Comparative Analysis of Glycoside Hydrolases Activities from Phylogenetically Diverse Marine Bacteria of the Genus Arenibacter

A total of 16 marine strains belonging to the genus Arenibacter, recovered from diverse microbial communities associated with various marine habitats and collected from different locations, were evaluated in degradation of natural polysaccharides and chromogenic glycosides. Most strains were affiliated with five recognized species, and some presented three new species within the genus Arenibacter. No strains contained enzymes depolymerizing polysaccharides, but synthesized a wide spectrum of glycosidases. Highly active β-N-acetylglucosaminidases and α-N-acetylgalactosaminidases were the main glycosidases for all Arenibacter. The genes, encoding two new members of glycoside hydrolyses (GH) families, 20 and 109, were isolated and characterized from the genomes of Arenibacter latericius. Molecular genetic analysis using glycosidase-specific primers shows the absence of GH27 and GH36 genes. A sequence comparison with functionally-characterized GH20 and GH109 enzymes shows that both sequences are closest to the enzymes of chitinolytic bacteria Vibrio furnissii and Cellulomonas fimi of marine and terrestrial origin, as well as human pathogen Elisabethkingia meningoseptica and simbionts Akkermansia muciniphila, gut and non-gut Bacteroides, respectively. These results revealed that the genus Arenibacter is a highly taxonomic diverse group of microorganisms, which can participate in degradation of natural polymers in marine environments depending on their niche and habitat adaptations. They are new prospective candidates for biotechnological applications due to their production of unique glycosidases.     

Phylogeny of Penicillium and the segregation of Trichocomaceae into three families

Species of Trichocomaceae occur commonly and are important to both industry and medicine. They are associated with food spoilage and mycotoxin production and can occur in the indoor environment, causing health hazards by the formation of β-glucans, mycotoxins and surface proteins. Some species are opportunistic pathogens, while others are exploited in biotechnology for the production of enzymes, antibiotics and other products. Penicillium belongs phylogenetically to Trichocomaceae and more than 250 species are currently accepted in this genus. In this study, we investigated the relationship of Penicillium to other genera of Trichocomaceae and studied in detail the phylogeny of the genus itself. In order to study these relationships, partial RPB1, RPB2 (RNA polymerase II genes), Tsr1 (putative ribosome biogenesis protein) and Cct8 (putative chaperonin complex component TCP-1) gene sequences were obtained. The Trichocomaceae are divided in three separate families: Aspergillaceae, Thermoascaceae and Trichocomaceae. The Aspergillaceae are characterised by the formation flask-shaped or cylindrical phialides, asci produced inside cleistothecia or surrounded by Hülle cells and mainly ascospores with a furrow or slit, while the Trichocomaceae are defined by the formation of lanceolate phialides, asci borne within a tuft or layer of loose hyphae and ascospores lacking a slit. Thermoascus and Paecilomyces, both members of Thermoascaceae, also form ascospores lacking a furrow or slit, but are differentiated from Trichocomaceae by the production of asci from croziers and their thermotolerant or thermophilic nature. Phylogenetic analysis shows that Penicillium is polyphyletic. The genus is re-defined and a monophyletic genus for both anamorphs and teleomorphs is created (Penicillium sensu stricto). The genera Thysanophora, Eupenicillium, Chromocleista, Hemicarpenteles and Torulomyces belong in Penicilliums. str. and new combinations for the species belonging to these genera are proposed. Analysis of Penicillium below genus rank revealed the presence of 25 clades. A new classification system including both anamorph and teleomorph species is proposed and these 25 clades are treated here as sections. An overview of species belonging to each section is presented. TAXONOMIC NOVELTIES: New sections, all in Penicillium: sect. Sclerotiora Houbraken & Samson, sect. Charlesia Houbraken & Samson, sect. Thysanophora Houbraken & Samson,sect. Ochrosalmonea Houbraken & Samson, sect. Cinnamopurpurea Houbraken & Samson, Fracta Houbraken & Samson, sect. Stolkia Houbraken & Samson, sect. Gracilenta Houbraken & Samson, sect. Citrina Houbraken & Samson, sect. Turbata Houbraken & Samson, sect. Paradoxa Houbraken & Samson, sect. Canescentia Houbraken & Samson. New combinations:Penicillium asymmetricum (Subramanian & Sudha) Houbraken & Samson, P. bovifimosum (Tuthill & Frisvad) Houbraken & Samson, P. glaucoalbidum (Desmazières) Houbraken & Samson, P. laeve (K. Ando & Manoch) Houbraken & Samson, P. longisporum (Kendrick) Houbraken & Samson, P. malachiteum (Yaguchi & Udagawa) Houbraken & Samson, P. ovatum (K. Ando & Nawawi) Houbraken & Samson, P. parviverrucosum (K. Ando & Pitt) Houbraken & Samson, P. saturniforme (Wang & Zhuang) Houbraken & Samson, P. taiwanense (Matsushima) Houbraken & Samson. New names:Penicillium coniferophilum Houbraken & Samson, P. hennebertii Houbraken & Samson, P. melanostipe Houbraken & Samson, P. porphyreum Houbraken & Samson.     

