Madalena Martins

Engineered Thermobifida fusca cutinase with increased activity on polyester substrates

A bacterial cutinase from Thermobifida fusca, named Tfu_0883, was genetically modified by site-directed mutagenesis to enhance its activity on poly(ethylene terephthalate) (PET). The new mutations tailored the catalytic site for PET, increasing the affinity of cutinase to this hydrophobic substrate and the ability to hydrolyze it. The mutation I218A was designed to create space and the double mutation Q132A/T101A was designed both to create space and to increase hydrophobicity. The activity of the double mutant on the soluble substrate p-nitrophenyl butyrate increased two-fold compared to wild-type cutinase, while on PET both single and double mutants exhibited considerably higher hydrolysis efficiency. The replacement of specific amino acids at the active site was an effective approach for the improvement of the Tfu_0883 cutinase capacity to hydrolyze polyester surfaces. Thus, this study provides valuable insight on how the function and stability of enzymes can be improved by molecular engineering for their application in synthetic fiber biotransformation.

Sonochemical and hydrodynamic cavitation reactors for laccase/hydrogen peroxide cotton bleaching ☆

The main goal of this work is to develop a novel and environmental-friendly technology for cotton bleaching with reduced processing costs. This work exploits a combined laccase-hydrogen peroxide process assisted by ultrasound. For this purpose, specific reactors were studied, namely ultrasonic power generator type K8 (850 kHz) and ultrasonic bath equipment Ultrasonic cleaner USC600TH (45 kHz). The optimal operating conditions for bleaching were chosen considering the highest levels of hydroxyl radical production and the lowest energy input. The capacity to produce hydroxyl radicals by hydrodynamic cavitation was also assessed in two homogenizers, EmulsiFlex®-C3 and APV-2000. Laccase nanoemulsions were produced by high pressure homogenization using BSA (bovine serum albumin) as emulsifier. The bleaching efficiency of these formulations was tested and the results showed higher whiteness values when compared to free laccase. The combination of laccase-hydrogen peroxide process with ultrasound energy produced higher whiteness levels than those obtained by conventional methods. The amount of hydrogen peroxide was reduced 50% as well as the energy consumption in terms of temperature (reduction of 40 °C) and operating time (reduction of 90 min).

In vitro and computational studies of transdermal perfusion of nanoformulations containing a large molecular weight protein

Transdermal perfusion of a large protein is reported for the first time, using a nanoemulsion of bovine serum albumin (66kDa) of 160nm prepared by a solid-in-oil (S/O) process. Molecular dynamics simulations confirmed skin permeation by these formulations, with integration of the protein into the lipid bilayers. These results demonstrate the real possibility of delivering large proteins transdermally for a range of medical and cosmetic applications.

Practical insights on enzyme stabilization

Abstract Enzymes are efficient catalysts designed by nature to work in physiological environments of living systems. The best operational conditions to access and convert substrates at the industrial level are different from nature and normally extreme. Strategies to isolate enzymes from extremophiles can redefine new operational conditions, however not always solving all industrial requirements. The stability of enzymes is therefore a key issue on the implementation of the catalysts in industrial processes which require the use of extreme environments that can undergo enzyme instability. Strategies for enzyme stabilization have been exhaustively reviewed, however they lack a practical approach. This review intends to compile and describe the most used approaches for enzyme stabilization highlighting case studies in a practical point of view.

Design of novel BSA/hyaluronic acid nanodispersions for transdermal pharma purposes.

A novel transdermal hyaluronic acid (HA) conjugated with bovine serum albumin (BSA) was developed in the form of solid-in-oil (S/O) nanodispersion (129.7 nm mean diameter). Ex vivo skin penetration analysis by fluorescence and confocal observation of histological skin sections revealed the ability of BSA/HA nanodispersions to cross the stratum corneum and penetrate into the dermis. Furthermore, no significant toxicity was found in fibroblast and keratinocyte cells in vitro. These results proved the potential of the developed nanodispersion for transdermal delivery of hyaluronic acid constituting a high value to biopharmaceutical and cosmetics industries.

PEGylation greatly enhances laccase polymerase activity

Laccase catalyzes the oxidation and polymerization of phenolic compounds in the presence of oxygen. Herein, we report for the first time that a previous PEGylation of laccase enhances the polymerase activity 3‐fold compared with the reaction of the native enzyme, as confirmed by UV/Vis spectroscopy. The polymerization of catechol increased only 1.5‐fold if poly(ethylene glycol) (PEG) was added to the medium reaction. Molecular‐dynamics simulations suggest the formation of a miscible complex of polycatechol and PEG, which is responsible to push the reaction forward. In a negative control experiment set, all catalysts were entrapped inside polyacrylamide gels and here the native laccase showed a relatively higher activity. These results suggest that the mobility of PEG is a key feature for the enhancement of the reaction.

Ultrasound‐assisted swelling of bacterial cellulose

Bacterial cellulose (BC) was obtained by static cultivation using commercial BC gel from scoby. BC membranes (oven dried and freeze‐dried) were swelled with 8% NaOH, in the absence and in the presence of ultrasound (US), for 30, 60, and 90 min. The influence of swelling conditions on both physico‐chemical properties and molecules entrapment was evaluated. Considering the highest levels of entrapment, an optimum swelling procedure was established: 8% NaOH for 30 min at room temperature in the presence of US. Native and PEGylated laccase from Myceliophthora thermophila was immobilized on BC membranes and a different catalytic behaviour was observed after immobilization. Native laccase presented activity values similar to published reports (5–7 U/gBC) after immobilization whereas PEGylated enzymes showed much lower activity (1–2 U/gBC). BC swelled membranes are presented herein as a potential support for the preparation of immobilized enzymes for industrial applications, like phenolics polymerization.

Protein Formulations for Emulsions and Solid-in-Oil Dispersions.

Needs from medical and cosmetic areas have led to the design of novel nanosized emulsions and solid-in-oil dispersions of proteins. Here, we describe the production of those emulsions and dispersions using high-energy methodologies such as high-pressure homogenization or ultrasound. Recent work has resulted in new mechanistic insights related to the formation of protein emulsions and dispersions. The production method and composition of these formulations can determine major parameters such as size, stability, and functionality, and therefore their final application. Aqueous nanoemulsions of proteins can be used for drug delivery, while solid-in-oil dispersions are often used in transdermal applications.

Molecular recognition of esterase plays a major role on the removal of fatty soils during detergency.

In this work it is describe for the first time, the use of an esterase with null activity (Tfu_0883 bacterial cutinase from Thermobifida fusca) on the removal of fat from the surface of a cotton substrate. Similar levels of fat removal were found for both null and wild-type proteins, despite that only wild type protein yielded fatty acids. Our results show that molecular recognition of esterase plays a major role on the removal of fatty soils, allowing important guidelines for the design of detergent enzymes. Furthermore, the advantage of using null esterase enzymes lies in the avoidance of the rancid smell of short chained fatty acids, typical after esterase treatment.

Effect of a peptide in cosmetic formulations for hair volume control

The capacity of hair to absorb water causes changes in its physical and cosmetic properties under different environmental conditions. Hence, the control of hair volume in variable relative humidity settings is an important topic in cosmetics. The behaviour of two types of hair, Caucasian and Asian, was studied regarding their volume change in different relative humidity conditions. The ability of a peptide as a hair volume treatment was evaluated in two climate control formulations.