纳豆激酶的深层发酵工艺及分离纯化的研究
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摘要
本论文对从日本纳豆中分离出的纳豆芽孢杆菌(Bacillus，natto)产生纳豆激酶的发酵工艺、分离纯化及酶学性质进行了研究。
利用纳豆芽孢杆菌(Bacillus Natto)所应具有的耐热性、耐盐性、蛋白酶活性及生物素缺陷等生物学特性设计了定向选育方案，从日本传统食品纳豆中筛选出产生纳豆激酶的8株纳豆芽孢杆菌，命名为NK-1～NK-8。其中，菌株NK-4产量最高，且遗传稳定性良好，确定为发酵生产菌株。
采用单因素及正交设计试验法优化出了种子培养基组成、发酵培养基组成及最佳摇瓶分批发酵培养条件；与初始条件的摇瓶发酵相比较，菌株NK-4在优化条件下纳豆激酶的产量(1865.59U／mL)比初始条件下产量(467.18U／mL)提高将近4倍。
采用MATLAB工具软件，利用全局收敛的修正高斯-牛顿算法，以误差平方和最小为目标，获得分批发酵动力学方程待估参数，建立了发酵动力学模型。通过数据比较，证明了得到的数学模型误差较小，能较好地反映纳豆激酶的分批发酵过程。
建立了纳豆激酶的分离提纯工艺，经过硫酸铵分级沉淀、超滤、CM
Sepharose Fast Flow离子交换层析及Sephadex G-75凝胶层析，得到电泳纯的纳豆激酶，纯化后的纳豆激酶比活力达到23251.9U／mg，纯度提高了35.8倍，回收率为59.8％。
通过SDS-PAGE电泳测得纳豆激酶的分子量为28000Da；有直接降解血纤维蛋白和激活血纤维蛋白溶酶原成纤溶酶的双重溶栓作用；纳豆激酶的最适温度为45℃，酶作用温度范围为25℃～50~C，超过55℃酶活迅速下降；纳豆激酶的最适pH为7.0～8.0；pH7稳定性最强，在pH6～8范围内，24h残余酶活不低于80％，而在pH＜6和pH＞8情况下，酶活迅速降低，且在酸性条件下降速更快。金属离子Hg~(2+)、Zn~(2+)及Al~(3+)对酶活性有抑制作用，Ca~(2+)是较好的酶活稳定剂，而Mg~(2+)对酶有激活作用。
The dissertation focuses on isolation of strains, optimization of liquid fermentation condition producing nattokinase by Bacillus natto, purification of nattokinase from liquid fermentation and enzyme characteristics.
Directional isolation on Bacillus natto strains from Japanese natto were conducted according to the Bacterium s properties such as heatproof ability, salt resistance, notable proteinase activities and (V_H)~-. Eight fibrinolytic enzyme producing strains were screened from Japanese natto, which were named NK-1～NK-8. With nattokinase activity as target, the high-procing NK-4 with highly genetic stability was selected by shaking flask liquid cultivation.
The seed medium composition, shaking flask batch fermentation medium compositions and culture conditions were determined through single factor experiment and orthogonal experiment. The maximum nattokinase yield was 1865.59U/mL, which was improved from 467.18U/mL in original fermentation conditions.
The batch fermentation kinetics of nattokinase produced by Bacillus Natto were studied based on the experimental data from fermentation in shaking flask liquid cultivation. The kinetic models of the fermentation were established by MATLAB software.
Purification process of nattokinase was explored and established. The nattokinase was purified using salting out, ultrafiltration, CM Sepharose Fast Flow ion exchange and Sephadex G-75.Based on the protocol 35.8-fold purification of the enzyme was obtained, with a final yield of 59.8 percents.
