Dynamic light scattering of cutinase in AOT reverse micelles

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.     

Analysis of protein-surfactant interactions--a titration calorimetric and fluorescence spectroscopic investigation of interactions between Humicola insolens cutinase and an anionic surfactant

We have studied interactions of cutinase (HiC) from Humicula insolens and sodium dodecyl sulphate (SDS) by parallel calorimetric and fluorescence investigations of systems in which the concentration of both components was changed systematically. Results from the two methods exhibit a number of synchronous characteristics, when plotted against the total SDS concentration, [SDS]tot. The molecular origin of several of these anomalies was assigned, and five intervals of [SDS]tot in which different modes of interactions dominated were identified. Going from low to high [SDS]tot, these modes were: binding of (a few) SDS to native HiC, formation of oligomeric protein aggregates, denaturation of HiC and adsorption of SDS on denatured protein. For [SDS]tot>3-6 mM (depending on the protein concentration), the adsorption saturated, and no further protein-detergent interaction could be detected. Two particularly conspicuous anomalies in the calorimetric data were ascribed to respectively denaturation and saturation. It was found that [SDS]tot at these points depended linearly on the (total) protein concentration, [HiC]. We suggest that this reflects the balance between bound and free SDS [SDS]tot=[SDS]aq+[HiC] Nb where [SDS]aq and Nb are, respectively, the aqueous ("free") concentration of SDS and the average number of SDS bound per protein. Interpretation of the results along these lines showed that at 22 degrees C and pH 7.0, HiC denatures with approximately 14 bound surfactant molecules at [SDS]aq=1.0 mM. Saturation is characterized by Nb approximately 39 and [SDS]aq=2.2 mM. The latter value is equal to CMC in the (protein free) buffer. These results are discussed with respect to the SDS-binding capacity of HiC and the origin and location of the saturation point.     

Determination and structural characterisation of dermatan sulfate in the presence of other galactosaminoglycans

Chondroitin sulfate and dermatan sulfate are galactosaminoglycans that have similar size and charge density thus making difficult their separation and accurate determination from tissue preparations. A procedure was developed, which was based on the specific action of chondroitinase B, that allowed the determination of dermatan sulfate content in a mixture of chondroitin sulfate/dermatan sulfate, its molecular mass (M_r), and iduronic acid content and distribution throughout the chain. According to this procedure, the galactosaminoglycan sample was treated with chondroitinase B and its profile, upon gel chromatography on Sepharose CL-6B, was compared to that of the initial sample. The differences in uronic acid content of the fractions of the gel chromatographies were plotted and a secondary profile was constructed, which corresponded to the elution profile of intact dermatan sulfate in the sample. From this profile, the size distribution of dermatan sulfate was obtained and its M_r was calculated. In addition, the accurate content of dermatan sulfate in the sample was determined. The digest contained oligosaccharides of variable size that were separated on BioGel P-10. From the separated oligosaccharides the distribution of iduronic acid throughout the dermatan sulfate chains was determined. The procedure was applied to the determination and partial characterisation of dermatan sulfate from sheep nasal cartilage, in which it is reported for the first time that it contains a significant proportion of dermatan sulfate chains of low iduronic acid content.     

Interaction of an anionic surfactant with a recombinant cutinase from Fusarium solani pisi: a spectroscopic study

Structural effects resulting from the interaction of the anionic surfactant sodium bis[2-ethylhexyl]ester sulfosuccinic acid (AOT) with a recombinant cutinase are studied and characterised by means of spectroscopic techniques. Levels of interaction are described in terms of surfactant to protein molar ratio (MR). Three major regions may be identified: MR=0–10, MR=10–30 and MR>30. The latter corresponds to co-operative binding of the surfactant to protein leading to overall denaturation as observed by far-UV circular dichroism (CD) and 8-anilino-1-naphtalene-sulfonic acid (1,8-ANS) fluorescence. For MR=10–30, steady-state fluorescence suggests slight conformational changes while near-UV CD shows almost complete loss of signal for the chromophore residues. Finally, the first level, MR=0–10, reveals two distinct effects of interaction. For very low MR (0–5), the protein seems to remain structurally intact. However, at MR=10, both near-UV CD and unfolding kinetics reveal a structurally disturbed protein contrary to steady-state fluorescence spectra. This suggests that AOT interacts specifically with cutinase at this level, through electrostatic interactions mostly. By promoting localised disruption or destabilisation of crucial native electrostatic interactions, the surfactant initiates conformational loss of tertiary structure, leading to higher denaturation as MR increases.     

