From the problem to the solution: Chitosan valorization cycle.

The problem of fisheries waste has increased in recent years and has become a global problem influenced by various biological, technical, operational and socioeconomic factors. In this context, the use of these residues as raw materials is a proven approach not only to reduce the crisis of unprecedented magnitude facing the oceans, but also to improve the management of marine resources and increase the competitiveness of the fisheries sector. However, the implementation of valorization strategies at the industrial level is being excessively slow, despite this great potential. Chitosan, a biopolymer extracted from shellfish waste, is a clear example of this because although countless chitosan-based products have been described for a wide variety of applications, commercial products are still limited. To address this drawback, it is essential to consolidate a "bluer" chitosan valorization cycle towards sustainability and circular economy. In this perspective we wanted to focus on the cycle of valorization of chitin, which allows to transform a waste product (chitin) into a material suitable for the development of useful products to solve the source of its origin as a waste product and pollutant; chitosan-based membranes for wastewater remediation.

Design of a biocatalytic cascade for the enzymatic sulfation of unsulfated chondroitin with in situ generation of PAPS.

Sulfation of molecules in living organisms is a process that plays a key role in their functionality. In mammals, the sulfation of polysaccharides (glycosaminoglycans) that form the proteoglycans present in the extracellular matrix is particularly important. These polysaccharides, through their degree and sulfation pattern, are involved in a variety of biological events as signal modulators in communication processes between the cell and its environment. Because of this great biological importance, there is a growing interest in the development of efficient and sustainable sulfation processes, such as those based on the use of sulfotransferase enzymes. These enzymes have the disadvantage of being 3'-phosphoadenosine 5'-phosphosulfate (PAPS) dependent, which is expensive and difficult to obtain. In the present study, a modular multienzyme system was developed to allow the in situ synthesis of PAPS and its coupling to a chondroitin sulfation system. For this purpose, the bifunctional enzyme PAPS synthase 1 (PAPSS1) from Homo sapiens, which contains the ATP sulfurylase and APS kinase activities in a single protein, and the enzyme chondroitin 4-O-sulfotransferase (C4ST-1) from Rattus norvegicus were overexpressed in E. coli. The product formed after coupling of the PAPS generation system and the chondroitin sulfation module was analyzed by NMR.

Fast and Sustained Axonal Growth by BDNF Released from Chitosan Microspheres.

Brain-derived neurotrophic factor (BDNF) regulates dendritic branching and dendritic spine morphology, as well as synaptic plasticity and long-term potentiation. Consequently, BDNF deficiency has been associated with some neurological disorders such as Alzheimer's, Parkinson's or Huntington's diseases. In contrast, elevated BDNF levels correlate with recovery after traumatic central nervous system (CNS) injuries. The utility of BDNF as a therapeutic agent is limited by its short half-life in a pathological microenvironment and its low efficacy caused by unwanted consumption of non-neuronal cells or inappropriate dosing. Here, we tested the activity of chitosan microsphere-encapsulated BDNF to prevent clearance and prolong the efficacy of this neurotrophin. Neuritic growth activity of BDNF release from chitosan microspheres was observed in the PC12 rat pheochromocytoma cell line, which is dependent on neurotrophins to differentiate via the neurotrophin receptor (NTR). We obtained a rapid and sustained increase in neuritic out-growth of cells treated with BDNF-loaded chitosan microspheres over control cells (p < 0.001). The average of neuritic out-growth velocity was three times higher in the BDNF-loaded chitosan microspheres than in the free BDNF. We conclude that the slow release of BDNF from chitosan microspheres enhances signaling through NTR and promotes axonal growth in neurons, which could constitute an important therapeutic agent in neurodegenerative diseases and CNS lesions.

Design and biocatalytic applications of genetically fused multifunctional enzymes.

