Isolation,purification, and enzymatic activity of cellulase components of the fungus <Emphasis Type="Italic">Aspergillus terreus</Emphasis>

The accumulation dynamics of cellulolytic enzymes in culture media of the basidiomycete fungi Panus tigrinus, Pleurotus ostreatus, Fomes fomentarus, and the micromycete Aspergillus terreus were studied during a long incubation period. It was found that A. terreus was the most active producer of cellulolytic enzymes among the studied fungi. Two protein fractions with cellulase activity were isolated using gel filtration and ion-exchange chromatography. PAAG electrophoresis showed that fraction-I consisted of four components; fraction-II, an electrophoretically homogeneous protein. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 489–491, September–October, 2007.     

Response of Cellulase Activity in pH-Controlled Cultures of the Filamentous Fungus <Emphasis Type="Italic">Acremonium cellulolyticus</Emphasis>

Cellulase production was investigated in pH-controlled cultures of Acremonium cellulolyticus. The response to culture pH was investigated for three cellulolytic enzymes, carbomethyl cellulase (CMCase), avicelase, and β-glucosidase. Avicelase and β-glucosidase showed similar profiles, with maximum activity in cultures at pH 5.5–6. The CMCase activity was highest in a pH 4 culture. At an acidic pH, the ratios of CMCase and avicelase activity to cellulase activity defined by filter paper unit were high, but at a neutral pH, the β-glucosidase ratio was high. The pH 6.0 culture showed the highest cellulase activity within the range of pH 3.5–6.5 cultures. The saccharification activity from A. cellulolyticus was compared to those of the cellulolytic enzymes from other species. The A. cellulolyticus culture broth had a saccharification yield comparable to those of the Trichoderma enzymes GC220 and Cellulosin T2, under conditions with the same cellulase activity. The saccharification yields from Solka floc, Avicel, and waste paper, measured as the percent of released reducing sugar to dried substrate, were greater than 80% after 96 h of reaction. The yields were 16% from carboxymethylcellulose and 26% from wood chip refiner. Thus, the A. cellulolyticus enzymes were suitable for converting cellulolytic biomass to reducing sugars for biomass ethanol production. This study is a step toward the establishment of an efficient system to reutilize cellulolytic biomass.     

Xanthothone,a new nematicidal N-compound from <Emphasis Type="Italic">Coprinus xanthothrix</Emphasis>

Coprinus xanthothrix was found to have nematicidal activity. Xanthothone was isolated from culture extract guided by activity assay, which was identified as a novel natural product. Two other compounds were also isolated. These compounds showed nematicidal activity, with LD₅₀ value of 125–250 ppm both against Panagrellus redivivus and Meloidogyne incognita. Published in Khimiya Prirodnykh Soedinenii, No. 2, pp. 161–162, March–April, 2008.     

Cellulolytic enzymes produced by the edible mushroom,Pleurotus sajor-caju

Pleurotus sajor-caju grows efficiently and degrades all the components present in lignocellulosic residues. Production of cellulase and xylanase enzymes in submerged culture and during solid state cultivation has been studied. An initial pH of 5.0 was found to be optimal for the production of cellulase in shake flasks; this was attained in about 6–8 d in a medium containing either cellulose or rice straw as the sole source of carbon. On the cellulose medium, the maximum filter paper activity attained was 0.15 IU/mL in 7 d whereas the endoglycanase activity of 1.0 IU/mL, xylanase activity of 1.55 IU/mL, and Β-glucosidase activity of 0.57 IU/mL were acheived after 9 d fermentation. The reducing sugars were absent in the culture medium. The cellulases (filter paper activity and endoglucanases) were most active at pH 5.0 and 45‡C. Xylanase had maximum activity at pH 4.8 and 45‡C, and Β-glucosidase at pH 5.5 and 40‡C. In shake cultures,P. sajor-caju produced dispersed suspension of short mycelial threads and various sizes of pellets. The profile and extent of enzyme biosynthesis during submerged cultivation on rice straw was found to be of the same nature as obtained on cellulose. During solid state cultivation ofP. sajor-caju on rice straw beds for 36 d, the elaboration of enzyme activities did not appear to follow any definite pattern. However, filter paper activity, which is representative of cellulase action in hydrolyzing cellulose, remained more or less constant during the period of about the first 20 d of cultivation after the appearance of fruit bodies on the surface of rice straw beds. All the activities attained their minimum values after 23 d of cultivation, during which approximately 1 kg of fresh fruit bodies had been harvested. The total fruit bodies harvested till 36th days were approx. 1.1 kg. ThroughT. sajor-caju elaborates cellulase and xylanse extracellularly, the activity values were not as high as those of other cellulase producers such asTrichoderma reesei.     

