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The increasing interest for environmentally friendly technologies is driving the transition from fossil-based economy to bioeconomy. A key enabler for circular bioeconomy is to valorize renewable biomasses as feedstock to extract high value-added chemicals. Within this transition the discovery and the use of robust biocatalysts to replace toxic chemical catalysts play a significant role as technology drivers. To meet both the demands, we performed microbial enrichments on two energy crops, used as low-cost feed for extremophilic consortia. A culture-dependent approach coupled to metagenomic analysis led to the discovery of more than 300 glycoside hydrolases and to characterize a new α-glucosidase from an unknown hyperthermophilic archaeon. Aglu1 demonstrated to be the most active archaeal GH31 on 4Np-α-Glc and it showed unexpected specificity vs. kojibiose, revealing to be a promising candidate for biotechnological applications such as the liquefaction/saccharification of starch.

The enzymes from hyperthermophilic microorganisms populating volcanic sites represent interesting cases of protein adaptation and biotransformations under conditions where conventional enzymes quickly denature. The difficulties in cultivating extremophiles severely limit access to this class of biocatalysts. To circumvent this problem, we embarked on the exploration of the biodiversity of the solfatara Pisciarelli, Agnano (Naples, Italy), to discover hyperthermophilic carbohydrate-active enzymes (CAZymes) and to characterize the entire set of such enzymes in this environment (CAZome). Here, we report the results of the metagenomic analysis of two mud/water pools that greatly differ in both temperature and pH (T = 85 °C and pH 5.5; T = 92 °C and pH 1.5, for Pool1 and Pool2, respectively). DNA deep sequencing and following in silico analysis led to 14 934 and 17 652 complete ORFs in Pool1 and Pool2, respectively. They exclusively belonged to archaeal cells and viruses with great genera variance within the phylum Crenarchaeota, which reflected the difference in temperature and pH of the two Pools. Surprisingly, 30% and 62% of all of the reads obtained from Pool1 and 2, respectively, had no match in nucleotide databanks. Genes associated with carbohydrate metabolism were 15% and 16% of the total in the two Pools, with 278 and 308 putative CAZymes in Pool1 and 2, corresponding to ~ 2.0% of all ORFs. Biochemical characterization of two CAZymes of a previously unknown archaeon revealed a novel subfamily GH5_19 β-mannanase/β-1,3-glucanase whose hemicellulose specificity correlates with the vegetation surrounding the sampling site, and a novel NAD+ -dependent GH109 with a previously unreported β-N-acetylglucosaminide/β-glucoside specificity. DATABASES: The sequencing reads are available in the NCBI Sequence Read Archive (SRA) database under the accession numbers SRR7545549 (Pool1) and SRR7545550 (Pool2). The sequences of GH5_Pool2 and GH109_Pool2 are available in GenBank database under the accession numbers MK869723 and MK86972, respectively. The environmental data relative to Pool1 and Pool2 (NCBI BioProject PRJNA481947) are available in the Biosamples database under the accession numbers SAMN09692669 (Pool1) and SAMN09692670 (Pool2).

In the field of biocatalysis and the development of a bio-based economy, hemicellulases have attracted great interest for various applications in industrial processes. However, the study of the catalytic activity of the lignocellulose-degrading enzymes needs to be improved to achieve the efficient hydrolysis of plant biomasses. In this framework, hemicellulases from hyperthermophilic archaea show interesting features as biocatalysts and provide many advantages in industrial applications thanks to their stability in the harsh conditions encountered during the pretreatment process. However, the hemicellulases from archaea are less studied compared to their bacterial counterpart, and the activity of most of them has been barely tested on natural substrates. Here, we investigated the hydrolysis of xyloglucan oligosaccharides from two different plants by using, both synergistically and individually, three glycoside hydrolases from Saccharolobus solfataricus: a GH1 β-gluco-/β-galactosidase, a α-fucosidase belonging to GH29, and a α-xylosidase from GH31. The results showed that the three enzymes were able to release monosaccharides from xyloglucan oligosaccharides after incubation at 65 °C. The concerted actions of β-gluco-/β-galactosidase and the α-xylosidase on both xyloglucan oligosaccharides have been observed, while the α-fucosidase was capable of releasing all α-linked fucose units from xyloglucan from apple pomace, representing the first GH29 enzyme belonging to subfamily A that is active on xyloglucan.

Understanding the formation of biogenic molecules in abiotic conditions is a prerequisite in the origin-of-life studies. Determining the conditions allowing an efficient one-pot synthesis of the largest possible panel of biogenic compounds may shed light on the plausible scenario in which the processes that kick-started life might have occurred. We report a set of experiments describing the syntheses taking place from formamide (NH2CHO) and thermal water in the presence of meteorites, in the hydrothermal physical-chemical environment of the Phlegrean Fields. The results show that meteorites catalyse the synthesis of a large panel of organic compounds of biological relevance, including carboxylic acids, nucleobases, amino acids and sugars. The simplicity of the system (a one-carbon molecule as starting compound, a volcanic hydrothermal environment, meteorites as catalysts) hints to a possible extension of the results to similar environments present in other planetary bodies and space objects.

The highly stereocontrolled de novo synthesis of L-NBDNJ (the unnatural enantiomer of the iminosugar drug Miglustat) and a preliminary evaluation of its chaperoning potential are herein reported. L-NBDNJ is able to enhance lysosomal α-glucosidase levels in Pompe disease fibroblasts, either when administered singularly or when co-incubated with the recombinant human α-glucosidase. In addition, differently from its D-enantiomer, L-NBDNJ does not act as a glycosidase inhibitor.

