Separation and Purification of Nattokinase Produced by Bacillus Subtilis with Ammonium Sulfate Precipitation and Chromatography

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1 20 1 No.1 Vol Journal of Chemical Engineering of Chinese Universities Feb (2006) , 1, 2, 3, 1, 1 (1., ; 2., ; 3., ) Bacillus subtilis 20 ~60 Superdex 75 SP Sepharose Fast Flow - (SDS-PAGE) SDS-PAGE 27.7 kd % Q814.1 A Separation and Purification of Nattokinase Produced by Bacillus Subtilis with Ammonium Sulfate Precipitation and Chromatography GAO Da-hai 1, MEI Le-he 1, SHENG Qing 2, XU Jing 3, LIN Dong-qiang 1, YAO Shan-jing 1 (1. Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou , China; 2. College of Life Science, Zhejiang University of Science and Technology, Hangzhou , China; 3. Hangzhou East-China Pharmaceutical Company, Hangzhou , China) Abstract: In order to obtain high purity nattokinase from the fermentation broth of Bacillus subtilis, some separation and purification techniques were explored. The optimized separation and purification process includes the following steps: removing cells by the centrifugation, 20%~60% saturation ammonium sulfate precipitation, gel filtration chromatography with Superdex 75 and ion-exchange chromatography with SP Sepharose Fast Flow. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to examine the purification effect, and the results indicate that the final product obtained is homogeneous and has a molecular weight around 27.7 kd. The fibrinolytic activity of the nattokinase was measured by means of fibrin plate method. The purification factor and activity recovery of the nattokinase are 8.4 and 49%, respectively. The specific activities of purified nattokinase could reach IU mg 1. The results demonstrate that the combination of bioseparation process of ammonium sulfate precipitation with gel filtration and ion exchange chromatography is suitable for the separation and purification of nattokinase from culture broth of Bacillus subtilis. Especially for lab scale, the process mentioned above can be easily performed as an efficient way to prepare pure nattokinase with high yield. Key words: nattokinase; purification; gel filtration; ion-exchange chromatography ( ) (2004C33036) (1980-)

2 [1] (nattokinase, NK) [2] NK (ELT) (t-pa) [3] Sephadex SDS-PAGE [4] Bacillus subtilis HL-1 (Fibrinogen) Sigma ÄKTA Explorer 100 XK16/20 Superdex 75 CM Sepharose Fast Flow SP Sepharose Fast Flow Amersham Biosciences Mini-PROTEAN 3 system Bio-RAD 2.2 LB 10 g L 1 5 g L 1 10 g L 1 NaCl 15 g L 1 ph7.0 LB 10 g L 1 5 g L 1 10 g L 1 NaCl ph g L 1 20 g L 1 Na 2 HPO g L 1 NaH 2 PO g L 1 CaCl g L 1 MgSO g L r min 1 12 h h [5] 2.3 [6] Astrup [7] ( IU ml 1 ) 10 µl 10 min 37 18h L r min 1 10 min (NH 4 ) 2 SO r min 1 30 min ( ) 60% 4

3 r min 1 30 min 10 mmol L 1 ph Superdex 75 ÄKTA Explorer ml min 1 Superdex Sepharose Fast Flow Superdex ml min mol L 1 NaCl SDS-PAGE 12 R kda, 66.0 kda, 45.0 kda, 30.0 kda, 20.1 kda, 14.4 kda mL 20% 60% 10mL 10mmol L 1 ph %~60% Table 1 The recovery of nattokinase and protein during salt-out with ammonium sulfate from 20%~60% saturation Culture broth Supernatant of the 20% Redissolved the precipitate in 60% saturation (NH 4 ) 2 SO 4 saturation(nh 4 ) 2 SO 4 NK activity / IU ml Amount of proteins / mg ml Volume / ml Total NK activity / IU Total protein / mg Recovery of active NK / % Recovery of proteins / % Specific activities / IU mg Fold Superdex 75 Superdex 75 24mL 1.0mL 1.0 ml min 1 10mmol L 1 ph nm SDS-PAGE ( 4) 1 Superdex 75 Fig.1 Superdex 75 gel filtration of nattokinase after salting-out with ammonium sulfate 2 Superdex 75 Table 2 Superdex 75 gel filtration of nattokinase Sample Eluent Total activities of NK/IU Total protein / mg Recovery of NK / % Recovery of protein / % Specific activities / IU mg Purified factor 1 3.9

