Purification and properties of β-glucosidase from a thermophilic fungus Humicola lanuginosa (Griffon and Maublanc) Bunce

An extracellular β-glucosidase (EC 3.2.1.21) has been purified to homogeneity from the culture filtrate of a thermophilic fungus, Humicola lanuginosa (Griffon and Maublanc) Bunce, using duplicating paper as the carbon source. The enzyme was purified 82-fold with a 43% yield by ion-exchange chromatography and gel filtration. The molecular weight of the protein was estimated to be 135,000 by gel filtration and 110,000 by electrophoresis. The sedimentation coefficient was 10.5 S. It was an acidic protein containing high amounts of acidic amino acid residues. It was poor in sulphur-containing amino acids. It also contained 9% carbohydrate. The enzyme activity was optimum at pH 4.5 and at 60°C. The enzyme was stable in the pH range 6–9 for 24 h at 25°C. The enzyme had similar affinities towards cellobiose and p-nitrophenyl-β-d-glucoside with Km values of 0.44 mM and 0.50 mM, respectively. The enzyme was capable of hydrolysing larchwood xylan, xylobiose and p-nitrophenyl-β-d-xyloside, though to a lesser extent. The enzyme was specific for the β-configuration and glucose moiety in the substrate.

Purification and characterization of endo-1,4-β-xylanase from Paecilomyces varioti Bainier

An endo-xylanase (1,4-β-d-xylanxylanohydrolase EC 3.2.1.8) was isolated from the culture filtrate of Paecilomyces varioti Bainier. The enzyme was purified 3.2 fold with a 60% yield by gel filtration and ion exchange chromatography. The purified enzyme had a molecular weight of 25,000 with a sedimentation coefficient of 2.2 S. The isoelectric point of the enzyme was 3.9. The enzyme was obtained in crystalline form. The optimum pH range was 5.5–7.0 and the temperature, 65°C. The Michaelis constant was 2.5 mg larchwood xylan/ml. The enzyme was found to degrade xylan by an endo mechanism producing arabinose, xylobiose, xylo- and arabinosylxylo-oligosaccharides, during the initial stages of hydrolysis. On prolonged incubation, xylotriose, arabinosylxylotriose and xylobiose were the major products with traces of xylotetraose, xylose and arabinose.

Studies in biogas technology. Part III. Thermal analysis of biogas plants

A thermal model for a conventional biogas plant has been developed in order to understand the heat transfer from the slurry and the gas holder to the surrounding earth and air respectively. The computations have been performed for two conditions : (i) when the slurry is at an ambient temperature of 20°C, and (ii) when it is at 35°C, the optimum temperature for anaerobic fermentation. Under both these conditions, the gas holder is the major “culprit” with regard to heat losses from the biogas plant. The calculations provide an estimate for the heat which has to be supplied by external means to compensate for the net heat losses which occur if the slurry is to be maintained at 35°C. Even if this external supply of heat is realised through (the calorific value of) biogas, there is a net increase in the biogas output, and therefore a net benefit, by operating the plant at 35°C. At this elevated temperature, the cooling effect of adding the influent at ambient temperature is not insignificant. In conclusion, the results of the thermal analysis are used to define a strategy for operating biogas plants at optimum temperatures, or at higher temperatures than the ambient.

A New Pathway for the Degradation of a Sesquiterpene Alcohol, Nerolidol by Alcaligenes eutrophus

An oxidative pathway hitherto unknown for tile degradation of a sesquiterpene alcohol, nerolidol (I) by Alcaligenes eutrophus is presented. Fermentation of nerolidol (I) by this organism in a mineral salts medium resulted in the formation of geranylacetone (II) and an optically active alcohol (S)-(+)-geranylacetol (III), as major metabolites. Nerolidol (I) induced cells readily transformed 1,2-epoxynerolidol (IV) and 1,2-dihydroxynerolidol (V) into geranylacetone (II). These cells also exhibited their ability to carry out stereospecific reduction of II into (S)-(+)-geranylacetol (III). Oxygen uptake studies clearly indicated that nerolidol induced cells oxidized compounds II, III, IV, V and ethyleneglycol. Based on these observations a new oxidative pathway for the degradation of I is suggested which envisages the epoxidation of the terminal double bond, opening of the epoxide and cleavage between C-2 and C-3 in a manner similar to the periodate oxidation of diol.

