Identification and characterization of emGalaseE, a ß-1,4 galactosidase from Elizabethkingia meningoseptica, and its application on living cell surface.

The biological function of terminal galactose on glycoprotein is an open field of research. Although progress had being made on enzymes that can remove the terminal galactose on glycoproteins, there is a lack of report on galactosidases that can work directly on living cells. In this study, a unique beta 1,4 galactosidase was isolated from Elizabethkingia meningoseptica (Em). It exhibited favorable stability at various temperatures (4-37 °C) and pH (5-8) levels and can remove ß-1, 4 linked galactoses directly from glycoproteins. Using Alanine scanning, we found that two acidic residues (Glu-468, and Glu-531) in the predicted active pocket are critical for galactosidase activity. In addition, we also demonstrated that it could cleave galactose residues present on living cell surface. As this enzyme has a potential application for living cell glycan editing, we named it emGalaseE or glycan-editing galactosidase I (csgeGalaseI). In summary, our findings lay the groundwork for further investigation by presenting a simple and effective approach for the removal of galactose moieties from cell surface.

Expression and Characterization of a ß-Galactosidase from the Pacific Oyster, Crassostrea gigas, and Evaluation of Strategies for Testing Substrate Specificity.

ß-Galactosidases (EC 3.2.1.23) are exoglycosidases that catalyze the cleavage of glycoconjugates with terminal ß-D-galactose residues in ß1,3-, ß1,4- or ß1,6-linkage. Although this family of exoglycosidases has been extensively studied in vertebrates, plants, yeast, and bacteria, little information is available for mollusks. Mollusks are a diverse and highly successful group of animals that play many different roles in their ecosystems, including filter feeders and detritivores. Here, the first ß-galactosidase from the Pacific oyster, Crassostrea gigas was discovered, biochemically characterized, and compared to our previously characterized slug enzyme from Arion vulgaris (UniProt Ref. Nr.: A0A0B7AQJ9). Overall, the mussel enzyme showed similar biochemical parameters to the snail enzyme. The enzyme from C. gigas was most active in an acidic environment (pH 3.5) and at a reaction temperature of 50 °C. Optimal storage conditions were up to 37 °C. In contrast to the enzyme from A. vulgaris, the supplementation of cations (Ni2+, Co2+, Mn2+, Mg2+, Ca2+, Cu2+, Ba2+) increased the activity of the enzyme from C. gigas. Substrate specificity studies of the ß-galactosidases from the mussel, C. gigas, and the slug, A. vulgaris, revealed activity towards terminal ß1,3- and ß1,4-linked galactose residues for both enzymes. Using the same substrates in labeled and unlabeled form, we were able to detect the effect of labeling on the ß-galactosidase activity using MALDI-TOF MS, HPTLC, and HPLC. While lactose was cleaved by the enzymes in an unlabeled or labeled state, galacto-N-biose was not cleaved as soon as a 2-amino benzoic acid label was added. In this study we present the biochemical characterization of the first recombinantly expressed ß-galactosidase from the Pacific oyster, C. gigas, and we compare different analytical methods for the determination of ß-galactosidase activity using the enzyme from C. gigas and A. vulgaris.

Dual 6Pß-Galactosidase/6Pß-Glucosidase GH1 Family for Lactose Metabolism in the Probiotic Bacterium Lactiplantibacillus plantarum WCFS1.

