电喷雾质谱法检测假单胞菌BS-03产鼠李糖脂及其自由酸前体(英文)

采用电喷雾质谱法可同时检测到假单胞菌BS-03发酵液提取物中的鼠李糖脂及其自由酸前体(3-羟基葵酸单体和二聚体)。根据ESI-MS/MS的双鼠李糖脂二级质谱图显示存在强度较高的m/z为205、247的特征碎片离子峰,而单鼠李糖脂中却不存在此特征碎片离子。同时自由酸前体的主要组分为C8,C10,C8C10/C10C8,C10C10。其同分异构体与鼠李糖脂同分异构体类似,其中短链位于羟基端的组分含量高于长链在羟基端的组分。     

不同结构配比的鼠李糖脂乳化活性与油泥清洗效果

以大庆油田原油和含油污泥为对象,研究不同结构配比鼠李糖脂表面活性剂乳化活性及其对含油污泥清洗效果的影响,并优化清洗工艺参数。结果表明:单鼠李糖脂比例越高,其表面活性越好;双鼠李糖脂比例越高,其对原油的乳化能力越强;临界胶束浓度随着双鼠李糖脂比例的增加而逐渐加大;单、双鼠李糖脂配比不同的表活剂对油泥的清洗效果也不同,质量比为50∶50时清洗效率最高;鼠李糖脂浓度为1.0 g·L^-1、热洗时间为1.5h、热洗温度为65℃、洗脱强度为220 r·min^-1、固液质量比为1∶5条件下,油泥的清洗效率最高,可达81.3%;含油率为29.6%的落地油泥,经一级洗涤后油泥残油率降至5.5%,原油回收率达到87.3%,清洗出的原油无明显乳化,易于分离。由此可知,鼠李糖脂的单、双糖脂比例不同对其理化性质和清洗含油污泥的效果均有不同程度的影响。     

9株海洋来源铜绿假单胞菌合成鼠李糖脂的比较分析

目的:从海洋来源的铜绿假单胞菌中筛选多株具有鼠李糖脂合成能力的菌株。方法:以9株分离自不同海洋环境的铜绿假单胞菌为研究对象,考察并比较其发酵合成鼠李糖脂生物表面活性剂的表面活性、产量和产物成分的差异,扩增并比对合成途径中的关键基因。结果:9株菌的发酵产物均具有表面活性,其中菌株1A01151发酵液的表面活性最强,表面张力值可降低至28 m N/m;9株菌的基因组中均含有鼠李糖脂合成途径中关键基因rhl AB和rhl C,都具有合成单、双鼠李糖脂的能力;菌株1A01151和1A00364的发酵产量最高(2.69 g/L),产物经LC-MS/MS检测,所合成的鼠李糖脂同系物组分不同,双糖双脂的含量最高(1A01151:75.96%;1A00364:61.01%)。结论:海洋来源的铜绿假单胞菌是具有鼠李糖脂高产潜力的菌株,可用于合成性能不同、组成多样的鼠李糖脂生物表面活性剂。     

以甘油为底物鼠李糖脂高产菌株的诱变选育

通过诱变选育,将铜绿假单胞菌(Pseudomonas aeruginosa)RG-14利用甘油发酵生产鼠李糖脂产量由13.6g/L提高到16.5 g/L。突变株经过5次连续传代培养,菌株仍维持稳定的鼠李糖脂产量,表明该菌株具有较好的遗传稳定性。利用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)技术分析诱变后菌株发酵甘油生产鼠李糖脂的组成,结果显示鼠李糖脂由Rha-C8-C8、Rha-C8-C10、Rha-C10-C10、Rha-C10-C12∶1、Rha-C10-C12、Rha2-C8-C10、Rha2-C10-C10、Rha2-C10-C12∶1和Rha2-C10-C12组成,其中单、双鼠李糖脂的相对丰度分别为54.8%和45.2%。当以工业粗甘油代替精甘油为底物时,该菌株鼠李糖脂产量达到14.2 g/L,表明其具有较好的应用潜力。     

