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Cartilage Acidic Protein 2 (CRTAC2) is a novel protein present from prokaryotes to vertebrates with abundant expression in the teleost fish pituitary gland and an isoform of CRTAC1, a chondrocyte marker in humans. The two proteins are non-integrins containing N-terminal integrin-like Ca(2+)-binding motifs and their structure and function remain to be assigned. Structural studies of recombinant sea bream (sb)CRTAC2 revealed it is composed of 8.8% α-helix, 33.4% β-sheet and 57.8% unordered protein. sbCRTAC2 bound Ca(2+) with high affinity (K(d)=1.46nM) and favourable Gibbs free energy (∆G=-12.4kcal/mol). The stoichiometry for Ca(2+) bound to sbCRTAC2 at saturation indicated six Ca(2+) ligand-binding sites exist per protein molecule. No conformational change in sbCRTAC2 occurred in the presence of Ca(2+). Fluorescence emission revealed that the tertiary structure of the protein is hyperthermostable between 25°C and 95°C and the fully unfolded state is only induced by chemical denaturing (4M GndCl). sbCRTAC has a widespread tissue distribution and is present as high molecular weight aggregates, although strong reducing conditions promote formation of the monomer. sbCRTAC2 promotes epithelial cell outgrowth in vitro suggesting it may share functional homology with mammalian CRTAC1, recently implicated in cell-cell and cell-matrix interactions.

The contribution of the disulfide bridge in CotA-laccase from Bacillus subtilis is assessed with respect to the enzyme’s functional and structural properties. The removal of the disulfide bond by site-directed mutagenesis, creating the C322A mutant, does not affect the spectroscopic or catalytic properties and, surprisingly, neither the long-term nor the thermodynamic stability parameters of the enzyme. Furthermore, the crystal structure of the C322A mutant indicates that the overall structure is essentially the same as that of the wild type, with only slight alterations evident in the immediate proximity of the mutation. In the mutant enzyme, the loop containing the C322 residue becomes less ordered, suggesting perturbations to the substrate binding pocket. Despite the wild type and the C322A mutant showing similar thermodynamic stability in equilibrium, the holo or apo forms of the mutant unfold at faster rates than the wild-type enzyme. The picosecond to nanosecond time range dynamics of the mutant enzyme was not affected as shown by acrylamide collisional fluorescence quenching analysis. Interestingly, copper uptake or copper release as measured by the stopped-flow technique also occurs more rapidly in the C322A mutant than in the wild-type enzyme. Overall the structural and kinetic data presented here suggest that the disulfide bridge in CotA-laccase contributes to the conformational dynamics of the protein on the microsecond to millisecond timescale, with implications for the rates of copper incorporation into and release from the catalytic centres.

Endoplasmic reticulum (ER) oxidation 1 (ERO1) transfers disulfides to protein disulfide isomerase (PDI) and is essential for oxidative protein folding in simple eukaryotes such as yeast and worms. Surprisingly, ERO1-deficient mammalian cells exhibit only a modest delay in disulfide bond formation. To identify ERO1-independent pathways to disulfide bond formation, we purified PDI oxidants with a trapping mutant of PDI. Peroxiredoxin IV (PRDX4) stood out in this list, as the related cytosolic peroxiredoxins are known to form disulfides in the presence of hydroperoxides. Mouse embryo fibroblasts lacking ERO1 were intolerant of PRDX4 knockdown. Introduction of wild-type mammalian PRDX4 into the ER rescued the temperature-sensitive phenotype of an ero1 yeast mutation. In the presence of an H2O2-generating system, purified PRDX4 oxidized PDI and reconstituted oxidative folding of RNase A. These observations implicate ER-localized PRDX4 in a previously unanticipated, parallel, ERO1-independent pathway that couples hydroperoxide production to oxidative protein folding in mammalian cells. EMBO [ALTF649-2008]; Fundacao para a Ciencia e Tecnologia, Portugal [SFRH/BSAB/922/2009, PTDC/QUI/73027/2006, IBB/CBME LA]; NIH [DK47119, DK075311, ES08681]; 100 Women In Hedge Funds Foundation; [NS050276]; [CA016087]; Medical Research Council [G0600717B] info:eu-repo/semantics/publishedVersion

