嗜盐古菌分类学研究进展

嗜盐古菌是一类需要高盐维持生长的古菌。到目前为止,已发现的嗜盐古菌都属于古菌域的广古菌门,主要包括:嗜盐甲烷古菌类群、嗜盐古菌纲的全部成员以及尚不能培养的纳米嗜盐古菌类群。嗜盐古菌是盐环境的土著类群,驱动着盐环境生态系统的生物地球化学循环。作为极端微生物,嗜盐古菌在理论研究和应用领域具有重要的研究价值。本文从嗜盐古菌分类学地位的变迁、分类学方法、分类学研究现状及我国的嗜盐古菌分类学研究等方面综述了嗜盐古菌分类学的最新研究进展。     

新疆罗布泊地区可培养嗜盐古菌多样性及其功能酶筛选

【目的】探索新疆罗布泊地区高盐环境可培养嗜盐古菌的多样性及其功能酶应用潜力。【方法】采集罗布泊地区13份土样,用纯培养并结合基于16S rRNA基因系统发育分析的方法来研究样品中嗜盐古菌的多样性。按系统进化树的聚类关系,挑选出一些菌株进行盐度耐受及淀粉酶、蛋白酶、酯酶的酶活检测。【结果】从13份土样中共分离到56株嗜盐古菌,经16S rRNA基因克隆测序,通过MEGA 4.0构建N-J树分析,56株菌分布于嗜盐古菌的10个生效发表属和5个潜在新属。运用Shannon-Wiener方法计算其多样性指数为1.820。挑选17株嗜盐古菌所测试盐浓度实验结果表明这一批嗜盐古菌的大部分生长范围在10%-35%之间,最适盐浓度在20%-25%之间。不同酶活检测结果为:淀粉酶酶活率为70.6%,蛋白酶酶活率为35.3%,酯酶酶活率为82.4%。【结论】新疆罗布泊周边地区由于气候及地理位置的独特性,蕴藏丰富的嗜盐古菌资源。本实验所设计的分离方法对嗜盐古菌的分离是极其有效的,为进一步研究新疆罗布泊及周边地区嗜盐古菌资源提供了技术基础。盐度耐受实验结果验证在低盐环境中分离嗜盐古菌新物种的可行性。同时,嗜盐古菌的酶活比率较高且活性较强为进一步开发利用嗜盐古菌资源提供了理论依据。     

中度嗜盐菌在生物技术中的应用

生存于盐环境下的中度嗜盐菌在生物技术方面具有很多潜在的应用价值。其中,中度嗜盐菌在盐发酵食品加工业和食品添加剂中已经被广泛应用;由中度嗜盐菌分泌的胞外酶如淀粉酶、脂肪酶等能够在高盐环境下继续保持较高的活力;中度嗜盐菌细胞内积累的多种类相容性溶质也可以作为生物大分子稳定剂以及抗冻剂等;其它如其产生的生物表面活性剂、多聚糖类物质能够在石油回收和生物修复中进行应用等。     

