Purification, physico-chemico-kinetic characterization and thermal inactivation thermodynamics of milk clotting enzyme from Bacillus subtilis MTCC 10422
Abstract
Extracellular milk clotting enzyme from Bacillus subtilis MTCC 10422, which has the capacity of forming milk curds, was purified 32.9 fold with 20.82% recovery using sequential chromatographic techniques. The molecular weight of the enzyme was found to be 27 kDa which is of monomeric nature. The optimum temperature of the enzyme was found to be 45 °C and it was quite stable in the temperature range of 35–65 °C. The enzyme showed the pH optima of 6.0, and quite stable in broad pH range of 5.0–8.0 and showed a typical hyperbolic velocity saturation curve with Km value of 5 mg mL−1 with skim milk as a substrate. Calcium chloride at the concentration of 10 mM was found to be the most effective stimulator, accelerating the enzyme activity by about 2.8 folds. Various kinetic parameters towards thermo-inactivation of milk clotting enzyme were studied. Thermal inactivation behaviour of the purified enzyme suggested its thermostability and also its sensitivity to duration of heat treatment. After purification, both yield and recovery of purified preparation were found appreciable. Since this enzyme has wider pH stability and thermostability, usage of this high activity microbial enzyme for various cheese preparations can be evaluated at commercial scale.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
The low thermal stability of milk-clotting enzymes enabled the denaturation of MCE residues through temperature control, which can minimize the adverse impact of milk-clotting activity residues on the texture and flavor of cheese throughout the aging process.
“In addition, the low thermal stability enabled the denaturation of MCE residues through temperature control, which can minimize the adverse impact of milk-clotting activity residues on the texture and flavor of cheese throughout the aging process (Kumari Narwal et al., 2016).”
From Invited review: Milk-clotting enzymes of microbial origin and their role in cheesemaking-A review. (2025) · cites this paper as Kumari Narwal et al., 2016
The milk-clotting enzyme from Bacillus subtilis exhibits its highest activity at pH 6.0, with only a modest decline in clotting efficiency at both lower and higher pH values.
“Kumari Narwal et al. (2016) reported that the milk-clotting enzyme from B. subtilis exhibits its highest activity at pH 6.0, with only a modest decline in clotting efficiency at both lower and higher pH values.”
From Invited review: Milk-clotting enzymes of microbial origin and their role in cheesemaking-A review. (2025) · cites this paper as Kumari Narwal et al., 2016
Cites
PROTEIN MEASUREMENT WITH THE FOLIN PHENOL REAGENT
Lowry, O. H., Rosebrough, N., Farr, A. L., & Randall, R. (1951) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(19)52451-6 · Cited by 315,771 · see citing works
Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4
LAEMMLI, U. K. (1970) · Nature · doi:10.1038/227680a0 · Cited by 249,961 · see citing works
The Determination of Enzyme Dissociation Constants
Lineweaver, H., & Burk, D. (1934) · Journal of the American Chemical Society · doi:10.1021/ja01318a036 · Cited by 12,031 · see citing works
CRYSTALLINE SOYBEAN TRYPSIN INHIBITOR
Kunitz, M. (1947) · The Journal of General Physiology · doi:10.1085/jgp.30.4.291 · Cited by 2,459 · see citing works
Recent Advances in Milk Clotting Enzymes
JACOB, M., JAROS, D., & ROHM, H. (2011) · International Journal of Dairy Technology · doi:10.1111/j.1471-0307.2010.00633.x · Cited by 295 · see citing works
Extracellular acid protease from Rhizopus oryzae: Purification and characterization
Kumar, S., Sharma, N. S., Saharan, M. R., & Singh, R. (2005) · Process Biochemistry · doi:10.1016/j.procbio.2004.06.047 · Cited by 187 · see citing works
[30] Milk-clotting enzyme from Mucor pusillus var. Lindt
Arima, Κ., Yu, J., & Iwasaki, S. (1970) · Methods in enzymology on CD-ROM/Methods in enzymology · doi:10.1016/0076-6879(70)19033-1 · Cited by 170 · see citing works
Production and characterization of a milk-clotting enzyme from Aspergillus oryzae MTCC 5341
Vishwanatha, K. S., Rao, A. G. A., & Singh, S. A. (2009) · Applied Microbiology and Biotechnology · doi:10.1007/s00253-009-2197-z · Cited by 102 · see citing works
Merheb‐Dini, C., Gomes, E., Boscolo, M., & da Silva, R. (2009) · Food Chemistry · doi:10.1016/j.foodchem.2009.09.075 · Cited by 98 · see citing works
Mechanisms of Incorporation and Release of Enzymes into Cheese during Ripening
Wilkinson, M. G., & Kilcawley, K. N. (2005) · International Dairy Journal · doi:10.1016/j.idairyj.2004.08.021 · Cited by 94 · see citing works
Partial purification of new milk-clotting enzyme produced by Nocardiopsis sp.
