Isolation and characterization of a surfactant produced byBacillus licheniformis 86

Summary Surfactant (BL86) was isolated from foam produced during growth ofBacillus licheniformis 86 by acid-precipitation followed by extraction into tetrahydrofuran or methanol. The surfactant is anionic and dissolves in tetrahydrofuran, methanol, chloroform, dichloromethane, xylene, toluene, and alkaline water. The surfactant lowers the surface tension of water to 27 dynes/cm, and achieves the critical micelle concentration with as little as 10 g surfactant/ml. Its interfacial tension can reach 0.36 dynes/cm when measured in 4% sodium chloride againstn-hexadecane. The surfactant is stable from pH 4.0 to 13.0, at temperatures ranging from 25 to 120°C, and in salt solutions ranging from 0 to 30% NaCl. Preliminary analytical results indicate that the surfactant is a mixture of lipopeptides different from previously reportedBacillus produced surfactants.     

Isolation and structural analysis of bamylocin A, novel lipopeptide from Bacillus amyloliquefaciens LP03 having antagonistic and crude oil-emulsifying activity

Bacillus amyloliquefaciens strain LP03 isolated from soil, produced an antagonistic compound that strongly inhibited the growth of plant-pathogenic fungi and a lipopeptide biosurfactant. Also, isolated strain LP03 had a marked crude oil-emulsifying activity as it developed a clear zone around the colony after incubation for 24 h at 37°C. LP03 was identified as Bacillus amyloliquefaciens by analysis of partial 16 S rRNA gene and partial gyrA gene sequence. The lipopeptide was purified by acid precipitation of cell-free culture broth, extraction of the precipitates with methanol, silica gel column chromatography, and reverse-phase, high-pressure liquid chromatography. The purified biosurfactant was analyzed biochemical structure by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and electrospray ionization mass spectrometry/mass spectrometry (ESI-MS/MS). The masses of the two peaks were observed by HPLC chromatography. Their masses were determined to be 1,044 and 1,058 m/z with MALDI-TOF mass spectrometry. As constituents of the peptide and lipophilic part of the m/z 1,022.6, seven amino acids (Glu-Leu-Met-Leu-Pro-Leu-Leu) and β-hydroxy-C13 fatty acid were determined by ESI-MS/MS. The lipopeptide of 1,022.6 Da differed from surfactins in the substitution of leucine, valine and aspartic acid in positions 3, 4, and 5 by methionine, leucine, and proline, respectively. Novel lipopeptide was designated as bamylocin A.     

生物表面活性剂脂肽的发酵生产及抑菌应用研究进展*

表面活性素(surfactin)是一种环脂肽型生物表面活性剂,具有卓越的表/界面活性,能够显著降低水的表面张力,表现出良好的抗真菌、抗病毒、抗肿瘤、杀虫和抗支原体等生物活性,在医药、农业、食品、日化、石油开采等领域具有很大的应用潜力,但高昂的成本和缺乏竞争力的应用领域使其难以真正地实际应用起来。多年来,大量的研究工作在于促进其工业化应用。综述了surfactin的结构、特性及发酵生产,同时系统的比较和总结了surfactin在抑菌方面的应用研究。     

纳豆芽孢杆菌Bna05菌株产抗霉脂肽的鉴定

【目的】分析枯草芽孢杆菌纳豆菌亚种Bna05菌株代谢产物中脂肽类物质的存在情况,并探讨它们在抗霉功能中所发挥的作用。【方法】利用特异性引物对Bna05菌株进行脂肽合成酶类基因片段扩增、测序和BLAST比对分析;通过平板抑菌圈区域取样法获得Bna05菌株的高抗霉活性代谢产物,对该产物进行反相高效液相色谱(RP-HPLC)分离;用琼脂微稀释法测定分离物的抗霉活性,并对活性分离物进行质谱鉴定。【结果】Bna05菌株含有sfp和srf AA基因,未检测到itu C、itu D、fen D、fen ACE、bym B、bym C基因;RP-HPLC分离得到3组抗霉活性物质F_2、F_3和F_4,F_2中未检测到脂肽类物质,从F_3和F_4中分别鉴定出两类Surfactin同系物:V_7-surfactin和I/L_7-surfactin。两类Surfactin分别与F_2组合使用时,均表现出抗霉协同作用;此外,与Surfactin单独使用相比,两类Surfactin混合物与F_2组合后的协同抗霉活性得到进一步增强。【结论】Bna05菌株所产脂肽类物质主要是V_7-surfactin和I/L_7-surfactin,Surfactin与Bna05菌株所产其它活性物质之间存在抗霉协同作用,而V_7-surfactin和I/L_7-surfactin的同时存在,对于增强这种协同抗霉作用是有利的。     

Production, isolation and characterization of [Leu4]- and [Ile4]surfactins from Bacillus subtilis

