Towards a cost effective strategy for cutinase production by a recombinant Saccharomyces cerevisiae: strain physiological aspects

Although the physiology and metabolism of the growth of yeast strains has been extensively studied, many questions remain unanswered where the induced production of a recombinant protein is concerned. This work addresses the production of a Fusarium solani pisi cutinase by a recombinant Saccharomyces cerevisiae strain induced through the use of a galactose promoter. The strain is able to metabolise the inducer, galactose, which is a much more expensive carbon source than glucose. Both the transport of galactose into the cell-required for the induction of cutinase production-and galactose metabolism are highly repressed by glucose. Different fermentation strategies were tested and the culture behaviour was interpreted in view of the strain metabolism and physiology. A fed-batch fermentation with a mixed feed of glucose and galactose was carried out, during which simultaneous consumption of both hexoses was achieved, as long as the glucose concentration in the medium did not exceed 0.20 g/l. The costs, in terms of hexoses, incurred with this fermentation strategy were reduced to 23% of those resulting from a fermentation carried out using a more conventional strategy, namely a fed-batch fermentation with a feed of galactose.     

Systematic study of high molecular weight compounds in Amazonian plants by high temperature gas chromatography-mass spectrometry

The fractions of hexane and dichloromethane extraction from marupá (Simaruba amara) and (Bertholletia excelsa) leaves were analyzed by HT-HRGC (high temperature high resolution gas chromatography) and HT-HRGC coupled to mass spectrometry (HT-HRGC-MS). Several compounds can be characterized including unusual high molecular weight compounds.     

Reverse micelles as reaction media for lipases

Reversed micelles are at the present time faced as common organic media to perform biocatalysis. They have been associated to the idea of a microreactor where the enzyme can be sheltered and protected from solvent detrimental effects. This simplistic idea led some investigators to ignore some basic understanding, such as the recognition of the enzyme-specific microenvironment and what the enzyme experiences inside the reversed micelle. To date the number of reactions catalyzed by lipases in reversed micelles is large. This review aims to highlight some of the fundamental aspects of the lipase microencapsulation as well as to resume the outstanding progress of the reversed micellar systems. The properties of the micellar microenvironment are reviewed and related to the lipases' performance both in terms of activity and stability. The heterogeneity of reversed micellar systems is discussed in relation to component distribution models and also to enzymatic kinetics. The new trends and the practical aspects where efforts should be centralized in order to spread out the micellar bioreactor technology over industrial processes are also discussed.     

Role of intrarenal alpha(2)-adrenoceptors in the renal responses to xylazine in rats

This study examined the contribution of intrarenal alpha(2)-adrenoceptor mechanisms to the enhanced urine flow rate (V) and urinary sodium excretion (U(Na)V) responses in ketamine-xylazine-anesthetized rats. Ten minutes after left renal artery (LRA) injection, the alpha(2)-adrenoceptor antagonist yohimbine (5 microg) significantly decreased V from 58 +/- 8 to 35 +/- 7 microl. min(-1). g kidney wt(-1) and U(Na)V from 2.8 +/- 0.4 to 2.1 +/- 0.4 microeq. min(-1). g kidney wt(-1) without altering right kidney function. The renal effects of the LRA injection of yohimbine were completely abolished in chronic bilaterally renal-denervated (RDNX) rats. In RDNX rats, a higher LRA dose of yohimbine (15 microg) significantly reduced left and right kidney V, with no effects on U(Na)V. In separate bladder-catheterized rats, yohimbine (0.5 mg/kg), 20 min after intravenous injection, significantly decreased V from 63 +/- 9 to 13 +/- 2 microl. min(-1). g kidney wt(-1 )and U(Na)V from 4.5 +/- 0.5 to 1.1 +/- 0.1 microeq. min(-1). g kidney wt(-1). In RDNX rats, this dose of yohimbine reduced V and U(Na)V, but the magnitude was blunted compared with intact rats. In contrast, 0.1 mg/kg iv yohimbine significantly reduced V and U(Na)V to similar magnitudes in intact and RDNX groups. Together, these findings indicate that intravenous xylazine acts by renal nerve-dependent and -independent mechanisms to enhance renal excretory function in ketamine-anesthetized rats. Because the effects of the LRA dose of yohimbine were abolished in renal-denervated animals, it appears that xylazine has a direct renal action to augment the renal excretion of water and sodium via a presynaptic alpha(2)-adrenoceptor pathway that inhibits the release of neurotransmitters from renal sympathetic nerve terminals.     

The increasing importance of Acanthamoeba infections

Free-living amebae belonging to the genus Acanthamoeba are the causative agents of granulomatous amebic encephalitis, a chronic progressive disease of the central nervous system, and of amebic keratitis, a chronic eye infection. Granulomatous amebic encephalitis occurs more frequently in immunocompromised patients while keratitis occurs in healthy individuals. The recent increased incidence in Acanthamoeba infections is due in part to infection in patients with acquired immune deficiency syndrome, while that for keratitis is due to the increased use of contact lenses. Understanding the mechanism of host resistance to Acanthamoeba is essential since the amebae are resistant to many therapeutic agents. Studies in our laboratory as well as from others have demonstrated that macrophages from immunocompetent animals are important effector cells against Acanthamoeba. We have demonstrated also that microglial cells, resident macrophages of the brain, elicit cytokines in response to A. castellanii. Neonatal rat cortical microglia from Sprague-Dawley rats co-cultured with A. castellanii produced mRNA for the inflammatory cytokines, interleukin 1alpha, interleukin 1beta, and tumor necrosis factor alpha. In addition, scanning and transmission electron microscopy revealed that microglia ingested and destroyed A. castellanii in vitro. These results implicate macrophages as playing an effector role against Acanthamoeba and suggest immune modulation as a potential alternative therapeutic mode of treatment for these infections.     