Cytological and structural changes in Pinus pines L. needles following the application of an anionic surfactant

In many coastal areas, synthetic surfactants are present as pollutants in the chemical composition of sea spray. Their phytotoxic action in synergism with sea salt is well known. This paper examines the early effects of an anionic surfactant (sodium alkylbenzene sulphonate = ABS) on the anatomy and ultrastructure of Pinus pinea L. needles. Three successive treatments in the open field on Pinus pinea trees about 2 m in height were carried out in the month of August. The crowns of the trees were sprayed with an aqueous solution containing 1000 g m^?3, and subsequently the current-year needles were observed by light microscopy, as well as by scanning and transimission electron microscopy. A few days after the treatment the needles had turned yellow. The results of the microscopical analysis show that the surfactant is absorbed prevalently through the stomata and that it causes deterioration of the epistomatal wax structures and induces alterations in the stomatal guard cell walls. Lastly, it causes degeneration of the protoplasm in cells of the mesophyll, beginning with the cells closest to the stomatal openings. The efficiency of both photosynthesis and gaseous exchange processes may be affected.     

Biochemical and structural characterisation of a haloalkane dehalogenase from a marine Rhodobacteraceae

A putative haloalkane dehalogenase has been identified in a marine Rhodobacteraceae and subsequently cloned and over-expressed in Escherichia coli. The enzyme has highest activity towards the substrates 1,6-dichlorohexane, 1-bromooctane, 1,3-dibromopropane and 1-bromohexane. The crystal structures of the enzyme in the native and product bound forms reveal a large hydrophobic active site cavity. A deeper substrate binding pocket defines the enzyme preference towards substrates with longer carbon chains. Arg136 at the bottom of the substrate pocket is positioned to bind the distal halogen group of extended di-halogenated substrates.     

Liquid-liquid extraction of a recombinant cutinase from fermentation media with AOT reversed micelles

This work reports the extraction and back-extraction of an intracellular recombinant cutinase from complex biological media using AOT reversed micelles in isooctane. Cutinase was recovered from different complex media namely, fermentation broths and supernatants after cell disruption by osmotic shock and sonication. The application of the AOT reversed micellar system to the extraction of cutinase allowed activity yields and purification factors ranging from about 5% to 50% and 1.2 to 10.2, respectively, depending on the biological medium.Maria das Graças Carneiro da Cunha, from ITEP-Instituto Tecnológico do Estado de Pernambuco, acknowledges a Ph.D fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Centro de Pesquisa Aggeu Magalhães, Recife — PE — Brasil. E. P. Melo thanks Junta Nacional de Investigação Científica, Lisboa, Portugal, for providing a Ph.D. fellowship. The scientific support given by Prof. Sílvia M. B. Costa for the spectroscopic data and further discussions are particularly acknowledged.This work was partly financed by the BRIDGE and BIOTECHNOLOGY Programmes (Contracts BIOT-CT91-0274(DTEE) and BIOT 2 CT-943016).     

A steady-state fluorescence study of cutinase microencapsulated in AOT reversed micelles at optimal stability conditions

A steady-state fluorescence study of cutinase was performed to evaluate the structure of cutinase in reversed micelles of AOT with the optimised conditions assigned by factorial design. The results obtained by two independent methods are compared. At a W₀ (water to surfactant ratio) value of 2.7, and in the presence of 500 mM hexanol, the fluorescence intensity maximum (_max) remained almost constant for a period of time longer than 30.5 h and a slight red-shift from 305 to 310 nm was verified changing the W₀ value to 6. Decreasing the amount of hexanol to 100 mM, the changes in _max were more significant, especially for W₀=6 indicating a noticeable unfolding process. Structural evidence is given reinforcing the role of hexanol as a stabiliser of microencapsulated cutinase and the effect of a drastic reduction in water content.     

