Characterization of commercial cellulases and their use in the saccharification of a sugarcane bagasse sample pretreated with dilute sulfuric acid

This study aimed to correlate the efficiency of enzymatic hydrolysis of the cellulose contained in a sugarcane bagasse sample pretreated with dilute H(2)SO(4) with the levels of independent variables such as initial content of solids and loadings of enzymes and surfactant (Tween 20), for two cellulolytic commercial preparations. The preparations, designated cellulase I and cellulase II, were characterized regarding the activities of total cellulases, endoglucanase, cellobiohydrolase, cellobiase, beta-glucosidase, xylanase, and phenoloxidases (laccase, manganese and lignin peroxidases), as well as protein contents. Both extracts showed complete cellulolytic complexes and considerable activities of xylanases, without activities of phenoloxidases. For the enzymatic hydrolyses, two 2(3) central composite full factorial designs were employed to evaluate the effects caused by the initial content of solids (1.19-4.81%, w/w) and loadings of enzymes (1.9-38.1 FPU/g bagasse) and Tween 20 (0.0-0.1 g/g bagasse) on the cellulose digestibility. Within 24 h of enzymatic hydrolysis, all three independent variables influenced the conversion of cellulose by cellulase I. Using cellulase II, only enzyme and surfactant loadings showed significant effects on cellulose conversion. An additional experiment demonstrated the possibility of increasing the initial content of solids to values much higher than 4.81% (w/w) without compromising the efficiency of cellulose conversion, consequently improving the glucose concentration in the hydrolysate.

Ethanol production process from banana fruit and its lignocellulosic residues: Energy analysis

Tropical countries, such as Brazil and Colombia, have the possibility of using agricultural lands for growing biomass to produce bio-fuels such as biodiesel and ethanol. This study applies an energy analysis to the production process of anhydrous ethanol obtained from the hydrolysis of starch and cellulosic and hemicellulosic material present in the banana fruit and its residual biomass. Four different production routes were analyzed: acid hydrolysis of amylaceous material (banana pulp and banana fruit) and enzymatic hydrolysis of lignocellulosic material (flower stalk and banana skin). The analysis considered banana plant cultivation, feedstock transport, hydrolysis, fermentation, distillation, dehydration, residue treatment and utility plant. The best indexes were obtained for amylaceous material for which mass performance varied from 346.5 L/t to 388.7 L/t, Net Energy Value (NEV) ranged from 9.86 MJ/L to 9.94 MJ/L and the energy ratio was 1.9 MJ/MJ. For lignocellulosic materials, the figures were less favorable: mass performance varied from 86.1 to 123.5 L/t, NEV from 5.24 10 8.79 MJ/L and energy ratio from 1.3 to 1.6 MJ/MJ. The analysis showed, however, that both processes can be considered energetically feasible. (C) 2010 Elsevier Ltd. All rights reserved.

Photodegradation of poly(3-hydroxybutyrate)

The effect of ultraviolet radiation on the properties of poly(3-hydroxybutyrate) (PHB) was studied. The PHB investigated is produced from microbial fermentation using saccharose from sugarcane as the carbon source to the bacteria. The material was exposed to artificial UV-A radiation for 3, 6, 9 and 12 weeks. The photodegradation effect was followed by changes of molecular weight, of chemical and crystalline structures, of thermal, morphological, optical and mechanical properties, as well as of biodegradability. The experimental results showed that PHB undergoes both chain scission and cross-linking reactions, but the continuous decrease in its mechanical properties and the low amount of gel content upon UV exposure indicated that the scission reactions were predominant. Molar mass, melting temperature and crystallinity measurements for two layers of PHB samples with different depth suggested that the material has a strong degradation profile, which was attributed to its dark colour that restricted the transmission of light. Previous photodegradation initially delayed PHB biodegradability, due to the superficial increase in crystallinity seen with UV exposure. The possible reactions taking place during PHB photodegradation were presented and discussed in terms of the infrared and nuclear magnetic resonance spectra. A reference peak (internal standard) in the infrared spectra was proposed for PHB photodegradation. (C) 2010 Elsevier Ltd. All rights reserved.

Phase equilibria of lysozyme precipitation with the volatile salt ammonium carbamate

Lysozyme precipitation induced by the addition of the volatile salt ammonium carbamate was studied through cloud-point measurements and precipitation assays. Phase equilibrium experiments were carried out at 5.0, 15.0 and 25.0 degrees C and the compositions of the coexisting phases were determined. A complete separation of the coexisting liquid and solid phases could not be achieved. Nevertheless it was possible to determine the composition of the solid precipitate through the extensions of experimental tie lines. The same precipitate was found at all temperatures. Lysozyme enzymatic activities of the supernatant and precipitate phases were also determined. The activity balance suggests that ammonium carbamate preserves lysozyme activity after the salting-out precipitation. (C) 2010 Elsevier B.V. All rights reserved.

