Purification and properties of a milk-clotting enzyme produced by Bacillus amyloliquefaciens D4

Xiaoling He; Fazheng Ren; Huiyuan Guo; Weibing Zhang; Xi Song; Bozhong Gan

Korean Journal of Chemical Engineering, Vol.28, No.1, 203-208, January, 2011

DOI10.1007/s11814-010-0347-8 Export Citation

Purification and properties of a milk-clotting enzyme produced by Bacillus amyloliquefaciens D4

Xiaoling He, Fazheng Ren¹, Huiyuan Guo¹, Weibing Zhang, Xi Song, and Bozhong Gan^†

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

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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.

Keywords:Milk-clotting Enzyme;Rennet;Purification;Enzyme Properties;Bacillus amyloliquefaciens

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[2] Cavalcanti MTH, Teixeira MFS, Filho JLL, Porto ALF, Bioresour. Technol., 93(1), 29 (2004)

[3] Raposo S, Domingos A, Process. Biochem., 43, 139 (2008)

[4] Ageitos JM, Vallejo JA, Sestelo ABF, Poza M, Villa TG, J. Appl. Microbiol., 103(6), 2205 (2007)

[5] Neelakantan S, Mohanty AK, Kaushik JK, Curr. Sci., 77, 143 (1999)

[6] Breccia JD, Sineriz F, Baigon MD, Castro GR, Hatti-Kaul R, Enzyme Microb. Technol., 22(1), 42 (1998)

[7] Chung H, Friedberg F, Biochem. J., 180, 387 (1985)

[8] Balaban NP, Malikova LA, Mardanova AM, Rudenskaya GN, Sharipova MR, Biochemistry (Moscow)., 4, 459 (2007)

[9] Lee YJ, Kim BK, Lee BH, Jo KI, Lee NK, Chung CH, Lee YC, Lee JW, Bioresour. Technol., 99(2), 378 (2008)

[10] Laemmli U, Nature., 227, 680 (1970)

[11] Bradford MM, Anal. Biochem., 72, 248 (1976)

[12] Arima K, Yu J, Iwasaki S, Methods. Enzymol., 19, 446 (1970)

[13] Lineweaver H, Burk D, J. Am. Chem. Soc., 56, 658 (1934)

[14] Nouani A, Belhamiche N, Slamani R, Belbraouet S, Fazouane F, Bellal MM, Int. J. Dairy. Technol., 112 (2009)

[15] Preetha S, Boopathy R, World. J. Microbiol. Biotechnol., 13, 573 (1997)

[16] Sardinas JL, Appl. Microbiol., 16, 248 (1968)

[17] Vishwanatha AG, Appu R, Singh SA, Appl. Microbiol.Biotechnol., 1, 1 (2009)

[18] Magda A, EI-Bendary, Maysa E, Moharam H, Thanaa HA, J. Appl. Sci. Res., 3, 695 (2007)

[19] Areces LB, Bonino MBDJ, Parry MAA, Fraile ER, Fernandez HM, Cascone O, Appl. Biochem. Biotechnol., 37, 283 (1992)

[20] Hashem AM, Bioresour. Technol., 75(3), 219 (2000)

[21] Yasuda M, Aoyama M, Sakaguchi M, Nakachi K, Kobamoto N, Appl. Microbiol. Biotechnol., 51(4), 474 (1999)

[22] Sternberg MZ, J. Dairy. Sci., 54, 159 (1971)

[23] Wang Y, Cheng Q, Ahmed Z, Jiang X, Bai X, Korean. J.Chem. Eng., 1, 1 (2009)

[24] Sousa MJ, Ardo Y, McSweeney PLH, Int. Dairy. J., 11, 327 (2001)

[25] Harboe M and Budtz P, Technology of Cheesemaking. In: Law,Barry A. Ed., New York, Academic Press (1999)

[26] Kim HS, Legoy MD, Thomas D, Korean J. Chem. Eng., 1, 35 (1989)

[27] Fernandez-Lahore HM, Auday RM, Fraile ER, Biscoglio de Jimenez Bonino M, Pirpignani L, Machalinski C, Cascone O, J. Peptide Res., 53, 599 (1999)

[28] Anema SG, Lee SK, Klostermeyer H, LWT-Food Sci.Technol., 40, 99 (2007)

[29] Merheb-Dini C, Gomes E, Boscolo M, da Silva R, Food Chem., 120, 87 (2010)

[30] Pires MS, Orellana GA, Gatti CA, Food Hydrocolloids., 13, 235 (1999)

[31] Matta H, Punj V, Int. J. Food Microbiol., 42, 139 (1998)

[32] Payne MR, Morison KR, Int. Dairy. J., 9, 887 (1999)

[33] Yamazaki H, Ohnishi Y, Horinouchi S. Published Only in Database of NCBI (1999)

[34] Gray GL, Hayenga K, Cullen D, Wilson LJ, Norton S, Gene., 48, 41 (1986)

[35] Shimada H, Honjo M, Mita I, Nakayama A, Akaoka A, Manabe K, Furutani Y, J. Biotechnol., 2, 75 (1985)