Anti-Herpes simplex virus (HSV-1) activity and antioxidant capacity of carrageenan-rich enzymatic extracts from Solieria filiformis (Gigartinales, Rhodophyta).

Solieria filiformis has been reported to have molecules with various biological activities. In this study we used environmentally friendly extraction methods, such as enzyme-assisted extraction (EAE), as a first step to obtain bioactive compounds from this species. Five combinations of protease (PRO) and carbohydrase (AMG) were utilized (1:0, 0:1, 2:1, 1:1, 1:2 PRO:AMG) to obtain Water Soluble Enzymatic Hydrolysates (WSEHs). Extraction yields, biochemical and structural characterization, as well as in vitro activity against Herpes simplex virus type 1 (HSV-1) and antioxidant capacities were determined. All PRO:AMG combinations significantly improved yields. EAE yielded heterogeneous extracts rich in iota-carrageenan and phenols, as confirmed by FTIR spectra. The highest antiherpetic activity (EC50 4.5 ± 0.4 µg mL-1) was found in the WSEHs obtained under 2:1 PRO:AMG. At this combination high antioxidant capacity was also obtained for ABTS (2,2'-Azino-Bis-3-ethylbenzoThiazoline-6-Sulfonic acid) radical scavenging activity and Ferric Reducing Antioxidant Power (FRAP). These could probably play a synergistic role associated to the strong antiviral activity obtained. These results suggest that 2:1 PRO:AMG could be effective in promoting the hydrolytic breakdown of high MW polysaccharides, contributing to the improvement of WSEHs bioactivity. Although Solieria filiformis WSEHs showed promising results, further research, including separation and purification techniques are needed.

Phycoerythrin-Derived Tryptic Peptide of a Red Alga Pyropia yezoensis Attenuates Glutamate-Induced ER Stress and Neuronal Senescence in Primary Rat Hippocampal Neurons.

SCOPE: Glutamate excitotoxicity has been observed in association with neurodegenerative disorders. This study aimed to investigate whether a phycoerythrin-derived tryptic peptide of Pyropia yezoensis (PYP) reduces glutamate-induced excitotoxicity and neuronal senescence in primary rat hippocampal neurons. METHODS AND RESULTS: Glutamate exposure (100 µm) decreased cell viability and increased expression of endoplasmic reticulum (ER) stress response protein glucose-regulated protein 78 (GRP78) starting at 60 min following glutamate exposure, which was prevented by pretreating the neurons with PYP (1 µg mL-1 ). The glutamate-induced increase in GRP78 expression was downregulated by blocking N-methyl-d-aspartate (NMDA) receptor with MK801 (10 µm) and inhibiting c-Jun N-terminal kinase (JNK) phosphorylation with SP600125 (10 µm). Moreover, phosphorylation of JNK was decreased by blockade of NMDA receptor. The PYP pretreatment downregulated glutamate-induced increase in GRP78 expression and JNK phosphorylation, and this effect was abolished by inhibiting tropomyosin-related kinase B (TrkB) receptor, phosphatidylinositiol 3-kinase, and extracellular signal-regulated kinase (ERK)1/2 using cyclotraxin B (200 nm), LY294002 (20 µm), and SL327 (10 µm), respectively. In addition, PYP downregulated increase in GRP78 expression, senescence-associated ß-galactosidase activity, and neurite degeneration in aging hippocampal neurons. CONCLUSION: These findings indicate that activation of TrkB receptor-mediated ERK1/2 by PYP attenuates glutamate-induced ER stress, which may improve the survival of hippocampal neurons with age.

One-Step Bioconversion of Fatty Acids into C8-C9 Volatile Aroma Compounds by a Multifunctional Lipoxygenase Cloned from Pyropia haitanensis.

The multifunctional lipoxygenase PhLOX cloned from Pyropia haitanensis was expressed in Escherichia coli with 24.4 mg·L-1 yield. PhLOX could catalyze the one-step bioconversion of C18-C22 fatty acids into C8-C9 volatile organic compounds (VOCs), displaying higher catalytic efficiency for eicosenoic and docosenoic acids than for octadecenoic acids. C20:5 was the most suitable substrate among the tested fatty acids. The C8-C9 VOCs were generated in good yields from fatty acids, e.g., 2E-nonenal from C20:4, and 2E,6Z-nonadienal from C20:5. Hydrolyzed oils were also tested as substrates. The reactions mainly generated 2E,4E-pentadienal, 2E-octenal, and 2E,4E-octadienal from hydrolyzed sunflower seed oil, corn oil, and fish oil, respectively. PhLOX showed good stability after storage at 4 °C for 2 weeks and broad tolerance to pH and temperature. These desirable properties of PhLOX make it a promising novel biocatalyst for the industrial production of volatile aroma compounds.

