Purification and characteristics of a new milk-clotting enzyme from Bacillus licheniformis BL312
Abstract
A new milk-clotting enzyme (MCE) was produced from Bacillus licheniformis BL312. The characteristics of BL312 MCE were compared with those of Calf rennet, R. miehei MCE, and Chymopapain. BL312 MCE showed a remarkable activity of 5291 SU/mg after purification by ammonium sulfate fractionation and DEAE-Sepharose Fast Flow, which yielded 5.09-fold purification with a recovery of 39.24%. The purified BL312 MCE gave a single protein band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel, corresponding to 27 kDa. The αₛ-casein (αₛ-CN) and β-casein (β-CN) hydrolysates generated by BL312 MCE were different from those by Calf rennet, R. miehei MCE, and Chymopapain through SDS-PAGE and reversed-phase HPLC (RP-HPLC) analyses. The main cleavage sites in κ-casein (κ-CN) were the Met106-Ala107 and Asn123-Thr124 bonds for BL312 MCE, as determined by mass spectrometry analysis. BL312 MCE hydrolyzed αₛ-CN to a greater level than β-CN. It was stable at a wide pH range of 5.5–11.0 and temperature below 45 °C. The milk-clotting activity (MCA) reached the maximum when the substrate contained 50 mM CaCl₂ at pH 5.5. The optimal temperature of MCE was 55 °C. The successful application in Monascus-fermented cheese production confirmed the usefulness of BL312 MCE in food and dairy processing.
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?
Bacteria-derived proteases hold considerable value in industrial applications because of the wide biochemical variability of bacterial species and their ease of genetic modification.
“Bacteria-derived proteases hold considerable value in industrial applications because of the wide biochemical variability of bacterial species and their ease of genetic modification (Zhang et al., 2019).”
From Invited review: Milk-clotting enzymes of microbial origin and their role in cheesemaking-A review. (2025) · cites this paper as Zhang et al., 2019
At pH levels below 5.0, the properties of BL312 closely resembled those of calf rennet.
“Moreover, at pH levels below 5.0, these properties closely resembled those of calf rennet.”
From Invited review: Milk-clotting enzymes of microbial origin and their role in cheesemaking-A review. (2025) · cites this paper as Zhang et al., 2019
The activity of the milk-clotting enzyme BL312 remained between 75% and 90% in the pH range of 5.0 to 6.0.
“The activity of the milk-clotting enzyme BL312 remained between 75% and 90% in the pH range of 5.0 to 6.0.”
From Invited review: Milk-clotting enzymes of microbial origin and their role in cheesemaking-A review. (2025) · cites this paper as Zhang et al., 2019
When the pH increased above 5.5, the milk-clotting activity of BL312 gradually decreased, with complete loss of activity at pH 8.5.
“When the pH increased above 5.5, the MCA gradually decreased, with complete loss of activity at pH 8.5.”
From Invited review: Milk-clotting enzymes of microbial origin and their role in cheesemaking-A review. (2025) · cites this paper as Zhang et al., 2019
The activity of BL312 decreased significantly at pH levels below 5.0 and above 11.0 and almost disappeared below pH 3.0.
“However, it decreased significantly at pH levels below 5.0 and above 11.0 and almost disappeared below pH 3.0.”
From Invited review: Milk-clotting enzymes of microbial origin and their role in cheesemaking-A review. (2025) · cites this paper as Zhang et al., 2019
The BL312 enzyme remained stable over a wider pH range (from pH 5.5 to pH 11.0), whereas that from GSBa-1 was less stable at different pH values.
“It was also observed that the BL312 enzyme remained stable over a wider pH range (from pH 5.5 to pH 11.0), whereas that from GSBa-1 was less stable at different pH values.”
