常温环境中产嗜热蛋白酶菌资源

通过计数、分离与筛选,对常温环境嗜热菌和产嗜热蛋白酶菌的分布及资源状况进行了研究。结果表明,常温环境中存在着一定数量的嗜热菌和产嗜热蛋白酶菌。土壤与水体相比,其嗜热菌资源相对丰富,且耕作肥沃的土壤中产嗜热蛋白酶菌多于贫瘠土壤;在水环境中,无论湖水、江水还是处理中的废水,在常温条件下均有一定比例的嗜热菌和产嗜热蛋白酶菌。在啤酒废水曝气阶段,产嗜热蛋白酶菌占嗜热菌的比例较大,达45％。本研究筛选的1株嗜热菌其产嗜热蛋白酶活性较高,该菌株在pH7．6、温度68℃条件下其蛋白酶活力达到642U·ml^-1。该项研究为开发产嗜热蛋白酶菌资源,在工业和环境治理等方面的应用提供重要科学依据。     

嗜酸嗜热浸矿微生物

随着人们对浸矿菌的研究不断加深,嗜热嗜酸菌的浸矿潜力及在微生物冶金中的作用和地位得到认识,利用嗜热菌对矿石进行高效浸出已成为微生物冶金领域的研究重点。嗜热微生物包括中度嗜热微生物和极端嗜热微生物,主要栖息于热泉、工厂高温废水排放区以及火山口等高温环境中。本综述总结了嗜热浸矿微生物种类,分析了嗜中温菌和极端嗜热菌等嗜酸菌种的生长习性、利用的能源物质、浸矿能力等,并进一步介绍了嗜热嗜酸微生物在高温生物冶金中的发展及应用。     

地微生物学研究热点之一

地球表层或地球内部的一切极端环境中生存微生物是一个潜在的资源宝库。曾报道在温泉、深海热液出口交接处有嗜热细菌的存在,源于这些细菌含有很强的嗜极酶、如耐热酶、蛋白质及其他细胞组分,有些嗜热酶进入实用化,商品化。有的嗜热细菌全部基因组测序工作的完成,对嗜极酶基因组的研究与探索以及对嗜热菌适应极端环境的生存和繁衍与分子机制的研究有重要价值。这是深海火山口嗜热微生物研发的一个热点,也是地微生物学研究的新领域。英国“自然”(2005．元月)报道,地中海深处含盐量极高的4个盐盆地区,有一群活着的微生物,该地区水的含盐量极高,比酱油成2倍,这些微生物照样维持它们的生命活动。     

一种有效的嗜热真菌杜邦嗜热菌靶向基因替代系统

以嗜热真菌杜邦嗜热菌Thermomyces dupontii NRRL 2155为研究材料,利用同源重组原理和真菌原生质体转化方法,以潮霉素抗性基因替换嗜热真菌目标基因,获得抗潮霉素的靶向基因敲除突变菌株。优化的遗传转化体系为：用15mg/mL裂解酶,在28℃下酶解2g杜邦嗜热菌菌丝5.5h以获得原生质体,经STC缓冲液洗涤重悬后,利用PEG（polyethylene glycol）介导的遗传转化方式,将10μg线性敲除全长片段转化至杜邦嗜热菌原生质体中,通过潮霉素筛选及PCR验证得到基因替换突变菌株,同源重组率达到20%。本研究首次将原生质体转化方法应用在杜邦嗜热菌,并成功建立稳定高效的基因替换体系,为快速构建杜邦嗜热菌的遗传转化体系和研究该嗜热真菌的基因功能提供有效方法。     

嗜热酶的特性及其应用

海洋微生物作为一类生长在特殊极端环境下的生物正日益引起人们的重视。其中嗜热微生物由于能在高温温泉及火山口附近的高热环境下生长而引起人们的极大关注[1] 。同时 ,人们也从许多人工高热环境 (如堆肥 )中分离得到这种嗜热菌。近年来 ,人们从这些嗜热菌中已分离得到多种嗜热酶 (5 5℃～80℃ )及超级嗜热酶 (80℃～ 1 1 3℃ ) [2 ] 。嗜热酶不仅具有化学催化剂无法比拟的优点 ,如催化效率高和底物专一性强 ,而且酶在高温条件下的稳定性极好[3 ] 。因而它可以克服中温酶 (2 0℃～ 5 5℃ )及低温酶 (-2℃～ 2 0℃ )在应用过程中常常出现的…     

