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Mucopolysaccharidosis type IIIB is a devastating neurological disease caused by a lack of the lysosomal enzyme, α‐N‐acetylglucosaminidase (NAGLU), leading to a toxic accumulation of heparan sulfate. Herein we explored a pharmacological chaperone approach to enhance the residual activity of NAGLU in patient fibroblasts. Capitalizing on the three‐dimensional structures of two modest homoiminosugar‐based NAGLU inhibitors in complex with bacterial homolog of NAGLU, CpGH89, we have synthesized a library of 17 iminosugar C‐glycosides mimicking N‐acetyl‐D‐glucosamine and bearing various pseudo‐anomeric substituents of both α‐ and β‐configuration. Elaboration of the aglycon moiety results in low micromolar selective inhibitors of human recombinant NAGLU, but surprisingly it is the non‐functionalized and wrongly configured β‐homoiminosugar that was proved to act as the most promising pharmacological chaperone, promoting a 2.4 fold activity enhancement of mutant NAGLU at its optimal concentration. [ABSTRACT FROM AUTHOR]

Gum arabic arabinogalactan (AG) protein (AGP) is a unique dietary fiber that is degraded and assimilated by only specific strains of Bifidobacterium longum subsp. longum. Here, we identified a novel 3-O-α-D-galactosyl-α-L-arabinofuranosidase (GAfase) from B. longum JCM7052 and classified it into glycoside hydrolase family 39 (GH39). GAfase released a-D-Galp-(1→3)-L-Ara and β-L-Arap-(1→3)-L-Ara from gum arabic AGP and β-L-Arap-(1→3)-L-Ara from larch AGP, and the a-D-Galp-(1→3)-L-Ara release activity was found to be 594-fold higher than that of β-L-Arap-(1→3)-L-Ara. The GAfase gene was part of a gene cluster that included genes encoding a GH36 a-galactosidase candidate and ABC transporters for the assimilation of the released a-D-Galp-(1→3)-L-Ara in B. longum. Notably, when a-D-Galp-(1→3)-L-Ara was removed from gum arabic AGP, it was assimilated by both B. longum JCM7052 and the nonassimilative B. longum JCM1217, suggesting that the removal of a-D-Galp-(1→3)-L-Ara from gum arabic AGP by GAfase permitted the cooperative action with type II AG degradative enzymes in B. longum. The present study provides new insight into the mechanism of gum arabic AGP degradation in B. longum. IMPORTANCE Bifidobacteria harbor numerous carbohydrate-active enzymes that degrade several dietary fibers in the gastrointestinal tract. B. longum JCM7052 is known to exhibit the ability to assimilate gum arabic AGP, but the key enzyme involved in the degradation of gum arabic AGP remains unidentified. Here, we cloned and characterized a GH39 3-O-α-D-galactosyl-α-L-arabinofuranosidase (GAfase) from B. longum JCM7052. The enzyme was responsible for the release of a-D-Galp-(1→3)-L-Ara and β-L-Arap-(1→3)-LAra from gum arabic AGP. The presence of a gene cluster including the GAfase gene is specifically observed in gum arabic AGP assimilative strains. However, GAfase carrier strains may affect GAfase noncarrier strains that express other type II AG degradative enzymes. These findings provide insights into the bifidogenic effect of gum arabic AGP. [ABSTRACT FROM AUTHOR]

Alpha galactosidase is an exoglycosidase that cleaves α-D-galactose and has numerous applications in medicine, biotechnology, food and pharma industries. In this study, a low molecular weight acidic α-galactosidase was identified from the seeds of custard apple. The purification of α-galactosidase from the crude extract of defatted seeds was achieved by employing ammonium sulphate fractionation, hydrophobic interaction and gel filtration chromatographic techniques. The purified custard apple α-galactosidase (Ca G) migrated as a single band in native PAGE corresponding to molecular weight of ~67 kDa and cleaved chromogenic, fluorogenic and natural substrates. Ca G was found to be a heterodimer with subunit masses of 40 and 30 kDa. The kinetic parameters such as K M and Vmax were found to be 0.67 mM and 1.5 U/mg respectively with p -nitrophenyl α-D-galactopyranoside. Galactose, methyl α-D-galactopyranoside and D-galacturonic acid inhibited Ca G activity in mixed mode. The CD spectral analysis at far UV region showed that purified Ca G exists predominantly as helix (35%), beta sheets (16.3%) and random coils (32.3%) in its secondary structure. These biochemical and biophysical properties of Ca G provide leads to understand its primary sequence and glycan structures which will eventually define its novel physiological roles in plants and potential industrial applications. • Acidic α-galactosidase has been purified to homogeneity from Custard apple seeds. • Custard apple α-galactosidase is heterodimeric in nature. • α-Galactosidase hydrolyzed chromogenic, fluorogenic and natural substrates. • Galactose inhibited custard apple α-galactosidase in mixed mode. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii is one of the most clinically important nosocomial pathogens. The World Health Organization refers it to its "critical priority" category to develop new strategies for effective therapy. This microorganism is capable of producing structurally diverse capsular polysaccharides (CPSs), which serve as primary receptors for A. baumannii bacteriophages carrying polysaccharide-degrading enzymes. In this study, eight novel bacterial viruses that specifically infect A. baumannii strains belonging to K2/K93, K32, K37, K44, K48, K87, K89, and K116 capsular types were isolated and characterized. The overall genomic architecture demonstrated that these viruses are representatives of the Friunavirus genus of the family Autographiviridae. The linear double-stranded DNA phage genomes of 41,105 to 42,402 bp share high nucleotide sequence identity, except for genes encoding structural depolymerases or tailspikes, which determine the host specificity. Deletion mutants lacking N-terminal domains of tailspike proteins were cloned, expressed, and purified. The structurally defined CPSs of the phage bacterial hosts were cleaved with the specific recombinant depolymerases, and the resultant oligosaccharides that corresponded to monomers or/and dimers of the CPS repeats (K units) were isolated. Structures of the derived oligosaccharides were established by nuclear magnetic resonance spectroscopy and high-resolution electrospray ionization mass spectrometry. The data obtained showed that all depolymerases studied were glycosidases that specifically cleave the A. baumannii CPSs by the hydrolytic mechanism, in most cases, by the linkage between the K units. IMPORTANCE Acinetobacter baumannii, a nonfermentative, Gram-negative, aerobic bacterium, is one of the most significant nosocomial pathogens. The pathogenicity of A. baumannii is based on the cooperative action of many factors, one of them being the production of capsular polysaccharides (CPSs) that surround bacterial cells with a thick protective layer. Polymorphism of the chromosomal capsule loci is responsible for the observed high structural diversity of the CPSs. In this study, we describe eight novel lytic phages which have different tailspike depolymerases (TSDs) determining the interaction of the viruses with corresponding A. baumannii capsular types (K types). Moreover, we elucidate the structures of oligosaccharide products obtained by cleavage of the CPSs by the recombinant depolymerases. We believe that as the TSDs determine phage specificity, the diversity of their structures should be taken into consideration as selection criteria for inclusion of certain phage candidates in the cocktail designed to control A. baumannii with different K types. [ABSTRACT FROM AUTHOR]

