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Listeria monocytogenes represents one of the main risks for food safety worldwide. Two enzyme-based antimicrobials (enzybiotics) have been combined in a novel treatment against this pathogenic bacterium, resulting in a powerful synergistic effect. One of the enzymes is an endolysin from Listeria phage vB_LmoS_188 with amidase activity (henceforth A10), and the other is an engineered version of glucose oxidase from Aspergillus niger (GOX). Both enzymes, assayed separately against Listeria innocua, showed antibacterial activity at the appropriate doses. The combination of the two enzybiotics resulted in a synergistic effect with a log reduction in viable cells (log N0/N) of 4, whereas, taken separately, the same dose of A10 and GOX caused only 1.2 and 0.2 log reductions, respectively. Flow cytometry and microscopy analyses revealed that A10 treatment alone induced the aggregation of dead cells. L. monocytogenes showed higher resistance to single treatment with GOX or A10 than L. innocua. However, the synergic combination of A10 and GOX resulted in a high lethality of L. monocytogenes with a log N0/N higher than 5 (below the detection limit in our analysis). Altogether, these results represent a novel efficient and eco-friendly antimicrobial treatment against the most lethal food-borne pathogen. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Extra and intracellular cellobiose hydrolysis by Saccharomyces was analyzed. • The bottleneck for efficient intracellular cellobiose fermentation is sugar intake. • Ten sugar permeases from Trichoderma reesei were assayed in Saccharomyces. • Only one assayed sugar permease had cellobiose transport activity in Saccharomyces. • Extracellular cellobiose hydrolysis outperformed intracellular fermentation. Abstract A prevalent procedure for the production of second generation bioethanol makes use of engineered yeast strains capable to hydrolyze cellobiose either in the cytosol or extracellularly. These two approaches have been compared in this study. For intracellular cellobiose hydrolysis, we initially tested three recombinant Saccharomyces cerevisiae strains that produced different cytosolic β-glucosidases and the cellodextrin transporter Po_CdtC from Penicillium oxalicum. The strain coexpressing Po_CdtC and the β-glucosidase from Neurospora crassa (NcBgl) showed the highest cellobiase activity but its growth in cellobiose was limited by sugar intake. A search of alternative cellobiose permeases was carried out among putative sugar transporters from Trichoderma reesei. Ten candidates were selected by sequence similarity with previously characterized fungal cellobiose permeases. Only one of them (Tr_StrC) was able to support yeast growth in cellobiose. Strains NcBgl/PoCdtC and NcBgl/TrStrC were compared to a yeast transformant (T500) that produced the extracellular β-glucosidase from Saccharomycopsis fibuligera , in terms of cellobiose fermentation. Extracellular hydrolysis resulted in faster cellobiose fermentation rates and higher ethanol yields. The strain producing extracellular β-glucosidase was also more efficient for simultaneous saccharification and fermentation of cellulose. The main targets to improve cellobiose fermentation through both strategies are discussed. [ABSTRACT FROM AUTHOR]

Glucoamylases, one of the main types of enzymes involved in starch hydrolysis, are exo-acting enzymes that release consecutive glucose units from the non-reducing ends of starch molecules. Glucoamylases are microbial enzymes, present in bacteria, archaea, and fungi but not in plants and animals. Structurally, they are classified in family 15 of glycoside hydrolases and characterised by the invariable presence of a catalytic domain with (α/α)-fold, often bound to a non-catalytic domain of diverse origin and function. Fungal glucoamylases are biotechnologically very important as they are used industrially in large amounts and have been extensively studied during the past 30 years. Prokaryotic glucoamylases are of biotechnological relevance for being generally thermophilic enzymes, active at elevated temperatures. [ABSTRACT FROM AUTHOR]

Human intestinal lactase-phlorizin hydrolase, LPH, encompasses four homologous domains, which presumably have evolved from two subsequent duplications of one ancestral gene. The profragment, LPHn, comprises homologous domains I and II and functions as an intramolecular chaperone in the context of the brush-border LPHβ region of LPH. Here, we analyze the inter-relationship between homologous domains III and IV of LPHβ and their implication in the overall structure, function, and trafficking of LPH. In silico analyses revealed potential domain boundaries for these domains as a basis for loop-out mutagenesis and construction of deletion or individual domain forms of LPH. Removal of domain IV, which contains lactase, results in a diminished phlorizin hydrolase activity, lack of dimerization in the endoplasmic reticulum (ER), but accelerated transport kinetics from the ER to the Golgi apparatus. By contrast, deletion of domain III, which harbors phlorizin hydro-lase, generates a malfolded protein that is blocked in the ER. Interestingly, homologous domain III is transport-competent per se and sorted to the apical membrane in polarized Madin-Darby canine kidney cells. Nevertheless, it neither dimerizes nor acquires complete phlorizin hydrolase activity. Our data present a hierarchical model of LPH in which the homologous domain III constitutes (i) a fully autonomous core domain within LPH and (ii) another intramolecular chaperone besides the profragment LPHa. Nevertheless, the regulation of the trafficking kinetics and activity of domain III and entire LPH including elevation of the enzymatic activities require the correct dimerization of LPH in the ER, an event that is accomplished by the non-autonomous domain IV. [ABSTRACT FROM AUTHOR]

Abstract: Bacteria species involved in degradation of cellulosic substrates produce a variety of enzymes for processing related compounds along the hydrolytic pathway. Paenibacillus polymyxa encodes two homologous β-glucosidases, BglA and BglB, presenting different quaternary structures and substrate specificities. We previously reported the 3D-structure of BglA, which is highly specific against cellobiose. Here, we present structural analysis of BglB, a monomeric enzyme that acts as an exo-β-glucosidase hydrolyzing cellobiose and cellodextrins of higher degree of polymerization. The crystal structure of BglB shows that several polar residues narrow the active site pocket and contour additional subsites. The structure of the BglB–cellotetraose complex confirms these subsites, revealing the substrate-binding mode, and shows the oligosaccharide-enzyme recognition pattern in detail. Comparison between BglA and BglB crystal structures suggests that oligomerization in BglA can assist in fine-tuning the specificity of the active centre by modulating the loops surrounding the cavity. We have solved the crystal structure of BglB with bound thiocellobiose, a competitive inhibitor, which together with the BglB–cellotetraose complex delineate the general features of the aglycon site. The detailed characterization of the atomic interactions at the aglycon site show a recognition pattern common to all bacterial β-glucosidases, and presents some differences with the aglycon site in plant β-glycosidases essentially by means of a different orientation of the basal Trp. The crystal structures of of BglB with a covalently bound inhibitor (derived from 2-fluoroglucoside) and glucose (produced by hydrolysis of the substrate in the crystal), provide additional pictures of the binding events and the intermediates formed during the reaction. Altogether, this information can assist in the understanding of subtle differences of the enzyme mechanism and substrate recognition within this family of enzymes, and consequently it can help in the development of new enzymes with improved activity or specificity. [Copyright &y& Elsevier]

