Optimal Conditions for Biomass and Recombinant Glycerol Kinase Production Using the Yeast Pichia pastoris

The extracellular glycerol kinase gene from Saccharomyces cerevisiae (GUT]) was cloned into the expression vector pPICZ alpha. A and integrated into the genome of the methylotrophic yeast Pichia pastoris X-33. The presence of the GUT1 insert was confirmed by PCR analysis. Four clones were selected and the functionality of the recombinant enzyme was assayed. Among the tested clones, one exhibited glycerol kinase activity of 0.32 U/mL, with specific activity of 0.025 U/mg of protein. A medium optimized for maximum biomass production by recombinant Pichia pastoris in shaker cultures was initially explored, using 2.31 % (by volume) glycerol as the carbon source. Optimization was carried out by response surface methodology (RSM). In preliminary experiments, following a Plackett-Burman design, glycerol volume fraction (phi(Gly)) and growth time (t) were selected as the most important factors in biomass production. Therefore, subsequent experiments, carried out to optimize biomass production, followed a central composite rotatable design as a function of phi(Gly) and time. Glycerol volume fraction proved to have a significant positive linear effect on biomass production. Also, time was a significant factor (at linear positive and quadratic levels) in biomass production. Experimental data were well fitted by a convex surface representing a second order polynomial model, in which biomass is a function of both factors (R(2)=0.946). Yield and specific activity of glycerol kinase were mainly affected by the additions of glycerol and methanol to the medium. The optimized medium composition for enzyme production was: 1 % yeast extract, 1 % peptone, 100 mM potassium phosphate buffer, pH=6.0, 1.34 % yeast nitrogen base (YNB), 4.10(-5) % biotin, 1 %, methanol and 1 %, glycerol, reaching 0.89 U/mL of glycerol kinase activity and 14.55 g/L of total protein in the medium after 48 h of growth.

Optimization of acid hydrolysis from the hemicellulosic fraction of Eucalyptus grandis residue using response surface methodology

Biotechnological conversion of biomass into fuels and chemicals requires hydrolysis of the polysaccharide fraction into monomeric sugars. Hydrolysis can be performed enzymatically and with dilute or concentrate mineral acids. The present study used dilute sulfuric acid as a catalyst for hydrolysis of Eucalyptus grandis residue. The purpose of this paper was to optimize the hydrolysis process in a 1.41 pilot-scale reactor and investigate the effects of the acid concentration, temperature and residue/acid solution ratio on the hemicellulose removal and consequently on the production of sugars (xylose, glucose and arabinose) as well as on the formation of by-products (furfural, 5-hydroxymethylfurfural and acetic acid). This study was based on a model composition corresponding to a 2 3 orthogonal factorial design and employed the response surface methodology (RSM) to optimize the hydrolysis conditions, aiming to attain maximum xylose extraction from hemicellulose of residue. The considered optimum conditions were: H2SO4 concentration of 0.65%, temperature of 157 degrees C and residue/acid solution ratio of 1/8.6 with a reaction time of 20 min. Under these conditions, 79.6% of the total xylose was removed and the hydrolysate contained 1.65 g/l glucose, 13.65 g/l xylose, 1.55 g/l arabinose, 3.10 g/l acetic acid, 1.23 g/l furfural and 0.20 g/l 5-hydroxymethylfurfural. (c) 2006 Published by Elsevier Ltd.

Morphology and bacterial colonisation of tooth/ceramic restoration interface after different cement excess removal techniques

Objectives: To evaluate the influence of different protocols for resin cement removal during cementation on biofilm formation.Methods: Twenty-eight ceramic blocks, which were injected under pressure, were placed over enamel blocks obtained from freshly extracted bovine incisors. The ceramic blocks were cemented to the enamel blocks using a dual-cured resin cement and the excess resin was removed according to the experimental group: TS: Teflon spatula; BR: brush; BR+: brush and polishing; SB+: scalpel blade and polishing. After autoclaving, the samples were colonised by incubation in a sucrose broth suspension standardised with Streptococcus mutans in microaerophilic stove. Specimens were quantitatively analysed for bacterial adherence at the adhesive interface using confocal laser scanning microscopy and counting the colony forming units, and qualitatively analysed using SEM. The roughness (Ra/Rz/RSm) was also analysed. Data were analysed by 1-way ANOVA and Tukey's test (5%).Results: The roughness values ranged from 0.96 to 1.69 mu m for Ra (p > 0.05), from 11.59 to 22.80 mu m for Rz (p = 0.02 < 0.05) and from 293.2 to 534.3 mu m for RSm (p = 0.00). Bacterial adhesion varied between 1,974,000 and 2,814,000 CFU/ml (p = 0.00). Biofilm mean thickness ranged from 0.477 and 0.556 mu m (p > 0.05), whilst the biovolume values were between 0.388 and 0.547 mu m(3)/mu m(2) (p = 0.04). Lower values for roughness, bacterial adhesion, biofilm thickness and biovolume were found with BR, whilst TS presented the highest values for most of the parameters. SEM images confirmed the quantitative values.Conclusions: The restoration margin morphology and interface roughness affects bacterial accumulation. The brush technique promoted less bacterial colonisation at the adhesive interface than did the other removal methods.Clinical significance: The brush technique seems to be a good option for removing the excess resin cement after adhesive cementation in clinical practice, as indicated by its better results with lower bacterial colonisation. (C) 2012 Elsevier Ltd. All rights reserved.

