Title:Recombinant Expression and Characterization of Lemon (Citrus limon) Peroxidase
Volume: 28
Issue: 4
Author(s): Veda P. Pandey, Apoorvi Tyagi, Shagoofa Ali, Kusum Yadav, Anurag Yadav, Ajit K. Shasany and Upendra N. Dwivedi*
Affiliation:
- Department of Biochemistry, University of Lucknow, Lucknow-226007,India
Keywords:
Functional annotation, in-vitro folding, in-silico analyses, molecular modeling, phenylpropanoid pathway, recombinant
expression.
Abstract: Background: Class III plant peroxidases play important role in a number of physiological
processes in plants such as lignin biosynthesis, suberization, cell wall biosynthesis, reactive oxygen
species metabolism and plant defense against pathogens. Peroxidases are also of significance
in several industrial applications. In view of this, the production and identification of novel peroxidases
having resistance towards temperature, pH, salts is desirable.
Objective: The objective of the present work was to clone and characterize a novel plant peroxidase
suitable for industrial application.
Methods: A full length cDNA clone of lemon peroxidase was isolated using PCR and RACE approaches,
characterized and heterologously expressed in Escherichia coli using standard protocols.
The expressed peroxidase was purified using Ni-NTA agarose column and biochemically characterized
using standard protocols. The peroxidase was also in-silico characterized at nucleotide as well
as protein levels using standard protocols.
Results: A full length cDNA clone of lemon peroxidase was isolated and expressed heterologously
in E. coli. The expressed recombinant lemon peroxidase (LPRX) was activated by in-vitro refolding
and purified. The purified LPRX exhibited pH and temperature optima of pH 7.0 and 50°C, respectively.
The LPRX was found to be activated by metal ions (Na+, Ca2+, Mg2+ and Mn2+) at lower
concentration. The expressional analysis of the transcripts suggested involvement of lemon peroxidase
in plant defense. The lemon peroxidase was in silico modelled and docked with the substrates
guaiacol, and pyrogallol and shown the favourability of pyrogallol over guaiacol, which is in agreement
with the in-vitro findings. The protein function annotation analyses suggested the involvement
of lemon peroxidase in the phenylpropanoid biosynthesis pathway and plant defense mechanisms.
Conclusion: Based on the biochemical characterization, the purified peroxidase was found to be resistant
towards the salts and thus, might be a good candidate for industrial exploitation. The in-silico
protein function annotation and transcript analyses highlighted the possible involvement of the
lemon peroxidase in plant defense response.
