Abstract: In this paper, R-phycoerythrin with relatively high purity (A565 nm/A280 nm = 5.4) was obtained from Porphyra haitanensis by ammonium sulfate fractionation and DEAE-Sepharose anion exchange chromatography. Prolyl endopeptidase (PEP) inhibitory peptides from R-phycoerythrin were prepared by two-step enzymatic digestion with gastrointestinal fluid proteases. Optimal hydrolysis conditions were determined as sequential hydrolysis with 0.5% pepsin at an enzyme-tosubstrate ratio of for 30 min at an initial pH of 1.2 and 37 ℃ followed by a 1:1 mixture of trypsin and chymotrypsin at an enzyme-to-substrate ratio of 0.25% for 30 min at pH 7.5 and 37 ℃. Tricine-SDS-PAGE showed that R-phycoerythrin was completely degraded by two-step enzymatic hydrolysis. Gel filtration chromatography analysis revealed that fractions with molecular mass lower than 3 kDa accounted for 86.06% of the total peptides. The half-maximum inhibitory concentration (IC50) of R-phycoerythrin hydrolysate toward PEP was 136.35 μg/mL. Inhibition kinetic study revealed that the hydrolysate acted as a noncompetitive inhibitor and the constant of inhibition (Ki) was 14 μg/mL. Successful preparation of R-phycoerythrin peptides with potential PEP inhibitory activity provides a theoretical rationale for the processing and utilization of P. haitanensis.

Key words: Porphyra haitanensis, R-phycoerythrin, PEP inhibitory activity, molecular mass distribution, inhibition kinetics