Tamlana laminarinivorans sp. nov. and Tamlana sargassicola sp. nov., two novel species isolated from Sargassum, show genomic and physiological adaptations for a Sargassum-associated lifestyle.

The genus Tamlana from the Bacteroidota currently includes six validated species. Two strains designated PT2-4T and 62-3T were isolated from Sargassum abundant at the Pingtan island coast in the Fujian Province of China. 16S rRNA gene sequence analysis showed that the closest described relative of strains PT2-4T and 62-3T is Tamlana sedimentorum JCM 19808T with 98.40 and 97.98% sequence similarity, respectively. The 16S rRNA gene sequence similarity between strain PT2-4T and strain 62-3T was 98.68 %. Furthermore, the highest average nucleotide identity values were 87.34 and 88.97 % for strains PT2-4T and 62-3T, respectively. The highest DNA-DNA hybridization (DDH) value of strain PT2-4T was 35.2 % with strain 62-3T, while the DDH value of strain 62-3T was 37.7 % with T. sedimentorum JCM 19808T. Growth of strains PT2-4T and 62-3T occurs at 15-40 °C (optimum, 30 °C) with 0-4 % (w/v) NaCl (optimum 0-1 %). Strains PT2-4T and 62-3T can grow from pH 5.0 to 10.0 (optimum, pH 7.0). The major fatty acids of strains PT2-4T and 62-3T are iso-C15 : 0 and iso G-C15 : 1. MK-6 is the sole respiratory quinone. Genomic and physiological analyses of strains PT2-4T and 62-3T showed corresponding adaptive features. Significant adaptation to the growth environment of macroalgae includes the degradation of brown algae-derived diverse polysaccharides (alginate, laminarin and fucoidan). Notably, strain PT2-4T can utilize laminarin, fucoidan and alginate via specific carbohydrate-active enzymes encoded in polysaccharide utilization loci, rarely described for the genus Tamlana to date. Based on their distinct physiological characteristics and the traits of utilizing polysaccharides from Sargassum, strains PT2-4T and 62-3T are suggested to be classified into two novel species, Tamlana laminarinivorans sp. nov. and Tamlana sargassicola sp. nov. (type strain PT2-4T=MCCC 1K04427T=KCTC 92183T and type strain 62-3T=MCCC 1K04421T=KCTC 92182T).

Winogradskyella endarachnes sp. nov., a marine bacterium isolated from the brown alga Endarachne binghamiae.

A Gram-negative, aerobic, rod-shaped, non-flagellated and motile by gliding bacterium HL2-2T, was isolated from the surface of the brown alga Endarachne binghamiae in China. The 16S rRNA gene sequence analysis showed that this strain was affiliated with the genus Winogradskyella in the family Flavobacteriaceae and presented great similarity with the type strain Winogradskyella litoriviva KMM 6491T (97.9 % sequence similarity). The whole genome of strain HL2-2T comprised 3.6 Mbp with a G+C content of 31.9 mol%. The average nucleotide identity between strain HL2-2T and Winogradskyella litoriviva KMM 6491T was 83.7 %. Growth of the isolated strain was observed from 20-40 °C (optimum, 30 °C), at pH ranged from 5.5 to 8.0 (optimum, pH 6.0) and in the presence of 0-5 % (w/v) NaCl (optimum, 0-2 %). The major fatty acids (>10 % of the total) were C16 : 0, iso-C15 : 0 and the predominant menaquinone was MK-6. The combined phylogenetic, physiological and chemotaxonomic analysis show that the strain HL2-2T represents a novel species belonging to the genus Winogradskyella, for which the name Winogradskyella endarachnes sp. nov. is proposed, and which the type strain is HL2-2T (=CICC 24857T=KCTC 72882T).

Characterization and activity enhancement of a novel exo-type agarase Aga575 from Aquimarina agarilytica ZC1.

