High-amylose starch (HAS) is gaining attention in biotechnology for its thermal stability, structural resilience and health benefits. Its dense crystalline structure hinders hydrolysis by human gut enzymes, making it a promising source of type 2 resistant starch for hydro-thermal and enzymatic upgrading. 4-α-Glucanotransferases (4αGTs) of glycoside hydrolase family 77 catalyse disproportionation of α-1,4-glucan chains in HAS, enhancing functionality and nutritional properties. Here, a 4αGT, ParGT from the hyperthermophilic archaeon Pyrobaculum arsenaticum, identified in a metagenomic dataset from Pisciarelli hot spring (85 °C, pH 5.5; Naples, Italy), showed highest activity at 100 °C and pH 5.5, and specific activity of maltotriose disproportionation at 75 °C of 1170 U/mg. ParGT effectively modified HAS granules under controlled heating (annealing) at 75 °C, altering crystallinity, surface order and chain length. Comparative analysis of native, heat-treated and ParGT-modified HAS granules from wheat, potato, maize, and barley revealed distinct effects of botanical source, enzymatic modification, and heating. Notably, ParGT increased the resistant starch (RS) contents in wheat and potato HASs subjected to in vitro digestion. Interfacial kinetics correlated the increased resistance to decreased density of glucoamylase attack sites. Overall, ParGT showed strong potential in enzyme- and hydro-thermal modifications developing starch-based ingredients for health and food applications.

α-L-Rhamnosidases are a class of glycosyl hydrolases (GHs) that catalyze the hydrolysis of terminal α-L-rhamnose residues from diverse glycoconjugates. While extensively characterized in bacterial and fungal sources, no archaeal α-L-rhamnosidases have been characterized to date. Herein, we report the identification and characterization of the first thermostable archaeal α-L-rhamnosidase (ArRha), derived from the metagenomic data set of Pisciarelli solfatara hot spring. ArRha, classified in glycoside hydrolase family GH78, efficiently hydrolyzes α-1,2 and α-1,6 rhamnosyl linkages in flavonoid glycosides with notable biological activities. The novel enzyme showed remarkable temperature stability, wide-range pH activity, organic solvent tolerance, and no metal dependence. Combined with a thermostable β-glucosidase, ArRha converts naringin to prunin and naringenin in sweet and blood orange juices, achieving >95% conversion within 2 h at 65 °C. This represents the first report of a hyperthermostable archaeal GH78 α-L-rhamnosidase with promising applications in industrial enzymatic juice debittering and sustainable flavonoid biotransformation.

Hydrogen is a fundamental electron donor in diverse microbial metabolisms and it is considered the energetic currency exchanged within microbial communities in anaerobic environments. Hydrogen is also the major actor in the transition to alternative low-carbon energy sources, primarily due to its dual role as energy source and energy carrier and to the production of water as a byproduct of its combustion. The geological storage of hydrogen gas produced from diverse sources in stable terrestrial reservoirs, known also as Underground Hydrogen Storage (UHS), is a key prerequisite to decouple production from utilization. UHS targets include depleted porous natural gas reservoirs, salt caverns and deep aquifers. Studies carried out in the past 30 years have unveiled a large subsurface ecosystem able to interact with the geochemical cycles and volatiles present in Earth’s crust. When hydrogen is stored underground, the microbial communities present in situ can interact with it, consuming it as electron donor, potentially producing undesired metabolic byproducts capable of affecting the success of UHS operations. Additionally, subsurface microbial communities might impact the geological production, migration and accumulation of hydrogen in natural reservoirs. Here, we review the current state of knowledge in hydrogenotrophic metabolisms capable of affecting UHS operations and natural hydrogen prospecting, and discuss how the microbiology of natural hydrogen-rich springs can be used as analog to model the state space of hydrogen operations. We discuss our current knowledge of the limits of life in the context of hydrogen economy, and the complex trophic network that hydrogen might sustain in the subsurface. While energy demands increase globally, the ability to effectively operate geological hydrogen storage and identify natural hydrogen deposits will become a key prerequisite to reduce the global carbon footprint. Understanding the potential for microbes to interact with hydrogen in the subsurface is therefore at the forefront of the ecological transition.

GH31 glycosidases are widespread across organisms, but remarkably, less than 1% of them have been biochemically characterised to date. Among them, human lysosomal acid α-glucosidase (GAA) stands out due to its link to Pompe disease, a rare lysosomal storage disorder caused by its deficiency. This disease results in glycogen accumulation, severe cellular damage, motor impairment, and premature death. Structural and functional studies of GAA mutants are challenging due to their instability and lack of activity, hindering their expression and purification. The GH31 enzyme MalA from a hyperthermophilic archaeon is explored here as a stable homolog of GAA. MalA is highly expressible, easy to purify, and structurally characterised. The R400H mutant in MalA, corresponding to the pathogenic GAA R600H mutation, revealed here a 1200-fold drop in specificity constant and >8 °C reduction in thermal stability. We propose MalA’s as a robust model for studying GAA mutations and developing therapeutic chaperones.