Recovery of lactoferrin and lactoperoxidase from sweet whey using colloidal gas aphrons (CGAs) generated from an anionic surfactant, AOT

The recovery of lactoferrin and lactoperoxidase from sweet whey was studied using colloidal gas aphrons (CGAs), which are surfactant-stabilized microbubbles (10-100 microm). CGAs are generated by intense stirring (8000 rpm for 10 min) of the anionic surfactant AOT (sodium bis-2-ethylhexyl sulfosuccinate). A volume of CGAs (10-30 mL) is mixed with a given volume of whey (1-10 mL), and the mixture is allowed to separate into two phases: the aphron (top) phase and the liquid (bottom) phase. Each of the phases is analyzed by SDS-PAGE and surfactant colorimetric assay. A statistical experimental design has been developed to assess the effect of different process parameters including pH, ionic strength, the concentration of surfactant in the CGAs generating solution, the volume of CGAs and the volume of whey on separation efficiency. As expected pH, ionic strength and the volume of whey (i.e. the amount of total protein in the starting material) are the main factors influencing the partitioning of the Lf.Lp fraction into the aphron phase. Moreover, it has been demonstrated that best separation performance was achieved at pH = 4 and ionic strength = 0.1 mol/L i.e., with conditions favoring electrostatic interactions between target proteins and CGAs (recovery was 90% and the concentration of lactoferrin and lactoperoxidase in the aphron phase was 25 times higher than that in the liquid phase), whereas conditions favoring hydrophobic interactions (pH close to pI and high ionic strength) led to lower performance. However, under these conditions, as confirmed by zeta potential measurements, the adsorption of both target proteins and contaminant proteins is favored. Thus, low selectivity is achieved at all of the studied conditions. These results confirm the initial hypothesis that CGAs act as ion exchangers and that the selectivity of the process can be manipulated by changing main operating parameters such as type of surfactant, pH and ionic strength.     

Renaturation and interaction of ribonuclease A with AOT surfactant in reverse micelles

This study extended works on effects of solute on the percolation of reverse micelles to the effects of interactions between protein and surfactants on protein refolding by reverse micelles. The changes in percolation behavior were identified and attributed to the position of solutes in the core aqueous phase and the interaction between the solute and the surfactants. The percolation behavior of reverse micelles with solutes was related to protein renaturation and the reverse micelle. This study aims to highlight the involvement of the interface and the interaction of the protein with the surfactant during protein refolding. Ribonuclease A and AOT reverse micelles together constitute a model system considered here. The systemic parameters of the reverse micelle, water content (W(o)) and pH value, were applied to modify the interaction between the denatured protein molecules and the surfactant interface. The interactions and the locations of the protein molecules were determined from changes in percolation temperature measured by conductivity. The percolation and protein activity show that a stronger interaction of the protein molecules with surfactant corresponds to superior recovery of protein activity. The investigation concludes that the refolding of protein by reverse micelles is not only facilitated by the isolation of reverse micelles but also by the interaction due to the interface of the reverse micelle.     

Biochemical and structural characterisation of dehydroquinate synthase from the New Zealand kiwifruit Actinidia chinensis

One of the novel aspects of kiwifruit is the presence of a high level of quinic acid which contributes to the flavour of the fruit. Quinic acid metabolism intersects with the shikimate pathway, which is responsible for the de novo biosynthesis of primary and secondary aromatic metabolites. The gene encoding the enzyme which catalyses the second step of the shikimate pathway, dehydroquinate synthase (DHQS), from the New Zealand kiwifruit Actinidia chinensis was identified, cloned and expressed. A. chinensis DHQS was activated by divalent metal ions, and was found to require NAD⁺ for catalysis. The protein was crystallised and the structure was solved, revealing a homodimeric protein. Each monomer has a NAD⁺ binding site nestled between the distinct N- and C-terminal domains. In contrast to other microbial DHQSs, which show an open conformation in the absence of active site ligands, A. chinensis DHQS adopts a closed conformation. This is the first report of the structure of a DHQS from a plant source.     

