Expression and enzymatic characterization of a cold-adapted β-agarase from Antarctic bacterium Pseudoalteromonas sp.NJ21

An agar-degrading bacterium,designated as Pseudoalteromonas sp. NJ21,was isolated from an Antarctic sediment sample. The agarase gene a ga1161 from Pseudoalteromonas sp. NJ21 consisting of a 2 382-bp coding region was cloned. The gene encodes a 793-amino acids protein and was found to possess characteristic features of the Glyco_hydro_42 family. The recombinant agarase(r Aga1161) was overexpressed in Escherichia coli and purified as a fusion protein. Enzyme activity analysis revealed that the optimum temperature and pH for the purified recombinant agarase were 30–40°C and 8.0,respectively. rAga1161 was found to maintain as much as 80% of its maximum activity at 10°C,which is typical of a coldadapted enzyme. The pattern of agar hydrolysis demonstrated that the enzyme is an β-agarase,producing neoagarobiose(NA2) as the final main product. Furthermore,this work is the first proof of an agarolytic activity in Antarctic bacteria and these results indicate the potential for the Antarctic agarase as a catalyst in medicine,food and cosmetic industries.     

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.     

Cloning and Characterization of a New κ-Carrageenase Gene from Marine Bacterium Pseudoalteromonas sp. QY203

κ-carrageenan oligosaccharides exhibit various biological activities. Enzymatic degradation by κ-carrageenase is safe and controllable. Therefore, κ-carrageenases have captured more and more attentions. In this study, a κ-carrageenase encoding gene, cgk X, was cloned from Pseudoalteromonas sp. QY203 with degenerate and inverse PCR. It comprised an ORF of 1194 bp in length, encoding a protein with 397 amino acid residues. Cgk X is a new member of glycoside hydrolase family 16. The deduced amino acid sequence shared a high similarity with Cgk X of Pseudoalteromonas κ-carrageenase; however, the recombinant Cgk X showed different biochemical characteristics. The recombinant enzyme was most active at p H 7.0 and 55℃ in the presence of 300 mmol L~(~(-1))Na Cl. It was stable in a broad range of acidity ranging from p H 3.0 to p H 10.0 when temperature was below 40℃. More than 80% of its activity was maintained after being incubated at p H 3.6–10.0 and 4℃ for 24 h. Cgk X retained more than 90% of activity after being incubated at 40℃ for 1 h. EDTA and SDS(1 mmol L~(-1)) did not inhibit its activity. Cgk X hydrolyzed κ-carrageenan into disaccharide and tetrasaccharide as an endo-cleaver. All these characteristics demonstrated that Cgk X is applicable to both κ-carrageenan oligosaccharide production and κ-carrageenase structure-function research.     

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.     

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.     

Screening and characterization of the alkaline protease isolated from PLI-1, a strain of Brevibacillus sp. collected from Indonesia’s hot springs

A total of 69 strains of thermophilic bacteria were isolated from water, soil and sediment samples from three Indonesia’s hot spring areas (Pantai cermin, Kalianda and Banyu wedang) by using Minimal Synthetic Medium (MSM). The extreme thermophile Brevibacillus sp. PLI-1 was found to produce extracellular thermophilic alkaline protease with optimal activity at 70℃ and pH 8.0-9.0. The molecular weight of the protease was estimated to be around 56 kD by SDS-PAGE. The maximum activity of the protease was 26.54 U mL-1. The protease activity did not decrease after 30 min and still retained more than 70% of relative activity after 60 min at 70℃ and pH 8.0. The ion Mg2+ was found to promote protease activity at both low and high concentrations, whereas Cu2+ and Zn2+ could almost completely inhibit the activity. Divalent cation chelator EDTA inhibited the enzyme activity by 55.06% ± 0.27%, while the inhibition caused by PMSF, Leupeptin, Pepstain A and Benzamidine were 66.78% ± 3.25%, 52.37% ± 0.25%, 62.47% ± 2.96% and 50.99% ± 0.24%, respectively. Based on these observations, the enzyme activity was conspicuously sensitive to the serine and cysteine protease inhibitors. All these results indicated that the protease isolated from the strain PLI-1 was a thermophilic protease and had a high-temperature stability and a pH stability.     

