Release of antimicrobial peptides from electrospun nanofibres as a drug delivery system.

Electrospinning is a very versatile technique, which holds great potentials for several clinical applications. The ability to produce biocompatible nanofibres mimicking the extracellular matrix of the body in combination with both the large surface area and the adsorption/release properties of nanofibres makes drug loaded electrospun fibres very promising for both drug delivery and tissue engineering purposes. An interesting type of molecules to incorporate into electrospun nanofibres are antimicrobial peptides (AMPs) due to their characteristic mode of action both as antimicrobial- and as immunological agents. The focus of the presented work was on the release properties and the loading density of the synthetic AMP fluorescein labelled inverse-Crabrolin (iCR-fluor) incorporated into electrospun nanofibres of poly(epsilon-caprolactone) (PCL). The release properties were compared to the release properties of fluorescein and tetracycline hydrochloride. Furthermore, the antimicrobial effect of the different loading agents was evaluated both before and after release from the fibres, where only tetracycline hydrochloride was found to retain its activity. The loading density of fluorescein and iCR-fluor was investigated with deconvolution fluorescence microscopy. iCR-fluor followed a linear release profile with a significantly slower release kinetics than tetracycline hydrochloride and fluorescein. After the first 60 min, approximately 85% of both fluorescein and tetracycline were released, whereas only 40% of iCR-fluor was released. Furthermore, iCR-fluor did not show uniform distribution within the fibres and had an overall lower loading density than fluorescein.

The degradation of arabinoxylan-rich cell walls in digesta obtained from piglets fed wheat-based diets varies depending on digesta collection site, type of cereal, and source of exogenous xylanase.

The objective of the present study was to compare the ability of experimental and commercial xylanases to degrade, in vitro, the arabinoxylan (AX) fraction in digesta from 28-d-old piglets fed a wheat (Triticum aestivum)-based diet (49% wheat). Pigs were euthanized at 1, 2, 3, or 4 h after feeding; stomach and ileum contents were isolated and frozen and later used for the in vitro studies. Xylan solubilization provided information regarding the ability of the enzymes to degrade AX during the harsh in vivo conditions prevailing in the gastrointestinal tract. The hydrolytic capacity of a commercial xylanase was compared with that of an experimental xylanase using stomach digesta (pH 1.8) obtained at 4 h after feeding. Relative to the control, both enzymes increased (P < 0.001) xylan solubilization 3-fold. In the ileal digesta (1 h), xylan solubilization was increased by 36% (P < 0.001). Inclusion of arabinofuranosidases (Ara f) with xylanases increased xylan solubilization in stomach samples (P = 0. 007 and P = 0. 030) but not in ileal samples (P = 0.873 and P = 0.997). Our results illustrate clearly the importance of using different conditions and substrates when enzyme performance is studied in vitro as a prescreening tool for setting up in vivo trials.

An atomic force microscopy study of the interactions between indolicidin and supported planar bilayers.

Indolicidin, a tryptophane-rich antimicrobial peptide, was used to investigate the interactions with a zwitterionic phosphatidylcholine as a model membrane system. In situ atomic force microscopy in liquid medium and phosphatidylcholine supported planar bilayers enabled the study of the interactions between indolicidin and the lipid membrane in real time. It was evident that indolicidin induced a continuous shrinking and thinning of the supported planar bilayers. The effect of indolicidin was dependent upon the composition and physical properties of the membrane bilayer. The interaction of indolicidine with the membrane could be best and most pronounced seen and studied at the boundary of the gel-fluid-domains. Dye leakage experiments with phosphatidylcholine vesicles encapsulated with calcein revealed that indolicidin induced fluidisation of the lipid membrane leading to dye release. The present study indicates that the mode of action for indolicidin can be best described by a stepwise interaction of the peptide with the membrane. Formation of pores however can not be supported on the basis of our experiments.

Atomic force microscopy study of the interaction of Fusarium solani pisi cutinase with lipid surfaces.

We present an atomic force microscopy (AFM) study of a supported triacylglyceride multilayer phase and its interaction with lipolytic enzyme cutinase from Fusarium solani pisi. The multilayer triacylglyceride phase of coconut oil showed a rippled surface structure in the AFM images. Upon enzymatic degradation of the triacylglyceride phase, the ripple structure vanished rapidly. The apparent catalytic rate constants could be estimated based on the AFM image information. Interestingly, in one sample we observed what we interpret as a recurrent structural collapse of the cavity dug out by the protein. We interpret the cavities seen in the AFM images as molten surfaces or surface holes filled with liquidified phase containing product molecules, which appear transparent during the image recording.

Engineering the pH-optimum of a triglyceride lipase: from predictions based on electrostatic computations to experimental results.