Nutritional evaluation of silage made from the toxic weed parthenium hysterophorus in animals

After ensilation, the toxic Compositae weed Parthenium hysterophorus was devoid of the toxic principle parthenin. Laboratory-scale ensilation indicated that no parthenin was detectable after 5 wk of anaerobic fermentation. For animal feeding studies, silage was made on a large scale from Parthenium mixed with maize or from Parthenium alone. Crossbred bull and buffalo bull calves were fed diets containing the silages, or control diet without silage, for 12 wk. The animals consumed both silages with relish, and body weight gains of silage-fed calves did not differ from those of the controls. The digestibilities of dry matter, fibre and nitrogen-free extract were greater with the control diet, but the biological value of proteins tended to be greater with the silage-containing diets. Haematological studies indicated no significant differences between experimental and control groups in selected parameters, except for a reduction in blood urea nitrogen in the animals fed silage. The possible causes for these biochemical alterations are discussed. Since the nutritive value of Parthenium silage compares favourably with the standard diet, and Parthenium seeds collected from the silage did not germinate, we suggest that ensilation can be used as an additional method in the containment and eradication of these plants, which grow wild in India.

Role of neutral metabolites in microbial conversion of 3beta-acetoxy-19-hydroxycholest-5-ene into estrone

Biotransformation of 3 beta-acetoxy-19-hydroxycholest-5-ene (19-HCA, 6 g) by Moraxella sp. was studied. Estrone (712 mg) was the major metabolite formed. Minor metabolites identified were 5 alpha-androst-1-en-19-ol-3,17-dione (33 mg), androst-4-en-19-ol-3,17-dione (58 mg), androst-4-en-9 alpha,19-diol-3,17-dione (12 mg), and androstan-19-ol-3,17-dione (1 mg). Acidic metabolites were not formed. Time course experiments on the fermentation of 19-HCA indicated that androst-4-en-19-ol-3,17-dione was the major metabolite formed during the early stages of incubation. However with continuing fermentation its level dropped, with a concomitant increase in estrone. Fermentation of 19-HCA in the presence of specific inhibitors or performing the fermentation for a shorter period (48 h) did not result in the formation of acidic metabolites. Resting-cell experiments carried out with 19-HCA (200 mg) in the presence of alpha,alpha'-bipyridyl led to the isolation of three additional metabolites, viz., cholestan-19-ol-3-one (2 mg), cholest-4-en-19-ol-3-one (10 mg), and cholest-5-en-3 beta,19-diol (12 mg). Similar results were also obtained when n-propanol was used instead of alpha,alpha'-bipyridyl. Resting cells grown on 19-HCA readily converted both 5 alpha-androst-1-en-19-ol-3,17-dione and androst-4-en-19-ol-3,17-dione into estrone. Partially purified 1,2-dehydrogenase from steroid-induced Moraxella cells transformed androst-4-en-19-ol-3,17-dione into estrone and formaldehyde in the presence of phenazine methosulfate, an artificial electron acceptor. These results suggest that the degradation of the hydrocarbon side chain of 19-HCA does not proceed via C-22 phenolic acid intermediates and complete removal of the C-17 side chain takes place prior to the aromatization of the A ring in estrone. The mode of degradation of the sterol side chain appears to be through the fission of the C-17-C-20 bond. On the basis of these observations, a new pathway for the formation of estrone from 19-HCA in Moraxella sp. has been proposed.

Biochemistry of water-logged soils. Part IV. Carbon and nitrogen transformations

1. During the fermentation of water-logged soil containing added substances with different carbon-nitrogen ratios, the reaction first turns slightly acid, but soon returns to the original hydrogen-ion concentration (pH 7·6). 2. The quantities of ammonia present in the medium increase up to a point, after which there is steady decrease. 3. There is nitrification only in the case of substances with narrow C/N ratios. The production of nitrate generally commences only after about a month, when the vigour of the initial fermentation has subsided and fairly large quantities of ammonia have accumulated in the medium. 4. The extent of mineralisation of nitrogen is determined chiefly by the C/N ratio, though in the cases of substances like mahua and lantana the presence of other constituents may also influence the processes. The quantities of mineralised nitrogen present in the soil system generally tend to decrease after about two months.