Biomedical applications of osmolytes, including stabilization of protein-based pharmaceutics, preservation of living biological material and potential therapeutic prescription in vivo, are intimately related to the fact that osmolytes favour the native structure of proteins. The shift towards the native structure is associated to the compaction of the protein by a non-specific mechanism. This compaction is observed mostly for the unfolded state but also for the transition state ensemble and even for the native state. In addition, more stable three-dimensional structures are more stabilized by osmolytes if the overall protein fold is the same indicating that point mutations and osmolytes should share a similar mechanism for protein stabilization. A synergistic effect to increase protein stability between accumulation of osmolytes and protein engineering strategies seems to have operated during evolution. However, the conformational pre-organization of the unfolded state (compaction) induced by osmolytes which increases the folding rate, might lead to the accumulation of off-folding pathway intermediates with non-native structure that delay folding. Also, osmolytes favor protein aggregation as an alternative way to shield protein surfaces from the solvent. The sometimes observed effect of osmolytes on the prevention of protein aggregation is apparent as they only decrease the accumulation of aggregation-competent partially unfolded states.

A simple method of assessing the viability of a lactic starter culture is presented. The method is based on the kinetics of growth of a culture during the lag and early exponential phases. The microorganisms used throughout this work were Lactobacillus spp. The method was tested with inocula samples of different ages and with samples taken during chemostat runs, at different dilution rates. The results obtained are similar to ones describes in the literature for similar situations. The method is very easy to operate once the intrinsic parameters have been established (χmax and μmax). It needs only standard laboratory equipment and is very economical.

The production of a flocculent strain of Lactobacillus plantarum was performed in a high cell density reactor: a fluidized bed reactor (FBR) with a settler and an external cell recirculation. Two variables were assessed, the recirculation rate (R) and the dilution rate (D). The effect of the latter is much more important than the effect of the former in ensuring a quick start up in the flocculation process. The cell volumetric productivities obtained with this system increase directly with dilution rate and recirculation rate. The values of cell volumetric productivities obtained are considerably higher than those obtained in continuous stirred tank reactors (CSTR) and much higher than in batch reactors.

Phytophthora cinnamomi has been reported to be regularly associated with cork and holm oak decline. This oomycete secretes elicitins, a group of unique highly conserved proteins that can enhance plant defence reactions. In our previous work it was shown that the absorption of one of these elicitins─ α-CIN─ by the roots of cork and holm oak at concentrations of 500 μg and 1 mg/ml reduced P. cinnamomi viability and its progression into internal tissues. The objectives of the present work were to study the restriction of host tissue colonization and the loss of pathogen viability in the roots treated with lower concentration of α-CIN, and to verify structural alterations at cellular level induced by this elicitin in aerial part of the plantlets. Roots of Quercus suber seedlings two month old were immersed in aqueous α-CIN solutions [250 µg/ml] and [100 µg/ml] during 24 h. Roots were then inoculated with P. cinnamomi. The samples pre-treated with α-CIN at the two concentrations and inoculated with P. cinnamoni revealed similar histological and cytological aspects. The hyphae were mainly intercellularly located, restricted to the external cortex (Figs.A and B). The treatment induced loss of pathogen viability and it was evident the pathogen membranar degradation, loss of organelles and reduction of

Trehalose has been widely used to stabilize cellular structures such as membranes and proteins. The effect of trehalose on the stability of the enzyme cutinase was studied. Thermal unfolding of cutinase reveals that trehalose delays thermal unfolding, thus increasing the temperature at the midpoint of unfolding by 7.2°. Despite this stabilizing effect, trehalose also favors pathways that lead to irreversible denaturation. Stopped-flow kinetics of cutinase folding and unfolding was measured and temperature was introduced as experimental variable to assess the mechanism and thermodynamics of protein stabilization by trehalose. The main stabilizing effect of trehalose was to delay the rate constant of the unfolding of an intermediate. A full thermodynamic analysis of this step has revealed that trehalose induces the phenomenon of entropy–enthalpy compensation, but the enthalpic contribution increases more significantly leading to a net stabilizing effect that slows down unfolding of the intermediate. Regarding the molecular mechanism of stabilization, trehalose increases the compactness of the unfolded state. The conformational space accessible to the unfolded state decreases in the presence of trehalose when the unfolded state acquires residual native interactions that channel the folding of the protein. This residual structure results into less hydrophobic groups being newly exposed upon unfolding, as less water molecules are immobilized upon unfolding. © 2008 Wiley Periodicals, Inc. Biopolymers 89: 538–547, 2008. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