The optimisation of enzymes for particular purposes or conditions remains an important target in virtually all protein engineering endeavours. Here, we present a successful strategy for altering the pH-optimum of the triglyceride lipase cutinase from Fusarium solani pisi. The computed electrostatic pH-dependent potentials in the active site environment are correlated with the experimentally observed enzymatic activities. At pH-optimum a distinct negative potential is present in all the lipases and esterases that we studied so far. This has prompted us to propose the "The Electrostatic Catapult Model" as a model for product release after cleavage of the ester bond. The origin of the negative potential is associated with the titration status of specific residues in the vicinity of the active site cleft. In the case of cutinase, the role of Glu44 was systematically investigated by mutations into Ala and Lys. Also, the neighbouring Thr45 was mutated into Proline, with the aim of shifting the spatial location of Glu44. All the charge mutants displayed altered titration behaviour of active site electrostatic potentials. Typically, the substitution of the residue Glu44 pushes the onset of the active site negative potential towards more alkaline conditions. We, therefore, predicted more alkaline pH optima, and this was indeed the experimentally observed. Finally, it was found that the pH-dependent computed Coulombic energy displayed a strong correlation with the observed melting temperatures of native cutinase.

How do lipases and esterases work: the electrostatic contribution.

This work explores the role of one of the factors explaining lipase/esterase activity: the contribution of electrostatic interactions to lipase/esterase activity. The electrostatic potential distribution on the molecular surface of an enzyme as a function of pH determines, to a large extent, the enzyme's pH activity profile. Other important factors include the presence and distribution of polar and hydrophobic residues in the active cleft. We have mapped the electrostatic potential distribution as a function of pH on the molecular surface of nine lipases/esterases for which the 3D structure is experimentally known. A comparison of these potential maps at different pH values with the corresponding pH-activity profile, pH optimum or pH range where the activity displayed by the enzyme is maximum, has revealed a considerable correlation. A negative potential in the active site appears correlated with maximum activity towards triglycerides, which has prompted us to propose a model for product release ('The electrostatic catapult model') after cleavage of an ester bond. At the same time as the bottom of the active site cleft becomes negatively charged, other nearby regions also titrate and become negatively charged when pH becomes more alkaline, for some of the studied lipases. If such lipases also show phospholipase activity (such as guinea pig lipase-related proteins 2 chimera) we raise the hypothesis that such other titratable regions after becoming negatively charged might stabilise the positive charge present in the polar head of phospholipids, such as phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. The distribution of polar, weak polar and non-polar residues on the molecular surface of each studied lipase, in particular the active site region, was compared for all the lipases studied. The combination of graphical visualisation of the electrostatic potential maps and the polarity maps combined with knowledge about the location of key residues on the protein surface allows us to envision atomic models for lipolytic activity.

What distinguishes an esterase from a lipase: a novel structural approach.

Esterases and lipases both hydrolyse ester bonds. Whereas the lipases display high activity towards the aggregated state of its substrate, the esterases typically show highest activity towards the soluble state of its substrate. We have compared the amino acid sequence, the 3D-structure as well as the pH-dependent electrostatic signature of selected members of the two families, for which 3D-structural information is publicly available. Lipases display a statistically significant enhanced occurrence of non-polar residues close to the surface, clustering around the active-site. Lid opening appears to strengthen this pattern further. As we have proposed earlier the active site of lipases displays negative potential in the pH-range associated with their maximum activity, typically at pH values above 8. The esterases show a very similar pattern, however, at pH values around 6 correlated with their usually lower pH-activity optimum.

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.

Protein engineering the surface of enzymes.

The protein surface is the interface through which a protein senses the external world. Its composition of charged, polar and hydrophobic residues is crucial for the stability and activity of the protein. The charge state of seven of the twenty naturally occurring amino acids is pH dependent. A total of 95% of all titratable residues are located on the surface of soluble proteins. In evolutionary related families of proteins such residues are particularly prone to substitutions, insertions and deletions. We present here an analysis of the residue composition of 4038 proteins, selected from 125 protein families with < 25% identity between core members of each family. Whereas only 16.8% of the residues were truly buried, 40.7% were > 30% exposed on the surface and the remainder were < 30% exposed. The individual residue types show distinct differences. The data presented provides an important new approach to protein engineering of protein surfaces. Guidelines for the optimization of solvent exposure for a given residue are given. The cutinase family of enzymes has been investigated. The stability of native cutinase has been studied as a function of pH, and has been compared with the cutinase activity towards tributyrin. Whereas the onset of enzymatic activity is linked with the deprotonation of the active site HIS188, destabilization of the 3D structure as determined by differential scanning calorimetry is coupled with the loss of activity at very basic pH values. A modeling investigation of the pH dependence of the electrostatic potentials reveals that the activity range is accompanied by the development of a highly significant negative potential in the active site cleft. The 3D structures of three mutants of the Fusarium solani pisi cutinase have been solved to high resolution using X-ray diffraction analysis. Preliminary X-ray data are presented.