利用分枝杆菌降解植物甾醇生产雄甾烯酮的研究
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摘要
本实验利用分枝杆菌M1与M2切除植物甾醇C_(17)位边链，制备ADD与AD，主要研究内容包括以下几个方面：
1．分析测定方法的建立：薄层层析法，以乙酸乙酯:石油醚(6:4)作为展层剂，以硅胶作为固定相，主要用于定性分析底物与产物；紫外吸光光度法，采用双波长(254nm与298nm)检测，主要用于快速定量分析ADD与AD；根据高效液相色谱与薄层层析相同的工作原理，采用硅胶柱，在薄层层析分析法基础上建立了高效液相色谱法，同时建立了两种定量测定方法：标准曲线法与内标法，高效液相色谱法主要用于准确定量测定产物ADD与AD；气相色谱法，采用FID检测器和大口径毛细管柱，主要用于分析底物甾醇，根据FID的工作原理，确定了各物质的相对校正因子，气相色谱法可用于定量分析底物的四种成份和产物AD(D)在发酵液中的相对含量。
2．生产菌种的性质：实验中发现菌体在培养过程中随着营养物质的消耗，细胞形态发生有规律的变化，从长杆状逐渐变为短杆状，最后变为球形；这种形态分化与菌体降解甾醇的活性有一定相关性：当菌体为长杆状时转化活性很高；当菌体变为短杆状时转化活性明显下降；菌体变为球形时转化活力基本消失。两株实验菌M1与M2在菌体形态上没有区别，但在生产能力上有较大差别，根据菌株对底物转化能力的大小，选择M2作为主要研究对象。
3．工艺参数的确定：详细研究了一级转化法与二级转化法两种工艺。一级转化法中：斜面种子用MYC/S2培养基29℃恒温培养60h，冷冻保藏不超过20d，转接发酵培养基MYC/02的接种量为7％；二级转化法中：液体种子的接种种龄为35h～40h，以7％的接种量接种发酵培养基MYC/02，并优化了发酵培养基的培养条件：pH=7.0、温度29℃、保证充足的溶氧。
4．投料的方式：通过对乙醇法、Tween80法、环糊精法及超声波法等的试验，确定以Tween80乳化底物的投料方式，即用0.5％～1.0％的Tween80悬浮甾醇，在120℃高温中乳化，然后用于配制发酵培养基MYC/02。
5．5L发酵罐转化工艺优化：在摇瓶实验的基础上，进行了5L罐扩大培养实验，在培养过程中保持转化温度为29℃，流加10％NaOH与2M盐酸控制pH为7.0，通过调节空气流量与搅拌转速保持充足的供氧。5L罐转化结果(转化168h)：①投料量为0.3％，底物转化率为100％，AD(D)产率为90％以上；②投料量为0.5％，底物转化率为95％左右，AD(D)产率为85％左右；③投料量为1.0％，底物转化率为50％～60％，产物生成率在50％左右。
In this experiment, phytosterols were cleaved successfully into ADD and AD through cleaving the C17side chain of sterols by Mycobacterium Ml and M2. The mainly researching contents are blow:
1. The methods of analysis and determination: The method of Layer Chromatography which employed silica gel and Ether-ethyl acetate-Petroleum
(6 : 4) was mainly used to qualitatively analyze the substrates and the products. The Ultraviolet Absorption Spectrometry which worked at two kinds of wave length (254nm and 298nm) was mainly used to quantitatively analyze ADD and AD. According to the similar work theory of the Layer Chromatography and the High Performance Liquid Chromatography, the method of High Performance Liquid Chromatography with silica gel column was established. Through the methods of Calibration curve and Internal standard, the products ADD and AD could be quantitatively determined. Using FID detector and large calibre capillary column, the Gas Chromatography could be used to analyze the four kinds of phytosterols in the substrate. And according to the work theory of FID detector, the relative emendation factors were determined. So the Gas Chromatography could be used to analyze the sterols and AD(D) in the fermentation sample.
