using wheat bran as substrate was optimized using statistical methods. The CDA production in SSF increased 1.79-fold in comparison to the unoptimized basal level medium. It was purified to a final purity of 3.94-fold by ammonium sulphate precipitation, ion-exchange chromatography, and gel-permeation chromatography (GPC) consecutively and further characterized. The molecular mass of the enzyme was estimated to be about 28?kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and GPC analysis. The optimum pH and temperature of the purified enzyme were pH 8.0 and 50?°C, respectively. Additionally, the effect of some cations and other chemical compounds on the CDA activity was studied. A marginal increase in enzyme activity was observed with metal ions mainly Mn²⁺ and Zn²⁺. No inhibition of the enzyme was observed by the end product, that is, acetate up to 70?mM concentration. The K_m and k_cat values of the enzyme were determined to be 9.45?mg mL^?1 and 26.72?s^?1 respectively, using colloidal chitin as substrate. Among various substrates tested, glycol chitin and colloidal chitin were deacetylated.     

Production of Aflatoxins B1 and G1 by Aspergillus flavus and Aspergillus parasiticus Isolated from Market Pecans

One hundred and forty-eight isolates of Aspergillus flavus and A. parasiticus were isolated from 5,608 pecans obtained from Chicago and Georgia markets. The percentage of internal contamination by these species was 7.3% in the Chicago market pecans and 1.7% in those from markets in Georgia. Of the 148 isolates, 93% of the A. parasiticus, but only 54% of the A. flavus, were capable of producing aflatoxin. Overall, 57% of the isolates were potentially aflatoxigenic. A. parasiticus isolates generally produced a greater amount of aflatoxins than A. flavus.     

Production and purification of a granular-starch-binding domain of glucoamylase 1 from Aspergillus niger

A domain of glucoamylase 1 from Aspergillus niger which binds to granular starch was produced by proteolytic digestion and purified to apparent homogeneity by extraction with corn starch followed by anion-exchange chromatography and gel filtration. The peptide has a molecular weight of 25,100, contains approximately 38% carbohydrate (w/w) and corresponds to residues 471-616 at the C-terminus of glucoamylase 1. The peptide bound to granular corn starch maximally at 1.08 nmol/mg starch. It inhibited the hydrolysis of granular starch by glucoamylase 1 but had no effect on the hydrolysis of starch in solution.     

Production,purification and characterization of a thermostable laccase from a tropical white-rot fungus

A thermostable laccase was isolated from a tropical white-rot fungus Polyporus sp. which produced as high as 69,738 units of laccase l⁻¹ in an optimized medium containing 20 g of malt extract l⁻¹, 2 g of yeast extract l⁻¹, 1.5 mM CuSO₄. The laccase was purified to electrophoretic purity with a final purification of 44.70-fold and a recovery yield of 21.04%. The purified laccase was shown to be a monomeric enzyme with a molecular mass of 60 kDa. The optimum temperature and pH value of the laccase were 75°C and pH 4.0, respectively, for 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS). The Michaelis–Menten constant (K _m ) of the laccase was 18 μM for ABTS substrate. The laccase was stable at pH values between 5.5 and 7.5. About 80% of the initial enzyme activity was retained after incubation of the laccase at 70°C for 2 h, indicating that the laccase was intrinsically highly thermostable and with valuable potential applications. The laccase activity was promoted by 4.0 mM of Mg²⁺, Mn²⁺, Zn²⁺ and Ca²⁺, while inhibited by 4.0 mM of Co²⁺, Al³⁺, Cu²⁺, and Fe²⁺, showing different profiles of metal ion effects.     

Production, purification, and characterization of human alpha1 proteinase inhibitor from Aspergillus niger

