Thermo- and Detergent-stable Alkaline Protease fromBacillus thuringiensisSubsp.kurstaki
Enzyme Technology Laboratory, Biotechnology Division, Department of Botany, University of Calicut, Kerala - 673635, India
Bt Research, 2015, Vol. 6, No. 6 doi: 10.5376/bt.2015.06.0006
Received: 19 Aug., 2015 Accepted: 20 Sep., 2015 Published: 13 Oct., 2015
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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, Bt Research, Vol.6, No.6, 1-10 (doi: 10.5376/bt.2015.06.0006)
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 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 Mn2+. 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..
Bacillus thringiensis subsp. kurstaki; Alkaline protease; Soybean flour; Zymogram