Sour-orange was selected as the source material for pectin esterase (s), as it in the cheapest of all other citrus fruits in the country and also no work had been undertaken.
Pectin esterase content of flavedo, albedo and whole peel, and other physical and chemical characteristics (weight) of fruit, juice content, diameter of fruit, thickness of peel, ascorbic acid were determined over five month and seven month period of maturity during 1967-68 and 1969-70 sesons. The distribution of the activity in the comment parts of fully mature sour oranges was in the increasing order of juice (1.13 x10â€”4 PEu/ml), seeds. (2.23 x 10â€”4 PE/ml), pulp (11.86 x 10â€”4 PEu/ml), and peels. (18.42 x 10-4 PEu/ml). PE activity in flovedo (18.72 x 10-4 PEu/ml).
Drying of sour orange peels was carried out by various drying techniques shade-drying, sun drying, and dehydration at low temperature (30 â€“ 35oc). The latter procedure was found most suitable in obtaining dried material.
Pectin methyl esterase was extracted from the powdered peel by successive extractions with dilute sodium chloride solution (0.25m) in the ratio of 1:10(v/v) at pH 7-8. PH activity was found to be highest in the first (43.51% activity)
Fractionation of PE by ammonium soleplate yielded the maximum activity (22.08% activity) at 0.6 saturation of the extract with ammonium sulphate. However, this method was not found feasible as most of the activity (345% activity) remained in the solution. While fractional precipitation with cold ( 0 â€“ 3oc) acetones proved to be the most convenient initial step for isolation of the enzyme from its crude extract at PH 6.5. The optimum PE activity (46.85%) was obtained at 60% acetone. Toluene was found satisfactory pre servitude for pE preparations.
Calcium phosphate-gel was found useful for further purification of the solvent fractionated enzyme. Almost completes elution of the adsorbed enzyme was made at room temperature (25oc), by employing 0.2m phosphate butter at ph 7.5.
Electrophoresis analysis of the elute showed one spot suggesting the enzyme protein to be homogenous which had PE activity of 7.8 x10-4 pEu/ml.
Km value of sour orange pe was found to be 0.45% by the method of line weaver and budk. Optima for ph and temperature of pe were found to be 7.5, and respectively.
Inhibitory effects of pâ€”chloromereuri â€“benzoic acid. Iodo acetate, 2:4-dinitro-1-flouro-benzene, and nitrorussid* test suggest the possibility of thiol group (s) which may from an integral part of the active site of the enzyme. It was also found that eyestone could not reverse the inhibition brought about by p-chloremeromercure benzoate.
pE activity was increased by the presence of sodium chlorides at pH 7.5. This was shown by the application of EDTA (disokium ethylene diamante terracotta) to pe which reduced its activity.
Pectin esterase preparation was applied to various juices namely : grape, apple, pear , sweat lime and pomegranate, at low (18oc) and high (45oc) temperatures. The action of PE, on the clarification of these juice incubate at higher temperature was faster than that at lower temperatures.