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.     

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.     

Enhancing effect of Fe2+ on the formaldehyde production from trimethylamine N-oxide decomposition catalyzed by the extract of Harpadon nehereus kidney

The effects of Fe2+ on the trimethylamine N-oxide (TMAO) demethylating activity of the Harpadon nehereus kidney extract were studied in this research.The activity of the kidney extract was presumably inhibited by ethylene diamine tetra-acetic acid (EDTA),which indicates that the kidney extract contains an enzyme or enzyme system with metal cations as activator.Activity of the kidney extract was enhanced significantly when Fe2+ was added into the model system in vitro.As the concentration of Fe2+ increased,the decomposing rate of TMAO increased rapidly until TMAO decomposed completely.The activity of the kidney extract was also enhanced by reductant such as ascorbic acid.Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) was employed to determine the content of total iron in a number of fishery products.Significant positive correlation between the contents of total iron and endogenous formaldehyde (FA) was found,especially in marine products.     

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.     

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 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.     

Impact of external factors on sperm motility of Sepiella maindroni

Sperm motility is important in reproductive biology. To understand the physiological ecology characteristics of sperm of Sepiella maindroni, we studied the activation rate, active time, and lifespan of sperm to evaluate the influence of external factors, including temperature, salinity, pH, glucose, NaCl, KCl, MgCl2, CaCl2, EDTA-2Na and artificial seawater on sperm motility. The results show that the appropriate activation and motility condition for sperm were: temperature 20?30°C (optimum 25°C), salinity 10.6?30.6 (optimum 15.9) and pH 8.0?8.6 (optimum 8.0-8.4). Sperm activity varied in different concentrations of glucose, NaCl, KCl, MgCl2 and CaCl2. None of the selected concentrations of EDTA-2Na solution could activate the sperm. The activation rate in artificial seawater devoid of Ca2+, Mg2+ or HCO3ˉ was low. The results should help further studies on the preservation and activation of squid sperm.     

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.     

Removal of Mercury(Hg(Ⅱ)) from Seaweed Extracts by Electrodialysis and Process Optimization Using Response Surface Methodology

In this work,response surface methodology(RSM)was employed to model and optimize electrodialysis process for mercury(Hg(II))removal from seaweed extracts.Box-Behnken design(BBD)was utilized to evaluate the effects and the interaction of influential variables such as operating voltage,influent flow rate,initial concentration of Hg(II)on the removal rate of Hg(II).The developed regression model for removal rate response was validated by analysis of variance,and presented a good agreement of the experimental data with the quadratic equation with high value coefficient of determination value(R2=0.9913,RAdj 2=0.9678).The optimum operating parameters were determined as 7.17V operating voltage,72.54L h−1 influent flow rate and 5.04mgL−1 initial concentration of mercury.Hg(II)removal rate of 76.45%was acquired under the optimum conditions,which showed good agreement with model-predicted(75.81%)result.The results revealed that electrodialysis can be considered as a promising strategy for removal of Hg(II)from seaweed extracts.     

Effects of UV-B irradiation on isoforms of antioxidant enzymes and their activities in red alga Grateloupiafilicina （Rhodophyta）

Macroalgae in a littoral zone are inevitably exposed to UV-B irradiance.We analyzed the effects of UV-B on isoenzyme patterns and activities of superoxide dismutase(SOD),peroxidase(POX),catalase(CAT),and ascorbate peroxidase(APX) of red algae Grateloupia filicina(Lamour.) C.Agardh.The activities of SOD,CAT,and APX changed in response to UV-B in a time- and dose-dependent manner.POX activity increased significantly under all three UV-B treatments.The enzymatic assay showed three distinct bands of SODⅠ(Mn-SOD),SODⅡ(Fe-SOD),and SODⅢ(CuZn-SOD) under a low(Luv) and medium(Muv) dose of UV-B irradiation,while SODⅠ and SODⅢ activities decreased significantly when exposed to a high dose of UV-B irradiation(Huv).The activity of POX isoenzymes increased significantly after exposure to UV-B,which is consistent with the total activity.In addition,a clear decrease in activity of CATIV was detected in response to all the three doses of UV treatments.Some bands of APX isoenzyme were also clearly influenced by UV-B irradiation.Correspondingly,the daily growth rate declined under all the three exposure doses,and was especially significant under Muv and Huv treatments.These data suggest that,although the protection mechanisms of antioxidant defense system are partly inducible by UV-B to prevent the damage,G.filicina has incomplete tolerance to higher UV-B irradiation stress.     

