• Title/Summary/Keyword: isolation and purification

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In situ isolation and characterization of the biosurfactants of B. Subtilis

  • Akthar, Wasim S.;Aadham, Mohamed Sheik;Nisha, Arif S.
    • Advances in environmental research
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    • v.9 no.3
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    • pp.215-232
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    • 2020
  • Crude oils are essential source of energy. It is majorly found in geographical locations beneath the earth's surface and crude oil is the main factor for the economic developments in the world. Natural crude oil contains unrefined petroleum composed of hydrocarbons of various molecular weights and it contains other organic materials like aromatic compounds, sulphur compounds, and many other organic compounds. These hydrocarbons are rapidly getting degraded by biosurfactant producing microorganisms. The present study deals with the isolation, purification, and characterization of biosurfactant producing microorganism from oil-contaminated soil. The ability of the microorganism producing biosurfactant was investigated by well diffusion method, drop collapse test, emulsification test, oil displacement activity, and blue agar plate method. The isolate obtained from the oil contaminated soil was identified as Bacillus subtilis. The identification was done by microscopic examinations and further characterization was done by Biochemical tests and 16SrRNA gene sequencing. Purification of the biosurfactant was performed by simple liquid-liquid extraction, and characterization of extracted biosurfactants was done using Fourier transform infrared spectroscopy (FTIR). The degradation of crude oil upon treatment with the partially purified biosurfactant was analyzed by FTIR spectroscopy and Gas-chromatography mass spectroscopy (GC-MS).

Isolation of Total RNA from a Freshwater Green Alga, Zygnema cruciatum, Containing High Levels of Pigments

  • Han, Jong-Won;Yoon, Min-Chul;Lee, Key-Pyoung;Kim, Gwang-Hoon
    • ALGAE
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    • v.22 no.2
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    • pp.125-129
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    • 2007
  • Conventional methods for the isolation and purification of mRNA from Zygnema were unsuccessful because of its high amount of pigments and RNA interactive molecules. In particular, pigments were difficult to remove using conventional protocols because they interacted with RNA during pulverization of the materials. This resulted in total degeneration of RNA in two to three hours. To alleviate this problem, we developed an isolation method that utilized DEAE-cellulose resin. The pigments bound to DEAE anion exchange resin and separated from the RNA. Purified total RNA showed an yield of 50 μg per 100 mg of tissue with this method. The amplified 2nd strand cDNA was distributed 300 bp and over.

Isolation and Purification of Hemicelluloses in Alkali Pre-extractives from Mixed Hardwood (활엽수 알칼리 선추출물 내 헤미셀룰로오스의 분리 및 정제)

  • Sim, Kyu-Jeong;Shin, Hee-Nae;Youn, Hye-Jung;Lee, Hak-Lae
    • Journal of Korea Technical Association of The Pulp and Paper Industry
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    • v.42 no.4
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    • pp.64-70
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    • 2010
  • In this study, hemicelluloses were pre-extracted from mixed hardwood chips using sodium hydroxide solution and then they were isolated from pre-extractives by using various solvents. Isolation was conducted by precipitating hemicelluloses with isopropanol, ethanol, 1,4-dioxane, dimethylsulfoxide (DMSO) and potassium hydroxide (KOH), respectively. The precipitate yield on alkali pre-extractives was the highest when hemicelluloses were isolated by DMSO and then precipitated with ethanol. Most precipitates were yellow colored. The efficiency of isolation was evaluated by analysing the characteristics of isolated hemicelluloses. Isolation using DMSO, KOH and 1,4-dioxane ($80^{\circ}C$) showed rather high efficiency. The highest total separation efficiency was about 77% when alkali pre-extractives were reacted with KOH and precipitated with ethanol. The quantity and purity of isolated hemicelluloses were affected by the solvent type.

Isolation and Purification of Methyl Mercaptan Oxidase from Rhodococcus rhodochrous for Mercaptan Detection

  • Kim, Sang-Joon;Shin, Hyun-Jae;Kim, Yeu-Chun;Lee, Dae-Sil;Yang, Ji-Won
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.5 no.6
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    • pp.465-468
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    • 2000
  • Methyl mercaptan oxidase was successfully induced from Rhodococcus rhodochrous IGTS8 using methyl mercaptan gas and purified to homogeneity for the detection of mercaptans. The purification procedure involved DEAE-Sephacel and Superose 12 column chromatography with recovery yields of 85.8 and 83.3%, and a specific activity of 92.7 and 303.4 units/mg-protein, respectively. The molecular weight of purified methyl mercaptan oxidase was determined to be 64.5 kDa by SDS-PAGE. The extract from gel filtration chromatography oxidizes methyl mercaptan to produce formaldehyde, which can be easily detected by the purpald-coloring method. Optimum temperature for activity was achieved at 60$^{\circ}C$. This enzyme was inhibited by both K$_2$SO$_4$and NaCl at concentration of less than 100mM and recovered to original activity at concentration of 200mM. In the presence of methanol, the activity decreased by 33%.

