Characterization of ACE Inhibitory Peptides Prepared from Pyropia pseudolinearis Protein

More than 7000 red algae species have been classified. Although most of them are underused, they are a protein-rich marine resource. The hydrolysates of red algal proteins are good candidates for the inhibition of the angiotensin-I-converting enzyme (ACE). The ACE is one of the key factors for cardiovascular disease, and the inhibition of ACE activity is related to the prevention of high blood pressure. To better understand the relationship between the hydrolysates of red algal proteins and the inhibition of ACE activity, we attempted to identify novel ACE inhibitory peptides from Pyropia pseudolinearis. We prepared water soluble proteins (WSP) containing phycoerythrin, phycocyanin, allophycocyanin, and ribulose 1,5-bisphosphate carboxylase/oxygenase. In vitro analysis showed that the thermolysin hydrolysate of the WSP had high ACE inhibitory activity compared to that of WSP. We then identified 42 peptides in the hydrolysate by high-performance liquid chromatography and mass spectrometry. Among 42 peptides, 23 peptides were found in chloroplast proteins. We then synthesized the uncharacterized peptides ARY, YLR, and LRM and measured the ACE inhibitory activity. LRM showed a low IC₅₀ value (0.15 μmol) compared to ARY and YLR (1.3 and 5.8 μmol). In silico analysis revealed that the LRM sequence was conserved in cpcA from Bangiales and Florideophyceae, indicating that the novel ACE inhibitory peptide LRM was highly conserved in red algae.     

Preparation,Identification, Molecular Docking Study and Protective Function on HUVECs of Novel ACE Inhibitory Peptides from Protein Hydrolysate of Skipjack Tuna Muscle

To prepare bioactive peptides with high angiotensin-I-converting enzyme (ACE)-inhibitory (ACEi) activity, Alcalase was selected from five kinds of protease for hydrolyzing Skipjack tuna (Katsuwonus pelamis) muscle, and its best hydrolysis conditions were optimized using single factor and response surface experiments. Then, the high ACEi protein hydrolysate (TMPH) of skipjack tuna muscle was prepared using Alcalase under the optimum conditions of enzyme dose 2.3%, enzymolysis temperature 56.2 °C, and pH 9.4, and its ACEi activity reached 72.71% at 1.0 mg/mL. Subsequently, six novel ACEi peptides were prepared from TMPH using ultrafiltration and chromatography methods and were identified as Ser-Pro (SP), Val-Asp-Arg-Tyr-Phe (VDRYF), Val-His-Gly-Val-Val (VHGVV), Tyr-Glu (YE), Phe-Glu-Met (FEM), and Phe-Trp-Arg-Val (FWRV), with molecular weights of 202.3, 698.9, 509.7, 310.4, 425.6, and 606.8 Da, respectively. SP and VDRYF displayed noticeable ACEi activity, with IC₅₀ values of 0.06 ± 0.01 and 0.28 ± 0.03 mg/mL, respectively. Molecular docking analysis illustrated that the high ACEi activity of SP and VDRYF was attributed to effective interaction with the active sites/pockets of ACE by hydrogen bonding, electrostatic force, and hydrophobic interaction. Furthermore, SP and VDRYF could significantly up-regulate nitric oxide (NO) production and down-regulate endothelin-1 (ET-1) secretion in HUVECs after 24 h treatment, but also abolish the negative effect of 0.5 μM norepinephrine (NE) on the generation of NO and ET-1. Therefore, ACEi peptides derived from skipjack tuna (K. pelamis) muscle, especially SP and VDRYF, are beneficial components for functional food against hypertension and cardiovascular diseases.     

Structural Analysis of a Heteropolysaccharide from Saccharina japonica by Electrospray Mass Spectrometry in Tandem with Collision-Induced Dissociation Tandem Mass Spectrometry (ESI-CID-MS/MS)

A fucoidan extracted from Saccharina japonica was fractionated by anion exchange chromatography. The most complex fraction F0.5 was degraded by dilute sulphuric acid and then separated by use of an activated carbon column. Fraction Y1 was fractionated by anion exchange and gel filtration chromatography while Fraction Y2 was fractionated by gel filtration chromatography. The fractions were determined by ESI-MS and analyzed by ESI-CID-MS/MS. It was concluded that F0.5 had a backbone of alternating 4-linked GlcA and 2-linked Man with the first Man residue from the nonreducing end accidentally sulfated at C6. In addition, F0.5 had a 3-linked glucuronan, in accordance with a previous report by NMR. Some other structural characteristics included GlcA 1→3 Man 1→4 GlcA, Man 1→3 GlcA 1→4 GlcA, Fuc 1→4 GlcA and Fuc 1→3 Fuc. Finally, it was shown that fucose was sulfated at C2 or C4 while galactose was sulfated at C2, C4 or C6.     

