Endopeptidase Activity in pH 7-OB
As shown in Fig. 1, the proteins of pH 7-OB were composed of OB intrinsic and extrins proteins. By analyzing the band intensity on three lanes C (Fig. 1), it was calculated that oleosins contributed approximately 30% to total proteins in pH 7-OB, while approximately 26% of total proteins were attributed to 11S globulins. As stated above, two oleosin bands respectively showed apparent MWs of 14 and 12.5 kDa on Tricine−SDS−PAGE gel, although their corresponding theoretical MWs were around 17 and 15 kDa. Therefore, the two oleosin bands were respectively deemed as oleosins with high MWs (oleosin-H) and oleosins with low MWs (oleosin-L) in this study. Caleosins and steroleosins showed the similar trends to oleosins.
By incubation at pH 3–9 and 50 °C for 2 h (Fig. 1a), it was found that the proteins in pH 7-OB could be mainly divided into three groups based on their hydrolysis behaviors. Oleosins were the most easily hydrolyzed by proteases, whereas 2S albumins were the most difficult to be hydrolyzed. 11S and 7S globulins and some other proteins were intermediate. At pH 4–5, all oleosin-H were hydrolyzed, and approximately 4% were remained at pH 6 (Fig. 1b). At pH 7–9, 60–93% of oleosin-H were remained, and approximately 54% at pH 3. As for 11S globulins, approximately 25% were still remained at pH 5. Different from oleosin-H, the residual 11S globulins sharply increased to 87% at pH 6, and gradually to 97% at pH 9. At pH 3–4, 44–46% were remained. Moreover, these results showed a trend that endopeptidases preferred 11S and 7S globulins at pH 3, but they preferred oleosins in a broad pH range of 4–9. The similar phenomenon was also found in the isolated peanut OBs: one aspartic endopeptidase optimally hydrolyzed the oleosins at pH 4, while it optimally acted on Ara h 1 at pH 3 (Chen et al., 2018). Steroleosins showed the similar trend to oleosins, while caleosins to 11S globulins. It was reported that 2S albumins were stable to proteolysis, being extremely resistant to pepsin, and relatively resistant to trypsin and chymotrypsin (Orruño & Morgan, 2011). This could be used to explain the hydrolysis behaviors of 2S albumins. It could be summarized that oleosins were hydrolyzed in a broad pH range of 3–9 with optimal pH of 4–6, whereas 11S and 7S were obviously hydrolyzed at pH 3–5.
Furthermore, the effects of temperature on endopeptidase activity were examined at pH 5 for 1 h (Fig. 1c). At 30 °C, the band intensity of oleosin-H greatly weakened down compared to control, but the band position for oleosin-L showed great intensity increase. Due to the slight changes of 11S and 7S globulins, and caleosin at 30 °C, it was suggested that the intensity increase at the position for oleosin-L was induced by the hydrolyzed products of oleosin-H, which had similar MWs to oleosin-L. This phenomenon was also occurred in isolated soybean OBs, in which P34 probable thiol protease hydrolyzed 24 kDa oleosins into hydrolyzed products with similar MWs to 16 kDa oleosins (Zhao et al., 2017). With increasing temperature, oleosin-H disappeared from 40 to 70 °C, and re-appeared at 80 °C. As for the band position for oleosin-L, its intensity greatly weakened down from 30 to 40 °C, and gradually weakened down from 40 to 70 °C. Then the oleosin-L band was obviously observed at 80 °C. In the case of 11S globulins, approximately 95% were remained at 30 °C, and gradually to 62–64% at 50–60 °C (Fig. 1d). Then the residual 11S globulins increased to 70–71% at 70–80 °C. Comprehensively speaking, these results revealed that endopeptidases showed optimal temperature at 50–60 °C, whereas 70 °C, especially 80 °C denatured endopeptidases.