Biochemical Properties of a New Polysaccharide Lyase Family 25 Ulvan Lyase TsUly25B from Marine Bacterium Thalassomonas sp. LD5

Marine macroalgae, contributing much to the bioeconomy, have inspired tremendous attention as sustainable raw materials. Ulvan, as one of the main structural components of green algae cell walls, can be degraded by ulvan lyase through the β-elimination mechanism to obtain oligosaccharides exhibiting several good physiological activities. Only a few ulvan lyases have been characterized until now. This thesis explores the properties of a new polysaccharide lyase family 25 ulvan lyase TsUly25B from the marine bacterium Thalassomonas sp. LD5. Its protein molecular weight was 54.54 KDa, and it was most active under the conditions of 60 °C and pH 9.0. The K_m and k_cat values were 1.01 ± 0.05 mg/mL and 10.52 ± 0.28 s⁻¹, respectively. TsUly25B was salt-tolerant and NaCl can significantly improve its thermal stability. Over 80% of activity can be preserved after being incubated at 30 °C for two days when the concentration of NaCl in the solution is above 1 M, while 60% can be preserved after incubation at 40 °C for 10 h with 2 M NaCl. TsUly25B adopted an endolytic manner to degrade ulvan polysaccharides, and the main end-products were unsaturated ulvan disaccharides and tetrasaccharides. In conclusion, our research enriches the ulvan lyase library and advances the utilization of ulvan lyases in further fundamental research as well as ulvan oligosaccharides production.     

Identification and Characterization of Three Chitinases with Potential in Direct Conversion of Crystalline Chitin into N,N′-diacetylchitobiose

Chitooligosaccharides (COSs) have been widely used in agriculture, medicine, cosmetics, and foods, which are commonly prepared from chitin with chitinases. So far, while most COSs are prepared from colloidal chitin, chitinases used in preparing COSs directly from natural crystalline chitin are less reported. Here, we characterize three chitinases, which were identified from the marine bacterium Pseudoalteromonas flavipulchra DSM 14401^T, with an ability to degrade crystalline chitin into (GlcNAc)₂ (N,N’-diacetylchitobiose). Strain DSM 14401 can degrade the crystalline α-chitin in the medium to provide nutrients for growth. Genome and secretome analyses indicate that this strain secretes six chitinolytic enzymes, among which chitinases Chia4287, Chib0431, and Chib0434 have higher abundance than the others, suggesting their importance in crystalline α-chitin degradation. These three chitinases were heterologously expressed, purified, and characterized. They are all active on crystalline α-chitin, with temperature optima of 45–50 °C and pH optima of 7.0–7.5. They are all stable at 40 °C and in the pH range of 5.0–11.0. Moreover, they all have excellent salt tolerance, retaining more than 92% activity after incubation in 5 M NaCl for 10 h at 4 °C. When acting on crystalline α-chitin, the main products of the three chitinases are all (GlcNAc)₂, which suggests that chitinases Chia4287, Chib0431, and Chib0434 likely have potential in direct conversion of crystalline chitin into (GlcNAc)₂.     

Overexpression and Characterization of a Novel Thermostable β-Agarase YM01-3, from Marine Bacterium Catenovulum agarivorans YM01T

Genome sequencing of Catenovulum agarivorans YM01^T reveals 15 open-reading frames (ORFs) encoding various agarases. In this study, extracellular proteins of YM01^T were precipitated by ammonium sulfate and separated by one-dimensional gel electrophoresis. The results of in-gel agarase activity assay and mass spectrometry analysis revealed that the protein, YM01-3, was an agarase with the most evident agarolytic activity. Agarase YM01-3, encoded by the YM01-3 gene, consisted of 420 amino acids with a calculated molecular mass of 46.9 kDa and contained a glycoside hydrolase family 16 β-agarase module followed by a RICIN superfamily in the C-terminal region. The YM01-3 gene was cloned and expressed in Escherichia coli. The recombinant agarase, YM01-3, showed optimum activity at pH 6.0 and 60 °C and had a K_m of 3.78 mg mL⁻¹ for agarose and a V_max of 1.14 × 10⁴ U mg⁻¹. YM01-3 hydrolyzed the β-1,4-glycosidic linkages of agarose, yielding neoagarotetraose and neoagarohexaose as the main products. Notably, YM01-3 was stable below 50 °C and retained 13% activity after incubation at 80 °C for 1 h, characteristics much different from other agarases. The present study highlights a thermostable agarase with great potential application value in industrial production.     

