A Novel Bifunctional Alginate Lyase and Antioxidant Activity of the Enzymatic Hydrolysates.

Alginate lyase Aly448, a potential new member of the polysaccharide lyase (PL) 7 family, which was cloned and identified from the macroalgae-associated bacterial metagenomic library, showed bifunctionality. The molecular docking results revealed that Aly448 has two completely different binding sites for alginate (polyMG), poly-α-l-guluronic acid (polyG), and poly-ß-d-mannuronic acid (polyM) substrates, respectively, which might be the molecular basis for the enzyme's bifunctionality. Truncational results confirmed that predicted key residues affected the bifunctionality of Aly448, but did not wholly explain. Besides, Aly448 presented excellent biochemical characteristics, such as higher thermal stability and pH tolerance. Degradation of polyMG, polyM, and polyG substrates by Aly448 produced tetrasaccharide (DP4), disaccharide (DP2), and galactose (DP1), which exhibited excellent antioxidant activity. These findings provide novel insights into the substrate recognition mechanism of bifunctional alginate lyases and pave a new path for the exploitation of natural antioxidant agents.

Ribosomal frameshifting at normal codon repeats recodes functional chimeric proteins in human.

Ribosomal frameshifting refers to the process that ribosomes slip into +1 or -1 reading frame, thus produce chimeric trans-frame proteins. In viruses and bacteria, programmed ribosomal frameshifting can produce essential trans-frame proteins for viral replication or regulation of other biological processes. In humans, however, functional trans-frame protein derived from ribosomal frameshifting is scarcely documented. Combining multiple assays, we show that short codon repeats could act as cis-acting elements that stimulate ribosomal frameshifting in humans, abbreviated as CRFS hereafter. Using proteomic analyses, we identified many putative CRFS events from 32 normal human tissues supported by trans-frame peptides positioned at codon repeats. Finally, we show a CRFS-derived trans-frame protein (HDAC1-FS) functions by antagonizing the activities of HDAC1, thus affecting cell migration and apoptosis. These data suggest a novel type of translational recoding associated with codon repeats, which may expand the coding capacity of mRNA and diversify the regulation in human.

Metagenomic Insights Reveal the Microbial Diversity and Associated Algal-Polysaccharide-Degrading Enzymes on the Surface of Red Algae among Remote Regions.

Macroalgae and macroalgae-associated bacteria together constitute the most efficient metabolic cycling system in the ocean. Their interactions, especially the responses of macroalgae-associated bacteria communities to algae in different geographical locations, are mostly unknown. In this study, metagenomics was used to analyze the microbial diversity and associated algal-polysaccharide-degrading enzymes on the surface of red algae among three remote regions. There were significant differences in the macroalgae-associated bacteria community composition and diversity among the different regions. At the phylum level, Proteobacteria, Bacteroidetes, and Actinobacteria had a significantly high relative abundance among the regions. From the perspective of species diversity, samples from China had the highest macroalgae-associated bacteria diversity, followed by those from Antarctica and Indonesia. In addition, in the functional prediction of the bacterial community, genes associated with amino acid metabolism, carbohydrate metabolism, energy metabolism, metabolism of cofactors and vitamins, and membrane transport had a high relative abundance. Canonical correspondence analysis and redundancy analysis of environmental factors showed that, without considering algae species and composition, pH and temperature were the main environmental factors affecting bacterial community structure. Furthermore, there were significant differences in algal-polysaccharide-degrading enzymes among the regions. Samples from China and Antarctica had high abundances of algal-polysaccharide-degrading enzymes, while those from Indonesia had extremely low abundances. The environmental differences between these three regions may impose a strong geographic differentiation regarding the biodiversity of algal microbiomes and their expressed enzyme genes. This work expands our knowledge of algal microbial ecology, and contributes to an in-depth study of their metabolic characteristics, ecological functions, and applications.

Characterization of a novel ß-agarase from Antarctic macroalgae-associated bacteria metagenomic library and anti-inflammatory activity of the enzymatic hydrolysates.

