Antifouling potential of bacteria isolated from a marine biofilm

Marine microorganisms are a new source of natural antifouling compounds. In this study, two bacterial strains, Kytococcus sedentarius QDG-B506 and Bacillus cereus QDG-B509, were isolated from a marine biofilm and identified. The bacteria fermentation broth could exert inhibitory effects on the growth of Skeletonema costatum and barnacle larvae. A procedure was employed to extract and identify the antifouling compounds. Firstly, a toxicity test was conducted by graduated pH and liquid-liquid extraction to determine the optimal extraction conditions. The best extraction conditions were found to be pH 2 and 100% petroleum ether. The EC50 value of the crude extract of K. sedentarius against the test microalgae was 236.7 ± 14.08 μg mL-1, and that of B. cereus was 290.6 ± 27.11 μg mL-1. Secondly, HLB SPE columns were used to purify the two crude extracts. After purification, the antifouling activities of the two extracts significantly increased: the EC50 of the K. sedentarius extract against the test microalgae was 86.4 ± 3.71 μg mL-1, and that of B. cereus was 92.6 ± 1.47 μg mL-1. These results suggest that the metabolites produced by the two bacterial strains are with high antifouling activities and they should be fatty acid compounds. Lastly, GC-MS was used for the structural elucidation of the compounds. The results show that the antifouling compounds produced by the two bacterial strains are myristic, palmitic and octadecanoic acids.     

Purification and characterization of iron-cofactored superoxide dismutase from Enteromorpha linza

A superoxide dismutase was purified from Enteromorpha linza using a simple and safe procedure, which comprised phosphate buffer extraction, ammonium sulphate precipitation, ion exchange chromatography on Q-sepharose column, and gel filtration chromatography on Superdex 200 10/300GL. The E. linza superoxide dismutase （E/SOD） was purified 103.6-fold, and a yield of 19.1% and a specific activity of 1 750 U/rag protein were obtained. The SDS-PAGE exhibited E/SOD a single band near 23 kDa and the gel filtration study showed E/SOD＇s molecular weight is near 46 kDa in nondenatured condition, indicating it＇s a homodimeric protein. E/SOD is an iron-cofactored superoxide dismutase （Fe-SOD） because it was inhibited by hydrogen peroxide, insensitive to potassium cyanide. The optimal temperature for its maximal enzyme activity was 35℃, and it still had 29.8% relative activity at 0℃, then E/SOD can be classified as a cold-adapted enzyme. E/SOD was stable when temperature was below 40℃ or the pH was within the range of 5 10. The first 11 N-terminal amino acids orE/SOD were ALELKAPPYEL, comparison of its N-terminal sequence with other Fe-SOD N-terminal sequences at the same position suggests it is possibly a chloroplastic Fe-SOD.     

Purification and characterization of an alkaline protease from Acetes chinensis

An alkaline protease from Acetes chinensis was purified and characterized in this study. The steps of purification include ammonium sulfate precipitation, ion-exchange chromatography with Q-sepharose Fast Flow, gel filtration chromatography with S300 and the second ion-exchange chromatography with Q-sepharose Fast Flow. The protease was isolated and purified, which was present and active on protein substrates (azocasein and casein). The specific protease activity was 17.15 folds and the recovery was 4.67. The molecular weight of the protease was estimated at 23.2 kD by SDS-PAGE. With azocasein as the susbstrate, the optimal temperature was 55°C and the optimal pH value was 5.5. Ion Ca²⁺ could enhance the proteolytic activity of the protease, while Cu²⁺, EDTA and PMSF could inhibit its activity.     

Purification and characterization of novel κ-carrageenase from marine Tamlana sp. HC4

We isolated a bacterial strain (HC4) that is able to degrade κ-carrageenan from a live specimen of the red alga Hyalosiphonia caespitosa. With 16S rRNA gene sequencing, we identified the strain as Tamlana sp., and then purified an extracellular κ-carrageenase from a culture of Tamlana sp. HC4 by ammonium sulfate precipitation, Sephadex G-200 gel filtration chromatography, and DE-cellulose 52 anion-exchange chromatography. The purified enzyme yields a single band on SDS-PAGE with a molecular mass of 66.4 kDa. The optimal pH and temperature for κ-carrageenase activity are at 8.0 and 30°C, respectively. The enzyme is stable over the range of pH 7.2–8.6 below 45°C. The enzyme activity is strongly inhibited by Zn²⁺ and Cu²⁺ at 1 mmol/L. The enzyme-catalyzed reaction follows Michaelis-Menten kinetics with the Michaelis constant (K _m ) at 7.63 mg/ml. Analysis of the degradation products of the κ-carrageenase by ESI-MS and ¹³C-NMR spectroscopy indicates that the enzyme degrades κ-carrageenan down to the level of κ-neocarrabiose sulfate.     

