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6mol/L HCI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 LH-20层析柱获得粗纯化物,经MALDI-TOF-MS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μPRC 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。     

Isolation and characterization of a halotolerant <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">subtilis</Emphasis> BBK-1 which produces three kinds of lipopeptides: bacillomycin L,plipastatin, and surfactin

Twenty-three halotolerant and biosurfactant producing strains were collected from salty conditions in central Thailand. One of the strains designated BBK-1 produced the biosurfactants with the highest activity. BBK-1 was isolated from fermented foods and was identified as B. subtilis based on its physiological characteristics and 16S rRNA gene sequence. We show that the strain grows in media containing NaCl up to 16% (w/v) and produces biosurfactants in NaCl up to 8%. We found that B. subtilis BBK-1 produces three kinds of surface-active lipopeptides simultaneously. By their respective molecular weights and amino acid compositions, it is indicated that these lipopeptides are bacillomycin L, plipastatin, and surfactin. In order to analyze the production mechanism of lipopeptides further in the strain, a generally important biosynthetic gene encoding 4'-phosphopantetheinyl transferase was cloned and sequenced. The gene existed in a single copy in the genome and the deduced amino acid sequence was almost identical to that of Lpa-14 from B. subtilis strain RB14, which co-produces iturin A and surfactin.     

Solid-Phase Synthesis of Surfactin,a Powerful Biosurfactant Produced by <Emphasis Type="Italic">Bacillus subtilis</Emphasis>, and of Four Analogues

Using the Fmoc methodology, we report the chemical synthesis of surfactin and of four of its analogues, by stepwise solid-phase peptide synthesis (SPPS) on Sasrin resin. Formation of depsipeptide bond was performed with EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. In developing our strategy, surfactin was used as a model and we synthesized both its racemic mixture and its R isoform. (R) 3-hydroxy fatty acid was obtained using Candida antarctica lipase from the racemic fatty acid, allowing a further identification of both R and S isoforms in the racemic mixture. Analogues were synthesized as racemic linear lipopeptides. Then, both enantiomers were separated and purified by adsorption chromatography on silicic acid, following cleavage from the resin. Linear R lipoheptapeptides were identified by TLC. They exhibit, in all cases, higher R_f values than those of the corresponding S isoforms. Cyclization was then performed independently for each enantiomer, using a HATU/DIEA coupling in solution. The yields were highly dependent on the position and on the nature of the modified amino acids.     

Anionic lipopeptides from Bacillus mojavensis I4 as effective antihypertensive agents: Production,characterization, and identification

A new isolated Bacillus mojavensis strain I4 was found as producer of biosurfactants by different screening methods, such as parafilm M test, hemolytic activity, oil displacement test, emulsification index, surface tension, and lipase production assay. Enhanced biosurfactants production was obtained using glucose and glutamic acid as carbon and nitrogen sources, respectively. The optimal production of the biosurfactants was obtained by using a C/N ratio of 17, pH of 7.0, and temperature of 37°C. The surface tension was reduced to 29 mN/m and the emulsification index E24 of 62% was achieved after 72 h of culture. The purified biosurfactants showed stability with regard to surface tension reduction and emulsification in a wide range of temperatures (4–120°C), pH (4–10), and salinity (2–12% of NaCl). The thin‐layer chromatography showed that the produced biosurfactants were lipopeptides. The biosurfactants were characterized as a group of anionic lipopeptides with zeta potential measurement. Chromatographic characterization using HPLC revealed that I4 lipopeptides contained numerous isoforms and surfactin was the major component. Moreover, the I4 lipopeptides showed interesting angiotensin‐converting enzyme‐inhibitory activity.     

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.     

Isolation and characterization of a C12-lipopeptide produced by Bacillus subtilis HSO 121.

