RESUMEN
Bacillus subtilis HSO121 was previously isolated from the formation water of an oil field and found to produce surfactin lipopeptides. Effects of 10 different components on surfactin production have been studied by Plackett- Burman design (PBD). Each component was screened on the basis of P-value, which was at 92% or above of confidence level. In PBD, significant factors influencing the surfactin production were selected for further optimization via response surface methodology approach (RSM). Four significant variables (calcium chloride, ferrous sulfate, maltose, and L-arginine) were selected for the optimization studies, and constructed via central composite design (CCD). Calcium chloride, maltose and L-arginine showed a significant positive effect on surfactin production, while ferrous sulfate had no significant effect. An overall 38.06-fold increase in surfactin production yield was achieved in the optimized medium as compared with the unoptimized basal medium. Surfactin production yield increased significantly with optimized medium (47.58 g/l) when compared with the unoptimized one (1.25 g/l).
Asunto(s)
Bacillus subtilis/metabolismo , Bioensayo , Medios de Cultivo , Microbiología Industrial/métodos , Lipopéptidos/química , Péptidos Cíclicos/química , Tensoactivos/metabolismo , Arginina/farmacología , Bacillus subtilis/efectos de los fármacos , Medios de Cultivo/química , Maltosa/farmacología , Tensoactivos/químicaRESUMEN
Brevibacillus brevis HOB1 was isolated from the formation water of an oil field and found to produce lipopeptides. The separation of lipopeptides was successfully achieved by reversed-phase high-performance liquid chromatography (HPLC) leading to nine separated peaks. The chemical structures of these lipopeptides were studied by means of electrospray ionization mass spectrometry (ESI-MS), gas chromatography-mass spectrometry (GC/MS), HPLC and electrospray ionization tandem mass spectrometry (ESI-MS/MS). As the results, all the lipopeptides had peptide parts with the same amino acid composition of Asp, Glu, Val, and Leu in the molar ratio 1:1:1:4, while the lipid part was composed of C(13)-C(15) beta-hydroxy fatty acids. As the sequence of fraction 1 was determined to be N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C, the same as surfactin, they were proposed to be surfactin isoforms. Fraction 4 (C(15) surfactin) exhibited a good surface activity of 26.8 mN/m with CMC of 9 x 10(-6 )M. Surfactin is a powerful biosurfactant possessing biological activities. As far as we know, Br. brevis is a new surfactin-producing species.
Asunto(s)
Bacillales/química , Lipopéptidos/química , Secuencia de Aminoácidos , Lipopéptidos/aislamiento & purificación , Lipopéptidos/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Tensión Superficial , Espectrometría de Masas en TándemRESUMEN
A biosurfactant-producing strain was isolated from the production water of an oil-field and was identified as Bacillus subtilis HOB2 by 16S rRNA gene sequencing. The production of biosurfactant by Bacillus subtilis HOB2 has been investigated using different carbon and nitrogen sources, under thermophilic and mesophilic conditions. The strain was able to grow and to produce surfactant, reducing the surface tension of medium to 27 mN/m on sucrose, and 28 mN/m on glucose after 24 h of cultivation. The strain was able to produce the maximum amount of biosurfactant when ammonium ions were used as nitrogen source. The surface-active compound was stable during exposure to elevate temperature (100degreesC), high salinity (25% NaCl) and a wide range of pH values (5.0-11.0). The biosurfactant was capable of forming a promising emulsification index (E(24)= 68%) with kerosene. The kinetic studies revealed that biosurfactant production is a cell growth-associated process. Preliminary chemical characterization revealed that the surfactant has a lipopeptide composition similar to surfactin as confirmed by TLC and IR analysis. Properties and characteristics of the biosurfactant produced by Bacillus subtilis HOB2 suggesting potential commercial applications, such as enhanced oil recovery, bioremediation of soil and marine environments, and food industries.