Cavalcanti, M. T. H. (2003) · Bioresource Technology · doi:10.1016/j.biortech.2003.10.003 · Cited by 85 · see citing works
Llorente, B. E., Brutti, C. B., & Caffini, N. O. (2004) · Journal of Agricultural and Food Chemistry · doi:10.1021/jf049006o · Cited by 76 · see citing works
Pal, A. K., & Khanum, F. (2010) · Process Biochemistry · doi:10.1016/j.procbio.2010.12.009 · Cited by 70 · see citing works
Raposo, S., & Domingos, A. (2008) · Process Biochemistry · doi:10.1016/j.procbio.2007.11.003 · Cited by 64 · see citing works
Production and properties of an alkaline protease by Aureobasidium pullulans
Donaghy, J., & McKay, A. M. (1993) · Journal of Applied Bacteriology · doi:10.1111/j.1365-2672.1993.tb05200.x · Cited by 61 · see citing works
Ageitos, J. M., Vallejo, J. A., Sestelo, A. B. F., Poza, M., & Villa, T. G. (2007) · Journal of Applied Microbiology · doi:10.1111/j.1365-2672.2007.03460.x · Cited by 56 · see citing works
Purification and properties of a milk-clotting enzyme produced by Penicillium oxalicum
Hashem, A. M. (2000) · Bioresource Technology · doi:10.1016/s0960-8524(00)00055-9 · Cited by 55 · see citing works
Purification and characterization of milk clotting enzyme from goat (Capra hircus)
Kumar, A., Sharma, J., Mohanty, A. K., Grover, S., & Batish, V. K. (2006) · Comparative Biochemistry and Physiology Part B Biochemistry and Molecular Biology · doi:10.1016/j.cbpb.2006.06.010 · Cited by 53 · see citing works
Hayaloglu, A. A., Karatekin, B., & Gurkan, H. (2014) · International Dairy Journal · doi:10.1016/j.idairyj.2014.04.001 · Cited by 46 · see citing works
Matta, H., & Punj, V. (1998) · International Journal of Food Microbiology · doi:10.1016/s0168-1605(98)00061-0 · Cited by 46 · see citing works
Kohlmann, K. L., Nielsen, S. S., & Ladisch, M. R. (1991) · Journal of Dairy Science · doi:10.3168/jds.s0022-0302(91)78607-4 · Cited by 46 · see citing works
Cho, S., Oh, S.-H., Pridmore, R. D., Juillerat, M. A., & Lee, C. (2003) · Journal of Agricultural and Food Chemistry · doi:10.1021/jf0259314 · Cited by 45 · see citing works
Wu, F., Chang, C.-W., & Shih, I. (2013) · SpringerPlus · doi:10.1186/2193-1801-2-33 · Cited by 38 · see citing works
Silva, B. L., Geraldes, F. M., Murari, C. S., Gomes, E., & Da-Silva, R. (2014) · Applied Biochemistry and Biotechnology · doi:10.1007/s12010-013-0655-7 · Cited by 35 · see citing works