Rutinosidases hydrolyze β-rutinosylated flavonoids. As retaining glycosidases they also have a transglycosylation activity. Here we show that two newly identified wild-type rutinosidases, which are members of the glycoside hydrolase family 5–23, are capable of glycosylation of an inorganic azide with rutin as a glycosyl donor, yielding rutinosyl β-azide. On the other hand, rutinosyl α-azide was synthesized by the catalytic nucleophile mutant of the rutinosidase from Aspergillus niger, which also belongs to GH5–23. Thus, we were able to synthesize at a preparatory scale both anomers of rutinosyl azide from rutin using either wild-type or mutant rutinosidases of GH5–23.

The human gut microbiota plays a central role in intestinal health and disease. Yet, many of its bacterial constituents are functionally still largely unexplored. A crucial prerequisite for bacterial survival and proliferation is the creation and/or exploitation of an own niche. For many bacterial species that are linked to human disease, the inner mucus layer was found to be an important niche. Allobaculum mucolyticum is a newly identified, IBD-associated species that is thought be closely associated with the host epithelium. To explore how this bacterium is able to effectively colonize this niche, we screened its genome for factors that may contribute to mucosal colonization. Up to 60 genes encoding putative Carbohydrate Active Enzymes (CAZymes) were identified in the genome of A. mucolyticum. Mass spectrometry revealed 49 CAZymes of which 26 were significantly enriched in its secretome. Functional assays demonstrated the presence of CAZyme activity in A. mucolyticum conditioned medium, degradation of human mucin O-glycans, and utilization of liberated non-terminal monosaccharides for bacterial growth. The results support a model in which sialidases and fucosidases remove terminal O-glycan sugars enabling subsequent degradation and utilization of carbohydrates for A. mucolyticum growth. A. mucolyticum CAZyme secretion may thus facilitate bacterial colonization and degradation of the mucus layer and may pose an interesting target for future therapeutic intervention.

Tetrachlorinated phthalimide analogues bearing a boron-pinacolate ester group were synthesised via two synthetic routes and evaluated in their glycosidase modulating and anticancer properties, with a view to use them in boron neutron capture therapy (BNCT), a promising radiation type for cancer, as this therapy does little damage to biological tissue. An unexpected decarbonylation/decarboxylation to five 2,3,4,5-tetrachlorobenzamides was observed and confirmed by X-ray crystallography studies, thus, giving access to a family of borylated 2,3,4,5-tetrachlorobenzamides. Biological evaluation showed the benzamide drugs to possess good to weak potencies (74.7–870 μM) in the inhibition of glycosidases, and to have good to moderate selectivity in the inhibition of a panel of 18 glycosidases. Furthermore, in the inhibition of selected glycosidases, there is a core subset of three animal glycosidases, which is always inhibited (rat intestinal maltase α-glucosidase, bovine liver β-glucosidase and β-galactosidase). This could indicate the involvement of the boron atom in the binding. These glycosidases are targeted for the management of diabetes, viral infections (via a broad-spectrum approach) and lysosomal storage disorders. Assays against cancer cell lines revealed potency in growth inhibition for three molecules, and selectivity for one of these molecules, with the growth of the normal cell line MCF10A not being affected by this compound. One of these molecules showed both potency and selectivity; thus, it is a candidate for further study in this area. This paper provides numerous novel aspects, including expedited access to borylated 2,3,4,5-tetrachlorophthalimides and to 2,3,4,5-tetrachlorobenzamides. The latter constitutes a novel family of glycosidase modulating drugs. Furthermore, a greener synthetic access to such structures is described.

