Immobilization of Bacillus licheniformis 5A1 milk-clotting enzyme and characterization of its enzyme properties
Abstract
Milk-clotting enzyme from Bacillus licheniformis 5A1 was immobilized on Amberlite IR-120 by ionic binding. Almost all the enzyme activity was retained on the support. The immobilized milk-clotting enzyme was repeatedly used to produce cheese in a batch reactor. The production of cheese was repeated 5 times with no loss of activity. The specific activity calculated on a bound-protein basis was slightly higher than that of free enzyme. The free and immobilized enzyme were highly tolerant to repeated freezing and thawing. The optimum temperature for milk-clotting activity was 70 °C with the free enzyme whereas, it was ranged from 70 to 80 °C with the immobilized milk-clotting enzyme. The activation energy (E A) of the immobilized milk-clotting enzyme was lower than the free enzyme (E A = 1.59 and 1.99 Kcal mol-¹ respectively). The immobilized milk-clotting enzyme exhibited great thermal stability. The milk-clotting optimum pH was 7.0 for both free and immobilized enzyme. The Michaelis constant K m of the immobilized milk-clotting enzyme was slightly lower than the free enzyme.
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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 Esawy and Combet-Blanc, 2006
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El-shishtawy, R., Ahmed, N., & Almulaiky, Y. Q. (2021) · Catalysts · doi:10.3390/catal11070820 · Cited by 63
“A similar decrease in Ea was reported by Lamb and Stuckey for the immobilization of β-galactosidase on colloidal liquid aphrons [39], and Esawy and Combet-Blanc for immobilized milk-clotting enzyme [41].”
Kumari Narwal, R., Bhushan, B., Pal, A., Panwar, A., & Malhotra, S. (2016) · LWT · doi:10.1016/j.lwt.2015.08.065 · Cited by 42 · see citing works
Kumari, A., Mahapatra, P., Garlapati, V. K., & Banerjee, R. (2007) · Bioprocess and Biosystems Engineering · doi:10.1007/s00449-007-0160-x · Cited by 41 · see citing works
“Ea was determined from the Arrhenius law: ln(V )=l nA‐Ea/(RT) where, A was a constant for particular reaction, V was relative enzyme activity, temperature (T) was in kelvin (K), gas constant (R = 1.987) in cal K ‐1 mol ‐1 and (Ea) in Kcal mol ‐1 [ 27 ].”
MethodInfluentialLiburdi, K., Emiliani Spinelli, S., Benucci, I., Lombardelli, C., & Esti, M. (2018) · Food Chemistry · doi:10.1016/j.foodchem.2017.06.093 · Cited by 34 · see citing works
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Comparative evaluation of Bacillus licheniformis 5A5 and Aloe variegata milk-clotting enzymes
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“The optimum temperature of the purified enzyme was similar to the values reported earlier for some MCE from other Bacillus strains [30, 31].”
ResultPurification 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
Bayramoglu, G., Altıntas, B., & Arica, M. (2011) · Bioprocess and biosystems engineering (Print) · doi:10.1007/s00449-010-0453-3 · Cited by 21
Bergamasco, J., de Araújo, M. V. D., Vasconcellos, A. D., Luizon, R. A., Hatanaka, R. R., Giotto, M., Aranda, D., & Nery, J. G. (2013) · doi:10.1016/j.biombioe.2013.09.006 · Cited by 20
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
Ding, Z., Wang, W.-F., Wang, B., Ouyang, A., Xiao, S., Wang, Y.-N., Liu, S.-P., Ding, M., Zhang, L., & Shi, G.-Y. (2012) · European Food Research and Technology · doi:10.1007/s00217-011-1650-2 · Cited by 17 · see citing works
“amyloliquefaciens JNU002 exhibited the maximum MCA at a relatively high temperature, which is comparable with enzymes from Bacillus licheniformis 5A1 [31] and E.”
MethodAlmaghrabi, O., & Almulaiky, Y. Q. (2022) · Biocatalysis and Biotransformation · doi:10.1080/10242422.2022.2082871 · Cited by 16
“2021), as also Esawy and CombetBlanc for immobilized milk-clotting enzyme (Esawy and Combet-Blanc 2006).”