The novel ß-agarase gene aga575 from the agarolytic bacterium Aquimarina agarilytica ZC1 is composed of 2142 bp, and the encoded protein Aga575 has the highest amino acid sequence homology of only 65.2% with known agarases. Though carrying a domain of glycoside hydrolase family 42 in the C-terminal, Aga575 should belong to glycoside hydrolase family 50 according to the phylogenetic analysis. Gene aga575 was successfully cloned and overexpressed in Escherichia coli Rosetta (DE3) cells. The recombinant protein had the maximal agarase activity at pH 8.0 and 37 °C. The values Km and Vmax toward agarose were 8.4 mg/mL and 52.2 U/mg, respectively. Aga575 hydrolyzed agarose and neoagarooligosaccharides to yield neoagarobiose as the sole product. The agarose hydrolysis pattern of Aga575 indicated that it was an exo-type ß-agarase. Random mutagenesis was carried out to obtain two beneficial mutants M1 (R534G) and M2 (S4R-R424G) with higher activities. The results showed that the agarase activity of mutant M1 and M2 reached 162% and 192% of the wild-type agarase Aga575, respectively. Moreover, the activity of the mixed mutant M1/M2 (S4R-R424G-R534G) increased to 227%. KEY POINTS: • Aga575 is a novel exo-type ß-agarase degrading agarose to yield neoagarobiose as the sole product. • Though owning a domain of glycoside hydrolase family GH42, Aga575 should belong to family GH50. • The agarase activity of one mutant increased to 227% of the wild-type Aga575.

Enteromorpha prolifera Diet Drives Intestinal Microbiome Composition in Siganus oramin.

Enteromorpha prolifera (E. prolifera) contains complex sulfated polysaccharides that are resistant to biological degradation. Most organisms cannot digest biomass of E. prolifera, except Siganus oramin (S. oramin). This study was conducted to identify the bacteria in the intestine of S. oramin facilitating the digestion of E. prolifera polysaccharides (EPP). Metagenomic sequencing analysis of the S. oramin intestinal microbiota revealed that E. prolifera diet increased the number of Firmicutes, replacing Proteobacteria to be the dominant bacteria. The proportion of Firmicutes increased from 38.8 to 58.6%, with Bacteroidetes increasing nearly fivefold from 5 to 23.7%. 16S rDNA high-throughput sequencing showed that EPP-induced Bacteroidetes increased significantly in the intestinal flora of S. oramin cultivated in vitro. Metatranscriptome analysis showed that EPP induced more transferase, polysaccharide hydrolase, glycoside hydrolase, and esterases expressed in vitro, and most of them were taxonomically annotated to Bacteroidetes. Compared with the aggregation of GH family genes in metagenomic sequencing analysis in vivo, EPP induced more CBM32, GH2, GT2, GT30, and GH30 families gene expression in vitro. In general, We found that the bacteria in intestinal tract of S. oramin responsible for digestion of E. prolifera were Firmicutes and Bacteroidetes, while Bacteroidetes was the dominant bacteria involved in EPP degradation in vitro cultures. Compared with in vivo experiments, only GH family genes were mostly involved, we detected a more complete and complex EPP degradation pathway in vitro. The results may benefit the further study of biodegradation of E. prolifera and has potential implications for the utilization of E. prolifera for biotechnology.

Bioconversion of bamboo shoot shells through the cultivation of the edible mushrooms Volvariella volvacea.

Bamboo shoot shell (BSS), as agricultural waste, is mostly burned or discarded, causing serious environment pollution. In this study, the degradation and utilization of BSS by the edible fungus Volvariella Volvacea was investigated. The composition of V. volvacea fruit body was determined by HPLC-MS, GC-MS and ICP-OES. The activities of CMCase and xylanase were monitored by DNS (3,5-dinitrosalicylic acid) method. Laccase activity was assayed by the oxidation reaction of ABTS [2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate)]. The degraded bamboo shoot shell powder was characterized by FTIR and SEM. The results showed that the mycelium of V. volvacea could degrade and utilize BSS for growth. The activities of carboxymethyl cellulase and laccase were increased during the cultivation. At the same time, the physical structure of the shell fiber becames porous and rough. Most of the products of decayed fibers contain alkanes, ethyl or methyl groups. Moreover, the biological efficiency (fruiting body yield) of V. volvacea cultivated on BSS was 1.52-fold higher than that of straw cultivation. The contents of total lipid, elaidic acid (C18:1n-9), total essential amino acids, total amino acids and iron in V. volvacea fruit bodies grown on BSS were 1.11, 1.66, 1.52, 1.60 and 1.30-fold higher than those of straw treatment, respectively. This study provides an effective method to solve the environmental pollution caused by BSS, and provides a new way for the potential utilization of BSS in edible fungi cultivation.

Tamlana fucoidanivorans sp. nov., isolated from algae collected in China.