以抽油烟机废油为原料发酵产鼠李糖脂的研究

鼠李糖脂是一种具有巨大潜力的阴离子生物表面活性剂,可应用于石油、食品、农业、日化工业等领域。探讨以抽油烟机废油为碳源发酵产鼠李糖脂的可能性,以铜绿假单胞菌WB505为出发菌体,在7 L发酵罐中鼠李糖脂的产量达到12.3±0.52 g/L。利用基质辅助激光解析飞行时间质谱(MALDI-TOF MS)分析出所产鼠李糖脂的组成,结果显示其主要含Rha-C_(10)-C_(10)和Rha_2-C_(10)-C_(10),其中单鼠李糖脂和双鼠李糖脂的总相对丰度分别为49.7%和50.3%。所产鼠李糖脂的临界胶束浓度(CMC)为45.0 mg/L,能将表面张力从60.5±0.81 mN/m降至25.3±0.68 mN/m,乳化系数E24均60%,并且对苯的乳化系数达到80.3±0.85%。以抽烟机废油为底物生产鼠李糖脂降低底物成本,为抽油烟机废油提供一种循环再利用处理方式。     

鼠李糖脂对微生物降解石油烃废水的影响

目的:研究鼠李糖脂对微生物降解石油烃废水的影响.方法:通过测定生物量和观察菌株表面来研究鼠李糖脂对菌株的影响;通过正交实验设计,确定石油烃降解率影响因素.通过石油烃降解率的测定,探讨鼠李糖脂与H2O2深度氧化协同作用对微生物降解石油烃的影响.结果:菌株对石油烃的降解率达53%,在相同条件下,添加鼠李糖脂的石油烃降解率提高了12%-20%.添加鼠李糖脂后菌株的生物量明显增多,菌株细胞表面疏水.正交设计表明,影响石油烃降解的主导因子是培养温度,其次是培养时间和鼠李糖脂的添加量.正交设计得到最佳组合为A3B2C1,即培养时间为7d;温度为35℃,鼠李糖脂浓度为60mg/L.3个因素的最佳组合下,石油烃降解率为82%.加入200 mg/L的H2O2时,降解率从82%提高到97%.结论:鼠李糖脂能促进菌株的生长.鼠李糖脂与H2O2深度氧化协同作用有助于微生物对石油烃类污染物降解效率的提高.     

铜绿假单胞杆菌O-2-2产鼠李糖脂的发酵培养基优化及LC-MS/MS分析

鼠李糖脂是一种性能优良的生物表面活性剂,在生物医药、环境保护、二次采油等方面具有很高的应用潜力.采用响应面分析法,对铜绿假单胞杆菌O-2-2的培养基进行了优化.Plackett-Burman(PB)实验设计表明,磷酸盐、硝酸盐和微量元素对鼠李糖脂的产量具有显著影响.Box-Behnke (BB)优化确定最佳培养基组成为磷酸盐、硝酸盐和微量元素用量分别为3.2g/L、13.76g/L和5.17ml,理论的最大产量为8.48g/L,与实测糖脂产量8.85g/L接近.摇瓶优化后的鼠李糖脂产量较优化前的6.24g/L提高了30.8％.最优化条件下采用10％的接种量逐级放大,并通过补料发酵,最终200L罐的鼠李糖脂产量达到70g/L,发酵时间仅为110h.采用新发明的二次蒸馏工艺,鼠李糖脂纯度达86.6％.液质联用(LC-MS)分析表明所生产的鼠李糖脂成分及含量为:双糖单脂32.9％、双糖双脂17.02％、单糖单脂3.16％、单糖双脂33.54％.     

产鼠李糖脂生物表面活性剂大肠杆菌的构建与优化

目前鼠李糖脂生物表面活性剂主要由条件致病的铜绿假单胞菌生产获得,从而影响工业应用。为了开发一种相对安全的鼠李糖脂生产菌,将带有不同强度组成型合成启动子的鼠李糖基转移酶基因(Rhamnosyltransferase gene,rhl AB)以单、中、高3种拷贝数分别在大肠杆菌ATCC 8739中异源表达,实现了不同产量的鼠李糖脂异源合成。对rhl AB基因和rha BDAC基因簇(TDP-L-鼠李糖合成的基因簇)进一步利用合成启动子进行组合调控,筛选获得了最优生产鼠李糖脂工程菌——大肠杆菌TIB-RAB226。对大肠杆菌TIB-RAB226进行发酵温度优化,鼠李糖脂产量达到124.3 mg/L,是优化前的1.17倍。通过分批补料发酵,12h时鼠李糖脂产量达到209.2 mg/L。对发酵产物进行高效液相色谱-质谱联用技术分析,共检出相对含量变化的5类质核比不同的鼠李糖脂同系物。本研究可为异源合成产鼠李糖脂提供重要参考。     