An agarase gene (aga1904) that codes a protein with 640 amino acids was obtained from the metagenomic library of macroalgae-associated bacteria collected from King George Island, Antarctica. Gene aga1904 was expressed in Escherichia coli BL21 (DE3) and recombinant Aga1904 was purified by His Bind Purification kit. The optimal temperature and pH for the activity of Aga1904 were 50°C and 6.0, respectively. Fe3+ and Cu2+ significantly inhibited the activity of Aga1904. The V max and K m values of recombinant Aga1904 were 108.70 mg/ml min and 6.51 mg/ml, respectively. The degradation products of Aga1904 against agarose substrate were mainly neoagarobiose, neoagarotetraose, and neoagarohexaose analyzed by thin layer chromatography. The cellular immunoassay of enzymatic hydrolysates was subsequently carried out, and the results showed that agaro-oligosaccharides dominated by neoagarobiose significantly inhibited key pro-inflammatory markers including, nitric oxide (NO), interleukins 6 (IL-6), and tumor necrosis factor α (TNF-α). This work provides a promising candidate for development recombinant industrial enzyme to prepare agaro-oligosaccharides, and paved up a new path for the exploitation of natural anti-inflammatory agent in the future.

Identification and Expression Analysis of an Interacting Protein (LvFABP) that Mediates Vibrio parahaemolyticus AHPND Toxin Action.

Acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus causing AHPND (VPAHPND) is the most serious disease affecting shrimp farming. The PirAvp and PirBvp toxins of VPAHPND are known virulence factors. However, the corresponding target protein in shrimp that mediates their action has not been identified. By screening yeast two-hybrid cDNA libraries from intestine, stomach, and hepatopancreas of Litopenaeus vannamei, the protein with the largest increase in gene expression in shrimp hepatopancreas in response to VPAHPND challenge was identified and designated LvFABP. Analysis revealed high sequence homology of the LvFABP gene and a lipocalin/cytosolic fatty acid binding gene. Yeast two-hybrid pairwise analysis, GST-pull down assay, and far-western blot assay were performed to determine the interaction between LvFABP and PirBvp. LvFABP was able to directly bind to PirBvp. The expression of LvFABP in the hepatopancreas was significantly higher at P23 and P27 developmental stages of L. vannamei. RNA interference (RNAi) of LvFABP reduced the mortality, histopathological signs of AHPND in the hepatopancreas, and the number of virulent VPAHPND bacteria in the intestine, stomach, and hepatopancreas after VPAHPND challenge. We concluded that the LvFABP was involved in AHPND pathogenesis and acted as a VPAHPND toxin interacting protein. This is the first identification of VPAHPND toxin interacting protein from the shrimp digestive system by yeast two-hybrid library screening and were confirmed by in vitro protein interaction verification and in vivo challenge experiments. This study provides novel insight into the contributions of LvFABP towards AHPND pathogenesis in shrimp. The findings could inform AHPND preventative measures in shrimp farming.

Adaptation of the Invasive Plant (Sphagneticola trilobata L. Pruski) to a High Cadmium Environment by Hybridizing With Native Relatives.

Invasive species can evolve rapidly in the invasion areas to adapt to new habitats. Sphagneticola trilobata L. Pruski, an invasive species, was studied for its tolerance to cadmium (Cd) in the soil and compared with its natural hybrid. From the perspective of photosynthetic physiology, antioxidant characteristics, and leaf hormone levels, the differences between the leaves of the two species before and after Cd treatment were compared. The results showed that the hybrid had stronger tolerance to Cd stress than invasive species. After Cd stress, the indexes of gas-exchange [net photosynthetic rate (Pn), intercellular CO2 concentration (Ci), stomatal conductance (Gs), and transpiration rate (Tr)] of the hybrid was higher than invasive species, while the content of non-enzymatic antioxidants (flavonoids and total phenols) and antioxidant enzyme activities [peroxidase (POD) and superoxide dismutase (SOD)] was lower in hybrid than in invasive species. The changes in the content of plant hormones [auxin (IAA) and abscisic acid (ABA)] under Cd stress showed that hybrid can still maintain growth and prevent leaf senescence. Furthermore, the differences in gene expression between hybrid and invasive species in photosynthetic physiology, the antioxidant capacity of leaves, and endogenous hormone (IAA and ABA) synthesis pathway also showed that hybrid has stronger Cd tolerance than invasive species. This suggests that invasive species will realize the invasion through hybridization with the native relatives to overcome the stress from environmental factors. The study implied that hybridization between invasive species and native relatives is an important way for invasive species to spread in a wider and new environment that invasive species have not experienced in the area of origin.

