枯草芽孢杆菌B2菌株产生的表面活性素变异体的纯化和鉴定

利用6 mol/L HCl沉淀枯草芽孢杆菌B2菌株的去细胞培养液,甲醇抽提获得脂肽类抗生素粗提物,过Sephadex LH20层析柱获得粗纯化物,经MALDITOFMS检测表明B2菌株仅含有表面活性素一种脂肽类抗生素。利用HPLC SMART SYSTEM,将粗纯化物过μRPC C2/C18层析柱对表面活性素变异体进行分离后获得纯化物。经MALDITOFPSDMS对纯化物的结构分析表明,B2菌株的表面活性素变异体由13、14和15个碳原子的脂肪酸链以及L-Glu-L-Leu-D-Leu-L-Val-L-Asp-D-Leu-L-Leu七环肽组成。     

解淀粉芽孢杆菌TF28抗菌脂肽芬芥素的分离鉴定及抑菌作用

对一株解淀粉芽孢杆菌TF28产生的抗菌脂肽进行分离鉴定及抑菌活性研究,采用酸沉淀、乙酸乙酯和甲醇萃取技术制备了抗菌脂肽粗提物,经过2次HPLC分离纯化,在保留时间32～42min内获得8个抗菌脂肽纯品,经MALDI-TOF-MS鉴定为芬芥素(fengycins),对尖孢镰刀菌和禾谷镰刀菌显示出较强的抑菌活性,该研究为提高菌株TF28抗菌脂肽产量的定向遗传改造奠定基础。     

芽胞杆菌产生的脂肽类抗生素的结构和应用

脂肽类抗生素不仅对细菌、真菌有抗菌作用,而且对支原体、寄生虫和病毒等也具有显著的抑制作用。其主要包括表面活性素(Surfactins)、伊枯草菌素(Iturins)和芬芥素(Fengycins),其中Iturins和Fengycins有抗真菌活性,Surfactins有抗支原体、抗病毒等活性。综述了芽胞杆菌产生的脂肽类抗生素的结构、功能和应用,为其在农业和医药的研究与应用提供参考。     

可可西里低温适生拮抗芽孢杆菌的筛选鉴定及脂肽化合物分析

极端环境特殊微生物资源研究和开发具有广阔的应用前景和研究意义.对分离筛选自青海可可西里境内植被根围的8株在4和10 ℃条件下生长良好的低温适生芽孢杆菌进行鉴定分析.结果表明: 通过生理生化特征分析、rep-PCR指纹图谱分析、16S rDNA及gyrB基因序列分析鉴定,8株供试菌株分别为莫哈韦芽孢杆菌(Bacillus mojavensis)3株,解淀粉芽孢杆菌(B. amyloliquefaciens)1株和简单芽孢杆菌(B. simplex)4株.采用平板对峙试验从中筛选到4株对油菜菌核病原真菌(Sclerotinia sclerotiorum)及水稻白叶枯病原细菌(Xanthomonas oryzae pv. oryzae)均具有显著拮抗活性的生防菌株;采用MALDI-TOF-MS质谱分析生防菌株的拮抗活性物质,结果显示菌株KKD-1 (B. mojavensis)产生脂肽类化合物泛革素和表面活性素,菌株KKD-2(B. amyloliquefaciens)产生脂肽类化合物伊枯草菌素A、泛革素和表面活性素,推断生防菌株的拮抗活性可能与脂肽化合物的合成及分泌有关.该研究为低温适生性芽孢杆菌生物肥料和生物农药的研发提供了菌株资源.     

可可西里低温适生拮抗芽孢杆菌的筛选鉴定及脂肽化合物分析

极端环境特殊微生物资源研究和开发具有广阔的应用前景和研究意义.对分离筛选自青海可可西里境内植被根围的8株在4和10℃条件下生长良好的低温适生芽孢杆菌进行鉴定分析.结果表明:通过生理生化特征分析、rep-PCR指纹图谱分析、16S rDNA及gyrB基因序列分析鉴定,8株供试菌株分别为莫哈韦芽孢杆菌(Bacillus mojavensis)3株,解淀粉芽孢杆菌(B.amyloliquefaciens)1株和简单芽孢杆菌(B.simplex)4株.采用平板对峙试验从中筛选到4株对油菜菌核病原真菌(Sclerotinia sclerotiorum)及水稻白叶枯病原细菌(Xanthomonasoryzae pv.oryzae)均具有显著拮抗活性的生防菌株;采用MALDI-TOF-MS质谱分析生防菌株的拮抗活性物质,结果显示菌株KKD1(B.mojavensis)产生脂肽类化合物泛革素和表面活性素,菌株KKD2(B.amyloliquefaciens)产生脂肽类化合物伊枯草菌素A、泛革素和表面活性素,推断生防菌株的拮抗活性可能与脂肽化合物的合成及分泌有关.该研究为低温适生性芽孢杆菌生物肥料和生物农药的研发提供了菌株资源.     

