Pseudoalteromonas Sp Research Articles

Tripeptides From Casein Are Signal Molecules to Induce the Expression of the Extracellular Protease MCP-01 Gene in Marine Bacterium Pseudoalteromonas sp. SM9913.

Microbial extracellular proteases play crucial roles in marine protein degradation and nitrogen recycling. Although a large number of marine bacteria are found to produce extracellular proteases, it is still unknown how marine bacteria respond to environmental proteins to activate the expression of genes encoding extracellular proteases. The inducing signal molecule for marine bacterial extracellular proteases has never been identified. In this study, we identified tripeptides as the inducing signal molecules for the extracellular protease MCP-01 of the deep-sea bacterium Pseudoalteromonas sp. SM9913. We found that casein, but not casamino acids, can induce the gene expression and synthesis of MCP-01, suggesting that peptides rather than amino acids derived from casein induce the gene expression and synthesis of MCP-01 in SM9913. Then, casein was hydrolyzed by SM9913 extracellular proteases, and the peptides with inducing effect were isolated and characterized. Finally, four tripeptides, SPP, RYP, RQF and FRQ, were shown to have significant inducing effect on the expression of MCP-01 gene, indicating that they are likely the inducing signal molecules for the expression of protease MCP-01 gene in SM9913. This study sheds light on the induction mechanism for the gene expression and biosynthesis of marine microbial extracellular proteases, which is helpful in better understanding the adaptation of bacteria to deep-sea sedimental environment.

Purification and characterization of a novel wild-type α-amylase from Antarctic sea ice bacterium Pseudoalteromonas sp. M175

A novel wild-type α-amylase named wtAmy175 from Pseudoalteromonas sp. M175 strain was purified through ammonium sulphate precipitation, DEAE cellulose, and Sephadex G-75 sequentially (25.83-fold, 7.67%-yield) for biochemical characterization. SDS-PAGE and zymographic activity staining of purified enzyme showed a single band with a predicted molecular mass of about 61 kDa. The optimum temperature and pH for enzyme activity were 30 °C and 7.5, respectively. Additionally, the enzyme exhibited high activity and remarkable stability in 0–10 mM SDS. The values of Km and Vmax for soluble starch as substrate were 2.47 mg/ml and 0.103 mg/ml/min, respectively. Analysis of hydrolysis products of soluble starch and maltooligosaccharides showed that wtAmy175 cleaved the interior and the terminal α-1,4-glycosidic linkage in starch, and had transglycosylation activity. The result of fluorescence spectroscopy showed that wtAmy175 had strong binding affinity with soluble starch. In brief, this study discovered the first wild-type α-amylase so far with several distinctive properties of cold activity, SDS-resistance, and the mixed activity of α-amylase and α-glucosidase, suggesting that wtAmy175 possess high adaptive capability to endure harsh industrial conditions and would be an excellent candidate in detergent and textile industries.

Enhancement of growth and paramylon production of Euglena gracilis by co-cultivation with Pseudoalteromonas sp. MEBiC 03485

This study investigated the putative effects of co-cultivation of Euglena gracilis with Pseudoalteromonas sp. MEBiC 03485 on the growth of E. gracilis and its paramylon production. The strain MEBiC 03485 had beneficial effects on the growth and paramylon contents of E. gracilis. To determine the optimal conditions for co-cultivation, the effects of algal to bacterial inoculum ratios and E. gracilis growth stages were examined. Under optimal conditions, the biomass productivity and paramylon production were increased by more than 23% and 34%, respectively. These effects were attributed to the extracellular polymeric substances (EPS) from the strain MEBiC 03485. GC–MS and HPAEC were carried out to analyze the composition of EPS. It was found that the EPS consisted of rhamnose, galactose, glucose, and mannose. These results suggest a novel approach for potentially enhancing the growth of E. gracilis as well as its paramylon production, via co-culturing with the symbiotic strain MEBiC 03485.

