本文選題：環(huán)氧化物水解酶　切入點：Talaromyces　出處：《浙江工業(yè)大學(xué)》2011年碩士論文

【摘要】：本課題研究以芐基縮水甘油醚為底物,采用兩輪篩選的模式,即先初篩后復(fù)篩,從各地采集的特殊土壤中篩選到31株具有環(huán)氧化物水解酶活性的菌株,其中有11株表現(xiàn)出對映選擇性。從中選擇對映選擇性最高的一株霉菌作為研究對象,對其進行了形態(tài)學(xué)觀察及ITS基因序列分析鑒定,確定其為黃藍(lán)狀菌(Talaromyces flavus),并命名為Talaromyces flavus ZJUTZQ159。 采用單因素實驗方法,對菌株Talaromyces flavus ZJUTZQ159的發(fā)酵條件進行了優(yōu)化,分別考察了碳源、氮源、金屬離子、發(fā)酵溫度、發(fā)酵時間、初始pH及底物誘導(dǎo)對生物量、酶活、e.e.s值及E值四個指標(biāo)的影響。研究發(fā)現(xiàn),當(dāng)溫度為30℃,初始pH為4.0時發(fā)酵66h,該菌的生物量及酶活都達到最佳狀態(tài),且發(fā)現(xiàn)該菌所含環(huán)氧化物水解酶不需要底物誘導(dǎo),為組成型酶。同時,通過正交試驗確定了碳源、氮源及金屬離子的最佳添加量,最終確定了該菌株的最佳發(fā)酵培養(yǎng)基為:可溶性淀粉20g/L、黃豆粉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。在最佳條件下,該菌的酶活達到18.66U/gDCM。 對菌株Talaromyces flavus ZJUTZQ159水解動力學(xué)拆分芐基縮水甘油醚的反應(yīng)條件進行了優(yōu)化,最終得知,該菌株在pH8.0的磷酸鉀緩沖液體系中,助溶劑為二甲基亞砜,底物濃度為15mmol/L,溫度為25℃的條件下反應(yīng)4h,可獲得最高e.e.s值,為96.4%,E值為7.16。同時研究該菌對底物的特異性發(fā)現(xiàn),對苯環(huán)上不同位置有取代基的底物,該菌的對映選擇性會大大降低。
[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，

本文編號：1678898