Comprehensive guide to acetyl-carboxylases in algae.

Lipids from microalgae have become an important commodity in the last 20 years, biodiesel and supplementing human diets with ω-3 fatty acids are just two of the many applications. Acetyl-CoA carboxylase (ACCase) is a key enzyme in the lipid synthesis pathway. In general, ACCases consist of four functional domains: the biotin carboxylase (BC), the biotin carboxyl binding protein (BCCP), and α-and ß-carboxyltransferases (α-and ß-CT). In algae, like in plants, lipid synthesis is another function of the chloroplast. Despite being well researched in plants and animals, there is a distinct lack of information about this enzyme in the taxonomically diverse algae. In plastid-containing organisms, ACCases are present in the cytosol and the plastid (chloroplasts) and two different forms exist, the heteromeric (prokaryotic) and homomeric (eukaryotic) form. Despite recognition of the existence of the two ACCase forms, generalized published statements still list the heteromeric form as the one present in algal plastids. In this study, the authors show this is not the case for all algae. The presence of heteromeric or homomeric ACCase is dependent on the origin of plastid. The authors used ACCase amino acid sequence comparisons to show that green (Chlorophyta) and red (Rhodophyta) algae, with the exception of the green algal class Prasinophyceae, contain heteromeric ACCase in their plastids, which are of primary symbiotic origin and surrounded by two envelope membranes. In contrast, algal plastids surrounded by three to four membranes were derived through secondary endosymbiosis (Heterokontophyta and Haptophyta), as well as apicoplast containing Apicomplexa, contain homomeric ACCase in their plastids. Distinctive differences in the substrate binding regions of heteromeric and homomeric α-CT and ß-CT were discovered, which can be used to distinguish between the two ACCase types. Furthermore, the acetyl-CoA binding region of homomeric α-CT can be used to distinguish between cytosolic and plastidial ACCase. The information provided here will be of fundamental importance in ACCase expression and activity research to unravel impacts of environmental and physicochemical parameters on lipid content and productivity.

A novel metabolic pathway for glucose production mediated by α-glucosidase-catalyzed conversion of 1,5-anhydrofructose.

α-Glucosidase is in the glycoside hydrolase family 13 (13AG) and 31 (31AG). Only 31AGs can hydrate the D-glucal double bond to form α-2-deoxyglucose. Because 1,5-anhydrofructose (AF), having a 2-OH group, mimics the oxocarbenium ion transition state, AF may be a substrate for α-glucosidases. α-Glucosidase-catalyzed hydration produced α-glucose from AF, which plateaued with time. Combined reaction with α-1,4-glucan lyase and 13AG eliminated the plateau. Aspergillus niger α-glucosidase (31AG), which is stable in organic solvent, produced ethyl α-glucoside from AF in 80% ethanol. The findings indicate that α-glucosidases catalyze trans-addition. This is the first report of α-glucosidase-associated glucose formation from AF, possibly contributing to the salvage pathway of unutilized AF.

Polyenoic fatty acid isomerase from the marine alga Ptilota filicina: protein characterization and functional expression of the cloned cDNA.

The recently described enzyme, polyenoic fatty acid isomerase (PFI), from the marine alga Ptilota filicina J. Argardh has been analyzed with respect to its protein structure and an associated cofactor. The enzyme was purified to homogeneity (as judged by SDS-PAGE and silver staining). By sedimentation equilibrium ultracentrifugation the mass of the native enzyme was estimated to be 125 kDa. The N-terminal peptide sequence derived from this protein was used to isolate two very similar cDNA clones encoding novel 500-amino acid proteins, both with calculated molecular masses of 55.9 kDa and pIs of 4.87. The data predict translation of a preprotein containing a signal peptide of 21 amino acids that is removed during maturation. Deglycosylation assays demonstrate that native PFI from P. filicina is a glycoprotein. The purified protein is chromophoric with a flavin-like UV spectrum and sequence analysis reveals the presence of a flavin-binding motif near the mature N-terminus. Heterologous expression of active PFI in Arabidopsis, using one of the cDNA clones, was successful as evidenced by conversion of arachidonic acid to a conjugated triene in an in vitro assay of the transgenic plant tissues.

Stable form of ascorbate peroxidase from the red alga Galdieria partita similar to both chloroplastic and cytosolic isoforms of higher plants.

