Cultures Of Aspergillus Terreus Research Articles

Photoclick reaction for rapid and simple fluorescence detection of itaconic acid and its derivatives in fungal cultures.

Itaconic acid (IA) and its derivatives produced by fungi have significant potential as industrial feedstocks. We recently developed a method for the detection of these compounds based on their terminal C-C double bonds. However, the presence of reducing agents, such as glucose and other fungal metabolites, leads to undesirable side reactions, and consequently, deteriorates thedetection specificity. Therefore, we developed a fluorescence detection method for IA and its derivatives underpinned by a photoclick reaction. The photoclick reaction between conjugated IA and 5-(4-methoxyphenyl)-2-phenyl-2H-tetrazole under UV irradiation affords a fluorescent product. No fluorescence was detected when succinic acid was subjected to the reaction, indicating that a terminal C-C double bond is required to induce fluorescence. Optimal reaction conditions were determined to be a combination of 80% final dimethyl sulfoxide concentration, 30-s UV irradiation, and a pH of 2. Two weeks after the reaction at 4°C, 89.0% of the initial intensity was retained, indicating that the reaction product was relatively stable. Glucose and kojic acid did not induce fluorescence after the reaction, indicating that these reducing agents did not affect fluorescence. IA was detected in a culture of Aspergillus terreus, and its quantification using the photoclick reaction was in agreement with the results obtained using high-performance liquid chromatography analysis. Interestingly, the IA derivative avenaciolide present in submillimolar quantities was also detectable in a culture of Aspergillus avenaceus using this method. The established method will enable the development of high-throughput screening methods to identify fungi that produce IA and its derivatives.

Hydroxylation of kaurenoic acid by Aspergillus terreus

In the present work the microbial transformation of kaurenoic acid (1) was performed using submerged shaken liquid culture of Aspergillus terreus (1.5×107 spores/mL). The microorganism was grown by a two-stage fermentation procedure. Compound 1 was added as a dimethylsulfoxide solution (0.1g/L) and incubated for 14 days. The culture was filtered and the aqueous layer was extracted with ethyl acetate to furnish the extract codified as AtKA. Chemical and NMR studies of AtKA allowed us to isolate and to identify four kaurenoic acid hydroxylated derivatives (Compounds 2–5).

Recombinant α-l-rhamnosidase from Aspergillus terreus in selective trimming of rutin

α-l-Rhamnosidase (EC 3.2.1.40) is a biotechnologically important enzyme used for derhamnosylation of many natural compounds. The extracellular α-l-rhamnosidase was purified from the culture of Aspergillus terreus grown on l-rhamnose-rich medium. This enzyme was found to be thermo- and alkali-tolerant, able to operate at 70°C and pH 8.0. The α-l-rhamnosidase cDNA was cloned from A. terreus, sequenced, and expressed in the yeast Pichia pastoris as a fully functional protein. The recombinant protein was purified to apparent homogeneity and biochemically characterized. Both the native and the recombinant α-l-rhamnosidases catalyzed the conversion of rutin into quercetin-3-glucopyranoside (isoquercitrin), a pharmacologically significant flavonoid usable in nutraceutics. This procedure has high volumetric productivity (up to 300g/L) and yields the product void of unwanted quercetin. The significant advantage of our expression system consists in shorter production times, up to fourfold increase in enzyme yields and the absence of unwanted β-d-glucosidase as compared to the native production system. Thanks to its unique properties, this enzyme is applicable in a selective synthesis/hydrolysis of various rhamnose containing structures.