Influence of Temperature on Growth and Production of Pectenotoxin-2 by a Monoclonal Culture of Dinophysis caudata

The effects of temperature on growth and production of Lipophilic Toxins (LT) by a monoclonal culture of Dinophysis caudata was studied. The cell density of D. caudata increased significantly with increasing temperature, and was the highest under 27, 30, and 32.5 °C. Temperature affected the average specific growth rate (µ) during the exponential growth phase (EG), which increased from 15 °C to 30 °C, and then decreased at 32.5 °C. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that this strain of D. caudata produced only pectenotoxin-2 (PTX-2) whose concentration increased significantly with incubation period, except at 32.5 °C. It was significantly different between temperatures ≤18 °C, ≥21 °C, and 32.5 °C. The cellular toxin production (CTP, pg·cell⁻¹·day⁻¹) showed variation with growth phase and temperature, except at 32.5 °C. The average net toxin production (R_tox) was not affected by temperature. During EG, the average specific toxin production rate (µ_tox) increased significantly with increase in temperature, reaching a peak of 0.66 ± 0.01 day⁻¹ at 30 °C, and then decreased. Over the entire growth span, µ_tox was significantly correlated to µ, and this correlation was most significant at 27 and 30 °C. During EG, µ_tox was affected by both temperature and growth. This study shows that temperature affects growth and toxin production of this strain of D. caudata during EG. In addition, a positive correlation was found between toxin production and growth.     

Supercritical Fluid Extraction of Fucoxanthin from the Diatom Phaeodactylum tricornutum and Biogas Production through Anaerobic Digestion

Phaeodactylum tricornutum is the marine diatom best known for high-value compounds that are useful in aquaculture and food area. In this study, fucoxanthin was first extracted from the diatom using supercritical fluid extraction (SFE) and then using the extracted diatom-like substrate to produce bioenergy through anaerobic digestion (AD) processes. Factors such as temperature (30 °C and 50 °C), pressure (20, 30, and 40 MPa), and ethanol (co-solvent concentration from 10% to 50% v/v) were optimized for improving the yield, purity, and recovery of fucoxanthin extracted using SFE. The highest yield (24.41% w/w) was obtained at 30 MPa, 30 °C, and 30% ethanol but the highest fucoxanthin purity and recovery (85.03mg/g extract and 66.60% w/w, respectively) were obtained at 30 MPa, 30 °C, and 40%ethanol. Furthermore, ethanol as a factor had the most significant effect on the overall process of SFE. Subsequently, P.tricornutum biomass and SFE-extracted diatom were used as substrates for biogas production through AD. The effect of fucoxanthin was studied on the yield of AD, which resulted in 77.15 ± 3.85 LSTP CH₄/kg volatile solids (VS) and 56.66 ± 1.90 LSTP CH₄/kg VS for the whole diatom and the extracted P.tricornutum, respectively. Therefore, P.tricornutuman can be considered a potential source of fucoxanthin and methane and both productions will contribute to the sustainability of the algae-biorefinery processes.     

Comparative Efficacy of CO2 and Ozone Gases Against Ephestia cautella (Lepidoptera: Pyralidae) Larvae Under Different Temperature Regimes

Comparative efficacy of three different modified atmospheres: 100% CO₂, 75% CO₂ + 25% N₂, and 22 ppm ozone were examined against larval mortality of the almond moth, Ephestia cautella (Walker) (Lepidoptera: Pyralidae) at temperature regimes of 25°C and 35 ± 2°C and 60 ± 5% relative humidity, and 9:15 dark and light. Wandering young larval instars, which are fast growing, large enough in size and considered as more tolerant to modified atmosphere, were collected directly from the rearing culture, placed inside pitted date fruits of vars.: “Khudri,” “Ruziz,” and “Saqie,” were treated with aforementioned gases for 24, 48, and 72 h. The immediate and delayed larval mortality was recorded after each exposure timing. Ozone possessed the strongest fumigant toxicity causing 100% mortality with all varieties, at 25 and 35°C after 24 h exposure and was more effective than 75% CO₂ that caused 83 and 100% immediate mortality with variety ruziz at 25 and 35°C, respectively. Extending the treatments exposure time to 72 h, 100% mortality was recorded by exposing larvae to any of the studied gases at 25 and 35°C. These results suggest that gases and temperature used in this study can be effectively used to control E. cautella in dates and stored grains.     

