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Carrageenans are sulfated polysaccharides found in the cell wall of certain red seaweeds. They are widely used in the food industry for their gelling and stabilizing properties. In nature, carrageenans undergo enzymatic modification and degradation by marine organisms. Characterizing these enzymes is crucial for understanding carrageenan utilization and may eventually enable the development of targeted processes to modify carrageenans for industrial applications. In our study, we characterized a κ-carrageenan sulfatase, AMOR_S1_16A, belonging to the sulfatase S1_16 subfamily, which selectively desulfates the nonreducing end galactoses of κ-carrageenan oligomers in an exomode. Notably, AMOR_S1_16A represents the first κ-carrageenan sulfatase within the S1_16 subfamily and exhibits a novel enzymatic activity. This study provides further understanding of the substrate specificity and characteristics of the S1_16 subfamily. Moreover, this research highlights that many processes and enzymes remain to be discovered to fully understand carrageenan utilization pathways and to develop enzymatic processes for carrageenan modification and processing.

Background: Brown macroalgae dominate temperate coastal ecosystems, and their productivity is typically limited by nitrate availability. As an economically important kelp, Saccharina japonica is the most productive farmed seaweed and needs to be supplemented with sufficient nitrate throughout the cultivation process. However, molecular characterization of genes involved in nitrogen assimilation has not been conducted in brown macroalgae., Results: Here, we described the identification of the nitrate reductase (NR) gene from S. japonica (SjNR). Using two different cloning methods for SjNR, i.e. rapid amplification of cDNA ends (RACE) and cDNA cloning alone, a single fragment was obtained respectively. According to results of sequence analysis between these two fragments, the tentative coding sequence in two clones, SjNR-L and SjNR-S, were suggested to represent two transcripts of the single copy SjNR, and the ATG of SjNR-S was located inside the third exon of SjNR-L. In the 5' upstream sequence of each transcript, promoter core elements, response elements, especially multiple N response elements which occurred in microalgal NR, were all predicted. Further sequence analysis revealed that both transcripts encoded all five domains conserved in eukaryotic plant NRs. RT-qPCR results showed that the transcription level of SjNR in juvenile sporophytes could be significantly induced by nitrate and inhibited by ammonium, which was in line with plant NRs. The recombinant SjNR-L and SjNR-S were all proved to have NR activity, suggesting that the single-copy gene SjNR might be regulated on transcription level based on alternative promoters and multiple transcriptional start sites. Moreover, both NADH and NADPH were found to be able to act as electron donors for SjNR alone, which is the first confirmation that brown algal NR has a NAD(P)H-bispecific form., Conclusion: These results will provide a scientific basis for understanding the N demand of kelp in various stages of cultivation and evaluating the environmental remediation potential of kelp in eutrophic sea areas., (© 2023. The Author(s).)

Various health beneficial outcomes associated with red seaweeds, especially their polysaccharides, have been claimed, but the molecular pathway of how red seaweed polysaccharides are degraded and utilized by cooperative actions of human gut bacteria has not been elucidated. Here, we investigated the enzymatic and metabolic cooperation between two human gut symbionts, Bacteroides plebeius and Bifidobacterium longum ssp. infantis, with regard to the degradation of agarose, the main carbohydrate of red seaweed. More specifically, B. plebeius initially decomposed agarose into agarotriose by the actions of the enzymes belonging to glycoside hydrolase (GH) families 16 and 117 (i.e., BpGH16A and BpGH117) located in the polysaccharide utilization locus, a specific gene cluster for red seaweed carbohydrates. Then, B. infantis extracted energy from agarotriose by the actions of two agarolytic β-galactosidases (i.e., Bga42A and Bga2A) and produced neoagarobiose. B. plebeius ultimately acted on neoagarobiose by BpGH117, resulting in the production of 3,6-anhydro-L-galactose, a monomeric sugar possessing anti-inflammatory activity. Our discovery of the cooperative actions of the two human gut symbionts on agarose degradation and the identification of the related enzyme genes and metabolic intermediates generated during the metabolic processes provide a molecular basis for agarose degradation by gut bacteria.

Alginates are one of the major polysaccharide constituents of marine brown algae in commercial manufacturing. However, the content and composition of alginates differ according to the distinct parts of these macroalgae and have a direct impact on the concentration of guluronate and subsequent commercial value of the final product. The Azotobacter vinelandii mannuronan C-5 epimerases AlgE1 and AlgE4 were used to determine their potential value in tailoring the production of high guluronate low-molecular-weight alginates from two sources of high mannuronic acid alginates, the naturally occurring harvested brown algae ( Ascophyllum nodosum, Durvillea potatorum , Laminaria hyperborea and Lessonia nigrescens ) and a pure mannuronic acid alginate derived from fermented production of the mutant strain of Pseudomonas fluorescens NCIMB 10,525. The mannuronan C-5 epimerases used in this study increased the content of guluronate from 32% up to 81% in both the harvested seaweed and bacterial fermented alginate sources. The guluronate-rich alginate oligomers subsequently derived from these two different sources showed structural identity as determined by proton nuclear magnetic resonance ( 1 H NMR), high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and size-exclusion chromatography with online multi-angle static laser light scattering (SEC-MALS). Functional identity was determined by minimum inhibitory concentration (MIC) assays with selected bacteria and antibiotics using the previously documented low-molecular-weight guluronate enriched alginate OligoG CF-5/20 as a comparator. The alginates produced using either source showed similar antibiotic potentiation effects to the drug candidate OligoG CF-5/20 currently in development as a mucolytic and anti-biofilm agent. These findings clearly illustrate the value of using epimerases to provide an alternative production route for novel low-molecular-weight alginates.

