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.     

Effect of isolation temperature on the characteristics of extracellular proteases produced by Antarctic bacteria

Protease-producing psychrotolerant bacteria were isolated from Antarctic biotopes on casein agar plates using different incubation temperatures. Most of the isolates were non-spore-forming Gram-negative motile rods with catalase activity, 30% were pigmented and none of them were glucose fermenters. All the strains were grown in liquid cultures at 20°C and protease secretion was tested using the azocasein method. Despite their capacity for production of a clear zone of hydrolysis in agar plates, some strains did not produce detectable levels of proteolytic activity in liquid cultures. The lowest apparent optimum temperature for protease activity found in culture supernatants was 40°C. Almost all the strains showed activation energy values about 10-20 kJ-mol⁻¹ lower than that observed for a mesophilic Subtilisin. Most of the proteases showed optimal activity at neutral or alkaline pH values and developed a multiple-band profile on gelatine-SDS-PAGE. It was observed that the lower the strain isolation temperature was, the more stongly cold-adapted–in terms of optimal temperature and activation energy–were the proteases produced by them. This dependence of the characteristics of the proteases on the isolation temperature is an important factor to take into account in the design of screening programmes directed towards the isolation of psychrotolerant bacteria able to produce proteases strongly or weakly adapted to work in the cold. The Antarctic area explored proved to be a promising source of proteolytic bacteria with potential use in industrial processes to be carried out at low or moderate temperatures.     

盐度对北极海冰细菌生长的影响

从中国第二次北极科学考察采集的海冰样品中分离到8种细菌,研究了盐度对其生长的影响。结果表明,8株海冰细菌对盐度有着广泛的适应性,在盐度为30-90的范围内均可较快生长,其中菌株BJ2、BJ3和BJ7的盐耐受性较强。进一步的研究表明,BJ2、BJ3和BJ7在0-150 的盐度范围内均有不同程度的生长,盐度为0-30时,菌株的生长最快;盐度为180时,这三株菌株也均有短暂的生长。随着盐度的增加,三株菌株生长的延迟期增加,到达生长稳定期的时间也相应增加。温度对菌株BJ2、BJ3和BJ7的盐耐受性也有着一定的影响,随着温度的升高, 菌株的生长速度变慢,但盐耐受性有所增加。     

北极斯瓦尔巴群岛冰川流域可培养细菌的系统发育多样性及固氮菌初步研究

从北极斯瓦尔巴群岛冰川流域的上、中、下游采集了5种代表性土壤和冰川融水样品(MT、AT、TN、BT、MS),采用常规LB和严格无氮培养基低温培养方法,对其中的可培养细菌和潜在固氮细菌进行了总菌计数以及菌株的富集、分离与纯化。经菌落菌体形态观察、16S r RNA基因初步测序排重、固氮基因nif H的PCR检测,分别选取48株可培养分离细菌和31株潜在固氮菌进行了基于16S r RNA基因全序列测定的系统发育分析。目的是初步了解该特殊生境中可培养细菌(包括潜在固氮菌)的多样性及其分布状况,为进一步研究它们在此生境中的生态功能奠定基础。实验结果表明,可培养细菌在5个采集样品中的总菌数为BTTNATMT=MS,严格无氮培养基分离菌总菌数为BTATMTTNMS。48株可培养代表菌分属于Proteobacteria、Actinobacteria、Bacteroidetes、Firmicutes 4个门的7纲、14属之中,它们在5个样品中的多样性程度为BT(6纲、6属)MT(4纲、6属)AT、MS(均为3纲、3属)TN(2纲、3属),优势菌群主要分布在γ-proteobacteria纲的Pseudomonas属(20.8%,集中在AT、BT、MS样品中)和β-proteobacteria纲的Janthinobacterium属(35.4%,集中在MS样品中),表现出一定程度的系统发育多样性。潜在固氮菌实验结果表明,能在严格无氮培养基上生长且可检测到nif H的31株代表菌具有极为单一的系统发育多样性。其中90.32%菌株集中在γ-proteobacteria纲的Pseudomonas属,仅在MT和MS样品中有3株菌分布在α-proteobacteria纲的Rhizobium属、β-proteobacteria纲的Janthinobacterium属和Actinobacteria纲的Arthrobacter属,它们与分离自其他极端环境的相关标准菌株具有高度16S r RNA基因同源性。另外,本实验还发现有8株菌的16S r RNA基因序列与相关标准菌株的相似度低于98.50%,推测它们具有成为该特殊生境下新种的潜质。     