From Invited review: Milk-clotting enzymes of microbial origin and their role in cheesemaking-A review. (2025) · cites this paper as Zhang et al., 2019
B. licheniformis MCE has potential use in producing soft or semi-hard cheeses.
“Bacillus licheniformis MCE preferentially hydrolyzes α S -casein, indicating its potential use in producing soft or semi-hard cheeses (Zhang et al., 2019).”
From Invited review: Milk-clotting enzymes of microbial origin and their role in cheesemaking-A review. (2025) · cites this paper as Zhang et al., 2019
The gel-forming mechanism of B. licheniformis MCE involves the hydrolysis of the Met106-Ala107 and Asn123-Thr124 bonds in κ-casein.
“The gel-forming mechanism of B. licheniformis MCE involves the hydrolysis of the Met106-Ala107 and Asn123-Thr124 bonds in κ-casein.”
From Invited review: Milk-clotting enzymes of microbial origin and their role in cheesemaking-A review. (2025) · cites this paper as Zhang et al., 2019
B. licheniformis MCE preferentially hydrolyzes αS-casein.
“Bacillus licheniformis MCE preferentially hydrolyzes α S -casein, indicating its potential use in producing soft or semi-hard cheeses (Zhang et al., 2019).”
From Invited review: Milk-clotting enzymes of microbial origin and their role in cheesemaking-A review. (2025) · cites this paper as Zhang et al., 2019
Cites
Bradford, M. M. (1976) · Analytical Biochemistry · doi:10.1016/0003-2697(76)90527-3 · Cited by 255,302 · see citing works
Kjeldahl Nitrogen Analysis as a Reference Method for Protein Determination in Dairy Products
Lynch, J. M., & Barbano, D. M. (1999) · Journal of AOAC International · doi:10.1093/jaoac/82.6.1389 · Cited by 345 · 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
Stability studies of protease from Bacillus cereus BG1
Ghorbel, B., Sellami-Kamoun, A., & Nasri, M. (2003) · Enzyme and Microbial Technology · doi:10.1016/s0141-0229(03)00004-8 · Cited by 205 · see citing works
Milk-clotting enzymes produced by culture of Bacillus subtilis natto.
Shieh, C.-J., Phan Thi, L.-A., & Shih, I.-L. (2009) · Biochemical Engineering Journal · doi:10.1016/j.bej.2008.09.003 · Cited by 99 · 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
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
Purification and characterization of a milk-clotting aspartic protease from Withania coagulans fruit
Salehi, M., Aghamaali, M. R., Sajedi, R. H., Asghari, S. M., & Jorjani, E. (2017) · International Journal of Biological Macromolecules · doi:10.1016/j.ijbiomac.2017.02.034 · Cited by 74 · see citing works
Arima, Κ., Iwasaki, S., & Tamura, G. (1967) · Agricultural and Biological Chemistry · Cited by 70 · see citing works
Voigt, D. D., Chevalier, F., Qian, M. C., & Kelly, A. L. (2009) · Innovative Food Science & Emerging Technologies · doi:10.1016/j.ifset.2009.10.009 · Cited by 69 · see citing works
Sousa, M. J., & Malcata, F. X. (1998) · Enzyme and Microbial Technology · doi:10.1016/s0141-0229(97)00173-7 · Cited by 64 · see citing works
Specificity of milk-clotting enzymes towards bovine κ-casein
Drøhse, H. B., & Foltmann, B. (1989) · Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology · doi:10.1016/0167-4838(89)90039-3 · Cited by 62 · see citing works
