dubium seeds were also shown to be highest at 50, 55 and 70 °C, respectively ( Ahmed et al., 2009, Lo Piero et al., 2002 and Teixeira
et al., 2000). Molecular rearrangements in protein structure can lead to increase of enzyme activity ( Purich, 2010). Caseinolytic activity was higher when PP was previously incubated at pH 4.0 and 7.0 (Table 2). A partially purified enzyme from S. dubium seeds also showed proteolytic activity towards azocasein at pH 4.0 but, unlike M. oleifera activity, the enzyme was highly active up to pH 11.0 ( Ahmed et al., 2009). It is known that pH affects the shape, charge Hydroxychloroquine molecular weight properties, the correct positioning of the substrate and the ionisation of side chains of amino acids, in both the active site and in the whole enzyme ( Purich, 2010). Heating of PP from 30 to 40 °C did not interfere in milk-clotting activity, which increased significantly after heating at 50 °C and was neutralised at 70 °C (Table 1). Milk-clotting
enzymes from Bromelia hieronymi, W. coagulans, Solanum esculentum and Solanum macrocarpon are stable proteins, remaining active after heating to 45, 70 and 70 °C, respectively ( Bruno et al., 2010, Guiama et al., 2010 and Naz et al., 2009). A milk-clotting enzyme called religiosin B, purified from Ficus religiosa stem latex, showed highest milk-clotting activity at temperatures of 55 and 60 °C ( Kumari, Sharma, & Jagannadham, 2012). Milk-clotting activity from M. OTX015 molecular weight oleifera flowers was highest after previous incubation of PP at pH 3.0 ( Table 2) and lost of activity was detected when PP was previously incubated at pH values higher than 8.0. Calf rennet showed similar behaviour, acting better in acid PTK6 than in alkaline reaction medium ( Richardson, Nelson, Lubnow, & Schwarberg, 1967). Differently, the milk-clotting enzyme religiosin B showed highest clotting ability at pH 6.0 ( Kumari et al., 2012). High thermal stability and ability to work in a wide pH range are
important criteria for the choice of proteases to be used in industrial processes (Vieille & Zeikus, 1996). In this sense, the milk-clotting enzymes present in PP are promising candidates for application in milk-clotting at an industrial large scale. Additionally, the traditional use of M. oleifera flowers in human diet, being eaten raw or after lightly blanched ( Makkar & Becker, 1996), is an indicative of PP safety for use in cheese production. The evaluation of enzyme activities from M. oleifera flowers in presence of protease inhibitors ( Table 3) showed that the caseinolytic activity on azocasein was not significantly (p > 0.05) altered in presence of PMSF, while milk-clotting activity was significantly (p < 0.05) reduced, by as much as 25%. E-64 significantly (p < 0.05) inhibited only milk-clotting activity (by 30%), while pepstatin A significantly reduced (p < 0.05) caseinolytic and milk-clotting activities, by 25% and 57.5%, respectively.
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