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.
Barhee and Zahdi dates (Phoenix dactylifera) contain polyphenol oxidase (PPO) at all maturity stages. The highest PPO activity is present in the medium green and late yellow stages of Barhee and Zahdi dates, respectively. The extracts of medium green stage of Barhee dates contained proteolytic activity. Four PPO forms were purified to homogeneity by acetone precipitation and DEAE‐cellulose chromatography. The total fold purification was 8.0, 10.0, 12.5, and 15.0 with 6.7, 0.7, 2.2, and 2.7% recovery of PPO activity. A single PPO form was observed when 1.0mM of phenylmethylsulfonyl chloride (PMSC) was used in extraction solutions. However, only a single PPO form was observed in Zahdi dates and purified 13.1 fold with 7.7% recovery of activity.
Purified polyphenol oxidase (PPO) from Barhee and Zahdi date cultivars had a molecular weight of 17,500 and 17,000, respectively, as determined by exclusion gel chromatography. The enzyme possessed activity on o‐diphenols but not on monophenols or m‐diphenols. The highest activity was on catechol with Km of 3.5 and 8.75 mM for PPO from Barhee and Zahdi dates, respectively. PPO from the two cultivars showed optimal pH for activity and stability around 6.0 and 7.0, respectively. The enzyme was completely inactivated when incubated at 70°C for 10 min. Activation energy for conversion of substrate to product was 3400 and 3600 cal/mole for PPO from Barhee and Zahdi dates, respectively. However, the activation energy for denaturation was 28000 and 27000 cal/mole for the enzyme from Barhee and Zahdi cultivars, respectively. Ascorbic acid, oxalic acid and sodium metabisulfite caused non‐competitive inhibition for PPO activity, while sodium chloride was an uncompetitive inhibitor. Sodium metabisulfite was the most potent inhibitor with Ki values of 0.025 and 0.030 mM for PPO from Barhee and Zahdi dates, respectively.
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