title: Analytical characterization and purification of a commercial extract of enzymes: a case study.
abstract: 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 a hydrolytic activity higher than the crude extract.
authors.affiliation: Centro de Investigación y Desarrollo en Ciencias Aplicadas-Dr. Jorge J. Ronco, Universidad Nacional de La Plata, CONICET, CCT La Plata, Calle 47 N(o) 257, B1900AJK La Plata, Buenos Aires, Argentina; Laboratorio de Investigación de Proteínas Vegetales (LIPROVE), Depto. de Cs. Biológicas, Fac. Cs. Exactas, Universidad Nacional de La Plata, Calle 47 y 115, La Plata, Argentina. Electronic address: carlollzz@yahoo.com.
authors.forename: Carlos R
authors.initials: CR
authors.lastname: Llerena-Suster
authors.affiliation: Centro de Investigación y Desarrollo en Ciencias Aplicadas-Dr. Jorge J. Ronco, Universidad Nacional de La Plata, CONICET, CCT La Plata, Calle 47 N(o) 257, B1900AJK La Plata, Buenos Aires, Argentina.
authors.forename: Laura E
authors.initials: LE
authors.lastname: Briand
authors.affiliation: Laboratorio de Investigación de Proteínas Vegetales (LIPROVE), Depto. de Cs. Biológicas, Fac. Cs. Exactas, Universidad Nacional de La Plata, Calle 47 y 115, La Plata, Argentina.
authors.forename: Susana R
authors.initials: SR
authors.lastname: Morcelle
mesh_terms: D000818:Animals; D002175:Candida / Q000201:enzymology*; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D008049:Lipase / Q000032:analysis* / Q000302:isolation & purification*; D012015:Reference Standards; D012710:Serum Albumin, Bovine / Q000032:analysis; D013056:Spectrophotometry, Ultraviolet
chemical_list: D012710:Serum Albumin, Bovine; C093666:Lipozyme; D008049:Lipase
keywords: CALB; Ion exchange chromatography; Protein purification; Protein quantification; Size exclusion chromatography; UV absorbance