【作者】 张正方；

【导师】 朱勍；

【作者基本信息】 浙江工业大学 ， 生物化工， 2010， 硕士

【摘要】 在环氧化物水解酶产生菌的筛选过程中,本文主要以间甲基苯基缩水甘油醚为底物,以高效液相为分析方法,获得一株具有高活力、高对映立体选择性的环氧化物水解酶产生菌。经过鉴定为巨大芽孢杆菌命名为Bacillus megaterium ZJUZQ-002。对Bacillus megaterium ZJUZQ-002进行发酵条件优化。发现菌株生长过程中pH值和温度对菌体的产量与环氧化物水解酶活力有重要影响。当发酵pH值为7.2左右,温度为30℃时,菌体产量与酶活力均较好。底物诱导对该菌的酶活力也有重要的影响,当底物诱导量为6 mmol/L时,所得菌的酶活力最好,当底物诱导的浓度高于12 mmol/L时该菌基本不生长。该菌产生环氧化物水解酶的最佳发酵条件为:蔗糖1.0 %,酵母膏1.0 %,NaCl 1.0 %, MgSO4·7H2O 5 mmol/L,K2HPO4·3H2O 0.3 %,KH2PO4 0.1 %,底物6 mmol/L,温度:30℃,pH 7.2;250 mL三角瓶装30 mL,转速200 r/min,时间30 h。在该条件下酶活可达58.5 U/L。对Bacillus megaterium ZJUZQ-002进行拆分条件,包括温度、缓冲液pH、助溶剂类型、时间及催化剂与底物的质量比等进行研究,并确定其最佳的拆分条件。缓冲液为pH 8.0、浓度为0.1 M的磷酸钾(PKB),转化温度30℃,转化时间16 h,助溶剂为DMSO,冻干细胞/底物(g/g) = 9/1,底物浓度为20 mmol/L;在该条件下底物的e.e.值为99.9 %,其E值为37。对底物特异性的研究,发现菌体对邻位和间位有甲基取代基的苯基缩水甘油醚类底物有较好的对映立体选择性,并催化水解(R)-型的环氧化物,保留(S)-型;而对于对位有甲基取代基的苯基缩水甘油醚类底物立体选择性稍差,且主要催化水解(S)-型的环氧化物,保留(R)-型;通过Docking研究已解释了这一现象。在最适的转化条件下,对反应体系进行了扩大化转化的研究,最终确定当体系放大到5倍时,对100 mmol的间甲基苯基缩水甘油醚进行转化,16 h后底物的e.e.值接近100 %,转化率为68.6 %;分离纯化后进一步胺解可得光学纯的β-受体阻滞剂类药物:(S)-型托利洛尔,e.e.值98 %,得率16.5 %。更多还原

【Abstract】 Strain which could enantiomeric stereoselectively hydrolysis the racemic glycidyl m-methylphenyl ether has been isolated from the soil and was named Bacillus megaterium ZJUZQ-002. The analysis of chiral purities was based on the chiral HPLC results.Optimization the fermentation conditions of the Bacillus megaterium ZJUZQ-002 revealed that the pH value and temperature have major impacts on biomass and epoxide hydrolase activity. The best result were obatined under the following conditions: pH 7.2, temperature 30℃. Substrate induction also has an impact on the enzyme activity, and substrate 6 mmol/L results the highest enzyme activity, whereas the 12 mmol/L of substrate would lead to the death of bacteria. The best fermentation condition: sucrose 1.0 %,yeast extract 1.0 %,NaCl 1.0 %, MgSO4·7H2O 5mmol/L,K2HPO4·3H2O 0.3 %,KH2PO4 0.1 %,substrate 6 mmol/L,temperature 30℃,pH 7.2,time 30 h, could give the activity of enzyme 58.5 U/L.The best resolution condition: 0.1 M pH 8.0 potassium phosphate buffer, temperature 30℃, time 16 h, cosolvent DMSO, the ration of catalyst/substrate (g/g) = 9/1, substrate concentration 20 mmol/L, could give the e.e. value of substrate is 99.9 %, the E value 37.The study towards the substrate specificity of this enzyme show that, by using Bacillus megaterium ZJUZQ-002, the position of the methyl group exerted effect not only on the enantioselectivities increasing from 2′≈3′> 4′, but also the configuration of the remained epoxides: for glycidyl o or m-methylphenyl ethers, the remaining epoxide was (R)-enantiomer, while p-methylphenyl ether gave (S)-configuration, and then we explain it with docking study.In the optimal resolution conditions, the effect of different magnification factor of the raction system on resolution was studied. It was found that the best magnification factor is five, and then the e.e. value of m-GMPE was obatined in 99.9 %, the conversion rate in 68.6 % after 16 h. After further purification and amination,β-blocks—(S)- Toliprolol was obtained in e.e. value of 98 % and yield of 16.5 % yield.更多还原