Characterization of the interdependency between residues that bind the substrate in a β-glycosidase

The manner by which effects of simultaneous mutations combine to change enzymatic activity is not easily predictable because these effects are not always additive in a linear manner. Hence, the characterization of the effects of simultaneous mutations of amino acid residues that bind the substrate can make a significant contribution to the understanding of the substrate specificity of enzymes. In the β-glycosidase from Spodoptera frugiperda (Sfβgly), both residues Q39 and E451 interact with the substrate and this is essential for defining substrate specificity. Double mutants of Sfβgly (A451E39, S451E39 and S451N39) were prepared by site-directed mutagenesis, expressed in bacteria and purified using affinity chromatography. These enzymes were characterized using p-nitrophenyl β-galactoside and p-nitrophenyl β-fucoside as substrates. The k cat/Km ratio for single and double mutants of Sfβgly containing site-directed mutations at positions Q39 and E451 was used to demonstrate that the effect on the free energy of ES‡ (enzyme-transition state complex) of the double mutations (∆∆G‡xy) is not the sum of the effects resulting from the single mutations (∆∆G‡x and ∆∆G‡y). This difference in ∆∆G‡ indicates that the effects of the single mutations partially overlap. Hence, this common effect counts only once in ∆∆G‡xy. Crystallographic data on β-glycosidases reveal the presence of a bidentate hydrogen bond involving residues Q39 and E451 and the same hydroxyl group of the substrate. Therefore, both thermodynamic and crystallographic data suggest that residues Q39 and E451 exert a mutual influence on their respective interactions with the substrate.

A biosensor based on immobilization of lactate oxidase in a PB-CTAB film for FIA determination of lactate in beer samples

An amperometric lactate biosensor with lactate oxidase immobilized into a Prussian Blue (PB) modified electrode was fabricated. The advantage of using cetyltrimethylammonium bromide (CTAB) in the electrodeposition step of PB films onto glassy carbon surfaces was confirmed taking into account both the stability and sensitivity of the measurements. The biosensor was used in the development of a FIA amperometric method for the determination of lactate. Under optimal operating conditions (pH = 6.9, E = -0.1 V), the linear response of the method was extended up to 0.28 µmol L-1 lactate with a limit of detection of 0.84 mmol L-1. The repeatability of the method for injections of a 0.28 mmol L-1 lactate solution was 2.2 % (n = 18). The usefulness of the method was demonstrated by determining lactate in beer samples and the results were in good agreement with those obtained by using a reference spectrophotometric enzyme method.

High gravity batch and continuous processes for beer production: Evaluation of fermentation performance and beer quality

This study deals with two innovative brewing processes, high gravity batch and complete continuous beer fermentation systems. The results show a significant influence of the variables such as concentration and temperature on the yield factor of the substrate into ethanol and consequently on the productivity of the high gravity batch process. The technological feasibility of continuous production of beer based on yeast immobilization on cheap alternative carriers was also demonstrated. The influence of process parameters on fermentation performance and quality of the obtained beers was studied by sensorial analysis. No significant difference in the degree of acceptance between the obtained products and some traditional market brands was found. (c) 2008 Institute of Chemistry, Slovak Academy of Sciences.

Banana as Adjunct in Beer Production: Applicability and Performance of Fermentative Parameters

Traditionally, the raw materials for beer production are barley, hops, water, and yeast, but most brewers use also different adjuncts. During the alcoholic fermentation, the contribution of aroma compounds from other ingredients to the final beer flavor depends on the wort composition, on the yeast strain, and mainly on the process conditions. In this context, banana can also be a raw material favorable to alcoholic fermentation being rich in carbohydrates and minerals and providing low acidity. In this work, the objective was to evaluate the performance of wort adjusted with banana juice in different concentrations. For this, static fermentations were conducted at 15 degrees C at pilot scale (140 L of medium). The addition of banana that changed the concentration of all-malt wort from 10 degrees P to 12 and 15 degrees P were evaluated (degrees P is the weight of the extract or the sugar equivalent in 100 g solution, at 20 degrees C). The results showed an increase in ethanol production, with approximately 0.4 g/g ethanol yield and 0.6 g/L h volumetric productivity after 84 h of processing when concentrated wort was used. Thus, it was concluded that banana can be used as an adjunct in brewing methods, helping in the development of new products as well as in obtaining concentrated worts.

