【作者】 李颖；

【导师】 陆兆新；

【作者基本信息】 南京农业大学 ， 发酵工程， 2007， 硕士

【摘要】 从降低烟草行业成本和减少环境污染的角度出发，降解上部烟叶和烟草废弃物中的烟碱已成为烟草行业的一个研究热点。本论文分离和纯化了一株新型的烟碱降解细菌中间苍白杆菌（Ochrobacterum intermedium）DN2所产的烟碱降解酶，对该酶的酶学性质进行了研究，并通过优化培养基和培养条件提高了酶产量。此外，还对烟碱降解酶在烟草薄片液中的应用进行了初步研究。论文的主要内容和结论如下：烟碱降解酶粗酶液经硫酸铵沉淀，凝胶过滤层析和离子交换柱层析分离纯化，得到了电泳纯的烟碱降解酶。分离纯化过程中酶被提纯了35.24倍，酶活回收率为6.4％。烟碱降解酶的分子量为40.06kDa。其最适反应pH为6.0，在pH5.0-8.0范围内都很稳定。烟碱脱氢酶的最适反应温度为55℃，50℃下酶活力较稳定，保温2.5h后仍有80％以上酶活，在温度超过60℃后酶活下降较快。EDTA和Na₂MoO₄对酶活有一定的促进作用，CoCl₂、MnCl₂对酶活抑制作用较显著。酶的Km为0.13mmol／L。采用Plackett-Burman设计法，对影响O．intermedium DN2产烟碱降解酶的20个因素进行了筛选。结果表明：影响该菌发酵产酶的显著因子为：烟碱、酵母膏、葡萄糖、Tween-80、初始pH、接种量、装液量和培养时间。在此基础上，采用响应曲面法分别对影响该菌发酵产酶的培养基组成和培养条件进行了优化。得到上述各显著因子的最佳水平为：烟碱2.183g／L、葡萄糖0.823g／L、酵母膏0.844g／L、Tween-80 0.976g／L、初始pH6.9、接种量8.6％、装液量413ml／1L三角瓶、培养时间32h。在此优化条件下得到实际酶活为9412U／L，与预测值9625U／L相近。实验确定利用烟碱降解酶降解烟草薄片液中的烟碱的最适反应条件为：烟碱含量1.21g／L，粗酶含量6％，pH7.0，45℃下反应，添加EDTA至终浓度为1mM／L。在此最适条件下，12h的烟碱降解率达到88％，薄片液中的烟碱浓度从最初的1.21g／L降至0.15g／L，从而大大提高了薄片液的可用性。更多还原

【Abstract】 Nicotine-degradation enzyme has attracted considerable research interest in recent yearsbecause of its potential application in the tobacco industry.In the paper, nicotine-degradation enzyme was purified by ammonium sulfateprecipitation, gel filtration and ion exchange chromatography. PAGE electrophoresis of thepurified fraction mitigated a simple band which revealed this enzyme was purified .Themolecular weight of the enzyme was 40.06KDa by SDS-PAGE. The optimal temperatureand pH of the enzyme was 55℃and pH6.0, respectively. This enzyme was stable atpH5.0-8.0 below 50℃. Kinetic studies demonstrated that it was an enzyme with Km of0.13mmol/L. EDTA and Mo⁶⁺ Can activate the enzyme although Mn²⁺ and Co²⁺ inhibited it.Plackett-Burman design was used to evaluate the importance of the selected twentyinternal and external factors on the activity of nicotine-degradation enzyme of DN2. Inaddition, the central composite design and response surface analysis were used to determinethe optimal levels of the main facters. The optimal levels of the main facters were asfollows: nicotine 2.183g/L、glucose 0.823g/L、Yeast extract 0.844g/L、Tween-80 0.976g/L、initial pH6.9、inoculum amount 8.6%、volume of medium 413ml/1L erlenmeyer、incubation time 32h. Under this condition, enzyme activity can reach to 9412U/L which isclose to the predicated value 9625U/L.The optimal condition for the enzyme to degradate nicotine in reconstituted tobaccoliquid was: nicotine 1.21g/L, enzyme 6%, EDTA 1mM/L, pH7.0, 45℃. Under thiscondition nicotine degradation ratio can reach to 88% after 12h, and the final nicotineconcentration got down to 0.15g/L from 1.21g/L.更多还原