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
与年龄相关的不确定潜能的克隆性造血(clonal hematopoiesis of indeterminate potential,CHIP)能通过激活炎症促进动脉粥样硬化的发生、发展,从而增加心脑血管疾病风险。作为最常见的CHIP相关突变的表观遗传调控基因之一,甲基胞嘧啶双...与年龄相关的不确定潜能的克隆性造血(clonal hematopoiesis of indeterminate potential,CHIP)能通过激活炎症促进动脉粥样硬化的发生、发展,从而增加心脑血管疾病风险。作为最常见的CHIP相关突变的表观遗传调控基因之一,甲基胞嘧啶双加氧酶2(ten-eleven translocation-2,TET2)基因突变可通过组蛋白去乙酰化作用增加巨噬细胞中IL-1β等促炎因子的转录,加速动脉粥样硬化斑块的形成,并与卒中风险增加及预后不良有关。未来通过靶向TET2基因及其相关机制通路可能有助于发现降低卒中风险或改善卒中预后的新途径。展开更多
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.
