Miguel A. Galán

Immobilized Metal‐Ion Affinity Chromatography: Status and Trends

Abstract The correct folding of solubilized recombinant proteins is of key importance for their production in industry. On‐column refolding of proteins is mainly achieved by three methods: size‐exclusion chromatography, ion exchange chromatography and affinity chromatography using immobilized metal chelates. The principles of these methods were first laid down in the 1990s, but many recent improvements have been made to these processes. Immobilized metal‐ion affinity chromatography (IMAC) represents a relatively new separation technique that is primarily appropriate for the purification of proteins with natural surface‐exposed histidine residues and for recombinant proteins with engineered histidine tags or histidine clusters. Because the method has gained broad popularity in recent years, the main recent developments in the field of new sorbents, techniques and possible applications are discussed in this article.

Molecular cloning of TvDAO1, a gene encoding a D-amino acid oxidase from Trigonopsis variabilis and its expression in Saccharomyces cerevisiae and Kluyveromyces lactis

The DAO1 gene of Trigonopsis variabilis encoding a D‐amino acid oxidase (EC 1.4.3.3) was isolated from genomic clones selected for their specific hybridization to synthetic oligodeoxyribonucleotide probes based on regions of the enzyme that have been conserved through evolution. The nucleotide sequence of the gene predicts a protein with similarities to human, pig, rabbit, mouse and Fusarium solani D‐amino acid oxidases. The open reading frame of the T. variabilis DAO1 gene was interrupted by an intron. The Dao1p sequence displays two regions, one in the N‐terminal section—the FAD binding site—and the other near the C‐terminal region that contains conserved signatures found in all the D‐amino acid oxidases. The three C‐terminal amino acids suggest that the enzyme may be located in peroxisomes. Northern blot experiments showed that no transcriptional activation occurred in the presence of D‐methionine. The cDNA encoding Dao1p was expressed in Saccharomyces cerevisiae and Kluyveromyces lactis. Both yeast species are able to synthesize a functional enzyme under the control of the GAL1 promoter. In K. lactis, up to six times more enzyme units per gram of dry weight are produced with a multicopy plasmid in comparison with the wild‐type strain of T. variabilis. The yeast expression system we describe may constitute an alternative source for the production of D‐amino acid oxidases at industrial level. The sequence presented here has been submitted to the EMBL data library under Accession Number Z50019.

Prediction of kLa in yeast broths

Abstract Oxygen transfer rate, can play an important role in the scale-up and economy of many microbial processes and values of the volumetric oxygen transfer coefficient (kLa) for specific fermentation culture media need to be evaluated and correlated to obtain the appropriate design tools. The purpose of this work was to determinate the values of kLa in yeast broths (Trigonopsis variabilis) in a mechanically-stirred, sparger-aerated and baffled reactor, the most common fermenter type, over a wide range of superficial air velocities impeller rotational speeds and geometric parameters. Three different mixing vessels (2, 5 and 15 litres) were used in order to consider the effect of the fermenter scale-up on kLa. Once kLa data are obtained, several empirical and theoretical correlations were used to fit the experimental data. A new correlation is proposed k L a=3.2·10 −3 P V 0.35 U sg 0.41 based on the power input per unit volume of liquid (P/V) and the superficial gas velocity (Usg). The correlation improves the prediction of kLa values in culture media with respect to other generic correlations, mainly because early correlations were developed for strong coalescent and non-coalescent fluids, whereas the medium used in this work and in most the yeast broths behaves as a typical Newtonian, slightly non-coalescence fluid, due to the moderate presence of mineral salts.

Mass transfer from oscillating bubbles in bioreactors

In bioreactors, the purpose of aeration is to transfer oxygen from air bubbles into the liquid phase where the biological reactions occur. In some cases, aeration is also used as a mixing tool. In all aeration devices there is a continuous liquid phase and, generally, a dispersed gas phase in the form of bubbles. The most common aeration devices are stirred tank reactors, bubble columns and sieve plate reactor-towers. Bubble shape, bubble volume, and associated liquid flow patterns are key aspects of bubble dynamics in sieve plates. The most prevalent bubble shapes are spherical, oscillating (wobbling), ellipsoidal, spherical-cap, and skirted. Bubble volume and bubble shapes determine the surface to volume ratios, a very important parameter in determining overall mass transfer rates. Mass transfer in sieve plate reactors takes place, predominantly, within the oscillating bubble regime. Mass transfer rates from oscillating bubbles can be orders of magnitude larger than mass transfer rates from spherical bubbles. A fundamental correlation for mass transfer from single, oscillating bubbles was developed based on a solution of the mass transfer equations following the domain perturbation technique first outlined by Joseph (1973) and the hydrodynamics results of Tsamopoulos and Brown (1983). The correlation derived here entirely from hydrodynamics and mass transfer concepts, introduces the effect of interfacial tension in bubble mass transfer from fundamental concepts, has no adjustable parameters, and agrees very well with experimental data.