We report herein the design and synthesis of a series of 5-C-alkyldeoxynojirimycins from l-sorbose, through an efficient and scalable method amenable to preparing a large variety of analogues. The interaction of this class of compounds with human acid α-glucosidase (GAA), the genetically defective enzyme in patients suffering from Pompe disease, was investigated to identify pharmacological chaperones exhibiting high selectivity for this enzyme. Crystallographic analyses provided a rationale for their binding mode to GAA and chaperone activity. The effects of 5-C-phenethyl-DNJ (4c) were evaluated on GAA activity enhancement in cells from Pompe disease patients and in vivo in GAA-KO mice. The significant increase of GAA activity in the presence of 4c in various tissues, particularly in the diaphragm, encourages further studies on this class of small molecules toward developing clinical drugs. Their chaperone activity and excellent selectivity may offer potential benefits over the current treatments for Pompe disease.

Trace metals play an essential role in the metabolism of all living organisms and many metal-containing enzymes contribute to key physiological and ecological processes such as aerobic and anaerobic respiration, photosynthesis, carbon and nitrogen fixation. Despite this, trace metals' potential to control functional diversity and metabolic shifts is unknown. Here we demonstrate that the availability of trace metals controls electron acceptors utilization in Escherichia coli. Physiological and proteomic data show that trace metals depleted cultures have significantly reduced growth, anticipate fermentation and increase energy expenditure for metal homeostasis, even when more energetically favourable electron acceptors are present. Overall these results suggest how evolutionary and competitive pressures arising from changes in biological trace metals availability in deep time have contributed to shaping evolution and competition. ### Competing Interest Statement The authors have declared no competing interest.

Abstract Background Spent coffee grounds (SCG) are the most abundant waste byproducts generated from coffee beverage production worldwide. Typically, these grounds are seen as waste and end up in landfills. However, SCG contain valuable compounds that can be valorized and used in different applications. Notably, they are rich in carbohydrates, primarily galactomannan, arabinogalactan type II, and cellulose. Within the framework of a circular bioeconomy, the targeted degradation of these polysaccharides via a tailored cocktail of carbohydrate-active enzymes offers a promising strategy for producing high-value saccharides from coffee waste. Results In this study, various mild pretreatments were evaluated to increase the enzyme accessibility of SCG-derived biomass, reduce lignin content, and minimize hemicellulose loss. Thermostable enzymes were selected to construct an enzymatic cocktail specifically targeting cellulose and hemicelluloses in pretreated SCGs. The approach used achieved a conversion of 52% of the polysaccharide content to oligo- and monosaccharides, producing 17.4 mg of reducing sugars and 5.1 mg of monosaccharides from 50 mg of SCG. Additionally, microwave pretreatment followed by the application of a thermostable endo β-mannanase resulted in the production of 62.3 mg of mannooligosaccharides from 500 mg of SCG. In vitro experiments demonstrated that the produced mannooligosaccharides exhibited prebiotic activity, promoting the growth and biofilm formation of five probiotic bacterial strains. Conclusions This study highlights an effective strategy for the valorization of SCG polysaccharides through mild pretreatment and customized enzymatic cocktails in a circular bioeconomic context. The production of both monosaccharides and oligosaccharides with prebiotic activity illustrates the versatility and commercial potential of SCG as a substrate for high-value saccharides. Furthermore, the use of mild pretreatment methods and thermostable enzymes minimizes chemical inputs and energy demands, aligning with sustainable processing practices. The ability to selectively target and degrade specific polysaccharides within SCG not only enhances the yield of desirable products, but also preserves key structural components, reducing waste and promoting resource efficiency.

Single-use plastics, such as polyethylene terephthalate (PET), have become an overwhelming environmental issue due to their persistence and accumulation. The discovery of a class of biocatalysts (PETase) capable of breaking down PET into its monomeric components has opened new possibilities for sustainable PET recycling. In this study, we identified a novel thermophilic PETase, called Depolymerising Isolated Enzyme from Geothermal Origin (DIEGO), from geothermal metagenomic samples. DIEGO was produced in recombinant form and its ability to hydrolyse PET and its analogues, including bis(2-hydroxyethyl)-TPA (BHET) and nanoPET, as well as post-consumer PET (PcPET) food trays, was proved. We observed a synergistic effect of DIEGO and temperature at 60 degrees C: the enzyme converts PET into ethylene glycol (EG), terephthalic acid (TPA), along with the intermediates mono(2-hydroxyethyl)-TPA (MHET) and BHET; while temperature promotes auto-hydrolysis of MHET and BHET units. Therefore, this combined effect allowed the complete degradation of PET in TPA and EG within four days, by-passing the use of an additional enzyme for the degradation of intermediates. These findings highlight DIEGO as a promising candidate for industrial-scale enzymatic PET recycling and contribute to the development of sustainable plastic waste management strategies.

