Relationships of crystallinity and reaction rates for enzymatic degradation of poly (ethylene terephthalate), PET.

Biocatalytic degradation of plastic waste is anticipated to play an important role in future recycling systems. However, enzymatic degradation of crystalline poly (ethylene terephthalate) (PET) remains consistently poor. Herein, we employed functional assays to elucidate the molecular underpinnings of this limitation. This included utilizing complementary activity assays to monitor the degradation of PET disks with varying crystallinity (XC), as well as determining enzymatic kinetic parameters for soluble PET fragments. The results indicate that an efficient PET-hydrolase, LCCICCG, operates through an endolytic mode of action, and that its activity is limited by conformational constraints in the PET polymer. Such constraints become more pronounced at high XC values, and this limits the density of productive sites on the PET surface. Endolytic chain-scissions are the dominant reaction type in the initial stage, and this means that little or no soluble organic product are released. However, endolytic cuts gradually and locally promote chain mobility and hence the density of attack sites on the surface. This leads to an upward concave progress curve; a behavior sometimes termed lag-phase kinetics.

Enzymatic degradation of poly(ethylene terephthalate) (PET): Identifying the cause of the hypersensitive enzyme kinetic response to increased PET crystallinity.

Plastic pollution poses a significant environmental challenge, with poly(ethylene terephthalate) (PET) being a major contributor due to its extensive use in single use applications such as plastic bottles and other packaging material. Enzymatic degradation of PET offers a promising solution for PET recycling, but the enzyme kinetics in relation to the degree of crystallinity (XC) of the PET substrate are poorly understood. In this study, we investigated the hypersensitive enzyme kinetic response on PET at XC from ∼8.5-12% at 50 °C using the benchmark PET hydrolysing enzyme LCCICCG. We observed a substantial reduction in the maximal enzymatic reaction rate (invVmax) with increasing XC, corresponding to a 3-fold reduction in invVmax when the XC of PET increased from 8.6% to 12.2%. The kinetic analysis revealed that the level of the Mobile Amorphous Fraction (XMAF) was a better descriptor for the enzymatic degradation rate response than XC (or (100%-XC)). By continuous monitoring of the enzymatic reaction progress, we quantified the lag phase prolongation in addition to the steady-state kinetic rates (vss) of the reactions and found that the duration of the lag phase of a reaction could be predicted from the vss and XC by multiple linear regression modeling. The linear correlation between the duration of the lag phase and the vss of the enzymatic PET degradation affirmed that the LCCICCG worked via a random/endo-type enzymatic attack pattern. The longer lag phase at increased XC of PET is proposed to be due to increased substrate entanglement density as well as unproductive enzyme binding to the crystalline regions of PET. The findings enhance our understanding of PET enzymatic degradation kinetics and its dependence on substrate composition, i.e., XMAF and XC.

Bioinformatics and functional selection of GH77 4-α-glucanotransferases for potato starch modification.

4-α-glucanotransferases (4αGTs, EC 2.4.1.25) from glycoside hydrolase family 77 (GH77) catalyze chain elongation of starch amylopectin chains and can be utilized to structurally modify starch to tailor its gelation properties. The potential relationship between the structural design of 4αGTs and functional starch modification is unknown. Here, family GH77 was mined in silico for enzyme candidates based on sub-grouping guided by Conserved Unique Peptide Patterns (CUPP) bioinformatics categorization. From + 12,000 protein sequences a representative set of 27 4αGTs, representing four different domain architectures, different bacterial origins and diverse CUPP groups, was selected for heterologous expression and further study. Most of the enzymes catalyzed starch modification, but their efficacies varied substantially. Five of the 4αGTs were characterized in detail, and their action was compared to that of the industrial benchmark enzyme, Tt4αGT (CUPP 77_1.2), from Thermus thermophilus. Reaction optima of the five 4αGTs ranged from ∼40-60 °C and pH 7.3-9.0. Several were stable for a minimum 4 h at 70 °C. Domain architecture type A proteins, consisting only of a catalytic domain, had high thermal stability and high starch modification ability. All five novel 4αGTs (and Tt4αGT) induced enhanced gelling of potato starch. One, At4αGT from Azospirillum thermophilum (CUPP 77_2.4), displayed distinct starch modifying abilities, whereas T24αGT from Thermus sp. 2.9 (CUPP 77_1.2) modified the starch similarly to Tt4αGT, but slightly more effectively. T24αGT and At4αGT are thus interesting candidates for industrial starch modification. A model is proposed to explain the link between the 4αGT induced molecular modifications and macroscopic starch gelation.

