Macrocystis pyrifera Extract Residual as Nutrient Source for the Production of Sophorolipids Compounds by Marine Yeast Rhodotorula rubra.

Seaweed processing generates liquid fraction residual that could be used as a low-cost nutrient source for microbial production of metabolites. The Rhodotorula strain is able to produce antimicrobial compounds known as sophorolipids. Our aim was to evaluate sophorolipid production, with antibacterial activity, by marine Rhodotorula rubra using liquid fraction residual (LFR) from the brown seaweed Macrocystis pyrifera as the nutrient source. LFR having a composition of 32% w/w carbohydrate, 1% w/w lipids, 15% w/w protein and 52% w/w ash. The best culture condition for sophorolipid production was LFR 40% v/v, without yeast extract, artificial seawater 80% v/v at 15 °C by 3 growth days, with the antibacterial activity of 24.4 ± 3.1 % on Escherichia coli and 21.1 ± 3.8 % on Staphylococcus aureus. It was possible to identify mono-acetylated acidic and methyl ester acidic sophorolipid. These compounds possess potential as pathogen controllers for application in the food industry.

Control of selectivity in the oxidation of 5-hydroxymethylfurfural to 5- formyl-2-furancarboxylic acid catalyzed by laccase in a multiphasic gas-liquid microbioreactor.

The selectivity of 5-formyl-2-furancarboxylic acid (FFCA) was studied in a batch bioreactor and microbioreactors with different internal diameters (ID). Using microbioreactors, the effect of the flow rate of the liquid and gas phase on the yield, space time yield (STYFFCA), and gas-liquid mixture velocity (UM) of the reaction was evaluated. The biooxidation in flow microbioreactors, a selectivity of 100 % for FFCA was achieved, while with the batch bioreactor at the same substrate concentration a selectivity of 6.7 % was obtained. The highest yield (30 %) with 15 mM of 5-hydroxymethylfurfural (HMF) was reached at a gas-liquid flow rate of 0.5 µL/min and the highest STYFFCA (0.07 mol m-3 min-1) was achieved at a gas-liquid flow rate of 1.5 µL/min with the microbioreactor with an ID of 0.5 mm. The UM values (0.5 to 1.6 cm min1) indicated that the reaction takes place under a kinetic regime without mass transfer limitations.

Purification of transthyretin as nutritional biomarker of selenium status.

Transthyretin has been proposed as nutritional biomarker of selenium intake. Previous transthyretin purification methods used different procedures to isolate transthyretin either from plasma or from pathological urine of humans. In general, the procedure for purification of transthyretin is laborious and expensive, and extensive sample recycling is necessary for purification in appreciable amounts. This work proposes a new, promissory, and cheap two-step process to purify transthyretin from blood plasma, composed by a first aqueous two-phase system fractionation followed by affinity chromatography, using thyroxine-immobilized on epoxy-activated Sepharose CL-6B. The aqueous two-phase system fractionation was demonstrated to perform better than commercial immunoaffinity-based kits for albumin depletion in blood plasma samples and is an effective first step for transthyretin purification. Thyroxine affinity chromatography was designed to bind transthyretin with high affinity, and was demonstrated to be useful to purify transthyretin, but was unable to completely resolve transthyretin from thyroxine-binding globulin and serum albumin, although the relative amount of albumin was lowered in the eluates. This purification process could be used in nutritional diagnosis tools or as a first step in structural and functional studies.

The growth of marine fungi on seaweed polysaccharides produces cerato-platanin and hydrophobin self-assembling proteins.

The marine fungi Paradendryphiela salina and Talaromyces pinophilus degrade and assimilate complex substrates from plants and seaweed. Additionally, these fungi secrete surface-active proteins, identified as cerato-platanins and hydrophobins. These hydrophobic proteins have the ability to self-assemble forming amyloid-like aggregates and play an essential role in the growth and development of the filamentous fungi. It is the first time that one cerato-platanin (CP) is identified and isolated from P. salina (PsCP) and two Class I hydrophobins (HFBs) from T. pinophilus (TpHYD1 and TpHYD2). Furthermore, it is possible to extract cerato-platanins and hydrophobins using marine fungi that can feed on seaweed biomass, and through a submerged liquid fermentation process. The propensity to aggregate of these proteins has been analyzed using different techniques such as Thioflavin T fluorescence assay, Fourier-transform Infrared Spectroscopy, and Atomic Force Microscopy. Here, we show that the formation of aggregates of PsCP and TpHYD, was influenced by the carbon source from seaweed. This study highlighted the potential of these self-assembling proteins generated from a fermentation process with marine fungi and with promising properties such as conformational plasticity with extensive applications in biotechnology, pharmacy, nanotechnology, and biomedicine.