Intestinal lactic acid bacteria can help alleviate lactose maldigestion by promoting lactose hydrolysis in the small intestine. This study shows that protein extracts from probiotic bacterium Lactiplantibacillus plantarum WCFS1 possess two metabolic pathways for lactose metabolism, involving ß-galactosidase (ß-gal) and 6Pß-galactosidase (6Pß-gal) activities. As L. plantarum WCFS1 genome lacks a putative 6Pß-gal gene, the 11 GH1 family proteins, in which their 6Pß-glucosidase (6Pß-glc) activity was experimentally demonstrated,, were assayed for 6Pß-gal activity. Among them, only Lp_3525 (Pbg9) also exhibited a high 6Pß-gal activity. The sequence comparison of this dual 6Pß-gal/6Pß-glc GH1 protein to previously described dual GH1 proteins revealed that L. plantarum WCFS1 Lp_3525 belonged to a new group of dual 6Pß-gal/6Pß-glc GH1 proteins, as it possessed conserved residues and structural motifs mainly present in 6Pß-glc GH1 proteins. Finally, Lp_3525 exhibited, under intestinal conditions, an adequate 6Pß-gal activity with possible relevance for lactose maldigestion management.

Biochemical characterization of a novel ß-galactosidase from Lacticaseibacillus zeae and its application in synthesis of lacto-N-tetraose.

Lacto-N-tetraose (LNT) is one of the most important components of human milk oligosaccharides, which has various beneficial health effects. ß-Galactosidase is an important enzyme used in dairy processing. The transglycosylation activity of ß-galactosidases offers an attractive approach for LNT synthesis. In this study, we reported for the first time the biochemical characterization of a novel ß-galactosidase (LzBgal35A) from Lacticaseibacillus zeae. LzBgal35A belongs to glycoside hydrolases (GH) family 35 and shared the highest identity of 59.9% with other reported GH 35 members. The enzyme was expressed as soluble protein in Escherichia coli. The purified LzBgal35A displayed optimal activity at pH 4.5 and 55°C. It was stable within the pH range of 3.5 to 7.0 and up to 60°C. Moreover, LzBgal35A could catalyze the synthesis of LNT via transferring the galactose residue from o-nitrophenyl-ß-galactopyranoside to lacto-N-triose II. Under optimal conditions, the conversion rate of LNT reached 45.4% (6.4 g/L) within 2 h, which was by far the highest yield of LNT synthesized through a ß-galactosidase-mediated transglycosylation reaction. This study demonstrated that LzBgal35A has great potential application in LNT synthesis.

Bifunctional and monofunctional α-neoagarooligosaccharide hydrolases from Streptomyces coelicolor A3(2).

Agar is a galactan and a major component of the red algal cell wall. Agar is metabolized only by specific microorganisms. The final step of the ß-agarolytic pathway is mediated by α-neoagarooligosaccharide hydrolase (α-NAOSH), which cleaves neoagarobiose to D-galactose and 3,6-anhydro-α-L-galactose. In the present study, two α-NAOSHs, SCO3481 and SCO3479, were identified in Streptomyces coelicolor A3(2). SCO3481 (370 amino acids, 41.12 kDa) and SCO3479 (995 amino acids, 108.8 kDa) catalyzed the hydrolysis of the α-(1,3) glycosidic bonds of neoagarobiose, neoagarotetraose, and neoagarohexaose at the nonreducing ends, releasing 3,6-anhydro-α-L-galactose. Both were intracellular proteins without any signal peptides for secretion. Similar to all α-NAOSHs reported to date, SCO3481 belonged to the glycosyl hydrolase (GH) 117 family and formed dimers. On the other hand, SCO3479 was a large monomeric α-NAOSH belonging to the GH2 family with a ß-galactosidase domain. SCO3479 also clearly showed ß-galactosidase activity toward lactose and artificial substrates, but SCO3481 did not. The optimum conditions for α-NAOSH were pH 6.0 and 25 °C for SCO3481, and pH 6.0 and 30 °C for SCO3479. Enzymatic activity was enhanced by Co2+ for SCO3481 and Mg2+ for SCO3479. The ß-galactosidase activity of SCO3479 was maximum at pH 7.0 and 50 °C and was increased by Mg2+. Many differences were evident in the kinetic parameters of each enzyme. Although SCO3481 is typical of the GH117 family, SCO3479 is a novel α-NAOSH that was first reported in the GH2 family. SCO3479, a unique bifunctional enzyme with α-NAOSH and ß-galactosidase activities, has many advantages for industrial applications. KEY POINTS: • SCO3481 is a dimeric α-neoagarooligosaccharide hydrolase belonging to GH117. • SCO3479 is a monomeric α-neoagarooligosaccharide hydrolase belonging to GH2. • SCO3479 is a novel and unique bifunctional enzyme that also acts as a ß-galactosidase.