Medium optimization for protopectinase production by batch culture of Aspergillus terreus

Turkey including its native wood species with high commercial value are recognised internationally. This work presents information about physical and mechanical properties and the use of lesser-known native wood species. This article’s aim is to familiarize wood expert outside Turkey with the wood of Silver Lime by comparing its physical and mechanical properties with some of the world’s most recognized hardwood species. The sample trees were harvested from a mixed oak-hornbeam-Silver Lime stand, western part of Turkey. Conventional destructive methods were followed on small clean specimens. Silver Lime wood’s air dry (526), oven dry (504) and basic (415 kgm-³) densities were determined. Fiber saturation point was calculated as 32.287%; volumetric shrinkage and swelling were found as 13.398 and 20.401%; while bending strength (MOR), modulus of elasticity (MOE), compression strength parallel to grain, impact bending, tensile strength parallel and perpendicular to grain, shear strength, cleavage strength, Janka hardness values (parallel and perpendicular to grain) and surface roughness (Ra) value were determined as 72.026 N/mm2, 5206.026 N/mm2, 50.531 N/mm2, 6.708 J/cm2, 90.521 N/mm2, 2.692 N/mm2, 11.742 N/mm2, 0.598 N/mm2, 0.636 and 0.569 kN, and 9.051 (µm), respectively. Further research should be used to determine the properties and suitable end uses of lesser known species that are likely to be beneficial to the country. Key words: Tilia argentea Desf., Silver Lime, wood properties, density, surface roughness.

Hydrolysis of galacto-oligosaccharides in soy molasses by α -galactosidases and invertase from Aspergillus terreus

Two α -galactosidase (P1 and P2) and one invertase present in the culture of Aspergillus terreus grown on wheat straw for 168 h at 28ºC were partially purified by gel filtration and hydrophobic interaction chromatographies. Optimum pH and temperatures for P1, P2 and invertase preparations were 4.5-5.0, 5.5 and 4.0 and 60, 55 and 65ºC, respectively. The K M app for Ï� -nitrophenyl-α -D-galactopyranoside were 1.32 mM and 0.72 mM for P1 and P2, respectively, while the K M app value for invertase, using sacarose as a substrate was 15.66 mM. Enzyme preparations P1 and P2 maintained their activities after pre-incubation for 3 h at 50ºC and invertase maintained about 90% after 6 h at 55 ºC. P1 and P2 presented different inhibition sensitivities by Ag+, D-galactose, and SDS. All enzyme preparations hydrolyzed galacto-ologosaccharides present in soymolasses.

Influence of ultrasound amplitude and duty cycle on fungal morphology and broth rheology of Aspergillus terreus

Effects of ultrasound amplitude and duty cycle on cultures of Aspergillus terreus are reported in a 25 l slurry bubble column sonobioreactor. Fermentations were carried out batchwise. A 2 k -factorial design with added central points was used. Sonication at any cycle and amplitude level did not affect biomass growth rate and yield relative to nonsonicated control, but did affect growth morphology. Ultrasound disrupted fungal pellets and caused the biomass to grow mainly as dispersed hyphae. Production of lovastatin was reduced by medium- and high-cycle sonication. Sonication affected broth rheology. In view of these results, sonication can be used to modify growth morphology and broth rheology without affecting growth rate and yield of filamentous fungi.

Enhancement of Lovastatin Production by Supplementing Polyketide Antibiotics to the Submerged Culture of Aspergillus terreus

Feedback inhibition existed in lovastatin biosynthesis from Aspergillus terreus. Exogenous lovastatin and other different polyketide antibiotics biosynthesized by polyketide synthase were supplemented to the cultures of A. terreus to investigate their influences on lovastatin production. Supplementing exogenous lovastatin of 100 mg l(-1) at the early stage of fermentation and the fast stage of its biosynthesis resulted in decreases of 76.4% and 20% in final lovastatin production, respectively. However, the fungal cell growth was not affected; the growing cycle was only prolonged in the submerged cultivation. Separate supplementation of the five kinds of polyketide antibiotics such as tylosin, erythromycin, tetracycline, daunorobin, and rifamycin to the cultures resulted in increases of about 20 approximately 25% in the final lovastatin production. Especially, supplementing tylosin of 50 mg l(-1) at the beginning of lovastatin biosynthesis led to the final lovastatin production of 952.7 +/- 24.3 mg l(-1), which was improved by 42% and 22% compared with that produced in the control and the original culture, respectively. These results are helpful to understand the regulations on lovastatin biosynthesis and improve the final desired metabolite contents in many antibiotics production.