Esawy, M. A., & Combet-Blanc, Y. (2006) · World Journal of Microbiology and Biotechnology · doi:10.1007/s11274-005-9018-z · Cited by 31 · see citing works
Li, Z., Liu, W., & Zheng, F. (2013) · SpringerPlus · doi:10.1186/2193-1801-2-s1-s3 · Cited by 31 · see citing works
So, S. A. T. O., TOKUDA, H., KOIZUMI, T., & NAKANISHI, K. (2004) · Food Science and Technology Research · doi:10.3136/fstr.10.44 · Cited by 30 · see citing works
Fazouane-Naimi, F., Mechakra, A., Abdellaoui, R., Nouani, A., Daga, S. M., Alzouma, A. M., Gais, S., & Penninckx, M. J. (2010) · Food Biotechnology · doi:10.1080/08905436.2010.507149 · Cited by 30 · see citing works
Extracellular protease from <i>Mucor pusillus</i>: purification and characterization
NOUANI, A., BELHAMICHE, N., SLAMANI, R., BELBRAOUET, S., FAZOUANE, F., & BELLAL, M. M. (2009) · International Journal of Dairy Technology · doi:10.1111/j.1471-0307.2008.00454.x · Cited by 26 · see citing works
Purification and characterization of a chymosin from Rhizopus microsporus var. rhizopodiformis.
Sun, Q., Wang, X.-P., Yan, Q.-J., Chen, W., & Jiang, Z.-Q. (2014) · Applied Biochemistry and Biotechnology · doi:10.1007/s12010-014-1044-6 · Cited by 25 · see citing works
Purification and characterization of two milk-clotting enzymes from Irpex lacteus.
Kobayashi, H., Kusakabe, I., & Murakami, K. (1983) · Agricultural and Biological Chemistry · doi:10.1271/bbb1961.47.551 · Cited by 20 · see citing works
Partial Purification and Characterization of a Yeast Extracellular Acid Protease
Alessandro, M., & Federico, F. (1980) · Journal of Dairy Science · doi:10.3168/jds.s0022-0302(80)83096-7 · Cited by 19 · see citing works
Further purification and characterization of a thermophilic rennet
Laxer, S., Pnsky, A., & BARTOOV, B. (1981) · Biotechnology and Bioengineering · doi:10.1002/bit.260231108 · Cited by 13 · see citing works
Thermostability of three ?-amylases of Streptomyces sp IMD 2679
McMahon, H. E. M., Kelly, C. T., & Fogarty, W. M. (1999) · Journal of Industrial Microbiology & Biotechnology · doi:10.1038/sj.jim.2900612 · Cited by 11 · see citing works
Krishna Rao, L., & Mathur, D. K. (1979) · Journal of Dairy Science · doi:10.3168/jds.s0022-0302(79)83255-5 · Cited by 10 · see citing works
Shah, R. K., & Mathur, D. K. (1990) · Cited by 1 · see citing works
Flagged first, then by citations
4 of the 40 references couldn’t be matched to a known paper.