Enzymes currently used in cheesemaking have various drawbacks, and there is a continual need to find new coagulants. This study describes the extraction and biochemical characterization of two proteases from the red alga Gracilaria edulis. The proteases were extracted with phosphate buffer and partially purified by ammonium sulphate precipitation and dialysis. The enzymes exhibited optimum caseinolytic activity at 60 °C and a pH range of 6-8. They showed a high ratio of milk-clotting over caseinolytic activity, indicating they had an excellent milk-clotting ability. The proteases were confirmed to be serine protease and metalloprotease with molecular weight (MW) of 44 and 108 kDa. They exhibited high hydrolytic activity on κ-caseins, cleaving κ-casein at four main sites, one of which being the same as that of calf rennet, which is the first reported for an algal protease. The findings demonstrated that the proteases could potentially be used as a milk coagulant in cheesemaking., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Marine macroalgae, seaweeds, are exceptionally prolific producers of halogenated natural products. Biosynthesis of halogenated molecules in seaweeds is inextricably linked to reactive oxygen species (ROS) signaling as hydrogen peroxide serves as a substrate for haloperoxidase enzymes that participate in the construction these halogenated molecules. Here, using red macroalga Asparagopsis taxiformis , a prolific producer of the ozone depleting molecule bromoform, we provide the discovery and biochemical characterization of a ROS-producing NAD(P)H oxidase from seaweeds. This discovery was enabled by our sequencing of Asparagopsis genomes, in which we find the gene encoding the ROS-producing enzyme to be clustered with genes encoding bromoform-producing haloperoxidases. Biochemical reconstitution of haloperoxidase activities establishes that fatty acid biosynthesis can provide viable hydrocarbon substrates for bromoform production. The ROS production haloperoxidase enzymology that we describe here advances seaweed biology and biochemistry by providing the molecular basis for decades worth of physiological observations in ROS and halogenated natural product biosyntheses.

Carotenoids are essential phytonutrients synthesized by all photosynthetic organisms. Acyclic lycopene is the first branching point for carotenoid biosynthesis. Lycopene β- and ε-cyclases (LCYB and LCYE, respectively) catalyze the cyclization of its open ends and direct the metabolic flux into different downstream branches. Carotenoids of the β,β-branch (e.g., β-carotene) are found in all photosynthetic organisms, but those of the β,ε-branch (e.g., lutein) are generally absent in cyanobacteria, heterokonts, and some red algae. Although both LCYBs and LCYEs have been characterized from land plants, there are only a few reports on LCYs from cyanobacteria and algae. Here, we cloned four LCY genes from Porphyra umbilicalis and Pyropia yezoensis (susabi-nori) of Bangiales, the most primitive red algal order that synthesizes lutein. Our functional characterization in both Escherichia coli and Arabidopsis thaliana demonstrated that each species has a pair of LCYB and LCYE. Similar to LCYs from higher plants, red algal LCYBs cyclize both ends of lycopene, and their LCYEs only cyclize a single end. The characterization of LCYEs from red algae resolved the first bifurcation step toward β-carotene and lutein biosynthesis. Our phylogenetic analysis suggests that LCYEs of the green lineage and the red algae originated separately during evolution.