Increased Galactosidase Beta 1 Expression as a Senescent Key Factor in ß-Cells Function Modulation at the Early Steps of Type 2 Diabetes.

BACKGROUND: In type 2 diabetes, insulin resistance is observed, and ß-cells are incapable of responding to glycemia demands, leading to hyperglycemia. Although the nature of ß-cells dysfunction in this disease is not fully understood, a link between the induction of pancreatic ß-cell premature senescence and its metabolic implications has been proposed. This study aimed to understand the relationship between diabetes and pancreatic senescence, particularly at the beginning of the disease. METHODS: C57Bl/6 J mice were fed two different diets, a normal diet and a high-fat diet, for 16 weeks. Pancreatic histomorphology analysis, insulin quantification, inflammation parameters, and senescence biomarkers for the experimental animals were assessed at weeks 12 and 16. RESULTS: The results proved that diabetes onset occurred at week 16 in the High Fat Diet group, supported by glycaemia, weight and blood lipid levels. Increased ß-cells size and number accompanied by increased insulin expression were observed. Also, an inflammatory status of the diabetic group was noted by increased levels of systemic IL-1ß and increased pancreatic fibrosis. Finally, the expression of galactosidase-beta 1 (GLB1) was significantly increased in pancreatic ß-cells. CONCLUSION: The study findings indicate that senescence, as revealed by an increase in GLB1 expression, is a key factor in the initial stage of diabetes.

Cloning, Expression, Purification and Characterization of the ß-galactosidase PoßGal35A from Penicillium oxalicum.

Galactosidases are industrially important enzymes that hydrolyze galactosidic bonds in carbohydrates. Identifying new galactosidases with distinct functional characteristics is of paramount importance. In this study, we report the finding of a novel ß-galactosidase PoßGal35A from the fungus Penicillium oxalicum. PoßGal35A belongs to the glycoside hydrolase family 35 (GH35), functions optimally at 70 °C and pH 5.0, and exhibits a specific high activity (191 ± 6.2 U/mg) towards pNPßgal. Ca2+, Fe3+and Ba2+ ions enhance the activity of the enzyme, whereas Cu2+ and Hg2+ significantly reduce it. This enzyme releases galactose from ß-1,3-galactan, ß-1,4-galactan, ß-1,6-galactan, as well as arabinogalactan from larchwood (LWAG). In addition, PoßGal35A acts synergistically with arabinosidase to degrade LWAG. These results suggest that PoßGal35A is a high activity exo-ß-1,3/4/6-galactanase that can be used to establish glycan blocks in glycoconjugates, and thus provides a new tool for biotechnological applications.

Ectopic expression of a grapevine alkaline α-galactosidase seed imbibition protein VvSIP enhanced salinity tolerance in transgenic tobacco plants.

