Wine aroma improvement using a β-glucosidase preparation from aureobasidium pullulans

Microbial β-glucosidases have been used for the enhancement of wine aroma. Nevertheless, few enzymes are active in the conditions of winemaking. In this work, the production of a β-glucosidase by an Aureobasidium pullulans strain (Ap-β-gl) isolated from grape ecosystems was evaluated. The maximum enzymatic synthesis using submerged fermentation was after 96 h of growth in complex media containing 20 g/L of cellobiose as the sole carbon source. The crude enzyme (Ap-β-gl) showed optimal pH at 5.5 and two peaks of optimum temperature (at 45 and 70 C). It showed a wide range of pH stability, stability at low temperatures, and tolerance to ethanol, showing suitable characteristics for winemaking conditions. The hydrolysis of glycosidic terpenes by Ap-β-gl was studied, and its ability to efficiently release free terpenols was demonstrated by gas chromatography/mass spectrometry. The enzymatic treatment notably increased the amount of monoterpenes, showing good prospects for its potential application for the development of aroma in wines. © 2012 Springer Science+Business Media New York.

Thermal denaturation and aggregation of hemoglobin of Glossoscolex paulistus in acid and neutral media

The thermal denaturation and aggregation of the HbGp, in the oxy- and cyanomet-forms, was investigated by DSC, AUC, DLS, optical absorption and CD, in the pH range from 5.0 to 7.0. Oxy-HbGp has a denaturation process partially reversible and dependent on the temperature. DSC melting curve is characterized by a single peak with Tc value of 333.4±0.2K for oxy-HbGp, while two peaks with Tc values of 332.2±0.1 and 338.4±0.2K are observed for cyanomet-HbGp, at pH 7.0. In acidic pH oxy- and cyanomet-HbGp are more stable showing higher Tc values and aggregation. AUC data show that, HbGp, at pH 7.0, upon denaturation, remains undissociated at 323K, presenting oligomeric dissociation at 333 (12±3% of tetramer and 88±5% of whole HbGp) and 343K (70±5% of monomer and 30±2% of trimer). DLS data show that the lag period before aggregation is dependent on the temperature and HbGp concentration. Optical absorption and CD results show that the increase of temperature leads to the oxy-HbGp oxidation and aggregation, above 331K, in acidic pH. CD data, for HbGp, present a greater thermal stability in acid medium than at neutral pH, with similar Tc values for both oxidation forms. Our data are consistent with previous studies and represents an advance in understanding the thermal stability of oligomeric HbGp structure. © 2012 Elsevier B.V.

Production, purification, and characterization of a major penicillium glabrum xylanase using brewer's spent grain as substrate

In recent decades, xylanases have been used in many processing industries. This study describes the xylanase production by Penicillium glabrum using brewer's spent grain as substrate. Additionally, this is the first work that reports the purification and characterization of a xylanase using this agroindustrial waste. Optimal production was obtained when P. glabrum was grown in liquid medium in pH 5.5, at 25 °C, under stationary condition for six days. The xylanase from P. glabrum was purified to homogeneity by a rapid and inexpensive procedure, using ammonium sulfate fractionation and molecular exclusion chromatography. SDS-PAGE analysis revealed one band with estimated molecular mass of 18.36 kDa. The optimum activity was observed at 60 °C, in pH 3.0. The enzyme was very stable at 50 °C, and high pH stability was verified from pH 2.5 to 5.0. The ion Mn2+ and the reducing agents β-mercaptoethanol and DTT enhanced xylanase activity, while the ions Hg2+, Zn2+, and Cu2+ as well as the detergent SDS were strong inhibitors of the enzyme. The use of brewer's spent grain as substrate for xylanase production cannot only add value and decrease the amount of this waste but also reduce the xylanase production cost. © 2013 Adriana Knob et al.