Characterization of Shrimp Oil from Pandalus borealis by High Performance Liquid Chromatography and High Resolution Mass Spectrometry

Northern shrimp (Pandalus borealis) oil, which is rich in omega-3 fatty acids, was recovered from the cooking water of shrimp processing facilities. The oil contains significant amounts of omega-3 fatty acids in triglyceride form, along with substantial long-chain monounsaturated fatty acids (MUFAs). It also features natural isomeric forms of astaxanthin, a nutritional carotenoid, which gives the oil a brilliant red color. As part of our efforts in developing value added products from waste streams of the seafood processing industry, we present in this paper a comprehensive characterization of the triacylglycerols (TAGs) and astaxanthin esters that predominate in the shrimp oil by using HPLC-HRMS and MS/MS, as well as ¹³C-NMR. This approach, in combination with FAME analysis, offers direct characterization of fatty acid molecules in their intact forms, including the distribution of regioisomers in TAGs. The information is important for the standardization and quality control, as well as for differentiation of composition features of shrimp oil, which could be sold as an ingredient in health supplements and functional foods.     

Structure of Fucoidan from Brown Seaweed Turbinaria ornata as Studied by Electrospray Ionization Mass Spectrometry (ESIMS) and Small Angle X-ray Scattering (SAXS) Techniques

The purpose of this study is to elucidate both the chemical and conformational structure of an unfractionated fucoidan extracted from brown seaweed Turbinaria ornata collected at Nha-trang bay, Vietnam. Electrospray ionization mass spectrometry (ESI-MS) was used for determining the chemical structure and small angle X-ray scattering (SAXS) provided conformational of the structure at the molecular level. The results showed that the fucoidan has a sulfate content of 25.6% and is mainly composed of fucose and galactose residues (Fuc:Gal ≈ 3:1). ESIMS analysis suggested that the fucoidan has a backbone of 3-linked α-l-Fucp residues with branches, →4)-Galp(1→ at C-4 of the fucan chain. Sulfate groups are attached mostly at C-2 and sometimes at C-4 of both fucose and galactose residues. A molecular model of the fucoidan was built based on obtained chemical structure and scattering curves estimated from molecular model and observed SAXS measurement were fitted. The results indicated that fucoidan under study has a rod-like bulky chain conformation.     

Angiotensin I Converting Enzyme Inhibitory Peptides Derived from Phycobiliproteins of Dulse Palmaria palmata

We examined the inhibitory activity of angiotensin I converting enzyme (ACE) in protein hydrolysates from dulse, Palmaria palmata. The proteins extracted from dulse were mainly composed of phycoerythrin (PE) followed by phycocyanin (PC) and allophycocyanin (APC). The dulse proteins showed slight ACE inhibitory activity, whereas the inhibitory activity was extremely enhanced by thermolysin hydrolysis. The ACE inhibitory activity of hydrolysates was hardly affected by additional pepsin, trypsin and chymotrypsin treatments. Nine ACE inhibitory peptides (YRD, AGGEY, VYRT, VDHY, IKGHY, LKNPG, LDY, LRY, FEQDWAS) were isolated from the hydrolysates by reversed-phase high-performance liquid chromatography (HPLC), and it was demonstrated that the synthetic peptide LRY (IC₅₀: 0.044 μmol) has remarkably high ACE inhibitory activity. Then, we investigated the structural properties of dulse phycobiliproteins to discuss the origin of dulse ACE inhibitory peptides. Each dulse phycobiliprotein possesses α-subunit (Mw: 17,477–17,638) and β-subunit (Mw: 17,455–18,407). The sequences of YRD, AGGEY, VYRT, VDHY, LKNPG and LDY were detected in the primary structure of PE α-subunit, and the LDY also exists in the APC α- and β-subunits. In addition, the LRY sequence was found in the β-subunits of PE, PC and APC. From these results, it was suggested that the dulse ACE inhibitory peptides were derived from phycobiliproteins, especially PE. To make sure the deduction, we carried out additional experiment by using recombinant PE. We expressed the recombinant α- and β-subunits of PE (rPEα and rPEβ, respectively), and then prepared their peptides by thermolysin hydrolysis. As a result, these peptides showed high ACE inhibitory activities (rPEα: 94.4%; rPEβ: 87.0%). Therefore, we concluded that the original proteins of dulse ACE inhibitory peptides were phycobiliproteins.     