Clinicopathologic and Diagnostic Imaging Characteristics of Systemic Aspergillosis in 30 Dogs

Systemic aspergillosis is a serious disease of dogs for which the clinical characteristics are poorly described. To describe the clinical and diagnostic imaging characteristics of dogs with systemic aspergillosis. Thirty dogs with systemic aspergillosis. Retrospective case review. Medical records were reviewed for signalment, clinical features, and results of clinicopathologic testing and diagnostic imaging. Diagnosis was confirmed by culture of Aspergillus terreus (n = 13), Aspergillus deflectus (n = 11), or other Aspergillus spp. (n = 6). Compared with the background hospital population, German Shepherd dogs and female dogs were overrepresented (odds ratio [OR] 43, 95% confidence interval [CI] 20-91, P < .0001, and OR 2.9, 95% CI 1.2-6.7, P= .02), respectively, with 20 of the 30 dogs being German Shepherd dogs and 77% (23 of 30) of the dogs being female. The median age was 4.5 years (range 2-8 years). Anemia, leukocytosis, hyperglobulinemia, azotemia, hypercalcemia, and hypoalbuminemia were present in 8, 21, 12, 9, 8, and 6 dogs, respectively. Diskospondylitis, osteomyelitis and thoracic lymphadenomegaly were present in 16, 10, and 5 dogs, respectively. Sonographic findings were enlarged hypoechoic lymph nodes (n = 12), mottled and irregular kidneys with or without masses (n = 12), pyelectasia, and an aggregate of echogenic material in the renal pelvis (n = 9). Thirteen dogs were treated with antifungal drugs, with survival times ranging from 0 to 25 months after diagnosis. Systemic aspergillosis typically involves young to middle-age female German Shepherd dogs, and there are characteristic abdominal ultrasound findings with the disease process. Infection with A. deflectus was as common as A. terreus, and in rare cases, long-term survival was associated with antifungal therapy.

Sesterterpenoids, Terretonins A−D, and an Alkaloid, Asterrelenin, from Aspergillus terreus

Four new sesterterpenoids, terretonins A-D (1-4), and a new alkaloid, asterrelenin (5), together with five known compounds, were isolated from the ethyl acetate extract of a solid-state fermented culture of Aspergillus terreus. Their structures were elucidated on the basis of spectroscopic analysis. The structures of 1, 2, and 5 were confirmed by X-ray crystallographic analysis.

Pellet morphology, culture rheology and lovastatin production in cultures of Aspergillus terreus

Pellet growth of Aspergillus terreus ATCC 20542 in submerged batch fermentations in stirred bioreactors was used to examine the effects of agitation (impeller tip speed u t of 1.01–2.71 m s −1) and aeration regimens (air or an oxygen-enriched mixture containing 80% oxygen and 20% nitrogen by volume) on the fungal pellet morphology, broth rheology and lovastatin production. The agitation speed and aeration methods used did not affect the biomass production profiles, but significantly influenced pellet morphology, broth rheology and the lovastatin titers. Pellets of ∼1200 μm initial diameter were reduced to a final stable size of ∼900 μm when the agitation intensity was ≥600 rpm ( u t ≥ 2.03 m s −1). A stable pellet diameter of ∼2500 μm could be attained in less intensely agitated cultures. These large fluffy pellets produced high lovastatin titers when aerated with oxygen-enriched gas but not with air. Much smaller pellets obtained under highly agitated conditions did not attain high lovastatin productivity even in an oxygen-enriched atmosphere. This suggests that both an upper limit on agitation intensity and a high level of dissolved oxygen are essential for attaining high titers of lovastatin. Pellet size in the bioreactor correlated equally well with the specific energy dissipation rate and the energy dissipation circulation function. The latter took into account the frequency of passage of the pellets through the high shear regions of the impellers. Pellets that gave high lovastatin titers produced highly shear thinning cultivation broths.