Charmonium-pion cross section from QCD sum rules

The J/psipi --> (D) over barD*, D (D) over bar*, (D) over bar *D* and (D) over barD cross sections as a function of roots are evaluated in a QCD sum rule calculation. We study the Borel sum rule for the four point function involving pseudoscalar and vector meson currents, up to dimension four in the operator product expansion. We find that our results are smaller than the J/psipi --> charmed mesons cross sections obtained with models based on meson exchange, but are close to those obtained with quark exchange models. (C) 2002 Elsevier B.V. B.V. All rights reserved.

Qcd Sum Rules for the G(KK*pi)(Q(2)) form factor

The QCD Sum Rules have been used to evaluate the form factor in the vertex KK*pi. The method of QCD Sum Rules is based on the duality principle in which it is assumed that the hadrons can simultaneously be described in two levels: quarks and hadrons. This work showed that the, axial current, used to describe the meson K is not appropriated to study the form factor.

Quark condensates and high density quark matter

We consider a Coulomb gauge quark model which includes an explicit construct for a nontrivial vacuum structure in QCD. The dynamics is described by a Hamiltonain that contains a linearly rising confining potential and longitudinal and transverse Coulomb-type interactions. The Coulomb potential gives rise to ultraviolate divergences which are non-perturbatively renormalized by adding appropriate counter terms to the Hamiltonian. The equation of state for u and d quark matter at zero temperature is derived in the Hartree-Fock approximation.

Studying nonlinear effects on the early stage of phase ordering using a decomposition method

Nonlinear effects on the early stage of phase ordering are studied using Adomian's decomposition method for the Ginzburg-Landau equation for a nonconserved order parameter. While the long-time regime and the linear behavior at short times of the theory are well understood, the onset of nonlinearities at short times and the breaking of the linear theory at different length scales are less understood. In the Adomians decomposition method, the solution is systematically calculated in the form of a polynomial expansion for the order parameter, with a time dependence given as a series expansion. The method is very accurate for short times, which allows to incorporate the short-time dynamics of the nonlinear terms in a analytical and controllable way. (c) 2005 Elsevier B.V. All rights reserved.

Optimization of temperature, sugar concentration, and inoculum size to maximize ethanol production without significant decrease in yeast cell viability

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Characterization of glycerol kinase from baker's yeast: Response surface modeling of the enzymatic reaction

The present study describes a methodology of dosage of glycerol kinase (GK) from baker's yeast. The standardization of the activity of the glycerol kinase from baker's yeast was accomplished using the diluted enzymatic preparation containing glycerol phosphate oxidase (GPO) and glycerol kinase. The mixture was incubated at 60 degrees C by 15 min and the reaction was stopped by the SDS solution addition. A first set of experiments was carried out in order to investigate the individual effect of temperature (7), pH and substrate concentration (S), on GK activity and stability. The pH and temperature stability tests showed that the enzyme presented a high stability to pH 6.0-8.0 and the thermal stability were completely maintained up to 50 degrees C during 1 h. The K(m) of the enzyme for glycerol was calculated to be 2 mM and V(max) to be 1.15 U/mL. In addition, modeling and optimization of reaction conditions was attempted by response surface methodology (RSM). Higher activity values will be attained at temperatures between 52 and 56 degrees C, pH around 10.2-10.5 and substrate concentrations from 150 to 170 mM.This low cost method for glycerol kinase dosage in a sequence of reactions is of great importance for many industries, like food, sugar and alcohol. RSM showed to be an adequate approach for modeling the reaction and optimization of reaction conditions to maximize glycerol kinase activity. (C) 2007 Elsevier B.V. All rights reserved.

