RESEARCH ARTICLE


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



Sayaka Miyazaki-Katamura1, 2, Mio Yoneta-Wada1, 3, Miyuki Kozuka4, 5, Tomohisa Sakaue6, Takuya Yamane5, 7, Junko Suzuki1, Yoshihito Arakawa1, 3, Iwao Ohkubo1, 8, *
1 Department of Nutrition, School of Nursing and Nutrition, Tenshi College, Sapporo 065-0013, Japan
2 Department of Food Science and Human Wellness, College of Agriculture, Food and Environmental Sciences, Rakuno Gakuen University, Ebetsu 069-0836, Japan
3 Department of Nutrition, School of Nursing and Nutrition, Sapporo University of Health Sciences, Sapporo 077-0894, Japan
4 Department of Health and Nutrition, Faculty of Human Science, Hokkaido Bunkyo University, Eniwa 061-1449, Japan
5 Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan
6 Department of Biochemistry and Molecular Genetics, Ehime University Graduate School of Medicine, Toon 791-0295, Japan
7 Center for Research and Development Bioresources, Research Organization for University-Community Collaborations, Osaka Prefecture University, Sakai 599-8570, Japan
8 Department of Pediatrics, Mikasa City Hospital, Mikasa 068-2156, Japan


© 2019 Miyazaki-Katamura et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Address correspondence to this author at the Department of Nutrition, School of Nursing and Nutrition, Tenshi College, Sapporo 065-0013, Japan;
Tel: +81 11 741 1051; Fax: +81 11 741 1077; E-mail: ohkubo@tenshi.ac.jp


Abstract

Background:

It has recently been reported that the fruit, stems and leaves of Actinidia arguta have 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 of A. arguta.

Methods:

Ion exchange chromatography to purify the cysteine protease from the fruit juice of A. arguta, and some synthetic substrates to determinate the enzyme activity were used.

Results:

Cysteine protease was purified to homogeneity from A. arguta fruit 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. The Km, Vmax, Kcat and Kcat/Km values for Z-Leu-Arg-MCA, the most preferentially cleaved by the enzyme, were 100 μM, 63.8 μmoles/mg/min, 27.26 sec-1 and 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 from Actinidia arguta was 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 from Acinidia deliciosa (95.0%) and actinidin from Actinidia eriantha (90%). These three cysteine proteases were thought to be common allied species.

Conclusion:

The biochemical properties of the enzyme purified from A. arguta fruit 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.

Keywords: Actinidia arguta, Cysteine protease, Purification, Biochemichal properties, Allied species, Chromatography.