Global economic growth often leads to depletion of raw materials and generation of greenhouse gases, as industry manufactures goods at ever increasing levels to keep up with the demand. The currently implemented production processes mostly rely on non-renewable resources, they suffer from high energy consumption, and generate waste that often has a negative environmental impact. Eco-friendly production methods are therefore intensely searched for. Among them, enzyme-based processes are appealing, because of their high substrate and reaction specificity and the relatively mild operation conditions required by these catalysts. In addition, renewable raw materials that allow sustainable production processes are also widely explored. Marine xylanases, which catalyze the hydrolysis of xylan, the major component of lignocellulose, are promising biocatalysts. Since they are produced by microorganisms that thrive in a wide variety of environmental conditions, the enzymes may be active at widely different ranges of pH, temperature, and salt concentrations. These properties are important for their successful application in various industrial processes, such as production of bioethanol, bleaching of paper and pulp, and in the food and feed sector. The present work gives a brief overview of marine sources of xylanases, their classification and features, and of the potential applications of these marine enzymes, especially in sustainable processes in the scope of circular economy., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Despite the high productivity and ecological importance of seaweeds in polar coastal regions, little is known about their carbon utilization mechanisms, especially the kinetics of the CO2-fixing enzyme Rubisco. We analyzed Rubisco carboxylation kinetics at 4 °C and 25 °C in 12 diverse polar seaweed species (including cold-temperate populations of the same species) and the relationship with their ability to use bicarbonate, by using 13C isotope discrimination and pH drift experiments. We observed a large variation in Rubisco carboxylation kinetics among the selected species, although no correlation was found between either the Michaelis-Menten constant for CO2 (Kc) or Rubisco content per total soluble protein ([Rubisco]/[TSP]) and the ability to use bicarbonate for non-green seaweeds. This study reports intraspecific Rubisco cold adaptation by means of either higher Rubisco carboxylation turnover rate (kcatc) and carboxylase efficiency (kcatc/Kc) at 4 °C or higher [Rubisco]/[TSP] in some of the analyzed species. Our data point to a widespread ability for photosynthetic bicarbonate usage among polar seaweeds, despite the higher affinity of Rubisco for CO2 and higher dissolved CO2 concentration in cold seawater. Moreover, the reported catalytic variation within form ID Rubisco might avert the canonical trade-off previously observed between Kc and kcatc for plant Rubiscos., (© The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Ethylene favors carposporogenesis in the red seaweed Grateloupia imbricata. Analyses of cystocarp development in vitro in thalli treated with ethylene suggest an interconnection between polyamine and ethylene biosynthesis pathways. Yet, little is known about molecular mechanisms underlying carposporogenesis. Here, we used droplet digital PCR to analyze genes encoding enzymes related to polyamine (Spermidine [Spd] synthase) and ethylene (ACC synthase) synthesis; a pivotal compound of both pathways (S-adenosyl methionine synthase, SAMS); the gene that encodes amine oxidase, which is involved in polyamine degradation, and a candidate gene involved in seaweed reproduction (ornithine decarboxylase, ODC). In addition, we analyzed genes encoding proteins related to stress and reactive oxygen species, ascorbate peroxidase (APX), cytochrome P450 and WD 40. We characterized gene expression in fertilized and fertile thalli from G. imbricata that were exposed to ethylene for 15 min at two time points after treatment (1 and 7 d). The differential gene expression of SAMS, Spd synthase, ACC synthase, and cytochrome P450 was related to disclosure and development of cystocarps in fertilized thalli that transitioned from having no visible cystocarps at 1 d to developing cystocarps at 7 d. Likewise, cytochrome P450 was associated with cystocarp disclosure and maturation. In addition, amine oxidase and APX were involved in fine-tuning polyamine and reactive oxygen species during carposporogenesis, respectively, whereas WD 40 did so in relation to ethylene signaling. Expression of the candidate gene ODC was increased when cystocarps were not visible (fertilized thalli, 1d), as previously described. This analysis suggests developmental stage-specific roles for these genes during carposporogenesis., (© 2018 Phycological Society of America.)

Some freshwater algae have lower (<130 osmol m-3) intracellular osmolarities than most others (>180 osmol m-3). Low osmolarities are related to the presence of flagella and the low energy cost of active water efflux following downhill water influx unconstrained by cell walls covering the plasmalemma, and the low resource cost of cell wall synthesis with the same mechanical degree of safety. One consequence of low intracellular osmolarity is limitation on the concentration of metabolites, that is, substrates and products of enzyme activity. Models of the flux through metabolic pathways, and hence the specific growth rate, using steady-state concentrations of enzymes and metabolites have involved organisms with intracellular metabolite osmolarities >280 osmol m-3, where the metabolite concentrations are much greater than the total osmolarity of some freshwater algae. Since the protein concentration (mol m-3) in the cells and the specific growth rates of freshwater cells with low and with higher intracellular osmolarity are highly similar, the models of trade-offs between enzyme and metabolite concentrations for cells with high intracellular osmolarity need modification for cells with low intracellular osmolarity. The soluble free-radical scavenger ascorbate can constitute as little as 0.2% of the low intracellular metabolite concentration (mol m-3) of low-intracellular-osmolarity cells.

We investigated the catalytic mechanism of α-1,4-glucan lyases using a full QM DFT approach based on the M06-2X functional. The reaction profile of the whole catalytic process can be divided into three phases: glycosylation, deglycosylation-elimination and tautomerization. Glycosylation is a highly asynchronous S N 1-like process with an energy barrier of 10.2 kcal mol -1 . A proton moves from the Asp 665 residue to the glycosidic oxygen. Asp 553 acts as a nucleophile and attacks the anomeric carbon causing the cleavage of the glycosidic bond. Deglycosilation-elimination is the rate-determining step of the entire process with an overall barrier of 18.3 kcal mol -1 . The final step (restoring the catalyst and tautomerization) occurs rather easily, since the Asp 553 carboxylate group "assists" the proton transfer in the tautomerization process. Our computations clearly indicate that tautomerization must occur inside the enzyme before leaving the active site rather than in the aqueous solution. Outside of the protein environment the enol-AF→keto-AF process "assisted" by a water molecule has a barrier of 35.8 kcal mol -1 ., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Vanadium-dependent haloperoxidases (V-HPO), able to catalyze the reaction of halide ions (Cl - , Br - , I - ) with hydrogen peroxide, have a great influence on the production of halocarbons, which in turn are involved in atmospheric ozone destruction and global warming. The production of these haloperoxidases in macroalgae is influenced by changes in the surrounding environment. The first reported vanadium bromoperoxidase was discovered 40 years ago in the brown alga Ascophyllum nodosum. Since that discovery, more studies have been conducted on the structure and mechanism of the enzyme, mainly focused on three types of V-HPO, the chloro- and bromoperoxidases and, more recently, the iodoperoxidase. Since aspects of environmental regulation of haloperoxidases are less well known, the present paper will focus on reviewing the factors which influence the production of these enzymes in macroalgae, particularly their interactions with reactive oxygen species (ROS).

