PURIFICATION AND PROPERTIES OF INVERTASE FROM MUSCAT-BAILEY-A GRAPES

A grape invertase was purified from Muscat Bailey A juice by salting o ut with ammonium sulfate and successive chromatographies on Sephadex G -100 and Con A-agarose to a homogeneous state as confirmed by polyacry lamide gel electrophoresis. The molecular weight of the purified enzym e was 72 kDa in gel filtration chromatography. However, SDS polyacryla mide gel electrophoresis revealed three bands with molecular weights o f 56 kDa, 25 kDa and 24 kDa. Affinity staining of Western blots with l ectins indicated that the enzyme was a glycoprotein. The optimum pH fo r the enzyme reaction was 3.5 and the optimum temperature 80 degrees C . The enzyme was stable from pH 2.7 to 6.4, and up to 80 degrees C. Th e transfructosylation reaction could not be observed. The K-m value of this enzyme for sucrose was 4.4 mM at pH 4.0, but as the pH of the re action mixture increased, the K-m value decreased sharply. From the de pendence of the V-max and K-m values on pH, the ionization constant (p K(e)) of one of the two essential ionizable groups of the free enzyme was determined to be 2.7, suggesting that this essential ionizable gro up was a carboxyl.