Purification and Biochemical Characterization of Cysteine Protease from Baby Kiwi (Actinidia arguta)

Abstract

Background:It has recently been reported that the fruit, stems and leaves ofActinidia argutahave various potential health effects including an antioxidant effect, anticancer effect, anti-allergic effect and α-glucosidase inhibitory effect. However, little is known about the biochemical properties of cysteine protease in the fruit juice ofA. arguta.Methods:Ion exchange chromatography to purify the cysteine protease from the fruit juice ofA. arguta, and some synthetic substrates to determinate the enzyme activity were used.Results:Cysteine protease was purified to homogeneity fromA. argutafruit juice by ion exchange chromatography. The molecular weight of the purified enzyme was calculated to be approximately 25,500 by SDS-PAGE in the presence of β-ME. The enzyme rapidly hydrolyzed the substrate Z-Leu-Arg-MCA and moderately hydrolyzed other substrates including Boc-Val-Leu-Lys-MCA, Z-Val-Val-Arg-MCA and Z-Phe-Arg-MCA. Kinetic parameters for these four substrates were determined. TheKm,Vmax,KcatandKcat/Kmvalues for Z-Leu-Arg-MCA, the most preferentially cleaved by the enzyme, were 100 μM, 63.8 μmoles/mg/min, 27.26 sec-1and 0.2726 sec-1μM-1, respectively. Furthermore, the activity of the enzyme was strongly inhibited by inhibitors including antipain, leupeptin, E-64, E-64c, kinin-free-LMW kininogen and cystatin C. Those biochemical data indicated that the enzyme was a cysteine protease. The amino acid sequence of the first 21 residues of cysteine protease purified fromActinidia argutawas Val1-Leu-Pro-Asp-Tyr5-Val-Asp-Trp-Arg-Ser10-Ala-Gly-Ala-Val-Val15-Asp-Ile-Lys-Ser-Qln20-Gly. This sequence showed high homology to the sequences of actinidin fromAcinidia deliciosa(95.0%) and actinidin fromActinidia eriantha(90%). These three cysteine proteases were thought to be common allied species.Conclusion:The biochemical properties of the enzyme purified fromA. argutafruit juice were determined. These basic data are expected to contribute to the maintenance and improvement of human health as well as to the promotion of protein digestion and absorption through its proteolytic functions.