Thermal study of TiO<Subscript>2</Subscript>–CeO<Subscript>2</Subscript> yellow ceramic pigment obtained by the Pechini method

TiO₂–CeO₂ oxides for application as ceramic pigments were synthesized by the Pechini method. In the present work the polymeric network of the pigment precursor was studied using thermal analysis. Results obtained using TG and DTA showed the occurrence of three main mass loss stages and profiles associated to the decomposition of the organic matter and crystallization. The kinetics of the degradation was evaluated by means of TG applying different heating rates. The activation energies (E _a) and reaction order (n) for each stage were determined using Horowitz–Metzger, Coats–Redfern, Kissinger and Broido methods. Values of E _a varying between 257–267 kJ mol^–1 and n=0–1 were found. According to the kinetic analysis the decomposition reactions were diffusion controlled.     

Enzyme Production by Solid Substrate Fermentation of <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> and <Emphasis Type="Italic">Trametes versicolor</Emphasis> on Tomato Pomace

A process of solid state fermentation (SSF) on tomato pomace was developed with the white-rot fungi Pleurotus ostreatus and Trametes versicolor, using sorghum stalks as support. Operative parameters (humidity, water activity, and size of substrate particles) guaranteeing a good colonization of tomato pomace by both fungi were defined and conditions for production at high titers of the industrially relevant enzymes laccase, xylanase and protease were identified. Significant laccase activity levels (up to 36 U g⁻¹ dry matter) were achieved without any optimization of culture conditions, neither by nutrient addition nor by O₂ enrichment. Furthermore, protease activity levels up to 34,000 U g⁻¹ dry matter were achieved, being higher than those reported for the fungi typically considered as the best protease producers such as Aspergillus strains. Moreover, as one of the most significant results of this study, analysis of P. ostreatus tomato SSF samples by zymogram revealed two bands with laccase activity which had not been detected so far.     

Lamiaceae carbohydrates. IV. Water-soluble polysaccharides from <Emphasis Type="Italic">Scutellaria baicalensis</Emphasis>

The composition of water-soluble polysaccharides from the aerial part of Scutellaria baicalensis Georgi (Lamiaceae) was studied. It was found that the dominant polymers were WSPS′-1, WSPS′-2, and WSPS′-3, which were characterized preliminarily as partially acetylated glucoarabinogalactans, for which antioxidant activity was found. Two homoglucans of the starch type were minor components. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 451–453, September-October, 2008.     

Induction and Purification by Three-Phase Partitioning of Aryl Alcohol Oxidase (AAO) from <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

Aryl alcohol oxidase (AAO) is an extracellular flavoenzyme involved in lignin degradation by white rot fungi. Screening of lignolytic and AAO activity from twenty different fungal species were carried out. Among them, seven species showed lignolytic activity and three of them (Pleurotus ostreatus, Pleurotus eous, and Pleurotus platypus) were found to be AAO positive. Maximal AAO activity was observed in batch cultures of P. ostreatus and was found to be induced by aromatic amino acids and aryl alcohols up to a level of 289 U/l. Purification of AAO was carried out by three-phase partitioning (TPP). The 67 kDa enzyme was purified up to 10.19-fold by TPP with an overall recovery of 10.95%. Optimum pH and temperature for P. ostreatus AAO activity was found to be around 6 and 40 °C, respectively. From the LB plot, K _m value of AAO for oxidizing veratryl alcohol was determined to be 0.6 mM. Results of the study indicate that P. ostreatus is the best producers of AAO, and they could be employed as promising fungal species for biotechnological applications.     

Characterization and Antineoplasic Effect of Extracts Obtained from <Emphasis Type="Italic">Pleurotus sajor</Emphasis>-<Emphasis Type="Italic">caju</Emphasis> Fruiting Bodies

Fungi of the Pleurotus genus present a great industrial interest due to their possibility of producing pharmacological compounds, pigments, aromas, organic acids, polysaccharides, enzymes, vitamins, amino acids, etc. Among the therapeutic products, we can highlight those with antineoplasic activity, attributed to the fungi cell wall components. Based on this, the objective of this work was to study the antineoplasic capacity of the polysaccharidic fractions obtained from Pleurotus sajor-caju fruiting bodies. Female Swiss mice were inoculated with the Ehrlich ascitic tumor (5 × 10⁶ cells/animal) in ascitic form. The polysaccharidic fractions were administered intraperitoneally, during a 6-day period. Fractions FI and FII presented a lower volume of ascitic liquid (3.1 and 1.8 mL, respectively) and a higher reduction in the number of neoplasic cells present in the ascitic liquid (86.2% and 85%, respectively), when compared to the positive control (group inoculated with the tumor but without treatment). These fractions were characterized in terms of monosaccharide composition. Glucose was the major component detected, followed by galactose and mannose. The anomeric carbon configuration of the β-glucan was confirmed by the ¹³C NMR (δ 103.7). Substituted and free C3 and C6 were also detected. Protein bands were confirmed through infrared analysis.     