Summary Bacillus subtilis coproduces several surfactin variants that are powerful biosurfactants and have potential applications in biology and industry. A single amino acid substitution in the heptapeptide moiety of surfactins strongly modifies their properties. To better establish structure-activity relationships and to search new variants with enhanced properties, Bacillus subtilis was grown into two modified culture media. Two new variants were isolated by chromatographic methods and studied by NMR spectroscopy. As planned, modifications consisted in the substitution of the l-valine residue at the fourth position by a more hydrophobic residue, i.e., leucine or isoleucine. These [Leu⁴]- and [Ile⁴]surfactins have a higher affinity for hydrophobic solvents and a twice improved surfactant power. Structure-property correlations were confirmed by analysis of the hydrophobic residue distribution in the three-dimensional model of the structure of surfactin in solution.Abbreviations DMSO dimethylsulfoxide - 1D, 2D, 3D one-, two-, three-dimensional - TLC thin layer chromatography - CMC critical micellar concentration - NMR nuclear magnetic resonance     

Antibiofilm Activity of Biosurfactant Producing Coral Associated Bacteria Isolated from Gulf of Mannar

Coral Associated Bacteria (CAB) (N = 22) isolated from the mucus of the coral Acropora digitifera were screened for biosurfactants using classical screening methods; hemolysis test, lipase production, oil displacement, drop collapse test and emulsifying activity. Six CAB (U7, U9, U10, U13, U14, and U16) were found to produce biosurfactants and were identified by 16S ribosomal RNA gene sequencing as Providencia rettgeri, Psychrobacter sp., Bacillus flexus, Bacillus anthracis, Psychrobacter sp., and Bacillus pumilus respectively. Their cell surface hydrophobicity was determined by Microbial adhesion to hydrocarbon assay and the biosurfactants produced were extracted and characterized by Fourier Transform Infrared spectroscopy. Since the biosurfactants are known for their surface modifying capabilities, antibiofilm activity of positive isolates was evaluated against biofilm forming Pseudomonas aeruginosa ATCC10145. Stability of the active principle exhibiting antibiofilm activity was tested through various temperature treatments ranging from 60 to 100 °C and Proteinase K treatment. CAB isolates U7 and U9 exhibited stable antibiofilm activity even after exposure to higher temperatures which is promising for the development of novel antifouling agents for diverse industrial applications. Further, this is the first report on biosurfactant production by a coral symbiont.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-014-0474-8) contains supplementary material, which is available to authorized users.     

Bacillus natto TK-1产脂肽的纯化、抑菌活性及其表面活性剂特性

利用酸沉、醇提和薄层层析等方法从Bacillus natto TK-1 发酵液中分离得到脂肽。TLC结果表明,在迁移值Rf 0.58-0.65处出现单一紫红色条带其为脂肽粗提物。脂肽的临界胶束浓度约115mg/L。在浓度为512mg/L时,脂肽能将水的表面张力显著地降低到30.1mN/m。同时,通过体外抗粘连实验表明,脂肽能显著抑制沙门氏菌、大肠杆菌和金黄色葡萄球菌对96孔板固体表面的粘附,其中,对沙门氏菌的抗粘连效果较为明显。通过平板扩散法考察脂肽抑菌活性,结果表明脂肽具有较广泛的抑菌谱,对灰霉和镰胞霉的抑菌能力较强。     

Brevibacillus brevis HOB1所产脂肽的分离纯化和鉴定

用常压反相色谱对从短短芽孢杆菌HOB1发酵液中提取到的脂肽类生物表面活性剂进行了分离纯化, 并用HPLC制备了其中的一个化合物。经电喷雾质谱分析得到该化合物的相对分子量为1035.7 D, GC/MS的分析结果显示其脂肪酸部分为C15 β-羟基脂肪酸, 由PITC柱前衍生法测得该化合物的氨基酸组成比例为: Asp:Glu:Val:Leu = 1:1:1:4。结果显示该脂肽的结构与表面活性素(C15 surfactin)类似。实验表明, 除芽孢杆菌属外, surfactin系列脂肽还能为短芽孢杆菌属所产生。     

Phenotypic variations in re-lysogenization ofBacillus licheniformis with temperate phage

A bacitracin-producing strainBacillus licheniformis ATCC 10716 harbors two types of inducible phages (LP52 and DLP 10716). 156 strains re-lysogenized with phage LP52 were independently isolated from a cured strain UM12 ofB. licheniformis. Those strains were divided into 12 groups based on colony morphology and pigment production. Some of the re-lysogenized strains grew faster than UM12 and others produced more bacitracin than the cured strain. For example, the production of bacitracin by one of the relysogenized strains, L89, was enhanced by about 70% in comparison with UM12. The phenotypic variations observed with re-lysogenized strains might be due to the re-insertion of the phage genome at different sites of the chromosome in addition to the pleiotropic effect assumed.Abbreviations ATCC American Type Culture Collection - DNA Deoxyribonucleic acid - MC Mitomycin C - OD Optical density - PFU Plaque forming unit - rpm Revolutions per minute - UOD Unit of optical density - UV Ultraviolet Definition Specific growth rate (h^-1) - t time (h) - X cell concentration (g/l)     

