A VIBRIO ANGUILIARUM STRAIN ASSOCIATED WITH SKIN ULCER ON CULTURED FLOUNDER, PARALICHTHYS OLIVACEUS

The characteristics of a bacterium strain M3, isolated from cultured flounderParalichthys olivaceus with remarkable external sign of skin ulcer during an epizootic outbreak, indicated that the bacterium belonged to the speciesVibrio anguillarum. Challenge by I.M. (intramuscular injection), bath, and oral administration with M3 showed that it was highly pathogenic forParalichthys olivacues. The LD₅₀ dose was 5.144×10³ CFU/ per fish infection by I.M. injection. Recovered inoculated bacteria from the surviving fish revealed that the asymptomatic carriers could be a latent contagious source. Study of the effect of bacterial culture CFS (cell-free-supernatant) showed that the exotoxins produced by M3 play an important role in its pathogenicity for flounder. The resistance of M3 to 36 out of 41 antibiotics indicated that the bacterial disease outbreak was mainly attributable to the frequent and excessive use of antimicrobial agents; and that vaccination would be an effective precaution against bacterial disease. Contribution No. 4107 from Institute of Oceanology, Chinese Academy of Science. Contribution No. 270 from Experimental marine Biology Laboratory, CAS. Funded by projects under the Major State Basic Research Development Program, Grant G1999012003.     

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.     

Identification and characterization of the Vibrio anguillarum prtV gene encoding a new metalloprotease

We cloned and sequenced a prtV-like gene from Vibrio anguillarum M3 strain. This prtV gene encodes a putative protein of 918 amino acids, and is highly homologous to the V. cholerae prtV gene. We found that a prtV insertion mutant strain displayed lower gelatinase activity on gelatin agar, lower protease activity against azocasein, and lower activity for four glycosidases. This prtV mutant strain also had increased activity for two esterases in its extracellular products, as analyzed by the API ZYM system. In addition, the prtV mutant strain exhibited decreased growth in turbot intestinal mucus and reduced hemolytic activity on turbot erythrocytes. Infection experiments showed that the LD₅₀ of the prtV mutant strain increased by at least 1 log compared to the wild-type in turbot fish. We propose that prtV plays an important role in the pathogenesis of V. anguillarum.     

Optimum production and characterization of an acid protease from marine yeast <Emphasis Type="Italic">Metschnikowia reukaufii</Emphasis> W6b

The marine yeast strain W6b isolated from sediment of the South China Sea was found to produce a cell-bound acid protease. The crude acid protease produced by this marine yeast showed the highest activity at pH 3.5 and 40 °C. The optimal pH and temperature for the crude acid protease were in agreement with those for acid protease produced by the terrestrial yeasts. The optimal medium of the acid protease production was seawater containing 1.0% glucose, 1.5% casein, and 0.5% yeast extract, and the optimal cultivation conditions of the acid protease production were pH 4.0, a temperature of 25 °C and a shaking speed of 140 rmin⁻¹. Under the optimal conditions, 72.5 UmL⁻¹ of acid protease activity could be obtained in cell suspension within 48 h of fermentation at shake flask level. The acid protease production was induced by high-molecular-weight nitrogen sources and repressed by low-molecular-weight nitrogen sources. Skimmed-milk-clotting test showed that the crude acid protease from the cell suspension of the yeast W6b had high skimmed milk coagulability. The acid protease produced by M. reukaufii W6b may have highly potential applications in cheese, food and fermentation industries.     

Oxygen consumption and ammonia-N excretion of Meretrix meretrix in different temperature and salinity

Effects of temperatures and salinities on oxygen consumption and ammonia-N excretion rate of clamMeretrix meretrix were studied in laboratory from Oct. 2003 to Jan. 2004. Two schemes were designed in incremented temperature at 10, 15, 20, 25°C at 31.5 salinity and in incremented salinity at 16.0, 21.0, 26.0, 31.5, 36.0, and 41.0 at 20°C, all for 8–10 days. From 10 to 25°C, both respiration and excretion rate were increased. One-way ANOVA analysis demonstrated significant difference (P<0.01) in physiological parameters in this temperature range except between 15 and 20°C. The highestQ ₁₀ thermal coefficient value (12.27) was acquired between 10 and 15°C, and about 1 between 15 and 20°C, indicatingM. meretrix could well acclimate to temperature changes in this range. Salinity also had significant effects on respiration and excretion rate (P<0.05). The highest values of respiration and excretion rate ofM. meretrix were recorded at 16.0 salinity (20°C). These two physiological parameters decreased as salinity increased until reached the minimumQ ₁₀ value at 31.5 (20°C), then again, these parameters increased with increasing salinity from 31.5 to 41.0.M. meretrix can catabolize body protein to cope with osmotic pressure stress when environmental salinity is away from its optimal range. No significant difference was observed between 26.0 and 36.0 in salinity (P>0.05), suggesting that a best metabolic salinity range for this species is between 26.0 and 36.0. This work is supported by National High-Tech R & D Program of China. (863 Program) (2002AA603014).     