Biochemical Characterization of a Carrageenase, Car1383, Derived From Associated Bacteria of Antarctic Macroalgae.

A carrageenase gene, car1383, was obtained from the metagenome of Antarctic macroalgae-associated bacteria. The amino acid sequence of its product showed up to 33% similarity with other carrageenases and contained a GH16-family motif. The recombinant Car1383 was heterologously expressed in Eschericia coli and exhibited maximal activity at 50°C and pH 6.0, with a Km of 6.51 mg/ml and a Vmax of 55.77 U/mg. Its activity was enhanced by some cations (Na+, K+, and Fe2+), but inhibited or inactivated by others (Sr2+, Ca2+, Ni2+, Ba2+, Mn2+, Cu2+, Fe3+, and Mg2+). Car1383 degraded carrageenan into neocarrabiose and neocarratetraose. Site-directed mutagenesis indicated that putative active sites, E190 and E195, conserved sites, W183 and G255, play important roles in Car1383 activity. This study provides a new candidate for the industrial preparation of bioactive algal oligosaccharides.

Multifunctional alkalophilic α-amylase with diverse raw seaweed degrading activities.

Uncultured microbes are an important resource for the discovery of novel enzymes. In this study, an amylase gene (amy2587) that codes a protein with 587 amino acids (Amy2587) was obtained from the metagenomic library of macroalgae-associated bacteria. Recombinant Amy2587 was expressed in Escherichia coli BL21 (DE3) and was found to simultaneously possess α-amylase, agarase, carrageenase, cellulase, and alginate lyase activities. Moreover, recombinant Amy2587 showed high thermostability and alkali resistance which are important characteristics for industrial application. To investigate the multifunctional mechanism of Amy2587, three motifs (functional domains) in the Amy2587 sequence were deleted to generate three truncated Amy2587 variants. The results showed that, even though these functional domains affected the multiple substrates degrading activity of Amy2587, they did not wholly explain its multifunctional characteristics. To apply the multifunctional activity of Amy2587, three seaweed substrates (Grateloupia filicina, Chondrus ocellatus, and Scagassum) were digested using Amy2587. After 2 h, 6 h, and 24 h of digestion, 121.2 ± 4 µg/ml, 134.8 ± 6 µg/ml, and 70.3 ± 3.5 µg/ml of reducing sugars were released, respectively. These results show that Amy2587 directly and effectively degraded three kinds of raw seaweeds. This finding provides a theoretical basis for one-step enzymatic digestion of raw seaweeds to obtain seaweed oligosaccharides.

Expression and Characterization of a Thermostable Carrageenase From an Antarctic Polaribacter sp. NJDZ03 Strain.

The complete genome of Polaribacter sp. NJDZ03, which was isolated from the surface of Antarctic macroalgae, was analyzed by next-generation sequencing, and a putative carrageenase gene Car3206 was obtained. Car3206 was cloned and expressed in Escherichia coli BL21(DE3). After purification by Ni-NTA chromatography, the recombinant Car3206 protein was characterized and the antioxidant activity of the degraded product was investigated. The results showed that the recombinant plasmid pet-30a-car3206 was highly efficiently expressed in E. coli BL21(DE3). The purified recombinant Car3206 showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular weight of 45 kDa. The optimum temperature of the recombinant Car3206 was 55°C, and it maintain 60-94% of its initial activity for 4-12 h at 55°C. It also kept almost 70% of the initial activity at 30°C, and more than 40% of the initial activity at 10°C. These results show that recombinant Car3206 had good low temperature resistance and thermal stability properties. The optimum pH of recombinant Car3206 was 7.0. Car3206 was activated by Na+, K+, and Ca2+, but was significantly inhibited by Cu2+ and Cr2+. Thin-layer chromatographic analysis indicated that Car3206 degraded carrageenan generating disaccharides as the only products. The antioxidant capacity of the degraded disaccharides in vitro was investigated and the results showed that different concentrations of the disaccharides had similar scavenging effects as vitamin C on O 2 • - , •OH, and DPPH•. To our knowledge, this is the first report about details of the biochemical characteristics of a carrageenase isolated from an Antarctic Polaribacter strain. The unique characteristics of Car3206, including its low temperature resistance, thermal stability, and product unity, suggest that this enzyme may be an interesting candidate for industrial processes.

Prokaryotic Diversity and Composition of Sediments From Prydz Bay, the Antarctic Peninsula Region, and the Ross Sea, Southern Ocean.