Purification and Refolding of a Novel β-Agarase from Inclusion Body of E. coli

β-agarase AgaB appears to represent a new family of glycoside hydrolase; it is structurally and functionally different from other known agarases. In the present study, AgaB was expressed with a temperature-inducible expression system in E. coli BL21 (DE3) as a fusion protein bearing a C-terminal hexahistidine tag. The protein existed mainly in the form of inclusion body.After being washed and solubilized, AgaB in inclusion body was denatured and purified to electrophoretic purity by immobilized metal affinity chromatography. The purified AgaB was then refolded using a simple pulse dilution method, and the refolded AgaB showed a high specific hydrolysis activity of about 1600 units/mg protein. Forty milligrams of refolded pure protein were obtained from 1L of culture.     

Purification and Refolding of a Novel β-Agarase from Inclusion Body of E. coli

1 Introduction Agarases produced by agar-degrading bacteria are classified into two groups based on their modes of action, namely, α-agarase and β-agarase. They hydrolyze α -1, 3 linkage and β-1, 4 linkage of agarose respectively (Duckworth and Turvey, 1969). Agarases are widely used in food, cosmetic and medical industries for the produc-tion of oligosaccharides from agar (Kobayashi et al., 1997; Yoshizawa et al., 1995). Neoagaro-oligo- saccharides produced by β-agarase inhibit the gro…     

Eualus heterodactylus sp. nov., a new hippolytid shrimp from Chinese coast of the Yellow Sea （Crustacea,Decapoda, Caridea）

A new species from the caridean family Hippolytidae, Eualus heterodactylus sp. nov. is described and illustrated based on the specimens collected from Chinese coast of the Yellow Sea. The new species is a part of an informal species group characterized by the possession of epipods on the anterior three pairs of pereopods, and is distinguished from other species of this group by the dactyli of the third to fifth pereopods possessing distinctly stair-like flexor margins in males.     

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 Apostichopusjaponicus.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 CFUg-1 diet of BC228 was significantly higher than that of those fed control diet （P〈0.05）.In addition,105 and 107 CFUg-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 NB 13 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.     

Effects of heavy metals （Pb^2＋ and Cd^2＋） on the ultrastructure, growth and pigment contents of the unicellular cyanobacterium Synechocystis sp. PCC 6803

The unicellular cyanobacterium Synechocystis sp. PCC 6803, a model organism known for its unique combination of highly desirable molecular genetic, physiological and morphological characteristics, was employed in the present study. The species was cultured in BG11 liquid medium contained various initial concentrations of Pb²⁺ and Cd²⁺ (0, 0.5, 1, 2, 4, 6 and 8 mg/L). The experiment was conducted for six days and the metal induced alterations in the ultrastructure, growth and pigment contents were assessed. Alterations in the ultrastructure of the Synechocystis sp. PCC 6803 cells became evident with the increased (>4 mg/L Pb²⁺) metal concentration. The photosynthetic apparatus (thylakoid membranes) were found to be the worst affected. Deteriorated or completely destroyed thylakoid membranes have made large empty spaces in the cell interior. In addition, at the highest concentration (8 mg/L Pb²⁺), the polyphosphate granules became more prominent both in size and number. Despite the initial slight stimulations (0.2, 3.8 and 6.5% respectively at 0.5, 1 and 2 mg/L Pb²⁺), both metals inhibited the growth in a dose-dependent manner as incubation progressed. Pigment contents (chlorophyll α, β carotene and phycocyanin) were also decreased with increasing metal concentration. Cells exposed to 6 mg/L Pb²⁺, resulted in 36.56, 37.39 and 29.34% reductions of chlorophyll α, β carotene and phycocyanin respectively over the control. Corresponding reductions for the same Cd²⁺concentrations were 57.83, 48.94 and 56.90%. Lethal concentration (96 h LC₅₀) values (3.47 mg/L Cd²⁺ and 12.11 mg/L Pb²⁺) indicated that Synechocystis sp. PCC 6803 is more vulnerable to Cd²⁺ than Pb²⁺. Supported by the Chinese Scholarship Council