Study On Fermentation Control Of Nattokinase And The Enzymatic Characteristics

Nattokinase(NK) is a fibrinolytic serine enzyme that is extracted from a traditional fermented food of Japan. It is reported that Nk have a strong thrombustic function. Compared with the thrombolytic agents such as urokinase and treptokinase, Nattokinase is safer ,easier to be absorbed by body because of its low molecular weight,and has more direct and more persistent effect on thrombosis,and it is more important that Nattokinase can be fermented by bacteria,which will lower the production cost. So nattokinase can be developed as a new fibrinolytic medicine.This paper study on the liquid fermentation of NK, purification,and character of nattokinase..At last ,the main results are as follows:1. The liquid fermentation optimization demonstrate : To the seminal medium, The best carbon source is glucose,the best nitrogen source is peptone . And the composition of the seminal medium is peptone 0.5%,Glu 1%,Yeast extract 0.1% ,NaCl 0.3%. To fermentative medium,the best carbon source is resolutive starch 2%,the best nitrogen source is tryptone 0.5%. inducer A and B can enhance the yield of NK. Ca2+ and Mg2+ are the positive ion which have the greatest effect on NK's synthesis.The bacteria grows the fast at 37℃,but 30℃ is the most suitable temperature for the production of NK; The bacteria has the biggest speed of growth and the biggest yield of NK under pH7.0.The volume of fermentation medium is 100ml/500ml,2%of 20h-old inoculum is the best condition for NK's synthesis.2. Purification experiment result: Purifying the enzyme by the ammonium sulfate fraction,DEAE-Sephrose, SephadexG-100 Chromatography and so on,the purified enzyme showed a single protein band in the SDS-PAGE.purified is 19,and the last yield is 42%,Specific activity is 22388.81 IU/mg.3. The character of nattokinase is the follows :the optimum reaction temperature of NK is 45-50℃ ,and the stability of NK is at 25-40℃,which is very good in 6-8 hours.when the temperature exceed 55 ℃,activity of NK drops very fast .when above 70℃ activity of NK forfeits completely.The pH7.0-7.5 is the optimal pH and pH6.0-9.0 is the best condition for NK's stability Na+ and K+ have less effect onthe NK's stability ,Cu2+ and Mn2+ have more effect on the NK's stability;but Hg2+ restraint the activity completely ,Ca2+ Mg2+ have positive effect on NK ,which is a fine activator of NK.