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
Author    Correspondence author
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

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.

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