Bromoperoxidase Producing Bacillus spp. Isolated from the Hypobranchial Glands of A Muricid Mollusc Are Capable of Tyrian Purple Precursor Biogenesis

The secondary metabolite Tyrian purple, also known as shellfish purple and royal purple, is a dye with historical importance for humans. The biosynthetic origin of Tyrian purple in Muricidae molluscs is not currently known. A possible role for symbiotic bacteria in the production of tyrindoxyl sulphate, the precursor to Tyrian purple stored in the Australian species, Dicathais orbita, has been proposed. This study aimed to culture bacterial symbionts from the purple producing hypobranchial gland, and screen the isolates for bromoperoxidase genes using molecular methods. The ability of bromoperoxidase positive isolates to produce the brominated indole precursor to Tyrian purple was then established by extraction of the culture, and analysis by liquid chromatography–mass spectrometry (LC–MS). In total, 32 bacterial isolates were cultured from D. orbita hypobranchial glands, using marine agar, marine agar with hypobranchial gland aqueous extracts, blood agar, thiosulphate citrate bile salts sucrose agar, and cetrimide agar at pH 7.2. These included 26 Vibrio spp., two Bacillus spp., one Phaeobacter sp., one Shewanella sp., one Halobacillus sp. and one Pseudoalteromonas sp. The two Bacillus species were the only isolates found to have coding sequences for bromoperoxidase enzymes. LC–MS analysis of the supernatant and cell pellets from the bromoperoxidase producing Bacillus spp. cultured in tryptone broth, supplemented with KBr, confirmed their ability to produce the brominated precursor to Tyrian purple, tyrindoxyl sulphate. This study supports a potential role for symbiotic Bacillus spp. in the biosynthesis of Tyrian purple.

The use ofPseudoalteromonassp. F15 in larviculture of the Yesso scallop,Patinopecten yessoensis

A 49-day rearing trial was conducted to investigate the effects of Pseudoalteromonas sp. F15 on growth, survival, digestive and immune-related enzyme activities in larval and juvenile Yesso scallop, Patinopecten yessoensis. Larvae (Day 2 post-fertilization) (d2 PF)) were provided with a diet only of microalgae (control) and supplemented with F15 at final concentrations of 1×104 and 1×106 cells/mL, respectively. Results indicated that the specific growth rates and survival rates of umbo larvae (d13 PF), crawling pediveliger larvae (d28 PF) and juveniles (d51 PF) fed the F15 supplementation diets at 104 and/or 106 cells/mL were significantly higher than those fed the diet of microalgae. Larvae and juveniles fed 104 and/or 106 cells/mL F15 supplementation diets showed a significant increase in pepsin, amylase and cellulase activities compared with the control group. The lysozyme, superoxide dismutase and catalase activities of larvae and juveniles fed the F15 supplementation diets at 104 and/or 106 cells/mL significantly increased compared to those fed the diet of microalgae. In addition, F15 protected juveniles against challenge with Vibrio splendidus. These data suggest that F15 can be useful in scallop larviculture.

Discovering, Characterizing, and Applying Acyl Homoserine Lactone-Quenching Enzymes to Mitigate Microbe-Associated Problems Under Saline Conditions.

Quorum quenching (QQ) is proposed as a new strategy for mitigating microbe-associated problems (e.g., fouling, biocorrosion). However, most QQ agents reported to date have not been evaluated for their quenching efficacies under conditions representative of seawater desalination plants, cooling towers or marine aquaculture. In this study, bacterial strains were isolated from Saudi Arabian coastal environments and screened for acyl homoserine lactone (AHL)-quenching activities. Five AHL quenching bacterial isolates from the genera Pseudoalteromonas, Pontibacillus, and Altererythrobacter exhibited high AHL-quenching activity at a salinity level of 58 g/L and a pH of 7.8 at 50°C. This result demonstrates the potential use of these QQ bacteria in mitigating microbe-associated problems under saline and alkaline conditions at high (>37°C) temperatures. Further characterizations of the QQ efficacies revealed two bacterial isolates, namely, Pseudoalteromonas sp. L11 and Altererythrobacter sp. S1-5, which could possess enzymatic QQ activity. The genome sequences of L11 and S1-5 with a homologous screening against reported AHL quenching genes suggest the existence of four possible QQ coding genes in each strain. Specifically, two novel AHL enzymes, AiiAS1-5 and EstS1-5 from Altererythrobacter sp. S1-5, both contain signal peptides and exhibit QQ activity over a broad range of pH, salinity, and temperature values. In particular, AiiAS1-5 demonstrated activity against a wide spectrum of AHL molecules. When tested against three bacterial species, namely, Aeromonas hydrophila, Pseudomonas aeruginosa, and Vibrio alginolyticus, AiiAS1-5 was able to inhibit the motility of all three species under saline conditions. The biofilm formation associated with P. aeruginosa was also significantly inhibited by AiiAS1-5. AiiAS1-5 also reduced the quorum sensing-mediated virulence traits of A. hydrophila, P. aeruginosa, and V. alginolyticus during the mid and late exponential phases of cell growth. The enzyme did not impose any detrimental effects on cell growth, suggesting a lower potential for the target bacterium to develop resistance over long-term exposure. Overall, this study suggested that some QQ enzymes obtained from the bacteria that inhabit saline environments under high temperatures have potential applications in the mitigation of microbe-associated problems.