Climate change is a global phenomenon that is considered an important threat to marine ecosystems. Ocean acidification and increased seawater temperatures are among the consequences of this phenomenon. The comprehension of the effects of these alterations on marine organisms, in particular on calcified macroalgae, is still modest despite its great importance. There are evidences that macroalgae inhabiting highly variable environments are relatively resilient to such changes. Thus, the aim of this study was to evaluate experimentally the effects of CO2-driven ocean acidification and temperature rises on the photosynthesis of calcified macroalgae inhabiting the intertidal region, a highly variable environment. The experiments were performed in a reef mesocosm in a tropical region on the Brazilian coast, using three species of frondose calcifying macroalgae (Halimeda cuneata, Padina gymnospora, and Tricleocarpa cylindrica) and crustose coralline algae. The acidification experiment consisted of three treatments with pH levels below those occurring in the region (-0.3, -0.6, -0.9). For the temperature experiment, three temperature levels above those occurring naturally in the region (+1, +2, +4°C) were determined. The results of the acidification experiment indicate an increase on the optimum quantum yield by T. cylindrica and a decline of this parameter by coralline algae, although both only occurred at the extreme acidification treatment (-0.9). The energy dissipation mechanisms of these algae were also altered at this extreme condition. Significant effects of the temperature experiment were limited to an enhancement of the photosynthetic performance by H. cuneata although only at a modest temperature increase (+1°C). In general, the results indicate a possible photosynthetic adaptation and/or acclimation of the studied macroalgae to the expected future ocean acidification and temperature rises, as separate factors. Such relative resilience may be a result of the highly variable environment they inhabit.

The catabolic fate of the major monomeric sugar of red macroalgae, 3,6-anhydro-L-galactose (AHG), is completely unknown in any organisms. AHG is not catabolized by ordinary fermentative microorganisms, and it hampers the utilization of red macroalgae as renewable biomass for biofuel and chemical production. In this study, metabolite and transcriptomic analyses of Vibrio sp., a marine bacterium capable of catabolizing AHG as a sole carbon source, revealed two key metabolic intermediates of AHG, 3,6-anhydrogalactonate (AHGA) and 2-keto-3-deoxy-galactonate; the corresponding genes were verified in vitro enzymatic reactions using their recombinant proteins. Oxidation by an NADP(+) -dependent AHG dehydrogenase and isomerization by an AHGA cycloisomerase are the two key AHG metabolic processes. This newly discovered metabolic route was verified in vivo by demonstrating the growth of Escherichia coli harbouring the genes of these two enzymes on AHG as a sole carbon source. Also, the introduction of only these two enzymes into an ethanologenic E. coli strain increased the ethanol production in E. coli by fermenting both AHG and galactose in an agarose hydrolysate. These findings provide not only insights for the evolutionary adaptation of a central metabolic pathway to utilize uncommon substrates in microbes, but also a metabolic design principle for bioconversion of red macroalgal biomass into biofuels or industrial chemicals., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Four isolates, belonging to the deep-branching phylum Planctomycetes, were recovered from the biofilm of two marine macroalgae, Ulva sp. and Laminaria sp., from the Northern coast of Portugal. These strains were light pink- or red-pigmented; the cells were variable in shape and usually organized in rosettes. They had a dimorphic cell cycle with budding reproduction. The organisms were chemoheterotrophic, strictly aerobic and mesophilic. The 16S rRNA gene sequence analysis showed that the strains belong to the family Planctomycetaceae with Rhodopirellula as the closest genus. The isolates form two separate branches (strain LF1(T) forms one branch and the strains UC8(T), UF3 and UF42 form a second branch) clearly separated from Rhodopirellula baltica with 94.2% and 93.8% 16S rRNA gene sequence similarity, respectively. Based on differential characteristics that distinguish the novel genera from R. baltica, such as cell size and shape, ultrastructure, enzymatic activities, substrate utilization pattern, fatty acid composition, phospholipid profiles and phylogeny we propose that the isolates represent two novel genera of the order Planctomycetales, Roseimaritima ulvae gen. nov., sp. nov. (type strain is UC8(T)=DSM 25454(T)=LMG 27778(T)) and Rubripirellula obstinata gen. nov., sp. nov. (type strain is LF1(T)=LMG 27779(T)=CECT 8602(T))., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Marine algae contribute approximately half of the global primary production. The large amounts of polysaccharides synthesized by these algae are degraded and consumed by microbes that utilize carbohydrate-active enzymes (CAZymes), thus creating one of the largest and most dynamic components of the Earth's carbon cycle. Over the last decade, structural and functional characterizations of marine CAZymes have revealed a diverse set of scaffolds and mechanisms that are used to degrade agars, carrageenan, alginate and ulvan-polysaccharides from red, brown and green seaweeds, respectively. The analysis of these CAZymes is not only expanding our understanding of their functions but is enabling the enhanced annotation of (meta)-genomic data sets, thus promoting an improved understanding of microbes that drive this marine component of the carbon cycle. Furthermore, this information is setting a foundation that will enable marine algae to be harnessed as a novel resource for biorefineries. In this review, we cover the most recent structural and functional analyses of marine CAZymes that are specialized in the digestion of macro-algal polysaccharides., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The presence and activation of MAPK-like proteins in intertidal macroalgae is described in the current study. Two MAPK-like proteins of 40 and 42 kDa in size similar to p38 and JNK, of mammalian cells have been identified in six representative species of intertidal macroalgae from the Strait of Gibraltar (Southern Spain), namely in the chlorophytes Ulva rigida and Chaetomorpha aerea, the rhodophytes Corallina elongata and Jania rubens, and the phaeophytes Dictyota dichotoma and Dilophus spiralis. Phosphorylation of MAPK-like proteins was studied during semi-tidal cycles. Analysis of p38-like and JNK-like MAPKs in macroalgae protein extracts was carried out by using specific antibodies against the phosphorylated forms of both MAPKs. Protein blot analysis of samples collected from 2009 to 2011 in natural growing sites on days when either low or high tide occurred at midday, indicated that MAPK-like proteins in all species were highly phosphorylated in response to desiccation imposed by low tide or high irradiance. Phosphorylation of p38-like MAPK always preceded that of JNK-like MAPK. In addition, phosphorylation of MAPKs was fastest in rhodophytes, followed by chlorophytes and then finally phaeophytes. In the first group, phosphorylation was mostly dependent on desiccation, whereas both high irradiance and desiccation were responsible for p38-like and JNK-like phosphorylation in chlorophytes. In phaeophytes, high irradiance was mostly responsible for MAPK-like activation., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