Significance of poly(ethylene terephthalate) (PET) substrate crystallinity on enzymatic degradation.

Poly(ethylene terephthalate) (PET) is a semi-crystalline plastic polyester material with a global production volume of 83 Mt/year. PET is mainly used in textiles, but also widely used for packaging materials, notably plastic bottles, and is a major contributor to environmental plastic waste accumulation. Now that enzymes have been demonstrated to catalyze PET degradation, new options for sustainable bio-recycling of PET materials via enzymatic catalysis have emerged. The enzymatic degradation rate is strongly influenced by the properties of PET, notably the degree of crystallinity, XC. The higher the XC of the PET material, the slower the enzymatic rate. Crystallization of PET, resulting in increased XC, is induced thermally (via heating) and/or mechanically (via stretching), and the XC of most PET plastic bottles and microplastics exceeds what currently known enzymes can readily degrade. The enzymatic action occurs at the surface of the insoluble PET material and improves when the polyester chain mobility increases. The chain mobility increases drastically when the temperature exceeds the glass transition temperature, Tg, which is ∼40 °C at the surface layer of PET. Since PET crystallization starts at 70 °C, the ideal temperature for enzymatic degradation is just below 70 °C to balance high chain mobility and enzymatic reaction activation without inducing crystal formation. This paper reviews the current understanding on the properties of PET as an enzyme substrate and summarizes the most recent knowledge of how the crystalline and amorphous regions of PET form, and how the XC and the Tg impact the efficiency of enzymatic PET degradation.

Structural Characterization and Cytotoxic Activity Evaluation of Ulvan Polysaccharides Extracted from the Green Algae Ulva papenfussii.

Ulvan, a sulfated heteropolysaccharide with structural and functional properties of interest for various uses, was extracted from the green seaweed Ulva papenfussii. U. papenfussii is an unexplored Ulva species found in the South China Sea along the central coast of Vietnam. Based on dry weight, the ulvan yield was ~15% (w/w) and the ulvan had a sulfate content of 13.4 wt%. The compositional constitution encompassed L-Rhamnose (Rhap), D-Xylose (Xylp), D-Glucuronic acid (GlcAp), L-Iduronic acid (IdoAp), D-Galactose (Galp), and D-Glucose (Glcp) with a molar ratio of 1:0.19:0.35:0.52:0.05:0.11, respectively. The structure of ulvan was determined using High-Performance Liquid Chromatography (HPLC), Fourier Transform Infrared Spectroscopy (FT-IR), and Nuclear Magnetic Resonance spectroscopy (NMR) methods. The results showed that the extracted ulvan comprised a mixture of two different structural forms, namely ("A3s") with the repeating disaccharide [→4)-ß-D-GlcAp-(1→4)-α-L-Rhap 3S-(1→]n, and ("B3s") with the repeating disaccharide [→4)-α-L-IdoAp-(1→4)-α-L-Rhap 3S(1→]n. The relative abundance of A3s, and B3s was 1:1.5, respectively. The potential anticarcinogenic attributes of ulvan were evaluated against a trilogy of human cancer cell lineages. Concomitantly, Quantitative Structure-Activity Relationship (QSAR) modeling was also conducted to predict potential adverse reactions stemming from pharmacological interactions. The ulvan showed significant antitumor growth activity against hepatocellular carcinoma (IC50 ≈ 90 µg/mL), human breast cancer cells (IC50 ≈ 85 µg/mL), and cervical cancer cells (IC50 ≈ 67 µg/mL). The QSAR models demonstrated acceptable predictive power, and seven toxicity indications confirmed the safety of ulvan, warranting its candidacy for further in vivo testing and applications as a biologically active pharmaceutical source for human disease treatment.

Polyphenol Oxidase Products Are Priming Agents for LPMO Peroxygenase Activity.

