Juan A. Asenjo

Aqueous two-phase systems for protein separation: Studies on phase inversion

Phase equilibrium studies were done with the PEG 400-phosphate system, obtaining equilibrium binodal lines, tie lines and phase inversion points. A method of calculation of the critical point on the binodal curve is described. The influence of the presence of NaCl in solution was studied, and the comparative results are presented. It was found that in some range of concentration the shift produced in the binodal line can be important. The rate of phase separation can be used as an indication of which of the phases is continuous. Using this method the phase inversion point can be determined in the system for each tie line. A range of ambiguity was found, where the continuity of the phases is affected not only by the composition of the mixture, but also by the fluid dynamics. Within this range, gentle agitation produces a bottom-continuous suspension. while strong agitation produces a top-continuous suspension. Two inversion points exist therefore on each tie line, delimiting on the phase equilibrium plane a region where the phase continuity depends on fluid dynamics. The convergence of this region towards the critical point can be used to control of the consistency of the experimental data.

Aqueous two-phase systems for protein separation: a perspective.

Aqueous two-phase systems (ATPS) that are formed by mixing a polymer (usually polyethylene glycol, PEG) and a salt (e.g. phosphate, sulphate or citrate) or two polymers and water can be effectively used for the separation and purification of proteins. The partitioning between both phases is dependent on the surface properties of the proteins and on the properties of the two phase system. The mechanism of partitioning is complex and not very easy to predict but, as this review paper shows, some very clear trends can be established. Hydrophobicity is the main determinant in the partitioning of proteins and can be measured in many different ways. The two methods that are more attractive, depending on the ATPS used (PEG/salt, PEG/polymer), are those that consider the 3-D structure and the hydrophobicity of AA on the surface and the one based on precipitation with ammonium sulphate (parameter 1/m*). The effect of charge has a relatively small effect on the partitioning of proteins in PEG/salt systems but is more important in PEG/dextran systems. Protein concentration has an important effect on the partitioning of proteins in ATPS. This depends on the higher levels of solubility of the protein in each of the phases and hence the partitioning observed at low protein concentrations can be very different to that observed at high concentrations. In virtually all cases the partition coefficient is constant at low protein concentration (true partitioning) and changes to a different constant value at a high overall protein concentration. Furthermore, true partitioning behavior, which is independent of the protein concentration, only occurs at relatively low protein concentration. As the concentration of a protein exceeds relatively low values, precipitation at the interface and in suspension can be observed. This protein precipitate is in equilibrium with the protein solubilized in each of the phases. Regarding the effect of protein molecular weight, no clear trend of the effect on partitioning has been found, apart from PEG/dextran systems where proteins with higher molecular weights partitioned more readily to the bottom phase. Bioaffinity has been shown in many cases to have an important effect on the partitioning of proteins. The practical application of ATPS has been demonstrated in many cases including a number of industrial applications with excellent levels of purity and yield. This separation and purification has also been successfully used for the separation of virus and virus-like particles.

Partitioning and purification of α-amylase in aqueous two-phase systems

The partition behavior of pure α-amylase (1,4-α-d-glucan glucanohydrolase, E.C. 3.2.1.1) in aqueous two-phase systems was examined in order to investigate the effects of changes in type of phase components on the partition coefficient K. Polyethylene glycol (PEG)/dextran, PEG/phosphate, and PEG/sulfate systems were evaluated. Factors such as PEG molecular weight (MW), pH, and concentration of NaCl were all found to influence K. At low values (<2 kUml−1 phase system), enzyme concentration had no effect on K. At higher concentrations (up to 20.40 kUml−1 phase system was used, corresponding to ca. 12.3 gl−1, the phases became saturated and a twofold increase in K was observed, but also lower recovery. Subsequently, the separation and purification of α-amylase from typical contaminants from supernatant and whole broth of Bacillus subtilis fermentation was examined. The best partition conditions were found in PEG 4000/phosphate systems with 8.8% w/w NaCl where the K can be increased 78 times from K = 1.3 to K = 100. These conditions were then used to study the effect of phase volume ratio (R) on the partition coefficient, the purification factor (PF), and recovery of α-amylase from industrial fermentations. R was found to influence the purification factor and the recovery, but not the partition coefficient. However, both α-amylase and contaminants partitioned to the top phase, leading to relatively poor separation (PF = 3.2 for R = 1). In a PEG/sulfate system, the addition of NaCl had an extreme effect on the partition behavior of α-amylase, giving an extreme K for α-amylase (K = 6,800) at a high concentration of NaCl 8.8% w/w) and an extremely low K (K < 0.005) at a lower concentration of NaCl. By exploiting this extreme partition behavior and manipulating R in a two-stage strategy, a 53-fold purification (with 86% w/w purity) could be calculated of a maximum possible of 63 at 100% w/w pure α-amylase.