盐渍海带盐中可培养嗜盐古菌多样性、胞外酶活及拮抗作用探究

盐渍海带盐是一种高盐环境,其中可能生存有嗜盐微生物,如嗜盐古菌。嗜盐古菌是一类生活于高盐环境的极端环境微生物。为了探究盐渍海带盐中嗜盐微生物的物种多样性,筛查分离了菌株的几种常见胞外功能酶活性和拮抗活性。采用纯培养技术,从盐渍海带盐样品中分离培养嗜盐微生物,并对其16S rRNA基因进行扩增和测序;基于16S rRNA基因序列的相似度分析,确定分离菌株在属级水平的分类地位;依据种属信息,挑选代表菌株,进行胞外常见功能酶活性和拮抗作用测定。从盐渍海带盐样品中分离到来自Haloarcula（盐盒菌属）、Halorubrum（盐红菌属）、Halarchaeum、Halobacterium（盐杆菌属）、Halococcus（盐球菌属）、Halolamina（盐尊片形菌属 ）和Haloplanus（盐扁平菌属）等7个属的131株嗜盐古菌;检测到产胞外蛋白酶菌株1株,产酯酶菌2株,产明胶酶菌7株,产氧化酶菌1株和产触菌酶5株;此外,筛选到6株具有拮抗活性的菌株,其中来自盐红菌属菌株Halorubrum sp. ZSA68较其他菌株生长快,产抑菌活性物质快,并显示出较强的抑菌活性和较广的抑菌谱,初步推测Halorubrum sp. ZSA68所产物质为多肽或蛋白类抑菌活性物质,其分子量大小约为30~50 kDa,该抑菌活性物质在60 ℃以上或NaCl浓度低于2%时容易失去活性。通过本研究认识了海带盐中可培养嗜盐古菌的分布,获得多株具有较高胞外酶活性及具拮抗作用的菌株,积累了丰富的嗜盐古菌菌株资源,这在高盐食品的防腐方面具有潜在的应用价值。     

微生物嗜盐酶的研究进展

嗜盐酶一般来自于嗜盐菌,它的主要特点是严格依赖体系中一定的盐离子浓度,可以在高盐环境中维持其结构稳定,并且能够抵抗高温、p H和有机溶剂存在下的变性,因此在高盐、水/有机和非水介质环境的催化中具有重要的应用价值。本综述从盐对嗜盐酶活性和稳定性的影响、金属离子和有机溶剂对嗜盐酶的影响几个方面介绍了嗜盐酶的特点。在总结蛋白质数据库(PDB)中已有嗜盐酶的结构和特点的基础上,对嗜盐酶的嗜盐机制进行了分析,认为嗜盐酶不同于非嗜盐酶的特点在于盐桥和氢键明显增多,含有一些特殊的盐离子结合位点并且常以低聚体的形式存在,表面酸性氨基酸含量明显增多。最后对嗜盐酶的分子改造和应用进行了简要的介绍。     

嗜盐菌耐盐机制相关基因的研究进展

嗜盐微生物能够在高盐环境中生存,其耐盐机制一直是微生物学家研究的热点。目前嗜盐微生物耐盐机制的研究主要集中在细胞吸K+排Na+作用、胞内积累小分子相容性溶质及嗜盐酶的氨基酸组成特性三个方面。文章从基因水平综述了嗜盐菌的耐盐机制,并对其在高盐废水处理上的应用进行讨论与展望。     

岱山盐场可培养嗜盐菌的多样性及其产酶活性筛选

从岱山盐场采集样品,利用选择性培养基分离培养嗜盐菌,对盐田环境中可培养嗜盐菌的多样性及产酶活性进行研究.共分离得到181株嗜盐菌菌株,通过真细菌和古生菌两对通用引物扩增其16S rRNA 基因,并采用限制性内切酶Hinf I进行ARDRA(amplified rDNA restriction analysis)多态性分析,共分为21个不同的操作分类单元(operation taxonomy units, OTUs),其中嗜盐细菌有12个OTUs,嗜盐古菌有9个OTUs.选取具有不同酶切图谱的代表菌株进行克隆测序,BLAST 比对及系统发育分析将嗜盐细菌归于7个属,其中嗜盐单胞菌属(Halomonas)的菌株数占优势,是嗜盐细菌总数的46.8%;嗜盐古菌归于4个属,盐盒菌属(Haloarcula)的菌株数占优势,是嗜盐古菌总数的49.1%.对分离菌株的产酶活性进行检测表明,岱山盐田环境蕴含丰富的产淀粉酶、蛋白酶和脂肪酶等生物活性酶的嗜盐菌, 其中盐盒菌属产酶菌株数最丰富.研究结果表明,岱山盐田环境中具有较为丰富的嗜盐菌多样性,是筛选产酶菌株的重要资源库.     