Gene Cloning,Expression and Characterization of a Novel Xylanase from the Marine Bacterium,Glaciecola mesophila KMM241

Marine xylanases are rather less studied compared to terrestrial xylanases. In this study, a new xylanase gene, xynB, was cloned from the marine bacterium, Glaciecola mesophila KMM241, and expressed in Escherichia coli. xynB encodes a multi-domain xylanase XynB of glycoside hydrolase (GH) family 8. The recombinant XynB comprises an N-terminal domain (NTD) with unknown function and a catalytic domain, which is structurally novel among the characterized xylanases of GH family 8. XynB has the highest identity (38%) to rXyn8 among the characterized xylanases. The recombinant XynB showed maximal activity at pH 6–7 and 35 °C. It is thermolabile and salt-tolerant. XynB is an endo-xylanase that demands at least five sugar moieties for effective cleavage and to hydrolyze xylohexaose and xylopentaose into xylotetraose, xylotriose and xylobiose. NTD was expressed in Escherichia coli to analyze its function. The recombinant NTD exhibited a high binding ability to insoluble xylan and avicel and little binding ability to chitosan and chitin. Since the NTD shows no obvious homology to any known carbohydrate-binding module (CBM) sequence in public databases, XynB may contain a new type of CBM.     

Pigmentation and spectral absorbance signatures in deep-water corals from the Trondheimsfjord, Norway

The pigmentation and corresponding in vivo and in vitro absorption characteristics in three different deep-water coral species: white and orange Lophelia pertusa, Paragorgia arborea and Primnoa resedaeformis, collected from the Trondheimsfjord are described. Pigments were isolated and characterized by High-Performance Liquid Chromatography (HPLC) analysis and High-Performance Liquid Chromatography Time-Of-Flight Mass Spectrometer (LC-TOF MS). The main carotenoids identified for all three coral species were astaxanthin and a canthaxanthin-like carotenoid. Soft tissue and skeleton of orange L. pertusa contained 2 times more astaxanthin g(-1) wet weight compared to white L. pertusa. White and orange L. pertusa were characterized with in vivo absorbance peaks at 409 and 473 nm, respectively. In vivo absorbance maxima for P. arborea and P. resedaeformis was typically at 475 nm. The shapes of the absorbance spectra (400-700 nm) were species-specific, indicated by in vivo, in vitro and the corresponding difference spectra. The results may provide important chemotaxonomic information for pigment when bonded to their proteins in vivo, bio-prospecting, and for in situ identification, mapping and monitoring of corals.     