Asunto(s)
Bacillus subtilis/aislamiento & purificación , Tensoactivos/aislamiento & purificación , Bacillus subtilis/crecimiento & desarrollo , Bacillus subtilis/metabolismo , Biodegradación Ambiental , Carbono/metabolismo , Concentración de Iones de Hidrógeno , Lipopéptidos , Nitrógeno/metabolismo , Microbiología del Suelo , Espectroscopía Infrarroja por Transformada de Fourier , Tensoactivos/metabolismoRESUMEN
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.
Asunto(s)
Bacterias/aislamiento & purificación , Lipopéptidos/biosíntesis , Tensoactivos/metabolismo , Bacterias/metabolismo , Tensión SuperficialRESUMEN
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.
Asunto(s)
Bacillus subtilis/metabolismo , Péptidos Cíclicos/química , Péptidos Cíclicos/aislamiento & purificación , Cromatografía Líquida de Alta Presión , LipopéptidosRESUMEN
Lipopeptides are amphiphilic compounds which contain both hydrophobic fatty acid moieties and amphiphilic peptide moieties. From the cell-free broth of Bacillus subtilis HSO121, eight cyclic lipopeptides were isolated by reversed-phase high performance liquid chromatography (RP-HPLC). The peptide part of each lipopeptide was elucidated according to electrospray ionization quadruple-time-of-flight mass spectrometry (ESI Q-TOF MS) and the fatty acid part was analyzed by electroionization gas chromatography/mass spectrometry (EI GC/MS). It showed that fractions 1-8 had molecular masses of 1007, 1021, 1021, 1035, 1035, 1035, 1063, and 1049, respectively. Analysis of hydrolyzed lipopeptides revealed that they had invariant amino acid compositions. The differences in molecular weights represent changes in the number of methylene groups and different types of branched chains in fatty acids. Peptide sequences of two of the eight lipopeptides appeared to be N-Asp-Leu-Leu-Val-Glu-Leu-Leu-C, which was different from previously reported lipopeptides. The remaining six had an identical peptide sequence of N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C. The fatty acid parts were found to be mixtures of iso C(12), iso C(13), anteiso C(13), iso C(14), n C(14), iso C(15), anteiso C(15), n C(15), anteiso C(16) and anteiso C(17) beta-hydroxy fatty acids. The structure of each lipopeptide was determined to be the beta-hydroxy fatty acid bonded to the peptide chain.
Asunto(s)
Bacillus subtilis/metabolismo , Lipoproteínas/química , Péptidos Cíclicos/química , Cromatografía Líquida de Alta Presión , Ácidos Grasos/química , Lipoproteínas/biosíntesis , Péptidos Cíclicos/biosíntesis , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
Copper-zinc superoxide dismutase (Cu,Zn SOD) has been extracted, purified and characterized from Radix lethospermi seed (RLS), a kind of Chinese traditional medicine. Before extraction, the lipid was removed by super critical fluid extraction (SCF). Partial protein fractionation in the crude extract was affected by using 50-75% (NH(4))(2)SO(2). Subsequently, superoxide dismutase was fractionated by column chromatographies on DEAE-52, Sephadex G-200 and DEAE-52 again. Pure Cu,Zn SOD had a specific activity of 4843 U/mg protein and was purified 267.2-fold, with a yield of 23.55%. The purified enzyme has a molecular weight of about 30,500+/-100 and is composed of two non-covalently joined equal subunits. Purity was confirmed by Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), HPLC and mass spectroscopy. Amino acid content has been investigated. The enzyme was found to remain stable in the pH range 6.0-9.0 at 25 degrees C and up to 45 degrees C at pH 7.8 for a 30 min incubation period. RLS Cu,Zn SOD appeared to have significant thermal stability lower than other Cu,Zn SODs, as revealed by irreversible heat inactivation at 60 degrees C. The enzyme was not inhibited by DTT, NaN(3) and beta-mercaptoethanol, but was inhibited by cyanide and hydrogen peroxide. Finally, in the presence of 2 mM ethylendiamine tetra acetic acid (EDTA) and sodium dodecyl sulphate (SDS), the enzyme showed approximately 18 and 34% activity loss.