In this study cutinases from Thermobifida cellulosilytica DSM44535 (Thc_Cut1 and Thc_Cut2) and Thermobifida fusca DSM44342 (Thf42_Cut1) hydrolyzing poly(ethylene terephthalate) (PET) were successfully cloned and expressed in E.coli BL21-Gold(DE3). Their ability to hydrolyze PET was compared with other enzymes hydrolyzing natural polyesters, including the PHA depolymerase (ePhaZmcl) from Pseudomonas fluorescens and two cutinases from T. fusca KW3. The three isolated Thermobifida cutinases are very similar (only a maximum of 18 amino acid differences) but yet had different kinetic parameters on soluble substrates. Their k cat and K m values on pNP–acetate were in the ranges 2.4–211.9 s–1 and 127–200 μM while on pNP–butyrate they showed k cat and K m values between 5.3 and 195.1 s–1 and between 1483 and 2133 μM. Thc_Cut1 released highest amounts of MHET and terephthalic acid from PET and bis(benzoyloxyethyl) terephthalate (3PET) with the highest concomitant increase in PET hydrophilicity as indicated by water contact angle (WCA) decreases. FTIR-ATR analysis revealed an increase in the crystallinity index A 1340/A 1410 upon enzyme treatment and an increase of the amount of carboxylic and hydroxylic was measured using derivatization with 2-(bromomethyl)naphthalene. Modeling the covalently bound tetrahedral intermediate consisting of cutinase and 3PET indicated that the active site His-209 is in the proximity of the O of the substrate thus allowing hydrolysis. On the other hand, the models indicated that regions of Thc_Cut1 and Thc_Cut2 which differed in electrostatic and in hydrophobic surface properties were able to reach/interact with PET which may explain their different hydrolysis efficiencies.

ABSTRACT:The in vitro biodegradation of Bombyx mori silk fibroin was studied by incubating fibers and films with proteolytic enzymes (collagenase type F, ␣-chymotrypsin type I-S, protease type XXI), for times ranging from 1 to 17 days. The changes in sample weight and degree of polymerization of silk fibers exposed to proteolytic attack were negligible. However, tensile properties were significantly affected, as shown by the drop of strength and elongation as a function of the degradation time. Upon incubation with proteolytic enzymes, silk films exhibited a noticeable decrease of sample weight and degree of polymerization, the extent of which depended on the type of enzyme, on the enzyme-to-substrate ratio, and on the degradation time. Protease was more aggressive than ␣-chymotrypsin or collagenase. Film fragments resistant to enzymatic degradation were enriched in glycine and alanine. FT-IR measurements showed that the degree of crystallinity of biodegraded films increased. Soluble degradation products of silk films consisted of a range of peptides widely differing in size, deriving from the amorphous sequences of the silk fibroin chains. Biodegraded fibers showed an increase of surface roughness, while films displayed surface cracks and cavities with internal voids separated by fiber-like elements.

The degeneration of photoreceptors in the retina is one of the major causes of adult blindness in humans. Unfortunately, no effective clinical treatments exist for the majority of retinal degenerative disorders. Here we report on the fabrication and functional validation of a fully organic prosthesis for long-term in vivo subretinal implantation in the eye of Royal College of Surgeons rats, a widely recognized model of Retinitis pigmentosa. Electrophysiological and behavioral analyses reveal a prosthesis-dependent recovery of light-sensitivity and visual acuity that persists up to 6-10 months after surgery. The rescue of the visual function is accompanied by an increase in the basal metabolic activity of the primary visual cortex, as demonstrated by positron emission tomography imaging. Our results highlight the possibility of developing a new generation of fully organic, highly biocompatible and functionally autonomous photovoltaic prostheses for subretinal implants to treat degenerative blindness.

Wool and silk were dissolved and used for the preparation of blended films. Two systems are proposed: (1) blend films of silk fibroin and keratin aqueous solutions and (2) silk fibroin and keratin dissolved in formic acid. The FTIR spectra of pure films cast from aqueous solutions indicated that the keratin secondary structure mainly consists of alpha-helix and random coil conformations. The IR spectrum of pure SF is characteristic of films with prevalently amorphous structure (random coil conformation). Pure keratin film cast from formic acid shows an increase in the amount of beta-sheet and disordered keratin structures. The FTIR pattern of SF dissolved in formic acid is characteristic of films with prevalently beta-sheet conformations with beta-sheet crystallites embedded in an amorphous matrix. The thermal behavior of the blends confirmed the FTIR results. DSC curve of pure SF is typical of amorphous SF and the curve of pure keratin show the characteristic melting peak of alpha-helices for the aqueous system. These patterns are no longer observed in the films cast from formic acid due to the ability of formic acid to induce crystallization of SF and to increase the amount of beta-sheet structures on keratin. The nonlinear trend of the different parameters obtained from FTIR analysis and DSC curves of both SF/keratin systems indicate that when proteins are mixed they do not follow additives rules but are able to establish intermolecular interactions. Degradable polymeric biomaterials are preferred candidates for medical applications. It was investigated the degradation behavior of both SF/keratin systems by in vitro enzymatic incubation with trypsin. The SF/keratin films cast from water underwent a slower biological degradation than the films cast from formic acid. The weight loss obtained is a function of the amount of keratin in the blend. This study encourages the further investigation of the type of matrices presented here to be applied whether in scaffolds for tissue engineering or as controlled release drug delivery vehicles.