A gene construct encoding a xylanase, which is active in extreme conditions of temperature and alkaline pH (90 °C, pH 10.5), has been transitorily expressed with high efficiency in Nicotiana benthamiana using a viral vector. The enzyme, targeted to the apoplast, accumulates in large amounts in plant tissues in as little as 7 days after inoculation, without detrimental effects on plant growth. The properties of the protein produced by the plant, in terms of resistance to temperature, pH, and enzymatic activity, are equivalent to those observed when Escherichia coli is used as a host. Purification of the plant-produced recombinant xylanase is facilitated by exporting the protein to the apoplastic space. The production of this xylanase by N. benthamiana, which avoids the hindrances derived from the use of E. coli, namely, intracellular production requiring subsequent purification, represents an important step for potential applications in the food industry in which more sustainable and green products are continuously demanded. As an example, the use of the enzyme producing prebiotic xylooligosdaccharides from xylan is here reported. [ABSTRACT FROM AUTHOR]

The formation and analysis of amyloid fibers by two β-glucosidases, BglA and BglB, belonging to the GH1 enzyme family, are reported. Both proteins have the (β/α)8 TIM-barrel fold, which is characteristic of this family and is also the most common protein structure. BglA is an octamer, whereas BglB is a monomer. Amyloid fibrillation using pH and temperature as perturbing agents was investigated using fluorescence spectroscopy as a preliminary approach and corroborated using wide-field optical microscopy, confocal microscopy, and field-emission scanning electron microscopy. These analyses showed that both enzymes fibrillate at a wide range of acidic and alkaline conditions and at several temperature conditions, particularly at acidic pH (3–4) and at temperatures between 45 and 65 °C. Circular dichroism spectroscopy corroborated the transition from an α-helix to a β-sheet secondary structure of both proteins in conditions where fibrillation was observed. Overall, our results suggest that fibrillation is a rather common phenomenon caused by protein misfolding, driven by a transition from an α-helix to a β-sheet secondary structure, that many proteins can undergo if subjected to conditions that disturb their native conformation. [ABSTRACT FROM AUTHOR]

Iron participates as a vital cofactor in multiple metabolic pathways. Despite its abundance, iron bioavailability is highly restricted in aerobic and alkaline environments. Therefore, living organisms have evolved multiple adaptive mechanisms to respond to iron scarcity. These strategies include a global remodeling of iron metabolism directed to optimize iron utilization. In the baker's yeast Saccharomyces cerevisiae, this metabolic reorganization is accomplished to a large extent by an mRNA-binding protein called Cth2. Yeast Cth2 belongs to a conserved family of tandem zinc finger containing proteins that specifically bind to transcripts with AU-rich elements and promote their turnover. A recent study has revealed that Cth2 also inhibits the translation of its target mRNAs (Ramos-Alonso et al., PLoS Genet 14:e1007476, 10.1371/journal.pgen.1007476, 2018). Interestingly, the mammalian Cth2 ortholog known as tristetraprolin (aka TTP/TIS11/ZFP36), which is also implicated in controlling iron metabolism, promotes the decay and prevents the translation of its regulated transcripts. These observations open the possibility to study the relative contribution of altering mRNA stability and translation to the physiological adaptation to iron deficiency, the function played by the different domains within the mRNA-binding protein, and the potential factors implicated in coordinating both post-transcriptional events. [ABSTRACT FROM AUTHOR]

We describe a simple, efficient process for the production of 6-kestose, a trisaccharide with well-documented prebiotic properties. A key factor is the use of a yeast transformant expressing an engineered version of Saccharomyces invertase with enhanced transfructosylating activity. When the yeast transformant was grown with 30 % sucrose as the carbon source, 6-kestose accumulated up to ca. 100 g/L in the culture medium. The 6-kestose yield was significantly enhanced (up to 200 g/L) using a two-stage process carried out in the same flask. In the first stage, the culture was grown in 30 % sucrose at physiological temperature (30 °C) to allow overexpression of the invertase. In the second stage, sucrose was added to the culture at high concentration (60 %) and the temperature shifted to 50 °C. In both cases, 6-kestose was synthesized with high specificity, representing more than 95 % of total FOS. [ABSTRACT FROM AUTHOR]

Xylanases can boost pulp bleachability in Elemental Chlorine Free (ECF) processes, but their industrial implementation for producing bleached kraft pulps is not straightforward. It requires enzymes to be active and stable at the extreme conditions of alkalinity and high temperature typical of this industrial process; most commercial enzymes are unable to withstand these conditions. In this work, a novel highly thermo and alkaline-tolerant xylanase from Pseudothermotoga thermarum was overproduced in E. coli and tested as a bleaching booster of hardwood kraft pulps to save chlorine dioxide (ClO2) during ECF bleaching. The extremozyme-stage (EXZ) was carried out at 90 °C and pH 10.5 and optimised at lab scale on an industrial oxygen-delignified eucalyptus pulp, enabling us to save 15% ClO2 to reach the mill brightness, and with no detrimental effect on paper properties. Then, the EXZ-assisted bleaching sequence was validated at pilot scale under industrial conditions, achieving 25% ClO2 savings and reducing the generation of organochlorinated compounds (AOX) by 18%, while maintaining pulp quality and papermaking properties. Technology reproducibility was confirmed with another industrial kraft pulp from a mix of hardwoods. The new enzymatic technology constitutes a realistic step towards environmentally friendly production of kraft pulps through industrial integration of biotechnology. [ABSTRACT FROM AUTHOR]

Efficient enzymatic synthesis of tailor-made prebiotic fructo-oligosaccharides (FOS) used in functional food formulation is a relevant biotechnological objective. We have engineered the Saccharomyces cerevisiae invertase (Suc2) to improve its transferase activity and to identify the enzymatic determinants for product specificity. Amino acid replacement (W19Y, N21S, N24S) within a conserved motif (β-fructosidase) specifically increased the synthesis of 6-kestose up to 10-fold. Mutants with lower substrate (sucrose) affinity produced FOS with longer half-lives. A mutation (P205V) adjacent to another conserved motif (EC) caused a 6-fold increment in 6-kestose yield. Docking studies with a Suc2 modeled structure defined a putative acceptor substrate binding subsite constituted by Trp 291 and Asn 228. Mutagenesis studies confirmed the implication of Asn 228 in directing the orientation of the sucrose molecule for the specific synthesis of β(2,6) linkages. [ABSTRACT FROM AUTHOR]

Abstract: Constructs carrying the Saccharomycopsis fibuligera β-glucosidase gene (BGL1) under the control of a constitutive actin or a galactose-inducible promoter were introduced into eleven Saccharomyces strains. In ten of these recombinant strains, BGL1 expression driven by the actin promoter was between 1.6- and 18-fold higher than that obtained with the galactose-inducible promoter. Strains carrying the actin promoter yielded ethanol concentrations from cellobiose of between 0.5% and 14%, depending on their ability to accumulate Bgl1 (between 30 and 250mU/mL) but also on their genetic background. Comparative analysis of a S. cerevisiae strain and its corresponding petite version showed similar ethanol yields, despite a 3-fold lower β-glucosidase production of the latter, suggesting that respiratory activity could be one of the factors influencing ethanol production when using carbon sources other than glucose. This study provides a selection of strains that may be good candidates as hosts for ethanol biosynthesis from cellulosic substrates. [Copyright &y& Elsevier]

