Aneli M. Barbosa

Xylanase production by Trichoderma harzianum rifai by solid state fermentation on sugarcane bagasse

Sugarcane bagasse was used as substrate for xylanase production by means of a strain of Trichoderma harzianum Rifai isolated from decaying Aspidosperma sp. (peroba) wood. The bagasse was washed, dried, milled and wetted with minimal salts medium and the cultures grown at 28 ± 2oC for 7 days. Two extraction methods were tested for enzyme recovery: (A) Tween 80, 0.1% (v/v), in physiological saline, and (B) 50mM sodium acetate buffer, pH 5.0, under agitation (180rpm) for 15, 30 and 60min. After a single extraction, both extraction methods recovered an average of 15U/ml of xylanase activity, independent on the time of shaking. A second and third extraction recovered 10.4 and 6.6U/ml xylanase, respectively. The effect of volume size for extraction, and sugarcane bagasse concentration, on xylanase production were also investigated. The growth profile of Trichoderma harzianum was followed over 20 days on 14% (w/v) bagasse, and highest xylanase activity (288U/ml) appeared on the seventh day. The enzymatic extract after precipitation with ammonium sulphate was submitted to electrophoresis on polyacrylamide gels and showed 4 protein-staining bands, one of which exhibited xylanase activity.

The effect of lignin-related compounds on the growth and production of laccases by the ascomycete, Botryosphaeria sp.

The concentration at which 50% inhibition (I50) of fungal growth occurred for various lignin-related compounds was evaluated on Botryosphaeria sp. MAMB-5 cultivated on agar medium. A trend for inhibition of growth on lignin-related aromatic acids and phenolic compounds was observed and was based upon chemical structural features: benzoic acids containing di-methoxyl substituents on the aromatic ring were less inhibitory than the parent unsubstituted compound; p-phenolic benzoates containing a single methoxyl group were less inhibitory than those containing a di-methoxyl group; methoxylated derivatives of p-phenolic cinnamic acids were less inhibitory than the unsubstituted parent compound; but un-methylated hydroxyls on di-phenols allowed growth compared to the mono- and di-O-methylated derivatives, which were inhibitory. Botryosphaeria sp. grew on basal media containing up to 10% (w/v) lignosulfonate and kraft lignin in stationary liquid culture. In submerged liquid cultivation, Botryosphaeria sp. grew on basal medium containing abietic acid, catechol, 4-chlorophenol, guaiacol, vanillyl alcohol, veratraldehyde and lignosulfonate, and in each case produced laccases. The production of laccases implicated a possible role for their involvement in the biodegradation of aromatic compounds by Botryosphaeria sp. 4-Chlorophenol was the most toxic of the compounds examined.

A new role for veratryl alcohol: Regulation of synthesis of lignocellulose-degrading enzymes in the ligninolytic ascomyceteous fungus, Botryosphaeria sp.; influence of carbon source

A new physiological role for veratryl alcohol in fungi important in the biodegradation of the lignified plant cell wall is presented. Botryosphaeria sp., grown on starch, pectin, cellulose or xylan produced amylase, pectinase, cellulase, xylanase and laccase, whereas glucose and xylose repressed the synthesis of cellulase and xylanase, but not laccase. When cultured on each of these substrates in the presence of veratryl alcohol, laccase activity increased but the activities of amylase, pectinase, cellulase and xylanase significantly decreased. Basal medium containing softwood kraft lignin in the presence of veratryl alcohol induced laccases above constitutive levels. Ethyl alcohol also stimulated laccase production.

Hypoglycemic and Hypocholesterolemic Effects of Botryosphaeran from Botryosphaeria rhodina MAMB-05 in Diabetes-Induced and Hyperlipidemia Conditions in Rats

Abstract Botryosphaeran, a water-soluble exopolysaccharide of the β-(1 → 3;1 → 6)-D-glucan type that has been isolated from the culture medium of Botryosphaeria rhodina MAMB-05 grown in submerged fermentation using glucose as the sole carbon source, was previously demonstrated to be non-genotoxic in peripheral blood and bone marrow, and exhibited strong anticlastogenic activity. In the present study, the effects of botryosphaeran were investigated in streptozotocin-induced diabetic rats as well as in high-fat diet-fed hyperlipidemic Wistar rats. The plasma glucose level was reduced by 52% in the diabetic group of rats after administration of 12 mg botryosphaeran/kg body weight of the rats (b.w.)/day by gavage over 15 days. A reduction in the median ration intake was accompanied by an increase in the median body weight gain, as well as the efficiency of food conversion. These results demonstrate that botryosphaeran has protective effects by reducing the symptoms of cachexia in Diabetes mellitus. Botryosphaeran administered by gavage at a concentration of 12 mg botryosphaeran/kg b.w./day over 15 days also reduced the plasma levels of total cholesterol and low density lipoprotein-cholesterol by 18% and 27%, respectively, in hyperlipidemic rats. Based on these findings, we conclude that botryosphaeran possesses hypoglycemic and hypocholesterolemic properties in conditions of diabetes mellitus and hyperlipidemia, respectively, and may be used as an oral anti-diabetic agent.

