From laboratory to pilot plant E. coli fed-batch cultures: optimizing the cellular environment for protein maximization.

For recombinant protein production in E. coli fed-batch cultures, post-induction conditions have great influence in the quantity and quality of the product. The present paper covers the effect of different factors affecting the cellular environment in recombinant aldolase (rhamnulose-1-phosphate aldolase, RhuA) production. An operational mode employing an exponential addition profile for constant specific growth rate has been analyzed, in order to understand and define possible modifications with influence on post-induction cellular behavior. A constant addition profile has been demonstrated to render higher specific aldolase production than the exponential addition profile, probably due to a more constant environment for the cells. On the other hand, amino acid (leucine) supplementation has proven to increase protein quality in terms of activity units (U) per unit mass of RhuA (U mg(-1) RhuA), alleviating metabolic overload. Based on the above, a production process was set up and scaled up to pilot plant. Resulting production was double that of a standard laboratory operation, 45,000 U L(-1), and almost all the protein retained the 6xHis-tag with the highest quality, 11.3 U mg(-1) RhuA.

Ideas sobrevaloradas: cuestiones psicopatológicas / Overvalued ideas: psychopathologic issues

Se revisa críticamente la posición atribuida en los últimos años a las ideas sobrevaloradas entre el delirio y la obsesión, y que deriva de la reducción del constructo mismo a una de sus características, el insight. A través de un caso con ideas dismorfofóbicas se plantearán las dificultades para delimitarlo y diferenciarlo del delirio y se esbozarán distintas aproximaciones psicopatológicas que permitan reconstruir el caso de modo distinto (AU)

The position attributed in recent years to overvalued ideas between delusion and obsession and that are derived from the reduction of the construct per se to one of its characteristics, that is, insight, has undergone a critical review. Using a case with dysmorphophobic ideas, the difficulties to define it and differentiate it from delusionare proposed and different psychopathological approaches are outlined that make it possible to reconstruct the case differently (AU)

Concept of representation and mental symptoms. The case of theory of mind.

BACKGROUND: Most current theories explaining theory of mind (ToM) rely on the concept of 'representation', as it is usually employed in cognitive science, and is thus affected by its epistemic shortcoming, namely its incapacity to use 'sub-signifier' level information. This shortcoming is responsible for the lack of specificity of ToM deficits, which are now found in very different syndromes, from schizophrenia to bipolar disorder or borderline personality disorder, in spite of its original formulation being restricted to childhood autism. METHOD: Representation, its shortcomings and the way they may affect clinical/research programs undergo a conceptual analysis, which shows how representational-founded semiology leave out information that is essential for symptom specificity and correct symptom assessment. Schizophrenic autism, delusional perception and axial syndromes are studied as examples of both the difficulties that have arisen and possible ways of dealing with them. RESULTS: Transfers of properties between different meanings of 'representation' together with a systematic ambiguity in the use of 'representation' are proposed as the main ways for representational approaches to assure stability to their proposals in spite of the violence exerted on clinical phenomena. CONCLUSIONS: It is exposed how systematic ambiguity and epistemic shortcomings both affect Leslie's formulation of ToM and, further, the importance of these characteristics of the concept of 'representation' for general issues in psychiatric semiology.

Origen y destino de la teoría de la mente: su afectación en trastornos distintos del espectro autista / Evolution of the theory of mind: its involvement in distinct autistic spectrum disorders

La disfunción de la habilidad para adscribir estados mentales a otros, esto es, la posesión de una teoría de la mente, ha sido propuesta por Frith como condición necesaria, suficiente y específica de la esquizofrenia. Este artículo revisa los datos a favor y en contra de tal asunción, teniendo en cuenta su relación hipotética con otras variables cognitivas, si la falta de una teoría de la mente es un marcador de rasgo o de estado y si su rol causal implica el trastorno psicótico básico o sólo algunos síntomas psicóticos. Igualmente se considera si los recientes hallazgos de disfunción de la teoría de la mente en el trastorno bipolar y en la demencia frontotemporal podrían modificar las asunciones teóricas básicas sobre la teoría de la mente, la esquizofrenia y los trastornos relacionados