An extracellular oligopeptide permease may be a potential virulence factor of Vibrio harveyi

An oligopeptide permease A(OppA)was purified from the extracellular product of Vibrio harveyi SF-1.The molecular weight of the purified protein was estimated to be 58 kDa on SDS-PAGE.The purified protein showed phospholipase C activity at the optimal values of temperature 50℃ and pH 8.0.The enzymatic activity decreased when the temperature increased to 40℃.The N-terminal sequence of the purified protein was determined as ADVPAGTKLA,which is similar to that of OppA.The OppA pre-cursor gene was cloned from th...     

Molecular Cloning and Expression ofPoIR2, a Novel Gene Involved in Immune Response in Japanese Flounder （Paralichthys olivaceus）

A novel immune-related gene was expressed in Japanese flounder (Paralichthys olivaceus) injected with Vibrio anguillarum. The complete cDNA contained a 169 bp 5’UTR, a 336 bp open reading frame (ORF) encoding 111 amino acids and a 556bp 3’UTR. Six exons and five introns were identified in the PoIR2 gene. Blastp similarity comparison showed its encoding protein had 50% similarity to Danio rerio neuromedin S (NMS), but further alignment indicated they did not have NMS C-terminal conservational signature domain. So it was not defined as an NMS homologue. Protein structure analysis indicated it had a 26aa signal peptide and was a secretory pathway protein. RT-PCR demonstrated that the expression of PoIR2 was quickly induced and drastically increased in liver, kidney, spleen, gills, intestine, heart, and skeletal muscle after infected with V. anguillarum. These results indicated that the PoIR2 might play some important role in Japanese flounder immune response system. This gene was named PoIR2 (P.olivaceus immune-related gene 2, GenBank accession number: EU224372). The mature PoIR2 peptide was expressed in BL21(DE3) pLysS using pET-32a(+) vector and a great part of the recombinant mature peptide existed as soluble type.     

Rapid, sensitive detection of Vibrio anguillarum using loop-mediated isothermal amplification

Vibrio anguillarum is an important bacterial pathogen of aquatic organisms and a significant problem in aquatic farming. The rapid detection and identification of V. anguillarum, and other pathogens that infect marine organisms, is crucial to effective disease management. In this study, we developed a loop-mediated amplification (LAMP) assay to detect V. anguillarum in an hour in a single tube without the need for thermal cycling. Conserved regions of the metalloproteinase (empA) gene of V. anguillarum served as the targets for primer design. A fragment of the empA gene was amplified at 65°C in the presence of the primer mixture and Bst DNA polymerase. In the optimized LAMP assay, 6.7 pg of V. anguillarum DNA could be detected. Six strains of V. anguillarum and 17 strains of non-V. anguillarum bacteria were used in this study to evaluate the species specificity of the primers. The six V. anguillarum strains gave a positive result in the LAMP assay. This method was also validated in V. anguillarum-infected fish. This LAMP method is more sensitive than PCR in the detection of V. anguillarum and shows good species specificity. The LAMP assay is therefore an effective method for the quick detection of V. anguillarum both in the laboratory and in the field.     

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.     

Alkaline protease production by a strain of marine yeasts

Yeast strain 10 with high yield of protease was isolated from sediments of saltern near Qingdao, China. The protease had the highest activity at pH 9.0 and 45℃. The optimal medium for the maximum alkaline protease production of strain 10 was 2.5 g soluble starch and 2.0 g NaNO3 in 100 mL seawater with initial pH6.0. The optimal cultivation conditions for the maximum protease production were temperature 24.5 ℃, aeration rate 8.0 L min^- 1 and agitation speed 150 r min^-1 . Under the optimal conditions, 623.1 Umg^-1 protein of alkaline protease was reached in the culture within 30 h of fermentation.     

Competition Among Dinoflagellate Alexandrium Tam—arense, Raphidophyte Heterosigma Carterae and Diatom Skeletonema Costatum under Combinations ofTwo Temperatures and Five Salinities

Competition among HAB (Harmful Algal Bloom) species Dinoflagellate Alexandrium tamarense, Raphidophyte Heterosigma carterae, and Diatom Skeletonema costatum was studied in the laboratory. Experiments with these three major HAB species under combinations of different salinities (10, 18, 25, 30, 35) and temperatures (19℃, 25℃) were carried out. The results showed that S. costatum successfully competed with the other two species at salinities of 18, 25,30, and 35 at temperatures of 19℃ and 25℃. However, H. carterae showed its advantage at low salinity of 10 and became the single dominant species at salinity 10 and 25℃. A. tamarense could not compete successfully with the other two species especially at low salinities. However, it could remain at low density in the presence of higher densities of other algae.     