Polyphenol oxidases catalyze the hydroxylation of monophenols to diphenols, which are reducing agents for lytic polysaccharide monooxygenases (LPMOs) in their degradation of cellulose. In particular, the polyphenol oxidase MtPPO7 from Myceliophthora thermophila converts lignocellulose-derived monophenols, and under the new perspective of the peroxygenase reaction catalyzed by LPMOs, we aim to differentiate the role of the catalytic products of MtPPO7 in priming and fueling of LPMO activity. Exemplified by the activity of MtPPO7 towards guaiacol and by using the benchmark LPMO NcAA9C from Neurospora crassa we show that MtPPO7 catalytic products provide the initial electron for the reduction of Cu(II) to Cu(I) but cannot provide the required reducing power for continuous fueling of the LPMO. The priming reaction is shown to occur with catalytic amounts of MtPPO7 products and those compounds do not generate substantial amounts of H2 O2 in situ to fuel the LPMO peroxygenase activity. Reducing agents with a low propensity to generate H2 O2 can provide the means for controlling the LPMO catalysis through exogenous H2 O2 and thereby minimize any enzyme inactivation.

Conserved unique peptide patterns (CUPP) online platform 2.0: implementation of +1000 JGI fungal genomes.

Carbohydrate-processing enzymes, CAZymes, are classified into families based on sequence and three-dimensional fold. Because many CAZyme families contain members of diverse molecular function (different EC-numbers), sophisticated tools are required to further delineate these enzymes. Such delineation is provided by the peptide-based clustering method CUPP, Conserved Unique Peptide Patterns. CUPP operates synergistically with the CAZy family/subfamily categorizations to allow systematic exploration of CAZymes by defining small protein groups with shared sequence motifs. The updated CUPP library contains 21,930 of such motif groups including 3,842,628 proteins. The new implementation of the CUPP-webserver, https://cupp.info/, now includes all published fungal and algal genomes from the Joint Genome Institute (JGI), genome resources MycoCosm and PhycoCosm, dynamically subdivided into motif groups of CAZymes. This allows users to browse the JGI portals for specific predicted functions or specific protein families from genome sequences. Thus, a genome can be searched for proteins having specific characteristics. All JGI proteins have a hyperlink to a summary page which links to the predicted gene splicing including which regions have RNA support. The new CUPP implementation also includes an update of the annotation algorithm that uses only a fourth of the RAM while enabling multi-threading, providing an annotation speed below 1 ms/protein.

Establishment of specific age-related macular degeneration relevant gene expression panels using porcine retinal pigment epithelium for assessing fucoidan bioactivity.

PURPOSE: Age-related macular degeneration (AMD) is the leading cause of severe vision loss in industrialized nations. Important factors in pathogenesis are oxidative stress, inflammation, and, in the wet form of AMD, angiogenesis. Fucoidans, sulfated polysaccharides from brown algae, may have antioxidant, anti-inflammatory, and antiangiogenic effects. In this study, we established specific gene expression panels for inflammation, oxidative stress and angiogenesis in porcine retinal pigment epithelium (RPE), and investigated the effect of fucoidans on gene expression under different noxious agents. METHODS: Primary porcine RPE cells cultured for at least 14 days were used. Using viability assays with tetrazolium bromide and real-time polymerase chain reaction of marker genes, positive controls were established for appropriate concentrations and exposure times of selected noxious agents (lipopolysaccharide (LPS), H2O2, CoCl2). Three different AMD relevant gene panels specific for porcine RPE for inflammation, oxidative stress, and angiogenesis were established, and the influence of fucoidans (mainly Fucus vesiculosus; FV) on gene expression was investigated. RESULTS: The following was shown by gene expression analyses: (1) Inflammation panel: Expression of 18 genes was affected under LPS (three days). Among them, LPS increased genes for interleukin 1 receptor 2, interleukin 8, cyclooxygenase-2 and vascular cell adhesion protein 1 expression which were diminished when FV was present. (2) Oxidative stress panel: Under stimulation of H2O2 (one day) and LPS (one day), expression of a total of 15 genes was affected. LPS induced increase in genes for superoxide dismutase-1, C-X-C motif chemokine 10, and CC chemokine ligand-5 expression was not detected when FV was present. (3) Angiogenesis panel: Under stimulation with CoCl2 (three days) expression of six genes was affected, with the increase of genes for angiopoietin 2, vascular endothelial growth factor receptor-1, and follistatin being diminished when FV was present. CONCLUSION: Three specific gene expression panels for porcine RPE that map genes for three of the major pathological factors of AMD, inflammation, oxidative stress, and angiogenesis, were established. Further, we demonstrated that fucoidans can reduce stress related gene activation in all of these three major pathogenic pathways. This study is another indication that fucoidans can act on different pathomechanisms of AMD simultaneously, which provides further evidence for fucoidans as a possible drug for treatment and prevention of AMD.

