HAEMAGGLUTINATION ACTIVITY OF THE EXTRACTS FROM SOME VIETNAM MARINE INVERTEBRATES

Aqueous extracts from 21 species of Vietnam marine invertebrates, including 11 bivalve and 10 gastropod species, were examined for haemagglutination activity using native and enzyme-treated different animal and human erythrocytes. The 8 bivalve and 10 gastropod species were found to have haemagglutinination activities toward at least one type of erythrocyte tested. A total of 86% of marine invertebrate species surveyed were active. Strong activity was detected in extracts from two bivalve species (Tridacna squamosa and Geloina coaxans) and three gastropod species (Tutufa rubeta, Pleuroploca trapezium and Tectus conus) with enzyme-treated rabbit, horse and human A, B, O erythrocytes. In a haemagglutination–inhibition test with various monosaccharides and glycoproteins, haemagglutination activities of two extracts from T. rubeta and P. trapezium had no affinity for any of the monosaccharides and glycoproteins tested, while activities of the extracts from T. squamosa and T. conus were strongly inhibited by porcine stomach mucin tested, suggesting the presence of lectins specific for O-glycans of these species. The activities of four marine invertebrate extracts were stable over a wide range ofpH and temperature. The haemagglutination activities of T. rubeta and P. trapezium extracts were independent of the presence of divalent cations, whereas the haemagglutination activity of extracts from T. squamosa and T. conus were slightly dependent on the presence of divalent cations. The results suggest that Vietnam marine invertebrates may be good sources of useful lectins for biochemical and biomedical applications

EFFECT OF CULTURE CONDITIONS FOR ANTIMICROBIAL ACTIVITY OF MARINE - DERIVED FUNGUS ASPERGILLUS FLOCCULOSUS 01NT.1.1.5

The biosynthesis of compounds with antibiotic activity produced by marine fungi, strongly depends on their growth conditions. A good understanding of the role of culture conditions in the biosynthesis of metabolites may lead to better exploitation of microbial metabolites. In this study, the influence of culture conditions including incubation period, initial pH and salinity on antimicrobial activity and secondary metabolites production of marine fungus 01NT.1.1.5 was investigated. This isolate, obtained from sponge Stylissa sp. in Nha Trang Bay, exhibited a broad spectrum of in vitro antimicrobial activity to Bacillus cereus ATCC 11778, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Listeria monocytogenes ATCC 19111, Streptococcus faecalis ATCC 19433 and Candida albicans ATCC 10231. According to morphological characteristics and sequence analysis of 28S rDNA, the fungus was identified as Aspergillus flocculosus. The results indicated that antimicrobial activity and metabolite amount were highest when the fungus was cultivated in rice medium with incubation period of 20 days. The optimum salinity of 35 g/L and initial pH of 6.0 were found for the maximum antibiotic production. The colony growth, antimicrobial activity and production of secondary metabolites of the strain A. flocculosus 01NT.1.1.5 varied depending on salt concentrations and initial pH of medium. Particularly, extract of this fungus only showed activity against C. albicans when it was cultured in medium with 30-35 g/L salinity and initial pH 4.0-8.0. The results  indicate that salinity and initial pH along with cultivation period are important factors influencing antimicrobial activity and secondary metabolites of A. flocculosus 01NT.1.1.5, and might be for other marine fungi

The Endo-α(1,4) Specific Fucoidanase Fhf2 From Formosa haliotis Releases Highly Sulfated Fucoidan Oligosaccharides

Fucoidanases are endo-fucoidanases (also known as endo-fucanases) that catalyze hydrolysis of α-glycosidic linkages in fucoidans, a family of sulfated fucose-rich polysaccharides primarily found in the cell walls of brown seaweeds. Fucoidanases are promising tools for producing bioactive fucoidan oligosaccharides for a range of biomedical applications. High sulfation degree has been linked to high bioactivity of fucoidans. In this study, a novel fucoidanase, Fhf2, was identified in the genome of the aerobic, Gram-negative marine bacterium Formosa haliotis. Fhf2 was found to share sequence similarity to known endo-α(1,4)-fucoidanases (EC 3.2.1.212) from glycoside hydrolase family 107. A C-terminal deletion mutant Fhf2∆484, devoid of 484 amino acids at the C-terminus, with a molecular weight of approximately 46 kDa, was constructed and found to be more stable than the full-length Fhf2 protein. Fhf2∆484 showed endo-fucoidanase activity on fucoidans from different seaweed species including Fucus evanescens, Fucus vesiculosus, Sargassum mcclurei, and Sargassum polycystum. The highest activity was observed on fucoidan from F. evanescens. The Fhf2∆484 enzyme was active at 20–45°C and at pH 6–9 and had optimal activity at 37°C and pH 8. Additionally, Fhf2∆484 was found to be calcium-dependent. NMR analysis showed that Fhf2∆484 catalyzed hydrolysis of α(1,4) linkages between L-fucosyl moieties sulfated on C2 (similar to Fhf1 from Formosa haliotis), but Fhf2∆484 in addition released oligosaccharides containing a substantial amount of 2,4-disulfated fucose residues. The data thus suggest that the Fhf2∆484 enzyme could be a valuable candidate for producing highly sulfated oligosaccharides applicable for fucoidan bioactivity investigations