Secondary structural changes of metmyoglobin and apomyoglobin in anionic and cationic surfactant solutions: Effect of the hydrophobic chain length of the surfactants on the structural changes

Secondary structural changes of metmyoglobin and apomyoglobin were examined in solutions of sodium alkylsulfates with hydrocarbon numbers of 8 and 12, and alkyltrimethylammonium bromides with hydrocarbon numbers of 10, 12, 14, and 16. The relative proportion ofa-helical structure was estimated by the curve-fitting method of circular dichroic spectrum. The helical proportions of metmyoglobin and apomyoglobin were 82 and 63%, respectively. The shorter the hydrocarbon chain the surfactant had, the higher the concentration necessary to disrupt the secondary structures of these proteins. However, the helical proportion had a tendency to decrease down to lower values in solutions of the cationic surfactants with short hydrophobic groups. On the other hand, thea-helical structure of apomyoglobin was disrupted in lower concentrations of each cationic surfactant than that of metmyoglobin, although the disruptions of the same structures in both the proteins occurred in the same concentration range of each anionic surfactant. It appeared likely that the removal of the heme group unstabilized the myoglobin conformation only in the cationic surfactant solutions.     

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.     

Optimization of the surfactant (AOT) concentration in a reverse micellar extraction process

Summary The surfactant concentration is an important parameter for optimizing protein extraction with microemulsions. Equilibrium and kinetic experiments show that the surfactant concentration can be reduced by a factor of 20 compared to published data. The mass-transfer coefficient is found to be only about 18% lower for small AOT concentrations.     

Kinetic and structural characterisation of Escherichia coli nitroreductase mutants showing improved efficacy for the prodrug substrate CB1954

Escherichia coli nitroreductase (NTR) is a flavoprotein that reduces a variety of quinone and nitroaromatic substrates. Among these substrates is the prodrug 5-[aziridin-1-yl]-2,4-dinitrobenzamide (CB1954) that is activated by NTR to form two products, one of which is highly cytotoxic. NTR in combination with CB1954 has entered clinical trials for virus-directed enzyme-prodrug therapy of cancer. Enhancing the catalytic efficiency of NTR for CB1954 is likely to improve the therapeutic potential of this system. We previously identified a number of mutants at six positions around the active site of NTR that showed enhanced sensitisation to CB1954 in an E. coli cell-killing assay. In this study we have purified improved mutants at each of these positions and determined their steady-state kinetic parameters for CB1954 and for the antibiotic nitrofurazone. We have also made a double mutant, combining two of the most beneficial single mutations. All the mutants show enhanced specificity constants for CB1954, and, apart from N71S, the enhancement is selective for CB1954 over nitrofurazone. One mutant, T41L, also shows an increase in selectivity for reducing the 4-nitro group of CB1954 rather than the 2-nitro group. We have determined the three-dimensional structures of selected mutants bound to the substrate analogue nicotinic acid, using X-ray crystallography. The N71S mutation affects interactions of the FMN cofactor, while mutations at T41 and F124 affect the interactions with nicotinic acid. The structure of double mutant N71S/F124K combines the effects of the two individual single mutations, but it gives a greater selective enhancement of activity with CB1954 over nitrofurazone than either of these, and the highest specificity constant for CB1954 of all the mutations studied.     

Anti-inflammatory and anti-viral actions of anionic pulmonary surfactant phospholipids

Pulmonary surfactant is a mixture of lipids and proteins, consisting of 90% phospholipid, and 10% protein by weight, found predominantly in pulmonary alveoli of vertebrate lungs. Two minor components of pulmonary surfactant phospholipids, phosphatidylglycerol (PG) and phosphatidylinositol (PI), are present within the alveoli at very high concentrations, and exert anti-inflammatory effects by regulating multiple Toll like receptors (TLR2/1, TLR4, and TLR2/6) by antagonizing cognate ligand-dependent activation. POPG also attenuates LPS-induced lung injury in vivo. In addition, these lipids bind directly to RSV and influenza A viruses (IAVs) and block interaction between host cells and virions, and thereby prevent viral replication in vitro. POPG and PI also inhibit RSV and IAV infection in vivo, in mice and ferrets. The lipids markedly inhibit SARS-CoV-2 infection in vitro. These findings suggest that both POPG and PI have strong potential to be applied as both prophylaxis and post-infection treatments for problematic respiratory viral infections.     