Fusion proteins, understood as those created by joining two or more genes that originally encoded independent proteins, have numerous applications in biotechnology, from analytical methods to metabolic engineering. The use of fusion enzymes in biocatalysis may be even more interesting due to the physical connection of enzymes catalyzing successive reactions into covalently linked complexes. The proximity of the active sites of two enzymes in multi-enzyme complexes can make a significant contribution to the catalytic efficiency of the reaction. However, the physical proximity of the active sites does not guarantee this result. Other aspects, such as the nature and length of the linker used for the fusion or the order in which the enzymes are fused, must be considered and optimized to achieve the expected increase in catalytic efficiency. In this review, we will relate the new advances in the design, creation, and use of fused enzymes with those achieved in biocatalysis over the past 20 years. Thus, we will discuss some examples of genetically fused enzymes and their application in carbon­carbon bond formation and oxidative reactions, generation of chiral amines, synthesis of carbohydrates, biodegradation of plant biomass and plastics, and in the preparation of other high-value products.

Chitosan sulfate-lysozyme hybrid hydrogels as platforms with fine-tuned degradability and sustained inherent antibiotic and antioxidant activities.

The control of the properties and biological activities of chitosan-lysozyme hybrid hydrogels to exploit their interesting biomedical applications depends largely on the chitosan acetylation pattern, a difficult parameter to control. Herein, we have prepared sulfated chitosan-lysozyme hydrogels as versatile platforms with fine-tuned degradability and persistent bactericidal and antioxidant properties. The use of chitosan sulfates instead of chitosan has the advantage that the rate and mechanisms of lysozyme release, as well as antibacterial and antioxidant activities, depend on the sulfation profile, a structural parameter that is easily controlled by simple chemical modifications. Thus, while 6-O-sulfated chitosan hydrogels allow the release of loaded lysozyme in a short time (60% in 24 h), due to a high rate of degradation that allows rapid antibiotic and antioxidant activities, in 3-O-sulfated systems there is a slow release of lysozyme (80% in 21 days), resulting in long-lasting antibiotic and antioxidant activities.

Preparation and Characterization of Aminoglycoside-Loaded Chitosan/Tripolyphosphate/Alginate Microspheres against E. coli.

Although aminoglycosides are one of the common classes of antibiotics that have been widely used for treating infections caused by pathogenic bacteria, the evolution of bacterial resistance mechanisms and their inherent toxicity have diminished their applicability. Biocompatible carrier systems can help sustain and control the delivery of antibacterial compounds while reducing the chances of antibacterial resistance or accumulation in unwanted tissues. In this study, novel chitosan gel beads were synthesized by a double ionic co-crosslinking mechanism. Tripolyphosphate and alginate, a polysaccharide obtained from marine brown algae, were employed as ionic cross-linkers to prepare the chitosan-based networks of gel beads. The in vitro release of streptomycin and kanamycin A was bimodal; an initial burst release was observed followed by a diffusion mediated sustained release, based on a Fickian diffusion mechanism. Finally, in terms of antibacterial properties, the particles resulted in growth inhibition of Gram-negative (E. coli) bacteria.

Heparanized chitosans: towards the third generation of chitinous biomaterials.

The functionalization of chitosans is an emerging research area in the design of solutions for a wide range of biomedical applications. In particular, the modification of chitosans to incorporate sulfate groups has generated great interest since they show structural similarity to heparin and heparan sulfates. Most of the biomedical applications of heparan sulfates are derived from their ability to bind different growth factors and other proteins, as through these interactions they can modulate the cellular response. This review aims to summarize the most recent advances in the synthesis, and structural and physicochemical characterization of heparanized chitosan, a remarkably interesting family of polysaccharides that have demonstrated the ability to mimic heparan sulfates as ligands for different proteins, thereby exerting their biological activity by mimicking the function of these glycosaminoglycans.

Deciphering Structural Determinants in Chondroitin Sulfate Binding to FGF-2: Paving the Way to Enhanced Predictability of their Biological Functions.