A new lipopeptide with C12 fatty acid has been isolated from the cell broth of Bacillus subtilis HSO121 by chromatographic methods, which is believed to be the homologue of lipopeptides. The fatty acid portion was methylated and analyzed by GC/MS, ESI Q-TOF MS and 1H-NMR. The peptide portion, of which the amino acid composition was obtained by HPLC combined with a phenyl isothiocyanate (PITC) derivatization methods, was analyzed by ESI Q-TOF MS. Comparing the obtained results with surfactin C13 showed that the new lipopeptide has a peptide moiety similar to that of surfactin and the difference exists in the fatty acid portion, which is an iso-C12 beta-hydroxy fatty acid. The critical micelle concentration (CMC) of this new homologue is estimated to be 6.27 x 10(-5) mol/l in 10 mmol/l phosphate buffer solution (PBS, pH 8.0) at 30 degrees C, and the surface tension at CMC (gamma CMC) achieved is as little as 27.71 mN/m. The hemolytic activities of the C12-lipopeptide on 2% human erythrocytes showed a HC50 of 26.5 micromol/l.     

Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectral Analysis of Marine Lipopeptides with Potential Therapeutic Implications

Marine Bacillus circulans DMS-2 (MTCC 8281) was found to produce lipopeptides, which reduced the surface tension of the medium from 69 to 28 mNm⁻¹. The methanol extracts of the lipopeptides were resolved in RP-HPLC, which resulted in the presence of two major surface active fractions corresponding to the peaks E and F were eluted at the retention times of 16.8 and 18 min, respectively. Fourier transform infrared spectroscopy and matrix assisted laser desorption ionization time of flight (MALDI-ToF) mass spectral analysis showed that the produced lipopeptides belonged to surfactin family. In the MALDI-ToF spectrum, the major molecular mass of the HPLC purified isoforms were identified at m/z 1,044 and 1,058 Da, respectively. In addition, this marine strain also produced new variants of surfactins with molecular weights ranging from m/z 1,066 to 1,098 Da, which have not been reported earlier. Both surface-active fractions were found to have potent antimicrobial activity against the Gram-positive and Gram-negative bacterial strains.     

Recombinant acylheptapeptide lichenysin: high level of production by Bacillus subtilis cells

Peptide synthetases are multi-domain proteins that catalyze the assembly, from amino acids and amino acid derivatives, of peptides and lipopeptides, some of which exhibit activities (pharmaceutical, surfactant, etc.) of considerable biotechnological importance. Although there is substantial interest in the generation of greater peptide diversity, in order to create new biotechnologically interesting products, attempts reported so far to exchange amino acid-activating minimal modules between enzymes have only yielded hybrid catalysts with poor activities. We report here the replacement of an entire first, L-Glu-, and fifth, L-Asp-incorporating modules of surfactin synthetase, to create a fully active hybrid enzyme that forms a novel peptide in high yields. Whole encoding regions of lichenysin A synthetase modules were introduced into surfactin biosynthesis operon between His140/His1185 of SrfAA and His1183/His2226 of SrfAB, the amino acid residues of a proposed active-site motif (HHXXXDG) of the condensation domains which is involved in the catalysis of nonribosomal peptide bond formation (Stachelhaus et al., 1998). When the lipopeptides produced by the recombinant Bacillus subtilis strains were purified and characterized, they appeared to be expressed approximately at the same level of the wild type surfactin and to be identical by their fatty acid profiles. We thereby demonstrate the utility of whole module swapping for designing novel peptides, for creating peptide diversity, and for redesigning existing peptides produced in performant production strains in high yields to correspond to desired peptides produced in low yields, or from strains unsuitable for production purposes.     

ANF (Arg 101--Tyr 126) is the peptide secreted by rat atrial cardiocytes in culture

The atrial natriuretic factor (ANF) secreted from rat cardiocytes in culture was purified and characterized. The purification procedure involves extraction of ANF by activated Vycor glass, followed by HPLC on C18 mu Bondapak and Vydac columns. The detection of ANF in column eluates was performed by a simple and sensitive radioimmunoassay. The amino acid composition and N-terminal amino acid sequencing appeared to be identical to the Arg 101 - Tyr 126 peptide. The isolated ANF showed biological activity, inhibiting basal and ACTH-stimulating aldosterone secretion from rat zona glomerulosa cells with the same potency as the synthetic peptide.     