Inhibitory activity of an extract from a marine bacterium Halomonas sp. HSB07 against the red-tide microalga Gymnodinium sp. （Pyrrophyta）

In recent years, red tides occurred frequently in coastal areas worldwide. Various methods based on the use of clay, copper sulfate, and bacteria have been successful in controlling red tides to some extent. As a new defensive agent, marine microorganisms are important sources of compounds with potent inhibitory bioactivities against red-tide microalgae, such as Gymnodinium sp. （Pyrrophyta）. In this study, we isolated a marine bacterium, HSB07, from seawater collected from Hongsha Bay, Sanya, South China Sea. Based on its 16S rRNA gene sequence and biochemical characteristics, the isolated strain HSB07 was identified as a member of the genus Halomonas. A crude ethyl acetate extract of strain HSB07 showed moderate inhibition activity against Gymnodinium sp. in a bioactive prescreening experiment. The extract was further separated into fractions A, B, and C by silica gel column chromatography. Fractions B and C showed strong inhibition activities against Gymnodinium. This is the first report of inhibitory activity of secondary metabolites of a Halomonas bacterium against a red-tide-causing microalga.     

Purification and identification of a clotting protein from the hemolymph of Chinese shrimp (Fenneropenaeus chinensis)

The clotting protein (CP) plays important and diverse roles in crustaceans, such as coagulation and lipid transportation. A clotting protein was purified from the hemolymph of Chinese shrimp Fenneropenaeus chinensis (named as Fc-CP) with Q sepharose HP anion-exchange chromatography and phenyl sepharose HP hydrophobic interaction chromatography. Fc-CP was able to form stable clots in vitro in the presence of hemocyte lysate and Ca²⁺, suggesting that the clotting reaction is catalyzed by a Ca²⁺-dependent transglutaminase in shrimp hemocytes. The molecular mass of Fc-CP was 380 kDa under non-reducing conditions and 190 kDa under reducing conditions as was determined with SDS-PAGE. CP exists as disulfide-linked homodimers and oligomers. The N-terminal amino acid sequence of Fc-CP was identical to that of shrimps including Penaeus monodon, Farfantepenaeus paulensis and Litopenaeus vannamei; and similar to that of other decapods. The purified Fc-CP was digested with trypsin and verified on an ABI 4700 matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry. Our results will aid to better understanding the coagulation mechanism of shrimp hemolymph.     

Purification and some properties of Fe protein of nitrogenase from

The Fe protein ofAnabaena cylindrica was first separated and purified by chromatography through DEAE-cellulose columns then by gel electrophoresis. The specific activity was up to 142.46 nmol C₂H₄/mg protein · min. It was homogeneous as shown by 1) a single band in the gel electrophorogram; 2) absence of Mo and tryptophan; 3) content of about 3.4 atoms of Fe per mole protein. The molecular weight of the Fe protein ofA. cylindrica was about 61,000 daltons as estimated by SDS-gel electrophoresis and calculated from the amino acid composition. The residues of aspartate and glutamate were about 2.6 times that of arginine and lysine in the Fe protein. Crossing Fe protein ofA. cylindrica with Mo?Fe protein ofAzotobacter vinelandii gave positive result. The reciprocal crossing also showed activity.     

Purification and characterization of an alkaline protease from <Emphasis Type="Italic">Micrococcus</Emphasis> sp. isolated from the South China Sea

Protease is wildly used in various fields, such as food, medicine, washing, leather, cosmetics and other industrial fields. In this study, an alkaline protease secreted by Micrococcus NH54PC02 isolated from the South China Sea was purified and characterized. The growth curve and enzyme activity curve indicated that the cell reached a maximum concentration at the 30th hour and the enzyme activity reached the maximum value at the 36th hour. The protease was purified with 3 steps involving ammonium sulfate precipitation, ion-exchange chromatography and hydrophobic chromatography with 8.22-fold increase in specific activity and 23.68% increase in the recovery. The molecular mass of the protease was estimated to be 25 kDa by SDS-PAGE analysis. The optimum temperature and pH for the protease activity were 50°C and pH 10.0, respectively. The protease showed a strong stability in a wide range of pH values ranging from 6.0–11.0, and maintained 90% enzyme activity in strong alkaline environment with pH 11.0. Inhibitor trials indicated that the protease might be serine protease. But it also possessed the characteristic of metalloprotease as it could be strongly inhibited by EDTA and strongly stimulated by Mn²⁺. Evaluation of matrix-assisted laser desorption ionization/time-of-flight MS (MALDI-TOF-TOF/MS) showed that the protease might belong to the peptidase S8 family.     