CHEMICAL CHARACTERISTICS OF FOUR KINDS OF WATER IN THE RONGBUK GLACIER BASIN, MOUNT QOMOLANGMA

ＤｕｒｉｎｇｔｈｅｔｈｒｅｅＭｔ．ＱｏｍｏｌａｎｇｍａＥｘｐｅｄｉｔｉｏｎｓｏｆ１９５９－１９６０，１９６６－１９６８ａｎｄ１９７５，ＣｈｉｎｅｓｅｓｃｉｅｎｔｉｓｔｓｈａｄｏｂｔａｉｎｅｄｍａｎｙｄａｔａｏｆｇｌａｃｉｅｒｓｉｎｔｈｅｄｉｓｔｒｉｃｔｏｆＭｔ．Ｑｏｍｏｌａｎｇｍａ（Ｗａｎｇｅｔａｌ．，１９８０；Ｘｉｅｅｔａｌ．，１９７５；Ｚｈａｎｇｅｔａｌ．，１９７５）．ＩｎＭａｙ１９９７，Ｐｒｏｆ．ＱｉｎＤａｈｅａｎｄＰｒｏｆ．ＰａｕｌＡ．Ｍａｙｅｗｓｋｉｏｒｇａｎｉｚｅｄａｎｏｔｈｅ…     

Bioaccumulation of Pb<Superscript>2+</Superscript> and its effects on growth,morphology and pigment contents of <Emphasis Type="Italic">Spirulina</Emphasis> (<Emphasis Type="Italic">Arthrospira</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis>

A laboratory experiment was conducted to assess the bioaccumulation of Pb²⁺ and its effects on growth, morphology and pigment contents of Spirulina (Arthrospira) platensis. The specimen cultured in Zarrouk liquid medium was treated with various initial metal concentrations (0, 5, 10, 30, 50 and 100 μg mL⁻¹). The growth of S. platensis was adversely affected by Pb²⁺ at high concentrations (30, 50 and 100 μg mL⁻¹). However, at low concentrations (5 μg mL⁻¹), Pb²⁺ could stimulate its growth slightly. The pigment contents (chlorophyll α and β carotene) were decreased in a dose-dependent manner. The highest reductions (67% and 53% respectively in chlorophyll α and β carotene) were observed in 100 μg mL⁻¹ treatment group. The LC₅₀ (96 h) of Pb²⁺ was measured as 75.34 μg mL⁻¹. Apart from a few cases of filament breakages at elevated concentrations (50 and 100 μg mL⁻¹), morphological abnormalities are not specific. Metal bioaccumulation increased with Pb²⁺ concentrations, but decreased with exposure time. The maximum accumulated amount was 188 mg g⁻¹ dry weight. The bioconcentration factor (BCF) reached to a peak at day 2, followed by a gradual reduction for all the exposure concentrations. S. platensis is able to tolerate considerably high Pb²⁺ concentrations. Consequently it can be used as a potential species to remove heavy metal from contaminated waters.     