A Gram-stain-negative, aerobic, non-motile and rod-shaped marine bacterium, CW2-9T, was isolated from algae collected from Fujian Province in PR China. 16S rRNA gene sequence analysis showed that this strain was affiliated with the genus Tamlana in the family Flavobacteriaceae of the class Flavobacteriia and was very similar to the type strain Tamlana sedimentorum MCCC 1A10799T (96.3 % sequence similarity). The whole genome of strain CW2-9T comprised 3 997 513 bp with a G+C content of 34.3 mol%. The average nucleotide identity value between strain CW2-9T and T. sedimentorum MCCC 1A10799T was 73.8 %. Growth was observed from 15 to 40 °C (optimum, 30 °C), at pH from pH 5.0 to 10.0 (pH 8.0) and in the presence of 0-4 % (w/v) NaCl (0-1 %). The major fatty acids (>10 % of the total) were iso-C15 : 0, iso G-C15 : 1, iso-C17 : 0 3-OH and anteiso-C15 : 0. The predominant menaquinone was MK-6. The combined phylogenetic, physiological and chemotaxonomic data indicate that strain CW2-9T represents a novel species in the genus Tamlana, for which the name Tamlana fucoidanivorans sp. nov. is proposed. The type strain is CW2-9T (=CICC 24749T=KCTC 72389T).

Glutamine Synthetase (GS): A Key Enzyme for Nitrogen Assimilation in The Macroalga Gracilariopsis lemaneiformis (Rhodophyta).

This study aimed to address the importance of glutamine synthetase II (GSII) during nitrogen assimilation in macroalga Gracilariopsis lemaneiformis. The cDNA full-length sequence of the three glGSII genes was revealed to have the 5' m7 G cap, 5'-untranslated region, open reading frame (ORF), 3'-untranslated region, and a 3' poly (A) tail. The three glGSIIs were classified into plastid glGS2 and cytosolic glGS1-1 and glGS1-2, having conserved GSII domains but different cDNA sequences. The complicated 5' end flanking region indicates complex function of glGS genes. glGS1 genes were significantly up-regulated under the different NH4+ : NO3- ratio (i.e., 40:10, 25:25, 10:40, and 0:50) except glGS2 which dramatically up-regulated under the low NH4+ : NO3- ratio (i.e., 10:40 and 0:50) during different cultivation times. These different expression patterns perhaps are due to the different biological roles of GS1 and GS2 in the gene family. Furthermore, hypothetical working model of nitrogen assimilation pathway exhibiting the role of glGS1 and glGS2 is proposed. Finally, glGS2 was expressed in Escherichia coli BL21 (DE3), and the optimal conditions for culture (15°C, overnight), purification (500 mM imidazole washing), and activity (pH 7.4, 37°C) were established. This study lays a very important foundation for exploring the role of GS in nitrogen assimilation in algae and plants.

Functional domains of Litopenaeus vannamei transglutaminase and their involvement in immunoregulation in shrimp.

Shrimps, which mainly rely on their innate immune system to response to infectious pathogens, have clottable proteins as an important component of this system. While transglutaminases (TGase) are found in Litopenaeus vannamei and constitute part of the coagulation system, the specific immune-related roles played by its functional domains in the immunoregulation of shrimp has not been well understood. In the present study, we report that the Ig-like domain of L. vannamei transglutaminase (TGase-C) is the main immune-related domain among the three functional domains, as it had higher bacterial agglutinative activity against Vibrio parahaemolyticus and Streptococcus iniae. Using Co-immunoprecipitation and LC-MS/MS analysis, TGase-C was shown to interact with 474 proteins, of which 52 proteins were annotated to L. vannamei. More than half of the L. vannamei annotated proteins have immune-related functions, including apoptosis. Further analysis using pull-down assay revealed that TGase-C interacted with CAP-3 (a homologue of caspase 3). In addition, siRNA-mediated knockdown of LvTGase significantly (p < 0.01) increased the expression level of LvCAP-3 coupled with a significant (p < 0.01) increase in caspase 3/7 activity, suggesting that probably LvTGase participates in shrimp immune response by modulating the activity of LvCAP-3. These findings thus suggest the Ig-like functional domain of L. vannamei's transglutaminase is the domain that is involved in immunoregulation in shrimp.

SNPs in the Toll1 receptor of Litopenaeus vannamei are associated with immune response.