Cited by
Sorted by citations
Rehman, S., Bhatti, H. N., Bilal, M., & Asgher, M. (2016) · International Journal of Biological Macromolecules · doi:10.1016/j.ijbiomac.2016.06.081 · Cited by 172 · see citing works
Ji, C., Nguyen, L. N., Hou, J., Hai, F. I., & Chen, V. (2017) · Separation and Purification Technology · doi:10.1016/j.seppur.2017.01.043 · Cited by 167 · see citing works
Aulitto, M., Fusco, S., Bartolucci, S., Franzén, C. J., & Contursi, P. (2017) · Biotechnology for Biofuels · doi:10.1186/s13068-017-0896-8 · Cited by 81 · see citing works
Advances in research on calf rennet substitutes and their effects on cheese quality
Liu, X., Wu, Y., Guan, R., Jia, G., Ma, Y., & Zhang, Y. (2021) · Food Research International · doi:10.1016/j.foodres.2021.110704 · Cited by 68 · see citing works
Gagaoua, M., Ziane, F., Rabah, S. N., Boucherba, N., Ait-Kaki, A., Bouanane‐Darenfed, A., & Hafid, K. (2017) · International Journal of Biological Macromolecules · doi:10.1016/j.ijbiomac.2017.04.060 · Cited by 50 · see citing works
Sun, F., Sun, Q., Zhang, H., Kong, B., & Xia, X. (2019) · International Journal of Biological Macromolecules · doi:10.1016/j.ijbiomac.2019.04.169 · Cited by 45 · see citing works
Siar, E.-H., Morellon‐Sterling, R., Zidoune, M. N., & Fernández‐Lafuente, R. (2019) · International Journal of Biological Macromolecules · doi:10.1016/j.ijbiomac.2019.12.140 · Cited by 42 · see citing works
Zhang, X., Tong, Y., Wang, J., Lyu, X., & Yang, R. (2021) · Journal of Dairy Science · doi:10.3168/jds.2020-19756 · Cited by 33 · see citing works
High Milk-Clotting Activity Expressed by the Newly Isolated Paenibacillus spp. Strain BD3526
Hang, F., Liu, P., Wang, Q., Han, J., Wu, Z., Gao, C., Liu, Z., Zhang, H., & Chen, W. (2016) · Molecules · doi:10.3390/molecules21010073 · Cited by 34 · see citing works
Gülmüş, E. Ö., & Görmez, A. (2019) · Archives of Microbiology · doi:10.1007/s00203-019-01728-7 · Cited by 31 · see citing works
Wang, H., Liu, J., Chen, Q., Kong, B., & Sun, F. (2021) · Food Bioscience · doi:10.1016/j.fbio.2021.101130 · Cited by 30 · see citing works
Zhao, X., Zheng, Z., Zhang, J., Sarwar, A., Aziz, T., & Yang, Z. (2019) · Food Science & Nutrition · doi:10.1002/fsn3.1003 · Cited by 29 · see citing works
The non-edible parts of fennel ( Fœniculum vulgare ) as a new milk-clotting protease source
Bey, N., Debbabi, H., Abidi, F., Marzouki, M. N., & Salah, A. B. (2017) · Industrial Crops and Products · doi:10.1016/j.indcrop.2017.10.050 · Cited by 25 · see citing works
Zhao, X., Cai, M., Yang, Z.-J., Luo, T.-Q., Sarwar, A., Megrous, S., Aziz, T., & Yang, Z.-N. (2019) · European Food Research and Technology · doi:10.1007/s00217-019-03361-6 · Cited by 24 · see citing works
Avwioroko, O. J., Anigboro, A. A., Ejoh, A. S., Atanu, F. O., Okeke, M. A., & Tonukari, N. J. (2019) · Biocatalysis and Agricultural Biotechnology · doi:10.1016/j.bcab.2019.101298 · Cited by 22 · see citing works
Wehaidy, H. R., Abdel Wahab, W. A., Kholif, A. M. M., Elaaser, M., Bahgaat, W. K., & Abdel-Naby, M. A. (2020) · Biocatalysis and Agricultural Biotechnology · doi:10.1016/j.bcab.2020.101589 · Cited by 21 · see citing works
Purification and characteristics of a new milk-clotting enzyme from Bacillus licheniformis BL312
Zhang, Y., Xia, Y., Ding, Z., H. Lai, P. F., Wang, G., Xiong, Z., Liu, X., & Ai, L. (2019) · LWT · doi:10.1016/j.lwt.2019.108276 · Cited by 21 · see citing works
