【作者】 王春辉；

【导师】 郑裕国；

【作者基本信息】 浙江工业大学 ， 生物化工， 2004， 硕士

【摘要】 井冈霉素是我国最大的一类农用抗生素，对纹枯病有好的防治作用，目前在我国大量使用，其市场价格低廉。井冈胺是井冈霉素的组成部分，是重要的医药中间体，其销售价格是井冈霉素的几十倍。因此以井冈霉素为原料生产井冈胺具有巨大的经济效益和社会效益。 本实验拟从井冈霉素出发，利用微生物培养和酶催化的基本原理和技术，研究微生物菌株的生理生化特性，实现井冈霉素的有效微生物酶降解。通过菌种的诱变和培养工艺的优化，提高微生物菌株的分解酶活力。 首先对本实验室已筛选到的菌株经过紫外诱变和亚硝基胍诱变后，得到了一株在生理特性上有较大区别的菌株，诱变后的菌株能使井冈胺的产量提高到一倍以上。 对新筛选到的菌株进行了培养工艺条件的优化，得到发酵培养基的最适初始pH值为8.0，最佳发酵温度为30℃，最适底物浓度为1％。在培养基优化过程中，研究了碳源、氮源、磷源和无机离子对发酵的影响。碳源以井冈霉素为好，最佳浓度为1％，氮源选择了1％的（NH₄）₂SO₄；选择了有促进作用的CaCl₂、KCl、MgSO₄和ZnSO₄，对其浓度进行了单因素试验和正交试验，结果表明，在最佳培养基条浙江工业大学硕士学位论文件下发酵液中井冈胺的含量达到了2.43mg/ml，井冈胺得率有71 .5%，比在原培养基基础上所得的结果l.6omg/ml，产量提高了51.9%。 最后对菌种产生的酶进行了分离纯化和酶的性质研究。经过一系列的纯化酶的纯化倍数达到了巧.37，产率为9.6%。对酶的性质经过初步研究，结果表明:酶的最适pH值为8.0，pH值稳定范围为7一8，最适反应温度为40℃，温度稳定范围在40℃以下，CaZ+和K+对该酶有强烈的激活作用，而FeZ十和SDS对该酶有明显的抑制作用，以井冈霉素为底物该酶的Km一22.smmol几，vm一59.17ogml一‘h一’。更多还原

【Abstract】 Validamycins, effective fungicide against the sheath blight of rice plants, is the most widely used homemade farm-antibiotics. It has a low price while valienamine, an important intermediate of medicine, is much more expensive. So it is potential in economical and social benefits to produce valienamine from validamycins.In this dissertation, we were expected to depend on the principium and skill of microorganism culture and enzyme catalyse to research the character of microorganism strain, furthermore, we were also expected to improve valienamine yield via mutation strain and optimizing the whole fermentation process.At first, a highly valienamine-producing variation strain was screened from its original strain by treating with UV as well as NTG., which was fairly different in physiological properties from former. In producing valienamine from validamycins, the new one was double than the old.After the optimizing fermentation process for new strain, the results showed that the optimum temperature was 30℃ ,the optimum pH was 8.0 and the substrate concentration was 1%. Furthermore, the optimum carbon and nitrogen resource were investigated, including inorganic compound which were promoting for fermentation. Validamycins was the optimum corbon resource and the optimum nitrogen resource was sulfate ammonium. Calcium chloride, potassium chloride, bitter salt and zine sulfate could improve the yield of valienamine. The last results showed that under the condition of the best culture medium, the yield of valienmine reached 2.43mg/ml, which compared with the former almost improved 51.9%.At last, the enzyme produced by strain was purified and the character of enzymewas researched. After a series of purification, the multiple of purified enzyme was 15.37 and the production rate was 9.6%, respectively. The analysis of enzymatic character showed that the optimum temperature was 40℃ and the optimum pH was 8.0. The enzyme was relatively stabilization in pH 7-8, and it was thermo-stability below 40℃. Most metal ion such as Ca2+and K+ had forceful activation whereas Fe2+and SDS had distinct restrain. The Michaelis constant （Km） was 22.8mmol/l and Vm was 59.17μgml^-1h^-1.更多还原