Tolls and Toll-like receptors (TLRs) are important regulators in the innate immune system and their genetic variations usually affect the host's susceptibility/resistance to pathogen infections. In this study, we report on the single nucleotide polymorphisms (SNPs) of Toll1 in Litopenaeus vannamei (LvToll1) and how this is associated with immune response. PCR-DGGE analysis revealed genetic polymorphisms in LvToll1 at both the genomic DNA (gDNA) and cDNA levels. Using high-throughput sequencing, 223 SNPs were identified at the gDNA level, of which 145 were non-synonymous SNP (nsSNP), with 3 nsSNPs having frequency over 1%. On the other hand, 60 SNPs were identified at the cDNA level including 38 nsSNPs and 4 nsSNPs with frequency over 1%. Upon challenging shrimps with Streptococcus iniae, Vibrio parahaemolyticus and white spot syndrome virus (WSSV), LvToll1 was shown to generate 6, 4 and 4 novel bands, respectively when analyzed with PCR-DGGE. Sequencing analysis of these bands showed that they contained 6, 4 and 2 nsSNPs, respectively. Moreover, the nsSNP C1526T was detected in S. iniae-resistant but not in susceptible shrimps. Most significantly, the C1526T mutation could shorten the α-helix of the LRR domain and was predicted to affect the function of LvToll1, indicating that SNP C1526T might be associated with shrimp's resistance to pathogen infections. In sum, our findings here reveal that the genetic polymorphisms of Toll receptor are linked with the immune response to pathogen infections in L. vannamei.

Comparative transcriptomic analysis of shrimp hemocytes in response to acute hepatopancreas necrosis disease (AHPND) causing Vibrio parahemolyticus infection.

The recent emergence of acute hepatopancreas necrosis disease (AHPND) in shrimps has posed a major challenge in the shrimp aquaculture industry. The Pir toxin proteins carried by some strains of Vibrio parahaemolyticus are believed to play essential roles in the pathogenesis of AHPND. However, few studies have so far explored how the host immune system responds to these bacteria. In this study, AHPND V. parahaemolyticus (with Pir) and non-AHPND V. parahaemolyticus (without Pir) were injected into two groups of shrimps, and the hemocytes collected for comparative transcriptomic analyses. A total of 1064 differentially expressed genes (DEGs) were identified, of which 910 were up-regulated and 154 were down-regulated. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that many DEGs were involved in a number of biological processes such as cellular process, metabolic process and single-organism process in the AHPND V. parahaemolyticus injected group than the non-AHPND V. parahaemolyticus injected group. Among these, major metabolic processes such as carbohydrate metabolism, lipid metabolism and amino acid metabolism were further identified as the major responsive gene groups. We observed that genes involved in cell growth and anti-apoptosis including src, iap2, cas2, cytochrome P450, gst and cytochromecoxidase were strongly activated in the AHPND V. parahaemolyticus group than in the non-AHPND V. parahaemolyticus group. Collectively, our results unveiled that shrimp hemocytes respond to AHPND related strain of Vibrio parahaemolyticus infection at the transcriptional level, which is useful in furthering our understanding of AHPND.

A Litopenaeus vannamei Hemocyanin-Derived Antimicrobial Peptide (Peptide B11) Attenuates Cancer Cells' Proliferation.

Antimicrobial peptides play important roles in the immune response to pathogens and tumor cells; for this reason, they are being exploited for therapeutic use. In this study, we describe a Litopenaeus vannamei hemocyanin-derived peptide, denoted B11, which shares similar features with other anticancer peptides and attenuates the proliferation of cancer cells. Cell viability assay revealed that B11 significantly inhibited the proliferation of human cervical (HeLa), human hepatocellular carcinoma (HepG2), and human esophageal cancer (EC109) cancer cell lines, but not normal liver cell lines (T-antigen-immortalized human liver epithelial (THLE) cells or THLE-3), by inducing morphological changes, nuclear condensation, and margination, features which are indicative of apoptosis. Besides, peptide B11-induced apoptosis was confirmed by isothiocyanate-labeled Annexin V/propidium iodide (Annexin V-FITC/PI) double staining of HeLa cells. Moreover, cell uptake studies, confocal microscopy, and Western blot analysis revealed that rhodamine-labeled B11 permeated HeLa cells and localized to the mitochondria, causing mitochondria dysfunction through lost mitochondrial membrane potential, which consequently triggered the induction of apoptosis. Increased expression levels of caspase-9, caspase-3, and Bax (Bcl-2-associated X) proteins, coupled with a decrease in Bcl-2 (B-cell lymphoma 2) protein, confirmed that peptide B11 induced apoptosis via the mitochondrial pathway. Thus, the hemocyanin-derived peptide, B11, inhibits the proliferation of cancer cells by causing mitochondrial dysfunction and inducing apoptotic cell death, for which reason it could be explored as an anticancer peptide.