Structural Characterization of New Peptide Variants Produced by Cyanobacteria from the Brazilian Atlantic Coastal Forest Using Liquid Chromatography Coupled to Quadrupole Time-of-Flight Tandem Mass Spectrometry

Cyanobacteria from underexplored and extreme habitats are attracting increasing attention in the search for new bioactive substances. However, cyanobacterial communities from tropical and subtropical regions are still largely unknown, especially with respect to metabolite production. Among the structurally diverse secondary metabolites produced by these organisms, peptides are by far the most frequently described structures. In this work, liquid chromatography/electrospray ionization coupled to high resolution quadrupole time-of-flight tandem mass spectrometry with positive ion detection was applied to study the peptide profile of a group of cyanobacteria isolated from the Southeastern Brazilian coastal forest. A total of 38 peptides belonging to three different families (anabaenopeptins, aeruginosins, and cyanopeptolins) were detected in the extracts. Of the 38 peptides, 37 were detected here for the first time. New structural features were proposed based on mass accuracy data and isotopic patterns derived from full scan and MS/MS spectra. Interestingly, of the 40 surveyed strains only nine were confirmed to be peptide producers; all of these strains belonged to the order Nostocales (three Nostoc sp., two Desmonostoc sp. and four Brasilonema sp.).     

Diversity of Peptides Produced by Nodularia spumigena from Various Geographical Regions

Cyanobacteria produce a great variety of non-ribosomal peptides. Among these compounds, both acute toxins and potential drug candidates have been reported. The profile of the peptides, as a stable and specific feature of an individual strain, can be used to discriminate cyanobacteria at sub-population levels. In our work, liquid chromatography-tandem mass spectrometry was used to elucidate the structures of non-ribosomal peptides produced by Nodularia spumigena from the Baltic Sea, the coastal waters of southern Australia and Lake Iznik in Turkey. In addition to known structures, 9 new congeners of spumigins, 4 aeruginosins and 12 anabaenopeptins (nodulapeptins) were identified. The production of aeruginosins by N. spumigena was revealed in this work for the first time. The isolates from the Baltic Sea appeared to be the richest source of the peptides; they also showed a higher diversity in peptide profiles. The Australian strains were characterized by similar peptide patterns, but distinct from those represented by the Baltic and Lake Iznik isolates. The results obtained with the application of the peptidomic approach were consistent with the published data on the genetic diversity of the Baltic and Australian populations.     

Two Novel Antioxidant Nonapeptides from Protein Hydrolysate of Skate (Raja porosa) Muscle

In the current study, the preparation conditions of neutrase hydrolysate (SMH) from skate (Raja porosa) muscle protein were optimized using orthogonal L₉(3)⁴ tests, and R values indicated that pH was the most important factor affecting HO· scavenging activity of SMH. Under the optimum conditions of pH 7.0, enzymolysis temperature 60 °C, enzyme/substrate ratio (E/S) 2%, and enzymolysis time 5 h, EC₅₀ of SMH on HO· was 2.14 ± 0.17 mg/mL. Using ultrafiltration, gel filtration chromatography, and RP-HPLC, two novel antioxidant nonapeptides (SP-A and SP-B) were isolated from SMH and their amino acid sequences were found to be APPTAYAQS (SP-A) and NWDMEKIWD (SP-B) with calculated molecular masses of 904.98 Da and 1236.38 Da, respectively. Both showed strong antioxidant activities. SP-A and SP-B exhibited good scavenging activities on HO· (EC₅₀ 0.390 and 0.176 mg/mL), DPPH· (EC₅₀ 0.614 and 0.289 mg/mL), and O₂⁻· (EC₅₀ 0.215 and 0.132 mg/mL) in a dose-dependent manner. SP-B was also effective against lipid peroxidation in the model system. The aromatic (2Trp), acidic (2Asp and Glu), and basic (Lys) amino acid residues within the sequences of SP-B might account for its pronounced antioxidant activity. The results of this study suggested that protein hydrolysate and peptides from skate muscle might be effective as food additives for retarding lipid peroxidation occurring in foodstuffs.     