Changes in human IgG galactosylation and sialylation have been associated with several inflammatory diseases which are a major burden on the health care system. A large body of work on well-established glycomic and glycopeptidomic assays has repeatedly demonstrated inflammation-induced changes in IgG glycosylation. However, these assays are usually based on specialized analytical instrumentation which could be considered a technical barrier for uptake by some laboratories. Hence there is a growing demand for simple biochemical assays for analyzing these glycosylation changes. We have addressed this need by introducing a novel glycosidase plate-based assay for the absolute quantification of galactosylation and sialylation on IgG. IgG glycoproteins are treated with specific exoglycosidases to release the galactose and/or sialic acid residues. The released galactose monosaccharides are subsequently used in an enzymatic redox reaction that produces a fluorescence signal that is quantitative for the amount of galactosylation and, in-turn, sialylation on IgG. The glycosidase plate-based assay has the potential to be a simple, initial screening assay or an alternative assay to the usage of high-end analytical platforms such as HILIC-FLD-MSn when considering the analysis of galactosylation and sialylation on IgG. We have demonstrated this by comparing our assay to an industrial established HILIC-FLD-MSn glycomic analysis of 15 patient samples and obtained a Pearson's r correlation coefficient of 0.8208 between the two methods. [ABSTRACT FROM AUTHOR]

Asparagine (N)-linked glycosylation is one of the most common co- and post-translational modifications of both intra- and extracellularly distributing proteins, which directly affects their biological functions, such as protein folding, stability and intercellular traffic. Production of the structural well-defined homogeneous N-glycans contributes to comprehensive investigation of their biological roles and molecular basis. Among the various methods, chemo-enzymatic approach serves as an alternative to chemical synthesis, providing high stereoselectivity and economic efficiency. This review summarizes some recent advances in the chemo-enzymatic methods for the production of N-glycans, including the preparation of substrates and sugar donors, and the progress in the glycosyltransferases characterization which leads to the diversity of N-glycan synthesis. We discuss the bottle-neck and new opportunities in exploiting the chemo-enzymatic synthesis of N-glycans based on our research experiences. In addition, downstream applications of the constructed N-glycans, such as automation devices and homogeneous glycoproteins synthesis are also described.

A novel hyperthermophilic, anaerobic filamentous archaeon, Thermofilum adornatum strain 1910bT, is capable of growing with cellulose as its sole carbon and energy source. This strain was isolated from a terrestrial hot spring in Kamchatka, Russia. The isolate 1910bT grew optimally at a temperature of 80°C and a pH of 5.5–6.0, producing cell-bound inducible cellulases. During genome analysis, genes, encoding various glycosidases (GHs) involved in oligo- and polysaccharide hydrolysis and genes for the fermentation of sugars were identified. No homologs of currently known cellulase families were found among the GHs encoded by the 1910bT genome, suggesting that novel proteins are involved. To figure this out, a proteomic analysis of cells grown on cellulose or pyruvate (as a control) was performed. Both in-depth genomic and proteomic analyses revealed four proteins (Cel25, Cel30, Cel40, and Cel45) that were the most likely to be involved in the cellulose hydrolysis in this archaeon. Two of these proteins (Cel30 and Cel45) were hypothetical according to genome analysis, while the other two (Cel25 and Cel40) have GH3 and GH1 domains, respectively. The respective genes were heterologously expressed in Escherichia coli BL21 (DE3), and enzymatic activities of recombinant proteins were measured with carboxymethyl cellulose (CMC), Avicel and cellobiose as substrates. It was revealed that the Cel30 and Cel25 proteins were likely exoglucanases with side beta-glucosidase and endoglucanase activities, that Cel40 was a multifunctional glucanase capable of hydrolyzing beta-1,4-glucosides of various lengths, and that Cel45 was an endoglucanase with side exoglucanase activity. Taking into account that the cellulolytic activity of T. adornatum 1910bT surface protein fractions was inducible, that recombinant Cel25 and Cel30 were much less active than Cel40 and Cel45, and that their gene expressions were (almost) non-induced by CMC, we suggest that Cel40 and Cel45 play a major role in the degradation of cellulose, while Cel25 and Cel30 act only as accessory enzymes.

Fucosidases are associated with several pathological conditions and play an important role in the health of the human gut. For example, fucosidases have been shown to be indicators and/or involved in hepatocellular carcinoma, breast cancer, and helicobacter pylori infections. A prerequisite for the detection and profiling of fucosidases is the formation of a specific covalent linkage between the enzyme of interest and the activity-based probe (ABP). The most commonly used fucosidase ABPs are limited to only one of the classes of fucosidases, the retaining fucosidases. New approaches are needed that allow for the detection of the second class of fucosidases, the inverting type. Here, we report an ortho-quinone methide-based probe with an azide mini-tag that selectively labels both retaining and inverting bacterial α-l-fucosidases. Mass spectrometry-based intact protein and sequence analysis of a probe-labeled bacterial fucosidase revealed almost exclusive single labeling at two specific tryptophan residues outside of the active site. Furthermore, the probe could detect and image extracellular fucosidase activity on the surface of live bacteria.

Glycosphingolipids (GSLs), the main topic of this review, are a subclass of sphingolipids. With their glycans exposed to the extracellular space, glycosphingolipids are ubiquitous components of the plasma membrane of cells. GSLs are implicated in a variety of biological processes including specific infections. Several pathogens use GSLs at the surface of host cells as binding receptors. In addition, lipid-rafts in the plasma membrane of host cells may act as platform for signaling the presence of pathogens. Relatively common in man are inherited deficiencies in lysosomal glycosidases involved in the turnover of GSLs. The associated storage disorders (glycosphingolipidoses) show lysosomal accumulation of substrate(s) of the deficient enzyme. In recent years compounds have been identified that allow modulation of GSLs levels in cells. Some of these agents are well tolerated and already used to treat lysosomal glycosphingolipidoses. This review summarizes present knowledge on the role of GSLs in infection and subsequent immune response. It concludes with the thought to apply glycosphingolipid-lowering agents to prevent and/or combat infections.