Silk fibroin membranes recently have been suggested as matrices for biomedical applications, such as guided tissue regeneration and burn wound dressings. The aim of this study was to evaluate the inflammatory potential of fibroin films and to compare the fibroin films with two model materials with completely different physico-chemical properties: poly(styrene) and poly(2-hydroxyethyl methacrylate). Fibroin bound lower levels of fibrinogen than did the two synthetic polymers while the same amounts of adsorbed human plasma complement fragment C3 and IgG were detected. Studies of the binding strength of C3 to fibroin, evaluated by a novel experimental procedure, indicated the occurrence of strong hydrophobic interactions at the interface. The activation of the mononuclear cells by fibroin, measured as interleukin 1beta production, was lower than the reference materials. Adhesion experiments showed the ability of the macrophages to adhere to fibroin by filopodia without a complete spreading of the cells. The results achieved in this study demonstrate that the interactions of fibroin with the humoral components of the inflammatory system were comparable with those of the two model surfaces while the degree of activation and adhesion of the immunocompetent cells appeared more limited.

Structural changes of native and regenerated silk fibroin membranes were induced by immersion in water‐methanol solutions and examined as a function of immersion time and methanol concentration. X‐ray diffractometry and infrared spectroscopy data showed that transition from random coil to β‐sheet structure occurred favorably when both native and regenerated silk fibroin membranes were immersed in water‐methanol solutions, regardless of the different immersion time. Only native silk membrane, treated for 2 min with pure methanol, maintained its original amorphous structure, as demonstrated by differential scanning calorimetric (DSC) curves. The degree of displacement, measured by thermomechanical analysis (TMA), was much greater for regenerated than for native silk fibroin membranes. SDS‐PAGE pattern showed that native silk fibroin has a molecular weight of 350, while the regenerated sample is formed by a large number of polypeptides in the range of 200‐50 KD. The amount of acidic and basic amino acids decreased slightly in regenerated silk fibroin. Physical properties of silk membranes treated with water‐methanol solutions are discussed in terms of membrane structure, treatment conditions, and chemical structure of starting material. © 1994 John Wiley & Sons, Inc.

This study focuses on the conformational characterization of differently processedBombyx mori silk fibroin samples by Raman spectroscopy. The Raman spectra of silk fibroin film and liquid silk are discussed in comparison with those of the crystalline fractions of Bombyx mori silk fibroin (Cp, chymotryptic precipitate) with Silk I (Silk I-Cp) and Silk II (Silk II-Cp) structures. The complete 1800-200 cm −1 Raman spectrum of Silk I-Cp is reported for the first time. The amide I and amide III modes were found to be scarcely suitable for the spectroscopic characterization of silk fibroin in the Silk I form in the presence of a random coil conformation. Raman marker bands for the Silk I form were identified in other spectral ranges at about 1415, 950, 930, 865, 260 and 230 cm −1 . On the basis of the above findings, the comparison of the Raman spectra of film, liquid silk and Silk I-Cp in the range 1000-800 cm −1 clearly indicates that in addition to random coil, both film and liquid silk contain local domains of Silk I structure; their amount is higher in liquid silk, as indicated by the relative intensity of the bands at about 950, 930 and 865 cm −1 and by the I 1415 /I 1455 intensity ratio.The assignments of the bands at about 1275 and 1107 cm −1 are also discussed. These bands were previously assigned to the presence of a-helical conformation in Bombyx mori silk but, from the results reported, they should rather be attributed to the Silk I form.

This study was focused on the conformational characterization of Bombyx mori silk Ðbroin in Ðlm, Ðber and powder form by means of Fourier transform Raman spectroscopy. Native and regenerated silk Ðbroin Ðlms prepared by casting dilute silk Ðbroin solutions (AE1% , w/v) display characteristic conformationally sensitive bands at 1660 cm-1 (amide I), in the range 1276-1244 cm-1 (a complex amide III region with multiple detectable maxima) and at 1107 and 938 cm-1. This spectral pattern can be related to a prevalently random coil conformation, with traces of a-helix. Liquid silk, prepared by casting the silk gland content (Ðbroin concentration 20-25% , w/v), shows almost the same wavenumbers in the amide I and III ranges, while di †erences appear below 1000 cm-1, where three bands at 952, 930 and 867 cm-1 increase in intensity. The spectral di †erences between Ðlms and liquid silk are discussed with a view to identifying possible markers for silk I structure, a crystalline modiÐ-cation of silk Ðbroin. The treatment of both native and regenerated Ðlms with 50% (v/v) methanol solution induces the conformational transition to a b-sheet structure, as demonstrated by the shift of amide I to 1665 cm-1 and the appearance of new maxima at 1262 and 1236 cm-1 (amide III) and at 1084 cm-1. When liquid silk is cast at above 50 ÄC, the prevailing conformation taken by silk Ðbroin is b-sheet, whatever the rate of drying. By comparing the Raman spectra of silk Ðbroin Ðber and powder, both having a b-sheet structure, a di †erence in the tyrosine doublet bands and in the amide I band can be observed. The value of the intensity ratio increases in I 853 /I 830 (R tyr ) the powder while amide I shifts to lower wavenumbers, suggesting that the hydrogen bonds involving the tyrosil residues are weaker in the powder than in the Ðber.