嗜盐古菌启动子的研究及应用

嗜盐古菌是古菌域的一个重要代表类群,在遗传与代谢、进化与适应、前沿生物技术及合成生物学领域都显示了其重要的研究价值。嗜盐古菌启动子的认识和利用,可以为嗜盐古菌的基础和应用研究提供必要的条件。本文从古菌启动子的结构与功能出发,就启动子的研究方法、嗜盐古菌启动子的特征及嗜盐古菌启动子的应用3个方面综述了嗜盐古菌启动子的研究现状,并对嗜盐古菌启动子未来研究的重点和方向进行了展望。     

安徽定远盐矿可培养嗜盐微生物多样性

【背景】嗜盐微生物多生活于高盐环境,具有独特的生理代谢特征,是一类重要的极端环境微生物资源。【目的】为更好地认识我国陆相盐矿的嗜盐微生物多样性组成,更好地开发利用嗜盐微生物资源积累丰富的微生物菌种。【方法】对安徽定远盐矿盐芯样品进行嗜盐微生物的纯培养分离,并对所分离菌株进行基于16SrRNA基因的测序和序列相似性分析,并对所分离菌株进行物种多样性分析。在此基础上,对代表菌株进行菌落形态和耐盐度及酶活测定。【结果】通过纯培养共分离获得了嗜盐微生物264株,其中嗜盐古菌150株,占56.8%;嗜盐细菌114株,占43.2%。嗜盐古菌物种分别来自于Halorubrum、 Halopenitus、 Haloterrigena、 Natrinema、 Natronoarchaeum和Natronomonas等6个属;嗜盐细菌物种分别来自于Pseudomonas、Aliifodinibius、Halobacillus、Halomonas和Halospina等5个属。通过代表菌株的酶活平板检测,发现产胞外蛋白酶菌株1株,酯酶1株,淀粉酶2株;能液化明胶菌株2株。在物种多样性组成方面,发现嗜盐古菌的物种多样性指数高于嗜盐细菌。【结论】本研究对我国安徽定远陆相盐矿的可培养嗜盐微生物多样性进行探究,积累了丰富的嗜盐微生物菌株资源。     

极端嗜盐古生菌(Natrinema sp.)R6-5胞外嗜盐蛋白酶的纯化和性质研究

采用bacitracin-Sepharose 4B亲和层析的方法得到凝胶电泳均一的来自极端嗜盐古生菌(Natrinema sp.)R6-5的胞外嗜盐蛋白酶。经SDS-PAGE分析该酶亚基分子量为62kDa。PMSF对它的活性完全抑制,表明它是一种丝氨酸蛋白酶,该酶反应的最适NaCl浓度为3mol/L,最适温度为45℃,最适pH值为8.0。在高盐条件下能维持高活性并十分稳定,具有重要的潜在应用价值。     

Haloarchaea: worth exploring for their biotechnological potential

Halophilic archaea are unique microorganisms adapted to survive under high salt conditions and biomolecules produced by them may possess unusual properties. Haloarchaeal metabolites are stable at high salt and temperature conditions that are useful for industrial applications. Proteins and enzymes of this group of archaea are functional under salt concentrations at which bacterial counterparts fail to be active. Such properties makes haloarchaeal enzymes suitable for salt-based applications and their use under dehydrating conditions. For example, bacteriorhodopsin or the purple membrane protein present in halophilic archaea has the most recognizable applications in photoelectric devices, artificial retinas, holograms etc. Haloarchaea are also useful for bioremediation of polluted hypersaline areas. Polyhydroxyalkanoates and exopolysccharides produced by these microorganisms are biodegradable and have the potential to replace commercial non-degradable plastics and polymers. Moreover, halophilic archaea have excellent potential to be used as drug delivery systems and for nanobiotechnology by virtue of their gas vesicles and S-layer glycoproteins. Despite of possible applications of halophilic archaea, laboratory-to-industrial transition of these potential candidates is yet to be established.     