Isolation and Characterization of Collagen and Antioxidant Collagen Peptides from Scales of Croceine Croaker (Pseudosciaena crocea)

Acid soluble collagen (ASC) from scales of croceine croaker (ASC-C) was successfully isolated with the yield of 0.37% ± 0.08% (dry weight basis), and characterized as type I collagen on the basis of amino acid analysis and electrophoretic pattern. The antioxidant hydrolysate of ASC-C (ACH) was prepared through a two-stage in vitro digestion (4-h trypsin followed by 4-h pepsin), and three antioxidant peptides (ACH-P1, ACH-P2, and ACH-P3) were further isolated from ACH using ultrafiltration, gel chromatography, and RP-HPLC, and their amino acid sequences were identified as GFRGTIGLVG (ACH-P1), GPAGPAG (ACH-P2), and GFPSG (ACH-P3). ACH-P1, ACH-P2, and ACH-P3 showed good scavenging activities on hydroxyl radical (IC₅₀ 0.293, 0.240, and 0.107 mg/mL, respectively), DPPH radical (IC₅₀ 1.271, 0.675, and 0.283 mg/mL, respectively), superoxide radical (IC₅₀ 0.463, 0.099, and 0.151 mg/mL, respectively), and ABTS radical (IC₅₀ 0.421, 0.309, and 0.210 mg/mL, respectively). ACH-P3 was also effectively against lipid peroxidation in the model system. The antioxidant activities of three collagen peptides were due to the presence of hydrophobic amino acid residues within the peptide sequences. The collagen peptides might be used as antioxidant for the therapy of diseases associated with oxidative stress, or reducing oxidative changes during storage.     

Angiotensin-I Converting Enzyme (ACE) Inhibitory and Anti-Hypertensive Effect of Protein Hydrolysate from Actinopyga lecanora (Sea Cucumber) in Rats

Food protein hydrolysates are known to exhibit angiotensin converting enzyme (ACE) inhibitory properties and can be used as a novel functional food for prevention of hypertension. This study evaluated the ACE inhibitory potentials of Actinopyga lecanora proteolysate (ALP) in vivo. The pre-fed rats with ALP at various doses (200, 400, 800 mg/kg body weight) exhibited a significant (p ≤ 0.05) suppression effect after inducing hypertension. To determine the optimum effective dose that will produce maximal reduction in blood pressure, ALP at three doses was fed to the rats after inducing hypertension. The results showed that the 800 mg/kg body weight dose significantly reduced blood pressure without noticeable negative physiological effect. In addition, there were no observable changes in the rats’ heart rate after oral administration of the ALP. It was concluded that Actinopyga lecanora proteolysate could potentially be used for the development of functional foods and nutraceuticals for prevention and treatment of hypertension.     

Anticancer Activity of a Hexapeptide from Skate (Raja porosa) Cartilage Protein Hydrolysate in HeLa Cells

In this study, the hexapeptide Phe-Ile-Met-Gly-Pro-Tyr (FIMGPY), which has a molecular weight of 726.9 Da, was separated from skate (Raja porosa) cartilage protein hydrolysate using ultrafiltration and chromatographic methods, and its anticancer activity was evaluated in HeLa cells. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay indicated that FIMGPY exhibited high, dose-dependent anti-proliferation activities in HeLa cells with an IC₅₀ of 4.81 mg/mL. Acridine orange/ethidium bromide (AO/EB) fluorescence staining and flow cytometry methods confirmed that FIMGPY could inhibit HeLa cell proliferation by inducing apoptosis. Western blot assay revealed that the Bax/Bcl-2 ratio and relative intensity of caspase-3 in HeLa cells treated with 7-mg/mL FIMGPY were 2.63 and 1.83, respectively, significantly higher than those of the blank control (p < 0.01). Thus, FIMGPY could induce apoptosis by upregulating the Bax/Bcl-2 ratio and caspase-3 activation. Using a DNA ladder method further confirmed that the anti-proliferation activity of FIMGPY was attributable to its role in inducing apoptosis. These results suggest that FIMGPY from skate cartilage protein hydrolysate may have applications as functional foods and nutraceuticals for the treatment and prevention of cancer.     

