氨苷霉素的菌种选育和发酵工艺的改进
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摘要
新型核苷类农用抗生素氨苷霉素(Anganmycin)对各种植物的粉状霉菌有强烈的拮抗作用，并具有很低的毒性。本文对氨苷霉素生产菌的选育及发酵生产进行了较系统的研究，其中包括氨苷霉素测定方法、诱变选育、发酵工艺和发酵放大和相关的动力学研究。
1．建立了通过坂口反应初步测定氨苷霉素的化学方法，以红酵母为敏感菌的生物快速测定法，以及高效液相色谱的精确测定法。
2．氨苷霉素高产菌的选育和发酵工艺优化。利用物理化学诱变，根据氨苷霉素生物合成途径选用代谢类似物抗性筛选，进行氨苷霉素高产菌的选育。最终筛选到高产菌D-62-2，其在较优化工艺条件下氨苷霉素的摇瓶发酵产量达1.5g／L，比初始菌提高4倍。
3．氨苷霉素的小型发酵罐试验。利用高产菌在国产10L发酵罐上进行分批补料发酵、葡萄糖流加发酵和淀粉补磷发酵等试验，并与进口B.Braun罐上间歇发酵相比较。试验结果，发酵罐氨苷霉素水平由原始菌(ZD615)0.38g／L的产量提高到0.8g／，但发酵水平只为摇瓶最高产量的53％。
4．对于氨苷霉素间歇发酵进行初步的动力学研究，根据实验测定数据以及假设模型，采用Matlab进行拟合，得到菌体生长、底物消耗、产物生成的动力学方程：。
5．初步进行氨苷霉素的中试放大研究，在250L、700L和2500L通气搅拌发酵罐中共进行了6批试验，最高氨苷霉素的发酵水平达到0.628g／L。并对放大工艺成在的问题进行探讨。
Anganmycin, a new nucleoside antibiotic, showed remarkable biological activity against powdery mildews of many plants with low toxicity. A systematic investigation was carried out to improve the productivity of one anganmycin-producing strain with mutation breeding and bioprocess optimization. Also, different anganmycin determination methods, primary fermentation kinetic and scaling up of fermentation process were included.
(1) The chemical method for the determination of anganmycin was developed with the aid of SAKAGUCHI reaction. The rapid determination method was built with red yeast as the test mico-organism. Further studies showed that HPLC was the best method to determinate the anganmycin precisely.
(2) Mutant breeding and optimization of fermentation bioprocess. According to the suggested biosynthesis pathway of anganmycin, high producing mutants were selected with the anti-metabolism activity to improve the synthesis of anganmycin after physical and chemical mutation. One highest producer, D-62-2, was obtained which could produce 1.5g/L anganmycin, about four times higher than that of the origin strain ZD615.
(3) Anganmycin fermentation in 10L and 15 liter bench-top fermentors. Three different types of batch and fed-batch fermentation were carried out in the 10 liter domestic fermentor, and further test was performed in 15 liter B.Braun fermentor. With the optimization of these fermentation bioprocess, the productivity of anganmycin was improved from 0.38g/L to 0.8g/L, which only showed 53% of the productivity in the shaking flask.
(4) Kinetic study of anganmycin fermentation. According to the experimental data and the supposed model, the kinetic equations including the growth of mycelium, the consumption of substrate and the production of anganmycin were obtained with the use of Matlab software:. These equations are as
(5) The anganmycin fermentation process was scaled up in a pharmaceutic factory. Six runs of experiments was performed on 250L, 700L and 2500L fermentor respectively. The highest productivity of anganmycin (0.628g/L) was achieved , and some important problem was also discussed here.
