Partial Purification and Characterization of Invertase from Flowers of Madhuca longifolia (Mi)

In ayurveda, fermentation processes have been used for ages to modify the potency of drugs and to extract and to preserve active constituents from medicinal plants. Therapeutic application of these fermented drugs are based on the properties of ingredients and the method of preparation. In general, these fermented ayurvedic preparations posses a moderate potency and administered in the convalescent phase of a disease (Namijoshi 1979, Caraka Samhita 1949). One such fermented preparations known as ‘Arishtas’ are widely used in Sri Lanka as well as in other South Asian countries. ‘Arishtas’ are weak alcoholic preparations used for the treatment of a wide variety of diseases and specially, prescribed for diseases which require long term treatments. Since, Arishtas are not perishable very often they are used as tonics (Kroes et al. 1992, Kroes 1990, Ayurvedic Pharmacopoeia 1976 &1979). In the preparation of many Arishtas, dried flowers of Woodfordia fruticosa (L.) Kurz ( Malitta in Sinhalese, Dataki in Sanskrit; Family, Lythraceae), or Madhuca Enzyme Invertase (β-D-fructofuranosidase, EC 3.2.1.26), present in the flowers of Madhuca longifolia plays an important role during the preparation of fermented Ayurvedic drugs known as ‘Arishta’. This enzyme was partially purified with a yield of 11.6%, using (NH4)2SO4 fractionation, followed by gel filtration through Sepharose 4B and DEAE cellulose chromatography at pH 6.5 and 4.2. The molecular mass of the enzyme as determined by elution through Sepharose 4B gel column was found to be about 210 kDa . The enzyme showed a broad pH optimum between 4 -7 . Optimum assay temperature was 37oC and above 45oC enzyme activity slowly declined and inactivated around 80oC. Approximate Km value for the enzyme for sucrose is 125 mM.