Paenibacillus barcinonensis is a soil bacterium bearing a complex set of enzymes for xylan degradation, including several secreted enzymes and Xyn10B, one of the few intracellular xylanases reported to date. The crystal structure of Xyn10B has been determined by x-ray analysis. The enzyme folds into the typical (β/α)8 barrel of family 10 glycosyl hydrolases (GH10), with additional secondary structure elements within the β/α motifs. One of these loops -L7- located at the β7 C terminus, was essential for xylanase activity as its partial deletion yielded an inactive enzyme. The loop contains residues His249-Glu250, which shape a pocket opened to solvent in close proximity to the +2 subsite, which has not been described in other GH10 enzymes. This wide cavity at the +2 subsite, where methyl-2,4-pentanediol from the crystallization medium was found, is a noteworthy feature of Xyn10B, as compared with the narrow crevice described for other GH10 xylanases. Docking analysis showed that this open cavity can accommodate glucuronic acid decorations of xylo-oligosaccharides. Co-crystallization experiments with conduramine derivative inhibitors supported the importance of this open cavity at the +2 subsite for Xyn10B activity. Several mutant derivatives of Xyn10B with improved thermal stability were obtained by forced evolution. Among them, mutant xylanases S15L and M93V showed increased half-life, whereas the double mutant S15L/M93V exhibited a further increase in stability, showing a 20-fold higher heat resistance than the wild type xylanase. All the mutations obtained were located on the surface of Xyn10B. Replacement of a Ser by a Leu residue in mutant xylanase S15L can increase hydrophobic packing efficiency and fill a superficial indentation of the protein, giving rise to a more compact structure of the enzyme. [ABSTRACT FROM AUTHOR]

RL5, a gene coding for a novel polyphenol oxidase, was identified through activity screening of a metagenome expression library from bovine rumen microflora. Characterization of the recombinant protein produced in Escherichia coil revealed a multipotent capacity to oxidize a wide range of substrates (syringaldazine > 2,6-dimethoxyphenol > veratryl alcohol > guaiacol > tetramethylbenzidine > 4-methoxybenzyl alcohol > 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) ⪢ phenol red) over an unusually broad range of pH from 3.5 to 9.0. Apparent Km and kcat values for ABTS, syringaldazine, and 2,6-dimetoxyphenol obtained from steady-state kinetic measurements performed at 40 °C, pH 4.5, yielded values of 26, 0.43, and 0.45 μ and 18, 660, and 1175 s-1, respectively. The Km values for syringaldazine and 2,6-dimetoxyphenol are up to 5 times lower, and the kcat values up to 40 times higher, than values previously reported for this class of enzyme. RL5 is a 4-copper oxidase with oxidation potential values of 745, 400, and 500 mV versus normal hydrogen electrode for the T1, T2, and T3 copper sites. A three-dimensional model of RL5 and site-directed mutants were generated to identify the copper ligands. Bioinformatic analysis of the gene sequence and the sequences and contexts of neighboring genes suggested a tentative phylogenetic assignment to the genus Bacteroides. Kinetic, electrochemical, and EPR analyses provide unequivocal evidence that the hypothetical proteins from Bacteroides thetaiotaomicron and from E. coli, which are closely related to the deduced protein encoded by the RL5 gene, are also multicopper proteins with polyphenol oxidase activity. The present study shows that these three newly characterized enzymes form a new family of functional multicopper oxidases with laccase activity related to conserved hypothetical proteins harboring the domain of unknown function DUF152 and suggests that some other of these proteins may also be laccases. [ABSTRACT FROM AUTHOR]

Lactose is a very important sugar because of its abundance in the milk of humans and domestic animals. Lactose is a valuable asset as a basic nutrient and the main substrate in fermentative processes that led to the production of fermented milk products, such as yogurt and kefir. In some instances, lactose also can be a problem as the causative agent of some diseases, such as lactose intolerance and galactosemia, or for being a by-product generated in huge amounts by the cheese industry. The study of the biochemical reactions leading to the synthesis and assimilation of lactose has provided valuable models for the understanding of biosynthetic and catabolic processes. Lactose-hydrolyzing enzymes are structurally and phylogenetically related to different types of beta-galactosidases and bacterial cellobiases involved in the enzymatic degradation of cellulose. Biotransformation of lactose, by either enzymatic or fermentative procedures, is important for different types of industrial applications in dairy and pharmaceutical industries. [ABSTRACT FROM AUTHOR]

Background: Xylanases are one of the most extensively used enzymes for biomass digestion. However, in many instances, their use is limited by poor performance under the conditions of pH and temperature required by the industry. Therefore, the search for xylanases able to function efficiently at alkaline pH and high temperature is an important objective for different processes that use lignocellulosic substrates, such as the production of paper pulp and biofuels. Results: A comprehensive in silico analysis of family GH11 sequences from the CAZY database allowed their phylogenetic classification in a radial cladogram in which sequences of known or presumptive thermophilic and alkalophilic xylanases appeared in three clusters. Eight sequences from these clusters were selected for experimental analysis. The coding DNA was synthesized, cloned and the enzymes were produced in E. coli. Some of these showed high xylanolytic activity at pH values > 8.0 and temperature > 80 °C. The best enzymes corresponding to sequences from Dictyoglomus thermophilum (Xyn5) and Thermobifida fusca (Xyn8). The addition of a carbohydrate-binding module (CBM9) to Xyn5 increased 4 times its activity at 90 °C and pH > 9.0. The combination of Xyn5 and Xyn8 was proved to be efficient for the saccharification of alkali pretreated rice straw, yielding xylose and xylooligosaccharides. Conclusions: This study provides a fruitful approach for the selection of enzymes with suitable properties from the information contained in extensive databases. We have characterized two xylanases able to hydrolyze xylan with high efficiency at pH > 8.0 and temperature > 80 °C. [ABSTRACT FROM AUTHOR]

Thermostable β-galactosidase (TmLac) has been immobilized as hybrid inorganic-protein nanoflowers using salts of Cu2+, Mn2+, Zn2+, Co2+ and Ca2+ as the inorganic component. The incorporation efficiency of enzyme into the nanoflowers was higher than 95% for a protein concentration of 0.05 mg/mL. The structure, activity and recyclability of the nanoflowers with different chemical composition were analyzed. Ca2+, Mn2+ and Co2+ nanoflowers showed a level of lactase activity equivalent to their same content of free enzyme. Cu2+nanoflowers showed only marginal enzyme activity in agreement with the inhibitory effect of this cation on the enzyme. TmLac nanoflowers provide an efficient methodology for enzyme immobilization and recyclability. TmLac-Ca2+ nanoflowers presented the best properties for lactose hydrolysis both in buffered and in milk, and could be reused in five consecutive cycles. [ABSTRACT FROM AUTHOR]

