Bt Research 2015, Vol.6, No.6, 1-10
1
Research Article
Open Access
Thermo- and Detergent-stable Alkaline Protease From Bacillus
Thuringiensis
Subsp.
Kurstaki
Jisha V.N., Sajith S., Priji P., Smitha R.B., Benjamin S.
Enzyme Technology Laboratory, Biotechnology Division, Department of Botany, University of Calicut, Kerala - 673635, India
Corresponding author email
Bt Research, 2015, Vol.6, No.6
doi: 10.5376/bt.2015.06.0006
Received: 19 Apr., 2015
Accepted: 20 Sep., 2015
Published: 10 Oct., 2015
Copyright
©
2015
Jisha et al., This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:
Jisha V.N., Sajith S., Priji P., Smitha R.B. and Benjamin S., 2015, Thermo- and Detergent-stable Alkaline Protease From Bacillus
Thuringiensis
Subsp.
Kurstaki
.
kurstaki
, Bt Research, Vol.6, No.6 1
-
8 (doi
Abstract
The alkaline protease produced by
Bacillus thuringiensis
subsp.
kurstaki (Btk)
as by-product during fermentation in LB
medium supplemented with 30% (w/v) soybean flour is demonstrated in this study. After 12 h fermentation under semi-solid state of
fermentation, the supernatant was collected as crude protease; which was subjected to three step purification, i.e., ammonium
sulphate fractionation; spin column partitioning and sephadex G-100 gel filtration; the resultant protein was with 12.8 folds
purification, 0.3% overall yield and 1766 U/mg specific activity. Sephadex G-100 gel elution profile showed two distinct peaks for
protease active fraction, which was further confirmed by SDS-PAGE with apparent molecular weights of 43 and 32 kDa. The
zymogram of protease with skim milk showed clear proteolytic zone. The optimized activity conditions of Btk protease were: 1.5%
casein as substrate, pH 9.0, 70
℃
temperature for 30 min incubation in the presence of 2 mM Mn
2+
. At this optimized condition, the
maximum protease activity was 11732 U/mleqv, i.e, 5.6 folds higher activity than the un-optimized conditions. The protease activity
was much stable (over 88% activity retained) in the presence of detergents (0.2%) like SDS and Triton X100; but of commercial
detergents tested, over 40% activity was retained with Ariel (0.7%). The Km and Vmax of the protease were 0.9 mg/ml and 879U/mg,
respectively. Briefly, the alkaline protease reported as a by-product from Btk is detergent- and thermo-tolerant, capable of working at
detergent conditions in washing machines, which offers higher potentials for using it as a cheaper biocatalyst in detergent industry.
.
Keywords
Bacillus thringiensis
subsp.
kurstaki
; Alkaline protease; Soybean flour; Zymogram
Background
Proteases or peptidases constitute the largest group of
enzymes in bio-industry with a long array of uses.
They play an inevitable role in industrial biotechnology,
especially in in the manufacture of detergents, foods
and pharmaceuticals. They differ in properties such as
substrate specificity, active site and catalytic mechanism,
pH and temperature optima, and stability profiles.
Studies relating to such properties are imperative for
the successful application of these enzymes in their
respective industries (Sumantha et al. 2005). Proteolytic
enzymes are classified on the basis of pH as acidic,
neutral or alkaline. Alkaline proteases encompass
commercially important proteases; they are further
categorized as serine proteases, aspartic proteases,
cysteine proteases or metallo proteases - depending on
their specific catalytic mechanism. Proteases have a
large variety of applications, mainly in the detergents,
leather processing, silver recovery, medicine, food
processing, feed making, and chemical industries, as
well as waste treatment (Jisha et al. 2013a). Proteases
and amylases are the predominant detergent enzymes,
being blended for removing proteins from clothes
soiled with blood, milk, sweat, grass,
etc
., and
enzyme-blended detergents are found far more
effective than non-enzyme detergents. Proteases
hydrolyze proteins and break them down into more
soluble polypeptides or free amino acids. Combined
effect of surfactants and enzymes facilitate fast removal
of bad stains intercalated in the fiber meshes; therefore,
detergents with enzymes are far more effectively than
non-enzyme detergents. Detergent enzymes must be
cost-effective and safe to use, and should display
optimal activities at flexible salt concentration,
temperature, and pH.
Bacillus thuringiensis
Berliner (
Bt
) is a ubiquitous
Gram-positive and sporulating bacterium producing
insecticidal crystal proteins (the
δ-
endotoxin) juxtaposed
to the endospores in the bacterium (sporangium) during