Rate Response of Poly(Ethylene Terephthalate)-Hydrolases to Substrate Crystallinity: Basis for Understanding the Lag Phase.

The rate response of poly(ethylene terephthalate) (PET)-hydrolases to increased substrate crystallinity (XC ) of PET manifests as a rate-lowering effect that varies significantly for different enzymes. Herein, we report the influence of XC on the product release rate of six thermostable PET-hydrolases. All enzyme reactions displayed a distinctive lag phase until measurable product formation occurred. The duration of the lag phase increased with XC . The recently discovered PET-hydrolase PHL7 worked efficiently on "amorphous" PET disks (XC ≈10 %), but this enzyme was extremely sensitive to increased XC , whereas the enzymes LCCICCG , LCC, and DuraPETase had higher tolerance to increases in XC and had activity on PET disks having XC of 24.4 %. Microscopy revealed that the XC -tolerant hydrolases generated smooth and more uniform substrate surface erosion than PHL7 during reaction. Structural and molecular dynamics analysis of the PET-hydrolyzing enzymes disclosed that surface electrostatics and enzyme flexibility may account for the observed differences.

Enzymatic cofactor regeneration systems: A new perspective on efficiency assessment.

Nowadays, the specificity of enzymatic processes makes them more and more important every year, and their usage on an industrial scale seems to be necessary. Enzymatic cofactors, however, play a crucial part in the prospective applications of enzymes, because they are indispensable for conducting highly effective biocatalytic activities. Due to the relatively high cost of these compounds and their consumption during the processes carried out, it has become crucial to develop systems for cofactor regeneration. Therefore, in this review, an attempt was made to summarize current knowledge on enzymatic regeneration methods, which are characterized by high specificity, non-toxicity and reported to be highly efficient. The regeneration of cofactors, such as nicotinamide dinucleotides, coenzyme A, adenosine 5'-triphosphate and flavin nucleotides, which are necessary for the proper functioning of a large number of enzymes, is discussed, as well as potential directions for further development of these systems are highlighted. This review discusses a range of highly effective cofactor regeneration systems along with the productive synthesis of many useful chemicals, including the simultaneous renewal of several cofactors at the same time. Additionally, the impact of the enzyme immobilization process on improving the stability and the potential for multiple uses of the developed cofactor regeneration systems was also presented. Moreover, an attempt was made to emphasize the importance of the presented research, as well as the identification of research gaps, which mainly result from the lack of available literature on this topic.

A new continuous assay for quantitative assessment of enzymatic degradation of poly(ethylene terephthalate) (PET).

Enzymatic degradation of poly(ethylene terephthalate) (PET) has emerged as a promising route for ecofriendly biodegradation of plastic waste. Several discontinuous activity assays have been developed for assessing PET hydrolyzing enzymes, usually involving manual sampling at different time points during the course of the enzymatic reaction. In this work, we present a novel, compartmentalized UV absorbance assay for continuous detection of soluble hydrolysis products released during enzymatic degradation of PET. The methodology is based on removal of the walls separating two diagonally adjacent wells in UV-transparent microplates, to ensure passage of soluble enzymatic hydrolysis products between the two adjacent wells: One well holds an insoluble PET disk of defined dimensions and the other is used for continuous reading of the enzymatic product formation (at 240 nm). The assay was validated by quantifying the rate of mixing of the soluble PET degradation product BHET (bis(2-hydroxyethyl) terephthalate) between the two adjacent wells. The assay validation also involved a simple adjustment for water evaporation during prolonged assays. With this new assay, we determined the kinetic parameters for two PET hydrolases, DuraPETase and LCCICCG, and verified the underlying assumption of steady-state reaction rates. This new continuous assay enables fast exploration and robust kinetic characterization of PET degrading enzymes.