The axial ligand of the catalytic mononuclear T1 copper site (Met(502)) of the CotA laccase was replaced by a leucine or phenylalanine residue to increase the redox potential of the enzyme. These mutations led to an increase in the redox potential by approx. 100 mV relative to the wild-type enzyme but the catalytic constant k(cat) in the mutant enzymes was severely compromised. This decrease in the catalytic efficiency was unexpected as the X-ray analysis of mutants has shown that replacement of methionine ligand did not lead to major structural changes in the geometry of the T1 Centre or in the overall fold of the enzyme. However, the mutations have a profound impact on the thermodynamic stability of the enzyme. The fold of the enzyme has become unstable especially with the introduction of the larger phenylalanine residue and this instability should be related to the decrease in the catalytic efficiency. The instability of the fold for the mutant proteins resulted in the accumulation of an intermediate state, partly unfolded, in-between native and unfolded states. Quenching of tryptophan fluorescence by acrylamide has further revealed that the intermediate state is partly unfolded. info:eu-repo/semantics/publishedVersion

The beta ->alpha transition of beta-lactoglobulin, a globular protein abundant in the milk of several mammals, is investigated in this work. This transition, induced by the cationic surfactant dodecyltrimethylammonium chloride (DTAC), is accompanied by partial unfolding of the protein. In this work, unfolding of bovine beta-lactoglobulin in DTAC is compared with its unfolding induced by the chemical denaturant guanidine hydrochloride (GnHCl). The final protein states attained in the two media have quite different secondary structure: in DTAC the alpha-helical content increases, leading to the so-called alpha-state; in GnHCl the amount of ordered secondary-structure decreases, resulting in a random coil-rich final state (denatured, or D, state). To obtain information on both mechanistic routes, in DTAC and GnHCl, and to characterize intermediates, the kinetics of unfolding were investigated in the two media. Equilibrium and kinetic data show the partial accumulation of an on-pathway intermediate in each unfolding route: in DTAC, an intermediate (I-1) with mostly native secondary structure but loose tertiary structure appears between the native (beta) and alpha-states; in GnHCl, another intermediate (I-2) appears between states beta and D. Kinetic rate constants follow a linear Chevron-plot representation in GnHCl, but show a more complex mechanism in DTAC, which acts like a stronger binding species. info:eu-repo/semantics/publishedVersion

Detailed analysis of the effects of UV and blue light illumination of horseradishperoxidase A2, a heme-containing enzyme that reduces H2O2 to oxidize organic andinorganic compounds, is presented. The effects of increasing illumination time on theprotein's enzymatic activity, Reinheitzahl value, fluorescence emission, fluorescencelifetime distribution, fluorescence mean lifetime and heme absorption are reported. UVillumination leads to an exponential decay of the enzyme activity followed by changes inheme group absorption. Longer UV illumination time leads to lower Tm values as well ashelical content loss. Prolonged UV illumination and Heme irradiation at 403nm haspronounced effect on the fluorescence quantum yield, correlated with changes in theprosthetic group pocket, leading to pronounced decrease in the heme's Soret absorbanceband. Analyzes of the picosecond resolved fluorescence emission of horseradishperoxidase A2 with streak camera shows that UV illumination induces an exponentialchange in the pre-exponential factors distribution associated to the protein's fluorescencelifetimes, leading to an exponential increase of the mean fluorescence lifetime.Illumination of aromatic residues and of the heme group leads to changes indicative ofheme leaving the molecule and/or that photo-induced chemical changes occur in the hememoiety. Our studies bring new insight into light induced reactions in proteins. We showhow streak camera technology can be of outstanding value to follow such ultra-fastprocesses and how streak camera data can be correlated with protein structural changes. Udgivelsesdato: MAR 15

