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

<正> 107 strains producing protease were screened from 260 strains of Ant-arctic psychrophilic bacteria,among which proteolytic activity of five strains wasmore than 45 U ml~(-1).The 16S rRNA gene sequences homology and phylogenet-ic analysis of five Antarctic psychrophilic bacteria showed that NJ276、NJ5-9、NJ16-70、NJ345 belonged to the described genus Pseudoalteromonas and NJ341belonged to the genus Colwellia.The growth and the protease characteristic offour Antarctic psychrophilic bacteria had been studied,and the result showedthat the optimal temperature for growth and protease-producing of four strainswas about 10℃.Their growth and protease-producing were still high during in-cubating 2-5 days.The maximum proteolytic activity occurred at pH 9 for fourAntarctic psychrophilic bacteria.The optimal temperature of protease action ofboth strains NJ276 and NJ5-9 was about 50℃,however,the optimal temperatureof protease action of both strains NJ341 and NJ345 was about 40℃,and theirproteolytic activity under 0℃ exhibited nearly 30% of the maximum activity,but their thermal stabilities were weaker.These results indicated that proteasesfrom NJ341 and NJ345 were low-temperature proteases.     

北极海冰细菌产胞外酶及主要环境因子的初步研究

覆盖在北极海洋上的广阔海冰为其内部生存的微生物群落提供了一个独特的生境。研究表明 ,大量海冰细菌能够分泌产生胞外酶 ,其中产蛋白或脂质水解酶细菌的比例远高于产多糖水解酶的细菌。温度、盐度是直接影响海冰细菌生存与活力的 2个主要环境因子。 76%的产酪蛋白酶海冰细菌为低温菌 ,菌株只能在 <35°C条件下生长 ;而 98%的酪蛋白酶最适作用温度≥ 35°C ,其中 62 %的酶最适作用温度≥ 45°C。几乎所有的产酪蛋白酶海冰细菌都耐盐或嗜盐     

北极海洋细菌BSw20353的胞外蛋白酶性质

从北极楚科奇海水中分离到一株产蛋白酶耐冷河豚毒素假交替单胞菌BSw20353。该菌可合成多种胞外蛋白酶,而培养温度影响蛋白酶的合成。分离纯化出其中一种在15℃、25℃培养时均有生成的酶:该酶最适作用温度55℃;最适pH8;金属离子Hg2+、Sr2+抑制酶活,Fe2+、Mn2+则具有激活作用;该酶属于金属蛋白酶,受螯合剂邻二氮杂菲、EDTA及EGTA抑制,基本不受丝氨酸蛋白酶抑制剂PMSF、TPCK及TLCK抑制,半胱氨酸蛋白酶抑制剂E-64对该酶无抑制作用,天冬氨酸蛋白酶抑制剂pepstatin则具有一定抑制作用;SDS、二硫苏糖醇、碘乙酸可抑制酶活;而在变性剂尿素、盐酸胍作用下,酶活反而有所增高。     

吉尔吉斯斯坦伊塞克湖一处土壤样品细菌多样性

采用纯培养方法从吉尔吉斯斯坦共和国伊塞克湖一处温泉旁的土壤中分离到32株细菌,通过ARDRA 分析后得到不同的类群,从各个类群中随机选取1个代表菌株进行16S rDNA 序列测定和系统发育的分析。结果表明：分离的菌株分布在假单胞菌属（Pseudomonas）、类芽孢杆菌属（Paenibacillus）、芽孢杆菌属（Bacillus）、寡养单胞菌属（Stenotrophomonas）、Lysinibacillus、溶杆菌属（Lysobacter）、短芽孢杆菌属（Brevibacillus）、根瘤菌属（Rhizobium）、变形杆菌属（Proteus）等9个属,且部分菌株的16S rDNA序列同源性低于97%,可能是潜在的新种。结果揭示了该样点可培养细菌种群的多样性及独特性。     

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.     

