Identification of ε-Poly-L-lysine as an Antimicrobial Product from an Epichloë Endophyte and Isolation of Fungal ε-PL Synthetase Gene.

The endophytic fungus Epichloë festucae is known to produce bioactive metabolites, which consequently protect the host plants from biotic and abiotic stresses. We previously found that the overexpression of vibA (a gene for transcription factor) in E. festucae strain E437 resulted in the secretion of an unknown fungicide. In the present study, the active substance was purified and chemically identified as ε-poly-L-lysine (ε-PL), which consisted of 28-34 lysine units. The productivity was 3.7-fold compared with that of the wild type strain E437. The isolated ε-PL showed inhibitory activity against the spore germination of the plant pathogens Drechslera erythrospila, Botrytis cinerea, and Phytophthora infestans at 1-10 µg/mL. We also isolated the fungal gene "epls" encoding ε-PL synthetase Epls. Overexpression of epls in the wild type strain E437 resulted in the enhanced production of ε-PL by 6.7-fold. Interestingly, overexpression of epls in the different strain E. festucae Fl1 resulted in the production of shorter ε-PL with 8-20 lysine, which exhibited a comparable antifungal activity to the longer one. The results demonstrate the first example of ε-PL synthetase gene from the eukaryotic genomes and suggest the potential of enhanced expression of vibA or/and epls genes in the Epichloë endophyte for constructing pest-tolerant plants.

Purification and Structural Analysis of the Effective Anti-TMV Compound ε-Poly-l-lysine Produced by Streptomyces ahygroscopicus.

Microbial secondary metabolites produced by actinomycetes are important natural products widely applied to control plant diseases. A variety of actinomycetes were isolated from soil samples collected from Tianzhu Mountain in Shenyang, China. A Streptomyces strain Shenyang Tianzhu (STZ) exhibits effective antiviral activity against Tobacco mosaic virus (TMV). The isolate was identified as Streptomyces ahygroscopicus based on its cultural, morphological, physiological, biochemical characteristics as well as the phylogenetic analysis using 16S rRNA sequences. To obtain the pure anti-TMV compound from Streptomyces STZ, the culture broth was subjected to Amberlite IRC-50 ion-exchange resin, SX-8 macroporous adsorption resin and Sephadex G-25 gel column chromatography. The purified active compound was confirmed to be ε-poly-l-lysine (ε-PL), with molecular mass in the range of 3454⁻4352 Da by structural analysis with infrared (IR), matrix-assisted laser desorption ionization-time-of-flight MS (MALDI-TOF), thin-layer chromatography (TLC) and high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR). The protective and curative effects of the purified compound ε-PL were tested and the results showed that the compound exhibited significant protective and curative activity against TMV. The potential application of ε-PL as an efficient anti-plant virus agent was expected.

Heronamides D-F, polyketide macrolactams from the deep-sea-derived Streptomyces sp. SCSIO 03032.

Three new macrolactams, heronamides D-F (1-3), were isolated from the deep-sea-derived Streptomyces sp. SCSIO 03032 upon changing cultivation conditions. The planar structures of heronamides D-F (1-3) were elucidated by extensive MS and NMR spectroscopic analyses and comparisons with the closely related heronamides A-C. The relative configurations of 1-3 were deduced by detailed analysis of (3)JHH values and NOESY data. The absolute configurations of 1 and 2 were determined by chemical modifications and application of the modified Mosher's method. None of the compounds exhibited obvious antimicrobial or cytotoxic activities.

Modeling the electrophoresis of highly charged peptides: application to oligolysines.

The "coarse-grained" bead modeling methodology, BMM, is generalized to treat electrostatics at the level of the nonlinear Poisson-Boltzmann equation. This improvement makes it more applicable to the important class of highly charged macroions and highly charged peptides in particular. In the present study, the new nonlinear Poisson-Boltzmann, NLPB-BMM procedure is applied to the free solution electrophoretic mobility of low molecular mass oligolysines (degree of polymerization 1-8) in lithium phosphate buffer at pH 2.5. The ionic strength is varied from 0.01 to 0.10 M) and the temperature is varied from 25 to 50°C. In order to obtain quantitative agreement between modeling and experiment, a small amount of specific phosphate binding must be included in modeling. This binding is predicted to increase with increasing temperature and ionic strength.

