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 Cd^(2+), Ca^(2+), Fe^(3+), and Mn^(2+), but inhibited by Cu^(2+), Si^(2+), Fe^(2+), and Ni^(2+). Thin-layer chromatographic analysis indicated that Al163 degraded sodium alginate, poly M, and poly G, 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.

Production and characterization of an extracellular polysaccharide of antarctic marine bacteria Pseudoalteromonas sp. S-15-13

Twenty-seven antarctic bacteria producing extracellular polysaccharide （EPS） were selected from 57 strains by staining technology. The effects of major environmental factors on the growth and EPS production of Pseudoalteromonas sp. S - 15 - 13 were investigated, and the EPS was separated and purified for characterization analysis. The results showed that the optimal conditions for the EPS production were culture period, 56 h; growth temperature, 8 ℃ ; carbon source, 1.0% glucose; NaCI concentration, 3.0% ; pH 6.0 - 7.0. The EPS was purified by cold ethanol precipitation, proteins removal, ion exchange chromatography and gel chromatography technology. The molecular mass of EPS - H was 62 kDa as determined by the high performance gel permeation chromatography. Its sugar composition was a homopolymer of marmose analyzed by gas chromatograph spectroscopy. After repeated freezing and thawing of the bacteria hiomass in the presence of EPS, the bacterial growth was much higher than that observed after freezing in the absence of EPS and the difference augmented with the increase of freeze-thaw cycles. It is hypothesized that the adaptation of Pseudoalteromonas sp. S- 15 - 13 to the antarctic marine conditions, characterized by low temperature, high NaCl concentration and repeated freeze-thaw cycles, might be related to the EPS production ability.

Gene cloning and sequence analysis of the cold-adapted chaperones DnaK and DnaJ from deep-sea psychrotrophic bacterium Pseudoalteromonas sp. SM9913

Pseudoalteromonas sp. SM9913 is a phychrotmphic bacterium isolated from the deep-sea sediment. The genes encoding chaperones DnaJ and DnaK of P. sp. SM9913 were cloned by normal PCR and TAIL - PCR （GenBank accession Nos DQ640312, DQ504163 ）. The chaperones DnaJ and DnaK from the strain SM9913 contain such conserved domains as those of many other bacteria, and show some cold-adapted characteristics in their structures when compared with those from psychro-, meso-and themophilic bacteria. It is indicated that chaperones DnaJ and DnaK of P. sp. SM9913 may be adapted to low temperature in deep-sea and function well in assisting folding, assembling and translocation of proteins at low temperature. This research lays a foundation for the further study on the cold-adapted mechanism of chaperones DnaJ and DnaK of cold-adapted microorganisms.

Isolation, chemical characteristics and immunity activity of an extracellular polysaccharide EPS I isolated from Antarctic bacterium Pseudoalteromonas sp.S-15-13

A new extracelluar polysaccharide （EPS） was isolated and purified from Antarctic bacterium S-15-13, identified as Pseudoalteromonas sp. After being separated and purified by DEAE-Sephadex A-50 ionexchange and Sephadex G-100 gel chromatography, two mains fractions （EPS I and EPS Ⅱ ） were ob-tained. EPS I was composed of mannose, glucose and galactose with a molecular weight of 23kDa and EPS Ⅱ was composed of mannose only with a molecular weight of 62kDa. The effect of the polysaccharide EPS I on the cellular immune response of mice was investigated. Results demonstrated that EPS I could markedly facilitate lymphocyte proliferation, and might be a strong immunomodulator.

Effects of Pseudoalteromonas sp. BC228 on Digestive Enzyme Activity and Immune Response of Juvenile Sea Cucumber(Apostichopus japonicus)

A marine bacterium, Pseudoalteromonas sp. BC228 was supplemented to feed in a feeding experiment aiming to determine its ability of enhancing the digestive enzyme activity and immune response of juvenile Apostichopus japonicus. Sea cucumber individuals were fed with the diets containing 0(control), 105, 107 and 109 CFU g-1 diet of BC228 for 45 days. Results showed that intestinal trypsin and lipase activities were significantly enhanced by 107 and 109 CFU g-1 diet of BC228 in comparison with control(P < 0.01). The phagocytic activity in the coelomocytes of sea cucumber fed the diet supplemented with 107 CFU g-1 diet of BC228 was significantly higher than that of those fed control diet(P < 0.05). In addition, 105 and 107 CFU g-1 diet of BC228 significantly enhanced lysozyme and phenoloxidase activities in the coelomic fluid of sea cucumber, respectively, in comparison with other diets(P < 0.01). Sea cucumbers, 10 each diet, were challenged with Vibrio splendidus NB13 after 45 days of feeding. It was found that the cumulative incidence and mortality of sea cucumber fed with BC228 containing diets were lower than those of animals fed control diet. Our findings evidenced that BC228 supplemented in diets improved the digestive enzyme activity of juvenile sea cucumber, stimulated its immune response and enhanced its resistance to the infection of V. splendidus.

