ORIGINAL RESEARCH PAPER Insights into the hydrolytic activity of Asclepias fruticosa L. protease Marı ´a J. Torres . Claudia Natalucci . Laura M. I. Lo ´pez . Sebastia ´n A. Trejo Received: 22 March 2019 / Accepted: 2 July 2019 Ó Springer Nature B.V. 2019 Abstract Objective To determine the enzymatic properties of asclepain f, a plant cysteine protease isolated and purified from the latex of Asclepias fruticosa, and to investigate its potential application to hydrolyze soybean proteins. Results Kinetic parameters were determined by hydrolysis of p-Glu-Phe-Leu-p-nitroanilide (PFLNA). The Km value for asclepain f was 6 to 8 times higher than those achieved for papain, bromelain and ficin, the main plant cysteine proteases. Asclepain f showed 12 cut-off points toward the oxidized B chain insulin, revealing that the enzyme possesses broad substrate specificity. The cut specificity was governed by the presence of hydrophobic residues (F, L, V) in the P2 position. Asclepain f was able to selectively hydrolyze soybean proteins at pH 10, employing an enzyme/sub- strate ratio of 0.2% (w/w). The enzymatic hydrolysis allowed a strong increase in the solubility, water and oil holding capacity. Conclusions Asclepain f was revealed as a success- ful enzyme for biocatalysis of protein hydrolysis processes at alkaline pH. This new plant protease has a broad substrate specificity and is capable of selectively degrading the fractions of soy proteins and improving its functional properties. Keywords Plant protease Á Soy proteins Á Enzymatic hydrolysis Á Asclepain f Á Specificity Á Cysteine peptidase Introduction The hydrolysis of proteins is an irreversible process that leads to significant changes in the behavior of proteins (Thomas and van der Hoorn 2018). Enzy- matic hydrolysis has been investigated intensively because it has advantages in relation to chemical hydrolysis (Deng et al. 2016). The proteases possess Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10529-019-02706-1) con- tains supplementary material, which is available to authorized users. M. J. Torres Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires, UNNOBA-CONICET, Junı ´n, Argentina C. Natalucci CIPROVE, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina L. M. I. Lo ´pez (&) CITEC, INTI-CICPBA, Gonnet, Buenos Aires, Argentina & Universidad Nacional Arturo Jauretche, Florencio Varela, Buenos Aires, Argentina e-mail: lmilopez@biol.unlp.edu.ar S. A. Trejo Sector Agro, Ambiente y Biotecnologia, YPF Tecnologı ´a S.A., Berisso, Buenos Aires, Argentina 123 Biotechnol Lett https://doi.org/10.1007/s10529-019-02706-1