Isolation of a gene essential for biosynthesis of the lipopeptide antibiotics plipastatin B1 and surfactin in Bacillus subtilis YB8

Bacillus subtilis YB8 was found to produce the lipopeptide antibiotics surfactin and plipastatin B1. A gene, lpa-8, required for the production of both lipopeptides was cloned from strain YB8. When this gene was inactivated in strain YB8, neither surfactin nor plipastatin B1 was produced. However, the defective strain transformed with an intact lpa-8 gene had restored ability to produce both peptides. Nucleotide sequence analysis of the region essential for the production of the peptides revealed the presence of a large open reading frame. The deduced amino acid sequence of lpa-8 (224 amino acid residues) showed sequence similarity to that of sfp (from surfactin-producing B. subtilis), lpa-14 (from iturin A- and surfactin-producing B. subtilis), psf-1 (from surfactin-producing Bacillus pumilus), gsp (from gramicidin-S-producing Bacillus brevis), and entD (from siderophore-enterobactin-producing Escherichia coli), which are able to complement a defect in the sfp gene and promote production of the lipopeptide antibiotic surfactin. The sequence similarity among these proteins and the product similarity of cyclic peptides suggests that they might be involved in the biosynthesis or secretion of the peptides. Received: 14 July 1995 / Accepted: 22 December 1995     

Kinetic studies on surfactant production byPseudomonas aeruginosa 44T1

Summary Biosurfactant accumulation occurred in the exponential and stationary phases. Production started when the nitrogen level was very low. Surfactant was produced with a diauxic pattern. Rhamnolipid concentration increased as nitrogen levels increased. Maximum product yield (Y _p/x) 2.9 was detected when C/N ratio was 6.6 and specific rate of product formation (p _q) was calculated. The examination of these kinetics parameters such as product yield and specific rate of product formation should be taken into account to develop a high efficient production process.     

双歧杆菌对哮喘儿童DC表达CD86和HLA-DR的影响

目的研究双歧杆菌对过敏性哮喘儿童外周血单个核细胞来源的树突状细胞（DC）表面表达CD86和HLA-DR的影响。方法从12例过敏性哮喘儿童和10例对照组儿童的外周血单个核细胞诱导生成未成熟DC,分别经双歧杆菌或细菌脂多糖（LPS）处理,用流式细胞仪检测各组DC表面CD86和HLA-DR分子表达。结果双歧杆菌刺激后,哮喘儿童DC表面CD86表达明显增高（P〈0．05）,HLA-DR表达无明显变化（P〉0．05）,对照组儿童CD86和HLA-DR表达无明显影响;LPS刺激可明显增加哮喘儿童和对照组儿童CD86和HLA-DR的表达。结论过敏性哮喘儿童DC表面CD86的表达可能存在缺陷,双歧杆菌能适度上调其表达,在DC的成熟过程中可能起调节作用。     

Production and structural characterization of surfactin (C14/Leu7) produced by Bacillus subtilis isolate LSFM-05 grown on raw glycerol from the biodiesel industry

The production of biosurfactant by Bacillus subtilis LSFM-05 was carried out using raw glycerol, obtained from a vegetable oil biodiesel plant in Brazil, as the sole carbon source. Production of the biosurfactant was carried out in a 15-L bench-top fermentor and the surfactant was obtained from the foam produced. The crude surfactant was purified by silica gel column chromatography with a yield of 230 mg of the purified biosurfactant per liter of foam. TLC, IR spectroscopy, ¹H and ¹³C NMR and Fourier transform ion cyclotron resonance mass spectrometry with electrospray ionization (ESI-FTMS) were used to characterize the purified surfactant. The isolated surfactant was identified as a surfactin lipopeptide. MS/MS data identified the amino acid sequence as GluOMe-Leu-Leu-Asp-Val-Leu-Leu and showed that the fatty acid moiety contained 14 carbons in iso, anteiso or normal configurations. The critical micelle concentration of the C₁₄/Leu₇ surfactin was 70 μM, with emulsification efficiency after 24 h (E₂₄) of 67.6% against crude oil. Raw glycerol represents an abundant and renewable carbon source and provides an opportunity for reducing the cost of biosurfactant production and may add value to biodiesel production by creating new commercial applications for this by-product.     