Carboxymethylation of (1 → 6)-β-glucan (lasiodiplodan): Preparation, characterization and antioxidant evaluation.

D-Glucans possess immunomodulatory activities and potential for the development of new therapeutic agents. Biological activities can be enhanced in these biopolymers through chemical derivatization, e.g., carboxymethylation. This work presents the carboxymethylation, characterization and the evaluation of antioxidant activities of the exocellular (1 → 6)-β-D-glucan produced by Lasiodiplodia theobromae MMPI. Thermal analysis indicated that the native and carboxymethylated polysaccharides presented four stages of mass-loss. The first stage occurred at 125°C (loss of water) with two consecutive events of mass loss (200-400°C) attributed to polymer degradation and the fourth stage between 425 and 620°C (final decomposition). Scanning electron microscopy analysis indicated that the gross morphological features of lasiodiplodan were ruptured following carboxymethylation. X-ray diffractometry analysis demonstrated that the native and carboxymethylated polysaccharides presented a non-crystalline structure. Carboxymethylation contributed to improving the polysaccharides water solubility and antioxidant capacity.

A simple method for monitoring chromatography column eluates for laccase activity during enzyme purification

A simple and economical method is described that allows rapid detection of laccase activity in chromatography column fractions during enzyme purification. Aliquots of column eluants are applied to filter paper coated with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) containing a numbered grid, and incubated at ambient temperature for 20xa0min. Indications of enzyme activity are simply observed by a colour change. This method avoids having to manually assay each fraction of a chromatographic run for enzyme activity.

Thermal and Rheological Properties of a Family of Botryosphaerans Produced by Botryosphaeria rhodina MAMB-05

Differential scanning calorimetry (DSC), thermogravimetry (TG) and Fourier-transform infra-red spectroscopy (FT-IR) analyses were performed to investigate changes in the physico-chemical properties of botryosphaerans, a family of exopolysaccharides (EPS) produced by the fungus Botryosphaeria rhodina MAMB-05 grown on glucose (EPSGLC), sucrose (EPSSUC) and fructose (EPSFRU). A slight endothermic transition and small mass loss attributable to the removal of water of hydration were observed in the DSC and TG analyses, respectively, for the three EPS samples. The FT-IR spectra confirmed no structural changes occurred during thermal treatment. Viscometry was utilized to obtain information on the rheological behaviour of the EPS in aqueous solutions. The Power Law and Cross Equations determined the natural pseudoplastic characteristics of the EPS. Comparatively, results obtained for EPS produced when B. rhodina MAMB-05 was grown on each of the three carbohydrate sources demonstrated similar apparent viscosity values for EPSGLC and EPSSUC, while EPSFRU displayed the lowest apparent viscosity of the three botryosphaerans, suggesting a higher degree of ramification and lower Mw. EPSGLC and EPSSUC possessed similar degrees of ramification. The slight differences found in their viscosities can be explained by the differences in the type of branching among the three botryosphaerans, thus varying the strength of intermolecular interactions and consequently, consistency and viscosity. The physico-chemical studies of botryosphaerans represent the originality of this work, and the knowledge of these properties is an important criterion for potential applications.

Production of β-(1,3)-glucanases by Trichoderma harzianum Rifai: Optimization and Application to Produce Gluco-oligosaccharides from Paramylon and Pustulan

β-(1→3)-Glucanases were produced by Trichoderma harzianum Rifai PAMB-86 cultivated on botryosphaeran in a bench-fermenter and optimised by the response surface method. Maximal enzyme titres occurred at 5 days, initial pH 5.5 and aeration of 1.5vvm. β-(1→3)-The β-glucanolytic enzyme complex produced by T. harzianum Rifai PAMB- 86 was fractionated by gel filtration into 2 fractions (F-I, F-II), and employed to produce gluco-oligosaccharides from algal paramylon ((1→3)-β-D-glucan) and lichen pustulan ((1→6)-β-D-glucan). Both enzymes attacked paramylon to the extent of ~15-20% in 30 min releasing glucose and laminaribiose as major end-products, and laminari- oligosaccharides of degree of polymerization (DP) ≥ 3. Only F-I degraded pustulan resulting in ~2% degradation at 30 min, with glucose, gentiobiose and gentio-oligosaccharides of DP ≥ 4 as major products. The difference in the nature of the hydrolysis products can be explained by the substrate specificities of each enzyme fraction, and the structural differences of the β-D-glucans attacked.