Chemo-enzymatic synthesis of oligosaccharides exploits the diversity of glycosidases and their ability to promote transglycosylation reactions in parallel with hydrolysis. Methods to increase the transglycosylation/hydrolysis ratio include site-directed mutagenesis and medium modification. The former approach was successful in several cases and has provided the best synthetic yields with glycosynthases-mutants at the catalytic nucleophile position that promote transglycosylation with high efficiency, but do not hydrolyze the oligosaccharide products. Several glycosidases have proven recalcitrant to this conversion, thus alternative methods to increase the transglycosylation/hydrolysis ratio by mutation would be very useful. Here we show that a mutant of a β-galactosidase from Alicyclobacillus acidocaldarius in an invariant residue in the active site of the enzymes of this family (glutamic acid 361) carries out efficient transglycosylation reactions on different acceptors only in the presence of external ions with yields up to 177-fold higher than that of the wild type. This is the first case in which sodium azide and sodium formate in combination with site-directed mutagenesis have been used to introduce transglycosylation activity into a glycosidase. These observations will hopefully guide further efforts to generate useful synthases.

Peach and apricot can cause allergic reactions with symptoms ranging from mild to very severe, including anaphylaxis. Sometimes subjects allergic to fruits of the Prunus genus have been reported to be also allergic to rubber latex products. The objective of this study is the characterization of a newly identified peach and apricot protein showing similarities with the allergens Hey b 5 from rubber latex and Man e 5 from manioc. This protein has been named ENEA on the basis of the single letter amino acid code of the first four N-terminal residues of the isolated molecule. It has been found in very variable amounts in different peach cultivars and batches. ENEA was isolated from peach pulp extracts by chromatographic separations and identified by direct protein sequencing. At that time, the full length sequence was available only for the homologous protein of the taxonomically closely related apricot, which was produced as a recombinant molecule in Escherichia coli. The following availability of the full length sequence of peach ENEA revealed a very high identity (97%) with the apricot homolog. Similarly to Hey b 5 and to Man e 5, the structural characterization indicated that ENEA is an intrinsically disordered protein. The immunological properties, investigated by dot blotting, the ABA system and the FABER test, showed that ENEA is recognized by specific IgE of allergic patients. In a selected population of 31 patients reporting allergic reactions to peach fruit and/or IgE positive to Hey b 5, 28 and 27 subjects resulted co-sensitized to rENEA and Hey b 5 in the ABA and ISAC test, respectively. In a random population of 3305 suspected allergic patients, analyzed with the FABER test, 17 of them were sensitized to rENEA and 10 of them were also positive to Hey b 5. In addition, both the natural molecule from peach and the recombinant protein of apricot partially inhibited the IgE binding to Hey b 5. In conclusion, a new peach and apricot IgE-binding protein, cross-reacting with the major latex allergen Hey b 5, has been identified. Its variable concentration in the fruit might explain some occasionally occurring allergic reactions. The apricot molecule has recently been registered by the WHO/ IUIS Allergen Nomenclature Sub-Committee with the allergen name Pru ar 5. The recombinant form of apricot ENEA, now available, will contribute to allergy diagnosis.

Sulfolobus solfataricus is an aerobic crenarchaeal hyperthermophile with optimum growth at temperatures greater than 80°C and pH 2 to 4. Within the crenarchaeal group of Sulfolobales, N-acetylglucosamine (GlcNAc) has been shown to be a component of exopolysaccharides, forming their biofilms, and of the N-glycan decorating some proteins. The metabolism of GlcNAc is still poorly understood in Archaea, and one approach to gaining additional information is through the identification and functional characterization of carbohydrate active enzymes (CAZymes) involved in the modification of GlcNAc. The screening of S. solfataricus extracts allowed the detection of a novel α-N-acetylglucosaminidase (α-GlcNAcase) activity, which has never been identified in Archaea. Mass spectrometry analysis of the purified activity showed a protein encoded by the sso2901 gene. Interestingly, the purified recombinant enzyme, which was characterized in detail, revealed a novel de-N-acetylase activity specific for GlcNAc and derivatives. Thus, assays to identify an α-GlcNAcase found a GlcNAc de-N-acetylase instead. The α-GlcNAcase activity observed in S. solfataricus extracts did occur when SSO2901 was used in combination with an α-glucosidase. Furthermore, the inspection of the genomic context and the preliminary characterization of a putative glycosyltransferase immediately upstream of sso2901 (sso2900) suggest the involvement of these enzymes in the GlcNAc metabolism in S. solfataricus. IMPORTANCE In this study, a preliminary screening of cellular extracts of S. solfataricus allowed the identification of an α-N-acetylglucosaminidase activity. However, the characterization of the corresponding recombinant enzyme revealed a novel GlcNAc de-N-acetylase, which, in cooperation with the α-glucosidase, catalyzed the hydrolysis of O-α-GlcNAc glycosides. In addition, we show that the product of a gene flanking the one encoding the de-N-acetylase is a putative glycosyltransferase, suggesting the involvement of the two enzymes in the metabolism of GlcNAc. The discovery and functional analysis of novel enzymatic activities involved in the modification of this essential sugar represent a powerful strategy to shed light on the physiology and metabolism of Archaea.

In the framework of the discovery of new thermophilic enzymes of potential biotechnological interest, we embarked in the characterization of a new thermophilic esterase from the thermophilic bacterium Geobacillus thermodenitrificans. The phylogenetic analysis of the GTNG_0744 esterase indicated that the sequence belongs to the enterochelin/enterobactin esterase group, which have never been recognized as a family in the lipases/esterase classification. These enzymes catalyze the last step in the acquisition of environmental Fe3+ through siderophore hydrolysis. In silico analysis revealed, for the first time, that the machinery for the uptake of siderophores is present in G. thermodenitrificans. The purified recombinant enzyme, EstGtA3, showed different substrate specificity from known enterochelin/enterobactin esterases, recognizing short chain esters with a higher specificity constant for 4-NP caprylate. The enzyme does not require cofactors for its activity, is active in the pH range 7.0–8.5, has highest activity at 60 °C and is 100% stable when incubated for 16 h at 55 °C. DTT, β-mercaptoethanol and Triton X-100 have an activating effect on the enzymatic activity. Organic solvents have in general a negative effect on the enzyme, but n-hexane is a strong activator up to 150, making EstGtA3 a good candidate for applications in biotechnology.

