Bt Research 2015, Vol.6, No.3, 1-10
8
10% glacial acetic acid. Broad range protein MW
marker (Genei, Bangalore) was sued to judge the
unknown MW of the protein in the sample.
Native gel electrophoresis
Non-denaturing (native) PAGE was carried out according
to the modified method of (Kazan et al., 2005).
Electrophoresis was performed on 10% (w/v) gel
containing 1% skim milk at 50 V and 100 V, respectively
for stacking and resolving gels. After thorough wash
in sterile ddH
2
O, the gel was incubated for 3 h at
37
℃
in 0.1 M Glycine-NaOH buffer (pH 10.0) for
the proteolysis of skim milk protein impregnated into
the gel. Subsequently, the gel was stained with 0.2%
CBB solution, and destained with 50% methanol and
10% glacial acetic acid mixture. A clear zone on the
gel would indicate the presence of alkaline protease
activity.
Calculation of
Km
and
Vmax
The
Km
and
Vmax
values were calculated using the
effect of casein on enzyme activity using the software
Hyper 32 and Graph pad prism. Protease activity
(U/ml or U/ml
eqv
)
=
. Where, ΔE =
absorbance at 540 nm, V
f
= final volume of reaction
mixture including DNS, V
s
= crude supernatant (ml)
containing cellulase used, Δt = incubation time for of
hydrolysis, ∑ = extinction coefficient of glucose
(0.0026), d = diameter of cuvette.
Characterization of protease
The protease active fraction obtained from Sephadex
G-100 chromatography was used for various assays.
Effects of pH, temperature, substrate concentration,
different metal ions, inhibitors, surfactants and detergents
on the activity of protease activity were studied.
Effect of pH on the protease activity and
stability
Effect of pH on protease activity was tested using buffers
of different pH ranging from pH 7 to 12. Stability of the
enzyme at various pH values was studied by
pre-incubating the enzyme in buffers of different pH
(7-12) for 1 h; buffers used were 0.2 M sodium
phosphate (pH 7-8), 0.2 M glycine-NaOH (pH 9-10) and
0.2 M NaOH-Na
2
HPO
4
(pH 1-12). Casein (10 mg/ml)
was used as substrate, and incubated for 20 min
incubation at 37
℃
,
i.e
., with changing pH (substrate
concentration and temperature fixed).
Effect of temperature on the enzyme activity
and stability
Effect of temperature on the enzyme activity was
determined by incubating the reaction mixture at
different temperatures (60-80
℃
). To determine the
temperature stability, the enzyme was pre-incubated at
different temperatures (60-80
℃
) for 1 h, and the
activity was assayed under standard assay conditions
(10 mg/ml casein as substrate in phosphate buffer (pH
9) for 20 min incubation,
i.e
., with changing temperature
(substrate concentration and pH fixed).
Effect of substrate concentration
Protease was incubated (from 5 min to 60 min at 70
℃
)
in phosphate buffer (pH 9) with different concentrations
(0.5, 1, 5, 10, 15, 20 mg/ ml) of casein,
i.e
., with
changing substrate concentration (pH and temperature
fixed).
Effect of metallic salts
Protease was pre-incubated for 1 h with various
concentrations (1, 2 and 2.5 mM) of different metallic
salts (Hg
2+
, Zn
2+
,
Ca
2+
, Cu
2+
, Fe
2+
, Mg
2+
, Mn
2+
,
Na
2+
and K
+
), then the activity was measured under
the reaction conditions; 15 mg/ml casein as substrate
for 30 min at pH 9 and 70
℃
(
i.e
., the optimized
conditions).
Inhibitors on protease activity
Protease was pre-incubated for 1 h with varying
concentrations (1-30 mM) of different protease
inhibitors like ethylene-diamenetetraacetic acid (EDTA),
β
-mercaptoethanol, and then assayed for protease
activity at standardized conditions.
Effect of surfactants and commercial
detergents
Efficacy of protease from
Btk
as an additive in
detergent was determined by testing its stability with
surfactants, and also in commercial detergents. The
protease was incubated with different concentrations
of surfactants like SDS (0.1, 0.4, 0.8, and 1%), Triton
X-100 (0.1, 0.4, 0.8, and 1%) for 30 min, and the
activity of the protease was measured by the standard
assay procedure. Stability of the protease in the
presence of commercial detergents was also tested by
incubating (1 h) it with measured quantity of the
protease with different commercial detergents such
as Ariel, Tide, Surf excel or Sunlight. The activity was
