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Received April 6, 2010
Accepted June 6, 2010
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Purification and properties of a milk-clotting enzyme produced by Bacillus amyloliquefaciens D4
College of Food Science and Technology Engineering, Gansu Agricultural University, Lanzhou 730070, China 1College of Food Science and Nutritional Engineering, China Agricultural University, Key Laboratory of Functional Dairy,Beijing Higher Institution Engineering Research Center of Animal Product, Beijing 100083, China
ganbz@126.com
Korean Journal of Chemical Engineering, January 2011, 28(1), 203-208(6), 10.1007/s11814-010-0347-8
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Abstract
The milk-clotting enzyme from Bacillus amyloliquefaciens D4 was purified to 17.2-fold with 20% recovery by precipitation in ammonium sulfate and ion-exchange chromatography. The molecular mass of the enzyme was 58.2 kDa as determined by SDS-PAGE, and it was proved to be a metalloprotease by EDTA inhibition. The enzyme was active in the pH range 5.5-7.0 and was inactivated completely by heating at 55 ℃ for 20 min. The highest level of enzyme activity was obtained at 65 ℃, pH 5.5, in the presence of 25mM CaCl2. The milk-clotting activity was inhibited only slightly by Na+ and K+ and significantly by Cu2+, Zn2+ and Sn2+. The Km value of this enzyme was 0.471 mg/mL. The high level of milk-clotting activity coupled with a low level of thermal stability suggested that the milk-clotting enzyme from B. amyloliquefaciens D4 should be considered as a potential substitute for calf rennet.
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Sardinas JL, Appl. Microbiol., 16, 248 (1968)
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Areces LB, Bonino MBDJ, Parry MAA, Fraile ER, Fernandez HM, Cascone O, Appl. Biochem. Biotechnol., 37, 283 (1992)
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Kim HS, Legoy MD, Thomas D, Korean J. Chem. Eng., 1, 35 (1989)
Fernandez-Lahore HM, Auday RM, Fraile ER, Biscoglio de Jimenez Bonino M, Pirpignani L, Machalinski C, Cascone O, J. Peptide Res., 53, 599 (1999)
Anema SG, Lee SK, Klostermeyer H, LWT-Food Sci.Technol., 40, 99 (2007)
Merheb-Dini C, Gomes E, Boscolo M, da Silva R, Food Chem., 120, 87 (2010)
Pires MS, Orellana GA, Gatti CA, Food Hydrocolloids., 13, 235 (1999)
Matta H, Punj V, Int. J. Food Microbiol., 42, 139 (1998)
Payne MR, Morison KR, Int. Dairy. J., 9, 887 (1999)
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Gray GL, Hayenga K, Cullen D, Wilson LJ, Norton S, Gene., 48, 41 (1986)
Shimada H, Honjo M, Mita I, Nakayama A, Akaoka A, Manabe K, Furutani Y, J. Biotechnol., 2, 75 (1985)