Carotenoid β-ring hydroxylase and ketolase from marine bacteria-promiscuous enzymes for synthesizing functional xanthophylls

Marine bacteria belonging to genera Paracoccus and Brevundimonas of the α-Proteobacteria class can produce C₄₀-type dicyclic carotenoids containing two β-end groups (β rings) that are modified with keto and hydroxyl groups. These bacteria produce astaxanthin, adonixanthin, and their derivatives, which are ketolated by carotenoid β-ring 4(4′)-ketolase (4(4′)-oxygenase; CrtW) and hydroxylated by carotenoid β-ring 3(3′)-hydroxylase (CrtZ). In addition, the genus Brevundimonas possesses a gene for carotenoid β-ring 2(2′)-hydroxylase (CrtG). This review focuses on these carotenoid β-ring-modifying enzymes that are promiscuous for carotenoid substrates, and pathway engineering for the production of xanthophylls (oxygen-containing carotenoids) in Escherichia coli, using these enzyme genes. Such pathway engineering researches are performed towards efficient production not only of commercially important xanthophylls such as astaxanthin, but also of xanthophylls minor in nature (e.g., β-ring(s)-2(2′)-hydroxylated carotenoids).     

Insights into the Toxicological Properties of a Low Molecular Weight Fraction from Zoanthus sociatus (Cnidaria)

The phylum Cnidaria is an ancient group of venomous animals, specialized in the production and delivery of toxins. Many species belonging to the class Anthozoa have been studied and their venoms often contain a group of peptides, less than 10 kDa, that act upon ion channels. These peptides and their targets interact with high affinity producing neurotoxic and cardiotoxic effects, and even death, depending on the dose and the administration pathway. Zoanthiniaria is an order of the Subclass Hexacorallia, class Anthozoa, and unlike sea anemone (order Actiniaria), neither its diversity of toxins nor the in vivo effects of the venoms has been exhaustively explored. In this study we assessed some toxicological tests on mice with a low molecular weight fraction obtained by gel filtration in Sephadex G-50 from Zoanthus sociatus crude extract. The gel filtration chromatogram at 280 nm revealed two major peaks, the highest absorbance corresponding to the low molecular weight fraction. The toxicological effects seem to be mostly autonomic and cardiotoxic, causing death in a dose dependent manner with a LD₅₀ of 792 μg/kg. Moreover, at a dose of 600 μg/kg the active fraction accelerated the KCl-induced lethality in mice.     

Helicusin E,Isochromophilone X and Isochromophilone XI: New Chloroazaphilones Produced by the Fungus Bartalinia robillardoides Strain LF550

Microbial studies of the Mediterranean sponge Tethya aurantium led to the isolation of the fungus Bartalinia robillardoides strain LF550. The strain produced a number of secondary metabolites belonging to the chloroazaphilones. This is the first report on the isolation of chloroazaphilones of a fungal strain belonging to the genus Bartalinia. Besides some known compounds (helicusin A (1) and deacetylsclerotiorin (2)), three new chloroazaphilones (helicusin E (3); isochromophilone X (4) and isochromophilone XI (5)) and one new pentaketide (bartanolide (6)) were isolated. The structure elucidations were based on spectroscopic analyses. All isolated compounds revealed different biological activity spectra against a test panel of four bacteria: three fungi; two tumor cell lines and two enzymes.     

Heterofucans from the brown seaweed Canistrocarpus cervicornis with anticoagulant and antioxidant activities

Fucan is a term used to denominate a family of sulfated polysaccharides rich in sulfated l-fucose. We extracted six fucans from Canistrocarpus cervicornis by proteolytic digestion followed by sequential acetone precipitation. These heterofucans are composed mainly of fucose, glucuronic acid, galactose and sulfate. No polysaccharide was capable of prolonging prothrombin time (PT) at the concentration assayed. However, all polysaccharides prolonged activated partial thromboplastin time (aPTT). Four sulfated polysaccharides (CC-0.3/CC-0.5/CC-0.7/CC-1.0) doubled aPTT with only 0.1 mg/mL of plasma, only 1.25-fold less than Clexane, a commercial low molecular weight heparin. Heterofucans exhibited total antioxidant capacity, low hydroxyl radical scavenging activity, good superoxide radical scavenging efficiency (except CC-1.0), and excellent ferrous chelating ability (except CC-0.3). These results clearly indicate the beneficial effect of C. cervicornis polysaccharides as anticoagulants and antioxidants. Further purification steps and additional studies on structural features as well as in vivo experiments are needed to test the viability of their use as therapeutic agents.     