荒漠灌木内生固氮菌对环境因子的适应性研究

 以塔里木河下游荒漠灌木多枝柽柳(Tamarix ramosissima Ledeb.)、黑果枸杞(Lycium ruthenicum Murr.)组织中分离获得的5个内生固氮菌株为材料,研究了pH值、温度、含盐量、化合态氮含量、干旱对其生长和固氮能力的影响。结果表明,测定菌株对酸碱、温度适应范围广,在pH值5.0～10.0和15～40 ℃温度范围内均能生长并表达固氮酶活性; 菌株能耐受0.1%～5.0%NaCl、0.5～4 mmol·L-1NH+4、-0.3～-0.15 mPa渗透势,表现出较强的抗逆性能,但各菌株之间存在一定差异。     

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.)。     

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.     

Phylogenetic analysis of cultivable bacteria isolated from Arctic sea-ice

Phylogenetic analysis based on 16S rDNA of 8 strains of cultivable bacteria isolated from Arctic sea-ice was studied.The results showed that strain BJ1 belonged to genus Planococcus,which was a genus of low mole percent G+C gram-positive bacteria;strain BJ6 belonged to genus Burkholderia of β-proteobacteria and the rest 6 strain all belonged to γ-proteobacteria,of which strain BJ8 was a species of Pseudoalteromonas,strain BJ2-BJ5 and BJ7 were members of genus Psychrobacter.Phylogenetic analysis also indicated that bacteria of genus Psychrobacter of the isolates formed a relatively independent phylogenetic cluster in comparison with other bacteria belonged to genus Psychrobacter.     

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。     

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     

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。     

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.     

北极海洋放线菌R-527F的抗菌活性筛选、鉴定及其抗菌物质合成条件研究

对分离自北冰洋７３°Ｎ以北海域沉积物中的一株放线菌Ｒ５２７Ｆ进行分子鉴定及生物活性研究。通过对菌株的形态特征、培养性状、生理生化特征以及１６ＳｒＤＮＡ序列进行综合分析,确定该株菌为链霉菌属,暂定名为Ｓｔｒｅｐｔｏｍｙｃｅｓ ｓｐ．Ｒ５２７Ｆ。其含有聚酮类Ⅰ型（ＰＫＳⅠ）和非核糖体含硫多肽类（ｎｏｎｒｉｂｏｓｏｍａｌｐｅｐｔｉｄｅｓｙｎｔｈｅｔａｓｅ,ＮＲＰＳ）化合物的合成基因。对其抗菌活性及活性物质发酵条件进行了研究,实验结果表明,该菌株所产生的抗菌物质对枯草芽孢杆菌具有显著抑制效果,抑菌圈直径达到１８．２ｍｍ。适合其抗菌物质的发酵条件：种子液接种量为４％,培养基初始ｐＨ为８．０,培养温度为２８℃,培养时间为６ｄ,摇床转速为１８０ｒ?ｍｉｎ－１,装液量为４０％,为该菌株抗菌物质的进一步研究奠定发酵条件参数基础。     

一株分离自北极海水红球菌(Rhodococcus sp.)B7740的生长条件及其粗类胡萝卜素提取方法的优化

红球菌(Rhodococcus sp.)B7740分离自北冰洋海水样品,前期实验表明其能产生大量黄色素,但是该菌生物量很低。本实验通过碳源、氮源、培养温度、发酵液起始pH值、菌种接种量等单因素和正交实验优化了该菌株的培养条件,在此基础上,通过对提取剂种类及pH、提取时间、料液比等的研究,优化了色素的提取条件。实验结果表明,1L陈海水中,添加5g酵母粉,5g葡萄糖,3g乳糖,7g牛肉膏;起始pH7,培养温度25℃,菌种接种量1.0%,160r/min摇床培养7d时,生物量从6.1mg/mL上升到23.08mg/mL,提高了278.4%。以上述培养条件下得到的干菌体为材料,采用pH7的无水乙醇,当料液比1:30,浸提温度75℃,浸提1h重复提取2次时,色素含量为1160.625μg/g干菌体。对色素的分析表明,该色素为类胡萝卜素,实验结果为进一步研究极地微生物红球菌(Rhodococcus sp.)B7740产类胡萝卜素的提取工艺提供了基础。     

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.     