Alpha-galactosidase seed imbibition protein (VvSIP) isolated from Vitis vinifera is up-regulated upon salt stress and mediates osmotic stress responses in a tolerant grapevine cultivar. So far, little is known about the putative role of this stress-responsive gene. In the present study, VvSIP function was investigated in model tobacco plants via Agrobacterium-mediated genetic transformation. Our results showed that overexpression of VvSIP exhibited increased tolerance to salinity at germination and late vegetative stage in transgenic Nicotiana benthamiana compared to the nontransgenic plants based on the measurement of the germination rate and biomass production. High salt concentrations of 200 and 400 mM NaCl in greenhouse-grown pot assay resulted in better relative water content, higher leaf osmotic potential, and leaf water potential in transgenic lines when compared to the wild-type (WT) plants. These physiological changes attributed to efficient osmotic adjustment improved plant performance and tolerance to salinity compared to the WT. Moreover, the VvSIP-expressing lines SIP1 and SIP2 showed elevated amounts of chlorophyll with lower malondialdehyde content indicating a reduced lipid peroxidation required to maintain membrane stability. When subjected to high salinity conditions, the transgenic tobacco VvSIP exhibited higher soluble sugar content, which may suggest an enhancement of the carbohydrate metabolism. Our findings indicate that the VvSIP is involved in plant salt tolerance by functioning as a positive regulator of osmotic adjustment and sugar metabolism, both of which are responsible for stress mitigation. Such a candidate gene is highly suitable to alleviate environmental stresses and thus could be a promising candidate for crop improvement.

[Isolation, culture and inflammatory characteristics of astrocytes in aged rats nervous tissue].

Objective: To establish an optimized method for the isolation and purification of astrocytes from the neural tissues of young and aged rats. Then, the morphological and functional differences of astrocytes between young and aged rats were compared to explore the functional changes of astrocytes after aging and its possible mechanism in the aging process. Methods: Young (2 months old) and aged (20 months old) SD rats were used. Astrocytes in brain and spinal cord tissue were purified by 50% - 35% percoll density gradient centrifugation. Each group of cells was set up with three duplicate wells. After 72 h of culture, Glial fibrillary acidic protein (GFAP) which was astrocyte specific marker were detected by immunofluorescence to evaluate the morphological characteristics. Cell senescence markers (p16 and p21) and ß- Galactosidase were detected by qPCR and staining respectively. The expressions of pro-inflammatory cytokines (IL-1ß, TNF-α) and anti-inflammatory cytokines were detected by qPCR. Results: Using 50%-35% percoll gradient separation, astrocytes were obtained with large number, good activity and purity of more than 95%, which could be used in subsequent experiments. Compared with the astrocytes in the nerve tissue of young rats, the astrocytes in the nervous tissue of the aged rats had fewer protrusions and tended to be activated in cell morphology; the positive rate of ß -galactosidase staining was increased significantly and the expressions of p16 and p21 were increased (P<0.01). The expressions of pro-inflammatory cytokines (IL-1ß, TNF-α) were increased (P<0.05), and the expression of anti-inflammatory cytokine (IL-10) was decreased (P<0.05) in astrocytes of the aged rats nervous tissue. Conclusion: The percoll gradient of 50% - 35% could be used as a method for separation, purification and primary culture of astrocytes. With the increase of age, astrocytes undergo cellular senescence, showing a pro-inflammatory phenotype, promoting inflammaging of the nervous system, which may be one of the mechanisms of nervous system aging and neurodegenerative diseases.

A Novel Carrageenan Metabolic Pathway in Flavobacterium algicola.