Identification and characterization of an 18.4kDa antimicrobial truncation from shrimp Litopenaeus vannamei hemocyanin upon Vibrio parahaemolyticus infection.

Hemocyanin (HMC) is a multifunctional protein which plays many essential roles in invertebrate organism. Recently more and more immune-related functions have been discovered on this protein. Here the shrimp was infected with Vibrio parahaemolyticus and the shrimp sera were analyzed by two-dimensional gel electrophoresis. Totally 15 spots were identified as significantly up-regulated spots and further analyzed by MALDI-TOF/TOF mass spectrometry (MS). Four of them were identified as HMC derived truncations (HMCS1, HMCS3, HMCS4 and HMCS5). The HMCS4 primary sequence was further determined via Edman N terminal sequencing, MALDI-TOF MS and amino acid sequence alignment. The result indicated that the HMCS4 was a 165aa fragment from shrimp HMC small subunit C-terminal. The HMCS4 immunological activities were further analyzed by agglutination experiment and antibacterial assay in vitro. The results showed that the recombinant HMCS4 (rHMCS4) had strong agglutination and antibacterial activities against pathogenic bacteria at the optimum bacteriostasis concentration. In addition, the HMCS4 immunological activities were explored via mortality assay in vivo. The shrimp was challenged with V. parahaemolyticus and rHMCS4 V. parahaemolyticus mixture separately. The shrimp mortality rate was significantly decreased at 96 h post-infection with rHMCS4 injection. Our data showed that shrimp HMC truncation generation upon infection was an effective immune response against invaded pathogens. Moreover, these findings may have some potential applications in shrimp industry.

Cloning and characterization of a novel hemocyanin variant LvHMCV4 from shrimp Litopenaeus vannamei.

Recently, we found 3 variants of hemocyanin subunit with higher molecular weight in shrimp Litopenaeus vannamei (Named as LvHMCV1-3). In this study, a novel L. vannamei hemocyanin variant (Named as LvHMCV4) was further cloned and characterized. Bioinformatic analysis predicted that LvHMCV4 contains one open reading frame of 2137 bp and encodes a polypeptide of 678 amino acids. It shares 84-99% cDNA sequences identity to that of the classical form of L. vannamei hemocyanin (LvHMC, AJ250830.1) and LvHMCV1-3. LvHMCV4 possesses a conserved structure characteristic of the hemocyanin family and can be clustered into one branch along with other arthropod hemocyanins in a phylogenetic tree. Further, the full-length DNA of LvHMCV4 contains 2660 bp and two introns, which are located at the 80-538 bp and 2063-2227 bp regions, respectively. In addition, the mRNA transcript of LvHMCV4 was expressed highly in the hepatopancreas, lymphoid, brain and hemocytes, and weakly in the heart, intestine and gill, while no expression was found in the muscle, stomach and gut. Infection by Escherichia coli K12, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio fluvialis, Streptococcus pyogenes or Staphylococcus aureus up-regulated significantly LvHMCV4 mRNA expression in the hepatopancreas. Furthermore, the recombinant protein of LvHMCV4 (rLvHMCV4) was prepared, which showed agglutination activities against six pathogenic bacteria at concentrations ranging from 15.6 to 125 µg/ml. When co-injected with V. parahaemolyticus in L.vannamei, rLvHMCV4 significantly increased the survival rate after 48 h injection. Together, these studies suggested that hemocyanin variant, LvHMCV4, might be involved in shrimp resistance to pathogenic infection.

An overview of microbial enzymatic approaches for pectin degradation.

