Bt Research 2015, Vol.6, No.3, 1-10
6
and 4.25 folds purification (Guangrong et al., 2006).
Sephadex G-100 gel filtration gives rise to comparatively
pure fractions of the enzyme with a significant
increase in its specific activity. A variety of MWs for
proteases from
Bacillus
species was reported: 30.9
kDa protease from a thermophilic
Bacillus
strain
HS08 (Guangrong et al., 2006); 27.0 kDa from
B.
megaterium
; 75.0 kDa from
Bacillus
sp. S17110 (Seong
and Choi 2007);34.0 kDa from
B. thuringiensis
(Kunitate et al. 1989); 38.0 kDa from
B. cereus
KCTC
3674 (Kim et al., 2001); 15.0 kDa from
B. subtilis
PE-11(Adinarayan and Ellaiah 2002); 34.0 kDa from
B. cereus
BG1(Ghorbel-frikha et al., 2005); 66.2 kDa,
31.0 kDa and 20.1 kDa from
B. licheniformis
strains
BLP1, BLP2 and BLP3, respectively (Cheng et al.
2006).Thus, the MW of proteases secreted by
Bacillus
spp. range is between 20 and 75 kDa, while the MW
of proteases secreted by
Btk
were of medium size.
Most of the commercially available proteases are also
active in the pH range of 8 and 12 (Gupta et al., 2002).
The results obtained in this study indicated that the
protease from
Btk
is fit for use in laundry and dishwashing
detergents, which indicates the commercial significance
of the protease; because the pH optimum in the present
study was 9-11. Similar results were obtained with
protease from
B. stearothermophilus
(Dhandapani and
Vijayaragavan 1994),
Bacillus
sp. strain MPTK 712
(Kumar et al., 2012),
Bacillus
sp. (Agarwal et al.
2012), and
Bt
(Kunitate 1989).Alkaline proteases of
the genus
Bacillus
showed an optimum activity and a
good stability at higher (alkaline) pH values (Margesin et
al. 1992). The optimum pH range of alkaline proteases
from
Bacillus
lies generally between pH 9 and 11,
with a few exceptions of higher pH optima like 11.5
(Fujiwara and Yamamoto 1987), 11 and 12 (Kumar
and Takagi 1999), 12 and 13 (Takami et al. 1989;
Ferraro et al., 1996). In general, all currently used
detergent-compatible proteases are alkaline in nature,
and optimally acting at high pH (8-12).
Two temperature optima were determined for protease
from
Btk
,
i.e
., 70 and 75
℃
; the temperature in
washing machines (laundry, dishwasher,
etc
.) is
normally less than this temperature. Normally, the
optimum temperatures of reported alkaline proteases
from
Bacillus
range from 50 to 70
℃
(Zhang et al
2010).
Bt
exhibited the optimum activity at a broad
temperature range with the maximum (70-75
℃
) at
alkaline pH (Kunitate et al., 1989). In addition, their
thermal stability is the most important parameter
regarding their utility in different sectors like detergent
industry.
In addition, protease requires divalent cations like
Ca
2+
and Mn
2+
or combination of these cations for the
maximum activity (Kumar and Takagi 1999). Most of
the previous studies showed that the role of sodium,
calcium and manganese ions in increasing the activity
(Adinarayana et al., 2003; Nascimento and Martins
2004), which was similar to the present results. In
addition, these cations would enhance the stability of
protease
produced by
Bacillus
spp. (Durham et al.,
1987). These metal ions may protect the enzyme from
thermal denaturation and maintain its active
conformation at high temperature by enabling proper
folding.
The protease from
Btk
was characterized further for its
Km
and
Vmax
using casein as a substrate, which was
0.9 mg/ml and 879.3 U/mg
,
respectively. The
Km
value represents the dissociation constant (affinity for
substrate) of the enzyme-substrate (ES) complex. Low
values of
Km
indicate that the ES complex is held
together tightly and dissociates rarely before the
substrate is converted to product, and
Vmax
indicates
the speed of its catalysis. Using casein as a substrate,
(Kaur et al
.,
1998) reported a
Km
of 3.7 mg/ml for the
protease
from
B.
polymyxa
, while Thangam and
Rajkumar (2002) reported a
Km
and
Vmax
of 1.66
mg/ml and 526 U/mg/min, respectively for the
alkaline protease from
Alcaligenes faecalis.
A
Km
value of 0.4 toward casein (mg/ml) was reported for
the alkaline proteases from
B. alcalophilus
var.
halodurans
(Rao et al., 1998). The
Km
and
Vmax
were
determined to be 0.5 mg/ml and 230 U/mg/min,
respectively for the protease from
B. sphaericus
strain,
in which the optimum reaction condition was at pH 10
and 55
℃
(Aboul-Soud et al., 2011). The
Km
and
Vmax
of protease produced by a haloalkaliphilic
Bacillus
sp. were found to be 2 mg/ml and 289.8 μg/min,
respectively.In another haloalkaliphilic
Bacillus
sp. AH-6,
the respective
Km
and
Vmax
were 2.5 mg/ml and 625
U/min (Dodia et al. 2008). The
Km
(0.7614 mg/ml)
and
Vmax
(2582 U/min) of
B. subtilis
DKMNR was
determined at 70
℃
(Kezia et al., 2011b).