Optimization of the purification methods for recovery of recombinant growth hormone from Paralichthys olivaceus

This study aimed to optimize the purification of recombinant growth hormone from Paralichthys olivaceus. Recombinant flounder growth hormone (r-fGH) was expressed by Escherichia coli in form of inclusion body or as soluble protein under different inducing conditions. The inclusion body was renatured using two recovery methods, i.e., dilution and dialysis. Thereafter, the refolded protein was purified by Glutathione Sepharase 4B affinity chromatography and r-fGH was obtained by cleavage of thrombin. For soluble products, r-fGH was directly purified from the lysates by Glutathione Sepharase 4B affinity chromatography. ELISA-receptor assay demonstrated that despite its low receptor binding activity, the r-fGH purified from refolded inclusion body had a higher yield (2.605 mgL-1) than that from soluble protein (1.964 mgL-1). Of the tested recovery methods, addition of renaturing buffer (pH 8.5) into denatured inclusion body yielded the best recovery rate (17.9%). This work provided an optimized purification method for high recovery of r-fGH, thus contributing to the application of r-fGH to aquaculture.     

Heterotrophic bacterial floras in industrial area and unpolluted marine environments around Qingdao

From Oct. 1999 to Oct. 2000, the heterotrophic bacterial floras in the industrial marine environment around the Qingdao Power Plant (QPP) and in the unpolluted marine environments were investigated. The results showed that the numbers of the heterotrophic bacteria around QPP were much higher than those in unpolluted environments, and the average numbers in QPP Seawater, QPP Sediment, Unpolluted Seawater and Unpolluted Sediment were 5.4×10⁴cfu(mL)⁻¹, 5.0×10⁵cfug⁻¹, 3.0×10²cfu(mL)⁻¹ and 1.3×10⁵cfug⁻¹ respectively. Totally, 118 strains were isolated from QPP and 99 of them were Gram-negative. One hundred and twenty one strains were isolated from the unpolluted environments and 104 of them were Gram-negative. All the Gram-negative bacteria belonged to 13 genera. The distribution of the bacteria was varied in different marine environments. The results showed that the unpolluted marine environments contained much more Vibrio than seawater and sediment around QPP.     

Purification and Characterization of a Novel Lipase from Antarctic Krill

Lipase from Antarctic krill,with a molecular weight of 71.27kDa,was purified with ammonium sulfate precipitation and a series of chromatographic separations over ion exchange(DEAE)and gel filtration columns(Sephacryl S-100),resulting in 5.2%recovery with a 22.4-fold purification ratio.The optimal pH and temperature for enzyme activity were 8.0 and 45℃,respectively.Purified lipase had Km and Vmax values of 3.27mmolL−1 and 2.4Umg−1,respectively,using p-nitrophenyl laurate as the substrate.Lipase activity was enhanced by adding Ca2+and Mg2+ions in the concentration ranges of 0–0.5mmolL−1 and 0–0.3mmolL−1,respectively,while the activity was inhibited by a further increase in these ion concentrations.Fe3+and Cu2+ions showed obvious inhibitory effects on enzyme activity,and the inhibition rates were 71.8%and 53.3%when the ion concentrations were 0.5mmolL−1.     

Purification and characterization of cold-active endo-1,4-β-glucanase produced by Pseudoalteromonas sp. AN545 from Antarctica

A bacterium hydrolyzing carboxymethylcellulose, isolated from Antarctic sea ice, was identified as Pseudoalteromonas sp. based on 16S rDNA gene sequences and named as Pseudoalteromonas sp. AN545. The extracellular endo-1,4-β-glucanase AN-1 was purified successively by ammonium sulfate precipitation, DEAE-Sepharose ion exchange chromatography and Sephadex G-75 gel filtration chromatography. The molecular mass of AN-1 was estimated to be 47.5 kDa utilizing SDS-PAGE and gel chromatography analysis. AN-1 could hydrolyze caboxymethylcellulose, avicel and β-glucan, but not cellobiose, xylan and p-Nitrophenyl-β-D-glucopyranoside. The optimal temperature and pH for the β-glucanase activity of AN-1 were determined to be at 30°C and pH 6.0, respectively. AN-1 was stable at acidic solutions of pH 5.0-6.5 and temperatures below 30°C for 1 h. Moreover, the specific activity was enhanced by Ca2+ and Mg2+, and inhibited by Cu2+. The kinetic parameters Michaelis constant (Km) and maximum velocity (Vmax) of AN-1 were 3.96 mg/mL and 6.06×10-2 mg/(min·mL), respectively.     