废弃食用油脂生物合成鼠李糖脂研究进展

碳源的成本过高限制了鼠李糖脂的工业化生产及应用,废弃食用油脂作为一种廉价易得的碳源,越来越多的研究者开始关注用它发酵生产鼠李糖脂.废弃食用油脂的种类、投加量对鼠李糖脂的产量、结构、性质均会产生影响,目前研究中用废弃食用油脂作碳源,鼠李糖脂产量最高可达24.61g/L、表面张力最低达到24mN/m、产物CMC最低可达40.19mg/L.此外,本文还总结了菌株、氮源、微量元素、pH、溶氧及培养方式等因素对废弃食用油脂生产鼠李糖脂的影响,并展望了利用废弃食用油脂生产鼠李糖脂实现产业化的重点研究方向.     

强磁场重力环境对Pseudomonas aeruginosa N1207的影响

旨在研究由超导磁体产生的强磁场重力环境(HMGE)对Pseudomonas aeruginosa N1207的影响。该磁体是经特殊设计的大梯度超导磁体,可产生多个重力梯度(从失重到超重)以及相应的磁场强度,分别为(0 g,12 T)、(1 g,16 T)和(2 g,12 T)。Pseudomonas aeruginosa N1207在HMGE中分别诱变24h、48h和72h之后,从磁场强度最高(16 T)组别中筛选得到最佳的突变株M14808。结果表明M14808的鼠李糖脂产量提高了30%以上并且遗传特性稳定。比较生长周期发现,突变株到达指数生长期早于原始菌株。诱变实验结果表明强磁场是引起诱变的主要因素从而导致鼠李糖脂产量变化和生长周期改变。抗肿瘤实验结果显示双鼠李糖脂可抑制四株肿瘤细胞,分别为MCF-7、H460、HepG2和A549,双鼠李糖脂对MCF-7的效果最佳,其IC₅₀为125.13μg/ml。     

Identification and production of a rhamnolipidic biosurfactant by a Pseudomonas species

 A glycolipid-producing bacterium, Pseudomonas aeruginosa GL1, was isolated from the soil contaminated with polycyclic aromatic hydrocarbons (PAH) from a manufactured gas plant. The glycolipid produced was characterized in detail by chromatographic procedures as a mixture of four rhamnolipids, consisting of different associations of rhamnose and hydroxy fatty acids: the main component was monorhamnosyl di-3-hydroxydecanoic acid. The rhamnolipid composition presented marked analogies with a defined part of P. aeruginosa outer membrane lipopolysaccharides (lipopolysaccharide band A). Rhamnolipid production was stimulated under conditions of nitrogen limitation. Glycerol yielded higher productions than did hydrophobic carbon sources. Cell hydrophobicity decreased during growth on glycerol and on n-hexadecane whereas glycolipid production increased. P. aeruginosa GL1 was found to be unable to grow on a variety of 2, 3 and 4 cycle PAH. However, it was shown to persist after at least 12 subcultures in a bacterial population growing on a mixture of pure PAH, suggesting a physiological role for rhamnolipid as a means to enhance PAH availability in a mutualistic PAH-degrading bacterial community. Received: 4 July 1995/Received revision: 7 September 1995/Accepted: 13 September 1995     

Characterization of an extracellular glycolipid from <Emphasis Type="Italic">Lentinus edodes</Emphasis> (Berk.) Sing [<Emphasis Type="Italic">Lentinula edodes</Emphasis> (Berk.) Pegler]

Submerged mycelium of a xylotrophic basidiomycete Lentinus edodes produces an extracellular glycolipid, S3, associated with a lectin. Galactose glycan residue, as well as the lipid pool composition, which includes nonhydroxylated short-chain fatty acids, is uncommon for basidiomycetes. The glycolipid consists of D-galactopyranose (15% of S3 contains galactose sulfate) acylated by octadecanoic and nonadecanoic fatty acid residues (28 and 72%, respectively). The glycolipid structure and composition are confirmed by physicochemical analysis. The glycolipid is assumed to be a regulator of lectin activity.     