Ethnobotany of the Monpa ethnic group at Arunachal Pradesh, India

Background: The present paper documents the uses of plants in traditional herbal medicine for human and veterinary ailments, and those used for dietary supplements, religious purpose, local beverage, and plants used to poison fish and wild animals. Traditional botanical medicine is the primary mode of healthcare for most of the rural population in Arunachal Pradesh. Materials and methods: Field research was conducted between April 2006 and March 2009 with randomly selected 124 key informants using semi-structured questionnaire. The data obtained was analyzed through informant consensus factor (F(IC)) to determine the homogeneity of informant's knowledge on medicinal plants. Results: We documented 50 plants species belonging to 29 families used for treating 22 human and 4 veterinary ailments. Of the medicinal plants reported, the most common growth form was herbs (40%) followed by shrubs, trees, and climbers. Leaves were most frequently used plant parts. The consensus analysis revealed that the dermatological ailments have the highest F(IC) (0.56) and the gastro-intestinal diseases have F(IC) (0.43). F(IC) values indicated that there was high agreement in the use of plants in dermatological and gastro-intestinal ailments category among the users. Gymnocladus assamicus is a critically rare and endangered species used as disinfectant for cleaning wounds and parasites like leeches and lice on livestocks. Two plant species (Illicium griffithii and Rubia cordifolia) are commonly used for traditional dyeing of clothes and food items. Some of the edible plants recorded in this study were known for their treatment against high blood pressure (Clerodendron colebrookianum), diabetes mellitus (Momordica charantia), and intestinal parasitic worms like round and tape worms (Lindera neesiana, Solanum etiopicum, and Solanum indicum). The Monpas of Arunachal Pradesh have traditionally been using Daphne papyracea for preparing hand-made paper for painting and writing religious scripts in Buddhist monasteries. Three plant species (Derris scandens, Aesculus assamica, and Polygonum hydropiper) were frequently used to poison fish during the month of June-July every year and the underground tuber of Aconitum ferrox is widely used in arrow poisoning to kill ferocious animals like bear, wild pigs, gaur and deer. The most frequently cited plant species; Buddleja asiatica and Hedyotis scandens were used as common growth supplements during the preparation of fermentation starter cultures. Conclusion: The traditional pharmacopoeia of the Monpa ethnic group incorporates a myriad of diverse botanical flora. Traditional knowledge of the remedies is passed down through oral traditions without any written document. This traditional knowledge is however, currently threatened mainly due to acculturation and deforestation due to continuing traditional shifting cultivation. This study reveals that the rural populations in Arunachal Pradesh have a rich knowledge of forest-based natural resources and consumption of wild edible plants is still an integral part of their socio-cultural life. Findings of this documentation study can be used as an ethnopharmacological basis for selecting plants for future phytochemical and pharmaceutical studies.

Anaerobic retting of banana and arecanut wastes in a plug flow digester for recovery of fiber, biogas and compost

Leaves and leaf sheath of banana and areca husk (Areca catechu) constitute an important component of urban solid waste (USW) in India which are difficult to degrade under normal windrow composting conditions. A successful method of anaerobic digestion built around the fermentation properties of these feedstock has been evolved which uses no moving parts, pretreatment or energy input while enabling recovery of four products: fiber, biogas, compost and pest repellent. An SRT of 27 d and 35 d was found to be optimum for fiber recovery for banana leaf and areca husk, respectively. Banana leaf showed a degradation pattern different from other leaves with slow pectin-1 degradation (80%) and 40% lignin removal in 27 d SRT. Areca husk however, showed a degradation pattern similar to other plant biomass. Mass recovery levels for banana leaf were fiber-20%, biogas-70% (400 ml/g TS) and compost-10%. For areca husk recovery was fiber-50%, biogas-45% (250 ml/g TS) and compost-5%. (C) 2012 Elsevier Inc. All rights reserved.