Narwal, R., Bhushan, B., Pal, A., Malhotra, S., Kumar, S., & Saharan͙, V. (2016) · International Journal of Biological Macromolecules · doi:10.1016/j.ijbiomac.2016.05.025 · Cited by 18 · see citing works
Salim, N., Santhiagu, A., & Joji, K. (2020) · 3 Biotech · doi:10.1007/s13205-020-02494-w · Cited by 18 · see citing works
Bouazizi, A., Felfoul, I., Attia, H., & Karoui, R. (2021) · Colloids and Surfaces B Biointerfaces · doi:10.1016/j.colsurfb.2021.112312 · Cited by 18 · see citing works
Lemes, A. C., Gautério, G. V., da Rosa, C. A., Brandelli, A., & Kalil, S. J. (2023) · Processes · doi:10.3390/pr11030803 · Cited by 14 · see citing works
Mamo, J., Kangwa, M., Orellana, J. F. S., Yelemane, V., Fernández‐Lahore, M., & Assefa, F. (2021) · Catalysis Letters · doi:10.1007/s10562-021-03777-4 · Cited by 13 · see citing works
Wang, X., Zhao, Q., Li, H., Shi, Y., Fan, J., Chen, Y., & Huang, A. (2022) · Journal of Dairy Science · doi:10.3168/jds.2021-21415 · Cited by 12 · see citing works
Wang, Y., Aziz, T. A., Hu, G., Liu, J., Zhang, M., Yang, Z., Alharbi, M., Alshammari, A., & Alasmari, A. F. (2023) · LWT · doi:10.1016/j.lwt.2023.115080 · Cited by 12 · see citing works
Putranto, W. S., Suhartono, M. T., Kusumaningrum, H. D., Giriwono, P. E., & Mustopa, A. Z. (2020) · Biocatalysis and Agricultural Biotechnology · doi:10.1016/j.bcab.2020.101818 · Cited by 12 · see citing works
Novel <i>Bacillus</i> Milk-Clotting Enzyme Produces Diverse Functional Peptides in Semihard Cheese
Meng, F., Zhao, H., Lu, F., Bie, X., Lu, Z., & Lu, Y. (2021) · Journal of Agricultural and Food Chemistry · doi:10.1021/acs.jafc.0c08120 · Cited by 11 · see citing works
Wehaidy, H. R., Abdel–Naby, M. A., Kholif, A. M. M., Elaaser, M. M., Bahgaat, W. K., & Wahab, W. A. A. (2023) · Journal of Genetic Engineering and Biotechnology · doi:10.1186/s43141-023-00513-w · Cited by 10 · see citing works
Zhang, Y., Xia, Y., Liu, X., Xiong, Z., Wang, S., Zhang, N., & Ai, L. (2019) · Journal of Agricultural and Food Chemistry · doi:10.1021/acs.jafc.9b06114 · Cited by 11 · see citing works
Purification and characterization of chalcone isomerase from fresh-cut Chinese water-chestnut
Fengping, H., & Pan, Y. (2017) · LWT · doi:10.1016/j.lwt.2017.01.034 · Cited by 10 · see citing works
Zhang, Y., Hu, J., Wang, J., Liu, C., Liu, X., Sun, J., Song, X., & Wu, Y. (2023) · Journal of Dairy Science · doi:10.3168/jds.2023-23450 · Cited by 10 · see citing works
Wehaidy, H. R., Abdel–Naby, M. A., Shousha, W. G., Mallah, M. Y. E., & Shawky, M. M. (2016) · Egyptian Pharmaceutical Journal · doi:10.4103/1687-4315.197584 · Cited by 9 · see citing works
Bensmail, S., Boudjema, K., Fazouane–Naimi, F., Bensmail, S., Djouahra-Fahem, D., Ferhoum, F., & Bourfis, N. (2023) · Biotechnologia · doi:10.5114/bta.2023.132770 · Cited by 8 · see citing works
The Purification Of Rennin-Like Protease From Lactobacillus Paracasei Isolated From Ettawa Goat Milk
Putranto, W. S., Mustopa, A. Z., Kusumawati, A., & Prastyowati, A. (2021) · ANNALES BOGORIENSES · doi:10.14203/ann.bogor.2020.v24.n2.74-80 · Cited by 8 · see citing works
Zhang, Y., Wang, J., He, J., Liu, X., Sun, J., Song, X., & Wu, Y. (2023) · Food Research International · doi:10.1016/j.foodres.2023.113202 · Cited by 8 · see citing works
Invited review: Milk-clotting enzymes of microbial origin and their role in cheesemaking-A review.
