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Production of Milk-Clotting Protease from Bacillus subtilis

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Abstract

An indigenous Bacillus subtilis strain isolated from soil was found to be a potent milk-clotting protease (mcp) producer. Production optimized using response surface methodology (RSM) yielded 1,190 U/ml of enzyme in medium containing 6% fructose, 1% casein, 0.3% NH4NO3, 10 mM CaCl2, pH 6.0 and inoculated with 3% inoculum and incubated at 250 rpm for 72 h. Solid-state fermentation resulted in 1,080 and 952.3 U/gds of milk-clotting protease using soybean meal and rice bran, respectively, with higher proteolytic values of 18.97 and 9.1 IU/gds. Production in a biphasic system using an overlay of RSM-optimized medium on solid layer of 6% fructose and 1% casein with 1.5% agar resulted in significant enzyme production. Maximum mcp was obtained using a biphasic system where solid: liquid ratio of 3.0 resulted in a final yield of 1,276.65 U/ml with a yield index of 1.80 as compared to static liquid culture. However, significant increase or difference was noted as compared to yield obtained after RSM. This is the first report on the use of RSM for production of mcp from a bacterial species.

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Abbreviations

MCA:

milk-clotting activity

PA:

proteolytic activity

milk-clotting protease:

mcp

SmF:

submerged fermentation

SSF:

solid-state fermentation

RSM:

response surface methodology

FCCCD:

face-centered central composite design

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Acknowledgement

The authors would like to thank Mrs. Rekha Kaushik, Dr. Ruchi Gulati, Ms. Pinki, and Mr. Shailendra for their critical evaluation of the manuscript. The financial assistance provided by the Council of Scientific and Industrial Research is also acknowledged by the authors.

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Authors and Affiliations

  1. Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India

    Kakoli Dutt, Pritesh Gupta, Saurabh Saran, Swati Misra & Rajendra Kumar Saxena

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  1. Kakoli Dutt
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  2. Pritesh Gupta
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  3. Saurabh Saran
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  4. Swati Misra
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  5. Rajendra Kumar Saxena
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Correspondence to Rajendra Kumar Saxena.

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Dutt, K., Gupta, P., Saran, S. et al. Production of Milk-Clotting Protease from Bacillus subtilis . Appl Biochem Biotechnol 158, 761–772 (2009). https://doi.org/10.1007/s12010-008-8504-9

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  • DOI: https://doi.org/10.1007/s12010-008-8504-9

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