Glycoside hydrolases (GHs) have been employed for industrial and biotechnological purposes and often play an important role in new applications. The red blood cell (RBC) antigen system depends on the composition of oligosaccharides on the surface of erythrocytes, thus defining the ABO blood type classification. Incorrect blood transfusions may lead to fatal consequences, making the availability of the correct blood group critical. In this regard, it has been demonstrated that some GHs may be helpful in the conversion of groups A and B blood types to produce group O universal donor blood. GHs belonging to the GH109 family are of particular interest for this application due to their ability to convert blood from group A to group O. This work describes the biochemical characterisation of three novel GH109 enzymes (NAg68, NAg69 and NAg71) and the exploration of their ability to produce enzymatically converted RBCs (ECO-RBC). The three enzymes showed superior specificity on pNP-α-N-acetylgalactosamine compared to previously reported GH109 enzymes. These novel enzymes were able to act on purified antigen-A trisaccharides and produce ECO-RBC from human donor blood. NAg71 converted type A RBC to group O with increased efficiency in the presence of dextran compared to a commercially available GH109, previously used for this application.

On August 26, 2023, we bid farewell to Professor Mosè Rossi, a distinguished figure in the field of enzymology and a beloved member of the International Society for Extremophiles since 1993. Born in Castellabate (Salerno) in 1938, Professor Rossi embarked on a remarkable journey in the world of science.

Polyethylene terephthalate (PET) degradation by enzymatic hydrolysis is significant for addressing plastic pollution and fostering sustainable waste management practices. Identifying thermophilic and thermostable PET hydrolases is particularly crucial for industrial bioprocesses, where elevated temperatures may enhance enzymatic efficiency and process kinetics. In this study, we present the discovery of a novel thermophilic and thermostable PETase enzyme named Sis, obtained through metagenomic sequence-based analysis. Sis exhibits robust activity on nanoPET substrates, demonstrating effectiveness at temperatures up to 70 °C and displaying exceptional thermal stability with a melting temperature (Tm) of 82 °C. Phylogenetically distinct from previously characterised PET hydrolases, Sis represents a valuable addition to the repertoire of enzymes suitable for PET degradation.

The space race is entering a new era of exploration, in which the number of robotic and human missions to various places in our solar system is rapidly increasing. Despite the recent advances in propulsion and life support technologies, there is a growing need to perform analytical measurements and laboratory experiments across diverse domains of science, while keeping low payload requirements. In this context, lab-on-a-chip nanobiosensors appear to be an emerging technology capable of revolutionizing space exploration, given their low footprint, high accuracy, and low payload requirements. To date, only some approaches for monitoring astronaut health in spacecraft environments have been reported. Although non-invasive molecular diagnostics, like lab-on-a-chip technology, are expected to improve the quality of long-term space missions, their application to monitor microbiological and environmental variables is rarely reported, even for analogous extreme environments on Earth. The possibility of evaluating the occurrence of unknown or unexpected species, identifying redox gradients relevant to microbial metabolism, or testing for specific possible biosignatures, will play a key role in the future of space microbiology. In this review, we will examine the current and potential roles of lab-on-a-chip technology in space exploration and in extreme environment investigation, reporting what has been tested so far, and clarifying the direction toward which the newly developed technologies of portable lab-on-a-chip sensors are heading for exploration in extreme environments and in space.

Archaea represents the third domain of life, displaying a closer relationship with eukaryotes than bacteria. These microorganisms are valuable model systems for molecular biology and biotechnology. In fact, nowadays, methanogens, halophiles, thermophilic euryarchaeota, and crenarchaeota are the four groups of archaea for which genetic systems have been well established, making them suitable as model systems and allowing for the increasing study of archaeal genes' functions. Furthermore, thermophiles are used to explore several aspects of archaeal biology, such as stress responses, DNA replication and repair, transcription, translation and its regulation mechanisms, CRISPR systems, and carbon and energy metabolism. Extremophilic archaea also represent a valuable source of new biomolecules for biological and biotechnological applications, and there is growing interest in the development of engineered strains. In this review, we report on some of the most important aspects of the use of archaea as a model system for genetic evolution, the development of genetic tools, and their application for the elucidation of the basal molecular mechanisms in this domain of life. Furthermore, an overview on the discovery of new enzymes of biotechnological interest from archaea thriving in extreme environments is reported.

(Hyper)thermophilic archaeal glycosidases are enzymes that catalyze the hydrolysis of glycosidic bonds to break down complex sugars and polysaccharides at high temperatures. These enzymes have an unique structure that allows them to remain stable and functional in extreme environments such as hot springs and hydrothermal vents. This review provides an overview of the current knowledge and milestones on the structures and functions of (hyper)thermophilic archaeal glycosidases and their potential applications in various fields. In particular, this review focuses on the structural characteristics of these enzymes and how these features relate to their catalytic activity by discussing different types of (hyper)thermophilic archaeal glycosidases, including β-glucosidases, chitinase, cellulases and α-amylases, describing their molecular structures, active sites, and mechanisms of action, including their role in the hydrolysis of carbohydrates. By providing a comprehensive overview of (hyper)thermophilic archaeal glycosidases, this review aims to stimulate further research into these fascinating enzymes.