南极罗斯海沉积物中可培养菌株的分离及胞外水解酶活性检测

研究了南极罗斯海地区沉积物中可培养微生物多样性,并检测了菌株胞外酶活性,为开发利用南极资源奠定基础。采用Zobell2216E培养基、土豆培养基(PDA)及稀释平板划线法分离并获得纯培养的细菌和真菌,分别通过16SrDNA、18S/ITS序列鉴定分析了可培养微生物的多样性和系统发育。从南极罗斯海沉积物样品中获得78株细菌和54株真菌,细菌、真菌的优势菌株分别为嗜冷杆菌属(Psychrobacter)、枝孢属(Cladosporium);78株细菌菌株中有2株与模式菌株的16SrDNA基因序列相似性小于97%,可能是潜在的细菌新物种。细菌API20NE生理生化和真菌产胞外酶活性结果表明,获得的细菌和真菌大多数可检测到胞外水解酶活性。本研究丰富了南极可培养微生物资源,为南极罗斯海地区可培养微生物的多样性研究以及南极微生物资源的应用研究奠定了基础。     

Effect of solar radiation and the subsequent dark periods on two newly isolated and characterized Antarctic marine bacteria

Two strains of psychrotolerant Antarctic marine bacteria were isolated and characterized using biochemical and molecular techniques. Sequencing of 16S rRNA gene showed that UVvi strain belongs to the genus Arthrobacter whereas UVps strain is related to the Flexibacter-Cytophaga-Bacteroides (FCB) group. Response of the strains to solar radiation was studied during the summer of 1999 in Potter Cove, near Jubany station (South Shetland Island, Antarctica). The effect of photosynthetically available radiation (PAR, 400-700 nm), ultraviolet-A (UV-A, 320-400 nm) and ultraviolet-B radiation (UV-B, 280-320 nm) on cell viability was studied using mixed cultures in quartz bottles covered with interferential filters and exposed to solar radiation. In all experiments, four treatments were used: dark (with light screened out), PAR (with UV radiation screened out), PAR+UV-A (UV-B screened out) and PAR+UV-A+UV-B. Under the assayed conditions, PAR+UV-A and PAR+UV-A+UV-B radiation showed similar negative effects on the viability of the studied strains. However, at the end of the exposure time, mortality values in PAR+UV-A+UV-B treatments were higher than those observed under PAR+UV-A treatments. In both PAR+UV-A and PAR+UV-A+UV-B treatments we observed high levels of hydrogen peroxide compared with the dark control. The Arthrobacter UVvi strain showed significant recovery in dark conditions after exposure to the PAR+UV-A but not after the PAR+UV-A+UV-B treatment. This strain proved to be more resistant to UV radiation than the FCB group-related UVps strain. The results showed that UV radiation has a deleterious effect on these Antarctic marine bacteria and also revealed that the analysed components of the Antarctic bacterioplankton may have different responses when they are exposed to the same irradiance conditions.     

Diesel oil and PCB-degrading psychrotrophic bacteria isolated from Antarctic seawaters (Terra Nova Bay, Ross Sea)

Fifty-seven Antarctic marine bacteria were examined for their ability to degrade commercial diesel oil as the sole organic substrate at both 4 °C and 20 °C. Based on the preliminary screening, two isolates (B11 and B15) with high capacity to degrade diesel oil were selected and their biodegradation efficiency was quantified by gas chromatographic analysis. As expected for psychrotrophs, diesel oil biodegradation was slower at 4 °C than at 20 °C. The two strains also mineralized the C₂₈ n-paraffin octacosane at 20 °C and polychlorinated biphenyls (PCBs) at 4 °C and 20 °C.     

