Screening Of Producer Strain With High Yield, Optimization Of Fermentation Conditions And Purification Of Fibrinolysin From Bacillus Subtilis

The thrombus was one of the causes that resulted in the high death rate among patientsat present. Dissolving thrombus was considered as the most important therapy for thisdisease. Fibrinolysin can catalyze the hydrolysis of fibrin that was the main component ofthrombus. So fibrinolysin became the focus of research for its special therapy effect forthrombus. Microorganism was the important source of the enzyme with fibrinolytic activity.The nattokinase discovered from natto by Japanese researchers was proved to possess goodfibrinolytic activity and one of relevant fibrinolysins was also isolated from fermentedliquid of Bacillus subtilis by Chinese and Korean researchers.Due to the low yield of enzyme from wildness strains, the Bacillus subtilis XZ3â‘£which can produce fibrinolysin was mutagenized by N⁺ ion beam implantation to obtain astrain with high yield of fibrinolysin which named Bacillus subtilis XZI125. Thefermentation optimization of B. subtilis XZI125 and the purification of fibrinolysin fromthis strain were carried out in this paper. The objective of this research was to offertheoretical basis for exploitation and application of fibrinolysin of B. subtilis. The mainresults were as follows:1. A strain, B. subtilis XZ3â‘£, producing fibrinolysin of B. subtilis was mutagenizedby 20 keV N⁺ ion implantation, which dose ranged from 20×2.6×10¹³ion/cm²-200×2.6×10¹³ ion/cm². The survival curve was typical "saddle-shaped" dosageeffect curve. A strain, B. subtilis XZI125, which yielded high fibrinolysin activity of B.subtilis was screened out from 284 alive strains. The fibrinolysin activity of B. subtilisincreased by 160 %, achieved 1230.45 u.ml^-1.2. In the present investigation, the methodology of Plackett-Burman (PB) wasundertaken to screen the key factors rapidly from the related 26 factors. By analyzing thestatistical regression and the prediction profiler, bran, glycerin, (NH₄)₂SO₄, defattedsoybean, phytic acid, dimethylsulfoxide, fermentation temperature, fermentation time, volume of medium were found to be the most important 9 factors for fibrinolysin of B. subtilis produced by B.subtilis XZI125.3. Based on the results of our previous Plackett-Burman design, five-level six-factorcentral composite rotatable design (CCRD) was employed in the present study to optimizethe levels of the chosen variables, which significantly affect fibrinolysin activity of B.subtilis in submerged fermentation using B. subtilis XZI125, i.e. bran, glycerin, (NH₄)₂SO₄,defatted soybean, phytic acid, dimethylsulfoxide. By analysis of the response surfacecontour plots generated from the quadratic regression equation, the effects of the individualvariable as well as the mutual interactions between variables were extensively studied. Bysolving the model equation using appropriate statistic methods, the optimum concentrationlevels were determined: under conditions of bran, 1.70%; glycerin, 1.30%; (NH₄)₂SO₄,0.07%; defatted soybean,3.94%; phytic acid, 282.64ppm and dimethylsulfoxide, 5.84ppm,the predicted maximum of fibrinolysin activity of B. subtilis was 2382.00 u-ml^-1. Thevalidation data under different experimental levels have confirmed the correlation betweenthe experimental and theoretical values.4. Based on the results of Plackett-Burman design and CCRD design, a three-levelthree-factor Box-Behnken design was executed to optimize fibrinolysin activity of B.subtilis in submerged fermentation using B. subtilis XZI125. The critical factors selectedfor the investigation were fermentation temperature, fermentation time and volume ofmedium. By analysis of the 3-D plots and their corresponding plots, the optimum ranges offermentation temperature, fermentation time and volume of medium were obviously clear.By solving the inverse matrix from the second-order polynomial equations, the optimalconditions were determined: under conditions of fermentation temperature, 31.58℃;fermentation time, 48.54h and volume of medium, 53.81ml, the predicted maximum, offibrinolysin activity of B. subtilis was 2665.16 u·ml^-1.5. Based on the results of fermentation in shaking flask, the effect of dissolved oxygen(DO) concentrateion and pH on the batch production of fibrinolysin of B. subtilis in a 100Lstirred fermenter by B. subtilis XZI125 was studied. When the air flow rate, agitation rateand pH were respectively controlled at 1.0vvm, 250rpm and 7.0, fibrinolysin activity of B.subtiIis in fermentation mash increased by 211.78 u·ml^-1 to 2876.94 u·ml^-1 in comparisonwith fermentation in shaking flask.6. The enzyme in the fermented liquid of B. subtilis XZI125 was purified tohomogeneity by a procedure involving MF (0.1μm inorganic ceramic membrane), UF(hollow fiber membrane module) and HZD-2 (cation exchange and adsorption resin )column. The procedure described here purified fibrinolysin of B. subtilis 18.34-fold with24.58 % yield, 4291.03 u·mg^-1 specific activity. Relevant molecular weight of fibrinolysinof B. subtilis was estimated to be 27.TKDa by SDS- polyacrylamide gel electrophoresis.