Exopeptidase Activity in pH 7-OB
As shown in Table 2, the major exopeptidases were tripeptidyl-peptidase
2 and serine carboxypeptidases, while aminopeptidases (proline
iminopeptidase and leucine aminopeptidase) showed extremely low relative
abundances. The hydrolyzed products of carboxypeptidases and
aminopeptidases are free amino acids, while those of
tripeptidyl-peptidase 2 are tripeptides. Different from
carboxypeptidases and aminopeptidases, it was difficult to directly
estimate the activity of tripeptidyl-peptidase 2 in isolated sesame OBs.
However, the tripeptides would be hydrolyzed into free amino acids by
aminopeptidases and carboxypeptidases. Therefore, the determination of
free amino acids could be used to quantitatively explain the global
activity of exopeptidases.
As shown in Table 3, the total free amino acid content in pH 7-OB showed
a very low value of 5.59 mg/L (just 0.06% of total proteins), which was
induced by the preparation method of pH 7-OB (floated up from water
extract, and then washed by DI water). By incubation at pH 3–9 and 50
°C for 2 h, the total free amino acid content peaked at pH 4 with a
value of 868.48 mg/L (9.04% of total proteins), followed by pH 5
(415.02 mg/L) and 3 (404.79 mg/L). At pH 6, the total free amino acid
content was 118.41 mg/L, and gradually decreased to 48.18 mg/L at pH 9.
By comparing the free amino acids produced at pH 4, it was found that
leucine, phenylalanine, valine, alanine, isoleucine, and tyrosine were
greatly produced, which agreed with the optimal pH and cleavage
specificity of serine carboxypeptidases (Granat, Wilson, & Tan-Wilson,
2003). Together with the results in Table 2, it could be concluded that
in the acidic pH range, especially at pH 3–5, the exopeptidase activity
was attributed to serine carboxypeptidases. Generally, aminopeptidases
exert optimal activity at neutral and alkaline pH (Walling & Gu, 1996;
Simpson, 2001). Therefore, the low total free amino acid contents at
neutral and alkaline pH should be produced by these aminopeptidases with
low relative abundances.
TCA–NSI analysis was used to quantitatively estimate the combined
activity of endopeptidases and exopeptidases, and to compare the
endopeptidase activity with exopeptidase activity together with the
results of total free amino acids (Table 3). It was found that the
combined activity peaked at pH 4 with a high TCA–NSI value of 41.47%,
closely followed by pH 5 (38.67%). With increasing pH from 5, the
TCA–NSI gradually decreased to 4.67% at pH 9. At pH 3, the TCA–NSI
was 31.18%. By subtracting the percentage of total free amino acids
from TCA–NSI, it was found that TCA-soluble peptides peaked at pH 5
with a value of 34.35%, closely followed by pH 4 (32.43%). With
increasing pH from 5, the TCA-soluble peptides gradually decreased to
4.17% at pH 9. At pH 3, the TCA-soluble peptides showed a value of
26.97%. These results showed that 1) the activity of endopeptidases
(aspartic endopeptidases and subtilisin-like proteases) peaked at pH 5,
2) the activity of exopeptidases (serine carboxypeptidases) peaked at pH
4, and 3) the combined activity of endopeptidases and exopeptidases was
optimal at pH 4–5.