Kinetic studies of urban solid residues and leachate from sanitary landfill

Urban solid residues are constituted of food remaining, grass leaves, fruit peelings, paper, cardboard, rubber, plastic, etc. The organic fraction formed represents about 50% during the decomposition yields biogas and leachate, which are sources of pollution. Residue samples were collected from the landfill in different and cells from several ages and the corresponding leachate, both after treatments, were submitted to thermal analysis. Kinetic parameters were determined using Flynn-Wall-Ozawa method. The linear relation between the two kinetic parameters (ln A and E) was verified for organic residue urban`s samples, but not for leachate`s sample. The occurred difference can be attributed to the constituents present in leachate.

Using of the residues and wood composites for production of an electric guitar

The residues generation is a quite serious problem in several industrial areas and also in the lumbering area. The search for the elimination or reduction of the volume of generated residues is endless, however limited, resulting in the search for a proper destination or better use, instead of simply burning it. A lot of uses and services are commonly proposed, but with low aggregated value to the residue. This work shows the usage viability of different discarded residues and wood composites in the production of an electric guitar. Cupiuba, ipe and jatoba residues have been used besides wood composites of pinus. The residues and wood composites have shown appropriate resistance, surfacing quality and design terms, and could be used to substitute the traditionally wood used in the production of the instrument as well as in other products of similar characteristics and with larger aggregated value.

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.

OPTIMIZATION OF THE TREATMENT PROCESS FOR RESIDUAL WATER FROM A BEER INDUSTRY

The optimization of the treatment process for residual waters from a brewery operating under the modality of an anaerobic reactor and activated sludge combination was studied in two phases. In the first stage, lasting for six months, the characteristics and parameters of the plant operation were analyzed, wherein a diversion rate of more than 50% to aerobic treatment, the use of two aeration tanks and a high sludge production prevailed. The second stage comprised four months during which the system worked under the proposed operational model, with the aim of improving the treatment: reduction of the diversion rate to 30% and use of only one aeration tank At each stage, TSS, VSS and COD were measured at the entrance and exit of the anaerobic reactor mid the aeration tanks. The results were compared with the corresponding design specifications and the needed conditions were applied to reduce the diversion rate towards the aerobic process through monitoring the volume and concentration of the affluent, while applying the strategic changes in reactor parameters needed to increase its efficiency. A diversion reduction from 53 to 34% was achieved, reducing the sludge discharge generated in the aerobic system from 3670mg TSS/l. with two aeration tanks down to 2947mf TSS/l using one tank keeping the same relation VSS:TSS (0.55) and an efficiency of total removal of 98% in terms of COD.

Wine industry residues extracts as natural antioxidants in raw and cooked chicken meat during frozen storage

The effect of Isabel (IGE) and Niagara (NGE) grape seed and peel extracts on lipid oxidation, instrumental colour, pH and sensory properties of raw and cooked processed chicken meat stored at -18 degrees C for nine months was evaluated. The pH of raw and cooked samples was not affected by the addition of grape extracts. IGE and NGE were effective in inhibiting the lipid oxidation of raw and cooked chicken meat, with results comparable to synthetic antioxidants. The extracts caused alterations in colour, as evidenced by the instrumental (darkening and lower intensity of red and yellow colour) and sensory results of cooked samples. In the sensory evaluation of odour and flavour, IGE produced satisfactory results, which did not differ from synthetic antioxidants. These findings suggest that the ICE and NGE are effective in retarding lipid oxidation of raw and cooked chicken meat during frozen storage. (c) 2011 Elsevier Ltd. All rights reserved.