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%。     

Characterization of the surfactin synthetase isolated from the Bacillus subtilis strain producing iturin

Summary The lipopeptides, surfactin and iturin, are co-produced by B. subtilis. In this work, the three subunits of surfactin synthetase have been characterized by affinity chromatography on affigel columns where the ligand is one of the amino acid components of surfactin.     

Surfactin/iturin A interactions may explain the synergistic effect of surfactin on the biological properties of iturin A.

Iturin A and surfactin are two lipopeptides extracted from a same strain of Bacillus subtilis. Iturin A possesses antibiotic and antifungal activities and surfactin is a strong surfactant. The presence of surfactin, at a concentration at which, alone, it is inactive, increases to a very large extent the haemolysis percent induced by iturin A. This synergistic effect seems to be in relation with interactions between iturin A and surfactin. Iturin A adsorbs to and penetrates into surfactin monolayers. Iturin A and surfactin are miscible and interact specifically in mixed monolayers.     

Isolation of a new laccase isoform from the white-rot fungi Pycnoporus cinnabarinus strain ss3

Two extracellular laccase isoforms (Lac I and Lac II) produced by the white-rot fungus Pycnoporus cinnabarinus from the monokaryotic strain ss3 were purified from ferulic-acid-induced liquid culture medium using ammonium sulphate precipitation, followed by anion-exchange chromatography on a Mono Q column. Strain ss3 is the first generation of the parental strain P. cinnabarinus I-937. The new isolated isoform, Lac II, consists of an 86,000 molecular weight protein as determined by SDS polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of both isoforms were determined, and compared to known laccase protein sequences of other organisms.     

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.     

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.     

Anabaenolysins, novel cytolytic lipopeptides from benthic Anabaena cyanobacteria

Two novel cyclic lipopeptides, anabaenolysin A and anabaenolysin B, were isolated from two benthic cyanobacterial strains of the genus Anabaena. This novel class of cyanobacterial lipopeptides has a general structure of a small peptide ring consisting of four amino acids from which two are proteinogenic and two unusual; glycine(1), glycine(2), 2-(3-amino-5-oxytetrahydrofuran-2-yl)-2-hydroxyacetic acid(3) and a long unsaturated C(18) β-amino acid(4) with a conjugated triene structure. They are distinguished by the presence of a conjugated dienic structure in the C18 β-amino acid present in anabaenolysin A but not in anabaenolysin B. Conjugated triene structure generates a typical UV spectrum for anabaenolysins for easy recognition. Anabaenolysin A constituted up to 400 ppm of the cyanobacterial dry weight. We found evidence of thirteen variants of anabaenolysins in one cyanobacterial strain. This suggests that the anabaenolysins are an important class of secondary metabolites in benthic Anabaena cyanobacteria. Both anabaenolysin A and B had cytolytic activity on a number of mammalian cell lines.     

产表面活性素和伊枯草菌素A菌株的筛选及其脂肽类产物的特性

采用血琼脂平板法, 从菜园土壤中分离到8株代谢表面活性剂的菌株, 比较各菌株的排油性、抑菌性, 根据合成脂肽类物质表面活性素(Surfactin)和伊枯草菌素A (Iturin A)必需的sfp、ituD和lpa-14基因设计引物, 结合PCR的方法筛选到一株具广谱抗菌性且含有sfp、ituD和lpa-14 3个关键基因的细菌XZ-173。经过生理生化试验测定和16S rDNA序列系统发育学分析, 将其鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。通过红外光谱(FT-IR)分析该菌株代谢产物, 初步鉴定为脂肽类物质, 并对照高效液相色谱(HPLC)与标准品比对结果, 确定含有Surfactin和Iturin A组分。该菌株产生的脂肽粗品能使纯水的表面张力降低至26.6 mN/m, 临界胶束浓度(CMC)为500 mg/L, 具有很好的乳化性能, 对立枯丝核菌和青枯菌表现出很好的拮抗活性。因此, 产脂肽细菌XZ-173是一株应用前景广阔的功能菌。     

Similarity of the yellow chromophores isolated from human cataracts with those from ascorbic acid-modified calf lens proteins: evidence for ascorbic acid glycation during cataract formation

