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mmbrane was washed once in Tris NnCl
and
twice in Tris NaCI, 0.05% Tween-20
(10 min. each) and incubated for 30 min. in
n
1 5 0 0 0 dilution of ~~lkilline phosph;it:lse-
1:lklled goat anti-rabhit IgG. Detection was achicvcd with the chrun~ogcnic
,~ibrtrates. 5-bromo-4-chloro-3-inllolyl phosph;~le ;11111 nitroblue
tetr;rsolil~ln
(DCIPINBT tnhlets,
Sigma Che~nic;~l Co, h10.
USA.),
according to the
n1:lnufacturer's instructions.
3.2.3.3.11. Chitinase activity assay
Chitinase activity was determined by measuring the reducing end group
N -
;tcetaniino-glucose produced from colloidal chitin. Reaction mixture con.risting of I
nil enzyme solution. and I ml of 1% (wlv) colloidal chitin (pH
5.4)
was incubated at
50
"C for 60 min. Termination of the reaction was done by adding
2
ml of
dinitrosalicylic acid reagent and heating in boiling water for 5 min. The reaction wuq
then cooled to room temperature and centrifuged ill 6.MK) g for 10 min. The
~upernntant was subjected to spectrophotornetric measurement at 530 nm. One unit of
chitinilse activity was defined as the amount of enzyme that liberates I pg
N-
ace[amino-glucose per min. at pH 5.4 and 50 "C.
3.2.3.3.12. Determination of protein solubility a n d Factor Xa cleavage
assay
In order to determine the solubility percentage of protein when overexpressed
in
E.
coli, Wilkinson and Harrison model was used (1991). Factor Xa (New England
Biolabs Inc.) cleavage was carried out using 1 pg of Factor Xa and 50 pg of LTP
fusion protein at 4 OC as
per
the recommendation of manufacturers. The reactions
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