The selection of alkaline protease-producing yeasts fi＇om marine environments and evaluation of their bioactive peptide production

A total of 400 yeast strains from seawater, sediments, saltern mud, marine fish guts, and marine algae were obtained. The protease activity of the yeast cultures was estimated, after which four strains (HN3.11, N11b, YF04C and HN4.9) capable of secreting extracellular alkaline protease were isolated. The isolated strains were identified as Aureobasidium pullulans, Yarrowia lipolytica, Issatchenkia orientalis and Cryptococcus cf. aureus. The optimal pH of the protease activity produced by strains HN3.11, YF04C, and HN4.9 was 9.0, while that of the protease produced by strain N11b was 10.0. The optimal temperature for protease activity was 45°C for strains HN3.11, N11b, and YF04C, and 50°C for strain HN4.9. After digestion of shrimp (Penaeus vannamei) protein and spirulina (Arthospira platensis) protein with the four crude alkaline proteases, the filtrate from spirulina (Arthrospira platensis) powder digested by the crude alkaline protease of strain HN3.11 was found to have the highest antioxidant activity (61.4%) and the highest angiotensin I converting enzyme (ACE)-inhibitory activities (68.4%). The other filtrates had much lower antioxidant activity and ACE-inhibitory activities.     

Isolation of protoplasts from <Emphasis Type="Italic">undaria pinnatifida</Emphasis> by alginate lyase digestion

The aim of this study is to isolate protoplasts from Undaria pinnatifida. Protoplasts of the alga were isolated enzymatically by using alginate lyase, which was prepared by fermenting culture of a strain Vibrio sp. 510. Monofacterial method was applied for optimizing digestion condition. The optimum condition for protoplast preparation is enzymatic digestion at 28°C for 2h using alginate lyase at the concentration of 213.36 U (8 mL) every 0.5g fresh thalline with NaCl 50 and at the shaking speed of 150 r min⁻¹ during digestion. The protoplast yield can reach 2.62±0.09 million per 0.5 g fresh leave under the optimum condition. The enzyme activity is inhibited by Ca²⁺ and slightly enhanced by Fe²⁺ and Mn²⁺ at concentrations of 0.05, 0.08 and 0.10 mol L⁻¹.     

Physiological responses of Chinese longsnout catfish to water temperature

We evaluated the effect of water temperature on the growth and physiology of the Chinese longsnout catfish (Leiocassis longirostris Günther). The fish were reared at four temperatures (20, 25, 30, and 35°C) and sampled on days 7, 20, and 30. We measured plasma levels of insulin, free thyroxine (FT4), free 3,5,3′-triiodothyronine (FT3), lysozyme and leukocyte phagocytic activity. The optimum water temperature for growth was 27.7°C. The plasma levels of insulin and FT4 declined significantly (P<0.05) on day 30 at temperatures above 20°C. Lysozyme activity was significantly (P<0.05) lower at 25°C than at other temperatures. We conclude that final weight, insulin, FT4, and lysozyme were significantly affected by water temperature.     

Study on the Tripolyphosphatase （TPPase） Property of Bighead Carp （Aristichthys nobilis） Myosin Subfragment-1

Myosin subfragment-1 was prepared from the myofibrils of bighead carp (Aristichthys nobilis). The myosin subfrag- ment-1 was proved to have the activity of tripolyphosphatase (TPPase) responding to the hydrolysis of sodium tripolyphosphate (STPP). The optimum temperature and pH for the TPPase of myosin subfragment-1 were 30℃ and pH 5.0, and at pH 8.0 the TPPase also showed a high activity. Mg2 was necessary to TPPase. The TPPase activity of myosin subfragment-1 was activated by Mg2 under low concentrations, but was inhibited when the concentration was over 17 mmolL-1. The TPPase activity was also affected by KCl. The optimum concentration of KCl for TPPase was 0.3 molL-1 under the condition of 17 mmolL-1 Mg2 . The TPPase activity was significantly inhibited by EDTA-Na2. Reagents such as KBr, KI and KIO3 could inhibit the TPPase effectively. K2Cr2O7 as well as KMnO7 and KNO3 exhibited weak inhibiting effects. The TPPase converted STPP to pyrophosphate (PP) and orthophosphate (Pi) stoichiometrically with a KM of 3.2 mmolL-1.     