Human alpha one proteinase inhibitor (alpha1-PI) was cloned and expressed in Aspergillus niger, filamentious fungus that can grow in defined media and can perform glycosylation. Submerged culture conditions were established using starch as carbon source, 30% dissolved oxygen concentration, pH 7.0 and 28 degrees C. Eight milligrams per liter of active alpha1-PI were secreted to the growth media in about 40 h. Controlling the protein proteolysis was found to be an important factor in the production. The effects of various carbon sources, pH and temperature on the production and stability of the protein were tested and the product was purified and characterized. Two molecular weights variants of the recombinant alpha1-PI were produced by the fungus; the difference is attributed to the glycosylated part of the molecule. The two glycoproteins were treated with PNGAse F and the released glycans were analyzed by HPAEC, MALDI/TOF-MS, NSI-MS(n), and GC-MS. The MALDI and NSI- full MS spectra of permethylated N-glycans revealed that the N-glycans of both variants contain a series of high-mannose type glycans with 5-20 hexose units. Monosaccharide analysis showed that these were composed of N-acetylglucos-amine, mannose, and galactose. Linkage analysis revealed that the galactosyl component was in the furanoic conformation, which was attaching in a terminal non-reducing position. The Galactofuranose-containing high-mannnose type N-glycans are typical structures, which recently have been found as part of several glycoproteins produced by Aspergillus niger.     

Antioxidant-related catalase CTA1 regulates development,aflatoxin biosynthesis,and virulence in pathogenic fungus Aspergillus flavus

Reactive oxygen species (ROS) induce the synthesis of a myriad of secondary metabolites, including aflatoxins. It raises significant concern as it is a potent environmental contaminant. In Aspergillus flavus., antioxidant enzymes link ROS stress response with coordinated gene regulation of aflatoxin biosynthesis. In this study, we characterized the function of a core component of the antioxidant enzyme catalase (CTA1) of A. flavus. Firstly, we verified the presence of cta1 corresponding protein (CTA1) by Western blot analysis and mass-spectrometry based analysis. Then, the functional study revealed that the growth, sporulation and sclerotia formation significantly increased, while aflatoxins production and virulence were decreased in the cta1 deletion mutant as compared with the WT and complementary strains. Furthermore, the absence of the cta1 gene resulted in a significant rise in the intracellular ROS level, which in turn added to the oxidative stress level of cells. A further quantitative proteomics investigation hinted that in vivo, CTA1 might maintain the ROS level to facilitate the aflatoxin synthesis. All in all, the pleiotropic phenotype of A. flavus CTA1 deletion mutant revealed that the antioxidant system plays a crucial role in fungal development, aflatoxins biosynthesis and virulence.     

Production of Aflatoxins B1 and G1 by Aspergillus flavus in a Semisynthetic Medium

Isolates of Aspergillus flavus produced 0.2 to 63 mg of aflatoxins B(1) and G(1) per 100 ml in a nutrient solution consisting of 20% sucrose and 2% yeast extract. Various factors influencing the fermentation were studied. The maximal amount of toxin was produced by ATCC culture 15548 in 1-liter flasks containing 100 ml of medium incubated as stationary cultures for 6 days at 25 C.     

Gene cloning, purification, and characterization of a novel peptidoglutaminase-asparaginase from Aspergillus sojae

Glutaminase is an enzyme that catalyzes the hydrolysis of l-glutamine to l-glutamate, and it plays an important role in the production of fermented foods by enhancing the umami taste. By using the genome sequence and expressed sequence tag data available for Aspergillus oryzae RIB40, we cloned a novel glutaminase gene (AsgahA) from Aspergillus sojae, which was similar to a previously described gene encoding a salt-tolerant, thermostable glutaminase of Cryptococcus nodaensis (CnGahA). The structural gene was 1,929 bp in length without introns and encoded a glutaminase, AsGahA, which shared 36% identity with CnGahA. The introduction of multiple copies of AsgahA into A. oryzae RIB40 resulted in the overexpression of glutaminase activity. AsGahA was subsequently purified from the overexpressing transformant and characterized. While AsGahA was located at the cell surface in submerged culture, it was secreted extracellularly in solid-state culture. The molecular mass of AsGahA was estimated to be 67 kDa and 135 kDa by SDS-PAGE and gel filtration chromatography, respectively, indicating that the native form of AsGahA was a dimer. The optimal pH of the enzyme was 9.5, and its optimal temperature was 50°C in sodium phosphate buffer (pH 7.0). Analysis of substrate specificity revealed that AsGahA deamidated not only free l-glutamine and l-asparagine but also C-terminal glutaminyl or asparaginyl residues in peptides. Collectively, our results indicate that AsGahA is a novel peptidoglutaminase-asparaginase. Moreover, this is the first report to describe the gene cloning and purification of a peptidoglutaminase-asparaginase.     