产高温蛋白酶微生物菌种资源的研究

对嗜温及嗜热微生物在不同环境中的分布及其产蛋白酶的情况进行了调查。研究结果显示从常温环境中也有可能分离到具有应用前景的嗜热菌菌株。     

超嗜热古菌基因组的热稳定性

超嗜热古菌能够生活在80℃以上的高温环境中,它们的耐热性已经成为当前研究的热点之一。以往对超嗜热菌的认识多集中于蛋白质的耐热性,而很少有关于基因组热稳定性的综述文章。综述了当前对超嗜热古菌的基因组稳定性以及DNA损伤识别机制的研究进展,以期更好地了解超嗜热古菌的耐热机制。     

极端嗜热菌的酶应用进展

极端嗜热菌一般在６０℃以上的环境中生长,其研究历史已持续一百多年。极端嗜热菌所产生的酶,因其耐高温（５０℃─９４℃）,酶活力性能稳定,已被广泛应用在聚合酶链反应,糖发酵,以及蛋白质、淀粉、纤维素和脂肪的分解等工艺技术方面,显示出了可喜的苗头。有关极端嗜热菌的基因结构及其嗜热机理正在探索之中,可以预见人们将采用酶工程技术,开发出更多的嗜热菌酶制剂市场。     

嗜热性古核菌的研究进展

古核菌是有区别于细菌,真菌独特的生理,生化特性。特别是嗜热古核菌的发现,扩大了生物酶资源,使许多高温反应得以实现。但是由于野生菌培养条件苛刻,生长周期长,限制了对其的进一步应用,目前主要采用定向进化的方法进行体外重组来获得大量的目标蛋白。来自于嗜热菌的嗜热酸通常对酸,碱,有机溶剂等有较好的抗性,特别是热稳定性好,其稳定机制与结构密切相关,一级结构中疏水,带电荷及芳香族氨基酸相对较多,二级结构中α螺旋结构更加稳定,从高级结构看,嗜热蛋白质含有大量的盐桥,氢键,离子对等与稳定性相关的因素。     

超嗜热羧酸酯酶的性质和应用研究

摘要 来源于超嗜热菌的超嗜热羧酸酯酶,结构上与激素敏感性脂肪酶(HSL)家族近似,属于α/β-水解酶,但是其空间结构比HSL更加紧密而有韧性,具有很强的热稳定性。性质研究表明,温度和有机溶剂对超嗜热羧酸酯酶的活性和对映选择性影响显著;其最适底物一般是对硝基苯酯,部分酯酶的基因含有GGGX基序,能够水解叔醇酯结构。由于超嗜热羧酸酯酶的独特结构和性质,其应用潜力巨大,尤其在拆分手性的外消旋酯方面独具优势。     

The dynamic nature of thermophily

1. Evidence for a close relation between thermophilic and mesophilic bacteria is discussed. 2. It is shown that in the absence of nutrients thermophilic bacteria at 55°C. die as rapidly as mesophilic bacteria, and that enzyme systems of the thermophils are rapidly inactivated at this temperature. 3. It is concluded that the thermophils can live at high temperatures because they can synthesize enzymes and other cellular constituents faster than these are destroyed by heat. 4. In order to account for this great synthetic capacity at high temperatures, and for the high minimum temperatures observed for many thermophils, it is postulated that these organisms have a higher temperature coefficient of enzyme synthesis than mesophils.     

Thermophilic,lignocellulolytic bacteria for ethanol production: current state and perspectives

Lignocellulosic biomass contains a variety of carbohydrates, and their conversion into ethanol by fermentation requires an efficient microbial platform to achieve high yield, productivity, and final titer of ethanol. In recent years, growing attention has been devoted to the development of cellulolytic and saccharolytic thermophilic bacteria for lignocellulosic ethanol production because of their unique properties. First of all, thermophilic bacteria possess unique cellulolytic and hemicellulolytic systems and are considered as potential sources of highly active and thermostable enzymes for efficient biomass hydrolysis. Secondly, thermophilic bacteria ferment a broad range of carbohydrates into ethanol, and some of them display potential for ethanologenic fermentation at high yield. Thirdly, the establishment of the genetic tools for thermophilic bacteria has allowed metabolic engineering, in particular with emphasis on improving ethanol yield, and this facilitates their employment for ethanol production. Finally, different processes for second-generation ethanol production based on thermophilic bacteria have been proposed with the aim to achieve cost-competitive processes. However, thermophilic bacteria exhibit an inherent low tolerance to ethanol and inhibitors in the pretreated biomass, and this is at present the greatest barrier to their industrial application. Further improvement of the properties of thermophilic bacteria, together with the optimization production processes, is equally important for achieving a realistic industrial ethanol production.     

