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Page 34
Biochemistry & Molecular Biology Journal
ISSN: 2471-8084
Internat i ona l Conference on
Biotechnology, Biomarkers
& Systems Biology
M a r c h 0 4 - 0 5 , 2 0 1 9
Am s t e r d a m , N e t h e r l a n d s
Biotechnology, Biomarkers & Systems Biology 2019
Cysteine is an intriguing and enigmatic amino acid; while it is one of the
least abundant amino acids in the structure of proteins, but it is frequently
observed in functionally important sites of proteins such as catalytic, regulatory
and cofactor binding sites. Moreover, its physio-chemical classification as a
hydrophobic or polar residue is arguable. Whereas it has polar thiol group, free
Cys in proteins is often buried and surrounded by a hydrophobic environment.
It has been shown that both removal and insertion of Cys can lead to increase
protein thermo-stability. Since Selenomonas ruminantium β-D-xylosidase
(SXA) has four free cysteines, it used as a model. To characterize the role of
cysteine residues in the structure, function and stability of SXA, we prepared
and evaluated wild-type and four cysteines de cient SXA proteins. Buried
cysteine residues, were replaced with valine using QuikChange site-directed
mutagenesis. In comparison with the wild-type, the Km values remained
relatively constant while the kcat decreased in mutants. The optimal pH and
temperature were similar in the wild-type enzyme and its variants. The C101V
and C286V displayed higher thermal stability than the wild-type at 55 and 60
°C. Secondary and tertiary conformational changes using circular dichroism
and fluorescence spectroscopy revealed that changing a buried cysteine
to a hydrophobic residue could lead to an increase in thermal stability with
minimal perturbation of the overall wild-type protein structure. In addition to
experimental methods, the stability of WT SXA and C101V and C286V mutants
at 333 K was also studied by MD simulation. Our theoretical data had a good
agreement with the experimental results.
Biography
E Dehnavi has graduated in Biochemistry in 2015 from Tarbiat
Modares University, Tehran Iran. His doctoral dissertation was
conducted in consultation with Prof Khajeh and examines the
use of protein engineering methodology for improving kinetic
properties of hemicellulosic enzymes. He has been working
on the expression of Selenomonas ruminantiumXylosidase
in yeast Pichia pastoris. Moreover, he worked to improve the
thermal stability of some industrial enzyme by site-directed
mutagenesis. Currently, he is a Team Leader in Gene Transfer
Pioneers research group, the company is active in the field of
biotechnology where optimization of protein expression of
some industrial enzymes such as endoglucanase, xylosidase
and phytase through protein engineering and producing more
effective expression vectors is done. His current research is
increasing enzymatic saccharification yields through cellulase
and hemicellulase enzymes protein engineering. He has
published 10 papers in reputed journals.
Improvement of Selenomonas ruminantiumβ-xylosidase
thermal stability by replacement of free buried cysteines
E Dehnavi, M Aghaeepoor and A Akbarzadeh
Gene Transfer Pioneers (GTP) research group, Iran
E Dehnavi et al., Biochem Mol biol J 2019, Volume:5
DOI: 10.21767/2471-8084-C1-023