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Rapid and Efficient Purification of Nucleic Acids from the Macroalga Porphyra(Rhodophyta) (홍조류인 한국산 김종에서의 염색체 DNA 분리방법)

  • 류태형;최학선;최경희;이춘환
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.27 no.6
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    • pp.1166-1172
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    • 1998
  • A method for the isolation and purification of DNA from a red algae, Porphyra was innovated. The innovation of the method consists mainly of three steps that include sodium acetate treatment, chloroform extraction, and 0.2 volume isopropanol precipitation step. The sodium acetate treatment was designed to remove polysaccharide contamination, and the isopropanol step to remove proteins and salts contaminents. Genomic DNA,s of several species(for example, P. tenera, P. yezoensis, P. seriata, and P. pseudolinearis) was successfully isolated by the innovated method. The amount of DNA purified from one g of sample material with the innovated method was 53 g in average. The resulting DNA was characterized to include high molecular weight and showed no nuclease activity. The DNA was pure enough to be digested directly by various restriction enzymes without any difficulties. Porphyra DNA was pure enough and adequate for amplification reaction through the polymerase chain reaction (small nuclear rDNA PCR amplification).

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Extraction and Purification of Bioactive Materials from Agaricus blazei Fruiting Bodies (아가리쿠스 버섯에서 생리활성물질의 추출 및 정제)

  • 최정우;류동열;김영기;홍억기;권명상;한진수
    • KSBB Journal
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    • v.15 no.3
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    • pp.293-298
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    • 2000
  • ${\beta}$-Glucan a kind of polysaccharide which is particularly abundant in Agaricus blazei is known as the bioactive materials especially anticancer agents. The process development of the isolation and the purification process of water soluble ${\beta}$-glucans from A. blazei was achieved. and the process operation variables were optimized. Crude polysaccharides (CR.PS) were obtained from A. blazei by hot water extraction filtration solvent precipitation dialysis and freeze drying. Neutral and acidic fraction of polysaccharides were separated from crude polysaccharides by ion chromatography and then high molecular weight and low molecular weight fraction were separated from neutral fraction by gel chromatography. Quantitative and qualitative analysis of each compounds were performed with FT-IR NMR spectroscopy. Based on these analysis the optimal conditions of temperatures operating time organic solvent volume for precipitation and dialysis time were determined.

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Prepurification of paclitaxel by micelle and precipitation

  • Seong, Ju-Ri;An, Hui-Bun;Kim, Jin-Hyeon
    • 한국생물공학회:학술대회논문집
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    • 2003.04a
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    • pp.501-504
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    • 2003
  • A novel prepurification method was developed aiming at increasing yield and purity, also reducing solvent usage for purification of paclitaxel. This method was a simple and efficient procedure, for the isolation and prepurification of paclitaxel from the biomass of Taxus chinensis, consisting of micelle formation, followed by two steps of precipitation. The use of a micelle and precipitation in the prepurification process allows for rapid separation of paclitaxel from interfering compounds and dramatically reduces solvent usage compared to alternative methodologies. This prepurification process serves to minimize the size and complexity of the HPLC operations for paclitaxel purification. This process is readily scalable to a pilot plant and eventually to a production environment where multikilogram quantities of material are expected to be produced. As much as possible, the process has been optimized to minimize solvent usage, complexity, and operating costs.

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Isolation, Purification and Characterization of Chitosanase from Bacillus subtilis CH1

  • Oh, Chul-Hong;Lee, Je-Hee
    • Journal of Aquaculture
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    • v.19 no.1
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    • pp.40-46
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    • 2006
  • Bacillus subtilis strain with highly active chitosanase was isolated from the intestine of Sebastiscus marmoratus (scorpion fish). It was named as Bacillus subtilis CH1 by morphological, biochemical and 165 rRNA gene analysis. The optimal conditions for chitosanase production were investigated. The optimum carbon and nitrogen sources for Bacillus stibtilis CH1 were 2% starch and 1% yeast extract respectively. Unlike other chitosanases, the expression of this chitosanase was not induced or slightly induced with chitosan. The chitosanase secreted into the medium were concentrated with ammonium sulfate precipitation and purified by gel permeation chromatography. The molecular weight of purified chitosanase was 30 kDa. The optimum pH and temperature of purified chitosanase were 5.5 and $60^{\circ}C$ respectively. The purified chitosanase was continuously thermostable at $40^{\circ}C$ and showed stable activity between pH 6.0 and 8.0. Chitosanase activity of Bacillus subtilis CH1 under optimum condition was 4.1 units/ml.