Identification and Characterization of Bacillus cereus SW7-1 in Bombyx mori (Lepidoptera: Bombycidae)

The bacterial diseases of silkworms cause significant reductions in sericulture and result in huge economic loss. This study aimed to identify and characterize a pathogen from diseased silkworm. SW7-1, a pathogenic bacterial strain, was isolated from the diseased silkworm. The strain was identified on the basis of its bacteriological properties and 16S rRNA gene sequence. The colony was round, slightly convex, opaque, dry, and milky on a nutrient agar medium, the colony also exhibited jagged edges. SW7-1 was Gram-positive, without parasporal crystal, and 0.8–1.2 by 2.6–3.4 µm in length, resembling long rods with rounded ends. The strain was positive to most of the physiological biochemical tests used in this study. The strain could utilize glucose, sucrose, and maltose. The results of its 16S rRNA gene sequence analysis revealed that SW7-1 shared the highest sequence identity (>99%) with Bacillus cereus strain 14. The bacterial strain was highly susceptible to gentamycin, streptomycin, erythromycin, norfloxacin, and ofloxacin and moderately susceptible to tetracycline and rifampicin. It exhibited resistance to other antibiotics. SW7-1 had hemolytic activity and could produce extracellular casease, lipase, and amylase. SW7-1 could reproduce septicemia-like symptoms with high mortality rate when re-fed to healthy silkworm. .The median lethal concentration (LC₅₀) was 5.45 × 10⁴ cfu/ml. Thus, SW7-1 was identified as B. cereus, which is a pathogen for silkworm and human infections are possible.     

Characterization of Protein Hydrolysates from Fish Discards and By-Products from the North-West Spain Fishing Fleet as Potential Sources of Bioactive Peptides

Fish discards and by-products can be transformed into high value-added products such as fish protein hydrolysates (FPH) containing bioactive peptides. Protein hydrolysates were prepared from different parts (whole fish, skin and head) of several discarded species of the North-West Spain fishing fleet using Alcalase. All hydrolysates had moisture and ash contents lower than 10% and 15%, respectively. The fat content of FPH varied between 1.5% and 9.4% and had high protein content (69.8–76.6%). The amino acids profiles of FPH are quite similar and the most abundant amino acids were glutamic and aspartic acids. All FPH exhibited antioxidant activity and those obtained from Atlantic horse mackerel heads presented the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, reducing power and Cu²⁺ chelating activity. On the other hand, hydrolysates from gurnard heads showed the highest ABTS radical scavenging activity and Fe²⁺ chelating activity. In what concerns the α-amylase inhibitory activity, the IC₅₀ values recorded for FPH ranged between 5.70 and 84.37 mg/mL for blue whiting heads and whole Atlantic horse mackerel, respectively. α-Glucosidase inhibitory activity of FPH was relatively low but all FPH had high Angiotensin Converting Enzyme (ACE) inhibitory activity. Considering the biological activities, these FPH are potential natural additives for functional foods or nutraceuticals.     

Purification of Antioxidant Peptides by High Resolution Mass Spectrometry from Simulated Gastrointestinal Digestion Hydrolysates of Alaska Pollock (Theragra chalcogramma) Skin Collagen

In this study, the stable collagen hydrolysate was prepared by alcalase hydrolysis and twice simulated gastrointestinal digestion from Alaska pollock skin. The characteristics of hydrolysates and antioxidant activities in vitro, including 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS^•+) scavenging activity, ferric-reducing antioxidant power (FRAP) and hydroxyl radical (OH·) scavenging activity, were determined. After twice simulated gastrointestinal digestion of skin collagen (SGI-2), the degree of hydrolysis (DH) reached 26.17%. The main molecular weight fractions of SGI-2 were 1026.26 and 640.53 Da, accounting for 59.49% and 18.34%, respectively. Amino acid composition analysis showed that SGI-2 had high content of total hydrophobic amino acid (307.98/1000). With the simulated gastrointestinal digestion progressing, the antioxidant activities increased significantly (p < 0.05). SGI-2 was further purified by gel filtration chromatography, ion exchange chromatography and high performance liquid chromatography, and the A_1a3c–p fraction with high hydroxyl radical scavenging activity (IC50 = 7.63 μg/mL) was obtained. The molecular weights and amino acid sequences of key peptides of A_1a3c–p were analyzed using high resolution mass spectrometry (LC-ESI-LTQ-Orbitrap-MS) combined with de novo software and UniProt of MaxQuant software. Four peptides were identified from A_1a3c–p, including YGCC (444.1137 Da) and DSSCSG (554.1642 Da) identified by de novo software and NNAEYYK (900.3978 Da) and PAGNVR (612.3344 Da) identified by UniProt of MaxQuant software. The molecular weights and amino acid sequences of four peptides were in accordance with the features of antioxidant peptides. The results indicated that different peptides were identified by different data analysis software according to spectrometry mass data. Considering the complexity of LC-ESI-LTQ-Orbitrap-MS, it was necessary to use the different methods to identify the key peptides from protein hydrolysates.     