Characterization and DNA methylation modulatory activity of gold nanoparticles synthesized by Pseudoalteromonas strain

Marine extremophiles are shown to tolerate extreme environmental conditions and have high metal reducing properties. Here, we report intracellular synthesis of gold nanoparticles (AuNP) by marine extremophilic bacteria Pseudoalteromonas sp. Bac178 which was isolated from the OMZ of Arabian Sea. Preliminary observations suggest that these bacteria use different pathways which may involves the membrane as well as intracellular proteins for the gold salt reduction. Characterization of the biosynthesised nanoparticles by various techniques such as Scanning electron microscopy (SEM), Transmission electron microscopy (TEM), X-ray diffraction (XRD) and Energy-dispersive X-ray spectroscopy (EDS) confirmed the presence of crystalline gold. These biologically synthesized AuNP were investigated for cytotoxicity and oxidative stress generation in human normal fibroblast and melanoma cells (A375). As AuNP are envisaged to find many applications in the medical field, it was of interest to study the effect of AuNP at the epigenetic level. They were found to be non-cytotoxic, non-genotoxic and non-oxidative stress generating over a range of concentrations. Exposure to these AuNP is observed to cause alterations in global DNA methylation as well as in the expression of DNA methyltransferase (DNMT) genes. Since biosynthesized AuNP are being used in various applications and therapies, their epigenetic modulatory activity needs careful consideration.

Identification and biochemical characterization of a novel exo-type β-agarase Aga3463 from an Antarctic Pseudoalteromonas sp. strain.

An agar-degrading Antarctic bacterium was isolated and identified as Pseudoalteromonas sp. NJ21. This strain showed strong agarolytic activity and could use agar as the sole carbon source for growth. A novel β-agarase gene aga3463 was cloned and identified from the genomic library of Pseudoalteromonas sp. NJ21. It was predicted to encode a peptide of 366 amino acids, including a 21-amino-acid signal peptide. BLAST analysis revealed the deduced amino acid sequence of aga3463 shared less than 36% similarity with any characterized agarase, and phylogenetic analysis showed it belonged to the glycoside hydrolase GH86 family. The recombinant Aga3463 protein had optimal temperature and pH of 50 °C and 7.0, respectively, and retained more than 60% activity from 10 to 50 °C. The metal ions Cd2+, Ca2+, Fe3+, and Mn2+ increased Aga3463 activity, whereas Cu2+, Si2+, Fe2+, and Ni2+ decreased Aga3463 activity. Notably, Aga3463 exhibited good thermostability in the presence of Ca2+; however, Ca2+ was not necessary for its catalytic activity. The Km and Vmax values for Aga3463 were 6.17 mg/ml and 557 U/mg, respectively. Thin layer chromatography, liquid chromatography-mass spectrometry, nuclear magnetic resonance spectroscopy, and enzyme assay using p-nitrophenyl-α/β-d-galactopyranoside revealed Aga3463 as an exo-type β-agarase with the ability to degrade agarose to neoagarobiose as the major end product.

Purification and Characterization of A New Cold-Adapted and Thermo-Tolerant Chitosanase from Marine Bacterium Pseudoalteromonas sp. SY39.