一株北极海洋细菌及其蛋白酶的生理生化特征研究

从北极海洋环境中筛选分离出一株产胞外碱性蛋白酶的革兰氏阴性杆菌 Ar/ W/ b/ 75°2 5′ N/ 1。该菌能在 7～ 30℃范围内生长 ,并且在 30℃条件下生长良好 ,但不能在 40℃条件下生长。该菌在 0 .5%～ 1 0 % Na Cl的盐浓度范围内能良好生长 ,表现出一定的嗜盐性。葡萄糖、蔗糖及可溶性淀粉等碳源物质可被该菌株利用以供生长之需 ,其中以葡萄糖最佳。氮源物质中 ,蛋白胨是最有利于菌株生长、产酶的有机氮源 ,而 NH4 NO3则是效果最好的无机氮源。对该菌蛋白酶的酶学特性研究表明 :所产胞外蛋白酶占其蛋白酶总量的 75.7%左右 ;蛋白酶反应的最适温度为40℃ ,酶活力在温度不高于 40℃条件下保持稳定 ;该菌蛋白酶具有较宽的 p H活性范围 ,在 p H7～ 1 1均表现出较高酶活力 ,其最适 p H在 8左右     

产低温超氧化物歧化酶南极菌株的筛选、鉴定及其部分酶学性质

从421株南极细菌中筛选出产超氧化物歧化酶南极细菌NJ062、NJ379、NJ522和NJ548。它们的最适生长温度在10-15℃之间,在0℃均能生长,属于嗜冷菌。4株南极嗜冷菌均含有一种类型的SOD同工酶。菌株NJ062的超氧化物歧化酶为Mn-SOD;菌株NJ379,NJ522和NJ548的超氧化物歧化酶均为Fe-SOD。它们所产的超氧化物歧化酶最适酶活温度大约在40-50℃,在0℃仍有较高的活性,为最高活性的20%-35%,热稳定性较差,属低温酶。通过16S rDNA序列对4株产低温超氧化物歧化酶的南极嗜冷菌NJ062、NJ379、NJ522和NJ548进行鉴定。结果表明,它们均属于细菌域(Bacteria)中的紫细菌类群(Proteobacteria)中的γ-亚群。其中菌株NJ062属于假交替单胞菌属(Pseudoalteromonassp.);NJ379属于希瓦氏菌属(Shewanellasp.);NJ522和NJ548属于海单胞菌属(Marinomonassp.)。     

Isolation and characterization of a marine bacterium producing protease from Chukchi Sea, Arctic

A Gram negative bacterium Ar/W/b/75°25′N/1 producing extracellular alkaline protease was isolated from surface water of latitude 75°25′N, and longitude 162°25′W in Chukchi sea, Arctic. The strain can grow at the temperature range from 7℃ to 30℃, and grow better at 30℃. It can not grow at 40℃. Keeping certain salinity concentration in medium is necessary for cell growth. It grows well in medium containing salinity concentration from 0.5% to 10% sodium chloride. Glucose, sucrose and soluble starch can be utilized by the strain, among which glucose is the optimal carbon source. Peptone is the optimal organic nitrogen source for cell growth and protease producing, and ammonium nitrate is the optimal inorganic nitrogen source. About 75 7% of total protease of the strain are extracellular enzyme. Optimal temperature for proteolytic activity is at 40℃. Protease of the strain keeps stable below 40℃, and shows high proteolytic activity within the pH range from 7 to 11.     

南极阿德雷岛不同基底中真菌的分离培养及初步鉴定

为了探索南极真菌的多样性,进一步揭示其生态学角色和筛选有特定酶活性菌株,本研究对中国第27次南极科学考察采集自阿德雷岛的6份样品进行了真菌的分离培养及其胞外酶活性测定。从6份样品中共分离得到168株真菌,其中大型海藻腐烂物样品中分离到的真菌种类和数量最多,占所分离菌株总数的34.6%;对选取的15株代表菌株进行了真菌ITS1-5.8S-ITS2序列鉴定,结果表明它们分属于4个纲,8个属,其中青霉属5株,假散囊菌属、地丝菌属和枝孢属各2株,生赤壳属、曲霉属、出芽短梗霉属和unclassified Onygenales各 1株,该结果表明南极地区具有丰富的真菌物种多样性,其优势类群青霉菌。胞外酶测定显示产淀粉酶、蛋白酶和纤维素酶产生菌分别占供试菌株的64.6%、45.1%、14.6%,产纤维素酶的菌株可能在南极物质能量循环中扮演着重要角色。     

极地寡营养细菌AT52(Alteromonas stellipolarissp.nov.)超微结构、生长生理特性及液态富营养发酵培养条件优化

寡营养细菌是一类能利用低浓度有机营养基质的细菌,通过研究1株分离自南极海洋的极地寡营养细菌AT52,发现富营养条件下AT52菌体细胞周身附肢,附肢长短不一,具鞭毛,菌体出芽生殖,由一层原生质膜包围.寡营养饥饿生存的菌体细胞相对较小,近球形,边缘不平滑,无鞭毛附肢等超微结构,细胞体眠未见出芽生殖过程.实验中采用寡营养环境...     