CAZypedia was initiated in 2007 to create a comprehensive, living encyclopedia of the carbohydrate-active enzymes (CAZymes) and associated carbohydrate-binding modules involved in the synthesis, modification, and degradation of complex carbohydrates. CAZypedia is closely connected with the actively-curated CAZy database, which provides a sequence-based foundation for the biochemical, mechanistic, and structural characterization of these diverse proteins. Now celebrating its 10th anniversary online, CAZypedia is a successful example of dynamic, community-driven, and expert-based biocuration. CAZypedia is an open-access resource available at URL http://www.cazypedia.org.

We report here on the preparation of a novel ?-thioglycoligase that can be used for the fast and efficient synthesis of ?-N-acetylglucosamine-based glycosides. Using the ?-N-acetyl-glucosaminidase from Clostridium perfringens of family GH89 (according to the Carbohydrate Active Enzymes classification) as starting point, we prepared mutants in the acid/base residue glutamic acid 483 that were found to have different synthetic efficiencies (maximal yields >80% after 24 h) in the presence of an activated donor and suitable acceptors. The synthetic potential of the Glu483 alanine mutant as an ?-thioglycoligase - only the third biocatalyst with this stereospecificity reported to date, and the first selective for the N-acetylglucosamine moiety - was demonstrated by producing for the first time N-acetyl-?-d-glucosaminyl azide and N-acetylglucosamine ?-thioglycosides in high yields. To showcase the application of such compounds, we show that they offer the wild-type CpGH89 protection from thermal unfolding, demonstrating their potential as pharmacological chaperones for the treatment of mucopolysaccharidosis IIIB (Sanfilippo syndrome). (Figure presented.).

The field of thermophilic microbiology was born in the late 1970s with the pioneering work of Brock (Thermophiles biodiversity, ecology, and evolution. Springer, Boston, pp. 1–9, 2001) and dramatically expanded through the ’80s with the isolation of hyperthermophiles by Stetter (FEMS Microbiol Rev 18:149–158, 1996). The development of SSU rRNA phylogenetics revealed the complexity and diversity of prokaryotic phylotypes on biotopes widely differing in extreme conditions (e.g. spanning gradients of pH between 0 and 10 and temperatures from 60 °C to over 120 °C, respectively). Sites of volcanic activity all over the Earth’s surface and under the sea provide a variety of different environments for extremophilic microorganisms. Hot springs populated by hyperthermophiles (Topt > 65 °C), the majority of which belonging to the domain of Archaea, are very diverse and some of them show combinations of other extreme conditions, for example, acidic, alkaline, high pressure, and high concentrations of salts and heavy metals (Cowan et al. in Curr Opin Microbiol 25:97–102, 2015). Archaea inhabiting hot springs are considered to be the closest living descendants of the earliest living forms on Earth and their study provide insights into the origin and evolution of life (Woese et al. in Proc Natl Acad Sci USA 87:4576–4579, 1990; Olsen et al. in J Bacteriol 176:1–6, 1994). As with all studies of environmental microbiology, our understanding of the function of (hyper)thermophilic microbial consortia has lagged substantially behind. However, recent advances in ‘omics’ technologies, particularly within a system biology context, have made significant progresses into the prediction of in situ functionality (Cowan et al. in Curr Opin Microbiol 25:97–102, 2015). Most extremophilic microorganisms are recalcitrant to cultivation-based approaches (Amann et al. in Microbiol Rev 59:143–69, 1995; Lorenz et al. in Curr Opin Biotechnol 13:572–577, 2002); therefore, culture-independent metagenomic strategies are promising approaches to assess the phylogenetic composition and functional potential of microbial communities living in extreme environments (Lopez-Lopez et al. in Life 3:308–320, 2013). In addition, these approaches implement tremendously the access to enzymes from (hyper)thermophilic microorganisms that have important potential applications in several biotechnological processes. We report here on the state-of-the-art of the metagenomic surveys of different hot springs (T > 65 °C) (Table 5.1) and on the recent advance in the discovery of new hyperthermostable biocatalysts of biotechnological interest from metagenomic studies of these extreme environments.

Geothermally heated regions of Earth, such as terrestrial volcanic areas (fumaroles, hot springs, and geysers) and deep-sea hydrothermal vents, represent a variety of different environments populated by extremophilic archaeal and bacterial microorganisms. Since most of these microbes thriving in such harsh biotopes, they are often recalcitrant to cultivation; therefore, ecological, physiological and phylogenetic studies of these microbial populations have been hampered for a long time. More recently, culture-independent methodologies coupled with the fast development of next generation sequencing technologies as well as with the continuous advances in computational biology, have allowed the production of large amounts of metagenomic data. Specifically, these approaches have assessed the phylogenetic composition and functional potential of microbial consortia thriving within these habitats, shedding light on how extreme physico-chemical conditions and biological interactions have shaped such microbial communities. Metagenomics allowed to better understand that the exposure to an extreme range of selective pressures in such severe environments, accounts for genomic flexibility and metabolic versatility of microbial and viral communities, and makes extreme- and hyper-thermophiles suitable for bioprospecting purposes, representing an interesting source for novel thermostable proteins that can be potentially used in several industrial processes.

