【作者】 盛艳旻；

【导师】 朱勍； 王宇光；

【作者基本信息】 浙江工业大学 ， 发酵工程， 2010， 硕士

【摘要】 手性环氧化物及其水解产物邻二醇是合成许多生物活性物质和手性目标产物的高价值中间体。本实验以邻甲氧基苯基缩水甘油醚为唯一碳源进行多轮富集,从土壤中获得380多株能利用该环氧化物的微生物。之后用TLC法定性筛选出25株具有环氧化物水解酶活性的菌株,之后再通过HPLC法筛选出5株具有立体选择性的菌株,从中选取最好的一个菌株进行下一步研究并进行菌株鉴定,经过形态观察及ITS基因序列分析,确定该菌株为Botryosphaeria dothidea,编号为ZJUZQ007。以单因素方法,对发酵培养基的配方及发酵条件进行了优化,考察了不同的碳源、氮源、金属离子及浓度、发酵培养基初始pH值、菌体培养温度以及底物诱导对菌株的生长及酶活的影响。最终确立了菌株Botryosphaeria d.ZJUZQ007的最优发酵培养基配方:以2%乳糖为碳源,2%玉米粉为氮源,金属离子为2mmol/L的Fe2+,1%的NaCl,最适培养条件为30℃,最适pH在4.0-7.0之间,发酵时间为72h,该酶为非诱导型酶,无需加入底物做诱导剂。对Botryosphaeria d.环氧化物水解酶拆分邻甲氧基苯醚(EMPP)、邻甲基苯醚(o-GMPE)、间甲基苯醚(m-GMPE)、对甲基苯醚(p-GMPE)四种环氧化物的反应条件进行了优化。确定了最佳反应温度为30℃,最适缓冲液pH为7.6及最适反应时间为40min,在此反应条件下,(R)-EMPP的e.e.s和E值分别达到了99.9%和50.4,(R)-o-GMPE则为99.9%和48.6,而(R)-m-GMPE是91.2%和18.1,(S)-p-GMPE则是98.0%和29.5。同时发现,该菌株环氧化物水解酶对底物的选择性及活性受底物苯环上取代基的类型及取代基位置的影响。用分子模拟对接软件(Discovery Studio 2.1),通过模拟酶与底物的结合情况对这个实验结果成功地进行了解释。更多还原

【Abstract】 Optically pure epoxides and their corresponding vicinal diols are recognized as important building blocks for the synthesis of various bioactive and chiral products.More than 380 strains which could use 1,2-Epoxy-3-(2-methoxyphenyloxy)-propane(EMPP) as the sole carbon source were obtained from soil samples. Then 25 strains with epoxide hydrolase were screened by TLC and 5 with enantioselective activity by HPCL. A fungi with best enantioselectivity was further studied and identified as Botryosphaeria dothidea based on morphology and ITS sequence. It’s named ZJUZQ007.Based on single factor experiments, the fermentation conditions for the strain ZJUZQ007 were investigated, including carbon and nitrogen sources, metal ions and the concentrations, the initial pH of fermentation medium, temperature and inducement. The best fermentation conditions for ZJUZQ007 were: lactose 2.0%, corn flour 2.0%, NaCl 1.0%, FeSO4·7H2O 2mmol/L,temperature 30℃, pH 4.0-7.0, time 30h. The epoxide hydrolase is not inducible.Furthermore, the conditions for kinetic resolution of racemic EMPP and glycidyl (o, m, p)-methylphenyl ethers by ZJUZQ007 were studied, including reaction temperature, pH of potassium phosphate buffer and reaction time. It showed that the best resolution conditions as follows: racemic epoxides (12 mg) in DMSO (1:10 v/v) was hydrolyzed in 10ml KPB (100 mmol/L, pH 7.6) using wet mycelium of ZJUZQ007 (0.8 g) by shaking the mixture (220 rpm) at 30℃for 40 minutes.Under this optimized conversion conditions, the e.e.s and E value for (R)-EMPP was 99.9% and 50.4, 99.9% and 48.6 for (R)-o-GMPE, 91.2 and 18.1 for (R)-m-GMPE, 98.0% and 29.5 for (S)-p-GMPE.We found that by using Botryosphaeria dothidea ZJUZQ007, the sort of substituent group had an effect on the enantioselectivity that the e.e.s and E value for (R)-EMPP were higher than those for (R)-o-GMPE. While the position of the methyl group effected not only on the enantioselectivity but also the configuration of the remained epoxides: the (R)-enantiomer was obtained from rac-glycidyl (o or m)-methylphenyl ethers whereas the (S)-epoxides was obtained from glycidyl p-methylphenyl ether. The observations are explained by enzyme-substrate docking studies.更多还原