This study was carried out in order to understand the lipid and biochemical alterations resulting from different nutritional regimes of nitrate and phosphate in Ulva lactuca. The algal thalli cultured in artificial seawater (ASW) showed higher levels of carbohydrates and non-polar lipids and increased phosphatase activities, accompanied by degradation of polar lipids, proteins and pigments. Further, higher levels of lipid hydroperoxides indicated reative oxygen species (ROS)-mediated non-enzymatic lipid peroxidation due to nutritional limitation-induced oxidative stress. Those thalli cultured in ASW supplemented with nitrate showed responses corresponding to nitrate addition, such as an increase in pigments, monogalactosyldiacylglycerols, polyunsaturated fatty acids and nitrate reductase. In addition, these thalli showed partial induction of phosphatases, low phospholipids, and high sulfolipid and 1,2-diacylglyceryl-3-O-4'-(N,N,N-trimethyl)-homoserine (DGTS) due to phosphate limitation. Similarly, algal thalli cultured in ASW supplemented with phosphate showed down-regulation of phosphatases, an increase in phospholipids due to availability of phosphate as well as a decrease in nitrate reductase, pigment, monogalactosyldiacylglycerols and polyunsaturated fatty acids due to nitrate limitation. On the other hand, algal thalli cultured in ASW supplemented with both nitrate and phosphate showed recovery of lost pigments and proteins, a high monogalactosyldiacylglycerol/digalactosyldiacylglycerol ratio, high unsaturation and high oxylipin levels (both C18 and C20). Further, the accumulation of indole-3-acetic acid in nutrient-limited thalli and of kinetin and kinetin riboside in nutrient-supplemented thalli indicated their antagonistic roles under nutrient stress. Thus, U. lactuca copes with nitrate and phosphate nutritional stress by altering the metabolic pathways involved in lipid biosynthesis including a shift in lipid classes, fatty acids, oxylipins and indole-3-acetic acid/kinetin cross-talk.

α-1,4-Glucan lyase (EC 4.2.2.13) from the red seaweed Gracilariopsis lemaneiformis cleaves α-1,4-glucosidic linkages in glycogen, starch, and malto-oligosaccharides, yielding the keto-monosaccharide 1,5-anhydro-D-fructose. The enzyme belongs to glycoside hydrolase family 31 (GH31) but degrades starch via an elimination reaction instead of hydrolysis. The crystal structure shows that the enzyme, like GH31 hydrolases, contains a (β/α)8-barrel catalytic domain with B and B' subdomains, an N-terminal domain N, and the C-terminal domains C and D. The N-terminal domain N of the lyase was found to bind a trisaccharide. Complexes of the enzyme with acarbose and 1-dexoynojirimycin and two different covalent glycosyl-enzyme intermediates obtained with fluorinated sugar analogues show that, like GH31 hydrolases, the aspartic acid residues Asp(553) and Asp(665) are the catalytic nucleophile and acid, respectively. However, as a unique feature, the catalytic nucleophile is in a position to act also as a base that abstracts a proton from the C2 carbon atom of the covalently bound subsite -1 glucosyl residue, thus explaining the unique lyase activity of the enzyme. One Glu to Val mutation in the active site of the homologous α-glucosidase from Sulfolobus solfataricus resulted in a shift from hydrolytic to lyase activity, demonstrating that a subtle amino acid difference can promote lyase activity in a GH31 hydrolase.

Conversion of fibrinogen to fibrin inside blood vessels results in thrombosis, leading to myocardial infarction and other cardiovascular diseases. In general, there are four therapy options: surgical operation, intake of antiplatelets, anticoagulants, or fibrinolytic enzymes. Microbial fibrinolytic enzymes have attracted much more attention than typical thrombolytic agents because of the expensive prices and the side effects of the latter. The fibrinolytic enzymes were successively discovered from different microorganisms, the most important among which is the genus Bacillus. Microbial fibrinolytic enzymes, especially those from food-grade microorganisms, have the potential to be developed as functional food additives and drugs to prevent or cure thrombosis and other related diseases. There are several assay methods for these enzymes; this may due to the insolubility of substrate, fibrin. Existing assay methods can be divided into three major groups. The first group consists of assay of fibrinolytic activity with natural proteins as substrates, e.g., fibrin plate methods. The second and third groups of assays are suitable for kinetic studies and are based on the determination of hydrolysis of synthetic peptide esters. This review will deal primarily with the microorganisms that have been reported in literature to produce fibrinolytic enzymes and the first review discussing the methods used to assay the fibrinolytic activity.

