Extracellular enzymatic activities of cold-adapted bacteria from polar oceans and effect of temperature and salinity on cell growth

The potential of 324 bacteria isolated from different habitats in polar oceans to produce a variety of extracellular enzymatic activities at low temperature was investigated. By plate assay, lipase, protease, amylase, gelatinase, agarase, chitinase or cellulase were detected. Lipases were generally present by bacteria living in polar oceans. Protease-producing bacteria held the second highest proportion in culturable isolates. Strains producing amylase kept a relative stable proportion of around 30% in different polar marine habitats. All 50 Arctic sea-ice bacteria producing proteases were cold-adapted strains, however, only 20% were psychrophilic. 98% of them could grow at 3% NaCl, and 56% could grow without NaCl. On the other hand, 98% of these sea-ice bacteria produced extracellular proteases with optimum temperature at or higher than 35℃, well above the upper temperature limit of cell growth. Extracellular enzymes including amylase, agarase, cellulase and lipase released by bacteria from seawater or sediment in polar oceans, most expressed maximum activities between 25 and 35℃. Among extracellular enzymes released by bacterial strain BSw20308, protease expressed maximum activity at 40℃, higher than 35℃ of polysaccharide hydrolases and 25℃ of lipase.     

Isolation and characteristics of one marine psychrotrophic cellulase-generating bacterium Ar/w/b/75°/10/5 from Chuckchi Sea,Arctic

Microorganisms living in polar zones play an important part as the potential source of organic activity materials with low temperature characteristics in the bio-technological applications. A psychrotrophic bacterium (strain Ar/w/b/75°/10/5) , producing cellulose at low temperatures during late-exponential and early-stationary phases of cell growth, was isolated from sea ice-covered surface water in Chuckchi Sea, Arctic. This bacterium, with rod cells, was Gram-negative, slightly halophilic. Colony growing on agar plate was in black. Optimum growth temperature was 15℃. No cell growth was observed at 351 or above. Optimum salt concentration for cell growth was between 2 and 3 % of sodium chloride in media. Maximal cellulase activity was detected at a temperature of 35℃ and pH8. Cellulase was irreversibly inactivated when incubated at 55℃ within 30 min. Enzyme can be kept stable at the temperature no higher than 25℃. Of special interest was that this bacterium produced various extracellular enzymes i     

Cold-adaptive alkaline protease from the psychrophilic Planomicrobium sp.547: enzyme characterization and gene cloning

A psychrophilic bacterium strain 547 producing cold-adaptive alkaline protease was isolated from the deep sea sediment of Prydz Bay, Antarctica. The organism was identified as a Planomicrobium species by 16S rRNA analysis. The optimal and highest growth temperatures for strain 547 were 15℃ and 30℃, respectively. The extracellular protease was purified by ammonium sulfate precipitation and DEAE cellulose-52 chromatography. The optimal temperature and pH for the activity of the purified enzyme were 35 ℃ and pH 9.0, respectively. The enzyme retained approximately 40% of its activity after 2 h of incubation at 50℃. The enzymatic activity was inhibited by 1 mmol/L phenylmethyl sulfonylfluoride (PMSF) and hydrochloride 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), indicating that it was a serine protease. The presence of Ca2+ and Mn2+ increased the activity of the enzyme. The protease gene with a size of 1 269 bp was cloned from Planomicrobium sp. 547 using primers designed based on the conserved sequences of proteases in GenBank. The Planomicrobium sp. 547 protease contained a domain belonging to the peptidase S8 family, which has a length of 309 amino acid (AA) residues. The alignment and phylogenetic analysis of the AA sequence indicated that the protease belonged to the subtilisin family.     

Screening and molecular classification of low-temperature protease from Antarctic microorganism and its characterization

107 strains producing protease were screened from 260 strains of Antarctic psychrophilic bacteria, among which proteolytic activity of five strains was more than 45 U ml^-1. The 16S rRNA gcne sequences homology and phylogcnetic analysis of five Antarctic psychrophillc bacteria showed that NJ276, NJS-9, NJ16-70,NJ345 belonged tO the described genus Pseudoalteromonas and NJ341 belonged to the genus Colwellia. The growth and the protease characteristic of four Antarctic psychrophilic bacteria had been studied, and the result showed that the 6ptimal temperature for growth and protease-produeing of four strains was about 10℃. Their growth and protease-produeing were still high during incubatlng 2-5 days. The maximum proteolytic activity occurred at pH 9 for four Antarctic psychrophilic bacteria. The optimal temperature of protease action of both strains NJ276 and NJ5-9 was about 50℃, however, the optimal temperature of protease aetlon of both strains NJ341 and NJ345 was about 40 ℃, and their proteolytic activity under 0℃ exhibited nearly 30% of the maximum activity, but their thermal stabilities were weaker. These results indicated that proteases from NJ341 and NJ345 were low-temperature proteases.     

