Biochemical Engineering Journal 43 (2009) 85–91 Contents lists available at ScienceDirect Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej Milk-clotting enzymes produced by culture of Bacillus subtilis natto Chwen-Jen Shieh a , Lan-Anh Phan Thi b , Ing-Lung Shih b,∗ a Biotechnology Center, National Chung-Hsing University, Taichung, Taiwan b Department of Environmental Engineering, Da-Yeh University, Chang-Hwa, Taiwan article info Article history: Received 7 July 2008 Received in revised form 20 August 2008 Accepted 1 September 2008 Keywords: Bacillus subtilis natto Milk-clotting enzyme Milk-clotting activity Proteolytic activity Cheese making abstract Factors affecting the production of milk-clotting enzyme (MCE) by Bacillus subtilis (natto) Takahashi, a ready available commercial natto starter, were studied. Remarkable milk-clotting activity (MCA), 685.7 SU/ml or 12,000 SU/g, was obtained when the bacteria were cultivated in the medium contain- ing sucrose (50 g/L) and basal salts at pH 6, 37 ◦ C with shaking at 175 rpm for 1 day. The MCA and MCA/PA ratio of the crude enzyme obtained are comparable with those of Pfizer microbial rennin and Mucor ren- nin. The crude enzyme showed excellent pH and thermal stability; it retained 96% of MCA after incubation for 40 min at 40 ◦ C and retained more than 80% of its activity between pH 4 and pH 7 for more than 30 min at 30 ◦ C. The MCE of B. subtilis (natto) Takahashi has potential as calf rennet substitutes. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Milk coagulation is a basic step in cheese manufacturing. For a long time calf rennet, the conventional milk-clotting enzyme obtained from the fourth stomach of suckling calves [1] is the most widely used coagulant in cheese making all over the world to manu- facture most of the cheese varieties. The worldwide reduced supply of calf rennet and the ever increase of cheese production and con- sumption have stimulated the research for milk-clotting enzyme (MCE) from alternative sources to be used as calf rennet substitutes [2–5]. Various animals, plants and microbial proteases have been suggested as milk coagulants [6–13]. However, attention has been focused on the production of milk-clotting enzymes (MCEs) from microbial sources for use as rennin substitutes [8,14–26]. Although there are many microorganisms that produce MCEs [3], only the MCEs produced by strains of Rhizomucor miehei, Rhizomucor pusil- lus var. Lindt, Aspergillus oryzae and Enthothia parasitica are widely used [27–29]. Bacillus subtilis (natto) Takahashi, a commercial natto starter, is commonly used to prepare fermented soybean product-natto, which is a traditional Japanese food for more than 1000 years. Recently, we have shown that B. subtilis (natto) Takahashi produced simultaneously a mixture of poly(-glutamic acid) and levan when ∗ Corresponding author. Present address: 112 Shan-Jiau Road, Da-Tsuen, Chang- Hwa 51505, Taiwan. Fax: +886 4 8511344. E-mail address: ils@mail.dyu.edu.tw (I.-L. Shih). it was grown in a basal medium containing sucrose and l-glutamate [30]. Furthermore, it can produce poly(-glutamic acid) and levan selectively under chosen conditions in that poly(-glutamic acid) was mainly produced in a medium containing l-glutamic acid and without sucrose; in contrast, levan was the only product when the bacteria were cultivated in a medium containing 20% (w/w) sucrose and without l-glutamate. Nonetheless, B. subtilis (natto) was never investigated for the production of MCE(s). B. subtilis is one of the most investigated microbial groups, because they can produce vari- eties of biotechnological interesting substances [31]; it is known to secrete several proteases during the fermentation process [32]. The capacity of selected Bacillus strains to produce and secrete large quantities of extracellular enzymes has led them to be among the most important industrial enzyme producers. While B. subtilis (natto) produces many enzymes, including amylases and cellulases, the most important enzymes in the production of natto are pro- teases; many of them have been characterized [33]. The proteases are responsible for the main flavor, through hydrolysis of soybean protein. It is conceivable that B. subtilis (natto) may produce MCE(s). In fact, we have recently discovered that the fermentation product of B. subtilis (natto) Takahashi displayed strong milk-clotting activ- ity (MCA) in the process of making bakery products [34]. To further investigate the possibility of the use of B. subtilis (natto) Takahashi for the efficient production of the rennin-like enzymes for cheese making, the present work was undertaken to investigate the pat- tern of milk-clotting and proteolytic activities of B. subtilis (natto) in extracelluar bacterial enzyme preparations under different con- ditions of culturing. 1369-703X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2008.09.003