Saccharification of Brown Macroalgae Using an Arsenal of Recombinant Alginate Lyases: Potential Application in the Biorefinery Process.

Alginate lyases (endo and exo-lyases) are required for the degradation of alginate into its constituting monomers. Efficient bioethanol production and extraction of bioactives from brown algae requires intensive use of these enzymes. Nonetheless, there are few commercial alginate lyase preparations, and their costs make them unsuitable for large scale experiments. A recombinant expression protocol has been developed in this study for producing seven endo-lyases and three exo-lyases as soluble and highly active preparations. Saccharification of alginate using 21 different endo/exo-lyase combinations shows that there is complementary enzymatic activity between some of the endo/exo pairs. This is probably due to favorable matching of their substrate biases for the different glycosidic bonds in the alginate molecule. Therefore, selection of enzymes for the best saccharification results for a given biomass should be based on screens comprising both types of lyases. Additionally, different incubation temperatures, enzyme load ratios, and enzyme loading strategies were assessed using the best four enzyme combinations for treating Macrocystis pyrifera biomass. It was shown that 30°C with a 1:3 endo/exo loading ratio was suitable for all four combinations. Moreover, simultaneous loading of endo-and exo-lyases at the beginning of the reaction allowed maximum alginate saccharification in half the time than when the exo-lyases were added sequentially.

The effect of a lytic polysaccharide monooxygenase and a xylanase from Gloeophyllum trabeum on the enzymatic hydrolysis of lignocellulosic residues using a commercial cellulase.

Hydrolysis of lignocellulosic biomass depends on the concerted actions of cellulases and accessory proteins. In this work we examined the combined action of two auxiliary proteins from the brown rot fungus Gloeophyllum trabeum: a family AA9 lytic polysaccharide monooxygenase (GtLPMO) and a GH10 xylanase (GtXyn10A). The enzymes were produced in the heterologous host Pichia pastoris. In the presence of an electron source, GtLPMO increased the activity of a commercial cellulase on filter paper, and the xylanase activity of GtXyn10A on beechwood xylan. Mixtures of GtLPMO, GtXyn10A and Celluclast 1.5L were used for hydrolysis of pretreated wheat straw. Results showed that a mixture of 60% Celluclast 1.5L, 20% GtXyn10A and 20% GtLPMO increased total reducing sugar production by 54%, while the conversions of glucan to glucose and xylan to xylose were increased by 40 and 57%, respectively. This suggests that GtLPMO can contribute to lignocellulose hydrolysis, not only by oxidative activity on glycosidic bonds, but also to hemicellulose through the oxidation of xylosyl bonds in xylan. The concerted action of these auxiliary enzymes may significantly improve large-scale recovery of sugars from lignocellulose.

Interaction between trypsin and alginate: An ITC and DLS approach to the formation of insoluble complexes.

Trypsin is a protease widely used in several industrial areas for leather and meat softening and to produce enzymatic detergents, among others applications. The high demand for this enzyme has motivated the development of purification, stabilization and immobilization methods Formation of insoluble complexes between proteins and polyelectrolytes is a methodology that may include these features. The aim of this paper is to give evidence for a novel methodology that combines precipitation of the insoluble trypsin-alginate complex and hydrophobic interaction chromatography. This methodology allows the interaction between trypsin and alginate and their separation when necessary. It could be applied to isolation, stabilization and/or immobilization of trypsin. Isothermal titration calorimetry experiments showed that 232µmol of trypsin interacts electrostatically with 1g of alginate to form an insoluble complex that can be separated from soluble contaminants by decantation. Dynamic light scattering experiments confirmed the calorimetric results and allowed measuring the Rh of the soluble complex at pH 3.5 (185nm). When the optimal conditions were applied to precipitate commercially available trypsin, the recovery of the precipitation was around 92%. Finally, hydrophobic interaction chromatography allowed separating alginate from trypsin in order to obtain a polymer-free enzyme.