Produção de glicose e frutose por invertase de Saccharomyces cerevisiae imobilizada em suporte MANAE-agarose

Invertase from Saccharomyces cerevisiae was immobilized on agarose beads, activated with various groups (glyoxyl, MANAE or glutaraldehyde), and on some commercial epoxy supports (Eupergit and Sepabeads). Very active and stable invertase derivatives were produced by the adsorption of the enzyme on MANAE-agarose, MANAE-agarose treated with glutaraldhyde and glutaraldehyde-agarose supports. At pH 5.0, these derivatives retained full activity after 24h at 40°C and 50 °C. When assayed at 40°C and 50°C, with the pH adjusted to 7.0, the invertase-MANAE-agarose derivative treated with glutaraldehyde retained 80% of the initial activity. Recovered activities of the derivatives produced with MANAE, MANAE treated with glutaraldehyde and glutaraldehyde alone were 73.5%, 44.4% and 36.8%, respectively. These three preparations were successfully employed to produce glucose and fructose in 3 cycles of sucrose hydrolysis.

Produção, purificação e propriedades bioquímicas de lipase ácida de Candida viswanathii

Pós-graduação em Ciências Biológicas (Microbiologia Aplicada) - IBRC

Imobilização da enzima álcool desidrogenase em suportes alginato-quitosana e glioxil-agarose

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Production, purification, and characterization of an extracellular acid protease from the marine Antarctic yeast Rhodotorula mucilaginosa L7

The production, purification, and characterization of an extracellular protease released by Rhodotorula mucilaginosa L7 were evaluated in this study. This strain was isolated from an Antarctic marine alga and previously selected among others based on the capacity to produce the highest extracellular proteolytic activity in preliminary tests. R. mucilaginosa L7 was grown in Saboraud-dextrose medium at 25 °C, and the cell growth, pH of the medium, extracellular protease production and the glucose and protein consumption were determined as a function of time. The protease was then purified, and the effects of pH, temperature, and salt concentration on the catalytic activity and enzyme stability were determined. Enzyme production started at the beginning of the exponential phase of growth and reached a maximum after 48 h, which was accompanied by a decrease in the pH as well as reductions of the protein and glucose concentrations in the medium. The purified protease presented optimal catalytic activity at pH 5.0 and 50 °C. Finally, the enzyme was stable in the presence of high concentrations of NaCl. These characteristics are of interest for future studies and may lead to potential biotechnological applications that require enzyme activity and stability under acidic conditions and/or high salt concentrations.

Sviluppo di biosensori: modifiche di superfici elettrodiche e sistemi di immobilizzazione enzimatica

An amperometric glucose biosensor was developed using an anionic clay matrix (LDH) as enzyme support. The enzyme glucose oxidase (GOx) was immobilized on a layered double hydroxide Ni/Al-NO3 LDH during the electrosynthesis, which was followed by crosslinking with glutaraldehyde (GA) vapours or with GA and bovine serum albumin (GABSA) to avoid the enzyme release. The electrochemical reaction was carried out potentiostatically, at -0.9V vs. SCE, using a rotating disc Pt electrode to assure homogeneity of the electrodeposition suspension, containing GOx, Ni(NO3)2 and Al(NO3)3 in 0.3 M KNO3. The mechanism responsible of the LDH electrodeposition involves the precipitation of the LDH due to the increase of pH at the surface of the electrode, following the cathodic reduction of nitrates. The Pt surface modified with the Ni/Al-NO3 LDH shows a much reduced noise, giving rise to a better signal to noise ratio for the currents relative to H2O2 oxidation, and a linear range for H2O2 determination wider than the one observed for bare Pt electrodes. We pointed out the performances of the biosensor in terms of sensitivity to glucose, calculated from the slope of the linear part of the calibration curve for enzimatically produced H2O2; the sensitivity was dependent on parameters related to the electrodeposition in addition to working conditions. In order to optimise the glucose biosensor performances, with a reduced number of experimental runs, we applied an experimental design. A first screening was performed considering the following variables: deposition time (30 - 120 s), enzyme concentration (0.5 - 3.0 mg/mL), Ni/Al molar ratio (3:1 or 2:1) of the electrodeposition solution at a total metals concentration of 0.03 M and pH of the working buffer solution (5.5-7.0). On the basis of the results from this screening, a full factorial design was carried out, taking into account only enzyme concentration and Ni/Al molar ratio of the electrosynthesis solution. A full factorial design was performed to study linear interactions between factors and their quadratic effects and the optimal setup was evaluated by the isoresponse curves. The significant factors were: enzyme concentration (linear and quadratic terms) and the interaction between enzyme concentration and Ni/Al molar ratio. Since the major obstacle for application of amperometric glucose biosensors is the interference signal resulting from other electro-oxidizable species present in the real matrices, such as ascorbate (AA), the use of different permselective membranes on Pt-LDHGOx modified electrode was discussed with the aim of improving biosensor selectivity and stability. Conventional membranes obtained using Nafion, glutaraldehyde (GA) vapours, GA-BSA were tested together with more innovative materials like palladium hexacyanoferrate (PdHCF) and titania hydrogels. Particular attention has been devoted to hydrogels, because they possess some attractive features, which are generally considered to favour biosensor materials biocompatibility and, consequently, the functional enzyme stability. The Pt-LDH-GOx-PdHCF hydrogel biosensor presented an anti-interferant ability so that to be applied for an accurate glucose analysis in blood. To further improve the biosensor selectivity, protective membranes containing horseradish peroxidase (HRP) were also investigated with the aim of oxidising the interferants before they reach the electrode surface. In such a case glucose determination was also accomplished in real matrices with high AA content. Furthermore, the application of a LDH containing nickel in the oxidised state was performed not only as a support for the enzyme, but also as anti-interferant sistem. The result is very promising and it could be the starting point for further applications in the field of amperometric biosensors; the study could be extended to other oxidase enzymes.

