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 2-Haloacid Dehalogenasefrom Marine Bacterium Paracoccus sp. DEH99, Isolatedfrom Marine Sponge Hymeniacidonperlevis

2-haloacid dehalogenases constitute a group of dehalogenases which are capable of dehalogenating the halogenatedorganic compounds. So far, the 2-haloacid dehalogenases have been found in many bacteria, but not in Paracoccus genus. In thepresent study, one enzyme 2-haloacid dehalogenase （designated as Deh99）, induced by DL-2-chloropropionate （DL-2-CPA）, waspurified from the marine bacterium Paracoccus sp. DEH99, isolated from marine sponge Hymeniacidon perlevis. The enzyme ofDeh99 was purified to homogeneity by ammonium sulfate precipitation, ion exchange chromatography （Q-Sepharose HP）, and Su-perdex 200 gel filtration chromatography. The molecular weight of Deh99 was estimated to be 25.0 kDa by sodium dodecyl sul-fate-polyacrylamide gel electrophoresis （SDS-PAGE）, and 50.0 kDa natively by gel filtration chromatography. The enzyme of Deh99stereospecifically dehalogenated L-2-CPA to produce D-lactate, with an apparent Michaelis-Menten constant （Kin） value of0（21 mmolL-1 for L-2-CPA. The optimal pH and temperature for Deh99 activity were 10.0 and 40°C, respectively. The enzyme ofDeh99 acted on short-carbon-chain 2-haloacids, with the highest activity towards monochloroacetate. The activity of Deh99 wasslightly affected by DTT and EDTA, but strongly inhibited by Cu2＋ and Zn2＋. The enzyme of Deh99 shows unique substrate specific-ity and inhibitor sensitivities compared to previously characterized 2-haloacid dehalogenases and is the reported one about purified 2-haloacid dehalogenase isolated from the bacteria of Paracoccus genus.     

Purification and characterization of 2-haloacid dehalogenase from marine bacterium Paracoccus sp. DEH99, isolated from marine sponge Hymeniacidon perlevis

2-haloacid dehalogenases constitute a group of dehalogenases which are capable of dehalogenating the halogenated organic compounds. So far, the 2-haloacid dehalogenases have been found in many bacteria, but not in Paracoccus genus. In the present study, one enzyme 2-haloacid dehalogenase(designated as Deh99), induced by DL-2-chloropropionate(DL-2-CPA), was purified from the marine bacterium Paracoccus sp. DEH99, isolated from marine sponge Hymeniacidon perlevis. The enzyme of Deh99 was purified to homogeneity by ammonium sulfate precipitation, ion exchange chromatography(Q-Sepharose HP), and Superdex 200 gel filtration chromatography. The molecular weight of Deh99 was estimated to be 25.0 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), and 50.0 kDa natively by gel filtration chromatography. The enzyme of Deh99 stereospecifically dehalogenated L-2-CPA to produce D-lactate, with an apparent Michaelis-Menten constant(Km) value of 0.21 mmol L-1 for L-2-CPA. The optimal pH and temperature for Deh99 activity were 10.0 and 40℃, respectively. The enzyme of Deh99 acted on short-carbon-chain 2-haloacids, with the highest activity towards monochloroacetate. The activity of Deh99 was slightly affected by DTT and EDTA, but strongly inhibited by Cu2+ and Zn2+. The enzyme of Deh99 shows unique substrate specificity and inhibitor sensitivities compared to previously characterized 2-haloacid dehalogenases and is the reported one about purified 2-haloacid dehalogenase isolated from the bacteria of Paracoccus genus.     

Purification and Characterization of an Alkaline Protease from Micrococcus 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 30 th hour and the enzyme activity reached the maximum value at the 36 th 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 k Da by SDS-PAGE analysis.The optimum temperature and p H for the protease activity were 50℃ 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 p H 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 Mn~(2+).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.     

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 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.     

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 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 23.2kD by SDS-PAGE. With azocasein as the susbstrate, the optimal temperature was 55℃ and the optimal pH value was 5.5. Ion Ca^2＋ could enhance the proteolytic activity of the protease, while Cu^2＋, EDTA and PMSF could inhibit its activity.     

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.     

PURIFICATION AND SOME PROPERTIES OF Fe PROTEIN OF NITROGENASE FROM Anabaena cylindrica

The Fe protein of Anabaena 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 ofMo and tryptophan ;3)content of about 3.4 atoms ofFe per mole protein.The molecular weight of the Fe protein of A . 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 of A . cylindrica with Mo-Fe protein of Azotobacter vinelandii gave positive result. The reciprocal crossing also showed activity.     

