Confirmation on the Catalytic Centers of Endopeptidases
As shown in Table 2, the dominant endopeptidases were aspartic endopeptidases, which had obviously higher relative abundance than metalloendopeptidase and subtilisin-like proteases. Cysteine endopeptidases showed extremely low relative abundance. Therefore, pepstatin A, AEBSF, and EDTA-2Na were selected for the protease inhibitor assay. Without inhibitors (Fig. 2a), the Tricine–SDS–PAGE profiles of pH 7-OB incubated at pH 3–9 were similar to the corresponding results in Fig. 1a. In the presence of pepstatin A (Fig. 2b), the hydrolysis of oleosin-H was obviously inhibited at pH 3–6, while the hydrolysis of 11S globulins was obviously inhibited at pH 3–4. However, the hydrolysis of oleosin-H was still obvious at pH 4–6, especially at pH 5. In the presence of AEBSF (Fig. 2c), the Tricine–SDS–PAGE profiles were almost the same as those in Fig. 2a, which supported the results that aspartic endopeptidases were the dominant endopeptidases. Generally, aspartic endopeptidases are sensitive to pepstatin A (Simoes & Faro, 2004), but some ones are insensitive to it (Toogood, Prescott, & Daniel, 1995; Kocabıyık & Özel, 2007). Therefore, it was suggested that there were at least two types of aspartic endopeptidases in pH 7-OB. One type (optimal at pH 3) could be inhibited by pepstatin A, but the other (optimal at pH 5) could not. This could be well supported by the three kinds of aspartic endopeptidases (aspartic proteinase-like, 2.938‰; aspartyl protease 2-like, 0.205‰; aspartic proteinase A1-like, 0.177‰) with high relative abundances in Table 2.
In one previous study, it was clarified that one aspartic endopeptidase, which was bound to peanut OBs, could be greatly washed from OBs by alkaline pH (Chen et al., 2018). Therefore, pH 9.5-OB was prepared to examine whether AEBSF could exert obvious inhibition activity. In the absence of inhibitors (Fig. 2d), it was found that the hydrolysis of oleosin-L at pH 4 and 6 and 11S globulins at pH 3 was obviously weaker than that in pH 7-OB (Fig. 2a), indicating that aspartic endopeptidases were greatly removed from OBs by pH 9.5 washing. However, the hydrolysis of oleosin-H at pH 5 was still obvious. In the presence of pepstatin A (Fig. 2e), the hydrolysis of oleosin-H at pH 5 was inhibited to some extent, which could be attributed to the residual aspartic endopeptidases in pH 9.5-OB. In the presence of AEBSF (Fig. 2f), the hydrolysis of oleosin-H at pH 5 was greatly inhibited. These results revealed that subtilisin-like proteases showed higher resistance to pH 9.5 washing than aspartic endopeptidases, and they exhibited sharp optimum at pH 5. One subtilisin-like protease from soybean also showed sharp optimum at pH 5 (Boyd, Barnaby, Tan-Wilson, & Wilson, 2002).
Until now, all examined plant metalloendopeptidases had optimal activity at neutral and alkaline pH (Marino & Funk, 2012). Therefore, the effects of EDTA-2Na on proteolytic activity in isolated sesame OBs was conducted at pH 8 and 50 °C. It was found that hydrolysis of oleosin-H could be inhibited by EDTA-2Na (data not shown). Thus, it was suggested that metalloendopeptidase contributed to the low proteolytic activity at neutral and alkaline pH, while the proteolytic activity in the pH range of 3–6 was dominantly attributed to aspartic endopeptidases. In addition, it should be noted that subtilisin-like proteases also contributed to the proteolytic activity at pH 5.