Controlling chondroitin sulfates (CSs) biological functions to exploit their interesting potential biomedical applications requires a comprehensive understanding of how the specific sulfate distribution along the polysaccharide backbone can impact in their biological activities, a still challenging issue. To this aim, herein, we have applied an "holistic approach" recently developed by us to look globally how a specific sulfate distribution within CS disaccharide epitopes can direct the binding of these polysaccharides to growth factors. To do this, we have analyzed several polysaccharides of marine origin and semi-synthetic polysaccharides, the latter to isolate the structure-activity relationships of their rare, and even unnatural, sulfated disaccharide epitopes. SPR studies revealed that all the tested polysaccharides bind to FGF-2 (with exception of CS-8, CS-12 and CS-13) according to a model in which the CSs first form a weak complex with the protein, which is followed by maturation to tight binding with k D ranging affinities from ~ 1.31 µM to 130 µM for the first step and from ~ 3.88 µM to 1.8 nM for the second one. These binding capacities are, interestingly, related with the surface charge of the 3D-structure that is modulated by the particular sulfate distribution within the disaccharide repeating-units.

Unraveling the Structural Landscape of Chitosan-Based Heparan Sulfate Mimics Binding to Growth Factors: Deciphering Structural Determinants for Optimal Activity.

Chitosan sulfates have demonstrated the ability to mimic heparan sulfate (HS) function. In this context, it is crucial to understand how the specific structural properties of HS domains determine their functionalities and biological activities. In this study, several HS-mimicking chitosans have been prepared to mimic the structure of HS domains that have proved to be functionally significant in cell processes. The results presented herein are in concordance with the hypothesis that sulfated chitosan-growth factor (GF) interactions are controlled by a combination of two effects: the electrostatic interactions and the conformational adaptation of the polysaccharide. Thus, we found that highly charged O-sulfated S-CS and S-DCS polysaccharides with a low degree of contraction interacted more strongly with GFs than N-sulfated N-DCS, with a higher degree of contraction and a low charge. Finally, the evidence gathered suggests that N-DCS would be able to bind to an allosteric zone and is likely to enhance GF signaling activity. This is because the bound protein remains able to bind to its cognate receptor, promoting an effect on cell proliferation as has been shown for PC12 cells. However, S-CS and S-DCS would sequester the protein, decreasing the GF signaling activity by depleting the protein or locally blocking its active site.

Simple and Practical Multigram Synthesis of d-Xylonate Using a Recombinant Xylose Dehydrogenase.

An efficient multienzyme system for the preparative synthesis of d-xylonate, a chemical with versatile industrial applications, is described. The multienzyme system is based on d-xylose oxidation catalyzed by the xylose dehydrogenase from Calulobacter crescentus and the use of catalytic amounts of NAD+. The cofactor is regenerated in situ by coupling the reduction of acetaldehyde into ethanol catalyzed by alcohol dehydrogenase from Clostridium kluyveri. Excellent conversions (>95%) were obtained in a process that allows easy product isolation by simple evaporation of the volatile buffer and byproducts.

Assembly of glycoamino acid building blocks: a new strategy for the straightforward synthesis of heparan sulfate mimics.

A new strategy that enables a modular straightforward synthesis of heparan sulfate oligosaccharide mimics by the assembly of simple glycoamino acid building blocks is described. The coupling between units is readily carried out by an amidation reaction. Several glycoamino acid oligomers were prepared and their interaction with the FGF2 protein was analyzed.

A holistic approach to unravelling chondroitin sulfation: Correlations between surface charge, structure and binding to growth factors.

Chondroitin sulfate (CS) is a relevant family of polysaccharides that participates in a large variety of biological events that are related to neural processes by regulating various growth factors through the pattern and degree of sulfation of the polysaccharide. However, their own complexity makes their optimization for biomedical applications a difficult undertaking. Thus, a different perspective has to be taken. Herein, we show that the particular sulfate distribution within the disaccharide repeating-unit plays a key role in the binding of growth factors (GFs). In particular, this disposition modulates the surface charge of the helical structure that, interestingly, has a significant influence on the binding capacity of CSs with several GFs. This fact should be carefully considered in the design of new ligands with improved activity as GFs ligands.

Biochemical profiling of rat embryonic stem cells grown on electrospun polyester fibers using synchrotron infrared microspectroscopy.