Standardized method for controlled modification of poly (ethylene terephthalate) (PET) crystallinity for assaying PET degrading enzymes.

Poly(ethylene terephthalate) (PET) is a polyester plastic, which is widely used, notably as a material for single-use plastic bottles. Its accumulation in the environment now poses a global pollution threat. A number of enzymes are active on PET providing new options for industrial biorecycling of PET materials. The enzyme activity is strongly affected by the degree of PET crystallinity (XC), and the XC is therefore a relevant factor to consider in enzyme catalyzed PET recycling. Here, we present a new experimental methodology, based on systematic thermal annealing for controlled preparation of PET disks having different XC, to allow systematic quantitative evaluation of the efficiency of PET degrading enzymes at different degrees of PET substrate crystallinity. We discuss the theory of PET crystallinity and compare PET crystallinity data measured by differential scanning calorimetry and attenuated Fourier transform infrared spectroscopy.•This study introduces a simple method for controlling the crystallinity of PET samples via annealing in a heat block.•The present methodology is not limited to the analytical methods included in the methods details.

Iron can be microbially extracted from Lunar and Martian regolith simulants and 3D printed into tough structural materials.

In-situ resource utilization (ISRU) is increasingly acknowledged as an essential requirement for the construction of sustainable extra-terrestrial colonies. Even with decreasing launch costs, the ultimate goal of establishing colonies must be the usage of resources found at the destination of interest. Typical approaches towards ISRU are often constrained by the mass and energy requirements of transporting processing machineries, such as rovers and massive reactors, and the vast amount of consumables needed. Application of self-reproducing bacteria for the extraction of resources is a promising approach to reduce these pitfalls. In this work, the bacterium Shewanella oneidensis was used to reduce three different types of Lunar and Martian regolith simulants, allowing for the magnetic extraction of iron-rich materials. The combination of bacterial treatment and magnetic extraction resulted in a 5.8-times higher quantity of iron and 43.6% higher iron concentration compared to solely magnetic extraction. The materials were 3D printed into cylinders and the mechanical properties were tested, resulting in a 400% improvement in compressive strength in the bacterially treated samples. This work demonstrates a proof of concept for the on-demand production of construction and replacement parts in space exploration.

Bioremediation of lignin derivatives and phenolics in wastewater with lignin modifying enzymes: Status, opportunities and challenges.

Lignin modifying enzymes from fungi and bacteria are potential biocatalysts for sustainable mitigation of different potentially toxic pollutants in wastewater. Notably, the paper and pulp industry generates enormous amounts of wastewater containing high amounts of complex lignin-derived chlorinated phenolics and sulfonated pollutants. The presence of these compounds in wastewater is a critical issue from environmental and toxicological perspectives. Some chloro-phenols are harmful to the environment and human health, as they exert carcinogenic, mutagenic, cytotoxic, and endocrine-disrupting effects. In order to address these most urgent concerns, the use of oxidative lignin modifying enzymes for bioremediation has come into focus. These enzymes catalyze modification of phenolic and non-phenolic lignin-derived substances, and include laccase and a range of peroxidases, specifically lignin peroxidase (LiP), manganese peroxidase (MnP), versatile peroxidase (VP), and dye-decolorizing peroxidase (DyP). In this review, we explore the key pollutant-generating steps in paper and pulp processing, summarize the most recently reported toxicological effects of industrial lignin-derived phenolic compounds, especially chlorinated phenolic pollutants, and outline bioremediation approaches for pollutant mitigation in wastewater from this industry, emphasizing the oxidative catalytic potential of oxidative lignin modifying enzymes in this regard. We highlight other emerging biotechnical approaches, including phytobioremediation, bioaugmentation, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based technology, protein engineering, and degradation pathways prediction, that are currently gathering momentum for the mitigation of wastewater pollutants. Finally, we address current research needs and options for maximizing sustainable biobased and biocatalytic degradation of toxic industrial wastewater pollutants.

Building a Resilient, Sustainable, and Healthier Food Supply Through Innovation and Technology.