Hydrogen Peroxide in Biocatalysis. A Dangerous Liaison

Hydrogen peroxide is a substrate or side-product in many enzyme-catalyzed reactions. For example, it is a side-product of oxidases, resulting from the re-oxidation of FAD with molecular oxygen, and it is a substrate for peroxidases and other enzymes. However, hydrogen peroxide is able to chemically modify the peptide core of the enzymes it interacts with, and also to produce the oxidation of some cofactors and prostetic groups (e.g., the hemo group). Thus, the development of strategies that may permit to increase the stability of enzymes in the presence of this deleterious reagent is an interesting target. This enhancement in enzyme stability has been attempted following almost all available strategies: site-directed mutagenesis (eliminating the most reactive moieties), medium engineering (using stabilizers), immobilization and chemical modification (trying to generate hydrophobic environments surrounding the enzyme, to confer higher rigidity to the protein or to generate oxidation-resistant groups), or the use of systems capable of decomposing hydrogen peroxide under very mild conditions. If hydrogen peroxide is just a side-product, its immediate removal has been reported to be the best solution. In some cases, when hydrogen peroxide is the substrate and its decomposition is not a sensible solution, researchers coupled one enzyme generating hydrogen peroxide “in situ” to the target enzyme resulting in a continuous supply of this reagent at low concentrations thus preventing enzyme inactivation. This review will focus on the general role of hydrogen peroxide in biocatalysis, the main mechanisms of enzyme inactivation produced by this reactive and the different strategies used to prevent enzyme inactivation caused by this “dangerous liaison”.

The chromatographic separation of carbohydrate mixtures

The separation performance of a semicontinuous counter-current chromatographic refiner (SCCR7), consisting of twelve 5.4 cm id x 75cm long columns packed with calcium charged cross-linked polysytrene resin (KORELA VO7C), was optimised. An industrial barley syrup was used containing 42% fructose, 52% glucose and 6% maltose and oligosaccharides. The effects of temperature, flow rates and concentration on the distribution coefficients were evaluated and quantified by deriving general relationships. The effects of flow rates, feed composition and concentration on the separation performance of the SCCR7 were identified and general relationships between them and the switch time, which was found to be the controlling parameter, were developed. Fructose rich (FRP) and glucose rich (GRP) product purities of 99.9% were obtained at 18.6% w/v feed concentrations. When a 66% w/v feed concentration was used and product splitting technique was employed, the throughput was 32.1 kg sugar solids/m3 resin/hr. The GRP contained less than 4.5% fructose, the FRP was over 95% pure, and the respective concentrations were 22.56 and 11.29% w/v. Over 94% of the glucose and 95.78% of the fructose in the feed were recovered in the GRP and FRP respectively. By recycling the dilute product split fractions, the GRP and FRP concentrations were increased to 25.4 and 12.96% w/v; the FRP was 90.2% pure and the GRP contained 6.69% w/v fructose. A theoretical link between batch and semicontinuous chromatographic equipments has been determined. A computer simulation was developed predicting successfully the purging concentration profiles at `pseudo-equilibrium', and also certain system design parameters. An important further aspect of the work has been to study the behaviour of chromatographic bioreactor-separators. Such batch systems of 5.4cm id and lengths varying between 30 and 230cm, were used to investigate the effect of scaling up on the conversion of sucrose into dextran and fructose in the presence of the dextransucrase enzyme. Conversions of over 80% were achieved at 4 hr sucrose residence times. The crude dextransucrase was purified using centrifugation, ultrafiltration and cross-flow microfiltration techniques. Better enzyme stability was obtained by first separating the non-solid impurities using cross-flow microfiltration, and then removing the cells from the enzyme immediately before use by continuous centrifugation.