Malfunction of the ability to ascribe mental states to others, that is, possession of a Theory of Mind (ToM), was proposed by CD Frith as a necessary, sufficient and specific condition of schizophrenia. The present article reviews the evidence for and against this assumption, bearing in mind its putative relationship with other cognitive variables, whether the lack of a ToM is trait marker or rather a state marker and whether its causal role involves the underlying psychotic disorder or only some psychotic symptoms. Equally, whether the recent findings of ToM dysfunction in bipolar disorder and frontotemporal dementia could modify basic theoretical assumptions about ToM, schizophrenia and related conditions is also discussed

Influence of phosphate on rhamnose-containing exopolysaccharide rheology and production by Klebsiella I-714.

Physiological conditions enhancing rhamnose-containing polysaccharide synthesis by Klebsiella I-714 were studied in batch culture (0.3-l and 2-l bioreactors). The four carbon sources tested, sucrose, sorbitol, Neosorb and Cerelose, allowed exopolysaccharide production. Larger amounts of polymer were produced when high carbon/nitrogen ratios and complex nitrogen sources were used. Exopolysaccharide synthesis was greatest at 30 degrees C, which was a suboptimal growth temperature. A reduction in the phosphate content of the medium enhanced rhamnose-containing polysaccharide production. When the initial carbon source concentration was augmented, byproducts other than exopolysaccharide were formed. Rhamnose-containing polysaccharide rheology can be modulated by changing the phosphate content of the medium.

Reaction engineering for consecutive enzymatic reactions in peptide synthesis: application to the synthesis of a pentapeptide.

A single-pot enzymatic synthesis of Z-CCK5 (4-8) is presented in this work, employing Z-Gly-Trp-OBzl as acyl donor, under kinetic control. The first goal of the work is the development of a synthetic strategy allowing the use of the same medium for two reactions catalyzed by immobilized alpha-chymotrypsin, discriminating between simultaneous and consecutive addition systems. The second goal is the maximization of the pentapeptide yield as a function of the molar excess of both nucleophiles employed. A maximum yield of 36% was obtained, and the addition strategy as well as the optimal initial concentrations of substrates have been determined.

Enzymatic condensation of cholecystokinin CCK-8 (4-6) and CCK-8 (7-8) peptide fragments in organic media.

The kinetically controlled condensation reaction of Z-Gly-Trp-Met-OR(1) (R(1): Et, Al, Cam) and H-Asp-(OR(2))-Phe-NH(2) (R(2): H, Bu(t)) catalyzed by alpha-chymotrypsin deposited onto polyamide in organic media was studied. The effect of the drying process of the enzyme-support preparation, substrate concentrations, reaction medium, acyl donor, and nucleophile structure on both enzymatic activity and pentapeptide yield was investigated. The immobilized preparation directly equilibrated at a(w) = 0.113, gave higher enzymatic activities than dried with vacuum first, and then equilibrated at a(w) = 0.113. The addition of triethylamine to the reaction medium increased dramatically the enzymatic activity. However, the pentapeptide yield was affected neither by the drying procedure nor by the addition of triethylamine. The donor ester Z-Gly-Trp-Met-OAl gave initial reaction rates 2.6 times higher than the conventional ethyl ester derivative but rendered similar yields. The best results were obtained using Z-Gly-Trp-Met-OCam as acyl-donor ester; 80% yield and initial reaction rates 4 times higher than the ethyl ester derivative. In all cases, acetonitrile containing Tris-HCl 50 mM pH 9 buffer (0.5% v/v) and triethylamine (0.5% v/v) was found to be the best reaction system. Under these conditions, it was possible to use the nucleophile H-Asp-Phe-NH(2) with beta-unprotected aspartic acid residue. In this case, 50% yield was obtained, but economic considerations could lead to select it as nucleophile. Finally, the fragment condensation reaction was carried out at gram scale, obtaining a 39% yield which included the reaction, removal of protecting groups and purification steps. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 456-463, 1997.

Enzymatic synthesis of a CCK-8 tripeptide fragment in organic media.