The modern food supply faces many challenges. The global population continues to grow and people are becoming wealthier, so the food production system must respond by creating enough high-quality food to feed everyone with minimal damage to our environment. The number of people suffering or dying from diet-related chronic diseases, such as obesity, diabetes, heart disease, stroke, and cancer, continues to rise, which is partly linked to overconsumption of highly processed foods, especially high-calorie or rapidly digestible foods. After falling for many years, the number of people suffering from starvation or malnutrition is rising, and thishas been exacerbated by the global COVID-19 pandemic. The highly integrated food supply chains that spread around the world are susceptible to disruptions due to policy changes, economic stresses, and natural disasters, as highlighted by the recent pandemic. In this perspective article, written by members of the Editorial Committee of the Annual Review of Food Science and Technology, we highlight some of the major challenges confronting the modern food supply chain as well as how innovations in policy and technology can be used to address them. Pertinent technological innovations include robotics, machine learning, artificial intelligence, advanced diagnostics, nanotechnology, biotechnology, gene editing, vertical farming, and soft matter physics. Many of these technologies are already being employed across the food chain by farmers, distributors, manufacturers, and consumers to improve the quality, nutrition, safety, and sustainability of the food supply. These innovations are required to stimulate the development and implementation of new technologies to ensure a more equitable, resilient, and efficient food production system. Where appropriate, these technologies should be carefully tested before widespread implementation so that proper risk-benefit analyses can be carried out. They can then be employed without causing unforeseen adverse consequences. Finally, it is important to actively engage all stakeholders involved in the food supply chain throughout the development and testing of these new technologies to support their adoption if proven safe and effective.

Functional Characterization of a New GH107 Endo-α-(1,4)-Fucoidanase from the Marine Bacterium Formosa haliotis.

Fucoidans from brown macroalgae are sulfated fucose-rich polysaccharides, that have several beneficial biological activities, including anti-inflammatory and anti-tumor effects. Controlled enzymatic depolymerization of the fucoidan backbone can help produce homogeneous, defined fucoidan products for structure-function research and pharmaceutical uses. However, only a few endo-fucoidanases have been described. This article reports the genome-based discovery, recombinant expression in Escherichia coli, stabilization, and functional characterization of a new bacterial endo-α-(1,4)-fucoidanase, Fhf1, from Formosa haliotis. Fhf1 catalyzes the cleavage of α-(1,4)-glycosidic linkages in fucoidans built of alternating α-(1,3)-/α-(1,4)-linked l-fucopyranosyl sulfated at C2. The native Fhf1 is 1120 amino acids long and belongs to glycoside hydrolase (GH) family 107. Deletion of the signal peptide and a 470 amino acid long C-terminal stretch led to the recombinant expression of a robust, minimized enzyme, Fhf1Δ470 (71 kDa). Fhf1Δ470 has optimal activity at pH 8, 37-40 °C, can tolerate up to 500 mM NaCl, and requires the presence of divalent cations, either Ca2+, Mn2+, Zn2+ or Ni2+, for maximal activity. This new enzyme has the potential to serve the need for controlled enzymatic fucoidan depolymerization to produce bioactive sulfated fucoidan oligomers.

Effect of Enzymatically Extracted Fucoidans on Angiogenesis and Osteogenesis in Primary Cell Culture Systems Mimicking Bone Tissue Environment.

Angiogenesis, the formation of new blood vessels from existing ones, is an essential process for successful bone regeneration. Further, angiogenesis is a key factor for the development of bone-related disorders like osteosarcoma or arthritis. Fucoidans, sulfated polysaccharides from brown algae, have been shown to affect angiogenesis as well as a series of other physiological processes including inflammation or infection. However, the chemical properties of fucoidan which define the biological activity vary tremendously, making a prediction of the bioactivity or the corresponding therapeutic effect difficult. In this study, we compare the effect of four chemically characterized high molecular weight fucoidan extracts from Fucus distichus subsp. evanescens (FE_crude and fractions F1, F2, F3) on angiogenic and osteogenic processes in bone-related primary mono- and co-culture cell systems. By determining the gene expression and protein levels of the regulatory molecules vascular endothelial growth factor (VEGF), angiopoietin-1 (ANG-1), ANG-2 and stromal-derived factor 1 (SDF-1), we show that the extracted fucoidans negatively influence angiogenic and osteogenic processes in both the mono- and co-culture systems. We demonstrate that purer fucoidan extracts with a high fucose and sulfate content show stronger effects on these processes. Immunocytochemistry of the co-culture system revealed that treatment with FE_F3, containing the highest fucose and sulfate content, impaired the formation of angiogenic tube-like structures, indicating the anti-angiogenic properties of the tested fucoidans. This study highlights how chemical properties of fucoidan influence its bioactivity in a bone-related context and discusses how the observed phenotypes can be explained on a molecular level-knowledge that is indispensable for future therapies based on fucoidans.