Lamiaceae carbohydrates. 1. Pectinic substances and hemicelluloses from <Emphasis Type="Italic">Mentha x piperita</Emphasis>

Pectinic substances from the aerial part of Mentha x piperita were isolated and characterized and found to be a mixture of β-(1→4)-glucogalactan (MPG) and two α-(1→4)-rhamnopolygalacturonans (MPP′-1 and MPP′-2). It was shown that the pectin and its components exhibited membrane-stabilizing, antiatherogenic, and antioxidant activity. Hemicelluloses from M. piperita were a lignocarbohydrate complex. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 411–416, September–October, 2007.     

Glucofructans from <Emphasis Type="Italic">Cousinii radians</Emphasis> roots

Structures of glucofructans isolated from Cousinii radians roots were studied by methylation, periodate oxidation, and IR and ¹³C NMR spectroscopies. It was found that fructofuranose units were bonded to each other by inulin-type β-(2→1)-gylcoside bonds. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 3–5, January–February, 2007.     

Quantitative study of antioxidant properties of phenolcarboxylic acids from <Emphasis Type="Italic">Larix sibirica</Emphasis> bark

Antioxidant properties of caffeic, ferulic, vanillic, and 4-hydroxybenzoic acids isolated from Larix sibirica Ledeb. bark were studied using model radical-chain reactions for oxidation of propan-2-ol and 1,4-dioxane under kinetic control. Quantitative characteristics of the antioxidant activities were determined as effective rate constants fk_In for oxidation chain termination of the phenolcarboxylic acids. It was found that caffeic acid had the highest antioxidant activity among the investigated natural compounds. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 124–127, March–April, 2008.     

TG–FTIR analysis applied to the study of thermal behaviour of some edible mushrooms

The article is devoted to the study on the thermal behaviour of three species of edible mushrooms: Boletus edulis (foot and cap), Pleurotus ostreatus (foot and cap), Lactarius deterrimus (cap) by the TG–FTIR-coupled technique, in air, over the 30–900 °C temperature range. The analysis of the TG–DTG–DTA curves reveals the thermal degradation mechanism to be complex and specific to every species under the recording conditions applied. A similar degradation mechanism is noticed for the foot and cap of Pleurotus ostreatus in comparison with the Boletus edulis and Lactarius deterrimus species where the mechanisms are different. The TG–FTIR analysis, combustion heats and IR spectra of the starting samples also support these results. The initial degradation temperatures from TG–DTG indicate the temperature range where these species are thermally stable and their nutrient features maintained making them proper for food. The TG–FTIR analysis gives information on the gaseous species evolved by the thermal degradation bringing thus a contribution to the elucidation of the changes developing by processing the edible mushrooms (industrialization, conservation, culinary preparations, etc.) at temperatures above the initial degradation temperature. At the same time, the environmental impact, when the mushroom failed cultures are burned, is also important.     

Glucofructans from <Emphasis Type="Italic">Taraxacum officinale</Emphasis> roots

The structure of glucofructans from Taraxacum officinale roots growing in Buryatia was studied by chemical, chromatographic, and spectral methods. It was found that fructose,glucose, saccharose, 1-kestose, and nystose were present in the free state. The structures of the two dominant polymeric compounds, TGf-1 (5.7 kDa) and TGf-2 (2.6 kDa), which were linear inulin-type macromolecules consisting of fructofuranose units bonded through β-(2→1)-bonds, were studied. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 125–127, March–April, 2009.     

Lipids from <Emphasis Type="Italic">Crambe amabilis</Emphasis> and <Emphasis Type="Italic">C. kotschyana</Emphasis> seeds grown with saline irrigation water

Lipids from Crambe amabilis Butk. et Majlun and C. kotschyana Boiss. grown in the open in Uzbekistan in soil imported from the Aral Sea region were studied. It was found that the seed oil content decreased with increasing salinity of irrigation water with EC 1.5 up to 9.0 dS/m. The content of polar lipids increased. The content of unsaturated acids from C. amabilis neutral lipids was 94.74–97.46%; C. kotschyana, 95.76– 96.78%.     