Site-directed mutagenesis has been used to replace Met502 in CotA laccase by the residues leucine and phenylalanine. X-ray structural comparison of M502L and M502F mutants with the wild-type CotA shows that the geometry of the T1 copper site is maintained as well as the overall fold of the proteins. The replacement of the weak so-called axial ligand of the T1 site leads to an increase in the redox potential by approximately 100 mV relative to that of the wild-type enzyme (E 0=455 mV). However the M502L mutant exhibits a twofold to fourfold decrease in the k cat values for the all substrates tested and the catalytic activity in M502F is even more severely compromised; 10% activity and 0.15–0.05% for the non-phenolic substrates and for the phenolic substrates tested when compared with the wild-type enzyme. T1 copper depletion is a key event in the inactivation and thus it is a determinant of the thermodynamic stability of wild-type and mutant proteins. Whilst the unfolding of the tertiary structure in the wild-type enzyme is a two-state process displaying a midpoint at a guanidinium hydrochloride concentration of 4.6 M and a free-energy exchange in water of 10 kcal/mol, the unfolding for both mutant enzymes is clearly not a two-state process. At 1.9 M guanidinium hydrochloride, half of the molecules are in an intermediate conformation, only slightly less stable than the native state (approximately 1.4 kcal/mol). The T1 copper centre clearly plays a key role, from the structural, catalytic and stability viewpoints, in the regulation of CotA laccase activity.

The effect of osmolyte sucrose on the stability and compaction of the folded and unfolded states of ribosomal protein S6 from Thermus thermophilus was analyzed. Confirming previous results obtained with sodium sulfate and trehalose, refolding stopped-flow measurements of S6 show that sucrose favors the conversion of the unfolded state ensemble to a highly compact structure (75% as compact as the folded state). This conversion occurs when the unfolded state is suddenly placed under native conditions and the compact state accumulates in a transient off-folding pathway. This effect of sucrose on the compaction of the unfolded state ensemble is counteracted by guanidinium hydrochloride. The compact state does not accumulate at higher guanidinium concentrations and the unfolded state ensemble does not display increased compaction in the presence of 6 M guanidinium as evaluated by collisional quenching of tryptophan fluorescence. In contrast, accessibility of the tryptophan residue of folded S6 above 1 M sucrose concentration decreased as a result of an increased compaction of the folded state. Unfolding stopped-flow measurements of S6 reflect this increased compaction of the folded state, but the unfolding pathway is not affected by sucrose. Compaction of folded and unfolded S6 induced by sucrose occurs under native conditions indicating that decreased protein conformational entropy significantly contributes to the mechanism of protein stabilization by osmolytes.

The crystal structure of the elicitin h-cinnamomin (h-CIN) was determined in complex with ergosterol at 1.1 Au resolution. h-CIN/ergosterol complex crystallized in the monoclinic space group P21, with unit cell parameters of a=31.0, b=62.8, c=50.0 Au and b=93.4- and two molecules in the asymmetric unit. Ligand extraction with chloroform followed by crystallographic analysis yielded a 1.35 Au structure of h-CIN (P43212 space group) where the characteristic elicitin fold was kept. After incubation with cholesterol, a new complex structure was obtained, showing that the protein retains, after the extraction procedure, its ability to complex sterols. The necrotic effect of h-CIN on tobacco was also shown to remain unchanged. Theoretical docking studies of the triterpene lupeol to h-CIN provided an explanation for the apparent inability of h-CIN to bind this ligand, as observed experimentally. D 2005 Elsevier B.V. All rights reserved.

The folding of cutinase, an enzyme displaying lipolytic activity, has been studied in the presence of trehalose. Equilibrium unfolding data show that trehalose increases the free energy change between folded and unfolded states. Unfolding kinetics reveal the presence of an intermediate which is ca. 60% folded in terms of solvent exposure. Trehalose stabilizes this intermediate relative to the folded state. In contrast, the intermediate revealed by folding kinetics is more compact than the transition state, as shown by the positive slope observed at low denaturant concentration in the chevron plot, as well as the decrease in the observable rate constant for folding with the increase in trehalose concentration. This intermediate displays more than 50% of area buried from the solvent (relative to the native state) compared to around 40% for the transition state for folding and therefore appears to be off the folding pathway. Trehalose stabilizes and guanidine hydrochloride destabilizes this compact intermediate. Both unfolding and folding kinetics show that compact conformational states are stabilized by trehalose, in agreement with current models on the effect of compatible solutes. This effect occurs even for compact states that decelerate the folding as in the case of the intermediate revealed by folding kinetics.