Laminarin from Irish Brown Seaweeds Ascophyllum nodosum and Laminaria hyperborea: Ultrasound Assisted Extraction,Characterization and Bioactivity

Ultrasound assisted extraction (UAE), purification, characterization and antioxidant activity of laminarin from Irish brown seaweeds Ascophyllum nodosum and Laminarina hyperborea were investigated. UAE was carried out using 60% ultrasonic power amplitude and 0.1 M hydrochloric acid for 15 min. Separately, solid-liquid extraction was carried in an orbital shaker using 0.1 M hydrochloric acid at 70 °C for 2.5 h. UAE with hydrochloric acid resulted in the highest concentration of laminarin, 5.82% and 6.24% on dry weight basis from A. nodosum and L. hyperborea, respectively. Purification of all extracts was carried out using molecular weight cut off dialysis at 10 kDa. Characterization of the laminarin fraction was carried out using matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Antioxidant activity of A. nodosum and L. hyperborea extracts had 2,2-diphenyl-1-picrylhydrazyl (DPPH) inhibition levels of 93.23% and 87.57%, respectively. Moreover, these extracts have shown inihibition of bacterial growth of Staphylcoccus aureus, Listeria monocytogenes, Escherichia coli and Salmonella typhimurium.     

Contact Toxicity and Repellency of the Main Components From the Essential Oil of Clausena anisum-olens Against Two Stored Product Insects

The essential oil of Clausena anisum-olens (Blanco) Merr. showed strong contact toxicity and repellency against Lasioderma serricorne and Liposcelis bostrychophila adults. The components of the essential oil obtained by hydrodistillation were determined by gas chromatography-mass spectrometry. It was found that the main components were myristicin (36.87%), terpinolene (13.26%), p-cymene-8-ol (12.38%), and 3-carene (3.88%). Myristicin and p-cymene-8-ol were separated by silica gel column chromatography, and their molecular structures were confirmed by means of physicochemical and spectrometric analysis. Myristicin and p-cymene-8-ol showed strong contact toxicity against L. serricorne (LD₅₀ = 18.96 and 39.68 μg per adult) and Li. bostrychophila (LD₅₀ = 20.41 and 35.66 μg per adult). The essential oil acting against the two grain storage insects showed LD₅₀ values of 12.44 and 74.46 μg per adult, respectively. Myristicin and p-cymene-8-ol have strong repellent toxicity to Li. bostrychophila.     

Yessotoxins, a group of marine polyether toxins: an overview

Yessotoxin (YTX) is a marine polyether toxin that was first isolated in 1986 from the scallop Patinopecten yessoensis. Subsequently, it was reported that YTX is produced by the dinoflagellates Protoceratium reticulatum, Lingulodinium polyedrum and Gonyaulax spinifera. YTXs have been associated with diarrhetic shellfish poisoning (DSP) because they are often simultaneously extracted with DSP toxins, and give positive results when tested in the conventional mouse bioassay for DSP toxins. However, recent evidence suggests that YTXs should be excluded from the DSP toxins group, because unlike okadaic acid (OA) and dinophyisistoxin-1 (DTX-1), YTXs do not cause either diarrhea or inhibition of protein phosphatases. In spite of the increasing number of molecular studies focused on the toxicity of YTX, the precise mechanism of action is currently unknown. Since the discovery of YTX, almost forty new analogues isolated from both mussels and dinoflagellates have been characterized by NMR or LC-MS/MS techniques. These studies indicate a wide variability in the profile and the relative abundance of YTXs in both, bivalves and dinoflagellates. This review covers current knowledge on the origin, producer organisms and vectors, chemical structures, metabolism, biosynthetic origin, toxicological properties, potential risks to human health and advances in detection methods of YTXs.     

