Metabolism of biphenyl by Aspergillus toxicarius: induction of hydroxylating activity and accumulation of water-soluble conjugates.

Biphenyl metabolism in Aspergillus toxicarius occurs by successive hydroxylations in the 4- and 4'-positions, followed by conjugation with sulfate to produce 4-hydroxybiphenyl-O-sulfonic acid and 4,4'-dihydroxybiphenyl-O-sulfonic acid. The hydroxylation reactions normally occur only after a prolonged lag period after which the appearance of the monohydroxylated compound precedes the dihydroxylated compound. The accumulation of the monohydroxy compound is transient; therefore, it is an intermediate in the hydroxylating pathway. The onset of hydroxylating activity can be greatly accelerated when the culture is primed with the intermediate or product of the reaction (4-hydroxybiphenyl or 4,4'-dihydroxybiphenyl) at the time of biphenyl addition; a concentration of 0.05 mg 4,4'-dihydroxybiphenyl per ml produces optimal induction. Water-soluble conjugates of 4-hydroxybiphenyl and 4,4'-dihydroxybiphenyl were found in cultures of A. toxicarius grown in the presence of biphenyl plus inducer. The conjugate was shown to be the sulfate ester; no glucuronide or other conjugate species was found in any phase of the transformation. As with hydroxylating activity, the sulfotransferase activity appeared to be induced by the products of biphenyl metabolism.     

Hydroxylation of biphenyl by Aspergillus parasiticus: Approaches to yield improvment in fermenter cultures

We carried out experiments designed to increase the rate of production of 4,4'-dihydroxybiphenyl (biphenol) from biphenyl by Aspergillus parasiticus. We show that 0.5 mg/ml biphenyl, the substrate for the reaction, significantly inhibits growth of the organism and that at 0.04 mg/ml, 2-hydroxybiphenyl or 4-hydroxybiphenyl (an intermediate of the reaction) strongly inhibit oxygen uptake, probably by inhibition of mitochondrial electron transport. Both factors may contribute to the low hydroxylation rates observed previously [J. H. Golbeck and J. C. Cox, Biotechnol. Bioeng., 26, 434 (1984)]. We therefore adapted the organism to the presence of 0.08 mg/ml 2- and 4-hydroxybiphenyl in the growth medium and found that cultures of adapted strains hydroxylated biphenyl at rates ca. three-fold faster than control cultures. Once the fungal mycelia were grown, they could be recycled at least twice into fresh fermentation broth. Recycled organisms were capable of hydroxylating biphenyl more rapidly than cells in the primary fermentation culture and there was no lag period between introduction of biphenyl and the onset of hydroxylation. Cell recycle thus results in a considerable saving in carbon costs and fermentation time.     

The fluorescence of some biphenyl derivatives

1. The variation with pH of the excitation and fluorescence wavelengths and fluorescence intensity of several hydroxy- and amino-biphenyls has been investigated with a view to using the data for the determination of hydroxybiphenyls in biological material. 2. 2- and 3-Hydroxy-, 2,2'-dihydroxy-, and 2-, 3- and 4-amino-biphenyl exhibited excited-state ionization. 4-Hydroxy- and 4,4'-dihydroxybiphenyl did not, and showed normal fluorescence changes with pH. 3. Biphenyl and 2- and 4-methoxybiphenyl showed no changes in fluorescence in the range pH0-14. 4. 4,4'-Dihydroxybiphenyl showed fluorescent peaks corresponding to the un-ionized form, the monoanion and dianion at the appropriate pH values, but 2,2'-dihydroxybiphenyl showed fluorescent peaks corresponding to the un-ionized form and the monoanion only, suggesting that it did not form a dianion. 5. 3- and 4-Nitrobiphenyl were non-fluorescent. 6. These results are discussed.     

Enzymatic degradation of 2,6-dichlorophenol by horseradish peroxidase: UV-visible and mass spectrometry characterization of the reaction products [corrected

The reaction mechanism of the oxidation of 2,6-dichlorophenol (2,6-DCP) by horseradish peroxidase (HRP) and H2O2 has been investigated and the reaction products have been characterized by UV-visible and mass spectrometry. Evidence for the dimerization of 2,6-DCP to 3,3',5,5'-tetrachloro-4,4'-dihydroxybiphenyl and the subsequent fast oxidation of this product to the corresponding 3,3',5,5'-tetrachlorodiphenoquinone have been collected. The reaction rate was found to decrease markedly as soon as the pH was raised, with a clear inflection point at pH congruent with 6.6-6.9; it also resulted independent from H2O2 concentration. Since the pK(a) for 2,6-DCP is 6.80, the reaction rate might be influenced by the protonation state of the substrate.     

Detection of Bioactive Exometabolites Produced by the Filamentous Marine Cyanobacterium Geitlerinema sp.

