Analytical characterization and purification of a commercial extract of enzymes: a case study

Authors
Llerena Suster, Carlos Rafael; Briand, Laura Estefania; Morcelle del Valle, Susana Raquel
Publication Year
2014
Language
English
Format
article
Status
Published version
Description
This paper presents a rational strategy to identify and quantify the components of a commercial extract of the lipase B of Candida antarctica that can be extended to the analytical investigation of other crude extracts of enzymes. These information provided the fundamental knowledge for the development of a methodology to obtain highly pure and catalytically active CALB enzyme. The commercial extract Lipozyme® was subjected to a series of analytical techniques that allowed determining the presence of a non-soluble fraction; nucleic acids; benzoate and sorbate species and a mixture of three proteins. Particularly, it is worth noticing that the Bradford assay using CALB as standard instead of BSA proved to be a more reliable and accurate methodology to quantify the protein content of the assayed enzymatic samples. Size exclusion chromatography coupled with anionic exchange chromatography using a non conventional, easy to remove buffer system such as ammonia-ammonium acetate afforded a sample that retains 47% of the proteins (being CALB the only enzymatic component of the purified sample) with an hydrolytic activity higher than the crude extract.
Fil: Llerena Suster, Carlos Rafael. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biologicas. Laboratorio de Investigacion de Proteinas Vegetales; Argentina
Fil: Briand, Laura Estefania. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Ciencias Aplicadas "Dr. Jorge J. Ronco". Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Ciencias Aplicadas; Argentina
Fil: Morcelle del Valle, Susana Raquel. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biologicas. Laboratorio de Investigacion de Proteinas Vegetales; Argentina
Subject
Protein purification
Protein quantification
CALB
UV absorbance
Size exclusion chromatography
Ion exchange chromatography
Otras Ciencias Biológicas
Ciencias Biológicas
CIENCIAS NATURALES Y EXACTAS
Access level
Restricted access
License
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
Repository
CONICET Digital (CONICET)
Institution
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identifier
oai:ri.conicet.gov.ar:11336/29267