Carbohydrate-active enzymes are important components of the polysaccharide metabolism system in marine bacteria. Carrageenase is indispensable for forming carrageenan catalytic pathways. Here, two GH16_13 carrageenases showed likely hydrolysis activities toward different types of carrageenans (e.g., κ-, hybrid ß/κ, hybrid α/ι, and hybrid λ), which indicates that a novel pathway is present in the marine bacterium Flavobacterium algicola to use κ-carrageenan (KC), ι-carrageenan (IC), and λ-carrageenan (LC). A comparative study described the different features with another reported pathway based on the specific carrageenans (κ, ι, and λ) and expanded the carrageenan metabolic versatility in F. algicola. A further comparative genomic analysis of carrageenan-degrading bacteria indicated different distributions of carrageenan metabolism-related genes in marine bacteria. The crucial core genes encoding the GH127 α-3,6-anhydro-d-galactosidase (ADAG) and 3,6-anhydro-d-galactose (d-AHG)-utilized cluster have been conserved during evolution. This analysis further revealed the horizontal gene transfer (HGT) phenomenon of the carrageenan polysaccharide utilization loci (CarPUL) from Bacteroidetes to other bacterial phyla, as well as the versatility of carrageenan catalytic activities in marine bacteria through different metabolic pathways. IMPORTANCE Based on the premise that the specific carrageenan-based pathway involved in carrageenan use by Flavobacterium algicola has been identified, another pathway was further analyzed, and it involved two GH16_13 carrageenases. Among all the characterized carrageenases, the members of GH16_13 accounted for only a small portion. Here, the functional analysis of two GH16_13 carrageenases suggested their hydrolysis effects on different types of carrageenans (e.g., κ, hybrid ß/κ, hybrid α/ι-, and hybrid λ-), which led to the identification of another pathway. Further exploration enabled us to elucidate the novel pathway that metabolizes KC and IC in F. algicola successfully. The coexistence of these two pathways may provide improved survivability by F. algicola in the marine environment.

Generation of the induced pluripotent stem cell line UKWNLi005-A derived from a patient with the GLA mutation c.376A > G of unknown pathogenicity in Fabry disease.

Human dermal fibroblasts (HDF) were obtained by skin punch biopsy from a 51-year old man with suspected Fabry disease (FD), carrying the hemizygous c.376A > G variant in the α-galactosidase A gene (GLA). Cultured HDF were reprogrammed to induced pluripotent stem cells (iPSC) using a non-modified RNA-based transfection protocol. GLA-S126G-iPSC exhibit typical embryonic stem cell-like morphology, normal karyotype, expression of all tested pluripotency markers, and three germ layer differentiation potential. We provide a novel patient-specific cell line that can be used to investigate a genetic variation of yet unknown significance.

Infant-gut associated Bifidobacterium dentium strains utilize the galactose moiety and release lacto-N-triose from the human milk oligosaccharides lacto-N-tetraose and lacto-N-neotetraose.

Much evidence suggests a role for human milk oligosaccharides (HMOs) in establishing the infant microbiota in the large intestine, but the response of particular bacteria to individual HMOs is not well known. Here twelve bacterial strains belonging to the genera Bifidobacterium, Enterococcus, Limosilactobacillus, Lactobacillus, Lacticaseibacillus, Staphylococcus and Streptococcus were isolated from infant faeces and their growth was analyzed in the presence of the major HMOs, 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 2',3-difucosyllactose (DFL), lacto-N-tetraose (LNT) and lacto-N-neo-tetraose (LNnT), present in human milk. Only the isolated Bifidobacterium strains demonstrated the capability to utilize these HMOs as carbon sources. Bifidobacterium infantis Y538 efficiently consumed all tested HMOs. Contrarily, Bifidobacterium dentium strains Y510 and Y521 just metabolized LNT and LNnT. Both tetra-saccharides are hydrolyzed into galactose and lacto-N-triose (LNTII) by B. dentium. Interestingly, this species consumed only the galactose moiety during growth on LNT or LNnT, and excreted the LNTII moiety. Two ß-galactosidases were characterized from B. dentium Y510, Bdg42A showed the highest activity towards LNT, hydrolyzing it into galactose and LNTII, and Bdg2A towards lactose, degrading efficiently also 6'-galactopyranosyl-N-acetylglucosamine, N-acetyl-lactosamine and LNnT. The work presented here supports the hypothesis that HMOs are mainly metabolized by Bifidobacterium species in the infant gut.

A Shuttle-Vector System Allows Heterologous Gene Expression in the Thermophilic Methanogen Methanothermobacter thermautotrophicus ΔH.