Therapeutic options for spinal cord injuries are severely limited; current treatments only offer symptomatic relief and rehabilitation focused on educating the individual on how to adapt to their new situation to make best possible use of their remaining function. Thus, new approaches are needed, and interest in the development of effective strategies to promote the repair of neural tracts in the central nervous system inspired us to prepare functional and highly anisotropic polymer scaffolds. In this work, an initial assessment of the behavior of rat neural progenitor cells (NPCs) seeded on poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) fiber scaffolds using synchrotron-based infrared microspectroscopy (SIRMS) is described. Combined with a modified touch imprint cytology sample preparation method, this application of SIRMS enabled the biochemical profiles of NPCs on the coated polymer fibers to be determined. The results showed that changes in the lipid and amide I-II spectral regions are modulated by the type and coating of the substrate used and the culture time. SIRMS studies can provide valuable insight into the early-stage response of NPCs to the morphology and surface chemistry of a biomaterial, and could therefore be a useful tool in the preparation and optimization of cellular scaffolds. Graphical abstract Synchrotron IR microspectroscopy can provide insight into the response of neural progenitor cells to synthetic scaffolds.

Synthesis, physicochemical characterization and biological evaluation of chitosan sulfate as heparan sulfate mimics.

Despite the relevant biological functions of heparan sulfate (HS) glycosaminoglycans, their limited availability and the chemical heterogeneity from natural sources hamper their use for biomedical applications. Chitosan sulfates (ChS) exhibit structural similarity to HSs and may mimic their biological functions. We prepared a variety of ChS with different degree of sulfation to evaluate their ability to mimic HS in protein binding and to promote neural cell division and differentiation. The structure of the products was characterized using various spectroscopic and analytical methods. The study of their interaction with different growth factors showed that ChS bound to the proteins similarly or even better than heparin. In cell cultures, a transition effect on cell number was observed as a function of ChS concentration. Differences in promoting the expression of the differentiation markers were also found depending on the degree of sulfation and modification in the chitosan.

Analytical Validation of a New Enzymatic and Automatable Method for d-Xylose Measurement in Human Urine Samples.

Hypolactasia, or intestinal lactase deficiency, affects more than half of the world population. Currently, xylose quantification in urine after gaxilose oral administration for the noninvasive diagnosis of hypolactasia is performed with the hand-operated nonautomatable phloroglucinol reaction. This work demonstrates that a new enzymatic xylose quantification method, based on the activity of xylose dehydrogenase from Caulobacter crescentus, represents an excellent alternative to the manual phloroglucinol reaction. The new method is automatable and facilitates the use of the gaxilose test for hypolactasia diagnosis in the clinical practice. The analytical validation of the new technique was performed in three different autoanalyzers, using buffer or urine samples spiked with different xylose concentrations. For the comparison between the phloroglucinol and the enzymatic assays, 224 urine samples of patients to whom the gaxilose test had been prescribed were assayed by both methods. A mean bias of -16.08 mg of xylose was observed when comparing the results obtained by both techniques. After adjusting the cut-off of the enzymatic method to 19.18 mg of xylose, the Kappa coefficient was found to be 0.9531, indicating an excellent level of agreement between both analytical procedures. This new assay represents the first automatable enzymatic technique validated for xylose quantification in urine.

Development of a new method for d-xylose detection and quantification in urine, based on the use of recombinant xylose dehydrogenase from Caulobacter crescentus.

The gene xylB from Caulobacter crescentus has been cloned and expressed in Escherichia coli providing a high yield of xylose dehydrogenase (XylB) production and excellent purity (97%). Purified recombinant XylB showed an absolute dependence on the cofactor NAD(+) and a strong preference for d-xylose against other assayed mono and disaccharides. Additionally, XylB showed strong stability when stored as freeze-dried powder at least 250days both at 4°C and room temperature. In addition, more than 80% of the initial activity of rehydrated freeze-dried enzyme remained after 150days of incubation at 4°C. Based on these characteristics, the capability of XylB in d-xylose detection and quantification was studied. The linearity of the method was maintained up to concentrations of d-xylose of 10mg/dL and the calculated limits of detection (LoD) and quantification (LoQ) of xylose in buffer were 0.568mg/dL and 1.89mg/dL respectively. Thus, enzymatic detection was found to be an excellent method for quantification of d-xylose in both buffer and urine samples. This method can easily be incorporated in a new test for the diagnosis of hypolactasia through the measurement of intestinal lactase activity.