Conserved unique peptide patterns (CUPP) online platform: peptide-based functional annotation of carbohydrate active enzymes.

The CUPP platform includes a web server for functional annotation and sub-grouping of carbohydrate active enzymes (CAZymes) based on a novel peptide-based similarity assessment algorithm, i.e. protein grouping according to Conserved Unique Peptide Patterns (CUPP). This online platform is open to all users and there is no login requirement. The web server allows the user to perform genome-based annotation of carbohydrate active enzymes to CAZy families, CAZy subfamilies, CUPP groups and EC numbers (function) via assessment of peptide-motifs by CUPP. The web server is intended for functional annotation assessment of the CAZy inventory of prokaryotic and eukaryotic organisms from genomic DNA (up to 30MB compressed) or directly from amino acid sequences (up to 10MB compressed). The custom query sequences are assessed using the CUPP annotation algorithm, and the outcome is displayed in interactive summary result pages of CAZymes. The results displayed allow for inspection of members of the individual CUPP groups and include information about experimentally characterized members. The web server and the other resources on the CUPP platform can be accessed from https://cupp.info.

The structural basis of fungal glucuronoyl esterase activity on natural substrates.

Structural and functional studies were conducted of the glucuronoyl esterase (GE) from Cerrena unicolor (CuGE), an enzyme catalyzing cleavage of lignin-carbohydrate ester bonds. CuGE is an α/ß-hydrolase belonging to carbohydrate esterase family 15 (CE15). The enzyme is modular, comprised of a catalytic and a carbohydrate-binding domain. SAXS data show CuGE as an elongated rigid molecule where the two domains are connected by a rigid linker. Detailed structural information of the catalytic domain in its apo- and inactivated form and complexes with aldouronic acids reveal well-defined binding of the 4-O-methyl-a-D-glucuronoyl moiety, not influenced by the nature of the attached xylo-oligosaccharide. Structural and sequence comparisons within CE15 enzymes reveal two distinct structural subgroups. CuGE belongs to the group of fungal CE15-B enzymes with an open and flat substrate-binding site. The interactions between CuGE and its natural substrates are explained and rationalized by the structural results, microscale thermophoresis and isothermal calorimetry.

Phenolic and fatty acid profiles, α-tocopherol and sucrose contents, and antioxidant capacities of understudied Portuguese almond cultivars.

Almonds have recognized health benefits, which are largely attributed to their chemical composition, including fatty acids, phenolics, vitamin E, and sucrose. This study was carried with the aim of providing information on the levels of the aforementioned bioactive compounds and antioxidant activities in six understudied Portuguese cultivars (Amendoão, Bonita, Casanova, Molar, Pegarinhos-Moncorvo, Pegarinhos-Murça and Refêgo), in comparison with two foreign cultivars (Ferragnès and Glorieta). A cultivar effect was observed for all the parameters evaluated, with some Portuguese cultivars comparing well and even favorably with the foreign ones. A multivariate analysis of the data allowed a clear discrimination of cultivars and that statistical tool could be used for authenticity purposes, especially for cultivars included in the Protected Designation of Origin "Amêndoa Douro." PRACTICAL APPLICATIONS: Almonds are among the most consumed nuts worldwide, with a considerable number of cultivars recorded around the world, although research has been neglecting the local cultivars. This work studies the chemical composition of several understudied cultivars and compares them to two widespread commercial ones. The results not only provide new information about these neglected cultivars, but also provide data for stakeholders to select more interesting cultivars with particular characteristics/or rich in compounds of interest.