ABSTRACT
Purple membrane (PM) is a part of cytoplasmic membrane in certain extreme halophilic microorganisms belonging to Domain Archaea. It transduces light energy to generate proton gradient for ATP synthesis in the microorganisms. Bacteriorhodopsin (BR) is the only protein in PM responsible for the generation of proton gradient. Generally, PM was purified from Halobacterium salinarum via a tedious and lengthy sucrose density gradient ultracentrifugation (SGU). In this work, a facile method based on polyethyleneglycol (PEG)-phosphate aqueous-two- phase extraction system (ATPS) was employed to purify PM from cell lysate of H. salinarum. The results showed that PM could be completely recovered from the interface of PEG-phosphate ATPS with BR purity ca 94.1% as measured by UV-visible absorption spectra. In comparison with PM obtained by SGU, the PM isolated by ATPS could achieve the same level of purity and photocurrent activity (ca 177.2nA/µgBR/cm(2)) as analyzed by SDS-PAGE and photocurrent measurement, respectively. The easily scalable and straightforward ATPS procedure demonstrated that PM can be purified and recovered more cost-effectively with a significantly reduced operation time that should lead to broader range applications of PM possible.
Subject(s)
Archaeal Proteins/isolation & purification , Archaeal Proteins/metabolism , Bacteriorhodopsins/isolation & purification , Bacteriorhodopsins/metabolism , Halobacterium salinarum/metabolism , Purple Membrane/metabolism , Cell Fractionation , Electrophoresis, Polyacrylamide Gel , Equipment Design , Halobacterium salinarum/chemistry , Phosphates/chemistry , Photochemistry/instrumentation , Polyethylene Glycols/chemistry , Purple Membrane/chemistry , UltracentrifugationABSTRACT
The production of fatty acid methyl esters (FAMEs) by a two-step in-situ transesterification from two kinds of rice bran was investigated in this study. The method included an in-situ acid-catalyzed esterification followed by an in-situ base-catalyzed transesterification. Free fatty acids (FFAs) level was reduced to less than 1% for both rice bran A (initial FFAs content=3%) and rice bran B (initial FFAs content=30%) in the first step under the following conditions: 10 g rice bran, methanol to rice bran ratio 15 mL/g, H(2)SO(4) to rice bran mass ratio 0.18, 60 degrees C reaction temperature, 600 rpm stirring rate, 15 min reaction time. The organic phase of the first step product was collected and subjected to a second step reaction by adding 8 mL of 5N NaOH solution and allowing to react for 60 and 30 min for rice bran A and rice bran B, respectively. FAMEs yields of 96.8% and 97.4% were obtained for rice bran A and rice bran B, respectively, after this two-step in-situ reaction.
