María Elena Lienqueo

Optimal synthesis of protein purification processes.

There has been an increasing interest in the development of systematic methods for the synthesis of purification steps for biotechnological products, which are often the most difficult and costly stages in a biochemical process. Chromatographic processes are extensively used in the purification of multicomponent biotechnological systems. One of the main challenges in the synthesis of purification processes is the appropriate selection and sequencing of chromatographic steps that are capable of producing the desired product at an acceptable cost and quality. This paper describes mathematical models and solution strategies based on mixed integer linear programming (MILP) for the synthesis of multistep purification processes. First, an optimization model is proposed that uses physicochemical data on a protein mixture, which contains the desired product, to select a sequence of operations with the minimum number of steps from a set of candidate chromatographic techniques that must achieve a specified purity level. Since several sequences that have the minimum number of steps may satisfy the purity level, it is possible to obtain the one that maximizes final purity. Then, a second model that may use the total number of steps obtained in the first model generates a solution with the maximum purity of the product. Whenever the sequence does not affect the final purity or more generally does not impact the objective function, alternative models that are of smaller size are developed for the optimal selection of steps. The models are tested in several examples, containing up to 13 contaminants and a set of 22 candidate high‐resolution steps, generating sequences of six operations, and are compared to the current synthesis approaches.

An Expert System for the selection and synthesis of multistep protein separation processes

A hybrid Expert System that combines expert rules and mathematical correlations to manipulate databases for selecting the sequence of operations for the downstream purification of proteins was built. This paper describes how the physicochemical data on the protein product and the other proteins present (contaminants) was used to select a sequence of operations with a minimum number of steps to achieve a defined level of purity. An algorithm was developed to model the amount of each protein contaminat eliminated after each step. The system was tested in a practical example and it was found that process synthesis was sufficiently sensitive to important changes in the physicochemical parameters of the product and rather robust to small variations or errors in the measurement of such parameters (e.g. 10%).

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 (α‐chymotrypsin and α‐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 6 × 10−4 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. Copyright

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.

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.

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.

Bioenergy II: Biological Pretreatment with Fungi as a Tool for Improvement of the Enzymatic Saccharification of Eucalyptus globulus Labill to Obtain Bioethanol

This study is focused on the effect of the application of biological pretreatment of Eucalyptus globulus Labill wood pieces on the cellulose digestibility by depolymerizing enzymes. Wood chips were incubated with five different white-rot fungi (WRF) for 30, 45 and 60 days at 25°C. The effect of the fungal action was determined as weight losses, changes in chemical composition and released sugars in the wood chips. Enzymatic hydrolysis was conducted in a discontinuous reactor at 37°C, using a commercial cellulase preparation from Trichoderma reesei. The enzyme/substrate ratio was 0.04 g/g. The highest weight losses were obtained using Stereum hirsutum and Lentinus edodes for all the incubation times, reaching 27.2% and 25.8% at 60 days, respectively. The lowest weight losses were produced by Coriolus versicolor and Pleurotus ostreatus, reaching 6.0 and 9.1%, respectively after 60 days. For all the tested fungi, the yield of reducing sugars and glucose increased significantly over the untreated controls, with Stereum hirsutum producing after 30 days pretreatment the best values (140 mM reducing sugars and 53 mM glucose); after this time no additional increments were observed. Pre-treated wood pieces showed changes in chemical composition in comparison to control samples. Total extractable substances contents are higher in wood pieces subjected to fungal action. However, lignin and alpha-cellulose values are lower in comparison to control samples. Some net holocellulose consumption could be observed, mainly during pretreatment with S. hirsutum by 45 and 60 days. Altogether these results support the potential of Eucalyptus globulus pre-treatment with S. hirsutum and L. edodes by periods not longer than 30 days, as a tool to increase the wood accessibility to depolymerizing action of hydrolytic enzymes. This preliminary study contributes to the identification of fungal pretreatment conditions for more effective cellulose degradation, a vital step in the utilization of monomer sugars process from cellulose to produce ethanol. Additional analysis of the solid and liquid fractions after saccharification is necessary to complement this information.

Design of an Expert System for Selection of Protein Purification Processes: Comparison Between Different Selection Criteria

Abstract We have implemented a hybrid Expert System, that combines experts rules and mathematical correlations to manipulate physicochemical databases for selecting the optimum sequence of operations for purification of proteins. Two criteria were implemented to select the optimum sequence of purification. One of these uses the selection separation coefficient (SSC) and the other uses the final level of purity. The sequences suggested by the expert system have been investigated experimentally using both criteria and it has been shown that both are valid for practical application, but the sequences suggested by the Purity criteria have fewer steps than those suggested by the SSC criteria.

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.