Amino Acid Profiles and Biopotentiality of Hydrolysates Obtained from Comb Penshell (Atrina pectinata) Viscera Using Subcritical Water Hydrolysis

The recovery of amino acids and other important bioactive compounds from the comb penshell (Atrina pectinata) using subcritical water hydrolysis was performed. A wide range of extraction temperatures from 140 to 290 °C was used to evaluate the release of proteins and amino acids. The amount of crude protein was the highest (36.14 ± 1.39 mg bovine serum albumin/g) at 200 °C, whereas a further increase in temperature showed the degradation of the crude protein content. The highest amount of amino acids (74.80 mg/g) was at 230 °C, indicating that the temperature range of 170–230 °C is suitable for the extraction of protein-rich compounds using subcritical water hydrolysis. Molecular weights of the peptides obtained from comb penshell viscera decreased with the increasing temperature. SDS-PAGE revealed that the molecular weight of peptides present in the hydrolysates above the 200 °C extraction temperature was ≤ 1000 Da. Radical scavenging activities were analyzed to evaluate the antioxidant activities of the hydrolysates. A. pectinata hydrolysates also showed a particularly good antihypertensive activity, proving that this raw material can be an effective source of amino acids and marine bioactive peptides.     

Characterization of Neoagarooligosaccharide Hydrolase BpGH117 from a Human Gut Bacterium Bacteroides plebeius

α-Neoagarobiose (NAB)/neoagarooligosaccharide (NAO) hydrolase plays an important role as an exo-acting 3,6-anhydro-α-(1,3)-L-galactosidase in agarose utilization. Agarose is an abundant polysaccharide found in red seaweeds, comprising 3,6-anhydro-L-galactose (AHG) and D-galactose residues. Unlike agarose degradation, which has been reported in marine microbes, recent metagenomic analysis of Bacteroides plebeius, a human gut bacterium, revealed the presence of genes encoding enzymes involved in agarose degradation, including α-NAB/NAO hydrolase. Among the agarolytic enzymes, BpGH117 has been partially characterized. Here, we characterized the exo-acting α-NAB/NAO hydrolase BpGH117, originating from B. plebeius. The optimal temperature and pH for His-tagged BpGH117 activity were 35 °C and 9.0, respectively, indicative of its unique origin. His-tagged BpGH117 was thermostable up to 35 °C, and the enzyme activity was maintained at 80% of the initial activity at a pre-incubation temperature of 40 °C for 120 min. K_m and V_max values for NAB were 30.22 mM and 54.84 U/mg, respectively, and k_cat/K_m was 2.65 s⁻¹ mM⁻¹. These results suggest that His-tagged BpGH117 can be used for producing bioactive products such as AHG and agarotriose from agarose efficiently.     

Chemical and Physical Culture Conditions Significantly Influence the Cell Mass and Docosahexaenoic Acid Content of Aurantiochytrium limacinum Strain PKU#SW8

Thraustochytrids are well-known unicellular heterotrophic marine protists because of their promising ability to accumulate docosahexaenoic acid (DHA). However, the implications of their unique genomic and metabolic features on DHA production remain poorly understood. Here, the effects of chemical and physical culture conditions on the cell mass and DHA production were investigated for a unique thraustochytrid strain, PKU#SW8, isolated from the seawater of Pearl River Estuary. All the tested fermentation parameters showed a significant influence on the cell mass and concentration and yield of DHA. The addition of monosaccharides (fructose, mannose, glucose, or galactose) or glycerol to the culture medium yielded much higher cell mass and DHA concentrations than that of disaccharides and starch. Similarly, organic nitrogen sources (peptone, yeast extract, tryptone, and sodium glutamate) proved to be beneficial in achieving a higher cell mass and DHA concentration. PKU#SW8 was found to grow and accumulate a considerable amount of DHA over wide ranges of KH₂PO₄ (0.125–1.0 g/L), salinity (0–140% seawater), pH (3–9), temperature (16–36 °C), and agitation (140–230 rpm). With the optimal culture conditions (glycerol, 20 g/L; peptone, 2.5 g/L; 80% seawater; pH 4.0; 28 °C; and 200 rpm) determined based on the shake-flask experiments, the cell mass and concentration and yield of DHA were improved up to 7.5 ± 0.05 g/L, 2.14 ± 0.03 g/L, and 282.9 ± 3.0 mg/g, respectively, on a 5-L scale fermentation. This study provides valuable information about the fermentation conditions of the PKU#SW8 strain and its unique physiological features, which could be beneficial for strain development and large-scale DHA production.     