Cloning,expression, and efficient purification in Escherichia coli of a halophilic nucleoside diphosphate kinase from the moderate halophile Halomonas sp. #593

Most typical halophilic enzymes from extremely halophilic archaea require high concentrations of salt for their activity and stability. These enzymes are inactive in Escherichia coli unless refolded in the presence of salts in vitro. In this report, we describe cloning of the ndk gene of nucleoside diphosphate kinase from a moderately halophilic eubacterium and overexpression of the protein in E. coli as an N-terminal hexa-His fusion to facilitate its purification on Ni-NTA affinity resin. We demonstrate evidence that the protein is properly folded and exhibits the same specific activity and stability as the native protein from Halomonas cells.     

Halophilic enzymes: proteins with a grain of salt

Halophilic enzymes, while performing identical enzymatic functions as their non-halophilic counterparts, have been shown to exhibit substantially different properties, among them the requirement for high salt concentrations, in the 1-4 M range, for activity and stability, and a high excess of acidic over basic amino residues. The following communication reviews the functional and structural properties of two proteins isolated from the extremely halophilic archaeon Haloarcula marismortui: the enzyme malate-dehydrogenase (hMDH) and the 2Fe-2S protein ferredoxin. It is argued that the high negative surface charge of halophilic proteins makes them more soluble and renders them more flexible at high salt concentrations, conditions under which non-halophilic proteins tend to aggregate and become rigid. This high surface charge is neutralized mainly by tightly bound water dipoles. The requirement of high salt concentration for the stabilization of halophilic enzymes, on the other hand, is due to a low affinity binding of the salt to specific sites on the surface of the folded polypeptide, thus stabilizing the active conformation of the protein.     

Characterization and regulation of the osmolyte betaine synthesizing enzymes GSMT and SDMT from halophilic methanogen Methanohalophilus portucalensis

The halophilic methanoarchaeon Methanohalophilus portucalensis can synthesize the osmolyte betaine de novo in response to extracellular salt stress. Betaine is generated by the stepwise methylation of glycine to form sarcosine, N, N-dimethylglycine and betaine by using S-adenosyl-L-methionine (AdoMet) as the methyl donor. The complete gene cluster of Mpgsmt-sdmt was cloned from Southern hybridization and heterologous expressed in E. coli respectively. The recombinant MpGSMT and MpSDMT both retained their in vivo functional activities in E. coli BL21(DE3)RIL to synthesize and accumulate betaine and conferred elevated survival ability in betaine transport deficient mutant E. coli MKH13 under high salt stress. The dramatic activating effects of sodium and potassium ions on the in vitro methyltransferase activities of MpGSMT, but not MpSDMT or bacterial GSMT and SDMT, revealed that GSMT from halophilic methanoarchaeon possesses novel regulate mechanism in betaine biosynthesis pathway. The circular dichroism spectra showed the fluctuated peaks at 206 nm were detected in the MpGSMT under various concentrations of potassium or sodium ions. This fluctuated difference may cause by a change in the β-turn structure located at the conserved glycine- and sarcosine-binding residue Arg167 of MpGSMT. The analytical ultracentrifugation analysis indicated that the monomer MpGSMT switched to dimeric form increased from 7.6% to 70% with KCl concentration increased from 0 to 2.0 M. The level of potassium and sodium ions may modulate the substrate binding activity of MpGSMT through the conformational change. Additionally, MpGSMT showed a strong end product, betaine, inhibitory effect and was more sensitive to the inhibitor AdoHcy. The above results indicated that the first enzymatic step involved in synthesizing the osmolyte betaine in halophilic archaea, namely, GSMT, may also play a major role in coupling the salt-in and compatible solute (osmolyte) osmoadaptative strategies in halophilic methanogens for adapting to high salt environments.     