The V3-V4 hypervariable regions of the 16S ribosomal RNA gene were analyzed to assess prokaryotic diversity and community compositions within 19 surface sediment samples collected from three different regions (depth: 250-3,548 m) of Prydz Bay, the Antarctic Peninsula region, and the Ross Sea. In our results, we characterized 1,079,709 clean tag sequences representing 43,227 operational taxonomic units (OTUs, 97% similarity). The prokaryotic community distribution exhibited obvious geographical differences, and the sequences formed three distinct clusters according to the samples' origins. In general, the biodiversity of Prydz Bay was higher than those of the Antarctic Peninsula region and the Ross Sea, and there were similar prokaryotic communities in different geographic locations. The most dominant clades in the prokaryotic communities were Proteobacteria, Bacteroidetes, Thaumarchaeota, Oxyphotobacteria, Deinococcus-Thermus, Firmicutes, Acidobacteria, Fusobacteria, and Planctomycetes, but unique prokaryotic community compositions were found in each of the sampling regions. Our results also demonstrated that the prokaryotic diversity and community distribution were mainly influenced by geographical and physicochemical factors, such as Zn, V, Na, K, water depth, and especially geographical distance (longitude variation of sample location) and Ba ion content. Moreover, geochemical factors such as nutrient contents (TC, P, and Ca) also played important roles in prokaryotic diversity and community distribution. This represents the first report that Ba ion content has an obvious effect on prokaryotic diversity and community distribution in Southern Ocean sediments.

The hybridization between Sphagneticola trilobata (L.) Pruski and Sphagneticola calendulacea (L.) Pruski improved the tolerance of hybrid to cadmium stress.

Hybridization is common between invasive and native species and may be accompanied by invasive evolution. The hybrid of Sphagneticola trilobata (alien invasive species) and Sphagneticola calendulacea (indigenous congener) was found in South China. According to previous studies, the hybrid performed weak environmental adaptability in comparison with parents. However, based on the results from this study, the hybridization significantly improved the tolerance of the hybrid to cadmium (Cd) stress (200 µmol L-1). Under Cd stress, the hybrid lines showed lowest level of oxidative damage and the highest level of photosynthetic efficiency. Compared with the parents, the hybrid utilized more active detoxification strategies, such as the cell walls of the leaves and roots adsorbed 88% and 95% Cd, respectively, reducing the amount of Cd entering cells; moreover, most of the Cd that entered cells was transformed into less toxic chemical forms through the reduction of the highly toxic chemical forms; furthermore, it accumulated a large number of phytochelatins to bind Cd2+ and reduced the damage of organelles by Cd2+. The results demonstrate that hybridization between S. trilobata and S. calendulacea improved the adaptability of the new hybrid species to Cd stress and may pose a greater threat to the survival of the native parent species in the presence of serious water and soil pollution.

Biosynthesis, characterization and antibacterial activity of silver nanoparticles by the Arctic anti-oxidative bacterium Paracoccus sp. Arc7-R13.

Silver nanoparticles (AgNPs) have attracted wide interest due to its broad range of applications. This study aims to describe the biosynthesis of AgNPs using an Arctic anti-oxidative bacterium Arc7-R13 and to study its characteristics and antibacterial activity. The biosynthesis of AgNPs was verified using UV-Vis spectrum with the maximum absorption at 416 nm. The morphology of the silver nanoparticles was characterized by TEM and its characterization were investigated by EDX and FTIR. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain Arc7-R13 was affiliated with genus Paracoccus. TEM analysis revealed that the AgNPs synthesized by strain Arc7-R13 were spherical and ellipsoidal in shape with size ranging from 2 to 25 nm. The optimal concentration of AgNO3 and temperature for the biosynthesis were 4 mmol/L and 37 °C, respectively. EDX analysis verified the presence of the element silver in the biosynthesized AgNPs. FTIR analysis revealed that the specific functional groups, OH, CH3 and C≡N, might be responsible for reduction and stabilization of AgNPs. Antimicrobial test showed that the AgNPs had strong antimicrobial activity against all kinds of strains investigated, including Gram-positive Bacillus subtilis, Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa, Escherichia coli.

Characterization of a thermostable κ-carrageenase from a hot spring bacterium and plant protection activity of the oligosaccharide enzymolysis product.

