| Summary: | 碩士 === 國立臺灣海洋大學 === 食品科學系 === 98 === Nattokinase (NK), from a fermented soybean product by Bacillus subtilis natto, is a strong fibrinolytic enzyme. Porphyra dentate is abundant in carbohydrates and protein. Optimal conditions for the production of nattokinase from Porphyra dentate by shaking flask method has been evaluated previously. In this study, factors of aeration and pH control in a ten-liter fermentor for nattokinase production was further evaluated. Then nattokinase from fermented Porphyra dentate substrate by B. subtilis natto was purified and characterized. The NK activity was increased with increasing of aeration, with maximal of 986.83 U/mL at 2 vvm. A homogeneous nattokinase was obtained after DEAE-Sepharose FF anion exchange and Sephadex G-50 gel filtration chromatography. The NK specific activity was 269.44 U/mg with purification of 5.73-fold. The molecular mass and pI of the enzyme were 46.54 kDa and pH 8.35. It’s kinetic parameters of Km and Vmax were 0.044 mg/mL and 9.5 mM/min, respectively. The optimal temperature and pH of nattokinase were 55 oC and pI 8.0, respectively. The residual activity of nattokinase at 55 oC for 1 hr was 80.33 %. The enzyme activities remained over 89.22% at pH 5-9 for 1 hour. The enzyme activity was increased in the presence of 10 mM Cu2+, Zn2+ (136 and 123 %) and 2 % Triton X-100 (122 %). Besides of fibrinogen, NK also hydrolyzed fibrin, hemoglobin and gelatin. PMSF, leupeptin and EDTA greatly inhibited the activity of nattokinase. These results indicated that the purified enzyme is a serine-like proteas.
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