Marine cyanobacteria are noted for their ability to excrete metabolites with biotic properties. This paper focuses on such exometabolites obtained from the culture of the marine filamentous cyanobacterium Geitlerinema sp. strain, their purification and subsequent analyses. By this means the recoveries of the active compounds, a prerequisite for properly determining their concentration, are quantified here for the first time. We demonstrate a new procedure using Amberlite XAD-1180 resin in combination with the eluent isopropanol for extraction of the culture media and gas chromatography as simplified chemical analysis. This procedure reduced necessary bacteria cultivation time (from 150 to 21?days) at low volumes of culture media (300?mL) required for identification of two selected bioactive compounds: 4,4'-dihydroxybiphenyl and harmane.     

Hydroxylations of Biphenyl by Fungi

Sixty-six species of fungi were screened for their ability to hydroxylate biphenyl. Thin-layer and gas chromatographic analyses indicated that seven organisms actively hydroxylated this substrate. Of particular interest were Cunninghamella echinulata ATCC 9244 which produced 4-hydroxybiphenyl as the major metabolite, Helicostylum piriforme QM 6945 which produced exclusively 2-hydroxybiphehyl, and Aspergillus parasiticus ATCC 15517 which produced a good yield of 4, 4'-dihydroxybiphenyl. These results were confirmed by isolation of the phenolic metabolites following preparative-scale conversions and comparisons of the isolated phenols with authentic reference materials.     

Anaerobic dehalogenation of hydroxylated polychlorinated biphenyls by Desulfitobacterium dehalogenans

Ten years after reports on the existence of anaerobic dehalogenation of polychlorinated biphenyls (PCBs) in sediment slurries, we report here on the rapid reductive dehalogenation of para-hydroxylated PCBs (HO-PCBs), the excreted main metabolites of PCB in mammals, which can exhibit estrogenic and antiestrogenic activities in humans. The anaerobic bacterium Desulfitobacterium dehalogenans completely dehalogenates all flanking chlorines (chlorines in ortho position to the para-hydroxyl group) from congeners such as 3,3',5, 5'-tetrachloro-4,4'-dihydroxybiphenyl.     

Antialgal, antibacterial and antifungal activity of two metabolites produced and excreted by cyanobacteria during growth

The antimicrobial activity of two cyanobacterial exometabolites, norharmane (9H-pyrido(3,4-b)indole) and 4,4'-dihydroxybiphenyl, was determined in suspension assays. Good anticyanobacterial activities (concentrations of 8-80 microg ml(-1)) and moderate antibacterial (16-160 microg ml(-1)) and antifungal (32-40 microg ml(-1)) activities were found for both compounds. The natural function as allelopathic chemicals and a possible applicability as antifouling agents or leads for the development of new antifouling chemicals are discussed.     

Metabolism of 2,2'- and 3,3'-dihydroxybiphenyl by the biphenyl catabolic pathway of Comamonas testosteroni B-356

The purpose of this investigation was to examine the capacity of the biphenyl catabolic enzymes of Comamonas testosteroni B-356 to metabolize dihydroxybiphenyls symmetrically substituted on both rings. Data show that 3,3'-dihydroxybiphenyl is by far the preferred substrate for strain B-356. However, the dihydrodiol metabolite is very unstable and readily tautomerizes to a dead-end metabolite or is dehydroxylated by elimination of water. The tautomerization route is the most prominent. Thus, a very small fraction of the substrate is converted to other hydroxylated and acidic metabolites. Although 2,2'-dihydroxybiphenyl is a poor substrate for strain B-356 biphenyl dioxygenase, metabolites were produced by the biphenyl catabolic enzymes, leading to production of 2-hydroxybenzoic acid. Data show that the major route of metabolism involves, as a first step, a direct dehydroxylation of one of the ortho-substituted carbons to yield 2,3,2'-trihydroxybiphenyl. However, other metabolites resulting from hydroxylation of carbons 5 and 6 of 2,2'-dihydroxybiphenyl were also produced, leading to dead-end metabolites.     

Production of calcium gluconate by fermentation.

Calcium gluconate production by Aspergillus niger was investigated in shake flask, rolling shaker, air-lift reactor and stirred reactor. Growth pattern of the organism and fermentation conditions determined the yield of the product. High calcium gluconate production was achieved in air-lift reactor with pellet form of cell growth at moderate specific growth rate and biomass concentration. In another variation of air-lift reactor, when calcium carbonate was confined to a cellulose membrane, calcium gluconate production was maximum (149 g/L). At higher specific growth rate, obtained in shake flask, despite the formation of cell pellets, product formation was low. Physical separation of particulate calcium carbonate and growing cells favoured product formation. In stirred reactor pulpy mycelial growth was obtained and calcium gluconate production was poor.     