Enzymatic lysis of microbial cells.

Cell wall lytic enzymes are valuable tools for the biotechnologist, with many applications in medicine, the food industry, and agriculture, and for recovering of intracellular products from yeast or bacteria. The diversity of potential applications has conducted to the development of lytic enzyme systems with specific characteristics, suitable for satisfying the requirements of each particular application. Since the first time the lytic enzyme of excellence, lysozyme, was discovered, many investigations have contributed to the understanding of the action mechanisms and other basic aspects of these interesting enzymes. Today, recombinant production and protein engineering have improved and expanded the area of potential applications. In this review, some of the recent advances in specific enzyme systems for bacteria and yeast cells rupture and other applications are examined. Emphasis is focused in biotechnological aspects of these enzymes.

Diversity of culturable actinomycetes in hyper-arid soils of the Atacama Desert, Chile

The Atacama Desert presents one of the most extreme environments on Earth and we report here the first extensive isolations of actinomycetes from soils at various locations within the Desert. The use of selective isolation procedures enabled actinomycetes to be recovered from arid, hyper-arid and even extreme hyper-arid environments in significant numbers and diversity. In some cases actinomycetes were the only culturable bacteria to be isolated under the conditions of this study. Phylogenetic analysis and some phenotypic characterisation revealed that the majority of isolates belonged to members of the genera Amycolatopsis, Lechevalieria and Streptomyces, a high proportion of which represent novel centres of taxonomic variation. The results of this study support the view that arid desert soils constitute a largely unexplored repository of novel bacteria, while the high incidence of non-ribosomal peptide synthase genes in our isolates recommend them as promising material in screening for new bioactive natural products.

Hydrophobic partitioning of proteins in aqueous two-phase systems

The hydrophobicity of five proteins was estimated by reversed-phase chromatography (RPC), hydrophobic-interaction chromatography (HIC), and precipitation with ammonium sulfate. These data were correlated to partition behavior in aqueous two-phase systems. A parameter (1m∗) that was derived from the precipitation curves is based on the solubility of proteins in an electrolyte solution. The correlation between 1m∗ and the retention times in HIC and RPC was poor due to interaction effects with the chromatography matrices and probably partial unfolding of the proteins; however, this parameter (1m∗) was expected to be a measure of hydrophobicity of proteins that relates better than the chromatographic data to experiments where the hydrophobic behavior of proteins in an aqueous solution is used for their separation. The partition behavior of the five proteins in aqueous two-phase systems (ATPS) in the absence and presence of NaCl was investigated. A poor correlation was found between log K (K is the partition coefficient) in ATPS and the hydrophobicity values measured by RPC and HIC; however, a very good correlation was found between log 1m∗ which is a measure of protein hydrophobicity based on the solubility of the protein during precipitation and log K, particularly in PEGPO4 systems with added NaCl. The parameter (1m∗) also demonstrated a good correlation with log K in PEG/dextran systems. A simple correlation for the prediction of partitioning in specific ATPS based on this parameter has been evaluated. An expression describing its resolution power, R, and a parameter describing the hydrophobicity of the system, P0, was determined making the correlation potentially predictive for other proteins in the ATPSs used. Hydrophobicity of proteins was better exploited in PEGPO4 systems than in PEG/dextran ones as a much higher resolution (R) is obtained in the former.

Use of chemically modified proteins to study the effect of a single protein property on partitioning in aqueous two-phase systems: Effect of surface hydrophobicity.

Two different series of hydrophobically modified proteins were partitioned in a number of aqueous two‐phase systems (ATPS) to investigate the effect of hydrophobicity as a single property on partitioning. The modified proteins were derived from β‐lactoglobulin and bovine serum albumin (BSA). Measurement of the surface hydrophobicity of the proteins is important; hydrophobic interaction chromatography (HIC) was used for this purpose. The resolution of the systems (R) in terms of protein surface hydrophobicity and the intrinsic hydrophobicity (log P0) of the systems was established. The effect of the addition of NaCl to PEG/phosphate and PEG/dextran systems was analyzed in terms of the hydrophobicity difference between the phases and their ability to promote hydrophobic interactions between the protein surface and the PEG molecules. The values for R and log P0 differed somewhat depending on which group of modified proteins was used for partitioning. The addition of NaCl to PEG/phosphate systems promoted an increase in the values of R, showing an important effect on the resolution of the systems for protein surface hydrophobicity (twice as high when compared with systems without NaCl). For PEG/dextran systems, the addition of 9% NaCl (w/w) promoted an improvement in the resolution toward surface hydrophobicity with an increase of 60% on the value of R.