Pectin, a complex natural macromolecule present in primary cell walls, exhibits high structural diversity. Pectin is composed of a main chain, which contains a high amount of partly methyl-esterified galacturonic acid (GalA), and numerous types of side chains that contain almost 17 different monosaccharides and over 20 different linkages. Due to this peculiar structure, pectin exhibits special physicochemical properties and a variety of bioactivities. For example, pectin exhibits strong bioactivity only in a low molecular weight range. Many different degrading enzymes, including hydrolases, lyases and esterases, are needed to depolymerize pectin due to its structural complexity. Pectin degradation involves polygalacturonases/rhamnogalacturonases and pectate/pectin lyases, which attack the linkages in the backbone via hydrolytic and ß-elimination modes, respectively. Pectin methyl/acetyl esterases involved in the de-esterification of pectin also play crucial roles. Many α-L-rhamnohydrolases, unsaturated rhamnogalacturonyl hydrolases, arabinanases and galactanases also contribute to heterogeneous pectin degradation. Although numerous microbial pectin-degrading enzymes have been described, the mechanisms involved in the coordinated degradation of pectin through these enzymes remain unclear. In recent years, the degradation of pectin by Bacteroides has received increasing attention, as Bacteroides species contain a unique genetic structure, polysaccharide utilization loci (PULs). The specific PULs of pectin degradation in Bacteroides species are a new field to study pectin metabolism in gut microbiota. This paper reviews the scientific information available on pectin structural characteristics, pectin-degrading enzymes, and PULs for the specific degradation of pectin.

Evidences of SNPs in the variable region of hemocyanin Ig-like domain in shrimp Litopenaeus vannamei.

Single nucleotide polymorphisms (SNPs) are the commonest mode of genetic variation in invertebrate immune-related genes. Hemocyanin presents in the hemolymph of both mollusks and arthropods and functions as an important antigen non-specific immune protein. But people know very little about its gene polymorphism so far. In current study, bioinformatics, molecular biology and environmental challenge approaches were used to identify the SNPs within hemocyanin Ig-like domain in shrimp Litopenaeus vannamei. A total of 11 SNPs were found in a variable region of Ig-like domain from L. vannamei hemocyanin large subunit (1258-1460 bp, HcLV1), 5 of which (1272, 1315, 1380, 1410 and 1450) were confirmed present in both genomic DNA and cDNA by clone sequencing. Furthermore, HcLV1 showed 3, 5 and 5 SSCP bands, respectively, in 16, 25 and 30 °C-treated shrimps, suggesting that the SSCP pattern of HcLV1 could be modulated by environmental stress. In addition, HcLV1 displayed two extra bands with different mobility when shrimps treated with Vibrio parahaemolyticus for 6-24 h, which was not observed in the control group. In conclusion, our data suggest that shrimp L. vannamei hemocyanin Ig-like domain possesses SNPs, which may be associated with environmental stress or pathogenic challenge.

Mutation of conserved residues in the laminarinase Lam1092 increases the antioxidant activity of the laminarin product hydrolysates.

Laminarinases from the glycoside hydrolase 16 (GH16) family are hydrolases that break ß-1,3-glycosidic bonds in laminarin, which is the major storage polysaccharide present in brown algae or microalgae. We explored a laminarinase from the marine Flavobacteriaceae species Tamlana sp. PT2-4 at the structural and functional levels. Based on a homology model of Lam1092-substrate interactions, the large active groove crossing Lam1092 was deemed a reasonable pathway for the bent substrates for hydrolysis. Eight residues (Gly361, Asn364, Arg400, His466, Asp449, Glu452, Ser477 and Thr538) were selected for mutagenesis based on the interactions of Lam1092 in complex with Lam4/Lam6. Ultimately, we generated eight mutants of Lam1092, and the antioxidant activities of the hydrolysates of two mutants (G361A and H466A) showed significant improvement. These results show that the antioxidant activity of laminarin can be improved by laminarinase mutation, which will be beneficial for developing efficient approaches to engineer the substrate specificity of laminarinases and improve the application of bioactive laminarioligosaccharides.

Identification and agglutination properties of hemocyanin from the mud crab (Scylla serrata).

Infectious diseases have significantly delayed the growth of crab aquaculture. Identification of the immune molecules and characterization of the defense mechanisms will be pivotal to the reduction of these diseases. Hemocyanin is an important non-specific immune protein present in the hemolymph of both mollusks and arthropods. However, little is known about the hemocyanin from the mud crab Scylla serrata. In this study, we identified the S. serrata hemocyanin using affinity proteomics and investigated its agglutinative properties. The results showed that S. serrata hemocyanin consists of five subunits with molecular weights of 70, 72, 75, 76 and 80 kDa, respectively. It demonstrated agglutination activities against seven bacterial species at concentrations ranging from 7.5 to 30 µg/ml. Agglutination was inhibited by 50-200 mM of N-acetylneuraminic acid, α-d-glucose, d-galactose and d-xylose. The 76 kDa subunit was identified as the protein that primarily binds bacterial cells and we speculate that it functions as the agglutinating subunit. We showed that outer membrane proteins (Omp) of bacteria could completely inhibit agglutination and that the agglutination activities of hemocyanin against Escherichia coli ▵OmpA and ▵OmpX mutants were significantly decreased, suggesting that these two Omps may be important ligands of hemocyanin. Together, the data collectively suggests that the 76 kDa subunit of S. serrata hemocyanin mediates agglutination through recognition of OmpA and OmpX proteins in bacteria.

