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Isolation, Purification and Characterization of a Surfactants-, Laundry Detergents- and Organic Solvents-Resistant Alkaline Protease from Bacillus sp. HR-08

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Abstract

Bacillus sp. HR-08 screened from soil samples of Iran, is capable of producing proteolytic enzymes. 16S rDNA analysis showed that this strain is closely related to Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus mojavensis, and Bacillus atrophaeus. The zymogram analysis of the crude extract revealed the presence of five extracellular proteases. One of the proteases was purified in three steps procedure involving ammonium sulfate precipitation, DEAE-Sepharose ionic exchange and Sephacryl S-200 gel filtration chromatography. The molecular mass of the enzyme on SDS-PAGE was estimated to be 29 kDa. The protease exhibited maximum activity at pH 10.0 and 60 °C and was inhibited by PMSF but it was not affected by cysteine inhibitors, suggesting that the enzyme is a serine alkaline protease. Irreversible thermoinactivation of enzyme was examined at 50, 60, and 70 °C in the presence of 10 mM CaCl2. Results showed that the protease activity retains more than 80% and 50% of its initial activity after incubation for 30 min at 60 and 70 °C, respectively. This enzyme had good stability in the presence of H2O2, nonionic surfactant, and local detergents and its activity was enhanced in the presence of 20% of dimethyl sulfoxide (DMSO), dimethyl formamide (DMF) and isopropanol. The enzyme retained more than 90% of its initial activity after pre-incubation 1 h at room temperature in the presence of 20% of these solvents. Also, activation can be seen for the enzyme at high concentration (50%, v/v) of DMF and DMSO.

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Abbreviations

SDS:

sodium dodecyl sulfate

PMSF:

phenyl methyl sulphonyl fluoride

EDTA:

ethylenediamine tetra acetic acid

DMSO:

dimethyl sulfoxide

DMF:

N,N-dimethyl formamide

DTT:

dithiotheritol

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Acknowledgment

The authors express their gratitude to the research council of Tarbiat Modares University and Hi Tech Center, Ministry of Mines and Industries for the financial support during the course of this project.

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Authors and Affiliations

  1. Department of Biochemistry and Biophysics, Faculty of Sciences, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran

    Fatemeh Moradian, Khosro Khajeh & Hossein Naderi-Manesh

  2. Department of Basic Science, Faculty of Agronomy Engineering, Sari Agricultural Sciences and Natural Resources University, Sari, Iran

    Fatemeh Moradian

  3. Department of Genetics, Faculty of Sciences, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran

    Majid Sadeghizadeh

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  1. Fatemeh Moradian
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  2. Khosro Khajeh
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Correspondence to Khosro Khajeh.

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Moradian, F., Khajeh, K., Naderi-Manesh, H. et al. Isolation, Purification and Characterization of a Surfactants-, Laundry Detergents- and Organic Solvents-Resistant Alkaline Protease from Bacillus sp. HR-08. Appl Biochem Biotechnol 159, 33–45 (2009). https://doi.org/10.1007/s12010-008-8402-1

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