Bt Research 2015, Vol.6, No.3, 1-10
2
the stationary phase of its growth cycle (Jisha et al.
2013b).
B. thuringiensis
subsp
. kurstaki
(
Btk)
is shown
to produce an extracellular, metal chelator-sensitive
protease during the early stages of sporulation (Li and
Yousten 1975), while Hotha and Banik (1997) showed
that
Bt
strain H14 produced an alkaline protease in an
aqueous two-phase system comprising polyethylene
glycol and potassium phosphate. In fact, because of
overwhelming focus on
Bt-
toxin, exploitation of the
potentials of
Bt
for the production of extracellular protease
with an industrial perspective was found totally
neglected.
Conventionally, commercial production of
Bt
toxin
has been achieved by submerged or liquid fermentation
(SmF), or by batch or fed-batch process (Vu et al.
2010), but advantages of solid-state fermentation (SSF)
for the production of both primary and secondary
metabolites of microbial origin have well been
appreciated by many investigators (Benjamin and
Pandey 1998; Jisha et al. 2013a). Compared to SmF,
SSF received more attention recently, as it uses
simpler fermentation medium, requires smaller space,
easier to aerate, higher productivity, lower waste water
out-put, lower energy requirement, and less
contamination (Benjamin et al. 2013). Combination of
these strategies can yield higher titers of proteases in
the fermentation medium. The product so obtained can
be recovered in highly concentrated form, as against
the dilute form obtained by SmF. However, only very
little is known about the about the enzymes produced
by
Bt
, which demonstrate industrial potentials.
Our group already reported the efficacy of SSF for the
production of
δ
-endotoxin by
Btk
on potato flour (Smitha
et al., 2013a, Smitha et al., 2015a) or soybean flour
(Jisha et al., 2014), and concomitant production of
amylase as a by-product with the enhanced production
of
δ
-endotoxin (Smitha et al., 2013b). Based upon this,
the present study is focused on the purification and
characterization of a detergent stable extracellular
alkaline protease produced by
Btk
upon its growth on
soybean flour supplemented solid medium.
Results
The LB supplemented with 30% (w/v) soybean flour
showed the maximum production of protease at 12 h
incubation; hence, the crude protein (superna tant)
obtained from this modified LB medium was used for
the purification and characterization of protease.
Purification of extracellular protease
The active protease fractions obtained by (NH
₄
)
₂
SO
₄
fractionation (60-80% fraction), Viva spin column
(below 45kDa fraction) partitioning, and Sephadex
G-100 gel filtration unequivocally showed that it
contained two proteases with MW 43 and 32 kDa, as
judged by SDS-PAGE profile (Figure 1); with 12.8
folds purification and 0.3% yield upon Sephadex
G-100 gel filtration (Table 1). The distinct peaks of
the elution profile (fractions between 8 and 17 of
G-100 gel filtration chromatography) also confirmed
the existence of 2 fractions (Figure 2).
Initial activity of protease
Initial assay conditions for protease were: 10 mg/ml
casein (substrate), 7.6 pH (phosphate buffer) at 37
℃
,
and incubated for 20 min. At this condition, the
protease active fraction obtained from sephadex G-100
gel filtration showed 2097 U/ml
eqv
, which was designated
as
initial activity
(for the calculation of fold increase). The
unit of activity was expressed in terms of actual LB
medium (
i.e
., U/ml
eqv
) used for preparing the LB
supplemented with 30% (w/v) soybean flour. The
Figure 1A and B Jisha et al. (Color in web edition only).
1A.
SDS-PAGE profiles showing protease active bands with
approximate MWs of 43 and 32 kDa. (NH
4
)
2
SO
4
fraction
(60-80%) of crude protein harvested from soybean flour
supplemented (30%, w/v) LB at 12 h fermentation was
subjected to spin column MW cut-off (Vivaspin 6 column,
Sweden); subsequently, the lower fraction (lane 3) containing
proteins below 45 kDa was subjected to sephadex G-100 gel
filtration so as to obtain clear protease active fraction (lane 2),
and lane 1 is the profile of standard protein MW marker. Lane 4
is the profile of 40-60% (NH
4
)
2
SO
4
fraction from soybean flour
supplemented (30%, w/v) LB after spin column cut-off. B.
Native PAGE profile of Protease