Thermal inactivation of polyphenoloxidase and peroxidase in green coconut (Cocos nucifera) water

P>Coconut water is an isotonic beverage naturally obtained from the green coconut. After extracted and exposed to air, it is rapidly degraded by enzymes peroxidase (POD) and polyphenoloxidase (PPO). To study the effect of thermal processing on coconut water enzymatic activity, batch process was conducted at three different temperatures, and at eight holding times. The residual activity values suggest the presence of two isoenzymes with different thermal resistances, at least, and a two-component first-order model was considered to model the enzymatic inactivation parameters. The decimal reduction time at 86.9 degrees C (D(86.9 degrees C)) determined were 6.0 s and 11.3 min for PPO heat labile and heat resistant fractions, respectively, with average z-value = 5.6 degrees C (temperature difference required for tenfold change in D). For POD, D(86.9 degrees C) = 8.6 s (z = 3.4 degrees C) for the heat labile fraction was obtained and D(86.9 degrees C) = 26.3 min (z = 6.7 degrees C) for the heat resistant one.

Phase equilibria for salt-induced lysozyme precipitation: Effect of salt type and temperature

The salt-induced precipitation of lysozyme from aqueous solutions was studied through precipitation assays in which the equilibrium compositions of the coexisting phases were determined. Lysozyme precipitation experiments were carried out at 5, 15 and 25 degrees C and pH 7.0 with ammonium sulfate, sodium sulfate and sodium chloride as precipitating agents. In these experiments a complete separation of the coexisting phases (liquid and solid) could not be achieved. Nevertheless it was possible to determine the composition of the precipitate. The enzymatic activity of lysozyme in the supernatant phase as well as in the precipitate phase was also determined. The activity balance suggests that there is a relationship between the composition of the true precipitate and the total activity recovery. (C) 2009 Elsevier B.V. All rights reserved.

QUALITY EVALUATION OF MINIMALLY PROCESSED `REGIS` PEACH

The aim of this work was evaluate the effect of citric acid at 1% and 2% in the maintenance of the quality of minimally processed `Regis` peaches, during 9 days storage under refrigeration (5+/-1 degrees C, 85-87% RU) and passive modified atmospheres. The following analyses were realized: color, firmness, texture, soluble solids, titratable acidity, pH, CO(2) and O(2). Increasing in a value and decreasing in b and L values on minimally processed peaches were observed, independent of the citric acid treatment, indicating a browning on the product mainly on the 9(th) day of storage. Overall, the levels of titratable acidity and pH changed at the storage period and at the concentrations studied, unlike the content of soluble solids that remained unchanged. The texture, CO(2) and O(2) changed only for the storage period unlike texture that was higher with the treatment of citric acid 2%. It can be concluded that neither tested concentration of citric acid was effective in preventing enzymatic browning of minimally processed peaches stored the 5+/-1 degrees C and 85-87% RU and passive modified atmosphere.

Structural and physicochemical characterization of RS prepared using hydrolysis and heat treatments of chickpea starch

The aim of this study was to evaluate the production and the structural and physicochemical properties of RS obtained by molecular mass reduction (enzyme or acid) and hydrothermal treatment of chickpea starch. Native and gelatinized starch were submitted to acid (2 M HCl for 2.5 h) or enzymatic hydrolysis (pullulanase, 40 U/g per 10 h), autoclaved (121 degrees C/30 min), stored under refrigeration (4 degrees C/24 h), and lyophilized. The hydrolysis of starch increased the RS content from 16% to values between 20 and 32%, and the enzymatic treatment of the gelatinized starch was the most efficient. RS showed an increase in water absorption and water solubility indexes due to hydrolytic and thermal process. The processes for obtaining RS changed the crystallinity pattern from C to B. Hydrolysis treatments caused an increase in relative crystallinity due to the greater retrogradation caused by the reduction in MW. RS obtained from hydrolysis showed a reduction in viscosity, indicating the rupture of molecules. The viscosity seemed to be inversely proportional to the RS content in the sample.