PURIFICATION AND CHARACTERIZATION OF A PROTEINASE FROM THE PROBIOTICLactobacillus rhamnosusOXY
Waśko, A. D., Kieliszek, M., & Targoński, Z. (2012) · Preparative Biochemistry & Biotechnology · doi:10.1080/10826068.2012.656869 · Cited by 61 · see citing works
Lee, K. D., Lo, C. G., & WARTHESEN, J. J. (1996) · Journal of Dairy Science · doi:10.3168/jds.s0022-0302(96)76512-8 · Cited by 60 · see citing works
Short communication: A comparative analysis of recombinant chymosins
Vallejo, J. A., Ageitos, J. M., Poza, M., & Villa, T. G. (2012) · Journal of Dairy Science · doi:10.3168/jds.2011-4445 · Cited by 60 · see citing works
Majumder, R., Banik, S. P., & Khowala, S. (2015) · Food Chemistry · doi:10.1016/j.foodchem.2014.10.027 · Cited by 59 · 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
Characterization of the purified actinidin as a plant coagulant of bovine milk
Piero, A. R. L., Puglisi, I., & Petrone, G. (2011) · European Food Research and Technology · doi:10.1007/s00217-011-1543-4 · Cited by 54 · 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
Mobile Phases in Reverse‐Phase HPLC for the Determination of Bitter Peptides in Cheese
LEE, K. D., & WARTHESEN, J. J. (1996) · Journal of Food Science · doi:10.1111/j.1365-2621.1996.tb14178.x · Cited by 45 · see citing works
Jacaratia corumbensis O. Kuntze a new vegetable source for milk-clotting enzymes
Duarte, A. R., Duarte, D. M. R., Moreira, K. A., Cavalcanti, M. T. H., de Lima-Filho, J. L., & Porto, A. L. F. (2009) · Brazilian Archives of Biology and Technology · doi:10.1590/s1516-89132009000100001 · Cited by 43 · see citing works
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
Influence of Australian Native Herbs on the Maturation of Vacuum-packed Cheese
Agboola, S. O., & Radovanovic-Tesic, M. (2002) · LWT · doi:10.1016/s0023-6438(02)90917-5 · Cited by 40 · see citing works
An, Z., He, X., Gao, W., Zhao, W., & Zhang, W. (2014) · Journal of Food Science · doi:10.1111/1750-3841.12340 · Cited by 39 · 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
Dutt, K., Meghwanshi, G. K., Gupta, P., & Saxena, R. K. (2008) · Letters in Applied Microbiology · doi:10.1111/j.1472-765x.2008.02324.x · Cited by 32 · 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
Hang, F., Wang, Q., Hong, Q., Liu, P., Wu, Z., Liu, Z., Zhang, H., & Chen, W. (2016) · International Journal of Biological Macromolecules · doi:10.1016/j.ijbiomac.2016.01.028 · Cited by 27 · 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
Li, Y., Liang, S., Zhi, D.-J., Chen, P., Su, F., & Li, H.-Y. (2012) · European Food Research and Technology · doi:10.1007/s00217-012-1663-5 · Cited by 22 · see citing works
Yu, X., Li, H., Li, J., & Chi, Z. (2010) · Biotechnology and Bioprocess Engineering · doi:10.1007/s12257-009-3081-5 · Cited by 20 · 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
Yu, H., Liu, Z., Hang, F., & Bei-hong, M. (2016) · Food Science and Biotechnology · doi:10.1007/s10068-016-0133-4 · Cited by 13 · 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
Flagged first, then by citations
3 of the 38 references couldn’t be matched to a known paper.
Cited by
Sorted by citations
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
Invited review: Role of Bacillus licheniformis in the dairy industry—Friends or foes?