Characterization of Biofilm Forming Marine Pseudoalteromonas spp

Biofilm forming bacteria are omnipresent in the marine environment.Pseudoalteromonas is one of the largest within the γ-proteobacteria class,and a member of marine bacteria.Species of Pseudoalteromonas are generally found in association with marine eukaryotes.In this work,we present the isolation and characterization of two strains forming biofilm on rock surface and associated with marine sponge.They were identified using 16SrDNA as Pseudoalteromonas prydzensis alex,and Pseudoalteromonas sp.alex.They showed the highest titer in biofilm formation quantified using the test tube method using crystal violet.

Agarase Production by Marine Pseudoalteromonas sp.MHS:Optimization,and Purification

Agar is an essential polysaccharide that has been utilized in numerous fields.Many kinds of literature have been published regarding agarolytic microorganisms’isolation and agarases biochemical studies.In this search,a local marine agarolytic bacterium associated with marine alga Ulva lactuca surface was isolated and identified as Pseudoalteromonas sp.MHS.The agarase production was parallel to the growth of Pseudoalteromonas sp.MHS as cells displayed a lag phase(2 h),subsequently an exponential growth that prolonged till 10 h where maximum growth(OD550nm=3.9)was achieved.The enzyme activity increased rapidly as cells increased exponentially where the maximum activity of 0.22 U/mL was achieved after 8h and remained constant till 12 h during the stationary phase of growth.Agarase production was optimized using Plackett-Burman statistical design by measuring enzyme activity as a response and the design was validated using a verification experiment;the activity of the enzyme increased from 0.22 U/mL to 0.29 U/mL.Pseudoalteromonas sp.MHS agarase was partially purified and its molecular weight(MW)was determined by SDS-PAGE(15-25 kDa).Agarase showed approximately 94% of its activity at 40℃.The enzyme stability decreased as the temperature increased;the enzyme could retain about 98,90,80,75,and 60% of its activity at 20,30,40,50,and 60℃,respectively.Biomass of the red alga Pterocladia capillacea proved to be a suitable substrate for agarase production using Pseudoalteromonas sp.MHS;the enzyme activity recorded after 24 h of incubation was 0.35 U/mL compared to 0.29 U/mL from the optimized medium.

Biofilm Formation by Marine Cobetia marina alex and Pseudoaltero­monas spp: Development and Detection of Quorum Sensing N-Acyl Homoserine Lactones (AHLs) Molecules

Surfaces submerged in seawater are colonized by various microorganisms,resulting in the formation of heterogenic marine biofilms.This work aims to evaluate the biofilm formation by Cobetia marina alex and doing a comparative study between this promising strain with the two bacterial strains isolated previously from the Mediterranean seawater,Alexandria,Egypt.Three strains;Cobetia marina alex,Pseudoalteromonas sp.alex,and Pseudoalteromonas prydzensis alex were screened for biofilm formation using the crystal violet(CV)quantification method in a single culture.The values of biofilm formed were OD600=3.0,2.7,and 2.6,respectively leading to their selection for further evaluation.However,factors affecting biofilm formation by C.marina alex were investigated.Biofilm formation was evaluated in single and multispecies consortia.Synergistic and antagonistic interactions proved in this work lead to the belief that these bacteria have the capability to produce some interesting signal molecules N-acyl Homoserine Lactones(AHLs).

Screening and Identification of a Bacterial Strain Antagonistic to Vibrio parahaemolyticus

In the present study, bacterial strains were isolated from the shrimp （Penaeus orientalis） ponds in different regions of Hainan Province, and tested for their antagonistic activity against Vibrio species on 2216E plates, using V. parahaemolyticus, V. harveyi and V. anguillarum as the indicator bacteria. As a result, an antagonistic strain was screened out and numbered 20160522Z-10 （referred to as Z-10）. Then, the antibacterial activity and minimum inhibitory concentration （MIG） of Z-10 against the indicator bacteria, and its antibacterial effect against other six Vibrio species were determined. The results proved that Z-10 was antagonistic to all the three indicator bacteria, and its MIC against V. parahaemolyticus, V. harveyi and V. anguillarum was 2.5× 10^4, 2.5× 10^5 and 2.5× 10^5 CFU/ml. Additionally, Z-10 had a certain antibacterial effect against other Vibrio species. Finally, the strain was identified as a Pseudoalteromonas sp. by 16S rDNA sequence analysis. The bacterium as a new probiotic, is expected to be used in disease prevention and control in marine aquaculture.