南极菲尔德斯半岛土壤氮循环微生物类群数量与功能活性的初步研究

南极乔治王岛南端的菲尔德斯半岛被认为是研究南极生态与生物资源的理想之地。本研究从该半岛全岛范围采集了3类不同类型的土壤样品,采用传统最大或然法(MPN)对其所含氮循环各类群微生物的总量进行了测定,同时利用常规Griess试剂、纳氏试剂等显色法初步估算了各菌群的功能活性强度,并结合土壤理化性质和采样点生态等进行了初步分析。目的是对该半岛土壤氮循环微生物进行先期初步了解,为进一步深入研究它们的其他特性奠定基础。结果表明:13个土样中所含氮循环微生物总量趋势为氨化菌反硝化菌硝化菌和固氮菌(活菌数),除极个别样品外均具有不同程度的硝化、反硝化、氨化活性。其中,所有土壤样品均具有比较高的氨化菌含量及氨化活性强度;丘陵山坡土壤的反硝化菌含量与反硝化活性强度要高于动、植物区土壤(极个别样品除外);动物区和植物区土壤的硝化菌含量较少,但硝化活性强度较高,预示着可能存在高效功能菌株;而丘陵山坡土壤的硝化菌含量及硝化活性强度则表现得高低不一。本结果为进一步深入研究该地区氮循环微生物提供了前期参考。     

Screening on DHA-producing Antarctic bacteria N-6 and its cultural conditions

In Antarctic, the geography and climate differs from those in other places, and the bacteria there have adapted well to the environment there. Two hundred strains of bacteria were isolated from the sea ice in Antarctica. The bacteria were screened for DHA by means of GC, with fish oil as the standard. Seven strains containing DHA or EPA were obtained, among which the strain of No. N-6 was outstanding. And the component of DHA was identified by GC-MS. The relative content of DHA in N-6 was 8.72%, and total lipids in dry bacteria was 22.54%. The effects of some factors, including temperature, salinity and pH value, on the growth and DHA-content of strain N-6 were studied. The results show that the DHA-content is relatively high when in low temperature and high pH, and the bacterium is psychrophilic, alkalophilic.     

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 impor- tance in basic research and potential in biotechnological application.This paper in- vestigated the culture condition and extracellular 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 pro- ducing amylase,protcase,cellulase 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.     

2003年夏季东南印度洋上层海洋的水文特征

本文利用 2 0 0 3年 2月我国第 1 9次南极考察队在南极普里兹湾到澳大利亚弗里曼特尔南印度洋断面上获得的XBT和XCTD数据 ,分析了该断面上层的水团分布和锋面的特征 ,并讨论了极锋以南表层的淡水输入和热量输入与海冰、降水及平流之间的关系。南极夏季表层水(AASSW)、亚南极表层水 (SASW)、亚热带表层水 (STSW)分布于温盐跃层之上的混合层中 ,由南向北依次分布。AASSW之下是向北延伸的温度最小值层 ,即冬季水 (WW) ,其下则是涌升的上层南极饶及深层水 (UCDW)。南侵的高温高盐的亚热带表层水 (STSW)之下是温度和盐度相对均匀的亚南极模式水 (SAMW)。本航次发现 ,在 63.5°S ,79.7°E的 60 0m以深有低盐水体 (相对其周围水体 )存在 ,核心盐度 34.5 8,位于 80 0- 90 0m水深之间。这是历次考察在该断面所不曾观测到的现象。断面上的锋面从南向北依次为南极陆坡锋 (ASF)、极锋 (PF)、亚南极锋 (SAF)和亚热带锋 (STF)。ASF位于 65°S以南的 1 0 0m以深 ,等温线和等盐线向南倾斜 ;PF位于 5 4°S ,90 .4°E ;SAF位于 45 .3°- 47.5°S ,1 0 2 .5°- 1 0 4 .4°E之间 ,深达整个测量深度 ,温度具有两个高梯度核心 ;首次在该断面观测到双STF结构 ,位于 41 .9°- 42 .6°S,1 0 6.7°- 1 0 7.3°E和 37.7°- 38     