The enzymatic synthesis of the tripeptide derivative Z-Gly-Trp-Met-OEt is reported. This tripeptide is a fragment of the cholecystokinin C-terminal octapeptide CCK-8. Studies on the alpha-chymotrypsin catalyzed coupling reaction between Z-Gly-Trp-R(1) and Met-R(2) have focused on low water content media, using deposited enzyme on inert supports such as Celite and polyamide. The effect of additives (polar organic solvents), the acyl-donor ester structure, the C-alpha protecting group of the nucleophile, enzyme loading, and substrate concentration were tested. The best reaction medium found was acetonitrile containing buffer (0.5%, v/v) and triethylamine (0.5%, v/v) using the enzyme deposited on Celite as catalyst (8 mg of alpha-chymotrypsin/g of Celite). A reaction yield of 81% was obtained with Z-Gly-Trp-OCam as acyl donor, at an initial concentration of 80 mM. The tripeptide synthesis was scaled up to the production of 2 g of pure tripeptide with an overall yield of 71%, including reaction and purification steps.

Production of 5'-ribonucleotides by enzymatic hydrolysis of RNA.

Study of optimal operational conditions for RNA enzymatic hydrolysis to obtain 5'-ribonucleotides has been carried out. RNA from brewer's yeasts, obtained by ammonium extraction, was hydrolysed by a partially purified 5'-phosphodiesterase from barley rootlets. Temperature of 60 degrees C and pH 7 have been determined as the best operational conditions. Low RNA initial concentration (approximately 0.1%) and reaction time (approximately 1 h) have been identified as necessary to obtain a good yield of 5'-ribonucleotides.

An approach in mathematical modeling of an upflow packed-bed reactor for enzymatic hydrolysis of wheat straw.

The behavior of a packed-bed reactor for enzymatic hydrolysis of pretreated wheat straw has been described by means of a mathematical model. The flow pattern has been evaluated by residence time distribution experiments. Small deviations from ideal plug flow behavior were found using the dispersion model. The kinetic model proposed for enzymatic hydrolysis of cellulosic fraction of pretreated wheat straw has been derived from batch experimental data. Variations of enzyme concentration throughout the straw bed have been approximately described using a ramp variation of adsorbed enzyme. The final explains qualitatively the experimental results.

Application of extended Kalman filter to identification of enzymatic deactivation.

A recursive estimation scheme, the Extended Kalman Filter (EKF) technique, was applied to study enzymatic deactivation in the enzymatic hydrolysis of pretreated cellulose using a model previously developed by the authors. When no deactivation model was assumed, the results showed no variation with time for all the model parameters except for the maximum rate of cellobiose-to-glucose conversion (r'(m)).The r'(m) variation occurred in two zones with a grace period. A new model of enzymatic hydrolysis of pretreated cellulose deactivation was proposed and validated showing better behavior than the old deactivation model. This approach allows one to study enzyme deactivation without additional experiments and within operational conditions.

Dilute acid hydrolysis of wheat straw hemicellulose at moderate temperature: a simplified kinetic model.

Wheat straw has been hydrolized with sulfuric acid at 34 and 90 degrees C. The treatment at 90 degrees C yields complete solubilization of hemicellulose to xylose and arabinose without significant amounts of furfural. The influence of acid concentration was studied and the kinetics of the acid-catalyzed hydrolysis has been modeled suggesting a two-consecutive reactions mechanism. This model is useful to explain the different behavior of the concentration of the two main sugars produced. The enhanced cellulose accessibility to enzymatic attack is also reported.

Kinetic modeling of the enzymatic hydrolysis of pretreated cellulose.

The production of sugars by the enzymatic hydrolysis of cellulose is a two-step process that includes conversion of the intermediate cellobiose to glucose by beta-glucosidase. The hydrolysis was followed by analyzing the two sugar products (cellobiose and glucose). The enzyme showed maximum activity at pH 4.8. Thermal deactivation was significant at temperatures above 45 degrees C. At 50 degrees C (optimum temperature) thermal deactivation was found to follow first-order kinetics. Several models were tested by modeling the kinetics of the reaction. Their parameter values were determined by numerical optimization, including temperature dependence. The best fitting model was a competitive product inhibition for the two reactions in the operational range.