原生质体融合选育高产脂肽LI-F多粘类芽胞杆菌及融合菌株表达差异分析

本研究以多粘类芽胞杆菌JSa-9前期诱变获得的两株带有营养缺陷型标记的菌株N1-37(Phe–)和N2-27(His–)作为亲本菌株,采用聚乙二醇作为促融剂,进行原生质体融合,筛选出高产LI-F类抗菌脂肽的融合菌株。通过HPLC对融合菌株和原始菌株产LI-F类抗菌脂肽进行定量检测,并利用实时荧光定量PCR对菌株JSa-9和融合菌株中LI-F类抗菌脂肽合成酶的关键基因fus A1、fus A2、fus C1和fus C2的差异性表达进行分析。结果表明,经过原生质融合获得一株LI-F类抗菌脂肽高产菌株F5-15。菌株F5-15的LI-F类抗菌脂肽产量为原始菌株产量的3.1倍,LI-F类抗菌脂肽合成酶的4个关键基因在融合菌株F5-15中的表达量分别是其在原始菌株JSa-9中的10.48、2.48、2.1和11.8倍。     

脂肽-糖脂混合生物表面活性剂产生菌筛选和优化培养

结合脂肽和糖脂的性能优势,致力于产脂肽-鼠李糖脂混合型生物表面活性剂的新菌株选育和培养条件优化。采用血平板溶血圈法初筛菌株、改进排油圈法快速检测产量以及飞行时间质谱鉴定产物结构。对优选菌株的碳源、氮源和磷酸盐缓冲液、重要金属离子浓度等进行了单因子和正交试验,优化了培养基和培养条件。采用高压液相色谱和蒽酮比色法定量分析了产物组成。筛选获得了同时积累糖脂和脂肽的新菌株,鉴定命名为芽胞杆菌Bacillus subtilis THY-7。摇瓶分批培养48 h,细胞OD600为37.0,产物浓度2.4 g/L,分别是优化前的3.4倍和3.1倍。发酵罐补料分批培养,泡沫中产物浓度达到4.5 g/L,且74％为表面活性素,22％为鼠李糖脂。B. subtilis THY-7是具有脂肽-鼠李糖脂高产潜力的优选菌株。     

枯草芽孢杆菌JA产生的脂肽类抗生素－iturin A的纯化 及电喷雾质谱鉴定

枯草芽孢杆菌JA产生的抗生素对植物病原真菌具有广谱抗性,明确抗生素的种类是进一步研究的基础.用6mol/L盐酸沉淀JA菌株的去菌体培养基,再用甲醇抽提获得抗生素的粗提物.利用反相HPLC系统,将粗提物过Diamonsil C18柱,收集有抗小麦赤霉病等病原真菌活性的化合物1、2.运用电喷雾质谱法(ESI/MS)测得其分子量分别为1042.4D和1056.5D.再利用碰撞诱导解离(CID)技术获得化合物的典型结构特征离子碎片,结果表明分子量为1042.4D的化合物一级结构为Pro-Asn-Tyr-βAA-Asn-Tyr-Asn-Gln(βAA为14个碳原子的氨基脂肪酸),属于脂iturin A.化合物1、2为相差一个亚甲基(-CH2)的iturin A同系物.研究结果提供了一种从枯草芽孢杆菌发酵液中快速分离纯化和鉴定脂肽类抗生素iturin A的新方法.     