The green credentials of ionic liquids (ILs) are being increasingly questioned due to the growing evidence of their toxicity to aquatic ecosystems, although the mechanisms of toxicity are unknown. This study provides insights into the mechanism of toxicity and biological effects of 1-alkyl-3-methylimidazolium bromide [C(n)mim]Br (n = 4 to 16) on the marine macroalga Ulva lactuca. The cell viability of this alga during IL exposure was found to be negatively correlated to the chain length of the alkyl group. The IL ([C(12)mim]Br) exposure triggers the generation of reactive oxygen species (ROS viz. O(2)(•-), H(2)O(2), and OH(•)), damage of the membrane and DNA, and inhibition of antioxidant systems in the alga. The enhanced production of ROS and lipid peroxidation in the alga subjected to LC(50) concentration for 4 days was largely attributed to lipoxygenase (LOX) activity coupled with the induction of two LOX isoforms (~80 kDa and ~55 kDa). Pretreatment of the algal thallus with enzyme inhibitors such as diphenylene iodonium, sodium azide, cantharidin, and oxadiazoloquinoxalin-1-one, prior to [C(12)mim]Br exposure showed the regulation of ROS by the activation of membrane bound NADPH-oxidase and cytochrome oxidase. The IL exposure resulted in the accumulation of n-3 and n-6 fatty acids at 0.5 LC(50) concentration indicating the induction of desaturase enzymes. Furthermore, antioxidant enzyme activities such as superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR) were enhanced by 1.3-2.0-fold, while glutathione peroxidase (GSH-Px) diminished, together with a higher regeneration rate of reduced ascorbate and glutathione. The isoforms of antioxidant enzymes, namely, Mn-SOD (~85 kDa), APX (~125 and 45 kDa), and GR (~135 kDa) regulated differentially to IL exposure. The comet assay performed for the first time for seaweeds revealed the significant induction of DNA damage (>50-70% increase in % tail DNA over control) in alga exposed to ≥ LC(50) concentration.

This paper describes the cloning of the ornithine decarboxylase gene from a red seaweed, Grateloupia imbricata (Rhodophyta), the characterization of its expression throughout the reproductive process, and demonstrates how polyamines are involved in seaweed reproduction. In addition, the data indicate that the basal perennial and non-spore-forming thalli behave physiologically and genetically differently from the distal reproductive tissue. The common polyamines putrescine, spermidine and spermine have been associated with carposporogenesis in red seaweeds. Ornithine decarboxylase (ODC, EC 4.1.1.17) produces the diamine putrescine from the non-protein amino acid, ornithine. ODC is predominant in the synthesis of polyamines in G. imbricata. The gene encoding the ornithine decarboxylase in G. imbricata was cloned by genomic polymerase chain reaction (PCR) using degenerate primers against conserved motives, followed by chromosome walking using inverse PCR (iPCR). The encoded protein (GiODC, accession # FJ223132) was very similar to other ODCs, bearing the characteristic conserved domain of pyridoxal-dependent decarboxylases. The expression of the GiODC gene was investigated by real-time PCR and in situ hybridization (ISH), and was observed to vary according to cystocarp differentiation. It was weakly transcribed in apical parts of fertile tissue where the cystocarps are located, while the transcript levels were comparatively high in the basal part. This expression pattern correlated with the levels of free polyamines, which were higher at the basal part.

Brown algal kelp species are the most efficient iodine accumulators among all living systems, with an average content of 1.0% of dry weight in Laminaria digitata, representing a ca. 30,000-fold accumulation of this element from seawater. Like other marine macroalgae, kelps are known to emit volatile short-lived organo-iodines, and molecular iodine which are believed to be a main vector of the iodine biogeochemical cycle as well as having a significant impact on atmospheric chemistry. Therefore, radioactive iodine can potentially accumulate in seaweeds and can participate in the biogeochemical cycling of iodine, thereby impacting human health. From a radioecological viewpoint, iodine-129 (129I, half-life of 1.6 x 10(7) years) is one of the most persistent radionuclide released from nuclear facilities into the environment. In this context, the speciation of iodine by seaweeds is of special importance and there is a need to further understand the mechanisms of iodine uptake and emission by kelps. Recent results on the physiological role and biochemistry of the vanadium haloperoxidases of brown algae emphasize the importance of these enzymes in the control of these processes.

The catalytic properties of a new class of chiral vanadium compounds--[(S,S,S)-VO(OMe)L1] (5), [(S,S)-VO(OMe)L2] (6), [(S,S)-VO(OMe)L3] (7), and [(R,R,R)-VO(OMe)L4] (8), as well as the system VO(OiPr)(3)/(R,R,R)-H(2)L4 [H(2)L1=(S,S)-bis(2-hydroxypropyl)-(S)-1-phenylethylamine, 1; H(2)L2=(S,S)-bis(2-hydroxypropyl)benzylamine, 2; H(2)L3=(S,S)-bis(2-hydroxypropyl)isopropylamine), 3; (H(2)L4)=(R,R)-bis(2-phenylethanol)-(R)-1-phenylethylamine, 4]--in the asymmetric oxidation of prochiral sulfides by organic hydroperoxides have been investigated. Particular attention has been paid to the factors that guide the discrimination between the two prochiral faces of the sulfides (methyl p-tolyl sulfide and benzyl phenyl sulfide), to steric implications stemming from the oxidant (cumyl hydroperoxide and tert-butyl hydroperoxide), and to the specific complex used. As an example, (S)-methyl p-tolyl sulfoxide was obtained in a 31 % enantiomeric excess by use of cumyl hydroperoxide as oxidant and complex 5 as the catalyst, after 150 min at 0 degrees C and with 100 % conversion of the sulfide. The crystal and molecular structures of 5 and 6 reveal the close relationship between these complexes and the active center of vanadate-dependent haloperoxidases: the vanadium is in a slightly distorted trigonal-bipyramidal environment with the nitrogen and the methoxy group in the axial positions, and the oxo and alkoxide functions of L2 and L3 are the plane. The presence and equilibrium situation of isomers of the catalysts in solution has been investigated by (51)V EXSY and variable-temperature multinuclear NMR spectroscopy. An intermediately formed peroxo (ROO(-)) vanadium complex was detected by (51)V NMR spectroscopy.