Depletion of the electron donor ascorbate causes rapid inactivation of chloroplastic ascorbate peroxidase (APX) of higher plants, while cytosolic APX is stable under such conditions. Here we report the cloning of cDNA from Galdieria partita, a unicellular red alga, encoding a novel type of APX (APX-B). The electrophoretic mobility, Km values, kcat and absorption spectra of recombinant APX-B produced in Escherichia coli were measured. Recombinant APX-B remained active for at least 180 min after depletion of ascorbate. The amino-terminal half of APX-B, which forms the distal pocket of the active site, was richer in amino acid residues conserved in chloroplastic APXs of higher plants rather than cytosolic APXs. In contrast, the sequence of the carboxyl-terminal half, which forms the proximal pocket, was similar to that of the cytosolic isoform. The stability of APX-B might be due to its cytosolic isoform-like structure of the carboxyl-terminal half.

Topical antiedematous activity of the organic extract of Liagora farinosa algae (Rhodophyta, Nemaliales)

Liagora farinosa is a red alga found in tropical around the world. The topical antiedematous activity of its organic extract was studied by means of two experimental models: the in vivo mouse ear edema and the in vitro inhibition of bee venom-derived PLA2. The results showed that the polar and apolar fractions of L. farinosa extract were able to inhibir respectively 52.53 and 63.61 percent of the ear edema induced by croton oil. In vitro tests showed thath both fractions also inhibited the activity of PLA2, indicating that the possible machanism of action for the topical antiedematous activity is through the inactivation of this enzyme that releases arachidonic acid during the beginning of the infalmatory mediator cascade.

Hexose oxidase from the red alga Chondrus crispus. Purification, molecular cloning, and expression in Pichia pastoris.

Hexose oxidase from Chondrus crispus catalyzes the oxidation of a variety of mono- and disaccharides including D-glucose, D-galactose, maltose, and lactose. The enzyme has previously been partially purified and was reported to be a highly glycosylated, copper-containing protein with a relative molecular mass of approximately 130,000 (Sullivan, J. D., and Ikawa, M. (1973) Biochim. Biophys. Acta 309, 11-22). We report here the purification to homogeneity of hexose oxidase from C. crispus. The purified enzyme was cleaved with cyanogen bromide and endoproteinase Lys-C and the peptide fragments were subjected to amino acid sequence analysis. Oligonucleotides were designed on the basis of the peptide sequences and a cDNA clone encoding C. crispus hexose oxidase was obtained using polymerase chain reaction on reverse transcribed cDNA. The nucleotide sequence of the hexose oxidase cDNA contained an open reading frame of 546 amino acid residues with a predicted relative molecular mass of 61,898. No significant sequence similarity was found between hexose oxidase and other protein sequences available in data bases. Expression of the hexose oxidase cDNA in Pichia pastoris as an active enzyme confirmed the identity of the DNA sequence. Native hexose oxidase from C. crispus was characterized and compared with purified, recombinant enzyme.

Amino acid sequence homology between N- and C-terminal halves of a carbonic anhydrase in Porphyridium purpureum, as deduced from the cloned cDNA.

Carbonic anhydrase (CA) from Porphyridium purpureum, a unicellular red alga, was purified >209-fold to a specific activity of 1,147 units/mg protein. cDNA clones for this CA were isolated. The longest clone, comprising 1,960 base pairs, contained an open reading frame which encoded a 571-amino acid polypeptide with a calculated molecular mass of 62,094 Da. The N- and C-terminal halves of the putative mature Porphyridium CA have amino acid sequence homology to each other (>70%) and to other prokaryotic-type CAs. Both regions contain, at equivalent positions, one set of three possible zinc-liganding amino acid residues conserved among prokaryotic-type CAs. CA purified from Porphyridium contained two atoms of zinc per molecule. We propose that the Porphyridium CA has evolved by duplication of an ancestral CA gene followed by the fusion of the duplicated CA gene. The CA truncated into the putative mature form was overexpressed in Escherichia coli, and the expressed protein was active. Clones expressing separately the N- and C-terminal halves of the CA were constructed. CA activity was present in extracts of E. coli cells expressing the N-terminal half, while no detectable activity was found in cells expressing the C-terminal half.

Isolation of a gametophyte-specific cDNA encoding a lipoxygenase from the red alga Porphyra purpurea.

A cDNA clone encoding a lipoxygenase was obtained from a subtracted cDNA library specific for the gametophyte of Porphyra purpurea. The amino acid sequence of the P. purpurea lipoxygenase is most similar to the sequences of animals and plants in the iron-binding, catalytic region located in the C-terminal half of the enzymes. Northern hybridization confirmed that the gene represented by this cDNA is expressed only in the gametophyte.