Chitosanases play an important role in chitosan degradation, forming enzymatic degradation products with several biological activities. Although many chitosanases have been discovered and studied, the enzymes with special characteristics are still rather rare. In this study, a new chitosanase, CsnM, with an apparent molecular weight of 28 kDa was purified from the marine bacterium Pseudoalteromonas sp. SY39. CsnM is a cold-adapted enzyme, which shows highest activity at 40 °C and exhibits 30.6% and 49.4% of its maximal activity at 10 and 15 °C, respectively. CsnM is also a thermo-tolerant enzyme that recovers 95.2%, 89.1% and 88.1% of its initial activity after boiling for 5, 10 and 20 min, respectively. Additionally, CsnM is an endo-type chitosanase that yields chitodisaccharide as the main product (69.9% of the total product). It’s cold-adaptation, thermo-tolerance and high chitodisaccharide yield make CsnM a superior candidate for biotechnological application to produce chitooligosaccharides.

Bioactive Compounds of Pseudoalteromonas sp. IBRL PD4.8 Inhibit Growth of Fouling Bacteria and Attenuate Biofilms of Vibrio alginolyticus FB3.

Biofouling is a phenomenon that describes the fouling organisms attached to man-made surfaces immersed in water over a period of time. It has emerged as a chronic problem to the oceanic industries, especially the shipping and aquaculture fields. The metal-containing coatings that have been used for many years to prevent and destroy biofouling are damaging to the ocean and many organisms. Therefore, this calls for the critical need of natural product-based antifoulants as a substitute for its toxic counterparts. In this study, the antibacterial and antibiofilm activities of the bioactive compounds of Pseudoalteromonas sp. IBRL PD4.8 have been investigated against selected fouling bacteria. The crude extract has shown strong antibacterial activity against five fouling bacteria, with inhibition zones ranging from 9.8 to 13.7 mm and minimal inhibitory concentrations of 0.13 to 8.0 mg/ml. Meanwhile, the antibiofilm study has indicated that the extract has attenuated the initial and pre-formed biofilms of Vibrio alginolyticus FB3 by 45.37 ± 4.88% and 29.85 ± 2.56%, respectively. Moreover, micrographs from light and scanning electron microscope have revealed extensive structural damages on the treated biofilms. The active fraction was fractionated with chromatographic methods and liquid chromatography-mass spectroscopy analyses has further disclosed the presence of a polyunsaturated fatty acid 4,7,10,13-hexadecatetraenoic acid (C16H24O2). Therefore, this compound was suggested as a potential bioactive compound contributing to the antibacterial property. In conclusion, Pseudoalteromonas sp. IBRL PD4.8 is a promising source as a natural antifouling agent that can suppress the growth of five fouling bacteria and biofilms of V. alginolyticus FB3.

Effect of Pentacyclic Guanidine Alkaloids from the Sponge Monanchora pulchra on Activity of α-Glycosidases from Marine Bacteria.

The effect of monanchomycalin B, monanhocicidin A, and normonanhocidin A isolated from the Northwest Pacific sample of the sponge Monanchora pulchra was investigated on the activity of α-galactosidase from the marine γ-proteobacterium Pseudoalteromonas sp. KMM 701 (α-PsGal), and α-N-acetylgalactosaminidase from the marine bacterium Arenibacter latericius KMM 426T (α-NaGa). All compounds are slow-binding irreversible inhibitors of α-PsGal, but have no effect on α-NaGa. A competitive inhibitor d-galactose protects α-PsGal against the inactivation. The inactivation rate (kinact) and equilibrium inhibition (Ki) constants of monanchomycalin B, monanchocidin A, and normonanchocidin A were 0.166 ± 0.029 min−1 and 7.70 ± 0.62 μM, 0.08 ± 0.003 min−1 and 15.08 ± 1.60 μM, 0.026 ± 0.000 min−1, and 4.15 ± 0.01 μM, respectively. The 2D-diagrams of α-PsGal complexes with the guanidine alkaloids were constructed with “vessel” and “anchor” parts of the compounds. Two alkaloid binding sites on the molecule of α-PsGal are shown. Carboxyl groups of the catalytic residues Asp451 and Asp516 of the α-PsGal active site interact with amino groups of “anchor” parts of the guanidine alkaloid molecules.

Pseudane-VII Regulates LPS-Induced Neuroinflammation in Brain Microglia Cells through the Inhibition of iNOS Expression.

