Cross-Linked Enzyme Aggregates and Their Application in Enzymatic Pretreatment of Microalgae: Comparison Between CLEAs and Combi-CLEAs

Carrier-free immobilization is a key process to develop efficient biocatalysts able to catalyze the cell wall degradation in microalgae where the traditional solid supports cannot penetrate. Thus, the insolubilization of commercial Celluclast®, Alcalase®, and Viscozyme® enzymes by carrier-free immobilization and their application in microalgae pretreatment was investigated. In this study, different precipitants at different ratios (ethanol, acetone, and polyethylene glycol 4000) were tested in the first part of the method, to establish the precipitation conditions. The screening of the best precipitant is needed as it depends on the nature of the enzyme. The best results were studied in terms of immobilization yield, thermal stability, and residual activity and were analyzed using scanning electron microscopy. Moreover, a novel strategy was intended including the three enzymes (combi-CLEAs) to catalyze the enzymatic degradation of Nannochloropsis gaditana microalgal cell wall in one pot. The carrier-free immobilized derivatives were 10 times more stable compared to soluble enzymes under the same. At the best conditions showed its usefulness in the pretreatment of microalgae combined with ultrasounds, facilitating the cell disruption and lipid recovery. The results obtained suggested the powerful application of these robust biocatalysts with great catalytic properties on novel and sustainable biomass such as microalgae to achieve cost-effective and green process to extract valuable bioactive compounds.

The Amino Acids Motif -32GSSYN36- in the Catalytic Domain of E. coli Flavorubredoxin NO Reductase Is Essential for Its Activity

Flavodiiron proteins (FDPs) are a family of modular and soluble enzymes endowed with nitric oxide and/or oxygen reductase activities, producing N2O or H2O, respectively. The FDP from Escherichia coli, which, apart from the two core domains, possesses a rubredoxin-like domain at the C-terminus (therefore named flavorubredoxin (FlRd)), is a bona fide NO reductase, exhibiting O2 reducing activity that is approximately ten times lower than that for NO. Among the flavorubredoxins, there is a strictly conserved amino acids motif, -G[S,T]SYN-, close to the catalytic diiron center. To assess its role in FlRd’s activity, we designed several site-directed mutants, replacing the conserved residues with hydrophobic or anionic ones. The mutants, which maintained the general characteristics of the wild type enzyme, including cofactor content and integrity of the diiron center, revealed a decrease of their oxygen reductase activity, while the NO reductase activity—specifically, its physiological function—was almost completely abolished in some of the mutants. Molecular modeling of the mutant proteins pointed to subtle changes in the predicted structures that resulted in the reduction of the hydration of the regions around the conserved residues, as well as in the elimination of hydrogen bonds, which may affect proton transfer and/or product release.

Biosynthesis of a Novel Antibacterial Dipeptide, Using Proteases From South American Native Fruits, Useful as a Food Preservative