Gene cloning, purification, and characterization of a heat-stable phytase from the fungus Aspergillus fumigatus.

The finding of heat-stable enzymes or the engineering of moderately thermostable enzymes into more stable ones by random or site-directed mutagenesis has become a main priority of modern biotechnology. We report here for the first time a heat-stable phytase able to withstand temperatures up to 100 degrees C over a period of 20 min, with a loss of only 10% of the initial enzymatic activity. The gene (phyA) encoding this heat-stable enzyme has been cloned from Aspergillus fumigatus and overexpressed in Aspergillus niger. The enzyme showed high activity with 4-nitrophenyl phosphate at a pH range of 3 to 5 and with phytic acid at a pH range of 2.5 to 7.5.     

Production of SCP and cellulase by Aspergillus terreus from bagasse substrate

The fermentation of 1.0% untreated bagasse under optimum cultural and nutritional conditions with Aspergillus terreus GN1 indicated that the maximum rate of protein and cellulase production could be obtained during three days of submerged fermentation. Even though 16.4% protein recovery, 0.55 units CMCase/mL, and 0.027 FPase units/mL were obtained on the seventh day, the rates of increase in protein recovery and cellulase production were slower than those obtained up to these days, which were 14.3% protein recovery, 0.45 units CMCase/mL, and 0.019 units FPase/mL. There was an initial lag in the utilization of cellulose up to two days due to the utilization of the water-soluble carbohydrate present in untreated bagasse. Cellulose utilization and water-soluble carbohydrate content during fermentation were correlated with protein recovery and enzyme production. The protein and cellulase production during three days fermentation with 1.0% untreated and treated bagasse were compared and the protein content of the total biomass was calculated and treated bagasse were compared and the protein content of the biomass was calculated into constituent protein contributed by the fungal mycelium and the under graded bagasse. The total biomass recovered with untreated and treated bagasse was 1020 and 820 mg/g bagasse substrate, respectively, and contained 14.3 and 20.6% crude protein, respectively. The contribution of fungal biomass and under graded bagasse was 309 and 711, and 373 and 447 mg/g untreated and treated bagasse substrates, respectively. In an 8-L-flask trial during three days of fermentation, the recovery of SCP and cellulase were 66 g and 32,400 units (Sigma) for treated bagasse and 82 g and 8200 units (Sigma) for untreated bagasse, respectively.     

Production, partial purification and characterization of feruloyl esterase by Aspergillus awamori in submerged fermentation

Microbial feruloyl esterases acting on plant cell wall polymers represent key tools for the degradation of plant cell wall. In this paper, we describe in detail the microbial production, partial purification and characterization of feruloyl esterase from a culture medium of Aspergillus awamori strain IFO4033 obtained from a crude hemicellulose preparation of wheat straw, corncobs and wheat germ. Feruloyl esterase was extracted using centrifugation and dialysis, and then purified by ion exchange chromatography and microfiltration to homogeneity, which was checked by SDSPAGE and isoelectric focusing-PAGE. Protein content and activity of the enzyme were measured in each step of extraction and purification. Biomass was determined by the dry weight method. pH and temperature optima of feruloyl esterase enzyme were also determined. The effects of culturing time, and carbon and nitrogen sources on enzyme production were systematically investigated. Finally, enzyme activities under different storage conditions were examined.     

Production, purification, and characterization of xylanase from a hyperxylanolytic mutant of Aspergillus ochraceus

Enhancement of the productivity of xylanase and beta-xy-losidase of Aspergillus ochraceus was investigated by multistep mutagenesis. The spores of the wild strain were subjected to UV and N-methyl-N-nitro-N-nitro-soguanidine (NTG). The hyperxylanolytic mutant (NG-13), which showed good clearing on the surface of the xylan-agar plate, secretes xylanase and beta-xylosidase at high levels during growth on commercial xylan and on agricultural wastes. Both liquid and solid state cultures were employed in the study for enzyme production. The xylanase from NG-13 was purified to homogeneity by ammonium sulfate precipitation and gel filtration. This purified enzyme showed a pH optimum of 6.0 and was stable in the range of pH 5 to 10. Prolonged stability of the enzyme was observed at 45 degrees C though its activity was maximal at 50 degrees C. The molecular weight of the enzyme was estimated to be 4.3 x 10(4) by SDS-polyacrylamide gel electrophoresis and 5 x 10(4) by gel filtration on Sephadex G-75. The kinetic data showed that the K(m) and V(max) values for xylan were 1 x 10(-3)M and 19.6 mumol/ min/mg protein, respectively. The enzyme was both more active and thermostable in the presence of K(+)and was inactivated by thiol reagents such as Hg(2+), p-hydroxymercuribenzoate (PHMB), 3', 5'-dithiobis (2'-nitrobenzoic acid) (DTNB), and N-ethylmaleimide (NEM).     

