PURIFICATION AND CHARACTERIZATION OF INVERTASE FROM DATES (PHOENIX DACTYLIFERA L., VAR. ZAHDI)

Abstract

Zahdi dates (Phoenix dactylifera) contain invertase at all development stages; the highest specific activity is present in the late yellow stage. The enzyme was purified to homogeneity, as determined by disc gel electrophoresis and isoelectric focusing, by a combination of techniques including ammonium sulfate precipitation, DEAE-cellulose chromatography and gel filtration on Sepharose 4B and Sephadex G-150 columns. A complex of invertase with a high molecular weight pectic substance of the date could not be dissociated by ammonium sulfate or DEAE-cellulose chromatography but the complex was dissociated by gel filtration on a Sepharose 4B column at pH 4.0 and ionic strength of 0.5 M. The enzyme contained 8.2% carbohydrate covalently linked probably via an amide linkage to aspartic acid. Molecular weight determination by exclusion gel chromatography and sedimentation equilibrium gave values of 130,000 and 97,100 ± 1,300, respectively. The enzyme is probably composed of two identical subunits as shown by SDS polyacrylamide gel electrophoresis. Amino acid analyses showed the enzyme to be low in sulfur-containing amino acids. Date invertase is an acid β-fructofuranosidase with a pH optimum between 3–4 and with a Km and kcat for sucrose of 6mM and 49 sec-1, respectively. Activation energies for denaturation of enzyme and conversion of substrate to product were determined to be 48.7 and 17.6 kcal/mole, respectively. Chemical modification indicated that sulfhydryl groups are probably not essential for activity while carboxyl groups may be involved in the active site of the enzyme.