Abstract
A cowpea class I chitinase (VuChil) was expressed in the methylotrophic
yeast P. pastoris. The recombinant protein was secreted into the culture
medium and purified by affinity chromatography on a chitin matrix. The
purified chitinase migrated on SDS-polyacrylamide gel electrophoresis as
two closely-related bands with apparent molecular masses of 34 and 37
kDa. The identity of these bands as VuChiI was demonstrated by mass
spectrometry analysis of tryptic peptides and N-terminal amino acid
sequencing. The recombinant chitinase was able to hydrolyze colloidal
chitin but did not exhibit enzymatic activity toward synthetic
substrates. The highest hydrolytic activity of the cowpea chitinase
toward colloidal chitin was observed at pH 5.0. Furthermore, most VuChil
activity (approximately 92%) was retained after heating to 50 degrees C
for 30 min, whereas treatment with 5 mM Cu2+ caused a reduction of 67%
in the enzyme's chitinolytic activity. The recombinant protein had
antifungal activity as revealed by its ability to inhibit the spore
germination and mycelial growth of Penicillium herquei. The
three-dimensional structure of VuChit was resolved at a resolution of
1.55 angstrom by molecular replacement. The refined model had 245 amino
acid residues and 381 water molecules, and the final R-factor and R-free
values were 14.78 and 17.22%, respectively. The catalytic domain of
VuChil adopts an alpha-helix-rich fold, stabilized by 3 disulfide
bridges and possessing a wide catalytic cleft. Analysis of the
crystallographic model and molecular docking calculations using
chito-oligosaccharides provided evidences about the VuChil residues
involved in sugar binding and catalysis, and a possible mechanism of
antifungal action is suggested. (C) 2017 Elsevier B.V. and Societe
Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights
reserved.
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