层析法分离井冈霉素A和B
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摘要
井冈霉素是我国最大的一类农用抗生素，它对水稻纹枯病有很好的防治效果。我们对层析法分离井冈霉素各组分进行了研究，所用的树脂分别是阳离子交换树脂和阴离子交换树脂。
首先我们考察了从井冈霉素发酵液中提取井冈霉素的工艺，主要比较了几种树脂，其中以凝胶型的001×4树脂效果最好。我们进行了强酸性001×4树脂层析法分离井冈霉素A和B的研究，在最优条件下，井冈霉素A和B可以被分离出来。同时还进行了阴离子树脂层析法分离井冈霉素各组分的研究，所用的树脂是201×4。我们优化了工艺条件，并考察了洗脱流速、上样量、柱床高度和洗脱液pH对井冈霉素A、B保留行为的影响，优化后的10×400mm层析柱上的工艺为：上样量0.7克，柱床高度33.0cm，线速度19.1cm／h，以去离子水作为洗脱剂。在该工艺条件下，井冈霉素A、B、D和E可以被分离出来。然后对该工艺进行了放大，放大后收率为53.4％。对该分离方法建立了动力学模型，求出了两个参数K_(bds[R+])和K_(bds[ROH])，这两个参数均比较大，说明吸附力是比较强的。最后对井冈霉素B的酶解工艺进行了初步的研究。
Jinggangmycins, which is effective fungicide against the sheath blight of rice plants, is the most widely used homemade farm-antibiotics. Experiments about separation of validamycin A and validamycin B with anion-exchange resin and cation-exchange resin were carried out.
Cation-exchange resin 001X4 was used in our experiment. Optimum separation conditions were obtained when separation was carried out in 20 X 400 mm column: flow rate was 0.5mL/min, adder quantity was 0.7g and eluent was 0.1mol/L pyridine-acetic acid at pH value of 6.0.
As to anion-exchange resin A600, the influences of elute flow rate, adder quantities, bed height, eluate pH value on separation of validamycin A and validamycin B were investigated. It was found that the influence of bed height on retention volume was the strongest, compared with other factors. The retention volume could decrease with increase of elute flow rate and adder quantity or decrease of bed height. Validamycin A and
validamycin B could be separated effectively in the 10X400 mm column under the optimized conditions: elution flow rate of 19.1 cm/h, adder quantity of 0.7 g, bed height of 33.0 cm and used de-ion water as eluent. Except for Validamycin A and B, other minor components, Validamycin D and E were also separated from crude validamycin powder. The separation was also scaled up. Then a kinetic model was established to describe this procedure, two adsorption coefficients, KbdS[R+] and Kbds[ROH], were calculated. The value of these two coefficients was very high, so the main mechanism that contributed to separation was adsorption. At last, hydrolyzation validamycin B by enzymes were also carried out.
首先我们考察了从井冈霉素发酵液中提取井冈霉素的工艺，主要比较了几种树脂，其中以凝胶型的001×4树脂效果最好。我们进行了强酸性001×4树脂层析法分离井冈霉素A和B的研究，在最优条件下，井冈霉素A和B可以被分离出来。同时还进行了阴离子树脂层析法分离井冈霉素各组分的研究，所用的树脂是201×4。我们优化了工艺条件，并考察了洗脱流速、上样量、柱床高度和洗脱液pH对井冈霉素A、B保留行为的影响，优化后的10×400mm层析柱上的工艺为：上样量0.7克，柱床高度33.0cm，线速度19.1cm／h，以去离子水作为洗脱剂。在该工艺条件下，井冈霉素A、B、D和E可以被分离出来。然后对该工艺进行了放大，放大后收率为53.4％。对该分离方法建立了动力学模型，求出了两个参数K_(bds[R+])和K_(bds[ROH])，这两个参数均比较大，说明吸附力是比较强的。最后对井冈霉素B的酶解工艺进行了初步的研究。
Jinggangmycins, which is effective fungicide against the sheath blight of rice plants, is the most widely used homemade farm-antibiotics. Experiments about separation of validamycin A and validamycin B with anion-exchange resin and cation-exchange resin were carried out.
Cation-exchange resin 001X4 was used in our experiment. Optimum separation conditions were obtained when separation was carried out in 20 X 400 mm column: flow rate was 0.5mL/min, adder quantity was 0.7g and eluent was 0.1mol/L pyridine-acetic acid at pH value of 6.0.
As to anion-exchange resin A600, the influences of elute flow rate, adder quantities, bed height, eluate pH value on separation of validamycin A and validamycin B were investigated. It was found that the influence of bed height on retention volume was the strongest, compared with other factors. The retention volume could decrease with increase of elute flow rate and adder quantity or decrease of bed height. Validamycin A and
validamycin B could be separated effectively in the 10X400 mm column under the optimized conditions: elution flow rate of 19.1 cm/h, adder quantity of 0.7 g, bed height of 33.0 cm and used de-ion water as eluent. Except for Validamycin A and B, other minor components, Validamycin D and E were also separated from crude validamycin powder. The separation was also scaled up. Then a kinetic model was established to describe this procedure, two adsorption coefficients, KbdS[R+] and Kbds[ROH], were calculated. The value of these two coefficients was very high, so the main mechanism that contributed to separation was adsorption. At last, hydrolyzation validamycin B by enzymes were also carried out.