Thermus thermophilus as biological model

Thermus spp is one of the most wide spread genuses of thermophilic bacteria, with isolates found in natural as well as in man-made thermal environments. The high growth rates, cell yields of the cultures, and the constitutive expression of an impressively efficient natural competence apparatus, amongst other properties, make some strains of the genus excellent laboratory models to study the molecular basis of thermophilia. These properties, together with the fact that enzymes and protein complexes from extremophiles are easier to crystallize have led to the development of an ongoing structural biology program dedicated to T. thermophilus HB8, making this organism probably the best so far known from a protein structure point view. Furthermore, the availability of plasmids and up to four thermostable antibiotic selection markers allows its use in physiological studies as a model for ancient bacteria. Regarding biotechnological applications this genus continues to be a source of thermophilic enzymes of great biotechnological interest and, more recently, a tool for the over-expression of thermophilic enzymes or for the selection of thermostable mutants from mesophilic proteins by directed evolution. In this article, we review the properties of this organism as biological model and its biotechnological applications.     

Lipase activity of thermophilic bacteria from icelandic hot springs

Summary Several bacteria strains were choosen from pre-selected strains for further testing and characterisation. Hydrolytic activity of lipases from thermophilic bacteria was examined using olive oil as a substrate at different reaction temperatures. Alcoholytic activity was also investigated. Lipases from thermophilic bacteria have been successfully produced on a large scale. To be able to predict if these lipases can be used for transesterification reactions, these preparations need to be purified further or to be cloned.     

Thermophilic bacteria: Ecology, physiology and technology

Thermophilic bacteria are common in soil and volcanic habitats and have a limited species composition. Yet they possess all the major nutritional categories and metabolize the same substrates as mesophilic bacteria. The ability to proliferate at growth temperature optima well above 60°C is associated with extremely thermally stable macromolecules. As a consequence of growth at high temperature and unique macromolecular properties, thermophilic bacteria can possess high metabolic rates, physically and chemically stable enzymes, and lower growth but higher end product yields than similar mesophilic species. Thermophilic processes appear more stable, rapid and less expensive, and facilitate reactant activity and product recovery. Thermophilic bacteria have application in chemical feedstock and fuel production, bioconversion of wastes, enzyme technology, and single cell protein production. This paper reviews the fundamental and applied aspects of thermophilic bacteria that are of potential industrial interest.     

Elucidation of the taxonomic status of industrial strains of thermophilic lactic acid bacteria by sequencing of 16S rRNA genes

Phenotypic characteristics and results of PCR tests for the presence of species-specific genes indicate that a number of strains of thermophilic lactic acid bacteria previously considered as belonging to Streptococcus thermophilus are actually closely related to enterococci. In the present study, partial (over 500 nucleotides) sequencing of 16S rRNA genes from 12 strains of thermophilic lactic acid bacteria used as starters for manufacturing sour milk products on the territory of the Commonwealth of Independent States (CIS) has been performed. According to the results of the sequencing, seven of the strains have been classified with Enterococcus durans. The earlier classification (based on PCR tests) of two of the strains as S. thermophilus and three of the strains as E. faecium has been confirmed. The data obtained demonstrate that the enterococci E. durans and E. faecium are widely used as thermophilic starters for manufacturing sour milk products on the territory of the CIS.     

Protein surface amino acid compositions distinctively differ between thermophilic and mesophilic bacteria

One of the well-known observations of proteins from thermophilic bacteria is the bias of the amino acid composition in which charged residues are present in large numbers, and polar residues are scarce. On the other hand, it has been reported that the molecular surfaces of proteins are adapted to their subcellular locations, in terms of the amino acid composition. Thus, it would be reasonable to expect that the differences in the amino acid compositions between proteins of thermophilic and mesophilic bacteria would be much greater on the protein surface than in the interior. We performed systematic comparisons between proteins from thermophilic bacteria and mesophilic bacteria, in terms of the amino acid composition of the protein surface and the interior, as well as the entire amino acid chains, by using sequence information from the genome projects. The biased amino acid composition of thermophilic proteins was confirmed, and the differences from those of mesophilic proteins were most obvious in the compositions of the protein surface. In contrast to the surface composition, the interior composition was not distinctive between the thermophilic and mesophilic proteins. The frequency of the amino acid pairs that are closely located in the space was also analyzed to show the same trend of the single amino acid compositions. Interestingly, extracellular proteins from mesophilic bacteria showed an inverse trend against thermophilic proteins (i.e. a reduced number of charged residues and rich in polar residues). Nuclear proteins from eukaryotes, which are known to be abundant in positive charges, showed different compositions as a whole from the thermophiles. These results suggest that the bias of the amino acid composition of thermophilic proteins is due to the residues on the protein surfaces, which may be constrained by the extreme environment.     

