Cited by 10Enzyme Production and CharacterizationProteins in Food SystemsEnzyme Catalysis and Immobilization

Purification and characteristics of a novel milk-clotting metalloprotease from Bacillus velezensis DB219

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Zhang, Yao, Hu, Jiayun, Wang, Jiaxin, Liu, Chen, Liu, Xiaofeng, Sun, Juan, Song, Xinjie, Wu, Yuanfeng·2023·Journal of Dairy Science

Abstract

Milk-clotting enzyme (MCE) is the essential active agents in dairy processing. The traditional MCE is mainly obtained from animal sources, in which calf rennet is the most widely used in cheese industry. Traditional MCE substitute is becoming necessary due to its limited production and increased cheese consumption. A novel traditional MCE substitute was produced from Bacillus velezensis DB219 in this study. DB219 MCE exhibited a notable specific activity of 6110 SU/mg and 3.16-fold purification yield with 28.87% recovery through ammonium sulfate fractionation and DEAE-Sepharose Fast Flow. The purified DB219 MCE was a metalloprotease with a molecular weight of 36 kDa. DB219 MCE was weak acid resistance and stable at pH 6.0–10.0 and temperature <45°C. The highest milk-clotting activity (MCA) was observed in substrate at pH 5.5 added with 20–30 mM of CaCl 2 . The Michaelis constant ( K m ) and maximal velocity ( V m ) for casein were 0.31 g/L and 14.22 μmol/min. DB219 MCE preferred to hydrolyze β-casein (β-CN) instead of α-casein (α-CN). DB219 MCE hydrolyzed α-CN, β-CN and κ-casein (κ-CN) to generate significantly different peptides in comparison with calf rennet and ES6023 MCE (fungal MCE) through SDS-PAGE and RP-HPLC analysis. DB219 MCE mainly cleaved Thr124-Ile125 and Ser104-Phe105 bonds in κ-CN and had unique casein cleavage sites and peptide composition through LC-MS/MS analysis. DB219 MCE was potential to be a new milk coagulant and enriched kinds of traditional MCE substitute.

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Registry

Crossref · 10.3168/jds.2023-23450

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Authors
Zhang, Y., Hu, J., Wang, J., Liu, C., Liu, X., Sun, J., Song, X., & Wu, Y.
Affiliations
  • Zhejiang University of Science and Technology (CN)
  • Zhejiang University of Technology (CN)
Venue
Journal of Dairy Science
Published
2023-10
Volume
106
Issue
10
Pages
6688-6700
ISSN
0022-0302 (ISSN-L)
OpenAlex
W4385617598
Cited by
10
References
40
Citations by year
2026: 5 · 2025: 3 · 2024: 2
FWCI
1.29
Type
journal article
Language
en
Open access
Locations
  • Journal of Dairy Science (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
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, metalloproteinases, Bacillus velezensis, rennet substitute, casein hydrolysis, kappa-casein, alpha-casein, cheese coagulation, enzyme purification, enzyme stability
MeSH terms
Animals, Bacillus, Caseins, Cheese, Chromatography, Liquid, Milk, Peptides, Metalloproteases, Tandem Mass Spectrometry

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