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Phylogenetic analysis of newly isolated protease producing salt tolerant psychrophilic bacteria from Tirich Mir glacier, Pakistan

Wasim Sajjad, Muhammad Rafiq, Sahib Zada, Neelam Zeb, Imran Khan, Aamer Ali Shah, Fariha Hasan

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Int. J. Biosci.7(2), 159-169, August 2015

DOI: http://dx.doi.org/10.12692/ijb/7.2.159-169


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Potent protease producing cold adapted bacteria were isolated from Tirich Mir glacier, Chitral, the highest mountain of the Hindu Kush range, Pakistan. Sediment, surface ice and melt water samples were collected and number of cells (CFU/ml) in samples were calculated. Casein medium was used to screen the protease producers.Three protease producing isolates; TG1-MRL, TG3-MRL and TG4-MRL were identified as Bacillus sp., Serratia sp. and Exiguobacterium sp. through 16S rRNA gene sequencing and their sequences were submitted to NCBI Gen Bank (Accession numbers; KF471118, KF550058 and KF550059, respectively). Optimum growth temperature and pH of all isolates were 15-25°C and 7-9, respectively. Serratia sp. TG3-MRL and Exiguobacterium sp. TG4-MRL showed growth in presence of high salt concentration, 5% and 9%, respectively. Maximum specific activity of protease was reported from Serratia sp. TG3-MRL (4.40 U/mg), followed byBacillus sp. TG1-MRL (3.69 U/mg) and Exiguobacterium sp. TG4-MRL (2.42 U/mg), after 96-120 h of incubation at 15°C. Effect of pH, temperature, metal ions, inhibitors and modulators was studied on the activity of crude enzyme. All the enzymes were stable at pH 7-9. Activity of protease from Serratia sp. TG3-MRLwas greatly affected by Zn+2. Most of the enzymes were stable in presence of EDTA, mercaptoethanol, tri-sodium citrate and PMSF. Protease by Exiguobacterium sp. TG4-MRL was sensitive to EDTA and PMSF. Activity of protease produced by Bacillus sp.TG1-MRL reduced to 20% by 1% phenyl-acetaldehyde. Stability results for protease signify their immense potential for various industrial applications such as in laundry detergent and food industries.


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Phylogenetic analysis of newly isolated protease producing salt tolerant psychrophilic bacteria from Tirich Mir glacier, Pakistan

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