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Purification of the Penicillium citrinum Lipase Using AOT Reversed Micelles
Author(s) -
Krieger Nadia,
Taipa M. Angela,
AiresBarros M. Raquel,
Melo Eduardo H. M.,
LimaFilho Jorge L.,
Cabral Joaquim M. S.
Publication year - 1997
Publication title -
journal of chemical technology and biotechnology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.64
H-Index - 117
eISSN - 1097-4660
pISSN - 0268-2575
DOI - 10.1002/(sici)1097-4660(199705)69:1<77::aid-jctb666>3.0.co;2-v
Subject(s) - chromatography , chemistry , lipase , extraction (chemistry) , penicillium citrinum , aqueous two phase system , micelle , ionic strength , aqueous solution , yield (engineering) , enzyme , biochemistry , organic chemistry , materials science , food science , metallurgy
This work describes the extraction and back‐extraction of a lipase from crude extract of Penicillium citrinum using AOT reversed micelles in isooctane. The effect of pH, ionic strength, AOT concentration on the protein forward and backward transfer at 20°C was studied. The maximum protein forward extraction (32·0%) was achieved at pH 4·0 with a 50 mmol dm −3 acetate buffer containing 100 mmol dm −3 KCl and 100 mmol dm −3 AOT in isooctane. Proteins were back‐extracted (82·7%) to a new aqueous phase containing 100 mmol dm −3 pH 8·0 phosphate buffer and 1000 mmol dm −3 KCl. No enzyme activity could be detected either in the micellar phase or in the aqueous phase after protein back‐extraction. However, the lipolytic activity was recovered after hydrophobic interaction chromatography on a Phenyl Superose column. The yield obtained for the overall process was 68% for activity, 26·4% for protein recovery and the purification factor was 810‐fold. A single protein band at 33000 Da was obtained for SDS–PAGE analysis for the recovered and purified enzyme. © 1997 SCI.