Inhibition of cancer cell proliferation and apoptosis-inducing activity of fungal taxol and its precursor baccatin III purified from endophytic Fusarium solani

Background: Taxol (generic name paclitaxel), a plant-derived antineoplastic agent, used widely against breast, ovarian and lung cancer, was originally isolated from the bark of the Pacific yew, Taxus brevifolia. The limited supply of the drug has prompted efforts to find alternative sources, such as chemical synthesis, tissue and cell cultures of the Taxus species both of which are expensive and yield low levels. Fermentation processes with microorganisms would be the methods of choice to lower the costs and increase yields. Previously we have reported that F. solani isolated from T. celebica produced taxol and its precursor baccatin III in liquid grown cultures J Biosci 33: 259-67, 2008. This study was performed to evaluate the inhibition of proliferation and induction of apoptosis of cancer cell lines by the fungal taxol and fungal baccatin III of F. solani isolated from T. celebica. Methods: Cell lines such as HeLa, HepG2, Jurkat, Ovcar3 and T47D were cultured individually and treated with fungal taxol, baccatin III with or without caspase inhibitors according to experimental requirements. Their efficacy on apoptotic induction was examined. Results: Both fungal taxol and baccatin III inhibited cell proliferation of a number of cancer cell lines with IC50 ranging from 0.005 to 0.2 mu M for fungal taxol and 2 to 5 mu M for fungal baccatin III. They also induced apoptosis in JR4-Jurkat cells with a possible involvement of anti-apoptotic Bcl2 and loss in mitochondrial membrane potential, and was unaffected by inhibitors of caspase-9,-2 or -3 but was prevented in presence of caspase-10 inhibitor. DNA fragmentation was also observed in cells treated with fungal taxol and baccatin III. Conclusions: The cytotoxic activity exhibited by fungal taxol and baccatin III involves the same mechanism, dependent on caspase-10 and membrane potential loss of mitochondria, with taxol having far greater cytotoxic potential.

Expression of Bioactive Callithrix jacchus Follicle-Stimulating Hormone in Pichia pastoris

Callithrix jacchus (common marmoset) is a New World primate monkey, used as an animal model in biomedical research. Marmoset-specific follicle-stimulating hormone (FSH) preparation is required to improve superovulation protocols and to develop homologous FSH monitoring assays in these monkeys. In this study, we document the large-scale expression of recombinant marmoset FSH in methylotropic yeast, Pichia pastoris. The recombinant preparation was found to be immunologically active in Western blotting and radioimmunoassay. The preparation displayed receptor binding ability in radioreceptor assay. Based on the receptor binding ability, the yield of fermentation was estimated to be 7.2 mg/L. FSH-induced cAMP assay and estradiol assay revealed that the recombinant hormone is able to induce signal transduction. Both immunological and in vitro biological activity of marmoset FSH was found to be comparable to purified human pituitary FSH, which served as reference hormone for these assays. Thus, the study suggests that a Pichia expression system can be used for large-scale expression of bioactive recombinant marmoset FSH.

Folivory as a Constraint on Social Behaviour of Langurs in South India

Folivory, being a dietary constraint, can affect the social time of colobines. In the present study, we compared food items and activity budgets of two closely related species of colobines inhabiting South India, i.e. the Hanuman langur (Semnopithecus hypoleucos) and Nilgiri langur (Semnopithecus johnii), to determine whether folivory had an impact on social time in these species. Our study established that Nilgiri langurs were more folivorous than Hanuman langurs. Nilgiri langurs spent much less time on social activities, but more time on resting, although the social organization of S. hypoleucos was similar to that of the Nilgiri langur. The enforced resting time for fermentation of leafy food items may have reduced the time available for social interactions, which in turn affected the social time in Nilgiri langurs. By comparing the data from previous studies on other Hanuman langur species, we found that S. hypoleucos spent a similar amount of time on social activities as Semnopithecus entellus. Hence, the social behaviour of S. entellus and S. hypoleucos is phylogenetically highly conservative. (C) 2015 S. Karger AG, Basel