Development of enzyme linked immunosorbent assays to measure Bm86 antigen of Boophilus microplus (cattle tick) and to detect anti-Bm86 antibodies in serum samples

The immunization of cattle with the Boophilus microplus Bm86 antigen has been successful for the control of cattle tick infestations. To monitor the Bm86 production process and to measure the anti-Bm86 antibody titers in vaccinated cattle, mAb-based ELISA were developed and validated. The development of both immunological methods is essential to obtain a product with high quality and immunogenic properties and to monitor the immunological protection induced in vaccinated cattle against B. microplus.     

Atrazine degradation by encapsulated Rhodococcus erythropolis NI86/21

AIMS: To develop an encapsulation procedure for Rhodococcus erythropolis NI86/21 and demonstrate its use as a slow-release inoculant for reducing atrazine levels in aquatic and terrestrial environments. METHODS AND RESULTS: Alginate encapsulation procedures were developed for the atrazine-degrading bacteria R. erythropolis NI86/21. Several bead amendments, including bentonite, powdered activated carbon (PAC) and skimmed milk (SM), were evaluated for slow release of R. erythropolis NI86/21 and efficacy of atrazine degradation. All bead types demonstrated a capacity to degrade atrazine in basal minimal nutrient buffer whilst continually releasing viable bacterial cells. We found that the addition of bentonite hastened cell release whilst SM sustained cell viability in bead formulations. Reducing the percentage of SM to 1% (w/v) resulted in faster rates of atrazine degradation in both liquid and soil, and was found to prolong cell survival upon bead storage. Limited oxygen transfer affects the capacity of the encapsulated R. erythropolis cells to degrade atrazine. CONCLUSIONS: Degradation studies have demonstrated the efficacy of R. erythropolis encapsulated cells to degrade atrazine in amended liquid and soil. However, in their current formulation, the wet alginate-based beads are impractical for field application because of their poor cell viability during storage. SIGNIFICANCE AND IMPACT OF THE STUDY: R. erythropolis NI86/21-encapsulated cells have the potential to reduce atrazine residues in a number of soil and water environments, possibly ensuring the continued registration and use of atrazine in agriculture by minimizing or eliminating nontarget effects.     

Thermostable phenylalanine dehydrogenase from a mesophilic Microbacterium sp. strain DM 86-1

Bacteria that produced NAD⁺-dependent phenylalanine dehydrogenase (EC 1.4.1.20) were selected among l-methionine utilizers isolated from soil. A bacterial strain showing phenylalanine dehydrogenase activity was chosen and classified in the genus Microbacterium. Phenylalanine dehydrogenase was purified from the crude extract of Microbacterium sp. strain DM 86-1 (TPU 3592) to homogeneity as judged by SDS-polyacrylamide disc gel electrophoresis. The enzyme has an isoelectric point of 5.8 and a relative molecular weight (M _r) of approximately 330,000. The enzyme is composed of eight identical subunits with an M _r of approximately 41,000. The apparent K _m values for l-phenylalanine and NAD⁺ were calculated to be 0.10 mM and 0.20 mM, respectively. No loss of the enzyme activity was observed upon incubation at 55° C for 10 min. Received: 30 July 1997 / Accepted: 4 November 1997     

Effect of Inducible Expressed Human Cytomegalovirus Immediate Early 86 Protein on Cell Apoptosis

Human cytomegalovirus is a common human pathogen that can cause life-threatening disease under certain conditions. During infection of host cells, the virus expresses regulatory proteins such as IE72 and IE86 that are important for viral propagation. IE86 plays a critical role in the modulation of viral replication as well as host cell cycle control and apoptosis. In this study, a Tet-On system was used to quantify the effect of IE86 on apoptosis and p53 expression. Our results indicate that IE86 inhibits tumor necrosis factor (TNF)-α induced apoptosis and that the anti-apoptotic activity of this viral protein correlates with its expression levels. In addition, IE86 did not alter the mRNA level of p53. The system developed should provide a method for functional analysis of human cytomegalovirus (HCMV) IE86 protein.     

Inhibition of Human Cytomegalovirus Infection by IE86-Specific Short Hairpin RNA-Mediated RNA Interference

To develop a gene therapeutic method for human cytomegalovirus (HCMV), the IE86 specific short hairpin (sh) RNA expressing vector was constructed and subsequently transfected into MRC-5 cells. After infection of these cells with HCMV AD169, expression of IE86 was reduced strikingly as compared to the control. In addition, the inhibitory effect corresponded to a decrease in viral DNA replication and the virus-induced cytopathic effect. Measurement of the virus yield demonstrated that infection of cells expressing IE86-specific shRNA resulted in suppression of the formation of infectious viral progeny. These observations indicate that IE86 can be used as an effective target against HCMV infection using RNA interference (RNAi) technology, which provides new possibilities for anti-HCMV studies.