Cutinase encapsulated in dioctyl sulfosuccinate reverse micelles displays very low stability, undergoing fast denaturation due to an anchoring at the micellar interface. The denaturation process and the structure of the reverse micelle were characterized using biophysical techniques. The kinetics of denaturation observed from fluorescence match the increase of the hydrodynamic radius of reverse micelles. Denaturation in reverse micelles is mainly the unfolding of the three-dimensional structure since the decrease in the circular dichroism ellipticity in the far-UV range is very small. The process is accompanied by an increase in the steady-state anisotropy, as opposed to what happens for denaturation in aqueous solution. Since 1-hexanol used as co-surfactant in dioctyl sulfosuccinate reverse micelles slows or even prevents cutinase denaturation, its effect on cutinase conformation and on the size of reverse micelles was analyzed. When 1-hexanol is present, cutinase is encapsulated in a large reverse micelle, as deduced from dynamic light scattering. The large reverse micelle filled with cutinase was built from the fusion of reverse micelles according to a pseudo-unimolecular process ranging in time from a few minutes to 2 h depending on the reverse micellar concentration. This slow equilibrium driven by the encapsulated cutinase has not been reported previously. The encapsulation of cutinase in dioctyl sulfosuccinate reverse micelles establishes a completely new equilibrium characterized by a bimodal population of empty and filled reverse micelles, whose characteristics depend greatly on the interfacial characteristics, that is, on the absence or presence of 1-hexanol.

Cutinase in aqueous solution at pH 4.5 deactivates following a parallel pathway. At 53 degrees C, 88% of the cutinase molecules are in the unfolded conformation, which can aggregate with a reaction order of 3 if the protein concentration is high (>/=12 microM). The aggregates show a sixfold increase in size as determined by dynamic light scattering. This aggregation process is the first phase observed during a deactivation experiment; however, after significant cutinase depletion and maturation of the aggregates, a first-order step starts to dominate and a second phase independent of the protein concentration is observed. Kinetic partitioning between aggregation and first-order irreversible changes of the unfolded conformation can occur during enzyme deactivation when the equilibrium between the native and the unfolded conformation is shifted and kept toward the unfolded conformation.

High-molecular-weight suspended organic matter of soil and aquatic origins competes with water for the dissolution of relatively water insoluble organic substances. The same happens with microalgae and other organisms present in natural waters. Several pheromones, which play a specific role in the reproductive cycle of fish, are secreted to the water and are generally, if not always, molecules with hydrophobic or amphiphilic characteristics. The natural tendency of these pheromones to dissolve in suspended or deposited organic matter may cause their signaling function to be adversely affected. In this work we study the partition constants between water and organic reservoirs in suspension, Kh, of two fish pheromones, 4-pregnene-17 alpha, 20beta-diol-3-one (17,20beta-P) and prostaglandin F2alpha (PGF2alpha), and also of 4-pregnene-11beta,21-diol-3,20-dione 21-sulfate (21-P-sulfate), used as a proxy for sulfated steroid pheromones. Two types of organic reservoirs are employed: aggregates of suspended humic substances and negatively charged phospholipid vesicles. We find that the three compounds have high affinities for both types of aggregates. However, 17,20beta-P, with pKh = 4.4 +/- 0.2, is the only one for which we may predict a significant decrease in availability in consideration of the normal content of dissolved organic matter in natural waters. Also to be considered is the fact that a relatively large amount of pheromones is retained and may be released at an inopportune moment. How significant these phenomena are in nature is not as yet clear, considering the variety of habitats in which fish spawn and the little that is known about the nature and mode of action of pheromones.

Horseradish peroxidase A1 thermal stability was studied by steady-state fluorescence, circular dichroism and differential scanning calorimetry at pH values of 4, 7 and 10. Changes in the intrinsic protein probes, tryptophan fluorescence, secondary structure, and heme group environment are not coincident. The T(m) values measured from the visible CD data are higher than those measured from Trp fluorescence and far-UV CD data at all pH values showing that the heme cavity is the last structural region to suffer significant conformational changes during thermal denaturation. However ejection of the heme group leads to an irreversible unfolding behavior at pH 4, while at pH 7 and 10 refolding is still observed. This is putatively correlated with the titration state of the heme pocket. Thermal transitions of HRPA1 showed scan rate dependence at the three pH values, showing that the denaturation process was kinetically controlled. The denaturation process was interpreted in terms of the classic scheme, N--U--D and fitted to far-UV CD ellipticity. A good agreement was obtained between the experimental and theoretical T(m) values and percentages of irreversibility. However the equilibrium between N and U is probably more complex than just a two-state process as revealed by the multiple T(m) values.