Thermophilic Methanothermobacter spp. are used as model microbes to study the physiology and biochemistry of the conversion of molecular hydrogen and carbon dioxide into methane (i.e., hydrogenotrophic methanogenesis). Yet, a genetic system for these model microbes was missing despite intensive work for four decades. Here, we report the successful implementation of genetic tools for Methanothermobacter thermautotrophicus ΔH. We developed shuttle vectors that replicated in Escherichia coli and M. thermautotrophicus ΔH. For M. thermautotrophicus ΔH, a thermostable neomycin resistance cassette served as the selectable marker for positive selection with neomycin, and the cryptic plasmid pME2001 from Methanothermobacter marburgensis served as the replicon. The shuttle-vector DNA was transferred from E. coli into M. thermautotrophicus ΔH via interdomain conjugation. After the successful validation of DNA transfer and positive selection in M. thermautotrophicus ΔH, we demonstrated heterologous gene expression of a thermostable ß-galactosidase-encoding gene (bgaB) from Geobacillus stearothermophilus under the expression control of four distinct synthetic and native promoters. In quantitative in-vitro enzyme activity assay, we found significantly different ß-galactosidase activity with these distinct promoters. With a formate dehydrogenase operon-encoding shuttle vector, we allowed growth of M. thermautotrophicus ΔH on formate as the sole growth substrate, while this was not possible for the empty-vector control. IMPORTANCE The world economies are facing permanently increasing energy demands. At the same time, carbon emissions from fossil sources need to be circumvented to minimize harmful effects from climate change. The power-to-gas platform is utilized to store renewable electric power and decarbonize the natural gas grid. The microbe Methanothermobacter thermautotrophicus is already applied as the industrial biocatalyst for the biological methanation step in large-scale power-to-gas processes. To improve the biocatalyst in a targeted fashion, genetic engineering is required. With our shuttle-vector system for heterologous gene expression in M. thermautotrophicus, we set the cornerstone to engineer the microbe for optimized methane production but also for production of high-value platform chemicals in power-to-x processes.

Secreted Expression of mRNA-Encoded Truncated ACE2 Variants for SARS-CoV-2 via Lipid-Like Nanoassemblies.

The transfer of foreign synthetic messenger RNA (mRNA) into cells is essential for mRNA-based protein-replacement therapies. Prophylactic mRNA COVID-19 vaccines commonly utilize nanotechnology to deliver mRNA encoding SARS-CoV-2 vaccine antigens, thereby triggering the body's immune response and preventing infections. In this study, a new combinatorial library of symmetric lipid-like compounds is constructed, and among which a lead compound is selected to prepare lipid-like nanoassemblies (LLNs) for intracellular delivery of mRNA. After multiround optimization, the mRNA formulated into core-shell-structured LLNs exhibits more than three orders of magnitude higher resistance to serum than the unprotected mRNA, and leads to sustained and high-level protein expression in mammalian cells. A single intravenous injection of LLNs into mice achieves over 95% mRNA translation in the spleen, without causing significant hematological and histological changes. Delivery of in-vitro-transcribed mRNA that encodes high-affinity truncated ACE2 variants (tACE2v mRNA) through LLNs induces elevated expression and secretion of tACE2v decoys, which is able to effectively block the binding of the receptor-binding domain of the SARS-CoV-2 to the human ACE2 receptor. The robust neutralization activity in vitro suggests that intracellular delivery of mRNA encoding ACE2 receptor mimics via LLNs may represent a potential intervention strategy for COVID-19.

Enzymatically synthesized α-galactooligosaccharides attenuate metabolic syndrome in high-fat diet induced mice in association with the modulation of gut microbiota.