海洋放线菌Y18的分子鉴定、生物活性及其抗菌物质发酵条件优化

从北冰洋楚科奇海海洋沉积物中分离到一株放线菌Y18,其良好生长需要海水。经16S rRNA基因序列分析确定该菌株属于拟诺卡氏菌属(Nocardiopsis),次级代谢产物合成酶基因及抗菌活性筛选发现,它含有聚酮类Ⅱ型(PKSⅡ)和非核糖体含硫多肽类(non ribosome-bound sulfur-containing peptides,NRPS)化合物合成酶基因,并且对枯草芽孢杆菌和白色念珠菌有抑制作用。以枯草芽孢杆菌为指示菌对Y18的发酵条件进行了初步优化,适合的发酵条件为:发酵培养基(蛋白胨2 g,酪素水解物2 g,酵母膏2 g,葡萄糖1 g,海水1 L),种子液接种量4%,发酵温度28℃,发酵培养基初始pH值8.0,培养时间6天,1 000 mL三角瓶装液量20%。并对Y18发酵液稳定性进行了测定,结果显示:发酵液对酸碱、热的稳定性都较强,4℃下贮藏发酵液的抗菌活性保留时间较长。     

基于GAM模型分析夏秋季南奥克尼群岛南极磷虾渔场时空分布及与环境因子之间的关系

基于中国南极磷虾渔业科学观察员收集的数据,利用广义可加模型（GAM）,对2009—2010渔季和2010—2011渔季夏秋季南奥克尼群岛周边水域南极磷虾渔场时空分布及其与表温和海况之间的关系进行了研究。结果表明,GAM模型对单位捕捞努力量渔获量（CPUE）总偏差解释率为41.78%,其中贡献最大的为月份,贡献率为15.53%;奥克尼群岛水域CPUE分布模式均有明显的月份差异。1月份平均CPUE值最低,而4月份平均CPUE值最高,各月份平均CPUE存在显著性差异。渔场主要集中在60°12′—60°30′S,45°30′—47°30′W。南极磷虾作业渔场的适宜海水表温范围为0.1—1.8℃,最适表温范围为0.5—1.5℃。各海况等级间及不同渔船的平均CPUE均存在显著性差异。逐步GAM模型分析结果表明,影响CPUE的因子按重要性从大到小依次为月份、渔船、经度、海表温度、纬度和海况。     

ERA-Interim再分析和NCEP FNL分析资料在东南极中山站至Dome A断面的适用性研究

利用2008年南极中山站至Dome A断面上观测站的近地面气象观测资料对ECMWF ERA-Interim再分析和NCEP FNL分析资料在东南极地区的适用性进行了验证。结果表明,ERA-Interim再分析资料的气温表现明显优于FNL分析资料,其与观测的年均绝对偏差在南极大陆沿岸地区1℃,在内陆高原2℃;而FNL分析资料的气温在南极内陆高原地区较观测明显偏暖,尤其在冬季偏暖达8—10℃,表明其不能直接用于南极内陆高原气温的变化分析。FNL的地面气压与观测比较接近,逐月平均偏差仅约1 h Pa,其精度明显高于ERA-Interim再分析资料的地面气压,而后者在沿岸地区存在明显的系统性偏低。ERA-Interim和FNL的近地表风速、风向差异不显著,其与观测的年平均、季节平均绝对风速偏差在沿岸和下降风区1 m·s-1,在内陆高原约2—4 m·s-1,年平均风向绝对偏差10°。