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Reverse micelles in protein separation: The use of silica for the back‐transfer process
Author(s) -
Leser Martin E.,
Mrkoci Kristina,
Luisi Pier Luigi
Publication year - 1993
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260410413
Subject(s) - chemistry , aqueous solution , micelle , chromatography , adsorption , extraction (chemistry) , biomolecule , pulmonary surfactant , trypsin , protein purification , micellar solutions , chymotrypsin , aqueous two phase system , yield (engineering) , chemical engineering , organic chemistry , enzyme , materials science , biochemistry , engineering , metallurgy
In order to use reverse micellar solutions successfully for the separation of proteins, good methods are needed to recover the biomolecules into an aqueous environment after solubilization into organic micellar media. Usually the recovery is accomplished by equilibrating the protein‐loaded reverse micellar solution with a water phase containing an appropriate salt (back‐transfer). In this article we describe an alternative “back extraction” procedure which is based on the addition of silica to the protein‐containing reverse micellar solution. In this way, the water is stripped from the reverse micellar solution. [i.e., bis(2‐ethylhexyl) sodium sulfosuccinate (AOT)/isooctane/water] and the proteins adsorb to the silica particles. The adsorption process is shown to be practically quantitative. The subsequent recovery of the proteins form the silica into an aqueous solution turns out to be most efficient at alkaline pH (pH 8); 60–80 of the total protein (α‐chymotrypsin or trypsin) could be recovered. The specific enzyme activity at the end of the whole cycle can be as high as 80−100%. The procedure is applied also for the back extraction from micellar solutions in which, instead of AOT, a biocompatible surfactant such as a synthetic short‐chain lecithin was used. It is shown that the recovery of a α‐chymotrypsin and trypsin is also achievable under these conditions in quite good yield and under good maintenance of the enzyme's catalytic activity. © 1993 John Wiley & Sons, Inc.