Carbon and Nitrogen Dynamics in an Oxisol as Affected by Liming and Crop Residues under No-Till

The short-term effects of surface lime application and black oat (Avena strigosa Schreb.) residues, with or without N fertilization, were evaluated in a long-term no-till (NT) system on a sandy clay loam, a kaolinitic, thermic Typic Hapludox from the state of Parana, Brazil. The main plot treatments were: control and dolomitic lime applied on soil surface at 8 Mg ha(-1). Three treatments with crop residues were evaluated on the subplots: (i) fallow, (ii) black oat residues, and (iii) black oat residues aft er N fertilization at 180 kg ha(-1). Black oat dry biomass was not affected by the treatments during 3 yr. Surface liming increased soil pH, microbial biomass, microbial activity, and bacterial/fungal ratio at the soil surface (0-5 cm), resulting in increased amino acid turnover, water-soluble humic substances formation, and N mineralization and nitrification. While the application of black oat did increase the soil pH, overall it had much less effect on soil biological processes and C and N pools than did lime. We concluded that black oat cannot replace the need for lime to optimize crop production in these tropical NT systems. In the long term, however, black oat should aid in the amelioration of acidity and replenishment of soil organic C pools and should help reduce erosion. Overall, this study suggests that overapplication of inorganic fertilizer N may occur in some tropical NT systems. Further experiments are required in NT systems to investigate the use of slow-release N fertilizers in combination with lime and black oat as a mechanism to reduce acidification and promote sustainability.

Sterilization of Medical Devices by Ethylene Oxide, Determination of the Dissipation of Residues, and Use of Green Fluorescent Protein as an Indicator of Process Control

Ethylene oxide (EO) is used to sterilize Oxygenator and Tubing applied to heart surgery. Residual levels of EO and its derivatives, ethylene chlorohydrin (ECH) and ethylene glycol (EG), may be hazardous to the patients. Therefore, it must be removed by the aeration process. This study aimed to estimate the minimum aeration time for these devices to attain safe limits for use (avoiding excessive aeration time) and to evaluate the Green Fluorescent Protein (GFP) as a biosensor capable of best indicating the distribution and penetration of EO gas throughout the sterilization chamber. Sterilization cycles of 2, 4, and 8 h were monitored by Bacillus atrophaeus ATCC 9372 as a biological indicator (131) and by the GFP. Residual levels of EO, ECH, and EG were determined by gas chromatography (GC), and the residual dissipation was studied. Safe limits were reached right after the sterilization process for Oxygenator and after 204 h of aeration for Tubing. In the 2 h cycle, the GFP concentration decreased from 4.8 (+/- 3.2)% to 7.5 (+/- 2.5)%. For the 4 h cycle, the GFP concentration decreased from 17.4 (+/- 3.0)% to 21.5 (+/- 6.8)%, and in the 8 h cycle, it decreased from 22.5 (+/- 3.2)% to 23.9 (+/- 3.9)%. This finding showed the potentiality for GFP applications as an EO biosensor. (C) 2009 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 9113: 626-630, 2009

Production of beta-fructofuranosidases by Aspergillus niveus using agroindustrial residues as carbon sources: Characterization of an intracellular enzyme accumulated in the presence of glucose

The production of beta-fructofuranosidases by Aspergillus niveus, cultivated under submerged fermentation using agroindustrial residues, was investigated. The highest productivity of beta-fructofuranosidases was obtained in Khanna medium supplemented with sugar cane bagasse as carbon source. Glucose enhanced the production of the intracellular enzyme, whereas that of the extracellular one was decreased. The intracellular beta-fructofuranosidase was a trimeric protein of approximately 141 kDa (gel filtration) with 53.5% carbohydrate content, composed of 57 kDa monomers (SDS-PAGE). The optimum temperature and optimum pH were 60 degrees C and 4.5, respectively. The purified enzyme showed good thermal stability and exhibited a half-life of 53 min at 60 degrees C. beta-Fructofuranosidase activity was slightly activated by Cu(2+), Mn(2+), Mg(2+), and Na(+) at 1 mM concentration. The enzyme hydrolyzed sucrose, raffinose, and inulin, with K(d) values of 5.78 mM, 5.74 mM, and 1.74 mM, respectively. (C) 2008 Elsevier Ltd. All rights reserved.