Influence of the interaction of temperature and water activity on the production of ochratoxin A and the growth of Aspergillus niger, Aspergillus carbonarius and Aspergillus ochraceus on coffee-based culture medium

In the present study, the effect of temperature and water activity on fungal growth and ochratoxin production on coffee-based medium was assessed. Optimal growth of three Aspergillus strains was observed in the same ecological conditions, namely 30 degrees C and 0.99 water activity. Maximal daily growth is 11.2, 6.92, and 7.22 mm/day for Aspergillus niger, Aspergillus carbonarius, and Aspergillus ochraceus, respectively. However, ecological conditions for optimal ochratoxin production vary according to the toxinogenic strain, with water activity as a limiting factor. Such an ochratoxin A production is inhibited at 42 degrees C and 0.75 water activity. Correspondence between laboratory tested water activity and that measured on a sun-dried ripe cherry batch shows that the first 5 days of drying are critical for fungal growth and ochratoxin A production. Accordingly, artificial drying of cherries at temperatures above 42 degrees C will impede not only fungal growth but also contamination with ochratoxin A.     

Enzyme formation during solid-substrate fermentation in rotating vessels

Aspergillus awamori NRRL 4869 was cultured on the solid substrate, wheat bran, in a modified Rollacell apparatus to produce alpha-galactosidase and invertase. The swivel cap on the elongated bottle permits the introduction of air while the bottle rotates. Parameters of air flow rate (0.05-0.2 liter/kg/min), rpm (0.15-15 rpm), and weight of solids (150 and 300 g) were varied. At low air flow rates (0.05 liter/kg solids/min), alpha-galactosidase production was minimal independent of the rotation rate. At 0.15 rpm and 0.2 liter/kg solids/min air flow rate, invertase production ceased after five days; whereas alpha-galactosidase production continued. The modified Rollacell can be a useful apparatus for studying solid-substrate cultures.     

Rotating simplex method of optimization of physical parameters for higher production of extracellular pectinases in bioreactor

Rotating simplex method of optimization has been successfully used to determine the optimal conditions for extracellular pectinase production by Aspergillus niger in batch bioreactor. Maximum production of 1.132 U of polymethylgalacturonase, 1.313 U of polygalacturonase and 0.0091 U of pectinlyase was obtained at pH (controlled). 3.38; agitation rate, 483 rpm and aeration rate, 2.12 vvm. These conditions were found to be more suitable for pectinase production.     

Use of CAS-agar plate modified to study the effect of different variables on the siderophore production by Aspergillus

AIMS: To evaluate the suitability of chrome azurol S (CAS) agar plate assay as a quantitative methodology for siderophore production. METHODS AND RESULTS: Aspergillus species (A. flavus, A. niger, A. tamarii) were inoculated in the CAS-agar plates and the siderophores production was determined and expressed as CAS-reaction rate (mm per day). All the species showed positive CAS reaction with different rates depending on culture conditions and A. flavus showed the highest CAS-reaction rate. The siderophore production in solid medium expressed as CAS-reaction rate was correlated with siderophore production in liquid medium. CONCLUSIONS: The use of CAS-agar plate assay was modified and the evaluation of CAS reaction in mm per day made it possible to study and quantify the effect of several variables on the siderophore production by Aspergillus fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: We describe the CAS-agar plate assay as a quantitative methodology, which make it possible to select and evaluate the siderophore production by several microorganisms (fungi and bacteria) according to different culture conditions.     

Lignan compounds and 4,4'-dihydroxybiphenyl protect C2C12 cells against damage from oxidative stress

Lignan compounds are known to have various biological activities, especially antioxidative effects. We investigated whether lignan compounds show antioxidative activity in myoblast C2C12 cells. Among 14 lignan compounds investigated, two lignans containing two phenolic functional groups, namely Gomisin J and GR-12, prevented hydrogen peroxide (H(2)O(2))-induced cell death. A simple compound, 4,4'-dihydroxybiphenyl, which was found to be a common component of Gomisin J and GR-12, also largely prevented H(2)O(2)-induced cell death and almost completely prevented H(2)O(2)-induced increases in p38 MAPK phosphorylation. Our present results provide a useful in vitro system for clarifying the molecular mechanisms of lignan-mediated antioxidative effects and evaluating lead molecules toward the development of therapeutic drugs.     