1．建立了通过坂口反应初步测定氨苷霉素的化学方法，以红酵母为敏感菌的生物快速测定法，以及高效液相色谱的精确测定法。
2．氨苷霉素高产菌的选育和发酵工艺优化。利用物理化学诱变，根据氨苷霉素生物合成途径选用代谢类似物抗性筛选，进行氨苷霉素高产菌的选育。最终筛选到高产菌D-62-2，其在较优化工艺条件下氨苷霉素的摇瓶发酵产量达1.5g／L，比初始菌提高4倍。
3．氨苷霉素的小型发酵罐试验。利用高产菌在国产10L发酵罐上进行分批补料发酵、葡萄糖流加发酵和淀粉补磷发酵等试验，并与进口B.Braun罐上间歇发酵相比较。试验结果，发酵罐氨苷霉素水平由原始菌(ZD615)0.38g／L的产量提高到0.8g／，但发酵水平只为摇瓶最高产量的53％。
4．对于氨苷霉素间歇发酵进行初步的动力学研究，根据实验测定数据以及假设模型，采用Matlab进行拟合，得到菌体生长、底物消耗、产物生成的动力学方程：。
5．初步进行氨苷霉素的中试放大研究，在250L、700L和2500L通气搅拌发酵罐中共进行了6批试验，最高氨苷霉素的发酵水平达到0.628g／L。并对放大工艺成在的问题进行探讨。
Anganmycin, a new nucleoside antibiotic, showed remarkable biological activity against powdery mildews of many plants with low toxicity. A systematic investigation was carried out to improve the productivity of one anganmycin-producing strain with mutation breeding and bioprocess optimization. Also, different anganmycin determination methods, primary fermentation kinetic and scaling up of fermentation process were included.
(1) The chemical method for the determination of anganmycin was developed with the aid of SAKAGUCHI reaction. The rapid determination method was built with red yeast as the test mico-organism. Further studies showed that HPLC was the best method to determinate the anganmycin precisely.
(2) Mutant breeding and optimization of fermentation bioprocess. According to the suggested biosynthesis pathway of anganmycin, high producing mutants were selected with the anti-metabolism activity to improve the synthesis of anganmycin after physical and chemical mutation. One highest producer, D-62-2, was obtained which could produce 1.5g/L anganmycin, about four times higher than that of the origin strain ZD615.
(3) Anganmycin fermentation in 10L and 15 liter bench-top fermentors. Three different types of batch and fed-batch fermentation were carried out in the 10 liter domestic fermentor, and further test was performed in 15 liter B.Braun fermentor. With the optimization of these fermentation bioprocess, the productivity of anganmycin was improved from 0.38g/L to 0.8g/L, which only showed 53% of the productivity in the shaking flask.
(4) Kinetic study of anganmycin fermentation. According to the experimental data and the supposed model, the kinetic equations including the growth of mycelium, the consumption of substrate and the production of anganmycin were obtained with the use of Matlab software:. These equations are as
(5) The anganmycin fermentation process was scaled up in a pharmaceutic factory. Six runs of experiments was performed on 250L, 700L and 2500L fermentor respectively. The highest productivity of anganmycin (0.628g/L) was achieved , and some important problem was also discussed here.
引文
[1]．张兴，马志卿等，生物农药评述，西北农林科技大学学报(自然科学版)，2002，30(2)，142—148。
[2]．陈万义，浅议生物农药取代化学农药，农药市场信息2003．07。
[3]．郭景福，郭艳红等．生物农药的发展思路．陕西农业科学．2002(11)28-30。
[4]．国内外生物农药现状及发展趋势，河北化工，2003(2)，51－52。
[5]．刘长令，新农药研究开发文集，1999，8～11。
[6]．顾宝根，姜辉，我国生物农药的现状及问题，微生物农药及其产业化，2000。
[7]. Takashi Iwasa,Keishiro Suetomi et al Taxomomic study and fermentation of producing organism and antimicrobial activity of mildiomycin. The journal of antibiotics(1978)6:511-517。
[8]. Summary of toxicological studies on mildiomycin(technical information), Journal of pesticide science, 1994,19(3), 135-140。