Cloning of novel cellulases from cellulolytic fungi: heterologous expression of a family 5 glycoside hydrolase from Trametes versicolor in Pichia pastoris.

Total cDNA isolated from cellulolytic fungi cultured in cellulose was examined for the presence of sequences encoding for endoglucanases. Novel sequences encoding for glycoside hydrolases (GHs) were identified in Fusarium oxysporum, Ganoderma applanatum and Trametes versicolor. The cDNA encoding for partial sequences of GH family 61 cellulases from F. oxysporum and G. applanatum shares 58 and 68% identity with endoglucanases from Glomerella graminicola and Laccaria bicolor, respectively. A new GH family 5 endoglucanase from T. versicolor was also identified. The cDNA encoding for the mature protein was completely sequenced. This enzyme shares 96% identity with Trametes hirsuta endoglucanase and 22% with Trichoderma reesei endoglucanase II (EGII). The enzyme, named TvEG, has N-terminal family 1 carbohydrate binding module (CBM1). The full length cDNA was cloned into the pPICZαB vector and expressed as an active, extracellular enzyme in the methylotrophic yeast Pichia pastoris. Preliminary studies suggest that T. versicolor could be useful for lignocellulose degradation.

Optimization of hydrophobic interaction chromatography using a mathematical model of elution curves of a protein mixture.

This paper describes a methodology for optimizing performance of hydrophobic interaction chromatography (HIC) for protein mixtures in which Rate Model simulations and evaluation of cost function are used. The system under study was HIC of a two-protein mixture (alpha-chymotrypsin and alpha-amylase), carried out with different conditions (gradient steepness, salt, concentration, volume of sample, pH, type of matrix, and flow rates). Parameters in the rate model were obtained from different sources. Mass transfer parameters (Reynolds, Peclet, and Biot numbers) were calculated using empirical correlations. Under the experimental conditions Re number was small (0.23) and axial dispersion negligible (PeL>300). Mass transfer was controlled by intraparticle diffusion (Bi>50). The model assumes that equilibrium constant (b) in the Langmuir isotherm was salt concentration (I) dependent [b(I)]. Parameters in the relationship for b(I) were estimated from experimental single protein elution curves and used to simulate protein mixtures. Rate model simulations showed that if protein sample load to the column was below 1 mg, displacement effects were negligible; in other cases protein interactions would limit the proposed mathematical description of HIC. The calibrated Rate Model successfully predicted elution curves of the protein mixture with deviations lower than 6x10(-4) absorbance units. The model was also able to predict that the Butyl Sepharose--NaCl 4 M system allowed to obtain the highest resolution (>1) for the two-protein mixture evaluated. The cost function built for optimizing the performance of HIC considers yield, purity, concentration, and the time needed to accomplish the separation. This function contains two types of parameters that have to be determined. The ones dependent on the HIC system and process conditions were obtained from simulations of elution curves of the two-protein mixture, by the calibrated Rate Model. The other parameters are dependent on characteristic and quality of the protein product; these were assumed for illustration purpose. Minimization of the cost function allows determination of flow rate, gradient steepness, and the fraction of eluted peak that has to be collected. Novelty of the present work is in showing how parameters in the fundamentally based Rate Model for HIC can be calibrated and how simulations can be used for the optimization of process conditions for the separation of a protein mixture.

A practical culture technique for enhanced production of manganese peroxidase by Anthracophyllum discolor Sp4

In this study, different growth conditions of Anthracophyllum discolor Sp4 including the effect of agitation, additions of lignocellulosic support, inducer and surfactant were evaluated on the MnP production in Kirk medium using a culture system made up of the tubes containing the glass bead . The highest MnP production (1,354 U/L on day 13) was obtained when the medium was supplemented with wheat grain and 0.25 mM MnSO4 as inducer, under static conditions at 30°C. Two isoenzymes were purified (35 and 38 kDa respectively). MnP presented a maximal activity in the pH range between 4.5 and 5.5, a relatively high temperature tolerance (50ºC) and a high catalytic activity for 2,6-dimethoxyphenol and hydrogen peroxide.