Reverse micelles are nanometer-sized (1-10 nm) water droplets dispersed in organic media obtained by the action of surfactants. Surfactant molecules organize with the polar part to the inner side able to solubilize water and the apolar part in contact with the organic solvent. Proteins can be solubilized in the water pool of reverse micelles. Studies on the structure-function relationships of proteins in reverse micelles are very important since the microenvironment in which the protein is solubilized has physico-chemical properties distinct from a bulk aqueous solution. Some of the unique characteristics of reverse micelles make them very useful for biotechnological applications. Charge and hydrophilic/hydrophobic characteristics of the protein and the selection of surfactant can be used to achieve selective solubilization of proteins. This has been used to extend the classical liquid-liquid extraction with solvents to protein bioseparation. For biocatalysis the presence of a bulk organic solvent allow synthetic reactions to be performed via the control of water content and the solubilization of hydrophobic substrates. This is accomplished with a higher interfacial area (about 100 m2/mL) than the conventional biphasic systems, minimizing mass transfer problems.

The thermal stability of the lipase from Chromobacterium viscosum was assessed by deactivation (loss of activity), fluorescence, circular dichroism (CD) and static light scattering (SLS) measurements. Lipase fluorescence emission is dominated by the tryptophyl contribution. An increase in the tyrosyl contribution from 2 to 16% was only observed upon prolonged incubation at 60 degrees C. The effect of temperature on the tryptophyl quantum yield was studied and two activation energies were calculated. Tryptophan residues in the native structure have an activation energy of 1.9 kcal mol(-1) for temperature-dependent non-radiative deactivation of the excited state. A structural change occurs at approximately 66.7 degrees C and the activation energy increases to 10.2 kcal mol(-1). This structural change is not characterized by tryptophan exposure on the surface of the protein. The deactivation and the evolution of structural changes with time after lipase incubation at 60 degrees C were assessed by fluorescence, CD and SLS measurements. CD spectra show that both secondary and tertiary structures remain native-like after incubation at 60 degrees C in spite of the fluorescence changes observed (red-shift from 330 to 336 nm on the trytophyl emission). SLS measurements together with the CD data show that deactivation may be due to protein association between native molecules. Deactivation and the decrease on the fraction of non-associated native lipase evaluated by changes in fluorescence intensity with time, show apparent first order kinetics. According to the rate constants, fluorescence changes precede deactivation pointing to an underestimation of the deactivation. Reactivation upon dilution during the activity assay and substrate-induced reactivation due to lipase interfacial adsorption are possible causes for this underestimation.

The fungal lipolytic enzyme cutinase, incorporated into sodium bis-(2ethylhexyl) sulfosuccinate reversed micelles has been investigated using dynamic light scattering. The reversed micelles form spontaneously when water is added to a solution of sodium bis-(2ethylhexyl) sulfosuccinate in isooctane. When an enzyme is previously dissolved in the water before its addition to the organic phase, the enzyme will be incorporated into the micelles. Enzyme encapsulation in reversed micelles can be advantageous namely to the conversion of water insoluble substrates and to carry out synthesis reactions. However protein unfolding occurs in several systems as for cutinase in sodium bis-(2ethylhexyl) sulfosuccinate reversed micelles. Dynamic light scattering measurements of sodium bis-(2ethylhexyl) sulfosuccinate reversed micelles with and without cutinase were taken at different water to surfactant ratios. The results indicate that cutinase was attached to the micellar wall and that might cause cutinase unfolding. The interactions between cutinase and the bis-(2ethylhexyl) sulfosuccinate interface are probably the driving force for cutinase unfolding at room temperature. Twenty-four hours after encapsulation, when cutinase is unfolded, a bimodal distribution was clearly observed. The radii of reversed micelles with unfolded cutinase were determined and found to be considerable larger than the radii of the empty reversed micelles. The majority of the reversed micelles were empty (90–96% of mass) and the remainder (4–10%) containing unfolded cutinase were larger by 26–89 A.

