Abstract:

Objective: To extract and purify black soybean lipoxygenase (bsLOX) containing β-glucosidase activity and
investigate its anthocyanins-degrading properties and enzymatic activity. Methods: Purification of bsLOX was carried
out by buffer extraction, ammonium sulfate precipitation, dialysis, cation exchange chromatography and gel filtration
chromatography. The activities of lipoxygenase and β-glucosidase in bsLOX were analyzed by using sodium linoleate and
p-nitrophenyl β-D-glucopyranoside (pNPG) as substrates, respectively. An LOX from black soybean seeds was obtained,
which showed a single band in SDS-PAGE, with approximate molecular weight of 95 kD. The purified protein had a
lipoxygenase activity of approximately 13 U/mL, and could produce nitro phenol using pNPG as substrate as detected at
405 nm, suggesting that bsLOX had β-glucosidase activity. HPLC results showed that bsLOX could only degrade part of
anthocyanins (cyanidin-3-O-glucoside and petunidin-3-O-glucoside) into aglycone anthocyanidins (cyanidin and petunidin)
and glucose; in turn, the residual anthocyanidins could inhibit the activity of bsLOX. Conclusion: LOX in black soybean
may be a kind of protein with a variety of enzymatic functions, while anthocyanidins may be a natural bsLOX inhibitor.

Key words: black soybean, lipoxygenase, β-glucosidase activity, anthocyanin, anthocyanidins