Antiacanthain and granulosain are the partially purified proteolytic extracts from the South American native fruits of Bromelia antiacantha (Bertol. ) and Solanum granuloso leprosum, respectively. The aim of this work was to compare the ability of both soluble and immobilized antiacanthain and granulosain f or the synthesis of Z-Tyr-Val-OH, a novel antibacterial dipeptide, in different reaction systems formed by almost anhydrous organic solvents (Xw: 1 × 10−5) and several percentages of immiscible organic solvents in 100 mM Tris(hydroxymethyl)aminomethane hydrochloride buffer pH 8.0. Soluble antiacanthain in half of the 24 different organic biphasic media showed higher catalytic potential than in 100 mM Tris(hydroxymethyl)aminomethane hydrolchloride buffer pH 8.0. Soluble granulosain showed lower catalytic potential in all liquid-liquid biphasic media than in the same buffer. However, 50% (v/v) ethyl ethanoate in 100 mM Tris(hydroxymethyl)aminomethane hydrolchloride buffer pH 8.0 allowed to express the highest catalytic potential of both soluble enzymes. In 50% v/v ethyl ethanoate, soluble antiacanthain and granulosain catalyzed the synthesis of Z-Tyr-Val-OH with 72 ± 0.15 and 60 ± 0.10% maximal peptide yields, respectively. Multi-point immobilization in glyoxyl-silica did not lead to better peptide yields than soluble enzymes, in that liquid-liquid biphasic medium under the same reaction conditions. Soluble and glyoxyl-silica immobilized antiacanthain in almost anhydrous ethyl ethanoate (Xw: 1 × 10−5) were able to retain 17.3 and 45% of the initial proteolytic activity of antiacanthain in 100 mM Tris hydrolchloride buffer pH 8.0, respectively, at 40°C under agitation (200 rpm). Soluble and glyoxyl-silica immobilized granulosain were inactivated under the same reaction conditions. Glyoxyl-silica immobilized antiacanthain showed to be a robust biocatalyst in almost anhydrous ethyl ethanoate (Xw: 1 × 10−5), eliciting the best peptide yield (75 ± 0.13%). The synthesis reaction of Z-Tyr-Val-OH could not proceed when soluble antiacanthain was used under the same conditions. Both peptidases only catalyzed the synthesis reaction under kinetic control, using activated acyl donor substrates. Finally, this work reports a novel broad-spectrum antibacterial peptide that significantly decreased (p ≤ 0.05) the specific growth rates of Gram positive and Gram negative microorganisms at very low concentrations (≥15 and 35 μg/ml, respectively); contributing with a new safe food preservative of applying for different food systems.

Trypsin-Like Proteases and Their Role in Muco-Obstructive Lung Diseases

Trypsin-like proteases (TLPs) belong to a family of serine enzymes with primary substrate specificities for the basic residues, lysine and arginine, in the P1 position. Whilst initially perceived as soluble enzymes that are extracellularly secreted, a number of novel TLPs that are anchored in the cell membrane have since been discovered. Muco-obstructive lung diseases (MucOLDs) are characterised by the accumulation of hyper-concentrated mucus in the small airways, leading to persistent inflammation, infection and dysregulated protease activity. Although neutrophilic serine proteases, particularly neutrophil elastase, have been implicated in the propagation of inflammation and local tissue destruction, it is likely that the serine TLPs also contribute to various disease-relevant processes given the roles that a number of these enzymes play in the activation of both the epithelial sodium channel (ENaC) and protease-activated receptor 2 (PAR2). More recently, significant attention has focused on the activation of viruses such as SARS-CoV-2 by host TLPs. The purpose of this review was to highlight key TLPs linked to the activation of ENaC and PAR2 and their association with airway dehydration and inflammatory signalling pathways, respectively. The role of TLPs in viral infectivity will also be discussed in the context of the inhibition of TLP activities and the potential of these proteases as therapeutic targets.

Reconstitution of GABA, Glycine and Glutamate Transporters

In contrast to water soluble enzymes which can be purified and studied while in solution, studies of solute carrier (transporter) proteins require both that the protein of interest is situated in a phospholipid membrane and that this membrane forms a closed compartment. An additional challenge to the study of transporter proteins has been that the transport depends on the transmembrane electrochemical gradients. Baruch I. Kanner understood this early on and first developed techniques for studying plasma membrane vesicles. This advanced the field in that the experimenter could control the electrochemical gradients. Kanner, however, did not stop there, but started to solubilize the membranes so that the transporter proteins were taken out of their natural environment. In order to study them, Kanner then had to find a way to reconstitute them (reinsert them into phospholipid membranes). The scope of the present review is both to describe the reconstitution method in full detail as that has never been done, and also to reveal the scientific impact that this method has had. Kanner’s later work is not reviewed here although that also deserves a review because it too has had a huge impact.