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.     

Studies on culture condition and extracellular hydrolase of psychrophilic bacteria from Arctic sea ice

Arctic sea ice in the polar region provides a cold habitat for microbial community. Arctic sea ice microorganisms are revealed to be of considerable importance in basic research and potential in biotechnological application. This paper investigated the culture condition and extraceIlular hydrolase of 14 strains of different Arctic sea ice bacteria. The results showed that optimal growth temperature of strains is 15 ℃ or 20 ℃. The optimal pH is about 8.0. They hardly grow at acid condition. 3 % NaCl is necessary for better growth. These strains have different abilities in producing amylase, protease, eellulase and lipase. Pseudoalteronomas sp. Bsi429 and Pseudoalteronomas sp. Bsi539 produced both cellulose, protease and lipase. These results provide a basis for further developing and exploiting the cold adapted marine enzyme resources.     

生物采油解堵剂中产蛋白酶菌株的筛选及培养条件优化

为研究生物酶采油解堵剂中产蛋白酶菌株的初、复筛选及培养条件优化,从大庆原油样品中筛选菌种,通过水解酪素的透明圈实验及福林酚测蛋白酶酶活的方法进行菌株的初、复筛选;以蛋白酶酶活为优化指标,采用单因素实验对筛选的产蛋白酶菌株的培养基及培养条件进行优化,优化最适培养基:可溶性淀粉为15g/L,蛋白胨为20g/L,酵母膏为20g/L,NaCl为1.0g/L,CaCl2为0.02g/L,Na2HPO4为0.2g/L,NaH2PO4为0.1g/L;在初始pH为6.0、接种量为5%(体积分数)、温度为31℃、摇床转速为160r/min的条件下,培养72h后,菌株的蛋白酶酶活为551.0U/mL,为复筛选菌株的蛋白酶酶活的22.92倍,即为菌株生长繁殖及代谢的最佳条件,能够获得更高的蛋白酶酶活,有利于后续实验的进行.结果表明:菌株产蛋白酶对原油作用效果为发酵液表面张力从作用前的56.2mN/m降低到作用后的30.5mN/m,表面张力显著降低,还有降解降黏原油等效果,具有一定的研究价值.     

Primary productivity and chlorophyll a in Prydz Bay and its mouth in Antarctica during the austral summer of 1999/2000

The investigation of phytoplankton standing stock, euphotic layer and photosynthesis rate were carried out in 19 - 27 of January, 2000 at three longitudinal sections (70°30＇E, 73°00＇E and 75°30＇E). The results showed that the high value of chlorophyll a concentration was in inshore bay, polynya and the continental slope of the investigated sea area. At various investigated stations, average chlorophyll a concentration at sub-surface layer (25 m) was higher than that at surface layer; its concentration at the deeper layers of over 50 m decreased with increasing depth. At anchor station, the maximum chlorophyll az concentration appeared at surface layer in Antarctic summer's afternoon while the minimum value appeared in the morning; chlorophyll a concentration at water layer of 0 - 25 m was obviously higher than that at deep water layer, being related to the releasing of ice-algae. High productivity was in inshore bay and polynya of continental shelf. Chlorophyll a concentration at surface layer is closely correlated to the dissolved oxygen concentration in seawater. The sea ares with chlorophyll a concentration of over 1.0ag/dms may be the convergence of CO2 and that of below 1.0 μg/dms may be the source of CO2.     

Effects of culture conditions on growth and docosahexaenoic acid production from Schizochytrium limacinum

The effects of temperature, initial pH, salinity of culture medium, and carbon and nitrogen sources on growth and docosahexaenoic acid （C22：6 n-3, DHA） production from Schizochytrium limacinum OUC88 were investigated in the present study. The results revealed that the optimal temperature, initial pH and salinity level of the medium for DHA production were 23 ℃, 7.0 and 18, respectively. Glucose was proved the best carbon source for the growth and DHA production from S. limacinum. Among the nitrogen sources tested, soybean cake hydrolysate, a cheap by-product, was found to be effective for the accumulation of DHA in S. limacinum cells. In addition, increasing the concentration of carbon sources in the medium caused a significant increase in cell biomass; however, accumulation of DHA in cells was mainly stimulated by the ratio of C/N in the medium. Under the optimal culture conditions, the maximum DHA yield achieved in flasks was 4.08 g L^-1 after 5 d of cultivation     

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.     

