L-脯氨酸发酵条件的优化
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摘要
L-脯氨酸是含有亚氨基的中性氨基酸,属构成蛋白质的20种基本氨基酸之一,为人体非必需氨基酸。L-脯氨酸具有一定的生理活性,在医药、农业、化工及食品等方面有着广泛的用途,尤其在医药上是许多新药合成的中间体。随着其研究的不断深入,应用范围也将进一步扩大。
本论文重点研究了L-脯氨酸摇瓶和5L发酵罐发酵条件的优化。
(1)应用“薄层层析法”和“酸性茚三酮比色法”对发酵液中L-脯氨酸进行了定性和定量分析研究。确定了L-脯氨酸的定量测定条件和计算方法。
(2)研究了菌株AP117的摇瓶分批发酵最优条件。应用正交设计试验法优化出种子培养基,并研究了种子培养条件,最佳种子培养基配方为(%):蔗糖3.0;硫酸铵4.0;L-异亮氨酸300mg/L;生物素500μg/L;硫胺素500μg/L;MgSO_4·7H_2O 1.0;KH_2PO_4 0.1;CaCO_3 3.0;ZnSO_4·7H_2O 0.001; FeSO_4·7H_2O 0.001;MnSO_4·5H_2O 0.001。种子的最佳培养条件:培养基初始pH7.0-7.2;装液量为30mL/500mL三角瓶;种龄为16-18h。
(3)在发酵培养基单因素影响试验研究的基础上,应用正交设计试验法优化出摇瓶分批发酵培养基的最佳组成,并研究了发酵培养条件,摇瓶分批发酵培养基的最佳组成为(%):蔗糖16.0;乙酸铵4.0;L-异亮氨酸300mg/L;生物素400μg/L;硫胺素600μg/L;MgSO_4·7H_2O 2.0;KH_2PO_4 0.2;CaCO_3 6.0;ZnSO_4·7H_2O 0.001;FeSO_4·7H_2O 0.001;MnSO_4·5H_2O 0.001;最佳摇瓶分批发酵培养条件:发酵培养基初始pH8.0;装液量为30mL/500mL三角瓶;摇瓶转速为240r/min;发酵周期为72h。在优化条件下,菌株AP117摇瓶分批发酵72h,产L-脯氨酸52.17g/L。
(4)以摇瓶分批发酵最优条件为基础,对菌株AP117进行了5L发酵罐分批发酵试验。试验结果表明,菌株AP117在优化条件下的L-脯氨酸产量为55.08g/L。
(5)以摇瓶分批发酵优化条件为基础,对菌株AP117进行了摇瓶补料分批发酵研究。摇瓶补料分批发酵的初糖浓度为80g/L,于发酵过程第24h,36h,48h,60h分四次补加补料液,发酵72h产L-脯氨酸55.28g/L。由于采用补糖补铵工艺,发酵72h产L-脯氨酸55.38g/L,比摇瓶分批发酵提高了6.15%,比初始条件提高了49.51%。
L-proline is a neutral amino acid with imono. It is one of 20 types of essential amino acids which constitute the protein. It is a non-essential amino acid for people. L-proline has certain physiological activities. It is widely used in medical, agricultural, chemical industrial and food areas, especially in medical area since it is the intermediate of synthesis for many new medicines. Along with the study of it, the application area will enlarge.
The dissertation focuses on the shake-flask culture conditions and 5-Liter fermentor of L–proline fermentation.
(1) L-proline concentration in fermented broth was qualitative determined with methods of thin layer chromatography and quantitative determined with methods of acidity triketohydrindene hydrate colorimetry. The quantitative determination condition and computing method were obtained.
(2) The batch fermentation condition of strain AP117 in shake flask was studied in this dissertation. The seed medium was optimized by orthogonal experiment, and the seed culture conditions were also studied. The optimization seed conditions for Brevibacterium flavum AP117 as follows (%): sucrose 3.0; (NH4)2SO_4 4.0; L-Ile 300mg/L; biotin 500μg/L; thiamin 500μg/L; MgSO_4·7H_2O 1.0; KH_2PO_4 0.1; CaCO_3 3.0; ZnSO_4·7H_2O 0.001; FeSO_4·7H_2O 0.001; MnSO_4·5H_2O 0.001.
(3) Based on the single factor experiment of fermentation medium, the fermentation medium was optimized by orthogonal experiment, and the fermentation condition was studied. The optimization fermentation conditions for Brevibacterium flavum AP117 are as follows (%): sucrose 16.0; ammonium acetate 4.0; L- Ile 300mg/L; biotin 400μg/L; thiamin 600μg/L; MgSO_4·7H_2O 2.0; KH_2PO_4 0.2; CaCO_3 6.0; ZnSO_4·7H_2O 0.001; FeSO_4·7H_2O 0.001; MnSO_4·5H_2O 0.001; and the optimal culture conditions such as medium initial pH is 7.0-7.2, 30mL medium in 500mL shake-flask, 16-18h for seed growth. Under the optimum condition, the strain AP117 could produce L-proline 52.17g/L after fermentation for 72 hours by flask-shaking batch fermentation.
(4) Results of 5-Liter fermentor experiment show that the strain AP117 could produce L-proline 55.08g/L after fermentation for 72 hours, and the optimal culture conditions such as medium initial pH, amount of inoculation, rotation rate are 8.0, 10%, 240r/min, respectively.
(5) Fed batch fermentation was studied with AP117 according to the optimum condition of batch fermentation. The optimum initial sucrose concentration was 80g/L in shake flask fed batch fermentation. The intermittent feeding was performed at the 24, 36, 48, 60 hour respectively. The production of L-proline reached 55.28g/L after fermentation for 72 hours. Because of the technology of low glucose, low amino and fed batch, production of L-proline reached 55.38g/L after fermentation for 72 hours. The production was improved by 6.15% than that of fermentation in shake flask fed batch. The production was improved by 49.51% than that of initial condition.
