摘要
本实验研究目的是:通过对香菇菌种的选取,培养基配方和培养条件的优化,最终得到实验室条件下最高的香菇漆酶活性,然后计算过程中的相关动力学参数。香菇漆酶活性测定底物采用邻联甲苯胺(3,3'-二甲基联苯胺)醋酸-醋酸钠缓冲溶液当酸碱度为4时是最佳反应缓冲体系,漆酶活性的最小变化值为0.2。铜离子对香菇漆酶活性测得值最高可提升50%,亚铁离子可以完全抑制香菇漆酶活性。对最初六种香菇菌种的初步培养表明,在完全不优化的情况下,香菇漆酶的活性在40 U/ml到100 U/ml之间,最佳菌种的漆酶活性约为120U/ml,选取香菇菌种A和E进一步优化,振荡培养条件提前了香菇漆酶活性最高值的出现时间但不利于香菇菌丝体产酶,250ml培养体系效果明显好于500ml培养体系。利用八种简单液体培养基对香菇菌种A、E(重新命名为B)和华香8号进行培养,最后选择香菇菌种B为接下来的实验菌种,并以培养基配方葡萄糖、蛋白胨、酵母粉、牛肉浸膏加微量元素为基础培养基进一步优化。培养基的均匀设计优化实验得到的最佳培养基配方为:葡萄糖22.57克,牛肉浸膏2.8克,酵母粉3.0克,蛋白胨5.12克,微量元素1.17ml。最高漆酶活性增加到180 U/ml左右。培养基中铜离子浓度在19mg/l到20mg/l之间时最适合香菇菌丝体产酶,利用数学方法除去了铜离子对测定结果的影响,诱导效应使香菇漆酶活性提高到270 U/ml。运用响应面分析优化法选择了培养基起始酸碱度、培养温度和培养天数三因素继续优化并最终利用分析图确立了各自的最优值为初始酸碱度为3.6左右,过程中香菇菌丝体的培养温度为26.1℃左右,香菇菌丝体培养到46到47天。通过所以的优化部分后,测得的香菇漆酶活性最高值为362.6(U/ml),比最初平均值大约提高了四倍。动力学部分计算了香菇漆酶反应动力学、香菇生长和产酶动力学的相关参数。
The aim of this experiment is by selecting different kinds of Lentinula edodes strain, optimizing the fermentation formula and fermentation process, and finally obtaining the highest activity value of Lentinula edodes Laccase, then calculating the kinetic parameters during the process. The o-tolidine(3,3'-dimethylbenzidine) is used as substrate to determine the optimal pH values in varies kind of conditions resulted in the acetic acid-natrium aceticum and its pH value 4 as the optimal buffer solution. The minimum changeable value of Laccase is 0.2. The Laccase activity was enhanced by about 50% when the copper ion concentration was between 19 and 20 mg/L and ferrous ion could entirely inhibit the activity of Laccase.First cultivation of six kinds of Lentinula edodes indicate, without any kind of optimization, the average activity value of Laccase is between 40 U/ml and 100 U/ml, the highest is about 120 U/ml. Strain A and E is selected to enter next step. Shaking culture condition can advance the appearing time of highest activity value of Lentinula edodes Laccase but ultimately, it reduce the Laccase activity value, and 500ml cultivation system reduce the Laccase activity value by about 50% compared to 250ml cultivation system. Eight basic liquid cultures are used to cultivate Lentinula edodes strain A, E (renamed B) and Huaxiang 8 resulted in strain B is the best candidate. In the process to find out the best fermentation formula by using homogeneous design resulted in glucose 22.57 g, beef extract 2.8 g, yeast powder 3.0g, peptone 5.12 g, microelement 1.17ml is the best formula to culture Lentinula edodes strain. The Laccase activity value increase to 180 U/ml. Copper ions as an inducer increase the value to 270 U/ml. Response surface method indicate that the pH 3.6, cultivation temperature 26.1℃and cultivation process time 46 are the best conditions for fermentation. 362.6(U/ml) is the highest Laccase activity value in the whole experiment, it is about 4 fold compared to original average values. In the dynamic part lots of kinetic parameters are calculated which are related to the whole optimization process.
引文
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