Expression and Characterization of a Novel Cold-Adapted and Stable β-Agarase Gene agaW1540 from the Deep-Sea Bacterium Shewanella sp. WPAGA9

The neoagaro-oligosaccharides, degraded from agarose by agarases, are important natural substances with many bioactivities. In this study, a novel agarase gene, agaW1540, from the genome of a deep-sea bacterium Shewanella sp. WPAGA9, was expressed, and the recombinant AgaW1540 (rAgaW1540) displayed the maximum activity under the optimal pH and temperature of 7.0 and 35 °C, respectively. rAgaW1540 retained 85.4% of its maximum activity at 0 °C and retained more than 92% of its maximum activity at the temperature range of 20–40 °C and the pH range of 4.0–9.0, respectively, indicating its extensive working temperature and pH values. The activity of rAgaW1540 was dramatically suppressed by Cu²⁺ and Zn²⁺, whereas Fe²⁺ displayed an intensification of enzymatic activity. The K_m and V_max of rAgaW1540 for agarose degradation were 15.7 mg/mL and 23.4 U/mg, respectively. rAgaW1540 retained 94.7%, 97.9%, and 42.4% of its maximum activity after incubation at 20 °C, 25 °C, and 30 °C for 60 min, respectively. Thin-layer chromatography and ion chromatography analyses verified that rAgaW1540 is an endo-acting β-agarase that degrades agarose into neoagarotetraose and neoagarohexaose as the main products. The wide variety of working conditions and stable activity at room temperatures make rAgaW1540an appropriate bio-tool for further industrial production of neoagaro-oligosaccharides.     

Purification and Characterization of a Fucoidanase (FNase S) from a Marine Bacterium Sphingomonas paucimobilis PF-1

The Search for enzyme activities that efficiently degrade marine polysaccharides is becoming an increasingly important area for both structural analysis and production of lower-molecular weight oligosaccharides. In this study, an endo-acting fucoidanase that degrades Miyeokgui fucoidan (MF), a sulfated galactofucan isolated from the sporophyll (called Miyeokgui in Korean) of Undaria pinnatifida, into smaller-sized galactofuco-oligosaccharides (1000–4000 Da) was purified from a marine bacterium, Sphingomonas paucimobilis PF-1, by ammonium sulfate precipitation, diethylaminoethyl (DEAE)-Sepharose column chromatography, and chromatofocusing. The specific activity of this enzyme was approximately 112-fold higher than that of the crude enzyme, and its molecular weight was approximately 130 kDa (FNase S), as determined by native gel electrophoresis and 130 (S1), 70 (S2) and 60 (S3) kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature of FNase S were pH 6.0–7.0 and 40–45 °C, respectively. FNase S activity was enhanced by Mn²⁺ and Na⁺ (115.7% and 131.2%), but it was inhibited by Ca²⁺, K⁺, Ba²⁺, Cu²⁺ (96%, 83.7%, 84.3%, and 89.3%, respectively), each at 1 mM. The K_m, V_max and K_cat values of FNase S on MF were 1.7 mM, 0.62 mg·min⁻¹, and 0.38·S⁻¹, respectively. This enzyme could be a valuable tool for the structural analysis of fucoidans and production of bioactive fuco-oligosaccharides.     