Studies on the community structures of meiofauna and marine nematode at six stations in the Southern Yellow Sea,China

1Introduction As part of a comprehensive investigation of ecosys-tem dynamics in the Yellow Sea and East China Sea,astudy has been made of meiofauna,defined here asmetazoans passing a0.5mm sieve but retained by a0.031mm sieve.Meiofauna is an important energeticgroup in benthic small food web due to their smallsize,high abundance and fast turnover rates.Theproduction of meiofauna is equal to or higher than thatof macrofauna in estuaries,shallow waters and deepseas(Gerlach,1971;Platt and Warw…     

De-eutrophication of effluent wastewater from fish aquaculture by using marine green alga Ulva pertusa

The de-eutrophication abilities and characteristics of Ulva pertusa, a marine green alga, were investigated in Qingdao Yihai Hatchery Center from spring to summer in 2005 by analyzing the dynamic changes in NH ₄ ⁺ , NO ₃ ^? , NO ₂ ^? as well as the total dissolved inorganic nitrogen (DIN). The results show that the effluent wastewater produced by fish aquaculture had typical eutrophication levels with an average of 34.3 μmol L^?1 DIN. This level far exceeded the level IV quality of the national seawater standard and could easily lead to phytoplankton blooms in nature if discarded with no treatment. The de-eutrophication abilities of U. pertusa varied greatly and depended mainly on the original eutrophic level the U. pertusa material was derived from. U. pertusa used to living in low DIN conditions had poor DIN removal abilities, while materials cultured in DIN-enriched seawater showed strong de-eutrophication abilities. In other words, the de-eutrophication ability of U. pertusa was evidently induced by high DIN levels. The de-eutrophication capacity of U. pertusa seemed to also be light dependent, because it was weaker in darkness than under illumination. However, no further improvement in the de-eutrophication capacity of U. pertusa was observed once the light intensity exceeded 300 μmol M² S^?1. Results of semi-continuous wastewater replacement experiments showed that U. pertusa permanently absorbed nutrients from eutrophicated wastewater at a mean rate of 299 mg/kg fresh weight per day (126 mg/kg DIN during the night, 173 mg/kg in daytime). Based on the above results, engineered de-eutrophication of wastewater by using a U. pertusa filter system seems feasible. The algal quantity required to purify all the eutrophicated outflow wastewater from the Qingdao Yihai Hatchery Center into oligotrophic level I clean seawater was also estimated using the daily discharged wastewater, the average DIN concentration released and the de-eutrophication capacity of U. pertusa.     

Optimization of ultrasonic extraction and clean-up protocol for the determination of polycyclic aromatic hydrocarbons in marine sediments by high-performance liquid chromatography coupled with fluorescence detection

The procedures of ultrasonic extraction and clean-up were optimized for the determination of polycyclic aromatic hydrocarbons (PAHs) in marine sediments. Samples were ultrasonically extracted, and the extracts were purified with a miniaturized silica gel chromatographic column and analyzed with high performance liquid chromatography (HPLC) with a fluorescence detector. Ultrasonication with methanol-dichloromethane (2:1, v/v) mixture gave higher extraction efficiency than that with dichloromethane. Among the three elution solvents used in clean-up step, dichloromethane-hexane (2:3, v/v) mixture was the most satisfactory. Under the optimized conditions, the recoveries in the range of 54.82% to 94.70% with RSDs of 3.02% to 23.22% for a spiked blank, and in the range of 61.20% to 127.08% with RSDs of 7.61% to 26.93% for a spiked matrix, were obtained for the 15 PAHs studied, while the recoveries for a NIST standard reference SRM 1941b were in the range of 50.79% to 83.78% with RSDs of 5.24% to 21.38%. The detection limits were between 0.75 ng L-1 and 10.99 ng L-1for different PAHs. A sample from the Jiaozhou Bay area was examined to test the established methods.     