Spore-forming halophilic bacteria isolated from Arctic terrains: Implications for long-range transportation of microorganisms

Organisms living in the Arctic terrains such as Greenland have to deal with low temperature conditions. The mechanisms by which bacteria resist to low temperature are largely unknown; however, a well-known survival strategy of the microorganisms inhabiting the Arctic is spore forming. Moreover, halophilic bacteria are often resistant to various stresses. We have attempted isolation of spore-forming halophilic bacteria from Arctic terrains. We isolated 10 strains of spore-forming halophilic bacteria from the samples collected from a glacial moraine in Qaanaaq, Greenland in July 2007. Identification based on 16S rRNA gene sequence similarities showed that the isolates were closely related to the Oceanobacillus, Ornithinibacillus, Virgibacillus, Gracilibacillus, and Bacillus genera. In addition, the 16S rRNA sequences of some isolates were extremely similar to those of strains from the desert sand in China (100% identity, near full length), the source of the so-called “yellow dust.” Previous research indicated that yellow dust had been transported to Greenland by the wind. Our research implies the long-range transportation of these microorganisms to locations such as the Arctic.     

1981-2019年全球气温变化特征

1981—2019年全球气温变化特征是揭示全球气温变化的空间差异性以及实现全球共同应对气候变化的关键。本文基于7套再分析数据,采用气候变化速率及空间插值等分析方法,分析了1981—2019年全球气温变化时空特征及主要国家气温变化。结果表明：1981—2019年全球陆地气温以0.320 ℃/10a的速率呈极显著升高趋势,年平均气温增加了0.835 ℃;南、北半球陆地气温变化速率分别为0.147 ℃/10a、0.362 ℃/10a,均呈极显著增加趋势,分别增加了0.874 ℃、0.828 ℃。全球陆地80%面积上气温呈现显著增加趋势,年平均气温升高速率最大的区域位于80°N~90°N,其次是70°N~80°N、60°N~70°N,高纬大于中、低纬,格陵兰地区、乌克兰、俄罗斯等中高纬度国家或地区增温速率较快,尤以格陵兰地区增加速率最快,气温变化速率为0.654 ℃/10a;增温最慢的地区主要位于新西兰和赤道附近的南美洲、东南亚、非洲南部等地,气温变化速率不足0.15 ℃/10a。本文统计的146个国家中,年平均气温呈显著增加趋势的国家136个,占93%;气温无显著变化的国家10个,占6.849%。1981—2019年全球增温2.0 ℃以上、1.5 ℃以上、1.0 ℃以上的国家分别为4个、34个、68个,分别约占统计国家的2.740%、23.288%、46.575%。本文认为1998年以来全球并没有出现气温变暖停滞的现象。     

北极海洋放线菌R-527F的抗菌活性筛选、鉴定及其抗菌物质发酵条件研究

对分离自北冰洋73°N以北海域沉积物中的一株放线菌R-527F进行分子鉴定及生物活性研究.通过对菌株的形态特征、培养性状、生理生化特征以及16S rDNA序列进行综合分析,确定该株菌为链霉菌属,暂定名为Streptomyces sp.R-527F.其含有聚酮类Ⅰ型(PKS Ⅰ)和非核糖体含硫多肽类(nonribosomal peptide synthetase,NRPS)化合物的合成基因.对其抗菌活性及活性物质发酵条件进行了研究,实验结果表明,该菌株所产生的抗菌物质对枯草芽孢杆菌具有显著抑制效果,抑菌圈直径达到18.2 mm.适合其抗菌物质的发酵条件:种子液接种量为4％,培养基初始pH为8.0,培养温度为28℃,培养时间为6d,摇床转速为180 r·min-1,装液量为40％,为该菌株抗菌物质的进一步研究奠定发酵条件参数基础.     

北极Ny-Ålesund地区真菌多样性分析及龙胆苦苷转化菌株的筛选

采用多种培养基对北极高纬地区（土壤及海洋沉积物）真菌进行分离纯化,共得真菌65株。通过分析菌株的18S rDNA序列研究了它们的系统发育多样性,18S rDNA序列分析表明,65株真菌分属25个属,其中数量上位居第一的是青霉属（Penicillium）,有菌株20株,其次为丛赤壳属（Nectria）和节枝孢属（Articulospora）,各有菌株6株。同时对这65株真菌开展转化龙胆苦苷阳性菌株的筛选。以β-葡萄糖苷酶作为筛选转化菌株的标志酶于65株真菌中筛选到阳性菌株28株,其中产酶能力强的9株菌作为转化实验的备选菌株,以龙胆苦苷为底物进行转化,将转化粗提物进行TLC及HPLC分析,最终确定将C-5作为转化龙胆苦苷的菌株。根据菌株形态特征和核酸序列分析结果,初步鉴定菌株C-5为青霉属真菌。暂定名为Penicillium sp.C-5。     

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.