Phenolic compounds in pig diets, originating either from feed ingredients or additives, may occur as glycosides, that is conjugated to sugar moieties. Upon ingestion, their bioavailability and functionality depend on hydrolysis of the glycosidic bond by endogenous or microbial glycosidases. Hence, it is essential to map the glycosidase activities towards phenolic glycosides present along gut. Therefore, the activity of three key glycosidases, that is α‐glucosidase (αGLU), β‐glucosidase (βGLU) and β‐galactosidase (βGAL), was quantified in small intestinal mucosa and digesta of piglets at different gastrointestinal sites (stomach, three parts of small intestine, caecum and colon) and at different ages around weaning (10 days before and 0, 2, 5, 14 and 28 days after weaning). Activity assays were performed with p‐nitrophenyl glycosides at neutral pH. The αGLU activities in mucosa and digesta were low (overall means 1.4 and 60 U respectively) as compared to βGLU (15.2 and 199 U) and βGAL (23.4 and 298 U; p <.001). Moreover, αGLU activity in mucosa was unaffected by age. Conversely, βGLU and βGAL activities dropped significantly after weaning. Minimal levels, ranging between 18% and 54% of the pre‐weaning values, were reached at 5 days post‐weaning. Similarly, in small intestinal digesta, reductions from 60% up to 90% were observed for the three enzyme activities on day five post‐weaning as compared to pre‐weaning levels. In caecal contents, activities were lowest at 14 days post‐weaning, while in stomach and colon no clear weaning‐induced effects were observed. Our data suggest that weaning affects the glycosidase activity in mucosa (mainly endogenous origin) and digesta (primarily bacterial origin) with the most pronounced effects occurring 5 days post‐weaning. Moreover, differences in activities exist between different glycosidases and between gut locations. These insights can facilitate the prediction of the fate of existing and newly synthetized glycosides after oral ingestion in piglets. [ABSTRACT FROM AUTHOR]

Inhibitor protonation of azasugars of the isofagomine type when bound to enzyme can be investigated using photon induced electron transfer (PET) quenching of an attached fluorophore. For this purpose, Isofagomine, iso‐d‐galacto‐fagomine, and iso‐l‐gulo‐fagomine were converted to N‐(10‐chloroanthracenenyl‐9‐alkyl) derivatives where the alkyl group contained one, two, or three methylene groups. The new derivatives displayed pH dependent fluorescence; as the ammonium forms they were fluorescent, while 90–99 % of the fluorescence was quenched in the amine forms. The 3 isofagomine derivatives were competitive inhibitors of T. Maritima Ι‐glucosidase with Ki values from 0.37–4.6ΤM. Similarly, the iso‐d‐galacto‐fagomines inhibited A. Niger Ι‐galactosidase with Ki values from 63–2000 nm. When bound to the enzymes the inhibitors displayed between 1–15 % fluorescence. [ABSTRACT FROM AUTHOR]

A lysosomal glycosidase, β-glucuronidase, has been purified to homogeneity, from the soluble extracts of a freshwater mussel, L. corrianus , by a series of chromatography techniques involving phenyl-Sepharose, ion exchange, affinity and gel filtration chromatography. In native PAGE, β-glucuronidase resolved into a single band and the molecular mass determined by gel filtration chromatography was found to be 250 kDa. Zymogram analysis with 4-methyl umbelliferyl β-glucuronide substrate validated the purified enzyme as β-glucuronidase. In SDS-PAGE, the purified enzyme was resolved into four sub-units with molecular weights around 90, 75, 65, and 50 kDa, respectively, and two of the subunits (90 and 50 kDa) cross-reacted with human β-glucuronidase antiserum. The optimum pH and temperature of the purified glycosidase were 5.0 and 70 °C, respectively. The enzyme kinetics parameters, substrate affinity (K M) and maximum velocity (V max) of the purified protein estimated with p -nitrophenyl β-D-glucuronide were 0.457 mM and 0.11867 μmol−1 min−1 mL−1, respectively. The secondary structure of β-glucuronidase was determined in the far-UV range (190 nm to 230 nm) using CD spectroscopy. Heat denaturation plots determined by CD spectroscopy showed that the purified enzyme was stable up to 70 °C. • Fresh water mussel, mollusc exhibited significant β-glucuronidase activity • Purified enzyme exhibited a pH optima of 5.0. • Purified enzyme is a heterotetramer and cross-reacts with human enzyme antibody. • Enzyme is thermally stable. • Studies conducted suggest enzyme to be lysosomal. [ABSTRACT FROM AUTHOR]

The glycoconjugates, herein glyco-proteins, existing in animal skins are closely related to the effectiveness of unhairing and fiber opening-up. Glycosidases have been used in leather making processes to reduce pollutants and improve leather quality. But the selection of glycosidases is still blind because the related mechanisms are not well understood yet. Hence, the animal skin structures and glycoconjugates components, the advances in the methods and mechanisms of removing glycoconjugates related to unhairing and fiber opening-up in leather manufacture, the kinds, compositions, structures and functions of typical glycoconjugates in skin are summarized. Then the approaches to destroy them, possible glycosidases suitable for leather making and their acting sites are analyzed based on the recognition of glycoconjugates in skin and the specificities of glycosidases toward substrates. It is expected to provide useful information for the optimization of glycosidases and the development of new enzymes and the cleaner technologies of unhairing and opening up fiber bundles assisted by glycosidases. [ABSTRACT FROM AUTHOR]