The composition and structure of gut microbiota plays an important role in obesity induced by a high-fat diet (HFD) and related metabolic syndrome (MetS). Previous studies have shown that galacto-oligosaccharides (GOSs) have an effective anti-obesity effect. In this study, we aimed to investigate the effect of enzymatically synthesized α-galacto-oligosaccharides (ES-α-GOSs) on MetS and gut microbiota dysbiosis in HFD-fed mice, and to further investigate whether the attenuation of MetS is associated with the modulation of gut microbiota. Our results indicated that ES-α-GOS could notably ameliorate obesity-related MetS, including hyperlipidemia, insulin resistance and mild inflammation. The subsequent analysis of gut microbiota further showed that ES-α-GOS supplements can significantly modulate the overall composition of the gut microbiota and reverse the gut microbiota disorder caused by HFD feeding. Moreover, Spearman correlation analysis showed that 40 key bacteria reversed by ES-α-GOS were highly associated with metabolic parameters. These results suggested that ES-α-GOSs could serve as a potential candidate for preventing obesity-induced MetS in association with the modulation of gut microbiota.

Molecular Characterization of a Novel 1,3-α-3,6-Anhydro-L-Galactosidase, Ahg943, with Cold- and High-Salt-Tolerance from Gayadomonas joobiniege G7.

1,3-α-3,6-anhydro-L-galactosidase (α-neoagarooligosaccharide hydrolase) catalyzes the last step of agar degradation by hydrolyzing neoagarobiose into monomers, D-galactose, and 3,6-anhydro-Lgalactose, which is important for the bioindustrial application of algal biomass. Ahg943, from the agarolytic marine bacterium Gayadomonas joobiniege G7, is composed of 423 amino acids (47.96 kDa), including a 22-amino acid signal peptide. It was found to have 67% identity with the α-neoagarooligosaccharide hydrolase ZgAhgA, from Zobellia galactanivorans, but low identity (< 40%) with the other α-neoagarooligosaccharide hydrolases reported. The recombinant Ahg943 (rAhg943, 47.89 kDa), purified from Escherichia coli, was estimated to be a monomer upon gel filtration chromatography, making it quite distinct from other α-neoagarooligosaccharide hydrolases. The rAhg943 hydrolyzed neoagarobiose, neoagarotetraose, and neoagarohexaose into D-galactose, neoagarotriose, and neoagaropentaose, respectively, with a common product, 3,6- anhydro-L-galactose, indicating that it is an exo-acting α-neoagarooligosaccharide hydrolase that releases 3,6-anhydro-L-galactose by hydrolyzing α-1,3 glycosidic bonds from the nonreducing ends of neoagarooligosaccharides. The optimum pH and temperature of Ahg943 activity were 6.0 and 20°C, respectively. In particular, rAhg943 could maintain enzyme activity at 10°C (71% of the maximum). Complete inhibition of rAhg943 activity by 0.5 mM EDTA was restored and even, remarkably, enhanced by Ca2+ ions. rAhg943 activity was at maximum at 0.5 M NaCl and maintained above 73% of the maximum at 3M NaCl. Km and Vmax of rAhg943 toward neoagarobiose were 9.7 mg/ml and 250 µM/min (3 U/mg), respectively. Therefore, Ahg943 is a unique α-neoagarooligosaccharide hydrolase that has cold- and high-salt-adapted features, and possibly exists as a monomer.

Mechanistic Insights into the Chaperoning of Human Lysosomal-Galactosidase Activity: Highly Functionalized Aminocyclopentanes and C-5a-Substituted Derivatives of 4-epi-Isofagomine.

Glycosidase inhibitors have shown great potential as pharmacological chaperones for lysosomal storage diseases. In light of this, a series of new cyclopentanoid ß-galactosidase inhibitors were prepared and their inhibitory and pharmacological chaperoning activities determined and compared with those of lipophilic analogs of the potent ß-d-galactosidase inhibitor 4-epi-isofagomine. Structure-activity relationships were investigated by X-ray crystallography as well as by alterations in the cyclopentane moiety such as deoxygenation and replacement by fluorine of a "strategic" hydroxyl group. New compounds have revealed highly promising activities with a range of ß-galactosidase-compromised human cell lines and may serve as leads towards new pharmacological chaperones for GM1-gangliosidosis and Morquio B disease.