Antibacterial and anticancer activity of ε-poly-L-lysine (ε-PL) produced by a marine Bacillus subtilis sp.

A marine Bacillus subtilis SDNS was isolated from sea water in Alexandria and identified using 16S rDNA sequence analysis. The bacterium produced a compound active against a number of gram negativeve bacteria. Moreover, the anticancer activity of this bacterium was tested against three different human cell lines (Hela S3, HepG2 and CaCo). The highest inhibition activity was recorded against Hela S3 cell line (77.2%), while almost no activity was recorded towards CaCo cell line. HPLC and TLC analyses supported evidence that Bacillus subtilis SDNS product is ε-poly-L-lysine. To achieve maximum production, Plackett-Burman experimental design was applied. A 1.5 fold increase was observed when Bacillus subtilis SDNS was grown in optimized medium composed of g/l: (NH(4))(2) SO(4), 15; K(2)HPO(4), 0.3; KH(2)PO(4), 2; MgSO(4) · 7 H(2)O, 1; ZnSO(4) · 7 H(2)O, 0; FeSO(4) · 7 H(2)O, 0.03; glucose, 25; yeast extract, 1, pH 6.8. Under optimized culture condition, a product value of 76.3 mg/l could be obtained. According to available literature, this is the first announcement for the production of ε-poly-L-lysine (ε-PL) by a member of genus Bacillus.

Production of ε-poly-L-lysine by Streptomyces sp. using resin-based, in situ product removal.

Resin-based, in situ product removal (ISPR) was used to increase production of ε-poly-L-lysine (PL) by Streptomyces sp. GIM8. D152 resin was selected over Amberlite IRC-50, Amberlite IRC-76 and Amberlite IR-120 to develop ISPR using adsorption capacity and desorption ratio as bases. The yield of PL in response to external PL was unaffected in shake-flask culture; however, the production of PL increased to 2.9 from 0.8 g l(-1) shake-flasks using ISPR. In a 5 l fermentor, 23.4 g PL l(-1) was achieved compared to 3.76 g PL l(-1), in the controls by attaching two bags of D152 resin to the probes and baffles of the fermentor.

Metal-Chelate Affinity Precipitation with Thermo-Responsive Polymer for Purification of ε-Poly-L-Lysine.

ε-Poly-L-lysine (ε-PL) is a natural preservative for food processing industry. A thermo-responsive polymer, attached with Cu2+ or Ni2+, was prepared for metal-chelate affinity precipitation for purification of ε-PL. The low critical solution temperatures (LCSTs) of these polymers were close to the room temperature (31.0-35.0 °C). The optimal adsorption conditions were as follows: pH 4.0, 0 mol/L NaCl, ligand density 75.00 µmol/g, and 120 min. The ligand Cu2+ showed a stronger affinity interaction with ε-PL and the highest adsorption amount reached 251.93 mg/g polymer. The elution recovery of ε-PL could be 98.42% with 0.50 mol/L imidazole (pH = 8.0) as the eluent. The method could purify ε-PL from fermentation broth and the final product was proved as electrophoretic pure by SDS-PAGE. Moreover, these affinity polymers could be recycled after the purification of ε-PL and the recoveries were above 95.00%. Graphical Abstract Scheme for affinity precipitation of ε-PL.

Synthesis of thermo-responsive polymers recycling aqueous two-phase systems and phase formation mechanism with partition of ε-polylysine.

Aqueous two-phase systems (ATPS) have the potential application in bioseparation and biocatalysis engineering. In this paper, a recyclable ATPS was developed by two thermo-responsive copolymers, PVBAm and PN. Copolymer PVBAm was copolymerized using N-vinylcaprolactam, Butyl methacrylate and Acrylamide as monomers, and PN was synthesized by N-isopropylacrylamide. The lower critical solution temperature (LCST) of PVBAm and PN were 45.0°C and 33.5°C, respectively. The recoveries of both polymers could achieve over 95.0%. The phase behavior and formation mechanism of PVBAm/PN ATPS was studied. Low-field nuclear magnetic resonance (LF-NMR) was applied in the phase-forming mechanism study in ATPS. In addition, combining the analysis results of surface tension, transmission electron microscopy and dynamic light scattering, the phase-forming of the PVBAm/PN ATPS was proved. The application was performed by partition of ε-polylysine in the 2% PVBAm/2% PN (w/w) ATPS. The results demonstrated that ε-polylysine was extracted into the PN-rich phase, the maximal partition coefficient (1/K) and extraction recovery of pure ε-polylysine were 6.87 and 96.36%, respectively, and 7.41 partition coefficient and 97.85% extraction recovery for ε-polylysine fermentation broth were obtained in the presence of 50mM (NH4)2SO4 at room temperature. And this method can effectively remove the most impurities from fermentation broth when (NH4)2SO4 exists in the ATPS. It is believed that the thermo-responsive recycling ATPS has a good application prospect in the field of bio-separation.