Cefixime–tellurite rhamnose MacConkey agar for isolation of Vero cytotoxin-producing Escherichia coli serogroup O26 from Scottish cattle and sheep faeces

Aims:  To compare rhamnose MacConkey agar supplemented with cefixime and tellurite (CT-RMac) and tryptone bile X-glucuronide (TBX) agars as isolation media for Vero cytotoxin-producing Escherichia coli (VTEC) serogroup O26 from animal faeces.
Methods and Results:  Nine VTEC O26 were isolated from sheep faeces; out of which six were isolated only on CT-RMac and one was isolated only on TBX. One hundred and twelve VTEC O26 were isolated from calf faeces; out of which 97% were from CT-RMac and 52% were from TBX. In a study of E. coli O26 strains, 84% of VT-positive O26 did not ferment rhamnose when compared with 16% of VT-negative O26. VT-positive (19%) and VT-negative (39%) E. coli O26 strains did not grow on CT-RMac agar.
Conclusions:  It is important to consider that VTEC O26 strains either may ferment rhamnose or may be sensitive to the CT supplement of CT-RMac agar.
Significance and Impact of the Study:  This work compares CT-RMac and TBX agars as isolation medium for VTEC O26 from Scottish animal faeces and highlights that VTEC O26 may be missed if only CT-RMac agar is used.     

Characterization of Pseudomonas paucimobilis FP2001 Which Forms Flagella Depending upon the Presence of Rhamnose in Liquid Medium

A bacterial strain FP2001 isolated from the exudate of land reclaimed for municipal waste was identified as Pseudomonas paucimobilis. Cells of strain FP2001 were mobile by means of polar monotrichous flagellum, only when rhamnose was added as a carbon source in the liquid medium. The replacement of rhamnose by arabinose, galactose, glucose or xylose did not lead to the formation of flagella.     

Pseudomonas aeruginosa rhamnolipids: biosynthesis and potential applications

Pseudomonas aeruginosa produces and secretes rhamnose-containing glycolipid biosurfactants called rhamnolipids. This review describes rhamnolipid biosynthesis and potential industrial and environmental applications of rhamnolipids. Rhamnolipid production is dependent on central metabolic pathways, such as fatty acid synthesis and dTDP-activated sugars, as well as on enzymes participating in the production of the exopolysaccharide alginate. Synthesis of these surfactants is regulated by a very complex genetic regulatory system that also controls different P. aeruginosa virulence-associated traits. Rhamnolipids have several potential industrial and environmental applications including the production of fine chemicals, the characterization of surfaces and surface coatings, as additives for environmental remediation, and as a biological control agent. Realization of this wide variety of applications requires economical commercial-scale production of rhamnolipids. Received: 4 February 2000 / Received revision: 9 June 2000 / Accepted: 9 June 2000     

Rapid analysis of Listeria monocytogenes cell wall teichoic acid carbohydrates by ESI-MS/MS

We report the application of electrospray ionization (ESI) mass spectrometry for compositional characterization of wall teichoic acids (WTA), a major component of gram-positive bacterial cell walls. Tandem mass spectrometry (ESI-MS/MS) of purified and chemically hydrolyzed monomeric WTA components provided sufficient information to identify WTA monomers and their specific carbohydrate constituents. A lithium matrix was used for ionization of uncharged WTA monomers, and successfully applied to analyze the WTA molecules of four Listeria strains differing in carbohydrate substitution on a conserved polyribitol-phosphate backbone structure. Carbohydrate residues such as N-acetylglucosamine or rhamnose linked to the WTA could directly be identified by ESI-MS/MS, circumventing the need for quantitative analysis by gas chromatography. The presence of a terminal N-acetylglucosamine residue tethered to the ribitol was confirmed using fluorescently labeled wheat-germ agglutinin. In conclusion, the mass spectrometry method described here will greatly facilitate compositional analysis and characterization of teichoic acids and similar macromolecules from diverse bacterial species, and represents a significant advance in the identification of serovar-specific carbohydrates and sugar molecules on bacteria.     

Identification of the glycosyl transferase required for synthesis of the principal glycolipid characteristic of heterocysts of Anabaena sp. strain PCC 7120

Nitrogenase is oxygen-labile. Cyanobacterial heterocysts can fix N(2) in an oxic milieu because their interior is micro-oxic, for which the glycolipid layer of the heterocyst envelope is required. ORF all5341 of the Anabaena sp. genome predicts a glycosyl transferase. An insertional mutant of all5341 synthesized only a nonglycosylated form of heterocyst envelope glycolipid, and lacked a glycolipid layer. All5341 appears to be the transferase required to glycosylate the glycolipid aglycone.     

Lipid changes during development of Blastocladiella emersonii

The lipid composition of swimming spores, cysts and five hour germlings was established. Spores utilized triglycerides first, then phospholipids. Upon encystment all glycolipid components decreased, while in germlings the phospholipids, monoglycerides and sterol esters exhibited a marked increase.