Endo-inulinase Stabilization by Pyridoxal Phosphate
Modification: A Kinetics, Thermodynamics,
and Simulation Approach
Homa Torabizadeh
&
Mehran Habibi-Rezaei
&
Mohammad Safari
&
Ali Akbar Moosavi-Movahedi
&
Ahmad Sharifizadeh
&
Homa Azizian
&
Massoud Amanlou
Received: 29 August 2010 /Accepted: 9 September 2011 /
Published online: 30 September 2011
#
Springer Science+Business Media, LLC 2011
Abstract The structural and storage and functional thermostabilization of endo-inulinase
(EC 3.2.1.7) through semi-rational modification of surface accessible lysine residues by
pyridoxal-5′-phosphate (PLP) and ascorbate reduction have been explored. Improved
stability was observed on modifications in the absence or presence of inulin, which
indicates storage or functional thermostabilization, respectively. Comparisons have been
made between non-modified and modified enzyme by the determination of Tmasan
indicator of structural stability, temperature-dependent half-lives (t1/2), energy barrier of the
inactivation process, and thermodynamic parameters (ΔH*, ΔG*, and ΔS*) in a storage
thermostability approach. These parameters coincided well with the observed stabilization
of the engineered enzyme. Moreover, relative activities with sucrose and inulin were
determined for non-modified and modified endo-inulinases at different temperatures. A
comparison of the sucrose-to-inulin ratios of the initial rate of hydrolysis as an indicator of
substrate specificity revealed about twofold improvement in inulinase versus sucrose
activity by enzyme modification. Molecular dynamics simulations and molecular docking
approaches were employed to explain the observed structural and functional thermo-
stabilization of endo-inulinase upon modification. We hypothesize the establishment of
Appl Biochem Biotechnol (2011) 165:1661–1673
DOI 10.1007/s12010-011-9385-x
H. Torabizadeh
:
M. Safari
Department of Food Science and Engineering, Faculty of Biosystem Engineering, University of Tehran,
Tehran, Iran
H. Torabizadeh
e-mail: htoraby@ut.ac.ir
M. Habibi-Rezaei (*)
School of Biology, College of Science, University of Tehran, Tehran, Iran
e-mail: mhabibi@khayam.ut.ac.ir
A. A. Moosavi-Movahedi
:
A. Sharifizadeh
Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran
H. Azizian
:
M. Amanlou
Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences,
Tehran, Iran