Microbial production of testosterone and testololactone in the culture of Aspergillus terreus

Two metabolites were obtained by microbial transformation of androstendione in the culture of Aspergillus terreus PTCC 5283, a fungus isolated from soil. Their structures were established as testosterone and testololactone on the basis of the spectral data including 1H NMR, 13C NMR, FTIR, MS and physical constants such as melting point and optical rotation. Aspergillus terreusproduced both metabolites after 3 days incubation at 27 °C. The bioconversion reactions observed were 17-carbonyl reduction and biological Baeyer–Villiger oxidation.

Fermentation optimization for the production of lovastatin by Aspergillus terreus: use of response surface methodology

AbstractA Box–Behnken experimental design was used to investigate the effects of five factors—ie oxygen content in the gas phase; concentrations of C, N and P; and fermentation time—on the concentrations of biomass and lovastatin produced in batch cultures of Aspergillus terreus. The values of the various factors in the experiment ranged widely, as follows: 20–80% (v/v) oxygen in the aeration gas; 8–48 g dm−3 C‐concentration; 0.2–0.6 g dm−3 N‐concentration; 0.5–2.5 g dm−3 phosphate‐concentration; and 7–11 days fermentation time. No previous work has used statistical analysis in documenting the interactions between oxygen supply and nutrient concentrations in lovastatin production. The Box–Behnken design identified the oxygen content in the gas phase as the principal factor influencing the production of lovastatin. Both a limitation and excess of oxygen reduced lovastatin titers. A medium containing 48 g dm−3 C supplied as lactose, 0.46 g dm−3 N supplied as soybean meal, and 0.79 g dm−3 phosphate supplied as KH2PO4, was shown to support high titers (∼230 mg dm−3) of lovastatin in a 7‐day fermentation in oxygen‐rich conditions (80% v/v oxygen in the aeration gas). Under these conditions, the culture medium had excess carbon but limiting amounts of nitrogen. The optimized fermentation conditions raised the lovastatin titer by four‐fold compared with the worst‐case scenario within the range of factors investigated. Copyright © 2004 Society of Chemical Industry

Biosynthesis of ML-236C and the hypocholesterolemic agents compactin by Penicillium aurantiogriseum and lovastatin by Aspergillus terreus: determination of the origin of carbon, hydrogen and oxygen atoms by 13C NMR spectrometry and observation of unusual labelling of acetate-derived oxygens by 18O2

Sodium [1-13C, 2-2H3]-, [2-13C, 2-2H3]- and [1-13C, 18O2]-acetate are incorporated in separate experiments into ML-236C 2 and the hypocholesterolemic agent compactin 3 by cultures of Penicillium aurantiogriseum, and the regiochemical distribution of 2H, 13C and 18O is determined by 13C NMR spectrometry. In addition, sodium [1-13C, 18O2]acetate and 18O2 are incorporated into lovastatin (mevinolin) 4 by cultures of Aspergillus terreus to re-examine the origin of oxygen atoms. The results show that the main-chain oxygen atoms of 2–4 originate from acetate, and the C-8 oxygen atom of 3 and 4 is derived from molecular oxygen. However, detailed mass spectral analysis shows that significant amounts of aerobic oxygen can be incorporated at sites normally labelled by acetate oxygen, presumably through generation of [18O]acetate by ω-oxidation of fats. On the basis of labelling results, a mechanism is proposed to account for the formation of the bicyclic ring system in these compounds.

Acetylcholinesterase Inhibition by Territrem B Derivatives

Five derivatives of territrem B [2], a potent acetylcholinesterase inhibitor isolated from a rice culture of Aspergillus terreus, were prepared from 2 as well as from its major metabolite, 4 beta-hydroxymethyl-4 beta-demethylterritrem B [4]. The inhibitory activities of these derivatives against electric eel acetylcholinesterase (E.C. 3.1.1.7) were tested and it was concluded that the enone and the pyrone groups present in 2 may play an important biological role.