Preparation and Characterization of Nano-Selenium Decorated by Chondroitin Sulfate Derived from Shark Cartilage and Investigation on Its Antioxidant Activity

In the present study, a selenium-chondroitin sulfate (SeCS) was synthesized by the sodium selenite (Na₂SeO₃) and ascorbic acid (Vc) redox reaction using chondroitin sulfate derived from shark cartilage as a template, and characterized by SEM, SEM-EDS, FTIR and XRD. Meanwhile, its stability was investigated at different conditions of pH and temperatures. Besides, its antioxidant activity was further determined by the DPPH and ABTS assays. The results showed the SeCS with the smallest particle size of 131.3 ± 4.4 nm and selenium content of 33.18% was obtained under the optimal condition (CS concentration of 0.1 mg/mL, mass ratio of Na₂SeO₃ to Vc of 1:8, the reaction time of 3 h, and the reaction temperature of 25 °C). SEM image showed the SeCS was an individual and spherical nanostructure and its structure was evidenced by FTIR and XRD. Meanwhile, SeCS remained stable at an alkaline pH and possessed good storage stability at 4 °C for 28 days. The results on scavenging free radical levels showed that SeCS exhibited significantly higher antioxidant activity than SeNPs and CS, indicating that SeCS had a potential antioxidant effect.     

Preparation and Characterization of Thermally Stable Collagens from the Scales of Lizardfish (Synodus macrops)

Marine collagen is gaining vast interest because of its high biocompatibility and lack of religious and social restrictions compared with collagen from terrestrial sources. In this study, lizardfish (Synodus macrops) scales were used to isolate acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC). Both ASC and PSC were identified as type I collagen with intact triple-helix structures by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and spectroscopy. The ASC and PSC had high amino acids of 237 residues/1000 residues and 236 residues/1000 residues, respectively. Thus, the maximum transition temperature (T_max) of ASC (43.2 °C) was higher than that of PSC (42.5 °C). Interestingly, the T_max of both ASC and PSC was higher than that of rat tail collagen (39.4 °C) and calf skin collagen (35.0 °C), the terrestrial collagen. Solubility tests showed that both ASC and PSC exhibited high solubility in the acidic pH ranges. ASC was less susceptible to the “salting out” effect compared with PSC. Both collagen types were nontoxic to HaCaT and MC3T3-E1 cells, and ASC was associated with a higher cell viability than PSC. These results indicated that ASC from lizardfish scales could be an alternative to terrestrial sources of collagen, with potential for biomedical applications.     

Increased Osteogenic Potential of Pre-Osteoblasts on Three-Dimensional Printed Scaffolds Compared to Porous Scaffolds for Bone Regeneration

Background:One of the main challenges with conventional scaffold fabrication methods is the inability to control scaffold architecture. Recently, scaffolds with controlled shape and architecture have been fabricated using 3D-printing. Herein, we aimed to determine whether the much tighter control of microstructure of 3DP PLGA/β-TCP scaffolds is more effective in promoting osteogenesis than porous scaffolds produced by solvent casting/porogen leaching. Methods:Physical and mechanical properties of porous and 3DP scaffolds were studied. The response of pre-osteoblasts to the scaffolds was analyzed after 14 days. Results:The 3DP scaffolds had a smoother surface (R_a: 22 ± 3 µm) relative to the highly rough surface of porous scaffolds (R_a: 110 ± 15 µm). Water contact angle was 112 ± 4° on porous and 76 ± 6° on 3DP scaffolds. Porous and 3DP scaffolds had the pore size of 408 ± 90 and 315 ± 17 µm and porosity of 85 ± 5% and 39 ± 7%, respectively. Compressive strength of 3DP scaffolds (4.0 ± 0.3 MPa) was higher than porous scaffolds (1.7 ± 0.2 MPa). Collagenous matrix deposition was similar on both scaffolds. Cells proliferated from day 1 to day 14 by fourfold in porous and by 3.8-fold in 3DP scaffolds. ALP activity was 21-fold higher in 3DP scaffolds than porous scaffolds. Conclusion:The 3DP scaffolds show enhanced mechanical properties and ALP activity compared to porous scaffolds in vitro, suggesting that 3DP PLGA/β-TCP scaffolds are possibly more favorable for bone formation. Key Words: Alkaline phosphatase, β-tricalcium phosphate, Poly(lactic-co-glycolic) acid copolymer     

Spasmolytic Effect of Caulerpine Involves Blockade of Ca2+ Influx on Guinea Pig Ileum