Dai, H., Ma, L., Xu, Z., Soteyome, T., Yuan, L., Yang, Z., & Jiao, X. (2024) · Journal of Dairy Science · doi:10.3168/jds.2024-24826 · Cited by 20 · see citing works
Artifon, S. E. S., Sumny, E. H., Fabris, T., de Jesus, B. A. P., Magalhães, M. D. L. B., Silva, G. F. D., Dognini, J., Andrade, N. C., Moroni, L. S., & Kempka, A. P. (2023) · Food Bioscience · doi:10.1016/j.fbio.2023.102879 · Cited by 15 · see citing works
Simultaneous Hydrolysis and Extraction Increased Erucin Yield from Broccoli Seeds
Lv, C., Zhang, Y., Zou, L., Sun, J., Song, X., Mao, J., & Wu, Y. (2021) · ACS Omega · doi:10.1021/acsomega.0c06319 · Cited by 14 · 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
Yang, X., Zhang, Z., Zhang, W., Qiao, H., Wen, P., & Zhang, Y. (2022) · Journal of Functional Foods · doi:10.1016/j.jff.2022.104944 · Cited by 12 · 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
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
Kuerman, M., Liu, Y., Liu, Y., Li, B., Hou, B., Li, B., Zhang, L., & Liu, T. (2024) · Food Hydrocolloids · doi:10.1016/j.foodhyd.2024.110044 · Cited by 8 · see citing works
Ren, Q., Zhao, H., Hu, H., Zhou, Z., Yang, Z., & Yang, Z. (2023) · International Dairy Journal · doi:10.1016/j.idairyj.2023.105866 · Cited by 8 · see citing works
Ferreira, A. N., da Silva, A. T., Nascimento, J., de Souza, C. B., Silva, M. D. C., Grillo, L. A. M., da Luz, J. M. R., & Pereira, H. J. V. (2023) · Biocatalysis and Biotransformation · doi:10.1080/10242422.2023.2196362 · Cited by 9 · 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
Zhao, Q., Huang, A., Wu, G., Guo, Q., Li, M., & Wang, X. (2022) · Food Research International · doi:10.1016/j.foodres.2022.111598 · 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
Role of Clay Substrate Molecular Interactions in Some Dairy Technology Applications
Azzouz, A., Aruș, V., & Platon, N. (2024) · International Journal of Molecular Sciences · doi:10.3390/ijms25020808 · Cited by 4 · see citing works
Çelik, S. Y. (2021) · International Food Research Journal · doi:10.47836/ifrj.28.4.04 · Cited by 2 · see citing works
Dahdah, K., Zikiou, A., Zaamta, M. F., Mechati, F., Fiala, S., & Saci, F. (2026) · World Journal of Microbiology and Biotechnology · doi:10.1007/s11274-026-05043-4 · Cited by 1 · see citing works
Dışhan, A., & Gönülalan, Z. (2024) · GIDA / THE JOURNAL OF FOOD · doi:10.15237/gida.gd23139
Wehaidy, H. R., Abdel-Naby, M. A., Kholif, A. M. M., Elaaser, M. M., Bahgaat, W. K., & Wahab, W. A. A. (2023) · Figshare · doi:10.6084/m9.figshare.22914682
Yang, W., Gan, L., Zhang, Y., Wang, Y., Zhang, W., Wang, T., & Qiao, H. (2026) · Journal of Agricultural and Food Chemistry · doi:10.1021/acs.jafc.6c07588
- Authors
- Zhang, Y., Xia, Y., Ding, Z., H. Lai, P. F., Wang, G., Xiong, Z., Liu, X., & Ai, L.
- Affiliations
- University of Shanghai for Science and Technology (CN)
- Jiangnan University (CN)
- Corresponding author
- Ai, Lianzhong
- ORCID
- Venue
- LWT
- Publisher
- Elsevier BV
- Published
- 2019-10
- Volume
- 113
- Pages
- 108276
- ISSN
- 0023-6438 (ISSN-L)
- OpenAlex
- W2952103111
- Cited by
- 21
- References
- 38
- Citations by year
- 2026: 2 · 2025: 1 · 2024: 4 · 2023: 8 · 2022: 2 · 2021: 3 · 2019: 1
- FWCI
- 0.83
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- LWT (publishedVersion)
- Funders
- Talent Program (14QB1400200)
- Science and Technology Commission of Shanghai Municipality
- National “Twelfth Five-Year” Plan for Science and Technology (2013BAD18B02)
- 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
- milk-clotting enzyme, Bacillus licheniformis, enzyme purification, casein hydrolysis, alpha-casein, β-casein, enzyme stability, calcium dependence
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
