Purification and characterization of a novel milk-clotting enzyme produced by Bacillus amyloliquefaciens GSBa-1
Abstract
A novel milk-clotting enzyme (MCE) produced by Bacillus amyloliquefaciens GSBa-1 was purified and identified to belong to the peptidase M4 family, and the mature peptide with milk-clotting activity (MCA) was a neutral metalloproteinase with molecular mass of about 38 kDa. The optimal pH and temperature were determined to be at 5.5 and 57 °C for MCA, and at 7.0 and 57 °C for the proteolytic activity, respectively. The MCE exhibited slight autolysis that could be inhibited by Ca²⁺ and Na⁺. Hydrolysis of caseins revealed that κ-casein exhibited higher sensitivity to the MCE action than α- and β-casein. By in-gel tryptic digestion and LC–MS/MS analysis of the major peptide (about 13 kDa) generated from hydrolysis of κ-casein by the MCE, the cleavage site was identified to be at Lys 111–Lys 112, which was different from those of other MCEs reported earlier. The MCE from B. amyloliquefaciens GSBa-1 could serve as a novel milk coagulant for potential application in making cheese with desired proteolysis.
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GSBa-1 (milk-clotting enzyme produced by Bacillus amyloliquefaciens) exhibited relatively high heat resistance, with maximum milk-clotting activity and proteolytic activity observed at 57°C, whereas a decrease in both was noted at 62°C.
“The GSBa-1 (MCE produced by B. amyloliquefaciens) exhibited relatively high heat resistance, with maximum MCA and PA observed at 57°C, whereas a decrease in both MCA and PA was noted at 62°C (Zhao et al., 2019).”
From Invited review: Milk-clotting enzymes of microbial origin and their role in cheesemaking-A review. (2025) · cites this paper as Zhao et al., 2019
The milk-clotting activity of the enzyme from Bacillus subtilis (BY-1) gradually increased with increasing temperature from 25°C to 65°C, reaching maximum activity at 65°C.
“They demonstrated that the MCA of the enzyme from B. subtilis (BY-1) gradually increased with increasing temperature from 25°C to 65°C, reaching a maximum activity at 65°C.”
From Invited review: Milk-clotting enzymes of microbial origin and their role in cheesemaking-A review. (2025) · cites this paper as Zhao et al., 2019
The optimal temperature for milk-clotting activity of enzymes produced by Bacillus amyloliquefaciens falls within the 65°C to 70°C range.
“Additionally, Zhao et al. (2019) determined that the optimal temperature for MCA of enzymes produced by B. amyloliquefaciens falls within the 65°C to 70°C range.”
From Invited review: Milk-clotting enzymes of microbial origin and their role in cheesemaking-A review. (2025) · cites this paper as Zhao 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 Zhao et al., 2019
MCE isolated from rice wine, used in Cheddar production and compared with a commercial rennet, did not lead to significant differences in sensory tests for those cheeses.
“Zhao et al. (2019), in their study, showed that MCE isolated from rice wine, used in Cheddar production and compared with a commercial rennet, did not lead significant differences in sensory test for those cheeses.”
From Invited review: Milk-clotting enzymes of microbial origin and their role in cheesemaking-A review. (2025) · cites this paper as Zhao et al., 2019
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- Authors
- Zhao, X., Cai, M., Yang, Z.-J., Luo, T.-Q., Sarwar, A., Megrous, S., Aziz, T., & Yang, Z.-N.
- Affiliations
- Beijing Technology and Business University (CN)
- Venue
- European Food Research and Technology
- Published
- 2019-09-14
- Volume
- 245
- Issue
- 11
- Pages
- 2447-2457
- ISSN
- 0373-0174 (ISSN-L)
- 1438-2377
- 1438-2385
- OpenAlex
- W2972361318
- Cited by
- 24
- References
- 39
- Citations by year
- 2026: 1 · 2025: 2 · 2024: 7 · 2023: 3 · 2022: 3 · 2021: 5 · 2020: 3
- FWCI
- 1.52
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- European Food Research and Technology (publishedVersion)
- Funders
- Beijing Talent Cultivation Quality Construction - First-class Professional Construction (Municipal Level) - Food Science and Engineering (PXM2019_014213_000010)
- National Key Research and Development Program of China (2018YFC1604302)
- National Natural Science Foundation of China (31871823)
- 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 amyloliquefaciens, casein hydrolysis, milk coagulants, cheese proteolysis, enzyme purification, proteolytic activity
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