We previously isolated pseudane-VII from the secondary metabolites of Pseudoalteromonas sp. M2 in marine water, and demonstrated its anti-inflammatory efficacy on macrophages. However, the molecular mechanism by which pseudane-VII suppresses neuroinflammation has not yet been elucidated in brain microglia. Microglia is activated by immunological stimulation or brain injury. Activated microglia secrete proinflammatory mediators which damage neurons. Neuroinflammation appears to be associated with certain neurological diseases, including Parkinson’s disease and Alzheimer’s disease. Natural compounds that suppress microglial inflammatory responses could potentially be used to prevent neurodegenerative diseases or slow their progression. In the present study, we found that pseudane-VII suppresses neuroinflammation in lipopolysaccaride (LPS)-stimulated BV-2 microglial cells and brain. Pseudane-VII was shown to inhibit the LPS-stimulated NO, ROS production and the expression of iNOS and COX-2. To identify the signaling pathway targeted by pseudane-VII, we used western blot analysis to assess the LPS-induced phosphorylation state of p38, ERK1/2, JNK1/2, and nuclear factor-kappaB (NF-κB). We found that pseudane-VII attenuated LPS-induced phosphorylation of MAPK and NF-κB. Moreover, administration of pseudane-VII in mice significantly reduced LPS-induced iNOS expression and microglia activation in brain. Taken together, our findings suggest that pseudane-VII may represent a potential novel target for treatment for neurodegenerative diseases.

On-board saline black water treatment by bioaugmentation original marine bacteria with Pseudoalteromonas sp. SCSE709-6 and the associated microbial community

To reduce fresh water load on ships, seawater can be used for toilet flushing on-board. And saline black water was treated on-site by bioaugmentation original marine bacteria with Pseudoalteromonas sp. SCSE709-6 (P. sp. SCSE709-6) to prevent marine pollution. In the batch experiments, P. sp. SCSE709-6 was effective in nutrient removal, which was not closely related to the amount of inoculation. In the on-board continuous experiments, the systems inoculated with P. sp. SCSE709-6 possessed excellent TP removal ability (removal rate: 80.93% for T3 and 88.39% for T4). The inoculum of P. sp. SCSE709-6 changed the microbial community structure and increased the similarity of microbial communities. P. sp. SCSE709-6 had a significant influence on the performance and microbial community of the systems. This study strongly proposes that the P. sp. SCSE709-6 is a promising alternative in saline black water treatment, which has great significance to the practice of on-board seawater flushing toilet.

Biodegradation of metal complex Naphthol Green B and formation of iron–sulfur nanoparticles by marine bacterium Pseudoalteromonas sp CF10-13

Treatment of metal complex dye wastewater has attracted growing attention due to the degradation-resistant, high cost and potential hazards of current techniques. This study reported a marine bacterium (Pseudoalteromonas sp CF10-13) with potential performance in decolorization and degradation of a metal complex dye—Naphthol Green B (NGB) at wide ranges of salinity, dye concentration and alkalinity under anaerobic conditions. It was inferred that the secretion of electron mediators in soluble extracellular metabolites by P. sp CF10-13 played important roles in NGB decolorization and degradation through extracellular electron transfer. Naphthalenesulfonate, the major structure in NGB molecule, was further degraded into low-toxic benzamide. Black stable iron–sulfur nanoparticles were formed endogenously avoiding H2S releasing, exogenous sulfur addition and metal sludge in accumulation. Accordingly, this study provided a cost-effective and eco-friendly biodegradation method to refractory NGB, further promoting the understanding of dye resources recovery.

Rapid and efficient protocol to introduce exogenous DNA in Vibrio harveyi and Pseudoalteromonas sp.

Vibrio campbellii BAA-1116 is renowned for its bioluminescence properties, and genetic tools are available to genetically track this strain. However, many other ecologically important V. harveyi strains exist, for which only few genetic tools are available. In this study, a rapid electroporation protocol was developed to transform replicative plasmids in various environmental V. harveyi and Pseudoalteromonas strains. Moreover, a mini-Tn7 delivery system was modified to site-specifically integrate mini-transposons in the genome of V. harveyi ORM4. As a proof-of-principle, replicative plasmids carrying bioreporters were introduced by electroporation in V. harveyi ORM4 cells, and gene expression was followed at the single cell level. We could demonstrate that a flagellar gene is subjected to bimodal gene expression in V. harveyi ORM4, being highly expressed in 10% of the population in stationary phase. This study extends the possibilities to study environmental Vibrio strains and uncovers the occurrence of phenotypic heterogeneity in flagellar expression in Vibrio.