Eugenol-Functionalized Magnetite Nanoparticles Modulate Virulence and Persistence in Pseudomonas aeruginosa Clinical Strains

Efficient antibiotics to cure Pseudomonas aeruginosa persistent infections are currently insufficient and alternative options are needed. A promising lead is to design therapeutics able to modulate key phenotypes in microbial virulence and thus control the progression of the infectious process without selecting resistant mutants. In this study, we developed a nanostructured system based on Fe3O4 nanoparticles (NPs) and eugenol, a natural plant-compound which has been previously shown to interfere with microbial virulence when utilized in subinhibitory concentrations. The obtained functional NPs are crystalline, with a spherical shape and 10–15 nm in size. The subinhibitory concentrations (MIC 1/2) of the eugenol embedded magnetite NPs (Fe3O4@EUG) modulate key virulence phenotypes, such as attachment, biofilm formation, persister selection by ciprofloxacin, and the production of soluble enzymes. To our knowledge, this is the first report on the ability of functional magnetite NPs to modulate P. aeruginosa virulence and phenotypic resistance; our data highlights the potential of these bioactive nanostructures to be used as anti-pathogenic agents.

Explorations of the Nonheme High-Valent Iron-Oxo Landscape: Crystal Structure of a Synthetic High-valent Complex with an [FeIV2(mu-O)2] Diamond Core Relevant to the Chemistry of sMMOH

Methanotrophic bacteria utilize methane monooxygenase (MMO) to carry out the first step in metabolizing methane. The soluble enzymes employ a hydroxylase component (sMMOH) with a nonheme diiron active site that...

Using structural biology of methanogen enzymes to aid our understanding of methane formation

Methane is a potent greenhouse gas (28-fold more potent than carbon dioxide) and is a significant gas contributing to global climate change. Approximately a billion tons of methane are produced each year by methanogenic archaea in ruminants. These archaea possess a number of unusual traits such as isoprenoid-based lipids, unusual cell wall chemistry and a unique energy metabolism (methanogenesis) that requires six methanogen-specific cofactors. Many of the enzymes involved in these processes have no direct analogues in the host animal. To gain insights into the fundamental biology of rumen methanogens we have determined crystal structures of key enzymes with archaeal-specific traits. Over 600 enzymes were targeted for structure determination which produced approximately 200 purified soluble enzymes for crystallographic screening. More than 50 different enzymes have produced crystals and 30 structures have been solved for individual enzymes to date. The results have helped illuminate our understanding of methane formation at this critical juncture in the world’s history.

EnzymeMiner: automated mining of soluble enzymes with diverse structures, catalytic properties and stabilities

Millions of protein sequences are being discovered at an incredible pace, representing an inexhaustible source of biocatalysts. Despite genomic databases growing exponentially, classical biochemical characterization techniques are time-demanding, cost-ineffective and low-throughput. Therefore, computational methods are being developed to explore the unmapped sequence space efficiently. Selection of putative enzymes for biochemical characterization based on rational and robust analysis of all available sequences remains an unsolved problem. To address this challenge, we have developed EnzymeMiner—a web server for automated screening and annotation of diverse family members that enables selection of hits for wet-lab experiments. EnzymeMiner prioritizes sequences that are more likely to preserve the catalytic activity and are heterologously expressible in a soluble form in Escherichia coli. The solubility prediction employs the in-house SoluProt predictor developed using machine learning. EnzymeMiner reduces the time devoted to data gathering, multi-step analysis, sequence prioritization and selection from days to hours. The successful use case for the haloalkane dehalogenase family is described in a comprehensive tutorial available on the EnzymeMiner web page. EnzymeMiner is a universal tool applicable to any enzyme family that provides an interactive and easy-to-use web interface freely available at https://loschmidt.chemi.muni.cz/enzymeminer/.

The function of lysosomes and their role in Parkinson’s disease

Lysosomes are cellular organelles that are important for the degradation and recycling of various biomolecules. Specialized lysosomal membrane proteins, as well as soluble enzymes, are important for the efficient turn-over of lysosomal substrates. A deficiency in the degradative capacity of lysosomes leads to severe pathologies referred to as lysosomal storage disorders. There is increasing evidence for the importance of lysosomal function in neurodegenerative disorders, including Parkinson’s disease. One reason for this might be the vulnerability of neuronal cells. Since neurons do not undergo further cell division, non-degraded substrates accumulate in aging cells, causing a buildup of toxicity. Recent genomic screenings identified a number of lysosome-associated genes as potential risk factors for Parkinson’s disease, which are discussed in this review. Moreover, it is outlined how targeting lysosomal function might help in developing novel therapeutic strategies.