Trehalose delays the reversible but not the irreversible thermal denaturation of cutinase

The effect of trehalose (0.5 M) on the thermal stability of cutinase in the alkaline pH range was studied. The thermal unfolding induced by increasing temperature was analyzed in the absence and in the presence of trehalose according to a two-state model (which assumes that only the folded and unfolded states of cutinase were present). Trehalose delays the reversible unfolding. The midpoint temperature of the unfolding transition (Tm) increases by 4.0 degrees C and 2. 6 degrees C at pH 9.2 and 10.5, respectively, in the presence of trehalose. At pH 9.2 the thermal unfolding occurs at higher temperatures (Tm is 52.6 degrees C compared to 42.0 degrees C at pH 10.5) and a refolding yield of around 80% was obtained upon cooling. This pH value was chosen to study the irreversible inactivation (long-term stability) of cutinase. Temperatures in the transition range from folded to unfolded state were selected and the rate constants of irreversible inactivation determined. Inactivation followed first-order kinetics and trehalose reduced the observed rate constants of inactivation, pointing to a stabilizing effect on the irreversible inactivation step of thermal denaturation. However, if the contribution of reversible unfolding on the irreversible inactivation of cutinase was taken into account, i.e., considering the fraction of cutinase molecules in the reversible unfolded conformation, the intrinsic rate constants can be calculated. Based on the intrinsic rate constants it was concluded that trehalose does not delay the irreversible inactivation. This conclusion was further supported by comparing the activation energy of the irreversible inactivation in the absence and in the presence of trehalose. The apparent activation energy in the absence and in the presence of trehalose were 67 and 99 Kcal/mol, respectively. The activation energy calculated from intrinsic rate constants was higher in the absence (30 Kcal/mol) than in the presence of trehalose (16 Kcal/mol), showing that kinetics of the irreversible inactivation step increased in the presence of trehalose. In fact, trehalose stabilized only the reversible step of thermal denaturation of cutinase.     

Enzymatic esterification of ethanol by an immobilised Rhizomucor miehei lipase in a perforated rotating disc bioreactor

A perforated rotating disc bioreactor was developed to perform the esterification of ethanol with oleic acid, catalyzed by a lipase from Rhizomucor miehei immobilized by adsorption on to a hydrophobic support-Accurel EP700. The bioreactor with total recirculation operated at an optimum agitation rate of 400 rev./min. The experimental results, in this condition, were predict by a kinetic model using the constants obtained in the batch (Erlenmeyer flasks) assays: a catalytic constant, k(cat) = 5.78 mmol/h. mg protein; a Michaelis constant for ethanol, K(m(Et)) = 1.20 M; a Michaelis constant for oleic acid, K(m(Ol)) = 1.16 x 10(-8) M, and a dissociation constant of the ethanol-lipase complex, K((Et)) = 9.46 x 10(7) M. The efficiency of conversion gradually decreased during continuous operation of the reactor. The enzymatic activity decayed according to a first order deactivation model and the integrated equations of a continuous stirred tank reactor (CSTR) and a plug flow reactor (PFR). A half-life time of the lipase of about 10 days and a deactivation constant of 0.003 h(-1) were obtained in the present system.     

Downstream processing of plasmid DNA for gene therapy and DNA vaccine applications

Interest in producing large quantities of supercoiled plasmid DNA has recently increased as a result of the rapid evolution of gene therapy and DNA vaccines. Owing to the commercial interest in these approaches, the development of production and purification strategies for gene-therapy vectors has been performed in pharmaceutical companies within a confidential environment. Consequently, the information on large-scale plasmid purification is scarce and usually not available to the scientific community. This article reviews downstream operations for the large-scale purification of plasmid DNA, describing their principles and the strategy used to attain a final product that meets specifications.     

Kinetics of cutinase catalyzed transesterification in AOT reversed micelles: modeling of a batch stirred tank reactor

A transesterification process is analyzed in its multiple kinetic components that include the determination of the kinetic constants for both substrates, butyl acetate (BAc) and hexanol (H), involved in the alcoholysis reaction and for the products formed (hexyl acetate (HAc) and butanol (B)), participating into the reverse reaction. The order of magnitude of these constants is discussed in relation with the AOT/isooctane reverse micellar system under study. The values of the equilibrium conversion (X(e)) and constant (K(eq)) were also determined. Diffusional limitations were detected for H concentrations lower than 450 mM and the correspondent effectiveness factors were calculated. Above 450 mM H the reaction is kinetically controlled. The operation of a batch stirred tank reactor (BSTR) was modeled considering the integrated rate equation for reversible kinetics.