Desempenho produtivo de alevinos de tilápia-do-nilo (Oreochromis niloticus) arraçoados com farinha de soja

This experiment was carried out in the Research Laboratory on Nutrition of Aquatic Organisms, Aquaculture Center UNESP, FMVZ - Botucatu, SP, Brazil. The study aimed to evaluate the effect of raw full-fat soybean meal on feeding in fingerlings of the Nile tilapia (Oreochromis niloticus). The suitable level of consumption of raw full-fat soybean meal (RSM) was determined by means of productive performance. The experimental design was a Multivariate Profile Analyses, with four treatments (four replicates each one). Each isoproteic (26% CP) and isoenergetic (3000 Kcal ME/ kg) diet received raw full-fat soybean meal at 6.0%, 12.0% and 18.0%. A treatment without soybean meal was used as control. Forty eight fingerlings with similar size (mean Wt ± SD: 6.47 ± 0.64 g; and mean Lt ± SD: 54.94 ± 7.79 mm) at the beginning of the experiments were used. No significant difference (p>0.05%) in weight gain among the four treatments was found. However, the treatments with 12.0 and 18.0% RSM showed a reduction in the coefficient of digestibility in these treatments. This shows a deleterious effect of the raw full-fat soybean meal at these considered levels (12.0% and 18.0% RSM).

Optimization of a synbiotic formulation of soy yoghurt containing yacon extract by response surface methodology

A synbiotic yoghurt based on a combination of soymilk and yacon water extract (from yacon root tubers) was developed as a novel food product fermented with a probiotic culture of Enterococcus faecium CRL 183 and Lactobacillus helveticus ssp jugurti 4l6. Response surface methodology (RSM) was used to optimize the independent variables soymilk protein concentration and percentage of yacon extract in the formulation through a Central Composite Rotatable Design (CCRD), consisting of a 22 factorial design with two levels (-1, +1), two central points (0) and four axial points (± a, 0) (0, ± α). The responses were assessed by consumer acceptance tests. The optimization indicated that a formulation with a soymilk protein concentration of 1.74g/L and 25.86% of yacon extract gave the best average values, 5.91 for the taste and 6.00 for the overall impression responses. The formulation with 40% of yacon extract and the same concentration of soymilk protein achieved similar acceptance values: taste (5.94) and overall impression (5.87), however, with the extra yacon, it probably had a greater content of prebiotic fructooligosaccharides. Consequently, both formulations may give useful functional foods, with sensory properties comparable with those of soy yoghurt (control formulation). Copyright © 2010 by New Century Health Publishers.

Agaricus blazei as a Substrate for the Production of β-1,3-Glucanase by Trichoderma harzianum Rifai

Extracellular β-1,3-glucanase was produced by Trichoderma harzianum Rifai cultivated in the Agaricus blazei (Agaricus brasiliensis) extract as a substrate in submerged fermentation. A 22-central composite factorial design was developed using the time of culture (x1/day) and Agaricus blazei extract concentration (x2/(g/L)) as variables, and the results were analyzed using response surface methodology (RSM). The results showed that the Agaricus blazei extract concentration was the most important variable in the production of β-1,3-glucanase, and the maximum β-1,3-glucanase activity (0.77 U/mL) was obtained in one day of cultivation. The β-glucan present in the cell wall of Agaricus blazei mushroom proved to be a good substrate for inducing the production of specific β-1,3-glucanase by Trichoderma harzianum Rifai.

Optimization of the surimi production from Mechanically Recovered Fish Meat (MRFM) using response surface methodology

The by-products generated from industrial filleting of tilapia surimi can be used for the manufacture of surimi. The surimi production uses large amounts of water, which generates a wastewater rich in organic compounds (lipids, soluble proteins and blood). Optimizing the number of washing cycles will contribute to a more sustainable production. A mathematical model of mechanically recovered tilapia meat (Oreochromis niloticus) for the processing of surimi (minced fish washing cycles and tapioca starch addition) based on two quality parameters (texture and moisture) was constructed by applying the response surface methodology (RSM). Each factor had an important effect on the moisture and texture of surimi. This study found that the optimal formulation for producing the best surimi using the by-products of tilapia filleting in manufacturing fish burger were the addition of 10% tapioca starch and three minced fish washing cycles. A microstructural evaluation supported the findings of the mathematical model. Practical Applications: The use of mechanically recovered fish meat (MRFM) for the production of surimi enables the utilization of the by-products of filleting fish. However, the inferior quality of the surimi produced from MRFM in relation to that produced with fillets necessitates the addition of starch; secondly, surimi production consumes a large volume of water. RSM provides a valuable means for optimizing the number of washing cycles and starch amounts utilized in fish burger production. Tapioca starch, widely produced in Brazil, has desirable characteristics (surface sheen, smooth texture, neutral taste and clarity in solution) for use in MRFM-produced surimi. © 2013 Wiley Periodicals, Inc.

Glicerol quinase de levedura de panificação

Pós-graduação em Alimentos e Nutrição - FCFAR

Produção otimizada de alginato e plástico biodegradável (poli-hidroxibutirato) por Azotobacter vinelandii

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)