[9]. Suzuki et al. Mildiomycin analogs and a method of production, United States Patent 4296107, Aμg. 19, 1980。
[10]. S.Harada,T Kishi Isolation and characterization of mildiomycin,a new nucleoside antibiotic, The journal of antibiotic, (1978)6:519-524
[11]. Tyler J. Avis, Richard R. Be¤langer, Mechanisms and means of detection of biocontrol activity of Pseudozyma yeasts against plant-pathogenic fungi, FEMS Yeast Research 2 (2002) 5～8。
[12]. Dale Walters a;Barbara Meurer-Grimes;Ivelisse Rovira, Antifungal activity of three spermidine conjμgates, FEMS Microbiology Letters 201 (2001) 255～258。
[13]. Mujeebur Rahman Khan. M. Wajid Khan, M. Jahangir Pasha, Effects of sulfur dioxide on the development of powdery mildew of cucumber, Environmental and Experimental Botany 40 (1998) 265～273。
[14]. Katsumitsu Kishimoto, Syun-ichi Akiyama Stimulatory effect of ferrous ion on
mildiomycin product by Streptoverticillium rimofaciens, Biotechnology Letters, (1997)7:699-702
[15]. K.Kishimoto, Y.S.Park et al.Effect of ferrous ion on amino acid metabolism in mildiomycin production by Streptoverticillium rimofaciens, The journal of antibiotics, (1997)3:206-211
[16]. K.Kishimoto, Y.S.Park et al Effect of ferrous ion on mildiomycin production by Streptoverticillium rimofaciens, The journal of antibiotics, (1996)8:770-773
[17]．施巧琴，吴松刚，工业微生物育种学，福建科学技术出版社
[18]. Hidekazu Sawada, Takashi Suzuki et al, Strain improvement of a mildiomycin producer, Streptoverticillium rimofaciens B-98891, App Microbiol Biotechnol (1986)25:132-136
[19]. Suzuki et al United States Patent (1982)4334022
[20]．孙彦，生物分离工程，化学工业出版社，1998。
[21]. Katsumitsu Kishimoto, Hidekazu Sawada, Large-Scale culture of the mildiomycin producer Streptoverticillium rimofaciens, Seibutsu-kogaku Kaishi, 1997, 75 (4), 245-253
[22]. K.Kishimoto, Y.S.Park et al Effect of phosphate ion on mildiomycin production by Streptoverticillium rimofaciens, The journal of antibiotics, (1996)8:775-779
[23]．陈秀齐，新型生物农药氨苷霉素的分离工艺及其结构鉴定，浙江大学硕士学位论文，2003．3
[24]．姚汝华，微生物工程工艺学，华南理工大学出版社(1996)
[25]. Weber CJ(1930), A modification of sakaguchi's reaction for the quantitative determination of arginine. J Biol Chem 86:217—222
[26]．吴梧桐，生物制药工艺学，中国医药科技出版社，1992
[27]．杜洪达，蒸氨废液中氨的测定——甲醛法的完善及应用，2001(4)
[28]. McCULLOUGH,H: A simple micro technique for the determination of blood ammonia and a note on the effect of exercise. Clinica Chemica Acta 1968, 19: 101-105
[29]．张惟杰，糖复合物生化研究技术，浙江大学出版社(1994)
[30]．刘刚，抗生素发酵动力学，浙江大学博士学位论文，1997
[31]．周德庆，微生物学教程，高等教育出版社，1991
[32]．张琴，利用啤酒厂废酵母生物合成谷光甘肽及其分离过程的研究，浙江大学硕士学位论文，2001．12
[33]. Katsumitsu Kishimoto, Yong Soo Park et al. Effect of phosphate ion on mildiomycin product by Streptoverticillium rimofaciens, The Journal of Antibiotics, 1996, 49 (8): 775-780.
[34]．陈俊英，马晓建等，SM，型气升式发酵罐中利福霉素发酵动力学研究，郑州大学学报(理学版)，2002，34(4)，73-77
[35]．宋文军，陈宁等，色氨酸产生菌分批发酵动力学模型，无锡轻工大学学报，2002，21(4)，340-34：3，356
[36]．陈剑锋，郭养浩等，抗生素JI-20A分批发酵过程动力学研究，福州大学学报(自然科学版)，2002，30(5)，641-644
[2]．陈万义，浅议生物农药取代化学农药，农药市场信息2003．07。
[3]．郭景福，郭艳红等．生物农药的发展思路．陕西农业科学．2002(11)28-30。
[4]．国内外生物农药现状及发展趋势，河北化工，2003(2)，51－52。
[5]．刘长令，新农药研究开发文集，1999，8～11。
[6]．顾宝根，姜辉，我国生物农药的现状及问题，微生物农药及其产业化，2000。
