生物法制备光学纯苄基缩水甘油醚的初步研究

发布时间：2018-03-29 01:15

  本文选题：环氧化物水解酶　切入点：Talaromyces　出处：《浙江工业大学》2011年硕士论文

【摘要】：本课题研究以苄基缩水甘油醚为底物,采用两轮筛选的模式,即先初筛后复筛,从各地采集的特殊土壤中筛选到31株具有环氧化物水解酶活性的菌株,其中有11株表现出对映选择性。从中选择对映选择性最高的一株霉菌作为研究对象,对其进行了形态学观察及ITS基因序列分析鉴定,确定其为黄蓝状菌(Talaromyces flavus),并命名为Talaromyces flavus ZJUTZQ159。 采用单因素实验方法,对菌株Talaromyces flavus ZJUTZQ159的发酵条件进行了优化,分别考察了碳源、氮源、金属离子、发酵温度、发酵时间、初始pH及底物诱导对生物量、酶活、e.e.s值及E值四个指标的影响。研究发现,当温度为30℃,初始pH为4.0时发酵66h,该菌的生物量及酶活都达到最佳状态,且发现该菌所含环氧化物水解酶不需要底物诱导,为组成型酶。同时,通过正交试验确定了碳源、氮源及金属离子的最佳添加量,最终确定了该菌株的最佳发酵培养基为:可溶性淀粉20g/L、黄豆粉15g/L、NaNO_3 1g/L、K_2HPO_4·3H_2O 1.0g/L、KCl 0.5g/L、MgSO_4·7H_2O 1.476g/L。在最佳条件下,该菌的酶活达到18.66U/gDCM。 对菌株Talaromyces flavus ZJUTZQ159水解动力学拆分苄基缩水甘油醚的反应条件进行了优化,最终得知,该菌株在pH8.0的磷酸钾缓冲液体系中,助溶剂为二甲基亚砜,底物浓度为15mmol/L,温度为25℃的条件下反应4h,可获得最高e.e.s值,为96.4%,E值为7.16。同时研究该菌对底物的特异性发现,对苯环上不同位置有取代基的底物,该菌的对映选择性会大大降低。
[Abstract]:Using benzyl glycidyl ether as substrate, 31 strains with epoxide hydrolase activity were screened from special soil by two rounds of screening model. Among them, 11 strains showed enantioselectivity, one of them had the highest enantioselectivity. Morphological observation and ITS gene sequence analysis showed that the fungus was Talaromyces flavusus and named as Talaromyces flavus ZJUTZQ159. The fermentation conditions of the strain Talaromyces flavus ZJUTZQ159 were optimized by single factor method. The biomass was determined by carbon source, nitrogen source, metal ion, fermentation temperature, fermentation time, initial pH and substrate induction. The results showed that the biomass and enzyme activity of the strain reached the optimal state when the temperature was 30 鈩，

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