Chromatographic evidence supporting the similarity of the yellow chromophores isolated from aged human and brunescent cataract lenses and calf lens proteins ascorbylated in vitro is presented. The water-insoluble fraction from early stage brunescent cataract lenses was solubilized by sonication (WISS) and digested with a battery of proteolytic enzymes under argon to prevent oxidation. Also, calf lens proteins were incubated with ascorbic acid for 4 weeks in air and submitted to the same digestion. The percent hydrolysis of the proteins to amino acids was approximately 90% in every case. The content of yellow chromophores was 90, 130 and 250 A(330) units/g protein for normal human WISS, cataract WISS and ascorbate-modified bovine lens proteins respectively. Aliquots equivalent to 2.0 g of digested protein were subjected to size-exclusion chromatography on a Bio-Gel P-2 column. Six peaks were obtained for both preparations and pooled. Side by side thin-layer chromatography (TLC) of each peak showed very similar R(f) values for the long wavelength-absorbing fluorophores. Glycation with [U-(14)C]ascorbic acid, followed by digestion and Bio-Gel P-2 chromatography, showed that the incorporated radioactivity co-eluted with the A(330)-absorbing peaks, and that most of the fluorescent bands were labeled after TLC. Peaks 2 and 3 from the P-2 were further fractionated by preparative Prodigy C-18 reversed-phase high-performance liquid chromatography. Two major A(330)-absorbing peaks were seen in peak 2 isolated from human cataract lenses and 5 peaks in fraction 3, all of which eluted at the same retention times as those from ascorbic acid glycated calf lens proteins. HPLC fractionation of P-2 peaks 4, 5 and 6 showed many A(330)-absorbing peaks from the cataract WISS, only some of which were identical to the asorbylated proteins. The major fluorophores, however, were present in both preparations. These data provide new evidence to support the hypothesis that the yellow chromophores in brunescent lenses represent advanced glycation endproducts (AGEs) probably due to ascorbic acid glycation in vivo.     

Dopamine-β-Hydroxylase: Structural Comparisons of Membrane-Bound Versus Soluble Forms from Adrenal Medulla and Pheochromocytoma

Dopamine-beta-hydroxylase (DBH) in membrane-bound (mDBH) and water-soluble (sDBH) forms was isolated from chromaffin granules of bovine adrenal medullae and a human pheochromocytoma tumor. sDBH was purified by concanavalin A-agarose column chromatography followed by DEAE-Sepharose column chromatography. The final bovine preparation had a specific activity of 16.27 IU/mg; the human preparation had a specific activity of 9.16 IU/mg. mDBH was isolated in enzymatically inactive form by preparative polyacrylamide gel electrophoresis. The proteins were subjected to amino acid analysis, as well as digestion with trypsin, followed by separation of the resulting peptides by two-dimensional TLC/electrophoresis. No intraspecies differences between sDBH and mDBH were found from comparisons of amino acid composition or peptide maps. Thus the basis of the difference between sDBH and mDBH cannot easily be explained by differences in primary structure, within the resolution of these techniques.     

Purification and characterization of ADP-ribosyltransferases (exoenzyme C3) of Clostridium botulinum type C and D strains

By cation-exchange column chromatography followed by gel filtration or hydroxylapatite column chromatography, ADP-ribosyltransferases (exoenzyme C3) were isolated from culture supernatants of Clostridium botulinum type C strains Stockholm (CST) and 6813 (C6813) and from type D strains South African (DSA) and 1873 (D1873), and their molecular properties were compared. The purified C3 enzymes were homogeneous in polyacrylamide gel electrophoresis. The C3 enzymes existed as single-chain polypeptides with molecular masses of 25.0 to 25.5 kDa and transferred ADP-riboses to the same substrates in rat brain membrane extract. The C3 enzymes could be roughly classified into two groups with respect to amino acid composition, amino-terminal sequence, and antigenicity. One group contains the C3 enzymes of strains C6813 and DSA, and the other contains those of strains CST and D1873. The specific activity of the C3 enzyme of strain C6813 was about 15 times higher than that of the C3 enzyme of strain CST. These results indicate that the classification of the C3 molecules differs from that of the neurotoxin molecules.