Abstract:
The aim of the present work was to purify cysteine
protease inhibitors from rapeseed
(
Brassica napus
L.), with potential activity on digestive protease
of Colorado Potato Beetle
(CPB),
Leptinotarsa decemlineata
(Say). Ammonium sulfate precipitated proteinaceous
fractions; 30, 50, 70, and 100% showed 39.07, 57.03
, 51.47, and 22.44% inhibition on the
fourth instar larval gut general protease activity,
respectively. Fraction 50% showed the
highest inhibitory effect on digestive general prot
ease activity of all developmental stages.
Gel assays approved the inhibition of the enzyme ac
tivity. Fraction 50% was purified by
using various chromatography techniques; ion-exchan
ge using DEAE, gel filtration and
affinity using SiO
2
-CPB larval gut homogenate. Three peaks of protein
were eluted from
ion exchange chromatography using NaCl step gradien
t, also from gel filtration
chromatography. When Z-Ala-Arg-Arg-4mßNA was used as
cysteine protease substrate,
the purification fold of second fraction of ion exc
hange chromatography was obtained
24.80, also the yield was 59.09%, the third fractio
n of gel permeation resulted in a 25.60
fold purification with 28.53% of recovery, and the
fraction of affinity chromatography
obtained a 22.72 fold purification and yielded 36.3
5%. In the SDS-PAGE, apparent
molecular mass of purified proteins were 34 and 32
kDa by ion-exchange and 24 and 22
kDa by affinity. However, gel filtration was not an
appropriate method in this study,
because the purified protein band(s) were not obser
ved on the gel. Consequently, these
chromatography methods were appropriate methods to
purification of inhibitor cystatins,
specially affinity which was prepared by using CPB
gut enzyme as ligand and obtained
specific inhibitor proteins of CPB gut protease act
ivity.
Keywords:
Chromatography, Colorado potato beetle, Enzyme inhi
bition, Protein, Rapesee