A Novel Cold-Adapted Leucine Dehydrogenase from Antarctic Sea-Ice Bacterium Pseudoalteromonas sp. ANT178.

l-tert-leucine and its derivatives are useful as pharmaceutical active ingredients, in which leucine dehydrogenase (LeuDH) is the key enzyme in their enzymatic conversions. In the present study, a novel cold-adapted LeuDH, psleudh, was cloned from psychrotrophic bacteria Pseudoalteromonas sp. ANT178, which was isolated from Antarctic sea-ice. Bioinformatics analysis of the gene psleudh showed that the gene was 1209 bp in length and coded for a 42.6 kDa protein containing 402 amino acids. PsLeuDH had conserved Phe binding site and NAD+ binding site, and belonged to a member of the Glu/Leu/Phe/Val dehydrogenase family. Homology modeling analysis results suggested that PsLeuDH exhibited more glycine residues, reduced proline residues, and arginine residues, which might be responsible for its catalytic efficiency at low temperature. The recombinant PsLeuDH (rPsLeuDH) was purified a major band with the high specific activity of 275.13 U/mg using a Ni-NTA affinity chromatography. The optimum temperature and pH for rPsLeuDH activity were 30 °C and pH 9.0, respectively. Importantly, rPsLeuDH retained at least 40% of its maximum activity even at 0 °C. Moreover, the activity of rPsLeuDH was the highest in the presence of 2.0 M NaCl. Substrate specificity and kinetic studies of rPsLeuDH demonstrated that l-leucine was the most suitable substrate, and the catalytic activity at low temperatures was ensured by maintaining a high kcat value. The results of the current study would provide insight into Antarctic sea-ice bacterium LeuDH, and the unique properties of rPsLeuDH make it a promising candidate as a biocatalyst in medical and pharmaceutical industries.

Characterization of Properties and Transglycosylation Abilities of Recombinant α-Galactosidase from Cold-Adapted Marine Bacterium Pseudoalteromonas KMM 701 and Its C494N and D451A Mutants.

A novel wild-type recombinant cold-active α-d-galactosidase (α-PsGal) from the cold-adapted marine bacterium Pseudoalteromonas sp. KMM 701, and its mutants D451A and C494N, were studied in terms of their structural, physicochemical, and catalytic properties. Homology models of the three-dimensional α-PsGal structure, its active center, and complexes with D-galactose were constructed for identification of functionally important amino acid residues in the active site of the enzyme, using the crystal structure of the α-galactosidase from Lactobacillus acidophilus as a template. The circular dichroism spectra of the wild α-PsGal and mutant C494N were approximately identical. The C494N mutation decreased the efficiency of retaining the affinity of the enzyme to standard p-nitrophenyl-α-galactopiranoside (pNP-α-Gal). Thin-layer chromatography, matrix-assisted laser desorption/ionization mass spectrometry, and nuclear magnetic resonance spectroscopy methods were used to identify transglycosylation products in reaction mixtures. α-PsGal possessed a narrow acceptor specificity. Fructose, xylose, fucose, and glucose were inactive as acceptors in the transglycosylation reaction. α-PsGal synthesized -α(1→6)- and -α(1→4)-linked galactobiosides from melibiose as well as -α(1→6)- and -α(1→3)-linked p-nitrophenyl-digalactosides (Gal2-pNP) from pNP-α-Gal. The D451A mutation in the active center completely inactivated the enzyme. However, the substitution of C494N discontinued the Gal-α(1→3)-Gal-pNP synthesis and increased the Gal-α(1→4)-Gal yield compared to Gal-α(1→6)-Gal-pNP.