// Giancarlo Vecchio 1, 2, * , Alessia Parascandolo 3, * , Chiara Allocca 1 , Clara Ugolini 4 , Fulvio Basolo 5 , Marco Moracci 6, 7 , Andrea Strazzulli 6, 7 , Beatrice Cobucci-Ponzano 6 , Mikko O. Laukkanen 3 , Maria Domenica Castellone 8 , Nobuo Tsuchida 9 1 Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universita di Napoli Federico II, Via Sergio Pansini, 5, Naples, Italy 2 Istituto Superiore di Oncologia, Via Sergio Pansini, 5, Naples and Via Balbi 5, Genoa, Italy 3 IRCCS SDN, Naples, Italy 4 Dipartimento di Medicina di Laboratorio, Azienda Ospedaliero-Universitaria Pisana, Pisa, Italy 5 Dipartimento di Patologia Chirurgica, Medica, Molecolare e dell’Area Critica, University of Pisa, Italy 6 Institute of Biosciences and Bioresources (IBBR), National Research Council of Italy (CNR), Via P. Castellino, 111, Naples, Italy 7 Department of Biology, University of Naples “Federico II”, Complesso Universitario di Monte S. Angelo, Naples, Italy 8 Istituto di Endocrinologia ed Oncologia Sperimentale “G. Salvatore” (IEOS), CNR, Naples, Italy 9 Graduate School of Medical and Dental Sciences Tokyo Medical and Dental University, Tokyo, Japan * These authors have contributed equally to this work Correspondence to: Maria Domenica Castellone, email: mcastell@unina.it Nobuo Tsuchida, email: ntutid.eme@tmd.ac.jp Keywords: glycosylation, fucosidase, thyroid carcinoma, metastasis Received: November 24, 2016 Accepted: January 24, 2017 Published: February 23, 2017 ABSTRACT Glycans containing α-L-fucose participate in diverse interactions between cells and extracellular matrix. High glycan expression on cell surface is often associated with neoplastic progression. The lysosomal exoenzyme, α-L-fucosidase-1 (FUCA-1) removes fucose residues from glycans. The FUCA-1 gene is down-regulated in highly aggressive and metastatic human tumors. However, the role of FUCA-1 in tumor progression remains unclear. It is speculated that its inactivation perturbs glycosylation of proteins involved in cell adhesion and promotes cancer. FUCA-1 expression of various thyroid normal and cancer tissues assayed by immunohistochemical (IHC) staining was high in normal thyroids and papillary thyroid carcinomas (PTC), whereas it progressively decreased in poorly differentiated, metastatic and anaplastic thyroid carcinomas (ATC). FUCA-1 mRNA expression from tissue samples and cell lines and protein expression levels and enzyme activity in thyroid cancer cell lines paralleled those of IHC staining. Furthermore, ATC-derived 8505C cells adhesion to human E-selectin and HUVEC cells was inhibited by bovine α-L-fucosidase or Lewis antigens, thus pointing to an essential role of fucose residues in the adhesive phenotype of this cancer cell line. Finally, 8505C cells transfected with a FUCA-1 containing plasmid displayed a less invasive phenotype versus the parental 8505C. These results demonstrate that FUCA-1 is down-regulated in ATC compared to PTC and normal thyroid tissues and cell lines. As shown for other human cancers, the down-regulation of FUCA-1 correlates with increased aggressiveness of the cancer type. This is the first report indicating that the down-regulation of FUCA-1 is related to the increased aggressiveness of thyroid cancer.

Translational recoding includes a group of events occurring during gene translation, namely stop codon readthrough, programmed +/- 1 frameshifting, and ribosome bypassing, which have been found in organisms from all domains of life. They serve to regulate protein expression at translational level and represent a relatively less known exception to the traditional central 'dogma' of biology that information flows as DNA -> RNA -> protein and that it is stored in a co-linear way between the 5'-> 3' of nucleic acids and N -> C-terminal of polypeptides. In archaea, in which translational recoding regulates the decoding of the 21st and the 22nd amino acids selenocysteine and pyrrolysine, respectively, only one case of programmed -1 frameshifting has been reported so far and further examples, although promising, have not been confirmed yet. We here summarize the current state-of-the-art of this field that, especially in archaea, has relevant implications for the physiology of life in extreme environments and for the origin of life.

Glycosynthases, engineered glycoside hydrolases that are able to synthesize glycans in quantitative yields without hydrolyzing them, are among the most interesting tools for the chemoenzymatic synthesis of carbohydrates that have been made available so far. From their invention in 1998, these enzymes have been developed enormously and demonstrated to be convenient alternatives to the more expensive glycosyltransferase. Glycosynthases have been proved to be efficient catalysts for the synthesis of oligosaccharides, polysaccharides, and glycoconjugates of various lengths containing different monosaccharides bound through alpha- and beta-linkages, which have interesting applications. In this review we describe the impact that glycosynthases have had on the biocatalysis and biosynthesis of carbohydrates through the description of the novel activities produced and of the development of the glycosynthase-based process. In addition, we summarize the approaches that, according to the current literature, can be adopted to produce novel glycosynthase activities and to improve the existing process.

Carbohydrate active enzymes (CAZymes) are a large class of enzymes, which build and breakdown the complex carbohydrates of the cell. On the basis of their amino acid sequences they are classified in families and clans that show conserved catalytic mechanism, structure, and active site residues, but may vary in substrate specificity. We report here the identification and the detailed molecular characterization of a novel glycoside hydrolase encoded from the gene sso1353 of the hyperthermophilic archaeon Sulfolobus solfataricus. This enzyme hydrolyzes aryl β-gluco- and β-xylosides and the observation of transxylosylation reactions products demonstrates that SSO1353 operates via a retaining reaction mechanism. The catalytic nucleophile (Glu-335) was identified through trapping of the 2-deoxy-2-fluoroglucosyl enzyme intermediate and subsequent peptide mapping, while the general acid/base was identified as Asp-462 through detailed mechanistic analysis of a mutant at that position, including azide rescue experiments. SSO1353 has detectable homologs of unknown specificity among Archaea, Bacteria, and Eukarya and shows distant similarity to the non-lysosomal bile acid β-glucosidase GBA2 also known as glucocerebrosidase. On the basis of our findings we propose that SSO1353 and its homologs are classified in a new CAZy family, named GH116, which so far includes β-glucosidases (EC 3.2.1.21), β-xylosidases (EC 3.2.1.37), and glucocerebrosidases (EC 3.2.1.45) as known enzyme activities.

