International network for natural sciences – research journal
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Production, purification and characterization of detergent-stable, halotolerant alkaline protease for eco-friendly application in detergents’ industry

By: Maliha Ahmed, Ramla Rehman, Aisha Siddique, Fariha Hasan, Naeem Ali, Abdul Hameed

Key Words: Alkaline Protease, Bacillus cereus, 16s rRna sequencing, Halotolerant, Washing Test.

Int. J. Biosci. 8(2), 47-65, February 2016.



An alkalophilic, halotolerant bacterial strain ASM1 isolated from agricultural soil was found to be capable of producing extracellular protease enzyme. Proteolytic strain was identified as Bacillus cereus and nucleotide sequence has been submitted in NCBI database under accession number KJ600795. Optimum enzyme production in terms of specific activity 9.58 U/mg of total protein was obtained at 35°C; pH, 9.0; 1 % glucose as C-source and 35 g/l beef extract as N-source after 48 hours of incubation in a defined medium inoculated with 2% inoculum size. Bacterial isolate was capable of tolerating up to 12.5% NaCl without requiring salt for physiological activities. Bacterial crude enzyme was purified by 6 folds with 25% yield and specific activity of 57.9 U/mg protein by two step purification i.e. ammonium sulfate precipitation and gel-filtration chromatography. Thermostability studies revealed retention of 60% proteolytic activity upto 55°C. Moreover enzyme remained stable in the pH range of 6-11. PMSF (phenylmethylsulfonyl fluoride) inhibited enzyme activity categorizing the enzyme as a serine protease. Enzyme remained stable in presence of 8 different metals, however activity declined in the presence of 20 mM Fe2+ ions. Enzyme retained substantial stability in the presence of solvents, surfactants, commercially available detergents, and NaCl. Enzyme exhibited efficacious de-staining of fixed blood stains in the washing test at room temperature, without requiring additional energy. This particular type of protease enzyme is of immense importance due to its alkaline-halotolerant profile at mesophilic temperature range which is a great deal for revolutionizing detergents’ industry.


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