The molecular weight of the enzyme was 28000Da, determined by SDS-PAGE, on which the isolated enzyme showed a single sharp band. The enzyme hydrolyzed fibrin, and it also activated plasminogen to plasmin. The enzyme had an optimal temperature of 45℃, it is stability in the temperature range from 25 ℃ to 50℃. and it rapidly declined by treating the enzyme at 55℃. The enzyme had an optimal pH of 7～8, kept 80% of the initial activity during pH6～8, out of which fibrinolytic activity reduced quickly. Hg~(2+)、 Zn~(2+)、 Al~(3+)could inhibit the activity, Ca~(2+) and Mg~(2+) could
利用纳豆芽孢杆菌(Bacillus Natto)所应具有的耐热性、耐盐性、蛋白酶活性及生物素缺陷等生物学特性设计了定向选育方案，从日本传统食品纳豆中筛选出产生纳豆激酶的8株纳豆芽孢杆菌，命名为NK-1～NK-8。其中，菌株NK-4产量最高，且遗传稳定性良好，确定为发酵生产菌株。
采用单因素及正交设计试验法优化出了种子培养基组成、发酵培养基组成及最佳摇瓶分批发酵培养条件；与初始条件的摇瓶发酵相比较，菌株NK-4在优化条件下纳豆激酶的产量(1865.59U／mL)比初始条件下产量(467.18U／mL)提高将近4倍。
采用MATLAB工具软件，利用全局收敛的修正高斯-牛顿算法，以误差平方和最小为目标，获得分批发酵动力学方程待估参数，建立了发酵动力学模型。通过数据比较，证明了得到的数学模型误差较小，能较好地反映纳豆激酶的分批发酵过程。
建立了纳豆激酶的分离提纯工艺，经过硫酸铵分级沉淀、超滤、CM
Sepharose Fast Flow离子交换层析及Sephadex G-75凝胶层析，得到电泳纯的纳豆激酶，纯化后的纳豆激酶比活力达到23251.9U／mg，纯度提高了35.8倍，回收率为59.8％。
通过SDS-PAGE电泳测得纳豆激酶的分子量为28000Da；有直接降解血纤维蛋白和激活血纤维蛋白溶酶原成纤溶酶的双重溶栓作用；纳豆激酶的最适温度为45℃，酶作用温度范围为25℃～50~C，超过55℃酶活迅速下降；纳豆激酶的最适pH为7.0～8.0；pH7稳定性最强，在pH6～8范围内，24h残余酶活不低于80％，而在pH＜6和pH＞8情况下，酶活迅速降低，且在酸性条件下降速更快。金属离子Hg~(2+)、Zn~(2+)及Al~(3+)对酶活性有抑制作用，Ca~(2+)是较好的酶活稳定剂，而Mg~(2+)对酶有激活作用。
The dissertation focuses on isolation of strains, optimization of liquid fermentation condition producing nattokinase by Bacillus natto, purification of nattokinase from liquid fermentation and enzyme characteristics.
Directional isolation on Bacillus natto strains from Japanese natto were conducted according to the Bacterium s properties such as heatproof ability, salt resistance, notable proteinase activities and (V_H)~-. Eight fibrinolytic enzyme producing strains were screened from Japanese natto, which were named NK-1～NK-8. With nattokinase activity as target, the high-procing NK-4 with highly genetic stability was selected by shaking flask liquid cultivation.
The seed medium composition, shaking flask batch fermentation medium compositions and culture conditions were determined through single factor experiment and orthogonal experiment. The maximum nattokinase yield was 1865.59U/mL, which was improved from 467.18U/mL in original fermentation conditions.
The batch fermentation kinetics of nattokinase produced by Bacillus Natto were studied based on the experimental data from fermentation in shaking flask liquid cultivation. The kinetic models of the fermentation were established by MATLAB software.
Purification process of nattokinase was explored and established. The nattokinase was purified using salting out, ultrafiltration, CM Sepharose Fast Flow ion exchange and Sephadex G-75.Based on the protocol 35.8-fold purification of the enzyme was obtained, with a final yield of 59.8 percents.
The molecular weight of the enzyme was 28000Da, determined by SDS-PAGE, on which the isolated enzyme showed a single sharp band. The enzyme hydrolyzed fibrin, and it also activated plasminogen to plasmin. The enzyme had an optimal temperature of 45℃, it is stability in the temperature range from 25 ℃ to 50℃. and it rapidly declined by treating the enzyme at 55℃. The enzyme had an optimal pH of 7～8, kept 80% of the initial activity during pH6～8, out of which fibrinolytic activity reduced quickly. Hg~(2+)、 Zn~(2+)、 Al~(3+)could inhibit the activity, Ca~(2+) and Mg~(2+) could
引文
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