This report describes an efficient procedure for enzyme encapsulation and its application for the hydrolysis of lactose. The enzymatic material that has been developed consists of hydrogel particles (ca. 3–4 mm of diameter) composed of either alginate or an alginate-agarose combination, in which bacterial cells loaded with a thermostable β-galactosidase are embedded. The cells were rendered fully permeable to the substrate, either chromogenic p -nitrophenyl galactose or lactose, by thermal treatment at 75 °C. Hydrogel particles made of a mixture of alginate and agarose displayed high catalytic activity (i.e. 1 g of beads hydrolyze the lactose equivalent of 100 mL of milk in 15 min) and thermal stability: they could be reused at 75 °C for complete hydrolysis of 5% (w/v) lactose solution at least six consecutive times without significant loss of activity. Image 1 • Agarose-alginate hydrogel beads with lactose hydrolysis activity were prepared. • Bacterial cells loaded with a thermostable β-galactosidase were the core material. • Zymoactive beads can be operated at high temperature (75 °C). • Hydrogel beads can be reused at least six times without significant loss of activity. • 1 g of beads hydrolyze the lactose equivalent of 100 mL of milk in 15 min. [ABSTRACT FROM AUTHOR]

Enzymatically-active bacterial cellulose (BC) was prepared by non-covalent immobilization of a hybrid enzyme composed by a β-galactosidase from Thermotoga maritima (TmLac) and a carbohydrate binding module (CBM2) from Pyrococcus furiosus . TmLac-CBM2 protein was bound to BC, with higher affinity at pH 6.5 than at pH 8.5 and with high specificity compared to the non-engineered enzyme. Both hydrated (HBC) and freeze-dried (DBC) bacterial cellulose showed equivalent enzyme binding efficiencies. Initial reaction rate of HBC-bound enzyme was higher than DBC-bound and both of them were lower than the free enzyme. However, enzyme performance was similar in all three cases for the hydrolysis of 5% lactose to a high extent. Reuse of the immobilized enzyme was limited by the stability of the β-galactosidase module, whereas the CBM2 module provided stable attachment of the hybrid enzyme to the BC support, after long incubation periods (3 h) at 75 °C. [ABSTRACT FROM AUTHOR]

Artemia is extensively used in aquaculture to feed early stages of cultured marine species. A problem associated with this practice is that Artemia fails to supply some essential nutrients. As a possible solution, we have devised a procedure to make Artemia a vehicle for exogenous nutrients and other bioactive compounds. It consists of the construction of chimeric proteins composed of a chitin-binding domain, which binds to the cuticle of Artemia , and a carrier domain that conveys a functional property. As confirmatory examples, we describe the successful fixation to Artemia 's metanauplii of two hybrid proteins: a β-galactosidase from the thermophilic bacterium Thermotoga maritima and the jellyfish green fluorescent protein (GFP), both linked to the CBM2 chitin-binding domain from the hyperthermophilic archaeon Pyrococcus furiosus . Positive results of experiments carried out ex vivo and in vivo show the validity of this approach. The methodology used could become a general procedure for the attachment of different kinds of bioactive compounds, such as enzymes, hormones, antibiotics, etc. , to the cuticle of Artemia as well as other arthropods. Statement of relevance Our results overcome shortcomings of Artemia as a feedstock. [ABSTRACT FROM AUTHOR]

In this work we report a detailed analysis of the topology and phylogenetics of family 2 glycoside hydrolases (GH2). We distinguish five topologies or domain architectures based on the presence and distribution of protein domains defined in Pfam and Interpro databases. All of them share a central TIM barrel (catalytic module) with two β-sandwich domains (non-catalytic) at the N-terminal end, but differ in the occurrence and nature of additional non-catalytic modules at the C-terminal region. Phylogenetic analysis was based on the sequence of the Pfam Glyco_hydro_2_C catalytic module present in most GH2 proteins. Our results led us to propose a model in which evolutionary diversity of GH2 enzymes is driven by the addition of different non-catalytic domains at the C-terminal region. This model accounts for the divergence of β-galactosidases from β-glucuronidases, the diversification of β-galactosidases with different transglycosylation specificities, and the emergence of bicistronic β-galactosidases. This study also allows the identification of groups of functionally uncharacterized protein sequences with potential biotechnological interest. [ABSTRACT FROM AUTHOR]

Metagenomics has opened up a vast pool of genes for putative, yet uncharacterized, enzymes. It widens our knowledge on the enzyme diversity world and discloses new families for which a clear classification is still needed, as is exemplified by glycoside hydrolase family-3 (GH3) proteins. Herein, we describe a GH3 enzyme (GlyA1) from resident microbial communities in strained ruminal fluid. The enzyme is aβ-glucosidase/β-xylosidase that also shows β-galactosidase, β-fucosidase, α-arabinofuranosidase, and α-arabinopyranosidase activities. Short cello- and xylo-oligosaccharides, sophorose and gentibiose, are among the preferred substrates, with the large polysaccharide lichenan also being hydrolyzed by GlyA1. The determination of the crystal structure of the enzyme in combination with deletion and site-directed mutagenesis allowed identification of its unusual domain composition and the active site architecture. Complexes of GlyA1 with glucose, galactose, and xylose allowed picturing the catalytic pocket and illustrated the molecular basis of the substrate specificity. A hydrophobic platform defined by residues Trp-711 and Trp-106, located in a highly mobile loop, appears able to allocate differently β-linked bioses. GlyA1 includes an additional C-terminal domain previously unobserved in GH3 members, but crystallization of the full-length enzyme was unsuccessful. Therefore, small angle x-ray experiments have been performed to investigate the molecular flexibility and overall putative shape. This study provided evidence that GlyA1 defines a new subfamily of GH3 proteins with a novel permuted domain topology. Phylogenetic analysis indicates that this topology is associated with microbes inhabiting the digestive tracts of ruminants and other animals, feeding on chemically diverse plant polymeric materials. [ABSTRACT FROM AUTHOR]

Glucose oxidase is one of the most conspicuous commercial enzymes due to its many different applications in diverse industries such as food, chemical, energy and textile. Among these applications, the most remarkable is the manufacture of glucose biosensors and in particular sensor strips used to measure glucose levels in serum. The generation of ameliorated versions of glucose oxidase is therefore a significant biotechnological objective. We have used a strategy that combined random and rational approaches to isolate uncharacterized mutations of Aspergillus niger glucose oxidase with improved properties. As a result, we have identified two changes that increase significantly the enzyme's thermal stability. One (T554M) generates a sulfur-pi interaction and the other (Q90R/Y509E) introduces a new salt bridge near the interphase of the dimeric protein structure. An additional double substitution (Q124R/L569E) has no significant effect on stability but causes a twofold increase of the enzyme's specific activity. Our results disclose structural motifs of the protein which are critical for its stability. The combination of mutations in the Q90R/Y509E/T554M triple mutant yielded a version of A. niger glucose oxidase with higher stability than those previously described. [ABSTRACT FROM AUTHOR]