Aerobic gram-negative bacterium Acinetobacter baumannii has recently become one of the most relevant pathogens associated with hospital-acquired infections worldwide. A. baumannii produces a capsule around the cell, which represents a thick viscous layer of structurally variable capsular polysaccharide (CPS). The capsule protects the bacteria against unfavorable environmental factors and biological systems, including bacteriophages and host immune system. Many A. baumannii phages have structural depolymerases (tailspikes) that specifically recognize and digest bacterial CPS. In this work, we studied the interaction of tailspike proteins of four lytic depolymerase-carrying phages with A. baumannii CPS. Depolymerases of three bacteriophages (Fri1, AS12, and BS46) were identified as specific glycosidases that cleave the CPS of A. baumannii strains 28, 1432, and B05, respectively, by the hydrolytic mechanism. The gp54 depolymerase from bacteriophage AP22 was characterized as a polysaccharide lyase that cleaves the CPS of A. baumannii strain 1053 by β-elimination at hexuronic acid (ManNAcA) residues. [ABSTRACT FROM AUTHOR]

The activity of glycosidases (maltase activity and amylolytic activity) in the intestinal mucosa of juvenile common carp Cyprinus carpio (L.) under a sharp increase in water temperature depends on its physiological state (satiety or starvation). The fish are acclimated to a temperature of 22°С during the summer (young-of-the-year) and winter (yearling) seasons. An increase in water temperature at a rate of 8.0°C/h leads to an increase in amylolytic activity in well-nourished fish, but the maltase activity does not change. Starving for 3 or 10 days changes the response of glycosidase to temperature stress: the maltase activity increases, but amylolytic activity does not change. Short-term starvation does not affect the thermal tolerance of juvenile common carp; i.e., the values of the critical thermal maximum in hungry and well-nourished specimens do not differ. [ABSTRACT FROM AUTHOR]

A novel hyperthermophilic, anaerobic filamentous archaeon, Thermofilum adornatum strain 1910bT, is capable of growing with cellulose as its sole carbon and energy source. This strain was isolated from a terrestrial hot spring in Kamchatka, Russia. The isolate 1910bT grew optimally at a temperature of 80°C and a pH of 5.5–6.0, producing cell-bound inducible cellulases. During genome analysis, genes, encoding various glycosidases (GHs) involved in oligo- and polysaccharide hydrolysis and genes for the fermentation of sugars were identified. No homologs of currently known cellulase families were found among the GHs encoded by the 1910bT genome, suggesting that novel proteins are involved. To figure this out, a proteomic analysis of cells grown on cellulose or pyruvate (as a control) was performed. Both in-depth genomic and proteomic analyses revealed four proteins (Cel25, Cel30, Cel40, and Cel45) that were the most likely to be involved in the cellulose hydrolysis in this archaeon. Two of these proteins (Cel30 and Cel45) were hypothetical according to genome analysis, while the other two (Cel25 and Cel40) have GH3 and GH1 domains, respectively. The respective genes were heterologously expressed in Escherichia coli BL21 (DE3), and enzymatic activities of recombinant proteins were measured with carboxymethyl cellulose (CMC), Avicel and cellobiose as substrates. It was revealed that the Cel30 and Cel25 proteins were likely exoglucanases with side beta-glucosidase and endoglucanase activities, that Cel40 was a multifunctional glucanase capable of hydrolyzing beta-1,4-glucosides of various lengths, and that Cel45 was an endoglucanase with side exoglucanase activity. Taking into account that the cellulolytic activity of T. adornatum 1910bT surface protein fractions was inducible, that recombinant Cel25 and Cel30 were much less active than Cel40 and Cel45, and that their gene expressions were (almost) non-induced by CMC, we suggest that Cel40 and Cel45 play a major role in the degradation of cellulose, while Cel25 and Cel30 act only as accessory enzymes. [ABSTRACT FROM AUTHOR]

High glycosidase-producing strains of Aspergillus luchuensis were isolated from 2-deoxyglucose (2-DG) resistant mutants. α-Amylase, exo-α-1,4-glucosidase, β-glucosidase and β-xylosidase activity in the mutants was ~3, ~2, ~4 and ~2.5 times higher than the parental strain RIB2604 on koji-making conditions, respectively. Citric acid production and mycelia growth of the mutants, however, approximately halved to that of the parent. Compared to the parent, the alcohol yield from rice and sweet potato shochu mash of the mutant increased ~5.7% and 3.0%, respectively. The mutant strains showed significantly low glucose assimilability despite the fructose one was almost normal, and they had a single missense or nonsense mutation in the glucokinase gene glkA. The recombinant strain that was introduced at one of the mutations, glkA Q300K, demonstrated similar but not identical phenotypes to the mutant strain. This result indicates that glkA Q300K is one of the major mutations in 2-DG resistant strains. 2-deoxyglucose resistant-black koji mold shows high glycosidase and low citric acid production with poor mycelia growth. These are mainly caused by glucokinase mutation. [ABSTRACT FROM AUTHOR]