A sensitive assay for creatine kinase in serum samples dried on paper : enhanced thermal stability of the dried enzyme

A new bioluminescent creatine kinase (CK) assay using purified luciferase was used to analyse CK activity in serum samples dried on filter paper. Enzyme activity was preserved for over 1 wk on paper stored at room temperature. At 60°C, CK activity in liquid serum samples was rapidly inactivated, but the activity of enzyme stored on paper was preserved for at least 2 days.

Engineered dimer interface mutants of triosephosphate isomerase: the role of inter-subunit interactions in enzyme function and stability

The role of inter-subunit interactions in maintaining optimal catalytic activity in triosephosphate isomerase (TIM) has been probed, using the Plasmodium falciparum enzyme as a model. Examination of subunit interface contacts in the crystal structures suggests that residue 75 (Thr, conserved) and residue 13 (Cys, variable) make the largest number of inter-subunit contacts. The mutants Cys13Asp (C13D) and Cys13Glu (C13E) have been constructed and display significant reduction in catalytic activity when compared with wild-type (WT) enzyme (similar to 7.4-fold decrease in k(cat) for the C13D and similar to 3.3-fold for the C13E mutants). Analytical gel filtration demonstrates that the C13D mutant dissociates at concentrations < 1.25 mu M, whereas the WT and the C13E enzymes retain the dimeric structure. The order of stability of the mutants in the presence of chemical denaturants, like urea and guanidium chloride, is WT > Cys13Glu > Cys13Asp. Irreversible thermal precipitation temperatures follow the same order as well. Modeling studies establish that the Cys13Asp mutation is likely to cause a significantly greater structural perturbation than Cys13Glu. Analysis of sequence and structural data for TIMs from diverse sources suggests that residues 13 and 82 form a pair of proximal sites, in which a limited number of residue pairs may be accommodated.

Structural basis for branching-enzyme activity of glycoside hydrolase family 57: Structure and stability studies of a novel branching enzyme from the hyperthermophilic archaeon Thermococcus Kodakaraensis KOD1

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

Extracellular β-fructofuranosidase from Fusarium graminearum: Stability of the spray-dried enzyme in the presence of different carbohydrates

Microbial enzymes have been used for various biotechnological applications; however, enzyme stabilization remains a challenge for industries and needs to be considered. This study describes the effects of spray-drying conditions on the activity and stability of β-fructofuranosidase from Fusarium graminearum. The extracellular enzyme β-fructofuranosidase was spray dried in the presence of stabilizers, including starch (Capsul) (SC), microcrystalline cellulose (MC), trehalose (TR), lactose (LC) and β-cyclodextrin (CD). In the presence of TR (2% w/v), the enzymatic activity was fully retained. After 1 year of storage, 74% of the enzymatic activity was maintained with the CD stabilizer (10% w/v). The residual activity was maintained as high as 80% for 1 h at 70°C when MC, SC and CD (5% w/v) stabilizers were used. Spray drying with carbohydrates was effective in stabilizing the F. graminearum β-fructofuranosidase, improved enzymatic properties compared to the soluble enzyme and demonstrated a potential use in future biotechnology applications. © 2013 Informa UK Ltd. All rights reserved.