Thermoresistant, recombinant β-galactosidase from Thermotoga maritima was purified and immobilized on the surface of epoxy-coated magnetic beads. The enzyme, which has hexameric quaternary structure as shown by gel filtration chromatography, attaches to the resin through multiple covalent linkages that involve different subunits. The bound enzyme shows higher stability than the free form. The immobilized enzyme showed to be efficient for the hydrolysis of lactose and the biosynthesis of galactooligosaccharides (GOS). The chemical structure of synthesized GOS has been determined by NMR revealing that the main product was β-3′-galactosyl lactose. Although β-galactosidases from different sources have been used for the same purposes, the distinct advantage of the methodology described in this communication is that the enzyme can be easily produced, purified and immobilized in large quantities. [ABSTRACT FROM AUTHOR]

Pectin methylesterases (PMEs) are a family of enzymes involved in plant reproductive processes such as pollen development and pollen tube growth. We have isolated and characterized PsPMEP, a pea ( Pisum sativum L.) pollen-specific gene that encodes a protein with homology to PMEs. Sequence analysis showed that PsPMEP belongs to group 2 PMEs, which are characterized by the presence of a processable amino-terminal PME inhibitor domain followed by the catalytic PME domain. Moreover, PsPMEP contains several motifs highly conserved among PMEs with the essential amino acid residues involved in enzyme substrate binding and catalysis. Northern blot and in situ hybridization analyses showed that PsPMEP is expressed in pollen grains from 4 days before anthesis till anther dehiscence and in pollinated carpels. In the PsPMEP promoter region, we have identified several conserved cis-regulatory elements that have been associated with gene pollen-specific expression. Expression analysis of PsPMEP promoter fused to the uidA reporter gene in Arabidopsis thaliana plants showed a similar expression pattern when compared with pea, indicating that this promoter is also functional in a non-leguminous plant. GUS expression was detected in mature pollen grains, during pollen germination, during pollen tube elongation along the transmitting tract, and when the pollen tube reaches the embryo sac in the ovule. [ABSTRACT FROM AUTHOR]

The esterases and lipases from the α/β hydrolase superfamily exhibit an enormous sequence diversity, fold plasticity, and activities. Here, we present the comprehensive sequence and biochemical analyses of seven distinct esterases and lipases from the metagenome of Lake Arreo, an evaporite karstic lake in Spain (42°46′N, 2°59′W; altitude, 655 m). Together with oligonucleotide usage patterns and BLASTP analysis, our study of esterases/lipases mined from Lake Arreo suggests that its sediment contains moderately halophilic and cold-adapted proteobacteria containing DNA fragments of distantly related plasmids or chromosomal genomic islands of plasmid and phage origins. This metagenome encodes esterases/lipases with broad substrate profiles (tested over a set of 101 structurally diverse esters) and habitat-specific characteristics, as they exhibit maximal activity at alkaline pH (8.0 to 8.5) and temperature of 16 to 40°C, and they are stimulated (1.5 to 2.2 times) by chloride ions (0.1 to 1.2 M), reflecting an adaptation to environmental conditions. Our work provides further insights into the potential significance of the Lake Arreo esterases/lipases for biotechnology processes (i.e., production of enantiomers and sugar esters), because these enzymes are salt tolerant and are active at low temperatures and against a broad range of substrates. As an example, the ability of a single protein to hydrolyze triacylglycerols, (non)halogenated alkyl and aryl esters, cinnamoyl and carbohydrate esters, lactones, and chiral epoxides to a similar extent was demonstrated. [ABSTRACT FROM AUTHOR]

The assembly and nuclear transport of RNA polymerase II (RNA pol II) are processes that require the participation of many auxiliary factors. In a yeast genetic screen, we identified a previously uncharacterized gene, YMR185w (renamed RTP1), which encodes a protein required for the nuclear import of RNA pol II. Using protein affinity purification coupled to mass spectrometry, we identified interactions between Rtp1p and members of the R2TP complex. Rtp1p also interacts, to a different extent, with several RNA pol II subunits. The pattern of interactions is compatible with a role for Rtp1p as an assembly factor that participates in the formation of the Rpb2/Rpb3 subassembly complex and its binding to the Rpb1p-containing subcomplex. Besides, Rtp1p has a molecular architecture characteristic of karyopherins, composed of HEAT repeats, and is able to interact with phe-nylalanine-glycine-containing nucleoporins. Our results define Rtp1p as a new component of the RNA pol II biogenesis machinery that plays roles in subunit assembly and likely in transport through the nuclear pore complex. [ABSTRACT FROM AUTHOR]

Invertase is an enzyme that is widely distributed among plants and microorganisms and that catalyzes the hydrolysis of the disaccharide sucrose into glucose and fructose. Despite the important physiological role of Saccharomyces invertase (SInv) and the historical relevance of this enzyme as a model in early biochemical studies, its structure had not yet been solved. We report here the crystal structure of recombinant SInv at 3.3 Å resolution showing that the enzyme folds into the catalytic β-propeller and β-sandwich domains characteristic of GH32 enzymes. However, SInv displays an unusual quaternary structure. Monomers associate in two different kinds of dimers, which are in turn assembled into an octamer, best described as a tetramer of dimers. Dimerization plays a determinant role in substrate specificity because this assembly sets steric constraints that limit the access to the active site of oligosaccharides of more than four units. Comparative analysis of GH32 enzymes showed that formation of the SInv octamer occurs through a β-sheet extension that seems unique to this enzyme. Interaction between dimers is determined by a short amino acid sequence at the beginning of the β-sandwich domain. Our results highlight the role of the non-catalytic domain in fine-tuning substrate specificity and thus supplement our knowledge of the activity of this important family of enzymes. In turn, this gives a deeper insight into the structural features that rule modularity and protein-carbohydrate recognition. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae invertase (ScInv) is an enzyme encoded by the SUC2 gene that releases β-fructose from the nonreducing termini of various β-D-fructofuranoside substrates. Its ability to produce 6-kestose by transglycosylation makes this enzyme an interesting research target for applications in industrial biotechnology. The native enzyme, which presents a high degree of oligomerization, was crystallized by vapour-diffusion methods. The crystals belonged to space group P3121, with unit-cell parameters a=268.6, b=268.6, c=224.4 Å. The crystals diffracted to 3.3 Å resolution and gave complete data sets using a synchrotron X-ray source. [ABSTRACT FROM AUTHOR]

Previously, we applied in vitro evolution to generate the thermoresistant triple mutant H62R/N223Y/M319I of β-glucosidase B (BglB) from Paenibacillus polymyxa. In order to dissect the energetic contributions to protein stabilization achieved by these mutations, we measured the kinetic constants of the heat denaturation of wild type BglB, the triple mutant and the three single mutants (H62R, N223Y, M319I) by circular dichroism at various temperatures. Our results show that all four mutants delayed the denaturation process. Based on the Transition State theory, the increase of the activation barrier for the thermal denaturation of the triple mutant ( ΔΔG) is equivalent to that produced by the sum of the contributions from the three single mutants, whose C s are located at least 18 Å apart. This analysis provides a formal demonstration of the generally accepted idea that protein thermal stability can be increased through sequential addition of individual mutations. Each of the mutations described here contribute in part to the overall effect, which in this case affects the unfolding barrier. [ABSTRACT FROM AUTHOR]

