Optimization of fibrinolytic enzyme production by Bacillus subtilis DC-2 in aqueous two-phase system (poly-ethylene glycol 4000 and sodium sulfate) Onesmus K. Ashipala, Qian He * Jiangnan University, School of Food Science, Research Centre of Functional Food Engineering, Ministry of Education, No. 170, Hui He Road, Wuxi, PR China Received 17 June 2007; received in revised form 29 August 2007; accepted 1 September 2007 Available online 5 November 2007 Abstract The central composite rotable design (CCRD) was used to determine optimal conditions for fibrinolytic enzyme production by Bacil- lus subtilis DC-2 in poly-ethylene glycol 4000 (PEG 4000) and sodium sulfate (Na 2 SO 4 ) aqueous two-phase system (ATPS). PEG 4000 and Na 2 SO 4 concentration, fermentation time and temperature, and pH were selected as variables to evaluate the fibrinolytic activity in PEG phase. Using response surface methodology (RSM), a second-order polynomial equation was obtained by multiple regression anal- ysis. The predicted maximal fibrinolytic activity in PEG phase was 1241.02 IU/ml with 9.05% PEG 4000 concentration, 5.06% Na 2 SO 4 concentration, 118.77 h fermentation time, 37.57 °C fermentation temperature and pH 6.52. The validity of the response model was ver- ified by a good agreement between predicted and experimental results. The fibrinolytic activity obtained from experimental results in PEG phase (1223.61 IU/ml) was higher than that produced in homogeneous fermentation (1165.58 IU/ml). Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Bacillus subtilis; Fibrinolytic activity; PEG-Na 2 SO 4 ATPS; Response surface methodology 1. Introduction Microbial fibrinolytic enzymes are agents that dissolve fibrin clots and have considerable potential to be developed into thrombolytic agents (Peng et al., 2005). Over the past decades, many derived microbial fibrinolytic enzymes have been discovered in various traditional fermented foods (Kim and Choi, 2000; Kim et al., 2006; Ko et al., 2004; Lee et al., 2001; Peng et al., 2003; Wang et al., 2006). Nattokinase (isolated from natto, a Japanese fermented food) not only directly cleaves cross-linked fibrin, but also activates the production of t-PA, resulting in the transfor- mation of inactive plasminogen to activate plasmin. Fur- thermore, natattokinase enhances its fibrinolysis through cleavage and inactivation of PAI-1, which is the primary inhibitor of fibrinolysis and regulates total fibrinolytic activity by its relative ratio with t-PA (Peng et al., 2005). The extraction and purification of nattokinase from fer- mentation broth such as chromatography, organic solvent fractionation, salting out, expanded bed adsorption, reverse micelle extraction and superparamagnetic poly (methyl methacrylate) beads have been reported (Yang et al., 2006). Optimization of culture medium for nattokin- ase production has been studied by Liu et al. (2005). In contrast, there are no reports concerning production and optimization of fibrinolytic activity in aqueous two-phase system (ATPS). An aqueous two-phase system (ATPS) is formed when combinations of hydrophilic solutes (polymers or polymer and certain salts) display incompatibility in aqueous solu- tion above critical concentrations (Rito-Palomares, 2004). Fermentation systems based on ATPS offer the possibility 0960-8524/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.09.029 * Corresponding author. E-mail address: amtf168@126.com (Q. He). Available online at www.sciencedirect.com Bioresource Technology 99 (2008) 4112–4119