Purification and properties of Saccharomyces cerevisiae acetolactate synthase from recombinant Escherichia coli.
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, Mg2+, and FAD.
Poulsen
C
C
Danisco A/S, Biotechnology Research Division, Copenhagen, Denmark.
Stougaard
P
P
D000095:Acetolactate Synthase / Q000096:biosynthesis / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D007652:Oxo-Acid-Lyases / Q000302:isolation & purification*; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D000095:Acetolactate Synthase; D007652:Oxo-Acid-Lyases