Proteomic identification of the related immune-enhancing proteins in shrimp Litopenaeus vannamei stimulated with vitamin C and Chinese herbs.

Recently, strong interest has been focused on immunostimulants to reducing the diseases in shrimp aquaculture. However, information regarding to the related immune-enhancing proteins in shrimps is not available yet. In this study, vitamin C (Vc), Chinese herbs (CH), and the mixture of vitamin C and Chinese herbs (Mix) were tested for their enhancement on shrimp's immune activity. Compared with those in the control group, values of phenoloxidase (PO), superoxide dismutase (SOD) and antibacterial (Ua) activity in the Mix-treated group were improved significantly 12 or 24 days after the treatment. The cumulative mortality was also lower in the Mix-treated group after infection with Vibrio parahemolyticus. Furthermore, comparative proteomic approach was used to assess the protein expression profile in shrimps. Approximately 220-290 and 300-400 protein spots were observed in the 2-DE gels. Among them, 29 and 28 altered proteins from hemocytes and hepatopancreas, respectively, were subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) analysis. The results revealed that the main altered proteins showed high homologies with Litopenaeus vannamei hemocyanin, hemolymph clottable protein, hemoglobin beta, cytosolic MnSOD, trypsin, cathepsin I(L) and zinc proteinase Mpc1. Together, these studies found Vc and CH were suitable immunostimulants to shrimp L. vannamei, and 7 altered proteins could be involved in the enhanced immune activities.

Isolation of marine benzo[a]pyrene-degrading Ochrobactrum sp. BAP5 and proteins characterization.

A bacterial strain BAP5 with a relatively high degradation ability of benzo[a]pyrene (BaP) was isolated from marine sediments of Xiamen Western Sea, China and identified as Ochrobactrum sp. according to 16S rRNA gene sequence as well as Biolog microbial identification system. Strain BAP5 could grow in mineral salt medium with 50 mg/L of BaP and degrade about 20% BaP after 30 d of incubation. Ochrobactrum sp. BAP5 was able to utilize other polycyclic aromatic hydrocarbons (PAHs) (such as phenanthrene, pyrene and fluoranthene) as the sole carbon source and energy source, suggesting its potential application in PAHs bioremediation. The profile of total soluble protein from Ochrobactrum sp. BAP5 was also investigated. Some over- and special-expressed proteins of strain BAP5 when incubated with the presence of BaP were detected by two-dimensional polyacrylamide gel electrophoresis, and found to be related with PAHs metabolism, DNA translation, and energy production based on peptide fingerprint analysis through matrix-assisted laser desorption/ionization-time of flight mass spectrometry.

Enhanced Fermentable Sugar Production from Enteromorpha Polysaccharides by the Crude Enzymes of Vibrio sp. H11.

In recent years, large-scale outbreaks of the green alga Enteromorpha prolifera in China's offshore waters have posed a serious threat. This study aimed to improve Enteromorpha polysaccharide (EP) enzymatic sugar production using the hydrolase system of Vibrio sp. H11, an EP-utilizing microbial strain. Strain H11 was found to contain 711 carbohydrate-related genes, and 259 genes belong to glycoside hydrolases that have the potential to hydrolyze EP. To maximize the capability of strain H11 to hydrolyze EP, both the culture medium and the composition were optimized. Response surface methodology analysis showed that maximal enzymatic production from strain H11 was 8.43 U/mL after 26-h incubation. When 50 g/L of EP were treated with crude H11 enzyme, the concentration of fermentation sugars increased by 36.12%. Under these conditions, the hydrolysates were capable of generating 3,217 mL/L of biogas and 6.74 g/L of biosolvents, with increases of 28.17 and 7.29%, respectively, compared to controls. The combined application of the H11 enzymatic system and anaerobic fermentation has the potential to improve the comprehensive application of EP.