Tuning the Phosphoryl Donor Specificity of Dihydroxyacetone Kinase from ATP to Inorganic Polyphosphate. An Insight from Computational Studies.

Dihydroxyacetone (DHA) kinase from Citrobacter freundii provides an easy entry for the preparation of DHA phosphate; a very important C3 building block in nature. To modify the phosphoryl donor specificity of this enzyme from ATP to inorganic polyphosphate (poly-P); a directed evolution program has been initiated. In the first cycle of evolution, the native enzyme was subjected to one round of error-prone PCR (EP-PCR) followed directly (without selection) by a round of DNA shuffling. Although the wild-type DHAK did not show activity with poly-P, after screening, sixteen mutant clones showed an activity with poly-phosphate as phosphoryl donor statistically significant. The most active mutant presented a single mutation (Glu526Lys) located in a flexible loop near of the active center. Interestingly, our theoretical studies, based on molecular dynamics simulations and hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) optimizations, suggest that this mutation has an effect on the binding of the poly-P favoring a more adequate position in the active center for the reaction to take place.

Hyperthermophilic aldolases as biocatalyst for C-C bond formation: rhamnulose 1-phosphate aldolase from Thermotoga maritima.

The TM1072 gene from Thermotoga maritima codifies for a putative form of a rhamnulose-1-phosphate aldolase (Rha-1PA Tm). To investigate this enzyme further, its gene was cloned and expressed in Escherichia coli. The purified enzyme was activated by Co(2+) as a divalent metal ion cofactor, instead of Zn(2+) as its E. coli homologue, and exhibited a maximum of activity at 95 °C. Furthermore, the enzyme displayed a high stability against extreme reaction conditions, retaining 90 % of its activity in the presence of 40 % of acetonitrile and showing a half-life greater than 3 h at 115 °C. The kinetic parameters at room temperature (R/T) were also studied; the K M was calculated to be 3.6 ± 0.33 mM, while k cat/K M was found to be 0.7 × 10(3) s(-1) M(-1). Given these characteristics, Rha-1PA Tm is an attractive enzyme for use as a biocatalyst for industrial applications, offering intriguing possibilities for practical biocatalysis.

Enzyme catalysed tandem reactions.

To transfer to the laboratory, the excellent efficiency shown by enzymes in Nature, biocatalysis, had to mimic several synthetic strategies used by the living organisms. Biosynthetic pathways are examples of tandem catalysis and may be assimilated in the biocatalysis field for the use of isolated multi-enzyme systems in the homogeneous phase. The concurrent action of several enzymes that work sequentially presents extraordinary advantages from the synthetic point of view, since it permits a reversible process to become irreversible, to shift the equilibrium reaction in such a way that enantiopure compounds can be obtained from prochiral or racemic substrates, reduce or eliminate problems due to product inhibition or prevent the shortage of substrates by dilution or degradation in the bulk media, etc. In this review we want to illustrate the developments of recent studies involving in vitro multi-enzyme reactions for the synthesis of different classes of organic compounds.

Synthesis and evaluation of xylopyranoside derivatives as "decoy acceptors" of human ß-1,4-galactosyltransferase 7.

Proteoglycans (PGs), including heparan sulfate forms, are important regulators of tumor progression. In the PGs biosynthetic process, the core protein is synthesized on a ribosomal template and the sugar chains are assembled post-translationally, one sugar at a time, starting with the linkage of xylose to a serine residue of the core protein and followed by galactosidation of the xylosylprotein. Hydrophobic xylopyranosides have been previously shown to prime heparan sulfate synthesis, a property that was required to cause growth inhibition of tumor cells. To know if the antiproliferative activity of synthetic xylopyranosides is related to their ability to act as "decoy acceptors" of xylosylprotein 4-ß-galactosyltransferase, we have heterologously expressed the catalytic domain of the human ß-1,4-GalT 7 and studied the ability of a variety of synthetic xylopyranoside derivatives to act as substrates or inhibitors of the recombinant enzyme.