在催化剂过氧磷钨酸十二烷基吡啶盐(Cat-PW4)的作用下,α-蒎烯与H2O2反应生成主要产物(3R,4R)-4,7,7-三甲基-6-氧杂二环[3.2.1]辛烷-3,4-二醇。不同反应条件对反应转化率和选择性的实验结果表明,最佳反应条件为:12.8 mmolα-蒎烯、5 m ...在催化剂过氧磷钨酸十二烷基吡啶盐(Cat-PW4)的作用下,α-蒎烯与H2O2反应生成主要产物(3R,4R)-4,7,7-三甲基-6-氧杂二环[3.2.1]辛烷-3,4-二醇。不同反应条件对反应转化率和选择性的实验结果表明,最佳反应条件为:12.8 mmolα-蒎烯、5 m L溶剂三氯甲烷、0.2 g催化剂、3.3 m L 30%H2O2,反应温度40℃,反应时间3 h,α-蒎烯转化率和产物的选择性分别为94.7%和39.8%。反应结束后,该产物存在于水相和有机相中,通过萃取和重结晶分离提纯,得率11%,纯度达到98%;其分子结构通过红外光谱、紫外光谱、1H核磁共振谱、13C核磁共振谱、低分辨率质谱及高分辨率质谱确证。展开更多
Catechol 2,3-dioxygenase(C23O)is the key enzyme of aromatic substance degradation by Pseudomonas sp..In order to establish a simple assay of C23O activity during the whole-cell catalysis of Pseudomonas putida mt-2,C23...Catechol 2,3-dioxygenase(C23O)is the key enzyme of aromatic substance degradation by Pseudomonas sp..In order to establish a simple assay of C23O activity during the whole-cell catalysis of Pseudomonas putida mt-2,C23O was induced by utilizing sodium benzoate acid as the sole carbon source,and its activity was determined in whole cells by the amended protocol of pure enzyme assay.After suspending the cells with potassium phosphate buffer,the substrate was added and the accumulation of 2-hydroxymuconic semialdehyde was measured by a UV757CRT spectrophotometer at 375 nm.The activity of C23O was evaluated by the climbing slope of time course curve of the UV absorption.By this means,the Km for catechol and C23O in whole cells was 34.67 μmol·L-1,while Vmax was 0.29 μmol·min-1·(mg dry cell)-1,both of which differed from those for pure enzyme by 2—3 orders of magnitude.To eliminate the cell wall barrier for substrate permeation,a cationic surfactant,n-dodecyltrimethylammonium bromide,was used to pre-treat the cells.With 0.1 g·L-1 dodecyl trimethyl ammonium bromide(DTAB) treated for 30 min,the maximum C23O activity could be achieved,which was consistent with the result of treated cells by beads milling.In the present study,a feasible and simple method was put forward for the apparent enzyme activity assay intracells which could be conveniently applied to the whole-cell biocatalysis or to environmental bioremediation.展开更多
文摘在催化剂过氧磷钨酸十二烷基吡啶盐(Cat-PW4)的作用下,α-蒎烯与H2O2反应生成主要产物(3R,4R)-4,7,7-三甲基-6-氧杂二环[3.2.1]辛烷-3,4-二醇。不同反应条件对反应转化率和选择性的实验结果表明,最佳反应条件为:12.8 mmolα-蒎烯、5 m L溶剂三氯甲烷、0.2 g催化剂、3.3 m L 30%H2O2,反应温度40℃,反应时间3 h,α-蒎烯转化率和产物的选择性分别为94.7%和39.8%。反应结束后,该产物存在于水相和有机相中,通过萃取和重结晶分离提纯,得率11%,纯度达到98%;其分子结构通过红外光谱、紫外光谱、1H核磁共振谱、13C核磁共振谱、低分辨率质谱及高分辨率质谱确证。
文摘Catechol 2,3-dioxygenase(C23O)is the key enzyme of aromatic substance degradation by Pseudomonas sp..In order to establish a simple assay of C23O activity during the whole-cell catalysis of Pseudomonas putida mt-2,C23O was induced by utilizing sodium benzoate acid as the sole carbon source,and its activity was determined in whole cells by the amended protocol of pure enzyme assay.After suspending the cells with potassium phosphate buffer,the substrate was added and the accumulation of 2-hydroxymuconic semialdehyde was measured by a UV757CRT spectrophotometer at 375 nm.The activity of C23O was evaluated by the climbing slope of time course curve of the UV absorption.By this means,the Km for catechol and C23O in whole cells was 34.67 μmol·L-1,while Vmax was 0.29 μmol·min-1·(mg dry cell)-1,both of which differed from those for pure enzyme by 2—3 orders of magnitude.To eliminate the cell wall barrier for substrate permeation,a cationic surfactant,n-dodecyltrimethylammonium bromide,was used to pre-treat the cells.With 0.1 g·L-1 dodecyl trimethyl ammonium bromide(DTAB) treated for 30 min,the maximum C23O activity could be achieved,which was consistent with the result of treated cells by beads milling.In the present study,a feasible and simple method was put forward for the apparent enzyme activity assay intracells which could be conveniently applied to the whole-cell biocatalysis or to environmental bioremediation.