Heterologous expression of an agarase gene in Bacillus subtilis, and characterization of the agarase

A β-agarase was identified from Pseudoalteromonas sp. Q30F and heterologously expressed in Bacillus subtilis WB800n. The β-agarase, Aga862 encoded by aga862 gene in an open reading frame of 1338 bp is 445 amino acids in length, and has a calculated molecular mass of 50.1 kDa and an estimated isoelectric point of 4.81. Protein sequence analysis showed that Aga862 belongs to family 16 of glycoside hydrolases (GH16) and carbohydrate-binding module family 13 (CBM13). The agarase was expressed in B. subtilis WB800n and purified by precipitation, anion exchange and gel filtration for a specific activity of 4.6 U/mg, a 27.8-fold improvement over the activity of the crude enzyme. Aga862 exhibited optimal activity at 45 °C and pH 6.5, and showed excellent pH stability with retention of over 80% relative activities after preincubation for the pH range of 3.0–10.0 at 4 °C for 3 h. The agarase exhibited a Km value of 14.15 mg/mL toward agarose and a Vmax of 256.41 U/mg. The mass spectrometry analysis revealed that the end products of agar degradation were neoagarotetraose and neoagarohexaose. Recombinant Aga862 has great potential for the manufacture of agaro-oligosaccharides for the non-pathogenic nature and safety of the B. subtilis WB800n.

Expression and characterization of a bifunctional alginate lyase named Al163 from the Antarctic bacterium Pseudoalteromonas sp. NJ-21

In this study, an endolytic alginate lyase, named Al163, was identified, cloned, and characterized from the Antarctic bacterium Pseudoalteromonas sp. NJ-21. Comparative sequence analysis showed that the predicted amino acid sequence encoded by al163 belongs to the polysaccharide lyase 6 (PL-6) family and has a molecular mass of about 80 kDa. Recombinant enzyme was purified by Ni-Sepharose affinity chromatography. Recombinant Al163 exhibited maximum activity (258 U/mg) at pH 7.0 and 40℃, and thermal stability assays showed retention of almost 90% activity after incubation at 30℃ for 30 min. Al163 activity was stimulated by Cd2+, Ca2+, Fe 3+, and Mn2+, but inhibited by Cu2+, Si2+, Fe2+, and Ni2+. Thin-layer chromatographic analysis indicated that Al163 degraded sodium alginate, polyM, and polyG, generating disaccharides and trisaccharides as the final products. Only a few bacterial strains that produce a bifunctional alginate lyase have been reported. Our results indicate that recombinant Al163 exhibits broad substrate specificity and its products exhibit low degrees of polymerization. Both properties imply high potential for use of the enzyme in several industrial fields, including cosmetics and pharmaceuticals, based on the high demand for biologically active oligosaccharides.

Survival improvement conferred by the Pseudoalteromonas sp. NC201 probiotic in Litopenaeus stylirostris exposed to Vibrio nigripulchritudo infection and salinity stress

This study aimed to investigate the potential protection conferred by the probiotic strain NC201 against biotic and abiotic stresses in Pacific blue shrimp Litopenaeus stylirostris that had received the probiotic throughout their lives. The presence of NC201 in shrimp hemolymph was investigated over the course of 24 h, before exposure to bacterial or physical stress. Results showed that NC201 had invaded the shrimp hemolymph 2 h following administration, but had completely disappeared by 48 h. NC201 identification through morphotype observation was confirmed by MALDI-TOF biotyping and results also indicated that NC201 and Pseudoalteromonas piscicida are closely related. A challenge by immersion was carried out on subadults using Vibrio nigripulchritudo at 105 CFU/mL. Cumulative mortality was two-fold lower in the treated group (24%) than in the control group (48%) at 144 h post infection. The probiotic in the shrimp hemolymph was diminished in infectious conditions compared with non-infectious ones and V. nigripulchritudo prevalence was simultaneously lower in animals treated with NC201. The relative expression of genes coding lysozyme and penaeidin 3 was evaluated 24 h post infection and their transcript numbers were found to be lower in probiotic animals than in control animals for both genes. Hyposaline stress was also used to evaluate the benefits of NC201 treatment on early juveniles and subadults. At low salinities, animals showed an increased survival rate when treated with NC201, by 10 and 17.5% at 48 h post stress, respectively. Moreover, in subadults treated with the probiotic, a better recovery of the plasmatic osmolality was observed. All these results confirm that NC201 is a good candidate probiotic for shrimp aquaculture.