The post-translational modification of proteins by O-linked N -acetylglucosamine (O-GlcNAc) dynamically programmes cellular physiology to maintain homoeostasis and tailor biochemical pathways to meet context-dependent cellular needs. Despite diverse roles of played by O-GlcNAc, only two enzymes act antagonistically to govern its cycling; O-GlcNAc transferase installs the monosaccharide on target proteins, and O-GlcNAc hydrolase removes it. The recent literature has exposed a network of mechanisms regulating these two enzymes to choreograph global, and target-specific, O-GlcNAc cycling in response to cellular stress and nutrient availability. Herein, we amalgamate these emerging mechanisms from a structural and molecular perspective to explore how the cell exerts fine control to regulate O-GlcNAcylation of diverse proteins in a selective fashion. [ABSTRACT FROM AUTHOR]

Within their native milieu of the cell, the activities of enzymes are controlled by a range of factors including protein interactions and post-translational modifications. The involvement of these factors in fundamental cell biology and the etiology of diseases is stimulating interest in monitoring enzyme activities within tissues. The creation of synthetic substrates, and their use with different imaging modalities, to detect and quantify enzyme activities has great potential to propel these areas of research. Here we describe the latest developments relating to the creation of substrates for imaging and quantifying the activities of glycoside hydrolases, focusing on mammalian systems. The limitations of current tools and the difficulties within the field are summarised, as are prospects for overcoming these challenges. [ABSTRACT FROM AUTHOR]

Abstract Background Genomic and proteomic analysis are potent tools for metabolic characterization of microorganisms. Although cellulose usually triggers cellulase production in cellulolytic fungi, the secretion of the different enzymes involved in polymer conversion is subjected to different factors, depending on growth conditions. These enzymes are key factors in biomass exploitation for second generation bioethanol production. Although highly effective commercial cocktails are available, they are usually deficient for β-glucosidase activity, and genera like Penicillium and Talaromyces are being explored for its production. Results This article presents the description of Talaromyces amestolkiae as a cellulase-producer fungus that secretes high levels of β-glucosidase. β-1,4-endoglucanase, exoglucanase, and β-glucosidase activities were quantified in the presence of different carbon sources. Although the two first activities were only induced with cellulosic substrates, β-glucosidase levels were similar in all carbon sources tested. Sequencing and analysis of the genome of this fungus revealed multiple genes encoding β-glucosidases. Extracellular proteome analysis showed different induction patterns. In all conditions assayed, glycosyl hydrolases were the most abundant proteins in the supernatants, albeit the ratio of the diverse enzymes from this family depended on the carbon source. At least two different β-glucosidases have been identified in this work: one is induced by cellulose and the other one is carbon source-independent. The crudes induced by Avicel and glucose were independently used as supplements for saccharification of slurry from acid-catalyzed steam-exploded wheat straw, obtaining the highest yields of fermentable glucose using crudes induced by cellulose. Conclusions The genome of T. amestolkiae contains several genes encoding β-glucosidases and the fungus secretes high levels of this activity, regardless of the carbon source availability, although its production is repressed by glucose. Two main different β-glucosidases have been identified from proteomic shotgun analysis. One of them is produced under different carbon sources, while the other is induced in cellulosic substrates and is a good supplement to Celluclast in saccharification of pretreated wheat straw.

Gut microbiota are significant to the host's nutrition and provide a flexible way for the host to adapt to extreme environments. However, whether gut microbiota help the host to colonize caves, a resource-limited environment, remains unknown. The nonobligate cave frog Oreolalax rhodostigmatus completes its metamorphosis within caves for 3-5 years before foraging outside. Their tadpoles are occasionally removed from the caves by floods and utilize outside resources, providing a contrast to the cave-dwelling population. For both cave and outside tadpoles, the development-related reduction in their growth rate and gut length during prometamorphosis coincided with a shift in their gut microbiota, which was characterized by decreased Lactobacillus and Cellulosilyticum and Proteocatella in the cave and outside individuals, respectively. The proportion of these three genera was significantly higher in the gut microbiota of cave-dwelling individuals compared with those outside. The cave-dwellers' gut microbiota harbored more abundant fibrolytic, glycolytic, and fermentative enzymes and yielded more short-chain fatty acids, potentially benefitting the host's nutrition. Experimentally depriving the animals of food resulted in gut atrophy for the individuals collected outside the cave, but not for those from inside the cave. Imitating food scarcity reproduced some major microbial features (e.g. abundant Proteocatella and fermentative genes) of the field-collected cave individuals, indicating an association between the cave-associated gut microbiota and resource scarcity. Overall, the gut microbiota may reflect the adaptation of O. rhodostigmatus tadpoles to resource-limited environments. This extends our understanding of the role of gut microbiota in the adaptation of animals to extreme environments., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

This study aimed to recover metagenome-assembled genomes (MAGs) from human fecal samples to characterize the glycosidase profiles of Bifidobacterium species exposed to different prebiotic oligosaccharides (galacto-oligosaccharides, fructo-oligosaccharides and human milk oligosaccharides, HMOs) as well as high-fiber diets. A total of 1806 MAGs were recovered from 487 infant and adult metagenomes. Unsupervised and supervised classification of glycosidases codified in MAGs using machine-learning algorithms allowed establishing characteristic hydrolytic profiles for B. adolescentis, B. bifidum, B. breve, B. longum and B. pseudocatenulatum, yielding classification rates above 90%. Glycosidase families GH5 44, GH32, and GH110 were characteristic of B. bifidum. The presence or absence of GH1, GH2, GH5 and GH20 was characteristic of B. adolescentis, B. breve and B. pseudocatenulatum, while families GH1 and GH30 were relevant in MAGs from B. longum. These characteristic profiles allowed discriminating bifidobacteria regardless of prebiotic exposure. Correlation analysis of glycosidase activities suggests strong associations between glycosidase families comprising HMOs-degrading enzymes, which are often found in MAGs from the same species. Mathematical models here proposed may contribute to a better understanding of the carbohydrate metabolism of some common bifidobacteria species and could be extrapolated to other microorganisms of interest in future studies.

