Purification and Characterization of Protopectinase Produced by Kluyveromyces marxianus

The crude protopectinase preparation obtained from Kluyveromycesmarxianus cultures was partially purified by fractionalprecipitation with ammonium sulphate. At 65% ammonium sulphate,the most active fractionation was obtained. Further purification by gelfiltration on CM-Sephadix C-50 followed by ion exchange chromatographyon Sephadix G-75 yielded 4 peaks of protopectinasecomponents. The second peak was the major one containing most ofthe recovered protein and all the protopectinase activity.The enzyme showed different hydrolytic activities on some protopectinsources. Characterization of the enzyme including, enzymeconcentration, amount of substrate, pH and temperature of the reactionmixture was carried out. The enzyme was stable up to 50ºC andat pH range between 4 and 7. The enzyme activity was respondeddifferently to the tested metal ions and some inhibitors. The aminoacids composition of the enzyme showed a high proportion of glycineand moderate amounts of glutamic acid, alanine and leucine but poorcontents of proline, cysteine and tyrosine.