N+注入选育黑曲霉益生菌及其突变菌株产酶条件的研究

以益生菌株黑曲霉AN01为材料,经N 多次诱变得突变益生菌株AN03。结果表明,出发益生菌株AN01酸性蛋白酶、纤维素酶和果胶酶的酶活分别由原来的71.6Ug、141.7Ug和264.8Ug相继提高到996.5Ug、940.4Ug和906.5Ug。突变益生菌株AN03经传5代培养,产酶特性稳定。试验还研究了变突变益生菌株AN03最佳产酶条件,培养基为每升含麸皮105g,玉米芯105g,豆粕105g,氯化铵16g,pH5.0。30℃培养4d。     

Optimization of culture conditions and bench-scale production of L-asparaginase by submerged fermentation of Aspergillus terreus MTCC 1782

Optimization of culture conditions for L-asparaginase production by submerged fermentation of Aspergillus terreus MTCC 1782 was studied using a 3-level central composite design of response surface methodology and artificial neural network linked genetic algorithm. The artificial neural network linked genetic algorithm was found to be more efficient than response surface methodology. The experimental L-asparaginase activity of 43.29 IU/ml was obtained at the optimum culture conditions of temperature 35 degrees C, initial pH 6.3, inoculum size 1% (v/v), agitation rate 140 rpm, and incubation time 58.5 h of the artificial neural network linked genetic algorithm, which was close to the predicted activity of 44.38 IU/ml. Characteristics of L-asparaginase production by A. terreus MTCC 1782 were studied in a 3 L bench-scale bioreactor.     

Physiological characterisation of recombinant Aspergillus nidulans strains with different creA genotypes expressing A. oryzae alpha-amylase.

The physiology of three strains of Aspergillus nidulans was examined--a creA deletion strain, a wild type creA genotype and a strain containing extra copies of the creA gene, all producing Aspergillus oryzae alpha-amylase. The strains were cultured in batch and continuous cultivations and the biomass formation and alpha-amylase production was characterised. Overexpression of the creA gene resulted in a lower maximum specific growth rate and a slightly higher repression of the alpha-amylase production during conditions with high glucose concentration. No expression of creA also resulted in a decreased maximum specific growth rate, but also in drastic changes in morphology. Furthermore, the expression of alpha-amylase was completely derepressed and creA thus seems to be the only regulatory protein responsible for glucose repression of alpha-amylase expression. The effect of different carbon sources on the alpha-amylase production in the creA deletion strain was investigated and it was found that starch was the best inducer. The degree of induction by starch increased almost linearly with the concentration of starch in starch/glucose mixtures. High-density batch cultivation was performed with the creA deletion strain and a final titre of 6.0 g l(-1) of alpha-amylase was reached after 162 h of cultivation.     

Continuous production of citric acid from dairy wastewater using immobilized<Emphasis Type="Italic">Aspergillus niger</Emphasis> ATCC 9142

The continuous production of citric acid from dairy wastewater was investigated using calcium-alginate immobilizedAspergillus niger ATCC 9142. The citric acid productivity and yield were strongly affected by the culture conditions. The optimal pH, temperature, and dilution rate were 3.0, 30°C, and 0.025 h⁻¹, respectively. Under optimal culture conditions, the maximum productivity, concentration, and yield of citric acid produced by the calcium-alginate immobilizedAspergillus niger were 160 mg L⁻¹ h⁻¹, 4.5 g/L, and 70.3% respectively. The culture was continuously perfored for 20 days without any apparent loss in citric acid productivity. Conversely, under the same conditions with a batch shake-flask culture, the maximum productivity, citric acid concentration, and yield were only 63.3 mg L⁻¹ h⁻¹, 4.7 g/L and 51.4%, respectively. Therefore, the results suggest that the bioreactor used in this study could be potentially used for continuous citric acid production from dairy wastewater by applying calcium-alginate immobilizedAspergillus niger.     

Effect of low oxygen concentrations on growth and alpha-amylase production of Aspergillus oryzae in model solid-state fermentation systems

Oxygen transfer in the fungal mat is a major concern in solid-state fermentation (SSF). Oxygen supply into the mycelial layers is hampered by diffusion limitation. For aerobic fungi, like Aspergillus oryzae, this oxygen depletion can be a severely limiting factor for growth and metabolite production. This paper describes the effects of a low oxygen concentration on growth at the levels of individual hyphae, colonies and overcultures, and on alpha-amylase production in overcultures. PDA medium was used to study the effect of a low oxygen concentration on hyphal elongation rate and branching frequency of hyphae, and radial extension rate of colonies of A. oryzae. We found similar saturation constants (K(O2)) of 0.1% (v/v in the gas phase) for oxygen concentration described with Monod kinetics, for branching frequency of hyphae and colony extension rate. When A. oryzae was grown as an over-culture on wheat-flour model substrate at 0.25% (v/v) oxygen concentration, the reduction in growth was more pronounced than as individual hyphae and a colony on PDA medium. Experimental results also showed that the specific alpha-amylase production rate under the condition of 0.25% (v/v) oxygen was reduced. Because the value of K(O2) is relatively low, it is reasonable to simplify the kinetics of growth of A. oryzae to zero-order kinetics in coupled diffusion/reaction models.