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.     

Amylase Production by the Marine Yeast Aureobasidium pullulans N13d

1 Introduction Amylases are enzymes which hydrolyze starch molecules to give diverse products including dextrin and pro-gressively smaller polymers composed of glucose units(Windish et al., 1965; Pandey et al., 2000; Chi et al.2001). Amylase is a kind of very important enzyme andconstitutes a class of industrial enzymes sharing approximately 25% of the enzyme market (Sindhu et al.1997; Rao et al., 1998). Amylases are universally distributed throughout the animal, plant and microbial kingdoms…     

COMBINED EFFECTS OF TEMPERATURE, IRRADIANCE AND SALINITY ON GROWTH OF DIATOM SKELETONEMA COSTATUM

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Production of polyhydroxybutyrate by the marine photosynthetic bacterium Rhodovulum sulfidophilum P5

The effects of different NaCl concentrations, nitrogen sources, carbon sources, and carbon to nitrogen molar ratios on biomass accumulation and polyhydroxybutyrate (PHB) production were studied in batch cultures of the marine photosynthetic bacterium Rhodovulum sulfidophilum P5 under aerobic-dark conditions. The results show that the accumulation of PHB in strain P5 is a growth-associated process. Strain P5 had maximum biomass and PHB accumulation at 2%-3% NaCl, suggesting that the bacterium can maintain growth and potentially produce PHB at natural seawater salinity. In the nitrogen source test, the maximum biomass accumulation (8.10±0.09 g/L) and PHB production (1.11±0.13 g/L and 14.62%±2.25% of the cell dry weight) were observed when peptone and ammonium chloride were used as the sole nitrogen source. NH 4 + -N was better for PHB production than other nitrogen sources. In the carbon source test, the maximum biomass concentration (7.65±0.05 g/L) was obtained with malic acid as the sole carbon source, whereas the maximum yield of PHB (5.03±0.18 g/L and 66.93%±1.69% of the cell dry weight) was obtained with sodium pyruvate as the sole carbon source. In the carbon to nitrogen ratios test, sodium pyruvate and ammonium chloride were selected as the carbon and nitrogen sources, respectively. The best carbon to nitrogen molar ratio for biomass accumulation (8.77±0.58 g/L) and PHB production (6.07±0.25 g/L and 69.25%±2.05% of the cell dry weight) was 25. The results provide valuable data on the production of PHB by R. sulfidophilum P5 and further studies are on-going for best cell growth and PHB yield.     

Screening and Characterization of Nitrite-Degrading Bacterial Isolates Using a Novel Culture Medium

In this study,a novel culture medium that simulates shrimp pond conditions was established to screen nitrite-degrading isolates.The medium was supplemented with nitrite as a nitrogen source and shrimp feed as the major carbon source,to achieve the high nitrogen and low carbon nutritional status found in shrimp farming ponds.Screening using this medium identified potent denitrifying Bacillus isolates,among which Bacillus subtilis M7-1 was considered best.M7-1 was able to completely degrade nitrite-N in 24 h without much consumption of dissolved oxygen.Efficient denitrification activity took place in liquid cultures within a set of non-stringent ranges of pH(5.0–9.0),salinity(0–30)and temperature(25–35℃).The denitrifying enzyme gene was amplified,sequenced and further identified as nirS type.In biosecurity assessments,M7-1 had no negative effects on shrimps at a dose of 106 cfu mL−1.M7-1 could therefore be used in aquaculture to reduce and control the nitrogen concentration,and to promote the development of sustainable and healthy culture systems.     

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.     

罗非鱼仔鱼开口率与温度、盐度间模型的建立与优化

采用中心复合设计及响应曲面法研究吉富罗非鱼仔鱼开口率与温度和盐度的量化关系,预实验确定温度范围为20~34℃,盐度为2~10,通过模型优化确定初孵仔鱼从出膜到开口这一阶段最适温度和盐度组合。结果表明,温度对仔鱼的开口率(First feeding rate,FFR)的一次效应有统计学意义(P0.01),盐度对仔鱼开口率的一次效应有统计学意义(P0.05),温度和盐度的交互作用对开口率的影响不具统计学意义(P0.05);温度对仔鱼开口率的二次效应有统计学意义(P0.01);盐度对仔鱼开口率的二次效应有统计学意义(P0.05);温度对开口率的影响较盐度更重要。建立开口率对温度和盐度的二次多项式回归方程,其决定系数为0.986 1,矫正决定系数为0.972 1,预测决定系数为0.911 2,表明所建模型拟合度极高,且可通过温度和盐度预测罗非鱼仔鱼的开口率。对模型方程优化的结果表明,当温度为29℃、盐度为5时,罗非鱼仔鱼的开口率达到最大值87.97%,可靠性达到0.972。     

