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.