Purification of recombinant His-tagged mesaconyl-CoA hydratase of C. aurantiacus and R. sphaeroides from 3 g of E. coli cellsa

Purification stepTotal enzyme activity (μmol min−1)Total protein (mg)Sp act (μmol min−1 mg protein−1)Recovery (%)Purification (fold)
Cell extract−/10,500106−/99−/100−/1
Heat precipitation9,300/−16.5/−560/−100/−1−
Ni-Sepharose high performance8,100/5,5006.4/4.01,300/1,40087/522.3/14.0
  • a The first number in each column refers to the C. aurantiacus enzyme, and the second number (boldface) in each column refers to the R. sphaeroides enzyme. For the enzyme assay, see Materials and Methods. −, not determined for the Chloroflexus enzyme or not applicable for the Rhodobacter enzyme.