Glycosynthases are mutant glycosidases, which in the presence of activated glycosides and suitable reaction conditions, synthesize oligosaccharides without hydrolysing them. This feature makes these catalysts promising tools for the large scale synthesis of carbohydrates. However, despite the popularity of the glycosynthetic approach, the number of enzymes effecting glycosynthetic reactions is still limited. We report here on the design of novel reaction conditions for a thermophilic α-l-fucosidase mutant, which might provide a route for the production of novel glycosynthases.

The study of the complex communities of hyperthermophiles, composed of Bacteria, Archaea, and viruses, populating volcanic sites is of interest to understand their adaptation and evolution to extreme conditions and as a rich source of biocatalysts [1, 2]. In fact, the intrinsic stability of their enzymes to common protein denaturants made them interesting tools for biotransformations in industrial applications. In the framework of the discovery of new enzymes for biocatalysis and biotransformations, we decided to explore the diversity of the solfataric field of Pisciarelli, Agnano (Naples, Italy), by performing a metagenomic analysis of the microbial community living in two mud/water pools that, although distant only 4.0 meters, greatly differ in both temperature and pH (T=85°C and pH 5.5; T=94°C and pH 1.5, for Pool1 and Pool2, respectively). In particular, DNA from Pool1 was extracted from the mud/water suspension while Pool2 DNA was obtained from the sediment. The deep sequencing by Illumina technology and the following in-silico analysis showed that the phylum Crenarcheaota was prevalent, but with great genera variance in the two pools according to the different environmental T and pH. The analysis of the functional categories revealed that genes involved in the carbohydrate metabolism were prevalent, followed by amino acid metabolism and by membrane transporters. The detailed phylogenetic and physiological analysis of the samples will be discussed together with the possible mechanisms of selection occurred in the two extremophilic environments. Bibliography 1. Cobucci-Ponzano B., Aurilia V., Riccio G., Henrissat B., Coutinho P.M., Strazzulli A., Padula A., Corsaro M.M., Pieretti G., Pocsfalvi G., Fiume I., Cannio R., Rossi M., and Moracci M.: J Biol Chem, 2010, 20691-20703. 2. Cobucci-Ponzano, B., Conte, F., Strazzulli, A., Capasso, C., Fiume, I., Pocsfalvi, G., Rossi, M., Moracci.: M. Biochimie, 2010, 92, 1895-907.

Background Biofuels production from plant biomasses is a complex multi-step process with important economic burdens. Several biotechnological approaches have been pursued to reduce biofuels production costs. The aim of the present study was to explore the production in tobacco plastome of three genes encoding (hemi)cellulolytic enzymes from thermophilic and hyperthermophilic bacterium and Archaea, respectively, and test their application in the bioconversion of an important industrially pretreated biomass feedstock (A. donax) for production of second-generation biofuels. Results The selected enzymes, endoglucanase, endo-β-1,4-xylanase and β-glucosidase, were expressed in tobacco plastome with a protein yield range from 2 % to more than 75 % of total soluble proteins (TSP). The accumulation of endoglucanase (up to 2 % TSP) gave altered plant phenotypes whose severity was directly linked to the enzyme yield. The most severe seedling-lethal phenotype was due to the impairment of plastid development associated to the binding of endoglucanase protein to thylakoids. Endo-β-1,4-xylanase and β-glucosidase, produced at very high level without detrimental effects on plant development, were enriched (fourfold) by heat treatment (105.4 and 255.4 U/mg, respectively). Both plastid-derived biocatalysts retained the main features of the native or recombinantly expressed enzymes with interesting differences. Plastid-derived xylanase and β-glucosidase resulted more thermophilic than the E. coli recombinant and native counterpart, respectively. Bioconversion experiments, carried out at 50 and 60 °C, demonstrated that plastid-derived enzymes were able to hydrolyse an industrially pretreated giant reed biomass. In particular, the replacement of commercial enzyme with plastid-derived xylanase, at 60 °C, produced an increase of both xylose recovery and hydrolysis rate; whereas the replacement of both xylanase and β-glucosidase produced glucose levels similar to those observed with the commercial cocktails, and xylose yields always higher in the whole 24–72 h range. Conclusions The very high production level of thermophilic and hyperthermophilic enzymes, their stability and bioconversion efficiencies described in this study demonstrate that plastid transformation represents a real cost-effective production platform for cellulolytic enzymes. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0569-z) contains supplementary material, which is available to authorized users.

We report here on the preparation of a novel a-thioglycoligase that can be used for the fast and efficient synthesis of a-N-acetylglucosamine- based glycosides. Using the a-N-acetyl-glucosamini- dase from Clostridium perfr ingens of family GH89 (according to the Carbohydrate Active Enzymes classification) as starting point, we prepare d mutants in the acid/base residue glutamic acid 483 that were found to have different synthetic efficiencies (maxi-mal yields > 80% after 24 h) in the presence of an activated donor and suitable acceptors. The synthetic potential of the Glu483 alanine mutant as an a-thio-glycoligase - only the third biocatalyst with this ste-reospecificity reported to date, and the first selectivefor the N-acetylglucosamine moiety - was demon- strated by producing for the first time N-acetyl-a-d-glucosaminyl azide and N-acetylglucosamine a-thio-glycosides in high yields. To showcase the application of such compounds, we show that they offer the wild-type CpGH89 protection from thermal unfold-ing, demonstrating their potential as pharmacological chaperones for the treatment of mucopolysacchari-dosis IIIB (Sanfilippo syndrome)

The identification of the determinants of protein thermal stabilization is often pursued by comparing enzymes from hyperthermophiles with their mesophilic counterparts while direct structural comparisons among proteins and enzymes from hyperthermophiles are rather uncommon. Here, oligomeric beta-glycosidases from the hyperthermophilic archaea Sulfolobus solfataricus (Ss beta-gly), Thermosphaera aggregans (Ta beta-gly), and Pyrococcus furiosus (Pf beta-gly), have been compared. Studies of FTIR spectroscopy and kinetics of thermal inactivation showed that the three enzymes had similar secondary structure composition, but Ss beta-gly and Ta beta-gly (temperatures of melting 98.1 and 98.4 degrees C, respectively) were less stable than Pf beta-gly, which maintained its secondary structure even at 99.5 degrees C. The thermal denaturation of Pf beta-gly, followed in the presence of SDS, suggested that this enzyme is stabilized by hydrophobic interactions. A detailed inspection of the 3D-structures of these enzymes supported the experimental results: Ss beta-gly and Ta beta-gly are stabilized by a combination of ion-pairs networks and intrasubunit S-S bridges while the increased stability of Pf beta-gly resides in a more compact protein core. The different strategies of protein stabilization give experimental support to recent theories on thermophilic adaptation and suggest that different stabilization strategies could have been adopted among archaea.