Background: A complete saccharification of plant polymers is the critical step in the efficient production of bio-alcohols. Beta-glucosidases acting in the degradation of intermediate gluco-oligosaccharides produced by cellulases limit the yield of the final product. Results: In the present work, we have identified and then successfully cloned, expressed, purified and characterised 4 highly active beta-glucosidases from fibre-adherent microbial community from the cow rumen. The enzymes were most active at temperatures 45-55°C and pH 4.0-7.0 and exhibited high affinity and activity towards synthetic substrates such as p-nitrophenyl-beta-D-glucopyranoside (pNPbetaG) and pNP-beta-cellobiose, as well as to natural cello-oligosaccharides ranging from cellobiose to cellopentaose. The apparent capability of the most active beta-glucosidase, herein named LAB25g2, was tested for its ability to improve, at low dosage (31.25 units g-1 dry biomass, using pNPbetaG as substrate), the hydrolysis of pre-treated corn stover (dry matter content of 20%; 350 g glucan kg-1 dry biomass) in combination with a beta-glucosidase-deficient commercial Trichoderma reseei cellulase cocktail (5 units g-1 dry biomass in the basis of pNPbetaG). LAB25g2 increased the final hydrolysis yield by a factor of 20% (44.5 ± 1.7% vs. 34.5 ± 1.5% in control conditions) after 96-120 h as compared to control reactions in its absence or in the presence of other commercial beta-glucosidase preparations. The high stability (half-life higher than 5 days at 50°C and pH 5.2) and 2-38000 fold higher (as compared with reported beta-glucosidases) activity towards cello-oligosaccharides may account for its performance in supplementation assays. Conclusions: The results suggest that beta-glucosidases from yet uncultured bacteria from animal digestomes may be of a potential interest for biotechnological processes related to the effective bio-ethanol production in combination with low dosage of commercial cellulases. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae Sta1 glucoamylase and Saccharomycopsis fibuligera Bgl1 β-glucosidase, two relevant enzymes from a biotechnological point of view, are proteins with multidomain structure. Starting with homology-based structural models of Sta1 and Bgl1, we have constructed a series of hybrid enzymes by interchanging domains of the two proteins. The first purpose of these constructs was to check available hypotheses about the uncertain biological functions of two domains: the serine/threonine-rich domain (STRD) of Sta1 and a β-sandwich domain present in Bgl1 that we have designated fibronectin-like domain (FLD). While, according to the initial hypothesis, proteins carrying the FLD tend to adhere to the cell wall, our results argued against the idea of an involvement of the STRD in protein secretion that stemmed from the presence of similar domains in different proteins secreted by yeast. The second objective of this work was to increase the enzymatic repertoire by generating enzymes with new structural and functional properties. [ABSTRACT FROM AUTHOR]

The fungus Phanerochaete flavido- alba is highly efficient in the oxidation of olive oil wastewater-derived polyphenols. This capability is largely due to the action of a multicopper-oxidase (MCO), encoded by the pfaL gene. We describe the sequence and organization of pfaL gene and the biochemical characterization and predicted 3D structural model of the encoded protein. pfaL gene organization and peptide sequence are highly similar to those of P. chrysosporium MCOs. However, PfaL is the first MCO in the Phanerochaete genus to show evident laccase activity. Phylogenetic analysis places PfaL in a differentiated sub-branch of ferroxidases. Protein structure analysis reveals close similarity of PfaL and ferroxidases and provides clues about the differences of activity between both types of enzymes. To summarize, P. flavido- alba laccase is the first enzyme in the novel and biochemically poorly defined group of 'ferroxidases/laccases' that shows efficacious oxidation of laccase substrates, biotechnologically exploitable in bioremediation approaches. [ABSTRACT FROM AUTHOR]

The guts and casts of earthworms contain microbial assemblages that process large amounts of organic polymeric substrates from plant litter and soil; however, the enzymatic potential of these microbial communities remains largely unexplored. In the present work, we retrieved carbohydrate-modifying enzymes through the activity screening of metagenomic fosmid libraries from cellulose-depleting microbial communities established with the fresh casts of two earthworm species, Aporrectodea caliginosa and Lumbricus terrestris, as inocula. Eight glycosyl hydrolases (GHs) from the A. caliginosa-derived community were multidomain endo-β-glucanases, β-glucosidases, β-cellobiohydrolases, β-galactosidase, and β-xylosidases of known Gil families. In contrast, two GHs derived from the L. terrestris microbiome had no similarity to any known GHs and represented two novel families of β-galactosidases/α-arabinopyranosidases. Members of these families were annotated in public databases as conserved hypothetical proteins, with one being structurally related to isomerases/dehydratases. This study provides insight into their biochemistry, domain structures, and active. site architecture. The two communities were similar in bacterial composition but significantly different with regard to their eukaryotic inhabitants. Further sequence analysis of fosmids and plasmids bearing the GH-encoding genes, along with oligonucleotide usage pattern analysis, suggested that those apparently originated from Gammaproteobacteria (pseudomonads and Cellvibrio-like organisms), Betaproteobacteria (Comamonadaceae), and Aiphaproteobacteria (Rhizobiales). [ABSTRACT FROM AUTHOR]

Carboxyl esterases (CE) exhibit various reaction specificities despite of their overall structural similarity. In present study we have exploited functional metagenomics, saturation mutagenesis and experimental protein evolution to explore residues that have a significant role in substrate discrimination. We used an enzyme, designated 3A6, derived from the earthworm gut metagenome that exhibits CE and feruloyl esterase (FAE) activities with p-nitrophenyl and cinnamate esters, respectively, with a [( kcat/ Km)]CE/[( kcat/ Km)]FAE factor of 17. Modelling-guided saturation mutagenesis at specific hotspots (Lys281, Asp282, Asn316 and Lys317) situated close to the catalytic core (Ser143/Asp273/His305) and a deletion of a 34-AA–long peptide fragment yielded mutants with the highest CE activity, while cinnamate ester bond hydrolysis was effectively abolished. Although, single to triple mutants with both improved activities (up to 180-fold in kcat/ Km values) and enzymes with inverted specificity (( kcat/ Km)CE/( kcat/ Km)FAE ratio of ∼0.4) were identified, no CE inactive variant was found. Screening of a large error-prone PCR-generated library yielded by far less mutants for substrate discrimination. We also found that no significant changes in CE activation energy occurs after any mutation (7.3 to −5.6 J mol−1), whereas a direct correlation between loss/gain of FAE function and activation energies (from 33.05 to −13.7 J mol−1) was found. Results suggest that the FAE activity in 3A6 may have evolved via introduction of a limited number of ‘hot spot’ mutations in a common CE ancestor, which may retain the original hydrolytic activity due to lower restrictive energy barriers but conveys a dynamic energetically favourable switch of a second hydrolytic reaction. [ABSTRACT FROM AUTHOR]