Identification and characterisation of structural maintenance of chromosome 1 (smc1) mutants of Coprinopsis cinerea

A Coprinopsis cinerea homokaryotic fruiting strain was mutagenised, identifying a mutant that exhibited a hyphal growth temperature sensitive defect and hyphal knot development defect at an early fruiting stage, even at the hyphal growth permissive temperature. Microscopic observation suggested that the mutant nuclei exhibited defects in the metaphase to anaphase transition at the restrictive temperature. The gene in which the mutation occurred was cloned, sequenced and determined to be homologous to smc1. Sequence analyses of the mutant revealed deletion of 28 base pairs in the 19th intron of the Cc.smc1 gene, resulting in complete failure of splicing of that intron and in insertion of 14 amino acids in the C-terminal region of the Cc.Smc1 protein. We isolated eight hyphal growth revertants and identified four intragenic suppressors. All were the result of amino acid substitutions in the C-terminal region. Three of the suppressors caused reversion of the arrest in an early fruiting stage. One of the suppressors exhibited cold sensitivity and failed to suppress the fruiting defect, suggesting that flexibility of a lobe in the C-terminal region is important for proper function of Cc.Smc1.     

Direct evidence for the presence of beta-hydroxyphenylalanine and beta-hydroxytyrosine in cutinase from Fusarium solani pisi

Extracellular cutinase induced by cutin hydrolysate in glucose-grown Fusarium solani f. pisi was isolated in electrophoretically homogeneous form. This enzyme was similar to cutinase I generated by cutin-grown cells in its catalytic properties such as pH optimum, substrate specificity, and inactivation by “active serine”-directed reagents. Its molecular weight was 26,300 and this enzyme had a much larger content of serine and threonine residues than that found in cutinase from the cutin-grown cells. The hydrolysate-induced enzyme was a glycoprotein containing 6% carbohydrates. Alkaline NaB³H₄ treatment of the protein generated labeled protein and labeled carbohydrates. Analyses of the hydrolysates of these labeled products showed that alanine, α-aminobutyrate, phenylalanine, and tyrosine in the protein were labeled strongly suggesting that serine, threonine, β-hydroxyphenylalanine, and β-hydroxytyrosine were involved in O-glycosidic linkages in this protein. The protein hydrolysate also contained labeled gulonic acid, suggesting that d-glucuronic acid was attached to the protein via a base stable linkage, presumably an amide linkage at the N-terminus. The labeled reduced carbohydrates were identified by ion-exchange, thin-layer, gas-liquid, and high-performance liquid chromatographic techniques as mannitol, arabitol, gulonic acid, and 2-aminosorbitol. Thus mannose, arabinose, glucuronic acid, and glucosamine (possibly N-acetylated) were attached O-glycosidically to the hydroxyamino acids. Induction of cutinase by cutin hydrolysate in the presence of tritiated phenylalanine gave labeled cutinase. Cleavage of the O-glycosidically attached carbohydrates by anhydrous HF, followed by enzymatic hydrolysis of the labeled protein, gave rise to labeled amino acids, which upon analysis with an amino acid analyzer revealed four radioactive components. Two of them were identified as phenylalanine and tyrosine, while the other two cochromatographed with authentic β-hydroxyphenylalanine and β-hydroxytyrosine not only by the amino acid analyzer but also upon thin-layer chromatography. These results constitute the first direct evidence for the presence of the novel β-hydroxyaromatic amino acids in a protein.     

Thermodynamic and structural investigation of the specific SDS binding of humicola insolens cutinase

The interaction of lipolytic enzymes with anionic surfactants is of great interest with respect to industrially produced detergents. Here, we report the interaction of cutinase from the thermophilic fungus Humicola insolens with the anionic surfactant SDS, and show the enzyme specifically binds a single SDS molecule under nondenaturing concentrations. Protein interaction with SDS was investigated by NMR, ITC and molecular dynamics simulations. The NMR resonances of the protein were assigned, with large stretches of the protein molecule not showing any detectable resonances. SDS is shown to specifically interact with the loops surrounding the catalytic triad with medium affinity (K_a ≈ 10⁵ M⁻¹). The mode of binding is closely similar to that seen previously for binding of amphiphilic molecules and substrate analogues to cutinases, and hence SDS acts as a substrate mimic. In addition, the structure of the enzyme has been solved by X-ray crystallography in its apo form and after cocrystallization with diethyl p-nitrophenyl phosphate (DNPP) leading to a complex with monoethylphosphate (MEP) esterified to the catalytically active serine. The enzyme has the same fold as reported for other cutinases but, unexpectedly, esterification of the active site serine is accompanied by the ethylation of the active site histidine which flips out from its usual position in the triad.