THE DENSITIES OF CHANGJIANG ESTUARINE WATERS

The International Eq一lation of State of Seawater,1980 and the PraeticalSalinity Scale,1978 have been adoPted by theUNESCO江CES沼COR八APSOJoint Panel ono‘eanogral,hie Tables and Standards(JPOTS),and endorsed bythese organizati6ns(Miller‘〕and Poisson,1981;Uneseo,1981).Th已new equa-tion and the Praetieal Salinity Seale are to be used for all values Published fromJan .1,1982 .The new equation 15 aeeurate for use in all oeeanie surfaee waters,but eannot be aeeurately aPPlied to…     

CHEMICAL CHARACTERISTICS OF WATER ENVIRONMENT IN LIMNIC WETLANDS IN THE WESTERN SONGNEN PLAIN, CHINA

Duetoregionalhydrogeologicandclimaticconditions,inthewesternSongnenPlain,themostdistinctcharacteristicofwaterenvironmentinlimnicwetlandsisthatmanywaterbodieshavehighsaltcontentsandpHvalues,andsomelimnicwetlandsbelongtosaltmarshes.Thelimnicwetlandsofthisregionarespecialnaturecomplex.Itisimportantfortheutilizationofwetlandresourcesandtheintegrateddevelopmentofsaline-alkalinesoiltostudychemicalcharacteristicsofwaterenvironmentinthisregion.1NATURALENVIRONMENTCHARACTERISTICSOFTHEWE…     

Simulating carbon sequestration and GHGs emissions in <Emphasis Type="Italic">Abies fabric</Emphasis> forest on the Gongga Mountains using a biogeochemical process model Forest-DNDC

The process-oriented model Forest-DNDC describing biogeochemical cycling of C and N and GHGs （greenhouse gases） fluxes （CO2, NO and N2O） in forest ecosystems was applied to simulate carbon sequestration and GHGs emissions in Abies fabric forest of the Gongga Mountains at southeastern edge of the Tibetan Plateau. The results indicated that the simulated gross primary production （GPP） of Abies fabric forest was strongly affected by temperature. The annual total GPP was 24,245.3 kg C ha^-1 yr^-1 for 2005 and 26,318.8 kg C ha^-1 yr^-1 for 2006, respectively. The annual total net primary production （NPP） was 5,935.5 and 4,882.2 kg C ha^-1 yr^-1 for 2005 and 2006, and the annual total net ecosystem production （NEP） was 4,815.4 and 3,512.8 kg C ha^-1 yr^-1 for 2005 and 2006, respectively. The simulated seasonal variation in CO2 emissions generally followed the seasonal variations in temperature and precipitation. The annual total CO2 emissions were 3,109.0 and 4,821.0 kg C ha^-1 yr^-1 for 2005 and 2006, the simulated annual total N2O emissions from forest soil were 1.47 and 1.36 kg N ha^-1 yr^-1 for 2005 and 2006, and the annual total NO emissions were 0.09 and o.12 kg N ha^-1 yr^-1 for 2005 and 2006, respectively.     

GROUNDWATER QUALITY AND CONTAMINATION INDEX MAPPING IN CHANGCHUN CITY, CHINA

1INTRODUCTIONGroundwater in Changchun City assures about 45% of total water supply. Drinking water supply of Chang-chun City was mostly served by surface water from Shitoukoumen and Xinlicheng reservoirs located in the east and south of the city (ZHANG, 1993). However, with the development of urban construction the ground-water especially from deep boreholes is also used for drinking purpose in suburban areas. The urbanization process in Changchun City threatens the groundwater quali…     

Biosorption of Cd (Ⅱ) and Pb (Ⅱ) Ions by Aqueous Solutions of Novel Alkalophillic Streptomyces VITSVK5 spp. Biomass