Purification and Characterization of a Novel Hydrolase That Can Specifically Degrade the Polysaccharide Isolated from Green Seaweed Ulva prolifera

The extracellular polysaccharide hydrolase-producing strain EP-1 was isolated from seawater and identified as Paenibacillus pabuli. Furthermore, a homogeneous extracellular polysaccharide hydrolase from Paenibacillus pabuli EP-1 was purified by combining ion-exchange chromatography and size exclusion chromatography with a purification fold of 90.69 and recovery of 16.23%. Characterization of the purified polysaccharide hydrolase revealed a molecular mass of 38 k Da and optimum activity at 45℃ and pH 6.0. The polysaccharide hydrolase maintained its stability within a wide range of pH(3.0–12.0) and thermal stability when the temperature was below 50℃. The presence of Hg~(2+), Fe~(2+), Mn~(2+), Co~(2+) and SDS notably decreased hydrolase activity, and organic solvents such as formaldehyde, acetone, DMF and acetonitrile completely inhibited hydrolase activity. The purified hydrolase had no activity on agar, carrageenan, gellan gum, sodium alginate, or starch, but effectively hydrolyzed the polysaccharide from Ulva prolifera. The Km and Vmax values of this hydrolase were 43.84 mg m L~(-1) and 4.33 mg m L~(-1) min~(-1), respectively. The sequence analysis with quantitative time-of-flight mass spectrometry indicated that the hydrolase was an endoglucanase.     

Optimization of sample pretreatment for determination of polycyclic aromatic hydrocarbons in estuarine sediments by gas chromatography

This study examined levels of polycyclic aromatic hydrocarbons (PAHs) in estuarine sediments in Licun (Qingdao, China) by gas chromatography under optimized conditions for sample pretreatment via ultrasonic extraction, column chromatography, and thin layer chromatography. Methanol and dichloromethane (DCM)/methanol (2:1, v/v) were used in ultrasonic extraction, and DCM was used as eluate for column chromatography. The developing system consisted of n-hexane and DCM at a ratio of 9:1 (v/v), with DCM as the extraction solvent for PAHs-containing silica gel scraped off the plate. When the spiking level is 100 ng, total recoveries of spiked matrices for four target PAHs (phenanthrene, anthracene, pyrene and chrysene) were 83.7%, 76.4%, 85.8%, and 88.7%, respectively, with relative standard deviation (RSD) between 5.0% and 6.5% (n = 4). When the spiking level is 1000 ng, associated total recoveries were 78.6%, 72.7%, 82.7% and 85.3%, respectively, with RSD between 4.4% and 5.3% (n = 4). The opti-mized method was advantageous for determination of PAHs in complex matrix due to its effective sample purification.     

Operational Laboratory Methods for GDGTs Groups Separation

Glycerol dialkyl glycerol tetraethers(GDGTs), specific membrane lipids synthesized mainly by bacteria and archaea, can be divided into isoprenoids and methyl branched alkyl GDGTs(i GDGTs and brGDGTs, respectively). Three GDGTs groups(i GDGTs, brGDGTs, and other membrane lipids) in a peat sample were separated and collected in this study using semi-preparative high-performance liquid chromatography(HPLC) and silica gel column chromatography. The obtained recoveries for the whole analytical procedure were 85% – 55% using semi-preparative HPLC and 70% – 20% using gel column chromatography. In addition, in each method, the recoveries of brGDGTs and iGDGTs were similar, regardless of the huge difference in their contents. High purity was found in the fractionated groups, determined based on ether cleavage and reduction. Moreover, the semi-preparative HPLC method could realize a better separation efficiency than the silica gel method, but it was time-consuming and required expensive equipment, while the silica gel chromatography method featured merits of time saving and convenient operation at the cost of a slight reduction in separation efficiency. The advantages of the silica gel method make it an operational laboratory method for batch experiments and isotopic studies.     

Optimization of Culturing Condition and Medium Composition for the Production of Alginate Lyase by a Marine Vibrio sp. YKW-34

Carbohydrases secreted by marine Vibrio sp. YKW-34 with strong Laminaria cell wall degrading ability were screened, and among them alginate lyase was found to be dominant. The effects of medium composition and culturing condition on the production of alginate lyase by marine Vibrio sp. YKW-34 in flask were investigated in this study. In the culture medium of marine broth, no alginate lyase was produced. The activity of the alginate lyase, after being induced, reached 5 UmL^-1. The best inoculum volume and inoculum age were 10% and 12h, respectively. The optimal temperature for alginate lyase production was 25℃. The fermentation medium was composed of 0.5% of Laminaria powder and 0.2% of KNO3 with an initial acidity of pH 8.0. Alginate could induce alginate lyase production but not as efficiently as Laminaria powder did. The addition of fucoidan, cellulose and glucose had negative effect on the alginate lyase production. Other kinds of nitrogen sources, such as yeast extract, beef extract and peptone, had positive effect on the growth of the microorganism and negative effect on alginate lyase production. In addition, the time course of alginate lyase production under the optimized condition was described. The optimal harvest time was 48 h.     