4 [2] ph 6~12 [7] 10 mmol L 1 ph 6.4 CM Sepharose Fast Flow 2 SP Sepharose Fast Flow 3 MAU %B Waste Waste ml 2 CM Sepharose Fast Flow Fig.2 CM Sepharose Fast Flow IEC of nattokinase after Superdex 75 gel filtration min 3 SP Sepharose Fast Flow Fig.3 SP Sepharose Fast Flow IEC of nattokinase after Superdex 75 gel filtration CM NaCl SP CM SP Sepharose Fast Flow SDS-PAGE ( 4) SDS-PAGE (1) (2) 3 SP Sepharose Fast Flow Table 3 SP Sepharose Fast Flow ion-exchange chromatography of nattokinase Sample Eluent Total activities of NK/IU Total protein / mg Recovery of NK / % Recovery of protein / % Specific activities / IU mg Purified factor kd Lane 1-molecular weight standard proteins; Lane 2-fermentation broth; 45.0 Lane 3-Superdex 75 gel filtration; kD Lane 4-SP Sepharose Fast Flow ion-exchange chromatography. 4 SDS-PAGE Fig.4 SDS-PAGE of purified nattokinase (3) 27.7 kd NK 27,782 [8] 4

5 Steps Total activities of NK / IU 4 Table 4 Purification of nattokinase Total protein / mg Specific activities / IU mg 1 Recovery of NK / % Culture broth Ammonium sulphate precipitation 20% saturation % saturation Gel filtration chromatography Ion exchange chromatography Purified factor 4 20%~60% 10mmol L 1 ph 6.4 Superdex 75 SP Sepharose Fast Flow IU mg 1 ( ) SDS-PAGE % [4] [9] [1] Sherry S. Recombinant tissue plasminogen activator(rt-pa): is it the thrombolytic agent of choice for an evolving acute myocardial infarction [J]. Am J Cardiol, 1987, 59(9): [2] Sumi H, Hamada H, Tsushima H, et al. A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto; a typical and popular soybean food in the Japanese diet [J]. Experientia, 1987, 43(10): [3] Sumi H, Hamada H, Nakanishi K, et al. Enhancement of the fibrinolytic activity in plasma by oral administration of nattokinase [J]. Acta Haematol, 1990, 84(3): [4] DING Gui-ping( ), CAI Zheng-sen( ), WANG Zheng-gang( ). Purification and characterization of nattokinase( ) [J]. Amino Acids & Biotic Resources( ), 2001, 23(3): [5] HU Sheng( ), MEI Le-he( ), YAO Shan-jing( ). Optimization of submerged fermentation of nattokinase production by Bacillus subtilis with response surface methodology( ) [J]. Food and Fermentation Industries ( ), 2003, 29(1): [6] Bradford M M. A rapid and sensitive method for the quantitation of microgramquantities of protein utilizing the principle of protein-dye binding [J]. Anal Biochem, 1976, 72(1-2): [7] Asturp T, Müllertz S. The fibrin plate method for estimating fibrinolytic activity [J]. Biochem Biophys, 1952, 40(2): [8] Fujita M, Nomura K, Hong K, et al. Purification and characterization of a strong fibrinolytic enzyme (nattokinase) in the vegetable cheese natto, apopular soybean fermented food in Japan [J]. Biochem Biophys Res Commun, 1993, 197(3): [9] MEI Le-he( ), HU Sheng( ), LIN Dong-qiang, et al ( ). Screening of Bacillus subtilit natto from traditional Japanese food Natto and separation of nattokinase( ) [J]. J Chem Eng of Chinese Univ ( ), 2005, 19(4):

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