Effects of Body Weight and Water Temperature on Maximum Food Consumption of Juvenile Sebastodes fuscescens （Houttuyn）

Maximum rate of food consumption ( Cmax ) was determined for juvenile Sebastodes fuscescens (Houttuyn) at water temperature of 10, 15, 20 and 25℃. The relationships of Cmax to the body weight (W) at each temperature were described by a power equation: lnCmax = a b lnW. Covariance analysis revealed significant interaction of the temperature and body weight. The relationship of adjusted Cmax to water temperature (T) was described by a quadratic equation: Cmax = -0.369 0.456T - 0.0117T^2. The optimal feeding temperature calculated from this equation was 19.5 ℃. The coefficients of the multiple regression estimation relating Cmax to body weight (W) and water temperature (T) were given in the Table 2.     

Response of Yesso scallop Patinopecten yessoensis to acute temperature challenge: physiological and biochemical parameters

Water temperature is generally considered to be a major factor affecting the physiological and biochemical activities of marine bivalves. Here, the physiological and biochemical responses of Yesso scallop, Patinopecten yessoensis, to acute water temperature changes in summer were studied. Scallops were transferred directly to a lower temperature (T_dec treatment) (from 23°C to 15°C) or to a higher temperature (T_dec treatment) (from 15°C to 23°C) for 72 h, respectively. Results showed that the oxygen consumption and ammonia-N excretion rates of P. yessoensis decreased significantly in the T_dec treatment but increased dramatically at 6 h in the T_dec treatment (P <0.05). In the T dec treatment, hepatopancreas antioxidant enzyme activities, superoxide dismutase (SOD) and catalase (CAT) activities, increased substantially within 72 h (P <0.05). However, a significant decrease in CAT activity was found at 12 h in the T_dec treatment (P <0.01). A significant enhancement of acid phosphatase (ACP) activity and malondialdehyde (MDA) content was detected when scallops were acutely exposed to a temperature of 15°C. The levels of Cu/Zn-SOD gene expression in their gills up-regulated significantly in response to acute temperature changes (P <0.01). These data suggest that acute temperature change affects physiological and biochemical functions, and improve our knowledge of P. yessoensis under conditions of thermal stress.

     

Anti-oxidant and anti-inflammatory activities of ultrasonic-assistant extracted polyphenol-rich compounds from Sargassum muticum

Polypehnol is an important, potentially bioactive component of Sargassum muticum. In this study, ultrasonic assisted extraction of polyphenol-rich substances was performed using a 38% ethanol solution at a solid:liquid ratio of 1:30 at 68℃ for 32 min, determined by single-factor and response surface methodology(RSM) optimization. The content of polyphenol was 5.66 mg/g in the crude extract. Further extraction showed that the polyphenol mainly distributed in ethyl acetate(SKEE) and water phases(SKEW).The anti-oxidation test by electron spin resonance(ESR) spectrum showed that the SKEE had the strongest scavenging activity on DPPH(1,1-diphenyl-2-picrylhydrazyl) and alkyl radicals. SKEE was shown noncytotoxic but could inhibit the generation of cellular ROS, showing protective effects in H_2O_2 and AAPHinduced Vero cells and UV-B irradiated HaCaT cells. SKEE also significantly inhibited the release of NO of LPS-induced RAW 264.7 cells. Therefore, the polyphenol-rich extracts in ethanol and ethyl acetate showed excellent anti-oxidant and anti-inflammatory activities, which is beneficial to the development of high-value bio-substances.     

Purification and characterization of a new thermostable κ-carrageenase from the marine bacterium Pseudoalteromonas sp. QY203

A new extracellular κ-carrageenase, namely CgkP, 34.0 kDa in molecular weight, was purified from Pseudoalteromonas sp. QY203. CgkP showed relatively high activity at acidities ranging from pH6.0 to pH9.0 and temperatures ranging from 30℃ to 50℃ with the highest activity at 45℃ and pH7.2. Sodium chloride increased its activity markedly, and KCl increased its activity slightly. The divalent and trivalent metal ions including Cu2+ , Ni2+ , Zn2+ , Mn2+ , Al3+ and Fe3+ significantly inhibited its activity, while Mg2+ did not. CgkP remained 70% of original activity after being incubated at 40℃ for 48 h, and remained 80% of the activity after being incubated at 45℃ for 1 h. It exhibited endo-κ-carrageenase activity, mainly depolymerizing the κ-carrageenan into disaccharide and tetrasaccharide. CgkP was more thermostable than most of previously reported κ-carrageenases with a potential of being used in industry.     

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.