Separation and Purification of ε-Poly-L-lysine with Its Colorimetric Determination Using Dipicrylamine.

A separation method for a biopolymer, ε-poly-L-lysine (εPL), with its colorimetric determination using a yellow anionic dye, dipicrylamine anion (DPA(-)), is presented. The εPL-producing culture broth was mixed with a NaDPA solution to precipitate the εPL in polycationic form with the DPA(-) anion. The precipitate was dissolved into acetonitrile (AN). The AN solution was yellowish, and gave an absorption maximum at around 420 nm. Thus, εPL in the culture broth can be assayed colorimetrically a with multiwell microtiter plate. By the addition of bis(triphenylphosphoranylidene)ammonium chloride, the polycationic εPL was precipitated from the AN solution as the hydrochloride salt. On the other hand, the DPA(-) anion remained in the AN solution as the quaternary ammonium salt. Thus, εPL larger than tetramer can be separated and purified after the high-throughput screening of the synthetic enzyme.

Separation and purification of ε-poly-L-lysine from the culture broth based on precipitation with the tetraphenylborate anion.

A biopolymer ε-poly-L-lysine (εPL) exists as a fully protonated form (εPLH(n+)) in solutions at pH < 4. Around pH 3.5, at which the tetraphenylborate (TPB(-)) anion is not decomposed immediately, the εPLH(n+) cation associates with the TPB(-) anion to form a precipitate of 1:n stoichiometry, εPLH(TPB)(n). By the addition of NaTPB to a culture broth containing εPL and NH(4)(+) and K(+) ions, not only the polycationic εPLH(n+) but also the monovalent cations would be precipitated with the TPB(-) anion. However, εPLH(TPB)(n) was purified by washing the mixed precipitate with acetone, in which NH(4)TPB and KTPB are soluble. By mixing the εPLH(TPB)(n) precipitate and a high-concentration HCl solution, the TPB(-) anion was decomposed immediately to hydrophobic molecules. By the addition of a much larger volume of acetone to the reaction mixture, the decomposition products dissolved in the solvent. Simultaneously, εPL was precipitated as the hydrochloride salt. Thus, εPL has been separated and purified from the culture broth. Also, the method has been successfully applied to the separation of oligomeric εPL species. The present chemical separation method is rapid, simple, and easy to carry out, and can be utilized in bioengineering studies of such basic peptides or polyamines.

Substantially monodispersed poly(epsilon-L-lysine)s frequently occurred in newly isolated strains of Streptomyces sp.

The presence of poly(epsilon-L-lysine) (epsilon-PL) was found quite frequently by screening various strains of Streptomyces sp. Most of the ten newly obtained epsilon-PLs, when they were produced from glucose, showed a polydispersity index of Mw/Mn = 1.01 using ion-pair chromatography analysis. The polymers were classified into five groups according to their chain lengths. The average numbers of residues in the five groups were 32, 28, 25, 19, and 16, respectively. The use of glycerol instead of glucose resulted in decreases of 10 to 20% in the Mn and slight increases in the Mw/Mn. These observations indicated the chain length and polydispersity of epsilon-PL were primarily determined by each producer strain. Proton and 13C NMR analysis revealed the signals of glycerol-derived ester at the C terminus of the polymer from several producers including the first discovered S. albulus strain, although the percentages of the ester were low under our culture conditions. These results, coupled with the previous observation that SO4(2-) was essential for the polymer production, led to discussion on the mechanistic aspects of monomer activation, elongation, and termination in the biosynthesis of epsilon-PL.

Production of epsilon-poly-L-lysine by newly isolated Kitasatospora sp. PL6-3.

