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Extraction, purification and characterization of a novel cysteine protease from the latex of plant Vallaris solanacea

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Abstract

Plant proteases with excellent catalytical properties perform many functions in biological systems. A novel plant protease Vallaris solanacea, was identified. Its proteolytic activity was screened using the substrate casein. This protein activity was specifically inhibited by p-chloromercuribenzoate, which showed that it is a cysteine protease. Preliminary investigations such as pH effect and temperature dependence on the caseinolytic activity of crude protease were done. Stability towards temperature and pH were also evaluated. The activity curves drawn in relation to pH, temperature and stability suggested the presence of one protease in the latex of Vallaris solanacea. In the present study, separation and purification of the latex cysteine protease solanain from Vallaris solanacea to a state of near homogeneity was also done using ion exchange and size exclusion chromatography. SDS PAGE was used to determine molecular weight of the solanain (28–29 kDa). The molecular weight was confirmed as 28.9 kDa using MALDI-TOF. Purified protease was named solanain and it was further characterized. An internal tryptic fragment was identified by MALDI-TOF, and this peptide showed a homology (66% sequence similarity) with target sequence of cysteine endopeptidase from Ricinus communis.

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Abbreviations

β-ME:

β-Mercapto ethanol

TCA:

Tri chloro acetate

EDTA:

Ethylene diamino tetra acetic acid

PCMB:

p-Chloromercuribenzoate

SDS:

Sodium dodecyl sulphate

DEAE:

Diethyl amino ethyl

PMSF:

Phenyl methyl sulfonyl fluoride

FC:

Folin–Ciocalteu reagent

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Acknowledgements

We thank the Department of Biochemistry, Gandhi Institute of Technology and Management, for providing the necessary facilities to conduct our research.

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Authors and Affiliations

  1. Department of Food Technology, Vikrama Simhapuri University, SPSR, Nellore, Andhra Pradesh, 524003, India

    Silpa Somavarapu

  2. Department of Biotechnology, Centre for Bioprocess Technology, K L E F University, Guntur, Andhra Pradesh, India

    Sandeep Vemula

  3. Department of Biochemistry, Gitam University, Visakhapatnam, Andhra Pradesh, India

    I. Bhaskar Reddy

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  1. Silpa Somavarapu
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Correspondence to Silpa Somavarapu.

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The article is entirely a study on plants. It does not include any animals or human participants.

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Somavarapu, S., Vemula, S. & Reddy, I.B. Extraction, purification and characterization of a novel cysteine protease from the latex of plant Vallaris solanacea . J. Plant Biochem. Biotechnol. 27, 186–198 (2018). https://doi.org/10.1007/s13562-017-0429-3

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