The four main classes of biological substances are carbohydrates, proteins, nucleic acids and lipids. The first three of these substances have been clearly defined on the basis of their structural features, whereas the property which is common to all lipids is a physicochemical one. Lipids are in fact a group of structurally heterogeneous molecules which are all insoluble in water but soluble in apolar and slightly polar solvents such as ether, chloroform and benzene.

Vaccinium mirtyllus peroxidase solubilized in reversed micelles was used for the oxidation of guaiacol. Some relevant parameters for the enzymatic activity, such as pH,w o (molar ratio water/surfactant), surfactant type and concentration, and cosurfactant concentration, were investigated. The peroxidase showed higher activities in reversed micelles than in aqueous solution. The stability of the peroxidase in reversed micelles was also studied, namely, the effect ofw o and temperature on enzyme deactivation. The peroxidase displayed higher stabilities in CTAB/hexanol in isooctane reversed micelles, with halflife times higher than 500 h.

A novel method to distribute proteins on solid surfaces is proposed. Proteins microencapsulated in the water pool of reverse micelles were used to coat a solid surface with well-individualized round spots of 1 to 3 µm in diameter. The number of spots per unit area can be increased through the concentration of reverse micelles, and networks of spots were obtained at high concentrations of large reverse micelles. Moreover, depending on the pool size of the water reverse micelles, proteins can be deposited far from each other or in close proximity within the range of 50 to 70 A. This proximity obtained with small reverse micelles was proved through fluorescence lifetime imaging microscopy and fluorescence resonance energy transfer (FLIM-FRET) measurements for the most relevant FRET pair in cell biology studies, the cyan and yellow fluorescent proteins. This novel procedure has several advantages and reveals the potential for study of protein-protein interactions on solid surfaces and for developing novel biomaterials and molecular devices based on biorecognition elements.

A lactic starter culture of a flocculent Lactobacillus plantarum was produced in a fluidized bed reactor with higher cell volumetric productivities than in a continuous stirred tank reactor. The fluidized bed reactor was operated at optimised parameters obtained in batch reactor performed with and without pH control.

Phytophthora cinnamomi and P. cambivora are considered as the causal agents of Castanea sativa ink disease. These soil-borne plant pathogens invade and destroy the root system leading to the death of the trees. Most Phytophthora species secrete elicitins, a group of unique highly conserved proteins that are able to enhance plant defence responses in a systemic acquired resistance manner against infection by several pathogens. A cluster of four elicitin genes was identified in P. cinnamomi. In previous works one of these elicitins, α- cinnamomin was shown to restrict the invasion of root cortical tissues by P. cinnamomi preventing vascular colonization in cork and holm oak. In the present work, roots of chestnut plantlets grown in vitro were allowed to absorb α-cinnamomin at 100 μg/ml for two days before being inoculated with P. cinnamomi. The effects of this elicitin on host-pathogen interaction were studied at histological and ultrastructural levels. P. cinnamomi was restricted to the outer cortex of 65% of the roots pre-treated with α-cinnamomin. In these roots, the vascular cylinders were free of pathogen. On the contrary, the pathogen reached the vascular cylinder, penetrating the phloem and xylem vessels in all non-treated assayed roots. The signs of pathogen degradation in the cortical parenchyma, mainly in the intercellular spaces, and the increase of a physical barrier in epidermal and sub-epidermal cell wall-media lamella and intercellular spaces by impregnation with phenol-like compounds strongly suggest that α-cinnamomin induced in chestnut defence reactions against P. cinnamomi.

The monomolecular film technique previously used to study the kinetics of lipase hydrolysis was adapted to synthesizing oleoyl glycerides (monoolein, diolein, and triolein). The water subphase was replaced by glycerol, and a film of oleic acid was initially spread on the glycerol surface. In this system a recombinant cutinase from Fusarium solani was able to catalyze oleoyl glyceride synthesis. More than 50% of the oleic acid film was acylated after 7 min of reaction. The surface pressure applied to the monomolecular film acts as a physical selectivity factor since glyceride synthesis can be steered so as to produce either diolein or triolein.