Greta Faccio

Crosslinking Food Proteins for Improved Functionality

Different possibilities for protein crosslinking are examined in this review, with special emphasis on enzymatic crosslinking and its impact on food structure. Among potential enzymes for protein crosslinking are transglutaminase (TG) and various oxidative enzymes. Crosslinking enzymes can be applied in cereal, dairy, meat, and fish processing to improve the texture of the product. Most of the current commercial applications are based on TG. The reaction mechanisms of the crosslinking enzymes differ, which in turn results in different technological properties.

Enzyme-catalyzed protein crosslinking

The process of protein crosslinking comprises the chemical, enzymatic, or chemoenzymatic formation of new covalent bonds between polypeptides. This allows (1) the site-directed coupling of proteins with distinct properties and (2) the de novo assembly of polymeric protein networks. Transferases, hydrolases, and oxidoreductases can be employed as catalysts for the synthesis of crosslinked proteins, thereby complementing chemical crosslinking strategies. Here, we review enzymatic approaches that are used for protein crosslinking at the industrial level or have shown promising potential in investigations on the lab-scale. We illustrate the underlying mechanisms of crosslink formation and point out the roles of the enzymes in their natural environments. Additionally, we discuss advantages and drawbacks of the enzyme-based crosslinking strategies and their potential for different applications.

TEMPO-Oxidized Nanofibrillated Cellulose as a High Density Carrier for Bioactive Molecules

Controlled and efficient immobilization of specific biomolecules is a key technology to introduce new, favorable functions to materials suitable for biomedical applications. Here, we describe an innovative and efficient, two-step methodology for the stable immobilization of various biomolecules, including small peptides and enzymes onto TEMPO oxidized nanofibrillated cellulose (TO-NFC). The introduction of carboxylate groups to NFC by TEMPO oxidation provided a high surface density of negative charges able to drive the adsorption of biomolecules and take part in covalent cross-linking reactions with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDAC) and glutaraldehyde (Ga) chemistry. Up to 0.27 μmol of different biomolecules per mg of TO-NFC could be reversibly immobilized by electrostatic interaction. An additional chemical cross-linking step prevented desorption of more than 80% of these molecules. Using the cysteine-protease papain as model, a highly active papain-TO-NFC conjugate was achieved. Once papain was immobilized, 40% of the initial enzymatic activity was retained, with an increase in kcat from 213 to >700 s(-1) for the covalently immobilized enzymes. The methodology presented in this work expands the range of application for TO-NFC in the biomedical field by enabling well-defined hybrid biomaterials with a high density of functionalization.

Tyrosinase-catalyzed site-specific immobilization of engineered C-phycocyanin to surface

Enzymatic crosslinking of proteins is often limited by the steric availability of the target residues, as of tyrosyl side chains in the case of tyrosinase. Carrying an N-terminal peptide-tag containing two tyrosine residues, the fluorescent protein C-phycocyanin HisCPC from Synechocystis sp. PCC6803 was crosslinked to fluorescent high-molecular weight forms with tyrosinase. Crosslinking with tyrosinase in the presence of L-tyrosine produced non fluorescent high-molecular weight products. Incubated in the presence of tyrosinase, HisCPC could also be immobilized to amino-modified polystyrene beads thus conferring a blue fluorescence. Crosslinking and immobilization were site-specific as both processes required the presence of the N-terminal peptide in HisCPC.

Light Harvesting Proteins for Solar Fuel Generation in Bioengineered Photoelectrochemical Cells

The sun is the primary energy source of our planet and potentially can supply all societies with more than just their basic energy needs. Demand of electric energy can be satisfied with photovoltaics, however the global demand for fuels is even higher. The direct way to produce the solar fuel hydrogen is by water splitting in photoelectrochemical (PEC) cells, an artificial mimic of photosynthesis. There is currently strong resurging interest for solar fuels produced by PEC cells, but some fundamental technological problems need to be solved to make PEC water splitting an economic, competitive alternative. One of the problems is to provide a low cost, high performing water oxidizing and oxygen evolving photoanode in an environmentally benign setting. Hematite, α-Fe2O3, satisfies many requirements for a good PEC photoanode, but its efficiency is insufficient in its pristine form. A promising strategy for enhancing photocurrent density takes advantage of photosynthetic proteins. In this paper we give an overview of how electrode surfaces in general and hematite photoanodes in particular can be functionalized with light harvesting proteins. Specifically, we demonstrate how low-cost biomaterials such as cyanobacterial phycocyanin and enzymatically produced melanin increase the overall performance of virtually no-cost metal oxide photoanodes in a PEC system. The implementation of biomaterials changes the overall nature of the photoanode assembly in a way that aggressive alkaline electrolytes such as concentrated KOH are not required anymore. Rather, a more environmentally benign and pH neutral electrolyte can be used.

Sulfhydryl oxidases: sources, properties, production and applications

The formation of disulfide bonds in proteins and small molecules can greatly affect their functionality. Sulfhydryl oxidases (SOXs) are enzymes capable of oxidising the free sulfhydryl groups in proteins and thiol-containing small molecules by using molecular oxygen as an electron acceptor. SOXs have been isolated from the intracellular compartments of many organisms, but also secreted SOXs are known. These latter enzymes are generally active on small compounds and their physiological role is unknown, whereas the intracellular enzymes prefer proteins as substrates and are involved in protein folding. An increasing number of scientific publications and patent applications on SOXs have been published in recent years. The present mini-review provides an up-to-date summary of SOXs from various families, their production and their actual or suggested applications. The sequence features and domain organisation of the characterised SOXs are reviewed, and special attention is paid to the physicochemical features of the enzymes. A review of patents and patent applications regarding this class of enzymes is also provided.