Hailei, Y., liang, S. Z., Hongbo, S., Cai, J., Wang, X., & Haixia, Y. (2013) · African Journal of Microbiology Research · doi:10.5897/ajmr12.1947 · Cited by 17 · see citing works
“The increase in thermal stability of the immobilized enzyme could be attributed to multipoint attachment of the enzyme to the carrier preventing them against the autolysis (Esawy and Combet-Blanc, 2006).”
BackgroundRecent Updates on Immobilization of Microbial Cellulase
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Hegde, K., & Veeranki, V. D. (2014) · doi:10.9734/bbj/2014/12558 · Cited by 13
“Similar results were observed for some other enzymes in previous studies [31-33].”
ResultSu, Z. (2012) · African Journal of Microbiology Research · doi:10.5897/ajmr11.922 · Cited by 15 · see citing works
“…2008), glass beads (Rojas-Melgarejo et al., 2004), poly (ethylene terephthalate) grafted acrylamide fiber (Temocin and Yigitoglu, 2009), chitosan (Bindhu and Abraham, 2003), eudragit (Gaur et al., 2005) and amberlite (Esawy and Combet-Blanc, 2006) have been used for the immobilization of enzyme.”
MethodEsawy, M., Kansoh, A., Kheiralla, Z., Ahmed, H., Kahil, T., & EL-Hameed, E. K. A. (2014) · doi:10.6000/1927-3037.2014.03.02.1 · Cited by 11
“Similar result was obtained by Esawy and Combet-Blanc [34].”
ResultSelf-citationImmobilization of Pepsin onto Chitosan Silica Nanobeads with Glutaraldehyde as Crosslink Agent
Cahyaningrum, S., & Sianita, M. M. (2014) · doi:10.9767/bcrec.9.3.7060.263-269 · Cited by 10
“Nowadays most reseachers use immobilized enzymes in industrial bioprocesses [6] by binding the enzymes with supports that are not dissolved in water.”
BackgroundProperties of chitosan‐immobilized cellulase in ionic liquid
Su, Z., Yu, Y., Liang, C., Li, L., & Yu, S.-T. (2013) · Biotechnology and applied biochemistry · doi:10.1002/bab.1057 · Cited by 10 · see citing works
Rangnoi, K., Phrommao, E., Yamabhai, M., & Yongsawatdigul, J. (2014) · International Journal of Dairy Technology · doi:10.1111/1471-0307.12145 · Cited by 8 · see citing works
Châari, F., Blibech, M., Bhiri, F., Maktouf, S., Ellouz-Ghorbel, R., & Ellouz-Chaabouni, S. (2012) · Applied Biochemistry and Biotechnology · doi:10.1007/s12010-012-9804-7 · Cited by 8
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
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
“The main problem for MCE reusability is the coagulation around the carrier which may result in blocking the active site of the enzyme [32, 33].”
BackgroundResultSelf-citationIsmail, A., Helal, G. E., El-Khouly, D. M. A., & Esawy, M. (2022) · International Journal of Biological Macromolecules · doi:10.1016/j.ijbiomac.2022.01.195 · Cited by 4
Self-citation
- Authors
- Esawy, M. A., & Combet-Blanc, Y.
- Affiliations
- Aix-Marseille Université (FR)
- Corresponding author
- Esawy, Mona A.
- Venue
- World Journal of Microbiology and Biotechnology
- Published
- 2006-03
- Volume
- 22
- Issue
- 3
- Pages
- 197-200
- ISSN
- 0959-3993 (ISSN-L)
- 1573-0972
- OpenAlex
- W2003711229
- Cited by
- 31
- References
- 0
- Citations by year
- 2025: 2 · 2024: 1 · 2022: 2 · 2021: 1 · 2019: 2 · 2017: 1 · 2016: 2 · 2015: 1 · 2014: 5 · 2013: 3 · 2012: 4
- FWCI
- 0.55
- Type
- journal article
- Language
- en
- Open access
- closed
- Licence
- www.springer.com/tdm (tdm)
- Locations
- World Journal of Microbiology and Biotechnology (publishedVersion)
- HAL (submittedVersion)
- Funders
- U.S. Department of State
- Ohio State University
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Enzyme Production and Characterization
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
- Life Sciences › Agricultural and Biological Sciences › Food Science › Proteins in Food Systems
- Keywords
- Bacillus licheniformis, milk-clotting enzyme, enzyme immobilization, Amberlite IR-120, ionic bonding, cheese production, batch reactor, enzyme reusability, thermal stability, activation energy, Michaelis constant, freeze-thaw resistance, enzyme characterization
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