The activity of alkaline phosphatase (ALP, EC 3.1.3.1.) was found in seaweeds, including five kinds of green alga, eighteen kinds of red alga, and six kinds of brown alga, collected from the seaside of Dalian in China. The enzyme was purified 1230-fold from Ulva pertusa Kjellm. It had a specific activity of 48.6 U/mg protein and was proven to be homogeneous by SDS-PAGE with a subunit molecular mass of 19.5 kDa. The activity of ALP peaked at pH9.8, and was completely inhibited by DTT and partly by NBS. The Michaelis-Menten constant Km and the maximum reaction velocity Vmax, at pH 9.8 and 37 degrees C were 0.950 mM and 5.00 microM/min, respectively.

Vanadium haloperoxidases (VHPO) catalyze the peroxidative halogenation of organic substrates. Crystallographic studies suggest that hydrogen bonding from a lysine side chain to the vanadium(V)-bound peroxo group may facilitate oxidation of halides (Cl(-), Br(-), I(-)). A ligand with pendant NH(2) functionality, N-(2-pyridylmethyl-6-amino) iminodiacetic acid (H(2)(NH)2pyg(2).2HCl) has been designed to explore the effects that H-bonding from Lys may have on peroxide activation. The first structural characterization of VBrPO model complexes [VO(O(2))((NH)2pyg(2))]K and [VO(O(2))((BrNH)2pyg(2))]K which demonstrate direct intramolecular H-bonding between an amine functionality and V(V)-bound peroxide is reported. The distances between NH(2) proton and bound peroxo moiety [(d(N(1)-H.O): 2.637(4) A in [VO(O(2))((NH)()2pyg(2))]K, and 2.640(8) and 2.6919(8) A in [VO(O(2))((BrNH)2pyg(2))]K] are indicative of intramolecular H-bonding. The intramolecular H-bond strength in [VO(O(2))((BrNH)2pyg(2))](-) is estimated at 6 kcal/mol by (1)H NMR studies and demonstrates that the H-bond interaction is also significant in solution.

We have previously shown that vanadium bromoperoxidase from Ascophyllum nodosum mediates production of the (R)-enantiomer of methyl phenyl sulfoxide with 91% enantiomeric excess. Investigation of the intrinsic selectivity of vanadium bromoperoxidase reveals that the enzyme catalyzes the sulfoxidation of methyl phenyl sulfide in a purely enantioselective manner. The K(m) of the enzyme for methyl phenyl sulfide was determined to be approximately 3.5 mM in the presence of 25% methanol or tert-butanol. The selectivity of the sulfoxidation of methyl phenyl sulfide is optimal in the temperature range 25-30 degrees C and can be further optimized by increasing the enzyme concentration, yielding selectivities with up to 96% enantiomeric excess. Furthermore, we established for the first time that vanadium bromoperoxidase is functional at temperatures up to 70 degrees C. A detailed investigation of the sulfoxidation activity of this enzyme using (18)O-labeled hydrogen peroxide shows that vanadium bromoperoxidase mediates the direct transfer of the peroxide oxygen to the sulfide. A schematic model of the vanadium haloperoxidase sulfoxidation mechanism is presented.

A new crystal form of the vanadium-dependent bromoperoxidase from Corallina officinalis has been obtained. The crystals exhibit a 'teardrop' morphology and are grown from 2 M ammonium dihydrogen phosphate pH and diffract to beyond 1.7 A resolution. They are in tetragonal space group P4222 with unit-cell dimensions of a = b = 201.9, c = 178.19 A, alpha = beta = gamma = 90 degrees. A 2.3 A resolution native data set has been collected at the Hamburg Synchrotron. A mercury derivative data set has also been collected, and the heavy-atom positions have been determined. The self-rotation function and the positions of the heavy atoms are consistent with the molecule being a dodecamer with local 23 symmetry.