Metagenomic analysis applied to specialized environments offers the possibility to understand the evolution of complex biotypes. In particular, metagenomics of extreme environments, which might resemble those of primitive Earth, is of interest for the study of the origin, evolution and distribution of extremophiles (organisms thriving at these conditions) as primitive forms of life populating our planet and, in an astrobiological perspective, other bodies in the universe. The Pisciarelli solfatara, located in the Phelgrean Fields (Naples, Italy), is characterized by a remarkable diversity in terms of temperature and pHs of the different mud pools and solfataric vents [1]. Here several thermoacidophilic species, such as Acidianus, Archaeoglobus spp, Pyrobaculum, Sulfolobus, and several viruses have been isolated [1]. In order to map the composition of this biotope in terms of number and abundance of the different species, we decided to carry out a metagenomic deep sequencing project on soil and water/mud pools of Pisciarelly solfatara. We will show our preliminary results derived by the sampling in the main water pool. Total DNA was extracted from the pellets obtained by centrifuging water samples and analysed by using Illumina paired-end sequencing technology. In a single run, we produced~3.0 Gb total nucleotides. These reads were assembled into 1,543 contigs with an N50 of 15863 bp，and a total of 17,652 Open Reading Frames (ORFs) were identified. These ORFs were subjected to BLAST similarity searches and annotated with KEGG, eggNOG and NR database respectively. The clean reads rate was 99.7% and a total of 17,652 Open Reading Frames (ORFs) were identified. The majority (77%) of the species found belonged to the Crenarcheaota phylum, which branches deeply in the phylogenetic tree of life, followed by Proteobacteria (22%), and, in much lesser amounts, Euryarchaeota, Actinobacteria, and others. The number of species identified is much higher than those found by a conventional PCR-based approach [2]. Details on the species composition and on the realtive abundance of each ORF will be described together with the astrobiological implications.

Summary Standard decoding of the genetic information into polypeptides is performed by one of the most sophisticated cell machineries, the translating ribo- some, which, by following the genetic code, ensures the correspondence between the mature mRNA and the protein sequence. However, the expression of a minority of genes requires programmed deviations from the standard decoding rules, globally named recoding . This includes ribosome programmed -/ + 1 frameshifting, ribosome hopping, and stop codon readthrough. Recoding in Archaea was unequivo- cally demonstrated only for the translation of the UGA stop codon into the amino acid selenocysteine. However, a new recoding event leading to the 22nd amino acid pyrrolysine and the preliminary reports on a gene regulated by programmed - 1 frameshifting have been recently described in Archaea. Therefore, it appears that the study of this phenomenon in Archaea is still at its dawn and that most of the genes whose expression is regulated by recoding are still uncharacterized.

Carbohydrates serve as structural components and en- ergy sources of cells. More interestingly, however, these biomolecules are involved in a variety of molecular recognition processes in intercellular communication and signal transduction such as cell adhesion, differ- entiation, development and regulation. For these rea- sons, great interest has arisen in carbohydrate-based pharmaceuticals and on the development of techniques for the analysis and synthesis of oligosaccharides. In this respect, enzymes involved in carbohydrates hydrolysis and modification are increasingly being utilised for the bioconversion of sugars, for the synthesis of oligosac- charides with potential application, and for the charac- terisation of carbohydrate compounds of unknown structure. In this review, the enzymology and the applications of three glycosyl hydrolases from the archaeon Sulfolobus solfataricus are described. In particular, we focus on the enzymological properties of a b-glycosidase, an a-xylo- sidase, and an a-fucosidase; their exploitation in oligo- saccharides synthesis will be also described.

The analysis of the complete genome of the thermoacidophilic Archaeon Sulfolobus solfataricus revealed two open reading frames (ORF), named SSO11867 and SSO3060, interrupted by a -1 frameshift and encoding for the N- and the C-terminal fragments, respectively, of an alpha-l-fucosidase. We report here that these ORFs are actively transcribed in vivo, and we confirm the presence of the -1 frameshift between them at the cDNA level, explaining why we could not find alpha-fucosidase activity in S. solfataricus extracts. Detailed analysis of the region of overlap between the two ORFs revealed the presence of the consensus sequence for a programmed -1 frameshifting. Two specific mutations, mimicking this regulative frameshifting event, allow the expression, in Escherichia coli, of a fully active thermophilic and thermostable alpha-l-fucosidase (EC ) with micromolar substrate specificity and showing transfucosylating activity. The analysis of the fucosylated products of this enzyme allows, for the first time, assigning a retaining reaction mechanism to family 29 of glycosyl hydrolases. The presence of an alpha-fucosidase putatively regulated by programmed -1 frameshifting is intriguing both with respect to the regulation of gene expression and, in post-genomic era, for the definition of gene function in Archaea.