Characterisation of an exo-(α-1,3)-3,6-anhydro-d-galactosidase produced by the marine bacterium Zobellia galactanivorans DsijT: Insight into enzyme preference for natural carrageenan oligosaccharides and kinetic characterisation on a novel chromogenic substrate.

Flavobacteriia are important degraders in the marine carbon cycle, due to their ability to efficiently degrade complex algal polysaccharides. A novel exo-(α-1,3)-3,6-anhydro-D-galactosidase activity was recently discovered from a marine Flavobacteriia (Zobellia galactanivorans DsijT) on red algal carrageenan oligosaccharides. The enzyme activity is encoded by a gene found in the first described carrageenan-specific polysaccharide utilization locus (CarPUL) that codes for a family 129 glycoside hydrolase (GH129). The GH129 family is a CAZy family that is strictly partitioned into two niche-based clades: clade 1 contains human host bacterial enzymes and clade 2 contains marine bacterial enzymes. Clade 2 includes the GH129 exo-(α-1,3)-3,6-anhydro-D-galactosidase from Z. galactanivorans (ZgGH129). Despite the discovery of the unique activity for ZgGH129, finer details on the natural substrate specificity for this enzyme are lacking. Examination of enzyme activity on natural carrageenan oligomers using mass spectrometry demonstrated that ZgGH129 hydrolyses terminal 3,6-anhydro-D-galactose from unsulfated non-reducing end neo-ß-carrabiose motifs. Due to the lack of chromogenic substrates to examine exo-(α-1,3)-3,6-anhydro-D-galactosidase activity, a novel substrate was synthesised to facilitate the first kinetic characterisation of an exo-(α-1,3)-3,6-anhydro-D-galactosidase, allowing determination of pH and temperature optimums and Michaelis-Menten steady state kinetic data.

A Pathogenic Galactosidase A Mutation Coexisting With an MYBPC3 Mutation in a Female Patient With Hypertrophic Cardiomyopathy.

The coexistence of GLA (Pro259Ser, c.775C>T) and MYBPC3 (c.1351+2T>C) mutations was found in a female patient with hypertrophic cardiomyopathy. Histology documented abundant vacuolisation with osmiophilic lamellar bodies and positive Gb3 immunohistochemistry. In the presence of a hypertrophic cardiomyopathy phenotype, the systematic search for unusual findings is mandatory to rule out a phenocopy.

Improvements in the production of Aspergillus oryzae ß-galactosidase crosslinked aggregates and their use in repeated-batch synthesis of lactulose.

Aspergillus oryzae ß-galactosidase was immobilized by aggregation and crosslinking, obtaining catalysts (CLAGs) well-endowed for lactulose synthesis. Type and concentration of the precipitating agent were determinants of immobilization yield, specific activity and thermal stability. CLAGs with specific activities of 64,007, 48,374 and 44,560 IUH g-1 were obtained using 50% v/v methanol, ethanol and propanol as precipitating agents respectively, with immobilization yields over 90%. Lactulose synthesis was conducted at 50 °C, pH 4.5, 50% w/w total sugars, 200 IUH g-1 of enzyme and fructose/lactose molar ratio of 8 in batch and repeated-batch operation. Lactulose yields were 0.19 g g-1 and 0.24 g g-1 for fructose to lactose molar ratios of 4 mol mol-1 and 8 mol mol-1 while selectivities were 3.3 mol mol-1 and 6.6 mol mol-1 respectively for CLAGs obtained by ethanol and propanol precipitation. Based on these results, both CLAGs were selected for the synthesis in repeated-batch mode. The cumulative mass of lactulose in repeated-batch was higher with CLAGs produced by ethanol and propanol precipitation than with the free enzyme. 86 and 93 repeated-batches could have been respectively performed with those CLAGs considering a catalyst replacement criterion of 50% of residual activity, as determined by simulation.