Acinetobacter baumannii, one of the most significant nosocomial pathogens, is capable of producing structurally diverse capsular polysaccharides (CPSs) which are the primary receptors for A. baumannii bacteriophages encoding polysaccharide-degrading enzymes. To date, bacterial viruses specifically infecting A. baumannii strains belonging to more than ten various capsular types (K types) were isolated and characterized. In the present study, we investigate the biological properties, genomic organization, and virus–bacterial host interaction strategy of novel myovirus TaPaz isolated on the bacterial lawn of A. baumannii strain with a K47 capsular polysaccharide structure. The phage linear double-stranded DNA genome of 93,703 bp contains 178 open reading frames. Genes encoding two different tailspike depolymerases (TSDs) were identified in the phage genome. Recombinant TSDs were purified and tested against the collection of A. baumannii strains belonging to 56 different K types. One of the TSDs was demonstrated to be a specific glycosidase that cleaves the K47 CPS by the hydrolytic mechanism.

Unique biodiversity and biotechnological potential of microorganisms in the Antarctic region contributes to the intensive search among them of enzymes producers with new properties and a wide range of stability. The aim of the work was to study the glycosidase activity in 26 strains of psychrotolerant UV-resistant yeast isolated from soil-plant сenosis of the Antarctic and evaluate their biotechnological potential as a producer of enzymes. Methods. Yeast cultures were grown in submerged conditions (15-42 °C). Enzymes activities were determined in the culture supernatant. Synthetic monosaccharide derivatives were used to measure glycosidase activity. Casein and elastin-congo red were used as substrates for the determination of proteolytic activity. Results. A spectrum of 12 glycosidase activities (α- and β-D-glucosidase, β-D-galactosidase, β-D-glucuronidase, α-L-fucosidase, α-L-ramnosidase, α-D-mannosidase, α- and β-D-xylosidase, α- and β-Nacetylglucosaminidase, β-N-acetylgalactosaminidase) was studied in Antarctic yeast. The most common were β-N-acetylglucosaminidase and β-glucosidase activities (65 and 58% of strains respectively). 4 strains (6p1s, 2299, 31s and 4p5s2) showed high activity of the complex of cellulose-degrading enzymes. 5p5s1 strain, isolated from the Deshampcia antarctica grass, showed high β-D-xylosidase activity (2.8 U/ml). Rhodotorula mucilaginosa 33c strain showed a spectrum of ten activities, including high α-L-rhamnosidase activity (6.2 U/ml). Conclusion. Yeast cultures from the Antarctic phytocenosis can be used for the directional search for microbial glycosidases with new properties and a wide range of activity. [ABSTRACT FROM AUTHOR]

In the present study, out of three isoforms of α-mannosidase identified in the crude extract of defatted Custard apple seed powder, isoform III has been purified to homogeneity by two-step chromatography: hydrophobic interaction and gel filtration. The purified Custard apple α-mannosidase isoform III (CAM) hydrolyzed both chromogenic (p -nitrophenyl-α-D-mannopyranoside) and fluorescent (4-methylumbelliferyl α-D-mannopyranoside) substrates. Custard apple α-mannosidase migrated as a single band in native PAGE, showed about 220 kDa molecular mass in gel filtration and in SDS PAGE, dissociated into four bands (Mr ~ 75, 68, 56 and 50 kDa respectively). Temperature and pH optima were found to be 50 °C and 4.0–5.0 respectively and CAM was stable up to 60–70 °C. The enzymatic activity of CAM was inhibited by EDTA, Ag+, Hg2+, Ni2+ and swainsonine (IC 50 value of 1.5 μM). CAM was observed to be a metallo enzyme requiring zinc for its activity. Kinetic parameters K M and Vmax were found to be 1.75 mM and 0.068 U/mL respectively. The CD spectral analysis at far UV region (190–300 nm) shows that purified CAM exists as helix (30.4%), β turns (18%) and random coils (29.7%) in its secondary structure. Chemical modification studies with N -Bromosuccinimide revealed the presence of tryptophan in its active site. • A vacuolar α-mannosidase was purified to homogeneity from Custard apple seeds • Custard apple α-mannosidase was inhibited by swainsonine and EDTA • CD spectral analysis revealed more content of α-helix in the secondary structure of Custard apple α-mannosidase • Chemical modification with NBS showed the involvement of tryptophan in the active site of Custard apple α-mannosidase [ABSTRACT FROM AUTHOR]

β-N-Acetylhexosaminidases (EC 3.2.1.52) are a unique family of glycoside hydrolases with dual substrate specificity and a particular reaction mechanism. Though hydrolytic enzymes per se, their good stability, easy recombinant production, absolute stereoselectivity, and a broad substrate specificity predestine these enzymes for challenging applications in carbohydrate synthesis. This mini-review aims to demonstrate the catalytic potential of β-N-acetylhexosaminidases in a range of unusual reactions, processing of unnatural substrates, formation of unexpected products, and demanding reaction designs. The use of unconventional media can considerably alter the progress of transglycosylation reactions. By means of site-directed mutagenesis, novel catalytic machineries can be constructed. Glycosylation of difficult substrates such as sugar nucleotides was accomplished, and the range of afforded glycosidic bonds comprises unique non-reducing sugars. Specific functional groups may be tolerated in the substrate molecule, which makes β-N-acetylhexosaminidases invaluable allies in difficult synthetic problems. [ABSTRACT FROM AUTHOR]