A novel epsilon-poly-L-lysine (epsilon-PL)-producing strain PL6-3 was isolated from soil, and was identified as a strain of Kitasatospora sp. This is the first detailed report of production of epsilon-PL by a strain in the genera of Kitasatospora. By controlling the culture pH at 4.0, the yield of epsilon-PL from PL6-3 reached 13.9 g/L after 120 h of cultivation in fed-batch fermentation. The morphological characteristics of Kitasatospora sp. PL6-3 in culture broth were different from those reported from strains of Streptomycetaceae, as no mycelium pellets were observed during the course of fermentation of PL6-3, which was beneficial to the assimilation of nutrition and secretion of the products. Furthermore, the molecular mass of the purified epsilon-PL from PL6-3 was determined to be 5.01 kDa by SDS-PAGE and 5.05 kDa by gel permeation chromatography, indicating that the epsilon-PL produced by this strain might be composed of 40 lysine residues. Usually, epsilon-PL with more lysine residues showed higher antimicrobial activity; however, it was difficult to obtain epsilon-PL with more than 36 lysine residues in this study. As a result, epsilon-PL from Kitasatospora sp. PL6-3, which contains more lysine residues than that from other strains, is more promising in the field of food preservatives.

Preparation of adenovirus-polylysine-DNA complexes.

This unit describes preparation of adenovirus-polylysine-DNA complexes, which is useful for transfection of DNA into a variety of cell types. A DNA complex is prepared with biotinylated adenovirus and streptavidin-polylysine, coupled to transferrin, and used to transfect cells. Several support protocols describe methods for adenovirus growth and purification, biotinylation, inactivation with psoralen, and quantitation of the adenovirus particles. Additional support protocols describes preparation of streptavidin-polylysine and transferrin-polylysine, necessary for the basic procedure. The DNA used for transfection must be free of lipopolysaccharide (LPS), and two methods for removing LPS are described. A more direct polylysine-virus linkage that is simple and requires no exotic reagents can be used for transfection. This protocol requires polylysine-modified adenovirus, prepared as described. An alternate protocol describes transfecting cells with free virus and DNA condensed with a polycation.

The interaction of calmodulin and polylysine as studied by 1H NMR spectroscopy and sedimentation equilibrium centrifugation.

The effects of polylysine on calmodulin were assessed using 1H NMR and sedimentation equilibrium centrifugation. Sedimentation equilibrium centrifugation measurements demonstrated that calmodulin associates with polylysine at calmodulin-polylysine molar ratios ranging from 10:1 to 2.5:1 and when polylysine is increased above the molar ratio of 1:1 a precipitate is formed. At a 1:2.5 calmodulin:polylysine molar ratio, 75% of the calmodulin precipitates from the solution and virtually no polylysine is present in the precipitate. 1H NMR studies of the aromatic region of calmodulin identified chemical shifts of three peaks at a calmodulin:polylysine molar ratio of 1:1. These studies suggest that polylysine associates with calmodulin in aqueous solution and can alter the structure of calmodulin to cause calmodulin self-aggregation.

DNA-binding transferrin conjugates as functional gene-delivery agents: synthesis by linkage of polylysine or ethidium homodimer to the transferrin carbohydrate moiety.

We have previously demonstrated that transferrin-polycation conjugates are efficient carrier molecules for the introduction of genes into eukaryotic cells. We describe here a more specific method for conjugation of transferrin with DNA-binding compounds involving attachment at the transferrin carbohydrate moiety. We used the polycation poly(L-lysine) or the DNA intercalator, ethidium homodimer as DNA-binding domains. Successful transferrin-receptor-mediated delivery and expression of the Photinus pyralis luciferase gene in K562 cells has been shown with these new transferrin conjugates. The activity of the transferrin-ethidium homodimer (TfEtD) conjugates is low relative to transferrin-polylysine conjugates; probably because of incomplete condensation of the DNA. However, DNA delivery with TfEtD is drastically improved when ternary complexes of the DNA with TfEtD and the DNA condensing agent polylysine are prepared. The gene delivery with the carbohydrate-linked transferrin-polylysine conjugates is equal or superior to described conjugates containing disulfide linkage. The new ligation method facilitates the synthesis of large quantities (greater than 100 mg) of conjugates.