Soybean Oil and Meal as Substrates for Lipase Production by Botryosphaeria ribis, and Soybean Oil to Enhance the Production of Botryosphaeran by Botryosphaeria rhodina

Soybean oil, a common vegetable (plant seed) oil, has traditionally been used as a food in cooking and salad dressings, and more recently, as a feedstock for the production of biodiesel fuels (Bajaj et al., 2010; Ghaly et al., 2010). After extracting the oil from soybean seeds, the residue constitutes an important by-product called soybean meal, or oil-seed cake. This edible by-product, a rich source of proteins and amino acids, especially tryptophan, threonine and lysine (Ramachandran et al., 2007), has been used as a livestock feed, as well as a nutrient source and fermentable substrate for producing microbial lipases (Ul-Haq et al., 2002), and other enzymes (Singhania et al., 2009). The fatty acid composition of soybean oil is typically: 53.8 % linoleic (C18:2), 20.8 % oleic (C18:1), 11.4 % palmitic (C16:1), 9.3 % linolenic (C18:3), 4.4 % stearic (C18:0), and 0.3 % arachidic acids (C20:0) (Ghaly et al., 2010). Besides the applications cited above, soybean oil was demonstrated an effective inducer for the production of fungal enzymes (lipases) that degrade plant seed oils (Messias et al., 2009). It has also been reported to enhance the synthesis of pleuromutilin by Pleurotus mutilis, an antibiotic effective against gram-positive bacterial pathogens (Hu et al., 2009), and promoted the production of laccases by Botryosphaeria rhodina MAMB-05 when added to nutrient medium (Dekker et al., 2007). Lipases (EC 3.1.1.3; triacylglycerol acylhydrolases) are hydrolytic enzymes, which catalyse the hydrolysis of the ester linkages of long-chain acylglycerols to glycerol and free fatty acids. These enzymes also conduct interesterification, transesterification and ammonolysis reactions. Lipases are ubiquitous among microorganisms being produced by bacteria, actinomycetes, filamentous fungi and yeasts, and have found applications in various sectors of commerce (Li & Zong, 2010). The main industrial application of lipase is still restricted to their use in laundry detergents to remove fats and oil stains (Hasan et al., 2010). They are also used in various food and agro-chemical industries, e.g., processing foods, treatment of fatty effluents, synthesis of biosurfactants, removal of resins (pitch) in processing paper from wood cellulose pulps, and as

Intracerebroventricular administration of the (1→6)-β-d-glucan (lasiodiplodan) in male rats prevents d-penicillamine-induced behavioral alterations and lipoperoxidation in the cortex

Abstract Context: Lasiodiplodan, an exocellular (1→6)-β-d-glucan of molecular weight >1.4u2009×u2009106u2009Da produced by MMPI strain of Lasiodiplodia theobromae (Pat.) Griffon & Maubl. (Brotyosphaeriaceae) is known to exhibit anti-proliferative activity on breast cancer cells (MCF-7), anticoagulant activity when sulfonylated, and reduction in transaminase activity when administered in rats. Objective: The effect of intracerebroventricular (I.C.V) injection of lasiodiplodan on neurotoxicity and behavioural changes induced by d-penicillamine was investigated. Materials and methods: Twenty-four male Wistar rats were initially separated in groups of six and treated with 0.15u2009μmol/μL of NaCl (Groups Ct and d-Pen) and 0.01u2009μg/μL of lasiodiplodan (Groups Las and Lasu2009+u2009d-Pen). After 15u2009min, they received 6u2009μmol/μL of NaCl (Groups Ct and Las) and 2u2009μmol/μL of d-penicillamine (Groups d-Pen and Lasu2009+u2009d-Pen). The animal behavior was observed in an open-field test for 60u2009min. Twenty-four h later, the animals were sacrificed and histopathological analysis and Thiobarbituric acid reactive substances (TBARS) production measurements were performed. Results: Lasiodiplodan prevented neurotoxicity induced by d-penicillamine significantly reducing the production of TBARS (308%; pu2009<u20090.05), and behavioural signs; convulsive and pre-convulsive. No histopathological alterations in the cerebral cortex were observed. Discussion and conclusion: The reduction of TBARS production and convulsive episodes suggests that the protector effect provided by lasiodiplodan passes thought an antioxidant path, possibly interfering in a cascade of neurochemical events, triggering cell death and convulsive episodes. These results demonstrated that lasiodiplodan can be effective in treating neurotoxicity, and reducing damage triggered by convulsions in neuropathies related to GABAergic system.