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Cited by 31Enzyme Production and CharacterizationAdvanced Scientific Research MethodsEnzyme Catalysis and Immobilization

Production of milk-clotting protease from Bacillus subtilis.

Dutt, Kakoli, Gupta, Pritesh, Saran, Saurabh, Misra, Swati, Saxena, Rajendra Kumar·2009·Applied Biochemistry and Biotechnology
DOI 10.1007/s12010-008-8504-9
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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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  • Solid-state fermentation fell short of the other two strategies despite yielding comparable amounts.

    “Meanwhile, despite yielding comparable amounts, SSF still fell short of the other 2 strategies.”

    From Invited review: Milk-clotting enzymes of microbial origin and their role in cheesemaking-A review. (2025) · cites this paper as Dutt et al., 2009

  • The highest yield of milk-clotting protease from Bacillus subtilis was obtained in a 2-phase system (1,276.65 U/mL), which was higher (2.23 times) than that of the single-stage process and higher (1.07 times) than that obtained using response surface methodology.

    “The highest yield of MCP was obtained in the 2-phase system (1,276.65U/ mL), which was higher (2.23×) than that of the singlestage process, and higher (1.07×) than that obtained using response surface methodology (RSM).”

    From Invited review: Milk-clotting enzymes of microbial origin and their role in cheesemaking-A review. (2025) · cites this paper as Dutt et al., 2009

  • Both response surface methodology and the 2-phase method significantly boosted milk-clotting protease yield compared with the initial value (571.43 U/mL).

    “As a result, both RSM and the 2-phase method significantly boosted the MCP yield compared with the initial value (571.43U/ mL).”

    From Invited review: Milk-clotting enzymes of microbial origin and their role in cheesemaking-A review. (2025) · cites this paper as Dutt et al., 2009

  • Microbial-origin milk-clotting enzymes remain stable at both acidic and alkaline pH, and milk-clotting activity and therefore MCA/PA values can be influenced by both fermentation pH and enzyme activity pH.

    “The results of many studies (Esawy and Combet-Blanc, 2006;El-Bendary et al., 2007;Dutt et al., 2009;Shieh et al., 2009;Deng et al., 2010;Fazouane-Naimi et al., 2010;El-Baky et al., 2011;Irfan et al., 2011;Yegin et al., 2012;Acha et al., 2014;Silva et al., 2014;Sun et al., 2014;Ayana et al., 2015;Guleria et al., 2016;Hang et al., 2016;Kumari Narwal et al., 2016;Lemes et al., 2016;Mehdi et al., 2017”

    From Invited review: Milk-clotting enzymes of microbial origin and their role in cheesemaking-A review. (2025) · cites this paper as Dutt et al., 2009

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Authors
Dutt, K., Gupta, P., Saran, S., Misra, S., & Saxena, R. K.
Affiliations
University of Delhi (IN)
Corresponding author
Saxena, Rajendra Kumar
ORCID
Venue
Applied Biochemistry and Biotechnology
Publisher
Springer Science and Business Media LLC
Published
2009-01-27
Volume
158
Issue
3
Pages
761-772
ISSN
  • 0273-2289 (ISSN-L)
  • 1559-0291
DOI
10.1007/s12010-008-8504-9
OpenAlex
W2093758309
Cited by
31
References
22
Citations by year
2025: 3 · 2023: 1 · 2021: 2 · 2020: 3 · 2019: 2 · 2017: 6 · 2016: 3 · 2014: 2 · 2013: 2 · 2012: 2
FWCI
1.63
Type
journal article
Language
en
Open access
closed
Licence
www.springer.com/tdm (tdm)
Locations
  • Applied Biochemistry and Biotechnology (publishedVersion)
  • PubMed (publishedVersion)
Funders
Council of Scientific and Industrial Research, India
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Enzyme Production and Characterization
  • Life Sciences › Agricultural and Biological Sciences › Food Science › Advanced Scientific Research Methods
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
Keywords
Bacillus subtilis, enzyme production optimization, response surface methodology, solid-state fermentation, soybean meal, rice bran, soil bacteria, casein hydrolysis
MeSH terms
Animals, Bacillus subtilis, Bacterial Proteins, Fermentation, Milk, Peptide Hydrolases, Bioreactors, Phase Transition

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