Endospores of thermophilic bacteria as tracers of microbial dispersal by ocean currents

Microbial biogeography is influenced by the combined effects of passive dispersal and environmental selection, but the contribution of either factor can be difficult to discern. As thermophilic bacteria cannot grow in the cold seabed, their inactive spores are not subject to environmental selection. We therefore conducted a global experimental survey using thermophilic endospores that are passively deposited by sedimentation to the cold seafloor as tracers to study the effect of dispersal by ocean currents on the biogeography of marine microorganisms. Our analysis of 81 different marine sediments from around the world identified 146 species-level 16S rRNA phylotypes of endospore-forming, thermophilic Firmicutes. Phylotypes showed various patterns of spatial distribution in the world oceans and were dispersal-limited to different degrees. Co-occurrence of several phylotypes in locations separated by great distances (west of Svalbard, the Baltic Sea and the Gulf of California) demonstrated a widespread but not ubiquitous distribution. In contrast, Arctic regions with water masses that are relatively isolated from global ocean circulation (Baffin Bay and east of Svalbard) were characterized by low phylotype richness and different compositions of phylotypes. The observed distribution pattern of thermophilic endospores in marine sediments suggests that the impact of passive dispersal on marine microbial biogeography is controlled by the connectivity of local water masses to ocean circulation.     

Crystal structure of histidine-containing phosphocarrier protein from <Emphasis Type="Italic">Thermoanaerobacter tengcongensis</Emphasis> MB4 and the implications for thermostability

Protein thermostability is an inherent characteristic of proteins from thermophilic microorganisms, and therefore enables these organisms to survive at extreme temperatures. Although it is well-known that thermostable proteins are critical for the growth of thermophilic organisms, the structural basis of protein thermostability is not yet fully understood. The histidine-containing phosphocarrier (HPr) protein, a phosphate shuttle protein in the phosphoenolpyruvate-dependent sugar transport system (PTS) of bacterial species, is an ideal model for investigating protein thermostability with respect to its small size and deficiency in disulphide bonds or cofactors. In this study, the HPr protein from Thermoanaerobacter tengcongensis (TtHPr) is cloned and purified. Crystal structure with good quality has been determined at 2.3 Å resolution, which provides a firm foundation for exploring the thermostable mechanism. However, it shows that the crystal structure is conserved and no clue can be obtained from this single structure. Furthermore, detailed comparison of sequence and structure with the homologs from meso- or thermophilic bacteria shows no obvious rule for thermostability, but the extra salt-bridge existing only in thermophilic bacteria might be a better explanation for thermostability of HPr. Thus, mutations are performed to interrupt the salt-bridge in HPrs in thermophilic bacteria. Using site-directed mutations and the circular dichroism method, thermostability is evaluated, and the mutational variations are shown to have a faster denaturing rate than for wild-type viruses, indicating that mutations cause instability in the HPrs. Understanding the higher-temperature resistance of thermophilic and hyperthermophilic proteins is essential to studies on protein folding and stability, and is critical in engineering efficient enzymes that can work at a high temperature.     

Isolation and Characterization of Thermophilic Bacteria from Gavmesh Goli Hot Spring in Sabalan Geothermal Field,Iran: Thermomonas hydrothermalis and Bacillus altitudinis Isolates as a Potential Source of Thermostable Protease

Abstract

Thermophilic bacteria have attracted great attention due to their ability to produce thermostable enzymes. The aim of this study was the isolation and characterization of thermophilic bacteria from Gavmesh Goli hot spring in Sareyn, North West of Iran. Of 10 water samples collected, 36 thermophilic bacteria were obtained. The thermophilic bacteria were tested for their ability to produce hydrolase enzymes. All the isolates were potentially protease producers. Lipase, DNase, and amylase activities were confirmed in 34 (94.4%), 8 (22.2%), and 3 (8.3%) isolates, respectively. Five isolates with higher levels of enzyme activity were selected for further studies. Morphological, biochemical, and molecular analysis by 16S rRNA gene sequencing revealed that four isolates (DH15, DH16, DH20, and DH29) could be identified as Thermomonas hydrothermalis and one (PA10) Bacillus altitudinis. The protease produced by these isolates was optimally active at 50–55?°C, pH 8–8.5, and 0–0.5?M NaCl. In this first time study, we isolated T. hydrothermalis and B. altitudinis from Iranian hot springs and demonstrated the characteristics of T. hydrothermalis protease. Accordingly, due to the valuable potential of these bacteria such as the production of protease with high temperature and pH stability, these isolates can be introduced as promising candidates for industrial applications.