引文
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[3] 殷向东．生物源杀虫剂研究应用进展及其在我国的发展思路，农药，1999，38(11)：45～46
[4] 陈在佴，吴继星，张志刚．我国苏云金杆菌液体深层发酵研究十年进展，中国生物防治，2002，18(1)：33～35
[5] 向红琼，冯志新，罗永俊．Pluerotusost reatus毒素产生的影响因素及粗毒素的杀线虫活性，中国植物保护学会农药分会，第二届全国植物农药暨第六届药剂毒理学术讨论会论文集，西安，陕西科学技术出版社，2001，468～469．
[6] 董锦艳，张克勤，赵智娴，等．杀线虫菌物毒素的研究进展(Ⅰ)，中国生物防治，2001，17(2)：92～95
[7] 王开运，姜兴印，仪美芹．梅岭霉素对3种叶螨和2种害虫的毒力评价，中国植物保护学会农药分会，第二届全国植物农药暨第六届药剂毒理学术讨论会论文集，西安，陕西科学技术出版社，2001，346～349
[8] 沈寅初，张一宾．生物农药，北京，化学工业出版社，2000，11-19
[9] Takashi Isawa, Yukiko Kameda, Mitsuku Asai. Studies on validaymcin, new antibiotics Ⅳ, J. Antibiot., 1971,24(2): 119～122
[10] Satoshi Horh, Yukihiko Kameda and Kunio Kawahara, Studies on validamycins, new antibiotics ⅤⅢ. Isolation and characterization of validamycins C, D, E, and F, J. Antibiot., 1972, 24: 48-53
[11] Kameda, Y., N. Asano, T. Yamaguichi, K. Matsui, S. Horh and H. Fukase, Validamycin G and Validoxylamine G, new members of the validamycins, J. Antibiot, 1986, 39: 1491-1494
[12] Asano N, Kameda Y, Matsui K, Horii S, Fukase H. Validamycin H, a new pseudo-tetrasaccharide antibiotic, J. Antibiot., 1990, 43(8): 1039-1041
[13] Kameda Y, Asano N, Yamaguchi T, Matsui K, Horii S, Fukase H. Validamycin G and validoxylamine G, new members of the validamycins, J. Antibiot., 1986, 39(10): 1491-1494.
[14] Naoki asano, Takuji yamaguchi, Yukihiko yameda. Effect of validamycins on glycohydrolases of rhizoctonia solani. J. Antibiot. 1987, (4): 526-532
[15] 上海市农药研究所农用抗菌素组编，井冈霉素，上海，上海人民出版社，1977，11-19，90-131，146-149
[16] 沈寅初，井冈霉素研究开发25年，植物保护，1996，22(4)：44-45
[17] 黄炳球，胡美英，黄瑞平，表面活性剂APS井冈霉素的增效作用研究，植物病理学报，1999，29(2)：169-173
[18] 彭绍裘，曾昭瑞，张志光，水稻纹枯病及其防治，上海，上海科技出版社，1986，2-18
[19] 徐辉，薄层—荧光法测定井冈霉素水剂，上海农学院学报，1998，16(2)：148-151
[20] Shigemoto R., Okuno T. and Matsura K, Effects of validamycin A on the growth of and trehalose content in mycelia of Rhizoctonia solani incubated in a medium containing several sugars as the sole carbohydrate, Ann. Phytopathil. Spc, Jpn, 1992, 58: 685-690
[21] 沈寅初，农用抗生素研究开发新进展，值保技术与推广，1997，17(6)：35-37
[22] 钱江生化所人员组编，钱江牌井冈霉素，农药，2001，40(1)：40
[23] 张穗，郭永霞，唐文华，牛灿芳．井冈霉素A对水稻纹枯病菌的毒力和作用机理研究，农药学学报，2001，3(4)：31-37
[24] Robson GD, Kuhn PJ, Trinci AP. Effects of validamycin A on the morphology, growth and sporulation of Rhizoctonia cerealis, Fusarium culmorum and other fungi. J. Gen. Microbiol., 1988, 134(12): 3187-3194
[25] 林延川，家鱼烂鳃病新疗法，内陆水产，1998，6：26
[26] Asano N, Yamaguchi T, Kameda Y, Matsui K. Effect of validamycins on glycohydrolase of Rhizoctonia salani, J. Antibiot., 1987, 40: 526-532
[27] Panek A. D., Adenosine triphosphate inhibition of yeast trehalas, Journal of bacteriology, 1969, 99(3): 904-905
[28] Deacon, J.W., In introduction of Modern Mycology, Blackwell Scientific Publication. Oxford, 1980, 91-105
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