Wiśniewski, P., Małkowska-Kowalczyk, M., Łobacz, A., Zadernowska, A., & Żulewska, J. (2025) · Journal of dairy science · doi:10.3168/jds.2025-26973 · Cited by 10 · see citing works
Peptidases used in dairy technology: Current knowledge and relevant applications
Paiva, V. N., Martins, E., Machado, S. G., & de Carvalho, A. F. (2022) · Research Society and Development · doi:10.33448/rsd-v11i7.30367 · Cited by 4 · see citing works
Effect of oxygen supply on milk-clotting activity expressed by Paenibacillus spp. strain BD3526
Hang, F., Wang, Q., & Chen, W. (2017) · LWT - Food Science and Technology · doi:10.1016/j.lwt.2017.04.006 · Cited by 3 · see citing works
Karam, E., Hassan, M. E., Elattal, N. A., Kansoh, A., & Esawy, M. (2024) · Microbial Cell Factories · doi:10.1186/s12934-024-02521-y · Cited by 4 · see citing works
Effect of pH on the coagulation properties of Tenebrio molitor coagulant
Song, Z., Zhang, Y., Wen, P., Wang, Y., Haijun, Q., Zhang, W., & Zhang, Z. (2021) · International Journal of Food Science & Technology · doi:10.1111/ijfs.15525 · Cited by 3 · see citing works
Mamo, J., Kangwa, M., Fernández‐Lahore, M., & Assefa, F. (2021) · Bacterial Empire · doi:10.36547/be.257
Zhang, Y., Wen, C., Yang, J., Wu, Y., Liu, X., Li, M., Song, X., & Zheng, L. (2025) · Food Bioscience · doi:10.1016/j.fbio.2025.107998
Saleem, S. S., Rahman, M. B. A., Leow, T. C., Noor, N. D. M., Salleh, A. B., & Oslan, S. N. (2026) · International Journal of Biological Macromolecules · doi:10.1016/j.ijbiomac.2026.150240
- Authors
- Kumari Narwal, R., Bhushan, B., Pal, A., Panwar, A., & Malhotra, S.
- Affiliations
- Central Institute of Post-Harvest Engineering and Technology (IN)
- Chaudhary Charan Singh Haryana Agricultural University (IN)
- Corresponding author
- Bhushan, Bharat
- Venue
- LWT
- Publisher
- Elsevier BV
- Published
- 2016-01
- Volume
- 65
- Pages
- 652-660
- ISSN
- 0023-6438 (ISSN-L)
- OpenAlex
- W2190240927
- Cited by
- 42
- References
- 40
- Citations by year
- 2026: 1 · 2025: 2 · 2024: 1 · 2023: 6 · 2022: 2 · 2021: 9 · 2020: 3 · 2019: 8 · 2017: 6 · 2016: 4
- FWCI
- 2.77
- Type
- journal article
- Language
- en
- Open access
- closed
- Licence
- Locations
- LWT (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Enzyme Production and Characterization
- Life Sciences › Agricultural and Biological Sciences › Food Science › Proteins in Food Systems
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
- Keywords
- milk-clotting enzyme, Bacillus subtilis, enzyme purification, thermal stability, thermal inactivation, enzyme kinetics, calcium chloride activation, cheese production, milk curdling, pH stability, molecular weight determination
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review