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Insights into the hydrolytic activity of Asclepias fruticosa L. protease

  • María J. Torres
  • Claudia Natalucci
  • Laura M. I. LópezEmail author
  • Sebastián A. Trejo
Original Research Paper
  • 15 Downloads

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/substrate 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 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.

Keywords

Plant protease Soy proteins Enzymatic hydrolysis Asclepain f Specificity Cysteine peptidase 

Notes

Acknowledgements

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.

Supporting information

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.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

10529_2019_2706_MOESM1_ESM.docx (13 kb)
Supplementary material 1 (DOCX 13 kb)
10529_2019_2706_MOESM2_ESM.tif (28.4 mb)
Supplementary material 2 Supplementary Fig. 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 (TIFF 29,043 kb)
10529_2019_2706_MOESM3_ESM.tif (3.3 mb)
Supplementary material 3 Supplementary Fig. 2. pH profile of asclepain f toward azocasein and PFLNA. All assays were performed by in triplicate; vertical bars correspond to standard deviation (TIFF 3333 kb)

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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires, UNNOBA-CONICETJunínArgentina
  2. 2.CIPROVE, Facultad de Ciencias ExactasUniversidad Nacional de La PlataLa PlataArgentina
  3. 3.CITEC, INTI-CICPBA, Gonnet, Buenos Aires, Argentina & Universidad Nacional Arturo JauretcheFlorencio VarelaArgentina
  4. 4.Sector Agro, Ambiente y Biotecnologia, YPF Tecnología S.A.BerissoArgentina

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