Immobilization of Mesenchymal Stem Cells and Monocytes in Biocompatible Microcapsules to Cell Therapy

The aim of cell therapy is to replace, repair, or enhance the function of damaged tissues or organs. Several factors complicate the development of cellular therapies. Of primary importance is protection of the implanted cells from the hostapos;s immune system. Cells are encapsulated in selectively semipermeable and biocompatible membranes that block entry of immune mediators but allow outward diffusion of active molecules produced by the cells. The immobilization of mesenchymal stem cells and monocytes, in micrometric (30–60 μm) alginate‐barium microcapsules based on atomization processes, has been achieved successfully. This size is necessary to the administration of microcapsules via injection (Hamilton syringe with a needle size of 100 μm) and aerosol. Microencapsulated cells survive at least 2 weeks after preparation in vitro.

Kinetics and heat-inactivation mechanisms of D-amino acid oxidase

Abstract A study was made of the stability of crude hog kidney d -amino acid oxidase ( d -AAO) under different experimental conditions of temperature, enzyme concentration, buffer, and in the presence of flavine adenine dinucleotide (FAD)_ and glycerol. A deactivation mechanism is proposed. The kinetic deactivation studies were performed in a buffer at pH over a temperature range of 277-327K (4–54°C). The activity for d -AAO at 1 U/ml at 277 K in potassium phosphate and potassium pyrophosphate buffer remains almost constant for 20 days. For d -AAO at 0·02 U/ml at 303 K (30°C) in phosphate buffer, the presence of FAD and glycerol causes the deactivation constant to decrease, while the initial activity and the half-life time increase, the latter doubling on passing from buffer alone to buffer with FAD and 10% glycerol. In the case of d -AAO at 0·2 U/ml, the stability increases considerably with respect to 0·02 U/ml. Both FAD and glycerol enhance this stabilization. For temperatures above 313 K (40°C), the deactivation of d -AAO at 0·2 U/ml showed values of the thermodynamic variables for the overall deactivation constant and for the deactivation constant due to the loss of FAD that indicate that the dissociation of FAD is the main deactivation mechanism. Finally, deactivation due to the loss of FAD is higher for d -AAO at 0·1 U/ml as compared with a concentration of 0·2 U/ml.

Rheological characterization of commercial highly viscous alginate solutions in shear and extensional flows

The rheological properties of sodium alginate in salt-free solutions were studied by steady shear, dynamic oscillatory and extensional measurements. This biopolymer consists of mannuronic and guluronic acid residues that give a polyelectrolyte character. We applied the scaling theories and checked their accordance with polyelectrolyte behaviour for low concentrations with a shift to neutral polymer behaviour at larger concentrations. This nature was supported by the effect of the concentration on the specific viscosity, the relaxation times from steady shear and the longest relaxation times from small amplitude oscillatory shear (SAOS) measurements. To analyze the extensional behaviour of the samples, we conducted a study of dimensionless numbers and time scales where filament thinning driven by viscous, capillary or elastic forces is at play. We conclude that an exponential filament thinning followed by breakup results in the best regimes that describe the experimental data. Besides, the data pointed out that alginate in salt-free concentrated solutions shows strain thinning of the extensional viscosity and chain rigidity, behaviours that cannot be inferred from the shear rheometry.

SUPERCRITICAL FLUID TECHNIQUE FOR PARTICLE ENGINEERING: DRUG DELIVERY APPLICATIONS

Supercritical Fluid (SCF) technology is now considered as a very innovative and promising way to design particles, especially for therapeutic drug formulation.(l) The advantages of SCF technology include use of mild conditions for pharmaceutical processing (which is advantageous for labile proteins and peptides), use of environmentally benign nontoxic materials (such as CO2), minimization of organic solvent use, and production of particles with controllable morphology, narrow size distribution, and low static charge(l). SCF technology is making inroads in several pharmaceutical industrial operations including crystallization, particle size reduction, and preparation of drug delivery systems, coating, and product sterilization. It has also been shown to be a viable option in the formulation of paniculate drug delivery systems, such as microparticles and nanoparticles, liposomes, and inclusion complexes, which control drug delivery and/or enhance the drug stability. This review describes the recent advances in the use of SCF technology for particle engineering and for the preparation of drug delivery systems.

Inhibition ofd-amino acid oxidase by α-keto acids analogs of amino acids

The inhibition of D-amino acid oxidase by certain alpha-keto acids products of the reaction with D-amino acids, in particular alpha-keto acids that are analogs of the amino acids alanine, valine, leucine, phenylanaline, phenylglycine, tyrosine and tryptophan, is reported. All the alpha-keto acids assayed behaved as substrate competitive inhibitors of the enzyme. The relationship between the degree of inhibition and the structure of the inhibitor is discussed.

Effect of nitrogen source on growth and lipid accumulation in Scenedesmus abundans and Chlorella ellipsoidea

Discovering microalgae strains containing a high lipid yield and adequate fatty acid composition is becoming a crucial fact in algae-oil factories. In this study, two unknown strains, named Scenedesmus abundans and Chlorella ellipsoidea, have been tested for their response to different nitrogen sources, in order to determine its influence in the production of lipids. For S. abundans, autotrophic culture with ammonium nitrate offers the maximum lipid yield, obtaining up to 3.55 mg L(-1) d(-1). For C. ellipsoidea, heterotrophic culture with ammonium nitrate has been shown to be the best condition, reaching a lipid production of 9.27 mg L(-1) d(-1). Moreover, fatty acid composition obtained from these cultures meets international biodiesel standards with an important amount of C18:1, achieving 70% of total fatty acids and thus representing a potential use of these two strains at an industrial scale.