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Purification and characterization of a novel milk-clotting enzyme produced by Bacillus amyloliquefaciens GSBa-1

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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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Acknowledgements

The National Key Research and Development Program of China (2018YFC1604302), National Natural Science Foundation of China (Grant no. 31871823), and Beijing Talent Cultivation Quality Construction—First-class Professional Construction (Municipal Level)—Food Science and Engineering (PXM2019_014213_000010) are kindly acknowledged.

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Authors and Affiliations

  1. Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology and Business University, Beijing, 100048, China

    Xiao Zhao, Miao Cai, Zhi-Jie Yang, Tian-Qi Luo, Abid Sarwar, Sarah Megrous, Tariq Aziz & Zhen-Nai Yang

  2. Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology and Business University, Beijing, 100048, China

    Xiao Zhao & Zhen-Nai Yang

  3. School of Food and Health, Beijing Technology and Business University, No. 11 Fu-Cheng Road, Hai-Dian District, Beijing, 100048, China

    Zhen-Nai Yang

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  1. Xiao Zhao
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Correspondence to Zhen-Nai Yang.

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Zhao, X., Cai, M., Yang, ZJ. et al. Purification and characterization of a novel milk-clotting enzyme produced by Bacillus amyloliquefaciens GSBa-1. Eur Food Res Technol 245, 2447–2457 (2019). https://doi.org/10.1007/s00217-019-03361-6

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  • DOI: https://doi.org/10.1007/s00217-019-03361-6

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