Discharge of heavy metals from metal processing industries is known to have adverse effects on the environment. Biosorption of heavy metals by metabolically inactive biomass of microbial organisms is an innovative and alternative technology for removal of these pollutants from aqueous solution. The search of marine actinobacteria with potential heavy metal biosorption ability resulted in the identification of a novel alkalophilic Streptomyces VITSVK5 species. The biosorption property of Streptomyces VITSVK5 spp. was investigated by absorbing heavy metals Cadmium (Cd) and Lead (Pb). Physiochemical characteristics and trace metal concentration analysis of the backwater showed the concentrations of different metals were lead 13±2.1 μg L⁻¹, cadmium 3.1±0.3μg L⁻¹, zinc 8.4±2.6μg L⁻¹ and copper 0.3±0.1μg L⁻¹, whereas mercury was well below the detection limit. The effect of pH and biomass dosage on removal efficiency of heavy metal ions was also investigated. The optimum pH for maximal biosorption was 4.0 for Cd (II) and 5.0 for Pb (II) with 41% and 84% biosorption respectively. The biosorbent dosage was optimized as 3 g L-1 for both the trace metals. Fourier transform infrared absorption spectrum results indicated the chemical interactions of hydrogen atoms in carboxyl (-COOH), hydroxyl (-CHOH) and amine (-NH₂) groups of biomass with the metal ions. This could be mainly involved in the biosorption of Cd (II) and Pb (II) onto Streptomyces VITSVK5 spp. The results of our study revealed Streptomyces metabolites could be used to develop a biosorbent for adsorbing metal ions from aqueous environments.     

Studies on culture conditions of benthic diatoms for feeding abalone

Effects of salinity, pH, nitrogenous and phosphate nutrients on the growth rate of four species of benthic diatoms were studied by using the method of in situ optical density measurement. The optimal culture conditions for the four species of diatoms are as follows: forAmphora coffeaeformis, s 35; pH 8.5; NO₃ ⁻−N 1.54, NH₄ ⁺−N, 7.0; CO (NH₂)₂−N, 2.5 (mg/L); PO₄ ³⁻−P 1.13 (mg/L); forCocconeis scutellum varparva, s 30; pH 8.5; NO ₃ ⁻ −N 3.08, NH ₄ ⁺ −N 3.5, CO(NH₂)₂−N 5.0 (mg/L); pO ₄ ³⁻ −P 0.283 (mg/L); forNavicula corymbosa s 25; pH 8.0; NO ₃ ⁻ −N 1.54, NH ₄ ⁺ −N 3.5 CO(NH₂)₂−N 5.0 (mg/L); PO ₄ ³⁻ −P 0.565 (mg/L); forNavicula mollis. s 25; pH 8.0; NO ₃ ⁻ −N 1.54, NH ₄ ⁺ −N 1.75, CO(NH₂)₂−N 1.25 (mg/L); PO ₄ ³⁻ −P 0.141 (mg/L). Part of the results of this paper were exchanged in the Fourth Asian Aquaculture Forum. Oct. 16–20. 1995. Beijing, P. R. China.     

Bioflocculant Produced by Klebsiella sp. MYC and Its Application in the li＇eatment of Oil-field Produced Water

Seventy-nine strains of bioflocculant-producing bacteria were isolated from 3 activated sludge samples. Among them, strain MYC was found to have the highest and stable flocculating rate for both kaolin clay suspension and oil-field produced water. The bacterial strain was identified as Klebsiella sp. MYC according to its morphological and biochemical characteristics and 16SrDNA sequence. The optimal medium for bioflocculant production by this bacterial strain was composed of cane sugar 20 g L^-1, KH2PO4 2 g L^-1, K2HPO4 5 g L^- 1, （ NH4）2SO4 0.2 g L^-1, urea 0.5 g L^- 1 and yeast extract 0.5 g L^- 1, the initial pH being 5.5. When the suspension of kaolin clay was treated with0.5% of Klebsiella sp. MYC culture broth, the flocculating rate reached more than 90.0% in the presence of 500mg L^-1 CaCI2, while the flocculating rate for oil-field produced water was near 80.0% in a pH range of 7.0 - 9.0 with the separation of oil and suspended particles from the oil-field produced water under similar conditions. The environment-friendly nature of the bioflocculant and high flocculating rate of the strain make the bioflocculant produced by Klebsiella sp. MYC an attractive bioflocculant in oil-field produced water treatment.