本论文重点研究了L-脯氨酸摇瓶和5L发酵罐发酵条件的优化。
(1)应用“薄层层析法”和“酸性茚三酮比色法”对发酵液中L-脯氨酸进行了定性和定量分析研究。确定了L-脯氨酸的定量测定条件和计算方法。
(2)研究了菌株AP117的摇瓶分批发酵最优条件。应用正交设计试验法优化出种子培养基,并研究了种子培养条件,最佳种子培养基配方为(%):蔗糖3.0;硫酸铵4.0;L-异亮氨酸300mg/L;生物素500μg/L;硫胺素500μg/L;MgSO_4·7H_2O 1.0;KH_2PO_4 0.1;CaCO_3 3.0;ZnSO_4·7H_2O 0.001; FeSO_4·7H_2O 0.001;MnSO_4·5H_2O 0.001。种子的最佳培养条件:培养基初始pH7.0-7.2;装液量为30mL/500mL三角瓶;种龄为16-18h。
(3)在发酵培养基单因素影响试验研究的基础上,应用正交设计试验法优化出摇瓶分批发酵培养基的最佳组成,并研究了发酵培养条件,摇瓶分批发酵培养基的最佳组成为(%):蔗糖16.0;乙酸铵4.0;L-异亮氨酸300mg/L;生物素400μg/L;硫胺素600μg/L;MgSO_4·7H_2O 2.0;KH_2PO_4 0.2;CaCO_3 6.0;ZnSO_4·7H_2O 0.001;FeSO_4·7H_2O 0.001;MnSO_4·5H_2O 0.001;最佳摇瓶分批发酵培养条件:发酵培养基初始pH8.0;装液量为30mL/500mL三角瓶;摇瓶转速为240r/min;发酵周期为72h。在优化条件下,菌株AP117摇瓶分批发酵72h,产L-脯氨酸52.17g/L。
(4)以摇瓶分批发酵最优条件为基础,对菌株AP117进行了5L发酵罐分批发酵试验。试验结果表明,菌株AP117在优化条件下的L-脯氨酸产量为55.08g/L。
(5)以摇瓶分批发酵优化条件为基础,对菌株AP117进行了摇瓶补料分批发酵研究。摇瓶补料分批发酵的初糖浓度为80g/L,于发酵过程第24h,36h,48h,60h分四次补加补料液,发酵72h产L-脯氨酸55.28g/L。由于采用补糖补铵工艺,发酵72h产L-脯氨酸55.38g/L,比摇瓶分批发酵提高了6.15%,比初始条件提高了49.51%。
L-proline is a neutral amino acid with imono. It is one of 20 types of essential amino acids which constitute the protein. It is a non-essential amino acid for people. L-proline has certain physiological activities. It is widely used in medical, agricultural, chemical industrial and food areas, especially in medical area since it is the intermediate of synthesis for many new medicines. Along with the study of it, the application area will enlarge.
The dissertation focuses on the shake-flask culture conditions and 5-Liter fermentor of L–proline fermentation.
(1) L-proline concentration in fermented broth was qualitative determined with methods of thin layer chromatography and quantitative determined with methods of acidity triketohydrindene hydrate colorimetry. The quantitative determination condition and computing method were obtained.
(2) The batch fermentation condition of strain AP117 in shake flask was studied in this dissertation. The seed medium was optimized by orthogonal experiment, and the seed culture conditions were also studied. The optimization seed conditions for Brevibacterium flavum AP117 as follows (%): sucrose 3.0; (NH4)2SO_4 4.0; L-Ile 300mg/L; biotin 500μg/L; thiamin 500μg/L; MgSO_4·7H_2O 1.0; KH_2PO_4 0.1; CaCO_3 3.0; ZnSO_4·7H_2O 0.001; FeSO_4·7H_2O 0.001; MnSO_4·5H_2O 0.001.
(3) Based on the single factor experiment of fermentation medium, the fermentation medium was optimized by orthogonal experiment, and the fermentation condition was studied. The optimization fermentation conditions for Brevibacterium flavum AP117 are as follows (%): sucrose 16.0; ammonium acetate 4.0; L- Ile 300mg/L; biotin 400μg/L; thiamin 600μg/L; MgSO_4·7H_2O 2.0; KH_2PO_4 0.2; CaCO_3 6.0; ZnSO_4·7H_2O 0.001; FeSO_4·7H_2O 0.001; MnSO_4·5H_2O 0.001; and the optimal culture conditions such as medium initial pH is 7.0-7.2, 30mL medium in 500mL shake-flask, 16-18h for seed growth. Under the optimum condition, the strain AP117 could produce L-proline 52.17g/L after fermentation for 72 hours by flask-shaking batch fermentation.
(4) Results of 5-Liter fermentor experiment show that the strain AP117 could produce L-proline 55.08g/L after fermentation for 72 hours, and the optimal culture conditions such as medium initial pH, amount of inoculation, rotation rate are 8.0, 10%, 240r/min, respectively.
(5) Fed batch fermentation was studied with AP117 according to the optimum condition of batch fermentation. The optimum initial sucrose concentration was 80g/L in shake flask fed batch fermentation. The intermittent feeding was performed at the 24, 36, 48, 60 hour respectively. The production of L-proline reached 55.28g/L after fermentation for 72 hours. Because of the technology of low glucose, low amino and fed batch, production of L-proline reached 55.38g/L after fermentation for 72 hours. The production was improved by 6.15% than that of fermentation in shake flask fed batch. The production was improved by 49.51% than that of initial condition.
引文
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