Production of fibrolytic enzymes by Aspergillus japonicus C03 using agro-industrial residues with potential application as additives in animal feed

Solid-state fermentation obtained from different and low-cost carbon sources was evaluated to endocellulases and endoxylanases production by Aspergillus japonicus C03. Regarding the enzymatic production the highest levels were observed at 30 degrees C, using soy bran added to crushed corncob or wheat bran added to sugarcane bagasse, humidified with salt solutions, and incubated for 3 days (xylanase) or 6 days (cellulase) with 70% relative humidity. Peptone improved the xylanase and cellulase activities in 12 and 29%, respectively. The optimum temperature corresponded to 60 degrees C and 50-55 degrees C for xylanase and cellulase, respectively, both having 4.0 as optimum pH. Xylanase was fully stable up to 40 degrees C, which is close to the rumen temperature. The enzymes were stable in pH 4.0-7.0. Cu(++) and Mn(++) increased xylanase and cellulase activities by 10 and 64%, respectively. A. japonicus C03 xylanase was greatly stable in goat rumen fluid for 4 h during in vivo and in vitro experiments.

Analysis of experimental interlocking blocks of concrete with addition of residues of process the tires retreading production

Analysis of experimental interlocking blocks of concrete with addition of residues of process the tires retreading production. With the population growth in recent years, industry in general has adjusted itself to resulting demand. the industry of tire retreading generates residues that have been discarded without any control. this adds to environmental pollution and promotes the proliferation of vectors harmful to health, aiming to find an application for this type of residues, this study presents experimental results to interlocking concrete block pavements, with addition of residues tires, interlocking blocks were built up and we determined, through laboratory tests, the need to set the mark that provide greater return regarding analyzed characteristics, there are four types of dosage of concrete with residues tires. We accomplished tests of compression strength, water absorption and resistance to impact. Through the preliminary results, we verified that are satisfactory, confirming the possibility of applying this type of interlocking block in environments with low demand, which would bring the economy of natural sources of aggregates, beyond ecological benefits through the reuse of residues from retreading of tires.

Sialic Acid Residues Are Essential for the Anaphylactic Activity of Murine IgG1 Antibodies

Glycosylation of the Ab molecule is essential for maintaining the functional structure of Fc region and consequently for Ab-mediated effector functions, such as binding to cells or complement system activation. Alterations in the composition of the sugar moiety can dramatically influence Ab activity; however, it is not completely clear how differences in the N-linked oligosaccharide structure impact the biological function of Abs. We have described that murine IgG1 Abs can be separated according to their ability to elicit in vivo anaphylaxis in a fraction of anaphylactic and other of non-anaphylactic molecules. Furthermore, we showed that the N-linked oligosaccharide chain is essential for the structural conformation of the anaphylactic IgG1, the binding to Fc gamma RIII on mast cells, and, consequently, for the ability to mediate anaphylactic reactions. In this study, we evaluated the contribution of individual sugar residues to this biological function. Differences in the glycan composition were observed when we analyzed oligosaccharide chains from anaphylactic or non-anaphylactic IgG1, mainly the presence of more sialic acid and fucose residues in anaphylactic molecules. Interestingly, the enzymatic removal of terminal sialic acid residues in anaphylactic IgG1 resulted in loss of the ability to trigger mast cell degranulation and in vivo anaphylactic reaction, similarly to the deglycosylated IgG1 Ab. In contrast, fucose removal did not affect the anaphylactic function. Therefore, we demonstrated that the ability of murine IgG1 Abs to mediate anaphylaxis is directly dependent on the amount of sialic acid residues associated to the oligosaccharide chain attached to the Fc region of these molecules. The Journal of Immunology, 2008, 181: 8308-8314.