枯草芽胞杆菌B006产表面活性素的培养条件优化

芽胞杆菌产生的环脂肽类生物表面活性素surfactin具有重要抗菌、促进生物膜形成等功能,其含量和同系物组分构成影响其功能。为了提高芽胞杆菌B006产生表面活性素的产量,通过单因素实验和正交实验,测定了碳源、氮源和无机盐等营养物质及接种量、培养时间、装液量和初始pH值对芽胞杆菌B006摇瓶发酵产生表面活性素的影响;采用排油圈法测定发酵液中表面活性素的产量,采用HPLC-MS方法比较培养基优化前后surfactin的产量和组分含量。结果表明,适合芽胞杆菌B006摇瓶发酵产生表面活性素的培养基组成为:牛肉膏10 g/L、玉米粉15 g/L、硝酸铵3 g/L和氯化钠3 g/L;适合的培养条件为:初始pH值7.0、接种量10%、装液量60 m L/500 m L,发酵周期64 h。优化后surfactin的产量约为314.73 mg/L,比优化前提高了74.88%;surfactin各组分比例发生改变,其中C16和C17组分的含量明显提高,为优化前相应组分含量的1.64和8.34倍。优化的培养基组分和培养条件可明显提高芽胞杆菌B006菌株产生surfactin的产量,改变surfactin同系物组分的构成,为利用芽胞杆菌B006进行高活性表面活性素的工业生产和代谢调控研究奠定了基础。     

地衣芽胞杆菌FJAT-4脂肽结构鉴定及其对尖孢镰刀菌的抑制作用

【目的】地衣芽胞杆菌FJAT-4产生的脂肽能够有效抑制尖孢镰刀菌的生长,本研究的目的在于探究地衣芽胞杆菌FJAT-4脂肽结构,分析培养基组分和培养温度对FJAT-4产抑菌脂肽的影响,阐述脂肽对尖孢镰刀菌的抑制作用,为菌株抑菌机理的阐释及其在枯萎病防治中的推广应用奠定基础。【方法】通过酸沉醇提法提取地衣芽胞杆菌FJAT-4产生的脂肽;利用液相色谱串联四极杆飞行时间质谱进行地衣芽胞杆菌FJAT-4脂肽组成分析及结构鉴定;以抑菌圈大小为指标评估地衣芽胞杆菌FJAT-4脂肽对尖孢镰刀菌的抑制效果;通过扫描电镜观察地衣芽胞杆菌FJAT-4粗脂肽对尖孢镰刀菌的抑制作用。【结果】地衣芽胞杆菌FJAT-4产生的抑菌脂肽由C_(17)fengycin A、C_(17)fengycin B、C_(17)fengycin B_2、C_(16)fengycin A衍生物、C_(16)fengycin B衍生物、C_(13)–C_(15)surfactin及C_(13)–C_(15)surfactin衍生物组成,其中C_(13)–C_(15) surfactin衍生物(m/z[M+Na]~+=1048.6/1062.6/1076.6)为新化合物。培养基成分不同对菌株FJAT-4脂肽组成影响较小,但温度对菌株FJAT-4产生抑菌脂肽的影响很大,该菌株在较低温度(20–25°C)下培养不产生脂肽,30–40°C下培养能产生抑菌脂肽,且高温有利于提高脂肽中surfactin的比例。该脂肽类物质对辣椒、番茄、香蕉和甜瓜尖孢镰刀菌等多种植物病原真菌均具有很好的抑制效果,且呈剂量依赖性。扫描电镜结果表明地衣芽胞杆菌FJAT-4所产的脂肽会严重影响辣椒、番茄、香蕉和甜瓜尖孢镰刀菌菌丝的正常生长,导致菌丝断裂变形、孢子变形或显著抑制了孢子的生长。【结论】地衣芽胞杆菌FJAT-4产生的抑菌脂肽为fengycin和surfactin类物质,该抑菌脂肽会致使尖孢镰刀菌菌丝体发育畸形,影响尖孢镰刀菌的正常生长。     

Isolation and characterization of a biosurfactant producing strain, <Emphasis Type="Italic">Brevibacilis brevis</Emphasis> HOB1

Biosurfactant-producing bacteria were isolated from the production water of an oil field. Isolates were screened for biosurfactant production using surface tension test. The highest reduction of surface tension was achieved with a bacterial strain which was identified by 16S rRNA gene sequencing as Brevibacilis brevis HOB1. It has been investigated using different carbon and nitrogen sources. It showed that the strain was able to grow and reduce the surface tension of the broth to 29 mN/m on commercial sugar and maltose, and to 32 mN/m on glucose after 72 h of growth. The maximum amount of biosurfactant was obtained when nitrate ions were supplied as nitrogen source. Biosurfactant produced by Brevibacilis brevis HOB1 was confirmed as a lipopeptide class of biosurfactant using TLC test and mass spectra. Lipopeptide isoforms were isolated from cell-free supernatants by acid-precipitation followed by one step of chromatographic separation on solid-phase ODS C18 column. The separation was confirmed by HPLC and ESI Q-TOF MS spectroscopy. Comparing the mass data obtained and the mass numbers reported for the lipopeptide complexes from other strains, it can be concluded that the major lipopeptide product of Brevibacilis brevis HOB1 is the surfactin isoform. This lipopeptide showed strong antibacterial and antifungal activity. It is a candidate for the biocontrol of pathogens in agriculture and other industries.     

