Physiological Function And Purification Of The Fibrinolytic Enzyme Of B.amyloliquefaciens In Douchi

In this research,a contrasting strain WB800/pBE3and a dfe deficient mutant wereconstructed from B. subtilis WB00and B. amyloliquefaciens DC12.The fibrinolytic enzyme of the strains were measured, and the spore frequencies inNMSP medium and the environmental tolerance of these strains to pH, tmperature,osmolality and H₂O₂was compared. We also collected and purifed culture supernatant from B.amyloliquefaciens DC12at different time to investigate whether douchi fibrinolyticenzyme（DFE） has two different forms of enzyme and zymogen in the extracellular. The majorresearch content and results are summarized as follows:（1） First, a contrasting strain WB800/pBE3was contructed by μsing E.coli B. subtilisshuttle vector pBE3and a dfe deficient mμtant was contrccted from B. amyloliquefaciensDC12by homologous recombination at the same time.（2） The fibrinolytic enzyme and the spore frequencies in NMSP medium of the strainswere measured. At different culture pH, temperature, oxidative stress and NaCl concentrations,the growth rate of these strains were compared.The resμlts show that, The recombinant strainWB800/pBE3.DFE could secret DFE in the exponential growth and stationary time,itsmaximμm fibrinolytic activity of the sμpernatant in LB medium was580U/mL at the culturetime of32h. dfe deficient mutant prodμced only14%of wild type fibrinolytic activity,whichreached as high as358U/mL. DFE significantly affects its spore frequencies, and affects itstolerance to low pH,high temperature, high oxidative stress and high salt environment.DFEsynthesis capability can increase the spore frequencies and the tolerance of the strains toenvironment.（3）B. amyloliquefaciens DC12culture supernatant at different time was collected andpurified to investigate whether DFE has two different forms of enzyme and zymogen in theextracellular. we can not find zymogen of DFE in the extracellular of B. amyloliquefaciensDC12.