Cloning,expression and characterization of a lipase gene from marine bacterium <Emphasis Type="Italic">Pseudoalteromonas lipolytica</Emphasis> SCSIO 04301

A lipase gene, lip1233, isolated from Pseudoalteromonas lipolytica SCSIO 04301, was cloned and expressed in E. coli. The enzyme comprised 810 amino acid residues with a deduced molecular weight of 80 kDa. Lip1233 was grouped into the lipase family X because it contained a highly conserved motif GHSLG. The recombinant enzyme was purified with Ni-NTA affinity chromatography. The optimal temperature and pH value of Lip1233 were 45°C and 8.0, respectively. It retained more than 70% of original activity after being incubated in pH ranging from 6.0 to 9.5 for 30 min. It was stable when the temperature was below 45°C, but was unstable when the temperature was above 55°C. Most metal ions tested had no significant effect on the activity of Lip1233. Lip1233 remained more than original activity in some organic solvents at the concentration of 30% (v/v). It retained more than 30% activity after incubated in pure organic solvents for 12 h, while in hexane the activity was nearly 100%. Additionally, Lip1233 exhibited typical halotolerant characteristic as it was active under 4M NaCl. Lip1233 powder could catalyze efficiently the synthesis of fructose esters in hexane at 40°C. These characteristics demonstrated that Lip1233 is applicable to elaborate food processing and organic synthesis.     

Isolation and characterization of a fucoidan-degrading bacterium from Laminaria japonica

Fucoidan, a polysaccharide containing abundant fucose and sulfate ester group, was prepared from Laminaria japonica. In order to obtain fucoidan-degrading enzyme, bacteria capable of degrading fucoidan were screened from kelp. A bacterial strain named RC2-3 was obtained, which degraded fucoidan by the maximum extent of 54% ± 1.3%, the highest among all bacterial isolates. High-performance size exclusion chromatography(HPSEC) showed that the molecular weight of fucoidan was gradually reduced by RC2-3 with culturing time, suggesting the production of fucoidan-degrading enzyme by RC2-3. Phylogenetic analysis of partial 16S ribosomal RNA gene(16S rDNA) sequence showed that RC2-3 belonged to the family Flavobacteriaceae. However, it showed different physiological and biochemical characteristics from the known Flavobacteriaceae members producing fucoidan-degrading enzyme, thus RC2-3 was proposed to be a new member of this family.     

The auxin concentration in sixteen Chinese marine algae

1 INTRODUCTION Auxins are plant hormones. They play impor- tant roles in regulating plant activities, including phototropism, fruit development, root initiation and so on. In early 1940, Thirmann and Scoog (1940) found that auxin was present in more than …     

Determination of Selenium in Marine Aquatic Products by Hydride Generation-atomic Fluorescence Spectrometry （HG-AFS）

A method for the analysis of selenium in marine aquatic products by HG-AFS has been investigated. The method is based on the reduction of inorganic selenium to volatile SeH2 which is bubbled out by carrier gas of pure argon, and then swept to Ar-H2 flame quarts atomizer to measure its fluorescence intensity. The hydride generation, transportation, atomization and some instrumental parameters were studied by a kind of orthogonal design. The optimum conditions selected are as follows： reactive acidity, 20% HC1; the amount of NaBH4, 4.9mL; gas flow of argon, 600mLmin^-1; atomizing temperature, 200 ℃ ; negative high voltage, - 300V; light current, 100 mA; integral time, 7s. The detection limit of the presented method is 0.072μgL^-1 for selenium. The calibration curve shows a satisfactory line inthe concentration range from 0.000 to 1.000μgL^-1 Se. The recovery is 95.8%-102.2%.     

Screening of marine fungus from Nanji Island and activity of their metabolites against pathogenic Vibrio from Pseudosciaena crocea

Seventy-eight marine fungal strains were isolated from sediment samples collected off the coast of Nanji Island,Wenzhou,Zhejiang Province,China.Antibacterial screening using the agar disc method showed that 19 of the isolated strains could inhibit at least one pathogenic Vibrio from Pseudosciaena crocea.Subsequent screening confirmed that nine strains produced antibacterial metabolites that had activity against one or several types of pathogenic Vibrio.Strain NJ0104 had the widest antimicrobial spectrum and strong activity,particularly against Vibrio parahaemolyticus-MM0810072.A preliminary study of NJ0104 antibacterial metabolites demonstrated that they had thermal stability up to 80°C,ultraviolet stability up to 40 min and pH stability between 4.0-7.0.In addition,the antibacterial metabolites were readily soluble in butanol.To identify the specific strain,the ITS-5.8S rDNA regions of NJ0104 were PCR amplified and sequenced.Based on the combination of phenotypic and genotypic data,the strain was identified as Arthrinium sp.