The surface exposed amino acid residues of monomeric proteins determine the partitioning in aqueous two-phase systems.

It is of great interest and importance how different amino acid residues contribute to and affect the properties of a protein surface. Partitioning in aqueous two-phase systems has the potential to be used as a rapid and simple method for studying the surface properties of proteins. The influence on partitioning of the surface exposed amino acid residues of eight structurally determined monomeric proteins has been studied. The proteins were characterized in terms of surface exposed residues with a computer program, Graphical Representation and Analysis of Surface Properties (GRASP), and partitioned in two EO30PO70-dextran aqueous two-phase systems, only differing in polymer concentrations (system I: 6.8% EO30PO70, 7.1% dextran; system II: 9% EO30PO70, 9% dextran). We show for the first time that the partitioning behaviour of different monomeric proteins can be described by the differences in surface exposed amino acid residues. The contribution to the partition coefficient of the residues was found to be best characterized by peptide partitioning in the aqueous two-phase system. Compared to hydrophobicity scales available in the literature, each amino acid contribution is characterized by the slope given by the graph of log K against peptide chain length, for peptides of different length containing only one kind of residue. It was also shown that each amino acid contribution is relative to the total protein surface and the other residues on the surface. Surface hydrophobicity calculations realized for systems I and II gave respectively correlation coefficients of 0.961 and 0.949 for the linear relation between log K and calculated hydrophobicity values. To study the effect on the partition coefficient of different amino acids, they were grouped into classes according to common characteristics: the presence of an aromatic group, a long aliphatic chain or the presence of charge. Using these groups it was possible to confirm that aromatic residues have the strongest effect on the partition coefficient, giving preference to the upper EO30PO70 phase of the system; on the other hand the presence of charged amino acids on the protein surface enhances the partition of the protein to the lower dextran phase. It is also important to note that the sensitivity of the EO30PO70-dextran system for the surface exposed residues was increased by increasing the polymer concentrations. The partition coefficient of a monomeric protein can thus be predicted from its surface exposed amino acid residues and the system can also be used to characterize protein surfaces of monomeric proteins in general.

Microbial Conversion of Methane into poly-β-hydroxybutyrate (PHB): Growth and intracellular product accumulation in a type II methanotroph

Abstract The biochemical pathways of methane to poly-β-hydroxybutyrate (PHB) in type II methanotrophs have been analyzed and used to propose stoichiometric equations for cell biomass and PHB. Conditions necessary for PHB accumulation in a batch culture of Methylocystis parvus OBBP were studied. In nitrogen limited cultures PHB started accumulating in the declining growth phase and maximum rate of PHB formation occurred during the late growth and early stationary phases. Inoculum age had an effect on maximum level of intracellular PHB, which increased with inoculum age up to ca. 70% of the cell dry weight using a 70 h inoculum. It has been shown that oxygen and methane requirements are high and pose a great stress on the mass transfer in the system; this limits the obtainment of high cell concentrations. When conditions for increased mass transfer of both CH 4 and O 2 were used, 5 g/ l of cells could be produced. Finally an enrichment strategy was investigated so that cells would start accumulating PHB earlier during the growth phase. Alternatives to increase cell mass concentrations are discussed.

Generation of an atlas for commodity chemical production in Escherichia coli and a novel pathway prediction algorithm, GEM-Path

The production of 75% of the current drug molecules and 35% of all chemicals could be achieved through bioprocessing (Arundel and Sawaya, 2009). To accelerate the transition from a petroleum-based chemical industry to a sustainable bio-based industry, systems metabolic engineering has emerged to computationally design metabolic pathways for chemical production. Although algorithms able to provide specific metabolic interventions and heterologous production pathways are available, a systematic analysis for all possible production routes to commodity chemicals in Escherichia coli is lacking. Furthermore, a pathway prediction algorithm that combines direct integration of genome-scale models at each step of the search to reduce the search space does not exist. Previous work (Feist et al., 2010) performed a model-driven evaluation of the growth-coupled production potential for E. coli to produce multiple native compounds from different feedstocks. In this study, we extended this analysis for non-native compounds by using an integrated approach through heterologous pathway integration and growth-coupled metabolite production design. In addition to integration with genome-scale model integration, the GEM-Path algorithm developed in this work also contains a novel approach to address reaction promiscuity. In total, 245 unique synthetic pathways for 20 large volume compounds were predicted. Host metabolism with these synthetic pathways was then analyzed for feasible growth-coupled production and designs could be identified for 1271 of the 6615 conditions evaluated. This study characterizes the potential for E. coli to produce commodity chemicals, and outlines a generic strain design workflow to design production strains.