Open AccessPurification and properties of Saccharomyces cerevisiae acetolactate synthase from recombinant Escherichia coli
Author(s) -
POULSEN Charlotte,
STOUGAARD Peter
Publication year - 1989
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1989.tb15133.x
Subject(s) - acetolactate synthase , size exclusion chromatography , biochemistry , escherichia coli , yeast , recombinant dna , molecular mass , enzyme , saccharomyces cerevisiae , biology , atp synthase , cofactor , ammonium sulfate precipitation , ammonium sulfate , microbiology and biotechnology , chromatography , chemistry , gene
The yeast ilv2 gene, encoding acetolactate synthase, was subcloned in an Escherichia coli expression vector. Although a major part of the acetolactate synthase synthesized by E. coli cells harbouring this vector was packaged into protein inclusion bodies, we used these recombinant E. coli cells to produce large quantities of the yeast enzyme. The yeast acetolactate synthase was purified to homogeneity using first streptomycin and ammonium sulfate precipitations, followed by T‐gel thiophilic interaction, Sephacryl S‐300 gel filtration, Mono Q anion exchange, and Superose 12 gel filtration chromatography. SDS/PAGE and gel filtration of the purified enzyme showed that it is a dimer composed of two subunits, each with the molecular mass of 75 kDa. The purified yeast acetolactate synthase was further characterized with respect to pH optimum, dependence of the substrate, pyruvate, and requirements of the cofactors, thiamin diphosphate, Mg 2+ , and FAD