Increase of salt dependence of halophilic nucleoside diphosphate kinase caused by a single amino acid substitution

Nucleoside diphosphate kinase (HsNDK) from an extremely halophilic archaea, Halobacterium salinarum, is composed of a homo hexamer, assembled as a trimer of basic dimeric units. It requires >2 M NaCl for refolding, although it does not require NaCl for stability or enzymatic activity below 30 °C. A HisN111L mutant with an N-terminal extension sequence containing hexa-His tag, in which Asn111 was replaced with Leu, was designed to be less stable between basic dimeric units. This mutant can lose between 6 and 12 hydrogen bonds between basic dimeric units in the hexamer structure. The HisN111L mutant had enhanced salt requirements for enzymatic activity and refolding even though the secondary structure of the HisN111L mutant was confirmed to be similar to the control, HisNDK, in low and high salt solutions using circular dichroism. We reported previously that G114R and D148C mutants, which had enhanced interactions between basic dimeric units, showed facilitated refolding and stabilization in low salt solution. The results of this study help to elucidate the process for engineering industrial enzymes by controlling subunit–subunit interactions through mutations.     

巴里坤湖和玛纳斯湖嗜盐菌的分离及功能酶的筛选

目的:了解新疆巴里坤湖与马纳斯湖中嗜盐菌及功能酶的多样性。方法:从两湖中采集水样进行菌种分离,采用PCR方法扩增出其16S rRNA基因(16S rDNA),并测定了基因的序列。对分离菌株进行了蛋白酶、淀粉酶、酯酶、脂肪酶、以及纤维素酶的筛选。结果:从两湖水样共分离得到51株嗜盐菌。基于16SrDNA序列的同源性比较和系统发育学分析,发现从两湖分离获得的中度嗜盐菌分别属于Planococcaceae、Bacillacea、Staphylococcus、Halomonadaceae、Salicolaceae以及Pseudomonadacaeae 6个属。分离得到的极端嗜盐古菌属于Halobacteriaceae属。功能酶筛选结果表明产蛋白酶的嗜盐菌共有15株,产酯酶的共有23株,产淀粉酶的共有8株,未获得产脂肪酶和纤维素酶的嗜盐菌。结论:新疆巴里坤湖和马纳斯湖中有丰富的嗜盐微生物资源及酶资源,有重要的研究意义和应用前景。     

乌勇布拉克盐湖嗜盐古菌絮凝效果筛选及活性检测

【目的】从嗜盐古菌中筛选可产生生物絮凝剂的菌株,对发酵液、上清液、菌悬液、胞外聚合物的絮凝作用进行检测,筛选能够适应高盐废水处理,且具有广谱盐度及pH作用范围的微生物絮凝剂。【方法】以新疆乌勇布拉克干盐湖沉积物为研究对象,利用纯培养方法对嗜盐古菌进行分离,对絮凝菌株进行初筛及16S rRNA基因测序,构建系统进化树,初步判断菌株分类地位;复筛检测不同生物材料的絮凝效果;选择絮凝效果较好的生物材料,检测其盐度、pH的絮凝效果稳定性。【结果】采用纯培养方法共分离到28株嗜盐古菌,絮凝初筛共筛选出16株嗜盐古菌,分布于碱线菌属(Natrinema)、盐缓长菌属(Halopiger)和盐土生菌属(Haloterrigena)。菌株发酵液、上清液、菌悬液、胞外聚合物具有不同程度的絮凝效果。菌株A279-1、A133、RP33、NGA0064、RM-152、A389的发酵液、上清液的絮凝效果较好,其中菌株A389的发酵液絮凝率为61.06%,上清液为67.92%。所有菌株菌悬液的絮凝率达到80%以上。菌株所产胞外聚合物表现出较好的絮凝效果,菌株RM-152所产胞外聚合物的絮凝率最高,达89.86%,其次是A389 (81.53%)。菌株A389所产胞外聚合物的产量最大,达12.53 g/L,具有广泛的盐度和pH适应性。【结论】乌勇布拉克干盐湖沉积物中蕴含丰富的可产生微生物絮凝剂的嗜盐古菌资源。嗜盐古菌菌株发酵液、上清液、菌悬液及胞外聚合物均具有良好的絮凝作用,尤其是胞外聚合物表现出较好的絮凝效果,具有广谱的盐度和pH耐受性。嗜盐古菌所产生物絮凝剂的发现对于后续高盐废水功能材料开发具有重要应用价值。