The biotransformation of lignocellulose biomasses into fermentable sugars is a very complex procedure including, as one of the most critical steps, the (hemi) cellulose hydrolysis by specific enzymatic cocktails. We explored here, the potential of stable glycoside hydrolases from thermophilic organisms, so far not used in commercial enzymatic preparations, for the conversion of glucuronoxylan, the major hemicellulose of several energy crops. Searches in the genomes of thermophilic bacteria led to the identification, efficient production, and detailed characterization of novel xylanase and a-glucuronidase from Alicyclobacillus acidocaldarius (G1-110-XA and GH67-GA, respectively) and a alpha-glucuronidase from Caldicellulosiruptor saccharolyticus (GH67-GC). Remarkably, GH10-XA, if compared to other thermophilic xylanases from this family, coupled good specificity on beechwood xylan and the best stability at 65 degrees C (3.5 days). In addition, GH67-GC was the most stable alpha-glucuronidases from this family and the first able to hydrolyse both aldouronic acid and aryl-alpha-glucuronic acid substrates. These enzymes, led to the very efficient hydrolysis of beechwood xylan by using 7- to 9-fold less protein (concentrations

The first, recently identified, archaeal alpha-xylosidase from Sulfolobus solfataricus (XylS) shows high specificity for hydrolysis of isoprimeverose [alpha-D-xylopyranosyl-(1,6)-D-glucopyranose, (X)], the p-nitrophenyl-beta derivative of isoprimeverose, and xyloglucan oligosaccharides and has transxylosidic activity, forming, in a retaining mode, interesting alpha-xylosides. This article describes the synthesis of isoprimeverose, the disaccharidic repeating unit of xyloglucan, of the p-nitrophenyl-beta derivative of isoprimeverose, and of a trisaccharide based on isoprimeverose that is one of the trisaccharidic building blocks of xyloglucan. A substrate structure-activity relationship is recognized for both the hydrolysis and the synthesis reactions of XylS, it being a biocatalyst (i) active hydrolytically only on X-ending substrates liberating a xylose molecule and (ii) capable of transferring xylose only on the nonreducing end glucose of p-nitrophenyl-(PNP)-beta-D-cellobioside. The compounds synthesized by this enzyme are a starting point for enzymological studies of other new enzymes (i.e., xyloglucanases) for which suitable substrates are difficult to synthesize. This study also allows us to define the chemical characteristics of the xylose-transferring activity of this new archaeal enzyme, contributing to building up a library of different glycosidases with high specific selectivity for oligosaccharide synthesis.

We here report the first molecular characterization of an alpha-xylosidase (XylS) from an Archaeon. Sulfolobus solfataricus is able to grow at temperatures higher than 80 degrees C on several carbohydrates at acidic pH. The isolated xylS gene encodes a monomeric enzyme homologous to alpha-glucosidases, alpha-xylosidases, glucoamylases and sucrase-isomaltases of the glycosyl hydrolase family 31. xylS belongs to a cluster of four genes in the S. solfataricus genome, including a beta-glycosidase, an hypothetical membrane protein homologous to the major facilitator superfamily of transporters, and an open reading frame of unknown function. The alpha-xylosidase was overexpressed in Escherichia coli showing optimal activity at 90 degrees C and a half-life at this temperature of 38 h. The purified enzyme follows a retaining mechanism of substrate hydrolysis, showing high hydrolytic activity on the disaccharide isoprimeverose and catalyzing the release of xylose from xyloglucan oligosaccharides. Synergy is observed in the concerted in vitro hydrolysis of xyloglucan oligosaccharides by the alpha-xylosidase and the beta-glycosidase from S. solfataricus. The analysis of the total S. solfataricus RNA revealed that all the genes of the cluster are actively transcribed and that xylS and orf3 genes are cotranscribed.

Here we suggest that the origin of the genetic code, that is to say, the birth of first mRNAs has been triggered by means of a widespread modification of all RNAs (proto-mRNAs and proto-tRNAs), as today observed in the RNA editing and in post-transcriptional modifications of RNAs, which are considered as fossils of this evolutionary stage of the genetic code origin. We consider also that other mechanisms, such as the trans-translation and ribosome frameshifting, could have favoured the transition from an ennuplet code to a triplet code. Therefore, according to our hypothesis all these mechanisms would be reflexive of this period of the evolutionary history of the genetic code. © 2014.

Chemoenzymatic synthesis of glycans as an alternative to classical chemical synthesis has great interest for the exquisite sterospecificity and improved processivity and regioselectivity of the biological catalysts and for the possibility of using reagents less toxic for the environment. Nonetheless, the limitations intrinsic to the natural enzymes promoting sugar synthesis, namely glycoside hydrolases and glycosyltransferases, prompted efforts to engineer the former catalysts obtaining glycosynthases that catalyze the synthesis of oligo-, polysaccharides and glycoconjugates in quantitative yields and from cheap substrates. In this Chapter we show a survey of methods that, exploiting glycosidases and glycosynthases, allow the efficient and reliable preparation of glycans of synthetic relevance.

The second generation bioethanol represents a main challenge in global efforts to utilize renewable resources rather than fossil fuels. However, the close association of cellulose and hemicelluloses to lignin in the plant cell wall makes it difficult to degrade lignocellulose into fermentable sugars. Consequently, pretreatments are necessary to make the polysaccharides more accessible to the enzymes, but the high temperature and extreme pH conditions required give rise to problems when using conventional enzymes in the saccharification step (Galbe and Zacchi 2002). Microorganisms thriving in habitats characterized by harsh conditions, and the enzymes derived therein, represent a helpful tool in the development of bioethanol production processes. In fact, they allow bioconversions at non-conventional conditions under which common biocatalysts are denatured. The use of high operational temperatures allows energy savings by reducing the cooling cost after high temperature pretreatments, and, in ethanol production, thermophilic conditions permit ethanol evaporation allowing harvest during fermentation.