Abstract: β-Fructofuranosidases are powerful tools in industrial biotechnology. We have characterized an extracellular β-fructofuranosidase from the yeast Schwanniomyces occidentalis. The enzyme shows broad substrate specificity, hydrolyzing sucrose, 1-kestose, nystose and raffinose, with different catalytic efficiencies (k cat/K m). Although the main reaction catalysed by this enzyme is sucrose hydrolysis, it also produces two fructooligosaccharides (FOS) by transfructosylation. A combination of 1H, 13C and 2D-NMR techniques shows that the major product is the prebiotic trisaccharide 6-kestose. The 6-kestose yield obtained with this β-fructofuranosidase is, to our concern, higher than those reported with other 6-kestose-producing enzymes, both at the kinetic maximum (76gl−1) and at reaction equilibrium (44gl−1). The total FOS production in the kinetic maximum was 101gl−1, which corresponded to 16.4% (w/w) referred to the total carbohydrates in the reaction mixture. [Copyright &y& Elsevier]

Abstract: An acetylxylan esterase (R.44), belonging to the carbohydrate esterase family 6 (CE6), retrieved from bovine rumen metagenome was analyzed. Molecular modelling and site-directed mutagenesis indicated that the enzyme possesses a catalytic triad formed by Ser14, His231 and Glu152. The catalytic Ser and His have been identified in highly conserved sequences GQSX and DXXH in the CE6 family, respectively, and the active-site glutamate was part of a highly conserved sequence HQGE. This motif is situated near to the so-called Block III in the CE6 family and its role in catalysis has not been identified so far. [Copyright &y& Elsevier]

Summary: Fungal laccases are remarkable green catalysts that have a broad substrate specificity and many potential applications in bioremediation, lignocellulose processing, organic synthesis, and more. However, most of these transformations must be carried out at high concentrations of organic cosolvents in which laccases undergo unfolding, thereby losing their activity. We have tailored a thermostable laccase that tolerates high concentrations of cosolvents, the genetic product of five rounds of directed evolution expressed in Saccharomyces cerevisiae. This evolved laccase—R2 variant—was capable of resisting a wide array of cosolvents at concentrations as high as 50% (v/v). Intrinsic laccase features such as the redox potential and the geometry of catalytic coppers varied slightly during the course of the molecular evolution. Some mutations at the protein surface stabilized the laccase by allowing additional electrostatic and hydrogen bonding to occur. [Copyright &y& Elsevier]

Abstract: Glucoamylase produced by amylolytic strains of Saccharomyces cerevisiae (var. diastaticus) lacks a starch binding domain that is present in homologous glucoamylases from Aspergillus niger and other filamentous fungi. The absence of the binding domain makes the enzyme inefficient against raw starch and hence unsuitable for most biotechnological applications. We have constructed a hybrid glucoamylase-encoding gene by in-frame fusion of the S. cerevisiae STA1 gene and DNA fragment that encodes the starch binding domain of A. niger glucoamylase. The hybrid enzyme resulting from expression of the chimeric gene in S. cerevisiae has substrate binding capability and hydrolyses insoluble starch, properties not present in the original yeast enzyme. [Copyright &y& Elsevier]

N-oligosaccharides of Saccharomyces cerevisiae glyco-proteins are classified as core and mannan types. The former contain 13–14 mannoses whereas mannan-type structures consist of an inner core extended with an outer chain of up to 200–300 mannoses, a process known as hyperglycosylation. The selection of substrates for hyperglycosylation poses a theoretical and practical question. To identify hyperglycosylation determinants, we have analyzed the influence of the second amino acid (Xaa) of the sequon in this process using the major exoglucanase as a model. Our results indicate that negatively charged amino acids inhibit hyperglycosylation, whereas positively charged counterparts promote it. On the basis of the tridimensional structure of Exg1, we propose that Xaa influences the orientation of the inner core making it accessible to mannan polymerase I in the appropriate position for the addition of α-1,6-mannoses. The presence of Glu in the Xaa of the second sequon of the native exoglucanase suggests that negative selection may drive evolution of these sites. However, a comparison of invertases secreted by S. cerevisiae and Pichia anomala suggests that hyperglycosylation signals are also subjected to positive selection. [ABSTRACT FROM AUTHOR]

Mucor miehei transformants resistant to geneticin have been obtained by treatment of protoplasts with different plasmids and by Agrobacterium-mediated DNA transfer. All transformants exhibited a very unstable phenotype whose maintenance required continuous selective pressure. Molecular analysis of transformants showed that the plasmid DNA was extensively modified and maintained in very low amount. Our results indicate that M. miehei reluctance to transformation is due to different causes, including the coenocytic nature of its mycelium and the existence of specific mechanisms for the detection and elimination of foreign DNA. [Copyright &y& Elsevier]

Extra-large pore zeolites are a small subset (21) among the whole list of 253 zeolites available. The discovery of new low-glycemic sugars is very attractive as new healthy additives in the food field. This is the case of the 6-kestose. In the present case, it appears in a mixture in aqueous solution together with sucrose, the separation of the mixture being necessary. For this, we have focused on using certain zeolites with adequate pore sizes that allow the separation of this mixture, considering that since the molecular size of 6-kestose is greater than sucrose, it is necessary to promote the sorption of the latter, so that the first can be purified. After a computational screening of micropores of the 253 IZA zeolites, 11 zeolites were selected. Of these, 3 extra-large pore zeolites (AET, DON, ETR) have been proposed, which were analyzed in-depth through a molecular dynamics study considering the external surface. The results show that DON presents the most promising theoretical results for a selective sucrose/6-kestose separation. [Display omitted] • Separation of dissacharide/trissacharide mixtures is important for agroalimentary process. • With 7.5/9.1 Å size of these sugars, extra-large pore zeolites might be used for separation. • Molecular dynamics of sucrose/kestose have been made on extra-large pore zeolites. • Models include large unit cells with: water, sugar, zeolite layers and reservoir (membranes). • Effects considered: external surface, terminal silanols, pore size, temperature, water. [ABSTRACT FROM AUTHOR]

In this work, enzymatically active polysaccharide-based hydrogels and aerogels have been developed. To this end, a thermostable β-galactosidase (TmLac) enzyme from Thermotoga maritima embedded in nanoflowers' format was used to evaluate the capacity of the hydrogel matrices to preserve the hydrolytic activity of the enzyme and the reusability of the hydrogels formed. Commercial agar, unpurified agar and agarose were compared as supporting materials. Although the developed hydrogel capsules can be used at high temperature (75 °C) and reused for the digestion of lactose to a greater extent than the free nanoflowers, loaded hydrogel capsules behaved differently depending on the type of polysaccharide used. Commercial agar was the most promising one since these hydrogel capsules could be reused, maintaining the structural integrity and reaching higher enzymatic activity (after seven cycles at 75 °C) than the free TmLac-Ca2+ nanoflowers. To facilitate handling and storage, aerogels were developed by freeze-drying the hydrogel capsules. Aerogels of agarose and unpurified agar underwent structural changes during freeze-drying that adversely affected their subsequent use, losing their integrity after being rehydrated. However, commercial agar aerogels were successfully developed and reused thanks to the existing interactions with TmLac-Ca2+ nanoflowers (confirmed by FTIR), which resulted in better capsule integrity and enzyme protection. The hydrolytic activity of enzymatically active aerogels based on commercial agar was in the same range of the free TmLac and TmLac-Ca2+ nanoflowers, being significantly higher to their counterparts in the hydrated form (hydrogels based on commercial agar). Image 1 • TmLac-nanoflowers were encapsulated in marine polysaccharide hydrogels and aerogels. • Enzymatic activity was more efficiently preserved in purified agar hydrogels/aerogels. • Active aerogels based on commercial agar showed the highest catalytic activity. • 0.5 g of aerogel beads hydrolyze the lactose equivalent of 100 mL of milk in 15 min. [ABSTRACT FROM AUTHOR]