Hexose oxidase from the red seaweed, Chondrus crispus was purified to homogeneity. The enzyme appeared to be encapsulated in particles obtained after mechanical disintegration of the fronds. Liberation of the enzyme in soluble form required either waiting for the spontaneous development of a suitable microbial flora in the suspension, or treatment with a mixture of proteases (pronase). As deduced from (SDS/)PAGE, the enzyme has a molecular mass of 87 kDa and probably consists of subunits of 36 kDa and 25 kDa. The low isoelectric point of 2.8 and the presence of 25% (by mass) sugars indicate that the enzyme is a strongly acidic glycoprotein. The absorption spectrum of isolated enzyme minus that of the substrate-reduced enzyme, and the EPR spectrum of the free radical observed in the reduced enzyme revealed the presence of a flavin. This cofactor is probably covalently bound since flavins were not released upon denaturation of the enzyme by heat or acid treatment. Taking free FAD as a reference compound, the enzyme contains 1 mol flavin/mol enzyme. EPR spectroscopy of the purified preparation showed the presence of Cu2+. However, since the amount was substoichiometric, substrate addition did not affect the signal, and the addition of chelator or Cu2+ did not affect the activity, the presence of this metal ion seems adventitious. It is concluded that the large discrepancies between the presently and the previously reported [Sullivan, J. D. & Ikawa, M. (1973) Biochim. Biophys. Acta 309, 11-22] characteristics of the enzyme probably originate from the characterization of a contaminating protein in the latter case.

Fucus serratus eggs were examined for evidence of the existence of a lysosome-like body using enzyme histochemical and vital staining techniques. Simultaneous coupling azo-dye techniques for lysosomal acid phosphatase proved inappropriate owing to endogenous phenolic binding artefacts. The large number of alginate polysaccharide and polyphenolic egg vesicles interfered with vital staining techniques for lysosomes. Lysosomal esterase activity was detected in the abundant egg lipid bodies. The role of the egg lipid body as an equivalent lysosome-like body of higher plants, the spherosome, is discussed in relation to egg fertilization and early zygote development.

Fucoïdans catalyse thrombin inhibition by antithrombin (AT) and heparin cofactor II (HCII); their affinity for each serpin varies according to the seaweed species from which they are extracted, as well as their chemical composition and molecular weight. We extracted a homogeneous fucoïdan fraction from Ascophyllum nodosum, a brown seaweed, and tested its anticoagulant and antithrombotic activities. At a fucoïdan concentration of 3.75 micrograms/ml, thrombin inhibition mediated by AT showed an apparent second-order rate constant (kapp) of 2 x 10(8) M-1 min-1, compared to 1.5 x 10(6) M-1 min-1 for the uncatalyzed reaction. The kapp value of thrombin inhibition via HCII was 1.17 x 10(9) M-1 min-1 at a fucoïdan concentration of 50 micrograms/ml, compared to 1.72 x 10(5) M-1 min-1 for the uncatalyzed reaction. In a Wessler model of venous thrombosis, the fucoïdan fraction, injected intravenously to rabbits 10 min before thrombosis induction, exhibited antithrombotic activity: 1.8 mg/kg was the dose which inhibited F Xa-induced thrombus formation by 80% (ED80), compared to a heparin ED80 of 0.1 mg/kg. At this ED80 the antithrombotic effect of the fucoïdan persisted longer than that of heparin (30 min versus 15 min). The thrombin clotting time (TCT) was significantly prolonged (73 s versus control 29 s, compared to 53 s with heparin) 10 min after a fucoïdan bolus infusion giving a plasma fucoïdan concentration of 14.6 +/- 2.7 micrograms/ml. The bleeding time was slightly increased after fucoïdan infusion at the ED80. Fucoïdan extracted from marine flora thus shows promise as an antithrombotic drug.

The vanadium-dependent haloperoxidase from the seaweed Corallina officinalis has been purified to homogeneity and crystallised. The protein is reported to be a hexamer of 12 x 64,000 Da, contains no haem, and is dependent on vanadium for activity. The crystals are grown from polyethylene glycol (PEG) 6,000 and 0.4 M potassium chloride. They are stable and diffract to better than 2 A resolution. They are of a cubic space group I23 (or 12(1)3) with cell dimensions a = b = c = 310 A.

In this article we report on the steady-state kinetics of the chlorination and the stability of the vanadium chloroperoxidase from the fungus Curvularia inaequalis. The data show that the kinetics of this enzyme resemble that of the vanadium bromoperoxidase from the seaweed Ascophyllum nodosum. At low pH, chloride inhibited the enzyme, but the inhibition was of a dual nature. At pH 4.1 a mixed type of inhibition by chloride with respect to hydrogen peroxide was observed whereas at pH 3.1 the nature of the inhibition became competitive. The log Km for hydrogen peroxide decreased linearly with pH with a slope of -1 in the pH range 3-5. A reaction mechanism is presented to explain the observed data. We also showed that this class of enzymes is inhibited by nitrate. At pH 5.5, nitrate inhibits the chlorination reaction competitively with respect to chloride (Ki = 2 mM) and uncompetitively with respect to hydrogen peroxide. Furthermore, we showed that the enzyme produces HOCl as a reaction product. The enzyme exhibited a high thermostability (tm of 90 degrees C) and displayed high stability in organic solvents (solutions of 40% methanol, ethanol or 2-propanol) and moderate stability in the presence of the chaotropic agent guanidine/HCl (G1/2, the concentration of guanidine/HCl at which the enzyme activity was half the original activity was 3.7 M).

A nonheme bromoperoxidase has been purified to homogeneity from the red seaweed Corallina officinalis. Like the corresponding enzyme previously reported from C. pilulifera, this bromoperoxidase contains a significant amount of nonheme iron. However, it is vanadate ion and not iron that activates the enzyme, and maximal activity is achieved with stoichiometric vanadium incorporation. The absence of competition between vanadium and iron suggests that they occupy distinct binding sites in the protein. A correlation between vanadium content and catalytic activity indicates that less than 12 percent of the maximal activity of the enzyme can be derived from metals other than vanadium.