Bt Research 2015, Vol.6, No.3, 1-10
7
The effect of inhibitors on protease activity was
examined after the protease was pre-incubated with
the inhibitor for 1 h at 70
℃
. The presence of the
chelating agent EDTA and
β
-mercaptoethanol in the
reaction mixture decreased the protease activity, which
may be due to the change in the protein conformation
induced by these agents. Some proteases are inhibited
by metal chelating agents like EDTA, which indicate
their metal ion dependency for activity (Steele et al.,
1992).
Stability patterns of protease in the presence of
surfactants and commercial detergents also disclosed
its promising commercial utility in detergent
formulations. Detergent-stable proteases have been
studied by several groups with varying levels of
activity in the presence of different detergents
(Kuddus and Ramteke 2009). Most of the manufacturers
recommend the use of detergents in the range 0.1 to
0.2% (w/v) for washing purposes, but in the present
study, the detergent concentration was 0.7%. Even at
this high concentration of detergent, over 40% of
activity was retained, which shows the efficiency of
the protease from
Btk
in detergent industry. Detergent
stability of an alkaline protease is an important
property for its industrial use, as they are currently
supplemented in detergent formulations for better
washing efficiency.
Briefly, the protease characterized from
Btk
(both
fractions) is best active at alkaline pH and higher
temperatures, which makes it suitable for use in
detergency and treatment of effluent rich in protease.
Materials and Methods
Source organism and medium
Bacillus thuringiensis
subspecies
kurstaki
(
Btk
) procured
from the Institute of Microbial Technology, Chandigarh,
India (strain: BA 83B; MTCC No. 868) was used in
this study, which was maintained on the Luria-Bertani
(LB)-agar medium. Five µl seed culture (12 h old)
was inoculated in 1 ml pre-sterilized modified LB
medium, which contained about 6.5 × 10
7
cfu. The
modified LB medium was made by supplementing 30
mg soybean flour (
i.e
., 30% w/v) per 1 ml LB; and
normal LB was used as the control. The medium
was incubated at 37
℃
with constant agitation (150
rpm, and initial pH 7.0) in a temperature controlled
shaker (Orbitek, India). After 12 h fermentation, the
culture was centrifuged (at slow speed 1000 ×
g
for 10
min, 4
℃
) to harvest the supernatant, as we described
previously (Smitha et al. 2013b). The crude supernatant
obtained as above was centrifuged at 9,440 ×
g
for 10
min and 5
℃
(Plastocrafts/Remi, India), and the clear
supernatant obtained so was used a crude protease for
further purification.
Purification of protease
The extracellular alkaline protease was purified by the
method of (Kunitate et al. 1989). The procedure consisted
of ammonium sulphate (NH
₄
)
₂
SO
₄
fractionation, spin
column purification by molecular weight (MW) cut-off,
and gel permeation chromatography. The purity of the
protease was judged by SDS-PAGE.
Ammonium sulphate fractionation
To the supernatant obtained as above, molecular biology
grade (NH
₄
)
₂
SO
₄
was added slowly (by continuous
stirring in a cold room) up to 80% saturation (0-20,
20-40, 40-60 and 60-80%). The precipitate formed in
each step was collected after centrifugation (9440 ×
g
for 10 min, 4
℃
), which was dialyzed separately
against 0.1M phosphate buffer (pH 7.6) for 24 h at
5
℃
with continuous stirring with two buffer changes.
Protein concentration and protease activity in the
dialyzate were determined at every step.
Spin column purification
The (NH
₄
)
₂
SO
₄
fraction which showed the highest
protease activity was subjected to spin column (Vivaspin 6,
Sweden) purification; the polyethersulfone semi-
permeable membrane contained in it facilitated the MW
cut-off of 45 kDa (lower fraction), and this fraction
was used for further purification.
Gel permeation chromatography
After concentration by dialysis, the spin column
fraction of the protein was subjected to Sephadex
G-100 (Sigma Aldrich, USA) gel permeation
chromatography (Riviera, India), which was performed
in a cold room.
Electrophoresis
Employing SDS–PAGE, the purity of protease was
checked at every stage of purification. SDS-PAGE
was performed on a vertical mini 12 % gel (8×7 cm)
slab system (BioTech, India); the running voltage for
stacking was 50 V, and 70 V for resolving (Smitha et
al. 2013b). Gel was stained using 0.1% Coomassie
Brilliant Blue (CBB) R-250 in 50% methanol and