Spatiotemporal evolution of SARS-CoV-2 Alpha and Delta variants during large nationwide outbreak of COVID-19, Vietnam, 2021

We analyzed 1,303 SARS-CoV-2 whole-genome sequences from Vietnam, and found the Alpha and Delta variants were responsible for a large nationwide outbreak of COVID-19 in 2021. The Delta variant was confined to the AY.57 lineage and caused >1.7 million infections and >32,000 deaths. Viral transmission was strongly affected by nonpharmaceutical interventions

Explorative discovery and characterization of new microbial GH107 endo-fucoidanases

Fucoidans are heterologous sulfated fucose-containing polysaccharides primarily found in the cell wall of brown seaweeds (Bilan and Usov, 2008; Ale and Meyer, 2013). Fucoidans were reported to possess various biological activities, including anti-oxidatant, anti-tumor, anti-coagulation, and anti-inflammatory (Ale et al., 2011; Li et al., 2008; Lim et al., 2014). Unfortunately, the complicated structures and too high molecular weight of fucoidans is a disadvantage for their applications in food, cosmetic, and pharmaceutical industry (Chollet et al., 2016; Flórez-Fernández et al., 2018). The modification of native fucoidans via enzymatic treatments able to specifically degrade fucoidan to produce chemical homogenous and well-defined bioactive oligosaccharides would be a valuable process.This thesis describes the discovery, stabilization, and characterization of four glycosyl hydrolase 107 (GH107) endo-fucoidanases all from marine bacteria, including Fhf2 from Formosa haliotis, Psf1 from Pseudoalteromonas sp. S3178, and Mef3 and Mef4 from Muricauda eckloniae. The recombinant fucoidanase enzymes were expressed and purified. Various fucoidan substrates were initially tested to determine the enzyme activity and specificity.A truncation strategy was used where parts of the C-terminal was removed from the Fhf2 sequence, since this protein was degraded during the expression. The substrate specificity of the truncated fucoidanase Fhf2Δ484 containing only the catalytic D1 domain characteristic of GH107 family members was investigated and the enzyme was biochemically characterized. The enzymatic reaction of Fhf2Δ484 on fucoidan from Fucus evanescens was carried out under the determined optimal conditions for production of oligosaccharides. Analysis of the structure of these hydrolyzed products using Nuclear Magnetic Resonance Spectroscopy (NMR) indicated that this enzyme is an α(1→4)- specific endo-fucoidanase and in addition it releases oligosaccharides with a substantial amount of C2 sulfated and C2, C4 disulfated fucose residues.The Psf1 gene found in the genomic sequence of the marine bacteria Pseudoalteromonas sp. S3178 isolated from a shrimp caught in the Southern Ocean in 2007 during the Galathea 3 global expedition was successfully cloned, expressed, and purified. Psf1 catalyzes the hydrolysis of α(1→3)-linked fucose residues containing fucoidan from Saccharina latissima and Undaria pinnatifida, and galactofucan type fucoidan from Sargassum oligocystum. The temperature optimum for Psf1 is 10-30 °C and interestingly, Psf1 shows activity down to even 1 °C and is therefore the first psychrophilic endofucoidanase described to date. Psf1 could be an interesting candidate for biotechnological applications that require low temperature condition.In the last phase of this PhD project, I focused on the C-terminal deletion strategy for stabilization and identification of the functional domains in the fucoidanases Mef3 and Mef4, as well as the characterization and determination of the substrate specificity and biochemical properties of the truncated enzymes. A galactose binding-like domain predicted in Mef3 and Mef4, might play an important role in fucoidanase function. Mef3 and Mef4 are likely endo-α(1→3)-specific fucoidanases. Besides sharing some similar characteristics, they also have distinct characteristics such as optimal temperature, pH, the influence of metal ions, and thermostability properties.The achievement of this PhD project reflect the diversity of biological characterizations and substrate specificity of fucoidanases from the GH107 family and extends the general knowledge of fucoidanases, which could be used to prepare chemically homogenous and well-defined bioactive oligosaccharides, a key requisite for application in the pharmaceutical industry