Bioinsecticidal activity of Talisia esculenta reserve protein on growth and serine digestive enzymes during larval development of Anticarsia gemmatalis

Plants synthesize a variety of molecules to defend themselves against an attack by insects. Talisin is a reserve protein from Talisia esculenta seeds, the first to be characterized from the family Sapindaceae. In this study, the insecticidal activity of Talisin was tested by incorporating the reserve protein into an artificial diet fed to the velvetbean caterpillar Anticarsia gemmatalis, the major pest of soybean crops in Brazil. At 1.5% (w/w) of the dietary protein, Talisin affected larval growth, pupal weight, development and mortality, adult fertility and longevity, and produced malformations in pupae and adult insects. Talisin inhibited the trypsin-like activity of larval midgut homogenates. The trypsin activity in Talisin-fed larvae was sensitive to Talisin, indicating that no novel protease-resistant to Talisin was induced in Talisin-fed larvae. Affinity chromatography showed that Talisin bound to midgut proteinases of the insect A. gemmatalis, but was resistant to enzymatic digestion by these larval proteinases. The transformation of genes coding for this reserve protein could be useful for developing insect resistant crops. (C) 2010 Elsevier Inc. All rights reserved.

Chitinase and peroxidase activity in different stages of eucalypt leaves after inoculation with Puccinia psidii and acibenzolar-S-metil

Chitinase and peroxidase activity in different stages of eucalypt leaves after inoculation with Puccinia psidii and acibenzolar-S-metil To elucidate some biochemical processes during infection in the pathosystem Puccinia psidii x eucalyptus, the defense metabolism in different-stage leaves was compared between rust-resistant and susceptible clones, respectively. In addition, chitinase and peroxidase activities were assayed. Each treatment consisted of 4 replicates, in a completely randomized design: 2 clones, inoculated and not inoculated with P. psidii; sprayed with acibenzolar-S-methyl (ASM) and distilled water; and represented by the 1(st) leaf pair (size equivalent to 1/5 total leaf development), 2(nd) pair (2/5 total development), and 4(th) pair (4/5 total leaf length). Leaves were harvested in 4 periods: 0, 24, 72 and 96 hours after inoculation. Results indicated that ASM treatment or P. psidii action led to higher chitinase and peroxidase activity level but did not alter the expression of these activities in developed leaves (4(th) pair) during the experiment. Alterations in enzyme levels after inoculation were only observed in developing leaves (1(st) and 2(nd) pairs), which suggests that the response to infection was concomitant to chitinase and peroxidase synthesis. The highest increases in enzymatic activities were observed in resistant clones at 72 hours after inoculation and in susceptible ones previously treated with ASM and later inoculated with the pathogen.

Evaluation of GFP Tag as a Screening Reporter in Directed Evolution of a Hyperthermophilic beta-Glucosidase

By applying a directed evolution methodology specific enzymatic characteristics can be enhanced, but to select mutants of interest from a large mutant bank, this approach requires high throughput screening and facile selection. To facilitate such primary screening of enhanced clones, an expression system was tested that uses a green fluorescent protein (GFP) tag from Aequorea victoria linked to the enzyme of interest. As GFP`s fluorescence is readily measured, and as there is a 1:1 molar correlation between the target protein and GFP, the concept proposed was to determine whether GFP could facilitate primary screening of error-prone PCR (EPP) clones. For this purpose a thermostable beta-glucosidase (BglA) from Fervidobacterium sp. was used as a model enzyme. A vector expressing the chimeric protein BglA-GFP-6XHis was constructed and the fusion protein purified and characterized. When compared to the native proteins, the components of the fusion displayed modified characteristics, such as enhanced GFP thermostability and a higher BglA optimum temperature. Clones carrying mutant BglA proteins obtained by EPP, were screened based on the BglA/GFP activity ratio. Purified tagged enzymes from selected clones resulted in modified substrate specificity.

Antioxidant response of Nicotiana tabacum cv. Bright Yellow 2 cells to cadmium and nickel stress

Plant cell cultures are a suitable model system for investigation of the physiological mechanisms of tolerance to environmental stress. We have determined the effects of Cd (0.1 and 0.2 mM CdCl(2)) and Ni (0.075 and 0.75 mM NiCl(2)) on Nicotiana tabacum L. cv. Bright Yellow (TBY-2) cell suspension cultures over a 72-h period. Inhibition of growth, loss of cell viability and lipid peroxidation occurred, in general, only when the TBY-2 cells were grown at 0.2 mM CdCl(2) and at 0.75 mM NiCl(2). At 0.1 mM CdCl(2), a significant increase in growth was determined at the end of the experiment. Increases in the activities of all of the four enzymatic antioxidant defence systems tested, were induced by the two concentrations of Cd and Ni, but at different times during the period of metal exposure. Overall, the cellular antioxidant responses to Cd and Ni were similar and were apparently sufficient to avoid oxidative stress at the lower concentrations of Cd and Ni. The activities of glutathione reductase and glutathione S-transferase increased early but transiently, whereas the activities of catalase and guaiacol peroxidase increased in the latter half of the experimental period. Therefore it is likely that the metabolism of reduced glutathione was enhanced during the initial onset of the stress, while catalase and guaiacol-type peroxidase appeared to play a more important role in the antioxidant response once the stress became severe.