Catalytic materials obtained by enzyme immobilization have multiple potential applications in the food industry. The choice of the immobilization method and support may be critical to define the properties of the immobilized enzyme compared to the soluble form. Although the use of immobilized enzymes shows multiple advantages, their catalytic efficiency is compromised in many instances. Molecular engineering techniques have been used to generate hybrid proteins where the enzyme of interest is fused to a module with affinity to a specific biopolymer. Binding of the hybrid TmLac-CBM2 protein, in which the β-galactosidase from Thermotoga maritima is fused to a carbohydrate-binding module from Pyrococcus furiosus , to cellulosic material from aquatic biomass wastes (such as Posidonia oceanica and Arundo donax) has been assayed. Both species generate environmental wastes that could be revalorized if converted into bioactive materials. Cellulose cryogels, but not films, from P. oceanica were able to bind the TmLac-CBM2 hybrid, with a higher immobilization yield (90%) than that from A. donax cellulose cryogels (60%). However, fractions containing also hemicellulose were less effective as immobilization supports in both cases, with yields of 47% and 30%, respectively. Cellulose cryogels loaded with β-galactosidase were able to hydrolyse lactose with the same efficiency as the free form of the enzyme. In contrast, enzyme-loaded cellulose films were inactive. This study represents a proof of concept for the valorisation of cellulosic wastes as bioactive materials. Furthermore, it provides information about the interaction specificity between the binding module and the cellulosic support, useful for other enzymes. Image 1 • CBM2 mediated binding of a hybrid enzyme was tested on cellulosic wastes. • The TmLac-CBM2 hybrid binds cellulose cryogels from P. oceanica and A. donax. • Binding affinity of hybrid enzyme was reduced in hemicellulose-containing fractions. • Immobilized enzyme in cellulose films is inactive. • Immobilized enzyme in cellulose cryogels shows the same activity as the free form. [ABSTRACT FROM AUTHOR]

• Efficient synthesis of multivalent pyrrolidine iminosugars via CuAAC. • Unexpected selectivity in the inhibition of two structurally related β-glucosidases. • A phenyl moiety in the spacer of the iminosugar was crucial for inhibition. • First co-crystal structure of a multivalent iminosugar with a GH1 β-glucosidase. The synthesis of multivalent pyrrolidine iminosugars via CuAAC click reaction between different pyrrolidine-azide derivatives and tri- or hexavalent alkynyl scaffolds is reported. The new multimeric compounds, together with the monomeric reference, were evaluated as inhibitors against two homologous GH1 β-glucosidases (BglA and BglB from Paenibacillus polymyxa). The multivalent inhibitors containing an aromatic moiety in the linker between the pyrrolidine and the scaffold inhibited the octameric BglA (µM range) but did not show affinity against the monomeric BglB, despite the similarity between the active site of both enzymes. A modest multivalent effect (rp/n = 12) was detected for the hexavalent inhibitor 12. Structural analysis of the complexes between the monomeric and the trimeric iminosugar inhibitors (4 and 10) and BglA showed the insertion of the inhibitors at the active site of BglA, confirming a competitive mode of inhibition as indicated by enzyme kinetics. Additionally, structural comparison of the BglA/ 4 complex with the reported BglB/2F-glucose complex illustrates the key determinants responsible for the inhibitory effect and explains the reasons of the inhibition of BglA and the no inhibition of BglB. Potential inhibition of other β-glucosidases with therapeutic relevance is discussed under the light of these observations. [ABSTRACT FROM AUTHOR]

The synthesis of a novel α-glucosylated derivative of pterostilbene was performed by a transglycosylation reaction using starch as glucosyl donor, catalyzed by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. The reaction was carried out in a buffer containing 20% (v/v) DMSO to enhance the solubility of pterostilbene. Due to the formation of several polyglucosylated products with CGTase, the yield of monoglucoside was increased by the treatment with a recombinant amyloglucosidase (STA1) from Saccharomyces cerevisiae (var. diastaticus). This enzyme was not able to hydrolyze the linkage between the glucose and pterostilbene. The monoglucoside was isolated and characterized by combining ESI-MS and 2D-NMR methods. Pterostilbene α-d-glucopyranoside is a novel compound. The α-glucosylation of pterostilbene enhanced its solubility in water to approximately 0.1 g/L. The α-glucosylation caused a slight loss of antioxidant activity towards ABTS˙+ radicals. Pterostilbene α-d-glucopyranoside was less toxic than pterostilbene for human SH-S5Y5 neurons, MRC5 fibroblasts and HT-29 colon cancer cells, and similar for RAW 264.7 macrophages. [ABSTRACT FROM AUTHOR]

Microbial communities from cow rumen are known for their ability to degrade diverse plant polymers at high rates. In this work, we identified 15 hydrolases through an activity-centred metagenome analysis of a fibre-adherent microbial community from dairy cow rumen. Among them, 7 glycosyl hydrolases (GHs) and 1 feruloyl esterase were successfully cloned, expressed, purified and characterised. The most striking result was a protein of GH family 43 (GHF43), hereinafter designated as R_09-02, which had characteristics very distinct from the other proteins in this family with mono-functional bxylosidase, α-xylanase, α-L-arabinase and a-L-arabinofuranosidase activities. R_09-02 is the first multifunctional enzyme to exhibit β-1,4 xylosidase, α-1,5 arabinofur(pyr)anosidase, β-1,4 lactase, α-1,6 raffinase, α-1,6 stachyase, b-galactosidase and α- 1,4 glucosidase activities. The R_09-02 protein appears to originate from the chromosome of a member of Clostridia, a class of phylum Firmicutes, members of which are highly abundant in ruminal environment. The evolution of R_09-02 is suggested to be driven from the xylose- and arabinose-specific activities, typical for GHF43 members, toward a broader specificity to the glucose- and galactose-containing components of lignocellulose. The apparent capability of enzymes from the GHF43 family to utilise xylose-, arabinose-, glucose- and galactose-containing oligosaccharides has thus far been neglected by, or could not be predicted from, genome and metagenome sequencing data analyses. Taking into account the abundance of GHF43-encoding gene sequences in the rumen (up to 7% of all GH-genes) and the multifunctional phenotype herein described, our findings suggest that the ecological role of this GH family in the digestion of ligno-cellulosic matter should be significantly reconsidered. [ABSTRACT FROM AUTHOR]