SERUM IMMUNOGLOBULIN OF THE MANDARIN FISH,SINIPERCA CHUATSI WITH DEVELOPMENT OF POLYCLONAL ANTIBODY

Serum immunoglobulin from the mandarin fish,or the so-called Chinese perch,Siniperca chuatsi(Basilewsky),was successfully purified using affinity chromatography.Heavy and light chains were detected on electrophoresis gel,with molecular weights being estimated at 72 and 29 kDa,respectively.The tetrameric IgM of S.chuatsi was calculated to be 808 kDa.The rabbit polyclonal antisera against the purifed immunoglobulin were developed and tested by Westem blot analysis.The antisera reacted strongly with the heavy chains of S.chuatsi immunoglobulin.Humoral immune responses of the mandarin fish can then be examined using the developed polyclonal antibody.     

Identification and Characterization of a Novel Alkali-and High Temperature-Tolerant Lipase(Lip4346) from a Macroalgae-Associated Bacterial Strain

A novel lipase gene(lip4346)encoding a primary translation product with 176 amino acids was screened from the genome fine mapping of the macroalgae-associated bacterial strain Microbulbifer sp.YNDZ01.Macroalgae were collected from the coast of the Halmahera Island of Indonesia.The lip4346 gene was cloned and heterologously expressed in Escherichia coli.The purified recombinant Lip4346 protein had a molecular mass of 19 k Da,a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),and a maximum enzyme activity of 31.2 U m L-1.The optimal temperature and pH for the lipase activity of Lip4346 were 70℃and 10.0,respectively.Lip4346 was tolerant with a number of organic solvents and detergents,and was active toward triacylglycerols and p-nitrophenyl esters with short-and medium-chain lengths.The unique characteristics of Lip4346 indicate that it is a promising nonaqueous biocatalyst for industrial applications.     

Antitumor and Antifungal Activities of Organic Extracts of SeacucumberHolothuria atra from the Southeast Coast of India

In phylum Echinodermata, the family Holothuridae is distinguished by its capacity of bioactive compounds. Sea cucumber Holothuria atra is commonly known as the lollyfish. The antifungal activity was detected using agar well diffusion method against the various fungal strains such as Trichoderma viride, Aspergillus niger, Aspergillus flavis, Candida albicans and Penicillium chrysogenum. Relatively high antifungal activity was seen against Candida albicans at 100 μL-1 concentration of extracts. Zone of inhibition was measured at 18 mm of diameter. The anti-tumor activities were detected against the Vero and Hep2 cell lines using MTT assay. The cells were treated with H. atra extract at concentrations 0.078-10 mg m L-1. The extract showed high proliferative activity against the Hep2 cells. The body wall extracts of sea cucumber(H. atra) showed effective antifungal and antitumor activities. All these findings suggest that the extracts could be used for the development of drugs.     

Preparation,Characteristics, and Formation Mechanism of Oyster Peptide-Zinc Nanoparticles

Oyster peptide–zinc nanoparticles(OPZNPs)(28–108 nm) were prepared in the presence of 0.5%–0.9% zinc sulfate at pH 6.0–11.0. The obtained nanoparticles exhibited uniform size distribution and spherical shapes. Nanoparticle characteristics, such as size, surface charge, and hydrophobicity, could be adjusted by controlling zinc sulfate concentration and environmental pH. Increasing pH value or decreasing zinc sulfate concentration tended to reduce nanoparticle size and increase nanoparticle surface charge and hydrophobicity. OPZNPs presented good stability at near-neutral pH and could be stored for at least 20 days at 4℃. The results of the peptide conformation study and nanoparticle dissociation test proved that zinc ions and carboxyl groups are the key factors that affect OPZNP formation. The intermolecular combinations of carboxyl groups via zinc bridging facilitated the aggregation of oyster peptides. Nanoparticle formation was accompanied by aggregate association and conformational changes. These changes included increments in β-sheets, especially intermolecular β-sheets, at the expense of α-helixes. Overall, this work provided a green alternative route for the synthesis of OPZNPs.     

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.     

Effects of temperature, pH and NaC1 on protease activity in digestive tract of young turbot, Scophthalmus maximus

The protease activity in digestive tract of young turbot Scophthalmus maximum was studied, and the optimal pH, temperature and NaCl concentration were determined for different portions of the fish＇s internal organs. The optimal activity in the fish＇s stomach was at pH of 2.2, while that in the intestinal extracts was within the alkaline range from 9.5 to 10.0. In hepatopancreas, the optimal pH was in low alkalinity at 8.5. The optimal reaction temperature was above 40℃ in stomach, intestine and hepatopancreas. With increasing temperature, the pH value increased in stomach, while in the intestine, an opposite tendency was observed due to combined effect of pH and temperature. NaCl concentration showed inhibitory impact on protein digestion in hepatopancreas. The main protease for protein digestion in turbot seemed to be pepsin. Moreover, the maximum protease activity in different segments of intestine existed in the hindgut.