不同盐度对龙虎斑生长和摄食的影响

在盐度5、10、15、20、25、30条件下,以全人工配合饵料饲喂龙虎斑[鞍带石斑鱼(Epinephelus lanceolatus♂)×棕点石斑鱼(E.fuscoguttatus♀)]60 d,研究不同盐度对龙虎斑生长和摄食的影响。结果表明,不同盐度对龙虎斑生长的影响有统计学意义(P0.05),盐度5~20组增长率、特定生长率均高于盐度25、30组,差异有统计学意义(P0.05),盐度10组的增长率最高,为55.08%,盐度15组的特定生长率最大,为2.30%/d,盐度和龙虎斑生长速率的回归方程为K=0.000 09 S 4-0.006 S 3+0.131 S 2-1.055 S+4.832(R 2=0.984);不同盐度对龙虎斑的摄食率和饵料系数影响有统计学意义(P0.05),盐度15组的摄食率最高,为1.64%/d,其次为盐度10组,为1.60%/d,饵料系数在盐度15组最低,为0.820,其次为盐度10组,为0.827。因此,龙虎斑在低盐度条件下生长比高盐度快,摄食率高,饵料利用率高,最佳生长盐度为10~15。     

Seasonal variations of the near surfacelayer parameters over the Antarctic ice sheet in Princess Elizabeth Land,East Antarctica

Analysis of sensible heat flux(Qh),latent heat flux(Qe),Richardson number(Ri),bulk transport coefficient(Cd) and katabatic winds are presented by using the meteorological data in the near surface layer from an automatic weather station(AWS) in Princess Elizabeth Land,East Antarctica ice sheet and the data of corresponding period at Zhongshan station in 2002.It shows that annual mean air temperature at LGB69 is-25.6°C,which is 16.4°C lower than that at Zhongshan,where the elevation is lower and located on the coast.The temperature lapse rate is about 1.0°C/110 m for the initial from coast to inland.The turbulence heat flux at LGB69 displays obvious seasonal variations with the average sensible heat flux-17.9 W/m2 and latent heat flux-0.9 W/m2.The intensity(Qh Qe) of coolling source is-18.8 W/m2 meaning the snow surface layer obtains heat from atmosphere.The near surface atmosphere is near-neutral stratified with bulk transport coefficients(Cd) around 2.8×10-3,and it is near constant when the wind speed higher than 8 m/s.The speed and the frequency of easterly Katabatic winds at LGB69 were higher than that at Zhongshan Station.     

温度、盐度对马氏珠母贝外套膜Hsp70基因表达量的联合影响

设定温度范围16～40°C,盐度范围10～50,采用复合设计和响应曲面分析法在实验室条件下研究不同温度、盐度对马氏珠母贝(P.fucata)外套膜Hsp70基因表达量的综合效应并建立模型。采用方差分析(ANOVA)、决定系数等对实验所得回归方程模型进行显著性及拟合度检验。结果表明,温度的一次、二次效应对马氏珠母贝外套膜Hsp70基因表达量影响显著(p值<0.05);盐度的一次效应对马氏珠母贝外套膜Hsp70基因表达量无显著影响(p值>0.05),二次效应对马氏珠母贝外套膜Hsp70基因表达量影响极显著(p值<0.05);温度、盐度之间的互作效应对马氏珠母贝外套膜Hsp70基因表达量无显著影响(p值>0.05)且温度的效应大于盐度的效应。经响应曲面分析法和优化,得到马氏珠母贝外套膜Hsp70基因表达量在适宜的温度、盐度范围内呈峰值变化,在温度26.85°C、盐度29.39时,马氏珠母贝外套膜Hsp70基因表达量达到最小值0.95,满意度达到99.36%。建立了马氏珠母贝外套膜Hsp70基因表达量模型方程,其决定系数98.69%,矫正决定系数97.38%,预测决定系数91.42%,模型的拟合度极高,可用于预测马氏珠母贝外套膜Hsp70基因表达量。