Purification and partial characterization of an elastinolytic proteinase from Aspergillus flavus culture filtrates

 A 23-kDa protein with elastinolytic activity was purified from Aspergillus flavus (NRRL 18543) culture filtrates by gel-filtration chromatography. Severe inhibition of the elastinolytic activity by 1,10-phenanthrolene (5 mM) and EDTA (0.8 mM) indicated that the protein belongs to the metallo class of proteases. The isoelectric point was 9.0. Natural substrates susceptible to cleavage by this protease, in addition to elastin, included cottonseed storage protein, collagen, ovalbumin and bovine serum albumin. The 23-kDa protein was thermostable to 70°C and retained its elastinolytic activity in concentrated form at 4°C for 6 months. Elastinolytic activity was initially secreted into the culture medium as a 35-kDa protein, which was subsequently converted to a 23-kDa protein, presumably through autolysis. This putative proteolytic degradation product appears to be identical to the 23-kDa protein recovered from the gel-filtration column. The 23-kDa protease may confer selective advantage to the fungus in the extracellular environment because of its temperature and pH stability and wide range of potential natural protein substrates. Received: 24 October 1995/Received last revision: 27 March 1996/Accepted: 30 March 1996     

黄曲酶尿酸氧化酶突变体的构建、表达、纯化及生物学活性分析

采用融合PCR的方法将黄曲霉尿酸氧化酶(UOX) 基因的307~309 bp的TGC(Cys) 突变为GCC(Ala),将所获得的突变体基因克隆到原核表达质粒pET-42a(+) 后转化大肠杆菌BL21(DE3)。经IPTG诱导,突变体蛋白 (UOX-Ala103) 得到高水平的可溶性表达,目的蛋白占总蛋白含量的45％。疏水柱及阴离子柱纯化后,UOX-Ala103蛋白纯度＞98％。Western blotting分析证实UOX-Ala103能与抗UOX单抗特异结合。与天然型相比较,其体外生物学活性增加约60％,在高尿酸血症小鼠模型体内也有良好的降解尿酸的活性。     

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     

Production of aflatoxin B1 from defined natural cultures of Aspergillus flavus (link)

Some more economical means of producing pure samples of aflatoxin B₁ from local materials is described. Our results indicate that there is, however, no correlation between the C/N ratio of the materials investigated and the amount of aflatoxin B₁ produced. Pawpaw (Carica papaya) appears the best substrate of those examined in this study.     

Production and partial purification of a peptide antibiotic from Staphylococcus epidermidis

When grown on solid or in liquid Brain Heart Infusion at 37°C, Staphylococcus epidermidis NCIB 11536 produced antibiotic activity against a wide range of Gram positive bacteria. Production was influenced by aeration, pH, glucose concentration and specific growth rate. Inhibitory activity could be concentrated by ammonium sulphate precipitation (30–55% saturation). On Sephadex G50 using 0.05 mol/1 sodium phosphate buffer, pH 6.0, two peaks of antibiotic activity were detected. The first peak eluted with the void volume (K_d= 0) and the second peak was retained by the gel (K_d= 0.73–0.77). These two substances did not represent the monomeric and polymeric forms of a staphylococcal bacteriocin. The low mol. wt inhibitor, which was responsible for over 95% of the recovered activity on Sephadex G50, could be partially purified by a combination of gel filtration on Biogel P2 and ion-exchange chromatography on Sephadex C-25. Yields were increased by combining these two steps into a single procedure (duocolumn). The semi-purified inhibitor was desalted using Sep-pak C₁₈ cartridges. Biological activity was resistant to enzymic denaturation except by high concentrations of trypsin (50 units/μg, 3 h, 25°C). This peptide antibiotic is different from any previously described staphylococcal inhibitors.