Itaconic acid production using an air-lift bioreactor in repeated batch culture of Aspergillus terreus

Repeated itaconic acid production using an air-lift bioreactor was carried out by three methods—two with cell recycling by means of centrifugation and filtration by a stainless steel filter set inside the bioreactor and one by repeated batch culture without cell recycling. In a flask culture, repeated itaconic acid production was stable for 9 cycles (45 d) and the production rate was 0.47 g/ l/h. However, in the air-lift bioreactor, it was difficult to produce itaconic acid in the repeated batch culture with cell recycling for a long period due to a decrease in fluidity resulting from an increase in mycelium concentration. In the method without cell recycling, however, repeated itaconic acid production was stable for 4 cycles (21 d) and the production rate was 0.37 g/ l/h.

Effects of territrem-B on cholinergic responses of snail neuron

Effects of territrem-B (TRB), a mycotoxin isolated from a rice culture of Aspergillus terreus, on the central neuron of the snail Achatina fulica were studied electrophysiologically. Territrem-B potentiated the acetylcholine (ACh) induced current of the neuron, while it had no effect on GABA or l-glutamate elicited currents. TRB and neostigmine increased the peak amplitude of the response elicited by the first perfusion of ACh and depressed the increase in current produced by a second perfusion. TRB and neostigmine showed different dose-dependent effects on ACh responses. The results suggested that TRB is a good tool for studying the physiological role of AChE in central neurons.

Mutants of a lovastatin-hyperproducingAspergillus terreus deficient in the production of sulochrin

A lovastatin-hyperproducing culture ofAspergillus terreus was shown to produce several co-metabolites extracted from whole broth. The predominant co-metabolite was the benzophenone, sulochrin, reported to arise from a polyketide biosynthetic pathway. This compound was targeted for elimination by classical mutagenesis and screening. A surface culture method employing microtiter, plates was used to ferment mutants for the primary screen. Qualitative determinations of lovastatin and sulochrin production were achieved by high-performance thin-layer chromatography. A mutant, strain AH6, which produced lovastatin titers equivalent to the parent culture and no detectable sulochrin was isolated. In addition, a lovastatin-hyperproducing mutant designated CB4 was capable of producing 16% more lovastatin and 30% less sulochrin than the parent culture in shake flask fermentations. In a pilot-scale 250-gallon fermentation, strain CB4 gave a 20% increase in lovastatin titer while producing 83% less sulochrin than the parent culture.

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.

Protein enrichment of sugar cane by-products using solid-state cultures of Aspergillus terreus

Solid state cultures of Aspergillus terreus were carried out at different temperatures using sugar cane trash blended with salts and diluted molasses. A phase of respiration related to spore germination was observed. The effect of temperature on mold growth was evaluated in terms of lag phase, μmax and Xmax values. The germination phase was more sensitive to temperature changes than the apical growth phase. Satisfactory protein content (9–10% dry matter) and protein yield (23% of spent sugars) were observed when the temperature was regulated within a medium range (25–35°C). Preliminary data for sealing-up studies regarding temperature distribution and CO 2 evolution are presented.

Application of stable isotope labelling methodology to the biosynthesis of the mycotoxin, terretonin, by aspergillus terreus: incorporation of 13C-labelled acetates and methionine, 2H- and 13C, 18O-labelled ethyl 3,5-dimethylorsellinate and oxygen-18 gas

Incorporation of 13C-labelled acetates and methionine, 14C and 2H-labelled ethyl 3,5-dimethylorsellinate into terretonin (5) by cultures of Aspergillus terreus indicated that its biosynthesis proceeds via a mixed polyketide-terpenoid (meroterpenoid) pathway. Incorporation of 18O2 gas and ethyl 3,5-dimethylorsellinate (7) doubly labelled with 13C and 18O in the carbonyl of the carboxyl group and at the C-6 position into terretonin (5) and observation of 18O isotope-induced shifts in the 13C n.m.r. spectra and GC/MS studies of the enriched metabolites determined the origin of all of the oxygen atoms in (5) and provided mechanistic insight into the biosynthetic pathway.