Regions with extreme environmental conditions can be considered as a system with unique biodiversity and potential. The aim was to determine resistance range to UVirradiation, temperature and concentration of NaCl, the glycosidase and proteolytic activity of bacteria isolated from terrestrial ecosystems of Antarctica, Ecuador and Israel. Methods. The objects of study were 19 extremе-tolerant bacteria. UV-irradiation of bacteria was performed with a BUF-15 lamp (λ = 254 nm) in the range of 37-1440 J/m². The temperature range of growth was determined at a temperature of 1-42 ºC, halotolerance -- at NaCl concentration of 1-100 g/l. Enzymes activities were studied in the culture supernatant. To determine glycosidase activities, p-nitrophenyl substrates, galactomannan guar and soluble starch were used. Casein and elastin were used as substrates for the determination of proteolytic activity. Results. Antarctic pigmented microorganisms showed a higher resistance to UV than non-pigmented. Cultures from highland biotopes and saline soils were resistant to UV-doses of 400-1440 J/m². Investigated strains mainly belonged to psychotolerant and moderately halophilic bacteria. Most of the strains showed α-amylase and/or β-mannanase activity. Two strains, 3271 and 3456, exhibited simultaneously four and five glycosidase activities respectively. Caseinolytic activity was observed in 17 cultures, elastase -- in one. Conclusions. Ecophysiological characteristics of the studied cultures reflect their adaptation to extreme environmental conditions. Both pigmented and non-pigmented microorganisms were resistant to UV-radiation, most of them are psychrotolerant moderate halophiles with α-amylase, β-mannanase, caseinolytic and elastase activity. [ABSTRACT FROM AUTHOR]

High biotechnology potential of marine microorganisms as producers of enzymes has been driving extensive research of their diversity and biological features in recent years, including the active screening of glycosidases of marine bacteria for commercial applications. The aim of this paper was to study the ability of bacteria isolated from the bottom sediments of the Black Sea to produce glycosidases of various specificities, as well as to investigate the possibility of using coordination compounds of germanium to increase the enzymatic activity of bacteria of the genus Bacillus. Methods. The cultivation of bacteria was carried out in submerged conditions; maltose and rhamnose were used as carbon sources. Glycosidases activities were determined by the Romero and Davis method, protein - by the Lowry method. Coordination compounds of germanium were used as effectors of biosynthesis and activity of α-L-rhamnosidase in concentrations of 0.1 and 0.01%. Results. Based on the screening of 12 glycosidases activities (β-D-galactosidase, β-D-glucuronidase, α-D-mannosidase, α-D-xylosidase, α-D-fucosidase, β-D-xylosidase, β-D-glucosidase, N-acetyl-β-D-glucosaminidase, N-acetyl-α-D-glucosaminidase, N-acetyl-β-D-galactosaminidase, α-D-glucosidase, α-L-rhamnosidase) among 10 cultures of Bacillus, α-L-rhamnosidase activity (0.03-0.18 U/mg protein) was detected in 8 strains. Culture of Bacillus sp. 19 was the most active. Decrease in α-L-rhamnosidase activity of a Bacillus sp. 19 was observed after introduction of germanium complexes (0.1 and 0.01%) into the growth medium. We showed that bis (bipyridine) chloromodynam (II) bis (citrato) germanate (IV) octahydrate and tris (bipyridine) nickel (II) bis (citrato) germanate(IV) monohydrate can be used to increase α-L-rhamnosidase activity of Bacillus sp. 19. Conclusions. α-L-Rhamnosidase activity of marine strains belonging to the species of Bacillus licheniformis, Bacillus atrophaus, Bacillus subtiliswas shown for the first time. Activation of α-L-rhamnosidase from Bacillus sp. 19 by coordination compounds of germanium was observed. [ABSTRACT FROM AUTHOR]

β-N-Acetylhexosaminidases (EC 3.2.1.52) are typical of their dual activity encompassing both N-acetylglucosamine and N-acetylgalactosamine substrates. Here we present the isolation and characterization of a selective β-N-acetylhexosaminidase from the fungal strain of Aspergillus versicolor. The enzyme was recombinantly expressed in Pichia pastoris KM71H in a high yield and purified in a single step using anion-exchange chromatography. Homologous molecular modeling of this enzyme identified crucial differences in the enzyme active site that may be responsible for its high selectivity for N-acetylglucosamine substrates compared to fungal β-N-acetylhexosaminidases from other sources. The enzyme was used in a sequential reaction together with a mutant β-N-acetylhexosaminidase from Talaromyces flavus with an enhanced synthetic capability, affording a bioactive disaccharide bearing an azido functional group. The azido function enabled an elegant multivalent presentation of this disaccharide on an aromatic carrier. The resulting model glycoconjugate is applicable as a selective ligand of galectin-3 — a biomedically attractive human lectin. These results highlight the importance of a general availability of robust and well-defined carbohydrate-active enzymes with tailored catalytic properties for biotechnological and biomedical applications. [ABSTRACT FROM AUTHOR]