Diversity and biotechnological potential of culturable bacteria from Brazilian mangrove sediment

Mangrove ecosystems are environments subject to substantial degradation by anthropogenic activities. Its location, in coastal area, interfacing the continents and the oceans makes it substantially important in the prospection for biotechnological applications. In this study, we assessed the diversity of culturable bacteria present over the seasons at two depths (0-10 and 30-40 cm) in a mangrove sediment and in a transect area from the land to the sea. In total, 238 bacteria were isolated, characterized by Amplified Ribosomal DNA Restriction Analysis (ARDRA) and further identified, by Fatty Acid Methyl Esther (FAME-MIDI), into the orders of Vibrionales, Actinomycetales and Bacillales. Also the ability of the isolates in producing economically important enzymes (amylases, proteases, esterases and lipases) was evaluated and the order Vibrionales was the main enzymatic source.

Changes in the genetic structure of Bacteria and microbial activity in an agricultural soil amended with tannery sludge

The application of tannery sludge to soils is a form of recycling; however, few studies have examined the impacts of this practice on soil microbial properties. We studied effects of two applications (2006 and 2007) of tannery sludge (with a low chromium content) on the structure of the bacterial community and on the microbial activity of soils. We fertilized an agricultural area in Rolandia, Parana state, Brazil with different doses of sludge based on total N content, which ranged from 0 to 1200 kg N ha(-1). Sludge remained on the soil surface for three months before being plowed. Soils were sampled seven times during the experiment. Bacterial community structure, assessed by denaturing gradient gel electrophoresis (DGGE), was modified by the application of tannery sludge. Soon after the first application, there was clear separation between the bacterial communities in different treatments, such that each dose of sludge was associated with a specific community. These differences remained until 300 days after application and also after the second sludge application, but 666 days after the beginning of the experiment no differences were found in the bacterial communities of the lowest doses and the control. The principal response curve (PRC) analysis showed that the first sludge application strongly stimulated biological activity even 300 days after application. The second application also stimulated activity, but at a lower magnitude and for a shorter time, given that 260 days after the second application there was no difference in biological activity among treatments. PRC also showed that the properties most influenced by the application of tannery sludge were enzymatic activities related to N cycling (asparaginase and urease). The redundancy analysis (RDA) showed that tannery sludge`s influence on microbial activity is mainly related to increases in inorganic N and soil pH. Results showed that changes in the structure of the bacterial community in the studied soils were directly related to changes of their biological activity. (C) 2010 Elsevier Ltd. All rights reserved.

Antioxidant Status in Humans after Consumption of Blackberry (Rubus fruticosus L.) Juices With and Without Defatted Milk

The present study was designed to evaluate the possible effect of the consumption of blackberry juices (BJ) prepared with water (BJW) and defatted milk (BJM) on the plasma antioxidant capacity and the. enzymatic and nonenzymatic antioxidants. A significant (p < 0.05) increase in the ascorbic acid content in the plasma was observed after intake of both BJs. However, no changes were observed in the plasma urate and a-tocopherol levels. An increase on the plasma antioxidant capacity, by ORAC assay, was observed only after consumption of BJW but not statistically significant. Plasma antioxidant capacity had a good positive correlation with ascorbic acid (r = 0.93) and a negative correlation with urate level (r = -0.79). No correlation was observed between antioxidant capacity and total cyanidin or total ellagic acid contents. Further, it was observed that plasma catalase increased following intake of BJ`s. No change was observed on the plasma and erythrocyte CAT and glutathione peroxidase activities. A significant decrease (p < 0.05) in the urinary antioxidant capacity between 1 and 4 h after intake of both BJs was observed. A good correlation was observed between total antioxidant capacity and urate and total cyanidin levels. These results suggested association between anthocyanin levels and CAT and a good correlation between antioxidant capacity and ascorbic acid in the human plasma after intake of BJs. Follow-up studies investigating the antioxidant properties and health benefits are necessary to demonstrate the health benefits of polyphenols.