Nature’s Lab for Derivatization: New and Revised Structures of a Variety of Streptophenazines Produced by a Sponge-Derived Streptomyces Strain

Eight streptophenazines (A–H) have been identified so far as products of Streptomyces strain HB202, which was isolated from the sponge Halichondria panicea from the Baltic Sea. The variation of bioactivities based on small structural changes initiated further studies on new derivatives. Three new streptophenazines (I–K) were identified after fermentation in the present study. In addition, revised molecular structures of streptophenazines C, D, F and H are proposed. Streptophenazines G and K exhibited moderate antibacterial activity against the facultative pathogenic bacterium Staphylococcus epidermidis and against Bacillus subtilis. All tested compounds (streptophenazines G, I–K) also showed moderate activities against PDE 4B.     

Pseudopterosin A: Protection of Synaptic Function and Potential as a Neuromodulatory Agent

Natural products have provided an invaluable source of inspiration in the drug discovery pipeline. The oceans are a vast source of biological and chemical diversity. Recently, this untapped resource has been gaining attention in the search for novel structures and development of new classes of therapeutic agents. Pseudopterosins are group of marine diterpene glycosides that possess an array of potent biological activities in several therapeutic areas. Few studies have examined pseudopterosin effects during cellular stress and, to our knowledge, no studies have explored their ability to protect synaptic function. The present study probes pseudopterosin A (PsA) for its neuromodulatory properties during oxidative stress using the fruit fly, Drosophila melanogaster. We demonstrate that oxidative stress rapidly reduces neuronal activity, resulting in the loss of neurotransmission at a well-characterized invertebrate synapse. PsA mitigates this effect and promotes functional tolerance during oxidative stress by prolonging synaptic transmission in a mechanism that differs from scavenging activity. Furthermore, the distribution of PsA within mammalian biological tissues following single intravenous injection was investigated using a validated bioanalytical method. Comparable exposure of PsA in the mouse brain and plasma indicated good distribution of PsA in the brain, suggesting its potential as a novel neuromodulatory agent.     

Apoptogenic Metabolites in Fractions of the Benthic Diatom Cocconeis scutellum parva

Benthic diatoms of the genus Cocconeis contain a specific apoptogenic activity. It triggers a fast destruction of the androgenic gland in the early post-larval life of the marine shrimp Hippolyte inermis, leading to the generation of small females. Previous in vitro investigations demonstrated that crude extracts of these diatoms specifically activate a dose-dependent apoptotic process in human cancer cells (BT20 breast carcinoma) but not in human normal lymphocytes. Here, a bioassay-guided fractionation has been performed to detect the apoptogenic compound(s). Various HPLC separation systems were needed to isolate the active fractions, since the apoptogenic metabolite is highly active, present in low amounts and is masked by abundant but non-active cellular compounds. The activity is due to at least two compounds characterized by different polarities, a hydrophilic and a lipophilic fraction. We purified the lipophilic fraction, which led to the characterization of an active sub-fraction containing a highly lipophilic compound, whose molecular structure has not yet been identified, but is under investigation. The results point to the possible medical uses of the active compound. Once the molecular structure has been identified, the study and modulation of apoptotic processes in various types of cells will be possible.     

