An unusual thermostable aspartic protease from the latex of Ficus racemosa (L.)

K. B. Devaraj, Lalitha R. Gowda, V. Prakash*

*Corresponding author for this work

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

The most extensively studied ficins have been isolated from the latex of Ficus glabrata and Ficus carica. However the proteases (ficins) from other species are less known. The purification and characterization of a protease from the latex of Ficus racemosa is reported. The enzyme purified to homogeneity is a single polypeptide chain of molecular weight of 44,500 ± 500 Da as determined by MALDI-TOF. The enzyme exhibited a broad spectrum of pH optima between pH 4.5-6.5 and showed maximum activity at 60 ± 0.5 °C. The enzyme activity was completely inhibited by pepstatin-A indicating that the purified enzyme is an aspartic protease. Far-UV circular dichroic spectra revealed that the purified enzyme contains predominantly β-structures. The purified protease is thermostable. The apparent T m , (mid point of thermal inactivation) was found to be 70 ± 0.5 °C. Thermal inactivation was found to follow first order kinetics at pH 5.5. Activation energy (E a ) was found to be 44.0 ± 0.3 kcal mol-1. The activation enthalpy (ΔH*), free energy change (ΔG*) and entropy (ΔS*) were estimated to be 43 ± 4 kcal mol-1 , -26 ± 3 kcal mol-1 and 204 ± 10 cal mol-1 K-1 , respectively. Its enzymatic specificity studied using oxidized B chain of insulin indicates that the protease preferably hydrolyzed peptide bonds C-terminal to glutamate, leucine and phenylalanine (at P1 position). The broad specificity, pH optima and elevated thermal stability indicate the protease is distinct from other known ficins and would find applications in many sectors for its unique properties.

Original languageEnglish
Pages (from-to)647-655
Number of pages9
JournalPhytochemistry
Volume69
Issue number3
DOIs
Publication statusPublished - 1 Feb 2008
Externally publishedYes

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Keywords

  • Aspartic protease
  • Endopeptidases
  • Ficin
  • Ficus racemosa
  • Moraceae
  • Plant proteases

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