Non-isothermal kinetics of the thermal decomposition of sodium oxalate Na<Subscript>2</Subscript>C<Subscript>2</Subscript>O<Subscript>4</Subscript>

The thermal transformation of Na₂C₂O₄ was studied in N₂ atmosphere using thermo gravimetric (TG) analysis and differential thermal analysis (DTA). Na₂C₂O₄ and its decomposed product were characterized using a scanning electron microscope (SEM) and the X-ray diffraction technique (XRD). The non-isothermal kinetic of the decomposition was studied by the mean of Ozawa and Kissinger–Akahira–Sunose (KAS) methods. The activation energies (E _α) of Na₂C₂O₄ decomposition were found to be consistent. Decreasing E _α at increased decomposition temperature indicated the multi-step nature of the process. The possible conversion function estimated through the Liqing–Donghua method was ‘cylindrical symmetry (R₂ or F_1/2)’ of the phase boundary mechanism. Thermodynamic functions (ΔH*, ΔG* and ΔS*), calculated by the Activated complex theory and kinetic parameters, indicated that the decomposition step is a high energy pathway and revealed a very hard mechanism.     

Co-cultured production of lignin-modifying enzymes with white-rot fungi

Co-cultivation was a potential strategy in lignocellulolytic biodegradation with producing high activity enzymes due to their synergistic action. The objective of this study was to investigate the rarely understood effects of co-culturing of two white-rot fungi on lignin-modifying enzymes (LMEs) production. Six species, Bjerkandera adusta, Phlebia radiata, Pleurotus ostreatus, Dichomitus squalens, Hypoxylon fragiforme and Pleurotus eryngii, were cultured in pairs to study the production of LMEs. The paired hyphal interaction observed showed that P. eryngii is not suitable for co-growth. The use of agar plates containing dye RBBR showed elevated decolourisation at the confrontation zone between mycelia. Laccase was significantly stimulated only in the co-culture of P. radiata with D. squalens under submerged cultivation; the highest value was measured after 4 days of incubation (120 U mg⁻¹). The improved productions of MnP and LiP were simultaneously observed at the co-culture of P. ostreatus and P. radiata (MnP = 800 nkat L⁻¹ after 4 days of incubation; LiP = 60 nkat L⁻¹ after 7 days of incubation), though it was not a good producer of laccase. P. ostreatus appeared to possess specific potential to be used in co-cultured production of LMEs. The phenotype of LMEs production was not only dependent on the species used but also regulated by different nutritions available in the culture medium. The present data will provide evidence for illustrating the regulatory roles of C/N on LMEs production under the co-cultures’ circumstances.     

New alkaloids from <Emphasis Type="Italic">Casimiroa edulis</Emphasis> fruits and their pharmacological activity

The extract of Casimiroa edulis was investigated for antihypertensive activity. The ethanol and total alkaloids (in chloroform) extracts were found to have antihypertensive properties at doses of 500 and 200 mg/kg, respectively. Four quinolinone alkaloids were isolated and identified as: 2-(2′-hydroxy-4′-methoxyphenyl)-5,8-dimethoxy-3-propyl-1H-quinolin-4-one (1), 5,8-dimethoxy-2-(3′-methoxyphenyl)-3-propyl-1H-quinolin-4-one (2), 5,8-dimethoxy-2-(3′,4′-dimethoxyphenyl)-3-propyl-1H-quinolin-4-one (3), and 5,6-dimethoxy-2-(2′,5′,6′-trimethoxyphenyl)-1H-quinolin-4-one (4). Interestingly, compounds 1, 2, and 3 were found to be new alkaloids. The four isolated alkaloids showed antihypertensive activity at doses of 50, 100, 200, and 300 mg/kg, respectively. Published in Khimiya Prirodnykh Soedinenii, No. 5, pp. 473–476, September–October, 2007.     

Non-isothermal decomposition kinetics of NiFe<Subscript>2</Subscript>O<Subscript>4</Subscript> nanoparticles synthesized using egg white solution route

The thermal decomposition kinetics of nickel ferrite (NiFe₂O₄) precursor prepared using egg white solution route in dynamical air atmosphere was studied by means of TG with different heating rates. The activation energy (E _α) values of one reaction process were estimated using the methods of Flynn–Wall–Ozawa (FWO) and Kissinger–Akahira–Sunose (KAS), which were found to be consistent. The dependent activation energies on extent of conversions of the decomposition reaction indicate “multi-step” processes. XRD, SEM and FTIR showed that the synthesized NiFe₂O₄ precursor after calcination at 773 K has a pure spinel phase, having particle sizes of ~54 ± 29 nm.     

Seasonal variations of the composition and structural characteristics of polysaccharides from the brown alga <Emphasis Type="Italic">Costaria costata</Emphasis>

Seasonal variations of the polysaccharide composition of the brown alga Costaria costata were studied. It was found that the alga synthesized in April-May a high-molecular-weight (200–800 kDa) low-sulfated heterofucan; in July, primarily a low-molecular-weight (20–300 kDa) sulfated and acetylated galactofucan. A small amount (<0.01% of total alga mass) of laminaran (1,3;1,6-β-D-glucan) was observed in mature alga.