Insights into the hydrolytic activity of Asclepias fruticosa L. protease
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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.
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/substrate ratio of 0.2% (w/w). The enzymatic hydrolysis allowed a strong increase in the solubility, water and oil holding capacity.
Asclepain f was revealed as a successful 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.
KeywordsPlant protease Soy proteins Enzymatic hydrolysis Asclepain f Specificity Cysteine peptidase
M.J. Torres, S.A. Trejo and L.M.I. López are members of the CONICET Researcher Career. The present work was supported by grants from CONICET (PIP 297) and CICPBA.
Supplementary Table 1—Kinetic parameters (PFLNA substrate) of asclepain f, comparison with the parameters of other plant cysteine proteases.
Supplementary Figure 1—a) SDS-PAGE: lane 1, latex of A. fruticosa; lane 2, molecular mass markers (97.0, 66.0, 45.0, 30.0, 20.1, and 14.4 kDa); lane 3, asclepain f. b) MALDI TOF MS of asclepain f.
Supplementary Figure 2—pH profile of asclepain f toward azocasein and PFLNA. All assays were performed by in triplicate; vertical bars correspond to standard deviation.
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Conflict of interest
The authors declare no conflict of interest.
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