In this work, we investigated the spasmolytic effect of caulerpine, a bisindole alkaloid isolated from marine algae of the Caulerpa genus, on guinea pig ileum. Our findings indicated that caulerpine inhibited phasic contractions induced by carbachol (IC₅₀ = 7.0 ± 1.9 × 10⁻⁵ M), histamine (IC₅₀ = 1.3 ± 0.3 × 10⁻⁴ M) and serotonin (IC₅₀ = 8.0 ± 1.4 × 10⁻⁵ M) in a non-selective manner. Furthermore, caulerpine concentration-dependently inhibited serotonin-induced cumulative contractions (pD′₂ = 4.48 ± 0.08), shifting the curves to the right with E_max reduction and slope of 2.44 ± 0.21, suggesting a noncompetitive antagonism pseudo-irreversible. The alkaloid also relaxed the ileum pre-contracted by KCl (EC₅₀ = 9.0 ± 0.9 × 10⁻⁵ M) and carbachol (EC₅₀ = 4.6 ± 0.7 × 10⁻⁵ M) in a concentration-dependent manner. This effect was probably due to inhibition of Ca²⁺ influx through voltage-gated calcium channels (Ca_V), since caulerpine slightly inhibited the CaCl₂-induced contractions in depolarizing medium without Ca²⁺, shifting the curves to the right and with E_max reduction. According to these results, the spasmolytic effect of caulerpine on guinea pig ileum seems to involve inhibition of Ca²⁺ influx through Ca_V. However, other mechanisms are not discarded.     

New Trichothecenes Isolated from the Marine Algicolous Fungus Trichoderma brevicompactum

Eight trichothecenes, including four new compounds 1–4 and four known entities 5–8, together with one known cyclonerane (9) were isolated from the solid-state fermentation of Trichoderma brevicompactum NTU439 isolated from the marine alga Mastophora rosea. The structures of 1–9 were determined by 1D/2D NMR (nuclear magnetic resonance), MS (mass spectrometry), and IR (infrared spectroscopy) spectroscopic data. All of the compounds were evaluated for cytotoxic activity against HCT-116, PC-3, and SK-Hep-1 cancer cells by the SRB assay, and compound 8 showed promising cytotoxic activity against all three cancer cell lines with the IC₅₀ values of 3.3 ± 0.3, 5.3 ± 0.3, and 1.8 ± 0.8 μM, respectively. Compounds 1–2, 4–6, and 7–8 potently inhibited LPS-induced NO production, and compounds 5 and 8 showed markedly inhibited gelatinolysis of MMP-9 in S1 protein-stimulated THP-1 monocytes.     

Epipolythiodiketopiperazines from the Marine Derived Fungus Dichotomomyces cejpii with NF-κB Inhibitory Potential

The Ascomycota Dichotomomyces cejpii was isolated from the marine sponge Callyspongia cf. C. flammea. A new gliotoxin derivative, 6-acetylmonodethiogliotoxin (1) was obtained from fungal extracts. Compounds 2 and 3, methylthio-gliotoxin derivatives were formerly only known as semi-synthetic compounds and are here described as natural products. Additionally the polyketide heveadride (4) was isolated. Compounds 1, 2 and 4 dose-dependently down-regulated TNFα-induced NF-κB activity in human chronic myeloid leukemia cells with IC_50s of 38.5 ± 1.2 µM, 65.7 ± 2.0 µM and 82.7 ± 11.3 µM, respectively. The molecular mechanism was studied with the most potent compound 1 and results indicate downstream inhibitory effects targeting binding of NF-κB to DNA. Compound 1 thus demonstrates potential of epimonothiodiketopiperazine-derived compounds for the development of NF-κB inhibitors.     

A Novel Agarase,Gaa16B,Isolated from the Marine Bacterium Gilvimarinus agarilyticus JEA5, and the Moisturizing Effect of Its Partial Hydrolysis Products

We recently identified a β-agarase, Gaa16B, in the marine bacterium Gilvimarinus agarilyticus JEA5. Gaa16B, belonging to the glycoside hydrolase 16 family of β-agarases, shows less than 70.9% amino acid similarity with previously characterized agarases. Recombinant Gaa16B lacking the carbohydrate-binding region (rGaa16Bc) was overexpressed in Escherichia coli and purified. Activity assays revealed the optimal temperature and pH of rGaa16Bc to be 55 °C and pH 6–7, respectively, and the protein was highly stable at 55 °C for 90 min. Additionally, rGaa16Bc activity was strongly enhanced (2.3-fold) in the presence of 2.5 mM MnCl₂. The K_m and V_max of rGaa16Bc for agarose were 6.4 mg/mL and 953 U/mg, respectively. Thin-layer chromatography analysis revealed that rGaa16Bc can hydrolyze agarose into neoagarotetraose and neoagarobiose. Partial hydrolysis products (PHPs) of rGaa16Bc had an average molecular weight of 88–102 kDa and exhibited > 60% hyaluronidase inhibition activity at a concentration of 1 mg/mL, whereas the completely hydrolyzed product (CHP) showed no hyaluronidase at the same concentration. The biochemical properties of Gaa16B suggest that it could be useful for producing functional neoagaro-oligosaccharides. Additionally, the PHP of rGaa16Bc may be useful in promoting its utilization, which is limited due to the gel strength of agar.     