Isolation and characterization of lipopeptide antibiotics produced by Bacillus subtilis

Aims:  Antibiotics from Bacillus subtilis JA show strong pathogen inhibition ability, which has potential market application; yet, the composition of these antibiotics has not been elucidated. The aim of this paper is to isolate and identify these antibiotics.
Methods and Results:  The antagonistic activity of JA was tested in vitro ; it exhibited strong inhibition against some important phytopathogens and postharvest pathogens. Crude antibiotic production was extracted with methanol from the precipitate by adding 6 mol l⁻¹ HCl to the bacillus-free culture broth. The crude extract was run on Diamonsil C₁₈ column (5  μ m, 250 × 4·6 mm) in HPLC system to separate the antibiotics. Major antibiotics were classified into three lipopeptide families according to electrospray ionization–mass spectrometry analysis. Subsequently, the classification of antibiotics was confirmed with typical collision-induced dissociation fragments.
Conclusions:  Three kinds of antibiotics were isolated from B. subtilis JA and were identified to the lipopeptide families, surfactin, iturin and fengycin. These compounds could function as biocontrol agents against a large spectrum of pathogens.
Significance and Impact of the Study:  This study provided a reliable and rapid method for isolation and structural characterization of lipopeptide antibiotics from B. subtilis .     

Identification and characterization of a mosquito pupicidal metabolite of a Bacillus subtilis subsp. subtilis strain

The culture supernatant of a strain of Bacillus subtilis subsp. subtilis isolated from mangrove forests of Andaman and Nicobar islands, India was found to kill larval and pupal stages of mosquitoes. A chloroform extract of the culture supernatant of the bacterium showed pupicidal effects at an LC₅₀ dose of 1 μg/ml. The mosquitocidal metabolite(s) produced by this strain were purified by gel permeation chromatography. The purified fraction was subjected to Fourier transform infrared (FTIR) spectroscopy and Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The FTIR spectrum of active fraction/CHCl3 residue showed strong band characteristic of peptides. MALDI-TOF spectrum of the sample showed well-resolved group of peaks at m/z values 1,030.6, 1,046.7, 1,044.6, 1,060.5, 1,058.6, 1,058.7, and 1,074.6. The results indicated production of different isoforms of surfactin, ranging from C13–C15. Further, the sfp gene responsible for the production of surfactin was amplified and sequenced. In conclusion, this study showed that the mosquito pupicidal metabolite(s), produced by B. subtilis subsp. subtilis is the cyclic lipopeptide, surfactin. The mode of action of surfactin on pupae of mosquitoes is discussed. This is the first report on the mosquito pupicidal activity of surfactin produced by B. subtilis subsp. subtilis.     

Surfactin isoforms from Bacillus subtilis HSO121: separation and characterization

Natural surfactin is a mixture of cyclic lipopeptides built from variants of a heptapeptide and a beta-hydroxy fatty acid. A biosurfactant-producing strain, Bacillus subtilis HSO121, was isolated from the production water of an oil field. The strain was able to produce eight surfactin isoforms which have been isolated by acid-precipitation followed by extraction into methanol. A novel procedure for the purification of surfactin was achieved. It consists of a solid-phase extraction on C(18) gel followed by reversed-phase high performance liquid chromatography using Prep. HiQ sil C18W, column. The surfactin isoforms were eluted by linear acetonitrile gradient from 80-100%. The peaks were analyzed by TLC on silica gel, and after acid hydrolysis their amino acid compositions were determined by HPLC analysis. Eight isoforms of surfactin had nearly the same amino acid composition and appeared a single spot in TLC. According to the R(f) values with the amino acid composition, these peaks belong to the surfactin group of lipopeptides. Infrared analysis of the purified samples also revealed a pattern similar to that of surfactin. This is a very effective method for isolating and fractionating lipopeptides, of the same or different nature.     

