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Substarte Specificty and Immobilization Studies of Purified Solanain from the Latex of Vallaris solanacea

  • Silpa SomavarapuEmail author
  • Sandeep Vemula
  • I. Bhaskar Reddy
Article
  • 42 Downloads

Abstract

A novel cysteine protease has been purified to electrophoretic homogeneity from apocyanacean member Vallaris solanacea. Molecular weight of the solanain was determined as 28.5 kDa using sodium dodecyl sulphate polyacrylamide gel electrophoresis. 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 identity) with the target sequence found as cysteine endopeptidase from Ricinus communis. The purpose of the present work is to examine the specificity towards synthetic peptide and ester substrates and also to compare with other cysteine proteases. In the present work protease solanain from V. solanacea was immobilized using various matrices such as calcium alginate, polyacrylamide, κ-carrageenan, chitosan and amberlite MB-150 employing entrapment and adsorption techniques. The purified solanain from the latex of V. solanacea exhibited broad specificity. Like other cysteine proteases it showed peptidase and amidase activity. However considerable difference was noticed in the rate of hydrolysis and also exhibited difference in specificity towards simple peptide substrates. Immobilization of solanain on amberlite MB-150 beads enhanced the enzyme stability against changes of pH and temperature. The immobilized enzyme retained 95, 89, 84 and 80% activity at the end of 2nd, 3rd, 4th and 5th cycle respectively which indicates that immobilized enzyme can be reused for 5 cycles.

Keywords

Cysteine protease Vallaris solanacea, p-nitrophenyl esters Immobilization Calcium alginate Polyacrylamide 

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

DEAE

Diethyl aminoethyl

TLC

Thin layer chromatography

EDTA

Ethylene diamino tetra acetic acid

CBZ

Carbo benzyloxy

Notes

Acknowledgements

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

Compliance with Ethical Standards

Conflict of interest

The authors have no conflicts of interest.

Ethical Approval

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

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2017

Authors and Affiliations

  1. 1.Department of Food TechnologyVikrama Simhapuri University, SPSRNelloreIndia
  2. 2.Department of Biotechnology, Centre for Bioprocess TechnologyK L E F UniversityGunturIndia
  3. 3.Department of BiochemistryGitam UniversityVisakhapatnamIndia

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