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Use of Dye Affinity Chromatography for the Purification of Aerococcus viridans Lactate Oxidase
Author(s) -
Streitenberger Sergio A.,
LópezMás José A.,
SánchezFerrer Álvaro,
GarcíaCarmona Francisco
Publication year - 2002
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp020022k
Subject(s) - fast protein liquid chromatography , chemistry , chromatography , size exclusion chromatography , biochemistry , oxidase test , molecular mass , affinity chromatography , ion chromatography , enzyme , high performance liquid chromatography
Lactate oxidase was purified from Aerococcus viridans ( A. viridans ) by dye affinity chromatography and FPLC ion exchange chromatography. The lactate oxidase could be purified by comparatively simple procedures, the purification achieved from a crude extract of A. viridans was 41‐fold with a specific activity of 143 units/(mg of protein). The purified enzyme was a l ‐ lactate oxidase, which catalyses the conversion of l ‐lactate in the presence of molecular oxygen to pyruvate and H 2 O 2 . This purified lactate oxidase showed an apparent molecular mass of 48 200 in SDS‐PAGE and the native molecular weight, as estimated by FPLC gel filtration, was 187 300. This molecular weight indicates that lactate oxidase exists in tetrameric form after gel filtration. To differing degrees, all the triazine dyes tested were inhibitors of lactate oxidase, solutions of free triazine dyes showing an inhibition mechanism which was both time‐ and pH‐dependent.