Variants of Lipopeptides Produced by <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> HSN221 in Different Medium Components Evaluated by a Rapid Method ESI-MS

A lipopeptide producing strain was isolated from an oil field and identified as Bacillus licheniformis HSN221. Nine different substrates were used to cultivate the strain under the same incubation conditions. Using a rapid method, Electrospray Ionization Mass Spectrometry (ESI-MS) combined with Thin Layer Chromatography (TLC), nine different lipopeptide homologues were found and identified. The strain produced four [Leu]surfactin homologues, surfactin C13, surfactin C14, surfactin C15 and surfactin C16, when cultivated in the medium with glucose, yeast extract and ammonium chloride, but it produced five lichenysin homologues, lichenysin C12, lichenysin C13, lichenysin C14, lichenysin C15 and lichenysin C16, when cultivated in the remaining eight media. Additionally, it showed that the type and relative content of each homologue were consistent with in each medium which is helpful for optimizing the medium components to cultivate the similar species.     

Identification and biochemical characteristics of lipopeptides from Bacillus mojavensis A21

This study reports the potential of a marine bacterium, Bacillus mojavensis A21, to produce lipopeptide biosurfactants. The crude lipopeptide mixture was found to be very effective in reducing surface tension to 31 mN m⁻¹. PCR experiments using degenerate primers revealed the presence of nonribosomal peptide synthetases genes implied in the biosyntheses of fengycin and surfactin. Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) performed on whole cells of B. mojavensis A21 confirmed the presence of lipopeptides identified as members of surfactin and fengycin families. Further, a detailed analysis performed by MALDI-TOF-TOF revealed the presence of pumilacidin compounds. The crude lipopeptide mixture was tested for its inhibitory activity against Gram-positive and Gram-negative bacteria, and fungal strains. It was found to display significant antimicrobial activity. Strain A21 lipopeptide mixture was insensitive to proteolytic enzymes, stable between pH 3.0 and 11.0, and resistant to high temperature. Production of lipopeptides is a characteristic of several Bacillus species, but to our knowledge this is the first report involving identification of pumilacidin, surfactin and fengycin isoforms in a B. mojavensis strain.     

Structural and immunological characterization of a biosurfactant produced by Bacillus licheniformis JF-2.

Bacillus licheniformis JF-2 produces a very active biosurfactant under both aerobic and anaerobic conditions. We purified the surface-active compound to homogeneity by reverse-phase C18 high-performance liquid chromatography and showed that it is a lipopeptide with a molecular weight of 1,035. Amino acid analysis, fast atom mass and infrared spectroscopy, and, finally, 1H, 13C, and two-dimensional nuclear magnetic resonance demonstrated that the biosurfactant consists of a heterogeneous C15 fatty acid tail linked to a peptide moiety very similar to that of surfactin, a lipopeptide produced by Bacillus subtilis. Polyclonal antibodies were raised against surfactin and shown to exhibit identical reactivity towards purified JF-2 lipopeptide in competition enzyme-linked immunosorbent assays, thus providing further evidence for the structural similarity of these two compounds. Under optimal conditions, the B. licheniformis JF-2 biosurfactant exhibits a critical micelle concentration of 10 mg/liter and reduces the interfacial tension against decane to 6 x 10(-3) dyne/cm, which is one of the lowest interfacial tensions ever reported for a microbial surfactant.     

Coproduction of surfactin and iturin A, lipopeptides with surfactant and antifungal properties, by Bacillus subtilis

Of the 13 strains of Bacillus subtilis tested for the coproduction of the lipopeptide surfactin and the antifungal lipopeptides of the iturin family, only 1 produced both lipopeptides with a high yield. The cultures were made in a synthetic medium. Several L-amino acids and various carbon sources were good substrates for the lipopeptide production. The maximum yield of surfactin was about 110 mg/liter and that of iturin A about 39 mg/liter/absorbance unit for the best strain, B. subtilis S 499.     

大豆根腐病生防菌KJB04-11的鉴定及其产生的脂肽类抗生素

从大豆根围筛选到1株对尖孢镰刀菌和立枯丝核菌都具有很好拮抗作用的菌株KJB04-11,经形态观察、生理生化特征和16SrDNA序列分析,属于枯草芽孢杆菌(Bacillussubtilis)。具有抗菌活性的KJB04-11发酵液无菌滤液对热和酸碱具有较强的稳定性。采用SephadexG-25柱层析、反相HPLC和冷冻干燥从KJB04-11发酵液中分离纯化了抗菌活性成分。由红外光谱、MALDI-TOF-MS、氨基酸组成及脂肽合成酶基因扩增结果推测该菌株产生的抗菌物质为C16、C17的mycosubtilin和C15的surfactin。田间试验表明,大豆种子经KJB04-11发酵液包衣处理对大豆根腐病防效为53.6%,大豆产量提高12.5%。