Screening Of Nattokinase Producing Bacterial Strain And Enzyme Characterization Of Nattokinase

Nattokinase is a serine protease produced by Bacillus natto or B. subtilis natto. It hasstrong fibrinolytic activity, which can direct effect on the fibrinolytic protein and activateplasminogen in vivo, to increase the amount and role of endogenous plasminogen. It comesfrom food, is expected to become the new thrombolytic agents with safe, nontoxic side effectsand orally active. In-depth study of strain produced nattokinase and characterization ofnattokinase has theoretical and applied value. A strain produced nattokinase from natto andfermented soy foods was screened and identified, fermentation conditions of nattokinase wereoptimized, and purification and enzyme characterization of nattokinase were studied in thispaper. The results and conclusions were as follows:1. Firstly14strains produced protease were screened though casein plate from natto andfermented soy foods, and then though shake flask liquid fermentation a strain producednattokinase was screened on fibrinogen plate, the strain’s enzyme activity can reach511.47IU/mL. Though colony morphology and cell morphology observation, physiological andbiochemical identification and16S rDNA moleclar identification, initially the strain wasidentified as Bacillus subtilis. It was preserved in Microbiological Culture Collection Centerof Shandong Agricultural University, with the number of the deposit for AMCC100051andGenbank accession for JQ267474.2. The culture medium as well as the fermentation conditions of nattokinase fromBacillus subitilis preserved in our laboratory was first optimized. Then three main factorsrelated to the nattokinase yield, tryptone, magnesium sulfate and fermentation temperature,were obtained using Plackett-Burman experiments. Based on these, the Box-Behnken designwas adopted and the response surface results were as follows: tryptone26.6g/L, lactose10g/L, Na₂HPO₄5.0g/L, NaH₂PO₄1.0g/L, CaCl₂0.2g/L, MgSO₄1.35g/L, bacteria age ofinoculation12h, inoculum size2%, fermentation temperature33℃, fermentation duration56h, liquid volume50mL/250mL. The optimization strategy enhanced the nattokinaseactivity1.5times than before.3. Purification and enzyme kinetics of nattokinase from the fermentation broth ofBacillus subtilis were studied. The target protein was purified by30%⁷0%saturationammonium sulfate precipitations and ion-exchange chromatography with Q Sepharose FastFlow. The purified nattokinase showed a single band on SDS-PAGE. The purification factorand activity recovery of the nattokinase were4.4and22.4%, respectively. The optimumtemperature and pH for nattokinase were50℃and9.0, the fibrinolytic activity of nattokinase was stable at below50℃and pH4.0¹2.0, inactivation at70℃and pH3.0. Ca²⁺and Mg²⁺could activate enzymatic activity, Fe²⁺, Zn²⁺, Cu²⁺and Al³⁺could inhibate the activity.