Extracellular Polymeric Substances Produced by the Thermophilic Cyanobacterium Gloeocapsa gelatinosa: Characterization and Assessment of Their Antioxidant and Metal-Chelating Activities

Cyanobacteria, particularly thermophilic strains, represent an important potential source of EPSs, harboring structural complexity that predicts diverse and specific bioactive potential. The thermophilic cyanobacteria Gloeocapsa gelatinosa, isolated from a natural hot source in Ain Echfa (Tunisia), was cultivated in a cylindrical reactor, and the production of biomass and EPSs was investigated. Results revealed that the strain is amongst the most efficient EPSs producers (0.89 g L⁻¹) and that EPSs production was not correlated with the growth phase. EPSs were sulfated heteropolysaccharides containing carbohydrates (70%) based on nine different monosaccharides, mainly mannose (22%), and with the presence of two uronic acids. EPSs were formed by two polymers moieties with a molecular weight of 598.3 ± 7.2 and 67.2 ± 4.4 kDa. They are thermostable in temperatures exceeding 100 °C and have an anionic nature (zeta potential of −40 ± 2 mV). Atomic force microscopy showed that EPSs formed multimodal lumps with 88 nm maximum height. EPSs presented high water holding capacity (70.29 ± 2.36%) and solubility index (97.43 ± 1.24%), and a strong bivalent metal sorption capacity especially for Cu²⁺ (91.20 ± 1.25%) and Fe²⁺ (75.51 ± 0.71%). The antioxidant activity of G. gelatinosa EPSs was investigated using four methods: the β-carotene-bleaching activity, DPPH assays, iron-reducing activity, and metal-chelating activity. EPS has shown high potential as free radicals’ scavenger, with an IC₅₀ on DPPH (0.2 g L⁻¹) three-fold lower than ascorbic acid (0.6 g L ⁻¹) and as a metal chelating activity (IC₅₀ = 0.4 g L⁻¹) significantly lower than EDTA. The obtained results allow further exploration of the thermophilic G. gelatinosa for several biotechnological and industrial applications.     

Spray Drying of Chitosan Acid Salts: Process Development,Scaling Up and Physicochemical Material Characterization

We investigated a spray drying process for preparing water-soluble salts of high molecular weight chitosan (CH) intended for pharmaceutical excipient applications. CH was derived from chitin of marine lobster origin (Panulirus argus). The effects of organic acid (acetic or lactic acid) and the ratio (difference) of inlet/outlet air temperature (140/90 °C or 160/100 °C) on spray drying were studied. The yield of spray-dried CH salt powders ranged from 50% to 99% in laboratory and industrial-scale processes. The spray-dried dry powder of CH salts consisted of spherical agglomerated particles with an average diameter of 36.2 ± 7.0 µm (CH acetate) and 108.6 ± 11.5 µm (CH lactate). After dispersing the spray-dried CH salt powder samples in purified water, the mean particle sizes obtained for the CH acetate salts were 31.4 nm (batch A001), 33.0 nm (A002) and 44.2 nm (A003), and for the CH lactate salts 100.8 nm (batch L001), 103.2 nm (L002) and 121.8 nm (L003). The optimum process conditions for spray drying were found: an inlet air temperature of 160 ± 5 °C, an outlet temperature of 100 ± 5 °C and an atomizer disk rotational speed of 18,200 min⁻¹. The X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC) results confirmed the amorphous state of the CH salts. The ¹H nuclear magnetic resonance (NMR) and Fourier transform infrared (FT-IR) spectra of CH acetate and lactate salts verified that the spray drying process does not affect the polymer backbone. In conclusion, both laboratory and industrial-scale spray drying methods for preparing water-soluble acid salts of CH are reproducible, and the physicochemical properties of the corresponding CH acid salts are uniform.