2. The quality of strains used in production: During the consuming of nutrition, the cell morph changed regularly from long bacilli to short bacilli and last to cocci. The strain capacity of cleaving sterols had some relationship to the cell morph. The transformation capacity was high when the cell was long bacilli, but the transformation capacity became low when the cell became to short bacilli, and last when the cell morph turn to cocci, the transformation disappeared. There were no difference in cell morph between Mycobacterium Ml and M2, but their production capability was different. According to their transformation capability, strain M2 was selected as the mainly researching object.
3. The parameters of processes: In this experiment one step process and two steps process were studied in detail. And the parameters of processes were determined, for instance: in one step process, the slant culture should be cultured in culture medium MYC/S2 under 29℃ and its preservative time should be no more than 20 days, the inoculation quantum to MYC/02 should be 7%, and in the two steps process, the liquid culture should be added to the fermentation medium in the period of 35h-40h by 7% of MYC/02. The optimal culture conditions of MYC/02 were also determined, for instance : pH=7.0, culture temperature was 29℃ and
the dissolved oxygen should be ample.
4. The methods of adding substrate : Among the methods of adding alcohol, cyclodextrin, Tween80 and ultrasonic, employing 0.3%-1% Tween80 to disperse substrate is the best method, In this method phytosterols were suspended in 0.5%-1.0% Tween80, and the suspending liquid was emulsified in 120℃, then it was used to prepare fermentation culture medium MYC/02.
5. The optimization of transformation process in 5L fermentation reactor : Based on the experiments of flask, the operation parameters of 5L fermentation reactor were established. During the course of cultivation, the temperature was controlled in 29℃, the pH=7 was adjusted through adding 10% NaOH and 2M hydrochloric acid, and the abundant oxygen could be acquired through adjusting the air flux and the stir rotate speed. The transformation effects (transformation 168h) of 5L fermentation reactor were below:
①When the adding rate was 0.3%, the substrate transformation rate was 100%, and AD(D) yield rate was above 90%. ②When the adding rate was 0.5%, the substrate transformation rate was about 95%, and AD(D) yield rate was about 85%. ③When the adding rate was 1.0%, the substrate transformation rate was 50%~60%, and AD(D) yield rate was about 50%.
1．分析测定方法的建立：薄层层析法，以乙酸乙酯:石油醚(6:4)作为展层剂，以硅胶作为固定相，主要用于定性分析底物与产物；紫外吸光光度法，采用双波长(254nm与298nm)检测，主要用于快速定量分析ADD与AD；根据高效液相色谱与薄层层析相同的工作原理，采用硅胶柱，在薄层层析分析法基础上建立了高效液相色谱法，同时建立了两种定量测定方法：标准曲线法与内标法，高效液相色谱法主要用于准确定量测定产物ADD与AD；气相色谱法，采用FID检测器和大口径毛细管柱，主要用于分析底物甾醇，根据FID的工作原理，确定了各物质的相对校正因子，气相色谱法可用于定量分析底物的四种成份和产物AD(D)在发酵液中的相对含量。
2．生产菌种的性质：实验中发现菌体在培养过程中随着营养物质的消耗，细胞形态发生有规律的变化，从长杆状逐渐变为短杆状，最后变为球形；这种形态分化与菌体降解甾醇的活性有一定相关性：当菌体为长杆状时转化活性很高；当菌体变为短杆状时转化活性明显下降；菌体变为球形时转化活力基本消失。两株实验菌M1与M2在菌体形态上没有区别，但在生产能力上有较大差别，根据菌株对底物转化能力的大小，选择M2作为主要研究对象。
3．工艺参数的确定：详细研究了一级转化法与二级转化法两种工艺。一级转化法中：斜面种子用MYC/S2培养基29℃恒温培养60h，冷冻保藏不超过20d，转接发酵培养基MYC/02的接种量为7％；二级转化法中：液体种子的接种种龄为35h～40h，以7％的接种量接种发酵培养基MYC/02，并优化了发酵培养基的培养条件：pH=7.0、温度29℃、保证充足的溶氧。
4．投料的方式：通过对乙醇法、Tween80法、环糊精法及超声波法等的试验，确定以Tween80乳化底物的投料方式，即用0.5％～1.0％的Tween80悬浮甾醇，在120℃高温中乳化，然后用于配制发酵培养基MYC/02。
5．5L发酵罐转化工艺优化：在摇瓶实验的基础上，进行了5L罐扩大培养实验，在培养过程中保持转化温度为29℃，流加10％NaOH与2M盐酸控制pH为7.0，通过调节空气流量与搅拌转速保持充足的供氧。5L罐转化结果(转化168h)：①投料量为0.3％，底物转化率为100％，AD(D)产率为90％以上；②投料量为0.5％，底物转化率为95％左右，AD(D)产率为85％左右；③投料量为1.0％，底物转化率为50％～60％，产物生成率在50％左右。
In this experiment, phytosterols were cleaved successfully into ADD and AD through cleaving the C17side chain of sterols by Mycobacterium Ml and M2. The mainly researching contents are blow:
1. The methods of analysis and determination: The method of Layer Chromatography which employed silica gel and Ether-ethyl acetate-Petroleum
(6 : 4) was mainly used to qualitatively analyze the substrates and the products. The Ultraviolet Absorption Spectrometry which worked at two kinds of wave length (254nm and 298nm) was mainly used to quantitatively analyze ADD and AD. According to the similar work theory of the Layer Chromatography and the High Performance Liquid Chromatography, the method of High Performance Liquid Chromatography with silica gel column was established. Through the methods of Calibration curve and Internal standard, the products ADD and AD could be quantitatively determined. Using FID detector and large calibre capillary column, the Gas Chromatography could be used to analyze the four kinds of phytosterols in the substrate. And according to the work theory of FID detector, the relative emendation factors were determined. So the Gas Chromatography could be used to analyze the sterols and AD(D) in the fermentation sample.
2. The quality of strains used in production: During the consuming of nutrition, the cell morph changed regularly from long bacilli to short bacilli and last to cocci. The strain capacity of cleaving sterols had some relationship to the cell morph. The transformation capacity was high when the cell was long bacilli, but the transformation capacity became low when the cell became to short bacilli, and last when the cell morph turn to cocci, the transformation disappeared. There were no difference in cell morph between Mycobacterium Ml and M2, but their production capability was different. According to their transformation capability, strain M2 was selected as the mainly researching object.
3. The parameters of processes: In this experiment one step process and two steps process were studied in detail. And the parameters of processes were determined, for instance: in one step process, the slant culture should be cultured in culture medium MYC/S2 under 29℃ and its preservative time should be no more than 20 days, the inoculation quantum to MYC/02 should be 7%, and in the two steps process, the liquid culture should be added to the fermentation medium in the period of 35h-40h by 7% of MYC/02. The optimal culture conditions of MYC/02 were also determined, for instance : pH=7.0, culture temperature was 29℃ and
the dissolved oxygen should be ample.
4. The methods of adding substrate : Among the methods of adding alcohol, cyclodextrin, Tween80 and ultrasonic, employing 0.3%-1% Tween80 to disperse substrate is the best method, In this method phytosterols were suspended in 0.5%-1.0% Tween80, and the suspending liquid was emulsified in 120℃, then it was used to prepare fermentation culture medium MYC/02.
5. The optimization of transformation process in 5L fermentation reactor : Based on the experiments of flask, the operation parameters of 5L fermentation reactor were established. During the course of cultivation, the temperature was controlled in 29℃, the pH=7 was adjusted through adding 10% NaOH and 2M hydrochloric acid, and the abundant oxygen could be acquired through adjusting the air flux and the stir rotate speed. The transformation effects (transformation 168h) of 5L fermentation reactor were below:
①When the adding rate was 0.3%, the substrate transformation rate was 100%, and AD(D) yield rate was above 90%. ②When the adding rate was 0.5%, the substrate transformation rate was about 95%, and AD(D) yield rate was about 85%. ③When the adding rate was 1.0%, the substrate transformation rate was 50%~60%, and AD(D) yield rate was about 50%.
引文
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