BACKGROUND: Seaweed oligosaccharides are environmentally-friendly natural products and their use for disease control in sustainable agriculture is extremely promising. Enzymatic digestion to prepare seaweed oligosaccharides has drawn considerable interest. However, the study of enzymatically degraded products of carrageenan is still in its infancy compared with that of other hydrocolloids such as agar and alginate. To prepare degraded carrageenan on a commercial scale, it is necessary to select superior producer bacterial strains to improve the yield and thermostability of carrageenases. RESULTS: The carrageenan-degrading bacterium Bacillus sp. HT19 was isolated from sediment of a hot spring in Indonesia, and a κ-carrageenase with high activity was purified from the culture supernatant. The purified enzyme, named Car19, had maximum activity (538 U mg-1 ) at 60 °C and pH 7.0. Notably, the enzyme retained >90% of its initial activity after incubation at 60 °C for 24 h. The Ca2+ obviously improved the thermostability of Car19 at 70 °C. The Km and Vmax values of purified Car19 were 0.061 mg mL-1 and 115.13 U mg-1 , respectively, with κ-carrageenan as substrate. Thin-layer chromatography and electrospray ionization mass-spectrometry analysis of hydrolysates indicated that the enzyme exolytically depolymerized κ-carrageenan to neo-carrabiose. The hydrolysate enhanced the resistance of cucumber to cucumber mosaic virus and increased the activity of antioxidant enzymes in infected plants. CONCLUSION: To our knowledge, Car19 is the most thermostable κ-carrageenase reported so far. Its high optimal reaction temperature and thermostability, and unitary hydrolysate constituent, makes Car19 a promising candidate for the preparation of carrageenan oligosaccharides with plant protection activity. © 2018 Society of Chemical Industry.

Multifunctional α-amylase Amy19 possesses agarase, carrageenase, and cellulase activities.

The open reading frame of an α-amylase coding gene, amy19, was obtained from a fosmid genomic library of hot spring bacterium Bacillus BI-19 by a plate-based assay of carrageenase activity. After heterologous expression of the gene, the recombinant Amy19 was found to possess α-amylase, agarase, carrageenase, and cellulase activities, and could degrade soluble starch, agarose, carrageen, and sodium cellulose into oligosaccharides with low degrees of polymerization. To explore the multifunctional mechanism of Amy19, three continuous glycosyl hydrolase 70 (GH70) motifs in the Amy19 encoding sequence were deleted one by one, then in pairs, then all at once. The GH70 motifs may play an important role in the multifunctionality of Amy19, but the multifunctionality was not determined by the GH70 motifs alone. To our knowledge, this is the first report of an α-amylase from a hot spring bacterium with additional agarase, carrageenase, and cellulase activities.

Phytotoxic effects of Cu, Cd and Zn on the seagrass Thalassia hemprichii and metal accumulation in plants growing in Xincun Bay, Hainan, China.

Seagrasses play an important role in coastal marine ecosystems, but they have been increasingly threatened by human activities. In recent years, seagrass communities have rapidly degenerated in the coastal marine ecosystems of China. To identify the reasons for the decline in seagrasses, the phytotoxic effects of trace metals (Cu, Cd and Zn) on the seagrass Thalassia hemprichii were investigated, and the environmental contents of the metals were analyzed where the seagrass grows. The results showed that leaf necrosis in T. hemprichii exposed to 0.01-0.1 mg L-1 of Cu2+ for 5 days was more serious than that in plants exposed to the same concentrations of Cd2+ and Zn2+. The chlorophyll content in T. hemprichii declined in a concentration-dependent manner after 5 days of exposure to Cu2+, Cd2+ and Zn2+. The evident reduction in ΔF/Fm' in T. hemprichii leaves was observed at day 1 of exposure to 0.01-1.0 mg L-1 of Cu2+ and at day 3 of exposure to 0.1-1.0 mg L-1 of Cd2+. The antioxidant enzyme activities (SOD, POD and CAT) in T. hemprichii leaves exposed to the three metal ions also showed significant changes. In seawater from Xincun Bay (Hainan, China), where T. hemprichii grows, Cu had reached a concentration (i.e., 0.01 mg L-1) that could significantly reduce chlorophyll content and ΔF/Fm' in T. hemprichii leaves. Our results indicate that Cu influences the deterioration of seagrasses in Xincun Bay.