[7]. Takashi Iwasa,Keishiro Suetomi et al Taxomomic study and fermentation of producing organism and antimicrobial activity of mildiomycin. The journal of antibiotics(1978)6:511-517。
[8]. Summary of toxicological studies on mildiomycin(technical information), Journal of pesticide science, 1994,19(3), 135-140。
[9]. Suzuki et al. Mildiomycin analogs and a method of production, United States Patent 4296107, Aμg. 19, 1980。
[10]. S.Harada,T Kishi Isolation and characterization of mildiomycin,a new nucleoside antibiotic, The journal of antibiotic, (1978)6:519-524
[11]. Tyler J. Avis, Richard R. Be¤langer, Mechanisms and means of detection of biocontrol activity of Pseudozyma yeasts against plant-pathogenic fungi, FEMS Yeast Research 2 (2002) 5～8。
[12]. Dale Walters a;Barbara Meurer-Grimes;Ivelisse Rovira, Antifungal activity of three spermidine conjμgates, FEMS Microbiology Letters 201 (2001) 255～258。
[13]. Mujeebur Rahman Khan. M. Wajid Khan, M. Jahangir Pasha, Effects of sulfur dioxide on the development of powdery mildew of cucumber, Environmental and Experimental Botany 40 (1998) 265～273。
[14]. Katsumitsu Kishimoto, Syun-ichi Akiyama Stimulatory effect of ferrous ion on
mildiomycin product by Streptoverticillium rimofaciens, Biotechnology Letters, (1997)7:699-702
[15]. K.Kishimoto, Y.S.Park et al.Effect of ferrous ion on amino acid metabolism in mildiomycin production by Streptoverticillium rimofaciens, The journal of antibiotics, (1997)3:206-211
[16]. K.Kishimoto, Y.S.Park et al Effect of ferrous ion on mildiomycin production by Streptoverticillium rimofaciens, The journal of antibiotics, (1996)8:770-773
[17]．施巧琴，吴松刚，工业微生物育种学，福建科学技术出版社
[18]. Hidekazu Sawada, Takashi Suzuki et al, Strain improvement of a mildiomycin producer, Streptoverticillium rimofaciens B-98891, App Microbiol Biotechnol (1986)25:132-136
[19]. Suzuki et al United States Patent (1982)4334022
[20]．孙彦，生物分离工程，化学工业出版社，1998。
[21]. Katsumitsu Kishimoto, Hidekazu Sawada, Large-Scale culture of the mildiomycin producer Streptoverticillium rimofaciens, Seibutsu-kogaku Kaishi, 1997, 75 (4), 245-253
[22]. K.Kishimoto, Y.S.Park et al Effect of phosphate ion on mildiomycin production by Streptoverticillium rimofaciens, The journal of antibiotics, (1996)8:775-779
[23]．陈秀齐，新型生物农药氨苷霉素的分离工艺及其结构鉴定，浙江大学硕士学位论文，2003．3
[24]．姚汝华，微生物工程工艺学，华南理工大学出版社(1996)
[25]. Weber CJ(1930), A modification of sakaguchi's reaction for the quantitative determination of arginine. J Biol Chem 86:217—222
[26]．吴梧桐，生物制药工艺学，中国医药科技出版社，1992
[27]．杜洪达，蒸氨废液中氨的测定——甲醛法的完善及应用，2001(4)
[28]. McCULLOUGH,H: A simple micro technique for the determination of blood ammonia and a note on the effect of exercise. Clinica Chemica Acta 1968, 19: 101-105
[29]．张惟杰，糖复合物生化研究技术，浙江大学出版社(1994)
[30]．刘刚，抗生素发酵动力学，浙江大学博士学位论文，1997
[31]．周德庆，微生物学教程，高等教育出版社，1991
[32]．张琴，利用啤酒厂废酵母生物合成谷光甘肽及其分离过程的研究，浙江大学硕士学位论文，2001．12
[33]. Katsumitsu Kishimoto, Yong Soo Park et al. Effect of phosphate ion on mildiomycin product by Streptoverticillium rimofaciens, The Journal of Antibiotics, 1996, 49 (8): 775-780.
[34]．陈俊英，马晓建等，SM，型气升式发酵罐中利福霉素发酵动力学研究，郑州大学学报(理学版)，2002，34(4)，73-77
[35]．宋文军，陈宁等，色氨酸产生菌分批发酵动力学模型，无锡轻工大学学报，2002，21(4)，340-34：3，356
[36]．陈剑锋，郭养浩等，抗生素JI-20A分批发酵过程动力学研究，福州大学学报(自然科学版)，2002，30(5)，641-644