Pompe disease (PD) is a metabolic myopathy due to the deficiency of the lysosomal enzyme alpha-glucosidase (GAA). The only approved treatment for this disorder, enzyme replacement with recombinant human GAA (rhGAA), has shown limited therapeutic efficacy in some PD patients. Pharmacological chaperone therapy (PCT), either alone or in combination with enzyme replacement, has been proposed as an alternative therapeutic strategy. However, the chaperones identified so far also are active site-directed molecules and potential inhibitors of target enzymes. We demonstrated that N-acetylcysteine (NAC) is a novel allosteric chaperone for GAA. NAC improved the stability of rhGAA as a function of pH and temperature without disrupting its catalytic activity. A computational analysis of NAC-GAA interactions confirmed that NAC does not interact with GAA catalytic domain. NAC enhanced the residual activity of mutated GAA in cultured PD fibroblasts and in COS7 cells overexpressing mutated GAA. NAC also enhanced rhGAA efficacy in PD fibroblasts. In cells incubated with NAC and rhGAA, GAA activities were 3.7-8.7-fold higher than those obtained in cells treated with rhGAA alone. In a PD mouse model the combination of NAC and rhGAA resulted in better correction of enzyme activity in liver, heart, diaphragm and gastrocnemia, compared to rhGAA alone. Received 12 May 2012; accepted 3 July 2012; advance online publication 18 September 2012. doi:10.1038/mt.2012.152

Glycosynthases are engineered glycoside hydrolases that in suitable reaction conditions promote the synthesis of oligosaccharides with exquisite stereoselectivity and enhanced regioselectivity, if compared to traditional chemical methods. This approach was demonstrated to be successful in a number of cases including β-glycosynthases acting at the termini or within an oligosaccharide chain (exo- and endo-glycosynthases, respectively) and, more recently, α-glycosynthases. This led to the production of a vast repertoire of products that include poly- and oligosaccharides, glycoconjugates, and glycopeptides. These molecules can be used as ligands of glycoside hydrolases, for the characterization of therapeutic enzymes, and as leads of drugs for the pharmaceutical industry. In this panorama, hyperthermophilic organisms, which thrive at temperatures as high as 80°C, which usually impede the growth of other living forms, have been used in the development of interesting novel glycosynthases. In fact, the extreme stability of these catalysts to extremes of pH and high concentrations of organics has allowed the exploration of novel reaction conditions, revealing new avenues for enzyme-catalyzed oligosaccharide synthesis.

Glycosynthases are engineered glycoside hydrolases that in suitable reaction conditions promote the synthesis of oligosaccharides with exquisite stereoselectivity and enhanced regioselectivity, if compared to traditional chemical methods. This approach was demonstrated to be successful in a number of cases including β-glycosynthases acting at the termini or within an oligosaccharide chain ( exo - and endo -glycosynthases, respectively) and, more recently, α-glycosynthases. This led to the production of a vast repertoire of products that include poly- and oligosaccharides, glycoconjugates, and glycopeptides. These molecules can be used as ligands of glycoside hydrolases, for the characterization of therapeutic enzymes, and as leads of drugs for the pharmaceutical industry. In this panorama, hyperthermophilic organisms, which thrive at temperatures as high as 80 °C, which usually impede the growth of other living forms, have been used in the development of interesting novel glycosynthases. In fact, the extreme stability of these catalysts to extremes of pH and high concentrations of organics has allowed the exploration of novel reaction conditions, revealing new avenues for enzyme-catalyzed oligosaccharide synthesis.

Covering: up to March 2012 Oligo-, polysaccharides, and glycoconjugates are a relevant part of the bioactive components of the natural products exploited in therapeutics, diagnostics, food additives, and biomaterials. Glycans are directly involved in important biological processes, such as immunostimulation, anti-inflammatory, antioxidant, and chemoprotectant actions and/or are crucial for their activity, by modulating target recognition, stability, and pharmacokinetics. On the other hand, carbohydrate extracts used for functional studies are rather heterogeneous and lack structural information because of their intrinsic complexity hampering purification and characterization. Therefore, methods for glycoside synthesis and modification are urgently needed. Recently, glycosynthases, engineered glycoside hydrolases with no hydrolytic activity that synthesize glycans in quantitative yields, were introduced. Here we will illustrate how the glycosynthases described so far might be exploited for the production of glycan analogs of natural products and their enormous potential in this field.

International audience; An interesting approach for the chemo-enzymatic synthesis of carbohydrates is the use of glycosynthases, a class of mutant glycosidases derived from β-glycoside hydrolases obtained by replacement of the active site nucleophile with a non-nucleophilic residue. However, the scarcity of α-glycosynthases has so far hampered access to the synthesis of a large class of oligosaccharides of biotechnological interest. We review here a new glycosynthetic methodology for the production of two retaining α-fucosynthases and an α-galactosynthase exploiting β-glycosyl azide derivatives. The general applicability of this approach, which opens new perspectives in the use of azide derivatives for the production of novel α-glycosynthases, is discussed.

Glycosynthases, engineered glycoside hydrolases that are able to synthesize glycans in quantitative yields without hydrolyzing them, are among the most interesting tools for the chemoenzymatic synthesis of carbohydrates that have been made available so far. From their invention in 1998, these enzymes have been developed enormously and demonstrated to be convenient alternatives to the more expensive glycosyltransferase. Glycosynthases have been proved to be efficient catalysts for the synthesis of oligosaccharides, polysaccharides, and glycoconjugates of various lengths containing different monosaccharides bound through α- and β-linkages, which have interesting applications. In this review we describe the impact that glycosynthases have had on the biocatalysis and biosynthesis of carbohydrates through the description of the novel activities produced and of the development of the glycosynthase-based process. In addition, we summarize the approaches that, according to the current literature, can be adopted to produce novel glycosynthase activities and to improve the existing process.

Glycosidases, the enzymes responsible in nature for the catabolism of carbohydrates, are well-studied catalysts widely used in industrial biotransformations and oligosaccharide synthesis, which are also attractive targets for drug development. Glycosidases from hyperthermophilic organisms (thriving at temperatures > 85 degrees C) are also interesting models to understand the molecular basis of protein stability and to produce robust tools for industrial applications. Here, we review the results obtained in the last two decades by our group on a beta-glycosidase from the hyperthermophilic Archaeon Sulfolobus solfataricus. Our findings will be presented in the general context of the stability of proteins from hyperthermophiles and of the chemo-enzymatic synthesis of oligosaccharides.