Production and Characterization of Antioxidant Properties of Exopolysaccharide(s) from Peanibacillus mucilaginosus TKU032

Natural polysaccharides have received much attention due to their wide range of applications. Although most microbial exopolysaccharides (EPSs) use sugars as the major carbon source, such as glucose or sucrose, in this study, EPSs were induced from a squid pen powder (SPP)-containing medium by Paenibacillus mucilaginosus TKU032, a bacterial strain isolated from Taiwanese soil. Under the optimal culture conditions, the maximum EPS yield (14.8 g/L) was obtained. MALDI-TOF MS analysis of an EPS fraction purified by gel filtration revealed two mass peaks with molecular weights of ∼1.05 × 10⁴ and ∼1.35 × 10⁴ Da, respectively. The analysis of the hydrolysates of TKU032 EPS with cellulase, pectinase or α-amylase indicated that the glycosidic bond of TKU032 EPS is most likely an α-1,4 glycosidic bond and the hydrolysates are similar to those of starch. In addition, the purified EPS demonstrated strong antioxidant abilities.     

Isolation and Structural Characterization of a Novel Antioxidant Mannoglucan from a Marine Bubble Snail,Bullacta exarata (Philippi)

Bullacta exarata is one of the most economically important aquatic species in China, noted for not only its delicious taste and nutritional value, but also for its pharmacological activities. In order to explore its potential in medical applications, a mannoglucan designated as BEPS-IB was isolated and purified from the foot muscle of B. exarata after papain digestion. Chemical composition analysis indicated BEPS-IB contained mainly d-glucose and d-mannose in a molar ratio of 1:0.52, with an average molecular weight of about 94 kDa. The linkage information was determined by methylation analysis, and the anomeric configuration and chain linkage were confirmed by IR and 2D NMR. The results indicated BEPS-IB was composed of Glcp₆Manp heptasaccharide repeating unit in the backbone, with occasional branch chains of mannose residues (14%) occurring in the backbone mannose. Further antioxidant assay indicated BEPS-IB exhibited positive antioxidant activity in scavenging superoxide radicals and reducing power. This is the first report on the structure and bioactivity of the mannoglucan from the B. exarata.     

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.     

Molecular and Chemical Analysis of the Lipopolysaccharide from Aeromonas hydrophila Strain AH-1 (Serotype O11)

A group of virulent Aeromonas hydrophila, A. sobria, and A. veronii biovar sobria strains isolated from humans and fish have been described; these strains classified to serotype O11 are serologically related by their lipopolysaccharide (LPS) O-antigen (O-polysaccharide), and the presence of an S-layer consisting of multiple copies of a crystalline surface array protein with a molecular weight of 52 kDa in the form of a crystalline surface array which lies peripheral to the cell wall. A. hydrophila strain AH-1 is one of them. We isolated the LPS from this strain and determined the structure of the O-polysaccharide, which was similar to that previously described for another strain of serotype O11. The genetics of the O11-antigen showed the genes (wb_O11 cluster) in two sections separated by genes involved in biosynthesis and assembly of the S-layer. The O11-antigen LPS is an example of an ABC-2-transporter-dependent pathway for O-antigen heteropolysaccharide (disaccharide) assembly. The genes involved in the biosynthesis of the LPS core (waa_O11 cluster) were also identified in three different chromosome regions being nearly identical to the ones described for A. hydrophila AH-3 (serotype O34). The genetic data and preliminary chemical analysis indicated that the LPS core for strain AH-1 is identical to the one for strain AH-3.