Simultaneous detection of pH value and glucose concentrations for wound monitoring applications

Aging population and longer life expectancy are the main reasons for an increasing number of patients with wound problems. Although the interest in wound care increases continuously, wound management still remains a challenge mainly due to the higher occurrence of chronic wounds, which require intensive care and constant monitoring. Here, we demonstrate a fluorescent sensing system to monitor the wound status and to distinguish between an autonomously healing and a chronic wound at an early stage. The system allows monitoring two of the most relevant fluctuating wound parameters during the healing process which are pH and glucose concentration. A fluorescent pH indicator dye, carboxynaphthofluorescein, and a metabolite-sensing enzymatic system, based on glucose oxidase and horseradish peroxidase, were immobilized on a biocompatible polysaccharide matrix to develop a functional hydrogel coating for wound monitoring. The changes in metabolite and enzyme concentration in artificial wound extract were converted into a fluorescent signal.

Experimental and bioinformatic investigation of the proteolytic degradation of the C-terminal domain of a fungal tyrosinase

Proteolytic processing is a key step in the production of polyphenol oxidases such as tyrosinases, converting the inactive proenzyme to an active form. In general, the fungal tyrosinase gene codes for a ~60 kDa protein that is, however, isolated as an active enzyme of ~40 kDa, lacking the C-terminal domain. Using the secreted tyrosinase 2 from Trichoderma reesei as a model protein, we performed a mutagenesis study of the residues in proximity of the experimentally determined cleavage site which are possibly involved in the proteolytic process. However, the mutant forms of tyrosinase 2 were not secreted in a full-length form retaining the C-terminal domain, but they were processed to give a ~45 kDa active form. Aiming at explaining this phenomenon, we analysed in silico the properties of the C-terminal domain of tyrosinase 2, of 23 previously retrieved homologous tyrosinase sequences from fungi (C. Gasparetti, G. Faccio, M. Arvas, J. Buchert, M. Saloheimo, K. Kruus, Appl. Microbiol. Biotechnol. 86 (2010) 213-226) and of nine well-characterised polyphenol oxidases. Based on the results of our study, we exclude the key role of specific amino acids at the cleavage site in the proteolytic process and report an overall higher sensitivity to proteolysis of the linker region and of the whole C-terminal domain of fungal tyrosinases.

Secreted fungal sulfhydryl oxidases: sequence analysis and characterisation of a representative flavin-dependent enzyme from Aspergillus oryzae.

BackgroundSulfhydryl oxidases are flavin-dependent enzymes that catalyse the formation of de novo disulfide bonds from free thiol groups, with the reduction of molecular oxygen to hydrogen peroxide. Sulfhydryl oxidases have been investigated in the food industry to remove the burnt flavour of ultraheat-treated milk and are currently studied as potential crosslinking enzymes, aiming at strengthening wheat dough and improving the overall bread quality.ResultsIn the present study, potential sulfhydryl oxidases were identified in the publicly available fungal genome sequences and their sequence characteristics were studied. A representative sulfhydryl oxidase from Aspergillus oryzae, AoSOX1, was expressed in the fungus Trichoderma reesei. AoSOX1 was produced in relatively good yields and was purified and biochemically characterised. The enzyme catalysed the oxidation of thiol-containing compounds like glutathione, D/L-cysteine, beta-mercaptoethanol and DTT. The enzyme had a melting temperature of 57°C, a pH optimum of 7.5 and its enzymatic activity was completely inhibited in the presence of 1 mM ZnSO4.ConclusionsEighteen potentially secreted sulfhydryl oxidases were detected in the publicly available fungal genomes analysed and a novel proline-tryptophan dipeptide in the characteristic motif CXXC, where X is any amino acid, was found. A representative protein, AoSOX1 from A. oryzae, was produced in T. reesei in an active form and had the characteristics of sulfhydryl oxidases. Further testing of the activity on thiol groups within larger peptides and on protein level will be needed to assess the application potential of this enzyme.

Enzymatic multi-functionalization of microparticles under aqueous neutral conditions

The immobilization of proteins and enzymes on microparticles enables innovative biofunctionalization and facilitates the recycling of, for example, catalysts in a biocatalytic process. When enzymes are used for the immobilization of proteins to material surfaces, site-specificity can be achieved, and the immobilization reaction can proceed under mild reaction conditions in an aqueous environment. By selectively oxidizing the exposed tyrosyl side chains of protein to reactive o-quinones using molecular oxygen as electron acceptor, tyrosinase promotes the formation of new covalent bonds via 1,4-addition of nucleophilic moieties to the o-quinones. The introduction of tyrosinase-susceptible tyrosine residues (Y-tag) to a protein by genetic engineering can thus enable site-specific crosslinking and site-specific protein immobilization. In this study, several variants of the fluorescent protein GFPuv were produced in order to investigate the tyrosinase-mediated crosslinking and immobilization reaction. The Y-tag was a target for rapid protein–protein crosslinking by tyrosinase catalysis. Moreover, low concentrations of tyrosinase were sufficient to obtain detectable fluorescent microparticles using the Y-tagged GFPuv variants, e.g. 0.4 μM (50 μg ml−1) protein concentrations. Eventually, we showed that this enzyme-based technology allows for the multiple functionalization of microparticles using different fluorescent proteins.