Phlorotannin Extracts from Fucales Characterized by HPLC-DAD-ESI-MSn: Approaches to Hyaluronidase Inhibitory Capacity and Antioxidant Properties

Purified phlorotannin extracts from four brown seaweeds (Cystoseira nodicaulis (Withering) M. Roberts, Cystoseira tamariscifolia (Hudson) Papenfuss, Cystoseira usneoides (Linnaeus) M. Roberts and Fucus spiralis Linnaeus), were characterized by HPLC-DAD-ESI-MSⁿ. Fucophloroethol, fucodiphloroethol, fucotriphloroethol, 7-phloroeckol, phlorofucofuroeckol and bieckol/dieckol were identified. The antioxidant activity and the hyaluronidase (HAase) inhibitory capacity exhibited by the extracts were also assessed. A correlation between the extracts activity and their chemical composition was established. F. spiralis, the species presenting higher molecular weight phlorotannins, generally displayed the strongest lipid peroxidation inhibitory activity (IC₅₀ = 2.32 mg/mL dry weight) and the strongest HAase inhibitory capacity (IC₅₀ = 0.73 mg/mL dry weight). As for superoxide radical scavenging, C. nodicaulis was the most efficient species (IC₅₀ = 0.93 mg/mL dry weight), followed by F. spiralis (IC₅₀ = 1.30 mg/mL dry weight). These results show that purified phlorotannin extracts have potent capabilities for preventing and slowing down the skin aging process, which is mainly associated with free radical damage and with the reduction of hyaluronic acid concentration, characteristic of the process.     

Characterization of seed proteome in Brachypodium distachyon

Brachypodium distachyon, an emerging model plant system for some economically important temperate grasses such as wheat, barley and switchgrass, has recently caught wide attention in modern biological research. In the current study, the glutenin, albumin and globulin components of 13 B. distachyon accessions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) followed by peptide mass finger printing (PMF) and MS/MS protein identification. Abundant wheat low molecular weight glutenin subunit (LMW-GS) like proteins and a few high molecular weight glutenin subunits (HMW-GS) with low expression level were detected in B. distachyon. A total of 18 storage proteins and 15 albumin proteins were identified through PMF and MS/MS. The results demonstrated that the major seed storage proteins in B. distachyon are wheat LMW-GS like proteins and globulins. The identified albumins and globulins were mostly various enzymes that were classified into five groups according to their functions. The 2-DE spot distribution and MS results suggested that post-translational modifications (PTMs) such as phosphorylations and glycosylations are common phenomena in B. distachyon seed proteome.     

Enzyme-Assisted Extraction of Bioactive Material from Chondrus crispus and Codium fragile and Its Effect on Herpes simplex Virus (HSV-1)

Codium fragile and Chondrus crispus are, respectively, green and red seaweeds which are abundant along the North Atlantic coasts. We investigated the chemical composition and antiviral activity of enzymatic extracts of C. fragile (CF) and C. crispus (CC). On a dry weight basis, CF consisted of 11% protein, 31% neutral sugars, 0.8% sulfate, 0.6% uronic acids, and 49% ash, while CC contained 27% protein, 28% neutral sugars, 17% sulfate, 1.8% uronic acids, and 25% ash. Enzyme-assisted hydrolysis improved the extraction efficiency of bioactive materials. Commercial proteases and carbohydrases significantly improved (p ≤ 0.001) biomass yield (40%–70% dry matter) as compared to aqueous extraction (20%–25% dry matter). Moreover, enzymatic hydrolysis enhanced the recovery of protein, neutral sugars, uronic acids, and sulfates. The enzymatic hydrolysates exhibited significant activity against Herpes simplex virus (HSV-1) with EC₅₀ of 77.6–126.8 μg/mL for CC and 36.5–41.3 μg/mL for CF, at a multiplicity of infection (MOI) of 0.001 ID₅₀/cells without cytotoxity (1–200 μg/mL). The extracts obtained from proteases (P1) and carbohydrases (C3) were also effective at higher virus MOI of 0.01 ID₅₀/cells without cytotoxity. Taken together, these results indicate the potential application of enzymatic hydrolysates of C. fragile and C. crispus in functional food and antiviral drug discovery.